sphingosine-kinase and sphingosine-1-phosphate

PF-543 is a sphingosine kinase 1（SPHK1）inhibitor developed by Pfizer and is currently considered the most potent selective SPHK1 inhibitor. SPHK1 catalyses the production of sphingosine 1-phosphate (S1P) from sphingosine. It is the rate-limiting enzyme of S1P production, and there is substantial evidence to support a very important role for sphingosine kinase in health and disease. This review is the first to summarize the role and mechanisms of PF-543 as an SPHK1 inhibitor in anticancer, antifibrotic, and anti-inflammatory processes, providing new therapeutic leads and ideas for future research and clinical trials.

Topics: Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine kinases type 1 (SphK1) is a key enzyme in the phosphorylation of sphingosine to sphingosine 1-phosphate (S1P). Different abnormalities in SphK1 functions may correspond with poor prognosis in various cancers. Additionally, upregulated SphK1/S1P could promote cancer cell proliferation, angiogenesis, mobility, invasion, and metastasis. MicroRNAs as conserved small noncoding RNAs play major roles in cancer initiation, progression, metastasis, etc. Their posttranscriptionally mechanisms could affect the development of cancer growth or tumorigenesis suppression. The growing number of studies has described that various microRNAs can be regulated by SphK1, and its expression level can also be regulated by microRNAs. In this review, the relationship of SphK1 and microRNA functions and their interaction in human malignancies have been discussed. Based on them novel treatment strategies can be introduced.

Topics: Humans; Lysophospholipids; MicroRNAs; Neoplasm Proteins; Neoplasms; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); RNA, Neoplasm; Sphingosine

Sphingosine 1-phosphate (S1P) is an important lipid biomolecule that exerts pleiotropic cellular actions as it binds to and activates its five G-protein-coupled receptors, S1P

Topics: Animals; Brain Damage, Chronic; Brain Ischemia; Clinical Trials as Topic; Disease Models, Animal; Drug Evaluation, Preclinical; Fingolimod Hydrochloride; Humans; Infarction, Middle Cerebral Artery; Inflammation; Ischemic Stroke; Lysophospholipids; Neovascularization, Physiologic; Nerve Tissue Proteins; Neuroprotective Agents; Phosphotransferases (Alcohol Group Acceptor); Rats; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Sphingosine 1-phosphates (S1Ps) are bioactive lipids that mediate a diverse range of effects through the activation of cognate receptors, S1P

Topics: Aldehyde-Lyases; Alzheimer Disease; Animals; Cerebrovascular Disorders; Clinical Trials as Topic; Dementia, Vascular; Drug Delivery Systems; Drug Evaluation, Preclinical; Fingolimod Hydrochloride; Humans; Infarction, Middle Cerebral Artery; Inflammation; Ischemic Stroke; Lysophospholipids; Mice; Mice, Knockout; Neurodegenerative Diseases; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Spontaneous segregation of cholesterol and sphingolipids as a liquid-ordered phase leads to their clustering in selected membrane areas, the lipid rafts. These specialized membrane domains enriched in gangliosides, sphingomyelin, cholesterol and selected proteins involved in signal transduction, organize and determine the function of multiprotein complexes involved in several aspects of signal transduction, thus regulating cell homeostasis. Sphingosine 1-phosphate, an important biologically active mediator, is involved in several signal transduction processes regulating a plethora of cell functions and, not only several of its downstream effectors tend to localize in lipid rafts, some of the enzymes involved in its pathway, of receptors involved in its signalling and its transporters have been often found in these membrane microdomains. Considering this, in this review we address what is currently known regarding the relationship between sphingosine 1-phosphate metabolism and signalling and plasma membrane lipid rafts.

Topics: Aldehyde-Lyases; Caveolin 1; Humans; Lysophospholipids; Membrane Microdomains; Membrane Proteins; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Receptor Cross-Talk; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Ovarian cancer is the most lethal gynaecological malignancy. It is commonly diagnosed at advanced stage when it has metastasised to the abdominal cavity and treatment becomes very challenging. While current standard therapy involving debulking surgery and platinum + taxane-based chemotherapy is associated with high response rates initially, the large majority of patients relapse and ultimately succumb to chemotherapy-resistant disease. In order to improve survival novel strategies for early detection and therapeutics against treatment-refractory disease are urgently needed. A promising new target against ovarian cancer is the sphingolipid pathway which is commonly hijacked in cancer to support cell proliferation and survival and has been shown to promote chemoresistance and metastasis in a wide range of malignant neoplasms. In particular, the sphingosine kinase 1-sphingosine 1-phosphate receptor 1 axis has been shown to be altered in ovarian cancer in multiple ways and therefore represents an attractive therapeutic target. Here we review the roles of sphingolipids in ovarian cancer progression, metastasis and chemoresistance, highlighting novel strategies to target this pathway that represent potential avenues to improve patient survival.

Topics: Animals; Female; Humans; Lysophospholipids; Neoplasm Proteins; Ovarian Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

For decades, lipids were confined to the field of structural biology and energetics as they were considered only structural constituents of cellular membranes and efficient sources of energy production. However, with advances in our understanding in lipidomics and improvements in the technological approaches, astounding discoveries have been made in exploring the role of lipids as signaling molecules, termed bioactive lipids. Among these bioactive lipids, sphingolipids have emerged as distinctive mediators of various cellular processes, ranging from cell growth and proliferation to cellular apoptosis, executing immune responses to regulating inflammation. Recent studies have made it clear that sphingolipids, their metabolic intermediates (ceramide, sphingosine-1-phosphate, and N-acetyl sphingosine), and enzyme systems (cyclooxygenases, sphingosine kinases, and sphingomyelinase) harbor diverse yet interconnected signaling pathways in the central nervous system (CNS), orchestrate CNS physiological processes, and participate in a plethora of neuroinflammatory and neurodegenerative disorders. Considering the unequivocal importance of sphingolipids in CNS, we review the recent discoveries detailing the major enzymes involved in sphingolipid metabolism (particularly sphingosine kinase 1), novel metabolic intermediates (N-acetyl sphingosine), and their complex interactions in CNS physiology, disruption of their functionality in neurodegenerative disorders, and therapeutic strategies targeting sphingolipids for improved drug approaches.

Topics: Alzheimer Disease; Central Nervous System; Ceramides; Eicosanoids; Forecasting; Homeostasis; Humans; Inflammation; Lipoxygenase; Lysophospholipids; Membrane Lipids; Models, Biological; Nerve Degeneration; Neurodegenerative Diseases; Neuroglia; Neurons; Parkinson Disease; Phosphotransferases (Alcohol Group Acceptor); Prostaglandin-Endoperoxide Synthases; Sphingolipids; Sphingosine

Sphingolipids are ubiquitous building blocks of eukaryotic cell membranes that function as signaling molecules for regulating a diverse range of cellular processes, including cell proliferation, growth, survival, immune-cell trafficking, vascular and epithelial integrity, and inflammation. Recently, several studies have highlighted the pivotal role of sphingolipids in neuroinflammatory regulation. Sphingolipids have multiple functions, including induction of the expression of various inflammatory mediators and regulation of neuroinflammation by directly effecting the cells of the central nervous system. Accumulating evidence points to sphingolipid engagement in neuroinflammatory disorders, including Alzheimer's and Parkinson's diseases. Abnormal sphingolipid alterations, which involves an increase in ceramide and a decrease in sphingosine kinase, are observed during neuroinflammatory disease. These trends are observed early during disease development, and thus highlight the potential of sphingolipids as a new therapeutic and diagnostic target for neuroinflammatory diseases. [BMB Reports 2020; 53(1): 28-34].

Topics: Alzheimer Disease; Central Nervous System; Ceramides; Humans; Inflammation; Lysophospholipids; Microglia; Parkinson Disease; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingolipids; Sphingosine

Sphingolipids are a class of complex lipids containing a backbone of sphingoid bases, namely the organic aliphatic amino alcohol sphingosine (Sph), that are essential constituents of eukaryotic cells. They were first described as major components of cell membrane architecture, but it is now well established that some sphingolipids are bioactive and can regulate key biological functions. These include cell growth and survival, cell differentiation, angiogenesis, autophagy, cell migration, or organogenesis. Furthermore, some bioactive sphingolipids are implicated in pathological processes including inflammation-associated illnesses such as atherosclerosis, rheumatoid arthritis, inflammatory bowel disease (namely Crohn's disease and ulcerative colitis), type II diabetes, obesity, and cancer. A major sphingolipid metabolite is ceramide, which is the core of sphingolipid metabolism and can act as second messenger, especially when it is produced at the plasma membrane of cells. Ceramides promote cell cycle arrest and apoptosis. However, ceramide 1-phosphate (C1P), the product of ceramide kinase (CerK), and Sph 1-phosphate (S1P), which is generated by the action of Sph kinases (SphK), stimulate cell proliferation and inhibit apoptosis. Recently, C1P has been implicated in the spontaneous migration of cells from some types of cancer, and can enhance cell migration/invasion of malignant cells through interaction with a Gi protein-coupled receptor. In addition, CerK and SphK are implicated in inflammatory responses, some of which are associated with cancer progression and metastasis. Hence, targeting these sphingolipid kinases to inhibit C1P or S1P production, or blockade of their receptors might contribute to the development of novel therapeutic strategies to reduce metabolic alterations and disease.

Topics: Animals; Cell Movement; Ceramides; Humans; Inflammation; Lysophospholipids; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingolipids; Sphingosine

Topics: Animals; Ceramides; Drug Development; Humans; Lysophospholipids; Molecular Targeted Therapy; Peripheral Nervous System Diseases; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysophosphatidic Acid; Sphingosine; Sphingosine 1 Phosphate Receptor Modulators

Topics: Aldehyde-Lyases; Antineoplastic Agents; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Lysophospholipids; Molecular Targeted Therapy; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Precision Medicine; Prognosis; Sphingosine; Survival Analysis

The world is currently experiencing the worst health pandemic since the Spanish flu in 1918-the COVID-19 pandemic-caused by the coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic is the world's third wake-up call this century. In 2003 and 2012, the world experienced two major coronavirus outbreaks, SARS-CoV-1 and Middle East Respiratory syndrome coronavirus (MERS-CoV), causing major respiratory tract infections. At present, there is neither a vaccine nor a cure for COVID-19. The severe COVID-19 symptoms of hyperinflammation, catastrophic damage to the vascular endothelium, thrombotic complications, septic shock, brain damage, acute disseminated encephalomyelitis (ADEM), and acute neurological and psychiatric complications are unprecedented. Many COVID-19 deaths result from the aftermath of hyperinflammatory complications, also referred to as the "cytokine storm syndrome", endotheliitus and blood clotting, all with the potential to cause multiorgan dysfunction. The sphingolipid rheostat plays integral roles in viral replication, activation/modulation of the immune response, and importantly in maintaining vasculature integrity, with sphingosine 1 phosphate (S1P) and its cognate receptors (SIPRs: G-protein-coupled receptors) being key factors in vascular protection against endotheliitus. Hence, modulation of sphingosine kinase (SphK), S1P, and the S1P receptor pathway may provide significant beneficial effects towards counteracting the life-threatening, acute, and chronic complications associated with SARS-CoV-2 infection. This review provides a comprehensive overview of SARS-CoV-2 infection and disease, prospective vaccines, and current treatments. We then discuss the evidence supporting the targeting of SphK/S1P and S1P receptors in the repertoire of COVID-19 therapies to control viral replication and alleviate the known and emerging acute and chronic symptoms of COVID-19. Three clinical trials using FDA-approved sphingolipid-based drugs being repurposed and evaluated to help in alleviating COVID-19 symptoms are discussed.

Topics: Betacoronavirus; Coronavirus Infections; COVID-19; Humans; Lysophospholipids; Pandemics; Phosphotransferases (Alcohol Group Acceptor); Pneumonia, Viral; SARS-CoV-2; Sphingolipids; Sphingosine; Sphingosine-1-Phosphate Receptors; Virus Replication

Sphingosine kinase 1 (SPHK1) is a crucial molecule that catalyzes sphingosine to synthesize sphingosine-1-phosphate (S1P), facilitating cell survival signaling. Pyroptosis is a perplexing inflammatory mode of cell death primarily triggered by caspase-1, evoked by the NLRP3 inflammasome. Sphingosine is identified as a danger-associated molecular pattern (DAMP), which activates the NLRP3 inflammasome assembly and induces the pyroptosis. It has been demonstrated that macrophages play a pro-tumorigenic role and are closely associated with tumor progression. Attenuation of SPHK1 activity contributes significantly to macrophage pyroptosis and tumor inhibition. Calcium and integrin-binding protein 1 (CIB1) plays an important role in the translocation of SPHK1 from the cytoplasm to the plasma membrane, whereas CIB2 blocks the subcellular trafficking of SPHK1. Therefore, knockout of CIB1 or over-expression of CIB2 will result in sphingosine accumulation and contribute significantly to cancer treatment by several approaches. First, it directly provokes cancer cell apoptosis or triggers robust anti-tumor immunity by pyroptosis-induced inflammation. Second, it could restrain SPHK1 translocation from the cytoplasm to the plasma membrane and further pyroptosis, which not only drive M2 macrophages death but also facilitate tumor microenvironment inflammation as well as the further release of sphingosine from damaged macrophages. The perspective might provide novel insight into the association between SPHK1 and pyroptosis and suggest the potential target for cancer therapy.

Topics: Alarmins; Animals; Antineoplastic Agents; Calcium-Binding Proteins; Humans; Lysophospholipids; Molecular Targeted Therapy; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Pyroptosis; Signal Transduction; Sphingosine; Tumor Microenvironment; Tumor-Associated Macrophages

Hepatocellular carcinoma (HCC) is primarily diagnosed in the latter stages of disease progression and is the third leading cause of cancer deaths worldwide. Thus, there is a need to find biomarkers of early HCC as well as the development of more effective treatments for the disease. Sphingosine-1-phosphate (S1P) is a pleiotropic lipid signaling molecule produced by two isoforms of sphingosine kinase (SphK1 and SphK2) that is involved in regulation of many aspects of mammalian physiology and pathophysiology, including inflammation, epithelial and endothelial barrier function, cancer, and metastasis, among many others. Abundant evidence indicates that SphK1 and S1P promote cancer progression and metastasis in multiple types of cancers. However, the role of SphK/S1P in HCC is less well studied. Here, we review the current state of knowledge of SphKs and S1P in HCC, including evidence for the correlation of SphK1 expression and S1P levels with progression of HCC and negative outcomes, and discuss how this information could lead to the design of more effective diagnostic and treatment modalities for HCC.

Topics: Animals; Carcinoma, Hepatocellular; Liver Neoplasms; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

The sphingosine 1-phosphate (S1P) metabolic pathway is a dynamic regulator of multiple cellular and disease processes. Identification of the immune regulatory role of the sphingosine analog FTY720 led to the development of the first oral therapy for the treatment of an autoimmune disease, multiple sclerosis. Furthermore, inhibitors of sphingosine kinase (SphK), which mediate S1P synthesis, are being evaluated as a therapeutic option for the treatment of cancer. In conjunction with these captivating discoveries, S1P and S1P-metabolizing enzymes have been revealed to display vital functions during virus infections. For example, S1P lyase, which is known for metabolizing S1P, inhibits influenza virus replication by promoting antiviral type I interferon innate immune responses. In addition, both isoforms of sphingosine kinase have been shown to regulate the replication or pathogenicity of many viruses. Pro- or antiviral activities of S1P-metabolizing enzymes appear to be dependent on diverse virus-host interactions and viral pathogenesis. This review places an emphasis on summarizing the functions of S1P-metabolizing enzymes during virus infections and discusses the opportunities for designing pioneering antiviral drugs by targeting these host enzymes.

Topics: Aldehyde-Lyases; Antiviral Agents; Host-Pathogen Interactions; Humans; Immunity, Innate; Interferon Type I; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Virus Diseases; Virus Replication; Viruses

The development and progression of colorectal cancer (CRC), a major cause of cancer-related death in the western world, is accompanied with alterations of sphingolipid (SL) composition in colon tumors. A number of enzymes involved in the SL metabolism have been found to be deregulated in human colon tumors, in experimental rodent studies, and in human colon cancer cells in vitro. Therefore, the enzymatic pathways that modulate SL levels have received a significant attention, due to their possible contribution to CRC development, or as potential therapeutic targets. Many of these enzymes are associated with an increased sphingosine-1-phosphate/ceramide ratio, which is in turn linked with increased colon cancer cell survival, proliferation and cancer progression. Nevertheless, more attention should also be paid to the more complex SLs, including specific glycosphingolipids, such as lactosylceramides, which can be also deregulated during CRC development. In this review, we focus on the potential roles of individual SLs/SL metabolism enzymes in colon cancer, as well as on the pros and cons of employing the current in vitro models of colon cancer cells for lipidomic studies investigating the SL metabolism in CRC.

Topics: Acid Ceramidase; Alkaline Ceramidase; Animals; Ceramides; Colonic Neoplasms; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Humans; Lactosylceramides; Lipid Metabolism; Lysophospholipids; Neutral Ceramidase; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Sphingolipids; Sphingosine; Sphingosine N-Acyltransferase; Tumor Cells, Cultured

Sphingolipid metabolites, such as ceramide, sphingosine and sphingosine-1-phosphate (S1P), play many important roles in cellular activities. Ceramide and sphingosine inhibit cell proliferation and induce cell apoptosis while S1P has the opposite effect. Maintaining a metabolic balance of sphingolipids is essential for growth and development of cells. Sphingosine kinase (SPHK) is an important regulator for keeping this balance. It controls the level of S1P and plays important roles in proliferation, migration, and invasion of cancer cells and tumor angiogenesis. There are two isoenzymes of sphingosine kinase, SPHK1 and SPHK2. SPHK1 is ubiquitously expressed in most cancers where it promotes survival and proliferation, while SPHK2 is restricted to only certain tissues and its functions are not well characterized. SPHK1 is currently considered as a novel target for the treatment of cancers. Targeting SPHK1 would provide new strategies for cancer treatment and improve the prognosis of cancer patients. Here we review and summarize the current research findings on the SPHK1-S1P axis in cancer from many aspects including structure, expression, regulation, mechanism, and potential inhibitors.

Topics: Animals; Drug Resistance, Neoplasm; Humans; Lysophospholipids; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine to form the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P). S1P is an important lipid mediator with a wide range of biological functions; it is also involved in a variety of diseases such as inflammatory diseases, Alzheimer's disease and cancer.. This review reports the recent advancement in the research of SphKs inhibitors. Our purpose is also to provide a complete overview useful for underlining the features needed to select a specific pharmacological profile.. Two distinct mammalian SphK isoforms have been identified, SphK1 and SphK2. These isoforms are encoded by different genes and exhibit distinct subcellular localizations, biochemical properties and functions. SphK1 and SphK2 inhibition can be useful in different pathological conditions.. SphK1 and SphK2 have many common features but different and even opposite biological functions. For this reason, several research groups are interested in understanding the therapeutic usefulness of a selective or non-selective inhibitor of SphKs. Moreover, a compensatory mechanism for the two isoforms has been demonstrated, thus leading to the development of dual inhibitors.

Topics: Animals; Drug Design; Enzyme Inhibitors; Humans; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Isoforms; Sphingosine

Breast cancer affects 1 out of 8 women in the US and is the second highest cause of death from cancer for women, leading to considerable research examining the causes, progression, and treatment of breast cancer. Over the last two decades, sphingosine-1-phosphate (S1P), a potent sphingolipid metabolite, has been implicated in many processes important for breast cancer including growth, progression, transformation and metastasis, and is the focus of this review. In particular, one of the kinases that produces S1P, sphingosine kinase 1 (SphK1), has come under increasing scrutiny as it is commonly upregulated in breast cancer cells and has been linked with poorer prognosis and progression, possibly leading to resistance to certain anti-cancer therapies. In this review, we will also discuss preclinical studies of both estrogen receptor (ER) positive as well as triple-negative breast cancer mouse models with inhibitors of SphK1 and other compounds that target the S1P axis and have shown good promise in reducing tumor growth and metastasis. It is hoped that in the future this will lead to development of novel combination approaches for effective treatment of both conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer.

Topics: Breast Neoplasms; Female; Humans; Lysophospholipids; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); Receptors, Estrogen; Sphingosine; United States

Preterm birth (PTB), defined as birth before 37 completed weeks of gestation, occurs in up to 18 percent of births worldwide and accounts for the majority of perinatal morbidity and mortality. While the single most common cause of PTB has been identified as inflammation, safe and effective pharmacotherapy to prevent PTB has yet to be developed.. Our group has used an in vivo model of inflammation-driven PTB, biochemical methods, pharmacological approaches, a novel endothelin receptor antagonist that we synthesized and RNA knockdown to help establish the role of endothelin-1 (ET-1) in inflammation-associated PTB. Further, we have used our in vivo model to test whether sphingosine kinase, which acts downstream of ET-1, plays a role in PTB.. We have shown that levels of endothelin converting enzyme-1 (ECE-1) and ET-1 are increased when PTB is induced in timed pregnant mice with lipopolysaccharide (LPS) and that blocking ET-1 action, pharmacologically or using ECE-1 RNA silencing, rescues LPS-induced mice from PTB. ET-1 activates the sphingosine kinase/sphingosine-1-phosphate (SphK/S1P) pathway. S1P, in turn, is an important signaling molecule in the proinflammatory response. Interestingly, we have shown that SphK inhibition also prevents LPS-induced PTB in timed pregnant mice. Further, we showed that SphK inhibition suppresses the ECE-1/ET-1 axis, implicating positive feedback regulation of the SphK/S1P/ECE-1/ET-1 axis.. The ET-1/SphK/SIP pathway is a potential pharmacotherapeutic target for the prevention of PTB.

Topics: Animals; Endothelin-1; Female; Humans; Inflammation; Lipopolysaccharides; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Pregnancy; Premature Birth; Sphingosine

Sphingosine kinases (SphKs) catalyze the conversion of the sphingosine to the promitogenic/migratory product, sphingosine-1-phosphate (S1P). SphK/S1P pathway has been linked to the progression of cancer and various other diseases including allergic inflammatory disease, cardiovascular diseases, rejection after transplantation, the central nervous system, and virus infections. Therefore, SphKs represent potential new targets for developing novel therapeutics for these diseases. The history and development of SphK inhibitors are discussed, summarizing SphK inhibitors by their structures, and describing some applications of SphK inhibitors. We concluded: i) initial SphK inhibitors based on sphingosine have low specificity with several important off-targets. Identification the off-targets that would work synergistically with SphKs, and developing compounds that target the unique C4 domain of SphKs should be the focus of future studies. ii) The modifications of SphK inhibitors, which are devoted to increasing the selectivity to one of the two isoforms, now focus on the alkyl length, the spacer between the head and linker rings, and the insertion and the position of lipidic group in tail region. iii) SphK/S1P signaling pathway holds therapeutic values for many diseases. To find the exact function of each isoform of SphKs increasing the number of SphK inhibitor clinical trials is necessary.

Topics: Animals; Cardiovascular Diseases; Drug Discovery; Humans; Hypersensitivity; Lysophospholipids; Molecular Targeted Therapy; Neoplasms; Patents as Topic; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Signal Transduction; Sphingosine

Huntington's disease (HD) is a single-gene inheritable neurodegenerative disorder with an associated complex molecular pathogenic profile that renders it the most 'curable incurable' brain disorder. Continuous effort in the field has contributed to the recent discovery of novel potential pathogenic mechanisms. Findings in preclinical models of the disease as well as in human post-mortem brains from affected patients demonstrate that alteration of the sphingosine-1-phosphate (S1P) axis may represent a possible key player in the pathogenesis of the disease and may act as a potential actionable drug target for the development of more targeted and effective therapeutic approaches. The relevance of the path of this new 'therapeutic route' is underscored by the fact that some drugs targeting the S1P axis are currently in clinical trials for the treatment of other brain disorders.

Topics: Animals; Humans; Huntington Disease; Lysophospholipids; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Sphingolipid metabolites, including ceramide, sphingosine, and their phosphorylates (ceramide-1-phosphonate [C1P] and sphingosine-1-phosphate [S1P]), regulate diverse cellular processes including apoptosis, the cell cycle, and cellular differentiation. Recent studies have shown that these sphingolipid metabolites are generated in response to ototoxic agents and play important roles in determining the fate of cochlear hair cells in ototoxic injury.. This review summarizes the current knowledge on the roles of sphingolipid mediators in cochlear ototoxicity.. During ototoxicity, ceramide is mainly generated via sphingomyelinase in the cochlea through a ceramide/sphingomyelin cycle from sphingomyelin. The generated ceramide is converted to other sphingolipid mediators. Ceramide and sphingosine accelerate cochlear hair cell death induced by ototoxic agents, while, C1P and S1P, on the other hand, protect cochlear hair cells. Hair cell protection of S1P is mediated by S1P receptor subtype 2 (S1PR2).. Sphingolipid mediators play important roles in cochlear hair cell survival or death in ototoxic injury.

Topics: Animals; Apoptosis; Cell Cycle; Ceramides; Cochlea; Hair Cells, Auditory; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingolipids; Sphingosine

Within the last 3 decades, there has been intense study of bioactive sphingolipids and the enzymes which metabolize those lipids. One enzyme is the critical lipid kinase sphingosine kinase 1 (SK1), which produces the potent and pleiotropic signaling lipid, sphingosine 1-phosphate (S1P). SK1 and S1P have been implicated in a host of different diseases including cancer, chronic inflammation, and metabolic diseases. However, while there is ample knowledge about the importance of these molecules in the development and progression of disease there is a dearth of knowledge of the molecular mechanisms which regulate SK1 function. In this review, we will cover some of the more recent and exciting findings about the different ways SK1 function can be regulated, from transcriptional regulation to protein stability. Finally, we will delve into recent structural insights into SK1 and how they might relate to function at cell membranes.

Topics: Animals; Cell Membrane; Enzyme Activation; Enzyme Stability; Gene Expression Regulation; Humans; Lysophospholipids; Models, Molecular; Phosphotransferases (Alcohol Group Acceptor); Protein Biosynthesis; Signal Transduction; Sphingosine; Transcription, Genetic

Over 20 years ago, sphingosine-1-phosphate (S1P) was discovered to be a bioactive signaling molecule. Subsequent studies later identified two related kinases, sphingosine kinase 1 and 2, which are responsible for the phosphorylation of sphingosine to S1P. Many stimuli increase sphingosine kinase activity and S1P production and secretion. Outside the cell, S1P can bind to and activate five S1P-specific G protein-coupled receptors (S1PR1-5) to regulate many important cellular and physiological processes in an autocrine or paracrine manner. S1P is found in high concentrations in the blood where it functions to control vascular integrity and trafficking of lymphocytes. Obesity increases blood S1P levels in humans and mice. With the world wide increase in obesity linked to consumption of high-fat, high-sugar diets, S1P is emerging as an accomplice in liver pathobiology, including acute liver failure, metabolic syndrome, control of blood lipid and glucose homeostasis, nonalcoholic fatty liver disease, and liver fibrosis. Here, we review recent research on the importance of sphingosine kinases, S1P, and S1PRs in liver pathobiology, with a focus on exciting insights for new therapeutic modalities that target S1P signaling axes for a variety of liver diseases.

Topics: Animals; Fatty Liver; Humans; Liver; Liver Diseases; Liver Failure; Lysophospholipids; Metabolic Syndrome; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine-1-phosphate signaling is emerging as a critical regulator of cellular processes that is initiated by the intracellular production of bioactive lipid molecule, sphingosine-1-phosphate. Binding of sphingosine-1-phosphate to its extracellular receptors activates diverse downstream signaling that play a critical role in governing physiological processes. Increasing evidence suggests that this signaling pathway often gets impaired during pathophysiological and diseased conditions and hence manipulation of this signaling pathway may be beneficial in providing treatment. In this review, we summarized the recent findings of S1P signaling pathway and the versatile role of the participating candidates in context with several disease conditions. Finally, we discussed its possible role as a novel drug target in different diseases.

Topics: Arthritis, Rheumatoid; Ceramidases; Diabetes Mellitus; Humans; Lysophospholipids; Molecular Targeted Therapy; Multiple Sclerosis; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Sphingosine kinase (SphK) is a lipid enzyme that maintains cellular lipid homeostasis. Two SphK isozymes, SphK1 and SphK2, are expressed from different chromosomes and several variant isoforms are expressed from each of the isozymes, allowing for the multi-faceted biological diversity of SphK activity. Historically, SphK1 is mainly associated with oncogenicity, however in reality, both SphK1 and SphK2 isozymes possess oncogenic properties and are recognized therapeutic targets. The absence of mutations of SphK in various cancer types has led to the theory that cancer cells develop a dependency on SphK signaling (hyper-SphK signaling) or "non-oncogenic addiction". Here we discuss additional theories of SphK cellular mislocation and aberrant "dicing and splicing" as contributors to cancer cell biology and as key determinants of the success or failure of SphK/S1P (sphingosine 1 phosphate) based therapeutics.

Topics: Animals; Disease Models, Animal; Evolution, Molecular; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Lysophospholipids; Multigene Family; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Protein Transport; Receptors, Lysosphingolipid; RNA Splicing; Signal Transduction; Sphingosine

Sphingosine kinase 1 (Sphk1) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). Growing studies have demonstrated that Sphk1 is overexpressed in various types of solid cancers and can be induced by growth factors, cytokines, and carcinogens, leading to the increase of S1P production. Subsequently, the increased Sphk1/S1P facilitates cancer cell proliferation, mobility, angiogenesis, invasion, and metastasis. Therefore, Sphk1/S1P signaling plays oncogenic roles. This review summarizes the features of Sphk1/S1P signaling and their functions in colorectal cancer cell growth, tumorigenesis, and metastasis, as well as the possible underlying mechanisms.

Topics: Animals; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Inflammation is part of our body's response to tissue injury and pathogens. It helps to recruit various immune cells to the site of inflammation and activates the production of mediators to mobilize systemic protective processes. However, chronic inflammation can increase the risk of diseases like cancer. Apart from cytokines and chemokines, lipid mediators, particularly sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), contribute to inflammation and cancer. S1P is an important player in inflammation-associated colon cancer progression. On the other hand, C1P has been recognized to be involved in cancer cell growth, migration, survival, and inflammation. However, whether C1P is involved in inflammation-associated cancer is not yet established. In contrast, few studies have also suggested that S1P and C1P are involved in anti-inflammatory pathways regulated in certain cell types. Ceramide is the substrate for ceramide kinase (CERK) to yield C1P, and sphingosine is phosphorylated to S1P by sphingosine kinases (SphKs). Biological functions of sphingolipid metabolites have been studied extensively. Ceramide is associated with cell growth inhibition and enhancement of apoptosis while S1P and C1P are associated with enhancement of cell growth and survival. Altogether, S1P and C1P are important regulators of ceramide level and cell fate. This review focuses on S1P and C1P involvement in inflammation and cancer with emphasis on recent progress in the field.

Topics: Animals; Biomarkers, Tumor; Ceramides; Humans; Inflammation; Inflammation Mediators; Lysophospholipids; Models, Biological; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Cellular level of sphingosine-1-phosphate (S1P), the simplest bioactive sphingolipid, is tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and degradation mediated by S1P phosphatases, lipid phosphate phosphatases, and S1P lyase. The pleotropic actions of S1P are attributed to its unique inside-out (extracellular) signaling via G-protein-coupled S1P1-5 receptors, and intracellular receptor independent signaling. Additionally, S1P generated in the nucleus by nuclear SphK2 modulates HDAC1/2 activity, regulates histone acetylation, and transcription of pro-inflammatory genes. Here, we present data on the role of S1P lyase mediated S1P signaling in regulating LPS-induced inflammation in lung endothelium. Blocking S1P lyase expression or activity attenuated LPS-induced histone acetylation and secretion of pro-inflammatory cytokines. Degradation of S1P by S1P lyase generates Δ2-hexadecenal and ethanolamine phosphate and the long-chain fatty aldehyde produced in the cytoplasmic compartment of the endothelial cell seems to modulate histone acetylation pattern, which is different from the nuclear SphK2/S1P signaling and inhibition of HDAC1/2. These in vitro studies suggest that S1P derived long-chain fatty aldehyde may be an epigenetic regulator of pro-inflammatory genes in sepsis-induced lung inflammation. Trapping fatty aldehydes and other short chain aldehydes such as 4-hydroxynonenal derived from S1P degradation and lipid peroxidation, respectively by cell permeable agents such as phloretin or other aldehyde trapping agents may be useful in treating sepsis-induced lung inflammation via modulation of histone acetylation. .

Topics: Acetylation; Acute Lung Injury; Aldehyde-Lyases; Aldehydes; Animals; Cytokines; Epigenesis, Genetic; Histone Deacetylase 1; Histone Deacetylase 2; Histones; Humans; Lipopolysaccharides; Lysophospholipids; Membrane Proteins; Mice; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Sphingosine kinase (SphK) is an important signalling enzyme that catalyses the phosphorylation of sphingosine (Sph) to form sphingosine‑1‑phosphate (S1P). The multifunctional lipid, S1P binds to a family of five G protein-coupled receptors (GPCRs). As an intracellular second messenger, S1P activates key signalling cascades responsible for the maintenance of sphingolipid metabolism, and has been implicated in the progression of cancer, and the development of other inflammatory and metabolic diseases. SphK and S1P are critical molecules involved in the regulation of various cellular metabolic processes, such as cell proliferation, survival, apoptosis, adhesion and migration. There is strong evidence supporting the critical roles of SphK and S1P in the progression of diabetes mellitus, including insulin sensitivity and insulin secretion, pancreatic β‑cell apoptosis, and the development of diabetic inflammatory state. In this review, we summarise the current state of knowledge for SphK/S1P signalling effects, associated with the development of insulin resistance, pancreatic β‑cell death and the vascular complications of diabetes mellitus.

Topics: Animals; Diabetes Complications; Diabetes Mellitus; Enzyme Activation; Extracellular Space; Humans; Insulin Resistance; Insulin-Secreting Cells; Intracellular Space; Isoenzymes; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Transport; Signal Transduction; Sphingolipids; Sphingosine

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid produced by the metabolism of sphingomyelin, regulates cell proliferation, migration, survival and cell-cell contacts. The sphingosine-1- phosphate signaling pathway can regulate the trafficking of lymphocyte, angiogenesis, the progress of cancer and many other cellular functions. The formation of S1P is catalyzed by sphingosine kinases (SPHK), and degraded by lyases(S1PL), therefore S1P level is subject to a dynamic balance in the physiological environment. S1P can act as a second messenger or couple with S1P receptors (S1PR) to exert effects. The targets in the S1P signaling pathway have received considerable attention. Here we review the physiological function and drug development of S1P signaling pathway.

Topics: Cell Movement; Cell Proliferation; Drug Design; Humans; Lysophospholipids; Neoplasms; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Second Messenger Systems; Signal Transduction; Sphingomyelins; Sphingosine

Breast cancer remains the most common malignant disease in women. The estrogen receptor-α (ERα) and its ligand 17β-estradiol (E2) play important roles in breast cancer. E2 elicits cellular effects by binding to ERα in the cytosol followed by receptor dimerization and translocation to the nucleus where it regulates gene expression by binding to ERE response elements. However, it has become apparent that E2 also exerts rapid non-genomic effects through membrane-associated receptors. There is emerging evidence that this induces formation of the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P). S1P in turn has been implicated in many processes important in breast cancer progression. One of the enzymes that produce S1P, sphingosine kinase 1 (SphK1), is upregulated in breast cancer and its expression has been correlated with poor prognosis. This review is focused on the role of the SphK/S1P axis in estrogen signaling and breast cancer progression and will discuss new therapeutic approaches targeting this axis for breast cancer treatment.

Topics: Animals; Breast Neoplasms; Estrogens; Female; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Sphingosine kinases (isoforms SK1 and SK2) catalyse the formation of a bioactive lipid, sphingosine 1-phosphate (S1P). S1P is a well-established ligand of a family of five S1P-specific G protein coupled receptors but also has intracellular signalling roles. There is substantial evidence to support a role for sphingosine kinases and S1P in health and disease. This review summarises recent advances in the area in relation to receptor-mediated signalling by S1P and novel intracellular targets of this lipid. New evidence for a role of each sphingosine kinase isoform in cancer, the cardiovascular system, central nervous system, inflammation and diabetes is discussed. There is continued research to develop isoform selective SK inhibitors, summarised here. Analysis of the crystal structure of SK1 with the SK1-selective inhibitor, PF-543, is used to identify residues that could be exploited to improve selectivity in SK inhibitor development for future therapeutic application.

Topics: Animals; Cardiovascular Diseases; Diabetes Mellitus; Humans; Inflammation; Lysophospholipids; Models, Molecular; Neoplasms; Neurodegenerative Diseases; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Receptors, G-Protein-Coupled; Signal Transduction; Sphingosine; Structure-Activity Relationship

Sphingosine kinases (SK1 and SK2) catalyse the conversion of sphingosine into sphingosine 1-phosphate and control fundamental cellular processes, including cell survival, proliferation, differentiation, migration, and immune function. In this review, we highlight recent breakthroughs in the structural and functional characterisation of SK1 and these are contextualised by analysis of crystal structures for closely related prokaryotic lipid kinases. We identify a putative dimerisation interface and propose novel regulatory mechanisms governing structural plasticity induced by phosphorylation and interaction with phospholipids and proteins. Our analysis suggests that the catalytic function and regulation of the enzymes might be dependent on conformational mobility and it provides a roadmap for future interrogation of SK1 function and its role in physiology and disease.

Topics: Alternative Splicing; Amino Acid Motifs; Catalytic Domain; Humans; Isoenzymes; Lysophospholipids; Molecular Docking Simulation; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Structure, Tertiary; Sphingosine; Structure-Activity Relationship; Substrate Specificity

Sphingolipids, such as ceramide, sphingosine and sphingosine 1-phosphate (S1P) are bioactive molecules that have important functions in a variety of cellular processes, which include proliferation, survival, differentiation and cellular responses to stress. Sphingolipids have a major impact on the determination of cell fate by contributing to either cell survival or death. Although ceramide and sphingosine are usually considered to induce cell death, S1P promotes survival of cells. Sphingosine kinases (SPHKs) are the enzymes that catalyze the conversion of sphingosine to S1P. There are two isoforms, SPHK1 and SPHK2, which are encoded by different genes. SPHK1 has recently been implicated in contributing to cell transformation, tumor angiogenesis and metastatic spread, as well as cancer cell multidrug-resistance. More recent findings suggest that SPHK2 also has a role in cancer progression. This review is an overview of our understanding of the role of SPHKs and S1P in hematopoietic malignancies and provides information on the current status of SPHK inhibitors with respect to their therapeutic potential in the treatment of hematological cancers.

Topics: Disease Progression; Hematologic Neoplasms; Humans; Lysophospholipids; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Sphingosine

Topics: Adenosine Triphosphate; Animals; Drug Design; Humans; Ligands; Lung Diseases; Lysophospholipids; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Sphingolipids are a diverse set of structurally and metabolically related lipids that have numerous functions in cell structure and signaling. The regulation of these lipids is critical for normal cell function and disregulation has been implicated in pathophysiological conditions such as cancer and inflammation. Here we examine control of the initiating, and rate limiting, enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT). We find that de novo synthesis of sphingolipid is stimulated by a number of cancer chemotherapeutics, suggesting that this may be an important aspect of their cytotoxic effects. The three ORMDL proteins are membrane proteins of the endoplasmic reticulum related to the yeast Orm proteins, which have been shown to be homeostatic regulators of SPT. We find that the ORMDL proteins are also negative regulators of SPT that transmit cellular levels of sphingolipids to SPT. The three isoforms have redundant functions in this system. The sphingosine kinases (sphingosine kinase-1 and -2) phosphorylate both sphingosine, which is released from ceramide, but also dihydrosphingosine, which is in the de novo biosynthetic pathway. We therefore examined the role of the sphingosine kinases in controlling de novo ceramide biosynthesis and find that sphingosine kinase-1 does indeed act as a negative regulator of this pathway. This establishes that sphingosine kinase, in addition to producing sphingosine-1-phosphate as a signaling molecule, also consumes dihydrosphingosine to regulate ceramide synthesis. Our studies demonstrate that there are multiple mechanisms of regulation of SPT and suggest that these regulators are important mediators of cell stress responses.

Topics: Animals; Ceramides; Humans; Lysophospholipids; Membrane Proteins; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Stress, Physiological

The transfer of the gamma phosphate from ATP to sphingosine (Sph) to generate a small signaling molecule, sphingosine 1-phosphate (S1P), is catalyzed by sphingosine kinases (SphK), which exist as two isoforms, SphK1 and SphK2. SphK is a key regulator of S1P and the S1P:Sph/ceramide ratio. Increases in S1P levels have been linked to diseases including sickle cell disease, cancer, and fibrosis. Therefore, SphKs are potential targets for drug discovery. However, the current chemical biology toolkit needed to validate these enzymes as drug targets is inadequate. With this review, we survey in vivo active SphK inhibitors and highlight the need for developing more potent and selective inhibitors.

Topics: Adenosine Triphosphate; Animals; Biocatalysis; Ceramides; Drug Discovery; Enzyme Inhibitors; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Topics: Animals; Antineoplastic Agents; Cell Transformation, Neoplastic; Ceramides; Humans; Lysophospholipids; Molecular Targeted Therapy; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysophospholipid; Signal Transduction; Sphingosine

Sphingolipids represent an important class of bioactive signaling lipids which have key roles in numerous cellular processes. Over the last few decades, the levels of bioactive sphingolipids and/or their metabolizing enzymes have been realized to be important factors involved in disease development and progression, most notably in cancer. Targeting sphingolipid-metabolizing enzymes in disease states has been the focus of many studies and has resulted in a number of pharmacological inhibitors, with some making it into the clinic as therapeutics. In order to better understand the regulation of sphingolipid-metabolizing enzymes as well as to develop much more potent and specific inhibitors, the field of sphingolipids has recently taken a turn toward structural biology. The last decade has seen the structural determination of a number of sphingolipid enzymes and effector proteins. In these terms, one of the most complete arms of the sphingolipid pathway is the sphingosine-1-phosphate (S1P) arm. The structures of proteins involved in the function and regulation of S1P are being used to investigate further the regulation of said proteins as well as in the design and development of inhibitors as potential therapeutics.

Topics: Aldehyde-Lyases; Animals; Binding Sites; Biological Transport; Enzyme Inhibitors; Humans; Ligands; Lysophospholipids; Membrane Transport Modulators; Models, Molecular; Molecular Conformation; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Second Messenger Systems; Sphingosine; Sphingosine-1-Phosphate Receptors

The development of chemotherapeutic resistance is a major challenge in oncology. Elevated sphingosine kinase 1 (SK1) levels is predictive of a poor prognosis, and SK1 overexpression may confer resistance to chemotherapeutics. The SK/sphingosine-1-phosphate (S1P)/sphingosine-1-phosphate receptor (S1PR) signaling pathway has been implicated in the progression of various cancers and in chemotherapeutic drug resistance. Therefore, SK1 may represent an important target for cancer therapy. Targeting the SK/S1P/S1PR signaling pathway may be an effective anticancer therapeutic strategy, particularly in the context of overcoming drug resistance. This review summarizes our current understanding of the role of SK/S1P/S1PR signaling in cancer and development of SK1 inhibitors.

Topics: Animals; Antineoplastic Agents; Apoptosis; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Lysophospholipids; Molecular Targeted Therapy; Neoplasm Metastasis; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Chronic kidney diseases including glomerulonephritis are often accompanied by acute or chronic inflammation that leads to an increase in extracellular matrix (ECM) production and subsequent glomerulosclerosis. Glomerulonephritis is one of the leading causes for end-stage renal failure with high morbidity and mortality, and there are still only a limited number of drugs for treatment available. In this MiniReview, we discuss the possibility of targeting sphingolipids, specifically the sphingosine kinase 1 (SphK1) and sphingosine 1-phosphate (S1P) pathway, as new therapeutic strategy for the treatment of glomerulonephritis, as this pathway was demonstrated to be dysregulated under disease conditions. Sphingosine 1-phosphate is a multifunctional signalling molecule, which was shown to influence several hallmarks of glomerulonephritis including mesangial cell proliferation, renal inflammation and fibrosis. Most importantly, the site of action of S1P determines the final effect on disease progression. Concerning renal fibrosis, extracellular S1P acts pro-fibrotic via activation of cell surface S1P receptors, whereas intracellular S1P was shown to attenuate the fibrotic response. Interference with S1P signalling by treatment with FTY720, an S1P receptor modulator, resulted in beneficial effects in various animal models of chronic kidney diseases. Also, sonepcizumab, a monoclonal anti-S1P antibody that neutralizes extracellular S1P, and a S1P-degrading recombinant S1P lyase are promising new strategies for the treatment of glomerulonephritis. In summary, especially due to the bifunctionality of S1P, the SphK1/S1P pathway provides multiple target sites for the treatment of chronic kidney diseases.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Fibrosis; Fingolimod Hydrochloride; Glomerulonephritis; Humans; Inflammation; Kidney Failure, Chronic; Lysophospholipids; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

The enzymes that catalyze formation of the bioactive sphingolipid, sphingosine 1-phosphate, sphingosine kinase 1 and 2, are predictive markers in inflammatory diseases and cancer as evidenced by data from patients, knockout mice and the use of available molecular and chemical inhibitors. Thus, there is a compelling case for therapeutic targeting of sphingosine kinase. In addition, there are several examples of functional interaction between sphingosine 1-phosphate receptors and sphingosine kinase 1 that can drive malicious amplification loops that promote cancer cell growth. These novel aspects of sphingosine 1-phosphate pathobiology are reviewed herein.

Topics: Animals; Humans; Inflammation; Lysophospholipids; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine kinase (SphK) is an oncogenic lipid kinase that regulates the sphingolipid metabolic pathway that has been shown to play a role in numerous hyperproliferative/inflammatory diseases. The SphK isoforms (SphK1 and SphK2) catalyze the conversion of the proapoptotic substrate d-erythrosphingosine to the promitogenic/migratory product sphingosine 1-phosphate (S1P). Accumulation of S1P has been linked to the development/progression of cancer and various other diseases including, but not limited to, asthma, inflammatory bowel disease, rheumatoid arthritis, and diabetic nephropathy. SphK therefore represents a potential new target for developing novel therapeutics for cancer and other diseases. This finding has stimulated the development and evaluation of numerous SphK inhibitors over the past decade or so. In this review, we highlight the recent advancement in the field of SphK inhibitors including SphK1 and SphK2 specific inhibitors. Both sphingolipid based and nolipidic small molecule inhibitors and their importance in treatment of cancer and other diseases are discussed.

Topics: Clofibrate; Drug Combinations; Enzyme Inhibitors; Humans; Isoenzymes; Lysophospholipids; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Practolol; Sphingosine

Sphingosine 1-phosphate (S1P) is a multifunctional signaling lipid generated from sphingosine by sphingosine kinases. S1P formation has been shown in numerous cells in the circulation, including platelets, vascular endothelial and smooth muscle cells and monocytes. S1P also exerts multiple effects on these cells, i.e. cell proliferation and migration, activation of proinflammatory signaling pathways and release of additional inflammatory mediators. Similar activities and targets have also been identified for activated clotting factors such as thrombin or the activated factor-X (FXa), suggesting a possible involvement of S1P in thrombus-associated cellular signaling and thrombin-induced inflammatory reactions. Several levels of S1P-mediated, thrombin /FXa-induced signaling have already been identified: regulation of sphingosine kinase expression and activity, stimulation of S1P release from platelets and other cells and, possibly regulation of S1P-receptors on target cells. This review summarizes the current state of knowledge about S1P as a clotting factor-regulated molecular link between blood coagulation and inflammation. It is concluded that S1P might represent an until now underestimated lipid mediator of inflammatory reactions following activation of the clotting system and, in this context, also involved in the development and progression of atherosclerosis.

Topics: Blood Coagulation; Blood Platelets; Endothelium, Vascular; Factor Xa; Humans; Inflammation; Lysophospholipids; Muscle, Smooth, Vascular; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Thrombin

Diabetic nephropathy (DN) is the major cause of end-stage renal disease worldwide and its prevalence continues to increase. Currently, therapies for DN provide only partial renoprotection; hence new targets for therapeutic intervention need to be identified. In this review, we summarized the new target, sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) pathway, explored its potential therapeutic role in the prevention and treatment of DN.. Most relevant articles were mainly identified by searching PubMed in English.. Mainly original articles and critical review articles by major pioneer investigators in this field were selected to be reviewed.. SphK1/S1P pathway can be activated by hyperglycemia, advanced glycation end products, and many pro-inflammatory cytokines, which leads to fibronectin, transforming growth factor-β1 up-regulation and AP-1 activation. And then it could promote glomerular mesangial cells proliferation and extracellular matrix accumulation, mediating the initiation and progression of diabetic renal fibrosis.. SphK1/S1P pathway is closely correlated with the pathogenesis of DN. The results suggest that SphK1/S1P pathway as a new target for clinically improving DN in future is of great prospect.

Topics: Diabetic Nephropathies; Extracellular Matrix; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Acute lung injury (ALI) attributable to sepsis or mechanical ventilation and subacute lung injury because of ionizing radiation (RILI) share profound increases in vascular permeability as a key element and a common pathway driving increased morbidity and mortality. Unfortunately, despite advances in the understanding of lung pathophysiology, specific therapies do not yet exist for the treatment of ALI or RILI, or for the alleviation of unremitting pulmonary leakage, which serves as a defining feature of the illness. A critical need exists for new mechanistic insights that can lead to novel strategies, biomarkers, and therapies to reduce lung injury. Sphingosine 1-phosphate (S1P) is a naturally occurring bioactive sphingolipid that acts extracellularly via its G protein-coupled S1P1-5 as well as intracellularly on various targets. S1P-mediated cellular responses are regulated by the synthesis of S1P, catalyzed by sphingosine kinases 1 and 2, and by the degradation of S1P mediated by lipid phosphate phosphatases, S1P phosphatases, and S1P lyase. We and others have demonstrated that S1P is a potent angiogenic factor that enhances lung endothelial cell integrity and an inhibitor of vascular permeability and alveolar flooding in preclinical animal models of ALI. In addition to S1P, S1P analogues such as 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720), FTY720 phosphate, and FTY720 phosphonates offer therapeutic potential in murine models of lung injury. This translational review summarizes the roles of S1P, S1P analogues, S1P-metabolizing enzymes, and S1P receptors in the pathophysiology of lung injury, with particular emphasis on the development of potential novel biomarkers and S1P-based therapies for ALI and RILI.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents; Biomarkers; Capillary Permeability; Fingolimod Hydrochloride; Humans; Lung; Lysophospholipids; Membrane Proteins; Nerve Tissue Proteins; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Propylene Glycols; Receptors, Lysosphingolipid; Sepsis; Sphingosine; Transferases (Other Substituted Phosphate Groups); Translational Research, Biomedical

The sphingosine kinases (SphKs) have relatively recently been implicated in contributing to malignant cellular processes with particular interest in the oncogenic properties of SPHK1. Whilst SPHK1 has received considerable attention as a putative oncoprotein, SPHK2 has been much more difficult to study, with often conflicting data surrounding its role in cancer. Initial studies focused on non-haemopoietic malignancies, however a growing body of literature on the role of sphingolipid metabolism in haemopoietic malignancies is now emerging. This review provides an overview of the current state of knowledge of the SphKs and the bioactive lipid sphingosine 1-phosphate (S1P), the product of the reaction they catalyse. It then reviews the current literature regarding the roles of S1P and the SphKs in haemopoietic malignancies and discusses the compounds currently available that modulate sphingolipid metabolism and their potential and shortcomings as therapeutic agents for the treatment of haematological malignancies.

Topics: Antineoplastic Agents; Drug Design; Drug Resistance, Neoplasm; Enzyme Inhibitors; Hematologic Neoplasms; Hematopoiesis; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

In this chapter, roles of bioactive sphingolipids, specifically sphingosine kinase 1 (SK1) and 2 (SK2) and their product-sphingosine 1-phosphate (S1P)-will be reviewed with respect to regulation of cancer growth, metastasis, chemotherapeutics, and drug resistance. Sphingolipids are known to be key bioeffector molecules that regulate cancer proliferation, angiogenesis, and cell death. Sphingolipid molecules such as ceramide and S1P have been shown to control cancer cell death and proliferation, respectively. Roles of S1P have been described with respect to their intracellular and extracellular pro-survival and drug resistance functions mostly through S1P receptor (S1PR1-5) engagement. Identification of novel intracellular SK/S1P targets has broadened the existing complex regulatory roles of bioactive sphingolipids in cancer pathogenesis and therapeutics. Thus, deciphering the biochemical and molecular regulation of SK/S1P/S1PR signaling could permit development of novel therapeutic interventions to improve cancer therapy and/or overcome drug resistance.

Topics: Animals; Antineoplastic Agents; Drug Design; Drug Resistance, Neoplasm; Humans; Lysophospholipids; Molecular Targeted Therapy; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Signal Transduction; Sphingosine

In this chapter, we review the latest developments concerning the role of sphingosine 1-phosphate (S1P) in cancer. Particular focus is paid to the role of sphingosine kinases 1 and 2, S1P lyase and S1P-dependent signalling networks in both solid tumours and haematological cancer. The potential of this S1P-dependent pathophysiology as a therapeutic target for the treatment of cancer is also discussed.

Topics: Aldehyde-Lyases; Animals; Antineoplastic Agents; Humans; Leukemia, Myeloid; Lysophospholipids; Molecular Targeted Therapy; Multiple Myeloma; Neoplasms; Oncogenes; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Sphingolipids have emerged as pleiotropic signaling molecules with roles in numerous cellular and biological functions. Defining the regulatory mechanisms governing sphingolipid metabolism is crucial in order to develop a complete understanding of the biological functions of sphingolipid metabolites. The sphingosine kinase/ sphingosine 1-phosphate pathway was originally thought to function in the irreversible breakdown of sphingoid bases; however, in the last few decades it has materialized as an extremely important signaling pathway involved in a plethora of cellular events contributing to both normal and pathophysiological events. Recognition of the SK/S1P pathway as a second messaging system has aided in the identification of many mechanisms of its regulation; however, a cohesive, global understanding of the regulatory mechanisms controlling the SK/S1P pathway is lacking. In this chapter, the role of the SK/S1P pathway as a second messenger is discussed, and its role in mediating TNF-α- and EGF-induced biologies is examined. This work provides a comprehensive look into the roles and regulation of the sphingosine kinase/ sphingosine 1-phosphate pathway and highlights the potential of the pathway as a therapeutic target.

Topics: Animals; Cytokines; Drug Design; Humans; Intercellular Signaling Peptides and Proteins; Lysophospholipids; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Studies performed in the last fifteen years have clearly established that the bioactive sphingolipid sphingosine 1-phosphate (S1P) affects various different biological properties of myogenic precursor cells as well as physiological features of adult skeletal muscle. Noticeably, in myogenic precursor cells multiple growth factors and cytokines cross-communicate with S1P axis and the engagement of distinct S1P receptor subtypes appears to be crucially implicated in transmitting specific biological effects. This paper summarizes current research findings and discloses the potential for new therapeutics designed to alter S1P signaling with the aim of improving skeletal muscle repair.

Topics: Animals; Cell Differentiation; Cell Movement; Cell Proliferation; Cytokines; Hormones; Humans; Intercellular Signaling Peptides and Proteins; Lysophospholipids; Muscle Development; Muscle Fibers, Skeletal; Muscle, Skeletal; Myoblasts, Skeletal; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

The recent success of FTY720 (Fingolimod, Gilenya(®)), which has been approved for the treatment of relapsing-remitting multiple sclerosis and is the first-in-class sphingosine-1-phosphate (S1P) receptor modulating drug, has boosted the interest in further drug development in this area. Several selective S1P1 receptor-modulating drugs are being investigated in clinical trials for the treatment of diverse autoimmune disorders. Sphingosine kinase inhibitors are under development for the treatment of cancer, aberrant angiogenesis and inflammatory diseases; an inhibitor of SK2 with relatively low affinity is being analysed in patients with advanced solid tumours. While an indirect S1P lyase inhibitor has just failed the proof of concept in patients with rheumatoid arthritis, S1P lyase is still a promising target for the treatment of inflammatory and autoimmune diseases. Another approach is the development of S1P-scavenging or -clearing agents, including a monoclonal S1P antibody that has successfully passed phase I clinical trials and will be further developed for age-related macular degeneration.

Topics: Aldehyde-Lyases; Animals; Enzyme Inhibitors; Fingolimod Hydrochloride; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Because of its highly bioactive properties sphingosine 1-phosphate (S1P) is an attractive target for the treatment of several diseases. Since the expression of sphingosine kinases as well as S1P receptors was demonstrated in the kidney, questions about the physiological and pathophysiological functions of S1P in this organ have been raised. In this review, we summarize the current state of knowledge about S1P-mediated functions in the kidney. A special focus is put on S1P modulated signal transduction in renal glomerular and tubular cells and consequences for the development and treatment of several kidney diseases, diabetic nephropathy, glomerulonephritis, ischemia-reperfusion injury, as well as for Wilms tumor progression.

Topics: Animals; Disease Models, Animal; Humans; Kidney Diseases; Kidney Glomerulus; Kidney Tubules; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine

Sphingolipids as an important regulator play a critical role in the cell biological functions. Among them, ceramide (Cer) and sphingosine (Sph) induce apoptosis and inhibit cell proliferation; on the contrary sphingosine 1-phosphate (S1P) promotes cell survival and proliferation. The balance between ceramide/sphingosine and S1P forms a so-called "sphingolipid-rheostat", which decides the cell fate. Sphingosine kinases, which catalyze the phosphorylation of sphingosine to S1P, are critical regulators of this balance. Here, we review the role of sphingosine kinase 1 (SphK1) in regulating fundamental biological processes and tumorigenesis and the potential of SphK1 as a new target for cancer therapeutics.

Topics: Amino Alcohols; Animals; Apoptosis; Cell Movement; Cell Proliferation; Ceramides; Enzyme Activation; Enzyme Inhibitors; Humans; Lysophospholipids; Neoplasms; Neovascularization, Pathologic; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Thiazoles

Sphingosine kinase 1 (SphK1) is an enzyme that phosphorylates the lipid sphingosine to generate sphingosine-1-phosphate (S1P). S1P can act intracellularly as a signaling molecule and extracellularly as a receptor ligand. The SphK1/S1P axis has well-described roles in cell signaling, the cell death/survival decision, the production of a pro-inflammatory response, immunomodulation, and control of vascular integrity. Agents targeting the SphK1/S1P axis are being actively developed as therapeutics for cancer and immunological and inflammatory disorders. Control of cell death/survival and pro-inflammatory immune responses is central to the pathology of infectious disease, and we can capitalize on the knowledge provided by investigations of SphK1/S1P in cancer and immunology to assess its application to selected human infections. We have herein reviewed the growing literature relating viral infections to changes in SphK1 and S1P. SphK1 activity is reportedly increased following human cytomegalovirus and respiratory syncytial virus infections, and elevated SphK1 enhances influenza virus infection. In contrast, SphK1 activity is reduced in bovine viral diarrhea virus and dengue virus infections. Sphingosine analogs that modulate S1P receptors have proven useful in animal models in alleviating influenza virus infection but have shown no benefit in simian human immunodeficiency virus and lymphocytic choriomeningitis virus infections. We have rationalized a role for SphK1/S1P in dengue virus, chikungunya virus, and Ross River virus infections, on the basis of the biology and the pathology of these diseases. The increasing number of effective SphK1 and S1P modulating agents currently in development makes it timely to investigate these roles with the potential for developing modulators of SphK1 and S1P for novel anti-viral therapies.

Topics: Animals; Disease Models, Animal; Humans; Immunologic Factors; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Virus Diseases

Activation of sphingosine kinase/sphingosine 1-phosphate (SK/S1P)-mediated signaling has emerged as a critical cardioprotective pathway in response to acute ischemia/reperfusion injury. S1P is released in both ischemic pre- and post-conditioning. Application of exogenous S1P to cultured cardiac myocytes subjected to hypoxia or treatment of isolated hearts either before ischemia or at the onset of reperfusion exerts prosurvival effects. Synthetic congeners of S1P such as FTY720 mimic these responses. Gene targeted mice null for the SK1 isoform whose hearts are subjected to ischemia/reperfusion injury exhibit increased infarct size and respond poorly either to ischemic pre- or postconditioning. Measurements of cardiac SK activity and S1P parallel these observations. Experiments in SK2 knockout mice have revealed that this isoform is necessary for survival in the heart. High density lipoprotein (HDL) is a major carrier of S1P, and studies of hearts in which selected S1P receptors have been inhibited implicate the S1P cargo of HDL in cardioprotection. Inhibition of S1P lyase, an endogenous enzyme that degrades S1P, also leads to cardioprotection. These observations have considerable relevance for future therapeutic approaches to acute and chronic myocardial injury. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Topics: Aldehyde-Lyases; Animals; Cardiotonic Agents; Humans; Lipoproteins, HDL; Lysophospholipids; Myocardium; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine kinases (SKs) catalyse the conversion of sphingosine to sphingosine 1-phosphate (S1P), a signalling lipid that is involved in a plethora of cellular processes including proliferation, apoptosis, calcium homeostasis, angiogenesis, vascular and neuronal maturation, cell migration and immune responses. Over the last few years, it has become clear that SKs are subject to various forms of post-translational regulation which play important roles in the function of these enzymes. Moreover, dysregulation of SKs has been implicated in many pathological conditions, such as cancer. Here we review the various mechanisms of post-translational regulation of the SKs with the view that such knowledge may lead to the development of therapeutic strategies to modulate the activities of these enzymes in the treatment of cancer and a range of other conditions. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Topics: Animals; Gene Expression Regulation; Humans; Isoenzymes; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Processing, Post-Translational; Sphingolipids; Sphingosine

Sphingosine 1-phosphate (S1P) is an important bioactive sphingolipid metabolite that has been implicated in numerous physiological and cellular processes. Not only does S1P play a structural role in cells by defining the components of the plasma membrane, but in the last 20 years it has been implicated in various significant cell signaling pathways and physiological processes: for example, cell migration, survival and proliferation, cellular architecture, cell-cell contacts and adhesions, vascular development, atherosclerosis, acute pulmonary injury and respiratory distress, inflammation and immunity, and tumorogenesis and metastasis [1,2]. Given the wide variety of cellular and physiological processes in which S1P is involved, it is immediately obvious why the mechanisms governing S1P synthesis and degradation, and the manner in which these processes are regulated, are necessary to understand. In gaining more knowledge about regulation of the sphingosine kinase (SK)/S1P pathway, many potential therapeutic targets may be revealed. This review explores the roles of the SK/S1P pathway in disease, summarizes available SK enzyme inhibitors and examines their potential as therapeutic agents. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Topics: Animals; Disease; Humans; Lysophospholipids; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Signal Transduction; Sphingosine

Sphingosine kinase 1 (SK1) is a lipid enzyme with oncogenic properties that converts the proapoptotic lipids ceramide and sphingosine into the antiapoptotic lipid sphingosine-1-phosphate and activates the signal transduction pathways that lead to cell proliferation, migration, the activation of the inflammatory response, and the impairment of apoptosis. There is compelling evidence that SK1 activation contributes to cancer progression leading to increased oncogenic transformation, tumor growth, resistance to therapies, tumor neovascularization, and metastatic spread. High levels of SK1 expression or activity have been associated with a poor prognosis in several human cancers. Recent studies using cancer cell and mouse models demonstrate a significant potential for SK1-targeting therapies to synergize with the effects of chemotherapy and radiotherapy; however, until recently the absence of clinically applicable SK1 inhibitors has limited the translation of these findings into patients. With the recent discovery of SK1 inhibiting properties of a clinically approved drug FTY720 (Fingolimod), SK1 has gained significant attention from both clinicians and the pharmaceutical industry and it is hoped that trials of newly developed SK1 inhibitors may follow soon. This review provides an overview of the SK1 signaling, its relevance to cancer progression, and the potential clinical significance of targeting SK1 for improved local or systemic control of human cancers.

Topics: Animals; Antineoplastic Agents; Humans; Lysophospholipids; Mice; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Hypoxia, defined as a poor oxygenation, has been long recognized as a hallmark of solid tumors and a negative prognostic factor for response to therapeutics and survival of patients. Cancer cells have evolved biochemical mechanisms that allow them to react and adapt to hypoxia. At the cellular level, this adaptation is under the control of two related transcription factors, HIF-1 and HIF-2 (hypoxia-inducible factor), that respond rapidly to decreased oxygen levels to activate the expression of a broad range of genes promoting neoangiogenesis, glycolysis, metastasis, increased tumor growth, and resistance to treatments. Recent studies have identified the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) signaling pathway-which elicits various cellular processes including cell proliferation, cell survival, or angiogenesis-as a new regulator of HIF-1 or HIF-2 activity. In this review, we will focus on how the inhibition/neutralization of the SphK1/S1P signaling could be exploited for cancer therapy.

Topics: Animals; Antineoplastic Agents; Drug Resistance, Neoplasm; Humans; Hypoxia; Lysophospholipids; Neoplasms; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

The role of sphingolipids as bioactive signaling molecules that can regulate cell fate decisions puts them at center stage for cancer treatment and prevention. While ceramide and sphingosine have been established as antigrowth molecules, sphingosine-1-phosphate (S1P) offers a progrowth message to cells. The enzymes responsible for maintaining the balance between these "stop" or "go" signals are the sphingosine kinases (SK), SK1 and SK2. While the relative contribution of SK2 is still being elucidated and may involve an intranuclear role, a substantial amount of evidence suggests that regulation of sphingolipid levels by SK1 is an important component of carcinogenesis. Here, we review the literature regarding the role of SK1 as an oncogene that can function to enhance cancer cell viability and promote tumor growth and metastasis; highlighting the importance of developing specific SK1 inhibitors to supplement current cancer therapies.

Topics: Animals; Antineoplastic Agents; Enzyme Inhibitors; Humans; Lysophospholipids; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

In many cell types metabolites of sphingomyelin have a profound role in cellular signalling. One particular field where these derivatives have obtained a crucial role is calcium signalling. This is an interesting aspect on how lipids may wield their physiological role, as calcium is probably one of the most versatile signalling molecules in the cell, and modulation of calcium signalling may have profound effects on cellular physiology. In this review we discuss a novel aspect of sphingolipid signalling, i.e. the autocrine role of sphingosine 1-phosphate (S1P) in regulating calcium entry in thyroid cells. Although many investigations have highlighted the importance of S1P as a regulator of both calcium release from the endoplasmic reticulum and calcium entry through plasma membrane channels, the autocrine mechanism presented here introduces a new aspect of S1P signalling in thyroid cells. This mechanism may be physiologically relevant in many other cell types, including cancer cells.

Topics: Animals; Autocrine Communication; Calcium; Calcium Channels; Calcium Signaling; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingolipids; Sphingomyelins; Sphingosine; Thyroid Gland

The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) is now recognized as a critical regulator of many physiological and pathophysiological processes, including cancer, atherosclerosis, diabetes and osteoporosis. S1P is produced in cells by two sphingosine kinase isoenzymes, SphK1 and SphK2. Many cells secrete S1P, which can then act in an autocrine or paracrine manner. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. More recently, it was shown that S1P also has important intracellular targets involved in inflammation, cancer and Alzheimer's disease. This suggests that S1P actions are much more complex than previously thought, with important ramifications for development of therapeutics. This review highlights recent advances in our understanding of the mechanisms of action of S1P and its roles in disease.

Topics: Animals; Biological Transport; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Sphingosine-1-phosphate (S1P), which is generated from the sphingosine kinase-catalyzed phosphorylation of sphingosine, is now recognized as a critical regulator of many kinds of physiological and pathological processes, including cancer, cardiovascular function, and diabetes. It can also trigger a wide variety of biological effect, such as cell movement, differentiation, survival, inflammation, immunity, calcium homeostasis, and angiogenesis. As we know, a number of the biological effects of S1P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface or intracellular targets. However, the synthesis and the secretion of S1P are regulated by various endogenetic or ectogenous stimuli and involve many kinds of enzymes and transporters. In this review, we discuss the regulation of S1P synthesis by many kinds of enzymes and mainly introduce the process of ceramide to S1P. Moreover, S1P deterioration is important balance in physiologic adjustment. We also describe the role of verified or potential transporters in S1P release in detail.

Topics: Aldehyde-Lyases; Animals; Biological Transport; Ceramidases; Ceramides; Humans; Lysophospholipids; Membrane Proteins; Membrane Transport Proteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

It is well established that the lysophospholipid and signalling molecule sphingosine 1-phosphate (S1P) has many important functions in immune surveillance. S1P is produced from sphingosine by two distinct sphingosine kinases, SphK1 and SphK2, and acts as an intracellular messenger and as an extracellular ligand of five G protein-coupled cell surface receptors designated S1P(1)-S1P(5). S1P not only regulates peripheral lymphocyte circulation, but also influences their differentiation, activation, infiltration, and local positioning. The therapeutic value of modulating S1P metabolism and S1P receptor function is currently tested in clinical trials and holds great promise for treatment of different autoimmune diseases. Despite its obvious contribution to immune regulation, the analysis of S1P is still challenging. A major obstacle is the difficulty to analyze S1P locally in tissues and within cells due to its high metabolic turnover and the limited resolution of current analytical techniques like liquid chromatography and mass spectrometry. This review focuses on recent advancements to our understanding how different sources of S1P contribute to immune function, and how changes in production, secretion, and degradation of S1P can influence immune responses.

Topics: Animals; Capillary Permeability; Chemokines; Humans; Immune System; Isoenzymes; Lymph Nodes; Lymphocytes; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Spleen

There is an increasing body of evidence demonstrating a critical role for the bioactive lipid S1P (sphingosine 1-phosphate) in cancer. S1P is synthesized and metabolized by a number of enzymes, including sphingosine kinase, S1P lyase and S1P phosphatases. S1P binds to cell-surface G-protein-coupled receptors (S1P1-S1P5) to elicit cell responses and can also regulate, by direct binding, a number of intracellular targets such as HDAC (histone deacetylase) 1/2 to induce epigenetic regulation. S1P is involved in cancer progression including cell transformation/oncogenesis, cell survival/apoptosis, cell migration/metastasis and tumour microenvironment neovascularization. In the present paper, we describe our research findings regarding the correlation of sphingosine kinase 1 and S1P receptor expression in tumours with clinical outcome and we define some of the molecular mechanisms underlying the involvement of sphingosine kinase 1 and S1P receptors in the formation of a cancer cell migratory phenotype. The role of sphingosine kinase 1 in the acquisition of chemotherapeutic resistance and the interaction of S1P receptors with oncogenes such as HER2 is also reviewed. We also discuss novel aspects of the use of small-molecule inhibitors of sphingosine kinase 1 in terms of allosterism, ubiquitin-proteasomal degradation of sphingosine kinase 1 and anticancer activity. Finally, we describe how S1P receptor-modulating agents abrogate S1P receptor-receptor tyrosine kinase interactions, with potential to inhibit growth-factor-dependent cancer progression.

Topics: Animals; Antineoplastic Agents; Drug Resistance, Neoplasm; Humans; Lysophospholipids; Neoplasms; Oncogenes; Phosphotransferases (Alcohol Group Acceptor); Receptor Protein-Tyrosine Kinases; Receptors, Estrogen; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Topics: Adaptive Immunity; Animals; Endothelium, Vascular; Fingolimod Hydrochloride; Humans; Immunity, Innate; Immunosuppressive Agents; Lysophospholipids; Membrane Microdomains; Nervous System Physiological Phenomena; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Second Messenger Systems; Sphingolipids; Sphingosine

Sphingolipids are components of all eukaryotic cells that play important roles in a wide variety of biological processes. Ceramides and sphingosine-1-phosphate (S1P) are signaling molecules that regulate cell fate decisions in a wide array of species including yeast, plants, vertebrates, and invertebrates. Ceramides favor anti-proliferative and cell death pathways such as senescence and apoptosis, whereas S1P stimulates cell proliferation and survival pathways. The control of cell fate by these two interconvertible lipids has been called the sphingolipid rheostat or sphingolipid biostat. Sphingosine kinase, the enzyme that synthesizes S1P, is a crucial enzyme in regulation of the balance of these sphingolipids. Sphingosine kinase has been shown to play dynamic roles in the responses of cells to stress, leading to modulation of cell fate through a variety of signaling pathways impinging on the processes of cell proliferation, apoptosis, autophagy and senescence. This review summarizes the roles of sphingosine kinase signaling in these processes and the mechanisms mediating these responses. In addition, we discuss the evidence tying sphingosine kinase-mediated stress responses to the process of aging.

Topics: Animals; Apoptosis; Autophagy; Cellular Senescence; Ceramides; Humans; Lysophospholipids; Oxidative Stress; Phosphotransferases (Alcohol Group Acceptor); Sphingolipids; Sphingosine

FTY720 is a recently approved first line therapy for relapsing forms of multiple sclerosis. In this context, FTY720 is a pro-drug, with its anti-multiple sclerosis, immunosuppressive effects largely elicited following its phosphorylation by sphingosine kinase 2 and subsequent modulation of G protein-coupled sphingosine 1-phosphate (S1P) receptor 1 that induces lymphopenia by altering lymphocyte trafficking. A number of other biological effects of FTY720 have, however, been described, including considerable evidence that this drug also has anti-cancer properties. These other effects of FTY720 are independent of S1P receptors, and appear facilitated by modulation of a range of other recently described protein targets by nonphosphorylated FTY720. Here, we review the direct targets of FTY720 that contribute to its anti-cancer properties. We also discuss other recently described protein effectors that, in combination with S1P receptors, appear to contribute to its immunosuppressive effects.

Topics: Antineoplastic Agents; Apoptosis; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Lymphopenia; Lysophospholipids; Multiple Sclerosis; Neoplasms; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine

Sphingosine-1-phosphate (S1P) is a potent sphingolipid metabolite that regulates a number of biological processes critical for cancer. S1P produced inside cancer cells is exported and exerts its extracellular functions by binding to its specific receptors in an autocrine, paracrine, and/or endocrine manner, which is known as inside-out signaling. S1P is also known to exert its intracellular functions especially in the inflammatory process, but its relevance to cancer biology remains to be elucidated. Recently, there have been growing interests in the role of S1P in breast cancer progression, including angiogenesis and lymphangiogenesis. Our group demonstrated that activation of sphingosine kinase 1, the enzyme that catalyzes the phosphorylation of sphingosine to S1P, is a key step of this process. In this review, we will cover our current knowledge on the role of S1P signaling pathway in breast cancer progression with an emphasis on its role in tumor-induced lymphangiogenesis.

Topics: Breast Neoplasms; Female; Humans; Lymph Nodes; Lymphangiogenesis; Lymphatic Metastasis; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite, which has emerged as an important signaling mediator participating in the regulation of multiple cellular processes. The discovery of a family of S1P receptors, together with the more recently identified intracellular targets, has provided fundamental understanding of the multi-faceted actions of S1P. Evidence from both in vitro and in vivo studies has implicated the S1P signaling system in the control of immunity, inflammation and many associated diseases. Enigmatically, S1P appears to have both pro- and anti-inflammatory effects depending on the cell context. Here, we review this emerging area and argue for a pivotal role for S1P, as a key mediator of the cytokine network, acting through juxtacrine signaling in the immune system.

Topics: Animals; Cell Communication; Cytokines; Gene Regulatory Networks; Humans; Lysophospholipids; Models, Biological; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Sphingolipids are amphiphatic molecules ubiquitously expressed in all eukaryotic cell membranes. Initially characterized as structural components of cell membranes, sphingolipids have emerged as sources of important signalling molecules over the past decade. Sphingolipid metabolites, such as ceramide and S1P (sphingosine 1-phosphate), have been demonstrated to have roles as potent bioactive messengers involved in cell differentiation, proliferation, apoptosis, migration and angiogenesis. The importance of SphK (sphingosine kinase) and S1P in inflammation has been demonstrated extensively. The prevalence of asthma is increasing in many developed nations. Consequently, there is an urgent need for the development of new agents for the treatment of asthma, especially for patients who respond poorly to conventional therapy. Recent studies have demonstrated the important role of SphK and S1P in the development of asthma by regulating pro-inflammatory responses. These novel pathways represent exciting potential therapeutic targets in the treatment of asthma and are described in the present review.

Topics: Animals; Anti-Asthmatic Agents; Asthma; Cytokines; Disease Models, Animal; Epithelial Cells; Fingolimod Hydrochloride; Humans; Inflammation; Lung; Lysophospholipids; Mast Cells; Mice; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Signal Transduction; Sphingolipids; Sphingosine

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, which has as its primary target, the synovial tissues and articular cartilage. The current pharmacological treatment of RA includes non-steroidal anti-inflammatory drugs, corticosteroids, and disease-modifying anti-rheumatic drugs. Newer biological agents that work by inactivation of proinflammatory cytokines are available for treatment of RA. Sphingosine-1-phosphate (S1P) is a bioactive lipid that is generated from phosphorylation of sphingosine by activation of sphingosine kinase, and has been implicated as an important mediator in pathophysiological processes, including cell growth, differentiation, migration and survival, and angiogenesis. Several studies have explored the role of S1P in the pathogenesis of RA. The aim of this article was to review the biology and distribution of S1P, together with its role in RA, and to discuss its potential as a therapeutic target for RA.

Topics: Animals; Arthritis, Rheumatoid; Humans; Lysophospholipids; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine-1-phosphate (S1P) is a bioactive lipid with important functions in the immune system. S1P levels are regulated by the balance between its synthesis through sphingosine kinases and its degradation by S1P lyase. S1P signals through plasma membrane G-protein-coupled receptors (S1PR1-S1PR5) or acts directly on intracellular targets. Although it has long been known that the S1P-S1PR1 axis mediates T cell egress from lymphoid organs, recent studies have revealed intrinsic functions of S1P and its receptors in both innate and adaptive immune systems that are independent of immune cell trafficking. Here I summarize recent advances in understanding of the roles of S1P and S1P receptors in inflammatory and allergic responses and lymphocyte differentiation, which directly contribute to the regulation of inflammatory and autoimmune diseases. I also describe strategies to target S1P and S1P receptors for immune-mediated diseases, particularly the immunosuppressant FTY720 (fingolimod), which has recently become the first oral therapy for relapsing multiple sclerosis.

Topics: Aldehyde-Lyases; Animals; Cell Differentiation; Humans; Immunity, Innate; Inflammation; Lymphocytes; Lysophospholipids; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Sphingosine-1-phosphate (S1P) is a pleiotropic sphingophospholipid generated from the phosphorylation of sphingosine by sphingosine kinases (SPHKs). S1P has been experimentally demonstrated to modulate an array of cellular processes such as cell proliferation, cell survival, cell invasion, vascular maturation, and angiogenesis by binding with any of the five known G-protein-coupled sphingosine 1 phosphate receptors (S1P1-5) on the cell surface in an autocrine as well as a paracrine manner. Recent studies have shown that the S1P receptors (S1PRs) and SPHKs are the key targets for modulating the pathophysiological consequences of various debilitating diseases, such as cancer, sepsis, rheumatoid arthritis, ulcerative colitis, and other related illnesses. In this article, we recapitulate these novel discoveries relative to the S1P/S1PR axis, necessary for the proper maintenance of health, as well as the induction of tumorigenic, angiogenic, and inflammatory stimuli that are vital for the development of various diseases, and the novel therapeutic tools to modulate these responses in oral biology and medicine.

Topics: Animals; Atherosclerosis; Autoimmune Diseases; Cell Proliferation; Gene Expression Regulation, Enzymologic; GTP-Binding Protein alpha Subunits; GTP-Binding Protein Regulators; Humans; Lymphatic Metastasis; Lysophospholipids; Mandibular Condyle; Neovascularization, Pathologic; Neurogenic Inflammation; Periodontitis; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Sphingosine 1-phosphate (S1P) is a bioactive lipid that has both physiological and pathophysiological roles. It regulates cellular processes such as proliferation, migration, survival and differentiation and affects all organ systems. S1P not only activates S1P-specific receptors to initiate cellular signalling pathways but also directly regulates specific intracellular target proteins. The therapeutic opportunities surrounding S1P signalling are numerous and exemplified by the recent approval of FTY720 (a sphingosine analogue, Gilenya™) for the treatment of relapsing multiple sclerosis. A major focus of research is to develop small-molecule antagonists/agonists/inhibitors that are specific to the different S1P receptor subtypes and the enzymes that regulate S1P levels. This review describes fundamental aspects of S1P biology with an emphasis on the translational potential of intervention therapeutics.

Topics: Animals; Gene Expression Regulation; Humans; Intracellular Space; Lysophospholipids; Membrane Proteins; Multiple Sclerosis, Relapsing-Remitting; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine; Translational Research, Biomedical

The potent lipid mediator sphingosine-1-phosphate (S1P) is produced inside cells by two closely related kinases, sphingosine kinase 1 (SPHK1) and SPHK2, and has emerged as a crucial regulator of immunity. Many of the actions of S1P in innate and adaptive immunity are mediated by its binding to five G protein-coupled receptors, designated S1PR1-5, but recent findings have also identified important roles for S1P as a second messenger during inflammation. In this Review, we discuss recent advances in our understanding of the roles of S1P receptors and describe the newly identified intracellular targets of S1P that are crucial for immune responses. Finally, we discuss the therapeutic potential of new drugs that target S1P signalling and functions.

Topics: Animals; Humans; Immunity; Inflammation; Lysophospholipids; Models, Immunological; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Second Messenger Systems; Sphingosine; Sphingosine-1-Phosphate Receptors

Hypoxia, defined as reduced tissue oxygen concentration, is a characteristic of solid tumors and is an indicator of unfavorable diagnosis in patients. At the cellular level, the adaptation to hypoxia is under the control of two related transcription factors, HIF-1α and HIF-2α (Hypoxia-Inducible Factor), which activate expression of genes promoting angiogenesis, metastasis, increased tumor growth and resistance to treatments. A role for HIF-1α and HIF-2α is also emerging in hematologic malignancies such as lymphoma and l eukemia. Recent studies have identified the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) signaling pathway - which elicits various cellular processes including cell proliferation, cell survival or angiogenesis - as a new regulator of HIF-1α or HIF-2α activity. This review will consider how targeting the SphK1/S1P signaling could represent an attractive strategy for therapeutic intervention in cancer.

Topics: Animals; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Hematologic Neoplasms; Humans; Hypoxia-Inducible Factor 1; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that regulates several processes important for hematologic cancer progression. S1P is generated by two sphingosine kinases, SphK1 and SphK2, and is exported outside the cell, where it activates specific cell surface S1P G-protein coupled receptors in autocrine/paracrine manner, coined "inside-out signaling". In this review, we highlight the importance of SphK1 and inside-out signaling by S1P in hematologic malignancy. We also summarize the results of studies targeting the SphK1/S1P/S1P receptor axis and the effects of the S1P receptor modulator, FTY720, in hematologic malignancy.

Topics: Animals; Fingolimod Hydrochloride; Hematologic Neoplasms; Humans; Immunosuppressive Agents; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine

For several decades, lipid biologists have investigated how sphingolipids contribute to physiology, cell biology, and cell fate. Foremost among these discoveries is the finding that the bioactive sphingolipids ceramide, sphingosine, and sphingosine-1-phosphate (S1P) have diverse and often opposing effects on cell fate. Interestingly, these bioactive sphingolipids can be interconverted by just a few enzymatic reactions. Therefore, much attention has been paid to the enzymes which govern these reactions with a disproportionate amount of focus on the enzyme sphingosine kinase 1 (SK1). Several studies have found that tissue expression of SK1 correlates with cancer stage, chemotherapy response, and tumor aggressiveness. In addition, overexpression of SK1 in multiple cancer cell lines increases their resistance to chemotherapy, promotes proliferation, allows for anchorage independent growth, and increases local angiogenesis. Inhibition of SK1 using either pharmacological inhibitors or by crossing SK1 null mice has shown promise in many xenograft models of cancer, as well as several genetic and chemically induced mouse models of carcinogenesis. Here, we review the majority of the evidence that suggests SK1 is a promising target for the prevention and/or treatment of various cancers. Also, we strongly advocate for further research into basic mechanisms of bioactive sphingolipid signaling, and an increased focus on the efficacy of SK inhibitors in non-xenograft models of cancer progression.

Topics: Animals; Cell Proliferation; Ceramides; Clinical Trials as Topic; Humans; Lysophospholipids; Mice; Neoplasms; Neoplasms, Experimental; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingolipids; Sphingosine

Members of the sphingosine kinase (SK) family of lipid signaling enzymes, comprising SK1 and SK2 in humans, are receiving considerable attention for their roles in a number of physiological and pathophysiological processes. The SKs are considered signaling enzymes based on their production of the potent lipid second messenger sphingosine-1-phosphate, which is the ligand for a family of five G-protein-linked receptors. Both SK1 and SK2 are intracellular enzymes and do not possess obvious membrane anchor domains within their primary sequences. The native substrates (sphingosine and dihydrosphingosine) are lipids, as are the corresponding products, and therefore would have a propensity to be membrane associated, suggesting that specific membrane localization of the SKs could affect both access to substrate and localized production of product. Here, we consider the emerging picture of the SKs as enzymes localized to specific intracellular sites, sometimes by agonist-dependent translocation, the mechanism targeting these enzymes to those sites, and the functional consequence of that localization. Not only is the signaling output of the SKs affected by subcellular localization, but the role of these enzymes as metabolic regulators of sphingolipid metabolism may be impacted as well.

Topics: Animals; Cell Membrane; Ceramides; Humans; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Protein Isoforms; Protein Transport; Signal Transduction; Sphingolipids; Sphingosine; Substrate Specificity

Sphingolipids form a broad class of lipids with diverse functions ranging from membrane constituents to intracellular second messengers and extracellular mediators. They can be rapidly generated or converted into each other and they play pivotal roles in various cellular processes, many of which are broadly associated with inflammation and apoptosis. Among the numerous sphingolipids, ceramide and sphingosine-1-phosphate (S1P) have received the greatest attention. Ceramide is a hydrophobic molecule that is increased in the lungs of patients with cystic fibrosis and chronic obstructive pulmonary disease (COPD). Ceramide is the eponym for ceramide-rich membrane platforms. that need to form as a prerequisite to the uptake of several microorganisms including Pseudomonas aeruginosa, and as a prerequisite to many signaling processes including apoptosis and increased vascular permeability. Accordingly, abnormal amounts of enzymes involved in the synthesis of ceramide, such as neutral or acid sphingomyelinase, are found in emphysematic smokers and in patients with severe sepsis, and are considered as novel pharmacological targets. S1P acts as an extracellular mediator that opposes several actions of ceramide and acts by binding to G-protein coupled S1P receptors (S1P(1)-S1P(5)). Of particular interest are S1P(1) receptors that enhance vascular barrier functions and are antiapoptotic. Therefore, S1P(1)-receptor ligands are suggested as novel drugs for COPD and acute lung injury. S1P is a potent chemotaxin for many leukocytes, it organizes lymphocyte trafficking and is involved in several key symptoms of asthma such as airway hyperresponsiveness and pulmonary eosinophil sequestration. S1P is formed by sphingosine kinases that have been identified as possible drug targets for the treatment of asthma. Based on these findings, several new drugs have recently been developed to specifically target sphingomyelinases, sphingosine kinases and S1P receptors for the treatment of COPD, cystic fibrosis, asthma and acute lung injury.

Topics: Animals; Drug Delivery Systems; Drug Design; Humans; Lung Diseases; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingolipids; Sphingomyelin Phosphodiesterase; Sphingosine

The pleiotropic sphingolipid mediator, sphingosine-1-phosphate, produced in cells by two sphingosine kinase isoenzymes, SphK1 and SphK2, regulates many cellular and physiological processes important for homeostasis and development and pathophysiology. Many of the actions of S1P are mediated by a family of five specific cell surface receptors that are ubiquitously and specifically expressed, although important direct intracellular targets of S1P have also recently been identified. S1P, SphK1, and or S1P receptors have been linked to onset and progression of numerous diseases, including many types of cancer, and especially inflammatory disorders, such as multiple sclerosis, asthma, rheumatoid arthritis, inflammatory bowel disease, and sepsis. S1P formation and signaling are attractive targets for development of new therapeutics. The effects of a number of inhibitors of SphKs and S1PRs have been examined in animal models of human diseases. The effectiveness of the immunosuppressant FTY720 (known as Fingolimod or Gilenya), recently approved for the treatment of multiple sclerosis, whose actions are mediated by downregulation of S1PR1, has become the gold standard for S1P-centric drugs. Here, we review S1P biology and signaling with an emphasis on potential therapeutic benefits of specific interventions and discuss recent development of small molecule antagonists and agonists that target specific subtypes of S1P receptors as well as inhibitors of SphKs.

Topics: Animals; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Drug Evaluation, Preclinical; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

There is considerable evidence that sphingosine kinases play a key role in cancer progression, which might involve positive selection of cancer cells that have been provided with a survival and growth advantage as a consequence of overexpression of the enzyme. Therefore, inhibitors of sphingosine kinase represent a novel class of compounds that have potential as anticancer agents. Poor inhibitor potency is a major issue that has precluded successful translation of these compounds into the clinic. However, recent discoveries have shown that sphingosine kinase 1 is an allosteric enzyme and that some inhibitors offer improved effectiveness by inducing proteasomal degradation of the enzyme or having nanomolar potency. Herein, we provide a perspective about these recent developments and highlight the importance of translating basic pharmacologic and biochemical findings on sphingosine kinase into new drug discovery programs for treatment of cancer.

Topics: Allosteric Regulation; Animals; Antineoplastic Agents; Cell Division; Dose-Response Relationship, Drug; Drug Design; Drug Screening Assays, Antitumor; Fingolimod Hydrochloride; Humans; Lysophospholipids; Mice; Molecular Targeted Therapy; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Organophosphonates; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Proteasome Endopeptidase Complex; Protein Kinase Inhibitors; Sphingosine; Vinyl Compounds

Sphingosine-1-phosphate (S1P) was first described as a signaling molecule over 20 years ago. Since then, great strides have been made to reveal its vital roles in vastly different cellular and disease processes. Initially, S1P was considered nothing more than the terminal point of sphingolipid metabolism; however, over the past two decades, a large number of reports have helped unveil its full potential as an important regulatory, bioactive sphingolipid metabolite. S1P has a plethora of physiological functions, due in part to its many sites of actions and its different pools, which are both intra- and extracellular. S1P plays pivotal roles in many physiological processes, including the regulation of cell growth, migration, autophagy, angiogenesis, and survival, and thus, not surprisingly, S1P has been linked to cancer. In this review, we will summarize the vast body of knowledge, highlighting the connection between S1P and cancer. We will also suggest new avenues for future research.

Topics: Aldehyde-Lyases; Animals; Biological Transport; Extracellular Fluid; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Humans; Lysophospholipids; Mitochondria; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Ubiquitin-Protein Ligases

Gene expression in eukaryotes depends on epigenetic changes that occur on both histones and DNA. Class I histone deacetylases (HDACs) are enzymes that remove acetyl groups from histones and other nuclear proteins, thereby inducing chromatin condensation and transcriptional repression. HDACs belong to a large family of enzymes that undergo posttranslational modifications after the activation of several intracellular pathways. However, the environmental stimuli that change nuclear HDAC functions remain largely unknown. New evidence has demonstrated that the lipid sphingosine-1-phosphate (S1P) inhibits the activity of HDAC1 and HDAC2. Both S1P and sphingosine kinase 2 (SphK2), the enzyme that synthesizes S1P, are assembled in corepressor complexes containing HDAC1 and HDAC2. S1P is among the few endogenous HDAC inhibitors that is synthesized in the nucleus in response to extracellular stimulation, and the first nuclear lipid associated with an epigenetic modification. The discovery of endogenous molecules that regulate HDAC activity in vivo has implications for the development of new therapeutic approaches for a host of human diseases, including cancer and neurodegenerative disorders.

Topics: Animals; Cell Nucleus; Chromatin Assembly and Disassembly; Epigenesis, Genetic; Histone Deacetylase 1; Histone Deacetylase 2; Humans; Lysophospholipids; Neoplasms; Neurodegenerative Diseases; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Simple bioactive sphingolipids include ceramide, sphingosine and their phosphorylated forms sphingosine 1-phosphate and ceramide 1-phosphate. These molecules are crucial regulators of cell functions. In particular, they play important roles in the regulation of angiogenesis, apoptosis, cell proliferation, differentiation, migration, and inflammation. Decoding the mechanisms by which these cellular functions are regulated requires detailed understanding of the signaling pathways that are implicated in these processes. Most importantly, the development of inhibitors of the enzymes involved in their metabolism may be crucial for establishing new therapeutic strategies for treatment of disease.

Topics: Animals; Ceramidases; Ceramides; Disease; Humans; Inflammation; Isoenzymes; Lysophospholipids; Macrophages; Molecular Structure; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingolipids; Sphingosine

Sphingolipid metabolites are critical to the regulation of a number of fundamental biological processes including cancer. Whereas ceramide and sphingosine mediate and trigger apoptosis or cell growth arrest, sphingosine 1-phosphate promotes proliferation, cell survival and angiogenesis. The delicate equilibrium between the intracellular levels of each of these sphingolipids is controlled by the enzymes that either produce or degrade these metabolites. Sphingosine kinase-1 is a crucial regulator of this two-pan balance, because its produces the pro-survival and pro-angiogenic sphingosine 1-phosphate and decreases the amount of both ceramide and sphingosine, the pro-apoptotic sphingolipids. Moreover, its gene is oncogenic, its mRNA is overproduced in several solid tumors, its overexpression protects cells from apoptosis, and its activity is down-regulated by anti-cancer treatments. Therefore, the sphingosine kinase-1/sphingosine 1-phosphate signaling pathway appears to be a target of interest for therapeutic manipulation.

Topics: Apoptosis; Ceramides; Humans; Lysophospholipids; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingolipids; Sphingosine

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator with crucial roles in a wide variety of cellular functions across a broad range of organisms. Though a simple molecule in structure, S1P functions are complex. The formation of S1P is catalyzed by one of two sphingosine kinases that have differential cellular distributions as well as both overlapping and opposing functions and which are activated by many different stimuli. S1P can act on a family of G protein-coupled receptors (S1PRs) that are also differentially expressed in different cell types, which influences the cellular responses to S1P. In addition to acting on receptors located on the plasma membrane, S1P can also function inside the cell, independently of S1PRs. It also appears that both the intracellular location and the isotype of sphingosine kinase involved are major determinants of inside-out signaling of S1P in response to many extracellular stimuli. This chapter is focused on the current literature on extracellular and intracellular actions of S1P.

Topics: Animals; Arabidopsis; Extracellular Space; Humans; Intracellular Space; Lysophospholipids; Mammals; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Saccharomyces cerevisiae; Sphingolipids; Sphingosine

Sphingolipids were once regarded as inert structural components of cell membranes. Now these metabolites are generally believed to be important bioactive molecules that control a wide repertoire of cellular processes such as proliferation and survival of cells. Along with these ubiquitous cell functions observed in many peripheral tissues sphingolipid metabolites, especially sphingosine 1-phosphate, exert important neuron-specific functions such as regulation of neurotransmitter release. This review summarizes physiological and pathological roles of sphingolipid metabolites emphasizing the role of sphingosine 1-phosphate in the central nervous system.

Topics: Animals; Apoptosis; Cell Survival; Central Nervous System; Exocytosis; Lysophospholipids; Neurotransmitter Agents; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is produced by the sphingosine kinase-catalysed phosphorylation of sphingosine. S1P is an important regulator of cell function, mediating many of its effects through a family of five closely related G protein-coupled receptors (GPCR) termed S1P(1-5) which exhibit high affinity for S1P. These receptors function to relay the effects of extracellular S1P via well-defined signal transduction networks linked to the regulation of cell proliferation, survival, migration etc. Diverse agonists (e.g. cytokines) also activate sphingosine kinase and the resulting S1P formed may bind to specific undefined intracellular targets to elicit cellular responses. The purpose of this review is to discuss some of the spatial/temporal aspects of intracellular S1P signalling and to define the function of sphingosine kinases and lipid phosphate phosphatases (which catalyse dephosphorylation of S1P) in terms of their regulation of cell function. Finally, we survey the function of S1P in relation to disease, where the major challenge is to dissect the role of intracellular versus extracellular actions of S1P in terms of association with defined diseased phenotypes.

Topics: Cell Differentiation; Cell Division; Cell Movement; Hypoxia; Lysophospholipids; Neoplasms; Phosphatidate Phosphatase; Phosphotransferases (Alcohol Group Acceptor); Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Activation of sphingosine kinase/sphingosine-1-phosphate (SK/S1P)-mediated signalling has been recognized as critical for cardioprotection in response to acute ischaemia/reperfusion injury. Incubation of S1P with cultured cardiac myocytes subjected to hypoxia or treatment of isolated hearts either before ischaemia or at the onset of reperfusion (pharmacologic pre- or postconditioning) results in reduced myocyte injury. Synthetic agonists active at S1P receptors mimic these responses. Gene-targeted mice null for the SK1 isoform whose hearts are subjected to ischaemia/reperfusion injury exhibit increased infarct size and respond poorly either to ischaemic pre- or postconditioning. Measurements of cardiac SK activity and S1P parallel these observations. Ischaemic postconditioning combined with sphingosine and S1P rescues the heart from prolonged ischaemia. These observations may have considerable relevance for future therapeutic approaches to acute and chronic myocardial injury.

Topics: Animals; Disease Models, Animal; Humans; Lysophospholipids; Mice; Myocardial Reperfusion Injury; Phosphotransferases (Alcohol Group Acceptor); Rats; Sphingosine

Activation of sphingosine kinase/sphingosine 1-phosphate-mediated signaling has emerged as a critical cardioprotective pathway in response to acute ischemia/reperfusion injury. Application of exogenous sphingosine 1-phosphate (S1P) in cultured cardiac myocytes subjected to hypoxia or treatment of isolated hearts either before ischemia or at the onset of reperfusion (pharmacologic preconditioning or postconditioning) exerts prosurvival effects. Synthetic congeners of S1P mimic these responses. Gene-targeted mice null for the sphingosine kinase 1 isoform whose hearts are subjected to ischemia/reperfusion injury exhibit increased infarct size and respond poorly either to ischemic preconditioning or to ischemic postconditioning. Measurements of cardiac sphingosine kinase activity and S1P parallel these observations. High-density lipoprotein is a major carrier of S1P, and studies of hearts in which selected S1P receptors have been deleted implicate the S1P cargo of high-density lipoprotein in cardioprotection. These observations have considerable relevance for future therapeutic approaches to acute and chronic myocardial injury.

Topics: Animals; Cardiotonic Agents; Cell Survival; Cells, Cultured; Fibroblasts; Humans; Lysophospholipids; Myocardial Reperfusion Injury; Myocytes, Cardiac; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), has emerged as a critical player in a number of fundamental biological processes and is important in cancer, angiogenesis, wound healing, cardiovascular function, atherosclerosis, immunity and asthma, among others. Activation of sphingosine kinases, enzymes that catalyze the phosphorylation of sphingosine to S1P, by a variety of agonists, including growth factors, cytokines, hormones, and antigen, increases intracellular S1P. Many of the biological effects of S1P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface in an autocrine and/or paracrine manner. Therefore, understanding the mechanism by which intracellularly generated S1P is released out of cells is both interesting and important. In this review, we will discuss how S1P is formed and released. We will focus particularly on the current knowledge of how the S1P gradient between tissues and blood is maintained, and the role of ABC transporters in S1P release.

Topics: ATP-Binding Cassette Transporters; Biological Transport; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Receptors, G-Protein-Coupled; Sphingosine

The reduction in the normal level of tissue oxygen tension or hypoxia is a characteristic of solid tumors that triggers the activation of signaling pathways promoting neovascularization, metastasis, increased tumor growth, and resistance to treatments. The activation of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha) has been identified as the master mechanism of adaptation to hypoxia. In a recent study, we identified the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) pathway, which elicits various cellular processes including cell proliferation, cell survival, or angiogenesis, as a new modulator of HIF-1alpha activity under hypoxic conditions. Here, we consider how the SphK1/S1P signaling pathway could represent a very important target for therapeutic intervention in cancer.

Topics: Animals; Cell Hypoxia; Drug Delivery Systems; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lysophospholipids; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Topics: Animals; Humans; Isoenzymes; Lysophospholipids; Neoplasms; Phosphatidate Phosphatase; Phosphotransferases (Alcohol Group Acceptor); Receptor Protein-Tyrosine Kinases; Signal Transduction; Sphingosine

The bioactive lipid sphingosine-1-phosphate (S1P) fulfils manifold tasks in the immune system acting in auto- and/or paracrine fashion. This includes regulation of apoptosis, migration and proliferation. Upon its generation by sphingosine kinases from plasma membrane sphingolipids, S1P can either act as a second messenger within cells or can be released from cells to occupy a family of specific G-protein-coupled receptors (S1P1-5). This diversity is reflected by the impact of S1P on macrophage biology and function. Over the last years it became apparent that the sphingosine kinase/S1P/S1P-receptor signalling axis in macrophages might play a central role in the pathogenesis of inflammatory diseases such as atherosclerosis, asthma, rheumatoid arthritis and cancer. Here, we summarize the current knowledge of the function of S1P in macrophage biology and discuss potential implications for pathology.

Topics: Animals; Arthritis, Rheumatoid; Asthma; Atherosclerosis; Humans; Inflammation; Lysophospholipids; Macrophage Activation; Macrophages; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

FTY720 (fingolimod) is a first-in-class sphingosine 1-phosphate (S1P) receptor modulator that was highly effective in Phase II clinical trials for Multiple Sclerosis (MS). FTY720 is phosphorylated in vivo by sphingosine kinase-2 to form the active moiety FTY720-phosphate that binds to four of the five G protein-coupled S1P receptor subtypes. Studies using conditional S1P1 receptor-deficient and sphingosine kinase-deficient mice showed that the egress of lymphocytes from lymph nodes requires signalling of lymphocytic S1P1 receptors by the endogenous ligand S1P. The S1P mimetic FTY720-phosphate causes internalization and degradation of cell membrane-expressed S1P1, thereby antagonizing S1P action at the receptor. In models of human MS and demyelinating polyneuropathies, functional antagonism of lymphocytic S1P1 slows S1P-driven egress of lymphocytes from lymph nodes, thereby reducing the numbers of autoaggressive TH17 cells that recirculate via lymph and blood to the central nervous system and the sciatic/ischiatic nerves. Based on its lipophilic nature, FTY720 crosses the blood-brain barrier, and ongoing experiments suggest that the drug also down-modulates S1P1 in neural cells/astrocytes to reduce astrogliosis, a phenomenon associated with neurodegeneration in MS. This may help restore gap-junctional communication of astrocytes with neurons and cells of the blood-brain barrier. Additional effects may result from (down-) modulation of S1P3 in astrocytes and of S1P1 and S1P5 in oligodendrocytes. In conclusion, FTY720 may act through immune-based and central mechanisms to reduce inflammation and support structural restoration of the central nervous system parenchyma. Beyond the autoimmune indications, very recent studies suggest that short-term, low-dose administration of FTY720 could help treat chronic (viral) infections. Differential effects of the drug on the trafficking of naïve, central memory and effector memory T cell subsets are discussed.

Topics: Animals; Astrocytes; Brain; Encephalomyelitis, Autoimmune, Experimental; Fingolimod Hydrochloride; Gap Junctions; Humans; Immune System; Inflammation; Lymph Nodes; Lysophospholipids; Multiple Sclerosis; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine; T-Lymphocytes

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes including proliferation, survival, and migration, as well as angiogenesis and allergic responses. S1P levels inside cells are tightly regulated by the balance between its synthesis by sphingosine kinases and degradation. S1P is interconvertible with ceramide, which is a critical mediator of apoptosis. It has been postulated that the ratio between S1P and ceramide determines cell fate. Activation of sphingosine kinase by a variety of agonists increases intracellular S1P, which in turn can function intracellularly as a second messenger or be secreted out of the cell and act extracellularly by binding to and signaling through S1P receptors in autocrine and/or paracrine manners. Recent studies suggest that this "inside-out" signaling by S1P may play a role in many human diseases, including cancer, atherosclerosis, inflammation, and autoimmune disorders such as multiple sclerosis. In this review we summarize metabolism of S1P, mechanisms of sphingosine kinase activation, and S1P receptors and their downstream signaling pathways and examine relationships to multiple disease processes. In particular, we describe recent preclinical and clinical trials of therapies targeting S1P signaling, including 2-amino-2-propane-1,3-diol hydrochloride (FTY720, fingolimod), S1P receptor agonists, sphingosine kinase inhibitors, and anti-S1P monoclonal antibody.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Enzyme Activation; Fingolimod Hydrochloride; Humans; Hypersensitivity; Immunosuppressive Agents; Lysophospholipids; Multiple Sclerosis; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sulfhydryl Compounds

In this chapter, roles of bioactive sphingolipids in the regulation of cancer pathogenesis and therapy will be reviewed. Sphingolipids have emerged as bioeffector molecules, which control various aspects of cell growth, proliferation, and anti-cancer therapeutics. Ceramide, the central molecule of sphingolipid metabolism, generally mediates anti-proliferative responses such as inhibition of cell growth, induction of apoptosis, and/or modulation of senescence. On the other hand, sphingosine 1-phosphate (S1P) plays opposing roles, and induces transformation, cancer cell growth, or angiogenesis. A network of metabolic enzymes regulates the generation of ceramide and S1P, and these enzymes serve as transducers of sphingolipid-mediated responses that are coupled to various exogenous or endogenous cellular signals. Consistent with their key roles in the regulation of cancer growth and therapy, attenuation of ceramide generation and/or increased S1P levels are implicated in the development of resistance to drug-induced apoptosis, and escape from cell death. These data strongly suggest that advances in the molecular and biochemical understanding of sphingolipid metabolism and function will lead to the development of novel therapeutic strategies against human cancers, which may also help overcome drug resistance.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Differentiation; Cellular Senescence; Ceramides; Chemoprevention; Drug Resistance, Neoplasm; Humans; Lysophospholipids; Neoplasms; Oxidoreductases; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingolipids; Sphingosine

Pharmacological interference with sphingolipid metabolizing enzymes promises to provide novel ways to modulate cellular pathways relevant in multiple diseases. In this review, we focus on two sphingolipid signaling molecules, sphingosine-1-phosphate (S1P) and ceramide, as they are involved in cell fate decisions (survival vs. apoptosis) and in a wide range of pathophysiological processes. For S1P, we will discuss sphingosine kinases and S1P lyase as the enzymes which are crucial for its production and degradation, respectively, emphasizing the potential therapeutic usefulness of inhibitors of these enzymes. For ceramide, we will concentrate on acid sphingomyelinase, and critically review the substantial literature which implicates this enzyme as a worthwhile target for pharmacological inhibitors. It will become clear that the task to validate these enzymes as drug targets is not finished and many questions regarding the therapeutic usefulness of their inhibitors remain unanswered. Still this approach holds promise for a number of totally new therapies, and, on the way, detailed insight into sphingolipid signaling pathways can be gained.

Topics: Aldehyde-Lyases; Anaphylaxis; Animals; Apoptosis; Atherosclerosis; Bacterial Infections; Ceramides; Cyclooxygenase 2; Dendritic Cells; Drug Design; Enzyme Inhibitors; Humans; Immunologic Factors; Leukocytes; Lysophospholipids; Macrophages; Mast Cells; Neoplasms; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); RNA, Small Interfering; Sphingolipids; Sphingomyelin Phosphodiesterase; Sphingosine

During the last several years, sphingolipids have been identified as a source of important signaling molecules. Particularly, the understanding of the distinct biological roles of ceramide, sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P) and lyso-sphingomyelin in the regulation of cell growth, death, senescence, adhesion, migration, inflammation, angiogenesis and intracellular trafficking has rapidly expanded. Additional studies have elucidated the biological roles of sphingolipids in maintaining a homeostatic environment in cells, as well as in regulating numerous cellular responses to environmental stimuli. This review focuses on the role of S1P and C1P in maintaining Ca2+ homeostasis. By studying changes in the metabolism of S1P and C1P in pathological conditions, it is hoped that altered sphingolipid-metabolizing enzymes and their metabolites can be used as therapeutic targets.

Topics: Animals; Calcium; Calcium Channels; Calcium Signaling; Ceramides; Homeostasis; Humans; Lysophospholipids; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

During the last few years, it has become clear that sphingolipids are sources of important signalling molecules. Particularly, the sphingolipid metabolites, ceramide and S1P, have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation. Sphingomyelin (SM) is the major membrane sphingolipid and is the precursor for the bioactive products. Ceramide is formed from SM by the action of sphingomyelinases (SMase), however, ceramide can be very rapidly hydrolysed, by ceramidases to yield sphingosine, and sphingosine can be phosphorylated by sphingosine kinase (SphK) to yield S1P. In immune cells, the sphingolipid metabolism is tightly related to the main stages of immune cell development, differentiation, activation, and proliferation, transduced into physiological responses such as survival, calcium mobilization, cytoskeletal reorganization and chemotaxis. Several biological effectors have been shown to promote the synthesis of S1P, including growth factors, cytokines, and antigen and G-protein-coupled receptor agonists. Interest in S1P focused recently on two distinct cellular actions of this lipid, namely its function as an intracellular second messenger, capable of triggering calcium release from internal stores, and as an extracellular ligand activating specific G protein-coupled receptors. Inhibition of SphK stimulation strongly reduced or even prevented cellular events triggered by several proinflammatory agonists, such as receptor-stimulated DNA synthesis, Ca(2+) mobilization, degranulation, chemotaxis and cytokine production. Another very important observation is the direct role played by S1P in chemotaxis, and cellular escape from apoptosis. As an extracellular mediator, several studies have now shown that S1P binds a number of G-protein-coupled receptors (GPCR) encoded by endothelial differentiation genes (EDG), collectively known as the S1P-receptors. Binding of S1P to these receptors trigger an wide range of cellular responses including proliferation, enhanced extracellular matrix assembly, stimulation of adherent junctions, formation of actin stress fibres, and inhibition of apoptosis induced by either ceramide or growth factor withdrawal. Moreover, blocking S1P1-receptor inhibits lymphocyte egress from lymphatic organs. This review summarises the evidence linking SphK signalling pathway to immune-cell activation and based on these data discuss the

Topics: Animals; Autoimmune Diseases; Calcium Signaling; Cell Adhesion Molecules; Ceramides; Humans; Hypersensitivity; Immune System; Inflammation; Lymphocytes; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingomyelin Phosphodiesterase; Sphingomyelins; Sphingosine

The recent identification of a cellular balance between ceramide and sphingosine 1-phosphate (S1P) as a critical regulator of cell growth and death has stimulated increasing research effort to clarify the role of ceramide and S1P in various diseases associated with dysregulated cell proliferation and apoptosis. S1P acts mainly, but not exclusively, by binding to and activating specific cell surface receptors, the so-called S1P receptors. These receptors belong to the class of G protein-coupled receptors that constitute five subtypes, denoted as S1P(1)-S1P(5), and represent attractive pharmacological targets to interfere with S1P action. Whereas classical receptor antagonists will directly block S1P action, S1P receptor agonists have also proven useful, as recently shown for the sphingolipid-like immunomodulatory substance FTY720. When phosphorylated by sphingosine kinase to yield FTY720 phosphate, it acutely acts as an agonist at S1P receptors, but upon prolonged presence, it displays antagonistic activity by specifically desensitizing the S1P(1) receptor subtype. This commentary will cover the most recent developments in the field of S1P receptor pharmacology and highlights the potential therapeutic benefit that can be expected from these novel drug targets in the future.

Topics: Animals; Fingolimod Hydrochloride; Humans; Lysophospholipids; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Sphingosine; Sulfhydryl Compounds; Thiazolidines; Thiophenes

Sphingosine-1-phosphate (S1P) is a lipid mediator involved in diverse biological processes, from vascular and neural development to the regulation of lymphocyte trafficking. Many of its functions are regulated by five widely expressed S1P G-protein-coupled receptors (S1P(1-5)). S1P is produced mostly intracellularly, thus, much of its potential as an autocrine and paracrine mediator depends on how, when, and where it is generated or secreted out of the cells. However, S1P can also have intracellular activity independent of its receptors, adding to the complexity of S1P function. The mast cell, a major effector cell during an allergic response, has proven instrumental towards understanding the complex regulation and function of S1P. Antigen (Ag) engagement of the IgE receptor in mast cells stimulates sphingosine kinases, which generate S1P and are involved in the activation of calcium fluxes critical for mast cell responses. In addition, mast cells secrete considerable amounts of S1P upon activation, thus affecting the surrounding tissues and recruiting inflammatory cells. Export of S1P is also involved in the autocrine transactivation of S1P receptors present in mast cells. The in vivo response of mast cells, however, is not strictly dependent on their ability to generate S1P, but they are also affected by changes in S1P in the environment previous to Ag challenge. This review will discuss the recent advances towards understanding the intricacies of S1P generation, secretion and regulation in mast cells. In addition, how S1P receptors are activated and their involvement in mast cell functions will also be covered, including new insights on the role of S1P in the mast cell-mediated allergic response of systemic anaphylaxis.

Topics: Anaphylaxis; Animals; Humans; Lysophospholipids; Mast Cells; Models, Biological; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine

Topics: Animals; Apoptosis; Ceramides; Humans; Lysophospholipids; Monoamine Oxidase; Myocardial Reperfusion Injury; Oxidative Stress; Phosphotransferases (Alcohol Group Acceptor); Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; Sphingosine

Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). Together with other sphingolipid metabolizing enzymes, SphKs regulate the balance of the lipid mediators, ceramide, sphingosine, and S1P. The ubiquitous mediator S1P regulates cellular functions such as proliferation and survival, cytoskeleton architecture and Ca(2+) homoeostasis, migration, and adhesion by activating specific high-affinity G-protein-coupled receptors or by acting intracellularly. In mammals, two isoforms of SphK have been identified. They are activated by G-protein-coupled receptors, receptor tyrosine kinases, immunoglobulin receptors, cytokines, and other stimuli. The molecular mechanisms by which SphK1 and SphK2 are specifically regulated are complex and only partially understood. Although SphK1 and SphK2 appear to have opposing roles, promoting cell growth and apoptosis, respectively, they can obviously also substitute for each other, as mice deficient in either SphK1 or SphK2 had no obvious abnormalities, whereas double-knockout animals were embryonic lethal. In this review, our understanding of structure, regulation, and functional roles of SphKs is updated and discussed with regard to their implication in pathophysiological and disease states.

Topics: Amino Acid Sequence; Animals; Humans; Isoenzymes; Lysophospholipids; Molecular Sequence Data; Phosphotransferases (Alcohol Group Acceptor); Sequence Homology, Amino Acid; Signal Transduction; Sphingosine

Increasing evidence suggests that High-density lipoproteins (HDL) are a direct cardioprotective agent in the setting of acute myocardial ischemia/reperfusion injury, and that this cardioprotection occurs independently of their atheroprotective effect. Studies on the involved mechanisms have revealed that the biologically active HDL-compound sphingosine-1-phosphate (S1P) is responsible for the beneficial effect of HDL on the myocardium. There appears to be an intricate interplay between known preconditioning agents and components of the S1P synthesis machinery in the heart, which makes S1P signalling an attractive downstream convergence point of preconditioning and cardioprotection at the level of its G protein-coupled receptors. While local S1P production has been known to protect the heart against ischemia/reperfusion injury and to mediate preconditioning, systemic S1P supply via HDL adds a novel aspect to the regulation of cardioprotection. Thus the S1P-content of HDL may serve both as a potential cardiovascular risk marker and a novel therapeutic target. Strategies for short-term "acute" HDL elevation as well as S1P analogues may prove beneficial not only in the high-risk patient but also in any patient at risk of myocardial ischemia.

Topics: Acute Disease; Cardiovascular Diseases; Creatine Kinase; Humans; Ischemic Preconditioning, Myocardial; Lipoproteins, HDL; Lysophospholipids; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Phosphotransferases (Alcohol Group Acceptor); Risk Factors; Signal Transduction; Sphingosine

Sphingolipids not only function as structural components of cell membranes but also act as signaling molecules to regulate fundamental cellular responses, such as cell death and differentiation, proliferation and certain types of inflammation. Particularly the cellular balance between ceramide and sphingosine 1-phosphate seems to be crucial for a cell's decision to either undergo apoptosis or proliferate, two events which are implicated in tumor development and growth. Whereas ceramide possesses proapoptotic capacity in many cell types, sphingosine 1-phosphate acts as a counterplayer able to induce cell proliferation and protect cells from undergoing apoptosis. Therefore, tipping the balance in favour of ceramide production, i.e. by inhibiting ceramidase or sphingosine kinase activities has potential to support its proapoptotic action and hence represents a promising rational approach to effective cancer therapy. This review highlights most recent data on the regulation of cellular sphingolipid formation and their potential implication in tumor development, and provides perspectives for their use as targets in molecular intervention therapy.

Topics: Animals; Ceramides; Enzyme Inhibitors; Humans; Lipid Metabolism; Lysophospholipids; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

The lysosphingolipid sphingosine 1-phosphate (S1P) is a component of HDL. Findings from a growing number of studies indicate that S1P is a mediator of many of the cardiovascular effects of HDL, including the ability to promote vasodilation, vasoconstriction, and angiogenesis, protect against ischemia/reperfusion injury, and inhibit/reverse atherosclerosis. These latter cardioprotective effects are being shown to involve the S1P-mediated suppression of inflammatory processes, including reduction of the endothelial expression of monocyte and lymphocyte adhesion molecules, decreased recruitment of polymorphonuclear cells to sites of infarction, and blocking of cardiomyocyte apoptosis after myocardial infarction. This review article summarizes the evidence that S1P as a component of HDL serves to regulate vascular cell and lymphocyte behaviors associated with cardiovascular (patho)physiology.

Topics: Animals; Atherosclerosis; ATP-Binding Cassette Transporters; Cardiovascular System; Cell Movement; Cystic Fibrosis Transmembrane Conductance Regulator; Endothelial Cells; Humans; Immunologic Factors; Lipoproteins, HDL; Lysophospholipids; Muscle, Smooth, Vascular; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Signal Transduction; Sphingosine; Vasoconstriction; Vasodilation

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has been implicated in many biological processes, including cell migration, survival, proliferation, angiogenesis and immune and allergic responses. S1P levels inside cells are regulated tightly by the balance between its synthesis by sphingosine kinases and degradation by S1P lyases and S1P phosphatases. Activation of sphingosine kinase by any of a variety of agonists increases S1P levels, which in turn can function intracellularly as a second messenger or in an autocrine and/or paracrine fashion to activate and signal through S1P receptors present on the surface of the cell. This review summarizes recent findings on the roles of S1P as a mediator of the actions of cytokines, growth factors and hormones.

Topics: Animals; Autocrine Communication; Breast Neoplasms; Gonadal Steroid Hormones; Humans; Inflammation Mediators; Intercellular Signaling Peptides and Proteins; Lysophospholipids; Models, Biological; Neovascularization, Pathologic; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

The sphingosine and diacylglycerol kinases form a superfamily of structurally related lipid signaling kinases. One of the striking features of these kinases is that although they are clearly involved in agonist-mediated signaling, this signaling is accomplished with only a moderate (and sometimes no) increase in the enzymatic activity of the enzymes. Here, we summarize findings that indicate that signaling by these kinases is strongly dependent on their localization to specific intracellular sites rather than on increases in enzyme activity. Both the substrates and products of these enzymes are bioactive lipids. Moreover, many of the metabolic enzymes that act on these lipids are found in specific organelles. Therefore, changes in the membrane localization of these signaling kinases have profound effects not only on the production of signaling lipid phosphates but also on the metabolism of the upstream signaling lipids.

Topics: Animals; Diacylglycerol Kinase; Enzyme Activation; Growth Substances; Humans; Isoenzymes; Lysophospholipids; Models, Chemical; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha

Sphingolipids have emerged as molecules whose metabolism is regulated leading to generation of bioactive products including ceramide, sphingosine, and sphingosine-1-phosphate. The balance between cellular levels of these bioactive products is increasingly recognized to be critical to cell regulation; whereby, ceramide and sphingosine cause apoptosis and growth arrest phenotypes, and sphingosine-1-phosphate mediates proliferative and angiogenic responses. Sphingosine kinase is a key enzyme in modulating the levels of these lipids and is emerging as an important and regulated enzyme. This review is geared at mechanisms of regulation of sphingosine kinase and the coming to light of its role in disease.

Topics: Animals; Atherosclerosis; Ceramides; Diabetes Mellitus; Enzyme Activation; Humans; Inflammation; Lysophospholipids; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine kinases, through the formation of the bioactive phospholipid sphingosine 1-phosphate, have been implicated in a diverse range of cellular processes, including cell proliferation, apoptosis, calcium homeostasis, angiogenesis and vascular maturation. The last few years have seen a number of significant advances in understanding of the mechanisms of action, activation, cellular localisation and biological roles of these enzymes. Here we review the current understanding of the regulation of and cellular signalling by sphingosine kinase and sphingosine 1-phosphate and discuss recent findings implicating sphingosine kinase as a potential therapeutic target for the control of cancer, inflammation and a number of other diseases. We suggest that, since the activation and subcellular localization of these enzymes appear to play critical roles in their biological functions, targeting these processes may provide more specific therapeutic options than direct catalytic inhibitors.

Topics: Animals; Forecasting; Humans; Lysophospholipids; Models, Biological; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Topics: Animals; Biological Transport; Cell Membrane; Drug Design; Fingolimod Hydrochloride; Humans; Immune System; Immunosuppressive Agents; Lysophospholipids; Membrane Proteins; Neovascularization, Physiologic; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Saccharomyces cerevisiae Proteins; Signal Transduction; Sphingosine

Sphingolipids are ubiquitous components of cell membranes and their metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. Cer and Sph are associated with growth arrest and apoptosis. Many stress stimuli increase levels of Cer and Sph, whereas suppression of apoptosis is associated with increased intracellular levels of S1P. In addition, extracellular/secreted S1P regulates cellular processes by binding to five specific G protein coupled-receptors (GPCRs). S1P is generated by phosphorylation of Sph catalyzed by two isoforms of sphingosine kinases (SphK), type 1 and type 2, which are critical regulators of the "sphingolipid rheostat", producing pro-survival S1P and decreasing levels of pro-apoptotic Sph. Since sphingolipid metabolism is often dysregulated in many diseases, targeting SphKs is potentially clinically relevant. Here we review the growing recent literature on the regulation and the roles of SphKs and S1P in apoptosis and diseases.

Topics: Animals; Apoptosis; Disease; Enzyme Activation; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

1. Sphingolipids are potent second messengers modulating biochemical intracellular events and acting as ligands to mediate extracellular systems. Sphingosine kinase (SPHK) is the enzyme that phosphorylates sphingosine into sphingosine-1-phosphate (S1P), a potent bioactive sphingolipid. 2. The fact that SPHK is highly conserved from protozoa to mammals and is ubiquitous in living tissues reveals important roles of the SPHK pathway for the maintenance of health maintenance. This is also supported by comprehensive reviews on features of its main product, S1P, as having intracellular as well as extracellular roles, inducing a wide range of physiological responses from triggering Ca2+ release from internal stores to promoting growth and cell motility. 3. Immune cell activities have been shown to be modulated by the dynamic balance between ceramide, sphingosine and S1P, conceptualized as a rheostat. Cell proliferation, differentiation, motility and survival have been attributed to the regulatory actions of S1P. The properties of SPHK activity in immune cells are linked to the functions of triggered growth and survival factors, phorbol esters, hormones, cytokines and chemokines, as well as antigen receptors, such as FcgammaRI and FcepsilonRI. 4. Mechanisms of the SPHK signalling pathway are explored as new targets for drug development to suppress inflammation and other pathological conditions.

Topics: Animals; Humans; Immune System; Lymphocytes; Lysophospholipids; Mast Cells; Monocytes; Neutrophils; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

It has become abundantly clear over the past decade that sphingolipids and their metabolites are key signaling molecules. Ceramide, the backbone of all sphingolipids, predominantly inhibits cell growth and induces apoptosis, while its metabolite, sphingosine-1-phosphate promotes growth and survival. Given the interconvertibility of these two opposing signaling molecules, it is essential that any study that examines the effects of one also look at the other. The newly available technology of liquid chromatography-tandem mass spectroscopy (LC-MS/MS) is increasingly being applied for this purpose, as it can quickly identify and measure many different sphingolipids simultaneously. An added benefit of LC-MS/MS is that it is several orders of magnitude more sensitive than enzymatic methods or more traditional chromatographic techniques, allowing smaller sample sizes and increased throughput. Here, we briefly discuss the importance of LC-MS/MS for measuring sphingolipid metabolites and some future directions researchers may take given the increasingly accessibility to this technology.

Topics: Animals; Apoptosis; Cell Proliferation; Cell Survival; Ceramides; Chromatography, Liquid; Humans; Lipids; Lysophospholipids; Mass Spectrometry; Models, Biological; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingolipids; Sphingosine

Topics: Animals; Biological Evolution; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Second Messenger Systems; Signal Transduction; Sphingosine

Sphingosine 1-phosphate (S1P), an evolutionarily conserved bioactive lipid mediator, is now recognized as a potent modulator of cell regulation. In vertebrates, S1P interacts with cell surface G protein-coupled receptors of the EDG family and induces profound effects in a variety of organ systems. Indeed, an S1P receptor agonist is undergoing clinical trials to combat immune-mediated transplant rejection. Recent information on S1P receptor biology suggests potential utility in the control of cardiovascular processes, including angiogenesis, vascular permeability, arteriogenesis, and vasospasm. However, studies from diverse invertebrates, such as yeast, Dictyostelium, Drosophila, and Caenorhabditis elegans have shown that S1P is involved in important regulatory functions in the apparent absence of EDG S1P receptor homologues. Metabolic pathways of S1P synthesis, degradation, and release have recently been described at the molecular level. Genetic and biochemical studies of these enzymes have illuminated the importance of S1P signaling systems both inside and outside of cells. The revelation of receptor-dependent pathways, as well as novel metabolic/intracellular pathways has provided new biological insights and may ultimately pave the way for the development of novel therapeutic approaches for cardiovascular diseases.

Topics: Aldehyde-Lyases; Animals; Cardiovascular Diseases; Cardiovascular System; Endothelium, Vascular; Fungi; Humans; Invertebrates; Lysophospholipids; Membrane Proteins; Muscle, Smooth, Vascular; Myocardium; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Plants; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Second Messenger Systems; Species Specificity; Sphingosine; Vertebrates

Sphingosine-1-phosphate (S1P) has diverse biological functions acting inside cells as a second messenger to regulate cell proliferation and survival, and extracellularly, as a ligand for a group of G protein-coupled receptors (GPCRs) named the endothelial differentiation gene (EDG) family. Five closely related GPCRs of EDG family (EDG1, EDG3, EDG5, EDG6, and EDG8) have recently been identified as high-affinity S1P receptors. These receptors are coupled via Gi, Gq, G12/13, and Rho. The signaling pathways are linked to vascular cell migration, proliferation, apoptosis, intracellular Ca2+ mobilization, and expression of adhesion molecules. The formation of an atherosclerotic lesion occurs through activation of cellular events that include monocyte adhesion to the endothelium and vascular smooth muscle cell (VSMC) migration and proliferation. Thus, S1P signaling may play an important role in the pathogenesis of atherosclerotic vascular disease. This review highlights S1P signalling in vascular cells and its involvement in the formation of atherosclerotic lesions.

Topics: Animals; Apoptosis; Arteriosclerosis; Cell Division; Cell Movement; Cytokines; Humans; Lysophospholipids; Muscle, Smooth, Vascular; Phosphotransferases (Alcohol Group Acceptor); Receptors, G-Protein-Coupled; Signal Transduction; Sphingosine

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphosphatidic acid (LPA) reduce mortality in hypoxic cardiac myocytes. S1P is also cardioprotective in both mouse and rat models of cardiac ischemia/reperfusion (I/R) injury. Although these results are consistent with prior work in other cell types, it is not known what signaling events are critical to cardioprotection, particularly with respect to ceramide and the preservation of mitochondrial function, which is essential for cardiac cell survival. Neither receptor regulation nor signaling has been studied during I/R in the heart with or without the application of S1P or LPA. The role of sphingosine kinase in I/R and in ischemic preconditioning (IPC) has not been defined, nor has the fate or function of S1P generated by this enzyme, particularly during preconditioning or I/R, been elucidated. Whether S1P infused systemically in animal models of myocardial infarction in which survival is an end-point will be hemodynamically tolerated has not been determined. If not, the substitution of agents such as the monosialoganglioside GM-1, which activates sphingosine kinase, or the development of alternative ligands for S1P receptors will be necessary.

Topics: Animals; Cell Survival; Heart; Humans; Ischemic Preconditioning; Lysophospholipids; Myocardium; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

The sphingolipid metabolites ceramide, sphingosine, and sphingosine 1-phosphate contribute to controlling cell proliferation and apoptosis. Ceramide and its catabolite sphingosine act as negative regulators of cell proliferation and promote apoptosis. Conversely, sphingosine 1-phosphate, formed by phosphorylation of sphingosine by a sphingosine kinase, has been involved in stimulating cell growth and inhibiting apoptosis. As the phosphorylation of sphingosine diminishes apoptosis, while dephosphorylation of sphingosine 1-phosphate potentiates it, the role of sphingosine as a messenger of apoptosis is of importance. Herein, the effects of sphingosine on diverse signaling pathways implicated in the apoptotic process are reviewed.

Topics: Amidohydrolases; Animals; Apoptosis; Caspases; Cell Division; Cell Line; Ceramidases; Humans; Lysophospholipids; Membrane Proteins; Mitochondria; Mitogen-Activated Protein Kinases; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Sphingomyelins; Sphingosine

The sphingolipid metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) play an important role in the regulation of cell proliferation, survival, and cell death. Cer and Sph usually inhibit proliferation and promote apoptosis, while the further metabolite S1P stimulates growth and suppresses apoptosis. Because these metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determines cell fate. The relevance of this "sphingolipid rheostat" and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations. A central finding of these studies is that Sph kinase (SphK), the enzyme that phosphorylates Sph to form S1P, is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S1P, but also decreases levels of pro-apoptotic Cer and Sph. Given the role of the sphingolipid rheostat in regulating growth and apoptosis, it is not surprising that sphingolipid metabolism is often found to be disregulated in cancer, a disease characterized by enhanced cell growth, diminished cell death, or both. Anticancer therapeutics targeting SphK are potentially clinically relevant. Indeed, inhibition of SphK has been shown to suppress gastric tumor growth [Cancer Res. 51 (1991) 1613] and conversely, overexpression of SphK increases tumorigenicity [Curr. Biol. 10 (2000) 1527]. Moreover, S1P has also been shown to regulate angiogenesis, or new blood vessel formation [Cell 99 (1999) 301], which is critical for tumor progression. Furthermore, there is intriguing new evidence that S1P can act in an autocrine and/or paracrine fashion [Science 291 (2001) 1800] to regulate blood vessel formation [J. Clin. Invest. 106 (2000) 951]. Thus, SphK may not only protect tumors from apoptosis, it may also increase their vascularization, further enhancing growth. The cytoprotective effects of SphK/S1P may also be important for clinical benefit, as S1P has been shown to protect oocytes from radiation-induced cell death in vivo [Nat. Med. 6 (2000) 1109]. Here we review the growing literature on the regulation of SphK and the role of SphK and its product, S1P, in apoptosis.

Topics: Animals; Anoikis; Apoptosis; Binding Sites; Cell Line; Cell Survival; Ceramides; Cloning, Molecular; Enzyme Activation; Humans; Lysophospholipids; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Proteins; Saccharomyces cerevisiae; Second Messenger Systems; Sphingosine; TNF Receptor-Associated Factor 2

Topics: Animals; Cell Membrane; Lysophospholipids; Membrane Microdomains; Membrane Proteins; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Saccharomyces cerevisiae; Second Messenger Systems; Signal Transduction; Sphingosine

Topics: Animals; Blood Platelets; Heparin; Humans; Isoenzymes; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Topics: Animals; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Conformation; Saccharomyces cerevisiae; Signal Transduction; Sphingosine; Structure-Activity Relationship

Sphingosine kinase (SPHK) catalyzes the formation of sphingosine-1-phosphate (S11). S1P plays an important role in regulation of a variety of biological processes through intracellular and extracellular actions. S1P has recently been shown to be the ligand for the EDG-1 family of G-protein-coupled receptors. To date, seven cloned SPHKs have been reported with confirmed SPHK activity, including human, mouse, yeast, and plant. A computer search of various databases suggests that a new SPHK family is emerging. The cloning and manipulation of SPHK genes will no doubt provide us with important information about the functions of S1P in a wide range of organisms.

Topics: Amino Acid Sequence; Animals; Cloning, Molecular; Conserved Sequence; Humans; Lysophospholipids; Mammals; Molecular Sequence Data; Phosphotransferases (Alcohol Group Acceptor); Phylogeny; Plants; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Sphingosine

Considerable attention has focused on identifying mediators of neovascularization at sites of growth and abnormal tissue development. By contrast, mediators of angiogenesis at sites of injury and wound repair are not well defined but factors generated during blood coagulation (haemostasis) are attractive candidates. In addition to proteins generated, activated and released during the activation of clotting cascades, platelet-derived lipid mediators are now known to play a key role in many aspects of the angiogenic response. The first indication of lipid mediator involvement in angiogenesis was the discovery that lysophosphatidate (LPA), phosphatidic acid (PA) and sphingosine 1-phosphate (SPP) are high affinity agonists for G-protein coupled EDG (endothelial differentiation gene) receptors. The prototype for this family, EDG-1, was cloned from genes expressed when endothelial cells were activated to assume an angiogenic phenotype in vitro. The subsequent finding that SPP is a high affinity ligand for EDG-1 led Spiegel, Hla and associates (Lee et al., Science 1998;279:1552-1555) to hypothesize that platelet-released phospholipids play an important role in angiogenesis. These investigators and others demonstrated that SPP, LPA and phosphatidate (PA) induce many important endothelial cell responses associated with angiogenesis, including liberation of endothelial cells from established monolayers, chemotactic migration, proliferation, adherens junction assembly and morphogenesis into capillary-like structures. Although these studies indicated the potential involvement of platelet-derived phospholipids in angiogenesis, their physiological importance was not established. However, recent work demonstrates that >80% of the potent endothelial cell chemoattractive activity generated in human serum during clotting--an activity necessary for optimal angiogenesis--results from platelet-derived SPP. Other factors released from platelets during clotting, including LPA and PA, exert profound effects on endothelial cells that contribute unique aspects to the angiogenic response. These combined studies establish that SPP and other platelet-derived lipid mediators provide a novel link between haemostasis and angiogenesis.

Topics: Apoptosis; Blood Platelets; Chemotaxis; Endothelium, Vascular; Growth Substances; Hemostasis; Humans; Ischemia; Lysophospholipids; Neovascularization, Physiologic; Phospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Wound Healing

Ample evidence indicates that sphingosine-1-phosphate (SPP) can serve as an intracellular second messenger regulating calcium mobilization, and cell growth and survival. Moreover, the dynamic balance between levels of the sphingolipids metabolites, ceramide and SPP, and consequent regulation of opposing signaling pathways, is an important factor that determines whether a cell survives or dies. SPP has also recently been shown to be the ligand for the EDG-1 family of G protein-coupled receptors, which now includes EDG-1, -3, -5, -6, and -8. SPP is thus a lipid mediator that has novel dual actions signaling inside and outside of the cell. This review is focussed on sphingosine kinase, the enzyme that regulates levels of SPP and thus plays a critical role in diverse biological processes.

Topics: Amino Acid Sequence; Isoenzymes; Lysophospholipids; Molecular Sequence Data; Phosphotransferases (Alcohol Group Acceptor); Sphingolipids; Sphingosine

Topics: Aldehyde-Lyases; Animals; DNA-Binding Proteins; Humans; I-kappa B Proteins; Immediate-Early Proteins; Lysophospholipids; Membrane Proteins; NF-KappaB Inhibitor alpha; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Phylogeny; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Second Messenger Systems; Sphingosine

Sphingosine 1-phosphate (S1P) is stored in and released from platelets in response to cell activation. However, recent studies show that it is also released from a number of cell types, where it can function as a paracrine/autocrine signal to regulate cell proliferation, differentiation, survival, and motility. This review discusses the role of S1P in cellular regulation, both at the molecular level and in terms of health and disease. The main biochemical routes for S1P synthesis (sphingosine kinase) and degradation (S1P lyase and S1P phosphatase) are described. The major focus is on the ability of S1P to bind to a novel family of G-protein-coupled receptors (endothelial differentiation gene [EDG]-1, -3, -5, -6, and -8) to elicit signal transduction (via G(q)-, G(i)-, G(12)-, G(13)-, and Rho-dependent routes). Effector pathways regulated by S1P are divergent, such as extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, phospholipases C and D, adenylyl cyclase, and focal adhesion kinase, and occur in multiple cell types, such as immune cells, neurones, smooth muscle, etc. This provides a molecular basis for the ability of S1P to act as a pleiotropic bioactive lipid with an important role in cellular regulation. We also give an account of the expanding role for S1P in health and disease; in particular, with regard to its role in atherosclerosis, angiogenesis, cancer, and inflammation. Finally, we describe future directions for S1P research and novel approaches whereby S1P signalling can be manipulated for therapeutic intervention in disease.

Topics: Aldehyde-Lyases; Animals; Arteriosclerosis; Cell Differentiation; Endothelium; Gene Expression Regulation; GTP-Binding Protein Regulators; Humans; Inflammation; Lysophospholipids; Neoplasms; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Topics: Animals; Humans; Lysophospholipids; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Cardiac remodeling is a common pathological change in various cardiovascular diseases and can ultimately result in heart failure. Thus, there is an urgent need for more effective strategies to aid in cardiac protection. Our previous work found that sphingosine-1-phosphate (S1P) could ameliorate cardiac hypertrophy. In this study, we aimed to investigate whether S1P could prevent cardiac fibrosis and the associated mechanisms in cardiac remodeling.. Eight-week-old male C57BL/6 mice were randomly divided into a sham, transverse aortic constriction (TAC) or a TAC+S1P treatment group.. We found that S1P treatment improved cardiac function in TAC mice and that the cardiac fibrosis ratio in the TAC+S1P group was significantly lower and was accompanied by a decrease in α-smooth muscle actin (α-SMA) and collagen type I (COL I) expression compared with the TAC group. We also found that one of the key S1P enzymes, sphingosine kinase 2 (SphK2), which was mainly distributed in cytoblasts, was downregulated in the cardiac remodeling case and recovered after S1P treatment in vivo and in vitro. In addition, our in vitro results showed that S1P treatment activated extracellular regulated protein kinases (ERK) phosphorylation mainly through the S1P receptor 2 (S1PR2) and spurred p-ERK transposition from the cytoplasm to cytoblast in H9c2 cells exposed to phenylephrine.. These findings suggest that SphK2 and the S1PR2/ERK pathway may participate in the anti-remodeling effect of S1P on the heart. This work therefore uncovers a novel potential therapy for the prevention of cardiac remodeling.

Topics: Animals; Fibrosis; Lysophospholipids; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Ventricular Remodeling

Studies in cell culture and mouse models of cancer have indicated that the soluble sphingolipid metabolite sphingosine 1-phosphate (S1P) promotes cancer cell proliferation, survival, invasiveness, and tumor angiogenesis. In contrast, its metabolic precursor ceramide is prodifferentiative and proapoptotic. To determine whether sphingolipid balance plays a significant role in glioma malignancy, we undertook a comprehensive analysis of sphingolipid metabolites in human glioma and normal gray matter tissue specimens. We demonstrate, for the first time, a systematic shift in sphingolipid metabolism favoring S1P over ceramide, which increases with increasing cancer grade. S1P content was, on average, 9-fold higher in glioblastoma tissues compared with normal gray matter, whereas the most abundant form of ceramide in the brain, C18 ceramide, was on average 5-fold lower. Increased S1P content in the tumors was significantly correlated with increased sphingosine kinase 1 (SPHK1) and decreased sphingosine phosphate phosphatase 2 (SGPP2) expression. Inhibition of S1P production by cultured glioblastoma cells, using a highly potent and selective SPHK1 inhibitor, blocked angiogenesis in cocultured endothelial cells without affecting VEGF secretion. Our findings validate the hypothesis that an altered ceramide/S1P balance is an important feature of human cancers and support the development of SPHK1 inhibitors as antiangiogenic agents for cancer therapy.

Topics: Angiogenesis Inhibitors; Animals; Brain Neoplasms; Ceramides; Enzyme Inhibitors; Follow-Up Studies; Glioblastoma; Humans; Lipid Metabolism; Lysophospholipids; Male; Membrane Proteins; Mice; Neoplasm Proteins; Neovascularization, Pathologic; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Vascular Endothelial Growth Factor A

Sphingosine 1-phosphate (S1P) is an important mediator of cancer cell growth and proliferation. Production of S1P is catalyzed by sphingosine kinase 1 (SphK). Safingol, (l-threo-dihydrosphingosine) is a putative inhibitor of SphK. We conducted a phase I trial of safingol (S) alone and in combination with cisplatin (C).. A 3 + 3 dose escalation was used. For safety, S was given alone 1 week before the combination. S + C were then administered every 3 weeks. S was given over 60 to 120 minutes, depending on dose. Sixty minutes later, C was given over 60 minutes. The C dose of 75 mg/m(2) was reduced in cohort 4 to 60 mg/m(2) due to excessive fatigue.. Forty-three patients were treated, 41 were evaluable for toxicity, and 37 for response. The maximum tolerated dose (MTD) was S 840 mg/m(2) over 120 minutes C 60 mg/m(2), every 3 weeks. Dose-limiting toxicity (DLT) attributed to cisplatin included fatigue and hyponatremia. DLT from S was hepatic enzyme elevation. S pharmacokinetic parameters were linear throughout the dose range with no significant interaction with C. Patients treated at or near the MTD achieved S levels of more than 20 μmol/L and maintained levels greater than and equal to 5 μmol/L for 4 hours. The best response was stable disease in 6 patients for on average 3.3 months (range 1.8-7.2 m). One patient with adrenal cortical cancer had significant regression of liver and lung metastases and another had prolonged stable disease. S was associated with a dose-dependent reduction in S1P in plasma.. Safingol, the first putative SphK inhibitor to enter clinical trials, can be safely administered in combination with cisplatin. Reversible dose-dependent hepatic toxicity was seen, as expected from preclinical data. Target inhibition was achieved with downregulation of S1P. The recommended phase II dose is S 840 mg/m(2) and C 60 mg/m(2), every 3 weeks.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Cisplatin; Dose-Response Relationship, Drug; Drug Administration Schedule; Fatigue; Female; Humans; Lymphopenia; Lysophospholipids; Male; Metabolic Clearance Rate; Middle Aged; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Treatment Outcome

Reciprocal interactions between breast cancer cells and the tumor microenvironment (TME) are important for cancer progression and metastasis. We report here that the deletion or inhibition of sphingosine kinase 2 (SphK2), which produces sphingosine-1-phosphate (S1P), markedly suppresses syngeneic breast tumor growth and lung metastasis in mice by creating a hostile microenvironment for tumor growth and invasion. SphK2 deficiency decreased S1P and concomitantly increased ceramides, including C16-ceramide, in stromal fibroblasts. Ceramide accumulation suppressed activation of cancer-associated fibroblasts (CAF) by upregulating stromal p53, which restrained production of tumor-promoting factors to reprogram the TME and to restrict breast cancer establishment. Ablation of p53 in SphK2-deficient fibroblasts reversed these effects, enabled CAF activation and promoted tumor growth and invasion. These data uncovered a novel role of SphK2 in regulating non-cell-autonomous functions of p53 in stromal fibroblasts and their transition to tumor-promoting CAFs, paving the way for the development of a strategy to target the TME and to enhance therapeutic efficacy.. Sphingosine kinase 2 (SphK2) facilitates the activation of stromal fibroblasts to tumor-promoting cancer-associated fibroblasts by suppressing host p53 activity, revealing SphK2 as a potential target to reprogram the TME.

Topics: Animals; Cancer-Associated Fibroblasts; Fibroblasts; Lung Neoplasms; Mammary Neoplasms, Animal; Mice; Phosphotransferases (Alcohol Group Acceptor); Tumor Microenvironment; Tumor Suppressor Protein p53

Inflammation is pathogenic to skin diseases, including atopic dermatitis (AD) and eczema. Treatment for AD remains mostly symptomatic with newer but costly options, tainted with adverse side effects. There is an unmet need for safe therapeutic and preventative strategies for AD. Resveratrol (R) is a natural compound known for its anti-inflammatory properties. However, animal and human R studies have yielded contrasting results. Mast cells (MCs) are innate immune skin-resident cells that initiate the development of inflammation and progression to overt disease. R's effects on MCs are also controversial. Using a human-like mouse model of AD development consisting of a single topical application of antigen ovalbumin (O) for 7 days, we previously established that the activation of MCs by a bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) initiated substantial skin remodeling compared to controls. Here, we show that daily R application normalized O-mediated epidermal thickening, ameliorated cell infiltration, and inhibited skin MC activation and chemokine expression. We unraveled R's multiple mechanisms of action, including decreased activation of the S1P-producing enzyme, sphingosine kinase 1 (SphK1), and of transcription factors Signal Transducer and Activator of Transcription 3 (Stat3) and NF-κBp65, involved in chemokine production. Thus, R may be poised for protection against MC-driven pathogenic skin inflammation.

Topics: Animals; Chemokines; Dermatitis, Atopic; Humans; Inflammation; Mast Cells; Mice; NF-kappa B; Resveratrol; Sphingosine; STAT3 Transcription Factor

To investigate the therapeutic role of calorie-restricted diet (CR) and raspberry ketone (RK) in non-alcoholic fatty liver disease (NAFLD) and the implication of sphingosine kinase-1 (SphK1)/sphingosine-1-phosphate (S1P) and toll-like receptor 4 (TLR4) signalling.. NAFLD was induced by feeding rats high-fat-fructose-diet (HFFD) for 6 weeks. Rats were then randomly assigned to three groups (n = 6 each); NAFLD group continued on HFFD for another 8 weeks. CR group was switched to CR diet (25% calorie restriction) for 8 weeks and RK group was switched to normal diet and received RK (55 mg/kg/day; orally) for 8 weeks. Another six rats were used as normal control.. HFFD induced a state of NAFLD indicated by increased fat deposition in liver tissue along with dyslipidemia, elevated liver enzymes, oxidative stress and inflammation. Either CR diet or RK reversed these changes and decreased HFFD-induced elevation of hepatic SphK1, S1P, S1PR1 and TLR4. Of notice, RK along with a normal calorie diet was even better than CR alone in most studied parameters.. SphK1/S1P and TLR4 are interconnected and related to the establishment of HFFD-induced NAFLD and can be modulated by RK. Supplementation of RK without calorie restriction to patients with NAFLD unable to follow CR diet to achieve their treatment goals would be a promising therapeutic modality.

Topics: Animals; Diet, High-Fat; Liver; Non-alcoholic Fatty Liver Disease; Phosphates; Rats; Sphingosine; Toll-Like Receptor 4

Microglia, the resident immune cells of the central nervous system, play important roles in brain homeostasis as well as in neuroinflammation, neurodegeneration, neurovascular diseases, and traumatic brain injury. In this context, components of the endocannabinoid (eCB) system have been shown to shift microglia towards an anti-inflammatory activation state. Instead, much less is known about the functional role of the sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) system in microglia biology. In the present study, we addressed potential crosstalk of the eCB and the S1P systems in BV2 mouse microglia cells challenged with lipopolysaccharide (LPS). We show that URB597, the selective inhibitor of fatty acid amide hydrolase (FAAH)-the main degradative enzyme of the eCB anandamide-prevented LPS-induced production of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β), and caused the accumulation of anandamide itself and eCB-like molecules such as oleic acid and

Topics: Animals; Anti-Inflammatory Agents; Endocannabinoids; Lipopolysaccharides; Mice; Microglia; Sphingosine; Tumor Necrosis Factor-alpha

Oxidative stress triggered by aging, radiation, or inflammation impairs ovarian function by inducing granulosa cell (GC) apoptosis. However, the mechanism inducing GC apoptosis has not been characterized. Here, we found that ovarian GCs from aging patients showed increased oxidative stress, enhanced reactive oxygen species activity, and significantly decreased expression of the known antiapoptotic factor sphingosine-1-phosphate/sphingosine kinase 1 (SPHK1) in GCs. Interestingly, the expression of Krüppel-like factor 12 (KLF12) was significantly increased in the ovarian GCs of aging patients. Furthermore, we determined that KLF12 was significantly upregulated in hydrogen peroxide-treated GCs and a 3-nitropropionic acid-induced in vivo model of ovarian oxidative stress. This phenotype was further confirmed to result from inhibition of SPHK1 by KLF12. Interestingly, when endogenous KLF12 was knocked down, it rescued oxidative stress-induced apoptosis. Meanwhile, supplementation with SPHK1 partially reversed oxidative stress-induced apoptosis. However, this function was lost in SPHK1 with deletion of the binding region to the KLF12 promoter. SPHK1 reversed apoptosis caused by hydrogen peroxide-KLF12 overexpression, a result further confirmed in an in vitro ovarian culture model and an in vivo 3-nitropropionic acid-induced ovarian oxidative stress model. Overall, our study reveals that KLF12 is involved in regulating apoptosis induced by oxidative stress in aging ovarian GCs and that sphingosine-1-phosphate/SPHK1 can rescue GC apoptosis by interacting with KLF12 in negative feedback.

Topics: Aging; Apoptosis; Feedback, Physiological; Female; Granulosa Cells; Humans; Hydrogen Peroxide; In Vitro Techniques; Kruppel-Like Transcription Factors; Lysophospholipids; Organ Culture Techniques; Oxidative Stress; Phosphotransferases (Alcohol Group Acceptor); Promoter Regions, Genetic; Reactive Oxygen Species; Sphingosine

The G protein-coupled receptor (GPCR) US28 encoded by the human cytomegalovirus (HCMV) is associated with accelerated progression of glioblastomas, aggressive brain tumors with a generally poor prognosis. Here, we showed that US28 increased the malignancy of U251 glioblastoma cells by enhancing signaling mediated by sphingosine-1-phosphate (S1P), a bioactive lipid that stimulates oncogenic pathways in glioblastoma. US28 expression increased the abundance of the key components of the S1P signaling axis, including an enzyme that generates S1P [sphingosine kinase 1 (SK1)], an S1P receptor [S1P receptor 1 (S1P

Topics: Glioblastoma; Humans; Lysophospholipids; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Glioblastoma patients commonly develop resistance to temozolomide chemotherapy. Hypoxia, which supports chemotherapy resistance, favors the expansion of glioblastoma stem cells (GSC), contributing to tumor relapse. Because of a deregulated sphingolipid metabolism, glioblastoma tissues contain high levels of the pro-survival sphingosine-1-phosphate and low levels of the pro-apoptotic ceramide. The latter can be metabolized to sphingosine-1-phosphate by sphingosine kinase (SK) 1 that is overexpressed in glioblastoma. The small molecule SKI-II inhibits SK and dihydroceramide desaturase 1, which converts dihydroceramide to ceramide. We previously reported that SKI-II combined with temozolomide induces caspase-dependent cell death, preceded by dihydrosphingolipids accumulation and autophagy in normoxia. In the present study, we investigated the effects of a low-dose combination of temozolomide and SKI-II under normoxia and hypoxia in glioblastoma cells and patient-derived GCSs.. Drug synergism was analyzed with the Chou-Talalay Combination Index method. Dose-effect curves of each drug were determined with the Sulforhodamine B colorimetric assay. Cell death mechanisms and autophagy were analyzed by immunofluorescence, flow cytometry and western blot; sphingolipid metabolism alterations by mass spectrometry and gene expression analysis. GSCs self-renewal capacity was determined using extreme limiting dilution assays and invasion of glioblastoma cells using a 3D spheroid model.. Temozolomide resistance of glioblastoma cells was increased under hypoxia. However, combination of temozolomide (48 µM) with SKI-II (2.66 µM) synergistically inhibited glioblastoma cell growth and potentiated glioblastoma cell death relative to single treatments under hypoxia. This low-dose combination did not induce dihydrosphingolipids accumulation, but a decrease in ceramide and its metabolites. It induced oxidative and endoplasmic reticulum stress and triggered caspase-independent cell death. It impaired the self-renewal capacity of temozolomide-resistant GSCs, especially under hypoxia. Furthermore, it decreased invasion of glioblastoma cell spheroids.. This in vitro study provides novel insights on the links between sphingolipid metabolism and invasion, a hallmark of cancer, and cancer stem cells, key drivers of cancer. It demonstrates the therapeutic potential of approaches that combine modulation of sphingolipid metabolism with first-line agent temozolomide in overcoming tumor growth and relapse by reducing hypoxia-induced resistance to chemotherapy and by targeting both differentiated and stem glioblastoma cells.

Topics: Antineoplastic Agents; Cell Death; Glioblastoma; Humans; Neoplasm Recurrence, Local; Neoplastic Processes; Sphingolipids; Temozolomide

Sphingosine-1-phosphate (S1P) is a pleiotropic, bioactive, lipid mediator, produced by sphingosine kinase 1 (SphK1). In this study, we evaluated the expression of phosphorylated SphK1 (pSphK1) in patients with pancreatic ductal adenocarcinoma (PDAC) and investigated its clinical significance.. A total of 111 patients who underwent curative-intent resection for PDAC were enrolled. We investigated pSphK1 (Ser-225) expression in surgically resected specimens of PDAC using immunohistochemistry. The patients were divided into two groups according to pSphK1 immunoreactive expression: a pSphK1-high group (n=63) and a pSphK1-low group (n=48).. Logistic regression analyses revealed that lymphatic invasion (p=0.007) was a significantly independent factor associated with high pSphK1 immunoreactive expression. The pSphK1-high group showed significantly worse disease-specific survival (DSS) than the pSphK1-low group (5-year DSS rate, 19.6% vs. 58.7%; p=0.001). High pSphK1 immunoreactive expression (hazard ratio=2.547; 95% confidence interval= 1.434-4.527; p=0.001) was an independent prognostic factor for DSS.. High pSphK1 expression is independently associated with lymphatic invasion and unfavorable prognosis in PDAC patients. Thus, the SphK1-S1P axis may be important in mechanisms of tumor progression, such as lymphatic invasion, in PDAC patients.

Topics: Carcinoma, Pancreatic Ductal; Clinical Relevance; Humans; Pancreatic Neoplasms

Sphingosine 1-phosphate (S1P) has been implicated in brown adipose tissue (BAT) formation and energy consumption; however, the mechanistic role of sphingolipids, including S1P, in BAT remains unclear. Here, we showed that, in mice, BAT activation by cold exposure upregulated mRNA and protein expression of the S1P-synthesizing enzyme sphingosine kinase 1 (SphK1) and S1P production in BAT. Treatment of wild-type brown adipocytes with exogenous S1P or S1P receptor subtype-selective agonists stimulated triglyceride (TG) breakdown only marginally, compared with noradrenaline. However, genetic deletion of Sphk1 resulted in hypothermia and diminished body weight loss upon cold exposure, suggesting that SphK1 is involved in thermogenesis through mechanisms different from receptor-mediated, extracellular action of S1P. In BAT of wild-type mice, SphK1 was localized largely in the lysosomes of brown adipocytes. In the brown adipocytes of Sphk1

Topics: Adipocytes, Brown; Adipose Tissue, Brown; Animals; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Signal Transduction; Sphingosine; Triglycerides

This study evaluated changes in the levels of Sphingosine-1-Phosphate (S1P) and Sphingosine Kinase (SPHK) activity in response to non-surgical periodontal treatment in humans.. Diseased (n = 65) and healthy sites (n = 72) were screened in 18 patients with localized periodontitis stage II or III. Periodontal clinical parameters were recorded, and the gingival crevicular fluid (GCF) collected at baseline, 30 and 90 days of non-surgical treatment. Internal control sites without attachment loss/bleeding were sampled at baseline and after 90 days of treatment. SPHK activity and S1P levels and SPHK 1/2 isoforms were determined in the GCF at different time points using ELISA.. Non-surgical treatment caused significant improvement in all periodontal clinical parameters (p < 0.01). Activity of SPHK and S1P levels was decreased (p < 0.05) 30 days after treatment and continued up to 90 days (p < 0.01); control sites remained unchanged throughout the study and resembled treated sites at 3 months (p > 0.05). SPHK1 levels presented decrease after periodontal treatment (p < 0.001). SPHK2 levels were lower than SPHK1 (p < 0.001) and remained unchanged.. S1P levels and SPHK activity decreased within 3 months of non-surgical periodontal treatment, which were correlated with improvements in periodontal parameters. Only SPHK1 levels varied significantly in the states of health and disease.

Topics: Humans; Lysophospholipids; Periodontitis; Periodontium; Phosphotransferases (Alcohol Group Acceptor)

The population with diabetes mellitus-induced erectile dysfunction is increasing rapidly, but current drugs are not effective in treating erectile dysfunction. Studies of the traditional Chinese medicine extract berberine on diabetes and its complications provide us with new ideas.. To evaluate the therapeutic effect and potential mechanism of berberine on the erectile function of diabetic rats.. Fifty male Sprague-Dawley rats were randomly grouped, and 42 rats were injected intraperitoneally with streptozotocin to establish a diabetes model. Erectile dysfunction rats were screened out through the apomorphine test and randomly divided into the diabetes mellitus and berberine groups, and these animals were administered berberine (200 mg/kg/day) and normal saline by gavage for 4 weeks. Primary corpus cavernous smooth muscle cells from healthy rats were cultured and treated with berberine.. Fasting blood glucose in the diabetes mellitus group was significantly increased, while berberine showed no significant effect on glucose. Erectile function was obviously impaired in the diabetes mellitus group, and berberine administration partially rescued this impairment. The expression of sphingosine kinase 1, S1PR2, and sphingosine-1-phosphate in the diabetes mellitus group was increased. Berberine partially inhibited the expression of sphingosine kinase 1 and S1PR2, but the decrease in sphingosine-1-phosphate was not significant. Moreover, mitogen-activated protein kinase pathway factor expression was upregulated and eNOS activity was decreased in the diabetes mellitus group. Berberine treatment could partially reverse these alterations. Severe fibrosis and apoptosis were detected in diabetic rats, accompanied by higher expression of TGFβ1, collagen I/IV, Bax/Bcl-2, and caspase 3 than in the other groups. However, supplementation with berberine inhibited the expression of these proteins and attenuated fibrosis and apoptosis.. Berberine ameliorated erectile dysfunction in rats with diabetes mellitus, possibly by improving endothelial function and inhibiting apoptosis and fibrosis by suppressing the sphingosine kinase 1/sphingosine-1-phosphate/S1PR2 and mitogen-activated protein kinase pathways.

Topics: Animals; Apoptosis; Berberine; Diabetes Mellitus, Experimental; Erectile Dysfunction; Lysophospholipids; Male; MAP Kinase Signaling System; Phosphotransferases (Alcohol Group Acceptor); Rats; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Streptozocin

Topics: CD4-Positive T-Lymphocytes; HIV Infections; HIV-1; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); SAM Domain and HD Domain-Containing Protein 1; Sphingosine; Virus Latency

Hypoxia facilitates an aggressive phenotype and immune evasion in solid tumors including bladder cancer (BC). Sphingosine kinase 1 (SphK1) is aberrantly expressed and correlated with poor prognosis in BC patients. However, its roles in hypoxia-evoked malignancies and immune evasion in BC remain elusive.. The expression of SphK1 in BC tissues was analysed using a bioinformatics database. BC cells were transfected with si-SphK1 or recombinant HIF-1α plasmids under hypoxic conditions. The mRNA level, activity and protein expression of SphK1 were determined. Transwell assay was performed to evaluate cell invasion. After co-culture with natural killer (NK) cells, NK cell cytotoxicity to BC cells was assessed. The involvement of sphingosine-1-phosphate (S1P)/HIF-1α signaling was analysed by ELISA, qRT-PCR and western blot.. UALCAN and GEPIA database confirmed high expression of SphK1 in BC tissues. Moreover, hypoxia increased the expression and activity of SphK1. Loss of SphK1 inhibited hypoxia-induced cell invasion. IL-2 induced NK cell activation by secreting TNF-α and IFN-γ. Hypoxia antagonized NK cell activation-evoked cytotoxicity to BC cells. Intriguingly, SphK1 knockdown reversed hypoxia-induced cell resistance to NK cell killing. Mechanically, SphK1 loss inhibited hypoxia-activated the S1P/HIF-1α signaling. However, S1P addition reversed the inhibitory effects of SphK1 down-regulation on hypoxia-activated S1P/HIF-1α signaling. Notably, reactivating HIF-1α overturned the suppressive roles of SphK1 loss in decreasing hypoxia-induced cell invasion and resistance to NK cell cytotoxicity.. Targeting SphK1 may inhibit hypoxia-evoked invasion and immune evasion via the S1P/HIF-1α signaling, indicating a promising therapeutic target for BC.

Topics: Carcinoma; Cell Death; Humans; Hypoxia; Interleukin-2; Killer Cells, Natural; Oncogenes; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Tumor Necrosis Factor-alpha; Urinary Bladder; Urinary Bladder Neoplasms

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Humans; Lung Neoplasms; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Pre-B-Cell Leukemia Transcription Factor 1; Proto-Oncogene Proteins c-akt; Sphingosine

Topics: Animals; Liver; Lysophospholipids; Microcirculation; Phosphotransferases (Alcohol Group Acceptor); Rats; Reperfusion Injury; Signal Transduction; Sphingosine

Influenza remains a world-wide health concern, causing 290,000-600,000 deaths and up to 5 million cases of severe illnesses annually. Noticing the host factors that control biological responses, such as inflammatory cytokine secretion, to influenza virus infection is important for the development of novel drugs. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite and has essential biological functions in inflammation. However, the kinetic effects of influenza virus infection on physiological S1P levels and their signaling in multiple tissues remain unknown. In this study, we utilized a mouse model intranasally infected with 50 or 500 plaque forming units (PFU) of A/Puerto Rico/8/34 (H1N1; PR8) virus to investigate how S1P levels and expression of its regulating factors are affected by influenza virus infection by the liquid-chromatography/mass spectrometry and real-time PCR, respectively. The S1P level was significantly high in the plasma of mice infected with 500 PFU of the virus than that in control mice at 6 day-post-infection (dpi). Elevated gene expression of sphingosine kinase-1 (Sphk1), an S1P synthase, was observed in the liver, lung, white adipose tissue, heart, and aorta of infected mice. This could be responsible for the increased plasma S1P levels as well as the decrease in the hepatic S1P lyase (Sgpl1) gene in the infected mice. These results indicate modulation of S1P-signaling by influenza virus infection. Since S1P regulates inflammation and leukocyte migration, it must be worth trying to target this signaling to control influenza-associated symptoms.

Topics: Aldehyde-Lyases; Animals; Chromatography, Liquid; Disease Models, Animal; Gene Expression Regulation; Influenza A Virus, H1N1 Subtype; Liver; Lung; Lysophospholipids; Male; Mass Spectrometry; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Intrinsic resistance to androgen receptor signalling inhibitors (ARSI) occurs in 20-30% of men with metastatic castration-resistant prostate cancer (mCRPC). Ceramide metabolism may have a role in ARSI resistance. Our study's aim is to investigate the association of the ceramide-sphingosine-1-phosphate (ceramide-S1P) signalling axis with ARSI resistance in mCRPC.. Lipidomic analysis (∼700 lipids) was performed on plasma collected from 132 men with mCRPC, before commencing enzalutamide or abiraterone. AR gene aberrations in 77 of these men were identified by deep sequencing of circulating tumour DNA. Associations between circulating lipids, radiological progression-free survival (rPFS) and overall survival (OS) were examined by Cox regression. Inhibition of ceramide-S1P signalling with sphingosine kinase (SPHK) inhibitors (PF-543 and ABC294640) on enzalutamide efficacy was investigated with in vitro assays, and transcriptomic and lipidomic analyses of prostate cancer (PC) cell lines (LNCaP, C42B, 22Rv1).. Men with elevated circulating ceramide levels had shorter rPFS (HR=2·3, 95% CI=1·5-3·6, p = 0·0004) and shorter OS (HR=2·3, 95% CI=1·4-36, p = 0·0005). The combined presence of an AR aberration with elevated ceramide levels conferred a worse prognosis than the presence of only one or none of these characteristics (median rPFS time = 3·9 vs 8·3 vs 17·7 months; median OS time = 8·9 vs 19·8 vs 34·4 months). SPHK inhibitors enhanced enzalutamide efficacy in PC cell lines. Transcriptomic and lipidomic analyses indicated that enzalutamide combined with SPHK inhibition enhanced PC cell death by SREBP-induced lipotoxicity.. Ceramide-S1P signalling promotes ARSI resistance, which can be reversed with SPHK inhibitors.. None.

Topics: Aged; Aged, 80 and over; Androstenes; Benzamides; Biomarkers, Tumor; Cell Line, Tumor; Ceramides; Circulating Tumor DNA; Drug Resistance, Neoplasm; Humans; Lysophospholipids; Male; Nitriles; Phenylthiohydantoin; Phosphotransferases (Alcohol Group Acceptor); Progression-Free Survival; Prostatic Neoplasms, Castration-Resistant; Receptors, Androgen; Signal Transduction; Sphingosine

Low plasma levels of the signaling lipid metabolite sphingosine 1-phosphate (S1P) are associated with disrupted endothelial cell (EC) barriers, lymphopenia and reduced responsivity to hypoxia. Total S1P levels were also reduced in 23 critically ill patients with coronavirus disease 2019 (COVID-19), and the two main S1P carriers, serum albumin (SA) and high-density lipoprotein (HDL) were dramatically low. Surprisingly, we observed a carrier-changing shift from SA to HDL, which probably prevented an even further drop in S1P levels. Furthermore, intracellular S1P levels in red blood cells (RBCs) were significantly increased in COVID-19 patients compared with healthy controls due to up-regulation of S1P producing sphingosine kinase 1 and down-regulation of S1P degrading lyase expression. Cell culture experiments supported increased sphingosine kinase activity and unchanged S1P release from RBC stores of COVID-19 patients. These observations suggest adaptive mechanisms for maintenance of the vasculature and immunity as well as prevention of tissue hypoxia in COVID-19 patients.

Topics: Aged; Cells, Cultured; COVID-19; Erythrocytes; Humans; Lipoproteins, HDL; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); SARS-CoV-2; Serum Albumin; Sphingosine

Pre-ovulatory mature follicles are not readily induced from gonadotropin (Gn)-independent early follicles in the poor ovarian response (POR) state, characterized by reduced number of retrieved oocytes. Bone morphogenetic protein (BMP), which is expressed in the ovary, contributes to early folliculogenesis, but its precise underlying mechanism remains unknown. The purpose of this study was to examine the effects of BMP-2 on granulosa cells (GCs) of Gn-independent early follicles.. Sphingosine kinase 1 (SPHK1) localization, which produces sphingosine 1-phosphate (S1P), was examined in human early follicles by immunohistochemistry. SPHK1 mRNA levels were examined in Gn-independent bovine GCs (bGCs) and human nonluteinized granulosa cell line (HGrC1) cells. Phosphorylated Yes-associated protein (YAP) expression was evaluated by Western blot, and its localization was evaluated immunocytochemically in bGCs. Verteporfin, a selective YAP inhibitor, was used to explore the influence of YAP on BMP-2-induced bGCs proliferation.. The expression of SPHK1 was observed in human GCs of primary and secondary follicles. BMP-2 significantly induced SPHK1 mRNA expression in bGCs and HGrC1 cells. Both BMP-2 and S1P decreased phosphorylated YAP protein levels and induced the nuclear translocation of YAP significantly, thereby increasing the number of bGCs by suppressing the Hippo pathway. This BMP-2-induced cell proliferation was completely blocked by verteporfin.. This is a first report showing that BMP-2 up-regulated SPHK1 mRNA expression in GCs and promoted GCs proliferation through Hippo pathway suppression. Thus, BMP-2 contributes to Gn-independent folliculogenesis via SPHK1, suggesting a potential therapeutic strategy for the POR patients with follicular dysgenesis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Bone Morphogenetic Protein 2; Cattle; Cell Proliferation; Cells, Cultured; Female; Gonadotropins; Granulosa Cells; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Transcription Factors; YAP-Signaling Proteins

The sphingolipid pool is key regulator of vital cellular functions in Plasmodium falciparum a causative agent for deadly malaria. Erythrocytes, the host for asexual stage of Plasmodium, are major reservoir for Sphingosine-1-phosphate (S1P). Erythrocyte possesses Sphingosine kinase (SphK) that catalyzed its biosynthesis from sphingosine (Sph). Since, Plasmodium lacks SphK homologous protein it can be envisaged that it co-opts sphingolipids from both intraerythrocytic as well as extracellular pools for its growth and development. Herein, by sphingosine-NBD probing, we report that infected erythrocytes imports Sph from extracellular pool, which is converted to S1P and thereby taken by P. falciparum. Next, by targeting of the SphK through specific inhibitor N,N-Dimethylsphingosine DMS, we show a reduction in erythrocyte endogenous S1P pool and SphK-phosphorylation that led to inhibition in growth and development of ring stage P. falciparum. Owing to the role of S1P in erythrocyte glycolysis we analyzed uptake of NBD-Glucose and production of lactate in DMS treated and untreated plasmodium. DMS treatment led to decreased glycolysis in Plasmodium. Interestingly the host free Plasmodium did not show any effect on glycolysis with DMS treatment indicating its host-mediated effect. Further to understand the in-vivo anti-plasmodial effects of exogenous and endogenous erythrocyte S1P level, Sphingosine-1-phosphate lyase (S1PL) inhibitor (THI), S1P and SphK-1 inhibitor (DMS), were used in Plasmodium berghei ANKA (PbA) mice model. DMS treatment led to reduction of endogenous S1P conferred significant decrease in parasite load, whereas the plasma level S1P modulated by (THI) and exogenous S1P have no effect on growth of Plasmodium. This suggested erythrocyte endogenous S1P pool is important for Plasmodium growth whereas the plasma level S1P has no effect. Altogether, this study provides insight on cellular processes regulated by S1P in P. falciparum and highlights the novel mechanistically distinct molecular target i.e. SphK-1.

Topics: Erythrocytes; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Plasmodium falciparum; Sphingosine

Collecting duct cells are physiologically subject to the hypertonic environment of the kidney. This condition is necessary for kidney maturation and function but represents a stress condition that requires active strategies to ensure epithelial integrity. Madin-Darby Canine Kidney (MDCK) cells develop the differentiated phenotype of collecting duct cells when subject to hypertonicity, serving as a model to study epithelial preservation and homeostasis in this particular environment. The integrity of epithelia is essential to achieve the required functional barrier. One of the mechanisms that ensure integrity is cell extrusion, a process initiated by sphingosine-1-phosphate (S1P) to remove dying or surplus cells while maintaining the epithelium barrier. Both types start with the activation of S1P receptor type 2, located in neighboring cells. In this work, we studied the effect of cell differentiation induced by hypertonicity on cell extrusion in MDCK cells, and we provide new insights into the associated molecular mechanism. We found that the different stages of differentiation influence the rate of apoptotic cell extrusion. Besides, we used a novel methodology to demonstrate that S1P increase in extruding cells of differentiated monolayers. These results show for first time that cell extrusion is triggered by the single-cell synthesis of S1P by sphingosine kinase 2 (SphK2), but not SphK1, of the extruding cell itself. Moreover, the inhibition or knockdown of SphK2 prevents cell extrusion and cell-cell junction protein degradation, but not apoptotic nuclear fragmentation. Thus, we propose SphK2 as the biochemical key to ensure the preservation of the epithelial barrier under hypertonic stress.

Topics: Animals; Apoptosis; Cell Differentiation; Dogs; Kidney; Lysophospholipids; Madin Darby Canine Kidney Cells; Phosphotransferases (Alcohol Group Acceptor); Single-Cell Analysis; Sphingosine

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is associated with high sphingosine kinase 1(SPHK1) expression in the colon, however its role in pathogenesis of UC is not clearly understood so, the aim of the present study was to clarify the role of SPHK1 and investigate whether the anti-inflammatory effects of metformin in UC is mediated by Sphingosine kinase 1/sphingosine 1 phosphate (S1P) signaling pathway.. Colitis was induced in adult male wistar rats by intra rectal administration of oxazolone in the fifth and seventh days from initial presensitization. Oxazolone treated rats were divided into untreated oxazolone group, metformin and mesalazine treated groups both in a dose of 100 mg/kg/day orally for 21 days. Along with these groups normal control and saline groups were used .Colitis was assessed by colon length, disease activity index (DAI) and histological examination of colontissue. Plasma samples were used to measure S1P.SPHK1 activity, signal transducer and activator of transcription -3(STAT-3), interleukin-6 (IL-6), nitric oxide (NO), myeloperoxidase activity (MPO), reduced glutathione (GSH) and tissue expression of intracellular cell adhesion molecule -1(ICAM-1) and caspase-3 genes were measured in tissue.. Metformin successfully attenuated oxazolone colitis by increasing colon length, decreasing DAI and improved colon histologic picture. Metformin also induced a significant decrease in Plasma SIP, SPHK1 activity, inflammatory, oxidative stress markers, ICAM-1 and Caspase-3 genes expression compared to oxazolone group.. It is revealed that metformin alleviated inflammation and underlying mechanism may result from inhibition of SPHK1/S1P signaling pathway.

Topics: Animals; Colitis, Ulcerative; Colon; Inflammation; Lysophospholipids; Male; Metformin; Oxazolone; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Signal Transduction; Sphingosine

Sphingosine kinase-1 (Sphk1) and its product, sphingosine-1-phosphate (S1P) are important regulators of cardiac growth and function. Numerous studies have reported that Sphk1/S1P signaling is essential for embryonic cardiac development and promotes pathological cardiac hypertrophy in adulthood. However, no studies have addressed the role of Sphk1 in postnatal cardiomyocyte (CM) development so far. The present study aimed to assess the molecular mechanism(s) by which

Topics: Animals; Animals, Newborn; Cell Differentiation; Cell Proliferation; Lysophospholipids; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Small Interfering; Signal Transduction; Sphingosine

Breast cancer MCF-7 cell-line-derived mammospheres were shown to be enriched in cells with a CD44+/CD24- surface profile, consistent with breast cancer stem cells (BCSC). These BCSC were previously reported to express key sphingolipid signaling effectors, including pro-oncogenic sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1P3). In this study, we explored intracellular trafficking and localization of SphK1 and S1P3 in parental MCF-7 cells, and MCF-7 derived BCSC-enriched mammospheres treated with growth- or apoptosis-stimulating agents. Intracellular trafficking and localization were assessed using confocal microscopy and cell fractionation, while CD44+/CD24- marker status was confirmed by flow cytometry. Mammospheres expressed significantly higher levels of S1P3 compared to parental MCF-7 cells (

Topics: Breast Neoplasms; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Lysophospholipids; MCF-7 Cells; Phosphotransferases (Alcohol Group Acceptor); Protein Transport; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Tumor Necrosis Factor-alpha

Topics: Blood Platelets; Carrier Proteins; Cell Shape; Dose-Response Relationship, Drug; Humans; Lysophospholipids; Peptide Fragments; Peptides; Phosphotransferases (Alcohol Group Acceptor); Platelet Activation; Platelet Aggregation; Receptor, PAR-1; Sphingosine; Sphingosine-1-Phosphate Receptors

Sphingosine kinase 1 (Sphk1) and the signaling molecule sphingosine-1-phosphate (S1P) are known to be key regulators of a variety of important biological processes, such as neovascularization. Nitric oxide (NO) is also known to play a role in vasoactive properties, whether Sphk1/S1P signaling is able to alter angiogenesis in the context of cerebral ischemia-reperfusion injury (IRI), and whether such activity is linked with NO production, however, remains uncertain.. We used immunofluorescence to detect the expression of Sphk1 and NOS in cerebral epithelial cells (EC) after IR or oxygen-glucose deprivation (OGDR). Western blotting was used to detect the Sphk1 and NOS protein levels in brain tissues or HBMECs. Adenovirus transfection was used to inhibit Sphk1 and NOS. An NO kit was used to detect NO contents in brain tissues and epithelial cells. Tube formation assays were conducted to measure angiogenesis.. We determined that EC used in a model of cerebral IRI expressed Sphk1, and that inhibiting this expression led to decreased expression of two isoforms of NO synthase (eNOS and iNOS), as well as to decrease neovascularization density and NO production following injury. In HBMECs, knocking down Sphk1 markedly reduced NO production owing to reduced eNOS activity, and inhibiting eNOS directly similarly decreased NO production in a manner which could be reversed via exogenously treating cells with S1P. We further found that knocking down Sphk1 reduced HBMEC eNOS expression, in addition to decreasing the adhesion, migration, and tube formation abilities of these cells under OGDR conditions.. Based on these results, we therefore postulate that Sphk1/S1P signaling is able to mediate angiogenesis following cerebral IRI via the regulation of eNOS activity and NO production. As such, targeting these pathways may potentially represent a novel means of improving patient prognosis in those suffering from cerebral IRI.

Topics: Animals; Brain Ischemia; Cells, Cultured; Humans; Lysophospholipids; Male; Neovascularization, Pathologic; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Reperfusion Injury; Sphingosine

Infections with the herpes simplex virus type 1 (HSV-1) are common and widespread. Most infections remain undetected but severe forms may develop in newborns and in immunocompromised patients. Moreover, HSV-1 might be involved in the pathogenesis of atherosclerosis, which may include viral infection of the endothelium. Antiviral therapy is efficient to treat symptomatic patients. However, an increasing accumulation of resistance-associated mutations has been observed in the viral genome. Thus, new antiviral strategies are focused on host factors. Among others, signaling of bioactive sphingolipids seems to be important in mediating HSV-1 replication. With the present study, regulation and function of sphingosine-1-phosphate (S1P)-based signaling were analyzed in HSV-1-infected human umbilical vein endothelial cells (HUVEC). Our data indicate that viral replication in endothelial cells relies on sphingosine kinase (SK) activity and S1P receptor (S1PR)

Topics: Cells, Cultured; Herpesvirus 1, Human; Host Microbial Interactions; Human Umbilical Vein Endothelial Cells; Humans; Lysophospholipids; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingolipids; Sphingosine; Virus Replication

Sphingosine-1-phosphate (S1P) is an important sphingolipid metabolite that regulates a wide range of physiological and pathophysiological processes. Our previous studies show that S1P selectively induces cell apoptosis in human breast cancer luminal A subtype cell line MCF7. In addition, S1P exhibits synergistic effects with chemotherapy drugs against both MCF7 and luminal B subtype cell line MDA-MB-361 at concentration in the high nM to low μM range. In the current study, we evaluated the effect of S1P on proliferation, apoptosis and cytotoxicity towards a panel of nine triple-negative breast cancer with basal-like morphology (TNBC-BL) cell lines (HCC1599, HCC1937, HCC1143, MDA-MB-468, HCC38, HCC70, HCC1806, HCC1187 and DU4475) in the same concentration range. S1P exhibited mild to moderate effects (<20% increase comparted to control) towards the TNBC-BL cell lines except HCC38, HCC70 and HCC1806. Furthermore, it increased cell apoptosis by ~15-20% in all the cell lines compared to the control, and elicited moderate to strong cytotoxic effect towards all cell lines except MDA-MB-468 and HCC1806. However, no synergistic/additive effect was observed between S1P and chemotherapy drug docetaxel for any TNBC-BL cell line.

Topics: Antineoplastic Agents; Apoptosis; Breast; Cell Line, Tumor; Cell Proliferation; Cell Survival; Docetaxel; Drug Interactions; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Sphingosine-1-Phosphate Receptors; Triple Negative Breast Neoplasms

Sphingolipid metabolism is increasingly recognised as a therapeutic target in cancer due to its regulation of cell proliferation and apoptosis. The sphingolipid rheostat is proposed to control cell fate through maintaining balance between pro-apoptotic and pro-survival sphingolipids. This balance is regulated by metabolising enzymes involved in sphingolipid production. One such enzyme, sphingosine kinase-2 (SPHK2), produces pro-survival sphingosine 1-phosphate (S1P) by phosphorylation of pro-apoptotic sphingosine. Elevated SPHK2 has been found in multiple cancer types and contributes to cell survival, chemotherapeutic resistance and apoptosis resistance. We have previously shown elevation of S1P in large granular lymphocyte (LGL) leukaemia serum and cells isolated from patients. Here, we examined SPHK2 expression in LGL leukaemia and found SPHK2 mRNA and protein upregulation in a majority of LGL leukaemia patient samples. Knockdown of SPHK2 with siRNA in LGL leukaemia cell lines decreased proliferation. Additionally, the use of ABC294640 or K145, both SPHK2-specific inhibitors, decreased viability of LGL leukaemia cell lines. ABC294640 selectively induced apoptosis in LGL cell lines and freshly isolated LGL leukaemia patient cells compared to normal controls. Mechanistically, SPHK2 inhibition downregulated pro-survival myeloid cell leukaemia-1 (Mcl-1) protein through proteasomal degradation. Targeting of SPHK2 therefore provides a novel therapeutic approach for the treatment of LGL leukaemia.

Topics: Adamantane; Adult; Aged; Apoptosis; Enzyme Induction; Female; Gene Expression Regulation, Leukemic; Humans; Leukemia, Large Granular Lymphocytic; Leukocytes, Mononuclear; Lysophospholipids; Male; Middle Aged; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Peptide Fragments; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Pyridines; RNA Interference; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Sphingosine; Thiazolidinediones; Up-Regulation

It is a great honor to be asked to write a "Reflections" article by one of the true icons of biochemistry, Herb Tabor. I felt humbled, especially since it follows many written by biochemists I admire and whose contributions have shaped major advances in biochemistry and molecular biology in the last century. Here I present my personal reflections on my adventure with the bioactive sphingolipid metabolite sphingosine-1-phosphate intertwined with those of my family life as a wife, mother, and grandmother. These reflections brought back many memories of events in my early career that played significant roles in determining the path I have taken for more than 40 years and that brought much fun and satisfaction into my life. It has been an exciting journey so far, with many surprises along the way, that still continues.

Topics: Autobiographies as Topic; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingolipids; Sphingosine; Sphingosine-1-Phosphate Receptors

Extremely low-frequency electromagnetic fields (ELF-EMFs) present a kind of common non-ionizing radiation in public and occupational environments. Previous studies have suggested that ELF-EMF exposure might have a potential impact on co-carcinogenesis and the progression of tumorigenesis by inducing cell proliferation. However, the underlying mechanisms remain largely unknown. In this study, we investigated the possible role of the sphingosine-1-phosphate (S1P)-related pathway in regulating cell proliferation induced by 50-Hz, 0.4-mT magnetic-field (MF) exposure. The results showed that MF exposure significantly promoted sphingosine kinase 1 (SphK1) activity, and that inhibition of the SphK1-S1P-S1P receptor (S1PR) pathway could remarkably reverse MF-induced cell proliferation. Additionally, we could infer indirectly from an exogenous-S1P experiment that MF-induced S1P might act on S1PR1/3 in a paracrine and/or autocrine manner to mediate the proliferation effect. Notably, although the MF activated the extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) pathways, the SphK1-S1P-S1PR1/3 cascade regulated MF-induced proliferation by activating the ERK rather than the Akt pathway. Taken together, the findings of this study indicated that the SphK1-S1P-S1PR1/3 cascade played an important role in MF-induced proliferation by mediating the ERK signaling pathway, which could bring new insights into understanding and preventing the adverse effects of MFs.

Topics: Amnion; Animals; Cell Line; Cell Proliferation; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Humans; Lysophospholipids; Magnetic Fields; MAP Kinase Signaling System; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Sphingosine; Sphingosine-1-Phosphate Receptors

Topics: Active Transport, Cell Nucleus; Adaptor Proteins, Signal Transducing; Alveolar Epithelial Cells; Animals; Bleomycin; Cell Cycle Proteins; Fibroblasts; Fibronectins; Gene Deletion; Gene Expression; Hippo Signaling Pathway; Humans; Idiopathic Pulmonary Fibrosis; Immunohistochemistry; Lysophospholipids; Methanol; Mice; Mitochondria; Phosphotransferases (Alcohol Group Acceptor); Protein Serine-Threonine Kinases; Pyrrolidines; Reactive Oxygen Species; Signal Transduction; Sphingosine; Sulfones; Transcription Factors; Transforming Growth Factor beta1; YAP-Signaling Proteins

Cancer stem cells (CSCs) represent rare tumor cell populations capable of self-renewal, differentiation, and tumor initiation and are highly resistant to chemotherapy and radiotherapy. Thus, therapeutic approaches that can effectively target CSCs and tumor cells could be the key to efficient tumor treatment. In this study, we explored the function of SPHK1 in breast CSCs and non-CSCs. We showed that RNAi-mediated knockdown of SPHK1 inhibited cell proliferation and induced apoptosis in both breast CSCs and non-CSCs, while ectopic expression of SPHK1 enhanced breast CSC survival and mammosphere forming efficiency. We identified STAT1 and IFN signaling as key regulatory targets of SPHK1 and demonstrated that an important mechanism by which SPHK1 promotes cancer cell survival is through the suppression of STAT1. We further demonstrated that SPHK1 inhibitors, FTY720 and PF543, synergized with doxorubicin in targeting both breast CSCs and non-CSCs. In conclusion, we provide important evidence that SPHK1 is a key regulator of cell survival and proliferation in breast CSCs and non-CSCs and is an attractive target for the design of future therapies.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Lineage; Cell Proliferation; Cell Survival; Doxorubicin; Female; HEK293 Cells; Humans; Interferons; Lysophospholipids; Neoplastic Stem Cells; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Proteomics; Signal Transduction; Spheroids, Cellular; Sphingosine; STAT1 Transcription Factor

Cardiac fibrosis and myocyte hypertrophy are hallmarks of the cardiac remodelling process in cardiomyopathies such as heart failure (HF). Dyslipidemia or dysregulation of lipids contribute to HF. The dysregulation of high density lipoproteins (HDL) could lead to altered levels of other lipid metabolites that are bound to it such as sphingosine-1- phosphate (S1P). Recently, it has been shown that S1P and its analogue dihydrosphingosine-1-phosphate (dhS1P) are bound to HDL in plasma. The effects of dhS1P on cardiac cells have been obscure. In this study, we show that extracellular dhS1P is able to increase collagen synthesis in neonatal rat cardiac fibroblasts (NCFs) and cause hypertrophy of neonatal cardiac myocytes (NCMs). The janus kinase/signal transducer and activator (JAK/STAT) signalling pathway was involved in the increased collagen synthesis by dhS1P, through sustained increase of tissue inhibitor of matrix metalloproteinase 1 (TIMP1). Extracellular dhS1P increased phosphorylation levels of STAT1 and STAT3 proteins, also caused an early increase in gene expression of transforming growth factor-β (TGFβ), and sustained increase in TIMP1. Inhibition of JAKs led to inhibition of TIMP1 and TGFβ gene and protein expression. We also show that dhS1P is able to cause NCM hypertrophy through S1P-receptor-1 (S1PR1) signalling which is opposite to that of its analogue, S1P. Taken together, our results show that dhS1P increases collagen synthesis in cardiac fibroblasts causing fibrosis through dhS1P-JAK/STAT-TIMP1 signalling.

Topics: Animals; Animals, Newborn; Biomarkers; Cell Differentiation; Collagen; Fibroblasts; Gene Expression Regulation; Hypertrophy; Janus Kinases; Lysophospholipids; Matrix Metalloproteinase 2; Models, Biological; Myocardium; Myocytes, Cardiac; Oxadiazoles; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Smad2 Protein; Sphingosine; STAT Transcription Factors; Thiophenes; Time Factors; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

There is thought to be a strong relationship between sphingosine-1-phosphate (S1P) signaling and pathophysiolosy of cerebral ischemia. We examined the change of expression and distribution of S1P receptors (S1PRs) and sphingosine kinases (SphKs) after cerebral ischemia in male C57BL6/J mice using immunohistochemical analysis at 1, 5, 14, and 28 days after 30 min of transient middle cerebral artery occlusion (tMCAO). S1PR1, 3, and 5 were transiently induced in the cells, which were morphologically similar to neurons in the peri-infarct lesion with a peak seen at 1 day after tMCAO (p < 0.01 vs. sham control). S1PR2 appeared in the inner layer of vessels in the ischemic core (p < 0.01 vs. sham control) and the peri-infarct lesion (p < 0.01 vs. sham control) at the acute phase after tMCAO. However, SphK1 was strongly induced at 1 and 5 days after tMCAO (p < 0.01 vs. sham control) in the peri-infarct lesion, whereas SphK2 expression did not change. Western blot analysis at 1 and 5 days after 30 min of tMCAO revealed that the expression of S1PRs were transiently enhanced at the acute phase, which was consistent with the immunohistochemical results. Double immunofluorescent analysis revealed S1PR2/NG2- and S1PR2/CD31-, S1PR3/CD31-, and S1PR5/CD31-double positive cells in the peri-infarct lesion 1 day after tMCAO. The present results suggest that S1PRs and SphK1 may be important therapeutic targets for rescuing the peri-infarct lesion.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Infarction, Middle Cerebral Artery; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Neurons; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Transcriptional Activation

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma. Despite being responsive to combination chemotherapy, median survival remains around 5 years due to high rates of relapse. Sphingolipid metabolism regulates MCL survival and proliferation and we found that sphingosine-1-phosphate (S1P) is upregulated in MCL cells. Therapeutic targeting of the S1P

Topics: Animals; Antigens, CD1d; Cardiolipins; Cell Line, Tumor; Humans; Lymphocyte Activation; Lymphoma, Mantle-Cell; Lysophospholipids; Mice, Inbred C57BL; Natural Killer T-Cells; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Rheumatoid arthritis (RA) is a common inflammatory autoimmune disease characterized by the formation of joint synovitis and pannus. Sphingosine 1-phosphate (S1P) is an important mediator related to angiogenesis, inflammation and autoimmunity. As Geniposide (GE) has potent immuno-modulation function, we investigated the effects on the dynamic balance of angiogenesis-related factors and Sphingosine kinase 1 (SphK1)-S1P-S1P receptor 1 (S1PR1) signal transduction in adjuvant-induced arthritis (AA) rats.. The model evaluation was performed from paw swelling degree, arthritis index and movement score. The immunohistochemistry and enzyme-linked immunosorbent assay were used to study the microvascular density (MVD) and pro/anti-angiogenic factors levels. The cell viability was examined by cell counting kit-8 assay. SphK1, S1PR1 mRNA and protein levels in fibroblast-like synoviocytes (FLSs) were detected by quantitative real-time polymerase chain reaction and Western blotting.. The results showed that GE can apparently suppressed the inflammatory pathological status. The arthritis index, paw swelling and MVD of AA rats were decreased with dose dependence (. It indicated that GE reduces the activity of SphK1 by restoring the dynamic balance between pro/anti-angiogenic factors, thereby interfering with SphK1-S1P-S1PR1 signal transduction, reducing the formation of synovial microvessels and exerting anti-angiogenesis effect of RA.

Topics: Animals; Cell Survival; Cells, Cultured; Drug Delivery Systems; Iridoids; Lysophospholipids; Male; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Random Allocation; Rats; Rats, Sprague-Dawley; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Lung alveolar epithelium is composed of alveolar type I (AT1) and type II (AT2) cells. AT1 cells mediate gas exchange, whereas AT2 cells act as progenitor cells to repair injured alveoli. Lung microvascular endothelial cells (LMVECs) play a crucial but still poorly understood role in regulating alveolar repair. Here, we studied the role of the LMVEC-derived bioactive lipid sphingosine-1-phosphate (S1P) in promoting alveolar repair using mice with endothelial-specific deletion of sphingosine kinase 1 (Sphk1), the key enzyme promoting S1P generation. These mutant lungs developed airspace-enlargement lesions and exhibited a reduced number of AT1 cells after Pseudomonas-aeruginosa-induced lung injury. We demonstrated that S1P released by LMVECs acted via its receptor, S1PR2, on AT2 cells and induced nuclear translocation of yes-associated protein (YAP), a regulator of AT2 to AT1 transition. Thus, angiocrine S1P released after injury acts via the S1PR2-YAP signaling axis on AT2 cells to promote AT2 to AT1 differentiation required for alveolar repair.

Topics: Adaptor Proteins, Signal Transducing; Alveolar Epithelial Cells; Animals; Cell Count; Cell Cycle Proteins; Down-Regulation; Female; Lung; Lysophospholipids; Male; Mice; Phenotype; Phosphotransferases (Alcohol Group Acceptor); Pseudomonas aeruginosa; Pulmonary Surfactant-Associated Protein C; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Regeneration; Signal Transduction; Sphingosine; YAP-Signaling Proteins

Recent studies suggested an important contribution of sphingosine-1-phospate (S1P) signaling via its specific receptors (S1PRs) in the production of pro-inflammatory mediators such as Interleukin (IL)-1β in cancer and inflammation. In an inflammation-driven cancer setting, we previously reported that myeloid S1PR1 signaling induces IL-1β production by enhancing NLRP3 (NOD-, LRR- and Pyrin Domain-Containing Protein 3) inflammasome activity. However, the autocrine role of S1P and enzymes acting on the S1P rheostat in myeloid cells are unknown. Using human and mouse macrophages with pharmacological or genetic intervention we explored the relative contribution of sphingosine kinases (SPHKs) in NLRP3 inflammasome activity regulation. We noticed redundancy in SPHK1 and SPHK2 activities towards macrophage NLRP3 inflammasome transcriptional induction and IL-1β secretion. However, pharmacological blockade of both kinases in unison completely abrogated NLRP3 inflammasome induction and IL-1β secretion. Interestingly, human and mouse macrophages demonstrate varied responses towards SPHKs inhibition and IL-1β secretion. Clinical datasets of renal cell carcinoma and psoriasis patients showed a positive correlation between enzymes affecting the S1P rheostat with NLRP3 inflammasome components expression, which corroborates our finding. Our data provide a better understanding on the role of SPHKs and de novo synthesized S1P in macrophage NLRP3 inflammasome activation.

Topics: Animals; Carrier Proteins; Caspase 1; Cells, Cultured; Humans; Inflammasomes; Inflammation; Lysophospholipids; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Cells; NLR Family, Pyrin Domain-Containing 3 Protein; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Nonalcoholic fatty liver (NAFL) is emerging as a leading risk factor of hepatic ischemia/reperfusion (I/R) injury lacking of effective therapy. Lipid dyshomeostasis has been implicated in the hepatopathy of NAFL. Herein, we investigate the bioactive lipids that critically regulate I/R injury in NAFL.. Lipidomics were performed to identify dysregulated lipids in mouse and human NAFL with I/R injury. The alteration of corresponding lipid-metabolizing genes was examined. The effects of the dysregulated lipid metabolism on I/R injury in NAFL were evaluated in mice and primary hepatocytes.. Sphingolipid metabolic pathways responsible for the generation of sphingosine-1-phosphate (S1P) were uncovered to be substantially activated by I/R in mouse NAFL. Sphingosine kinase 1 (Sphk1) was found to be essential for hepatic S1P generation in response to I/R in hepatocytes of NAFL mice. Sphk1 knockdown inhibited the hepatic S1P rise while accumulating ceramides in hepatocytes of NAFL mice, leading to aggressive hepatic I/R injury with upregulation of oxidative stress and increase of reactive oxygen species (ROS). In contrast, administration of exogenous S1P protected hepatocytes of NAFL mice from hepatic I/R injury. Clinical study revealed a significant activation of S1P generation by I/R in liver specimens of NAFL patients. In vitro studies on the L02 human hepatocytes consolidated that inhibiting the generation of S1P by knocking down SPHK1 exaggerated I/R-induced damage and oxidative stress in human hepatocytes of NAFL.. Generation of S1P by SPHK1 is important for protecting NAFL from I/R injury, which may serve as therapeutic targets for hepatic I/R injury in NAFL.

Topics: Animals; Hepatocytes; Humans; Ischemia; Lysophospholipids; Mice; Non-alcoholic Fatty Liver Disease; Phosphotransferases (Alcohol Group Acceptor); Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; Sphingosine

Although previous experiments have implicated sphingosine-1-phosphate (S1P) as a links between immune reactions and cancer progression, the exact mechanism of this interaction has not comprehensively studied in clinical human samples. This study sought to evaluate the S1P regulation by sphingosine kinase 1 (SPHK1), an S1P-producing enzyme, in the immunity/immuno-reactivity of clinical human breast cancer surgical specimens.. S1P levels were examined in tumor, peritumoral, and normal human breast samples using mass spectrometry. Genomics Data Commons data portal of The Cancer Genome Atlas cohort was used to assess the expression of S1P-related and immune-related genes.. S1P levels were significantly higher in tumor samples compared to peritumoral (P < 0.05) or normal human breast samples (P < 0.001). SPHK1 gene expression was elevated in tumoral samples compared to normal breast samples (P < 0.01). Furthermore, the elevated expression of SPHK1 in breast cancer tissue was associated with an increased expression of the different kinds of immune-related genes, such as CD68, CD163, CD4, and FOXP3 (forkhead box P3), in HER2-negative breast cancer. Network analysis showed the central role of SPHK1 in the interaction of S1P signaling and expression of immune cell-related proteins.. We demonstrated that S1P is mainly produced by tumor tissue, rather than peritumoral tissue, in breast cancer patients. Our data revealed the involvement of S1P signaling in the regulation of immune-related genes, suggesting the links between S1P and complicated immune-cancer interactions in breast cancer patients.

Topics: Breast; Breast Neoplasms; Cell Line, Tumor; Chromatography, High Pressure Liquid; Cohort Studies; Datasets as Topic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Interaction Maps; Signal Transduction; Spectrometry, Mass, Electrospray Ionization; Sphingosine; Tandem Mass Spectrometry

Triptolide (TP) exhibits effective activity against colon cancer in multiple preclinical models, but the mechanisms underlying the observed effects are not fully understood. Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid involved in the regulation of colon cancer progression. The aim of this study was to investigate the effect of TP on the sphingosine kinase (SPHK)-S1P signaling pathway in colitis-associated colon cancer.. An azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model and the THP-1 cell line were used to evaluate the therapeutic effects and mechanisms of TP in colitis-associated colon cancer (CACC). Various molecular cell biology experiments, including Western blotting, real-time PCR and immunofluorescence, were used to obtain relevant experimental data. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was also established to detect the levels of S1P in tissue and plasma.. In the AOM/DSS mouse model, TP treatment induced a dose-dependent decrease in tumor incidence and inhibited macrophage recruitment and M2 polarization in the tumors. TP also efficiently decreased the S1P levels and SPHK1/S1PR1/S1PR2 expression and significantly inhibited activation of the S1P-mediated phosphorylation of ERK protein in macrophages.. The results indicated that TP might influence the recruitment and polarization of tumor-associated macrophages by suppressing the SPHK-S1P signaling pathway.

Topics: Animals; Azoxymethane; Colitis; Colitis-Associated Neoplasms; Colon; Dextran Sulfate; Disease Models, Animal; Diterpenes; Epoxy Compounds; Female; Humans; Lysophospholipids; Male; Mice, Inbred BALB C; Mice, Inbred ICR; Mice, Nude; Phenanthrenes; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; THP-1 Cells; Tumor-Associated Macrophages

Sphingosine-1-phophate (S1P) is a sphingolipid-derived signaling molecule that controls diverse cellular functions including cell growth, homeostasis, and stress responses. In a variety of metazoans, cytosolic S1P is transported into the extracellular space where it activates S1P receptors in a concentration-dependent manner. In the free-living nematode

Topics: Animals; Anti-Bacterial Agents; Basic Helix-Loop-Helix Transcription Factors; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Escherichia coli; Host-Pathogen Interactions; Humans; Longevity; Lysophospholipids; Membrane Proteins; Mutation; p38 Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Pseudomonas aeruginosa; Signal Transduction; Sphingosine

There is substantial evidence that the enzymes, sphingosine kinase 1 and 2, which catalyse the formation of the bioactive lipid sphingosine 1-phosphate, are involved in pathophysiological processes. In this chapter, we appraise the evidence that both enzymes are druggable and describe how isoform-specific inhibitors can be developed based on the plasticity of the sphingosine-binding site. This is contextualised with the effect of sphingosine kinase inhibitors in cancer, pulmonary hypertension, neurodegeneration, inflammation and sickling.

Topics: Anemia, Sickle Cell; Binding Sites; Enzyme Inhibitors; Humans; Hypertension, Pulmonary; Inflammation; Lysophospholipids; Neoplasms; Neurodegenerative Diseases; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease, which often affects colon or rectum or both. It is now well recognized that sphingosine kinases-1/sphingosine-1-phosphate (S1P) signaling may have a very significant potential as targets for therapeutic intervention in UC. Compared with the pure dextran sodium sulfate group, administration of PF543 significantly reduced clinical symptoms with less weight loss, diarrhea, and shortening of the colon. The severity of colitis was improved with reduced disease activity index and degree of histological damage in colon. Moreover, treatment with PF543 not only decreased S1P but also inhibited mRNA expression of proinflammatory factors such as interleukin (IL)-1β and IL-6. This suggests that PF543 might exhibit an anti-inflammatory function against colitis through inhibition of expression of proinflammatory factors.

Topics: Animals; Colitis; Colon; Dextran Sulfate; Disease Models, Animal; Enzyme Inhibitors; Lysophospholipids; Male; Methanol; Mice; Mice, Inbred C57BL; Organ Size; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidines; Sphingosine; Spleen; Substrate Specificity; Sulfones

Topics: Antimicrobial Cationic Peptides; Cathelicidins; Cell Differentiation; Cells, Cultured; Endoplasmic Reticulum Stress; Humans; Keratinocytes; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Primary Cell Culture; Sphingosine

While the two mammalian sphingosine kinases, SK1 and SK2, both catalyze the generation of pro-survival sphingosine 1-phosphate (S1P), their roles vary dependent on their different subcellular localization. SK1 is generally found in the cytoplasm or at the plasma membrane where it can promote cell proliferation and survival. SK2 can be present at the plasma membrane where it appears to have a similar function to SK1, but can also be localized to the nucleus, endoplasmic reticulum or mitochondria where it mediates cell death. Although SK2 has been implicated in cancer initiation and progression, the mechanisms regulating SK2 subcellular localization are undefined. Here, we report that SK2 interacts with the intermediate chain subunits of the retrograde-directed transport motor complex, cytoplasmic dynein 1 (DYNC1I1 and -2), and we show that this interaction, particularly with DYNC1I1, facilitates the transport of SK2 away from the plasma membrane. DYNC1I1 is dramatically downregulated in patient samples of glioblastoma (GBM), where lower expression of DYNC1I1 correlates with poorer patient survival. Notably, low DYNC1I1 expression in GBM cells coincided with more SK2 localized to the plasma membrane, where it has been recently implicated in oncogenesis. Re-expression of DYNC1I1 reduced plasma membrane-localized SK2 and extracellular S1P formation, and decreased GBM tumor growth and tumor-associated angiogenesis in vivo. Consistent with this, chemical inhibition of SK2 reduced the viability of patient-derived GBM cells in vitro and decreased GBM tumor growth in vivo. Thus, these findings demonstrate a tumor-suppressive function of DYNC1I1, and uncover new mechanistic insights into SK2 regulation which may have implications in targeting this enzyme as a therapeutic strategy in GBM.

Topics: Animals; Apoptosis; Carcinogenesis; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Cytoplasmic Dyneins; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Glioblastoma; HEK293 Cells; Humans; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Xenograft Model Antitumor Assays

Topics: Animals; Biphenyl Compounds; Cell Line, Tumor; Cell Survival; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Inhibitors; Female; Humans; Lysophospholipids; Melanoma; Mice; Nitrophenols; Phosphotransferases (Alcohol Group Acceptor); Piperazines; Proto-Oncogene Proteins B-raf; Receptors, Lysosphingolipid; Sphingolipids; Sphingosine; Sphingosine-1-Phosphate Receptors; Sulfonamides; Vemurafenib; Xenograft Model Antitumor Assays

Extremely low frequency electromagnetic field (ELF-EMF) is a kind of physical stimulus in public and occupational environment. Numerous studies have indicated that exposure of cells to ELF-EMF could promote cell proliferation. But the detailed mechanisms implicated in these proliferative processes remain unclear. In the present experiment, the possible roles of sphingosine-1-phosphate (S1P) in 50-Hz magnetic field (MF)-induced cell proliferation were investigated. Results showed that exposure of human amniotic (FL) cells to a 50-Hz MF with an intensity of 0.4 mT significantly enhanced ceramide metabolism, increased S1P production, activated extracellular signal regulated kinase 1/2 (ERK1/2), and promoted cell proliferation. All of these effects induced by MF exposure could be inhibited by SKI II, an inhibitor of sphingosine kinase (SphK). In addition, both the cell proliferative response and the ERK1/2 activation induced by MF exposure were blocked completely by U0126, a specific inhibitor of MEK (ERK kinases 1 and 2). Taken together, the findings in present study suggested that S1P mediated 50-Hz MF-induced cell proliferation via triggering ERK1/2 signal pathway.

Topics: Cell Line, Tumor; Cell Proliferation; Electromagnetic Fields; Humans; Lysophospholipids; Magnetic Fields; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 3; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingolipid and cholesterol metabolism are closely associated at the structural, biochemical, and functional levels. Although HDL-associated sphingosine-1-phosphate (S1P) contributes to several HDL functions, and S1P signaling regulates glucose and lipid metabolism, no study has addressed the involvement of S1P in cholesterol efflux. Here, we show that sphingosine kinase (Sphk) activity was induced by the LXR agonist 22(R)-hydroxycholesterol and required for the stimulation of ABCA1-mediated cholesterol efflux to apolipoprotein A-I. In support, pharmacological Sphk inhibition and Sphk2 but not Sphk1 deficiency abrogated efflux. The involved mechanism included stimulation of both transcriptional and functional ABCA1 regulatory pathways and depended for the latter on the S1P receptor 3 (S1P3). Accordingly, S1P3-deficient macrophages were resistant to 22(R)-hydroxycholesterol-stimulated cholesterol efflux. The inability of excess exogenous S1P to further increase efflux was consistent with tonic S1P3 signaling by a pool of constitutively generated Sphk-derived S1P dynamically regulating cholesterol efflux. In summary, we have established S1P as a previously unrecognized intermediate in LXR-stimulated ABCA1-mediated cholesterol efflux and identified S1P/S1P3 signaling as a positive-feedback regulator of cholesterol efflux. This constitutes a novel regulatory mechanism of cholesterol efflux by sphingolipids.

Topics: Animals; Apolipoprotein A-I; ATP Binding Cassette Transporter 1; Biological Transport; Cholesterol; Homeostasis; Lysophospholipids; Macrophages; Mice; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

The role of phospholipid signaling in ovarian cancer is poorly understood. Sphingosine-1-phosphate (S1P) is a bioactive metabolite of sphingosine that has been associated with tumor progression through enhanced cell proliferation and motility. Similarly, sphingosine kinases (SPHK), which catalyze the formation of S1P and thus regulate the sphingolipid rheostat, have been reported to promote tumor growth in a variety of cancers. The findings reported here show that exogenous S1P or overexpression of SPHK1 increased proliferation, migration, invasion, and stem-like phenotypes in ovarian cancer cell lines. Likewise, overexpression of SPHK1 markedly enhanced tumor growth in a xenograft model of ovarian cancer, which was associated with elevation of key markers of proliferation and stemness. The diabetes drug, metformin, has been shown to have anticancer effects. Here, we found that ovarian cancer patients taking metformin had significantly reduced serum S1P levels, a finding that was recapitulated when ovarian cancer cells were treated with metformin and analyzed by lipidomics. These findings suggested that in cancer the sphingolipid rheostat may be a novel metabolic target of metformin. In support of this, metformin blocked hypoxia-induced SPHK1, which was associated with inhibited nuclear translocation and transcriptional activity of hypoxia-inducible factors (HIF1α and HIF2α). Further, ovarian cancer cells with high SPHK1 were found to be highly sensitive to the cytotoxic effects of metformin, whereas ovarian cancer cells with low SPHK1 were resistant. Together, the findings reported here show that hypoxia-induced SPHK1 expression and downstream S1P signaling promote ovarian cancer progression and that tumors with high expression of SPHK1 or S1P levels might have increased sensitivity to the cytotoxic effects of metformin. IMPLICATIONS: Metformin targets sphingolipid metabolism through inhibiting SPHK1, thereby impeding ovarian cancer cell migration, proliferation, and self-renewal.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Movement; Female; Humans; Hypoglycemic Agents; Hypoxia-Inducible Factor 1, alpha Subunit; Lysophospholipids; Metformin; Mice; Mice, Nude; Molecular Targeted Therapy; Ovarian Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Xenograft Model Antitumor Assays

Erythropoiesis is a highly coordinated stepwise process involving the progressive clearance of mitochondria via mitophagy. Based on the expression of several macroautophagy and mitophagy specific genes, we identified a sequential change in the transcriptional pattern during terminal erythroid differentiation. Because erythroid cells are a major source of serum sphingosine-1-phosphate, we analyzed the role of sphingolipid signaling in erythropoiesis and demonstrate that sphingosine kinase activity promotes terminal erythroid differentiation by regulating the expression of key mitophagy genes Pink1 and Bnip3l/Nix. Sphingosine kinase 1 (Sphk1) inhibition also disrupted Pink1-p62 mediated mitochondria clearance in late erythroblasts. Notably, we show that supplementing sphingosine-1-phosphate in vitro can promote erythroid differentiation. Our study clarifies the role of sphingolipid signaling in regulating mitophagy during terminal erythroid differentiation and highlights the potential utility of modulating sphingolipid signaling to facilitate the large-scale production of transfusable red blood cells.

Topics: Animals; Cell Differentiation; Erythropoiesis; Lysophospholipids; Membrane Proteins; Mice; Mice, Transgenic; Mitophagy; Phosphotransferases (Alcohol Group Acceptor); Protein Kinases; Proto-Oncogene Proteins; Signal Transduction; Sphingosine; Tumor Suppressor Proteins

Hypertension is considered the major modifiable risk factor for the development of cognitive impairment. Because increased blood pressure is often accompanied by an activation of the immune system, the concept of neuro-inflammation gained increasing attention in the field of hypertension-associated neurodegeneration. Particularly, hypertension-associated elevated circulating T-lymphocyte populations and target organ damage spurred the interest to understanding mechanisms leading to inflammation-associated brain damage during hypertension. The present study describes sphingosine-1-phosphate (S1P) as major contributor to T-cell chemotaxis to the brain during hypertension-associated neuro-inflammation and cognitive impairment. Using Western blotting, flow cytometry and mass spectrometry approaches, we show that hypertension stimulates a sphingosine kinase 1 (SphK1)-dependent increase of cerebral S1P concentrations in a mouse model of angiotensin II (AngII)-induced hypertension. The development of a distinct S1P gradient between circulating blood and brain tissue associates to elevated CD3+ T-cell numbers in the brain. Inhibition of S1P₁-guided T-cell chemotaxis with the S1P receptor modulator FTY720 protects from augmentation of brain CD3 expression and the development of memory deficits in hypertensive WT mice. In conclusion, our data highlight a new approach to the understanding of hypertension-associated inflammation in degenerative processes of the brain during disease progression.

Topics: Angiotensin II; Animals; Brain; Chemokines; Chemotaxis; Cognition Disorders; Female; Hypertension; Lysophospholipids; Male; Memory Disorders; Mice, Inbred C57BL; Mice, Transgenic; Models, Biological; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; T-Lymphocytes

A small molecule (1) with overlapping affinity for two microRNA (miRNA) precursors was used to inform design of a dimeric compound (2) selective for one of the miRNAs. In particular, 2 selectively targets the microRNA(miR)-515 hairpin precursor to inhibit production of miR-515 that represses sphingosine kinase 1 (SK1), a key enzyme in the biosynthesis of sphingosine 1-phosphate (S1P). Application of 2 to breast cancer cells enhanced SK1 and S1P levels, triggering a migratory phenotype. Knockout of SK1, forced overexpression of miR-515, and application of a small molecule SK1 inhibitor all ablated 2's effect on phenotype, consistent with its designed mode of action. Target profiling studies via Chem-CLIP showed that 2 bound selectively to the miR-515 hairpin precursor in cells. Global neoprotein synthesis upon addition of 2 to MCF-7 breast cancer cells demonstrated 2's selectivity and upregulation of cancer-associated proteins regulated by S1P. The most upregulated protein was human epidermal growth factor receptor 2 (ERBB2/HER2), which is regulated by the SK1/S1P pathway and is normally not expressed in MCF-7 cells. Like triple negative breast cancer (TNBC) cells, the lack of HER2 renders them insusceptible to Herceptin and its antibody-drug conjugate Kadcyla. In addition to proteomics, an RNA-seq study supports that 2 has limited off target effects and other studies support that 2 is more selective than an oligonucleotide. We therefore hypothesized that 2 could sensitize MCF-7 cells to anti-HER2 therapies. Indeed, application of 2 sensitized cells to Herceptin. These results were confirmed in two other cell lines that express miR-515 and are HER2-, the hepatocellular carcinoma cell line HepG2 and the TNBC line MDA-MB-231. Importantly, normal breast epithelial cells (MCF-10A) that do not express miR-515 are not affected by 2. These observations suggest a precision medicine approach to sensitize HER2- cancers to approved anticancer medicines. This study has implications for broadening the therapeutic utility of known targeted cancer therapeutics by using a secondary targeted approach to render otherwise insensitive cells, sensitive to a targeted therapeutic.

Topics: Ado-Trastuzumab Emtansine; Antineoplastic Agents; Base Sequence; Benzimidazoles; Cell Line, Tumor; Drug Design; Humans; Lysophospholipids; MicroRNAs; Phosphotransferases (Alcohol Group Acceptor); Proteome; Receptor, ErbB-2; RNA Precursors; Sphingosine; Trastuzumab; Triazoles

Ulcerative colitis (UC) is a chronic gastrointestinal disorder interfering with life quality. A total of 60 male Wistar rats were divided into four equal groups: Control (group I), hesperidin only (group II), UC untreated (group III), and UC treated with hesperidin (group IV). Hesperidin had modulatory effects on UC pathogenesis, which might be through alleviating colonic sphingosine phosphate phosphatase 2 messenger RNA expression and sphingosine kinase-1 levels, thus suppressing the subsequent downstream inflammatory and apoptotic cascades represented by decreased macrophage inflammatory protein-1α and enhancement of B-cell lymphoma 2 immunohistochemistry expression. Also, it improved mitochondrial biogenesis by increasing the peroxisome proliferator-activated receptor-gamma-coactivator 1-α level. It successfully restored redox potential as evidenced by marked alleviations of the nitric oxide and peroxynitrite levels, increasing total antioxidant capacity, and activating the superoxide dismutase enzyme. Also, hesperidin alleviated the UC disease activity index and improved the histopathological picture. These findings may offer a new therapeutic strategy for UC treatment.

Topics: Animals; Apoptosis; Colitis; Dextran Sulfate; Drug Delivery Systems; Hesperidin; Inflammation; Lysophospholipids; Male; Mitochondria; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Sphingosine

Epithelial-mesenchymal transition (EMT) is a critical process implicated in the initial stage of cancer metastasis, which is the major cause of tumor recurrence and mortality. Although key transcription factors that regulate EMT, such as snail family transcriptional repressor 2 (SNAI2), are well characterized, the upstream signaling pathways controlling these transcriptional mediators are largely unknown, which limits therapeutic strategies. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator, generated by sphingosine kinases (SPHK1 and SPHK2), that mainly exerts its effects by binding to the following 5 GPCRs: S1P

Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lysophospholipids; MCF-7 Cells; Neoplasm Invasiveness; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); RNA Interference; RNA Stability; RNA, Small Interfering; Snail Family Transcription Factors; Sphingosine; Sphingosine-1-Phosphate Receptors; Trans-Activators; Transcription Factors; YAP-Signaling Proteins

We have identified a mechanism to diminish the proliferative capacity of cells during cell expansion using human adiposederived stromal cells (hAD-SCs) as a model of replicative senescence. hAD-SCs of high-passage numbers exhibited a reduced proliferative capacity with accelerated cellular senescence. Levels of key bioactive sphingolipids were significantly increased in these senescent hAD-SCs. Notably, the transcription of sphingosine kinase 1 (SPHK1) was down-regulated in hAD-SCs at high-passage numbers. SPHK1 knockdown as well as inhibition of its enzymatic activity impeded the proliferation of hAD-SCs, with concomitant induction of cellular senescence and accumulation of sphingolipids, as seen in high-passage cells. SPHK1 knockdown-accelerated cellular senescence was attenuated by co-treatment with sphingosine-1-phosphate and an inhibitor of ceramide synthesis, fumonisin B1, but not by treatment with either one alone. Together, these results suggest that transcriptional down-regulation of SPHK1 is a critical inducer of altered sphingolipid profiles and enhances replicative senescence during multiple rounds of cell division. [BMB Reports 2019; 52(3): 220-225].

Topics: Apoptosis; Cell Proliferation; Cellular Senescence; Down-Regulation; Humans; Lysophospholipids; Mesenchymal Stem Cells; Phosphotransferases (Alcohol Group Acceptor); Sphingolipids; Sphingosine

Sphingosine kinase enzymes (SK1 and SK2) catalyze the conversion of sphingosine into sphingosine 1-phosphate and play a key role in lipid signaling and cellular responses. Mapping of isoform amino acid sequence differences for SK2 onto the recently available crystal structures of SK1 suggests that subtle structural differences exist in the foot of the lipid-binding "J-channel" in SK2, the structure of which has yet to be defined by structural biology techniques. We have probed these isoform differences with a ligand series derived from the potent SK1-selective inhibitor, PF-543. Here we show how it is possible, even with relatively conservative changes in compound structure, to systematically tune the activity profile of a ligand from ca. 100-fold SK1-selective inhibition, through equipotent SK1/SK2 inhibition, to reversed 100-fold SK2 selectivity, with retention of nanomolar potency.

Topics: Animals; Humans; Ligands; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Protein Isoforms; Sphingosine

Sphingolipids regulate several aspects of cell behavior and it has been demonstrated that cells adjust their sphingolipid metabolism in response to metabolic needs. Particularly, sphingosine-1-phosphate (S1P), a final product of sphingolipid metabolism, is a potent bioactive lipid involved in the regulation of various cellular processes, including cell proliferation, cell migration, actin cytoskeletal reorganization and cell adhesion. In previous work in rat renal papillae, we showed that sphingosine kinase (SK) expression and S1P levels are developmentally regulated and control de novo sphingolipid synthesis. The aim of the present study was to evaluate the participation of SK/S1P pathway in the triggering of cell differentiation by external hypertonicity. We found that hypertonicity evoked a sharp decrease in SK expression, thus activating the de novo sphingolipid synthesis pathway. Furthermore, the inhibition of SK activity evoked a relaxation of cell-cell adherens junction (AJ) with accumulation of the AJ complex (E-cadherin/β-catenin/α-catenin) in the Golgi complex, preventing the acquisition of the differentiated cell phenotype. This phenotype alteration was a consequence of a sphingolipid misbalance with an increase in ceramide levels. Moreover, we found that SNAI1 and SNAI2 were located in the cell nucleus with impairment of cell differentiation induced by SK inhibition, a fact that is considered a biochemical marker of epithelial to mesenchymal transition. So, we suggest that the expression and activity of SK1, but not SK2, act as a control system, allowing epithelial cells to synchronize the various branches of sphingolipid metabolism for an adequate cell differentiation program.

Topics: Adherens Junctions; Animals; Cell Differentiation; Cell Movement; Cell Proliferation; Dogs; Enzyme Inhibitors; Epithelial Cells; Hypertonic Solutions; Lysophospholipids; Madin Darby Canine Kidney Cells; Phosphotransferases (Alcohol Group Acceptor); RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Sphingolipids; Sphingosine

Sphingosine-1-phosphate (S1P) produced by sphingosine kinases (SPHK1 and SPHK2) is a signaling molecule involved in cell proliferation and formation of cellular junctions. In this study, we characterized the retinas of Sphk1 knockout (KO) mice by electron microscopy and immunocytochemistry. We also tested cultured Müller glia for their response to S1P. We found that S1P plays an important role in retinal and retinal pigment epithelial (RPE) structural integrity in aging mice. Ultrastructural analysis of Sphk1 KO mouse retinas aged to 15 months or raised with moderate light stress revealed a degenerated outer limiting membrane (OLM). This membrane is formed by adherens junctions between neighboring Müller glia and photoreceptor cells. We also show that Sphk1 KO mice have reduced retinal function in mice raised with moderate light stress. In vitro assays revealed that exogenous S1P modulated cytoskeletal rearrangement and increased N-cadherin production in human Müller glia cells. Aged mice also had morphological degeneration of the RPE, as well as increased lipid storage vacuoles and undigested phagosomes reminiscent of RPE in age-related macular degeneration. These findings show that SPHK1 and S1P play a vital role in the structural maintenance of the mammalian retina and retinal pigmented epithelium by supporting the formation of adherens junctions.

Topics: Adherens Junctions; Aging; Animals; Cadherins; cdc42 GTP-Binding Protein; Cell Membrane; Endothelium; Ependymoglial Cells; Humans; Lysophospholipids; Mice, Knockout; Phenotype; Phosphotransferases (Alcohol Group Acceptor); rac1 GTP-Binding Protein; Retina; Retinal Pigment Epithelium; Sphingolipids; Sphingosine; Vacuoles

Renin-angiotensin-aldosterone system (RAS) has been implicated in non-alcoholic fatty liver disease (NAFLD); the most common cause of chronic liver diseases. There is accumulating evidence that altered TLR4 and Sphingosine kinase 1(SphK1)/sphingosine1phosphate (S1P) signaling pathways are key players in the pathogenesis of NAFLD. Cross talk of the sphingosine signaling pathway, toll-4 (TLR4) receptors, and angiotensin II was reported in various tissues. Therefore, the aim of this study was to define the contribution of these two pathways to the hepatoprotective effects of telmisartan and/or chlorogenic acid (CGA) in NAFLD. CGA is a strong antioxidant that was previously reported to inhibit angiotensin converting enzyme. Male Wistar rats were treated with either high-fructose, with or without telmisartan, CGA, telmisartan + CGA for 8 weeks. Untreated NAFL rats showed characteristics of NAFLD, as evidenced by significant increase in the body weight, insulin resistance, and serum hepatotoxicity markers (Alanine and Aspartate transaminases) and lipids as compared to the negative control group, in addition to characteristic histopathological alterations. Treatment with either telmisartan and/or CGA improved aforementioned parameters, in addition to upregulation of antioxidant enzymes (Superoxide dismutase and Glutathione peroxidase). Effect of inhibiting RAS on both sphingosine pathway and TLR4 was evident by the suppressing effect of telmisartan and/or CGA on high fructose-induced upregulation of hepatic SPK1 and S1P, in addition to concomitant up-regulation of Sphingosine-1-Phosphate receptor (S1PR)3 protein level and increased expression of S1PR1 and TLR4. As TLR4 and SPK/S1P signaling pathways play important roles in the progression of liver inflammation, the effect on sphingosine pathway and TLR4 was associated with decreased concentrations of inflammatory markers, enzyme kB kinase (IKK), nuclear factor-kB and tumor necrosis factor-α as compared to untreated NAFL group. In conclusion, the present data strongly suggests the cross-talk between angiotensin, the Sphingosine SPK/S1P Axis and TLR4 Receptors, and their role in the pathogenesis of fructose-induced NAFLD, and the protection afforded by drugs inhibiting RAS.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensins; Animals; Chlorogenic Acid; Drug Therapy, Combination; Fructose; Lysophospholipids; Male; Non-alcoholic Fatty Liver Disease; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Signal Transduction; Sphingosine; Telmisartan; Toll-Like Receptor 4

Lung cancer is one of the most common and lethal types of oncological diseases. Despite the advanced therapeutic approaches, the prognosis for lung cancer still remains poor. Apparently, there is an imperative need for more efficient therapeutic strategies. In this work we report that concurrent treatment of human adenocarcinoma A549 cells with specific concentrations of two antitumor agents, the sphingosine kinase 1 inhibitor N, N dimethylsphingosine (DMS) and the alkylphosphocholine miltefosine, induced synergistic cytotoxic effect, which was confirmed by calculation of the combination index. The simultaneous action of these agents, induced significant decrease of A549 cell number, as well as pronounced morphological alterations. Combined drugs caused substantial apoptotic events, and significant reduction of the pro-survival marker sphingosine- 1-phosphate (S1P), when compared to the individual treatments with each of the anticancer drugs alone. Miltefosine is known to affect the synthesis of choline-containing phospholipids, including sphingomyelin, but we report for the first time that it also reduces S1P. Here we suggest a putative mechanism underlying the effect of miltefosine on sphingosine kinase 1, involving miltefosine-induced inhibition of protein kinase C. In conclusion, our findings provide a possibility for treatment of lung cancer cells with lower concentrations of the two antitumor drugs, DMS and miltefosine, which is favorable, regarding their potential cytotoxicity to normal cells.

Topics: A549 Cells; Adenocarcinoma of Lung; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Drug Synergism; Humans; Lysophospholipids; Phosphorylcholine; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Sphingosine

Wound healing starts with the recruitment of inflammatory cells that secrete wound-related factors. This step is followed by fibroblast activation and tissue construction. Sphingosine-1-phosphate (S1P) is a lipid mediator that promotes angiogenesis, cell proliferation, and attracts immune cells. We investigated the roles of S1P in skin wound healing by altering the expression of its biogenic enzyme, sphingosine kinase-1 (SphK1). The murine excisional wound splinting model was used. Sphingosine kinase-1 (SphK1) was highly expressed in murine wounds and that SphK1

Topics: Animals; Biomarkers; Cell Proliferation; Cicatrix; Disease Models, Animal; Gene Expression; Granuloma; Inflammation; Lysophospholipids; Mice; Mice, Knockout; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Skin; Sphingosine; Sphingosine-1-Phosphate Receptors; Wound Healing

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that modulates inflammation and angiogenesis. In this study, we investigated the possible involvement of S1P in the pathology of light-induced retinal degeneration in vivo and in vitro. The intracellular S1P and sphingosine kinase (SphK) activity in a photoreceptor cell line (661W cells) was significantly increased by exposure to light. The enhancement of SphK1 expression was dependent on illumination, and all-trans-retinal significantly promoted SphK1 expression. S1P treatment reduced protein kinase B (Akt) phosphorylation and increased the protein expression of cleaved caspase-3, and induced photoreceptor cell apoptosis. In vivo, light exposure enhanced the expression of SphK1 in the outer segments of photoreceptors. Intravitreal injection of a SphK inhibitor significantly suppressed the thinning of the outer nuclear layer and ameliorated the attenuation of the amplitudes of a-waves and b-waves of electroretinograms during light-induced retinal degeneration. These findings imply that light exposure induces the synthesis of S1P in photoreceptors by upregulating SphK1, which is facilitated by all-trans-retinal, causing retinal degeneration. Inhibition of this enhancement may be a therapeutic target of outer retinal degeneration, including age-related macular degeneration.

Topics: Animals; Apoptosis; Cell Line; Disease Models, Animal; Disease Susceptibility; Electroretinography; Humans; Light; Lysophospholipids; Macular Degeneration; Mice; Phosphotransferases (Alcohol Group Acceptor); Photoreceptor Cells; Retina; Retinal Degeneration; Sphingosine; Stress, Physiological; Tomography, Optical Coherence

Spondyloarthritis (SpA) is a common rheumatic disease characterized by enthesis inflammation (enthesitis) and ectopic ossification (enthesophytes). The current pathogenesis model suggests that inflammation and mechanical stress are both strongly involved in SpA pathophysiology. We have previously observed that the levels of sphingosine 1-phosphate (S1P), a bone anabolic molecule, were particularly high in SpA patients' serum compared to healthy donors. Therefore, we wondered how this deregulation was related to SpA molecular mechanisms. Mouse primary osteoblasts, chondrocytes, and tenocytes were used as cell culture models. The sphingosine kinase 1 (Sphk1) gene expression and S1P secretion were significantly enhanced by cyclic stretch in osteoblasts and chondrocytes. Further, TNF-α and IL-17, cytokines implicated in enthesitis, increased Sphk1 mRNA in chondrocytes in an additive manner when combined to stretch. The immunochemistry on mouse ankles showed that sphingosine kinase 1 (SK1) was localized in some chondrocytes; the addition of a pro-inflammatory cocktail augmented Sphk1 expression in cultured ankles. Subsequently, fingolimod was used to block S1P metabolism in cell cultures. It inhibited S1P receptors (S1PRs) signaling and SK1 and SK2 activity in both osteoblasts and chondrocytes. Fingolimod also reduced S1PR-induced activation by SpA patients' synovial fluid (SF), demonstrating that the stimulation of chondrocytes by SFs from SpA patients involves S1P. In addition, when the osteogenic culture medium was supplemented with fingolimod, alkaline phosphatase activity, matrix mineralization, and bone formation markers were significantly reduced in osteoblasts and hypertrophic chondrocytes. Osteogenic differentiation was accompanied by an increase in S1prs mRNA, especially S1P

Topics: Adolescent; Adult; Aged; Animals; Calcification, Physiologic; Cells, Cultured; Chondrocytes; Cytokines; Female; Fingolimod Hydrochloride; Humans; Lysophospholipids; Male; Metabolic Networks and Pathways; Mice; Middle Aged; Osteoblasts; Osteogenesis; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Spondylarthritis; Stress, Mechanical; Synovial Fluid; Tenocytes; Up-Regulation; Young Adult

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid that is found in relatively high concentration in human plasma. Erythrocytes, endothelial cells, and activated platelets are the main sources of circulating S1P. The majority of plasma S1P is transported bound to high-density lipoprotein (HDL) and albumin. In recent years, HDL-bound S1P attracted much attention due to its cardioprotective and anti-atherogenic properties. We have previously found that endurance-trained athletes are characterized by higher plasma S1P concentration compared to untrained individuals. This finding prompted us to examine the effect of endurance training on S1P metabolism in blood. Thirteen healthy, untrained, male subjects completed an 8-week training program on a rowing ergometer. Three days before the first, and 3 days after the last training session, blood samples were drawn from an antecubital vein. We found that total plasma S1P concentration was increased after the training. Further analysis of different plasma fractions showed that the training selectively elevated HDL-bound S1P. This effect was associated with activation of sphingosine kinase in erythrocytes and platelets and enhanced S1P release from red blood cells. We postulate that increase in HDL-bound S1P level is one of the mechanisms underlying beneficial effects of regular physical activity on cardiovascular diseases.

Topics: Blood Platelets; Erythrocytes; Exercise; Humans; Lipoproteins, HDL; Lysophospholipids; Male; Phosphotransferases (Alcohol Group Acceptor); Physical Endurance; Plasma; Sphingosine; Young Adult

Sphingosine kinases (SK) are the sole enzymes responsible for the production of sphingosine 1-phosphate (S1P). S1P is a signaling molecule with a plethora of targets, acting as both a second messenger intracellularly and extracellularly via a family of cell surface G-protein-coupled S1P receptors. The two sphingosine kinases, SK1 and SK2, arise from different genes and have some distinct and overlapping cellular functions that are regulated in part by differential cellular localization, developmental expression, and catalytic properties. Here, we describe an improved method for selectively detecting SK1 activity in vitro and cell lysates via the use of the zwitterionic detergent CHAPS, which effectively inhibits SK2 activity and thus allows selective analysis of SK1 activity in a range of cell samples. The assay measures the production of

Topics: Animals; Cholic Acids; Chromatography, Thin Layer; Detergents; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Sphingosine-1-phosphate (S1P) and the enzyme primarily responsible for its production, sphingosine kinase-1 (SphK-1), are dysregulated in multiple human diseases including cancer, multiple sclerosis (MS), diabetes, neurological diseases, fibrosis, and certain pathologies associated with impaired angiogenesis such as age-related macular degeneration (AMD). Antibody-based techniques to identify and localize S1P and SphK-1 within cells and tissue specimens represent a powerful tool, not only to understand biological role of these molecules but also to validate these unique in-class targets in multiple state diseases. Consequently, the potential applications of these molecules for therapy and diagnostic purposes are currently under investigation. Here, we describe a new improved technique, Agitated Low Temperature Epitope Retrieval (ALTER) for staining procedures, to identify expression of S1P and SphK-1 in human frozen tissue samples. The challenges encountered in the process of localization in tissue samples of lipid molecules such as S1P are discussed.

Topics: Cell Line; Frozen Sections; Humans; Immunohistochemistry; Liver; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Autism spectrum disorders (ASD) are a set of pervasive neurodevelopmental disorders that manifest in early childhood, and it is growing up to be a major cause of disability in children. However, the etiology and treatment of ASD are not well understood. In our previous study, we found that serum levels of sphingosine 1-phosphate (S1P) were increased significantly in children with autism, indicating that S1P levels may be involved in ASD.. The objective of this study was to identify a link between increased levels of S1P and neurobehavioral changes in autism.. We utilized a valproic acid (VPA) -induced rat model of autism to evaluate the levels of S1P and the expression of sphingosine kinase (SphK), a key enzyme for S1P production, in serum and hippocampal tissue. Furthermore, we assessed cognitive functional changes and histopathological and neurochemical alterations in VPA-exposed rats after SphK blockade to explore the possible link between increased levels of S1P and neurobehavioral changes in autism.. We found that SphK2 and S1P are upregulated in hippocampal tissue from VPA-exposed rats, while pharmacological inhibition of SphK reduced S1P levels, attenuated spatial learning and memory impairments, increased the expression of phosphorylated CaMKII and CREB and autophagy-related proteins, inhibited cytochrome c release, decreased the expression of apoptosis related proteins, and protected against neuronal loss in the hippocampus.. We have demonstrated that an increased level of SphK2/S1P is involved in the spatial learning and memory impairments of autism, and this signaling pathway represents a novel therapeutic target and direction for future studies.

Topics: Analysis of Variance; Animals; Apoptosis; Autistic Disorder; Autophagy; Biomarkers; Disease Models, Animal; Hippocampus; Humans; Lysophospholipids; Male; Memory Disorders; Neurons; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Signal Transduction; Spatial Learning; Sphingosine; Thiazoles; Valproic Acid

A pivotal role of sphingosine 1-phosphate (S1P) in cancer has been suggested based on the ceramide-S1P rheostat theory that the intracellular balance between prosurvival S1P and proapoptotic ceramide determines cell fate. Upregulation of S1P-generating sphingosine kinases (SKs) and downregulation of S1P-degrading S1P lyase (SPL) might increase intracellular S1P levels to exert a prosurvival effect in cancer in general, such as colon cancer. However, we recently observed a distinct S1P metabolism in hepatocellular carcinoma tissues that increased SPL mRNA levels with reduced S1P levels. Thus, we investigated S1P metabolism in colon cancer.. We enrolled 26 consecutive colon cancer patients, who had undergone surgical treatment.. Not only SK, but also SPL, mRNA levels were increased in colon cancer tissues compared with the adjacent nontumorous tissues. Furthermore, the mRNA levels of another S1P degrading enzyme, S1P phosphatase 1, S1P transporters, spinster homolog 2, adenosine triphosphate-binding cassette subfamily C member 1, and S1P receptors, S1P. In human colon cancer tissues, mRNA levels of S1P-generating and S1P-degrading enzymes, transporters from inside to outside the cells, and S1P receptors, S1P

Topics: Aged; Colonic Neoplasms; Extracellular Space; Female; Humans; Lysophospholipids; Male; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine

The bioactive lipid sphingosine-1-phosphate (S1P) regulates smooth muscle (SM) contractility predominantly via three G protein-coupled receptors. The S1P1 receptor is associated with nitric oxide (NO)-mediated SM relaxation, while S1P2 & S1P3 receptors are linked to SM contraction via activation of the Rho-kinase pathway. This study is to determine testosterone (T) modulating the expression and functional activity of S1P receptors in corpus cavernosum (CC). Adult male Sprague-Dawley rats were randomly divided into three groups: sham-operated controls, surgical castration and T supplemented group. Serum S1P levels were detected by high-performance liquid chromatography. The expression of S1P1-3 receptors and sphingosine kinases was detected by real-time RT-PCR. In vitro organ bath contractility and in vivo intracavernous pressure (ICP) measurement were also performed. T deprivation significantly decreased ICP rise. Meanwhile, surgical castration induced a significant increase in serum S1P level and the expression of S1P2-3 receptors by twofold (P < 0.05) but a decrease in the expression of S1P1 receptor. Castration also augmented exogenous phenylephrine (PE), S1P, S1P1,3 receptor agonist FTY720-P contractility and S1P2-specific antagonist JTE013 relaxation effect. T supplemented could restore the aforementioned changes. We provide novel data that castration increased serum S1P concentration and up-regulated the expression of S1P2-3 receptors in CC. Consistently, agonizing S1P receptors induced CCSM contraction and antagonizing mediated relaxation were augmented. This provides the first clear evidence that S1P system dysregulation may contribute to hypogonadism-related erectile dysfunction (ED), and S1P receptors may be expected as a potential target for treating ED.

Topics: Animals; Gene Expression Regulation; Lysophospholipids; Male; Muscle Contraction; Muscle, Smooth; Orchiectomy; Organ Culture Techniques; Organ Size; Organophosphates; Penile Erection; Penis; Phenylephrine; Phosphotransferases (Alcohol Group Acceptor); Prostate; Protein Isoforms; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Testis; Testosterone

Sphingosine kinase 1 (SK1) is required for production of sphingosine-1-phosphate (S1P) and thereby regulates many cellular processes, including cellular growth, immune cell trafficking, and inflammation. To produce S1P, SK1 must access sphingosine directly from membranes. However, the molecular mechanisms underlying SK1's direct membrane interactions remain unclear. We used hydrogen/deuterium exchange MS to study interactions of SK1 with membrane vesicles. Using the CRISPR/Cas9 technique to generate HCT116 cells lacking SK1, we explored the effects of membrane interface disruption and the function of the SK1 interaction site. Disrupting the interface resulted in reduced membrane association and decreased cellular SK1 activity. Moreover, SK1-dependent signaling, including cell invasion and endocytosis, was abolished upon mutation of the membrane-binding interface. Of note, we identified a positively charged motif on SK1 that is responsible for electrostatic interactions with membranes. Furthermore, we demonstrated that SK1 uses a single contiguous interface, consisting of an electrostatic site and a hydrophobic site, to interact with membrane-associated anionic phospholipids. Altogether, these results define a composite domain in SK1 that regulates its intrinsic ability to bind membranes and indicate that this binding is critical for proper SK1 function. This work will allow for a new line of thinking for targeting SK1 in disease.

Topics: Binding Sites; Cell Membrane; Deuterium Exchange Measurement; HCT116 Cells; Humans; Lipids; Lysophospholipids; Mass Spectrometry; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Sphingosine-1-phosphate, a pleiotropic bioactive lipid mediator, is an important player in cancer progression. Previous studies suggested that sphingosine-1-phosphate produced by sphingosine kinase 1, which is activated by phosphorylation, plays important roles in the progression of disease and metastasis. The association between phospho-sphingosine-1-phosphate produced by sphingosine kinase 1 and clinical parameters in human gastric cancer have not been fully investigated to date.. We created phospho-sphingosine-1-phosphate produced by sphingosine kinase expression profiles by immunohistochemistry for 136 patients who underwent operative intervention for gastric cancer in 2007-2009. Phospho-sphingosine-1-phosphate produced by sphingosine kinase expression and compared clinicopathologic factors by univariate and multivariate analyses.. The univariate analysis revealed that phospho-sphingosine-1-phosphate produced by sphingosine kinase expression was correlated significantly with depth of tumor invasion, lymph node metastasis, distant metastasis, histologic type, and lymphatic invasion. The multivariate analysis revealed that the diffuse type (odds ratio 2.210; 95% confidence interval, 1.045-4.671, P=.038) and the presence of lymphatic invasion (odds ratio 3.697; 95% confidence interval, 1.161-8.483, P=.002) were associated independently with phospho-sphingosine-1-phosphate produced by sphingosine kinase expression in patients with gastric cancer. The 5-year rate of disease-specific survival was 79.3% in patients with phospho-sphingosine-1-phosphate produced by sphingosine kinasephospho-sphingosine-1-phosphate produced by sphingosine kinase-positive expression and 98.3% in those with phospho-sphingosine-1-phosphate produced by sphingosine kinase-negative expression (P=.002). In multivariate analysis, however, high phospho-sphingosine-1-phosphate produced by sphingosine kinase expression was not an independent prognostic factor for disease-specific survival (hazard ratio 5.540; 95% confidence interval, 0.717-42.81, P=.100).. We provide the first evidence that diffuse histologic type and lymphatic invasion were independently associated with high phospho-sphingosine-1-phosphate produced by sphingosine kinase expression in gastric cancer patients, indicating a role of sphingosine-1-phosphate in disease progression among patients with gastric cancer. (Surgery 2017;160:XXX-XXX.).

Topics: Adult; Aged; Aged, 80 and over; Female; Gastrectomy; Humans; Japan; Lymph Node Excision; Lymphatic Metastasis; Lysophospholipids; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Retrospective Studies; Sphingosine; Stomach Neoplasms; Survival Rate

We show that glioblastoma multiform (GBM) cells overexpressing the constitutively active form of the epidermal growth factor receptor [epidermal growth factor receptor variant III (EGFRvIII) and U87MG human GBM cell line overexpressing EGFRvIII (EGFR+) cells] possess greater invasive properties and have higher levels of extracellular sphingosine-1-phosphate (S1P) and increased sphingosine kinase-1 (SK1) activity than the empty vector-expressing cells. Notably, the inhibition of SK1 or S1P receptors decreases the invasiveness of EGFR+ cells. Moreover, EGFR and MEK1 inhibitors reduce both SK1 activation and cell invasion, suggesting that the enhanced invasiveness observed in the EGFR+ cells depends on the increased S1P secretion, downstream of the EGFRvIII-ERK-SK1-S1P pathway. Altogether, the results of the present study indicate that, in GBM cells, EGFRvIII is connected with the S1P signaling pathway to enhance cell invasiveness and tumor progression.

Topics: Cell Line, Tumor; ErbB Receptors; Glioblastoma; Humans; Lysophospholipids; MAP Kinase Kinase 1; MAP Kinase Signaling System; Neoplasm Invasiveness; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid mediator, has been implicated in regulation of many processes important for breast cancer progression. Previously, we observed that S1P is exported out of human breast cancer cells by ATP-binding cassette (ABC) transporter ABCC1, but not by ABCB1, both known multidrug resistance proteins that efflux chemotherapeutic agents. However, the pathologic consequences of these events to breast cancer progression and metastasis have not been elucidated. Here, it is demonstrated that high expression of ABCC1, but not ABCB1, is associated with poor prognosis in breast cancer patients. Overexpression of ABCC1, but not ABCB1, in human MCF7 and murine 4T1 breast cancer cells enhanced S1P secretion, proliferation, and migration of breast cancer cells. Implantation of breast cancer cells overexpressing ABCC1, but not ABCB1, into the mammary fat pad markedly enhanced tumor growth, angiogenesis, and lymphangiogenesis with a concomitant increase in lymph node and lung metastases as well as shorter survival of mice. Interestingly, S1P exported via ABCC1 from breast cancer cells upregulated transcription of sphingosine kinase 1 (SPHK1), thus promoting more S1P formation. Finally, patients with breast cancers that express both activated SPHK1 and ABCC1 have significantly shorter disease-free survival. These findings suggest that export of S1P via ABCC1 functions in a malicious feed-forward manner to amplify the S1P axis involved in breast cancer progression and metastasis, which has important implications for prognosis of breast cancer patients and for potential therapeutic targets.

Topics: Animals; Breast Neoplasms; Female; Humans; Lysophospholipids; Mice; Mice, Inbred BALB C; Mice, Nude; Multidrug Resistance-Associated Proteins; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Survival Analysis

Sphingosine 1-phosphate (S1P) is an intercellular signaling molecule present in blood. Erythrocytes have a central role in maintaining the S1P concentration in the blood stream. We previously demonstrated that S1P is exported from erythrocytes by a glyburide-sensitive S1P transporter. However, the gene encoding the S1P transporter in erythrocytes is unknown. In this study, we found that the mouse erythroid cell line, MEDEP-E14, has S1P export activity and exhibits properties that are consistent with those of erythrocytes. Using microarray analysis of MEDEP-E14 cells and its parental cell line, E14TG2a, we identified several candidate genes for S1P export activity. Of those genes, only one gene, Mfsd2b, showed S1P transport activity. The properties of S1P release by MFSD2B were similar to those in erythrocytes. Moreover, knockout of MFSD2B in MEDEP-E14 cells decreased S1P export from the cells. These results strongly suggest that MFSD2B is a novel S1P transporter in erythroid cells.

Topics: Animals; Cell Line; CHO Cells; Cricetulus; Erythroid Cells; Gene Knockout Techniques; Lysophospholipids; Membrane Proteins; Mice; Microarray Analysis; Phosphotransferases (Alcohol Group Acceptor); Recombinant Proteins; Sphingosine

BACKGROUND Triple negative breast cancer (TNBC) has a more aggressive recurrence. Previous reports have demonstrated that sphingosine kinase 1 (SphK1) is a crucial regulator of breast cancer progression. However, the correlation of SphK1 with clinical prognosis has been poorly investigated. Thus, we aimed to elaborate the role of SphK1 in TNBC metastasis. MATERIAL AND METHODS We first determined the level of SphK1 in breast cancer tissue samples and breast cancer cells. Furthermore, the expression of HER2 and phosphor-SphK1 (pSphK1) in human breast cancer tissue samples was determined by immunohistochemical analysis. Associations between SphK1 and clinical parameters of tumors were analyzed. The activity of SphK1 was measured by fluorescence analysis. Extracellular sphingosine-1-phosphate (S1P) was detected using an ELISA kit. Associations between SphK1 and metastasis potential were analyzed by Transwell assay. RESULTS Levels of SphK1 in TNBC patients were significantly higher than levels in other patients with other breast tumors. The expression of SphK1 was positively correlated with poor overall survival (OS) and progression-free survival (PFS), as well as poor response to 5-FU and doxorubicin. The depression of SphK1 thus could repress the Notch signaling pathway, reduce migration, and invasion of TNBC cells in vivo and in vitro. Furthermore, silencing of SphK1 by Ad-SPHK1-siRNA or SphK1 inhibitor PF543 sensitized TNBCs to 5-FU and doxorubicin. Our results also indicated that SphK1 inhibition could effectively counteracts tumors metastasis via Notch signaling pathways, indicating a potentially anti-tumor strategy in TNBC. CONCLUSIONS We found that elevated levels of pSphK1 were positive correlation with high expression of S1P, which in turn promoted metastasis of TNBC through S1P/S1PR3/Notch signaling pathway.

Topics: Animals; Cell Line, Tumor; Doxorubicin; Drug Synergism; Female; Fluorouracil; Heterografts; Humans; Lysophospholipids; MCF-7 Cells; Methanol; Mice; Mice, Inbred NOD; Mice, SCID; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidines; Receptor, ErbB-2; Receptors, Lysosphingolipid; Receptors, Notch; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Sulfones; Triple Negative Breast Neoplasms; Up-Regulation

Alzheimer's disease (AD) is characterized by the accumulation of β-amyloid (Aβ) peptides and hyperphosphorylated tau protein accompanied by neuronal loss. Aβ accumulation has been associated with an impaired sphingosine 1-phosphate (S1P) metabolism. S1P is generated by sphingosine kinases (SphKs), of which there are two isoenzymes SphK1 and SphK2, and degraded by the sphingosine 1-phosphate lyase (SPL). We previously reported, that both a decrease in SphK1 expression and an increase in SPL expression, correlated with amyloid deposits in the entorhinal cortex of AD brains, suggesting a global loss of pro-survival S1P in AD neurons. SphK2 contribution has also been examined in AD yielding to conflicting results that may reflect the complexity of SphK2 regulation. The subcellular localization of SphK2, hence the compartmentalization of generated S1P, is recognized to play a crucial role in dictating either its pro-survival or pro-apoptotic functions. We therefore aimed at studying the expression of SphK2 and notably its subcellular localization in brain tissues from patients with AD.. We report that a decrease in SphK2 protein cytosolic expression correlated with the density of amyloid deposits in a cohort of 25 post-mortem brains. Interestingly, we observed that the equilibrium between cytoplasmic and nuclear SphK2 is disrupted and showed that SphK2 is preferentially localized in the nucleus in AD brain extracts as compared to control extracts, with a marked increase of cleaved SphK2.. Our results suggest that a shift in the subcellular localization of the S1P generating SphK2 may compromise the well established pro-survival cytosolic S1P by favoring the production of nuclear S1P associated with adverse effects in AD pathogenesis.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Brain; Female; Humans; Lysophospholipids; Male; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Subcellular Fractions

The possibility of sphingosine-1-phosphate production by induced pluripotent stem cells is examined to assess their potential in treatment of sepsis. The hematopoietic embryoid bodies were derived from the culture of 6-day-old differentiated induced pluripotent stem cells. These embryoid bodies secreted sphingosine-1-phosphate, an important bioactive lipid that regulates integrity of the pulmonary endothelial barrier, prevents elevation of its permeability, and impedes the formation of stress fibers in human endotheliocytes derived from umbilical vein. The data attest to potentiality of induced pluripotent stem cells in treatment of sepsis.

Topics: Animals; Cell Differentiation; Cell Membrane Permeability; Embryoid Bodies; GATA1 Transcription Factor; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Induced Pluripotent Stem Cells; Lipopolysaccharides; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Transcription Factors; Vascular Endothelial Growth Factor Receptor-2

Topics: Animals; Apoptosis; beta Catenin; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Glycogen Synthase Kinase 3 beta; HCT116 Cells; HT29 Cells; Humans; Lysophospholipids; Male; Mice; Mice, Nude; Neutral Ceramidase; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Sphingolipids; Sphingosine

The human cytomegalovirus (HCMV) is a common pathogen, which causes severe or even deadly diseases in immunocompromised patients. In addition, congenital HCMV infection represents a major health concern affecting especially the lung tissue of the susceptible individuals. Antivirals are a useful strategy to treat HCMV-caused diseases. However, all approved drugs target viral proteins but significant toxicity and an increasing resistance against these compounds have been observed. In infected cells, numerous host molecules have been identified to play important roles during HCMV replication. Among others, HCMV infection depends on the presence of bioactive sphingolipids. In this study, the role of sphingosine-1-phosphate (S1P) signaling in HCMV-infected human embryonal lung fibroblasts (HELF) was analyzed. Viral replication depended on the functional activity of sphingosine kinases (SK). During SK inhibition, addition of extracellular S1P restored HCMV replication. Moreover, neutralization of extracellular S1P by anti-S1P antibodies decreased HCMV replication as well. While the application of FTY720 as an functional antagonist of S1P receptor (S1PR)

Topics: Cells, Cultured; Cytomegalovirus; Fibroblasts; Humans; Lung; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Virus Replication

It has been well established that patients with diabetes or metabolic syndrome (MetS) have increased prevalence and severity of periodontitis, an oral infection initiated by bacteria and characterized by tissue inflammation and destruction. To understand the underlying mechanisms, we have shown that saturated fatty acid (SFA), which is increased in patients with type 2 diabetes or MetS, and LPS, an important pathogenic factor for periodontitis, synergistically stimulate expression of proinflammatory cytokines in macrophages by increasing ceramide production. However, the mechanisms by which increased ceramide enhances proinflammatory cytokine expression have not been well understood. Since sphingosine 1 phosphate (S1P) is a metabolite of ceramide and a bioactive lipid, we tested our hypothesis that stimulation of ceramide production by LPS and SFA facilitates S1P production, which contributes to proinflammatory cytokine expression. Results showed that LPS and palmitate, a major SFA, synergistically increased not only ceramide, but also S1P, and stimulated sphingosine kinase (SK) expression and membrane translocation in RAW264.7 macrophages. Results also showed that SK inhibition attenuated the stimulatory effect of LPS and palmitate on IL-6 secretion. Moreover, results showed that S1P enhanced the stimulatory effect of LPS and palmitate on IL-6 secretion. Finally, results showed that targeting S1P receptors using either S1P receptor antagonists or small interfering RNA attenuated IL-6 upregulation by LPS and palmitate. Taken together, this study demonstrated that LPS and palmitate synergistically stimulated S1P production and S1P in turn contributed to the upregulation of proinflammatory cytokine expression in macrophages by LPS and palmitate.

Topics: Animals; Apoptosis; Ceramides; Cytokines; Drug Synergism; Enzyme Activation; Gene Expression Regulation; Inflammation; Interleukin-6; Lipopolysaccharides; Lysophospholipids; Macrophages; Mice; Palmitates; Phosphotransferases (Alcohol Group Acceptor); RAW 264.7 Cells; Receptors, Lysosphingolipid; RNA Interference; Sphingosine

Idiopathic pulmonary fibrosis is an agnogenic chronic disorder with high morbidity and low survival rate. Quercetin is a flavonoid found in a variety of herbs with anti-fibrosis function. In this study, bleomycin was employed to induce a pulmonary fibrosis mouse model. The quercetin administration ameliorated bleomycin-induced pulmonary fibrosis, evidenced by the expression level changes of hydroxyproline, fibronectin, α-smooth muscle actin, Collagen I, and Collagen III. Similar results were observed in transforming growth factor (TGF)-β-treated human embryonic lung fibroblast (HELF). The bleomycin or TGF-β administration caused the increase of sphingosine-1-phosphate (S1P) level in pulmonary tissue and HELF cells, as well as its activation-required kinase, sphingosine kinase 1 (SphK1), and its degradation enzyme, sphinogosine-1-phosphate lyase (S1PL). However, the increase of S1P, SphK1, and S1PL was attenuated by application of quercetin. In addition, the effect of quercetin on fibrosis was abolished by the ectopic expression of SphK1. The colocalization of SphK1/S1PL and fibroblast specific protein 1 (FSP1) suggested the roles of fibroblasts in pulmonary fibrosis. In summary, we demonstrated that quercetin ameliorated pulmonary fibrosis in vivo and in vitro by inhibiting SphK1/S1P signaling.

Topics: Animals; Cells, Cultured; Humans; Idiopathic Pulmonary Fibrosis; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Quercetin; Signal Transduction; Sphingosine

Sphingosine 1-phosphate (S1P) is a bioactive phosphorylated product of sphingosine catalyzed by sphingosine kinase (SphK) and implicated in diverse cellular functions including vesicular trafficking. In the present study we have shown the importance of one of the subtypes of SphK, SphK2, in the regulation of cargo content in exosomes released from human myeloid leukemia K562 cells. First, SphK2 has been shown to localize with N-Rh-PE-positive late endosomes in the cells. Next, siRNA-mediated knockdown of Sphk2 but not SphK1 resulted in a reduction of cargo content in purified exosomes. The involvement of SphK2 in this phenomenon was further investigated by pharmacological approaches. When cells were treated with N,N-dimethylsphingosine (DMS), one of the most frequently used inhibitors for SphK, cargo contents in purified exosomes were enhanced unexpectedly. Finally, it has been shown that DMS has a potency to stimulate SphK2 activity depending on the substrate sphingosine- and the inhibitor-doses as estimated by in vitro assay systems using a purified SphK2. These findings suggest that SphK2/S1P signaling plays an important role in the regulation of cargo content in exosomes in K562 cells.

Topics: Exosomes; HEK293 Cells; Humans; K562 Cells; Lysophospholipids; Multivesicular Bodies; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Lymphatic metastasis is known to contribute to worse prognosis of biliary tract cancer (BTC). Recently, sphingosine-1-phosphate (S1P), a bioactive lipid mediator generated by sphingosine kinase 1 (SPHK1), has been shown to play an important role in lymphangiogenesis and lymph node metastasis in several types of cancer. However, the role of the lipid mediator in BTC has never been examined. Here we found that S1P is elevated in BTC with the activation of ceramide-synthetic pathways, suggesting that BTC utilizes SPHK1 to promote lymphatic metastasis. We found that S1P, sphingosine and ceramide precursors such as monohexosyl-ceramide and sphingomyelin, but not ceramide, were significantly increased in BTC compared to normal biliary tract tissue using LC-ESI-MS/MS. Utilizing The Cancer Genome Atlas cohort, we demonstrated that S1P in BTC is generated via de novo pathway and exported via ABCC1. Further, we found that SPHK1 expression positively correlated with factors related to lymphatic metastasis in BTC. Finally, immunohistochemical examination revealed that gallbladder cancer with lymph node metastasis had significantly higher expression of phospho-SPHK1 than that without. Taken together, our data suggest that S1P generated in BTC contributes to lymphatic metastasis.

Topics: Biliary Tract Neoplasms; Ceramides; Chromatography, High Pressure Liquid; Gene Expression; Humans; Lymphatic Metastasis; Lysophospholipids; Multidrug Resistance-Associated Proteins; Phosphotransferases (Alcohol Group Acceptor); Retrospective Studies; Sphingosine; Tandem Mass Spectrometry

Glioblastoma is one of the most malignant, angiogenic, and incurable tumors in humans. The aberrant communication between glioblastoma cells and tumor microenvironment represents one of the major factors regulating glioblastoma malignancy and angiogenic properties. Emerging evidence implicates sphingosine-1-phosphate signaling in the pathobiology of glioblastoma and angiogenesis, but its role in glioblastoma-endothelial crosstalk remains largely unknown. In this study, we sought to determine whether the crosstalk between glioblastoma cells and brain endothelial cells regulates sphingosine-1-phosphate signaling in the tumor microenvironment. Using human glioblastoma and brain endothelial cell lines, as well as primary brain endothelial cells derived from human glioblastoma, we report that glioblastoma-co-culture promotes the expression, activity, and plasma membrane enrichment of sphingosine kinase 2 in brain endothelial cells, leading to increased cellular level of sphingosine-1-phosphate, and significant potentiation of its secretion. In turn, extracellular sphingosine-1-phosphate stimulates glioblastoma cell proliferation, and brain endothelial cells migration and angiogenesis. We also show that, after co-culture, glioblastoma cells exhibit enhanced expression of S1P

Topics: Animals; Brain; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Proliferation; Coculture Techniques; Endothelial Cells; Glioblastoma; Humans; Lysophospholipids; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Rats; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Tumor Microenvironment

A concomitant action of multiple profibrotic mediators appears crucial in the development and progression of fibrosis. Sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways are both involved in pathogenesis of fibrosis in several organs by controlling differentiation of fibroblasts to myofibroblasts and the epithelial to-mesenchymal transition. However, their direct involvement in chronic colitis-associated fibrosis it is not yet known. In this study we evaluated the immunohistochemical expression of some proteins implicated in sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in Dextrane Sodium Sulphate (DSS)-induced colorectal fibrosis in mice. Compared to control mice, DSS-induced chronic colitis mice developed a marked intestinal fibrosis associated with a concomitant overexpression of TGF-β, p-Smad3, α-SMA, collagen I-III, SPHK1, RhoA, PI3K, Akt, p-Akt, p-mTOR. This study highlights the relationship between the two pathways and the possible role of SPHK1 in the intestinal fibrosis.  These results, if confirmed by in vitro studies, may have important clinical implications in the development of new therapeutical approaches in inflammatory bowel disease.

Topics: Animals; Colitis; Disease Models, Animal; Fibrosis; Immunohistochemistry; Intestines; Lysophospholipids; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Reference Standards; Signal Transduction; Smad3 Protein; Sphingosine; Transforming Growth Factor beta

Acute lymphoblastic leukemia (ALL) is the most common fatal cancer in people younger than 20 years of age. This study was designed to explore the anti-leukemia activity of physcion 8-O-β-glucopyranoside (PG) in B-cell ALL.. NALM6 and SupB15 cells were used as model cell lines. Cell viability, cell apoptosis, cell cycle distribution were determined by CCK-8 assay, DNA fragmentation assay and flow cytometry, and flow cytometry, respectively. Expression of proteins involved in cell apoptosis and cell cycle regulation was determined by western blot and the levels of ceramide and sphingosine 1-phosphate (S1P) were determined by ELISA. Activity of sphingosine kinase 1 (SphK1) was also determined with a Sphingosine Kinase Assay Kit. In the present study, both model cell lines were transfected with siRNA targeting SphK1 or an overexpression plasmid to examine the role of SphK1 in the anti-leukemia activity of PG. Moreover, the efficacy of PG was examined in vivo in a mouse model by measuring survival and spleen weight.. Our results provided experimental evidence that PG could significantly induce apoptosis and cell cycle arrest in vitro. Mechanistically, the anti-leukemia activity of PG was mediated by its ability to repress SphK1 and thus modulate ceramide-S1P rheostat. Moreover, the anti-leukemia activity of PG was also verified in a murine model.. Collectively, our results indicate that PG may be a promising agent for the treatment of B-cell leukemia.

Topics: Animals; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Ceramides; DNA Damage; Emodin; Glucosides; Humans; Lysophospholipids; Mice; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); RNA, Small Interfering; Sphingosine

The cutaneous wound healing process is tightly regulated by a range of cellular responses, including migration. Sphingosine-1-phosphate (S1P) is a signaling lipid produced in keratinocytes (KC) and it is known to stimulate skin wound repair through increased KC migration. Of the multifunctional triterpene ginsenosides, Rb1 enhances cutaneous wound healing process by increasing KC migration, but cellular mechanisms responsible for the Rb1-mediated increase in KC migration are largely unknown. Therefore, we hypothesized that, and assessed whether, Rb1 could stimulate KC migration through S1P-dependent mechanisms. Rb1 significantly increases S1P production by regulating the activity of metabolic conversion enzymes associated with S1P generation and degradation, sphingosine kinase 1 (SPHK1) and S1P lyase, respectively, in parallel with enhanced KC migration. However, blockade of ceramide to S1P metabolic conversion using a specific inhibitor of SPHK1 attenuated the expected Rb1-mediated increase in KC migration. Furthermore, a pan-S1P receptor inhibitor pertussis toxin significantly attenuated Rb1-induced stimulation of KC migration. Moreover, the Rb1-induced increases in KC migration required S1P receptor(s)-mediated activation of ERK1/2 and NF-κB, leading to production of key cutaneous migrating proteins, matrix metalloproteinase (MMP)-2 and MMP-9. Taken together, the results show that Rb1 stimulates KC migration through an S1P→S1P receptor(s)→ERK1/2→NF-κB→MMP-2/-9 pathway. This research revealed a previously unidentified cellular mechanism for Rb1 in enhancing KC migration and pointing to a new therapeutic approach to stimulate the cutaneous wound healing process.

Topics: Cell Movement; Cells, Cultured; Ginsenosides; Humans; Keratinocytes; Lysophospholipids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Membrane Proteins; Mitogen-Activated Protein Kinases; Panax; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Plant Extracts; Signal Transduction; Skin; Sphingosine

Sphingosine 1-phosphate (S1P) has a role in many cellular processes. S1P is involved in cell growth and apoptosis, regulation of cell trafficking, production of cytokines and chemokines. The kinases SphK1 and SphK2 (SphKs) phosphorilate Sphingosine (Sph) to S1P and several phosphatases revert S1P to sphingosine, thus assuring a balanced pool that can be depleted by a Sphingosine lyase in hexadecenal compounds and aldehydes. There are evidences that SphK1 and 2 may per se control cellular processes. Here, we report that Sph kinases regulate IL-17 expression in human T cells. SphKs inhibition impairs the production of IL-17, while their overexpression up-regulates expression of the cytokine through acetylation of IL-17 promoter. SphKs were up-regulated also in PBMCs of patients affected by IL-17 related diseases. Thus, S1P/S1P kinases axis is a mechanism likely to promote IL-17 expression in human T cells, representing a possible therapeutic target in human inflammatory diseases.

Topics: Cells, Cultured; Down-Regulation; Humans; Inflammation; Interleukin-17; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Sphingosine; T-Lymphocytes; Up-Regulation

Recently, sphingolipid derivatives, such as ceramide and sphingosine‑1‑phosphate (S1P), have emerged as key modulators in apoptotic cell death and cell proliferation. This study aimed to clarify the underlying signaling pathways of ceramide and S1P involved in breast cancer cell proliferation. Ceramide acyl chain length is determined by six mammalian ceramide synthases (CerS). We overexpressed CerS1 to 6 in MCF‑7 cells to examine whether ceramide signaling propagation varies as a function of acyl chain length. Among the six CerS, only CerS6 overexpression reduced phosphorylation of Akt, S6 kinase (S6K), and extracellular signal‑regulated kinases (ERK) as shown by western blotting. In addition, CerS6 overexpression reduced MCF‑7 cell proliferation. This effect was partially reversed by co‑treatment with MHY1485, an activator of mammalian target of rapamycin (mTOR), demonstrating an important role for the mTOR pathway in the CerS6‑mediated decrease in MCF‑7 cell proliferation. ERK inhibition, but not Akt inhibition, along with mTOR inhibition synergistically reduced MCF‑7 cell proliferation as measured by MTT assay. Notably, the expression of CerS6 and S1P receptor 2 (S1PR2), or CerS6 and sphingosine kinase 1 (SphK1), were negatively correlated according to the invasive breast carcinoma patient cohort in The Cancer Genome Atlas database. In addition, both SphK1 overexpression and S1P addition increased mTOR phosphorylation as shown by ELISA, while S1PR2 inhibition had the inverse effect. These data suggest that CerS6 and SphK1 regulate mTOR signaling in breast cancer cell proliferation. Moreover, mTOR activity can be regulated by the balance between S1P and C16‑ceramide, which is generated by CerS6.

Topics: Breast Neoplasms; Cell Proliferation; Ceramides; Female; Gene Expression Regulation, Neoplastic; Humans; Lysophospholipids; MAP Kinase Signaling System; MCF-7 Cells; Membrane Proteins; Morpholines; Oncogene Protein v-akt; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine N-Acyltransferase; Sphingosine-1-Phosphate Receptors; TOR Serine-Threonine Kinases; Triazines

Skeletal muscle (SkM) atrophy is caused by several and heterogeneous conditions, such as cancer, neuromuscular disorders and aging. In most types of SkM atrophy overall rates of protein synthesis are suppressed, protein degradation is consistently elevated and atrogenes, such as the ubiquitin ligase Atrogin-1/MAFbx, are up-regulated. The molecular regulators of SkM waste are multiple and only in part known. Sphingolipids represent a class of bioactive molecules capable of modulating the destiny of many cell types, including SkM cells. In particular, we and others have shown that sphingosine 1phosphate (S1P), formed by sphingosine kinase (SphK), is able to act as trophic and morphogenic factor in myoblasts. Here, we report the first evidence that the atrophic phenotype observed in both muscle obtained from mice bearing the C26 adenocarcinoma and C2C12 myotubes treated with dexamethasone was characterized by reduced levels of active phospho-SphK1. The importance of SphK1 activity is also confirmed by the specific pharmacological inhibition of SphK1 able to increase Atrogin-1/MAFbx expression and reduce myotube size and myonuclei number. Furthermore, we found that SkM atrophy was accomplished by significant increase of S1P transporter Spns2 and in changes in the pattern of S1P receptor (S1PRs) subtype expression paralleled by increased Atrogin-1/MAFbx expression, suggesting a role for the released S1P and of specific S1PR-mediated signaling pathways in the control of the ubiquitin ligase. Altogether, these findings provide the first evidence that SphK1/released S1P/S1PR axis acts as a molecular regulator of SkM atrophy, thereby representing a new possible target for therapy in many patho-physiological conditions.

Topics: Animals; Cell Line; Dexamethasone; Female; Lysophospholipids; Mice, Inbred BALB C; Muscle Fibers, Skeletal; Muscular Atrophy; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Sphingolipids, first described in the brain in 1884, are important structural components of biological membranes of all eukaryotic cells. In recent years, several lines of evidence support the critical role of sphingolipids such as sphingosine, sphingosine-1-phosphate (S1P), and ceramide as anti- or pro-inflammatory bioactive lipid mediators in a variety of human pathologies including pulmonary and vascular disorders. Among the sphingolipids, S1P is a naturally occurring agonist that exhibits potent barrier enhancing property in the endothelium by signaling via G protein-coupled S1P1 receptor. S1P, S1P analogs, and other barrier enhancing agents such as HGF, oxidized phospholipids, and statins also utilize the S1P/S1P1 signaling pathway to generate membrane protrusions or lamellipodia, which have been implicated in resealing of endothelial gaps and maintenance of barrier integrity. A better understanding of sphingolipids mediated regulation of lamellipodia formation and barrier enhancement of the endothelium will be critical for the development of sphingolipid-based therapies to alleviate pulmonary disorders such as sepsis-, radiation-, and mechanical ventilation-induced acute lung injury.

Topics: Acute Lung Injury; Endothelium, Vascular; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Pseudopodia; Reactive Oxygen Species; Signal Transduction; Simvastatin; Sphingolipids; Sphingosine

Graves' orbitopathy (GO) is an autoimmune orbital disorder associated with Graves' disease caused by thyrotropin receptor autoantibodies. Orbital fibroblasts (OFs) and CD40 play a key role in disease pathogenesis. The bioactive lipid sphingosine-1-phosphate (S1P) has been implicated in promoting adipogenesis, fibrosis, and inflammation in OFs. We investigated the role of CD40 signaling in inducing S1P activity in orbital inflammation.. OFs and T cells were derived from GO patients and healthy control (Ctl) persons. S1P abundance in orbital tissues was evaluated by immunofluorescence. OFs were stimulated with CD40 ligand and S1P levels were determined by ELISA. Further, activities of acid sphingomyelinase (ASM), acid ceramidase, and sphingosine kinase were measured by ultraperformance liquid chromatography. Sphingosine and ceramide contents were analyzed by mass spectrometry. Finally, the role for S1P in T-cell attraction was investigated by T-cell migration assays.. GO orbital tissue showed elevated amounts of S1P as compared to control samples. Stimulation of CD40 induced S1P expression in GO-derived OFs, while Ctl-OFs remained unaffected. A significant increase of ASM and sphingosine kinase activities, as well as lipid formation, was observed in GO-derived OFs. Migration assay of T cells in the presence of SphK inhibitor revealed that S1P released by GO-OFs attracted T cells for migration.. The results demonstrated that CD40 ligand stimulates GO fibroblast to produce S1P, which is a driving force for T-cell migration. The results support the use of S1P receptor signaling modulators in GO management.

Topics: Acid Ceramidase; CD40 Antigens; CD40 Ligand; Cell Movement; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fluorescent Antibody Technique, Indirect; Graves Ophthalmopathy; Humans; Inflammation; Lysophospholipids; Mass Spectrometry; Orbit; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingolipids; Sphingomyelin Phosphodiesterase; Sphingosine; T-Lymphocytes

The underlying molecular mechanisms involved in sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) mediation of platelet-derived growth factor (PDGF)-induced pulmonary arterial smooth muscle cell (PASMC) proliferation are still unclear, and the present study aims to address this issue.. Small interfering RNA (siRNA) and microRNA inhibitor transfection was performed to block the expression of SphK1, bone morphogenetic protein receptor II (BMPRII) and microRNA-21 (miR-21). Gene expression levels of SphK1, BMPRII and inhibitor of DNA binding 1 (Id1) were detected by immunoblotting, miR-21 expression level was examined with qRT-PCR, and S1P production was measured by ELISA. Additionally, PASMC proliferation was determined by BrdU incorporation assay.. Our results indicated that PDGF increased the expression of SphK1 protein and S1P production, up-regulated miR-21 expression, reduced BMPRII and Id1 expression, and promoted PASMCs proliferation. Pre-silencing of SphK1 with siRNA reversed PDGF-induced S1P production, miR-21 up-regulation, BMPRII and Id1 down-regulation, as well as PASMC proliferation. Pre-inhibition of miR-21 also blocked BMPRII and Id1 down-regulation as well as PASMC proliferation caused by PDGF. Knockdown of BMPRII down-regulated Id1 expression in PASMCs. We further found that inhibition of PI3K/Akt and ERK signaling pathways, particularly ERK cascade, suppressed PDGF-induced above changes.. Our study indicates that SphK1/S1P pathway plays an important role in PDGF-induced PASMC proliferation via miR-21/BMPRII/Id1 axis and targeting against SphK1/S1P axis might be a novel strategy in the prevention and treatment of pulmonary arterial hypertension (PAH).

Topics: Animals; Antagomirs; Bone Morphogenetic Protein Receptors, Type II; Cell Proliferation; Inhibitor of Differentiation Protein 1; Lysophospholipids; Male; MicroRNAs; Mitogen-Activated Protein Kinase 3; Myocytes, Smooth Muscle; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Pulmonary Artery; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; Signal Transduction; Sphingosine; Up-Regulation

Pancreatic cancer is a disease with poor prognosis, and development of new treatments is necessary. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinases (SphK1 and SphK2), plays a critical role in progression of many types of cancer. However, little is known about the role of sphingosine kinases in pancreatic cancer. This study investigated the roles of sphingosine kinases in pancreatic cancer progression.. S1P levels in pancreatic cancer and noncancerous pancreatic tissue were measured in 10 patients. We generated PAN02 murine pancreatic cancer cell lines with a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system genes 9 (Cas9)-mediated deletion of SphK1 or SphK2 and assessed cell growth and migration. In an animal model, we assessed the survival of mice injected with PAN02 cells intraperitoneally.. S1P levels in the pancreatic cancer tissue were significantly higher than those in noncancerous tissue. SphK1 knockout (KO) cells showed greater proliferation and migration than wild type (WT) cells, and SphK2 KO cells showed less proliferation and migration than WT cells. Animal experiments showed that the survival of mice injected with SphK1 KO cells was significantly shorter than those injected with WT cells, and the survival of mice injected with SphK2 KO cells was longer than those injected with WT cells. Surprisingly, cytotoxic assay using gemcitabine showed that SphK1 KO cells survived less than WT cells, and SphK2 KO cells survived more than WT cells.. S1P produced by SphK1 and SphK2 may have different functions in pancreatic cancer cells. Targeting both SphK1 and SphK2 may be a potential strategy for pancreatic cancer treatment.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Disease Progression; Humans; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Pancreas; Pancreatic Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine 1-phosphate (S1P) signaling regulates numerous biological processes including neurogenesis, inflammation and neovascularization. However, little is known about the role of S1P signaling in the eye. In this study, we characterize two sphingosine kinases (SPHK1 and SPHK2), which phosphorylate sphingosine to S1P, and three S1P receptors (S1PR1, S1PR2 and S1PR3) in mouse and rat eyes. We evaluated sphingosine kinase and S1P receptor gene expression at the mRNA level in various rat tissues and rat retinas exposed to light-damage, whole mouse eyes, specific eye structures, and in developing retinas. Furthermore, we determined the localization of sphingosine kinases and S1P receptors in whole rat eyes by immunohistochemistry. Our results unveiled unique expression profiles for both sphingosine kinases and each receptor in ocular tissues. Furthermore, these kinases and S1P receptors are expressed in mammalian retinal cells and the expression of SPHK1, S1PR2 and S1PR3 increased immediately after light damage, which suggests a function in apoptosis and/or light stress responses in the eye. These findings have numerous implications for understanding the role of S1P signaling in the mechanisms of ocular diseases such as retinal inflammatory and degenerative diseases, neovascular eye diseases, glaucoma and corneal diseases.

Topics: Animals; Cattle; Eye Proteins; Gene Expression Regulation; Immunohistochemistry; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Retina; Sphingosine

Topics: Animals; Brain; Brain Ischemia; Enzyme Inhibitors; Hemodynamics; Humans; Lysophospholipids; Male; Mice; Microscopy; Neuroprotection; Neuroprotective Agents; Oxygen; Phosphotransferases (Alcohol Group Acceptor); Photoacoustic Techniques; Sphingosine; Stroke

Sphingosine kinase 1 (SphK1) promotes cell proliferation and survival, and its abundance is often increased in tumors. SphK1 produces the signaling lipid sphingosine 1-phosphate (S1P), which activates signaling cascades downstream five G protein-coupled receptors (S1P

Topics: Animals; Blotting, Western; Cell Cycle Proteins; Cell Line, Tumor; Cells, Cultured; Chromosome Segregation; HeLa Cells; Humans; Lysophospholipids; Mice, Knockout; Microscopy, Confocal; Mitosis; Phosphotransferases (Alcohol Group Acceptor); Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Receptors, Lysosphingolipid; RNA Interference; Signal Transduction; Sphingosine; Time-Lapse Imaging

There is growing interest in the use of bone marrow cells to treat liver fibrosis, however, little is known about their antifibrotic efficacy or the identity of their effector cell(s). Sphingosine-1-phosphate (S1P) mediates egress of immune cells from the lymphoid organs into the lymphatic vessels; we investigated its role in the response of hematopoietic stem cells (HSCs) to liver fibrosis in mice.. Purified (c-kit+/sca1+/lin-) HSCs were infused repeatedly into mice undergoing fibrotic liver injury. Chronic liver injury was induced in BoyJ mice by injection of carbon tetrachloride (CCl. Infusions of HSCs into mice with liver injury reduced liver scarring based on picrosirius red staining (49.7% reduction in mice given HSCs vs control mice; P < .001), and hepatic hydroxyproline content (328 mg/g in mice given HSCs vs 428 mg/g in control mice; P < .01). HSC infusion also reduced hepatic expression of α-smooth muscle actin (0.19 ± 0.007-fold compared with controls; P < .0001) and collagen type I α 1 chain (0.29 ± 0.17-fold compared with controls; P < .0001). These antifibrotic effects were maintained with infusion of lymphoid progenitors that lack myeloid potential and were associated with increased numbers of recipient neutrophils and macrophages in liver. In studies of HSC cell lines, we found HSCs to recruit monocytes, and this process to require C-C motif chemokine receptor 2. In fibrotic liver tissue from mice and patients, hepatic S1P levels increased owing to increased hepatic sphingosine kinase-1 expression, which contributed to a reduced liver:lymph S1P gradient and limited HSC egress from the liver. Mice given the S1P antagonist (FTY720) with HSCs had increased hepatic retention of HSCs (1697 ± 247 cells in mice given FTY720 vs 982 ± 110 cells in controls; P < .05), and further reductions in fibrosis.. In studies of mice with chronic liver injury, we showed the antifibrotic effects of repeated infusions of purified HSCs. We found that HSCs promote recruitment of endogenous macrophages and neutrophils. Strategies to reduce SIP signaling and increase retention of HSCs in the liver could increase their antifibrotic activities and be developed for treatment of patients with liver fibrosis.

Topics: Actins; Aldehyde-Lyases; Animals; Cell Line; Cell Movement; Chemical and Drug Induced Liver Injury, Chronic; Collagen Type I; Collagen Type I, alpha 1 Chain; Female; Fingolimod Hydrochloride; Gene Expression; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Immunosuppressive Agents; Liver; Liver Cirrhosis; Lymph; Lysophospholipids; Macrophages; Male; Membrane Proteins; Mice; Monocytes; Neutrophils; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Graves' orbitopathy (GO) is initiated by excessive amount of various inflammatory mediators produced by orbital fibroblasts. This study aimed to assess the crucial role of sphingosine-1-phosphate (S1P) in the inflammatory process of GO.. Orbital adipose/connective tissue samples were obtained from 10 GO patients and 10 normal control individuals during surgery. Primary orbital fibroblast culture was done. After the expression of S1P receptors and sphingosine kinase (SphK) was assessed with the treatment of interleukin (IL)-1β, we evaluated the expression of pro-inflammatory factors [intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2) and IL-6] after treating S1P. S1P receptor antagonists and SphK 1 inhibitor were pretreated and the expression of the pro-inflammatory factors was assessed.. IL-1β exacerbated the inflammatory process by enhancing the expression of S1P receptors and SphK in GO orbital fibroblasts. IL-1β also induced the expressions of ICAM-1, COX-2, and IL-6 in GO orbital fibroblasts, and these expressions were effectively inhibited by S1P receptor antagonists and SphK1 inhibitor.. S1P has an important role in the pathological inflammatory process of GO, which is mediated through the SphK1-S1P- S1P receptor pathway. SphK1 inhibitors and S1P receptors or antagonists could be potential approaches for controlling the inflammatory process of GO.

Topics: Adult; Aged; Connective Tissue; Cyclooxygenase 2; Female; Fibroblasts; Graves Ophthalmopathy; Humans; Inflammation; Interleukin-1beta; Interleukin-6; Lysophospholipids; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine

To establish whether Sphingosine-1-phosphate (S1P) and sphingosine kinase 1 (SphK1) contribute to lymph node metastasis in esophageal squamous cell carcinoma.. Immunohistochemical analysis of SphK1 expression was performed using a tissue microarray containing 177 thoracic squamous cell esophageal cancer specimens resected at surgery, to investigate the association between intratumoral SphK1 expression and lymph node metastasis. Serum S1P levels and intratumoral SphK1 mRNA and protein expression were also evaluated in mice with vs. mice without lymph node metastasis in a murine lymph node metastasis model.. Among 177 esophageal cancer patients, 127 (72%) were defined as being SphK1-positive. In univariate and multivariate analyses, SphK1 expression status was a significant factor contributing to lymph node metastasis and poorer 5-year overall survival. In the murine lymph node metastasis model, there was no difference in tumor volume or weight between the lymph node metastasis-negative and lymph node metastasis-positive groups. However, levels of SphK1 mRNA and protein and serum S1P levels were all much higher in the metastasis-positive group.. S1P/SphK1 may be novel targets for inhibiting lymph node metastasis in esophageal squamous cell carcinoma, and may provide the basis for a therapeutic strategy to suppress lymph node metastasis.

Topics: Aged; Animals; Carcinoma, Squamous Cell; Disease Models, Animal; Esophageal Neoplasms; Female; Gene Expression; Humans; Lymphatic Metastasis; Lysophospholipids; Male; Mice; Middle Aged; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Sphingosine

Diabetes is considered a major public health problem affecting millions of individuals worldwide. Remarkably, scientific reports regarding salivary glands sphingolipid metabolism in diabetes are virtually non-existent. This is odd given the well-established link between the both in other tissues (e.g., skeletal muscles, liver) and the key role of these glands in oral health preservation. The aim of this paper is to examine sphingolipids metabolism in the salivary glands in (pre)diabetes (evoked by high fat diet feeding or streptozotocin). Wistar rats were allocated into three groups: control, HFD-, or STZ-diabetes. The content of major sphingolipid classes in the parotid (PSG) and submandibular (SMSG) glands was assessed via chromatography. Additionally, Western blot analyses were employed for the evaluation of key sphingolipid signaling pathway enzyme levels. No changes in ceramide content in the PSG were found, whereas an increase in ceramide concentration for SMSG of the STZ group was observed. This was accompanied by an elevation in SPT1 level. Probably also sphingomyelin hydrolysis was increased in the SMSG of the STZ-diabetic rats, since we observed a significant drop in the amount of SM. PSG and SMSG respond differently to (pre)diabetes, with clearer pattern presented by the later gland. An activation of sphingomyelin signaling pathway was observed in the course of STZ-diabetes, that is, metabolic condition with rapid onset/progression. Whereas, chronic HFD lead to an inhibition of sphingomyelin signaling pathway in the salivary glands (manifested in an inhibition of ceramide de novo synthesis and accumulation of S1P).

Topics: Animals; Ceramides; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diet, High-Fat; Insulin Resistance; Lysophospholipids; Male; Obesity; Parotid Gland; Phosphotransferases (Alcohol Group Acceptor); Rats, Wistar; Signal Transduction; Sphingolipids; Sphingomyelin Phosphodiesterase; Sphingosine; Sphingosine N-Acyltransferase; Streptozocin; Submandibular Gland

Epithelial ovarian carcinomas account for more than 90% of human ovarian cancers and have become the primary cause of death for gynecological malignancies. Unlimited cell proliferation and resistance to cell apoptosis contribute to the development of ovarian cancers. However, the underlying mechanisms involved in these processes in epithelial ovarian carcinomas are yet poorly understood. In the present study, we examined the Hippo signaling gene expression and investigated the effects of Sphingosine 1-phosphate (S1P) on cell proliferation and the underlying mechanisms in human ovarian cancer cell lines, OVCAR3 and SKOV3. Our results demonstrate that S1P disrupts Hippo signaling by reducing YAP phosphorylation and increasing the expression of CCN1 and CCN2 in both ovarian cancer cells. Furthermore, the increase in CCN1/CCN2 expression contributes to the S1P-induced increase in cancer cell proliferation.

Topics: Active Transport, Cell Nucleus; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Connective Tissue Growth Factor; Cysteine-Rich Protein 61; Female; Gene Expression Regulation, Neoplastic; Hippo Signaling Pathway; Humans; Lysophospholipids; Nuclear Proteins; Ovarian Neoplasms; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Protein Serine-Threonine Kinases; Signal Transduction; Sphingosine; Transcription Factors

Death receptor (DR5), a well-characterized death domain-containing cell surface pro-apoptotic protein, has been suggested to suppress cancer cell invasion and metastasis. However, the underlying mechanisms have not been fully elucidated. Our recent work demonstrates that DR5 suppression promotes cancer cell invasion and metastasis through caspase-8/TRAF2-mediated activation of ERK and JNK signaling and MMP1 elevation. The current study aimed at addressing the mechanism through which TRAF2 is activated in a caspase-8 dependent manner.. DR5 knockdown increased TRAF2 polyubiquitination, a critical event for TRAF2-mediated JNK/AP-1 activation. Suppression of sphingosine-1-phosphate (S1P) generation or depletion of casapse-8 inhibited not only enhancement of cell invasion, but also elevation and polyubiquitination of TRAF2, activation of JNK/AP-1 activation and increased expression of MMP1 induced by DR5 knockdown.. Both S1P and caspase-8 are critical for TRAF2 stabilization, polyubiquitination, subsequent activation of JNK/AP1 signaling and MMP1 expression and final promotion of cell invasion.

Topics: Caspase 8; Cell Line, Tumor; Enzyme Activation; Gene Knockdown Techniques; Humans; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; Matrix Metalloproteinase 1; Neoplasm Invasiveness; Phosphotransferases (Alcohol Group Acceptor); Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; Sphingosine; TNF Receptor-Associated Factor 2; Transcription Factor AP-1; Ubiquitination

Berberine (BBR) has shown neuroprotective properties. The present study aims to investigate the effects of BBR on experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), and SphK1/S1P signaling, which plays a key role in MS. EAE was induced in mice, followed by treatment with BBR at 50, 100, or 300 mg/kg/d. Neurophysiological function was evaluated daily; inflammation, cell infiltration, and the severity of demyelination were also examined. The SphK1, SphK2, and S1P levels in the animals and primary astrocyte culture were measured. We found that treatment with BBR reduced the loss of neurophysiological function and the degree of demyelination. Moreover, BBR was associated with a decrease in SphK1 and S1P levels both in the animals and in culture. These results indicated that BBR suppresses demyelination and loss of neurophysiological function by inhibiting the SphK1/S1P signaling pathway. The use of BBR as a treatment of MS warrant further exploration.

Topics: Animals; Berberine; Encephalomyelitis, Autoimmune, Experimental; Female; Lysophospholipids; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Neuroprotective Agents; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Spinal Cord

Spondyloarthritis (SpA) is a relatively common chronic inflammatory joint disorder, with a prevalence of about 0.2-0.5% worldwide. The primary target of the pathological process is the enthesis, where tendons and ligaments attach to underlying bone. These insertion sites are hotspots of bone formation (enthesophytes), which can lead to ankylosis. Unfortunately, the mechanisms causing the onset and progression of entheseal ossification remain largely unknown. Sphingosine 1-phosphate (S1P), a lipid generated after sphingosine phosphorylation by sphingosine kinases 1 and 2 (SK1/2), plays important roles in cell proliferation, differentiation and survival. S1P regulates fundamental biological processes such as cell cycle, inflammatory response or bone homeostasis. Indeed, S1P has been involved in some of most-spread skeletal diseases such as rheumatoid arthritis or osteoarthritis. On the other hand, the implication of S1P in SpA has not been explored yet. In the present work, we observed by ELISA that S1P content was significantly increased in the serum of SpA patients (6.1±4.2μM, n=21) compared to healthy donors (1.6±0.9μM, n=12). In vitro, gene expression of SK1 and SK2 as well as their activity were increased during differentiation of primary murine chondrocytes and osteoblasts into mineralizing cells. In addition, mRNA of the S1P-specific transporter Spns2 and S1P secretion were augmented. Using the pharmacological drugs SKi (SK pan-inhibitor), PF-543 (SK1 specific inhibitor) or K-145 (SK2 specific inhibitor), we showed that the inhibition of SK1 and/or SK2 decreased matrix mineralization, alkaline phosphatase activity and the mRNA expression of Runx2 and Bglap in chondrocytes and osteoblasts. To our knowledge, this is the first study indicating that S1P levels are significantly increased in serum from SpA patients. Moreover, we showed in vitro that SK activity was involved in the mineralization capacity of osteoblasts and chondrocytes. S1P metabolic pathway may represent an ingenious therapeutic target for SpA in the future.

Topics: Adolescent; Adult; Aged; Animals; Calcification, Physiologic; Female; Humans; Lysophospholipids; Male; Mice; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Spondylarthritis; Young Adult

Huntington's disease is characterized by a complex and heterogeneous pathogenic profile. Studies have shown that disturbance in lipid homeostasis may represent a critical determinant in the progression of several neurodegenerative disorders. The recognition of perturbed lipid metabolism is only recently becoming evident in HD. In order to provide more insight into the nature of such a perturbation and into the effect its modulation may have in HD pathology, we investigated the metabolism of Sphingosine-1-phosphate (S1P), one of the most important bioactive lipids, in both animal models and patient samples. Here, we demonstrated that S1P metabolism is significantly disrupted in HD even at early stage of the disease and importantly, we revealed that such a dysfunction represents a common denominator among multiple disease models ranging from cells to humans through mouse models. Interestingly, the in vitro anti-apoptotic and the pro-survival actions seen after modulation of S1P-metabolizing enzymes allows this axis to emerge as a new druggable target and unfolds its promising therapeutic potential for the development of more effective and targeted interventions against this incurable condition.

Topics: Aged; Aldehyde-Lyases; Animals; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Humans; Huntington Disease; Lysophospholipids; Male; Mice; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine

To delineate the role of Sphingolipids (SPLs) in the human cornea and their cross-talks with transforming growth factor beta (TGF-β) in order to develop novel, non-invasive therapies.. Human corneal fibroblasts (HCFs) were harvested from healthy donors, stimulated with Vitamin C to promote extracellular matrix assembly, treated with exogenous sphingosine-1-phosphate (S1P) or sphingosine kinase inhibitor 2 (SPHK I2) and isolated after 4 weeks for further analysis.. Data showed that S1P led to a significant decrease in cellular migration where SPHK I2 just delayed it for 24h. Significant modulation of the sphingolipid pathway was also noted. Sphingosine kinase-1 (SphK1) was significantly downregulated upon exogenous stimulation with S1P at a concentration of 5μM and Sphingosine kinase-2 (SphK2) was also significantly downregulated at concentrations of 0.01μM, 0.1μM, and 5μM; whereas no effects were observed upon stimulation with SPHK I2. S1PR3 was significantly downregulated by 0.1μM and 5μM S1P and upregulated by 5μM and 10μM SPHK I2. Furthermore, both S1P and SPHK I2 regulated corneal fibrosis markers such as alpha-smooth muscle actin, collagen I, III, and V. We also investigated the interplay between two TGF-β isoforms and S1P/SPHK I2 treatments and found that TGF-β1 and TGF-β3 were both significantly upregulated with the 0.1μM S1P but were significantly downregulated with the 5μM S1P concentration. When TGF-β1 was compared directly to TGF-β3 expression, we observed that TGF-β3 was significantly downregulated compared to TGF-β1 in the 5μM concentration of S1P. No changes were observed upon SPHK I2 treatment.. Our study delineates the role of sphingolipids in the human cornea and highlights their different activities based on the cell/tissue type.

Topics: Blotting, Western; Cell Extracts; Cell Movement; Corneal Stroma; Fibroblasts; Fibrosis; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Isoforms; Protein Kinase Inhibitors; Signal Transduction; Sphingolipids; Sphingosine; Transforming Growth Factor beta; Wound Healing

Sepsis mouse models revealed thymus atrophy, characterised by decreased thymus weight and loss of thymocytes due to apoptosis. Mice suffered from lymphopenia, a lack of T cells in the periphery, which attenuates their ability to fight against recurring and secondary infections during sepsis progression. Key players in thymus atrophy are IL-6, which is directly involved in thymus involution, and the sphingosine-1-phosphate - sphingosine-1-phosphate receptor 1 signaling, influencing thymocytes emigration. In healthy individuals a sphingosine-1-phosphate (S1P) gradient from lymphoid organs to the circulatory system serves as signal for mature T cell egress. In the present study we investigated, whether inhibition of S1P generation improves thymus involution. In sepsis, induced by cecal ligation and puncture (CLP), S1P in the thymus increased, while it decreased in serum, thus disrupting the naturally occurring S1P gradient. As a potential source of S1P we identified increased numbers of apoptotic cells in the thymic cortex of septic mice. Pharmacological inhibition of the S1P generating sphingosine kinases, by 4- [[4-(4-Chlorophenyl)-2-thiazolyl]amino]phenol (SK I-II), administered directly following CLP, prevented thymus atrophy. This was reflected by lymphocytosis, diminished apoptosis, decreased IL-6 expression, and an unaltered thymus weight. In addition SK I-II-treatment preserved the S1P balance and prevented S1P-dependent internalization of the sphingosine-1-phosphate receptor 1. Our data suggest that inhibition of sphingosine kinase and thus, S1P generation during sepsis restores thymic T cell egress, which might improve septic outcome.

Topics: Aminophenols; Animals; Apoptosis; Atrophy; Cecum; Disease Models, Animal; Interleukin-6; Lymphocytosis; Lymphopenia; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sepsis; Sphingosine; Thiazoles; Thymocytes; Thymus Gland

This study aimed to evaluate the effects of exosomes produced by human-induced pluripotent stem cell-derived mesenchymal stromal cells (hiPSC-MSCs-Exo) on hepatic ischemia-reperfusion (I/R) injury, as well as the underlying mechanisms.. Exosomes derived from hiPSC-MSCs were isolated and characterized both biochemically and biophysically. hiPSC-MSCs-Exo were injected systemically into a murine ischemia/reperfusion injury model via the inferior vena cava, and then the therapeutic effects were evaluated. The serum levels of transaminases (aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as histological changes were examined. Primary hepatocytes and human hepatocyte cell line HL7702 were used to test whether exosomes could induce hepatocytes proliferation in vitro. In addition, the expression levels of proliferation markers (proliferation cell nuclear antigen, PCNA; Phosphohistone-H3, PHH3) were measured by immunohistochemistry and Western blot. Moreover, SK inhibitor (SKI-II) and S1P1 receptor antagonist (VPC23019) were used to investigate the role of sphingosine kinase and sphingosine-1-phosphate-dependent pathway in the effects of hiPSC-MSCs-Exo on hepatocytes.. hiPSCs were efficiently induced into hiPSC-MSCs that had typical MSC characteristics. hiPSC-MSCs-Exo had diameters ranging from 100 to 200 nm and expressed exosome markers (Alix, CD63 and CD81). After hiPSC-MSCs-Exo administration, hepatocyte necrosis and sinusoidal congestion were markedly suppressed in the ischemia/reperfusion injury model, with lower histopathological scores. The levels of hepatocyte injury markers AST and ALT were significantly lower in the treatment group compared to control, and the expression levels of proliferation markers (PCNA and PHH3) were greatly induced after hiPSC-MSCs-Exo administration. Moreover, hiPSC-MSCs-Exo also induced primary hepatocytes and HL7702 cells proliferation in vitro in a dose-dependent manner. We found that hiPSC-MSCs-Exo could directly fuse with target hepatocytes or HL7702 cells and increase the activity of sphingosine kinase and synthesis of sphingosine-1-phosphate (S1P). Furthermore, the inhibition of SK1 or S1P1 receptor completely abolished the protective and proliferative effects of hiPSC-MSCs-Exo on hepatocytes, both in vitro and in vivo.. Our results demonstrated that hiPSC-MSCs-Exo could alleviate hepatic I/R injury via activating sphingosine kinase and sphingosine-1-phosphate pathway in hepatocytes and promote cell proliferation. These findings represent a novel mechanism that potentially contributes to liver regeneration and have important implications for new therapeutic approaches to acute liver disease.

Topics: Animals; Cell Line; Cell Proliferation; Cells, Cultured; Exosomes; Hepatocytes; Humans; Induced Pluripotent Stem Cells; Liver; Lysophospholipids; Male; Mesenchymal Stem Cells; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Signal Transduction; Sphingosine

Doxorubicin is one of the most commonly used chemotherapeutic drugs for breast cancer; however, its use is limited by drug resistance and side effects. We hypothesized that adding FTY720, a sphingosine-1-phosphate (S1P) receptor functional antagonist, to doxorubicin would potentiate its effects by suppression of drug-induced inflammation.. The Cancer Genome Atlas, Gene Expression Omnibus data sets, and National Cancer Institute-60 panel were used for gene expressions and gene set enrichment analysis. E0771 syngeneic mammary tumor cells were used. OB/OB mice fed with western high-fat diet were used as an obesity model.. STAT3 expression was significantly increased after doxorubicin treatment in human breast cancer that implicates that doxorubicin evokes inflammation. Expression of sphingosine kinase 1, the enzyme that produces S1P and links inflammation and cancer, tended to be higher in doxorubicin-resistant human cancer and cell lines. In a murine breast cancer model, sphingosine kinase 1, S1P receptor 1, interleukin 6, and STAT3 were overexpressed in the doxorubicin-treated group, whereas all of them were significantly suppressed with addition of FTY720. Combination therapy synergistically suppressed cancer growth both in vitro and in vivo. Furthermore, combination therapy showed higher efficacy in an obesity breast cancer model, where high body mass index demonstrated trends toward worse disease-free and overall survival, and high-serum S1P levels in human patients and volunteers.. We found that FTY720 enhanced the efficacy of doxorubicin by suppression of drug-induced inflammation, and combination therapy showed stronger effect in obesity-related breast cancer.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Body Mass Index; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; Female; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Lysophospholipids; Mammary Neoplasms, Experimental; Mice; Obesity; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Retrospective Studies; Sphingosine; STAT3 Transcription Factor

Sphingolipid metabolism pathway is essential in membrane homeostasis, and its dysfunction has been associated with favorable tumor microenvironment, disease progression, and chemotherapy resistance. Its major components have key functions on survival and proliferation, with opposing effects. We have profiled the components of the sphingolipid pathway on leukocytes of breast cancer (BC) patients undergoing chemotherapy treatment and without, including the five sphingosine 1-phosphate (S1P) receptors, the major functional genes, and cytokines, in order to better understand the S1P signaling in the immune cells of these patients. To the best of our knowledge, this is the first characterization of the sphingolipid pathway in whole blood of BC patients. Skewed gene profiles favoring high

Topics: Breast Neoplasms; Case-Control Studies; Female; Humans; Leukocytes; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tumor Necrosis Factor-alpha

Renal ischemia-reperfusion is a main cause of acute kidney injury (AKI), which is associated with high mortality. Here we show that extracellular vesicles (EVs) secreted from hiPSC-MSCs play a critical role in protection against renal I/R injury. hiPSC-MSCs-EVs can fuse with renal cells and deliver SP1 into target cells, subsequently active SK1 expression and increase S1P formation. Chromatin immunoprecipitation (ChIP) analyses and luciferase assay were used to confirm SP1 binds directly to the SK1 promoter region and promote promoter activity. Moreover, SP1 inhibition (MIT) or SK1 inhibition (SKI-II) completely abolished the renal protective effect of hiPSC-MSCs-EVs in rat I/R injury mode. However, pre-treatment of necroptosis inhibitor Nec-1 showed no difference with the administration of hiPSC-MSCs-EVs only. We then generated an SP1 knockout hiPSC-MSC cell line by CRISPR/Cas9 system and found that SP1 knockout failed to show the protective effect of hiPSC-MSCs-EVs unless restoring the level of SP1 by Ad-SP1 in vitro and in vivo. In conclusion, this study describes an anti-necroptosis effect of hiPSC-MSCs-EVs against renal I/R injury via delivering SP1 into target renal cells and intracellular activating the expression of SK1 and the generation of S1P. These findings suggest a novel mechanism for renal protection against I/R injury, and indicate a potential therapeutic approach for a variety of renal diseases and renal transplantation.

Topics: Acute Kidney Injury; Animals; Apoptosis; Cell Differentiation; Cell Line, Transformed; Epithelial Cells; Extracellular Vesicles; Gene Expression Regulation; Humans; Induced Pluripotent Stem Cells; Kidney; Lysophospholipids; Male; Mesenchymal Stem Cells; Necrosis; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Sp1 Transcription Factor; Sphingosine

The non-dioxin-like environmental toxicant 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), member of a group of persistent organic pollutants wide-spread throughout the environment, reduces gap junction intercellular communication (GJIC), an event possibly associated with tumor promotion. Since very few studies have investigated the signaling effectors and mode(s) of action of PCB153, and it is known that the gap junction (GJ) protein Cx43 can be regulated by the bioactive sphingolipid (SL) sphingosine 1-phosphate (S1P), this in vitro study mainly addresses whether SL metabolism is affected by PCB153 in rat liver epithelial WB-F344 cells. PCB153 treatment obtained significant changes in the S1P/ceramide (Cer) ratio, known to be crucial in determining cell fate. In particular, an increase in S1P at 30 min and a decrease of the bioactive lipid at 3 h were observed, whereas Cer level increased at 1 h and 24 h. Notably, a time-dependent modulation of sphingosine kinase (SphK), the enzyme responsible for S1P synthesis, and of its regulators, ERK1/2 and protein phosphatase PP2A, supports the involvement of these signaling effectors in PCB153 toxicity. Electrophysiological analyses, furthermore, indicated that the lipophilic environmental toxicant significantly reduced GJ biophysical properties, affecting both voltage-dependent (such as those formed by Cx43 and/or Cx32) and voltage-independent channels, thereby demonstrating that PCB153 may act differently on GJs formed by distinct Cx isoforms. SphK down-regulation alone induced GJIC impairment, and, when combined with PCB153, the acute effect on GJ suppression was additive. Moreover, after enzyme-specific gene silencing, the SphK1 isoform appears to be responsible for down-regulating Cx43 expression, while being the target of PCB153 at short-term exposure. In conclusion, we provide the first evidence of novel effectors in PCB153 toxic action in rat liver stem-like cells, leading us to consider SLs as potential markers for preventing GJIC deregulation and, thus, the tumorigenic action elicited by this environmental toxicant.

Topics: Animals; Cells, Cultured; Connexin 43; Dioxins; Electrophysiology; Gap Junctions; Liver; Lysophospholipids; Mitogen-Activated Protein Kinase 3; Phosphotransferases (Alcohol Group Acceptor); Polychlorinated Biphenyls; Protein Phosphatase 2; Rats; Signal Transduction; Sphingolipids; Sphingosine

During the last two decades the study of the sphingolipid anabolic, catabolic, and signaling pathways has attracted enormous interest. Especially the introduction of fingolimod into market as first p.o. therapeutic for the treatment of multiple sclerosis has boosted this effect. Although the complex regulation of sphingosine-1-phosphate (S1P) and other catabolic and anabolic sphingosine-related compounds is not fully understood, the influence on different (patho)physiological states from inflammation to cytotoxicity as well as the availability of versatile pharmacological tools that represent new approaches to study these states are described. Here, we have summarized various aspects concerning the many faces of sphingolipid function modulation by different pharmacological tools up to clinical candidates. Due to the immense heterogeneity of physiological or pharmacological actions and complex cross regulations, it is difficult to predict their role in upcoming therapeutic approaches. Currently, inflammatory, immunological, and/or antitumor aspects are discussed.

Topics: Animals; Eicosanoids; Humans; Lysophospholipids; Membrane Microdomains; Metabolic Networks and Pathways; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Small Molecule Libraries; Sphingosine

The sphingosine kinase (SphK)/sphingosine 1-phosphate (S1P) pathway is involved in multiple biological processes, including carcinogenesis. Melatonin shows beneficial effects in cell and animal models of hepatocellular carcinoma, but it is unknown if they are associated with the modulation of the SphK/S1P system, along with different downstream signaling pathways modified in cancer. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35 mg/kg body weight i.p) once a week for 8 weeks. Melatonin was given at 5 or 10 mg/kg/day i.p. beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Melatonin alleviated the distortion of normal hepatic architecture, lowered the incidence of preneoplastic/neoplastic lesions, and inhibited the expression of proliferative/cell cycle regulatory proteins (Ki67, PCNA, cyclin D1, cyclin E, CDK4, and CDK6). S1P levels and expression of SphK1, SphK2, and S1P receptors (S1PR1/S1PR3) were significantly elevated in DEN-treated mice. However, there was a decreased expression of S1P lyase. These effects were significantly abrogated in a time- and dose-dependent manner by melatonin, which also increased S1PR2 expression. Following DEN treatment, mice exhibited increased phosphorylation of PI3K, AKT, mTOR, STAT3, ERK, and p38, and a higher expression of NF-κB p50 and p65 subunits. Melatonin administration significantly inhibited those changes. Data obtained suggest a contribution of the SphK/S1P system and related signaling pathways to the protective effects of melatonin in hepatocarcinogenesis.

Topics: Animals; Blotting, Western; Carcinogens; Carcinoma, Hepatocellular; Diethylnitrosamine; Disease Models, Animal; Immunohistochemistry; Liver Neoplasms; Lysophospholipids; Male; Melatonin; Mice; Mice, Inbred ICR; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; Signal Transduction; Sphingosine

Topics: Animals; Carbon Tetrachloride; Cell Line; Gene Expression Regulation; Hepatic Stellate Cells; Humans; Indoles; Lipid Metabolism; Liver Cirrhosis; Lysophospholipids; Male; Melatonin; Mice; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Acute myeloid leukemia (AML) is an aggressive malignancy where despite improvements in conventional chemotherapy and bone marrow transplantation, overall survival remains poor. Sphingosine kinase 1 (SPHK1) generates the bioactive lipid sphingosine 1-phosphate (S1P) and has established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers. The role and targeting of SPHK1 in primary AML, however, has not been previously investigated. Here we show that SPHK1 is overexpressed and constitutively activated in primary AML patient blasts but not in normal mononuclear cells. Subsequent targeting of SPHK1 induced caspase-dependent cell death in AML cell lines, primary AML patient blasts, and isolated AML patient leukemic progenitor/stem cells, with negligible effects on normal bone marrow CD34

Topics: Amino Acid Chloromethyl Ketones; Amino Alcohols; Animals; Bone Marrow Cells; Caspase Inhibitors; Caspases; Cell Death; Cell Line, Tumor; Female; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Lysophospholipids; Mice; Mice, Inbred NOD; Molecular Targeted Therapy; Myeloid Cell Leukemia Sequence 1 Protein; Neoplastic Stem Cells; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Quinolines; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Survival Analysis; Xenograft Model Antitumor Assays

The balance between cholesterol and sphingolipids within the plasma membrane has long been implicated in endocytic membrane trafficking. However, in contrast to cholesterol functions, little is still known about the roles of sphingolipids and their metabolites. Perturbing the cholesterol/sphingomyelin balance was shown to induce narrow tubular plasma membrane invaginations enriched with sphingosine kinase 1 (SphK1), the enzyme that converts the bioactive sphingolipid metabolite sphingosine to sphingosine-1-phosphate, and suggested a role for sphingosine phosphorylation in endocytic membrane trafficking. Here we show that sphingosine and sphingosine-like SphK1 inhibitors induced rapid and massive formation of vesicles in diverse cell types that accumulated as dilated late endosomes. However, much smaller vesicles were formed in SphK1-deficient cells. Moreover, inhibition or deletion of SphK1 prolonged the lifetime of sphingosine-induced vesicles. Perturbing the plasma membrane cholesterol/sphingomyelin balance abrogated vesicle formation. This massive endosomal influx was accompanied by dramatic recruitment of the intracellular SphK1 and Bin/Amphiphysin/Rvs domain-containing proteins endophilin-A2 and endophilin-B1 to enlarged endosomes and formation of highly dynamic filamentous networks containing endophilin-B1 and SphK1. Together, our results highlight the importance of sphingosine and its conversion to sphingosine-1-phosphate by SphK1 in endocytic membrane trafficking.

Topics: Animals; Caveolins; Cell Line; Clathrin; Endocytosis; Endosomes; Gene Deletion; Humans; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

About 40,000 American women die from metastatic breast cancer each year despite advancements in treatment. Approximately, 15% of breast cancers are triple-negative for estrogen receptor, progesterone receptor, and HER2. Triple-negative cancer is characterized by more aggressive, harder to treat with conventional approaches and having a greater possibility of recurrence. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid signaling mediator has emerged as a key regulatory molecule in breast cancer progression. Therefore, we investigated whether cytosolic sphingosine kinase type 1 (SphK1) and nuclear sphingosine kinase type 2 (SphK2), the enzymes that make S1P are critical for growth and PI3K/AKT, ERK-MAP kinase mediated survival signaling of lung metastatic variant LM2-4 breast cancer cells, generated from the parental triple-negative MDA-MB-231 human breast cancer cell line. Similar with previous report, SphKs/S1P signaling is critical for the growth and survival of estrogen receptor positive MCF-7 human breast cancer cells, was used as our study control. MDA-MB-231 did not show a significant effect of SphKs/S1P signaling on AKT, ERK, and p38 pathways. In contrast, LM2-4 cells that gained lung metastatic phenotype from primary MDA-MB-231 cells show a significant effect of SphKs/S1P signaling requirement on cell growth, survival, and cell motility. PF-543, a selective potent inhibitor of SphK1, attenuated epidermal growth factor (EGF)-mediated cell growth and survival signaling through inhibition of AKT, ERK, and p38 MAP kinase pathways mainly in LM2-4 cells but not in parental MDA-MB-231 human breast cancer cells. Moreover, K-145, a selective inhibitor of SphK2, markedly attenuated EGF-mediated cell growth and survival of LM2-4 cells. We believe this study highlights the importance of SphKs/S1P signaling in metastatic triple-negative breast cancers and targeted therapies.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Down-Regulation; Epidermal Growth Factor; Female; Humans; Lysophospholipids; Neoplasm Metastasis; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; RNA, Small Interfering; Signal Transduction; Sphingosine; Triple Negative Breast Neoplasms

We have determined the gene expression of sphingosine-1-phosphate (S1P) metabolizing enzymes (SphK1, SphK2, SGPL1, SGPP1, SGPP2, PPAP2A, PPAP2B, and PPAP2C) by quantitative real-time polymerase chain reaction in tumor tissues and adjacent normal tissues of 50 oral squamous cell carcinoma (OSCC) patients. Expression of SphK1 and SGPP1 genes was up-regulated significantly in 70% and 75% OSCC tumors respectively. Importantly, expression of SphK2 and PPAP2B was down-regulated in the tumor tissues of 70% OSCC patients. Expression of SphK2 and PPAP2B negatively correlated with tumor-node-metastasis (TNM) staging and tumor volume respectively. Furthermore, LPP1 is an independent predictor of TNM staging and lymph node ratio.

Topics: Adult; Aged; Female; Humans; Lip Neoplasms; Lysophospholipids; Male; Membrane Proteins; Middle Aged; Mouth Neoplasms; Neoplasm Staging; Phosphatidate Phosphatase; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; RNA, Messenger; Sphingosine; Tongue Neoplasms; Young Adult

Pancreatic islet transplantation is a promising clinical treatment for type 1 diabetes, but success is limited by extensive β-cell death in the immediate posttransplant period and impaired islet function in the longer term. Following transplantation, appropriate vascular remodeling is crucial to ensure the survival and function of engrafted islets. The sphingosine kinase (SK) pathway is an important regulator of vascular beds, but its role in the survival and function of transplanted islets is unknown. We observed that donor islets from mice deficient in SK1 (

Topics: Animals; Cell Movement; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Endothelial Cells; Flow Cytometry; Islets of Langerhans; Islets of Langerhans Transplantation; Lysophospholipids; Mice; Mice, Knockout; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; Sphingosine; Transplants

Huntington disease (HD) is the most common inherited neurodegenerative disorder. It has no cure. The protein huntingtin causes HD, and mutations to it confer toxic functions to the protein that lead to neurodegeneration. Thus, identifying modifiers of mutant huntingtin-mediated neurotoxicity might be a therapeutic strategy for HD. Sphingosine kinases 1 (SK1) and 2 (SK2) synthesize sphingosine-1-phosphate (S1P), a bioactive lipid messenger critically involved in many vital cellular processes, such as cell survival. In the nucleus, SK2 binds to and inhibits histone deacetylases 1 and 2 (HDAC1/2). Inhibiting both HDACs has been suggested as a potential therapy in HD. Here, we found that SK2 is nuclear in primary neurons and, unexpectedly, overexpressed SK2 is neurotoxic in a dose-dependent manner. SK2 promotes DNA double-strand breaks in cultured primary neurons. We also found that SK2 is hyperphosphorylated in the brain samples from a model of HD, the BACHD mice. These data suggest that the SK2 pathway may be a part of a pathogenic pathway in HD. ABC294640, an inhibitor of SK2, reduces DNA damage in neurons and increases survival in two neuron models of HD. Our results identify a novel regulator of mutant huntingtin-mediated neurotoxicity and provide a new target for developing therapies for HD.

Topics: Animals; Cell Nucleus; Disease Models, Animal; Gene Expression Regulation; Histone Deacetylase 1; Histone Deacetylase 2; Humans; Huntingtin Protein; Huntington Disease; Lysophospholipids; Mice; Neurons; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

During development, progenitor expansion, lineage allocation, and implementation of differentiation programs need to be tightly coordinated so that different cell types are generated in the correct numbers for appropriate tissue size and function. Pancreatic dysfunction results in some of the most debilitating and fatal diseases, including pancreatic cancer and diabetes. Several transcription factors regulating pancreas lineage specification have been identified, and Notch signalling has been implicated in lineage allocation, but it remains unclear how these processes are coordinated. Using a combination of genetic approaches, organotypic cultures of embryonic pancreata, and genomics, we found that sphingosine-1-phosphate (S1p), signalling through the G protein coupled receptor (GPCR) S1pr2, plays a key role in pancreas development linking lineage allocation and specification. S1pr2 signalling promotes progenitor survival as well as acinar and endocrine specification. S1pr2-mediated stabilisation of the yes-associated protein (YAP) is essential for endocrine specification, thus linking a regulator of progenitor growth with specification. YAP stabilisation and endocrine cell specification rely on Gαi subunits, revealing an unexpected specificity of selected GPCR intracellular signalling components. Finally, we found that S1pr2 signalling posttranscriptionally attenuates Notch signalling levels, thus regulating lineage allocation. Both S1pr2-mediated YAP stabilisation and Notch attenuation are necessary for the specification of the endocrine lineage. These findings identify S1p signalling as a novel key pathway coordinating cell survival, lineage allocation, and specification and linking these processes by regulating YAP levels and Notch signalling. Understanding lineage allocation and specification in the pancreas will shed light in the origins of pancreatic diseases and may suggest novel therapeutic approaches.

Topics: Acinar Cells; Adaptor Proteins, Signal Transducing; Animals; Body Patterning; Cell Cycle Proteins; Cell Differentiation; Cell Lineage; Cell Survival; GTP-Binding Protein alpha Subunits, Gi-Go; Lysophospholipids; Mice; Models, Biological; Pancreas; Phosphoproteins; Phosphotransferases (Alcohol Group Acceptor); Protein Subunits; Receptors, Lysosphingolipid; Receptors, Notch; Signal Transduction; Sphingosine; Stem Cells; YAP-Signaling Proteins

Renal fibrosis is a progressive pathological change characterized by tubular cell apoptosis, tubulointerstitial fibroblast proliferation, and excessive deposition of extracellular matrix (ECM). miR-21 has been implicated in transforming growth factor-β (TGF-β)-stimulated tissue fibrosis. Recent studies showed that sphingosine kinase/sphingosine-1-phosphate (SphK/S1P) are also critical for TGF-β-stimulated tissue fibrosis; however, it is not clear whether SphK/S1P interacts with miR-21 or not. In this study, we hypothesized that SphK/S1P signaling is linked to upregulation of miR-21 by TGF-β. To verify this hypothesis, we first determined that miR-21 was highly expressed in renal tubular epithelial cells (TECs) stimulated with TGF-β by using qRT-PCR and Northern blotting. Simultaneously, inhibition of miR-21, mediated by the corresponding antimir, markedly decreased the expression and deposition of type I collagen, fibronectin (Fn), cysteine-rich protein 61 (CCN1), α-smooth muscle actin, and fibroblast-specific protein1 in TGF-β-treated TECs. ELISA and qRT-PCR were used to measure the S1P and SphK1 levels in TECs. S1P production was induced by TGF-β through activation of SphK1. Furthermore, it was observed that TGF-β-stimulated upregulation of miR-21 was abolished by SphK1 siRNA and was restored by the addition of exogenous S1P. Blocking S1PR2 also inhibited upregulation of miR-21. Additionally, miR-21 overexpression attenuated the repression of TGF-β-stimulated ECM deposition and epithelial-mesenchymal transition by SphK1 and S1PR2 siRNA. In summary, our study demonstrates a link between SphK1/S1P and TGF-β-induced miR-21 in renal TECs and may represent a novel therapeutic target in renal fibrosis.

Topics: Blotting, Northern; Cell Survival; Epithelial Cells; Humans; Lysophospholipids; MicroRNAs; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; Signal Transduction; Sphingosine; Transforming Growth Factor beta

Hyperglycemia induces all isoforms of transforming growth factor β (TGFβ), which in turn play key roles in inflammation and fibrosis that characterize diabetic nephropathy. Sphingosine 1-phosphate (S1P) is a signaling sphingolipid, derived from sphingosine by the action of sphingosine kinase (SK). S1P mediates many biological processes, which mimic TGFβ signaling. To determine the role of SK1 and S1P in inducing fibrosis and inflammation, and the interaction with TGFβ-1, 2 and 3 signalling in diabetic nephropathy, human proximal tubular cells (HK2 cells) were exposed to normal (5 mmol/L) or high (30 mmol/L) glucose or TGFβ-1, -2, -3 ± an SK inhibitor (SKI-II) or SK1 siRNA. Control and diabetic wild type (WT) and SK1(-/-) mice were studied. Fibrotic and inflammatory markers, and relevant downstream signalling pathways were assessed. SK1 mRNA and protein expression was increased in HK2 cells exposed to high glucose or TGFβ1,-2,-3. All TGFβ isoforms induced fibronectin, collagen IV and macrophage chemoattractant protein 1 (MCP1), which were reversed by both SKI-II and SK1 siRNA. Exposure to S1P increased phospho-p44/42 expression, AP-1 binding and NFkB phosphorylation. WT diabetic mice exhibited increased renal cortical S1P, fibronectin, collagen IV and MCP1 mRNA and protein expression compared to SK1(-/-) diabetic mice. In summary, this study demonstrates that inhibiting the formation of S1P reduces tubulointerstitial renal inflammation and fibrosis in diabetic nephropathy.

Topics: Animals; Biomarkers; Cell Line; Diabetic Nephropathies; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Gene Expression Regulation, Enzymologic; Gene Silencing; Glucose; Humans; Inflammation; Kidney Cortex; Kidney Tubules; Lysophospholipids; Male; Mice; NF-kappa B; Phosphoproteins; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Transcription Factor AP-1; Transforming Growth Factor beta

Sphingosine 1-phosphate (S1P) mediates multiple pathophysiological effects in the cardiovascular system. However, the role of S1P signaling in pathological cardiac remodeling following myocardial infarction (MI) remains controversial. In this study, we found that cardiac S1P greatly increased post-MI, accompanied with a significant upregulation of cardiac sphingosine kinase-1 (SphK1) and S1P receptor 1 (S1PR1) expression. In MI-operated mice, inhibition of S1P production by using PF543 (the SphK1 inhibitor) ameliorated cardiac remodeling and dysfunction. Conversely, interruption of S1P degradation by inhibiting S1P lyase augmented cardiac S1P accumulation and exacerbated cardiac remodeling and dysfunction. In the cardiomyocyte, S1P directly activated proinflammatory responses via a S1PR1-dependent manner. Furthermore, activation of SphK1/S1P/S1PR1 signaling attributed to β1-adrenergic receptor stimulation-induced proinflammatory responses in the cardiomyocyte. Administration of FTY720, a functional S1PR1 antagonist, obviously blocked cardiac SphK1/S1P/S1PR1 signaling, ameliorated chronic cardiac inflammation, and then improved cardiac remodeling and dysfunction in vivo post-MI. In conclusion, our results demonstrate that cardiac SphK1/S1P/S1PR1 signaling plays an important role in the regulation of proinflammatory responses in the cardiomyocyte and targeting cardiac S1P signaling is a novel therapeutic strategy to improve post-MI cardiac remodeling and dysfunction.

Topics: Animals; Animals, Newborn; Cytokines; Fingolimod Hydrochloride; Heart Diseases; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocarditis; Myocytes, Cardiac; Phosphotransferases (Alcohol Group Acceptor); Rats, Sprague-Dawley; Receptors, Lysosphingolipid; RNA, Small Interfering; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Transfection; Ultrasonography

Sphingolipid metabolites have emerged playing important roles in the pathogenesis of nonalcoholic fatty liver disease, whereas the underlying mechanism remains largely unknown. In the present study, we provide both in vitro and in vivo evidence showing a pathogenic role of sphingosine kinase 1 (SphK1) in hepatocellular steatosis. We found that levels of SphK1 expression were significantly increased in steatotic hepatocytes. Enforced overexpression of SphK1 or treatment with sphingosine 1-phosphate (S1P) markedly enhanced hepatic lipid accumulation. In contrast, the siRNA-mediated knockdown of SphK1 or S1P receptors, S1P2 and S1P3, profoundly inhibited lipid accumulation in hepatocytes. Moreover, Sphk1(-/-) mice exhibited a significant amelioration of hepatosteatosis under diet-induced obese (DIO) conditions, compared to wild-type littermates. In addition, DIO-induced up-regulation of PPARγ and its target genes were significantly reduced by SphK1 deficiency. Furthermore, treatment of hepatocytes with S1P induces a dose-dependent increase in PPARγ expression at the transcriptional level. Blockage of S1P receptors and the Akt-mTOR signaling profoundly inhibited S1P-induced PPARγ expression. Notably, down-regulation of PPARγ by using its siRNA significantly diminished the pro-steatotic effect of SphK1/S1P. Thus, the study demonstrates a new pathway connecting SphK1 and PPARγ involved in the pathogenesis of hepatocellular steatosis.

Topics: Animals; Diet, High-Fat; Dietary Fats; Fatty Liver; Gene Expression Regulation; Hepatocytes; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Obese; Obesity; Phosphotransferases (Alcohol Group Acceptor); PPAR gamma; Proto-Oncogene Proteins c-akt; Receptors, Lysosphingolipid; RNA, Small Interfering; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; TOR Serine-Threonine Kinases; Transcription, Genetic

Multiple sclerosis patients are treated with fingolimod (FTY720), a prodrug that acts as an immune modulator. FTY720 is first phosphorylated to FTY720-P and then internalizes sphingosine-1-phosphate receptors, preventing lymphocyte sequestration. IL-33 is released from necrotic endothelial cells and contributes to MS severity by coactivating T cells. Herein we analyzed the influence of FTY720, FTY720-P, and S1P on IL-33 induced formation of IL-2 and IFN-γ, by using IL-33 receptor overexpressing EL4 cells, primary CD8(+) T cells, and splenocytes. EL4-ST2 cells released IL-2 after IL-33 stimulation that was inhibited dose-dependently by FTY720-P but not FTY720. In this system, S1P increased IL-2, and accordingly, inhibition of S1P producing sphingosine kinases diminished IL-2 release. In primary CD8(+) T cells and splenocytes IL-33/IL-12 stimulation induced IFN-γ, which was prevented by FTY720 but not FTY720-P, independently from intracellular phosphorylation. The inhibition of IFN-γ by nonphosphorylated FTY720 was mediated via the SET/protein phosphatase 2A (PP2A) pathway, since a SET peptide antagonist also prevented IFN-γ formation and the inhibition of IFN-γ by FTY720 was reversible by a PP2A inhibitor. While our findings directly improve the understanding of FTY720 therapy in MS, they could also contribute to side effects of FTY720 treatment, like progressive multifocal leukoencephalopathy, caused by an insufficient immune response to a viral infection.

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Line, Tumor; DNA-Binding Proteins; Female; Fingolimod Hydrochloride; Histone Chaperones; Interferon-gamma; Interleukin-2; Interleukin-33; Lysophospholipids; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Oncogene Proteins; Organophosphates; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Phosphatase 2; Sphingosine; Spleen

Sphingosine kinase 1 (SPHK1), the enzyme that produces sphingosine 1 phosphate (S1P), is known to be highly expressed in many cancers. However, the role of SPHK1 in cells of the tumor stroma remains unclear. Here, we show that SPHK1 is highly expressed in the tumor stroma of high-grade serous ovarian cancer (HGSC), and is required for the differentiation and tumor promoting function of cancer-associated fibroblasts (CAFs). Knockout or pharmacological inhibition of SPHK1 in ovarian fibroblasts attenuated TGF-β-induced expression of CAF markers, and reduced their ability to promote ovarian cancer cell migration and invasion in a coculture system. Mechanistically, we determined that SPHK1 mediates TGF-β signaling via the transactivation of S1P receptors (S1PR2 and S1PR3), leading to p38 MAPK phosphorylation. The importance of stromal SPHK1 in tumorigenesis was confirmed in vivo, by demonstrating a significant reduction of tumor growth and metastasis in SPHK1 knockout mice. Collectively, these findings demonstrate the potential of SPHK1 inhibition as a novel stroma-targeted therapy in HGSC.

Topics: Animals; Apoptosis; Blotting, Western; Cell Differentiation; Cell Movement; Cell Proliferation; Cystadenocarcinoma, Serous; Female; Fibroblasts; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Lysophospholipids; Mice; Mice, Knockout; Myofibroblasts; Neoplasm Grading; Ovarian Neoplasms; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Real-Time Polymerase Chain Reaction; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sphingosine; Survival Rate; Transforming Growth Factor beta; Tumor Cells, Cultured

Alveolar macrophages from chronic obstructive pulmonary disease patients and cigarette smokers are deficient in their ability to phagocytose apoptotic bronchial epithelial cells (efferocytosis). We hypothesized that the defect is mediated via inhibition of sphingosine kinases and/or their subcellular mislocalization in response to cigarette smoke and can be normalized with exogenous sphingosine-1-phosphate or FTY720 (fingolimod), a modulator of sphingosine-1-phosphate signaling, which has been shown to be clinically useful in multiple sclerosis. Measurement of sphingosine kinase 1/2 activities by [(32)P]-labeled sphingosine-1-phosphate revealed a 30% reduction of sphingosine kinase 1 (P < 0.05) and a nonsignificant decrease of sphingosine kinase 2 in THP-1 macrophages after 1 h cigarette smoke extract exposure. By confocal analysis macrophage sphingosine kinase 1 protein was normally localized to the plasma membrane and cytoplasm and sphingosine kinase 2 to the nucleus and cytoplasm but absent at the cell surface. Cigarette smoke extract exposure (24 h) led to a retraction of sphingosine kinase 1 from the plasma membrane and sphingosine kinase 1/2 clumping in the Golgi domain. Selective inhibition of sphingosine kinase 2 with 25 µM ABC294640 led to 36% inhibition of efferocytosis (P < 0.05); 10 µM sphingosine kinase inhibitor/5C (sphingosine kinase 1-selective inhibitor) induced a nonsignificant inhibition of efferocytosis, but its combination with ABC294640 led to 56% inhibition (P < 0.01 vs. control and < 0.05 vs. single inhibitors). Cigarette smoke-inhibited efferocytosis was significantly (P < 0.05) reversed to near-control levels in the presence of 10-100 nM exogenous sphingosine-1-phosphate or FTY720, and FTY720 reduced cigarette smoke-induced clumping of sphingosine kinase 1/2 in the Golgi domain. These data strongly support a role of sphingosine kinase 1/2 in efferocytosis and as novel therapeutic targets in chronic obstructive pulmonary disease.

Topics: Bronchi; Cells, Cultured; Epithelial Cells; Fingolimod Hydrochloride; Gene Expression Regulation, Enzymologic; Humans; Immunosuppressive Agents; Lysophospholipids; Macrophages, Alveolar; Phagocytosis; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Disease, Chronic Obstructive; Signal Transduction; Smoking; Sphingosine

Capillaries of the external part of the normal arterial wall constitute the vasa vasorum network. In atherosclerotic lesions, neovascularization occurs in areas of intimal hyperplasia where it may promote plaque expansion, and intraplaque hemorrhage. Oxidized LDL that are present in atherosclerotic areas activate various angiogenic signaling pathways, including reactive oxygen species and the sphingosine kinase/sphingosine-1-phosphate pathway. We aimed to investigate whether oxidized LDL-induced angiogenesis requires neutral sphingomyelinase-2 activation and the neutral sphingomyelinase-2/sphingosine kinase-1 pathway. The role of neutral sphingomyelinase-2 in angiogenic signaling was investigated in Human Microvascular Endothelial Cells (HMEC-1) forming capillary tube on Matrigel and in vivo in the Matrigel plug assay in C57BL/6 mice and in the chicken chorioallantoic membrane model. Low concentration of human oxidized LDL elicits HMEC-1 capillary tube formation and neutral sphingomyelinase-2 activation, which were blocked by neutral sphingomyelinase-2 inhibitors, GW4869 and specific siRNA. This angiogenic effect was mimicked by low concentration of C6-Ceramide and was inhibited by sphingosine kinase-1 inhibitors. Upstream of neutral sphingomyelinase-2, oxidized LDL-induced activation required LOX-1, reactive oxygen species generation by NADPH oxidase and p38-MAPK activation. Inhibition of sphingosine kinase-1 blocked the angiogenic response and triggered HMEC-1 apoptosis. Low concentration of oxidized LDL was angiogenic in vivo, both in the Matrigel plug assay in mice and in the chorioallantoic membrane model, and was blocked by GW4869. In conclusion, low oxLDL concentration triggers sprouting angiogenesis that involves ROS-induced activation of the neutral sphingomyelinase-2/sphingosine kinase-1 pathway, and is effectively inhibited by GW4869.

Topics: Aniline Compounds; Animals; Apoptosis; Benzylidene Compounds; Ceramides; Endothelial Cells; Humans; Lipoproteins, LDL; Lysophospholipids; Mice; Muscle, Smooth, Vascular; NADPH Oxidases; Neovascularization, Pathologic; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Reactive Oxygen Species; Sphingomyelin Phosphodiesterase; Sphingosine; Transcriptional Activation

Dynamic interaction between cancer cells and the surrounding microenvironment is critical for cancer progression via changes in cellular behavior including alteration of secreted molecules. However, the molecular mechanisms underlying the influence exerted by the cancer microenvironment on secretion of molecules during cancer progression remain largely unknown. In this study, we report that secretion of spingsine-1-phosphate (S1P) and its regulator, SphK1 expression is dependent of the substrate rigidity, which is critical for the balance between cancer cell invasion and adhesion. Conditioned media (CM) of MDA-MB-231, an aggressive breast cancer cell obtained from soft substrate (~0.5 kPa) induced chemo-attractive invasion, while CM obtained from stiff substrate (~2.5 kPa) increased cell adhesion instead. We found that the expression of SphK1 is upregulated in the stiff substrate, resulting in an increase in S1P levels in the CM. We also found that upregulation of SphK1 expression in the stiff substrate is dominant in metastatic cancer cells but not in primary cancer cells. These results suggest that alterations in the mechanical environment of the ECM surrounding the tumor cells actively regulate cellular properties such as secretion, which in turn, may contribute to cancer progression.

Topics: Biomechanical Phenomena; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Culture Media, Conditioned; Extracellular Matrix; Female; Humans; Lysophospholipids; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tumor Microenvironment

Although sphingosine 1-phosphate (S1P) has been reported to play an important role in cancer pathophysiology, little is known about S1P and hepatocellular carcinoma (HCC). To clarify the relationship between S1P and HCC, 77 patients with HCC who underwent surgical treatment were consecutively enrolled in this study. In addition, S1P and its metabolites were quantitated by LC-MS/MS. The mRNA levels of sphingosine kinases (SKs), which phosphorylate sphingosine to generate S1P, were increased in HCC tissues compared with adjacent non-HCC tissues. Higher mRNA levels of SKs in HCC were associated with poorer differentiation and microvascular invasion, whereas a higher level of SK2 mRNA was a risk factor for intra- and extra-hepatic recurrence. S1P levels, however, were unexpectedly reduced in HCC compared with non-HCC tissues, and increased mRNA levels of S1P lyase (SPL), which degrades S1P, were observed in HCC compared with non-HCC tissues. Higher SPL mRNA levels in HCC were associated with poorer differentiation. Finally, in HCC cell lines, inhibition of the expression of SKs or SPL by siRNA led to reduced proliferation, invasion and migration, whereas overexpression of SKs or SPL enhanced proliferation. In conclusion, increased SK and SPL mRNA expression along with reduced S1P levels were more commonly observed in HCC tissues compared with adjacent non-HCC tissues and were associated with poor differentiation and early recurrence. SPL as well as SKs may be therapeutic targets for HCC treatment.

Topics: Aldehyde-Lyases; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Lysophospholipids; Metabolome; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Phosphotransferases (Alcohol Group Acceptor); Risk Factors; RNA, Messenger; Sphingosine

A signaling molecule which is involved in proliferation and migration of malignant cells is the lipid mediator sphingosine-1-phosphate (S1P). There are hints for a potential role of S1P signaling in malignant brain tumors such as glioblastoma multiforme (GBM) which is characterized by a poor prognosis. Therefore, a comprehensive expression analysis of S1P receptors (S1P1-S1P5) and S1P metabolizing enzymes in human GBM (n = 117) compared to healthy brain (n = 10) was performed to evaluate their role for patient´s survival. Furthermore, influence of S1P receptor inhibition on proliferation and migration were studied in LN18 GBM cells. Compared to control brain, mRNA levels of S1P1, S1P2, S1P3 and S1P generating sphingosine kinase-1 were elevated in GBM. Kaplan-Meier analyses demonstrated an association between S1P1 and S1P2 with patient´s survival times. In vitro, an inhibitory effect of the SphK inhibitor SKI-II on viability of LN18 cells was shown. S1P itself had no effect on viability but stimulated LN18 migration which was blocked by inhibition of S1P1 and S1P2. The participation of S1P1 and S1P2 in LN18 migration was further supported by siRNA-mediated silencing of these receptors. Immunoblots and inhibition experiments suggest an involvement of the PI3-kinase/AKT1 pathway in the chemotactic effect of S1P in LN18 cells.In summary, our data argue for a role of S1P signaling in proliferation and migration of GBM cells. Individual components of the S1P pathway represent prognostic factors for patients with GBM. Perspectively, a selective modulation of S1P receptor subtypes could represent a therapeutic approach for GBM patients and requires further evaluation.

Topics: Biomarkers, Tumor; Brain Neoplasms; Cell Movement; Glioblastoma; Humans; Kaplan-Meier Estimate; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

We recently identified a previously unidentified sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates NF-κB through formation of a previously unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP.

Topics: Animals; Antimicrobial Cationic Peptides; Blotting, Western; Cathelicidins; Cell Line; Cell Line, Tumor; Cells, Cultured; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Humans; Keratinocytes; Lysophospholipids; Mice, Knockout; Microscopy, Fluorescence; NF-kappa B; Nuclear Pore Complex Proteins; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; Signal Transduction; Sphingosine; TNF Receptor-Associated Factor 2

White matter lesions (WML) are thought to contribute to vascular cognitive impairment in elderly patients. Growing evidence show that failure of myelin formation arising from the disruption of oligodendrocyte progenitor cell (OPC) differentiation is a cause of chronic vascular white matter damage. The sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) signaling pathway regulates oligodendroglia differentiation and function, and is known to be altered in hypoxia. In this study, we measured SphK, S1P as well as markers of WML, hypoxia and OPC (NG2) in a mouse bilateral carotid artery stenosis (BCAS) model of chronic cerebral hypoperfusion. Our results indicated that BCAS induced hypoxia inducible factor (HIF)-1α, Sphk2, S1P, and NG2 up-regulation together with accumulation of WML. In contrast, BCAS mice treated with the SphK inhibitor, SKI-II, showed partial reversal of SphK2, S1P and NG2 elevation and amelioration of WML. In an in vitro model of hypoxia, SKI-II reversed the suppression of OPC differentiation. Our study suggests a mechanism for hypoperfusion-associated WML involving HIF-1α-SphK2-S1P-mediated disruption of OPC differentiation, and proposes the SphK signaling pathway as a potential therapeutic target for white matter disease.

Topics: Animals; Brain; Carotid Artery Diseases; Cells, Cultured; Chronic Disease; Enzyme Inhibitors; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Sphingosine; Thiazoles; White Matter

We previously demonstrated that activation of sphingosine kinase 1 (SphK1)- sphingosine 1- phosphate (S1P) signaling pathway by high glucose (HG) plays a pivotal role in increasing the expression of fibronectin (FN), an important fibrotic component, by promoting the DNA-binding activity of transcription factor activator protein 1 (AP-1) in glomerular mesangial cells (GMCs) under diabetic conditions. As a multi-target anti-oxidative drug, polydatin (PD) has been shown to have renoprotective effects on experimental diabetes. However, whether PD could resist diabetic nephropathy (DN) by regulating SphK1-S1P signaling pathway needs further investigation. Here, we found that PD significantly reversed the upregulated FN and ICAM-1 expression in GMCs exposed to AGEs. Simultaneously, PD dose-dependently inhibited SphK1 levels at the protein expression and kinase activity and attenuated S1P production under AGEs treatment conditions. In addition, PD reduced SphK activity in GMCs transfected with wild-type SphK(WT) plasmid and significantly suppressed SphK1-mediated increase of FN and ICAM-1 levels under normal conditions. Furthermore, we found that the AGEs-induced upregulation of phosphorylation of c-Jun at Ser63 and Ser73 and c-Fos at Ser32, DNA-binding activity and transcriptional activity of AP-1 were blocked by PD. In comparison with db/db model group, PD treatment suppressed SphK1 levels (mRNA, protein expression, and activity) and S1P production, reversed the upregulation of FN, ICAM-1, c-Jun, and c-Fos in the kidney tissues of diabetic mice, and finally ameliorated renal injury in db/db mice. These findings suggested that the downregulation of SphK1-S1P signaling pathway is probably a novel mechanism by which PD suppressed AGEs-induced FN and ICAM-1 expression and improved renal dysfunction of diabetic models.

Topics: Animals; Cells, Cultured; Diabetes Mellitus, Experimental; Drugs, Chinese Herbal; Female; Fibronectins; Glomerular Mesangium; Glucosides; Glycation End Products, Advanced; Humans; Hypoglycemic Agents; Intercellular Adhesion Molecule-1; Lysophospholipids; Male; Mice; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Signal Transduction; Sphingosine; Stilbenes; Up-Regulation

Microglial activation is one of the causative factors of neuroinflammation in cerebral ischemia/reperfusion (IR). Sphingosine kinase 1 (Sphk1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P), plays an important role in the regulation of proinflammatory cytokines in activated microglia. Recent research demonstrated that S1P increased IL-17A-secretion and then worsened CNS (central nervous system) inflammation. Thus, in the present study, we sought to use microglial cells as the object of study to discuss the molecular mechanisms in Sphk1/S1P-regulated IL-17A-secretion in IR.. We used immunofluorescence and confocal microscopy to detect whether Sphk1 is expressed in microglia after cerebral IR or oxygen-glucose deprivation (OGDR). Western blot analysis was used to estimate the total Sphk1 protein level at different time points after OGDR. To detect cytokine secretion in microglial supernatants in response to OGDR, we measured the concentration of IL-17A in the culture supernatants using an enzyme-linked immunosorbent assay (ELISA). To evaluate whether microglia subjected to OGDR exhibited neuronal injury, we used a commercially available terminal transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) kit to detect apoptotic neurons.. Sphk1 was expressed in microglia in response to cerebral IR or OGDR at appointed time. Pre-injection with PF-543, an inhibitor of Sphk1, before IR clearly reduced the expression of Sphk1 in microglia relative to brain IR alone. The number of TUNEL-positive neurons was also decreased in the PF-543-pretreated animals before IR compared to the animals with IR alone. When S1P was administered in OGDR microglia, IL-17A expression and neuronal apoptosis were increased compared to OGDR alone and the administration of S1P alone. ELISA further confirmed the above results. Moreover, the inhibition of Sphk1 by siRNA reduced IL-17A production and relieved neuronal apoptosis in OGDR microglia.. These results indicated that Sphk1/S1P regulates the expression of IL-17A in activated microglia, inducing neuronal apoptosis in cerebral ischemia/reperfusion. The microglial Sphk1/S1P pathway may thus be a potential therapeutic target to control neuroinflammation in brain IR.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Cells, Cultured; Glucose; Hypoxia, Brain; Infarction, Middle Cerebral Artery; Interleukin-17; Lysophospholipids; Male; Methanol; Microglia; Neurons; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidines; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Small Interfering; Sphingosine; Sulfones

The sphingosine kinase (SphK)1/sphingosine-1-phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10(4) hemagglutination units of a RHDV isolate and received 20 mg/kg of melatonin at 0, 12, and 24 hr postinfection. Liver mRNA levels, protein concentration, and immunohistochemical labeling for SphK1 increased in RHDV-infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll-like receptor (TLR)4, tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, nuclear factor-kappa B (NF-κB) p50 and p65 subunits, and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent.

Topics: Animals; Caliciviridae Infections; Hemorrhagic Disease Virus, Rabbit; Hepatitis, Viral, Animal; Liver Failure, Acute; Lysophospholipids; Male; Melatonin; Phosphotransferases (Alcohol Group Acceptor); Rabbits; Signal Transduction; Sphingosine

Oral squamous cell carcinoma (OSCC) is a lethal disease with a 5-year mortality rate of around 50%. Molecular targeted therapies are not in routine use and novel therapeutic targets are required. Our previous microarray data indicated sphingosine 1-phosphate (S1P) metabolism and signalling was deregulated in OSCC. In this study, we have investigated the contribution of S1P signalling to the pathogenesis of OSCC. We show that the expression of the two major enzymes that regulate S1P levels were altered in OSCC: SPHK1 was significantly upregulated in OSCC tissues compared to normal oral mucosa and low levels of SGPL1 mRNA correlated with a worse overall survival. In in vitro studies, S1P enhanced the migration/invasion of OSCC cells and attenuated cisplatin-induced death. We also demonstrate that S1P receptor expression is deregulated in primary OSCCs and that S1PR2 is over-expressed in a subset of tumours, which in part mediates S1P-induced migration of OSCC cells. Lastly, we demonstrate that FTY720 induced significantly more apoptosis in OSCC cells compared to non-malignant cells and that FTY720 acted synergistically with cisplatin to induce cell death. Taken together, our data show that S1P signalling promotes tumour aggressiveness in OSCC and identify S1P signalling as a potential therapeutic target.

Topics: Aldehyde-Lyases; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line; Cell Line, Tumor; Cell Movement; Cell Survival; Cisplatin; Drug Synergism; Female; Fingolimod Hydrochloride; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunosuppressive Agents; Kaplan-Meier Estimate; Lysophospholipids; Male; Middle Aged; Mouth Neoplasms; Phenotype; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors

The tumor microenvironment is a determining factor for cancer biology and progression. Sphingosine-1-phosphate (S1P), produced by sphingosine kinases (SphKs), is a bioactive lipid mediator that regulates processes important for cancer progression. Despite its critical roles, the levels of S1P in interstitial fluid (IF), an important component of the tumor microenvironment, have never previously been measured due to a lack of efficient methods for collecting and quantifying IF. The purpose of this study is to clarify the levels of S1P in the IF from murine mammary glands and its tumors utilizing our novel methods. We developed an improved centrifugation method to collect IF. Sphingolipids in IF, blood, and tissue samples were measured by mass spectrometry. In mice with a deletion of SphK1, but not SphK2, levels of S1P in IF from the mammary glands were greatly attenuated. Levels of S1P in IF from mammary tumors were reduced when tumor growth was suppressed by oral administration of FTY720/fingolimod. Importantly, sphingosine, dihydro-sphingosine, and S1P levels, but not dihydro-S1P, were significantly higher in human breast tumor tissue IF than in the normal breast tissue IF. To our knowledge, this is the first reported S1P IF measurement in murine normal mammary glands and mammary tumors, as well as in human patients with breast cancer. S1P tumor IF measurement illuminates new aspects of the role of S1P in the tumor microenvironment.

Topics: Activation, Metabolic; Animals; Antineoplastic Agents; Breast; Breast Neoplasms; Cell Line, Tumor; Extracellular Fluid; Female; Fingolimod Hydrochloride; Humans; Isoenzymes; Lysophospholipids; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice, Inbred BALB C; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Prodrugs; Random Allocation; Sphingosine; Tumor Microenvironment

Phyto-S1P and S1P induced stomatal closure in epidermis of pea ( Pisum sativum ) by raising the levels of NO and pH in guard cells. Phosphosphingolipids, such as phytosphingosine-1-phosphate (phyto-S1P) and sphingosine-1-phosphate (S1P), are important signaling components during drought stress. The biosynthesis of phyto-S1P or S1P is mediated by sphingosine kinases (SPHKs). Although phyto-S1P and S1P are known to be signaling components in higher plants, their ability to induce stomatal closure has been ambiguous. We evaluated in detail the effects of phyto-S1P, S1P and SPHK inhibitors on signaling events leading to stomatal closure in the epidermis of Pisum sativum. Phyto-S1P or S1P induced stomatal closure, along with a marked rise in nitric oxide (NO) and cytoplasmic pH of guard cells, as in case of ABA. Two SPHK inhibitors, DL-threo dihydrosphingosine and N',N'-dimethylsphingosine, restricted ABA-induced stomatal closure and prevented the increase of NO or pH by ABA. Modulators of NO or pH impaired both stomatal closure and increase in NO or pH by phyto-S1P/S1P. The stomatal closure by phyto-S1P/S1P was mediated by phospholipase D and phosphatidic acid (PA). When present, PA elevated the levels of pH, but not NO of guard cells. Our results demonstrate that stomatal closure induced by phyto-S1P and S1P depends on rise in pH as well as NO of guard cells. A scheme of signaling events initiated by phyto-S1P/S1P, and converging to cause stomatal closure, is proposed.

Topics: Abscisic Acid; Analysis of Variance; Fluorescent Dyes; Hydrogen-Ion Concentration; Lysophospholipids; Microscopy, Confocal; Nitric Oxide; Phosphotransferases (Alcohol Group Acceptor); Pisum sativum; Plant Epidermis; Plant Growth Regulators; Plant Stomata; Signal Transduction; Sphingosine; Time Factors

Sphingosine-1-phosphate (S1P) is a bioactive signalling lipid highly enriched in mature erythrocytes, with unknown functions pertaining to erythrocyte physiology. Here by employing nonbiased high-throughput metabolomic profiling, we show that erythrocyte S1P levels rapidly increase in 21 healthy lowland volunteers at 5,260 m altitude on day 1 and continue increasing to 16 days with concurrently elevated erythrocyte sphingonisne kinase 1 (Sphk1) activity and haemoglobin (Hb) oxygen (O2) release capacity. Mouse genetic studies show that elevated erythrocyte Sphk1-induced S1P protects against tissue hypoxia by inducing O2 release. Mechanistically, we show that intracellular S1P promotes deoxygenated Hb anchoring to the membrane, enhances the release of membrane-bound glycolytic enzymes to the cytosol, induces glycolysis and thus the production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific glycolytic intermediate, which facilitates O2 release. Altogether, we reveal S1P as an intracellular hypoxia-responsive biolipid promoting erythrocyte glycolysis, O2 delivery and thus new therapeutic opportunities to counteract tissue hypoxia.

Topics: 2,3-Diphosphoglycerate; Adaptation, Physiological; Adult; Altitude Sickness; Animals; Erythrocytes; Female; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Glycolysis; Humans; Hypoxia; Lysophospholipids; Male; Mice, Inbred C57BL; Mice, Mutant Strains; Oxygen; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Berberine exerts neuroprotective and modulates hypoxia inducible factor-1-alpha (HIF-1[Formula: see text]. Based on the role of HIF-1[Formula: see text] in hypoxia preconditioning and association between HIF-1[Formula: see text] and sphingosine-1-phosphate (S1P), we hypothesized that berberine preconditioning (BP) would ameliorate the cerebral injury induced by ischemia through activating the system of HIF-1[Formula: see text] and S1P. Adult male rats with middle cerebral artery occlusion (MCAO) and rat primary cortical neurons treated with oxygen and glucose deprivation (OGD) with BP at 24[Formula: see text]h (40[Formula: see text]mg/kg) and 2[Formula: see text]h (10[Formula: see text][Formula: see text]mol/L), respectively, were used to determine the neuroprotective effects. The HIF-1[Formula: see text] accumulation, and S1P metabolism were assayed in the berberine-preconditioned neurons, and the HIF-1[Formula: see text]-mediated transcriptional modulation of sphingosine kinases (Sphk) 1 and 2 was analyzed using chromatin immunoprecipitation and real-time polymerase chain reaction. BP significantly prevented cerebral ischemic injury in the MCAO rats at 24[Formula: see text]h and 72[Formula: see text]h following ischemia/reperfusion. In OGD-treated neurons, BP enhanced HIF-1[Formula: see text] accumulation with activation of PI3K/Akt, and induced S1P production by activating Sphk2 via the promotion of HIF-1[Formula: see text]-mediated Sphk2 transcription. In conclusion, BP activated endogenous neuroprotective mechanisms associated with the S1P/HIF-1 pathway and helped protect neuronal cells against hypoxia/ischemia.

Topics: Animals; Berberine; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemia; Lysophospholipids; Male; Neurons; Neuroprotective Agents; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Sphingosine

Although implicated in neurodegeneration, autophagy has been characterized mostly in yeast and mammalian non-neuronal cells. In a recent study, we sought to determine if SPHK1 (sphingosine kinase 1), implicated previously in macroautophagy/autophagy in cancer cells, regulates autophagy in neurons. SPHK1 synthesizes sphingosine-1-phosphate (S1P), a bioactive lipid involved in cell survival. In our study, we discovered that, when neuronal autophagy is pharmacologically stimulated, SPHK1 relocalizes to the endocytic and autophagic organelles. Interestingly, in non-neuronal cells stimulated with growth factors, SPHK1 translocates to the plasma membrane, where it phosphorylates sphingosine to produce S1P. Whether SPHK1 also binds to the endocytic and autophagic organelles in non-neuronal cells upon induction of autophagy has not been demonstrated. Here, we determined if the effect in neurons is operant in the SH-SY5Y neuroblastoma cell line. In both non-differentiated and differentiated SH-SY5Y cells, a short incubation of cells in amino acid-free medium stimulated the formation of SPHK1-positive puncta, as in neurons. We also found that, unlike neurons in which these puncta represent endosomes, autophagosomes, and amphisomes, in SH-SY5Y cells SPHK1 is bound only to the endosomes. In addition, a dominant negative form of SPHK1 was very toxic to SH-SY5Y cells, but cultured primary cortical neurons tolerated it significantly better. These results suggest that autophagy in neurons is regulated by mechanisms that differ, at least in part, from those in SH-SY5Y cells.

Topics: Animals; Apoptosis; Autophagosomes; Autophagy; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Membrane; Cell Survival; Cells, Cultured; Endocytosis; Endosomes; Green Fluorescent Proteins; Humans; Light; Lipids; Lysophospholipids; Lysosomes; Neuroblastoma; Neurons; Phagosomes; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats; Signal Transduction; Sphingosine

Clear cell renal cell carcinoma (ccRCC) is characterized by intratumoral hypoxia and chemoresistance. The hypoxia-inducible factors HIF1α and HIF2α play a crucial role in ccRCC initiation and progression. We previously identified the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) pathway as a new modulator of HIF1α and HIF2α under hypoxia in various cancer cell models. Here, we report that FTY720, an inhibitor of the S1P signaling pathway, inhibits both HIF1α and HIF2α accumulation in several human cancer cell lines. In a ccRCC heterotopic xenograft model, we show that FTY720 transiently decreases HIF1α and HIF2α intratumoral level and modifies tumor vessel architecture within 5 days of treatment, suggesting a vascular normalization. In mice bearing subcutaneous ccRCC tumor, FTY720 and a gemcitabine-based chemotherapy alone display a limited effect, whereas, in combination, there is a significant effect on tumor size without toxicity. Noteworthy, administration of FTY720 for 5 days before chemotherapy is not associated with a more effective tumor control, suggesting a mode of action mainly independent of the vascular remodeling. In conclusion, these findings demonstrate that FTY720 could successfully sensitize ccRCC to chemotherapy and establish this molecule as a potent therapeutic agent for ccRCC treatment, independently of drug scheduling. Mol Cancer Ther; 15(10); 2465-74. ©2016 AACR.

Topics: Animals; Antineoplastic Agents; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Fingolimod Hydrochloride; Gene Expression; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lysophospholipids; Mice; Neovascularization, Pathologic; Oxygen Consumption; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Vascular Endothelial Growth Factor A; Vascular Remodeling; Xenograft Model Antitumor Assays

Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect.

Topics: Animals; Cell Proliferation; Ceramidases; Exosomes; Hepatocytes; Lysophospholipids; Male; Mice; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); rab GTP-Binding Proteins; rab27 GTP-Binding Proteins; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Reperfusion Injury; Sphingomyelin Phosphodiesterase; Sphingosine

Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, has been implicated as a key regulatory molecule in cancer through its ability to promote cell proliferation, migration, angiogenesis, and lymphangiogenesis. Previous studies suggested that S1P produced by sphingosine kinase 1 (SphK1) in breast cancer plays important roles in progression of disease and metastasis. However, the associations between S1P and clinical parameters in human breast cancer have not been well investigated to date.. We determined levels of S1P and other sphingolipids in breast cancer tissue by electrospray ionization-tandem mass spectrometry. Associations between S1P levels and clinicopathologic features of the tumors were analyzed. Expression of phospho-SphK1 (pSphK1) in breast cancer tissues was determined by immunohistochemical scoring.. Levels of S1P in breast cancer tissues were significantly higher in patients with high white blood cell count in the blood than those patients without. S1P levels were lower in patients with human epidermal growth factor receptor 2 overexpression and/or amplification than those patients without. Furthermore, cancer tissues with high pSphK1 expression showed significantly higher levels of S1P than cancer tissues without. Finally, patients with lymph node metastasis showed significantly higher levels of S1P in tumor tissues than the patients with negative nodes.. To our knowledge, this is the first study to demonstrate that high expression of pSphK1 is associated with higher levels of S1P, which in turn is associated with lymphatic metastasis in breast cancer.

Topics: Breast Neoplasms; Female; Genes, erbB-2; Humans; Lymphatic Metastasis; Lysophospholipids; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Receptors, Estrogen; Receptors, Progesterone; Sphingosine; Triple Negative Breast Neoplasms

Lipid metabolism is crucially involved with the promotion of malignant progression and metastasis in various cancers. Growing evidence suggests that many types of cancers express high levels of sphingosine kinase 1 (Sphk1), which is known to mediate cell proliferation We hypothesized that Sphk1/sphingosine-1-phosphate (S1P) signaling contributes to tumor progression. In MCF10A and MCF10A-Sphk1 breast epithelial cells, we used TNF-α to activate the Sphk1/S1P pathway and the measured expression levels of NF-κBp65 and cyclin D1 mRNA and protein in the presence and absence of an NF-κB-p65 inhibitor. Chromatin immunoprecipitation assays were performed to determine whether NF-κB-p65 binds to the cyclin D1 promoter. We found that overexpression of Sphk1 induced NF-κB-p65 activation, increased expression of cyclin D1, shortened the cell division cycle, and thus promoted proliferation of breast epithelial cells. These findings provide insight into the mechanism by which an Sphk1/NF-κB-p65/cyclin D1 signaling pathway mediates cell proliferation.

Topics: Binding Sites; Cell Cycle; Cell Line; Cell Proliferation; Cyclin D1; Epithelial Cells; Female; Humans; Lysophospholipids; Mammary Glands, Human; Phosphotransferases (Alcohol Group Acceptor); Promoter Regions, Genetic; Signal Transduction; Sphingosine; Time Factors; Transcription Factor RelA; Transcription, Genetic

To investigate the role of the complement 5a (C5a)/C5a receptor (C5aR) pathway in the pathogenesis of acute liver failure (ALF) in a mouse model.. BALB/c mice were randomly assigned to different groups, and intraperitoneal injections of lipopolysaccharide (LPS)/D-galactosamine (D-GalN) (600 mg/kg and 10 μg/kg) were used to induce ALF. The Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase (ALT) levels, at different time points within a 1-wk period, were detected with a biochemistry analyzer. Pathological examination of liver tissue was performed 36 h after ALF induction. Serum complement 5 (C5), C5a, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, high-mobility group protein B1 (HMGB1) and sphingosine-1-phosphate levels were detected by enzyme-linked immunosorbant assay. Hepatic morphological changes at 36 h after ALF induction were assessed by hematoxylin and eosin staining. Expression of C5aR, sphingosine kinase 1 (SphK1), p38-MAPK and p-p38-MAPK in liver tissue, peripheral blood mononuclear cells (PBMCs) and peritoneal exudative macrophages (PEMs) of mice or RAW 264.7 cells was analyzed by western blotting. C5aR mRNA levels were detected by quantitative real-time PCR.. Activation of C5 and up-regulation of C5aR were observed in liver tissue and PBMCs of mice with ALF. Blockade of C5aR with a C5aR antagonist (C5aRa C5aRa) significantly reduced the levels of serum ALT, inflammatory cytokines (TNF-α, IL-1β and IL-6) and HMGB1, as well as the liver tissue damage, but increased the survival rates (. The C5a/C5aR pathway is essential for up-regulating SphK1 expression through p38 MAPK activation in ALF in mice, which provides a potential immunotherapeutic strategy for ALF in patients.

Topics: Animals; Blotting, Western; Complement C5a; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Galactosamine; HMGB1 Protein; Interleukin-1beta; Interleukin-6; Kaplan-Meier Estimate; Leukocytes, Mononuclear; Lipopolysaccharides; Liver; Liver Failure, Acute; Lysophospholipids; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; p38 Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Random Allocation; Real-Time Polymerase Chain Reaction; Receptor, Anaphylatoxin C5a; RNA, Messenger; Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha

Neovascularization occurring in atherosclerotic lesions may promote plaque expansion, intraplaque haemorrhage and rupture. Oxidized LDL (oxLDL) are atherogenic, but their angiogenic effect is controversial; both angiogenic and anti-angiogenic effects have been reported. The angiogenic mechanism of oxLDL is partly understood, but the role of the angiogenic sphingolipid, sphingosine 1-phosphate (S1P), in this process is not known. Thus, we investigated whether S1P is involved in the oxLDL-induced angiogenesis and whether an anti-S1P monoclonal antibody can prevent this effect.. Angiogenesis was assessed by capillary tube formation by human microvascular endothelial cells (HMEC-1) cultured on Matrigel and in vivo by the Matrigel plug assay in C57BL/6 mice.. Human oxLDL exhibited a biphasic angiogenic effect on HMEC-1; low concentrations were angiogenic, higher concentrations were cytotoxic. The angiogenic response to oxLDL was blocked by the sphingosine kinase (SPHK) inhibitor, dimethylsphingosine, by SPHK1-siRNA and by an anti-S1P monoclonal antibody. Moreover, inhibition of oxLDL uptake and subsequent redox signalling by anti-CD36 and anti-LOX-1 receptor antibodies and by N-acetylcysteine, respectively, blocked SPHK1 activation and tube formation. In vivo, in the Matrigel plug assay, low concentrations of human oxLDL or murine oxVLDL also triggered angiogenesis, which was prevented by i.p. injection of the anti-S1P antibody.. These data highlight the role of S1P in angiogenesis induced by oxLDL both in HMEC-1 cultured on Matrigel and in vivo in the Matrigel plug model in mice, and demonstrate that the anti-S1P antibody effectively blocks the angiogenic effect of oxLDL.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Cell Movement; Humans; Lipoproteins, LDL; Lysophospholipids; Mice, Inbred C57BL; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; RNA, Small Interfering; Sphingosine

Sphingosine-1-phosphate (S1P) has been widely associated with inflammation-based lung pathologies. Because B cells play a critical role as antigen-presenting and/or Ig-producing cells during asthmatic conditions, we wanted to dissect the role of these cells in S1P-dependent airway hyperreactivity and inflammation. Mice were sensitized to ovalbumin or exposed to S1P. Ovalbumin sensitization caused airway hyperreactivity coupled to an increased lung infiltration of B cells, which was significantly reduced after the inhibition of sphingosine kinases I/II. Similarly, the sole administration of S1P increased bronchial reactivity compared with vehicle and was accompanied by a higher influx of B cells in a time-dependent manner. This effect was associated with higher levels of IL-13, transforming growth factor-β, IL-10, and T regulatory cells. In addition, isolated S1P-derived lung B cells increased CD4(+) and CD8(+) T cell proliferation in vitro, and their suppressive nature at Day 14 was associated with the higher release of transforming growth factor-β and IL-10 when they were cocultured. Therefore, to prove the role of B cells in S1P-mediated airway inflammation, and because CD20 expression, contrary to major hystocompatibility complex I and major hystocompatibility complex II, was up-regulated at Day 14, CD20(+) B cells were depleted by means of a specific monoclonal antibody. The absence of CD20(+) B cells increased airway reactivity and inflammation in S1P-treated mice compared with control mice. These data imply that sphingosine kinase/S1P-mediated airway inflammation is countered by B cells via the induction of an immune-suppressive environment to reduce asthma-like outcomes in mice.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD20; B-Lymphocytes; Bronchial Hyperreactivity; Bronchoconstriction; Cell Proliferation; Chemotaxis, Leukocyte; Disease Models, Animal; Female; Inflammation Mediators; Interleukin-10; Interleukin-13; Lung; Lymphocyte Activation; Lysophospholipids; Mice, Inbred BALB C; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Protein Kinase Inhibitors; Sphingosine; T-Lymphocytes, Regulatory; Time Factors; Transforming Growth Factor beta

Recent preclinical and clinical studies have unfolded the potential of pharmacological modulation of activities of sphingosine-1-phosphate (S1P) receptors and S1P metabolizing enzymes for the development of therapeutic interventions against a variety of pathologies. An understanding of differential and temporal effects of hypoxia exposure on the key components of S1P signalling would certainly aid in designing improved drug development strategies in this direction. In view of this, the aim of the present study was to assess the effect of progressive hypobaric hypoxia exposure on expression of S1P receptors (S1PR1-5) and specific activities of S1P synthesizing enzymes--neutral sphingomyelinase (nSMase) and sphingosine kinase (Sphk) in pulmonary and cerebral tissues of rats exposed to simulated altitude of 21,000 feet in an animal decompression chamber. Along with this, development of cerebral and pulmonary edema and markers of inflammation were studied at 12, 24, and 48 h to validate our study model of hypobaric hypoxia-induced stress. The protein expression of S1PR1-5 and activities of Sphk and nSMase enzymes were observed to be dramatically affected by simulated hypobaric hypoxia exposure, concurrent with deterioration of pathology, with 12 h of exposure appearing to be the most critical of the various time points studied.

Topics: Animals; Brain; Capillary Permeability; Cytokines; Hypoxia; Lung; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Signal Transduction; Sphingomyelin Phosphodiesterase; Sphingosine; Sphingosine-1-Phosphate Receptors

The initiation and progression of heart failure is linked to adverse cardiac remodeling of the extracellular matrix (ECM) during disease mainly through the deregulation of myocardial metalloproteinases (MMPs). Relaxin (RLX), a peptide hormone acting as a physiological cardiac effector, is a key regulator of ECM remodeling in reproductive and nonreproductive tissues. Studying primary cultures of mouse cardiac muscle cells and rat H9c2 cardiomyoblasts, we have obtained evidence for a new signaling pathway activated by RLX to induce ECM remodeling that involves the bioactive sphingolipids sphingosine-1-phosphate (S1P) and ceramide. In both cell populations, recombinant human RLX increased sphingosine kinase activity and S1P formation, whereas sphingomyelin and ceramide content were decreased in [(3)H]serine-labeled cells. According to the literature, RLX promoted MMP-2 and MMP-9 expression/release. Pharmacological inhibition of sphingolipid metabolism and silencing of sphingosine kinase 1, the enzyme responsible for S1P formation, were able to prevent MMP expression/release elicited by the hormone and induce the expression of tissue inhibitor of MMPs. In addition, we found that sphingolipid signaling is required for the regulation of connective tissue growth factor, a member of the CCN 1-3 family of genes that are involved in cell proliferation and differentiation. Finally, the induction of cardiomyoblast maturation induced by RLX was also found to be counteracted by inhibition of S1P formation. In conclusion, these findings provide a novel mechanism by which RLX acts on cardiac ECM remodeling and cardiac cell differentiation and offer interesting therapeutic options to prevent heart fibrosis and to favor myocardial regeneration.

Topics: Aniline Compounds; Animals; Benzylidene Compounds; Cell Proliferation; Cells, Cultured; Ceramides; Enzyme Activation; Extracellular Matrix; Flavonoids; Imidazoles; Lysophospholipids; Matrix Metalloproteinases; Mice; Myocytes, Cardiac; p38 Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Pyridines; Rats; Relaxin; RNA Interference; RNA, Small Interfering; Signal Transduction; Sphingolipids; Sphingosine

Sphingosine 1-phosphate (S1P) is involved in multiple pathological processes, including fibrogenesis. S1P participates in mouse liver fibrogenesis via a paracrine manner. Herein, we investigated the involvement of S1P in human liver fibrosis. Human fibrotic samples were obtained from livers of patients undergoing liver transplantation. Expression of sphingosine kinase (SphK1), collagen (Col) α1(I), Col α1(III), α-smooth muscle actin, and p-Smad2/3 was characterized by immunofluorescence, real-time RT-PCR, high-content analysis, or Western blot analysis in the fibrotic liver, human bone marrow-derived mesenchymal stem cells, and human hepatogenic profibrotic cells. The effect of SphK1 was assessed using siSphK1 or SphK-specific inhibitor. SphK1, which was expressed in human fibrotic liver myofibroblasts, could be detected in human bone marrow-derived mesenchymal stem cells or human hepatogenic profibrotic cells activated by transforming growth factor β1 (TGF-β1). TGF-β1 evoked the activation of SphK1, increased intracellular S1P, and up-regulated expression of SphK1, Col α1(I), and Col α1(III) in a TGF-β receptor-dependent manner. TGF-β1 induced expression of Col α1(I) and Col α1(III) via SphK1, which was mediated by intracellular S1P, independent of S1P receptors. TGF-β1 evoked nuclear translocation of p-Smad2 and p-Smad3 in TGF-β receptor-dependent, but SphK1-independent, manner. In conclusion, intracellular S1P plays a crucial role in the TGF-β1-induced expression of Col α1(I) and Col α1(III), which is required for human fibrosis development. S1P exerts its effects in S1P receptor-independent manner.

Topics: Adult; Aged; Animals; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type III; Female; Gene Expression Regulation; Humans; Liver Cirrhosis; Lysophospholipids; Male; Mice; Middle Aged; Myofibroblasts; Phosphotransferases (Alcohol Group Acceptor); Smad2 Protein; Smad3 Protein; Sphingosine; Transforming Growth Factor beta1

Sepsis is a cluster of heterogeneous syndromes associated with progressive endotoxemic developments, ultimately leading to damage of multiple organs, including the heart. This study is to investigate the effects of apigenin on heart injury in lipopolysaccharide-induced endotoxemic rat model. Normal Wistar rats were randomly divided into four groups: control group, LPS group (15 mg/kg), LPS plus apigenin groups with different apigenin doses (50 mg/kg, 100 mg/kg). Serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) were measured after the rats were sacrificed. SphK1/S1P signaling pathway proteins, cleaved caspase-3, cleaved caspase-9, Bax and Bcl-2 in heart were measured by Western blot. In vitro, we evaluated the protective effect of apigenin on rat embryonic heart-derived myogenic cell line H9c2 induced by LPS. Apigenin decreased serum levels of CK-MB, LDH, TNF-α, IL-6, IL-1β. SphK1/S1P signaling pathway proteins, cleaved caspase-3, cleaved caspase-9, Bax in heart were found inhibited and Bcl-2 increased in the apigenin groups in vivo. In addition, apigenin inhibited intracellular calcium, the MAPK pathway and SphK1/S1P signaling pathway in vitro. Apigenin exerts pronounced cardioprotection in rats subjected to LPS likely through suppressing myocardial apoptosis and inflammation by inhibiting the SphK1/S1P signaling pathway.

Topics: Animals; Anti-Inflammatory Agents; Apigenin; Cell Line; Endotoxemia; Heart; Heart Injuries; Lipopolysaccharides; Lysophospholipids; Male; Myocardium; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Signal Transduction; Sphingosine

VEGFR2 tyrosine kinase inhibition (TKI) is a valuable treatment approach for patients with metastatic renal cell carcinoma (RCC). However, resistance to treatment is inevitable. Identification of novel targets could lead to better treatment for patients with TKI-naïve or -resistant RCC.. In this study, we performed transcriptome analysis of VEGFR TKI-resistant tumors in a murine model and discovered that the SPHK-S1P pathway is upregulated at the time of resistance. We tested sphingosine-1-phosphate (S1P) pathway inhibition using an anti-S1P mAb (sphingomab), in two mouse xenograft models of RCC, and assessed tumor SPHK expression and S1P plasma levels in patients with metastatic RCC.. Resistant tumors expressed several hypoxia-regulated genes. The SPHK1 pathway was among the most highly upregulated pathways that accompanied resistance to VEGFR TKI therapy. SPHK1 was expressed in human RCC, and the product of SPHK1 activity, S1P, was elevated in patients with metastatic RCC, suggesting that human RCC behavior could, in part, be due to overproduction of S1P. Sphingomab neutralization of extracellular S1P slowed tumor growth in both mouse models. Mice bearing tumors that had developed resistance to sunitinib treatment also exhibited tumor growth suppression with sphingomab. Sphingomab treatment led to a reduction in tumor blood flow as measured by MRI.. Our findings suggest that S1P inhibition may be a novel therapeutic strategy in patients with treatment-naïve RCC and also in the setting of resistance to VEGFR TKI therapy.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Cell Line, Tumor; Cluster Analysis; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Kidney Neoplasms; Lysophospholipids; Mice; Neoplasm Metastasis; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Receptors, Vascular Endothelial Growth Factor; Sphingosine; Transcriptome; Tumor Burden; Up-Regulation; Xenograft Model Antitumor Assays

Long-term glucocorticoid usage is a common cause of non-traumatic femoral head osteonecrosis. Glucocorticoids (i.e. dexamethasone (Dex)) could directly induce damages to osteoblasts. In the current study, we investigated the potential activity of K6PC-5 [N-(1,3-dihydroxyisopropyl)-2-hexyl-3-oxo-decanamide], a novel sphingosine kinase 1 (SphK1) activator, against this process. Our data revealed that both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts were responsible to K6PC-5. K6PC-5 activated SphK1, increased sphingosine-1-phosphate (S1P) production and induced Akt phosphorylation in cultured osteoblasts. Functionally, K6PC-5 protected osteoblasts from Dex-induced apoptosis and necrosis. Such signaling and functional effects by K6PC-5 were prevented by the SphK1 inhibitor N,N-dimethylsphingosine (DMS), and by SphK1-siRNAs. On the other hand, exogenously-added S1P activated Akt and reduced Dex-induced osteoblast damages. LY294002 and MK-2206, two established Akt inhibitors, alleviated K6PC-5- or S1P-mediated osteoblast protection against Dex. Together, our results suggest that K6PC-5 alleviates Dex-induced osteoblast injuries through activating SphK1-Akt signaling. K6PC-5 might be further investigated in animal or clinical studies for its anti-glucocorticoids-associated osteonecrosis potential.

Topics: Amides; Animals; Cell Line; Dexamethasone; Enzyme Activation; Glucocorticoids; Lysophospholipids; Mice; Osteoblasts; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protective Agents; Proto-Oncogene Proteins c-akt; Signal Transduction; Sphingosine

FTY720 (Fingolimod; Gilenya®) is an immune-modulatory prodrug which, after intracellular phosphorylation by sphingosine kinase 2 (SphK2) and export, mimics effects of the endogenous lipid mediator sphingosine-1-phosphate. Fingolimod has been introduced to treat relapsing-remitting multiple sclerosis. However, little has been published about the immune cell membrane penetration and subcellular distribution of FTY720 and FTY720-P. Thus, we applied a newly established LC-MS/MS method to analyze the subcellular distribution of FTY720 and FTY720-P in subcellular compartments of spleen cells of wild type, SphK1- and SphK2-deficient mice. These studies demonstrated that, when normalized to the original cell volume and calculated on molar basis, FTY720 and FTY720-P dramatically accumulated several hundredfold within immune cells reaching micromolar concentrations. The amount and distribution of FTY720 was differentially affected by SphK1- and SphK2-deficiency. On the background of recently described relevant intracellular FTY720 effects in the nanomolar range and the prolonged application in multiple sclerosis, this data showing a substantial intracellular accumulation of FTY720, has to be considered for benefit/risk ratio estimates.

Topics: Animals; Cells, Cultured; Female; Fingolimod Hydrochloride; Lysophospholipids; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Organophosphates; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Spleen; Tandem Mass Spectrometry

Sphingosine 1-phosphate (S1P) is a lysosphingolipid associated with high-density lipoproteins (HDL) that contributes to their anti-atherogenic potential. We investigated whether a reduction in S1P plasma levels affects atherosclerosis in low-density lipoprotein receptor deficient (LDL-R-/-) mice.. LDL-R-/- mice on Western diet containing low (0.25% w/w) or high (1.25% w/w) cholesterol were treated for 16 weeks with SKI-II, a sphingosine kinase 1 inhibitor that significantly reduced plasma S1P levels. SKI-II treatment increased atherosclerotic lesions in the thoracic aorta in mice on high but not low cholesterol diet. This compound did not affect body weight, blood cell counts and plasma total and HDL cholesterol, but decreased triglycerides. In addition, mice on high cholesterol diet receiving SKI-II showed elevated levels of tumor necrosis factor-α and endothelial adhesion molecules (sICAM-1, sVCAM-1).. Prolonged lowering of plasma S1P produces pro-atherogenic effects in LDL-R-/- mice that are evident under condition of pronounced hypercholesterolemia.

Topics: Animals; Aorta, Thoracic; Aortic Diseases; Atherosclerosis; Biomarkers; Cholesterol, Dietary; Cholesterol, HDL; Diet, Western; Disease Models, Animal; Enzyme Inhibitors; Female; Hypercholesterolemia; Intercellular Adhesion Molecule-1; Lysophospholipids; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Receptors, LDL; Risk Factors; Sphingosine; Thiazoles; Time Factors; Triglycerides; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Sphingosine kinase 1 (SK1), the enzyme responsible for sphingosine 1-phosphate (S1P) production, is overexpressed in many human solid tumors. However, its role in clear cell renal cell carcinoma (ccRCC) has not been described previously. ccRCC cases are usually associated with mutations in von Hippel-Lindau (VHL) and subsequent normoxic stabilization of hypoxia-inducible factor (HIF). We previously showed that HIF-2α up-regulates SK1 expression during hypoxia in glioma cells. Therefore, we hypothesized that the stabilized HIF in ccRCC cells will be associated with increased SK1 expression. Here, we demonstrate that SK1 is overexpressed in 786-0 renal carcinoma cells lacking functional VHL, with concomitant high S1P levels that appear to be HIF-2α mediated. Moreover, examining the TCGA RNA seq database shows that SK1 expression was ∼2.7-fold higher in solid tumor tissue from ccRCC patients, and this was associated with less survival. Knockdown of SK1 in 786-0 ccRCC cells had no effect on cell proliferation. On the other hand, this knockdown resulted in an ∼3.5-fold decrease in invasion, less phosphorylation of focal adhesion kinase (FAK), and an ∼2-fold decrease in angiogenesis. Moreover, S1P treatment of SK1 knockdown cells resulted in phosphorylation of FAK and invasion, and this was mediated by S1P receptor 2. These results suggest that higher SK1 and S1P levels in VHL-defective ccRCC could induce invasion in an autocrine manner and angiogenesis in a paracrine manner. Accordingly, targeting SK1 could reduce both the invasion and angiogenesis of ccRCC and therefore improve the survival rate of patients.

Topics: Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Renal Cell; Cell Line, Tumor; Down-Regulation; Focal Adhesion Kinase 1; Gene Knockdown Techniques; Humans; Kidney Neoplasms; Lysophospholipids; Mutation; Neoplasm Invasiveness; Neovascularization, Pathologic; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Up-Regulation; Von Hippel-Lindau Tumor Suppressor Protein

The endoplasmic reticulum (ER) is the principal organelle in the cell for protein folding and trafficking, lipid synthesis, and cellular calcium homeostasis. Perturbation of ER function results in activation of the unfolded protein response (UPR) and is implicated in abnormal lipid biosynthesis and development of insulin resistance. In this study, we investigated whether transcription of sphingosine kinase (Sphk)2 is regulated by ER stress-mediated UPR pathways. Sphk2, a major isotype of sphingosine kinase in the liver, was transcriptionally up-regulated by tunicamycin and lipopolysaccharides. Transcriptional regulation of Sphk2 was mediated by activation of activating transcription factor (ATF)4 as demonstrated by promoter assays, immunoblotting, and small interfering RNA analyses. In primary hepatocytes, adenoviral Sphk2 expression elevated cellular sphingosine 1 phosphate (S1P) and activated protein kinase B phosphorylation, with no alteration of insulin receptor substrate phosphorylation. Hepatic overexpression of Sphk2 in mice fed a high-fat diet (HFD) led to elevated S1P and reduced ceramide, sphingomyelin, and glucosylceramide in plasma and liver. Hepatic accumulation of lipid droplets by HFD feeding was reduced by Sphk2-mediated up-regulation of fatty acid (FA) oxidizing genes and increased FA oxidation in liver. In addition, glucose intolerance and insulin resistance were ameliorated by improved hepatic insulin signaling through Sphk2 up-regulation.. Sphk2 is transcriptionally up-regulated by acute ER stress through activation of ATF4 and improves perturbed hepatic glucose and FA metabolism.

Topics: Activating Transcription Factor 4; Animals; Cells, Cultured; Diet, High-Fat; Endoplasmic Reticulum Stress; Fatty Acids; Fatty Liver; Hepatocytes; Insulin Resistance; Lipid Droplets; Lipids; Liver; Lysophospholipids; Male; Mice, Inbred C57BL; Oxidation-Reduction; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Sphingosine; Unfolded Protein Response; Up-Regulation

Sphingosine-1-phosphate (S1P) participates in inflammation; however, its role in leukocyte rolling is still unclear. Here we use intravital microscopy in inflamed mouse cremaster muscle venules and human endothelial cells to show that S1P contributes to P-selectin-dependent leukocyte rolling through endothelial S1P receptor 3 (S1P3) and Gαq, PLCβ and Ca(2+). Intra-arterial S1P administration increases leukocyte rolling, while S1P3 deficiency or inhibition dramatically reduces it. Mast cells involved in triggering rolling also release S1P that mobilizes P-selectin through S1P3. Histamine and epinephrine require S1P3 for full-scale effect accomplishing it by stimulating sphingosine kinase 1 (Sphk1). In a counter-regulatory manner, S1P1 inhibits cAMP-stimulated Sphk1 and blocks rolling as observed in endothelial-specific S1P1(-/-) mice. In agreement with a dominant pro-rolling effect of S1P3, FTY720 inhibits rolling in control and S1P1(-/-) but not in S1P3(-/-) mice. Our findings identify S1P as a direct and indirect contributor to leukocyte rolling and characterize the receptors mediating its action.

Topics: Animals; Calcium; Endothelium, Vascular; Epinephrine; GTP-Binding Protein alpha Subunits, Gq-G11; Histamine; Human Umbilical Vein Endothelial Cells; Humans; Immunohistochemistry; Leukocyte Rolling; Lysophospholipids; Male; Mast Cells; Mice; Mice, Knockout; Muscle, Skeletal; P-Selectin; Phospholipase C beta; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Venules

Angiotensin II (AngII) plays a critical role in the regulation of vascular tone and blood pressure mainly via regulation of Ca(2+) mobilization. Several reports have implicated sphingosine kinase 1 (SK1)/sphingosine 1-phosphate (S1P) in the mobilization of intracellular Ca(2+) through a yet-undefined mechanism. Here we demonstrate that AngII-induces biphasic calcium entry in vascular smooth muscle cells, consisting of an immediate peak due to inositol tris-phosphate-dependent release of intracellular calcium, followed by a sustained transmembrane Ca(2+) influx through store-operated calcium channels (SOCs). Inhibition of SK1 attenuates the second phase of transmembrane Ca(2+) influx, suggesting a role for SK1 in AngII-dependent activation of SOC. Intracellular S1P triggers SOC-dependent Ca(2+) influx independent of S1P receptors, whereas external application of S1P stimulated S1P receptor-dependent Ca(2+) influx that is insensitive to inhibitors of SOCs, suggesting that the SK1/S1P axis regulates store-operated calcium entry via intracellular rather than extracellular actions. Genetic deletion of SK1 significantly inhibits both the acute hypertensive response to AngII in anaesthetized SK1 knockout mice and the sustained hypertensive response to continuous infusion of AngII in conscious animals. Collectively these data implicate SK1 as the missing link that connects the angiotensin AT1A receptor to transmembrane Ca(2+) influx and identify SOCs as a potential intracellular target for SK1.

Topics: Angiotensin II; Animals; Blood Pressure; Calcium; Calcium Channels; Cell Membrane; Chronic Disease; Enzyme Activation; Gene Deletion; HEK293 Cells; Humans; Hypertension; Lysophospholipids; Male; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sphingosine

Caveolae are plasma membrane invaginations enriched in sterols and sphingolipids. Sphingosine kinase 1 (SK1) is an oncogenic protein that converts sphingosine to sphingosine 1-phosphate (S1P), which is a messenger molecule involved in calcium signaling. Caveolae contain calcium responsive proteins, but the effects of SK1 or S1P on caveolar calcium signaling have not been investigated. We generated a Caveolin-1-Aequorin fusion protein (Cav1-Aeq) that can be employed for monitoring the local calcium concentration at the caveolae ([Ca²⁺]cav). In HeLa cells, Cav1-Aeq reported different [Ca²⁺] as compared to the plasma membrane [Ca²⁺] in general (reported by SNAP25-Aeq) or as compared to the cytosolic [Ca²⁺] (reported by cyt-Aeq). The Ca²⁺ signals detected by Cav1-Aeq were significantly attenuated when the caveolar structures were disrupted by methyl-β-cyclodextrin, suggesting that the caveolae are specific targets for Ca²⁺ signaling. HeLa cells overexpressing SK1 showed increased [Ca²⁺]cav during histamine-induced Ca²⁺ mobilization in the absence of extracellular Ca²⁺ as well as during receptor-operated Ca²⁺ entry (ROCE). The SK1-induced increase in [Ca²⁺]cav during ROCE was reverted by S1P receptor antagonists. In accordance, pharmacologic inhibition of SK1 reduced the [Ca²⁺]cav during ROCE. S1P treatment stimulated the [Ca²⁺]cav upon ROCE. The Ca²⁺ responses at the plasma membrane in general were not affected by SK1 expression. In summary, our results show that SK1/S1P-signaling regulates Ca²⁺ signals at the caveolae. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Topics: Aequorin; Calcium; Calcium Signaling; Caveolae; Caveolin 1; HeLa Cells; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Recombinant Fusion Proteins; Sphingosine

Cryptococcus neoformans is a fungal pathogen that causes pulmonary infections, which may progress into life-threatening meningitis. In commonly used mouse models of C. neoformans infections, fungal cells are not contained in the lungs, resulting in dissemination to the brain. We have previously reported the generation of an engineered C. neoformans strain (C. neoformans Δgcs1) which can be contained in lung granulomas in the mouse model and have shown that granuloma formation is dependent upon the enzyme sphingosine kinase 1 (SK1) and its product, sphingosine 1-phosphate (S1P). In this study, we have used four mouse models, CBA/J and C57BL6/J (both immunocompetent), Tgε26 (an isogenic strain of strain CBA/J lacking T and NK cells), and SK(-/-) (an isogenic strain of strain C57BL6/J lacking SK1), to investigate how the granulomatous response and SK1-S1P pathway are interrelated during C. neoformans infections. S1P and monocyte chemotactic protein-1 (MCP-1) levels were significantly elevated in the bronchoalveolar lavage fluid of all mice infected with C. neoformans Δgcs1 but not in mice infected with the C. neoformans wild type. SK1(-/-) mice did not show elevated levels of S1P or MCP-1. Primary neutrophils isolated from SK1(-/-) mice showed impaired antifungal activity that could be restored by the addition of extracellular S1P. In addition, high levels of tumor necrosis factor alpha were found in the mice infected with C. neoformans Δgcs1 in comparison to the levels found in mice infected with the C. neoformans wild type, and their levels were also dependent on the SK1-S1P pathway. Taken together, these results suggest that the SK1-S1P pathway promotes host defense against C. neoformans infections by regulating cytokine levels, promoting extracellular killing by phagocytes, and generating a granulomatous response.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cryptococcosis; Cryptococcus neoformans; Disease Models, Animal; Female; Gene Deletion; Granuloma; Lung; Lysophospholipids; Male; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

The interaction between endothelial cells and pericytes is crucial for the stabilization of newly formed vessels in angiogenesis. The comprehension of the mechanisms regulating pericyte recruitment might open therapeutical perspectives on vascular-related pathologies. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that derives from sphingomyelin catabolism and regulates biological functions in cell survival, proliferation, and differentiation. In this study, we aimed to identify the role of S1P axis in the intercellular communication between human mesenchymal progenitor mesoangioblasts (MAB) and endothelial cells (human microvascular endothelial cells (H-MVEC)) in the formation of capillary-like structures. We demonstrated that the S1P biosynthetic pathway brought about by sphingosine kinases (SK) SK1 and SK2 as well as spinster homolog 2 (SPNS2) transporter in H-MVEC is crucial for MAB migration measured by Boyden chambers and for the formation and stabilization of capillary-like structures in a 3D Matrigel culture. Moreover, the conditioned medium (CM) harvested from H-MVEC, where SK1, SK2, and SPNS2 were down-regulated, exerted a significantly diminished effect on MAB capillary morphogenesis and migration. Notably, we demonstrated that S1P1 and S1P3 receptors were positively involved in CM-induced capillary-like formation and migration, while S1P2 exerted a negative role on CM-induced migratory action of MAB. Finally, SK inhibition as well as MAB S1P1 and S1P3 down-regulation impaired H-MVEC-MAB cross-talk significantly reducing in vivo angiogenesis evaluated by Matrigel plug assay. These findings individuate novel targets for the employment of MAB in vascular-related pathologic conditions.. • Down-regulation of SK1/2 in H-MVEC impaired vessel formation when cultured with MAB. • H-MVEC SPNS2 is critical for morphogenesis and migration induced by H-MVEC CM of MAB. • CM from SK1- and SK2-siRNA H-MVEC impaired morphogenesis and migration of MAB. • S1P1/3 were involved on CM-induced morphogenesis and migration of MAB. • Matrigel plug assay showed the role of S1P axis in MAB-endothelial cell interaction.

Topics: Anion Transport Proteins; Cell Line; Cells, Cultured; Coculture Techniques; Endothelial Cells; Humans; Lysophospholipids; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Stem Cells

Sphingosine 1-phosphate (S1P) is a bioactive lipid that can function both extracellularly and intracellularly to mediate a variety of cellular processes. Using lipid affinity matrices and a radiolabeled lipid binding assay, we reveal that S1P directly interacts with the transcription factor peroxisome proliferator-activated receptor (PPAR)γ. Herein, we show that S1P treatment of human endothelial cells (ECs) activated a luciferase-tagged PPARγ-specific gene reporter by ∼12-fold, independent of the S1P receptors. More specifically, in silico docking, gene reporter, and binding assays revealed that His323 of the PPARγ ligand binding domain is important for binding to S1P. PPARγ functions when associated with coregulatory proteins, and herein we identify that peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1)β binds to PPARγ in ECs and their progenitors (nonadherent endothelial forming cells) and that the formation of this PPARγ:PGC1β complex is increased in response to S1P. ECs treated with S1P selectively regulated known PPARγ target genes with PGC1β and plasminogen-activated inhibitor-1 being increased, no change to adipocyte fatty acid binding protein 2 and suppression of CD36. S1P-induced in vitro tube formation was significantly attenuated in the presence of the PPARγ antagonist GW9662, and in vivo application of GW9662 also reduced vascular development in Matrigel plugs. Interestingly, activation of PPARγ by the synthetic ligand troglitazone also reduced tube formation in vitro and in vivo. To support this, Sphk1(-/-)Sphk2(+/-) mice, with low circulating S1P levels, demonstrated a similar reduction in vascular development. Taken together, our data reveal that the transcription factor, PPARγ, is a bona fide intracellular target for S1P and thus suggest that the S1P:PPARγ:PGC1β complex may be a useful target to manipulate neovascularization.

Topics: Animals; Carrier Proteins; CD36 Antigens; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Lysophospholipids; Mice; Mice, Knockout; Neovascularization, Physiologic; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Phosphotransferases (Alcohol Group Acceptor); Plasminogen Activator Inhibitor 1; PPAR gamma; Receptors, Lysosphingolipid; RNA-Binding Proteins; Serpin E2; Sphingosine; Transcription Factors; U937 Cells

To explore the effects of resveratrol in a human myelogenous leukemia cell line K562 and its potential molecular mechanisms. The anti-proliferation effect of resveratrol-induced apoptosis on K562 cells were detected using MTT assay. Western blotting was performed for detecting changes of SphK1 expression in total cell protein and membrane/cytosol protein in K562 cells respectively after exposure to resveratrol. A biochemical assay was used to measure the activity of SphK after treatment of resveratrol, and then S1P and ceramide levels were examined using ELISA kits. Hochest 33258 staining and flow cytometry were applied to detect the apoptosis condition of K562 cells treated with resveratrol. Resveratrol inhibited the proliferation and induced apoptosis in K562 cells in a dose and time-dependent manner. Western blotting revealed that resveratrol did not affect total SphK1 expression level in K562 cells, but significantly changed the translocation of SphK1, the membrane SphK1 was decreased while cytosol SphK1 level was elevated. The activity of SphK1 in resveratrol treated groups was decreased compared to control group with a significant decrease of S1P and increase of ceramide level. Furthermore, Hoechst 33258 staining and Annexin V-FITC analysis confirmed the notable apoptotic effect of resveratrol in its anti-leukemia process. Resveratrol-induced proliferation inhibition of K562 cells might be mediated through its modulation activity of SphK1 pathway by regulating S1P and ceramide levels, which then affected the proliferation and apoptosis process of leukemia cells. SphK1/S1P pathway represents a target of resveratrol in human leukemia.

Topics: Annexin A5; Antineoplastic Agents; Apoptosis; Cell Membrane; Cell Proliferation; Ceramides; Cytosol; Dose-Response Relationship, Drug; Humans; K562 Cells; Leukemia, Myeloid; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Transport; Resveratrol; Signal Transduction; Sphingosine; Stilbenes; Time Factors

The ceramide metabolite, sphingosine-1-phosphate (S1P), regulates multiple cellular functions in keratinocytes (KC). We recently discovered that production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), is stimulated via a NF-κB-dependent mechanism that is activated by S1P when S1P is generated by sphingosine kinase (SPHK) 1.. We investigated whether pharmacological modulation of SPHK1 activity, using a novel synthetic SPHK1 activator, (S)-methyl 2-(hexanamide)-3-(4-hydroxyphenyl) propanoate (MHP), stimulates CAMP expression.. MHP-mediated changes in both S1P and CAMP downstream mediators were analyzed in normal cultured human KC by qRT-PCR, Western immunoblot, ELISA, confocal microscopy for immunohistochemistry, HPLC and ESI-LC/MS/MS, and microbial pathogen invasion/colonization in a human epidermal organotypic model.. Treatment with MHP directly activated SPHK1 and increased cellular S1P content in normal cultured human KC. Because MHP did not inhibit S1P lyase activity, which hydrolyses S1P, augumented S1P levels could be attributed to increased synthesis rather than blockade of S1P degradation. Next, we found that exogenous MHP significantly stimulated CAMP mRNA and protein production in KC, increases that were significantly suppressed by siRNA directed against SPHK1, but not by a scrambled control siRNA. NF-κB activation, assessed by nuclear translocation of NF-κB, occurred in cells following incubation with MHP. Conversely, pretreatment with a specific inhibitor of SPHK1 decreased MHP-induced nuclear translocation of NF-κB, and significantly attenuated the MHP-mediated increase in CAMP production. Finally, topical MHP significantly suppressed invasion of the virulent Staphylococcus aureus into murine skin explants.. MHP activation of SPHK1, a target enzyme of CAMP production, can stimulate innate immunity.

Topics: Animals; Antimicrobial Cationic Peptides; Bacterial Physiological Phenomena; Cathelicidins; Cells, Cultured; Enzyme Activators; Enzyme Inhibitors; Epidermis; Humans; Immunity, Innate; Keratinocytes; Lysophospholipids; Mice; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; RNA, Small Interfering; Sphingosine; Staphylococcus aureus; Tyrosine

Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function.. To investigate the role of Sphk1- and Sphk2-derived S1P in the regulation of platelet function.. We found a 100-fold reduction in intracellular S1P levels in platelets derived from Sphk2(-/-) mutants compared with Sphk1(-/-) or wild-type mice, as analyzed by mass spectrometry. Sphk2(-/-) platelets also failed to secrete S1P on stimulation. Blood from Sphk2-deficient mice showed decreased aggregation after protease-activated receptor 4-peptide and adenosine diphosphate stimulation in vitro, as assessed by whole blood impedance aggregometry. We revealed that S1P controls platelet aggregation via the sphingosine 1-phosphate receptor 1 through modulation of protease-activated receptor 4-peptide and adenosine diphosphate-induced platelet activation. Finally, we show by intravital microscopy that defective platelet aggregation in Sphk2-deficient mice translates into reduced arterial thrombus stability in vivo.. We demonstrate that Sphk2 is the major Sphk isoform responsible for the generation of S1P in platelets and plays a pivotal intrinsic role in the control of platelet activation. Correspondingly, Sphk2-deficient mice are protected from arterial thrombosis after vascular injury, but have normal bleeding times. Targeting this pathway could therefore present a new therapeutic strategy to prevent thrombosis.

Topics: Animals; Arachidonic Acid; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Carotid Artery Injuries; Disease Models, Animal; Erythrocytes; Lysophospholipids; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Platelet Adhesiveness; Platelet Aggregation; Platelet Function Tests; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Thrombosis; Thromboxane A2; Vascular System Injuries

Acute lung injury (ALI) is a severe inflammatory disease for which no specific treatment exists. As glucocorticoids have potent immunosuppressive effects, their application in ALI is currently being tested in clinical trials. However, the benefits of this type of regimen remain unclear. Here we identify a mechanism of glucocorticoid action that challenges the long-standing dogma of cytokine repression by the glucocorticoid receptor. Contrarily, synergistic gene induction of sphingosine kinase 1 (SphK1) by glucocorticoids and pro-inflammatory stimuli via the glucocorticoid receptor in macrophages increases circulating sphingosine 1-phosphate levels, which proves essential for the inhibition of inflammation. Chemical or genetic inhibition of SphK1 abrogates the therapeutic effects of glucocorticoids. Inflammatory p38 MAPK- and mitogen- and stress-activated protein kinase 1 (MSK1)-dependent pathways cooperate with glucocorticoids to upregulate SphK1 expression. Our findings support a critical role for SphK1 induction in the suppression of lung inflammation by glucocorticoids, and therefore provide rationales for effective anti-inflammatory therapies.

Topics: Acute Lung Injury; Animals; Chromatin Immunoprecipitation; Cytokines; Flow Cytometry; Gene Expression Regulation; Glucocorticoids; Inflammation; Lysophospholipids; Macrophages; Mice; p38 Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; Receptors, Glucocorticoid; Ribosomal Protein S6 Kinases, 90-kDa; Sphingosine; Transcriptional Activation; Up-Regulation

Sphingosine 1-phosphate (S1P) levels are significantly higher in blood and lymph than in tissues. This S1P concentration difference is necessary for proper lymphocyte egress from secondary lymphoid tissue and to maintain endothelial barrier integrity. Studies with mice lacking either sphingosine kinase (SphK) type 1 and 2 indicate that these enzymes are the sole biosynthetic source of S1P, but they play different roles in setting S1P blood levels. We have developed a set of drug-like SphK inhibitors, with differing selectivity for the two isoforms of this enzyme. Although all SphK inhibitors tested decrease S1P when applied to cultured U937 cells, only those inhibitors with a bias for SphK2 drove a substantial increase in blood S1P in mice and this rise was detectable within minutes of administration of the inhibitor. Blood S1P also increased in response to SphK2 inhibitors in rats. Mass-labeled S1P was cleared more slowly after intravenous injection into SphK2 inhibitor-treated mice or mice lacking a functional SphK2 gene; thus, the increased accumulation of S1P in the blood appears to result from the decreased clearance of S1P from the blood. Therefore, SphK2 appears to have a function independent of generating S1P in cells. Our results suggest that differential SphK inhibition with a drug might afford a method to manipulate blood S1P levels in either direction while lowering tissue S1P levels.

Topics: Animals; Drug Design; Enzyme Inhibitors; Lysophospholipids; Male; Mice; Phosphotransferases (Alcohol Group Acceptor); Rats; Sphingosine; Substrate Specificity

The α-chemokine stromal-derived factor 1 (SDF-1), which binds to the CXCR4 receptor, directs migration and homing of CXCR4+ hematopoietic stem/progenitor cells (HSPCs) to bone marrow (BM) stem cell niches. Nevertheless, it is also known that CXCR4-/- fetal liver-derived hematopoietic stem cells engraft into BM and that blockade of CXCR4 by its antagonist AMD3100 does not prevent engraftment of HSPCs. Because of this finding of SDF-1-CXCR4-independent BM homing, the unique role of SDF-1 in HSPC homing has recently been challenged. While SDF-1 is the only chemokine that chemoattracts HSPCs, other chemoattractants for these cells have recently been described, including the bioactive phosphosphingolipid sphingosine-1-phosphate (S1P). To address the potential role of S1P in homing of HSPCs to BM, we performed hematopoietic transplants into mice deficient in BM-expressed sphingosine kinase 1 (Sphk1-/-) using hematopoietic cells from normal control mice as well as cells from mice in which floxed CXCR4 (CXCR4fl/fl) was conditionally deleted. We observed the presence of a homing and engraftment defect in HSPCs of Sphk1-/- mice that was particularly profound after transplantation of CXCR4-/- BM cells. Thus, our results indicate that BM-microenvironment-expressed S1P plays a role in homing of HSPCs. They also support the concept that, in addition to the SDF-1-CXCR4 axis, other chemotactic axes are also involved in homing and engraftment of HSPCs.

Topics: Animals; Bone Marrow Cells; Chemokine CXCL12; Female; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Lysophospholipids; Male; Mice; Phosphotransferases (Alcohol Group Acceptor); Receptors, CXCR4; Signal Transduction; Sphingosine; Stem Cell Niche; Transplantation Conditioning

Autophagy is an important homeostatic mechanism that eliminates long-lived proteins, protein aggregates and damaged organelles. Its dysregulation is involved in many neurodegenerative disorders. Autophagy is therefore a promising target for blunting neurodegeneration. We searched for novel autophagic pathways in primary neurons and identified the cytosolic sphingosine-1-phosphate (S1P) pathway as a regulator of neuronal autophagy. S1P, a bioactive lipid generated by sphingosine kinase 1 (SK1) in the cytoplasm, is implicated in cell survival. We found that SK1 enhances flux through autophagy and that S1P-metabolizing enzymes decrease this flux. When autophagy is stimulated, SK1 relocalizes to endosomes/autophagosomes in neurons. Expression of a dominant-negative form of SK1 inhibits autophagosome synthesis. In a neuron model of Huntington's disease, pharmacologically inhibiting S1P-lyase protected neurons from mutant huntingtin-induced neurotoxicity. These results identify the S1P pathway as a novel regulator of neuronal autophagy and provide a new target for developing therapies for neurodegenerative disorders.

Topics: Aldehyde-Lyases; Animals; Autophagy; Biomarkers; Cell Survival; Cytoplasm; Endoplasmic Reticulum; Endosomes; Enzyme Inhibitors; Gene Expression; Lysophospholipids; Neurons; Phagosomes; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Protein Transport; Rats; Signal Transduction; Sphingosine

We previously reported that alveolar macrophages from patients with chronic obstructive pulmonary disease (COPD) are defective in their ability to phagocytose apoptotic cells, with a similar defect in response to cigarette smoke. The exact mechanisms for this defect are unknown. Sphingolipids including ceramide, sphingosine and sphingosine-1-phosphate (S1P) are involved in diverse cellular processes and we hypothesised that a comprehensive analysis of this system in alveolar macrophages in COPD may help to delineate the reasons for defective phagocytic function.. We compared mRNA expression of sphingosine kinases (SPHK1/2), S1P receptors (S1PR1-5) and S1P-degrading enzymes (SGPP1, SGPP2, SGPL1) in bronchoalveolar lavage-derived alveolar macrophages from 10 healthy controls, 7 healthy smokers and 20 COPD patients (10 current- and 10 ex-smokers) using Real-Time PCR. Phagocytosis of apoptotic cells was investigated using flow cytometry. Functional associations were assessed between sphingosine signalling system components and alveolar macrophage phagocytic ability in COPD. To elucidate functional effects of increased S1PR5 on macrophage phagocytic ability, we performed the phagocytosis assay in the presence of varying concentrations of suramin, an antagonist of S1PR3 and S1PR5. The effects of cigarette smoking on the S1P system were investigated using a THP-1 macrophage cell line model.. We found significant increases in SPHK1/2 (3.4- and 2.1-fold increases respectively), S1PR2 and 5 (4.3- and 14.6-fold increases respectively), and SGPL1 (4.5-fold increase) in COPD vs. controls. S1PR5 and SGPL1 expression was unaffected by smoking status, suggesting a COPD "disease effect" rather than smoke effect per se. Significant associations were noted between S1PR5 and both lung function and phagocytosis. Cigarette smoke extract significantly increased mRNA expression of SPHK1, SPHK2, S1PR2 and S1PR5 by THP-1 macrophages, confirming the results in patient-derived macrophages. Antagonising SIPR5 significantly improved phagocytosis.. Our results suggest a potential link between the S1P signalling system and defective macrophage phagocytic function in COPD and advise therapeutic targets.

Topics: Adult; Aged; Aged, 80 and over; Female; Humans; Lysophospholipids; Macrophages, Alveolar; Male; Membrane Proteins; Middle Aged; Phagocytosis; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Disease, Chronic Obstructive; Receptors, Lysosphingolipid; Smoking; Sphingosine

Resistance to chemotherapy is common in gastroesophageal cancer. Mechanisms of resistance are incompletely characterised and there are no predictive biomarkers in clinical practice for cytotoxic drugs. We used new cell line models to characterise novel chemotherapy resistance mechanisms and validated them in tumour specimens to identify new targets and biomarkers for gastroesophageal cancer.. Cell lines were selected for resistance to oxaliplatin, cisplatin and docetaxel and gene expression examined using Affymetrix Exon 1.0 ST arrays. Leads were validated by qRT-PCR and HPLC of tumour metabolites. Protein expression and pharmacological inhibition of lead target SPHK1 was evaluated in independent cell lines, and by immunohistochemistry in gastroesophageal cancer patients.. Genes with differential expression in drug resistant cell lines compared to the parental cell line they were derived from, were identified for each drug resistant cell line. Biological pathway analysis of these gene lists, identified over-represented pathways, and only 3 pathways - lysosome, sphingolipid metabolism and p53 signalling- were identified as over-represented in these lists for all three cytotoxic drugs investigated. The majority of genes differentially expressed in chemoresistant cell lines from these pathways, were involved in metabolism of glycosphingolipids and sphingolipids in lysosomal compartments suggesting that sphingolipids might be important mediators of cytotoxic drug resistance in gastroeosphageal cancers . On further investigation, we found that drug resistance (IC50) was correlated with increased sphingosine kinase 1(SPHK1) mRNA and also with decreased sphingosine-1-phosphate lysase 1(SGPL1) mRNA. SPHK1 and SGPL1 gene expression were inversely correlated. SPHK1:SGPL1 ratio correlated with increased cellular sphingosine-1-phosphate (S1P), and S1P correlated with drug resistance (IC50). High SPHK1 protein correlated with resistance to cisplatin (IC50) in an independent gastric cancer cell line panel and with survival of patients treated with chemotherapy prior to surgery but not in patients treated with surgery alone. Safingol a SPHK1 inhibitor, was cytotoxic as a single agent and acted synergistically with cisplatin in gastric cancer cell lines.. Agents that inhibit SPHK1 or S1P could overcome cytotoxic drug resistance in gastroesophageal cancer. There are several agents in early phase human trials including Safingol that could be combined with chemotherapy or used in patients progressing after chemotherapy.

Topics: Aldehyde-Lyases; Antineoplastic Agents; Cell Line, Tumor; Drug Resistance, Neoplasm; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lysophospholipids; Male; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; RNA, Neoplasm; Signal Transduction; Sphingosine; Stomach Neoplasms

Initial and recurrent stroke produces central nervous system (CNS) damage, involving neuroinflammation. Receptor-mediated S1P signaling can influence neuroinflammation and has been implicated in cerebral ischemia through effects on the immune system. However, S1P-mediated events also occur within the brain itself where its roles during stroke have been less well studied. Here we investigated the involvement of S1P signaling in initial and recurrent stroke by using a transient middle cerebral artery occlusion/reperfusion (M/R) model combined with analyses of S1P signaling. Gene expression for S1P receptors and involved enzymes was altered during M/R, supporting changes in S1P signaling. Direct S1P microinjection into the normal CNS induced neuroglial activation, implicating S1P-initiated neuroinflammatory responses that resembled CNS changes seen during initial M/R challenge. Moreover, S1P microinjection combined with M/R potentiated brain damage, approximating a model for recurrent stroke dependent on S1P and suggesting that reduction in S1P signaling could ameliorate stroke damage. Delivery of FTY720 that removes S1P signaling with chronic exposure reduced damage in both initial and S1P-potentiated M/R-challenged brain, while reducing stroke markers like TNF-α. These results implicate direct S1P CNS signaling in the etiology of initial and recurrent stroke that can be therapeutically accessed by S1P modulators acting within the brain.

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Fingolimod Hydrochloride; Lysophospholipids; Male; Mice; Mice, Inbred ICR; Microinjections; Neuroglia; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Stroke; Tumor Necrosis Factor-alpha

The plant flavonoid luteolin exhibits different biological effects, including anticancer properties. Little is known on the molecular mechanisms underlying its actions in colorectal cancer (CRC). Here we investigated the effects of luteolin on colon cancer cells, focusing on the balance between ceramide and sphingosine-1-phosphate (S1P), two sphingoid mediators with opposite roles on cell fate. Using cultured cells, we found that physiological concentrations of luteolin induce the elevation of ceramide, followed by apoptotic death of colon cancer cells, but not of differentiated enterocytes. Pulse studies revealed that luteolin inhibits ceramide anabolism to complex sphingolipids. Further experiments led us to demonstrate that luteolin induces an alteration of the endoplasmic reticulum (ER)-Golgi flow of ceramide, pivotal to its metabolic processing to complex sphingolipids. We report that luteolin exerts its action by inhibiting both Akt activation, and sphingosine kinase (SphK) 2, with the consequent reduction of S1P, an Akt stimulator. S1P administration protected colon cancer cells from luteolin-induced apoptosis, most likely by an intracellular, receptor-independent mechanism. Overall this study reveals for the first time that the dietary flavonoid luteolin exerts toxic effects on colon cancer cells by inhibiting both S1P biosynthesis and ceramide traffic, suggesting its dietary introduction/supplementation as a potential strategy to improve existing treatments in CRC.

Topics: Apoptosis; Biological Transport; Caco-2 Cells; Ceramides; Colonic Neoplasms; Cytoprotection; Endoplasmic Reticulum; Enterocytes; Enzyme Activation; Golgi Apparatus; Humans; Luteolin; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Sphingosine

Despite progress in the understanding of the biology and genetics of melanoma, no effective treatment against this cancer is available. The adjacent microenvironment has an important role in melanoma progression. Defining the molecular signals that control the bidirectional dialog between malignant cells and the surrounding stroma is crucial for efficient targeted therapy. Our study aimed at defining the role of sphingosine-1-phosphate (S1P) in melanoma-stroma interactions. Transcriptomic analysis of human melanoma cell lines showed increased expression of sphingosine kinase-1 (SPHK1), the enzyme that produces S1P, as compared with normal melanocytes. Such an increase was also observed by immunohistochemistry in melanoma specimens as compared with nevi, and occurred downstream of ERK activation because of BRAF or NRAS mutations. Importantly, migration of melanoma cells was not affected by changes in SPHK1 activity in tumor cells, but was stimulated by comparable modifications of S1P-metabolizing enzymes in cocultured dermal fibroblasts. Reciprocally, incubation of fibroblasts with the conditioned medium from SPHK1-expressing melanoma cells resulted in their differentiation to myofibroblasts, increased production of matrix metalloproteinases and enhanced SPHK1 expression and activity. In vivo tumorigenesis experiments showed that the lack of S1P in the microenvironment prevented the development of orthotopically injected melanoma cells. Finally, local tumor growth and dissemination were enhanced more efficiently by coinjection of wild-type skin fibroblasts than by fibroblasts from Sphk1(-/-) mice. This report is the first to document that SPHK1/S1P modulates the communication between melanoma cells and dermal fibroblasts. Altogether, our findings highlight SPHK1 as a potential therapeutic target in melanoma progression.

Topics: Aldehyde-Lyases; Animals; Cell Communication; Cell Differentiation; Cell Movement; Cell Transformation, Neoplastic; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; Gene Expression Profiling; GTP Phosphohydrolases; HEK293 Cells; Humans; Lysophospholipids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanoma; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Myofibroblasts; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins B-raf; RNA Interference; RNA, Messenger; RNA, Small Interfering; Skin Neoplasms; Sphingosine

Sphingosine kinase (SPHK) is overexpressed by a variety of cancers, and its phosphorylation of sphingosine results in accumulation of sphingosine-1-phosphate (S1P) and activation of antiapoptotic signal transduction. Existing data indicate a role for S1P in viral pathogenesis, but roles for SPHK and S1P in virus-associated cancer progression have not been defined. Rare pathologic variants of diffuse large B-cell lymphoma arise preferentially in the setting of HIV infection, including primary effusion lymphoma (PEL), a highly mortal tumor etiologically linked to the Kaposi's sarcoma-associated herpesvirus (KSHV). We have found that ABC294640, a novel clinical-grade small molecule selectively targeting SPHK (SPHK2 >> SPHK1), induces dose-dependent caspase cleavage and apoptosis for KSHV(+) patient-derived PEL cells, in part through inhibition of constitutive signal transduction associated with PEL cell proliferation and survival. These results were validated with induction of PEL cell apoptosis using SPHK2-specific siRNA, as well as confirmation of drug-induced SPHK inhibition in PEL cells with dose-dependent accumulation of proapoptotic ceramides and reduction of intracellular S1P. Furthermore, we demonstrate that systemic administration of ABC294640 induces tumor regression in an established human PEL xenograft model. Complimentary ex vivo analyses revealed suppression of signal transduction and increased KSHV lytic gene expression within drug-treated tumors, with the latter validated in vitro through demonstration of dose-dependent viral lytic gene expression within PEL cells exposed to ABC294640. Collectively, these results implicate interrelated mechanisms and SPHK2 inhibition in the induction of PEL cell death by ABC294640 and rationalize evaluation of ABC294640 in clinical trials for the treatment of KSHV-associated lymphoma.

Topics: Adamantane; Apoptosis; Cell Line, Tumor; Cell Proliferation; Herpesvirus 8, Human; Humans; Lymphoma, Primary Effusion; Lysophospholipids; Molecular Targeted Therapy; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pyridines; Signal Transduction; Sphingosine

Our previous studies have confirmed that the sphingosine kinase 1 (SphK1)-sphingosine 1-phosphate (S1P) signaling pathway in the kidney under diabetic conditions is closely correlated with the pathogenesis of diabetic nephropathy (DN). The activation of SphK1-S1P pathway by high glucose (HG) can increase the expression of fibronectin (FN), an important fibrotic component, in glomerular mesangial cells (GMCs) by promoting the DNA-binding activity of transcription factor AP-1. However, the mechanism responsible for the sustained activation of SphK1-S1P pathway remains unclear. Given the binding motifs for AP-1 within the first intron of the SphK1 gene, we speculated that the activated AP-1 in the kidney under HG condition possibly regulates SphK1 expression in a positive feedback manner, thereby promoting the sustained activation of SphK1-S1P pathway and mediating the pathological progression of DN. Here, we observed the effect of AP-1 on SphK1 expression in GMCs and explored the molecular mechanism involved in the sustained activation of SphK1-S1P pathway. We found two consensus binding motifs for AP-1 in the promoter sequences and non-coding region downstream of the transcriptional initiation of the rat SphK1 gene by chromatin immunoprecipitation assay. The treatment of GMCs with both HG and S1P significantly increased the protein expression of c-Jun and c-Fos, and obviously enhanced the phosphorylation of c-Jun at Ser63 and Ser73, and c-Fos at Ser32. Knockdown of c-Jun and c-Fos with siRNAs substantially inhibited the expression of SphK1 and FN, whereas overexpression of c-Jun and c-Fos significantly increased the expression of SphK1 and FN. Curcumin treatment greatly decreased the levels of c-Jun, c-Fos, SphK1, and FN in the kidney tissues of diabetic rats. SiRNAs targeting SphK1 and S1P2 receptor respectively inhibited the phosphorylation of c-Jun (ser63 and ser73) and c-Fos (ser32), as well as FN expression under both normal and HG conditions. Our data demonstrated that the activated SphK1-S1P signaling pathway in GMCs under diabetic conditions is closely associated with AP-1 to form a positive feedback loop. This positive feedback loop functions as an important molecular basis for the sustained activation of SphK1-S1P pathway and increased FN expression that lead to the initiation and progression of DN.

Topics: Animals; Binding Sites; Cells, Cultured; Curcumin; Diabetes Mellitus; Diabetic Nephropathies; DNA-Binding Proteins; Enzyme Inhibitors; Fibronectins; Glucose; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; Male; Mesangial Cells; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-fos; Rats; Receptors, Lysosphingolipid; RNA Interference; RNA, Small Interfering; Signal Transduction; Sphingosine; Sweetening Agents; Transcription Factor AP-1

Sphingosine kinases (Sphk1/2) are crucial enzymes in regulation of the biostat between sphingosine-1-phosphate (S1P) and ceramide and play an important role in the pathogenesis/pathomechanism of Alzheimer's disease (AD). These enzymes synthesise S1P, which regulates neurotransmission, synaptic function and neuron cell proliferation, by activating five G protein-coupled receptors (S1P1-5). However, S1P synthesised by Sphk2 could be involved in amyloid β (Aβ) release by stimulation of Aβ precursor protein degradation. The significance of this bioactive sphingolipid in the pathogenesis of Parkinson's disease (PD) is unknown. The aim of our study was to investigate the expression level of Sphk1 and its role in human dopaminergic neuronal cell (SH-SY5Y) viability under oxidative stress, evoked by 1-methyl-4-phenylpyridinium (MPP+). Moreover, the mechanism of S1P action on the death signalling pathway in these experimental conditions was evaluated. Our study indicated marked downregulation of Sphk1 expression in this cellular PD model. Inhibition of Sphk1 decreased SH-SY5Y cell viability and concomitantly enhanced the reactive oxygen species (ROS) level. It was found that exogenous S1P (1 μM) exerted the neuroprotective effect by activation of Sphk1 and S1P1 receptor gene expression. Moreover, S1P downregulated Bax and harakiri, death protein 5 (Hrk/DP5) expression and enhanced cell viability in MPP+-treated cells. The neuroprotective mechanism of S1P is mainly dependent on S1P1 receptor signalling, which was indicated by using specific agonists and antagonists of S1P1 receptor. The results show that S1P and S1P1 receptor agonists protected a significant population of neuronal cells against death.

Topics: 1-Methyl-4-phenylpyridinium; Cell Line, Tumor; Ceramides; Dopaminergic Neurons; Humans; Lysophospholipids; Oxidative Stress; Parkinson Disease; Phosphotransferases (Alcohol Group Acceptor); Reactive Oxygen Species; Signal Transduction; Sphingosine

It has been postulated that disturbances in the sphingolipid metabolism play a key role in the pathogenesis of Alzheimer's disease (AD). An alteration in sphingosine kinases 1, 2 (SphK1/2) and sphingosine-1-phosphate (S1P) was recently reported in AD. However, the effect of AD-related amyloid beta (Aβ) peptides on SphK1/2 and the role of S1P in Aβ toxicity have not been fully elucidated. In this study the relationship between the Aβ concentration and SphK1/2 expression/activity was analysed in PC12 cells transfected with the Aβ precursor protein, wild-type (APPwt) or bearing a double Swedish mutation (APPsw). The role of SphK(s)/S1P in cell survival and death was also investigated. Our results indicated that endogenously liberated Aβ significantly decreases expression and activity of SphK1/2. The SphK(s) inhibitor (SKI II, 10 μM) decreased the viability of APPwt, APPsw as well as empty vector-transfected PC12 control cells. Our data demonstrated that expression of S1P receptor-1 (S1P1) was significantly reduced in APP-transfected cells. The effect of S1P applied exogenously was cell type-dependent. In control and APPwt cells S1P reduced the effect of the SphK1 inhibitor on death signalling. Conversely, it decreased the survival of APPsw cells and had no protective effect on cells treated with SKI II. Using the S1P1 agonist (SEW2871, 5 μM) and antagonist (W123, 20 μM), we demonstrated that the cytoprotective effect of S1P was receptor-independent. Summarising, we showed that Aβ peptides evoke down-regulation of gene expression and activity for SphK(s) and S1P1. Inhibition of SphK(s) significantly decreased cell survival. The effect of exogenous S1P depended on the concentration of Aβ peptides.

Topics: Amyloid beta-Protein Precursor; Animals; Cell Survival; Enzyme Inhibitors; Humans; Lysophospholipids; PC12 Cells; Phosphotransferases (Alcohol Group Acceptor); Rats; Signal Transduction; Sphingosine; Transfection

The greatest genetic risk factor for late-onset Alzheimer's disease (AD) is the ϵ4 allele of Apolipoprotein E (ApoE). ApoE regulates secretion of the potent neuroprotective signaling lipid Sphingosine 1-phosphate (S1P). S1P is derived by phosphorylation of sphingosine, catalysed by sphingosine kinases 1 and 2 (SphK1 and 2), and SphK1 positively regulates glutamate secretion and synaptic strength in hippocampal neurons. S1P and its receptor family have been subject to intense pharmacological interest in recent years, following approval of the immunomodulatory drug Fingolimod, an S1P mimetic, for relapsing multiple sclerosis.. We quantified S1P levels in six brain regions that are differentially affected by AD pathology, in a cohort of 34 post-mortem brains, divided into four groups based on Braak neurofibrillary tangle staging. S1P declined with increasing Braak stage, and this was most pronounced in brain regions most heavily affected by AD pathology. The S1P/sphingosine ratio was 66% and 64% lower in Braak stage III/IV hippocampus (p = 0.010) and inferior temporal cortex (p = 0.014), respectively, compared to controls. In accordance with this change, both SphK1 and SphK2 activity declined with increasing Braak pathology in the hippocampus (p = 0.032 and 0.047, respectively). S1P/sphingosine ratio was 2.5-fold higher in hippocampus of ApoE2 carriers compared to ApoE4 carriers, and multivariate regression showed a significant association between APOE genotype and hippocampal S1P/sphingosine (p = 0.0495), suggesting a new link between APOE genotype and pre-disposition to AD.. This study demonstrates loss of S1P and sphingosine kinase activity early in AD pathogenesis, and prior to AD diagnosis. Our findings establish a rationale for further exploring S1P receptor pharmacology in the context of AD therapy.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Apolipoproteins E; Brain; Ceramides; Disease Models, Animal; Disease Progression; Female; Gray Matter; Humans; Lysophospholipids; Male; Mice; Mice, Transgenic; Mutation; Phosphotransferases (Alcohol Group Acceptor); Regression Analysis; Sphingosine

We demonstrate that pre-treatment of estrogen receptor negative MDA-MB-231 breast cancer cells containing ectopically expressed HA-tagged sphingosine 1-phosphate receptor-2 (S1P2) with the sphingosine kinase 1/2 inhibitor SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) or the sphingosine kinase 2 selective inhibitor (R)-FTY720 methyl ether (ROMe) or sphingosine kinase 2 siRNA induced the translocation of HA-tagged S1P2 and Y416 phosphorylated c-Src to the nucleus of these cells. This is associated with reduced growth of HA-tagged S1P2 over-expressing MDA-MB-231 cells. Treatment of HA-S1P2 over-expressing MDA-MB-231 cells with the sphingosine 1-phosphate receptor-4 (S1P4) antagonist CYM50367 or with S1P4 siRNA also promoted nuclear translocation of HA-tagged S1P2. These findings identify for the first time a signaling pathway in which sphingosine 1-phosphate formed by sphingosine kinase 2 binds to S1P4 to prevent nuclear translocation of S1P2 and thereby promote the growth of estrogen receptor negative breast cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; CSK Tyrosine-Protein Kinase; Female; Fingolimod Hydrochloride; Humans; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Estrogen; Receptors, Lysosphingolipid; RNA Interference; RNA, Small Interfering; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; src-Family Kinases; Thiazoles; Tyrosine

Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes"), which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF-α via S1P receptors, and (iii) released TNF-α stimulates the production of inflammatory mediators such as IL-8.

Topics: Cells, Cultured; Endothelin-1; Humans; I-kappa B Proteins; Inflammation Mediators; Interleukin-8; Keratinocytes; Lysophospholipids; Neutral Ceramidase; NF-KappaB Inhibitor alpha; Phosphotransferases (Alcohol Group Acceptor); Pseudomonas; Receptors, Lysosphingolipid; Sphingosine; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Both sphingosine 1-phosphate (S1P) and estrogen have been documented to play endothelial protective roles. However, it remains unclear whether estrogen could regulate the anabolism of the bioactive molecule S1P and the underlying mechanisms. In this study, 108 healthy participants were separated into three age groups, and their plasma S1P levels were analyzed by liquid chromatography tandem mass spectrometry. Results showed that the plasma S1P levels were significantly higher in women than those in men within the age of 16-55years old and higher in pre-menopausal than post-menopausal women. The experiment in C57 BL/6 mice confirmed the gender difference of plasma S1P level. In vitro study demonstrated that after the stimulation of 17β-estradiol (E2), S1P levels both in EA.hy926 cells and the culture media were increased about 9 and 3 times, respectively; the mRNA expression, the protein level and the activity of sphingosine kinase (SphK) 1, not SphK2, were markedly increased; the mRNA and protein expression of ATP-binding cassette transporter (ABC) C1, G2 and S1P transporter spinster homolog 2 (Spns2) were significantly elevated; furthermore, the mRNA and protein expressions of S1P receptors (S1PRs) 1-2 were increased in a time-dependent manner. This study suggests that E2 markedly improves S1P synthesis by activating SphK1 and induces S1P export via activating ABCC1, G2 and Spns2 from endothelium system, which may consequently lead to the gender difference of plasma S1P in adult human and mouse. The results of this study suggest that E2 may exert its vasculoprotective function by activation of the SphK1-S1P-S1PR signaling axis.

Topics: Adolescent; Adult; Animals; Estradiol; Estrogens; Female; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Lysophospholipids; Male; Mice; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Postmenopause; Premenopause; Receptors, Lysosphingolipid; Sphingosine

Diabetes is one of the most prevalent human metabolic diseases. Wound healing in diabetes is frequently impaired and treatment remains challenging. Sphingolipid metabolites play important roles in the regulation of glucose metabolism. SPK1 is the key enzyme in the sphingolipid metabolic pathway. S1P/SPK plays a pivotal role in the signalling pathways of diverse cellular processes including proliferation, differentiation, migration, apoptosis in diverse cell types.. To investigate the role of sphingosine kinase 1 (SPK1) in skin injury, plasmids containing the SPK1 gene (pcDNA3-FLAG-SPK1) were applied to cutaneous wounds on a streptozotocin-induced diabetic rat model over a 21-day period. The wound area and rate of wound healing were determined. The histopathological features of the healed wounds were also observed, and SPK1 expression in the skin was detected by immunohistochemistry.. There was a significant decrease in wound area in diabetic rats treated with 125 and 60μg/wound pcDNA3-FLAG-SPK1 (P<0.001-0.01). The mean sizes of the wounds were 0.67±0.15cm(2), 0.83±0.18cm(2), and 1.09±0.23cm(2) in both treated and diabetic control group at the 7th day post-treatment respectively. In addition, wound healing in diabetic rats of test group was accelerated. At the 7th day, the mean rates of healing were 73.2±5.7% and 66±7.3% in test group of 125 and 60μg/wound respectively, and 55.4±9.9% in diabetic control group (P<0.001-0.01). Histology revealed that tissue sections from the treated diabetic rats contained more granulation tissue and capillaries than that of the control rats. There was high SPK1 expression in the skin of the treated diabetic rats.. SPK1 gene therapy may represent a novel approach to cutaneous wound healing.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Movement; Cell Proliferation; Diabetes Complications; Diabetes Mellitus, Experimental; Immunohistochemistry; Injections, Subcutaneous; Lysophospholipids; Male; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Skin; Sphingosine; Wound Healing

Ginsenoside compound K (CK) is a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer (Araliaceae), has long been used to treat against the development of cancer, inflammation, allergies, and diabetes. This study examined the anti-angiogenic properties of CK against sphingosine 1-phosphate (S1P)-induced cell migration via regulation of sphingosine kinase 1 (SPHK1) in human umbilical vein endothelial cells (HUVEC). Studies on S1P-induced cell migration, expression of SPHK1 and MMPs and analysis of sphingolipid metabolites by LC-MS/MS were examined after the treatment of CK (2.5, 5, 10 μg/mL) in HUVEC. S1P produced by SPHK1 is also involved in cell growth, migration, and protection of apoptosis; therefore, we sought to investigate whether ginsenosides are able to regulate SPHK1. For this purpose, we developed an inhibitory assay of SPHK1 activity and an analytical method for detection of S1P and other sphingolipid metabolites in HUVEC. Ginsenoside CK inhibited 100 nM S1P-induced cell migrations in a dose-dependent manner. Among tested ginsenosides, CK exclusively inhibited S1P production, SPHK1 activity and SPHK1 expression in HUVEC, whereas expression of the pro-apoptotic sphingolipids, sphingosine and ceramide, was increased in response to CK. The major subspecies of the increased ceramide was C24:0-ceramide. CK also disrupted the sphingolipid rheostat, which ultimately influences cell fate, and dose-dependently inhibited HUVEC migration by reducing expression of metalloproteinases (MMPs). Ginsenoside CK acts as a unique HUVEC migration inhibitor by regulating MMP expression, as well as the activity of SPHK1 and its related sphingolipid metabolites.

Topics: Angiogenesis Inhibitors; Anticarcinogenic Agents; Cell Movement; Cell Survival; Cells, Cultured; Ceramides; Endothelium, Vascular; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Ginsenosides; Human Umbilical Vein Endothelial Cells; Humans; Lysophospholipids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neovascularization, Pathologic; Osmolar Concentration; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Memo is a conserved protein that was identified as an essential mediator of tumor cell motility induced by receptor tyrosine kinase activation. Here we show that Memo null mouse embryonic fibroblasts (MEFs) are impaired in PDGF-induced migration and this is due to a defect in sphingosine-1-phosphate (S1P) signaling. S1P is a bioactive phospholipid produced in response to multiple stimuli, which regulates many cellular processes. S1P is secreted to the extracellular milieu where it exerts its function by binding a family of G-protein coupled receptors (S1PRs), causing their activation in an autocrine or paracrine manner. The process, termed cell-autonomous S1PR signaling, plays a role in survival and migration. Indeed, PDGF uses cell-autonomous S1PR signaling to promote cell migration; we show here that this S1P pathway requires Memo. Using vascular endothelial cells (HUVECs) with Memo knock-down we show that their survival in conditions of serum-starvation is impaired. Furthermore, Memo loss in HUVECs causes a reduction of junctional VE-cadherin and an increase in sprout formation. Each of these phenotypes is rescued by S1P or S1P agonist addition, showing that Memo also plays an important role in cell-autonomous S1PR signaling in endothelial cells. We also produced conventional and endothelial cell-specific conditional Memo knock-out mouse strains and show that Memo is essential for embryonic development. Starting at E13.5 embryos of both strains display bleeding and other vascular problems, some of the phenotypes that have been described in mouse strains lacking S1PRs. The essential role of Memo in embryonic vascular development may be due in part to alterations in S1P signaling. Taken together our results show that Memo has a novel role in the S1P pathway and that Memo is needed to promote cell-autonomous S1PR activation.

Topics: Animals; Apoptosis; Cell Movement; Cell Proliferation; Lysophospholipids; Mice; Mice, Knockout; Neovascularization, Physiologic; Nonheme Iron Proteins; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Cell therapy based on endothelial colony-forming cells (ECFCs) is a promising option for ischaemic cardiovascular diseases. A better understanding of the mechanisms by which these cells promote revascularization remains a critical challenge to improving their therapeutic potential. We aimed to identify the critical mechanisms involved in the revascularization activity of ECFCs by using the paracrine properties of mesenchymal stem cells (MSC).. Conditioned medium from human bone marrow-derived MSCs (MSC-CM) increased the angiogenic activity of cord blood ECFCs in vitro (proliferation, migration, and pseudo-tube formation), the survival of ECFCs in mice (Matrigel Plug assay), and the capacity of ECFCs to promote the recovery of blood perfusion in mice with hindlimb ischaemia. Furthermore, the capillary density in ischaemic gastrocnemius muscle was significantly increased in mice transplanted with the ECFCs pre-treated with the MSC-CM. The enhancement of ECFCs activity involved the up-regulation of sphingosine kinase 1 (SphK1) expression and activity. The inhibition of SphK1 in ECFCs by using an inhibitor or a siRNA knockdown of SphK1 prevented the stimulation of the ECFCs induced by the MSC-CM. The improvement of ECFC activity by MSC-CM also involved the up-regulation of sphingosine-1-phosphate receptor 1 (S1P1) and a S1P/S1P1/3-dependent mechanism. Finally, we showed that the stimulation of ECFCs with exogenous S1P increased angiogenesis and promoted blood perfusion in hindlimb ischaemia.. The up-regulation of SphK1 and S1P-dependent pathways is critical for the angiogenic/vasculogenic activity of ECFCs. The identification of this pathway provides attractive targets to optimize cell-based therapy for revascularization in ischaemic diseases.

Topics: Animals; Cells, Cultured; Colony-Forming Units Assay; Culture Media, Conditioned; Endothelial Cells; Gene Knockdown Techniques; Humans; Lysophospholipids; Mesenchymal Stem Cells; Mice; Mice, Nude; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Small Interfering; Sphingosine; Sphingosine-1-Phosphate Receptors

Sphingosine kinase 1 (SK1) is an FGF-inducible gene responsible for generation of sphingosine-1-phosphate, a critical lipid signaling molecule implicated in diverse endothelial cell functions. In this study, we identified SK1 as a target of the canonical FGF2/FGF receptor 1 activation pathway in endothelial cells and sought to identify novel transcriptional pathways that mediate lipid signaling. Studies using the 1.9-kb SK1 promoter and deletion mutants revealed that basal and FGF2-stimulated promoter activity occurred through two GC-rich regions located within 633 bp of the transcription start site. Screening for GC-rich binding transcription factors that could activate this site demonstrated that KLF14, a gene implicated in obesity and the metabolic syndrome, binds to this region. Congruently, overexpression of KLF14 increased basal and FGF2-stimulated SK1 promoter activity by 3-fold, and this effect was abrogated after mutation of the GC-rich sites. In addition, KLF14 siRNA transfection decreased SK1 mRNA and protein levels by 3-fold. Congruently, SK1 mRNA and protein levels were decreased in livers from KLF14 knock-out mice. Combined, luciferase, gel shift, and chromatin immunoprecipitation assays showed that KLF14 couples to p300 to increase the levels of histone marks associated with transcriptional activation (H4K8ac and H3K14ac), while decreasing repressive marks (H3K9me3 and H3K27me3). Collectively, the results demonstrate a novel mechanism whereby SK1 lipid signaling is regulated by epigenetic modifications conferred by KLF14 and p300. Thus, this is the first description of the activity and mechanisms underlying the function of KLF14 as an activator protein and novel regulator of lipid signaling.

Topics: Animals; Chromatin; Endothelial Cells; Epigenesis, Genetic; Fibroblast Growth Factor 2; HEK293 Cells; Histones; Human Umbilical Vein Endothelial Cells; Humans; Kruppel-Like Transcription Factors; Lipid Metabolism; Liver; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sp Transcription Factors; Sphingosine; Transcriptional Activation

Preanalytical standardization is required for a reliable quantification of the signaling molecules sphingosine-1-phosphate (S1P), sphinganine-1-phosphate (SA1P) and sphingosine (SPH).. Methanolic protein precipitation of 15μL EDTA-plasma was applied prior to analysis. Sphingolipids were separated in 3min by hydrophilic interaction liquid chromatography (HILIC, SeQuant™ ZIC®-HILIC column) followed by tandem mass spectrometry. Stability of analytes in whole blood and plasma was investigated. Sphingolipid concentrations were determined in human plasma (n=50) and mice deficient in sphingosine kinase 1 (SK1) and 2 (SK2) (n=5).. Storing EDTA whole blood >60min after blood withdrawal at room temperature resulted in an increase in S1P and SPH concentrations of ≥25%. Significant changes in SPH levels of +37% were observed after 60min of storage of EDTA plasma at room temperature. Repeated freeze-thaw cycles of EDTA plasma resulted in increased S1P and SPH levels. Concentrations in human EDTA plasma were between 55.5 and 145.2ng/mL for S1P and between 8.9 and 35.3ng/mL for SA1P. Concentrations of S1P were 36% lower and 96% higher in EDTA-plasma from SK1- and SK2-deficient mice, respectively, compared to the wild type.. Preanalytical standardization is a precondition for the analysis of sphingolipids in human blood.

Topics: Animals; Blood Chemical Analysis; Chromatography, Liquid; Female; Healthy Volunteers; Humans; Lysophospholipids; Male; Mice; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Reference Standards; Reproducibility of Results; Sphingosine; Tandem Mass Spectrometry; Time Factors

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates multicellular functions through interactions with its receptors on cell surfaces. S1P is enriched and stored in erythrocytes; however, it is not clear whether alterations in S1P are involved in the prevalent and debilitating hemolytic disorder sickle cell disease (SCD). Here, using metabolomic screening, we found that S1P is highly elevated in the blood of mice and humans with SCD. In murine models of SCD, we demonstrated that elevated erythrocyte sphingosine kinase 1 (SPHK1) underlies sickling and disease progression by increasing S1P levels in the blood. Additionally, we observed elevated SPHK1 activity in erythrocytes and increased S1P in blood collected from patients with SCD and demonstrated a direct impact of elevated SPHK1-mediated production of S1P on sickling that was independent of S1P receptor activation in isolated erythrocytes. Together, our findings provide insights into erythrocyte pathophysiology, revealing that a SPHK1-mediated elevation of S1P contributes to sickling and promotes disease progression, and highlight potential therapeutic opportunities for SCD.

Topics: Anemia, Sickle Cell; Animals; Antisickling Agents; Disease Models, Animal; Disease Progression; Enzyme Inhibitors; Erythrocytes, Abnormal; Gene Knockdown Techniques; Hemolysis; Humans; Lysophospholipids; Metabolomics; Methanol; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mice, Transgenic; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidines; Signal Transduction; Sphingosine; Sulfones

FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2(-/-) mice have lower levels of hippocampal sphingosine-1-phosphate, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, sphingosine-1-phosphate and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories.

Topics: Animals; Blotting, Western; Brain; Exploratory Behavior; Extinction, Psychological; Fear; Fingolimod Hydrochloride; Gene Expression; Hippocampus; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Immunosuppressive Agents; Isoenzymes; Learning Disabilities; Lysophospholipids; Maze Learning; Memory; Mice; Mice, Inbred C57BL; Mice, SCID; Models, Molecular; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Spectrometry, Mass, Electrospray Ionization; Sphingosine

Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor.

Topics: Animals; Blotting, Western; Cell Differentiation; Cell Proliferation; Cells, Cultured; Epidermal Growth Factor; Lysophospholipids; Mice; Muscle, Skeletal; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Satellite Cells, Skeletal Muscle; Signal Transduction; Sphingosine

A complicating factor in the translational pharmacology of sphingosine 1-phosphate agonists is that they exert their pharmacological effect through their respective phosphate metabolites, which are formed by the enzyme sphingosine kinase (S1PHK). In this investigation, we present a semimechanistic pharmacokinetic model for the interconversion of S1PHK substrates and their respective phosphates in rats and humans with the aim of investigating whether characterization of the rate of phosphorylation in blood platelets constitutes a basis for interspecies scaling using fingolimod as a model compound. Data on the time course of fingolimod and fingolimod-phosphate (fingolimod-P) blood concentrations after intravenous and oral administration of fingolimod and/or fingolimod-P in rats and after oral administration of fingolimod in doses of 0.5, 1.25, and 5 mg once daily in healthy volunteers were analyzed in conjunction with data on the ex vivo interconversion and blood-plasma distribution in rat and human blood, respectively. Integrating the data from the ex vivo and in vivo studies enabled simulation of fingolimod and fingolimod-P concentrations in plasma rather than blood, which are more relevant for characterizing drug effects. Large interspecies differences in the rate of phosphorylation between rats and humans were quantified. In human, phosphorylation of fingolimod in the platelets was four times slower compared with rat, whereas the dephosphorylation rates were comparable in both species. This partly explained the 10-12-fold overprediction of fingolimod-P exposure in human when applying a dose-by-factor approach on the developed rat model. Additionally, differences in presystemic phosphorylation should also be taken into account.

Topics: Administration, Intravenous; Administration, Oral; Animals; Blood Platelets; Double-Blind Method; Fingolimod Hydrochloride; Humans; Lysophospholipids; Male; Phosphates; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Randomized Controlled Trials as Topic; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Sphingosine

The tumor necrosis factor (TNF) receptor family member CD40 plays an essential role in the activation of antigen-presenting cells, B cell maturation, and immunoglobulin (Ig) class switching critical for adaptive immunity. Although the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) and the kinase that produces it, sphingosine kinase 1 (SphK1), have long been implicated in the actions of TNF mediated by engagement of TNFR1, nothing is yet known of their role in CD40-mediated events. We have now found that ligation of CD40 activates and translocates SphK1 to the plasma membrane, leading to generation of S1P. SphK1 inhibition in human tonsil B cells, as well as inhibition or deletion of SphK1 in mouse splenic B cells, significantly reduced CD40-mediated Ig class switching and plasma cell differentiation ex vivo. Optimal activation of downstream CD40 signaling pathways, including NF-κB, p38, and JNK, also required SphK1. In mice treated with a SphK1 inhibitor or in SphK1(-/-) mice, isotype switching to antigen-specific IgE was decreased in vivo by 70 and 55%, respectively. Our results indicate that SphK1 is important for CD40-mediated B cell activation and regulation of humoral responses and suggest that targeting SphK1 might be a useful therapeutic approach to control antigen-specific IgE production.

Topics: Animals; B-Lymphocytes; CD40 Antigens; Cell Differentiation; Cell Membrane; HEK293 Cells; Humans; Immunoglobulin Class Switching; Immunoglobulin E; Lysophospholipids; MAP Kinase Kinase 4; Mice; Mice, Inbred C57BL; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Protein Transport; Sphingosine

The sphingolipid metabolite sphingosine 1-phosphate (S1P) functions as a lipid mediator and as a key intermediate of the sole sphingolipid to glycerophospholipid metabolic pathway (S1P metabolic pathway). In this pathway, S1P is converted to palmitoyl-CoA through 4 reactions, then incorporated mainly into glycerophospholipids. Although most of the genes responsible for the S1P metabolic pathway have been identified, the gene encoding the trans-2-enoyl-CoA reductase, responsible for the saturation step (conversion of trans-2-hexadecenoyl-CoA to palmitoyl-CoA) remains unidentified. In the present study, we show that TER is the missing gene in mammals using analyses involving yeast cells, deleting the TER homolog TSC13, and TER-knockdown HeLa cells. TER is known to be involved in the production of very long-chain fatty acids (VLCFAs). A significant proportion of the saturated and monounsaturated VLCFAs are used for sphingolipid synthesis. Therefore, TER is involved in both the production of VLCFAs used in the fatty acid moiety of sphingolipids as well as in the degradation of the sphingosine moiety of sphingolipids via S1P.

Topics: Aldehyde-Lyases; Animals; Fatty Acids; Gene Expression Regulation, Enzymologic; HeLa Cells; Hep G2 Cells; Humans; Lysophospholipids; Metabolic Networks and Pathways; Mutation; Oxidoreductases Acting on CH-CH Group Donors; PC12 Cells; Phosphotransferases (Alcohol Group Acceptor); Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sphingolipids; Sphingosine

We previously reported that sphingosine kinase 1 (SphK1), an enzyme that catalyzes the production of sphingosine-1-phosphate (SIP), is upregulated in human gastric cancer and predicts poor clinical outcome. In the present study, we used known differential effects of UV irradiation on human MGC-803 gastric cancer cells to determine their effect on SphK1 activity. Ectopic expression of SphK1 in MGC-803 gastric cancer cells markedly enhanced their resistance to UV irradiation, whereas silencing endogenous SphK1 with shRNAs weakened this ability. Furthermore, these anti-apoptotic effects were significantly associated with decrease of Bim, an apoptosis-related protein. We further demonstrated that SphK1 could downregulate the transcriptional activity of forkhead box O3a (FoxO3a) by inducing its phosphorylation, which was found to be associated with the PI3K/Akt signaling. Taken together, our study supports the theory that SphK1 confers resistance to apoptosis in gastric cancer cells via the Akt/FoxO3a/Bim pathway.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Carcinoma; Cell Line, Tumor; Down-Regulation; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lysophospholipids; Membrane Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Sphingosine; Stomach Neoplasms; Ultraviolet Rays; Up-Regulation

Sphingosine kinase (SK), a key enzyme in sphingosine-1-phosphate (S1P) synthesis, is known to be overexpressed in various types of cancer cells. The effects of anticancer agents on SK1/S1P signaling have not yet been fully assessed in melanoma cells. In the present study, we investigated the effects of the combination of FTY720, an S1P receptor antagonist, and cisplatin, a DNA-damaging agent, on the induction of the death of human melanoma cells, as well as the molecular mechanisms involved. The viability of various human melanoma cell lines was examined following treatment with anticancer drugs. The cisplatin-resistant SK-Mel-28 and cisplatin-sensitive A375 cell lines were selected for this analysis. Protein expression and apoptotic rates were evaluated by western blot analysis following treatment with cisplatin and/or FTY720. Following treatment with a combination of FTY720 and cisplatin, cell viability significantly decreased and the expression of apoptosis-associated cleaved poly(ADP-ribose) polymerase (PARP) was significantly higher in comparison to treatment with cisplatin alone in the SK-Mel-28 cells. In addition, the combination of FTY720 and cisplatin reduced the protein expression of SK1 and the phosphorylation levels of phosphoinositide 3-kinase (PI3K), Akt and mTOR in the SK-Mel-28 cells; the expression of epidermal growth factor receptor (EGFR) was also markedly reduced. These findings suggest that FTY720 and cisplatin synergistically induce cell death through the downregulation of the PI3K/Akt/mTOR pathway and the decrease in EGFR expression in SK-Mel-28 cells. Thus, the combination of FTY720 and cisplatin may have therapeutic potential for chemotherapy-resistant melanoma, and the effects are likely exerted through the downregulation of S1P signaling.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Cisplatin; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; ErbB Receptors; Fingolimod Hydrochloride; Humans; Inhibitory Concentration 50; Lysophospholipids; Melanoma; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Signal Transduction; Sphingosine

Sphingosine-1-phosphate (S1P) is produced by sphingosine kinase 1 and is implicated in tumor growth, although the mechanisms remain incompletely understood. Pancreatic stellate cells (PSCs) reside within the tumor microenvironment and may regulate tumor progression. We hypothesized that S1P activates PSCs to release paracrine factors, which, in turn, increase cancer cell invasion and growth. We used a combination of human tissue, in vitro, and in vivo studies to mechanistically evaluate this concept. Sphingosine kinase 1 was overexpressed in human pancreatic tissue, especially within tumor cells. S1P activated PSCs in vitro and conditioned medium from S1P-stimulated PSCs, increased pancreatic cancer cell migration, and invasion, which was dependent on S1P2, ABL1 (alias c-Abl) kinase, and matrix metalloproteinase-9. In vivo studies showed that pancreatic cancer cells co-implanted with S1P2 receptor knockdown PSCs led to less cancer growth and metastasis in s.c. and orthotopic pancreatic cancer models compared with control PSCs. Pancreatic cancer cell-derived S1P activates PSCs to release paracrine factors, including matrix metalloproteinase-9, which reciprocally promotes tumor cell migration and invasion in vitro and cancer growth in vivo.

Topics: Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Lysophospholipids; Matrix Metalloproteinase 9; Pancreas; Pancreatic Neoplasms; Pancreatic Stellate Cells; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Tumor Microenvironment; Up-Regulation

Regulation of the motility of skeletal muscle precursor cells, such as satellite cells, is critically important for their proper recruitment at the site of tissue damage, and ultimately for its correct repair. Here we show that lysophosphatidic acid (LPA), which is well-recognized as a powerful bioactive agent, strongly stimulates cell migration of activated murine satellite cells. The biological effect exerted by LPA was found to be induced via activation of LPA1 and LPA3 , being abolished by cell treatment with the antagonist Ki16425, and severely impaired by siRNA-mediated down-regulation of the two receptor isoforms. In contrast, silencing of LPA2 potentiated the stimulation of cell motility by LPA, suggesting that it is negatively coupled to cell migration. Pharmacological inhibition of both sphingosine kinase (SK) isoforms using VPC96047, or the selective blocking of SK1 using VPC96091, abolished cell responsiveness to LPA; in agreement, gene silencing of SK1 or SK2 significantly reduced the biological effect of LPA. Moreover, the LPA-dependent stimulation of cell chemotaxis was found to be impaired by down-regulation of the sphingosine 1-phosphate (S1P) receptors S1P1 or S1P4 by specific siRNAs. In summary, the results obtained support the notion that the sphingosine kinase/sphingosine 1-phosphate (SK/S1P) axis is critically involved in the mechanism by which LPA elicits its pro-migratory action. This study provides compelling new information on the regulatory mechanisms of satellite cell motility, and reinforces the view that the SK/S1P signaling pathway plays a crucial role in the control of skeletal muscle precursor cell biology.

Topics: Animals; Cell Movement; Cells, Cultured; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Satellite Cells, Skeletal Muscle; Signal Transduction; Sphingosine

Sphingosine kinases (SphKs) 1 and 2 regulate the synthesis of the bioactive sphingolipid sphingosine-1-phosphate (S1P), an important lipid mediator that promotes cell proliferation, migration, and angiogenesis.. We aimed to examine whether SphKs and their product, S1P, play a role in the development of pulmonary arterial hypertension (PAH).. SphK1(-/-), SphK2(-/-), and S1P lyase heterozygous (Sgpl1(+/-)) mice, a pharmacologic SphK inhibitor (SKI2), and a S1P receptor 2 (S1PR2) antagonist (JTE013) were used in rodent models of hypoxia-mediated pulmonary hypertension (HPH). S1P levels in lung tissues from patients with PAH and pulmonary arteries (PAs) from rodent models of HPH were measured.. mRNA and protein levels of SphK1, but not SphK2, were significantly increased in the lungs and isolated PA smooth muscle cells (PASMCs) from patients with PAH, and in lungs of experimental rodent models of HPH. S1P levels were increased in lungs of patients with PAH and PAs from rodent models of HPH. Unlike SphK2(-/-) mice, SphK1(-/-) mice were protected against HPH, whereas Sgpl1(+/-) mice were more susceptible to HPH. Pharmacologic SphK1 and S1PR2 inhibition prevented the development of HPH in rodent models of HPH. Overexpression of SphK1 and stimulation with S1P potentially via ligation of S1PR2 promoted PASMC proliferation in vitro, whereas SphK1 deficiency inhibited PASMC proliferation.. The SphK1/S1P axis is a novel pathway in PAH that promotes PASMC proliferation, a major contributor to pulmonary vascular remodeling. Our results suggest that this pathway is a potential therapeutic target in PAH.

Topics: Animals; Humans; Hypertension, Pulmonary; Lysophospholipids; Male; Mice; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Signal Transduction; Sphingosine; Tissue Culture Techniques

Sphingosine kinase 1 (SK1) is a signaling enzyme that catalyzes the formation of sphingosine-1-phosphate. Overexpression of SK1 is causally associated with breast cancer progression and resistance to therapy. SK1 inhibitors are currently being investigated as promising breast cancer therapies. Two major transcriptional isoforms, SK143 kDa and SK151 kDa, have been identified; however, the 51 kDa variant is predominant in breast cancer cells. No studies have investigated the protein-protein interactions of the 51 kDa isoform and whether the two SK1 isoforms differ significantly in their interactions. Seeking an understanding of the regulation and role of SK1, we used a triple-labeling stable isotope labeling by amino acids in cell culture-based approach to identify SK1-interacting proteins common and unique to both isoforms. Of approximately 850 quantified proteins in SK1 immunoprecipitates, a high-confidence list of 30 protein interactions with each SK1 isoform was generated via a meta-analysis of multiple experimental replicates. Many of the novel identified SK1 interaction partners such as supervillin, drebrin, and the myristoylated alanine-rich C-kinase substrate-related protein supported and highlighted previously implicated roles of SK1 in breast cancer cell migration, adhesion, and cytoskeletal remodeling. Of these interactions, several were found to be exclusive to the 43 kDa isoform of SK1, including the protein phosphatase 2A, a previously identified SK1-interacting protein. Other proteins such as allograft inflammatory factor 1-like protein, the latent-transforming growth factor β-binding protein, and dipeptidyl peptidase 2 were found to associate exclusively with the 51 kDa isoform of SK1. In this report, we have identified common and isoform-specific SK1-interacting partners that provide insight into the molecular mechanisms that drive SK1-mediated oncogenicity.

Topics: Breast Neoplasms; Calcium-Binding Proteins; Cell Adhesion; Cell Line, Tumor; Cell Movement; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; DNA-Binding Proteins; Female; Humans; Latent TGF-beta Binding Proteins; Lysophospholipids; MCF-7 Cells; Microfilament Proteins; Phosphotransferases (Alcohol Group Acceptor); Protein Isoforms; Signal Transduction; Sphingosine

Transport of oxygen by red blood cells (rbc) is critical for life and embryogenesis. Here, we determined that provision of the lipid mediator sphingosine 1-phosphate (S1P) to the systemic circulation is an essential function of rbc in embryogenesis. Mice with rbc-specific deletion of sphingosine kinases 1 and 2 (Sphk1 and Sphk2) showed embryonic lethality between E11.5 and E12.5 due to defects in vascular development. Administration of an S1P1 receptor agonist to pregnant dams rescued early embryonic lethality. Even though rbc-specific Sphk1 Sphk2-KO embryos were anemic, the erythropoietic capacity of hematopoietic stem cells (HSCs) was not impaired, suggesting that rbc can develop in the absence of sphingosine kinase activity. Indeed, transplantation of HSCs deficient for Sphk1 and Sphk2 into adult mice produced rbc that lacked S1P and attenuated plasma S1P levels in recipients. However, in adult animals, both rbc and endothelium contributed to plasma S1P. Together, these findings demonstrate that rbc are essential for embryogenesis by supplying the lysophospholipid S1P, which regulates embryonic vascular development via its receptors.

Topics: Animals; Embryonic Development; Erythrocytes; Erythropoiesis; Female; Genes, Lethal; Hematopoietic Stem Cell Transplantation; Lysophospholipids; Mice; Mice, Knockout; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Many tumours originate from cancer stem cells (CSCs), which is a small population of cells that display stem cell properties. However, the molecular mechanisms that regulate CSC frequency remain poorly understood. Here, using microarray screening in aldehyde dehydrogenase (ALDH)-positive CSC model, we identify a fundamental role for a lipid mediator sphingosine-1-phosphate (S1P) in CSC expansion. Stimulation with S1P enhances ALDH-positive CSCs via S1P receptor 3 (S1PR3) and subsequent Notch activation. CSCs overexpressing sphingosine kinase 1 (SphK1), an S1P-producing enzyme, show increased ability to develop tumours in nude mice, compared with parent cells or CSCs. Tumorigenicity of CSCs overexpressing SphK1 is inhibited by S1PR3 knockdown or S1PR3 antagonist. Breast cancer patient-derived mammospheres contain SphK1(+)/ALDH1(+) cells or S1PR3(+)/ALDH1(+) cells. Our findings provide new insights into the lipid-mediated regulation of CSCs via Notch signalling, and rationale for targeting S1PR3 in cancer.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; Lysophospholipids; Mice; Mice, Inbred BALB C; Neoplastic Stem Cells; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Receptors, Notch; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration.

Topics: Animals; Bone Marrow Cells; Cell Proliferation; Cells, Cultured; Culture Media, Conditioned; Lysophospholipids; Mesenchymal Stem Cells; Mice; Myoblasts, Skeletal; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Regeneration; Signal Transduction; Sphingosine; Vascular Endothelial Growth Factor A; Wound Healing

Topics: Animals; Humans; Hypertension, Pulmonary; Lysophospholipids; Male; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

FTY720, a new immunomodulatory drug with low cytotoxicity, is currently used to treat multiple sclerosis. In this study, we investigated the effects of FTY720 on inflammatory cell infiltration in albumin overload-induced nephropathy of rats.. Male Wistar rats were subjected to right-side nephrectomy and divided into 3 groups. One week after the surgery, albumin overload (AO) group was treated with BSA (5 g·kg(-1)·d(-1), ip) for 9 weeks; AO+FTY720 group was given BSA (5 g·kg(-1)·d(-1), ip) plus FTY720 (0.5 g·kg(-1)·d(-1), ip) for 9 weeks; and control group received daily ip injection of equivalent volume of saline. All rats were killed 9 weeks after nephrectomy.. AO rats exhibited gradually increased urinary protein excretion accompanied by elevated urinary N-acetyl-β-O-glucosaminidase activity, and both reached their peak values at week 7. Furthermore, AO significantly increased lymphocytes and monocytes in circulation and the inflammatory cells recruited to tubulointerstitium, and the expression of inflammatory cytokines MCP-1, TNF-α and IL-6, as well as sphingosine 1-phosphate (S1P) receptors S1pr1 and S1pr3, and S1P-synthesizing enzyme sphingosine kinase 1 (Sphk1) in the kidney. Concomitant administration of FTY720 significantly attenuated all the AO-induced pathological changes.. FTY720 alleviates tubulointerstitium inflammation in an AO rat model of nephropathy via down-regulation of the Sphk1 pathway.

Topics: Acetylglucosaminidase; Albuminuria; Animals; Anti-Inflammatory Agents; Disease Models, Animal; Down-Regulation; Fingolimod Hydrochloride; Immunosuppressive Agents; Inflammation Mediators; Kidney Tubules; Lymphocytes; Lysophospholipids; Macrophages; Male; Nephritis, Interstitial; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Rats, Wistar; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Time Factors

Sphingosine kinase 1 (SK1), one of two SK enzymes, is highly regulated and has been shown to act as a focal point for the action of many growth factors and cytokines. SK1 leads to generation of sphingosine-1-phosphate (S1P) and potentially the activation of S1P receptors to mediate biologic effects. Our previous studies implicated SK1/S1P in the regulation of inflammatory processes, specifically in inflammatory bowel disease (IBD). These studies were conducted using a total body knockout mouse for SK1 and were unable to determine the source of SK1/S1P (hematopoietic or extra-hematopoietic) involved in the inflammatory responses. Therefore, bone marrow transplants were performed with wild-type (WT) and SK1-/- mice and colitis induced with dextran sulfate sodium (DSS). Irrespective of the source of SK1/S1P, bone marrow or tissue, DSS induced colitis in all mice; however, mice lacking SK1 in both hematopoietic and extra-hematopoietic compartments exhibited decreased crypt damage. Systemic inflammation was assessed, and mice with WT bone marrow demonstrated significant neutrophilia in response to DSS. In the local inflammatory response, mice lacking SK1/S1P in either bone marrow or tissue exhibited decreased induction of cytokines and less activation of STAT3 (signal transducer and activator of transcription 3). Interestingly, we determined that extra-hematopoietic SK1 is necessary for the induction of cyclooxygenase 2 (COX2) in colon epithelium in response to DSS-induced colitis. Taken together our data suggest that hematopoietic-derived SK1/S1P regulates specific aspects of the systemic inflammatory response, while extra-hematopoietic SK1 in the colon epithelium is necessary for the autocrine induction of COX2 in DSS-induced colitis.

Topics: Animals; Colitis; Hematopoietic System; Inflammation; Inflammatory Bowel Diseases; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine

Our previous study has shown that the activity and expression of sphingosine kinase (SPHK) regulated the sensitivity of human colon cancer cells to the chemotherapeutic oxaliplatin (L-OHP). In addition, the cancer stem cell marker CD44 increases cell resistance to anticancer drugs. Here, we use colon cancer cell lines to examine the relationship between SPHK1 activity and CD44 expression.CD44 expression was measured by western blotting and quantitative PCR in two human colon cancer cell lines: L-OHP-resistant RKO and L-OHP-sensitive HCT116. The regulation of CD44 by SPHK1 was examined by either blocking or overexpressing SPHK1 and by using an L-OHP-resistant HCT116 clone (HCT116-R).The levels of SPHK1, CD44, phosphorylated-Akt, and phosphorylated-extracellular signal-regulated kinase (ERK) were much higher in the RKO cells than in the HCT116 cells. The treatment of RKO cells with the SPHK inhibitor or SPHK1 silencing by RNA interference suppressed CD44 protein expression. SPHK1 and CD44 levels were much higher in HCT116-R cells compared with the parental HCT116 cells. Transfection of HCT116 cells with SPHK1 cDNA enhanced the expression of both CD44 and phosphorylated-ERK. The increase in the CD44 protein level was abolished by the inhibition of ERK phosphorylation. Treatment of RKO cells with the sphingosine-1-phosphate (S1P)2 receptor antagonist suppressed ERK phosphorylation and the expression of CD44 mRNA and protein. Exogenous stimulation with S1P increased ERK phosphorylation and CD44 protein expression in HCT116 cells, but treatment with an MEK inhibitor and S1P2 receptor antagonist blocked this effect.These findings indicate that SPHK1 and its product, S1P, contribute toward the regulation of CD44 protein expression through the ERK signaling pathway through S1P2 in human colon cancer cells.

Topics: Cell Line, Tumor; Colonic Neoplasms; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; HCT116 Cells; Humans; Hyaluronan Receptors; Lysophospholipids; Organoplatinum Compounds; Oxaliplatin; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Signal Transduction; Sphingosine; Up-Regulation

Sphingosine-1-phosphate (S1P), a bioactive lipid, has been reported to regulate inflammation processes. The onset of labor is thought to be related to inflammation. We therefore hypothesized that S1P might be involved in the onset of labor.. The expression of sphingosine kinase (SPHK)-1, which produces S1P, and S1P lyase (SPL)-1, which irreversibly inactivates S1P, were examined in the fetal membranes. The expression levels were compared between amnions from cases of elective Caesarean deliveries (pre-labor) and those from vaginal deliveries (post-labor). In primary cultured human amnion cells, the expression levels of prostaglandin-endoperoxide synthase (PTGS)-2 were examined in the presence or absence of S1P treatment.. SPHK-1 and SPL-1 were both expressed in the amnion. The expression of SPHK-1 in the post-labor amnions increased compared with that in the pre-labor amnions. The expression of PTGS-2, a key regulator of labor, also increased in the post-labor amnion. However, the SPL-1 expression in the pre-labor amnion was not significantly different from that in the post-labor amnion. S1P1-3 and 5, which were coupled with Gi protein, were consistently found in the amnion cells. The treatment with S1P increased the expression of PTGS-2, and this was completely suppressed by a Gi inhibitor in the amnion cells.. We are herein provide the first evidence of increased SPHK-1 expression in post-labor amnions, and that S1P increases the PTGS-2 expression in amnion cells.. Our results suggest that S1P might play a role in the onset of labor via the induction of PTGS-2.

Topics: Aldehyde-Lyases; Amnion; Cesarean Section; Cyclooxygenase 2; Female; Humans; Labor, Obstetric; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Pregnancy; Sphingosine

Sphingosine kinase (SphK)/sphingosine 1-phosphate (S1P)/S1P receptor (S1PR) axis is involved in multiple biological processes, including liver fibrosis. Angiogenesis is an important pathophysiological process closely associated with liver fibrosis; however, the functional role of SphK/S1P/S1PR in this process remains incompletely defined.. Bile duct ligation or carbon tetrachloride was used to induce liver fibrosis in mice. Human fibrotic samples were obtained from livers of patients undergoing liver transplantation. S1P levels in the liver were examined by HPLC. Expression of angiogenic markers, including angiopoietin 1, CD31, vascular cell adhesion molecule-1, and von Willebrand factor, was characterized by immunofluorescence, real-time RT-PCR, and Western blot in the fibrotic liver and primary mouse hepatic stellate cells (HSCs). SphK inhibitor (SKI) or S1PR antagonists were administered intraperitoneally in mice.. S1P levels in the liver were closely correlated with mRNA expression of angiogenic markers. Ang1 is expressed in activated HSCs of the fibrotic liver and in primary HSCs. In HSCs, by using specific antagonists or siRNAs, we demonstrated S1P stimulation induced Ang1 expression via S1PR1 and S1PR3. In vivo, S1P reduction by SKI inhibited angiogenesis in fibrotic mice. Furthermore, S1PR1/3 antagonist significantly blocked upregulation of angiogenic markers in the injured liver, and attenuated the extent of liver fibrosis, while S1PR2 antagonist had no effect on angiogenesis, supporting the key role of S1PR1 and S1PR3 in angiogenesis underlying liver fibrosis process.. SphK1/S1P/S1PR1/3 axis plays a crucial role in the angiogenic process required for fibrosis development, which may represent an effective therapeutic strategy for liver fibrosis.

Topics: Adult; Aged; Angiopoietin-1; Animals; Female; Hepatic Stellate Cells; Humans; Liver; Liver Cirrhosis; Liver Cirrhosis, Experimental; Lysophospholipids; Male; Mice; Mice, Inbred ICR; Middle Aged; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

We investigated the hypothesis that postconditioning by FTY720 (FTY) in isolated perfused mouse hearts is independent of the sphingosine 1-phosphate (S1P) pathway.. Ex vivo hearts were exposed to postconditioning (POST) by either ischemia or FTY720. Protection against ischemia/reperfusion (IR) injury was measured by recovery of left ventricular developed pressure (LVDP) and infarct size.. FTY effectively postconditioned (POST) ex vivo hearts against ischemia/reperfusion (IR) injury as measured by recovery of LVDP and a low infarct size. FTY protection, unlike S1P but like sphingosine (Sph), was insensitive to inhibition of S1P G-Protein Coupled Receptors (GPCRs) or inhibition of PI3 kinase. Protection by FTY and Sph was however blocked by inhibitors of PKA and PKG. Thus, FTY follows the same cardioprotective pathway as Sph. This was further supported by studies of FTY POST in knockout (KO) mice lacking the SphK2 form of Sph kinase that is needed for phosphorylation of FTY to an S1P analog. In the absence of SphK2, FTY (and Sph) POST was still cardioprotective. This differed from the effect of SphK2 KO on protection by ischemic POST (IPOST). IPOST was not effective in KO hearts. To see if the GPCR signaling pathway to protection is normal in KO hearts, we looked at POST by GPCR agonists S1P and adenosine. Both provided effective protection even in KO hearts suggesting that the problem with IPOST in KO hearts is a low level of S1P available for release during IPOST. Thus, pharmacologic POST with FTY or Sph, like adenosine and S1P, is unaffected in the KO.. FTY720 administered in vivo might behave in a dual manner showing both S1P-like effects and sphingosine-like effects. It appears that the latter may have been overlooked and may be the more important in aging hearts.

Topics: Adenosine; Algorithms; Animals; Fingolimod Hydrochloride; Immunosuppressive Agents; In Vitro Techniques; Ischemic Postconditioning; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Perfusion; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Reperfusion Injury; Sphingosine

Ezrin, radixin, and moesin (ERM) proteins link cortical actin to the plasma membrane and coordinate cellular events that require cytoskeletal rearrangement, including cell division, migration, and invasion. While ERM proteins are involved in many important cellular events, the mechanisms regulating their function are not completely understood. Our laboratory previously identified reciprocal roles for the sphingolipids ceramide and sphingosine-1-phosphate (S1P) in the regulation of ERM proteins. We recently showed that ceramide-induced activation of PP1α leads to dephosphorylation and inactivation of ERM proteins, while S1P results in phosphorylation and activation of ERM proteins. Following these findings, we aimed to examine known inducers of the SK/S1P pathway and evaluate their ability to regulate ERM proteins. We examined EGF, a known inducer of the SK/S1P pathway, for its ability to regulate the ERM family of proteins. We found that EGF induces ERM c-terminal threonine phosphorylation via activation of the SK/S1P pathway, as this was prevented by siRNA knockdown or pharmacological inhibition of SK. Using pharmacological, as well as genetic, knockdown approaches, we determined that EGF induces ERM phosphorylation via activation of S1PR2. In addition, EGF led to cell polarization in the form of lamellipodia, and this occurred through a mechanism involving S1PR2-mediated phosphorylation of ezrin T567. EGF-induced cellular invasion was also found to be dependent on S1PR2-induced T567 ezrin phosphorylation, such that S1PR2 antagonist, JTE-013, and expression of a dominant-negative ezrin mutant prevented cellular invasion toward EGF. In this work, a novel mechanism of EGF-stimulated invasion is unveiled, whereby S1P-mediated activation of S1PR2 and phosphorylation of ezrin T567 is required.

Topics: Cell Movement; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Epidermal Growth Factor; HeLa Cells; Humans; Immunoblotting; Lysophospholipids; Microscopy, Confocal; Mutation; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pyrazoles; Pyridines; Receptors, Lysosphingolipid; RNA Interference; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Stress-inducing agents, including oxidative stress, generate the sphingolipid mediators ceramide (Cer) and sphingosine-1-phosphate (S1P) that are involved in stress-induced cellular responses. The two redox-sensitive neutral sphingomyelinase-2 (nSMase2) and sphingosine kinase-1 (SK1) participate in transducing stress signaling to ceramide and S1P, respectively; however, whether these key enzymes are coordinately regulated is not known. We investigated whether a signaling link coordinates nSMase2 and SK1 activation by H2O2. In mesenchymal cells, H2O2 elicits a dose-dependent biphasic effect, mitogenic at low concentration (5μM), and anti-proliferative and toxic at high concentration (100μM). Low H2O2 concentration triggered activation of nSMase2 and SK1 through a nSMase2/Cer-dependent signaling pathway that acted upstream of activation of SK1. Further results implicated src and the trans-activation of PDGFRβ, as supported by the blocking effect of specific siRNAs, pharmacological inhibitors, and genetically deficient cells for nSMase2, src and SK1. The H2O2-induced src/PDGFRβ/SK1 signaling cascade was impaired in nSMase2-deficient fro/fro cells and was rescued by exogenous C2Cer that activated src/PDGFRβ/SK1. Thus, the results define a nSMase2/SK1 signaling pathway implicated in the mitogenic response to low oxidative stress. On the other hand, high oxidative stress induced inhibition of SK1. The results also showed that the toxicity of high H2O2 concentration was comparable in control and nSMase2-deficient cells. Taken together the results identify a tightly coordinated nSMase2/SK1 pathway that mediates the mitogenic effects of H2O2 and may sense the degree of oxidative stress.

Topics: Animals; Cell Line; Ceramides; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Hydrogen Peroxide; Lysophospholipids; Mice; Mice, Mutant Strains; Oxidants; Oxidative Stress; Phosphotransferases (Alcohol Group Acceptor); Receptor, Platelet-Derived Growth Factor beta; Signal Transduction; Sphingomyelin Phosphodiesterase; Sphingosine; src-Family Kinases

Sphingosine-1-phosphate (S1P) is a cellular signalling lipid generated by sphingosine kinase-1 (SPHK1). The aim of the study was to investigate whether the activated coagulation factor-X (FXa) regulates SPHK1 transcription and the formation of S1P and subsequent mitogenesis and migration of human vascular smooth muscle cells (SMC).. FXa induced a time- (3-6 h) and concentration-dependent (3-30 nmol/L) increase of SPHK1 mRNA and protein expression in human aortic SMC, resulting in an increased synthesis of S1P. FXa-stimulated transcription of SPHK1 was mediated by the protease-activated receptor-1 (PAR-1) and PAR-2. In human carotid artery plaques, expression of SPHK1 was observed at SMC-rich sites and was co-localized with intraplaque FX/FXa content. FXa-induced SPHK1 transcription was attenuated by inhibitors of Rho kinase (Y27632) and by protein kinase C (PKC) isoforms (GF109203X). In addition, FXa rapidly induced the activation of the small GTPase Rho A. Inhibition of signalling pathways which regulate SPHK1 expression, inhibition of its activity or siRNA-mediated SPHK1 knockdown attenuated the mitogenic and chemotactic response of human SMC to FXa.. These data suggest that FXa induces SPHK1 expression and increases S1P formation independent of thrombin and that this involves the activation of Rho A and PKC signalling. In addition to its key function in coagulation, this direct effect of FXa on human SMC may increase cell proliferation and migration at sites of vessel injury and thereby contribute to the progression of vascular lesions.

Topics: Aged; Aged, 80 and over; Carotid Stenosis; Cell Movement; Cells, Cultured; Factor Xa; Gene Knockdown Techniques; Humans; Lysophospholipids; Middle Aged; Mitosis; Myocytes, Smooth Muscle; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Receptor, PAR-1; Receptor, PAR-2; rhoA GTP-Binding Protein; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Sphingosine

Human megakaryocytes (MKs) release trillions of platelets each day into the circulation to maintain normal homeostatic platelet levels. We have previously shown that extracellular sphingosine 1-phosphate (S1P) plays a key role in thrombopoiesis via its receptor S1pr1. In addition to its role as an extracellular mediator, S1P can also function as a second messenger in the intracellular compartment. Although signaling via intracellular S1P is involved in various cellular processes, a role in thrombopoiesis has not been examined. Sphingosine kinases are the key enzymes that produce intracellular S1P. Here we report that sphingosine kinase 2 (Sphk2) is the major messenger RNA species present in MKs. Sphk2 predominantly localizes to the nucleus and is the major source of intracellular S1P in MKs. Loss of Sphk2 significantly reduced intracellular S1P in MKs and downregulated the expression and activity of Src family kinases (SFKs). Loss of Sphk2 and inhibition of SFK activity resulted in defective intravascular proplatelet shedding, the final stage of thrombopoiesis. Correspondingly, mice lacking Sphk2 in the hematopoietic system display thrombocytopenia. Together, our data suggest that Sphk2 provides the source of intracellular S1P that controls thrombopoiesis, which is associated with SFK expression and activity in MKs.

Topics: Animals; Blood Platelets; Cell Differentiation; Cell Survival; Homeostasis; Intracellular Space; Lysophospholipids; Megakaryocytes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; src-Family Kinases; Thrombopoiesis; Thrombopoietin

Sphingosine kinases (SphKs) and their product sphingosine-1-phosphate (S1P) have been reported to regulate apoptosis and survival of liver cells. Cholestatic liver diseases are characterized by cytotoxic levels of bile salts inducing liver injury. It is unknown whether SphKs and/or S1P play a role in this pathogenic process. Here, we investigated the putative involvement of SphK1 and S1P in bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA) to induce apoptosis. GCDCA-exposed hepatocytes were co-treated with S1P, the SphK1 inhibitor Ski-II and/or specific antagonists of S1P receptors (S1PR1 and S1PR2). Apoptosis and necrosis were quantified. Ski-II significantly reduced GCDCA-induced apoptosis in hepatocytes (-70%, P<0.05) without inducing necrosis. GCDCA increased the S1P levels in hepatocytes (P<0.05). GCDCA induced [Ca(2+)] oscillations in hepatocytes and co-treatment with the [Ca(2+)] chelator BAPTA repressed GCDCA-induced apoptosis. Ski-II inhibited the GCDCA-induced intracellular [Ca(2+)] oscillations. Transcripts of all five S1P receptors were detected in hepatocytes, of which S1PR1 and S1PR2 appear most dominant. Inhibition of S1PR1, but not S1PR2, reduced GCDCA-induced apoptosis by 20%. Exogenous S1P also significantly reduced GCDCA-induced apoptosis (-50%, P<0.05), however, in contrast to the GCDCA-induced (intracellular) SphK1 pathway, this was dependent on S1PR2 and not S1PR1. Our results indicate that SphK1 plays a pivotal role in mediating bile salt-induced apoptosis in hepatocytes in part by interfering with intracellular [Ca(2+)] signaling and activation of S1PR1.

Topics: Animals; Apoptosis; Bile Acids and Salts; Blotting, Western; Caspase 3; Cells, Cultured; Detergents; Gastrointestinal Agents; Glycochenodeoxycholic Acid; Hepatocytes; Lysophospholipids; Male; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sphingosine; Thiazoles

Glioblastomas are the most frequent and aggressive intracranial neoplasms in humans, and despite advances and the introduction of the alkylating agent temozolomide in therapy have improved patient survival, resistance mechanisms limit benefits. Recent studies support that glioblastoma stem-like cells (GSCs), a cell subpopulation within the tumour, are involved in the aberrant expansion and therapy resistance properties of glioblastomas, through still unclear mechanisms. Emerging evidence suggests that sphingosine-1-phosphate (S1P) a potent onco-promoter able to act as extracellular signal, favours malignant and chemoresistance properties in GSCs. Notwithstanding, the origin of S1P in the GSC environment remains unknown. We investigated S1P metabolism, release, and role in cell survival properties of GSCs isolated from either U87-MG cell line or a primary culture of human glioblastoma. We show that both GSC models, grown as neurospheres and expressing GSC markers, are resistant to temozolomide, despite not expressing the DNA repair protein MGMT, a major contributor to temozolomide-resistance. Pulse experiments with labelled sphingosine revealed that both GSC types are able to rapidly phosphorylate the long-chain base, and that the newly produced S1P is efficiently degraded. Of relevance, we found that S1P was present in GSC extracellular medium, its level being significantly higher than in U87-MG cells, and that the extracellular/intracellular ratio of S1P was about ten-fold higher in GSCs. The activity of sphingosine kinases was undetectable in GSC media, suggesting that mechanisms of S1P transport to the extracellular environment are constitutive in GSCs. In addition we found that an inhibitor of S1P biosynthesis made GSCs sensitive to temozolomide (TMZ), and that exogenous S1P reverted this effect, thus involving extracellular S1P as a GSC survival signal in TMZ resistance. Altogether our data implicate for the first time GSCs as a pivotal source of extracellular S1P, which might act as an autocrine/paracrine signal contributing to their malignant properties.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Separation; Cell Survival; Dacarbazine; Drug Resistance, Neoplasm; Extracellular Space; Glioblastoma; Humans; Intracellular Space; Lysophospholipids; Models, Biological; Neoplastic Stem Cells; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Temozolomide

The hepatic growth factor hepatopoietin Cn (HPPCn) prevents liver injury induced by carbon tetrachloride in rats. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid produced by sphingosine kinase (SphK). S1P and S1P receptors (S1PRs) are involved in liver fibrogenesis and oxidative injury. This work sought to understand the mechanism by which SphK/S1P/S1PRs are involved in the protective effects of HPPCn on ethanol-induced liver injury and fibrosis. Transgenic mice with liver-specific overexpression of HPPCn (HPPCn(liver) (+/+)) were generated. Two ethanol feeding protocols were used to assess the protective effect of HPPCn on acute and chronic liver injury in mice. Specific inhibitors of S1PR1, S1PR2 and S1PR3 and siRNA were used to examine the roles of S1PRs in hepatic stellate cell (HSC) activation and hepatocyte apoptosis. Increased HPPCn expression in transgenic mice attenuated fibrosis induced by ethanol and carbon tetrachloride (CCl4). Treatment with recombinant human HPPCn prevented human hepatocyte apoptosis and HSC activation. JTE-013 or S1PR2-siRNA attenuated the effect of HPPCn on HSC activation induced by tumour necrosis factor-α (TNF-α). Consistent with the effect of N,N-dimethylsphingosine (DMS), suramin or S1PR3-siRNA treatment blocked HPPCn-induced Erk1/2 phosphorylation in human hepatocytes. This study demonstrated that HPPCn attenuated oxidative injury and fibrosis induced by ethanol feeding and that the SphK1/S1P/S1PRs signalling pathway contributes to the protective effect of HPPCn on hepatocyte apoptosis and HSC activation.

Topics: Animals; Apoptosis; Cells, Cultured; Disease Models, Animal; Ethanol; Gene Expression Regulation; Hepatic Stellate Cells; Hepatocyte Growth Factor; Humans; Liver; Liver Cirrhosis, Alcoholic; Lysophospholipids; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nuclear Proteins; Oxidative Stress; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA Interference; RNA, Messenger; Signal Transduction; Sphingosine; Time Factors; Transfection; Tumor Necrosis Factor-alpha

Sphingosine 1-phosphate (S1P), a product of sphingomyelin metabolism, is generated via phosphorylation of sphingosine by sphingosine kinases (SphK). It acts via a family of G protein-coupled receptors or as an intracellular second messenger for agonists acting through the S1P receptors (S1P1-5). In our study, the expression of SphK1 and S1P1 was identified by immunohistochemistry and immunoblot. The concentration of S1P was measured using ELISA. The spontaneous contraction of isolated fallopian tube strips was determined by tension recording. Our results showed that SphK1 and S1P1 were localized in the fallopian tube epithelial cells. In addition, smooth muscle cells also contained S1P1. Compared with the intrauterine pregnancy group, SPHK1 and S1P1 were overexpressed in ectopic pregnancy. However, the S1P concentration within the human oviduct from ectopic pregnancy subjects was largely reduced than that from normal pregnancy subject. The results from tension recording indicated that exogenous and intracellularly generated S1P can regulate the spontaneous contraction of oviduct isolated from rats and human. In conclusion, the sphingolipid metabolism signal pathway functionally existed in the human fallopian tube. Aberrant sphingolipid metabolism in the human fallopian tube may be involved in ectopic pregnancy.

Topics: Animals; Blotting, Western; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Fallopian Tubes; Female; Gene Expression Regulation; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Pregnancy; Pregnancy, Ectopic; Rats; Signal Transduction; Sphingolipids; Sphingosine; Up-Regulation

To test the role of sphingosine-1-phosphate (S1P) signaling system in the in vivo setting of resuscitation and survival after cardiac arrest.. A mouse model of potassium-induced cardiac arrest and resuscitation was used to test the importance of S1P homeostasis in resuscitation and survival. C57BL/6 and sphingosine kinase-1 knockout (SphK1-KO) female mice were arrested for 8 min then subjected to 5 minute CPR with epinephrine bolus given at 90s after the beginning of CPR. Animal survival was monitored for 4h post-resuscitation. Upregulation of tissue and circulatory S1P levels were achieved via inhibition of S1P lyase by 2-acetyl-5-tetrahydroxybutyl imidazole (THI). Plasma and heart tissue S1P and ceramide levels were quantified by targeted ESI-LC/MS/MS.. Lack of SphK1 and low tissue/circulatory S1P levels in SphK1-KO mice led to poor animal resuscitation after cardiac arrest and to impaired survival post-resuscitation. Inhibition of S1P lyase in SphK1-KO mice drastically improved animal resuscitation and survival. Improved resuscitation and survival of THI-treated SphK1-KO mice were better correlated with cardiac dihydro-S1P (DHS1P) than S1P levels. The lack of SphK1 and the inhibition of S1P lyase by THI were accompanied by modulation in cardiac S1PR1 and S1PR2 expression and by selective changes in plasma N-palmitoyl- and N-behenoyl-ceramide levels.. Our data provide evidence for the crucial role for SphK1 and S1P signaling system in resuscitation and survival after cardiac arrest, which may form the basis for development of novel therapeutic strategy to support resuscitation and long-term survival of cardiac arrest patients.

Topics: Aldehyde-Lyases; Animals; Cardiopulmonary Resuscitation; Ceramides; Chromatography, Liquid; Disease Models, Animal; Female; Gene Expression Regulation; Heart Arrest; Imidazoles; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Spectrometry, Mass, Electrospray Ionization; Sphingosine; Sphingosine-1-Phosphate Receptors; Survival Rate; Tandem Mass Spectrometry

Bronchopulmonary dysplasia of the premature newborn is characterized by lung injury, resulting in alveolar simplification and reduced pulmonary function. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues; however, the role of increased S1P in the pathobiological characteristics of bronchopulmonary dysplasia has not been investigated. We hypothesized that an altered S1P signaling axis, in part, is responsible for neonatal lung injury leading to bronchopulmonary dysplasia. To validate this hypothesis, newborn wild-type, sphingosine kinase1(-/-) (Sphk1(-/-)), sphingosine kinase 2(-/-) (Sphk2(-/-)), and S1P lyase(+/-) (Sgpl1(+/-)) mice were exposed to hyperoxia (75%) from postnatal day 1 to 7. Sphk1(-/-), but not Sphk2(-/-) or Sgpl1(+/-), mice offered protection against hyperoxia-induced lung injury, with improved alveolarization and alveolar integrity compared with wild type. Furthermore, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells showed that exogenous S1P stimulated intracellular reactive oxygen species (ROS) generation, whereas SphK1 siRNA, or inhibitor against SphK1, attenuated hyperoxia-induced S1P generation. Knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling-regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of bronchopulmonary dysplasia.

Topics: Aldehyde-Lyases; Animals; Animals, Newborn; Bronchopulmonary Dysplasia; Disease Models, Animal; Down-Regulation; Endothelial Cells; Humans; Hyperoxia; Lysophospholipids; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidases; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Pulmonary Alveoli; rac1 GTP-Binding Protein; Reactive Oxygen Species; Signal Transduction; Sphingosine

Sphingosine kinase is a key enzyme in sphingolipid metabolism, catalysing the conversion of sphingosine or dihydrosphingosine into sphingosine-1-phosphate or dihydrosphingosine-1-phosphate respectively. In mammals, sphingosine-1-phosphate is a powerful signalling molecule regulating cell growth, differentiation, apoptosis and immunity. Functions of sphingosine kinase or sphingosine-1-phosphate in pathogenic protozoans are virtually unknown. While most organisms possess two closely related sphingosine kinases, only one sphingosine kinase homologue (SKa) can be identified in Leishmania, which are vector-borne protozoan parasites responsible for leishmaniasis. Leishmania SKa is a large, cytoplasmic enzyme capable of phosphorylating both sphingosine and dihydrosphingosine. Remarkably, deletion of SKa leads to catastrophic defects in both the insect stage and mammalian stage of Leishmania parasites. Genetic and biochemical analyses demonstrate that proper expression of SKa is essential for Leishmania parasites to remove toxic metabolites, to survive stressful conditions, and to cause disease in mice. Therefore, SKa is a pleiotropic enzyme with vital roles throughout the life cycle of Leishmania. The essentiality of SKa and its apparent divergence from mammalian counterparts suggests that this enzyme can be selectively targeted to reduce Leishmania infection.

Topics: Animals; Antifungal Agents; DNA Replication; DNA, Protozoan; Ethanolamine; Fatty Acids, Monounsaturated; Genes, Protozoan; Leishmania; Leishmaniasis; Lysophospholipids; Mice; Mice, Inbred BALB C; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Virulence

Angiogenesis is critical for many physiological and pathological processes. To identify molecules relevant to angiogenesis, we performed a proteomic screen comparing invading versus non-invading endothelial cells in three-dimensional collagen matrices. We found up-regulated levels of receptor for activated C kinase 1 (RACK1) and the intermediate filament protein vimentin that correlated with increased endothelial cell invasion. Because both RACK1 and vimentin have been linked to focal adhesion kinase (FAK), we investigated whether this pathway regulated invasion. RACK1 depletion reduced invasion responses, and this was associated with attenuated activation of FAK. Knockdown of vimentin significantly decreased levels of phosphorylated and total FAK. Treatment with a pharmacological inhibitor of FAK dose-dependently reduced invasion, indicating a crucial role for FAK activity during invasion. Because RACK1 and vimentin were both up-regulated with sphingosine 1-phosphate treatment, required for invasion, and regulated FAK, we tested whether they complexed together. RACK1 complexed with vimentin, and growth factors enhanced this interaction. In addition, RACK1, vimentin, and FAK formed an intermolecular complex in invading endothelial cultures in three dimensions in response to stimulation by sphingosine 1-phosphate and growth factors. Moreover, depletion of RACK1 decreased the association of vimentin and FAK, suggesting that RACK1 was required for stabilizing vimentin-FAK interactions during sprouting. Silencing of vimentin and RACK1 decreased cell adhesion and focal contact formation. Taken together, these results demonstrate that proangiogenic signals converge to enhance expression and association of RACK1 and vimentin, which regulated FAK, resulting in successful endothelial sprout formation in three-dimensional collagen matrices.

Topics: Collagen; Enzyme Activation; Extracellular Matrix; Focal Adhesion Kinase 1; Gene Expression Regulation, Enzymologic; Gene Knockdown Techniques; GTP-Binding Proteins; Human Umbilical Vein Endothelial Cells; Humans; Lysophospholipids; Multiprotein Complexes; Neoplasm Proteins; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Protein Stability; Proteomics; Receptors for Activated C Kinase; Receptors, Cell Surface; Signal Transduction; Sphingosine; Up-Regulation; Vimentin

Sphingosine kinases (Sphks), which catalyze the formation of sphingosine 1-phosphate (S1P) from sphingosine, have been implicated as essential intracellular messengers in inflammatory responses. Specifically, intracellular Sphk1-derived S1P was reported to be required for NFκB induction during inflammatory cytokine action. To examine the role of intracellular S1P in the inflammatory response of innate immune cells, we derived murine macrophages that lack both Sphk1 and Sphk2 (MΦ Sphk dKO). Compared with WT counterparts, MΦ Sphk dKO cells showed marked suppression of intracellular S1P levels whereas sphingosine and ceramide levels were strongly up-regulated. Cellular proliferation and apoptosis were similar in MΦ Sphk dKO cells compared with WT counterparts. Treatment of WT and MΦ Sphk dKO with inflammatory mediators TNFα or Escherichia coli LPS resulted in similar NFκB activation and cytokine expression. Furthermore, LPS-induced inflammatory responses, mortality, and thioglycolate-induced macrophage recruitment to the peritoneum were indistinguishable between MΦ Sphk dKO and littermate control mice. Interestingly, autophagic markers were constitutively induced in bone marrow-derived macrophages from Sphk dKO mice. Treatment with exogenous sphingosine further enhanced intracellular sphingolipid levels and autophagosomes. Inhibition of autophagy resulted in caspase-dependent cell death. Together, these data suggest that attenuation of Sphk activity, particularly Sphk2, leads to increased intracellular sphingolipids and autophagy in macrophages.

Topics: Animals; Autophagy; Caspases; Inflammation; Lipopolysaccharides; Lysophospholipids; Macrophages, Peritoneal; Mice; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

SphK1/S1P signaling pathway is involved in the development of hepatic inflammation and injury. But its role in high fructose-induced NAFLD has not yet been reported. The aim of this study was to elucidate the crucial role of SphK1/S1P signaling pathway in high fructose-induced hepatic inflammation and lipid accumulation in rats. Moreover, the hepatoprotective effects of morin, a flavonoid with anti-inflammatory and anti-hyperlipedimic activities, on these hepatic changes in rats were investigated. High fructose-fed rats were orally treated with morin (30 and 60mg/kg) and pioglitazone (4mg/kg) for 8 weeks, respectively. Fructose feeding induced hyperlipidemia, and activated SphK1/S1P signaling pathway characterized by the elevation of SphK1 activity, S1P production as well as SphK1, S1PR1 and S1PR3 protein levels, which in turn caused NF-κB signaling activation to produce IL-1β, IL-6 and TNF-α and inflammation in the liver of rats. Subsequently, hepatic insulin and leptin signaling impairment and lipid metabolic disorder were observed in this animal model, resulting in liver lipid accumulation. Morin restored high fructose-induced the activation of hepatic SphK1/S1P signaling pathway in rats. Subsequently, the reduced NF-κB signaling activation by morin decreased inflammatory cytokine production, recovered insulin and leptin signaling impairment to reduce lipid accumulation and injury in the rat liver. These effects of morin were confirmed in Buffalo rat liver (BRL3A) cell model stimulated with 5mM fructose. Thus, the inhibition of hepatic SphK1/S1P signaling pathway may be a novel mechanism by which morin exerts hepatoprotection in high fructose-fed rats, possibly involving liver inflammation inhibition and lipid accumulation recovery.

Topics: Animals; Dietary Carbohydrates; Flavonoids; Fructose; Inflammation; Lipid Metabolism; Liver; Lysophospholipids; Male; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Signal Transduction; Sphingosine

Concentrations of plasminogen activator inhibitor-1 (PAI-1) are increased in obese individuals. One source of PAI-1 is adipocytes. Hypoxia develops within adipose tissue as it expands, presumably contributing to increased levels of sphingosine-1-phosphate (S1P). S1P is a breakdown product of sphingosine, ubiquitous in cell membranes. We have shown previously that S1P increases the expression of PAI-1 in human liver-derived cell line. In the present study, we aimed to determine whether hypoxia induces S1P in adipocytes, thereby potentially contributing to an increase in PAI-1 and hence constraints on fibrinolysis associated with obesity.. Mouse 3T3-L1 adipocytes were exposed to CoCl2 to simulate hypoxia. Assays were performed for PAI-1 mRNA (quantitative PCR) and S1P (high-performance liquid chromatography).. The physiologic concentration of S1P increased PAI-1 mRNA expression. The S1P2 receptor antagonist attenuated the increase in PAI-1. Adipocytes expressed sphingosine kinase 1/2 (SPHK1/2) and S1P lyase, key enzymes involved in S1P production and degradation. Hypoxia increased SPHK activity and decreased S1P lyase mRNA. Hypoxia reduced cytosolic sphingosine and increased S1P release into conditioned medium. Inhibitors of ABCA1 and ABCC1 reduced the release of S1P into conditioned media. In obese patients with uncomplicated dyslipidemia and hypertension, plasma S1P was increased compared with that in nonobese and lean individuals.. Hypoxia in adipose tissue of obesity can promote elaboration of S1P that binds to S1P2 receptors in an autocrine or a paracrine manner. S1P potentially contributes toward increased expression of PAI-1 and consequent constraints on fibrinolysis. S1P production and extracellular transport provide an attractive target for therapy to attenuate impaired fibrinolysis associated with obesity.

Topics: 3T3-L1 Cells; Adipocytes; Aged; Aldehyde-Lyases; Animals; ATP Binding Cassette Transporter 1; Body Mass Index; Cell Hypoxia; Culture Media, Conditioned; Female; Humans; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Middle Aged; Multidrug Resistance-Associated Proteins; Obesity; Phosphotransferases (Alcohol Group Acceptor); Plasminogen Activator Inhibitor 1; Receptors, Lysosphingolipid; RNA, Messenger; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Time Factors; Up-Regulation

Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the mechanisms underlying this effect are still unknown. In this study, the ability of insulin to protect apoptotic cardiomyocytes from hypoxia/reoxygenation injury using the sphingosine kinase/sphingosine 1-phosphate axis was investigated.. Rat cardiomyocytes were isolated and subjected to hypoxia and reoxygenation. [γ-32P] ATP was used to assess sphingosine kinase activity. Insulin was found to increase sphingosine kinase activity. Immunocytochemistry and Western blot analysis showed changes in the subcellular location of sphingosine kinase 1 from cytosol to the membrane in cardiomyocytes. Insulin caused cardiomyocytes to accumulate of S1P in a dose-dependent manner. FRET efficiency showed that insulin also transactivates the S1P1 receptor. TUNEL staining showed that administration of insulin during reoxygenation could to reduce the rate of reoxygenation-induced apoptosis, which is a requirement for SphK 1 activity. It also reduced the rate of activation of the S1P receptor and inhibited hypoxia/reoxygenation-induced cell death in cardiomyocytes.. The sphingosine kinase 1/sphingosine 1-phosphate/S1P receptor axis is one pathway through which insulin protects rat cardiomyocytes from apoptosis induced by hypoxia/reoxygenation injury.

Topics: Animals; Cell Hypoxia; Cells, Cultured; In Situ Nick-End Labeling; Insulin; Lysophospholipids; Myocardial Reperfusion Injury; Myocytes, Cardiac; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptors, Lysosphingolipid; Sphingosine

Sphingosine kinase is an enzyme that catalyses the phosphorylation of sphingosine to form sphingosine 1-phosphate. Sphingosine 1-phosphate is a bioactive lipid, which has been shown to have an important role in promoting the survival, growth and invasiveness of cancer cells. Sphingosine 1-phosphate binds to five different plasma membrane sphingosine 1-phosphate receptors (S1P(1-5) ) and can regulate intracellular target proteins. We have used immunohistochemical analysis to determine the concurrent expression levels of sphingosine kinase 1 or S1P receptors and other signaling proteins in estrogen receptor-positive breast cancer tumors and have then assessed the impact of these combinations on clinical outcome. This approach has enabled identification of (i) novel biomarkers and (ii) several spatially controlled associations between either sphingosine kinase 1 or S1P(1-3) and other signaling proteins whose combination affect prognosis. For instance, the translocation of sphingosine kinase 1 to the plasma membrane has been shown to be a critical determinant in cancer progression. However, our findings identify an additional novel role for the nuclear localization of sphingosine kinase 1 combined with either ERK-1/2 or SFK or LYN or AKT or NF-κB, which significantly shortens disease-specific survival and/or recurrence. We also demonstrate that nuclear S1P(2) receptor and c-SRC are associated with improved prognosis and this is linked with a reduction in the nuclear localization of sphingosine kinase 1. These findings identify potential novel biomarker associations, which might serve as new targets for drug intervention designed to improve treatment of estrogen receptor-positive breast cancer.

Topics: Aged; Biomarkers, Tumor; Breast Neoplasms; Cell Membrane; Cell Nucleus; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Lysophospholipids; Middle Aged; NF-kappa B; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; src-Family Kinases; Tamoxifen

Sphingosine-1-phosphate (S1P), which is produced by 2 sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution, and the role that S1P plays in vivo in a mast cell- and IgE-dependent murine model of allergic asthma has not yet been examined.. We used an isoenzyme-specific SphK1 inhibitor, SK1-I, to investigate the contributions of S1P and SphK1 to mast cell-dependent airway hyperresponsiveness (AHR) and airway inflammation in mice.. Allergic airway inflammation and AHR were examined in a mast cell-dependent murine model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I before sensitization and challenge with OVA or only before challenge.. SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor κB (NF-κB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell-dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-13, IFN-γ, and TNF-α and the chemokines eotaxin and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation, as well as activation of NF-κB in the lungs.. S1P and SphK1 play important roles in mast cell-dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway.

Topics: Amino Alcohols; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cells, Cultured; Chemokine CCL2; Female; Goblet Cells; Humans; Hyperplasia; Immunoglobulin E; Inflammation; Interferon-gamma; Interleukins; Lung; Lysophospholipids; Mast Cells; Methacholine Chloride; Mice; Mice, Inbred C57BL; NF-kappa B; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tumor Necrosis Factor-alpha

Curcumin, a major polyphenol from the golden spice Curcuma longa commonly known as turmeric, has been recently discovered to have renoprotective effects on diabetic nephropathy (DN). However, the mechanisms underlying these effects remain unclear. We previously demonstrated that the sphingosine kinase 1-sphingosine 1-phosphate (SphK1-S1P) signaling pathway plays a pivotal role in the pathogenesis of DN. This study aims to investigate whether the renoprotective effects of curcumin on DN are associated with its inhibitory effects on the SphK1-S1P signaling pathway. Our results demonstrated that the expression and activity of SphK1 and the production of S1P were significantly down-regulated by curcumin in diabetic rat kidneys and glomerular mesangial cells (GMCs) exposed to high glucose (HG). Simultaneously, SphK1-S1P-mediated fibronectin (FN) and transforming growth factor-beta 1 (TGF-β1) overproduction were inhibited. In addition, curcumin dose dependently reduced SphK1 expression and activity in GMCs transfected with SphK(WT) and significantly suppressed the increase in SphK1-mediated FN levels. Furthermore, curcumin inhibited the DNA-binding activity of activator protein 1 (AP-1), and c-Jun small interference RNA (c-Jun-siRNA) reversed the HG-induced up-regulation of SphK1. These findings suggested that down-regulation of the SphK1-S1P pathway is probably a novel mechanism by which curcumin improves the progression of DN. Inhibiting AP-1 activation is one of the therapeutic targets of curcumin to modulate the SphK1-S1P signaling pathway, thereby preventing diabetic renal fibrosis.

Topics: Animals; Antioxidants; Cells, Cultured; Curcumin; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Drug Evaluation, Preclinical; Fibronectins; Gene Expression; Glucose; JNK Mitogen-Activated Protein Kinases; Kidney; Lysophospholipids; Male; Mesangial Cells; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Signal Transduction; Sphingosine; Transcription Factor AP-1; Transforming Growth Factor beta1

Inflammatory bowel disease is an important risk factor for colorectal cancer. We show that sphingosine-1-phosphate (S1P) produced by upregulation of sphingosine kinase 1 (SphK1) links chronic intestinal inflammation to colitis-associated cancer (CAC) and both are exacerbated by deletion of Sphk2. S1P is essential for production of the multifunctional NF-κB-regulated cytokine IL-6, persistent activation of the transcription factor STAT3, and consequent upregulation of the S1P receptor, S1PR1. The prodrug FTY720 decreased SphK1 and S1PR1 expression and eliminated the NF-κB/IL-6/STAT3 amplification cascade and development of CAC, even in Sphk2(-/-) mice, and may be useful in treating colon cancer in individuals with ulcerative colitis. Thus, the SphK1/S1P/S1PR1 axis is at the nexus between NF-κB and STAT3 and connects chronic inflammation and CAC.

Topics: Animals; Cell Transformation, Neoplastic; Colitis; Colon; Fingolimod Hydrochloride; Gene Deletion; Gene Expression Regulation, Neoplastic; Interleukin-6; Lysophospholipids; Mice; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Sphingosine; STAT3 Transcription Factor; Tumor Microenvironment

Two isoforms of sphingosine kinase, SK1 and SK2, catalyze the formation of the bioactive lipid sphingosine 1-phosphate (S1P) in mammalian cells. We have previously shown that treatment of androgen-sensitive LNCaP prostate cancer cells with a non-selective SK isoform inhibitor, 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi), induces the proteasomal degradation of SK1. This is concomitant with a significant increase in C22:0-ceramide and sphingosine levels and a reduction in S1P levels, resulting in the apoptosis of LNCaP cells. In contrast, we show here that a SK2-selective inhibitor, (R)-FTY720 methyl ether (ROME), increases sphingosine and decreases S1P levels but has no effect on ceramide levels and does not induce apoptosis in LNCaP cells. We also show that several glycolytic metabolites and (R)-S-lactoylglutathione are increased upon treatment of LNCaP cells with SKi, which induces the proteasomal degradation of c-Myc. These changes reflect an indirect antagonism of the Warburg effect. LNCaP cells also respond to SKi by diverting glucose 6-phosphate into the pentose phosphate pathway to provide NADPH, which serves as an antioxidant to counter an oxidative stress response. SKi also promotes the formation of a novel pro-apoptotic molecule called diadenosine 5',5'''-P(1),P(3)-triphosphate (Ap3A), which binds to the tumor suppressor fragile histidine triad protein (FHIT). In contrast, the SK2-selective inhibitor, ROME, induces a reduction in some glycolytic metabolites and does not affect oxidative stress. We conclude that SK1 functions to increase the stability of c-Myc and suppresses Ap3A formation, which might maintain the Warburg effect and cell survival, while SK2 exhibits a non-overlapping function.

Topics: Cell Line, Tumor; Ceramides; Cyclin D1; Dinucleoside Phosphates; Enzyme Inhibitors; Glycolysis; Humans; Lysophospholipids; Male; Metabolome; Microtubule-Associated Proteins; Oxidative Stress; Phosphotransferases (Alcohol Group Acceptor); Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; Sphingosine

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease, wherein transforming growth factor β (TGF-β) and sphingosine-1-phosphate (S1P) contribute to the pathogenesis of fibrosis. However, the in vivo contribution of sphingosine kinase (SphK) in fibrotic processes has not been documented. Microarray analysis of blood mononuclear cells from patients with IPF and SphK1- or SphK2-knockdown mice and SphK inhibitor were used to assess the role of SphKs in fibrogenesis. The expression of SphK1/2 negatively correlated with lung function and survival in patients with IPF. Also, the expression of SphK1 was increased in lung tissues from patients with IPF and bleomycin-challenged mice. Knockdown of SphK1, but not SphK2, increased survival and resistance to pulmonary fibrosis in bleomycin-challenged mice. Administration of SphK inhibitor reduced bleomycin-induced mortality and pulmonary fibrosis in mice. Knockdown of SphK1 or treatment with SphK inhibitor attenuated S1P generation and TGF-β secretion in a bleomycin-induced lung fibrosis mouse model that was accompanied by reduced phosphorylation of Smad2 and MAPKs in lung tissue. In vitro, bleomycin-induced expression of SphK1 in lung fibroblast was found to be TGF-β dependent. Taken together, these data indicate that SphK1 plays a critical role in the pathology of lung fibrosis and is a novel therapeutic target.

Topics: Aged; Animals; Bleomycin; Female; Gene Knockdown Techniques; Humans; Idiopathic Pulmonary Fibrosis; Lung; Lysophospholipids; Male; Mice; Mice, Knockout; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Transforming Growth Factor beta

Sphingolipids play a role in the development of emphysema and ceramide levels are increased in experimental models of emphysema; however, the mechanisms of ceramide-related pulmonary emphysema are not fully understood. Here we examine mechanisms of ceramide-induced pulmonary emphysema. Male Sprague-Dawley rats were treated with fenretinide (20 mg/kg BW), a synthetic derivative of retinoic acid that causes the formation of ceramide, and we postulated that the effects of fenretinide could be offset by administering sphingosine 1-phosphate (S1P) (100 µg/kg BW). Lung tissues were analyzed and mean alveolar airspace area, total length of the alveolar perimeter and the number of caspase-3 positive cells were measured. Hypoxia-inducible factor alpha (HIF-1α), vascular endothelial growth factor (VEGF) and other related proteins were analyzed by Western blot analysis. Immunohistochemical analysis of HIF-1α was also performed. Ceramide, dihydroceramide, S1P, and dihydro-S1P were measured by mass spectrometer. Chronic intraperitoneal injection of fenretinide increased the alveolar airspace surface area and increased the number of caspase-3 positive cells in rat lungs. Fenretinide also suppressed HIF-1α and VEGF protein expression in rat lungs. Concomitant injection of S1P prevented the decrease in the expression of HIF-1α, VEGF, histone deacetylase 2 (HDAC2), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein expression in the lungs. S1P injection also increased phosphorylated sphingosine kinase 1. Dihydroceramide was significantly increased by fenretinide injection and S1P treatment prevented the increase in dihydroceramide levels in rat lungs. These data support the concept that increased de novo ceramide production causes alveolar septal cell apoptosis and causes emphysema via suppressing HIF-1α. Concomitant treatment with S1P normalizes the ceramide-S1P balance in the rat lungs and increases HIF-1α protein expression via activation of sphingosine kinase 1; as a consequence, S1P salvages fenretinide induced emphysema in rat lungs.

Topics: Animals; Caspase 3; Ceramides; Emphysema; Fenretinide; Gene Expression Regulation; Histone Deacetylase 2; Humans; Hypoxia-Inducible Factor 1; Lysophospholipids; Male; NF-E2-Related Factor 2; Phosphotransferases (Alcohol Group Acceptor); Protective Agents; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Signal Transduction; Sphingosine; Vascular Endothelial Growth Factor A

FcεRI engagement in mast cells (MCs) induces the activation of two distinct sphingosine kinase isoforms (SphK1 and SphK2) to produce sphingosine-1-phosphate, a mediator essential for MC responses. Whereas embryonic-derived SphK2-null MCs showed impaired responses to Ag, RNA silencing studies on other MC types indicated a dominant role for SphK1. Given the known functional heterogeneity of MCs, we explored whether the reported differences in SphK1 or SphK2 usage could be reflective of phenotypic differences between MC populations. Using lentiviral-based short hairpin RNA to silence SphK1 or SphK2, we found that SphK2 is required for murine MC degranulation, calcium mobilization, and cytokine and leukotriene production, irrespective of the tissue from which the MC progenitors were derived, the stage of MC granule maturity, or the conditions used for differentiation. This finding was consistent with the lack of a full allergic response in SphK2-null mice challenged to undergo passive cutaneous anaphylaxis. A redundant role for both SphKs was uncovered, however, in chemotaxis toward Ag in all MC types tested and in TNF-α production in certain MC types. In contrast, human MC responses were dependent only on SphK1, associating with a more robust expression of this isoform and a more varied representation of SphK variants relative to murine MCs. The findings show that the function of SphK1 and SphK2 can be interchangeable in MCs; however, an important determinant of SphK isoform usage is the species of origin and an influencing factor, the tissue from which MCs may be derived and/or their differentiation state.

Topics: Animals; Calcium; Cell Degranulation; Cell Differentiation; Cells, Cultured; Cytokines; Enzyme Activation; Gene Expression; Gene Silencing; Genetic Vectors; Humans; Isoenzymes; Lentivirus; Leukotrienes; Lysophospholipids; Mast Cells; Mice; Organ Specificity; Phosphotransferases (Alcohol Group Acceptor); Receptors, IgE; RNA, Small Interfering; Signal Transduction; Sphingosine; Transduction, Genetic

Capillary electrophoresis (CE) is one among a number of highly sensitive chemical separation techniques used to characterize single or a small number of cells and to develop assays of enzymatic activity. Other commonly used techniques include mass spectrometry and electrochemistry; however, CE using laser-induced fluorescence detection (LIF) is the most sensitive of these techniques. In CE-LIF, fluorescently labeled proteins or lipids are normally separated based on their size to charge ratio in the interior of a small capillary filled with an electrolyte upon the application of an electric field. In this chapter, we describe the application of CE-LIF for the determination of the bioactivity of fluorescently lipids and sphingosine kinase activity.

Topics: Electrophoresis, Capillary; Enzyme Assays; Fluorescein; Fluorescent Dyes; Humans; Kinetics; Lipids; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Spectrometry, Fluorescence; Sphingosine

The mitochondrial permeability transition (MPT) and inflammation play important roles in liver injury caused by ischemia-reperfusion (IR). This study investigated the roles of sphingosine kinase-2 (SK2) in mitochondrial dysfunction and inflammation after hepatic IR.. Mice were gavaged with vehicle or ABC294640 (50 mg/kg), a selective inhibitor of SK2, 1 h before surgery and subjected to 1 h-warm ischemia to ~70% of the liver followed by reperfusion.. Following IR, hepatic SK2 mRNA and sphingosine-1-phosphate (S1P) levels increased ~25- and 3-fold, respectively. SK2 inhibition blunted S1P production and liver injury by 54-91%, and increased mouse survival from 28% to 100%. At 2 h after reperfusion, mitochondrial depolarization was observed in 74% of viable hepatocytes, and mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. SK2 inhibition decreased mitochondrial depolarization and prevented MPT onset. Inducible nitric oxide synthase, phosphorylated NFκB-p65, TNFα mRNA, and neutrophil infiltration, all increased markedly after hepatic IR, and these increases were blunted by SK2 inhibition. In cultured hepatocytes, anoxia/re-oxygenation resulted in increases of SK2 mRNA, S1P levels, and cell death. SK2 siRNA and ABC294640 each substantially decreased S1P production and cell death in cultured hepatocytes.. SK2 plays an important role in mitochondrial dysfunction, inflammation responses, hepatocyte death, and survival after hepatic IR and represents a new target for the treatment of IR injury.

Topics: Adamantane; Animals; Cell Death; Enzyme Inhibitors; Gene Knockdown Techniques; Hepatocytes; In Vitro Techniques; Inflammation; Liver; Lysophospholipids; Male; Mice; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Nitric Oxide Synthase Type II; Phosphotransferases (Alcohol Group Acceptor); Pyridines; Reperfusion Injury; RNA, Messenger; RNA, Small Interfering; Sphingosine

Sphingosine-1-phosphate-induced α1B-adrenergic receptor desensitization and phosphorylation were studied in rat-1 fibroblasts stably expressing enhanced green fluorescent protein-tagged adrenoceptors. Sphingosine-1-phosphate induced adrenoceptor desensitization and phosphorylation through a signaling cascade that involved phosphoinositide 3-kinase and protein kinase C activities. The autocrine/paracrine role of sphingosine-1-phosphate was also studied. It was observed that activation of receptor tyrosine kinases, such as insulin growth factor-1 (IGF-I) and epidermal growth factor (EGF) receptors increased sphingosine kinase activity. Such activation and consequent production of sphingosine-1-phosphate appear to be functionally relevant in IGF-I- and EGF-induced α1B-adrenoceptor phosphorylation and desensitization as evidenced by the following facts: a) expression of a catalytically inactive (dominant-negative) mutant of sphingosine kinase 1 or b) S1P1 receptor knockdown markedly reduced this growth factor action. This action of sphingosine-1-phosphate involves EGF receptor transactivation. In addition, taking advantage of the presence of the eGFP tag in the receptor construction, we showed that S1P was capable of inducing α1B-adrenergic receptor internalization and that its autocrine/paracrine generation was relevant for internalization induced by IGF-I. Four distinct hormone receptors and two autocrine/paracrine mediators participate in IGF-I receptor-α1B-adrenergic receptor crosstalk.

Topics: Animals; Autocrine Communication; ErbB Receptors; Fibroblasts; Humans; Lysophospholipids; Paracrine Communication; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptor, IGF Type 1; Receptors, Adrenergic, alpha-1; Receptors, Lysosphingolipid; Sphingosine

CXCL12 and VCAM1 retain hematopoietic stem cells (HSCs) in the BM, but the factors mediating HSC egress from the BM to the blood are not known. The sphingosine-1-phosphate receptor 1 (S1P(1)) is expressed on HSCs, and S1P facilitates the egress of committed hematopoietic progenitors from the BM into the blood. In the present study, we show that both the S1P gradient between the BM and the blood and the expression of S1P(1) are essential for optimal HSC mobilization by CXCR4 antagonists, including AMD3100, and for the trafficking of HSCs during steady-state hematopoiesis. We also demonstrate that the S1P(1) agonist SEW2871 increases AMD3100-induced HSC and progenitor cell mobilization. These results suggest that the combination of a CXCR4 antagonist and a S1P(1) agonist may prove to be sufficient for mobilizing HSCs in normal donors for transplantation purposes, potentially providing a single mobilization procedure and eliminating the need to expose normal donors to G-CSF with its associated side effects.

Topics: Adult; Aged; Animals; Anti-HIV Agents; Benzylamines; Blotting, Western; Cell Movement; Cell Proliferation; Cells, Cultured; Chemokine CXCL12; Cyclams; Cytokines; Drug Combinations; Drug Synergism; Female; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Heterocyclic Compounds; Humans; Immunoenzyme Techniques; Lysophospholipids; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Knockout; Mice, SCID; Mice, Transgenic; Middle Aged; Oligopeptides; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Real-Time Polymerase Chain Reaction; Receptors, CXCR4; Receptors, Lysosphingolipid; RNA, Messenger; Sphingosine; Sphingosine-1-Phosphate Receptors

Idiopathic pulmonary fibrosis (IPF) is characterised by the aberrant epithelial to mesenchymal transition (EMT) and myofibroblast accumulation. Sphingosine-1-phosphate (S1P) and sphingosine kinase 1 (SPHK1) have been implicated in lung myofibroblast transition, but their role in EMT and their expression in patients with IPF is unknown.. S1P levels were measured in serum (n=27) and bronchoalveolar lavage (BAL; n=15) from patients with IPF and controls (n=30 for serum and n=15 for BAL studies). SPHK1 expression was measured in lung tissue from patients with IPF (n=12) and controls (n=15). Alveolar type II transformation into mesenchymal cells was studied in response to S1P (10(-9)-10(-5) M). The median (IQR) of S1P serum levels was increased in patients with IPF (1.4 (0.4) μM) versus controls (1 (0.26) μM; p<0.0001). BAL S1P levels were increased in patients with IPF (1.12 (0.53) μM) versus controls (0.2 (0.5); p<0.0001) and correlated with diffusion capacity of the lung for carbon monoxide, forced expiratory volume in 1 s and forced vital capacity (Spearman's r=-0.87, -0.72 and -0.68, respectively) in patients with IPF. SPHK1 was upregulated in lung tissue from patients with IPF and correlated with α-smooth muscle actin, vimentin and collagen type I (Spearman's r=0.82, 0.85 and 0.72, respectively). S1P induced EMT in alveolar type II cells by interacting with S1P(2) and S1P(3), as well as by the activation of p-Smad3, RhoA-GTP, oxidative stress and transforming growth factor-β1 (TGF-β1) release. Furthermore, TGF-β1-induced EMT was partially conducted by the S1P/SPHK1 activation, suggesting crosstalk between TGF-β1 and the S1P/SPHK1 axis.. S1P is elevated in patients with IPF, correlates with the lung function and mediates EMT.

Topics: Adult; Aged; Aged, 80 and over; Bronchoalveolar Lavage Fluid; Case-Control Studies; Cells, Cultured; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Female; Humans; Idiopathic Pulmonary Fibrosis; Lung; Lysophospholipids; Male; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Alveoli; Reactive Oxygen Species; rhoA GTP-Binding Protein; Smad3 Protein; Sphingosine; Transforming Growth Factor beta1

Sphingosine kinase (SphK) has emerged as an attractive target for cancer therapeutics due to its role in cell survival. SphK phosphorylates sphingosine to form sphingosine 1-phosphate (S1P), which has been implicated in cancer growth and survival. SphK exists as two different isotypes, namely SphK1 and SphK2, which play different roles inside the cell. In this report, we describe SphK inhibitors based on the immunomodulatory drug, FTY720, which is phosphorylated by SphK2 to generate a S1P mimic. Structural modification of FTY720 provided a template for synthesizing new inhibitors. A diversity-oriented synthesis generated a library of SphK inhibitors with a novel scaffold and headgroup. We have discovered subtype selective inhibitors with K(i)'s in the low micromolar range. This is the first report describing quaternary ammonium salts as SphK inhibitors.

Topics: Antineoplastic Agents; Cell Line, Tumor; Drug Design; Enzyme Activation; Enzyme Inhibitors; Fingolimod Hydrochloride; Humans; Kinetics; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Sphingosine; Structure-Activity Relationship

Sphingosine and sphingosine-1-phosphate (S1P) are involved in regulating cell differentiation. This study postulated that changes in sphingolipid biosynthesis and metabolism are important in trophoblast syncytialization and therefore examined the production, metabolism and actions of sphingosine and S1P during spontaneous trophoblast differentiation and fusion in vitro. Significant declines in intracellular sphingosine concentration (P≤0.05) and sphingosine kinase 1 (SPHK1) expression (P≤0.01) were observed during trophoblast syncytialization. Secreted S1P concentrations dropped steeply after 72h, before rising to basal concentrations with syncytialization. Intracellular S1P concentrations were undetectable throughout. Treating cells with exogenous sphingosine (P≤0.01), S1P (P≤0.001) or a specific SPHK1 inhibitor (P≤0.05) for up to 72h in culture significantly inhibited trophoblast differentiation (measured as reduced human chorionic gonadotrophin production); effects on other biochemical and morphological markers of differentiation were absent or inconsistent. Phosphorylation of Akt, an established down-stream target of S1P that spontaneously declines with trophoblast differentiation, was markedly reduced by S1P (P≤0.05). In conclusion, changes in the sphingosine-S1P pathway are involved in the regulation of trophoblast differentiation in term human placenta. Dysregulation of sphingolipid homeostasis could, therefore, disrupt placental formation and function with deleterious consequences for pregnancy outcome.

Topics: Cell Differentiation; Cells, Cultured; Female; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Pregnancy; Proto-Oncogene Proteins c-akt; Sphingosine; Trophoblasts

Ophiopogon japonicus is a traditional Chinese medicine used to treat cardiovascular disease. Recent studies have confirmed the anti-ischemic properties of a water-soluble β-D-fructan (MDG-1) from O. japonicus. The sphingosine 1-phosphate (S1P) signaling pathway is involved in its cytoprotective effects. Herein, we explore the role of the S1P signaling pathway in the anti-ischemic effect of MDG-1 and assess one possible mechanism by which it induces S1P release and sphingosine 1-phosphate receptor 1 (S1P(1)) expression in human microvascular endothelial cells (HMEC-1) and cardiomyocytes. Our evidence demonstrates that MDG-1 promotes sphingosine kinase (SPHK) activity in HMEC-1 cells. An analytical method for measuring the mass of S1P using ESI/MS/MS was developed and we found that MDG-1 increases intracellular S1P levels. Meanwhile, MDG-1 is protective during hypoxia and ischemia through mechanisms that require S1P(1) receptor activation, which was confirmed both in oxygen glucose deprivation (OGD) and coronary artery ligation models by using transfection of cloned human S1P(1) receptor and RNA interference. These data indicate that the increase of intracellular S1P generation, particularly by activation of the SPHK enzyme, coupled with the autocrine and paracrine stimulation of cell surface S1P receptors, is a potential mechanism in the anti-ischemic and cell protective effect of MDG-1.

Topics: Animals; Cell Survival; Cytoprotection; Endothelial Cells; Gene Expression Regulation; Heart; Humans; Intracellular Space; Lysophospholipids; Male; Myocardial Ischemia; Phosphotransferases (Alcohol Group Acceptor); Polysaccharides; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; RNA Interference; Signal Transduction; Solubility; Sphingosine; Water

PPARγ agonists [thiazolidinediones (TZDs)] are known to exert anti-fibrotic effects in the kidney. In addition, we previously demonstrated that sphingosine kinase 1 (SK-1) and intracellular sphingosine-1-phosphate (S1P), by reducing the expression of connective tissue growth factor (CTGF), have a protective role in the fibrotic process.. Here, we investigated the effect of TZDs on intracellular sphingolipid levels and the transcriptional regulation of SK-1 in mesangial cells to evaluate potential novel aspects of the anti-fibrotic capacity of TZDs.. Stimulation with the TZDs, troglitazone and rosiglitazone, led to increased S1P levels in rat mesangial cells. This was paralleled by increased SK-1 activity as a consequence of direct effects of the TZDs on SK-1 expression. GW-9662, a PPARγ antagonist, inhibited the stimulating effect of TZDs on SK-1 mRNA and activity levels and intracellular S1P concentrations. Furthermore, SK-1 up-regulation by TZDs was functionally coupled with lower amounts of pro-fibrotic CTGF. SK-1 inhibition with SKI II almost completely abolished this effect in a dose-dependent manner. Moreover, the CTGF lowering effect of TZDs was fully blocked in MC isolated from SK-1 deficient mice (SK-1(-/-) ) as well as in glomeruli of SK-1(-/-) mice compared with wild-type mice treated with TRO and RSG.. These data show that TZD-induced SK-1 up-regulation results in lower amounts of CTGF, demonstrating novel facets for the anti-fibrotic effects of this class of drugs.

Topics: Animals; Blotting, Western; Cell Culture Techniques; Cells, Cultured; Connective Tissue Growth Factor; Enzyme Activation; Female; Fibrosis; Humans; Lysophospholipids; Mesangial Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutagenesis, Site-Directed; Phosphotransferases (Alcohol Group Acceptor); PPAR gamma; Promoter Regions, Genetic; Rats; Real-Time Polymerase Chain Reaction; Sphingosine; Thiazolidinediones; Up-Regulation

Resistance to therapies develops rapidly for melanoma leading to more aggressive disease. Therefore, agents are needed that specifically inhibit proteins or pathways controlling the development of this disease, which can be combined, dependent on genes deregulated in a particular patient's tumors. This study shows that elevated sphingosine-1-phosphate (S-1-P) levels resulting from increased activity of sphingosine kinase-1 (SPHK1) occur in advanced melanomas. Targeting SPHK1 using siRNA decreased anchorage-dependent and -independent growth as well as sensitized melanoma cells to apoptosis-inducing agents. Pharmacological SPHK1 inhibitors SKI-I but not SKI-II decreased S-1-P content, elevated ceramide levels, caused a G2-M block and induced apoptotic cell death in melanomas. Targeting SPHK1 using siRNA or the pharmacological agent called SKI-I decreased the levels of pAKT. Furthermore, SKI-I inhibited the expression of CYCLIN D1 protein and increased the activity of caspase-3/7, which in turn led to the degradation of PARP. In animals, SKI-I but not SKI-II retarded melanoma growth by 25-40%. Thus, targeting SPHK1 using siRNAs or SKI-I has therapeutic potential for melanoma treatment either alone or in combination with other targeted agents.

Topics: Animals; Apoptosis; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Down-Regulation; Fibroblasts; G1 Phase Cell Cycle Checkpoints; Humans; Lysophospholipids; Melanocytes; Melanoma; Mice; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Resting Phase, Cell Cycle; RNA, Small Interfering; Skin Neoplasms; Sphingosine; Staurosporine; Thiazoles; Up-Regulation; Xenograft Model Antitumor Assays

We showed previously that TNF-α down-regulates the Na+/K+ ATPase in HepG2 cells. This work was undertaken to study the role of ceramide and its metabolites in TNF-α action. Treating HepG2 cells with the cytokine in presence of an inhibitor of sphingomyelinase, abrogated the effect of TNF-α on the ATPase. To confirm the involvement of ceramide or its metabolites, cells were incubated with exogenous ceramide. Ceramide reduced time-dependently the activity of the ATPase and its effect disappeared in presence of CAY 10466 or SHKI, respective inhibitors of ceramidase and spingosine kinase, suggesting that ceramide acts via sphingosine or sphingosine-1-phosphate (S1P). However, HepG2 cells treated with exogenous sphingosine showed a higher Na+/K+ ATPase activity inferring that S1P is the one responsible for the down-regulatory effect of TNF-α and ceramide. This hypothesis was confirmed by the observed inhibitory effect of exogenous S1P on the pump, which was maintained when JNK and NF-κB were inhibited separately or simultaneously. The concurrent, but not individual inhibition of the kinase and transcription factor in the absence of S1P imitated the effect of S1P. It was concluded that S1P down-regulates the ATPase by inhibiting both JNK and NF-κB. This conclusion was supported by the observed decrease in the phosphorylation of c-jun and the enhanced protein expression of IκB and lower NK-KB activity.

Topics: Aniline Compounds; Anthracenes; Apoptosis; Benzylidene Compounds; Cell Line; Ceramides; Hep G2 Cells; Humans; I-kappa B Proteins; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Proline; Sodium-Potassium-Exchanging ATPase; Sphingomyelin Phosphodiesterase; Sphingosine; Thiocarbamates; Tumor Necrosis Factor-alpha

To investigate key genes expression of the sphingosine-1-phosphate pathway in endometriotic tissues.. A case-control laboratory study.. Tertiary care university hospital.. A total of 31 women, with (n = 16) and without (n = 15) endometriosis took part in the study.. After surgical excision with pathological analysis, endometrial specimens were obtained from women affected or not by endometriosis.. SPHK1-2, SGPP1-2, SGPL1, SPHKAP, and S1PR1-5 messenger RNA expression by quantitative real-time polymerase chain reaction (PCR) in the endometrium of 15 disease-free women, 16 eutopic and 16 ectopic endometrium of endometriosis-affected women. The S1PR1 and S1PR2 expression were further investigated by immunohistochemistry.. The SGPP2 expression was decreased in eutopic and ectopic endometrium of endometriosis-affected women (1.7- and 16.7-fold, respectively). The SGPP1, weakly expressed in healthy endometrium, is up-regulated in endometriosis-affected women (11.9- and 64.7-fold, respectively), but its expression remains low. The SGPL1 expression was decreased in ectopic endometrium (3.3-fold) and SPHKAP expression was increased in ectopic endometrium (112.6-fold) compared with endometrium of disease-free women. In endometriosis-affected women, S1PR3 expression was decreased in eutopic and ectopic endometrium (2.1- and 6.3-fold, respectively); S1PR2 and S1PR1 expression was increased in eutopic (2.5-fold) and ectopic endometrium (2.6-fold). These increases were confirmed at the protein levels by immunohistochemistry.. Expression of the enzymes implicated in the regulation of the sphingosine-1-phosphate level balance and of its receptors is overall heavily deregulated in endometriotic lesions in favor of a decreased sphingosine-1-phosphate catabolism. Our results plead for a role of the sphingosine pathway in establishing and survival of endometriotic lesions.

Topics: Aldehyde-Lyases; Analysis of Variance; Case-Control Studies; Endometriosis; Endometrium; Female; Hospitals, University; Humans; Immunohistochemistry; Lysophospholipids; Membrane Proteins; Ovarian Diseases; Paris; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine; Sphingosine-1-Phosphate Receptors

The mechanisms of hematopoietic progenitor cell egress and clinical mobilization are not fully understood. Herein, we report that in vivo desensitization of Sphingosine-1-phosphate (S1P) receptors by FTY720 as well as disruption of S1P gradient toward the blood, reduced steady state egress of immature progenitors and primitive Sca-1(+)/c-Kit(+)/Lin(-) (SKL) cells via inhibition of SDF-1 release. Administration of AMD3100 or G-CSF to mice with deficiencies in either S1P production or its receptor S1P(1), or pretreated with FTY720, also resulted in reduced stem and progenitor cell mobilization. Mice injected with AMD3100 or G-CSF demonstrated transient increased S1P levels in the blood mediated via mTOR signaling, as well as an elevated rate of immature c-Kit(+)/Lin(-) cells expressing surface S1P(1) in the bone marrow (BM). Importantly, we found that S1P induced SDF-1 secretion from BM stromal cells including Nestin(+) mesenchymal stem cells via reactive oxygen species (ROS) signaling. Moreover, elevated ROS production by hematopoietic progenitor cells is also regulated by S1P. Our findings reveal that the S1P/S1P(1) axis regulates progenitor cell egress and mobilization via activation of ROS signaling on both hematopoietic progenitors and BM stromal cells, and SDF-1 release. The dynamic cross-talk between S1P and SDF-1 integrates BM stromal cells and hematopoeitic progenitor cell motility.

Topics: Animals; Benzylamines; Bone Marrow; Cell Movement; Cells, Cultured; Chemokine CXCL12; Colony-Forming Units Assay; Cyclams; Female; Flow Cytometry; Fluorescent Antibody Technique; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cells; Heterocyclic Compounds; Lysophospholipids; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Reactive Oxygen Species; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Stromal Cells

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that promotes breast cancer progression by diverse mechanisms that remain somewhat unclear. Here we report pharmacologic evidence of a critical role for sphingosine kinase 1 (SphK1) in producing S1P and mediating tumor-induced hemangiogenesis and lymphangiogenesis in a murine model of breast cancer metastasis. S1P levels increased both in the tumor and the circulation. In agreement, serum S1P levels were significantly elevated in stage IIIA human breast cancer patients, compared with age/ethnicity-matched healthy volunteers. However, treatment with the specific SphK1 inhibitor SK1-I suppressed S1P levels, reduced metastases to lymph nodes and lungs, and decreased overall tumor burden of our murine model. Both S1P and angiopoietin 2 (Ang2) stimulated hemangiogenesis and lymphangiogenesis in vitro, whereas SK1-I inhibited each process. We quantified both processes in vivo from the same specimen by combining directed in vivo angiogenesis assays with fluorescence-activated cell sorting, thereby confirming the results obtained in vitro. Notably, SK1-I decreased both processes not only at the primary tumor but also in lymph nodes, with peritumoral lymphatic vessel density reduced in SK1-I-treated animals. Taken together, our findings show that SphK1-produced S1P is a crucial mediator of breast cancer-induced hemangiogenesis and lymphangiogenesis. Our results implicate SphK1 along with S1P as therapeutic targets in breast cancer.

Topics: Amino Alcohols; Animals; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cells, Cultured; Disease Progression; Enzyme Inhibitors; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Lymphangiogenesis; Lymphatic Metastasis; Lysophospholipids; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Staging; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sphingosine; Tumor Burden; Vascular Endothelial Growth Factor A

Current markers available for screening normal populations and for monitoring prostate cancer (PCa) treatment lack sensitivity and selectivity. Sphingosine-1-phosphate (S1P) is a circulating lipid second messenger involved in cell growth and migration, the immune response, angiogenesis, and malignant transformation.. Eighty-eight patients with localised, locally advanced, or metastatic PCa were recruited into this prospective single-centre study. Plasma S1P levels were measured and compared with age-matched controls with benign prostate hyperplasia (BPH) (n=110) or with young healthy males with the very small chance of having PCa foci (n=20).. Levels of circulating S1P were significantly higher in healthy subjects (10.36 ± 0.69 pmol per mg protein, P<0.0001) and patients with BPH (9.39 ± 0.75, P=0.0013) than in patients with PCa (6.89 ± 0.58, ANOVA, P=0.0019). Circulating S1P levels were an early marker of PCa progression to hormonal unresponsiveness and correlated with prostate-specific antigen (PSA) levels and lymph node metastasis. During the course of the study, nine patients have died of PCa. Importantly, their circulating S1P levels were significantly lower (5.11 ± 0.75) than in the surviving patients (7.02 ± 0.22, n=79, P=0.0439). Our data suggest that the decrease in circulating S1P during PCa progression may stem from a highly significant downregulation of erythrocyte sphingosine kinase-1 (SphK1) activity (2.14 ± 0.17 pmol per mg protein per minute in PCa patients vs 4.7 ± 0.42 in healthy individuals, P<0.0001), which may be a potential mechanism of cancer-induced anaemia.. This current study has provided a potential mechanism for cancer-related anaemia and the first evidence that plasma S1P and erythrocyte SphK1 activity are the potential markers for the diagnosis, monitoring, and predicating for PCa mortality.

Topics: Anemia; Biomarkers, Tumor; Cell Line, Tumor; Disease Progression; Early Detection of Cancer; Erythrocytes; Humans; Lysophospholipids; Male; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Prostate-Specific Antigen; Prostatic Neoplasms; Sphingosine

In this study, we examined the possible roles of ceramide/sphingosine-1-phosphate and ceramide/glucosyleceramide signaling in docetaxel-induced apoptosis by examining expression levels of the glucosyleceramide synthase and sphingosine kinase-1 and ceramide synthase gene family. As confirmed by isobologram analysis, docetaxel in combination with agents that increase intracellular ceramide levels increased the cytotoxic and apoptotic effects of docetaxel synergistically. More importantly, RT-PCR results revealed that expression levels of glucosyleceramide synthase and sphingosine kinase-1 were downregulated and ceramide synthase genes were upregulated in response to docetaxel. This study identifies mechanisms underlying the involvement of ceramide metabolizing genes in docetaxel-induced apoptosis in prostate cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Ceramides; Docetaxel; Down-Regulation; Drug Synergism; Gene Expression Regulation, Neoplastic; Glucosylceramides; Glucosyltransferases; Humans; Lysophospholipids; Male; Oxidoreductases; Phosphotransferases (Alcohol Group Acceptor); Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Sphingosine; Taxoids; Up-Regulation

The complement anaphylatoxin C5a has a pathogenetic role in endotoxin-induced lung inflammatory injury by regulating phagocytic cell migration and activation. Endotoxin and C5a activate the enzyme sphingosine kinase (Sphk) 1 to generate the signaling lipid sphingosine-1-phosphate (S1P), a critical regulator of phagocyte function. We assessed the function of Sphk1 and S1P in experimental lung inflammatory injury and determined their roles in anaphylatoxin receptor signaling and on the expression of the two C5a receptors, C5aR (CD88) and C5L2, on phagocytes. We report that Sphk1 gene deficient (Sphk1(-/-)) mice had augmented lung inflammatory response to endotoxin compared to wild type mice. Sphk1 was required for C5a-mediated reduction in cytokine and chemokine production by macrophages. Moreover, neutrophils from Sphk1(-/-) mice failed to upregulate the anaphylatoxin receptor C5L2 in response to LPS. Exogenous S1P restored C5L2 cell surface expression of Sphk1(-/-) mouse neutrophils to wild type levels but had no effect on cell surface expression of the other anaphylatoxin receptor, CD88. These results provide the first genetic evidence of the crucial role of Sphk1 in regulating the balance between expression of CD88 and C5L2 in phagocytes. S1P-mediated up-regulation of C5L2 is a novel therapeutic target for mitigating endotoxin-induced lung inflammatory injury.

Topics: Anaphylatoxins; Animals; Bone Marrow; Cytokines; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Lipopolysaccharides; Lysophospholipids; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Receptor, Anaphylatoxin C5a; Receptors, Chemokine; Sepsis; Signal Transduction; Sphingosine

Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS.

Topics: Animals; Cell Line, Tumor; Glucose; Glucose Intolerance; Glucose Tolerance Test; Injections, Intraperitoneal; Insulin; Insulin Secretion; Insulin-Secreting Cells; Insulinoma; Lysophospholipids; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); RNA, Small Interfering; Sphingosine

Our previous studies have indicated that de novo ceramide synthesis plays a critical role in ethanol-induced apoptotic neurodegeneration in the 7-day-old mouse brain. In this study, we examined whether the formation of sphingosine 1-phosphate (S1P), a ceramide metabolite, is associated with this apoptotic pathway. Analyses of basal levels of S1P-related compounds indicated that S1P, sphingosine, sphingosine kinase 2, and S1P receptor 1 increased significantly during postnatal brain development. In the 7-day-old mouse brain, sphingosine kinase 2 was localized mainly in neurons. Subcellular fractionation studies of the brain homogenates showed that sphingosine kinase 2 was enriched in the plasma membrane and the synaptic membrane/synaptic vesicle fractions, but not in the nuclear and mitochondrial/lysosomal fractions. Ethanol exposure in 7-day-old mice induced sphingosine kinase 2 activation and increased the brain level of S1P transiently 2-4 h after exposure, followed by caspase 3 activation that peaked around 8 h after exposure. Treatment with dimethylsphingosine, an inhibitor of sphingosine kinases, attenuated the ethanol-induced caspase 3 activation and the subsequent neurodegeneration. These results indicate that ethanol activates sphingosine kinase 2, leading to a transient increase in S1P, which may be involved in neuroapoptotic action of ethanol in the developing brain.

Topics: Animals; Apoptosis; Brain; Central Nervous System Depressants; Ethanol; Immunoblotting; Immunohistochemistry; Lysophospholipids; Mice; Mice, Inbred C57BL; Nerve Degeneration; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK1. PF-543 inhibits SphK1 with a K(i) of 3.6 nM, is sphingosine-competitive and is more than 100-fold selective for SphK1 over the SphK2 isoform. In 1483 head and neck carcinoma cells, which are characterized by high levels of SphK1 expression and an unusually high rate of S1P production, PF-543 decreased the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. In contrast with past reports that show that the growth of many cancer cell lines is SphK1-dependent, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 was effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.

Topics: Cell Line, Tumor; Cell Membrane Permeability; Humans; Lysophospholipids; Methanol; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidines; Sphingosine; Substrate Specificity; Sulfones

The pro-fibrotic connective tissue growth factor (CTGF) has been linked to the development and progression of diabetic vascular and renal disease. We recently reported that low-density lipoproteins (LDL) induced expression of CTGF in aortic endothelial cells. However, the molecular mechanisms are not fully defined. Here, we have studied the mechanism by which LDL regulates CTGF expression in renal mesangial cells. In these cells, treatment with pertussis toxin abolished LDL-stimulated activation of ERK1/2 and c-Jun N-terminal kinase (JNK), indicating the involvement of heterotrimeric G proteins in LDL signaling. Treatment with LDL promoted activation and translocation of endogenous sphingosine kinase 1 (SK1) from the cytosol to the plasma membrane concomitant with production of sphingosine-1-phosphate (S1P). Pretreating cells with SK inhibitor, dimethylsphinogsine or down-regulation of SK1 and SK2 revealed that LDL-dependent activation of ERK1/2 and JNK is mediated by SK1. Using a green fluorescent protein-tagged S1P₁ receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that LDL induced S1P receptor activation. Pretreating cells with S1P₁/S1P₃ receptor antagonist VPC23019 significantly inhibited activation of ERK1/2 and JNK by LDL, suggesting that LDL elicits G protein-dependent activation of ERK1/2 and JNK by stimulating SK1-dependent transactivation of S1P receptors. Furthermore, S1P stimulation induced expression of CTGF in a dose-dependent manner that was markedly inhibited by blocking the ERK1/2 and JNK signaling pathways. LDL-induced CTGF expression was pertussis toxin sensitive and inhibited by dimethylsphinogsine down-regulation of SK1 and VPC23019 treatment. Our data suggest that SK1-dependent S1P receptor transactivation is upstream of ERK1/2 and JNK and that all three steps are required for LDL-regulated expression of CTGF in mesangial cells.

Topics: Animals; Cell Membrane; Cells, Cultured; Connective Tissue Growth Factor; Diabetic Nephropathies; Dyslipidemias; Gene Silencing; Humans; Lipoproteins, LDL; Lysophospholipids; MAP Kinase Signaling System; Mesangial Cells; Phosphotransferases (Alcohol Group Acceptor); Protein Isoforms; Protein Transport; Rats; Receptors, Lysosphingolipid; Recombinant Fusion Proteins; RNA, Messenger; RNA, Small Interfering; Sphingosine; Sphingosine-1-Phosphate Receptors; Transcriptional Activation; Up-Regulation

Transplant arteriosclerosis is a major cause of late intestinal allograft dysfunction. However, little is known about the immunologic and molecular mechanisms underlying it, and no effective treatment is available. This study aimed to investigate the role of sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) in transplant arteriosclerosis and find out whether fish oil (FO) attenuates allograft arteriosclerosis through S1P signaling.. A rat model with orthotopic intestinal transplantation was conducted in this study. Animals received daily FO supplementation after intestinal transplant. The allogeneic recipients by phosphate-buffered saline or corn oil treatment served as controls. The allograft arteriosclerosis was characterized, and the expression of SPHK1 and S1P receptors (S1P₁, S1P₂, and S1P₃) was determined on day 190 posttransplant.. The allogeneic controls presented transplant vasculopathy in mesenteric vessels, including intimal thickening, fibrosis, and leukocyte infiltration. The transplant arteriosclerosis was markedly reduced in FO-fed animals. The pression of SPHK1 and its activity were significantly augmented, and the expression of S1P₁ and S1P₃ messenger RNA was up-regulated in the allogeneic controls. FO supplementation suppressed the activation of SPHK1 and led to a decrease in the expression of S1P₁ and S1P₃ in these tissues in transplant arteriosclerosis.. These results demonstrate that the activation of SPHK1/S1P signaling plays a possible role in the pathogenesis of transplant arteriosclerosis. The reduction of allograft arteriosclerosis by FO may be associated with down-regulation of SPHK1/S1P signaling. Understanding the role of FO for SPHK1/S1P may help us to identify considerable therapeutic targets for transplant arteriosclerosis.

Topics: Animals; Arteriosclerosis; Cell Movement; Cell Proliferation; Endothelial Cells; Fish Oils; Intestines; Lysophospholipids; Male; Phosphotransferases (Alcohol Group Acceptor); Postoperative Complications; Rats; Rats, Inbred F344; Rats, Inbred Lew; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Transplantation, Homologous

Isoflurane, a volatile anesthetic agent, has been recognized for its potential neuroprotective properties and has antiapoptotic effects. We examined whether isoflurane posttreatment is protective against early brain injury after subarachnoid hemorrhage and determined whether this effect needs sphingosine-related pathway activation.. Controlled in vivo laboratory study.. Animal research laboratory.. One hundred seventy-nine 8-wk-old male CD-1 mice weighing 30-38 g.. Subarachnoid hemorrhage was induced in mice by endovascular perforation. Animals were randomly assigned to sham-operated, subarachnoid hemorrhage-vehicle, and subarachnoid hemorrhage+2% isoflurane. Neurobehavioral function and brain edema were evaluated at 24 and 72 hrs. The expression of sphingosine kinase, phosphorylated Akt, and cleaved caspase-3 was determined by Western blotting and immunofluorescence. Neuronal cell death was examined by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling staining. Effects of a sphingosine kinase inhibitor N, N-dimethylsphingosine or a sphingosine 1 phosphate receptor inhibitor VPC23019 on isoflurane's protective action against postsubarachnoid hemorrhage early brain injury were also examined.. Isoflurane significantly improved neurobehavioral function and brain edema at 24 hrs but not 72 hrs after subarachnoid hemorrhage. At 24 hrs, isoflurane attenuated neuronal cell death in the cortex, associated with an increase in sphingosine kinase 1 and phosphorylated Akt, and a decrease in cleaved caspase-3. The beneficial effects of isoflurane were abolished by N, N-dimethylsphingosine and VPC23019.. Isoflurane posttreatment delays the development of postsubarachnoid hemorrhage early brain injury through antiapoptotic mechanisms including sphingosine-related pathway activation, implying its use for anesthesia during acute aneurysm surgery or intervention.

Topics: Animals; Apoptosis; Brain Edema; Brain Injuries; Isoflurane; Lysophospholipids; Male; Mice; Neuroprotective Agents; Phosphotransferases (Alcohol Group Acceptor); Random Allocation; Signal Transduction; Sphingosine; Subarachnoid Hemorrhage

Sphingosine kinases (SK) 1 and 2 are unique lipid kinases that phosphorylate sphingosine to form -sphingosine-1-phosphate (S1P). S1P is a bioactive molecule eliciting multiple effects both extracellularly via cell surface S1P receptors and intracellularly through a number of recently identified protein targets. The two enzymes arise from different genes, and differ in their cellular localisation, developmental expression, catalytic properties, and in at least some functional roles. Here, we describe methods for selectively detecting SK1 and SK2 activities in vitro, highlighting conditions that can discriminate between the activities of these two enzymes. The assays measure the production of (32)P-labelled S1P following the addition of exogenous sphingosine and [γ(32)P] adenosine-5'-triphosphate. The S1P product can be purified by Bligh-Dyer solvent extraction, separated by thin-layer chromatography (TLC), and the radiolabelled S1P quantified by exposing the TLC plate to a storage phosphor screen. This sensitive, reproducible assay can be used to selectively detect SK1 and SK2 activities in tissue, cell, and recombinant protein samples.

Topics: Biocatalysis; Chemical Fractionation; Chromatography, Thin Layer; Enzyme Assays; Isoenzymes; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingolipids (ceramide, sphingosine, and sphingosine-1-phosphate) are bioactive lipids with important biological functions in proliferation, apoptosis, angiogenesis, and inflammation. Herein, we describe easy and rapid biochemical methods with the use of radiolabeled molecules ((3)H, (32)P) for their mass determination. Quantitation of sphingosine kinase-1 activity, the most studied isoform, is also included.

Topics: Cell Extracts; Cell Line, Tumor; Ceramides; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Spectrophotometry; Sphingosine

Sphingosine-1-phosphate (S1P) and the enzyme primarily responsible for its production, sphingosine kinase-1 (SphK-1), are thought to be dysregulated in multiple human diseases including cancer, multiple sclerosis (MS), diabetes, neurological diseases, fibrosis, and certain pathologies associated with impaired angiogenesis such as, age-related macular degeneration (AMD). Antibody-based techniques to identify and localize S1P and SphK-1 within cells and tissue specimens represent powerful tools not only to understand the biological role of these molecules but also to validate these unique in-class targets in multiple state diseases. Consequently, the potential applications of these molecules for therapy and diagnostic purposes are currently under investigation. Here, we describe two staining procedures for identification of S1P and SphK-1 in human frozen tissue samples and the challenges encountered in the process of localization in tissue samples of lipid molecules, such as S1P.

Topics: Carcinoma, Hepatocellular; Humans; Immunohistochemistry; Liver Neoplasms; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. Sphingosine 1-phosphate is a bioactive sphingolipid that regulates crucial physiological processes. We have recently reported that sphingosine 1-phosphate and sphingosine kinase are involved in a novel signaling pathway leading to acrosomal exocytosis (Suhaiman L et al., J Biol Chem 285:1630-16314, 2010). Acrosomal exocytosis in mammalian sperm is a regulated secretion with unusual characteristics. We therefore employed biochemical functional assays to assess the sphingolipid signaling in both permeabilized and nonpermeabilized sperm. The exocytosis of the acrosomal content is regulated by Ca(2+). During exocytosis, changes in [Ca(2+)]i occur induced by either Ca(2+)-influx or Ca(2+)-mobilization from intracellular stores. By using single cell [Ca(2+)] measurements, we detected intracellular Ca(2+) changes after sphingosine 1-phosphate treatment. Additionally, measuring sphingosine kinase activity, we determined that sphingosine 1-phosphate levels increase after an exocytotic stimulus.This chapter is designed to provide the user with sufficient background to analyze sphingosine 1--phosphate signal transduction pathways during acrosomal exocytosis in human sperm.

Topics: Acrosome; Calcium; Exocytosis; Humans; Lysophospholipids; Male; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

The goal of the current study was to determine whether the sphingosine kinase 1 (SK1)/sphingosine-1-phosphate (S1P) pathway is involved in myogenic vasoconstriction under normal physiological conditions. In the present study, we assessed whether endogenous S1P generated by pressure participates in myogenic vasoconstriction and which signaling pathways are involved in SK1/S1P-induced myogenic response under normal physiological conditions.. We measured pressure-induced myogenic response, Ca(2+) concentration, and 20 kDa myosin light chain phosphorylation (MLC(20)) in rabbit posterior cerebral arteries (PCAs). SK1 was expressed and activated by elevated transmural pressure in rabbit PCAs. Translocation of SK1 by pressure elevation was blocked in the absence of external Ca(2+) and in the presence of mechanosensitive ion channel and voltage-sensitive Ca(2+) channel blockers. Pressure-induced myogenic tone was inhibited in rabbit PCAs treated with sphingosine kinase inhibitor (SKI), but was augmented by treatment with NaF, which is an inhibitor of sphingosine-1-phosphate phosphohydrolase. Exogenous S1P further augmented pressure-induced myogenic responses. Pressure induced an increase in Ca(2+) concentration leading to the development of myogenic tone, which was inhibited by SKI. Exogenous S1P further increased the pressure-induced increased Ca(2+) concentration and myogenic tone, but SKI had no effect. Pressure- and exogenous S1P-induced myogenic tone was inhibited by pre-treatment with the Rho kinase inhibitor and NADPH oxidase inhibitors. Pressure- and exogenous S1P-induced myogenic tone were inhibited by pre-treatment with S1P receptor blockers, W146 (S1P1), JTE013 (S1P2), and CAY10444 (S1P3). MLC(20) phosphorylation was increased when the transmural pressure was raised from 40 to 80 mmHg and exogenous S1P further increased MLC(20) phosphorylation. The pressure-induced increase of MLC(20) phosphorylation was inhibited by pre-treatment of arteries with SKI.. Our results suggest that the SK1/S1P pathway may play an important role in pressure-induced myogenic responses in rabbit PCAs under normal physiological conditions.

Topics: Animals; Calcium; Lysophospholipids; Muscle, Smooth, Vascular; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Posterior Cerebral Artery; Rabbits; Signal Transduction; Sphingosine; Vasoconstriction

Sphingosine 1-phosphate (S1P) is currently one of the most intensely studied lipid mediators. Interest in S1P has been propelled by the development of fingolimod, an S1P receptor agonist prodrug, which revealed both a theretofore unsuspected role of S1P in lymphocyte trafficking and that such modulation of the immune system achieves therapeutic benefit in multiple sclerosis patients. S1P is synthesized from sphingosine by two SphKs (sphingosine kinases) (SphK1 and SphK2). Manipulation of SphK levels using molecular biology and mouse genetic tools has implicated these enzymes, particularly SphK1, in a variety of pathological processes such as fibrosis, inflammation and cancer progression. The results of such studies have spurred interest in SphK1 as a drug target. In this issue of the Biochemical Journal, Schnute et al. describe a small molecule inhibitor of SphK1 that is both potent and selective. Such chemical tools are essential to learn whether targeting S1P signalling at the level of synthesis is a viable therapeutic strategy.

Topics: Humans; Lysophospholipids; Methanol; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidines; Sphingosine; Sulfones

Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.

Topics: Animals; Cell Proliferation; Female; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Mice, Knockout; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Regeneration; Satellite Cells, Skeletal Muscle; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; STAT3 Transcription Factor

Heart failure is associated with neurological deficits, including cognitive dysfunction. However, the molecular mechanisms underlying reduced cerebral blood flow in the early stages of heart failure, particularly when blood pressure is minimally affected, are not known.. Using a myocardial infarction model in mice, we demonstrate a tumor necrosis factor-α (TNFα)-dependent enhancement of posterior cerebral artery tone that reduces cerebral blood flow before any overt changes in brain structure and function. TNFα expression is increased in mouse posterior cerebral artery smooth muscle cells at 6 weeks after myocardial infarction. Coordinately, isolated posterior cerebral arteries display augmented myogenic tone, which can be fully reversed in vitro by the competitive TNFα antagonist etanercept. TNFα mediates its effect via a sphingosine-1-phosphate (S1P)-dependent mechanism, requiring sphingosine kinase 1 and the S1P(2) receptor. In vivo, sphingosine kinase 1 deletion prevents and etanercept (2-week treatment initiated 6 weeks after myocardial infarction) reverses the reduction of cerebral blood flow, without improving cardiac function.. Cerebral artery vasoconstriction and decreased cerebral blood flow occur early in an animal model of heart failure; these perturbations are reversed by interrupting TNFα/S1P signaling. This signaling pathway may represent a potential therapeutic target to improve cognitive function in heart failure.

Topics: Animals; Cerebral Arteries; Etanercept; Heart Failure; Immunoglobulin G; Lysophospholipids; Magnetic Resonance Imaging; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; Muscle Development; Muscle, Smooth, Vascular; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Receptors, Tumor Necrosis Factor; Regional Blood Flow; Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha; Vasoconstriction

Mechanisms by which cancer cells communicate with the host organism to regulate lung colonization/metastasis are unclear. We show that this communication occurs via sphingosine 1-phosphate (S1P) generated systemically by sphingosine kinase 1 (SK1), rather than via tumour-derived S1P. Modulation of systemic, but not tumour SK1, prevented S1P elevation, and inhibited TRAMP-induced prostate cancer growth in TRAMP(+/+) SK1(-/-) mice, or lung metastasis of multiple cancer cells in SK1(-/-) animals. Genetic loss of SK1 activated a master metastasis suppressor, Brms1 (breast carcinoma metastasis suppressor 1), via modulation of S1P receptor 2 (S1PR2) in cancer cells. Alterations of S1PR2 using pharmacologic and genetic tools enhanced Brms1. Moreover, Brms1 in S1PR2(-/-) MEFs was modulated by serum S1P alterations. Accordingly, ectopic Brms1 in MB49 bladder cancer cells suppressed lung metastasis, and stable knockdown of Brms1 prevented this process. Importantly, inhibition of systemic S1P signalling using a novel anti-S1P monoclonal antibody (mAb), Sphingomab, attenuated lung metastasis, which was prevented by Brms1 knockdown in MB49 cells. Thus, these data suggest that systemic SK1/S1P regulates metastatic potential via regulation of tumour S1PR2/Brms1 axis.

Topics: Animals; Disease Models, Animal; Humans; Lung Neoplasms; Lysophospholipids; Male; Mice; Mice, Knockout; Neoplasm Metastasis; Phosphotransferases (Alcohol Group Acceptor); Prostatic Neoplasms; Receptors, Lysosphingolipid; Repressor Proteins; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Urinary Bladder Neoplasms

Sphingosine-1-phosphate (S1P) is generated through phosphorylation of sphingosine by two sphingosine kinases (SPHK-1 and -2). As extra- and intracellular messenger S1P fulfils multiple roles in inflammation such as mediating proinflammatory inputs or acting as chemoattractant. In addition, S1P induces cyclooxygenase-2 (COX-2) expression and the synthesis of proinflammatory prostanoids in several cell types. Here, we analysed in vivo the regulation of S1P level as well as potential interactions between S1P and COX-dependent prostaglandin synthesis during zymosan-induced inflammation. S1P and prostanoid levels were determined in the blood and at the site of inflammation under basal conditions and during zymosan-induced inflammation using wild type and SPHK-1 and -2 knockout mice. We found that alterations in S1P levels did not correlate with changes in plasma- or tissue-concentrations of the prostanoids as well as COX-2 expression. In the inflamed tissue S1P and prostanoid concentrations were reciprocally regulated. Prostaglandin levels increased over 6h, while S1P and sphingosine level decreased during the same time, which makes an induction of prostanoid synthesis by S1P in zymosan-induced inflammation unlikely. Additionally, despite altered S1P levels wild type and SPHK knockout mice showed similar behavioural nociceptive responses and oedema sizes suggesting minor functions of S1P in this inflammatory model.

Topics: Animals; Cyclooxygenase 2; Edema; Inflammation; Lysophospholipids; Mice; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Prostaglandins; Sphingosine; Zymosan

S1P (sphingosine 1-phosphate) is a pleiotropic lipid mediator involved in numerous cellular and physiological functions. Of note among these are cell survival and migration, as well as lymphocyte trafficking. S1P, which exerts its effects via five GPCRs (G-protein-coupled receptors) (S1P1-S1P5), is formed by the action of two SphKs (sphingosine kinases). Although SphK1 is the more intensively studied isotype, SphK2 is unique in it nuclear localization and has been reported to oppose some of the actions ascribed to SphK1. Although several scaffolds of SphK1 inhibitors have been described, there is a scarcity of selective SphK2 inhibitors that are necessary to evaluate the downstream effects of inhibition of this isotype. In the present paper we report a cationic amphiphilic small molecule that is a selective SphK2 inhibitor. In the course of characterizing this compound in wild-type and SphK-null mice, we discovered that administration of the inhibitor to wild-type mice resulted in a rapid increase in blood S1P, which is in contrast with our SphK1 inhibitor that drives circulating S1P levels down. Using a cohort of F2 hybrid mice, we confirmed, compared with wild-type mice, that circulating S1P levels were higher in SphK2-null mice and lower in SphK1-null mice. Thus both SphK1 and SphK2 inhibitors recapitulate the blood S1P levels observed in the corresponding null mice. Moreover, circulating S1P levels mirror SphK2 inhibitor levels, providing a convenient biomarker of target engagement.

Topics: Animals; Drug Design; Enzyme Inhibitors; Humans; Isoenzymes; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Structure; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); Recombinant Proteins; Sphingolipids; Sphingosine; U937 Cells

Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Proliferation; Chromatography, Liquid; Cytokinesis; Enzyme Inhibitors; Female; Fluorescent Antibody Technique; Humans; Indoles; Lysophospholipids; Mitosis; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingosine; Tumor Cells, Cultured

Inflammation mediates/promotes graft injury after liver transplantation (LT). This study investigated the roles of sphingosine kinase-2 (SK2) in inflammation after LT. Liver grafts were stored in UW solution with and without ABC294640 (100 µM), a selective inhibitor of SK2, before implantation. Hepatic sphingosine-1-phosphate (S1P) levels increased ∼4-fold after LT, which was blunted by 40% by ABC294640. Hepatic toll-like receptor-4 (TLR4) expression and nuclear factor-κB (NF-κB) p65 subunit phosphorylation elevated substantially after transplantation. The pro-inflammatory cytokines/chemokines tumor necrosis factor-α, interleukin-1β and C-X-C motif chemokine 10 mRNAs increased 5.9-fold, 6.1-fold and 16-fold, respectively following transplantation, while intrahepatic adhesion molecule-1 increased 5.7-fold and monocytes/macrophage and neutrophil infiltration and expansion of residential macrophage population increased 7.8-13.4 fold, indicating enhanced inflammation. CD4+ T cell infiltration and interferon-γ production also increased. ABC294640 blunted TLR4 expression by 60%, NF-κB activation by 84%, proinflammatory cytokine/chemokine production by 45-72%, adhesion molecule expression by 54% and infiltration of monocytes/macrophages and neutrophils by 62-67%. ABC294640 also largely blocked CD4+ T cell infiltration and interferon-γ production. Focal necrosis and apoptosis occurred after transplantation with serum alanine aminotransferase (ALT) reaching ∼6000 U/L and serum total bilirubin elevating to ∼1.5 mg/dL. Inhibition of SK2 by ABC294640 blunted necrosis by 57%, apoptosis by 74%, ALT release by ∼68%, and hyperbilirubinemia by 74%. Most importantly, ABC294640 also increased survival from ∼25% to ∼85%. In conclusion, SK2 plays an important role in hepatic inflammation responses and graft injury after cold storage/transplantation and represents a new therapeutic target for liver graft failure.

Topics: Adamantane; Animals; Cell Adhesion Molecules; Chemokines; Enzyme Inhibitors; Inflammation; Leukocytes; Liver; Liver Transplantation; Lysophospholipids; Male; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Pyridines; Rats; Rats, Inbred Lew; Reactive Oxygen Species; Reperfusion Injury; Sphingosine; Toll-Like Receptor 4; Up-Regulation

Three bioactive sphingolipids, namely sphingosine-1-phosphate (S1P), ceramide (CER) and sphingosine (SPH) were shown to be involved in ischemia/reperfusion injury of the heart. S1P is a powerful cardioprotectant, CER activates apoptosis and SPH in a low dose is cardioprotective whereas in a high dose is cardiotoxic. The aim of the present study was to examine effects of experimental myocardial infarction on the level of selected sphingolipids in plasma, erythrocytes and platelets in the rat. Myocardial infarction was produced in male Wistar rats by ligation of the left coronary artery. Blood was taken from the abdominal aorta at 1, 6 and 24 h after the ligation. Plasma, erythrocytes and platelets were isolated and S1P, dihydrosphingosine-1-phosphate (DHS1P), SPH, dihydrosphingosine (DHS) and CER were quantified by means of an Agilent 6460 triple quadrupole mass spectrometer using positive ion electrospray ionization source with multiple reaction monitoring. The infarction reduced the plasma level of S1P, DHS1P, SPH and DHS but increased the level of total CER. In erythrocytes, there was a sharp elevation in the level of SPH and DHS early after the infarction and a reduction after 24 h whereas the level of S1P, DHS1P and total CER gradually increased. In platelets, the level of each of the examined compounds profoundly decreased 1 and 6 h after the infarction and partially normalized in 24 h. The results obtained clearly show that experimental heart infarction in rats produces deep changes in metabolism of sphingolipids in the plasma, platelets and erythrocytes.

Topics: Anesthesia; Animals; Ceramides; Coronary Vessels; Erythrocyte Count; Femoral Artery; Ligation; Lysophospholipids; Male; Myocardial Infarction; Phosphotransferases (Alcohol Group Acceptor); Platelet Count; Rats; Rats, Wistar; Sphingolipids; Sphingosine; Troponin T

Sphingosine-1-phosphate (S1P) is an important regulator of cancer development and progression. Its cellular concentration is controlled predominantly by sphingosine kinase (SK) and sphingosine-1-phosphate lyase (SPL). In the current study we showed that mRNA expressions for both SK and SPL were up-regulated throughout all four disease stages in human breast cancer patients. Exogenous administration of S1P produced a bell-shaped dose response for apoptosis in normal mammary gland MCF12A cells but a sigmoid-shaped apoptotic response in breast cancer MCF7 cells. Co-administration of S1P enhanced the cytotoxicity of anticancer drug docetaxel against MCF7 cells.

Topics: Aldehyde-Lyases; Apoptosis; Breast Neoplasms; Cell Proliferation; Docetaxel; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Sphingosine; Taxoids

The potent lipid mediator sphingosine-1-phosphate (S1P) regulates diverse physiological processes by binding to 5 specific GPCRs, although it also has intracellular targets. Here, we demonstrate that S1P, produced in the mitochondria mainly by sphingosine kinase 2 (SphK2), binds with high affinity and specificity to prohibitin 2 (PHB2), a highly conserved protein that regulates mitochondrial assembly and function. In contrast, S1P did not bind to the closely related protein PHB1, which forms large, multimeric complexes with PHB2. In mitochondria from SphK2-null mice, a new aberrant band of cytochrome-c oxidase was detected by blue native PAGE, and interaction between subunit IV of cytochrome-c oxidase and PHB2 was greatly reduced. Moreover, depletion of SphK2 or PHB2 led to a dysfunction in mitochondrial respiration through cytochrome-c oxidase. Our data point to a new action of S1P in mitochondria and suggest that interaction of S1P with homomeric PHB2 is important for cytochrome-c oxidase assembly and mitochondrial respiration.

Topics: Amino Acid Sequence; Animals; Cell Line; Electron Transport Complex IV; Gene Expression Regulation, Enzymologic; Humans; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Heart; Myocytes, Cardiac; Oxygen Consumption; Phosphotransferases (Alcohol Group Acceptor); Prohibitins; Repressor Proteins; Sphingosine

The sphingosine kinases (sphingosine kinase-1 and -2) have been implicated in a variety of physiological functions. Discerning their mechanism of action is complicated because in addition to producing the potent lipid second messenger sphingosine-1-phosphate, sphingosine kinases, both by producing sphingosine-1-phosphate and consuming sphingosine, have profound effects on sphingolipid metabolism. Sphingosine kinase-1 translocates to the plasma membrane upon agonist stimulation and this translocation is essential for the pro-oncogenic properties of this enzyme. Many of the enzymes of sphingolipid metabolism, including the enzymes that degrade sphingosine-1-phosphate, are membrane bound with restricted subcellular distributions. In the work described here we explore how subcellular localization of sphingosine kinase-1 affects the downstream metabolism of sphingosine-1-phosphate and the access of sphingosine kinase to its substrates. We find, surprisingly, that restricting sphingosine kinase to either the plasma membrane or the endoplasmic reticulum has a negligible effect on the rate of degradation of the sphingosine-1-phosphate that is produced. This suggests that sphingosine-1-phosphate is rapidly transported between membranes. However we also find that cytosolic or endoplasmic-reticulum targeted sphingosine kinase expressed at elevated levels produces extremely high levels of dihydrosphingosine-1-phosphate. Dihydrosphingosine is a proximal precursor in ceramide biosynthesis. Our data indicate that sphingosine kinase can divert substrate from the ceramide de novo synthesis pathway. However plasma membrane-restricted sphingosine kinase cannot access the pool of dihydrosphingosine. Therefore whereas sphingosine kinase localization does not affect downstream metabolism of sphingosine-1-phosphate, localization has an important effect on the pools of substrate to which this key signaling enzyme has access.

Topics: HEK293 Cells; HeLa Cells; Humans; Intracellular Membranes; Isoenzymes; Lysophospholipids; Molecular Structure; Phosphotransferases (Alcohol Group Acceptor); Sphingolipids; Sphingosine

Directed migration of hepatic myofibroblasts (hMFs) contributes to the development of liver fibrosis. However, the signals regulating the motility of these cells are incompletely understood. We have recently shown that sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) are involved in mouse liver fibrogenesis. Here, we investigated the role of S1P/S1PRs signals in human liver fibrosis involving motility of human hMFs.. S1P level in the liver was examined by high-performance liquid chromatography. Expression of S1PRs was characterized, in biopsy specimens of human liver and cultured hMFs, by immunofluorescence and real-time RT-PCR or Western blot analysis. Cell migration was determined in Boyden chambers, by using the selective S1P receptor agonist or antagonist and silencing of S1PRs expression with small interfering RNA.. S1P level in the human fibrotic liver was increased through up-regulation of sphingosine kinase (SphK), irrespective of the etiology of fibrosis. S1P receptors type 1, 2, and 3 (S1P(1,2,3)) were expressed in human hMFs in vivo and in vitro. Interestingly, S1P(1,3) were strongly induced in human fibrotic samples, whereas expression of S1P(2) was massively decreased. S1P exerted a powerful migratory action on human hMFs. Furthermore, the effect of S1P was mimicked by SEW2871 (an S1P(1) agonist), and blocked by suramin (an S1P(3) antagonist) and by silencing S1P(1,3) expression. In contrast, JTE-013 (an S1P(2) antagonist) and silencing of S1P(2) expression enhanced S1P-induced migration.. SphK/S1P/S1PRs signaling axis plays an important role in human liver fibrosis and is involved in the directed migration of human hMFs into the damaged areas.

Topics: Base Sequence; Cell Movement; Cells, Cultured; Humans; Liver Cirrhosis; Lysophospholipids; Myofibroblasts; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Suramin; Thiophenes

Sphingosine-1-phosphate (S1P) acts as high affinity agonist at specific G-protein-coupled receptors, S1P(1-5), that play important roles e.g. in the cardiovascular and immune systems. A S1P receptor modulating drug, FTY720 (fingolimod), has been effective in phase III clinical trials for multiple sclerosis. FTY720 is a sphingosine analogue and prodrug of FTY720-phosphate, which activates all S1P receptors except S1P(2) and disrupts lymphocyte trafficking by internalizing the S1P(1) receptor. Cis-4-methylsphingosine (cis-4M-Sph) is another synthetic sphingosine analogue that is readily taken up by cells and phosphorylated to cis-4-methylsphingosine-1-phosphate (cis-4M-S1P). Therefore, we analysed whether cis-4M-Sph interacted with S1P receptors through its metabolite cis-4M-S1P in a manner similar to FTY720. Indeed, cis-4M-Sph caused an internalization of S1P receptors, but differed from FTY720 as it acted on S1P(2) and S1P(3) and only weakly on S1P(1), while FTY720 internalized S1P(1) and S1P(3) but not S1P(2). Consequently, pre-incubation with cis-4M-Sph specifically desensitized S1P-induced [Ca(2+)](i) increases, which are mediated by S1P(2) and S1P(3), in a time- and concentration-dependent manner. This effect was not shared by sphingosine or FTY720, indicating that metabolic stability and targeting of S1P(2) receptors were important. The desensitization of S1P-induced [Ca(2+)](i) increases was dependent on the expression of SphKs, predominantly of SphK2, and thus mediated by cis-4M-S1P. In agreement, cis-4M-S1P was detected in the supernatants of cells exposed to cis-4M-Sph. It is concluded that cis-4M-Sph, through its metabolite cis-4M-S1P, acts as a S1P receptor modulator and causes S1P receptor internalization and desensitization. The data furthermore help to define requirements for sphingosine kinase substrates as S1P receptor modulating prodrugs.

Topics: Calcium; Fingolimod Hydrochloride; HEK293 Cells; Humans; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine; Stereoisomerism

Skeletal muscle regeneration is severely compromised in the case of extended damage. The current challenge is to find factors capable of limiting muscle degeneration and/or potentiating the inherent regenerative program mediated by a specific type of myoblastic cells, the satellite cells. Recent studies from our groups and others have shown that the bioactive lipid, sphingosine 1-phosphate (S1P), promotes myoblast differentiation and exerts a trophic action on denervated skeletal muscle fibres. In the present study, we examined the effects of S1P on eccentric contraction (EC)-injured extensor digitorum longus muscle fibres and resident satellite cells. After EC, skeletal muscle showed evidence of structural and biochemical damage along with significant electrophysiological changes, i.e. reduced plasma membrane resistance and resting membrane potential and altered Na(+) and Ca(2+) current amplitude and kinetics. Treatment with exogenous S1P attenuated the EC-induced tissue damage, protecting skeletal muscle fibre from apoptosis, preserving satellite cell viability and affecting extracellular matrix remodelling, through the up-regulation of matrix metalloproteinase 9 (MMP-9) expression. S1P also promoted satellite cell renewal and differentiation in the damaged muscle. Notably, EC was associated with the activation of sphingosine kinase 1 (SphK1) and with increased endogenous S1P synthesis, further stressing the relevance of S1P in skeletal muscle protection and repair/regeneration. In line with this, the treatment with a selective SphK1 inhibitor during EC, caused an exacerbation of the muscle damage and attenuated MMP-9 expression. Together, these findings are in favour for a role of S1P in skeletal muscle healing and offer new clues for the identification of novel therapeutic approaches to counteract skeletal muscle damage and disease.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Calcium; Caspase 3; Caspase 7; Cell Differentiation; Cell Membrane; Cell Survival; Extracellular Matrix; Lysophospholipids; Matrix Metalloproteinase 9; Membrane Potentials; Mice; Muscle Contraction; Muscle Fibers, Skeletal; Muscle, Skeletal; Phosphotransferases (Alcohol Group Acceptor); Regeneration; Satellite Cells, Skeletal Muscle; Signal Transduction; Sodium; Sphingosine; Wound Healing

Sphingosine kinases (SphKs) catalyze the transfer of phosphate from adenosine triphosphate (ATP) to sphingosine to generate sphingosine 1-phosphate (S1P), an important bioactive lipid molecule that mediates a diverse range of cell signaling processes. The conventional assay of SphK enzymatic activity uses [γ-(32)P]ATP and sphingosine as substrates, with the radiolabeled S1P product recovered by organic extraction, displayed by thin layer chromatography, and quantified by liquid scintillation counting. Although this assay is sensitive and accurate, it is slow and labor-intensive; thus, it precludes the simultaneous screening of more than a few inhibitor compounds. Here we describe a 96-well assay for SphKs that is rapid and reproducible. Our method, which takes advantage of the limited solubility of S1P, detects radioactive S1P adhering to the plate by scintillation proximity counting. Our procedure obviates extraction into organic solvents, postreaction transfers, and chromatography. Furthermore, our assay enables assessment of both inhibitors and substrates, and it can detect endogenous SphK activity in cell and tissue extracts. The SphK kinetic parameter, K(m), and the K(i) values of inhibitors determined with our assay and the conventional assay were indistinguishable. These results document that our assay is well-suited for the screening of chemical libraries of SphK inhibitors.

Topics: Adenosine Triphosphate; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Enzyme Inhibitors; High-Throughput Screening Assays; Kinetics; Lysophospholipids; Phosphorus Radioisotopes; Phosphotransferases (Alcohol Group Acceptor); Scintillation Counting; Small Molecule Libraries; Sphingosine; Substrate Specificity

The balance between the pro-apoptotic lipids ceramide and sphingosine and the pro-survival lipid sphingosine 1-phosphate (S1P) is termed the "sphingosine rheostat". Two isozymes, sphingosine kinase 1 and 2 (SK1 and SK2), are responsible for phosphorylation of pro-apoptotic sphingosine to form pro-survival S1P. We have previously reported the antitumor properties of an SK2 selective inhibitor, ABC294640, alone or in combination with the multikinase inhibitor sorafenib in mouse models of kidney carcinoma and pancreatic adenocarcinoma. Here we evaluated the combined antitumor effects of the aforementioned drug combination in two mouse models of hepatocellular carcinoma. Although combining the SK2 inhibitor, ABC294640, and sorafenib in vitro only afforded additive drug-drug effects, their combined antitumor properties in the mouse model bearing HepG2 cells mirrored effects previously observed in animals bearing kidney carcinoma and pancreatic adenocarcinoma cells. Combining ABC294640 and sorafenib led to a decrease in the levels of phosphorylated ERK in SK-HEP-1 cells, indicating that the antitumor effect of this drug combination is likely mediated through a suppression of the MAPK pathway in hepatocellular models. We also measured levels of S1P in the plasma of mice treated with two different doses of ABC294640 and sorafenib. We found decreases in the levels of S1P in plasma of mice treated daily with 100 mg/kg of ABC294640 for 5 weeks, and this decrease was not affected by co-administration of sorafenib. Taken together, these data support combining ABC294640 and sorafenib in clinical trials in HCC patients. Furthermore, monitoring levels of S1P may provide a pharmacodynamic marker of ABC294640 activity.

Topics: Adamantane; Animals; Antineoplastic Agents; Apoptosis; Benzenesulfonates; Blotting, Western; Carcinoma, Hepatocellular; Flow Cytometry; Hep G2 Cells; Humans; Liver Neoplasms; Lysophospholipids; Mice; Mitogen-Activated Protein Kinases; Niacinamide; Phenylurea Compounds; Phosphotransferases (Alcohol Group Acceptor); Pyridines; Sorafenib; Sphingosine; Xenograft Model Antitumor Assays

To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors used high-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract S1P from blood in 96- and 384-well plates prior to analyses using the RapidFire(®) mass spectrometry system.

Topics: Aminophenols; Dose-Response Relationship, Drug; Enzyme Inhibitors; High-Throughput Screening Assays; Humans; Kinetics; Lysophospholipids; Mass Spectrometry; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Thiazoles

Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P(1) receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility.. Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino)-4-(p-Chlorophenyl) Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1P(int), and attenuated S1P(ext) or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1P(int) and potentiated motility of HPAECs to S1P(ext) or serum. S1P(ext) mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting "inside-out" signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs). Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1P(ext) or 4-deoxypyridoxine-dependent endothelial cell motility.. These results suggest S1P(ext) mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL.

Topics: Aldehyde-Lyases; Cell Movement; Endothelial Cells; Humans; Lung; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); RNA, Small Interfering; Sphingosine

Internalized membrane proteins are either transported to late endosomes and lysosomes for degradation or recycled to the plasma membrane. Although proteins involved in trafficking and sorting have been well studied, far less is known about the lipid molecules that regulate the intracellular trafficking of membrane proteins. We studied the function of sphingosine kinases and their metabolites in endosomal trafficking using Drosophila melanogaster photoreceptors as a model system. Gain- and loss-of-function analyses show that sphingosine kinases affect trafficking of the G protein-coupled receptor Rhodopsin and the light-sensitive transient receptor potential (TRP) channel by modulating the levels of dihydrosphingosine 1 phosphate (DHS1P) and sphingosine 1 phosphate (S1P). An increase in DHS1P levels relative to S1P leads to the enhanced lysosomal degradation of Rhodopsin and TRP and retinal degeneration in wild-type photoreceptors. Our results suggest that sphingosine kinases and their metabolites modulate photoreceptor homeostasis by influencing endolysosomal trafficking of Rhodopsin and TRP.

Topics: Animals; Drosophila melanogaster; Drosophila Proteins; Endosomes; Lipid Metabolism; Lysophospholipids; Lysosomes; Phosphotransferases (Alcohol Group Acceptor); Photoreceptor Cells; Protein Transport; Sphingosine; Transient Receptor Potential Channels

We have recently reported that the bioactive lipid sphingosine-1-phosphate (S1P), usually signaling proliferation and anti-apoptosis induces neuronal death when generated by sphingosine-kinase2 and when accumulation due to S1P-lyase deficiency occurs. In the present study, we identify the signaling cascade involved in the neurotoxic effect of sphingoid-base phosphates. We demonstrate that the calcium-dependent cysteine protease calpain mediates neurotoxicity by induction of the endoplasmic reticulum stress-specific caspase cascade and activation of cyclin-dependent kinase5 (CDK5). The latter is involved in an abortive reactivation of the cell cycle and also enhances tau phosphorylation. Neuroanatomical studies in the cerebellum document for the first time that indeed neurons with abundant S1P-lyase expression are those, which degenerate first in S1P-lyase-deficient mice. We therefore propose that an impaired metabolism of glycosphingolipids, which are prevalent in the central nervous system, might be linked via S1P, their common catabolic intermediate, to neuronal death.

Topics: Aldehyde-Lyases; Animals; Apoptosis; Calcium; Calpain; Caspase 12; Caspase 9; Cell Cycle; Cerebellum; Cyclin-Dependent Kinase 5; Glycosphingolipids; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Nerve Degeneration; Neurons; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; tau Proteins

The sphingosine kinase (SPK)/sphingosine-1-phosphate (S1P) pathway recently has been associated with a variety of inflammatory-based diseases. The majority of these studies have been performed in vitro. Here, we have addressed the relevance of the SPK/S1P pathway in the acute inflammatory response in vivo by using different well known preclinical animal models. The study has been performed by operating a pharmacological modulation using 1) L-cycloserine and DL-threo-dihydrosphingosine (DTD), S1P synthesis inhibitors or 2) 2-undecyl-thiazolidine-4-carboxylic acid (BML-241) and N-(2,6-dichloro-4-pyridinyl)-2-[1,3-dimethyl-4-(1-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-hydrazinecarboxamide (JTE-013), specific S1P(2) and S1P(3) receptor antagonists. After local injection of carrageenan in mouse paw S1P release significantly increases locally and decreases during the resolution phase. Expression of SPKs and S1P(2) and S1P(3) receptors is increased in inflamed tissues. Administration of L-cycloserine or DTD caused a significant anti-inflammatory effect. By using different animal models we have also demonstrated that the SPK/S1P pathway contributes to changes in vascular permeability and promotes cell recruitment. The S1P effect on cell recruitment results is receptor-mediated because both JTE-013 and BML-241 inhibited zymosan-induced cell chemotaxis without effect on vascular leakage. Conversely, changes in vascular permeability involve mainly SPK activity, because compound 48/80-induced vascular leakage was significantly inhibited by DTD. In conclusion, the SPK/S1P pathway is involved in acute inflammation and could represent a valuable therapeutic target for developing a new class of anti-inflammatory drugs.

Topics: Animals; Capillary Permeability; Chemotaxis, Leukocyte; Cycloserine; Edema; Inflammation; Lysophospholipids; Male; Mice; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Thiazolidines

Sphingosine-1-phosphate (S1P) has been implicated in angiogenesis, inflammation, cancerogenesis, neurological excitability and immune regulation and is synthesized by two different sphingosine kinases (SphK). It was suggested that mice lacking the gene for SphK1 exhibit no obvious phenotype, because SphK2 compensates for its absence. However, recent investigations revealed that under challenge SphK1 contributed to pro-inflammatory processes favoring Th2 and Th17 rather than Th1-type reactions. To investigate the immune modulatory role of SphK1 as opposed to SphK2 specifically for the Th1 propagating IL-12p70 we compared WT and SphK1(-/-) splenocytes and Flt3-ligand differentiated BMCs of WT and SphK1(-/-), representing dendritic cells as major producers of IL-12p70, incubated with LPS. We determined the impact on IL-12p70 in comparison to other inflammatory cytokines, and on DC and macrophage surface marker expression, SphK mRNA, protein expression and enzymatic activity in splenocytes. Our data demonstrated that SphK1 deficiency enhanced LPS-induced IL-12p70 production although SphK2 was present. To further characterize SphK1-dependent IL-12p70 regulation we exogenously applied S1P, SEW2871 and the new potent S1P1 agonist CYM5442. Both S1P and S1P1-specific analogs fully compensated the increase of IL-12p70 production in SphK1-deficient splenocytes. The use of pertussis toxin, to block G(i)-coupled signaling downstream of S1P1, again increased IL-12p70 and neglected the compensation achieved by addition of S1P and S1P1 agonists pointing on the importance of this specific S1P-receptor. Given that, in parallel to a prominent IL-12p35 increase following LPS stimulation, LPS also enhanced SphK expression and total SphK activity, we concluded that SphK1-derived S1P acting via S1P1 is a major mechanism of this negative IL-12p70 feedback loop, which did not affect other cytokines. Moreover, our data showed that SphK2 activity failed to compensate for SphK1 deficiency. These findings clearly point to a divergent and cytokine-specific impact of immune cell SphK1 and SphK2 in chronic inflammation and cancer.

Topics: Animals; Bone Marrow Cells; CD8 Antigens; Cell Differentiation; Dendritic Cells; Enzyme Assays; Flow Cytometry; Gene Deletion; Gene Expression Regulation; Indans; Interleukin-12; Interleukin-12 Receptor beta 2 Subunit; Interleukin-12 Subunit p40; Lipopolysaccharides; Lymphocyte Activation; Lysophospholipids; Membrane Proteins; Mice; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Sphingosine; Spleen; Thiophenes; Toll-Like Receptors

Sphingosine kinase 1 (SK1) catalyzes the conversion of sphingosine to the bioactive lipid sphingosine 1-phosphate. We have previously demonstrated that FTY720 and (S)-FTY720 vinylphosphonate are novel inhibitors of SK1 activity. Here, we show that (S)-FTY720 vinylphosphonate binds to a putative allosteric site in SK1 contingent on formation of the enzyme-sphingosine complex. We report that SK1 is an oligomeric protein (minimally a dimer) containing noncooperative catalytic sites and that the allosteric site exerts an autoinhibition of the catalytic site. A model is proposed in which (S)-FTY720 vinylphosphonate binding to and stabilization of the allosteric site might enhance the autoinhibitory effect on SK1 activity. Further evidence for the existence of allosteric site(s) in SK1 was demonstrated by data showing that two new FTY720 analogues (a conjugate of sphingosine with a fluorophore and (S)-FTY720 regioisomer) increased SK1 activity, suggesting relief of autoinhibition of SK1 activity. Comparisons with the SK1 inhibitor, SKi or siRNA knockdown of SK1 indicated that (S)-FTY720 vinylphosphonate and FTY720 behave as typical SK1 inhibitors in preventing sphingosine 1-phosphate-stimulated rearrangement of actin in MCF-7 cells. These findings are discussed in relation to the anticancer properties of SK1 inhibitors.

Topics: Actins; Allosteric Regulation; Breast Neoplasms; Catalytic Domain; Cell Line, Tumor; Enzyme Inhibitors; Female; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Lysophospholipids; Models, Chemical; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Proteasome Endopeptidase Complex; Sphingosine

Sphingosine 1-phosphate (S1P) is a bioactive lipid that has been identified as an accelerant of cancer progression. The sphingosine kinases (SphKs) are the sole producers of S1P, and thus, SphK inhibitors may prove effective in cancer mitigation and chemosensitization. Of the two SphKs, SphK1 overexpression has been observed in a myriad of cancer cell lines and tissues and has been recognized as the presumptive target over that of the poorly characterized SphK2. Herein, we present the design and synthesis of amidine-based nanomolar SphK1 subtype-selective inhibitors. A homology model of SphK1, trained with this library of amidine inhibitors, was then used to predict the activity of additional, more potent, inhibitors. Lastly, select amidine inhibitors were validated in human leukemia U937 cells, where they significantly reduced endogenous S1P levels at nanomolar concentrations.

Topics: Amidines; Antineoplastic Agents; Cell Line, Tumor; Chemistry, Pharmaceutical; Drug Design; Enzyme Inhibitors; Gene Expression Regulation, Leukemic; Humans; Kinetics; Leukemia; Lysophospholipids; Models, Chemical; Models, Molecular; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; U937 Cells

Resveratrol, an important phytoalexin in many plants, has been reported to have cytotoxic effects on various types of cancer. Ceramide is a bioactive sphingolipid that regulates many signaling pathways, including cell growth and proliferation, senescence and quiescence, apoptosis, and cell cycle. Ceramides are generated by longevity assurance genes (LASS). Glucosylceramide synthase (GCS) and sphingosine kinase-1 (SK-1) enzymes can convert ceramides to antiapoptotic molecules, glucosylceramide, and sphingosine-1-phosphate, respectively. C8:ceramide, an important cell-permeable analogue of natural ceramides, increases intracellular ceramide levels significantly, while 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and SK-1 inhibitor increase accumulation of ceramides by inhibiting GCS and SK-1, respectively. Chronic myelogenous leukemia (CML) is a hematological disorder resulting from generation of BCR/ABL oncogene. In this study, we examined the roles of ceramide metabolizing genes in resveratrol-induced apoptosis in K562 CML cells. There were synergistic cytotoxic and apoptotic effects of resveratrol with coadministration of C8:ceramide, PDMP, and SK-1 inhibitor. Interestingly, there were also significant increases in expression levels of LASS genes and decreases in expression levels of GCS and SK-1 in K562 cells in response to resveratrol. Our data, in total, showed for the first time that resveratrol might kill CML cells through increasing intracellular generation and accumulation of apoptotic ceramides.

Topics: Apoptosis; Ceramides; Down-Regulation; Glucosylceramides; Glucosyltransferases; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lysophospholipids; Morpholines; Phosphotransferases (Alcohol Group Acceptor); Resveratrol; RNA, Neoplasm; Sphingosine; Stilbenes; Up-Regulation

The mechanisms by which sphingosine kinase-1 (SK-1)/sphingosine 1-phosphate (S1P) activation contributes to imatinib resistance in chronic myeloid leukemia (CML) are unknown. We show herein that increased SK-1/S1P enhances Bcr-Abl1 protein stability, through inhibition of its proteasomal degradation in imatinib-resistant K562/IMA-3 and LAMA-4/IMA human CML cells. In fact, Bcr-Abl1 stability was enhanced by ectopic SK-1 expression. Conversely, siRNA-mediated SK-1 knockdown in K562/IMA-3 cells, or its genetic loss in SK-1(-/-) MEFs, significantly reduced Bcr-Abl1 stability. Regulation of Bcr-Abl1 by SK-1/S1P was dependent on S1P receptor 2 (S1P2) signaling, which prevented Bcr-Abl1 dephosphorylation, and degradation via inhibition of PP2A. Molecular or pharmacologic interference with SK-1/S1P2 restored PP2A-dependent Bcr-Abl1 dephosphorylation, and enhanced imatinib- or nilotinib-induced growth inhibition in primary CD34(+) mononuclear cells obtained from chronic phase and blast crisis CML patients, K562/IMA-3 or LAMA4/IMA cells, and 32Dcl3 murine progenitor cells, expressing the wild-type or mutant (Y253H or T315I) Bcr-Abl1 in situ. Accordingly, impaired SK-1/S1P2 signaling enhanced the growth-inhibitory effects of nilotinib against 32D/T315I-Bcr-Abl1-derived mouse allografts. Since SK-1/S1P/S1P2 signaling regulates Bcr-Abl1 stability via modulation of PP2A, inhibition of SK-1/S1P2 axis represents a novel approach to target wild-type- or mutant-Bcr-Abl1 thereby overcoming drug resistance.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Cell Line, Tumor; Drug Resistance, Neoplasm; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lysophospholipids; Mice; Mice, SCID; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Piperazines; Protein Phosphatase 2; Pyrimidines; Receptors, Lysosphingolipid; RNA, Small Interfering; Signal Transduction; Sphingosine; Ubiquitination

To maintain an intact barrier, epithelia eliminate dying cells by extrusion. During extrusion, a cell destined for apoptosis signals its neighboring cells to form and contract a ring of actin and myosin, which squeezes the dying cell out of the epithelium. Here, we demonstrate that the signal produced by dying cells to initiate this process is sphingosine-1-phosphate (S1P). Decreasing S1P synthesis by inhibiting sphingosine kinase activity or by blocking extracellular S1P access to its receptor prevented apoptotic cell extrusion. Extracellular S1P activates extrusion by binding the S1P(2) receptor in the cells neighboring a dying cell, as S1P(2) knockdown in these cells or its loss in a zebrafish mutant disrupted cell extrusion. Because live cells can also be extruded, we predict that this S1P pathway may also be important for driving delamination of stem cells during differentiation or invasion of cancer cells.

Topics: Actomyosin; Animals; Antibodies, Monoclonal; Apoptosis; Cell Line; Dogs; Enzyme Inhibitors; Epithelial Cells; Gene Knockdown Techniques; Lysophospholipids; Mutation; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA Interference; Signal Transduction; Sphingosine; Zebrafish; Zebrafish Proteins

Metabolism of sphingolipids into downstream lipid mediators followed by signaling modulates tumor microenvironment and the cancer cells to influence tumor progression. As such, sphingolipid signaling represents a novel way to modulate tumor biology. Neuroblastoma (NB), the most common extracranial solid tumor of childhood, is highly angiogenic and often displays poor prognosis. However, the role of sphingolipid mediators is not known in NB. We found that NB expresses high levels of sphingosine kinase-2, which is essential for the formation of sphingosine-1-phosphate (S1P). S1P induced VEGF expression in SK-N-AS NB cells. The effect occurred at the transcriptional level. Hypoxia in combination with S1P had a synergistic effect on VEGF expression. Strong correlation was detected between S1P receptor-2 (S1P(2)) and VEGF mRNAs in 11 different cell lines and 17 NB tissues. Blockade of S1P(2) with the selective antagonist JTE-013 significantly inhibited S1P-induced VEGF expression. Overexpression and knockdown of S1P(2) in SK-N-AS cells increased or inhibited S1P-induced VEGF secretion, respectively. Interestingly, JTE-013 significantly inhibited tumor growth, VEGF mRNA expression, and induced apoptosis in the NB tumor xenografts. Taken together, our data suggest that enhanced formation of sphingolipid mediator S1P in NB profoundly influences tumor microenvironment by inducing VEGF expression via S1P(2). Modulation of sphingolipid signaling by inhibiting S1P(2) may constitute a novel strategy to control NB.

Topics: Angiogenesis Inducing Agents; Animals; Brain Neoplasms; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; In Situ Nick-End Labeling; Lysophospholipids; Male; Mice; Mice, Nude; Neuroblastoma; Phosphotransferases (Alcohol Group Acceptor); Platelet Endothelial Cell Adhesion Molecule-1; Sphingolipids; Sphingosine; Vascular Endothelial Growth Factor A

Sphingosine kinase 2 (SK2) catalyses the conversion of sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). We report here, the stereospecific synthesis of an analogue of FTY720 called (R)-FTY720-OMe, which we show is a competitive inhibitor of SK2. (R)-FTY720-OMe failed to inhibit sphingosine kinase 1 activity, thereby demonstrating specificity for SK2. Prolonged treatment of HEK 293 cells with (R)-FTY720-OMe also induced a reduction in SK2 expression. In addition, (R)-FTY720-OMe inhibited DNA synthesis and prevented S1P-stimulated rearrangement of actin in MCF-7 breast cancer cells. These findings demonstrate that SK2 functions as a pro-survival protein and is involved in promoting actin rearrangement into membrane ruffles/lamellipodia in response to S1P in MCF-7 breast cancer cells.

Topics: Actins; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cell Membrane; Enzyme Assays; Female; Fingolimod Hydrochloride; HEK293 Cells; Humans; Lysophospholipids; Microscopy, Fluorescence; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Pseudopodia; Sphingosine

The biological roles of sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) have been broadly investigated. However, at present pathophysiological roles of S1P/S1PRs axis in liver fibrosis are not well defined. Here, we investigated the functions of S1P/S1PRs axis in human hepatic stellate cells (HSC) line, LX-2 cells. We found that S1PR types 1, 2 and 3 (S1PR1-3) are clearly detected in LX-2 cells, as determined by RT-PCR, Western blot and immunocytochemistry analysis. S1P exerted a powerful migratory action on LX-2 cells, as determined in Boyden chambers, and stimulated fibrogenic activity of LX-2 cells, as demonstrated by increase of expression of smooth muscle α-actin, procollagen α1(I) and α1(III) and total hydroxyproline content. Moreover, the effects of S1P were mimicked by S1PR1 agonist SEW2871, and abrogated by W146 (S1PR1 antagonist) and/or silencing S1PR1, three expression with small interfering RNA, suggesting the main roles of S1PR1 and 3. However, studies with S1PR2 antagonist JTE-013 and silencing S1PR2 expression indicated that S1PR2 negatively regulated S1P-induced cell migration. Interestingly, exogenously added S1P induced significant up-regulation of sphingosine kinase-1 and the synthesis of additional S1P, and expression of S1PR1,3, but not S1PR2. In conclusion, our data have identified an additional function regulated by S1P/S1PR1,3 axis involving migration and fibrogenic activation of HSCs. These results suggest that selective modulation of S1PR activity may represent a new antifibrotic strategy.

Topics: Cell Line; Cell Movement; Gene Silencing; Hepatic Stellate Cells; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Sphingolipids are ubiquitous components of eukaryotic cells and sphingolipid metabolites, such as the long chain base phosphate (LCB-P), sphingosine 1 phosphate (S1P) and ceramide (Cer) are important regulators of apoptosis in animal cells. This study evaluated the role of LCB-Ps in regulating apoptotic-like programmed cell death (AL-PCD) in plant cells using commercially available S1P as a tool. Arabidopsis cell cultures were exposed to a diverse array of cell death-inducing treatments (including Cer) in the presence of S1P. Rates of AL-PCD and cell survival were recorded using vital stains and morphological markers of AL-PCD. Internal LCB-P levels were altered in suspension cultured cells using inhibitors of sphingosine kinase and changes in rates of death in response to heat stress were evaluated. S1P reduced AL-PCD and promoted cell survival in cells subjected to a range of stresses. Treatments with inhibitors of sphingosine kinase lowered the temperature which induced maximal AL-PCD in cell cultures. The data supports the existence of a sphingolipid rheostat involved in controlling cell fate in Arabidopsis cells and that sphingolipid regulation of cell death may be a shared feature of both animal apoptosis and plant AL-PCD.

Topics: Apoptosis; Arabidopsis; Cells, Cultured; Ceramides; Heat-Shock Response; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

The study of sphingosine and sphingosine-1-phosphate is now widespread due to their immense role as intra- and extracellular messenger molecules. The balance and interplay of these ceramide metabolites is dependent on the activities of kinase and phosphatase enzymes. Sphingosine and sphingosine-1-phosphate are found in very minute quantities in cells; thus, they require highly sensitive techniques for quantitative analysis. In this study, we developed a quantitative assay for the determination of sphingosine kinase 2 (SphK2) activity both in vitro and with cell lysates, using CE-LIF. Sphingosine fluorescein was used as the substrate. The K(M) of SphK2 for sphingosine fluorescein was 2.8 ± 0.8 μM with a V(max) of 2490 ± 520 μM/min and a k(cat) of 1920 ± 402/s. The inhibition of SphK2 was also investigated using four different inhibitors for which 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole inhibitor was the most potent for the in vitro inhibition of SphK2 while N,N-dimethylsphingosine (DMS) did not inhibit but rather increased SphK2 activity. The fluorescence-based approach for the determination of the enzymatic activity of SphK2 proves to be useful for the quantitative determination of SphK2 activity in vitro and in cell lysates, and could be extended to single-cell analysis or applied in drug screening.

Topics: Animals; Cell Line, Tumor; Electrophoresis, Capillary; Fluorescein; Humans; Lysophospholipids; Mice; NIH 3T3 Cells; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Reproducibility of Results; Sphingosine

Glial cell line-derived neurotrophic factor (GDNF) is important for the development and maintenance of dopamine neurons (Lin et al. [1993] Science 260: 1130-1132). GDNF is neuroprotective in animal models of Parkinson disease, where dopamine neurons show selective degeneration. We previously reported GDNF-induced SPHK1 gene expression in a neuroblastoma cell line, TGW (Murakami et al. [2007] J Neurochem 102: 1585-1594). In the present study, we focused on the regulatory mechanism of GAP43 (GDNF-induced neuronal phenotype) transcription to further elucidate physiological roles of GDNF-induced SPHK1 expression and activity. Stable wild-type (SPHK1-WT) but not dominant-negative SPHK1 (SPHK1-DN) overexpression increased both control- and GDNF-induced GAP43 expression. SPHK1-WT cells showed enhanced GDNF-induced sphingosine 1-phosphate (S1P) secretion compared with mock- and SPHK1-DN cells. Exogenous S1P also increased GAP43 expression. In TGW cells, PD98059, a MEK inhibitor, but not SB203580 (a p38 MAPK inhibitor) and LY294002 (a PI3K inhibitor) inhibited GDNF-induced GAP43 expression, suggesting the MEK/ERK pathway has a major role in GDNF-induced GAP43 transcription. A G-protein-coupled receptor inhibitor, pertussis toxin, and S1P(1) and S1P(3) receptor antagonists (VPC23019 and CAY10444) also inhibited ERK activation. Moreover, both S1P1 and S1P3 were serine-phosphorylated by GDNF, suggesting their activated states. C/EBPβ transcription factor was induced by GDNF, and DNA pull-down and chromatin immunoprecipitation assays revealed the C/EBP binding site between -131 bp and -98  bp from the first exon of GAP43. Taken together, our results showed that in TGW cells, GDNF increased SPHK1 transcription, leading to the production and secretion of S1P. Through MEK/ERK pathway, S1P stimulates GAP43 transcription with increased binding of C/EBPβ to the 5'-promoter.

Topics: Base Sequence; Blotting, Western; Cell Line, Tumor; Chromatin Immunoprecipitation; Cloning, Molecular; DNA Primers; GAP-43 Protein; Glial Cell Line-Derived Neurotrophic Factor; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Polymerase Chain Reaction; Promoter Regions, Genetic; Signal Transduction; Sphingosine; Transcription, Genetic

Sphingosine 1-phosphate (S1P), a bioactive lipid generated by sphingosine kinases (SphK1/2), initiates different signalling pathways involved in physiological and pathological processes. We previously demonstrated that in rat myometrium at late (day 19) gestation, SphK1 increases the expression of COX2 via S1P generation and release. In rat uterine leiomyoma cells (ELT3), SphK1/S1P axis controls survival and proliferation. In the present study we demonstrate that PDBu activates SphK1 but not SphK2. SphK1 activation requires PKC and MAPK ERK1/2. S1P produced by PDBu is released in the medium. PDBu-induced S1P export is abolished by Ro-318220 and BIM (PKC inhibitors), by U0126 and PD98059 (MEK inhibitors), SKI-II (SphKI/2 inhibitor) and SphK1-siRNA, suggesting the involvement of PKC, ERK and SphK1 respectively. The release of S1P is insensitive to inhibitors of ATP Binding Cassette (ABC)A1 and ABCB1 transporters, but is abolished when ABCC1 transporters are inhibited by MK571 or down-regulated by ABCC1-siRNA. PDBu increases COX2 expression that is blocked by the inhibition of PKC, ERK1/2, SphK1, and when cells are treated with MK571 or transfected with ABCC1-siRNA. The induction of COX2 by the S1P release due to PDBu or by exogenous S1P involves S1P2 receptors coupled to Gi. In myometrium from rat at late gestation, the release of S1P is also strongly reduced when SphK and ABCC1 are inhibited. The data reveal that in rat leiomyoma cells and late pregnant rat myometrium, the release of S1P involves a similar signalling pathway and occurs through ABCC1.

Topics: Animals; Cell Line, Tumor; Cyclooxygenase 2; Enzyme Assays; Female; Gene Expression; Gene Knockdown Techniques; GTP-Binding Protein alpha Subunits, Gi-Go; Leiomyoma; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Multidrug Resistance-Associated Proteins; Myometrium; Phosphotransferases (Alcohol Group Acceptor); Pregnancy; Propionates; Protein Kinase C; Quinolines; Rats; Rats, Wistar; Receptors, Lysosphingolipid; RNA Interference; Sphingosine; Uterine Neoplasms

S1P (sphingosine 1-phosphate) is a signalling molecule involved in a host of cellular and physiological functions, most notably cell survival and migration. S1P, which signals via a set of five G-protein-coupled receptors (S1P1-S1P5), is formed by the action of two SphKs (sphingosine kinases) from Sph (sphingosine). Interfering RNA strategies and SphK1 (sphingosine kinase type 1)-null (Sphk1-/-) mouse studies implicate SphK1 in multiple signalling cascades, yet there is a paucity of potent and selective SphK1 inhibitors necessary to evaluate the effects of rapid onset inhibition of this enzyme. We have identified a set of submicromolar amidine-based SphK1 inhibitors and report using a pair of these compounds to probe the cellular and physiological functions of SphK1. In so doing, we demonstrate that our inhibitors effectively lower S1P levels in cell-based assays, but we have been unable to correlate SphK1 inhibition with changes in cell survival. However, SphK1 inhibition did diminish EGF (epidermal growth factor)-driven increases in S1P levels and Akt (also known as protein kinase B)/ERK (extracellular-signal-regulated kinase) phosphorylation. Finally, administration of the SphK1 inhibitor to wild-type, but not Sphk1-/-, mice resulted in a rapid decrease in blood S1P levels indicating that circulating S1P is rapidly turned over.

Topics: Amidines; Animals; Caspase 3; Cell Line; Cell Survival; Extracellular Signal-Regulated MAP Kinases; Humans; Lysophospholipids; Mice; Mice, Inbred C57BL; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Pyrrolidines; Rats; Sphingolipids; Sphingosine; Stereoisomerism

Sphingosine kinase 1 phosphorylates sphingosine, which is converted to ceramide by ceramide synthetase. Ceramide triggers eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. Erythrocytes lack sphingosine phosphate-degrading enzymes and thus store large quantities of sphingosine phosphate. The present study explored the influence of sphingosine and sphingosine phosphate on eryptosis. [Ca(2+)](i), was estimated from Fluo3 fluorescence, cell volume from forward scatter and PS exposure from annexin V-binding in FACS analysis. Sphingosine (0.1 - 10 μM) but not sphingosine-1- phosphate (0.1 - 10 μM) increased [Ca(2+)](i), decreased cell volume and increased PS-exposure. The observations disclose sphingosine, but not sphingosine-1-phosphate, as a strong inducer of eryptosis.

Topics: Aniline Compounds; Apoptosis; Calcium; Calcium Channels; Erythrocytes; Fluorescent Dyes; Humans; Lysophospholipids; Patch-Clamp Techniques; Phosphatidylserines; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Xanthenes

Diabetic nephropathy is characterized by accumulation of glomerular extracellular matrix proteins, such as fibronectin (FN). Here, we investigated whether sphingosine kinase (SphK)1 pathway is responsible for the elevated FN expression in diabetic nephropathy. The SphK1 pathway and FN expression were examined in streptozotocin-induced diabetic rat kidney and glomerular mesangial cells (GMC) exposed to high glucose (HG). FN up-regulation was concomitant with activation of the SphK1 pathway as reflected in an increase in the expression and activity of SphK1 and sphingosine 1-phosphate (S1P) production in both diabetic kidney and HG-treated GMC. Overexpression of wild-type SphK1 (SphK(WT)) significantly induced FN expression, whereas treatment with a SphK inhibitor, N,N-dimethylsphingosine, or transfection of SphK1 small interference RNA or dominant-negative SphK1 (SphK(G82D)) abolished HG-induced FN expression. Furthermore, addition of exogenous S1P significantly induced FN expression in GMC with an induction of activator protein 1 (AP-1) activity. Inhibition of AP-1 activity by curcumin attenuated the S1P-induced FN expression. Finally, by inhibiting SphK1 activity, both N,N-dimethylsphingosine and SphK(G82D) markedly attenuated the HG-induced AP-1 activity. Taken together, these results demonstrated that the SphK1 pathway plays a critical role in matrix accumulation in GMC under diabetic condition, suggesting that the SphK1 pathway could be a potential therapeutic target for diabetic nephropathy.

Topics: Animals; Blood Glucose; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Extracellular Matrix; Fibronectins; Gene Expression; Gene Knockdown Techniques; Glucose; Kidney Glomerulus; Lysophospholipids; Male; Mesangial Cells; Phosphotransferases (Alcohol Group Acceptor); Proteinuria; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Lysosphingolipid; RNA Interference; Signal Transduction; Sphingosine; Transcription Factor AP-1

Although mitochondria are key determinants of myocardial injury during ischemia-reperfusion (I/R), their interaction with critical cytoprotective signaling systems is not fully understood. Sphingosine-1-phosphate (S1P) produced by sphingosine kinase-1 protects the heart from I/R damage. Recently a new role for mitochondrial S1P produced by a second isoform of sphingosine kinase, SphK2, was described to regulate complex IV assembly and respiration via interaction with mitochondrial prohibitin-2. Here we investigated the role of SphK2 in cardioprotection by preconditioning. Littermate (WT) and sphk2 (-/-) mice underwent 45 min of in vivo ischemia and 24 h reperfusion. Mice received no intervention (I/R) or preconditioning (PC) via 5 min I/R before the index ischemia. Despite the activation of PC-cytoprotective signaling pathways in both groups, infarct size in sphk2 (-/-) mice was not reduced by PC (42 ± 3% PC vs. 43 ± 4% I/R, p = ns) versus WT (24 ± 3% PC vs. 43 ± 3% I/R, p < 0.05). sphk2 (-/-) mitochondria exhibited decreased oxidative phosphorylation and increased susceptibility to permeability transition (PTP). Unlike WT, PC did not prevent ischemic damage to electron transport or the increased susceptibility to PTP. To evaluate the direct contribution to the resistance of mitochondria to cytoprotection, SphK2, PHB2 or cytochrome oxidase subunit IV was depleted in cardiomyoblasts. PC protection was abolished by each knockdown concomitant with decreased PTP resistance. These results point to a new action of S1P in cardioprotection and suggest that the mitochondrial S1P produced by SphK2 is required for the downstream protective modulation of PTP as an effector of preconditioning protection.

Topics: Animals; Blotting, Western; Ischemic Preconditioning; Lysophospholipids; Male; Mice; Mice, Transgenic; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Infarction; Phosphotransferases (Alcohol Group Acceptor); Prohibitins; RNA, Small Interfering; Signal Transduction; Sphingosine; Transfection

We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.

Topics: Cell Proliferation; Diglycerides; Enzyme Activation; Enzyme Activators; Gene Knockdown Techniques; Green Fluorescent Proteins; HEK293 Cells; Humans; Indoles; Insulin-Like Growth Factor II; Lysophospholipids; Maleimides; Mesangial Cells; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Protein Kinase C beta; Protein Transport; Receptor, IGF Type 1; Receptor, IGF Type 2; Recombinant Fusion Proteins; RNA Interference; Signal Transduction; Sphingosine; Tetradecanoylphorbol Acetate; Type C Phospholipases

Lysophospholipids such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are important signaling molecules that can regulate a wide range of cellular responses. We discovered that Sphingosine kinase 1 (Sphk1), a key enzyme that converts sphingosine to S1P, is expressed in neurons and progenitor cells in nascent trigeminal and dorsal root ganglia during mouse embryogenesis.. Sphk1 null mouse embryos do not display overt deficits owing to compensation by Sphk2. Thus, we analyzed embryos that are deficient in both Sphk1 and Sphk2 (which essentially eliminates S1P function) in order to investigate the role(s) of Sphk1 during sensory ganglia formation. While animals lacking 1-3 alleles of Sphk1 and Sphk2 had no obvious phenotype, embryos without both genes displayed clear developmental defects. The complete absence of Sphk1 and Sphk2 resulted in trigeminal and dorsal root ganglia with fewer neurons and progenitor cells. The profound loss in cell number could be attributed to a decrease in cell proliferation as well as an increase in apoptosis. Furthermore, Sphk1/2 double mutants displayed an overall reduction in other sphingolipids as well as an imbalance of S1P/sphingosine and S1P/ceramide ratio, thereby favoring cell death and reducing cell growth.. Together, these results provide strong in vivo evidence that sphingosine kinase/S1P signaling plays an important role in regulating early events during development of sensory ganglia.

Topics: Animals; Ganglia, Sensory; Genotype; In Situ Hybridization; Lysophospholipids; Mice; Mice, Mutant Strains; Neurons; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Stem Cells

Oxidative stress is involved in inducing apoptosis of photoreceptors in many retinal neurodegenerative diseases. It has been shown that oxidative stress increases in photoreceptors the synthesis of ceramide, a sphingolipid precursor that then activates apoptosis. In several cell types, ceramide is converted by ceramidases to sphingosine (Sph), another apoptosis mediator; hence, this study was undertaken to determine whether Sph participates in triggering photoreceptor apoptosis.. Rat retina neurons were incubated with [(3)H]palmitic acid and treated with the oxidant paraquat (PQ) to evaluate Sph synthesis. Sph was added to cultures with or without docosahexaenoic acid (DHA), the major retina polyunsaturated fatty acid and a photoreceptor survival factor, to evaluate apoptosis. Synthesis of Sph and sphingosine-1-phosphate (S1P), a prosurvival signal, were inhibited with alkaline ceramidase or sphingosine kinase inhibitors, respectively, before adding PQ, C(2)-ceramide, or Sph. Apoptosis, mitochondrial membrane polarization, cytochrome c localization, and reactive oxygen species (ROS) production were determined.. PQ increased [(3)H]Sph synthesis in photoreceptors and blocking this synthesis by inhibiting alkaline ceramidase decreased PQ-induced apoptosis. Addition of Sph induced photoreceptor apoptosis, increased ROS production, and promoted cytochrome c release from mitochondria. Although DHA prevented this apoptosis, inhibiting Sph conversion to S1P blocked DHA protection.. These results suggest that oxidative stress enhances formation of ceramide and its subsequent breakdown to Sph; ceramide and/or Sph would then trigger photoreceptor apoptosis. Preventing Sph synthesis or promoting its phosphorylation to S1P rescued photoreceptors, suggesting that Sph is a mediator of their apoptosis and modulation of Sph metabolism may be crucial for promoting photoreceptor survival.

Topics: Animals; Apoptosis; Cells, Cultured; Cytochromes c; Docosahexaenoic Acids; Enzyme Inhibitors; In Situ Nick-End Labeling; Lysophospholipids; Membrane Potential, Mitochondrial; Microscopy, Fluorescence; Oxidants; Oxidative Stress; Palmitic Acid; Paraquat; Phosphotransferases (Alcohol Group Acceptor); Photoreceptor Cells, Vertebrate; Rats; Rats, Wistar; Reactive Oxygen Species; Sphingosine

We recently reported that oxidized LDL (oxLDL) induces an oscillatory increase in intracellular calcium ([Ca(2+)](i)) levels in macrophages. Furthermore, we have shown that these [Ca(2+)](i) oscillations mediate oxLDL's ability to inhibit macrophage apoptosis in response to growth factor deprivation. However, the signal transduction pathways by which oxLDL induces [Ca(2+)](i) oscillations have not been elucidated. In this study, we show that these oscillations are mediated in part by intracellular mechanisms, as depleting extracellular Ca(2+) did not completely abolish the effect. Inhibiting sarco-endoplasmic reticulum ATPase (SERCA) completely blocked [Ca(2+)](i) oscillations, suggesting a role for Ca(2+) reuptake by the ER. The addition of oxLDL resulted in an almost immediate activation of sphingosine kinase (SK), which can increase sphingosine-1-phosphate (S1P) levels by phosphorylating sphingosine. Moreover, S1P was shown to be as effective as oxLDL in blocking macrophage apoptosis and producing [Ca(2+)](i) oscillations. This suggests that the mechanism in which oxLDL generates [Ca(2+)](i) oscillations may be 1) activation of SK, 2) SK-mediated increase in S1P levels, 3) S1P-mediated Ca(2+) release from intracellular stores, and 4) SERCA-mediated Ca(2+) reuptake back into the ER.

Topics: Animals; Biological Transport; Calcium; Calcium Channel Blockers; Cell Line; Cell Survival; Endoplasmic Reticulum; Enzyme Activation; Extracellular Space; Female; Humans; Lipoproteins, LDL; Lysophosphatidylcholines; Lysophospholipids; Macrophages; Mice; Phosphotransferases (Alcohol Group Acceptor); Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sphingosine; Thapsigargin; Type C Phospholipases

Vascular endothelial growth factor receptor (VEGFR) inhibition increases ceramides in lung structural cells of the alveolus, initiating apoptosis and alveolar destruction morphologically resembling emphysema. The effects of increased endogenous ceramides could be offset by sphingosine 1-phosphate (S1P), a prosurvival by-product of ceramide metabolism.. The aims of our work were to investigate the sphingosine-S1P-S1P receptor axis in the VEGFR inhibition model of emphysema and to determine whether stimulation of S1P signaling is sufficient to functionally antagonize alveolar space enlargement.. Concurrent to VEGFR blockade in mice, S1P signaling augmentation was achieved via treatment with the S1P precursor sphingosine, S1P agonist FTY720, or S1P receptor-1 (S1PR1) agonist SEW2871. Outcomes included sphingosine kinase-1 RNA expression and activity, sphingolipid measurements by combined liquid chromatography-tandem mass spectrometry, immunoblotting for prosurvival signaling pathways, caspase-3 activity and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays, and airspace morphometry.. Consistent with previously reported de novo activation of ceramide synthesis, VEGFR inhibition triggered increases in lung ceramides, dihydroceramides, and dihydrosphingosine, but did not alter sphingosine kinase activity or S1P levels. Administration of sphingosine decreased the ceramide-to-S1P ratio in the lung and inhibited alveolar space enlargement, along with activation of prosurvival signaling pathways and decreased lung parenchyma cell apoptosis. Sphingosine significantly opposed ceramide-induced apoptosis in cultured lung endothelial cells, but not epithelial cells. FTY720 or SEW2871 recapitulated the protective effects of sphingosine on airspace enlargement concomitant with attenuation of VEGFR inhibitor-induced lung apoptosis.. Strategies aimed at augmenting the S1P-S1PR1 signaling may be effective in ameliorating the apoptotic mechanisms of emphysema development.

Topics: Animals; Apoptosis; Blotting, Western; Cells, Cultured; Ceramides; Disease Models, Animal; Dose-Response Relationship, Drug; Fingolimod Hydrochloride; Indoles; Lysophospholipids; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Polymerase Chain Reaction; Propylene Glycols; Pulmonary Alveoli; Pulmonary Emphysema; Pyrroles; Receptors, Lysosphingolipid; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Sphingosine

An appropriate balance between uterine quiescence and activation during pregnancy is essential for a successful outcome. Sphingosine 1-phosphate (S1P), a bioactive lipid, increases cell survival, proliferation, and angiogenesis, all important to maintain the pregnancy. Indeed progesterone increases sphingosine kinase 1 (SPHK1) mRNA, which produces S1P. In contrast, induction of prostaglandin endoperoxide synthase 2 by S1P and stimulation of SPHK1 by estradiol and cytokines suggests a role for S1P in the termination of pregnancy. Human decidua is important for regulating the maintenance and termination of pregnancy with production of progesterone receptors, cytokines, and prostaglandins. We hypothesized that S1P is produced by and acts on the decidua to stimulate production of mediators that induce labor. Our objective was to investigate the metabolism of S1P and its receptors in human decidua during pregnancy. We found that SPHK1 protein and activity positively correlated with increasing gestational age in human decidua parietalis. This was accompanied at term by increased expression of the S1P lyase, which irreversibly degrades S1P. This implies increased S1P turnover in the decidua at term. Although the mRNA level of phosphatidic acid phosphatase type 2A and 2B (PPAP2A,B), which dephosphorylate extracellular S1P, were increased at term, PPAP2 activity did not change. Sphingosine 1-phosphate receptor 3 protein expression also increased at term, indicating increased signaling by S1P in the decidua. There were no differences in any parameter tested in decidua from women in labor compared to those who were not. This work provides the first evidence of increased S1P synthesis, degradation, and signaling in human decidua during gestation.

Topics: Adult; Decidua; Enzyme Activation; Enzymes; Female; Gene Expression Regulation, Developmental; Humans; Lysophospholipids; Phosphatidate Phosphatase; Phosphotransferases (Alcohol Group Acceptor); Pregnancy; Pregnancy Trimester, Third; Signal Transduction; Sphingosine; Up-Regulation; Young Adult

Sphingosine-1-phosphate and its receptors may be involved in vascular smooth muscle cell (VSMC) proliferation following vascular injury. Here, we evaluate the effect of d-erythro-N,N-dimethylsphingosine (DMS), a sphingosine kinase (SK) inhibitor, on VSMC proliferation, apoptosis and neointimal formation.. Growth responses in vitro to fetal calf serum (FCS) were measured by [(3)H]-thymidine incorporation and extracellular signal-regulated kinase-1/2 (ERK-1/2) activation in quiescent primary cultures of porcine VSMC in the presence and absence of various concentrations of the SK inhibitor DMS. In vivo treatment with DMS was delivered with a local endoluminal catheter, following balloon injury of coronary arteries. The artery intimal formation was investigated by angiography, myography and histomorphometry.. In vitro experiments indicated that DMS induced a dose-dependent reduction in [(3)H]-thymidine incorporation and ERK-1/2 activation via a protein kinase C (PKC) independent mechanism with an IC(50) value of 12 +/- 6 and 15 +/- 10 microM respectively. DMS also reduced Akt signalling. Four weeks following in vivo delivery of DMS, complete functional endothelial regeneration was observed in all treatment groups, with significant reduction of intimal formation (vehicle 23.7 +/- 4.6% vs. DMS infusion 8.92 +/- 2.9%, P < 0.05).. Taken together, these results demonstrate that local administration of the SK inhibitor, DMS, reduced neointimal formation, and this effect could involve inhibition of ERK-1/2 and Akt signalling, and modulation of smooth muscle growth.

Topics: Animals; Apoptosis; Catheterization; Cell Proliferation; Hyperplasia; Lysophospholipids; Male; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sphingosine; Sus scrofa; Tunica Intima

Lymphocyte egress from lymph nodes (LNs) is dependent on sphingosine-1-phosphate (S1P), but the cellular source of this S1P is not defined. We generated mice that expressed Cre from the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1) locus and that showed efficient recombination of loxP-flanked genes in lymphatic endothelium. We report that mice with Lyve-1 CRE-mediated ablation of sphingosine kinase (Sphk) 1 and lacking Sphk2 have a loss of S1P in lymph while maintaining normal plasma S1P. In Lyve-1 Cre+ Sphk-deficient mice, lymphocyte egress from LNs and Peyer's patches is blocked. Treatment with pertussis toxin to overcome Galphai-mediated retention signals restores lymphocyte egress. Furthermore, in the absence of lymphatic Sphks, the initial lymphatic vessels in nonlymphoid tissues show an irregular morphology and a less organized vascular endothelial cadherin distribution at cell-cell junctions. Our data provide evidence that lymphatic endothelial cells are an in vivo source of S1P required for lymphocyte egress from LNs and Peyer's patches, and suggest a role for S1P in lymphatic vessel maturation.

Topics: Animals; Cell Movement; Endothelial Cells; Glycoproteins; GTP-Binding Protein alpha Subunits, Gi-Go; Intercellular Junctions; Lymph Nodes; Lymphocytes; Lysophospholipids; Membrane Transport Proteins; Mice; Mice, Knockout; Pertussis Toxin; Peyer's Patches; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Microglial activation has been implicated as one of the causative factors for neuroinflammation in various neurodegenerative diseases. The sphingolipid metabolic pathway plays an important role in inflammation, cell proliferation, survival, chemotaxis, and immunity in peripheral macrophages. In this study, we demonstrate that sphingosine kinase1 (SphK1), a key enzyme of the sphingolipid metabolic pathway, and its receptors are expressed in the mouse BV2 microglial cells and SphK1 alters the expression and production of proinflammatory cytokines and nitric oxide in microglia treated with lipopolysaccharide (LPS). LPS treatment increased the SphK1 mRNA and protein expression in microglia as revealed by the RT-PCR, Western blot and immunofluorescence. Suppression of SphK1 by its inhibitor, N, N Dimethylsphingosine (DMS), or siRNA resulted in decreased mRNA expression of TNF-alpha, IL-1beta, and iNOS and release of TNF-alpha and nitric oxide (NO) in LPS-activated microglia. Moreover, addition of sphingosine 1 phosphate (S1P), a breakdown product of sphingolipid metabolism, increased the expression levels of TNF-alpha, IL-1beta and iNOS and production of TNF-alpha and NO in activated microglia. Hence to summarize, suppression of SphK1 in activated microglia inhibits the production of proinflammatory cytokines and NO and the addition of exogenous S1P to activated microglia enhances their inflammatory responses. Since the chronic proinflammatory cytokine production by microglia has been implicated in neuroinflammation, modulation of SphK1 and S1P in microglia could be looked upon as a future potential therapeutic method in the control of neuroinflammation in neurodegenerative diseases.

Topics: Animals; Cell Line; Cytokines; Encephalitis; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gliosis; Interleukin-1beta; Lipopolysaccharides; Lysophospholipids; Mice; Microglia; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphotransferases (Alcohol Group Acceptor); RNA Interference; RNA, Messenger; Sphingosine; Tumor Necrosis Factor-alpha; Up-Regulation

Sphingosine 1-phosphate (S1P), a potent sphingolipid mediator produced by sphingosine kinase isoenzymes (SphK1 and SphK2), regulates diverse cellular processes important for breast cancer progression acting in an autocrine and/or paracrine manner. Here we show that SphK1, but not SphK2, increased S1P export from MCF-7 cells. Whereas for both estradiol (E(2)) and epidermal growth factor-activated SphK1 and production of S1P, only E(2) stimulated rapid release of S1P and dihydro-S1P from MCF-7 cells. E(2)-induced S1P and dihydro-S1P export required estrogen receptor-alpha, not GPR30, and was suppressed either by pharmacological inhibitors or gene silencing of ABCC1 (multidrug resistant protein 1) or ABCG2 (breast cancer resistance protein). Inhibiting these transporters also blocked E(2)-induced activation of ERK1/2, indicating that E(2) activates ERK via downstream signaling of S1P. Taken together, our findings suggest that E(2)-induced export of S1P mediated by ABCC1 and ABCG2 transporters and consequent activation of S1P receptors may contribute to nongenomic signaling of E(2) important for breast cancer pathophysiology.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Estradiol; Estrogens; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrometry, Mass, Electrospray Ionization; Sphingosine

Execution of physiologic cell death known as apoptosis is tightly regulated and transfers immunologically relevant information. This ensures efficient clearance of dying cells and shapes the phenotype of their "captors" toward anti-inflammatory. Here, we identify a mechanism of sphingosine-1-phosphate production by apoptotic cells. During cell death, sphingosine kinase 2 (SphK2) is cleaved at its N-terminus in a caspase-1-dependent manner. Thereupon, a truncated but enzymatically active fragment of SphK2 is released from cells. This step is coupled to phosphatidylserine exposure, which is a hallmark of apoptosis and a crucial signal for phagocyte/apoptotic cell interaction. Our data link signaling events during apoptosis to the extracellular production of a lipid mediator that affects immune cell attraction and activation.

Topics: Animals; Apoptosis; Caspase 1; Humans; Jurkat Cells; Lysophospholipids; Mice; NIH 3T3 Cells; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine kinases (SKs) 1 and 2 produce high concentrations of sphingosine 1-phosphate (S1P) in blood and lymph. In contrast, S1P concentrations in lymphoid tissues are kept low by the S1P-degrading activity of the S1P-lyase. These differences in S1P concentrations drive lymphocyte circulation. Inhibition of the S1P-lyase prevents lymphocyte egress and causes lymphopenia because of increased S1P levels in lymphoid tissues. In this study, we investigated the source of this accumulating S1P in lymphoid tissues by using SK2-deficient (SK2(-/-)) mice. In contrast to wild-type mice, SK2(-/-) mice exhibited attenuated lymphopenia after S1P-lyase inhibition by 4-deoxypyridoxine (DOP). Consistently, S1P concentrations were only modestly increased in lymphoid tissues of SK2(-/-) mice compared with a significantly higher increase in wild-type mice after DOP treatment. Low S1P concentrations in lymphoid tissues of DOP-treated SK2(-/-) mice were accompanied by higher S1P concentrations in blood, suggesting that SK2(-/-) mice display defective S1P transport from blood into lymphoid tissues. To investigate this potential new role of SK2, RBCs loaded with traceable C17-S1P were transfused into wild-type and SK2(-/-) mice, resulting in much higher C17-S1P concentrations in blood of SK2(-/-) mice compared with wild-type mice 2 h after transfusion. Moreover, cocultures of RBCs with mouse splenocytes and endothelial cells demonstrated that SK2 regulated cellular uptake of S1P from RBCs. Collectively, our data suggest that S1P in lymphoid tissues derives from blood and point to an essential role of SK2 in S1P transport.

Topics: Animals; B-Lymphocyte Subsets; Catalysis; Cells, Cultured; Coculture Techniques; Endothelium, Vascular; Humans; Lymphoid Tissue; Lymphopenia; Lysophospholipids; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Transport; Sphingosine; T-Lymphocyte Subsets

We previously showed that the inhalational anesthetic isoflurane protects against renal ischemia reperfusion injury in part via sphingosine kinase (SK)-mediated synthesis of sphingosine-1-phosphate (S1P). In this study, we tested the hypothesis that isoflurane directly targets renal proximal tubule cells via SK activation, S1P synthesis and activation of S1P receptors to initiate cytoprotective signaling.. Isoflurane-mediated phosphorylation of extracellular signal-regulated kinase (ERK) and Akt and induction of HSP70 in human kidney proximal tubule (HK-2) cells were inhibited by dimethylsphingosine (DMS), an SK inhibitor, and VPC23019, an S1P(1/3) receptor selective antagonist, in HK-2 cells. A selective S1P(1) receptor agonist, SEW2781, mimicked isoflurane-induced phosphorylation of ERK and Akt and induction of HSP70. Moreover, isoflurane-mediated protection against H(2)O(2)-induced necrosis of HK-2 cells was significantly attenuated by an S1P(1/3) receptor antagonist, VPC23019, and by SK inhibitors DMS or 4-[[4- (4-chlorophenyl)-2-thiazolyl]amino]phenol. Finally, overexpression of the SK1 enzyme in HK-2 cells protected against H(2)O(2)-induced necrosis.. Collectively, our study demonstrates that S1P released via isoflurane-mediated SK1 stimulation produces direct anti-necrotic effects probably via S1P(1) receptor-mediated cytoprotective signaling (ERK/Akt phosphorylation and HSP70 induction) in HK-2 cells. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated renal protection and lead to new therapeutic applications of volatile anesthetics during the perioperative period.

Topics: Anesthetics, Inhalation; Cells, Cultured; Humans; Isoflurane; Kidney Tubules, Proximal; Lysophospholipids; Necrosis; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. Sphingosine 1-phosphate is a bioactive sphingolipid that regulates crucial physiological processes. Here we report that this lipid triggers acrosomal exocytosis in human sperm by a mechanism involving a G(i)-coupled receptor. Real-time imaging showed a remarkable increase of cytosolic calcium upon activation with sphingosine 1-phosphate and pharmacological experiments indicate that the process requires extracellular calcium influx through voltage and store-operated calcium channels and efflux from intracellular stores through inositol 1,4,5-trisphosphate-sensitive calcium channels. Sphingosine 1-phosphate-induced exocytosis requires phospholipase C and protein kinase C activation. We investigated possible sources of the lipid. Western blot indicates that sphingosine kinase 1 is present in spermatozoa. Indirect immunofluorescence showed that phorbol ester, a potent protein kinase C activator that can also trigger acrosomal exocytosis, redistributes sphingosine kinase 1 to the acrosomal region. Functional assays showed that phorbol ester-induced exocytosis depends on the activation of sphingosine kinase 1. Furthermore, incorporation of (32)P to sphingosine demonstrates that cells treated with the phorbol ester increase their sphingosine kinase activity that yields sphingosine 1-phosphate. We present here the first evidence indicating that human spermatozoa produce sphingosine 1-phosphate when challenged with an exocytic stimulus. These observations point to a new role of sphingosine 1-phosphate in a signaling cascade that facilitates acrosome reaction providing some clues about novel lipid molecules involved in exocytosis.

Topics: Acrosome; Enzyme Activation; Exocytosis; Female; Humans; Lysophospholipids; Male; Phorbol Esters; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

The mechanism underlying the protective effect of sphingosine kinase 1 (SphK1) in inflammatory injury is not clear. We demonstrated using SphK1-null mice (SphK1(-/-)) the crucial role of SphK1 in suppressing lipopolysaccharide-induced neutrophil oxidant production and sequestration in lungs and mitigating lung inflammatory injury. This effect of SphK1 was independent of the production of sphingosine 1-phosphate, the product of SphK1 activity. The anti-inflammatory effect of SphK1 in the lipopolysaccharide model was mediated through SphK1 interaction with JNK. SphK1 stabilization of JNK in turn inhibited JNK binding to the JNK-interacting protein 3 (JIP3) and thus abrogated the activation of NADPH oxidase and oxidant generation and resultant NF-kappaB activation. Therefore, SphK1-mediated down-regulation of JNK activity serves to dampen inflammation and tissue injury.

Topics: Adaptor Proteins, Signal Transducing; Animals; Down-Regulation; Enzyme Activation; Lipopolysaccharides; Lung; Lysophospholipids; MAP Kinase Kinase 4; Mice; Mice, Knockout; NADPH Oxidases; Nerve Tissue Proteins; Neutrophils; Oxidants; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Sphingosine

Sphingosine kinase (SphK) is a key enzyme in modulating the levels of sphingosine 1-phosphate (S1P) as well as an important enzyme in numerous biological responses. Using C17-sphingosine as a substrate, we established a rapid, sensitive and highly efficient method for determination of SphK activity by analyzing the product C17-sphingosine 1-phosphate (C17-S1P) using liquid chromatography-tandem mass spectrometry. The standard curve for C17-S1P was linear over a wide range (10-1000 ng/mL) with correlation coefficient (r(2)) greater than 0.999. The lower limit of quantification for C17-S1P was 10 ng/mL. The K(m) values for C17-sphingosine and ATP were determined to be 28.17 and 188.5  microM, respectively. More importantly, the SphK activity dramatically increased in cultured HEK 293 cells expressing wild-type SphK1 as well as cells treated with tumor necrosis factor-alpha, a sphingosine kinase activator. In contrast, the SphK activity decreased in cultured HEK 293 cells treated with dimethylsphngosine, a sphingosine kinase inhibitor. In conclusion, this method was sensitive and rapid in the determination of SphK acitivity, providing striking utilities in exploring the sphingosine kinase signaling pathway and screening active compounds targeting SphK activity.

Topics: Adenosine Triphosphate; Analysis of Variance; Cell Line; Chromatography, Liquid; Humans; Lysophospholipids; Nonlinear Dynamics; Phosphotransferases (Alcohol Group Acceptor); Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Sphingosine; Tandem Mass Spectrometry; Tumor Necrosis Factor-alpha

Neutral ceramidase (NCDase) and sphingosine kinases (SphKs) are key enzymes regulating cellular sphingosine-1-phosphate (S1P) levels. In this study we found that stress factor-induced apoptosis of rat renal mesangial cells was significantly reduced by dexamethasone treatment. Concomitantly, dexamethasone increased cellular S1P levels, suggesting an activation of sphingolipid-metabolizing enzymes. The cell-protective effect of glucocorticoids was reversed by a SphK inhibitor, was completely absent in SphK1-deficient cells, and was associated with upregulated mRNA and protein expression of NCDase and SphK1. Additionally, in vivo experiments in mice showed that dexamethasone also upregulated SphK1 mRNA and activity, and NCDase protein expression in the kidney. Fragments (2285, 1724, and 1126 bp) of the rat NCDase promoter linked to a luciferase reporter were transfected into rat kidney fibroblasts and mesangial cells. There was enhanced NCDase promoter activity upon glucocorticoids treatment that was abolished by the glucocorticoid receptor antagonist RU-486. Single and double mutations of the two putative glucocorticoid response element sites within the promoter reduced the dexamethasone effect, suggesting that both glucocorticoid response elements are functionally active and required for induction. Our study shows that glucocorticoids exert a protective effect on stress-induced mesangial cell apoptosis in vitro and in vivo by upregulating NCDase and SphK1 expression and activity, resulting in enhanced levels of the protective lipid second messenger S1P.

Topics: Animals; Apoptosis; Cell Differentiation; Cells; Dexamethasone; Fibroblasts; Glucocorticoids; Lysophospholipids; Mesangial Cells; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mifepristone; Neutral Ceramidase; Phosphotransferases (Alcohol Group Acceptor); Rats; Response Elements; RNA, Messenger; Sphingolipids; Sphingosine

Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artificial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PM-targeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an efficient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we find that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specific phosphatases are depleted, acute labeling of S1P is significantly reduced, indicative of a phosphatase-dependent recycling process. We conclude that the localization of SK1 influences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites.

Topics: Cell Membrane; Ceramides; Endoplasmic Reticulum; Gene Knockdown Techniques; HeLa Cells; Humans; Lysophospholipids; Membrane Proteins; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Recombinant Proteins; Sphingosine

Activated protein C (APC), the only FDA-approved biotherapeutic drug for sepsis, possesses anticoagulant, antiinflammatory, and barrier-protective activities. However, the mechanisms underlying its anti-inflammatory functions are not well defined. Here, we report that the antiinflammatory activity of APC on macrophages is dependent on integrin CD11b/CD18, but not on endothelial protein C receptor (EPCR). We showed that CD11b/CD18 bound APC within specialized membrane microdomains/lipid rafts and facilitated APC cleavage and activation of protease-activated receptor-1 (PAR1), leading to enhanced production of sphingosine-1-phosphate (S1P) and suppression of the proinflammatory response of activated macrophages. Deletion of the gamma-carboxyglutamic acid domain of APC, a region critical for its anticoagulant activity and EPCR-dependent barrier protection, had no effect on its antiinflammatory function. Genetic inactivation of CD11b, PAR1, or sphingosine kinase-1, but not EPCR, abolished the ability of APC to suppress the macrophage inflammatory response in vitro. Using an LPS-induced mouse model of lethal endotoxemia, we showed that APC administration reduced the mortality of wild-type mice, but not CD11b-deficient mice. These data establish what we believe to be a novel mechanism underlying the antiinflammatory activity of APC in the setting of endotoxemia and provide clear evidence that the antiinflammatory function of APC is distinct from its barrier-protective function and anticoagulant activities.

Topics: 1-Carboxyglutamic Acid; Animals; Endotoxemia; Integrins; Lipopolysaccharides; Lysophospholipids; Membrane Microdomains; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Protein C; Sepsis; Sphingosine

Sphingosine kinase-1 is known to mediate Mycobacterium smegmatis induced inflammatory responses in macrophages, but its role in controlling infection has not been reported to date. We aimed to unravel the significance of SphK-1 in controlling M. smegmatis infection in RAW 264.7 macrophages. Our results demonstrated for the first time that selective inhibition of SphK-1 by either D, L threo dihydrosphingosine (DHS; a competitive inhibitor of Sphk-1) or Sphk-1 siRNA rendered RAW macrophages sensitive to M. smegmatis infection. This was due to the reduction in the expression of iNOs, p38, pp-38, late phagosomal marker, LAMP-2 and stabilization of the RelA (pp-65) subunit of NF-kappaB. This led to a reduction in the generation of NO and secretion of TNF-alpha in infected macrophages. Congruently, overexpression of SphK-1 conferred resistance in macrophages to infection which was due to enhancement in the generation of NO and expression of iNOs, pp38 and LAMP-2. In addition, our results also unraveled a novel regulation of p38MAPK by SphK-1 during M. smegmatis infection and generation of NO in macrophages. Enhanced NO generation and expression of iNOs in SphK-1++ infected macrophages demonstrated their M-1(bright) phenotype of these macrophages. These findings thus suggested a novel antimycobacterial role of SphK-1 in macrophages.

Topics: Animals; Lipopolysaccharides; Lysophospholipids; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium Infections, Nontuberculous; Mycobacterium smegmatis; Nitric Oxide; p38 Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Reproducibility of Results; Sphingosine; Tumor Necrosis Factor-alpha

We demonstrate here a new concept termed "oncogene tolerance" whereby human EGF receptor 2 (HER2) increases sphingosine kinase 1 (SK1) expression in estrogen receptor-positive (ER(+)) MCF-7 HER2 cells and SK1, in turn, limits HER2 expression in a negative-feedback manner. The HER2-dependent increase in SK1 expression also limits p21-activated protein kinase 1 (p65 PAK1) and extracellular signal regulated kinase 1/2 (ERK-1/2) signaling. Sphingosine 1-phosphate signaling via S1P(3) is also altered in MCF-7 HER2 cells. In this regard, S1P binding to S1P(3) induces a migratory phenotype via an SK1-dependent mechanism in ER(+) MCF-7 Neo cells, which lack HER2. This involves the S1P stimulated accumulation of phosphorylated ERK-1/2 and actin into membrane ruffles/lamellipodia and migration. In contrast, S1P failed to promote redistribution of phosphorylated ERK-1/2 and actin into membrane ruffles/lamellipodia or migration of MCF-7 HER2 cells. However, a migratory phenotype in these cells could be induced in response to S1P when SK1 expression had been knocked down with a specific siRNA or when recombinant PAK1 was ectopically overexpressed. Thus, the HER2-dependent increase in SK1 expression functions to desensitize the S1P-induced formation of a migratory phenotype. This is correlated with improved prognosis in patients who have a low HER1-3/SK1 expression ratio in their ER(+) breast cancer tumors compared to patients that have a high HER1-3/SK1 expression ratio.

Topics: Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Estrogen Receptor alpha; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; p21-Activated Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Receptors, Estrogen; RNA, Small Interfering; Signal Transduction; Sphingosine

ENOX2 (tNOX), a tumor-associated cell surface ubiquinol (NADH) oxidase, functions as an alternative terminal oxidase for plasma membrane electron transport. Ubiquitous in all cancer cell lines studied thus far, ENOX2 expression correlates with the abnormal growth and division associated with the malignant phenotype. ENOX2 has been proposed as the cellular target for various quinone site inhibitors that demonstrate anticancer activity such as the green tea constituent epigallocatechin-3-gallate (EGCg) and the isoflavone phenoxodiol (PXD). Here we present a possible mechanism that explains how these substances result in apoptosis in cancer cells by ENOX2-mediated alterations of cytosolic amounts of NAD(+) and NADH. When ENOX2 is inhibited, plasma membrane electron transport is diminished, and cytosolic NADH accumulates. We show in HeLa cells that NADH levels modulate the activities of two pivotal enzymes of sphingolipid metabolism: sphingosine kinase 1 (SK1) and neutral sphingomyelinase (nSMase). Their respective products sphingosine 1-phosphate (S1P) and ceramide (Cer) are key determinants of cell fate. S1P promotes cell survival and Cer promotes apoptosis. Using plasma membranes isolated from cervical adenocarcinoma (HeLa) cells as well as purified proteins of both bacterial and human origin, we demonstrate that NADH inhibits SK1 and stimulates nSMase, while NAD(+) inhibits nSMase and has no effect on SK1. Additionally, intact HeLa cells treated with ENOX2 inhibitors exhibit an increase in Cer and a decrease in S1P. Treatments that stimulate cytosolic NADH production potentiate the antiproliferative effects of ENOX2 inhibitors while those that attenuate NADH production or stimulate plasma membrane electron transport confer a survival advantage.

Topics: Anticarcinogenic Agents; Apoptosis; Catechin; Cell Line; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Cell Survival; Ceramides; Chromatography, Thin Layer; Cytosol; Dose-Response Relationship, Drug; HeLa Cells; Humans; Isoflavones; Lysophospholipids; Multienzyme Complexes; NAD; NADH, NADPH Oxidoreductases; Phosphotransferases (Alcohol Group Acceptor); Sphingolipids; Sphingomyelin Phosphodiesterase; Sphingosine

Sphingosine 1-phosphate (S1P)-metabolizing enzymes regulate the level of sphingolipids and have important biological functions. However, the effects of S1P-metabolizing enzymes on host defense against invading viruses remain unknown. In this study, we investigated the role of S1P-metabolizing enzymes in modulating cellular responses to influenza virus infection. Overexpression of S1P lyase (SPL), which induces the degradation of S1P, interfered with the amplification of infectious influenza virus. Accordingly, SPL-overexpressing cells were much more resistant than control cells to the cytopathic effects caused by influenza virus infection. SPL-mediated inhibition of virus-induced cell death was supported by impairment of the upregulation of the proapoptotic protein Bax, a critical factor for influenza virus cytopathogenicity. Importantly, influenza virus infection of SPL-overexpressing cells induced rapid activation of extracellular signal-regulated kinase (ERK) and STAT1 but not of p38 mitogen-activated protein kinase (MAPK), Akt, or c-Jun N-terminal kinase (JNK). Blockade of STAT1 expression or inhibition of Janus kinase (JAK) activity elevated the level of influenza virus replication in the cells, indicating that SPL protects cells from influenza virus via the activation of JAK/STAT signaling. In contrast to that of SPL, the overexpression of S1P-producing sphingosine kinase 1 heightened the cells' susceptibility to influenza virus infection, an effect that was reversed by the inhibition of its kinase activity, representing opposed enzymatic activity. These findings indicate that the modulation of S1P-metabolizing enzymes is crucial for controlling the host defense against infection with influenza virus. Thus, S1P-metabolizing enzymes are novel potential targets for the treatment of diseases caused by influenza virus infection.

Topics: Aldehyde-Lyases; Cell Line; Cell Survival; Cytopathogenic Effect, Viral; Humans; Influenza A Virus, H1N1 Subtype; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Tumour-necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key component in NF-kappaB signalling triggered by TNF-alpha. Genetic evidence indicates that TRAF2 is necessary for the polyubiquitination of receptor interacting protein 1 (RIP1) that then serves as a platform for recruitment and stimulation of IkappaB kinase, leading to activation of the transcription factor NF-kappaB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyses the ubiquitination of RIP1 is lacking. TRAF2 binds to sphingosine kinase 1 (SphK1), one of the isoenzymes that generates the pro-survival lipid mediator sphingosine-1-phosphate (S1P) inside cells. Here we show that SphK1 and the production of S1P is necessary for lysine-63-linked polyubiquitination of RIP1, phosphorylation of IkappaB kinase and IkappaBalpha, and IkappaBalpha degradation, leading to NF-kappaB activation. These responses were mediated by intracellular S1P independently of its cell surface G-protein-coupled receptors. S1P specifically binds to TRAF2 at the amino-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, markedly increased recombinant TRAF2-catalysed lysine-63-linked, but not lysine-48-linked, polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a. Our data show that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing cofactor for TRAF2 E3 ubiquitin ligase activity, indicating a new paradigm for the regulation of lysine-63-linked polyubiquitination. These results also highlight the key role of SphK1 and its product S1P in TNF-alpha signalling and the canonical NF-kappaB activation pathway important in inflammatory, antiapoptotic and immune processes.

Topics: Animals; Biocatalysis; Cell Line; Enzyme Activation; Humans; I-kappa B Kinase; I-kappa B Proteins; Lysine; Lysophospholipids; Mice; Models, Molecular; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Protein Structure, Tertiary; Receptor-Interacting Protein Serine-Threonine Kinases; Sphingosine; Substrate Specificity; TNF Receptor-Associated Factor 2; Tumor Necrosis Factor-alpha; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Ubiquitination

Liver regeneration after partial hepatectomy (PH) is achieved by intense cells proliferation. Sphingosine-1-phosphate stimulates proliferation but ceramide and sphingosine induce apoptosis. The aim of the study was to investigate the influence of high-fat diet (HFD) on the sphingolipid metabolism during the first 24h of liver regeneration in rats. Rats were fed HFD or standard diet for 7 days prior to the PH. The content of sphingolipids and the activity of sphingomyelinases (n and aSMase), ceramidases (n and aCDase) and sphingosine kinase (SPHK) were measured. It has been found that HFD increased the activity of aSMase and nCDase at 4th hour after PH. The content of ceramide and sphingosine decreased in HFD group at each time point. This was accompanied by elevated content of sphingosine-1-phosphate and sphinganine-1-phosphate. Decrease in SPHK activity in cytosol after partial hepatectomy was inversely correlated (r=-0.7538) with increase in S1P, which suggest translocation of SPHK to plasma membrane. Shingosine-1-phosphate to ceramide ratio was higher in rats fed HFD. It is concluded that HFD stimulates the pro-mitotic action of the sphingolipid signaling in regenerating rat liver.

Topics: Animals; Ceramidases; Ceramides; Diet; Dietary Fats; Hepatectomy; Hydrogen-Ion Concentration; Linoleic Acid; Liver; Liver Regeneration; Lysophospholipids; Male; Oleic Acid; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Second Messenger Systems; Sphingolipids; Sphingomyelin Phosphodiesterase; Sphingosine

Sphingosine 1-phosphate (S1P) is involved in the local inflammatory response within the intestinal muscularis, which has been suggested to play a major role in the pathogenesis of postoperative ileus. The aim of the present study was to elucidate the role of S1P and the molecular mechanisms underlying regulation of inflammatory mediators in primary cultured rat intestinal smooth muscle (RISM) cells. Although our experimental data clearly show the mediatory role of sphingosine kinase (SK)-derived S1P in the TNF-alpha and the LPS induced activation of NF-kB, exogenously added S1P failed to trigger this transcription factor. Instead, exogenous S1P induced early growth response-1 (Egr-1), which was reported to play a proinflammatory role in postoperative ileus. Using RNA interference we found that Egr-1 is required primarily for S1P-induced expression of IL-1 and COX2. Conversely, IL-6 expression following S1P treatment was mediated by STAT3 (signal transducer and activator of transcription-3). In addition our data indicate that the proinflammatory effect of S1P is mediated by its receptors S1P(1-3) and requires activation of MAP-kinases. In conclusion, Egr-1 and STAT3 cooperatively mediate S1P-induced inflammatory responses in RISM cells, providing novel targets for attenuation of postoperative ileus.

Topics: Animals; Cells, Cultured; Cyclooxygenase 2; Early Growth Response Protein 1; Inflammation Mediators; Interleukin-1; Interleukin-6; Intestines; Lysophospholipids; Male; Myocytes, Smooth Muscle; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; RNA Interference; RNA, Small Cytoplasmic; Sphingosine; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha

Sphingosine kinase 1 (SphK1) is an enzyme that converts sphingosine to bioactive sphingosine-1-phosphate. Recent in vitro data suggest a potential role of SphK1 in TNF-alpha-mediated inflammation. Our aims in this study were to determine the in vivo significance of SphK1 in TNF-alpha-mediated chronic inflammation and to define which pathogenic mechanisms induced by TNF-alpha are SphK1 dependent. To pursue these aims, we studied the effect of SphK1 deficiency in an in vivo model of TNF-alpha-induced chronic inflammatory arthritis. Transgenic hTNF-alpha mice, which develop spontaneous inflammatory erosive arthritis beginning at 14-16 wk, were crossed with SphK1 null mice (SphK1(-/-)), on the C57BL6 genetic background. Beginning at 4 mo of age, hTNF/SphK1(-/-) mice had significantly less severe clinically evident paw swelling and deformity, less synovial and periarticular inflammation, and markedly decreased bone erosions as measured quantitatively through micro-CT images. Mechanistically, the mice lacking SphK1 had less articular cyclooxygenase 2 protein and fewer synovial Th17 cells than did hTNF/SphK1(+/+) littermates. Microarray analysis and real-time RT-PCR of the ankle synovial tissue demonstrated that hTNF/SphK1(-/-) mice had increased transcript levels of suppressor of cytokine signaling 3 compared with hTNF/SphK1(+/+) mice, likely also contributing to the decreased inflammation in the SphK1-deficient mice. Finally, significantly fewer mature osteoclasts were detected in the ankle joints of hTNF/SphK1(-/-) mice compared with hTNF/SphK1(+/+) mice. These data indicate that SphK1 plays a key role in hTNF-alpha-induced inflammatory arthritis via impacting synovial inflammation and osteoclast number.

Topics: Animals; Ankle Joint; Arthritis; Cyclooxygenase 2; Female; Foot Joints; Gene Expression Profiling; Humans; Immunoblotting; Immunohistochemistry; Joints; Lysophospholipids; Male; Mice; Mice, Knockout; Mice, Transgenic; Osteoclasts; Phosphotransferases (Alcohol Group Acceptor); Reverse Transcriptase Polymerase Chain Reaction; Severity of Illness Index; Sphingolipids; Sphingosine; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Synovial Membrane; Synovitis; Tumor Necrosis Factor-alpha

Berberine (BBR) was previously found to have beneficial effects on renal injury in experimental diabetic rats. However, the mechanisms underlying the effects are not fully understood. Sphingosine kinase-Sphingosine 1-phosphate (SphK-S1P) signaling pathway has been implicated in the pathogenesis of diabetic nephropathy (DN). The aim of this study was to investigate the effects of BBR on renal injury and the activation of SphK-S1P signaling pathway in alloxan-induced diabetic mice with nephropathy. Alloxan-induced diabetic mice were treated orally with BBR (300 mg/kg/day) or vehicle for 12 weeks. BBR inhibited the increases in fasting blood glucose, kidney/body weight ratio, blood urea nitrogen, serum creatinine and 24-h albuminuria in diabetic mice. It also prevented renal hypertrophy, TGF-beta1 synthesis, FN and Col IV accumulation. Moreover, BBR down-regulated the elevated staining, activity and levels of mRNA and protein of SphK1, and S1P production as well. These findings suggest that the inhibitory effect of BBR on the activation of SphK-S1P signaling pathway in diabetic mouse kidney is a novel mechanism by which BBR partly exerts renoprotective effects on DN.

Topics: Albuminuria; Animals; Berberine; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Glucose; Kidney; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Random Allocation; Signal Transduction; Sphingosine; Transforming Growth Factor beta1

Sphingosine-1-phosphate (S1P) is a significant lipid messenger modulating many physiological responses. S1P plays a critical role in autoimmune disease and is suggested to be involved in Sjögren's syndrome pathology. However, the mechanism of S1P signaling in salivary glands is unclear. Here we studied the effects of S1P on normal human submandibular gland cells. S1P increased levels of the intracellular Ca(2+) concentration ([Ca(2+)](i)), which was inhibited by pre-treatment with U73122 or 2-aminoethoxydiphenyl borate (2-APB). Pre-treated S1P did not inhibit subsequent carbachol-induced [Ca(2+)](i) increase, which suggests that S1P and muscarinic signaling are independent of each other. S1P1, S1P2, and S1P3 receptors SphK1 and SphK2 were commonly expressed in human salivary gland cells. S1P, but not carbachol, induces the expression of interleukin-6 and Fas. Our results suggest that S1P triggers Ca(2+) signaling and the apoptotic pathway in normal submandibular gland cells, which suggests in turn that S1P affects the progression of Sjögren's syndrome.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Boron Compounds; Calcium Signaling; Carbachol; Cell Culture Techniques; Cells, Cultured; Cholinergic Agonists; Estrenes; fas Receptor; Female; Humans; Interleukin-6; Lysophospholipids; Male; Middle Aged; Phosphodiesterase Inhibitors; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidinones; Receptors, Lysosphingolipid; Signal Transduction; Sjogren's Syndrome; Sphingosine; Submandibular Gland; Type C Phospholipases

Sphingolipid metabolism is driven by inflammatory cytokines. These cascade of events include the activation of sphingosine kinase (SK), and subsequent production of the mitogenic and proinflammatory lipid sphingosine 1-phosphate (S1P). Overall, S1P is one of the crucial components in inflammation, making SK an excellent target for the development of new anti-inflammatory drugs. We have recently shown that SK inhibitors suppress colitis and hypothesize here that the novel SK inhibitor, ABC294640, prevents the development of colon cancer. In an azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model, there was a dose-dependent decrease in tumor incidence with SK inhibitor treatment. The tumor incidence (number of animals with tumors per group) in the vehicle, ABC294640 (20 mg/kg) and ABC294640 (50 mg/kg) groups were 80, 40 and 30%, respectively. Tumor multiplicity (number of tumors per animal) also decreased from 2.1 ± 0.23 tumors per animal in the AOM + DSS + vehicle group to 1.2 ± 0 tumors per animal in the AOM + DSS + ABC294640 (20 mg/kg) and to 0.8 ± 0.4 tumors per animal in the AOM + DSS + ABC294640 (50 mg/kg) group. Importantly, with ABC294640, there were no observed toxic side effects. To explore mechanisms, we isolated cells from the colon (CD45-, representing primarily colon epithelial cells) and (CD45+, representing primarily colon inflammatory cells) then measured known targets of SK that control cell survival. Results are consistent with the hypothesis that the inhibition of SK activity by our novel SK inhibitor modulates key pathways involved in cell survival and may be a viable treatment strategy for the chemoprevention colitis-driven colon cancer.

Topics: Adamantane; Animals; Azoxymethane; Colitis; Colon; Colonic Neoplasms; Dextran Sulfate; Extracellular Signal-Regulated MAP Kinases; Lysophospholipids; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Pyridines; Sphingosine

Various studies in cell lines have previously demonstrated that sphingosine kinase 1 (SK1) and extracellular signal-regulated kinase 1/2 (ERK-1/2) interact in an estrogen receptor (ER)-dependent manner to influence both breast cancer cell growth and migration. A cohort of 304 ER-positive breast cancer patients was used to investigate the prognostic significance of sphingosine 1-phosphate (S1P) receptors 1, 2, and 3 (ie, S1P1, S1P2, and S1P3), SK1, and ERK-1/2 expression levels. Expression levels of both SK1 and ERK-1/2 were already available for the cohort, and S1P1, S1P2, and S1P3 levels were established by immunohistochemical analysis. High membrane S1P1 expression was associated with shorter time to recurrence (P=0.008). High cytoplasmic S1P1 and S1P3 expression levels were also associated with shorter disease-specific survival times (P=0.036 and P=0.019, respectively). Those patients with tumors that expressed high levels of both cytoplasmic SK1 and ERK-1/2 had significantly shorter recurrence times than those that expressed low levels of cytoplasmic SK1 and cytoplasmic ERK-1/2 (P=0.00008), with a difference in recurrence time of 10.5 years. Similarly, high cytoplasmic S1P1 and cytoplasmic ERK-1/2 expression levels (P=0.004) and high cytoplasmic S1P3 expression and cytoplasmic ERK-1/2 expression levels (P=0.004) were associated with shorter recurrence times. These results support a model in which the interaction between SK1, S1P1, and/or S1P3 and ERK-1/2 might drive breast cancer progression, and these findings, therefore, warrant further investigation.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; HEK293 Cells; Humans; Lysophospholipids; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphotransferases (Alcohol Group Acceptor); Protein Isoforms; Receptors, Estrogen; Receptors, Lysosphingolipid; RNA, Small Interfering; Sphingosine; Survival Rate; Tamoxifen

We sought to demonstrate that tumor necrosis factor (TNF)-α, via sphingosine-1-phosphate signaling, has the potential to alter cochlear blood flow and thus, cause ischemic hearing loss.. We performed intravital fluorescence microscopy to measure blood flow and capillary diameter in anesthetized guinea pigs. To measure capillary diameter ex vivo, capillary beds from the gerbil spiral ligament were isolated from the cochlear lateral wall and maintained in an organ bath. Isolated gerbil spiral modiolar arteries, maintained and transfected in organ culture, were used to measure calcium sensitivity (calcium-tone relationship). In a clinical study, a total of 12 adult patients presenting with typical symptoms of sudden hearing loss who were not responsive or only partially responsive to prednisolone treatment were identified and selected for etanercept treatment. Etanercept (25 mg s.c.) was self-administered twice a week for 12 weeks.. TNF-α induced a proconstrictive state throughout the cochlear microvasculature, which reduced capillary diameter and cochlear blood flow in vivo. In vitro isolated preparations of the spiral modiolar artery and spiral ligament capillaries confirmed these observations. Antagonizing sphingosine-1-phosphate receptor 2 subtype signaling (by 1 μmol/L JTE013) attenuated the effects of TNF-α in all models. TNF-α activated sphingosine kinase 1 (Sk1) and induced its translocation to the smooth muscle cell membrane. Expression of a dominant-negative Sk1 mutant (Sk1(G82D)) eliminated both baseline spiral modiolar artery calcium sensitivity and TNF-α effects, whereas a nonphosphorylatable Sk1 mutant (Sk1(S225A)) blocked the effects of TNF-α only. A small group of etanercept-treated, hearing loss patients recovered according to a 1-phase exponential decay (half-life=1.56 ± 0.20 weeks), which matched the kinetics predicted for a vascular origin.. TNF-α indeed reduces cochlear blood flow via activation of vascular sphingosine-1-phosphate signaling. This integrates hearing loss into the family of ischemic microvascular pathologies, with implications for risk stratification, diagnosis, and treatment.

Topics: Adult; Animals; Calcium; Cochlea; Etanercept; Gerbillinae; Guinea Pigs; Hearing Loss; Humans; Immunoglobulin G; Lysophospholipids; Microvessels; Models, Animal; Phosphotransferases (Alcohol Group Acceptor); Receptors, Tumor Necrosis Factor; Regional Blood Flow; Signal Transduction; Sphingosine; Spiral Ligament of Cochlea; Treatment Outcome; Tumor Necrosis Factor-alpha; Vasoconstriction

To determine if endogenously generated sphingosine-1-phosphate (S1P) is involved in the development of allergic bronchial asthma, the effects of systemic treatments with SKI-II, a specific inhibitor of sphingosine kinase, on antigen-induced bronchial smooth muscle (BSM) hyperresponsiveness and airway inflammation were examined in mice. Male BALB/c mice were actively sensitized with ovalbumin (OA) antigen and were repeatedly challenged with aerosolized antigen. Animals also received intraperitoneal injections with SKI-II (50 mg/kg) 1 h prior to each antigen challenge. The acetylcholine (ACh)-induced contraction of BSM isolated from the repeatedly antigen-challenged mice was significantly augmented, that is, BSM hyperresponsiveness, as compared with that from the control animals (P < 0.05). The BSM hyperresponsiveness induced by antigen exposure was ameliorated by the systemic treatment with SKI-II, whereas the treatments had no effect on BSM responsiveness to ACh in control animals. On the other hand, the systemic treatments with SKI-II had no effect on antigen-induced inflammatory signs, such as increase in cell counts in bronchoalveolar lavage fluids (BALFs) and change in airway histology; upregulation of BALF cytokines, such as interleukin-4 (IL-4) and IL-13; and elevation of total and OA-specific immunoglobulin E (IgE) in sera. These findings suggest that sphingosine kinase inhibitors such as SKI-II have an ability to prevent the development of BSM hyperresponsiveness, but not of allergic airway inflammation. The endogenously generated S1P might be one of the exacerbating factors for the airway hyperresponsiveness, one of the characteristic features of allergic bronchial asthma.

Topics: Animals; Antigens; Asthma; Bronchi; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Immunoglobulin E; Interleukin-13; Interleukin-4; Lysophospholipids; Male; Mice; Mice, Inbred BALB C; Muscle, Smooth; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Thiazoles

Although the hippocampus is a brain region involved in short-term memory, the molecular mechanisms underlying memory formation are not completely understood. Here we show that sphingosine 1-phosphate (S1P) plays a pivotal role in the formation of memory. Addition of S1P to rat hippocampal slices increased the rate of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) recorded from the CA3 region of the hippocampus. In addition long-term potentiation (LTP) observed in the CA3 region was potently inhibited by a sphingosine kinase (SphK) inhibitor and this inhibition was fully reversed by S1P. LTP was impaired in hippocampal slices specifically in the CA3 region obtained from SphK1-knockout mice, which correlates well with the poor performance of these animals in the Morris water maze test. These results strongly suggest that SphK/S1P receptor signaling plays an important role in excitatory synaptic transmission in the CA3 region of hippocampus and has profound effects on hippocampal function such as spatial learning.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Analysis of Variance; Animals; Cells, Cultured; Electric Stimulation; Embryo, Mammalian; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; Gene Expression Regulation; Glutamic Acid; Hippocampus; Humans; In Vitro Techniques; Long-Term Potentiation; Lysophospholipids; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Mossy Fibers, Hippocampal; Neurons; Patch-Clamp Techniques; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Receptors, Lysosphingolipid; RNA, Small Interfering; Sphingosine; Synapses; Transfection

Sphingosine kinase 1 (SK1) catalyses the generation of sphingosine 1-phosphate (S1P), a bioactive phospholipid that influences a diverse range of cellular processes, including proliferation, survival, adhesion, migration, morphogenesis and differentiation. SK1 is controlled by various mechanisms, including transcriptional regulation, and post-translational activation by phosphorylation and protein-protein interactions which can regulate both the activity and localisation of this enzyme. To gain a better understanding of the regulatory mechanisms controlling SK1 activity and function we performed a yeast two-hybrid screen to identify SK1-interacting proteins. Using this approach we identified that SK1 interacts with subunit 7 (eta) of cytosolic chaperonin CCT (chaperonin containing t-complex polypeptide, also called TRiC for TCP-1 ring complex), a hexadecameric chaperonin that binds unfolded polypeptides and mediates their folding and release in an ATP-dependent manner. Further analysis of the SK1-CCTeta interaction demonstrated that other CCT/TRiC subunits also associated with SK1 in HEK293T cell lysates in an ATP-sensitive manner, suggesting that the intact, functional, multimeric CCT/TRiC complex associated with SK1. Furthermore, pulse-chase studies indicated that CCT/TRiC binds specifically to newly translated SK1. Finally, depletion of functional CCT/TRiC through the use of RNA interference in HeLa cells or temperature sensitive CCT yeast mutants reduced cellular SK1 activity. Thus, combined this data suggests that SK1 is a CCT/TRiC substrate, and that this chaperonin facilitates folding of newly translated SK1 into its mature active form.

Topics: Chaperonin Containing TCP-1; Chaperonins; Fibroblasts; HeLa Cells; Humans; Leukocytes; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Conformation; Protein Folding; Saccharomyces cerevisiae; Sphingosine; Transcriptional Activation; Transfection; Two-Hybrid System Techniques

The bioactive lipid sphingosine-1-phosphate (S1P) is emerging as an important mediator of immune and inflammatory responses. S1P formation is catalyzed by sphingosine kinase (SK), of which the SK1 isoenzyme is activated by tumor necrosis alpha (TNF-alpha). SK1 has been shown to be required for mediating TNF-alpha inflammatory responses in cells, including induction of cyclooxygenase 2 (COX-2). Because TNF-alpha and COX-2 are increased in patients with inflammatory bowel disease (IBD), we investigated the role of SK1 in a murine model of colitis. SK1(-/-) mice treated with dextran sulfate sodium (DSS) had significantly less blood loss, weight loss, colon shortening, colon histological damage, and splenomegaly than did wild-type (WT) mice. In addition, SK1(-/-) mice had no systemic inflammatory response. Moreover, WT but not SK1(-/-) mice treated with dextran sulfate sodium had significant increases in blood S1P levels, colon SK1 message and activity, and colon neutrophilic infiltrate. Unlike WT mice, SK1(-/-) mice failed to show colonic COX-2 induction despite an exaggerated TNF-alpha response; thus implicating for the first time SK1 in TNF-alpha-mediated COX-2 induction in vivo. Inhibition of SK1 may prove to be a valuable therapeutic target by inhibiting systemic and local inflammation in IBD.

Topics: Animals; Body Weight; Colitis; Colitis, Ulcerative; Colon; Cyclooxygenase 2; Dextran Sulfate; Erythrocytes; Gene Expression Regulation; Humans; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Organ Size; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Spleen; Tumor Necrosis Factor-alpha

Sphingosine kinase 1 (SphK1) phosphorylates sphingosine to form sphingosine-1-phosphate (S1P) and is a critical regulator of sphingolipid-mediated functions. Cell-based studies suggest a tumor-promoting function for the SphK1/S1P pathway. Also, our previous studies implicated the SphK1/S1P pathway in the induction of the arachidonic acid cascade, a major inflammatory pathway involved in colon carcinogenesis. Therefore, we investigated whether the SphK1/S1P pathway is necessary for mediating carcinogenesis in vivo. Here, we report that 89% (42/47) of human colon cancer samples stained positively for SphK1, whereas normal colon mucosa had negative or weak staining. Adenomas had higher expression of SphK1 vs. normal mucosa, and colon cancers with metastasis had higher expression of SphK1 than those without metastasis. In the azoxymethane (AOM) murine model of colon cancer, SphK1 and S1P were significantly elevated in colon cancer tissues compared to normal mucosa. Moreover, blood levels of S1P were higher in mice with colon cancers than in those without cancers. Notably, SphK1(-/-) mice subjected to AOM had significantly less aberrant crypt foci (ACF) formation and significantly reduced colon cancer development. These results are the first in vivo evidence that the SphK1/S1P pathway contributes to colon carcinogenesis and that inhibition of this pathway is a potential target for chemoprevention.

Topics: Animals; Azoxymethane; Cell Transformation, Neoplastic; Colonic Neoplasms; Cyclooxygenase 2; Dextran Sulfate; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Lysophospholipids; Mice; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine-1-phosphate (S1P), produced by sphingosine kinase 1 (SphK1) or kinase 2 (SphK2), mediates biological effects through intracellular and/or extracellular mechanisms. Here we determined a role for these kinases in kidney injury of wild-type mice following ischemia-reperfusion. SphK1 but not SphK2 mRNA expression and activity increased in the kidney following injury relative to sham-operated animals. Although SphK1(-/-) mice had no alteration in renal function following injury, mice with a disrupted SphK2 gene (SphK2(tr/tr)) had histological damage and impaired function. The immune-modulating pro-drug, FTY720, an S1P agonist failed to provide protection in SphK2(tr/tr) mice. Injured kidneys of these mice showed increased neutrophil infiltration and neutrophil chemokine expression along with a 3- to 5-fold increase in expression of the G-protein-coupled receptor S1P(3) compared to heterozygous SphK2(+/tr) mice. Kidney function and reduced vascular permeability were preserved in S1P(3)(-/-) compared to S1P(3)(+/-) mice after ischemia-reperfusion injury, suggesting increased S1P(3) mRNA may play a role in the injury of SphK2(tr/tr) mice. Our study suggests that constitutive expression of SphK2 may contribute to reduced ischemia-reperfusion injury of the kidney, and its absence may enhance injury due to increased neutrophil infiltration and S1P(3) activation. We also confirm that SphK2 is necessary to mediate the protective effects of FTY720.

Topics: Animals; Fingolimod Hydrochloride; Gene Expression Regulation, Enzymologic; Kidney; Kidney Diseases; Lysophospholipids; Mice; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Reperfusion Injury; RNA, Messenger; Sphingosine

Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that exerts numerous biological activities both as a receptor ligand and as an intracellular second messenger. In the present study, we explored roles of sphingosine kinase (SphK), an S1P-producing enzyme, in adipose tissue. We utilized mouse 3T3-L1 cells as an in vitro model of adipogenesis, using a mixture of insulin/dexamethasone/3-isobutyl-1-methylxanthine (IBMX) to induce differentiation. Real-time quantitative PCR (qRT-PCR) assays revealed that the expression levels of transcripts encoding both isoforms of SphK-1 and SphK-2 are up-regulated during adipogenesis (37.6- and 6.6-fold vs. basal, P < 0.05, respectively). Concomitantly, SphK-1/SphK-2 protein abundance and S1P contents of these cells increased at 3 days after hormonal stimulation. Loss-of-function approaches by pharmacological inhibition of SphK activity as well as by transfection with small interfering RNA (siRNA) against SphK-1 led to significant attenuation of lipid droplet accumulation and adipocyte marker gene expression. We detected marked elevation of SphK-1 mRNA in adipose tissue derived from 13-week-old ob/ob mice with obese phenotype than their lean littermates. These results suggest that increased expression of SphK, an S1P-producing enzyme, plays a significant role during adipogenesis, potentially providing a novel point of control in adipose tissue.

Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Animals; Base Sequence; DNA Primers; Enzyme Induction; Gene Expression; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Phosphotransferases (Alcohol Group Acceptor); Rats; RNA Interference; RNA, Messenger; RNA, Small Interfering; Sphingosine; Transfection

Branching morphogenesis of the metanephric kidney is critically dependent on the delicate orchestration of diverse cellular processes including proliferation, apoptosis, migration, and differentiation. Sphingosine-1-phosphate (S1P) is a potent lipid mediator influencing many of these cellular events. We report increased expression and activity of both sphingosine kinases and S1P phosphatases during development of the mouse metanephric kidney from induction at embryonic day 11.5 to maturity. Sphingosine kinase activity exceeded S1P phosphatase activity in embryonic kidneys, resulting in a net accumulation of S1P, while kinase and phosphatase activities were similar in adult tissue, resulting in reduced S1P content. Sphingosine kinase expression was greater in the metanephric mesenchyme than in the ureteric bud, while the S1P phosphatase SPP2 was expressed at greater levels in the ureteric bud. Treatment of cultured embryonic kidneys with sphingosine kinase inhibitors resulted in a dose-dependent reduction of ureteric bud tip numbers and increased apoptosis. Exogenous S1P rescued kidneys from apoptosis induced by kinase inhibitors. Ureteric bud tip number was unaffected by exogenous S1P in kidneys treated with N,N-dimethylsphingosine, although tip number increased in those treated with d,l-threo-dihydrosphingosine. S1P1 and S1P2 were the predominant S1P receptors expressed in the embryonic kidney. S1P1 expression increased during renal development while expression of S1P2 decreased, and both receptors were expressed predominantly in the metanephric mesenchyme. These results demonstrate dynamic regulation of S1P homeostasis during renal morphogenesis and suggest that differential expression of S1P metabolic enzymes and receptors provides a novel mechanism contributing to the regulation of kidney development.

Topics: Animals; Apoptosis; Female; Homeostasis; Kidney; Lysophospholipids; Membrane Proteins; Mice; Morphogenesis; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Pregnancy; Receptors, Lysosphingolipid; Sphingosine

Sphingosine 1-phosphate (S1P) induces migration of the human thyroid follicular carcinoma cell line ML-1 by activation of S1P(1) and S1P(3) receptors, G(i) proteins, and the phosphatidylinositol 3-kinase-Akt pathway. Because sphingosine kinase isoform 1 (SK) recently has been implicated as an oncogene in various cancer cell systems, we investigated the functions of SK in the migration, proliferation and adhesion of the ML-1 cell line. SK overexpressing ML-1 cells show an enhanced secretion of S1P, which can be attenuated, by inhibiting SK activity and a multidrug-resistant transport protein (ATP-binding cassette transporter). Furthermore, overexpression of SK enhances serum-induced migration of ML-1 cells, which can be attenuated by blocking ATP-binding cassette transporter and SK, suggesting that the migration is mediated by autocrine signaling through secretion of S1P. Inhibition of protein kinase C alpha, with both small interfering RNA (siRNA) and small molecular inhibitors attenuates migration in SK overexpressing cells. In addition, SK-overexpressing cells show an impaired adhesion, slower cell growth, and an up-regulation of ERK1/2 phosphorylation, as compared with cells expressing a dominant-negative SK. Taken together, we present evidence suggesting that SK enhances migration of ML-1 cells by an autocrine mechanism and that the S1P-evoked migration is dependent on protein kinase C alpha, ERK1/2, and SK.

Topics: Autocrine Communication; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Oncogenes; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C-alpha; RNA, Small Interfering; Sphingosine; Thyroid Neoplasms; Transfection

Novel immunomodulatory molecule FTY720 is a synthetic analog of myriocin, but unlike myriocin FTY720 does not inhibit serine palmitoyltransferase. Although many of the effects of FTY720 are ascribed to its phosphorylation and subsequent sphingosine 1-phosphate (S1P)-like action through S1P(1,3-5) receptors, studies on modulation of intracellular balance of signaling sphingolipids by FTY720 are limited. In this study, we used stable isotope pulse labeling of human pulmonary artery endothelial cells with l-[U-(13)C, (15)N]serine as well as in vitro enzymatic assays and liquid chromatography-tandem mass spectrometry methodology to characterize FTY720 interference with sphingolipid de novo biosynthesis. In human pulmonary artery endothelial cells, FTY720 inhibited ceramide synthases, resulting in decreased cellular levels of dihydroceramides, ceramides, sphingosine, and S1P but increased levels of dihydrosphingosine and dihydrosphingosine 1-phosphate (DHS1P). The FTY720-induced modulation of sphingolipid de novo biosynthesis was similar to that of fumonisin B1, a classical inhibitor of ceramide synthases, but differed in the efficiency to inhibit biosynthesis of short-chain versus long-chain ceramides. In vitro kinetic studies revealed that FTY720 is a competitive inhibitor of ceramide synthase 2 toward dihydrosphingosine with an apparent K(i) of 2.15 microm. FTY720-induced up-regulation of DHS1P level was mediated by sphingosine kinase (SphK) 1, but not SphK2, as confirmed by experiments using SphK1/2 silencing with small interfering RNA. Our data demonstrate for the first time the ability of FTY720 to inhibit ceramide synthases and modulate the intracellular balance of signaling sphingolipids. These findings open a novel direction for therapeutic applications of FTY720 that focuses on inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P generation in cells.

Topics: Cells, Cultured; Ceramides; Chromatography, Liquid; Endothelium, Vascular; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Lung; Lysophospholipids; Oxidoreductases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Pulmonary Artery; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Serine C-Palmitoyltransferase; Sphingosine; Tandem Mass Spectrometry; Up-Regulation

Glioblastoma multiforme (GBM) is an aggressively invasive brain neoplasm with poor patient prognosis. We have previously shown that the bioactive lipid sphingosine-1-phosphate (S1P) stimulates in vitro invasiveness of GBM cells and that high expression levels of the enzyme that forms S1P, sphingosine kinase-1 (SphK1), correlate with shorter survival time of GBM patients. We also recently showed that S1P induces expression of CCN1 (also known as Cyr61), a matricellular protein known to correlate with poor patient prognosis, in GBM cells. In this study, we further explored the role of CCN1 as well as the urokinase plasminogen activator (uPA), a protein known to stimulate GBM cell invasiveness, in S1P-induced invasion using a spheroid invasion assay. We also investigated the roles of various S1P receptors in stimulating invasiveness through these pathways. S1P induced expression of uPA and its receptor, uPAR, in GBM cells. Whereas S1P(1), S1P(2), and S1P(3) receptors all contribute, at least partially, S1P(1) overexpression led to the most dramatic induction of the uPA system and of spheroid invasion, even in the absence of added S1P. Furthermore, neutralizing antibodies directed against uPA or CCN1 significantly decreased both basal and S1P-stimulated GBM cell invasiveness. Inhibition of SphK blocked basal expression of uPA and uPAR, as well as glioma cell invasion; however, overexpression of SphK did not augment S1P receptor-mediated enhancement of uPA activity or invasion. Thus, SphK is necessary for basal activity of the uPA system and glioma cell invasion, whereas S1P receptor signaling enhances invasion, partially through uPA and CCN1.

Topics: Cell Line, Tumor; Cell Survival; Cysteine-Rich Protein 61; Glioblastoma; Humans; Lysophospholipids; Neoplasm Invasiveness; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Receptors, Urokinase Plasminogen Activator; Sphingosine; Survivors; Urokinase-Type Plasminogen Activator

Two mammalian sphingosine kinase (SphK) isoforms, SphK1 and SphK2, possess identical kinase domains but have distinct kinetic properties and subcellular localizations, suggesting each has one or more specific roles in sphingosine-1-phosphate (S1P) generation. Although both kinases use sphingosine as a substrate to generate S1P, the mechanisms controlling SphK activation and subsequent S1P generation during lung injury are not fully understood. In this study, we established a murine lung injury model to investigate LPS-induced lung injury in SphK1 knockout (SphK1(-/-)) and wild-type (WT) mice. We found that SphK1(-/-) mice were much more susceptible to LPS-induced lung injury compared with their WT counterparts, quantified by multiple parameters including cytokine induction. Intriguingly, overexpression of WT SphK1 delivered by adenoviral vector to the lungs protected SphK1(-/-) mice from lung injury and attenuated the severity of the response to LPS. However, adenoviral overexpression of a SphK1 kinase-dead mutant (SphKKD) in SphK1(-/-) mouse lungs further exacerbated the response to LPS as well as the extent of lung injury. WT SphK2 adenoviral overexpression also failed to provide protection and, in fact, augmented the degree of LPS-induced lung injury. This suggested that, in vascular injury, S1P generated by SphK2 activation plays a distinctly separate role compared with SphK1-dependent S1P generation and survival signaling. Microarray and real-time RT-PCR analysis of SphK1 and SphK2 expression levels during lung injury revealed that, in WT mice, LPS treatment caused significantly enhanced SphK1 expression ( approximately 5x) levels within 6 h, which declined back to baseline levels by 24 h posttreatment. In contrast, expression of SphK2 was gradually induced following LPS treatment and was elevated within 24 h. Collectively, our results for the first time demonstrate distinct functional roles of the two SphK isoforms in the regulation of LPS-induced lung injury.

Topics: Adenoviridae; Animals; Gene Deletion; Gene Expression Regulation, Enzymologic; Gene Transfer Techniques; Lipopolysaccharides; Lung; Lung Injury; Lysophospholipids; Membrane Proteins; Mice; Mice, Inbred C57BL; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Pulmonary Edema; Sphingosine; Time Factors; Tumor Necrosis Factor-alpha

We show here that the endogenous sphingosine 1-phosphate 5 receptor (S1P(5), a G protein coupled receptor (GPCR) whose natural ligand is sphingosine 1-phosphate (S1P)) and sphingosine kinases 1 and 2 (SK1 and SK2), which catalyse formation of S1P, are co-localised in the centrosome of mammalian cells, where they may participate in regulating mitosis. The centrosome is a site for active GTP-GDP cycling involving the G-protein, G(i) and tubulin, which are required for spindle pole organization and force generation during cell division. Therefore, the presence of S1P(5) (which normally functions as a plasma membrane guanine nucleotide exchange factor, GEF) and sphingosine kinases in the centrosome might suggest that S1P(5) may function as a ligand activated GEF in regulating G-protein-dependent spindle formation and mitosis. The addition of S1P to cells inhibits trafficking of S1P(5) to the centrosome, suggesting a dynamic shuttling endocytic mechanism controlled by ligand occupancy of cell surface receptor. We therefore propose that the centrosomal S1P(5) receptor might function as an intracellular target of S1P linked to regulation of mitosis.

Topics: Animals; Cell Division; Cell Line; Centrosome; Guanine Nucleotide Exchange Factors; Humans; Lysophospholipids; Mice; Mitosis; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptors, Lysosphingolipid; RNA, Small Interfering; Sphingosine

Following injury, fibroblasts transform into myofibroblasts and produce extracellular matrix (ECM). Excess production of ECM associated with cardiac fibrosis severely inhibits cardiac function. Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, regulates the function of numerous cell types. In this study, we determined the role of S1P in promoting pro-fibrotic actions of cardiac fibroblasts (CFs).. S1P-mediated effects on myofibroblast transformation, collagen production, and cross-talk with transforming growth factor-beta (TGF-beta) using mouse CF were examined. S1P increased alpha-smooth muscle actin (a myofibroblast marker) and collagen expression in a S1P2 receptor- and Rho kinase-dependent manner. TGF-beta increased sphingosine kinase 1 (SphK1; the enzyme responsible for S1P production) expression and activity. TGF-beta-stimulated collagen production was inhibited by SphK1 or S1P2 siRNA, a SphK inhibitor, and an anti-S1P monoclonal antibody.. These findings suggest that TGF-beta-stimulated collagen production in CF involves 'inside-out' S1P signalling whereby S1P produced intracellularly by SphK1 can be released and act in an autocrine/paracrine fashion to activate S1P2 and increase collagen production.

Topics: Animals; Antibodies, Monoclonal; Cell Differentiation; Cells, Cultured; Collagen; Enzyme Inhibitors; Fibroblasts; Lymphotoxin-alpha; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Myocardium; Phosphotransferases (Alcohol Group Acceptor); rho-Associated Kinases; RNA, Small Interfering; Signal Transduction; Sphingosine

Sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which has potent pro-inflammatory and pro-angiogenic effects. We investigated the effects of raised SK1 levels on endothelial cell function and the possibility that this signaling pathway is activated in rheumatoid arthritis. Human umbilical vein endothelial cells with 3- to 5-fold SK1 (EC(SK)) overexpression were generated by adenoviral and retroviralmediated gene delivery. The activation state of these cells and their ability to undergo angiogenesis was determined. S1P was measured in synovial fluid from patients with RA and OA. EC(SK) showed an enhanced migratory capacity and a stimulated rate of capillary tube formation. The cells showed constitutive activation as evidenced by the induction of basal VCAM-1 expression, and further showed a more augmented VCAM-1 and E selectin response to TNF compared with empty vector control cells (EC(EV)). These changes had functional consequences in terms of enhanced neutrophil binding in the basal and TNFstimulated states in EC(SK). By contrast, over-expression of a dominant-negative SK inhibited the TNF-induced VCAM-1 and E selectin and inhibited PMN adhesion, confirming that the observed effects were specifically mediated by SK. The synovial fluid levels of S1P were significantly higher in patients with RA than in those with OA. Small chronic increases in SK1 activity in the endothelial cells enhance the ability of the cells to support inflammation and undergo angiogenesis, and sensitize the cells to inflammatory cytokines. The SK1 signaling pathway is activated in RA, suggesting that manipulation of SK1 activity in diseases of aberrant inflammation and angiogenesis may be beneficial.

Topics: Arthritis, Rheumatoid; Cell Adhesion; Cell Movement; Chronic Disease; E-Selectin; Endothelial Cells; Endothelium, Vascular; Humans; Lysophospholipids; Neovascularization, Physiologic; Osteoarthritis; Phenotype; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

The enzyme sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which is an important survival factor for endothelial cells (EC). Modest increases in intracellular SK1 activity in the EC are known to confer a survival advantage upon the cells. Here, we investigated the effects of more dramatic increases in intracellular SK1 in the EC. We found that these cells show reduced cell survival under conditions of stress, enhanced caspase-3 activity, cell cycle inhibition, and cell-cell junction disruption. We propose that alterations in the phosphorylation state of the enzyme may explain the differential effects on the phenotype with modest versus high levels of enforced expression of SK1. Our results suggest that SK1 activity is subject to control in the EC, and that this control may be lost in conditions involving vascular regression.

Topics: Apoptosis; Caspase 3; Cell Cycle; Cells, Cultured; Endothelial Cells; Humans; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Time Factors

Hypoxia and signaling via hypoxia-inducible factor-1 (HIF-1) is a key feature of solid tumors and is related to tumor progression as well as treatment failure. Although it is generally accepted that HIF-1 provokes tumor cell survival and induces chemoresistance under hypoxia, HIF-1-independent mechanisms operate as well. We present evidence that conditioned medium obtained from A549 cells, incubated for 24 h under hypoxia, protected naive A549 cells from etoposide-induced cell death. Lipid extracts generated from hypoxia-conditioned medium still rescued cells from apoptosis induced by etoposide. Specifically, the bioactive lipid sphingosine-1-phosphate (S1P) not only was essential for cell viability of A549 cells but also protected cells from apoptosis. We noticed an increase in sphingosine kinase 2 (SphK2) protein level and enzymatic activity under hypoxia, which correlated with the release of S1P into the medium. Knockdown of SphK2 using specific small interfering RNA relieved chemoresistance of A549 cells under hypoxia and conditioned medium obtained from SphK2 knockdown cells was only partially protective. Coincubations of conditioned medium with VPC23019, a S1P(1)/S1P(3) antagonist, reduced protection of conditioned medium, with the further notion that p42/44 mitogen-activated protein kinase transmits autocrine or paracrine survival signaling downstream of S1P(1)/S1P(3) receptors. Our data suggest that hypoxia activates SphK2 to promote the synthesis and release of S1P, which in turn binds to S1P(1)/S1P(3) receptors, thus activating p42/44 mitogen-activated protein kinase to convey autocrine or paracrine protection of A549 cells.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Hypoxia; Cell Line, Tumor; Culture Media; Drug Resistance, Neoplasm; Etoposide; Humans; Lung Neoplasms; Lysophospholipids; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Small Interfering; Sphingosine; Transfection

Cerebellar granule cells from sphingosine 1-phosphate (S1P) lyase-deficient mice were used to study the toxicity of this potent sphingolipid metabolite in terminally differentiated postmitotic neurons. Based on earlier findings with the lyase-stable, semi-synthetic, cis-4-methylsphingosine phosphate, we hypothesized that accumulation of S1P above a certain threshold induces neuronal apoptosis. The present studies confirmed this conclusion and further revealed that for S1P to induce apoptosis in lyase-deficient neurons it must also be produced by sphingosine-kinase2 (SK2). These conclusions are based on the finding that incubation of lyase-deficient neurons with either sphingosine or S1P results in a similar elevation in cellular S1P; however, only S1P addition to the culture medium induces apoptosis. This was not due to S1P acting on the S1P receptor but to hydrolysis of S1P to sphingosine that was phosphorylated by the cells, as described before for cis-4-methylsphingosine. Although the cells produced S1P from both exogenously added sphingosine as well as sphingosine derived from exogenous S1P, the S1P from these two sources were not equivalent, because the former was primarily produced by SK1, whereas the latter was mainly formed by SK2 (as also was cis-4-methylsphingosine phosphate), based on studies in neurons lacking SK1 or SK2 activity. Thus, these investigations show that, due to the existence of at least two functionally distinct intracellular origins for S1P, exogenous S1P can be neurotoxic. In this model, S1P accumulated due to a defective lyase, however, this cause of toxicity might also be important in other cases, as illustrated by the neurotoxicity of cis-4-methylsphingosine phosphate.

Topics: Animals; Apoptosis; Caspases; Cell Membrane; Cell Survival; Ceramides; Cyclin D1; Cyclin E; Lysophospholipids; Mass Spectrometry; Mice; Mice, Transgenic; Neurons; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

We investigated the existence and regional distribution of sphingosine-1-phosphate regulatory enzymes and receptors in the lower urinary tract and determined the functional role of sphingosine-1-phosphate receptors in the bladder.. Lower urinary tract tissue from 10 female rats was harvested for real-time reverse transcriptase-polymerase chain reaction or organ bath physiology, whereas blood serum was obtained for high-performance liquid chromatography determination of sphingosine-1-phosphate levels. Statistical analysis included the Student t test and analysis of variance.. All 3 sphingosine-1-phosphate receptors and major enzymes were expressed throughout the lower urinary tract, but expression and physiologic force generation varied among regions. Sphingosine-1-phosphate was detected in serum.. We provide novel data that the sphingosine-1-phosphate signaling pathway regulatory proteins exist throughout the female rat lower urinary tract, but that relative expression exhibits regional heterogeneity corresponding with lower urinary tract contractile response to sphingosine-1-phosphate. Our study suggests that sphingosine-1-phosphate signaling is important in the lower urinary tract and identifies this pathway as a possible target for altering bladder smooth muscle tone.

Topics: Animals; Enzymes; Female; Genetic Heterogeneity; Lyases; Lysophospholipids; Muscle Contraction; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; rho-Associated Kinases; Signal Transduction; Sphingosine; Urethra; Urinary Bladder; Urinary Bladder, Overactive

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in diverse cellular functions including survival, proliferation, tumorigenesis, inflammation, and immunity. Sphingosine kinase (SphK) contributes to these functions by converting sphingosine to S1P. We report here that the nonstructural protein NS3 from bovine viral diarrhea virus (BVDV), a close relative of hepatitis C virus (HCV), binds to and inhibits the catalytic activity of SphK1 independently of its serine protease activity, whereas HCV NS3 does not affect SphK1 activity. Uncleaved NS2-3 from BVDV was also found to interact with and inhibit SphK1. We suspect that inhibition of SphK1 activity by BVDV NS3 and NS2-3 may benefit viral replication, because SphK1 inhibition by small interfering RNA, chemical inhibitor, or overexpression of catalytically inactive SphK1 results in enhanced viral replication, although the mechanisms by which SphK1 inhibition leads to enhanced viral replication remain unknown. A role of SphK1 inhibition in viral cytopathogenesis is also suggested as overexpression of SphK1 significantly attenuates the induction of apoptosis in cells infected with cytopathogenic BVDV. These findings suggest that SphK is targeted by this virus to regulate its catalytic activity.

Topics: Animals; Cats; Cytopathogenic Effect, Viral; Diarrhea Viruses, Bovine Viral; Enzyme Inhibitors; Humans; Lysophospholipids; Peptide Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; RNA Helicases; RNA, Small Interfering; Sphingosine; Viral Nonstructural Proteins; Virus Replication

In the functional screening of a human heart cDNA library to identify a novel antiangiogenic factor, the prime candidate gene was "four-and-a-half LIM only protein-2" (FHL-2). The goal of this study is to clear the mechanism of antiangiogenic signaling of FHL-2 in endothelial cells (ECs).. Overexpressed FHL-2 strongly inhibited vascular endothelial growth factor (VEGF)-induced EC migration. In the angiogenic signaling, we focused on sphingosine kinase-1 (SK1), which produces sphingosine-1-phosphate (S1P), a bioactive sphingolipid, as a potent angiogenic mediator in ECs. Immunoprecipitation and immunostaining analysis showed that FHL-2 might bind to SK1. Importantly, overexpression of FHL-2 in ECs inhibited VEGF-induced SK1 activity, phosphatidylinositol 3-kinase activity, and phosphorylation of Akt and eNOS. In contrast, overexpression of FHL-2 had no effect on S1P-induced Akt phosphorylation. Interestingly, VEGF stimulation decreased the binding of FHL-2 and SK1. Depletion of FHL-2 by siRNA increased EC migration accompanied with SK1 and Akt activation, and increased the expression of VEGF receptor-2 which further enhanced VEGF signaling. Furthermore, injection of FHL-2 mRNA into Xenopus embryos resulted in inhibition of vascular network development, assessed by in situ hybridization with endothelial markers.. FHL-2 may regulate phosphatidylinositol 3-kinase/Akt via direct suppression of the SK1-S1P pathway in ECs.

Topics: Animals; Cattle; Cell Movement; Cells, Cultured; Endothelial Cells; Gene Expression Regulation, Developmental; Homeodomain Proteins; Humans; LIM-Homeodomain Proteins; Lysophospholipids; Muscle Proteins; Neovascularization, Physiologic; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Recombinant Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; Sphingosine; Time Factors; Transcription Factors; Transfection; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenopus laevis

Identifying the cues required for the survival and development of photoreceptors is essential for treating retinal neurodegeneration. The authors previously established that glial-derived neurotrophic factor (GDNF) stimulates proliferation and that docosahexaenoic acid (DHA) promotes photoreceptor survival and differentiation. Later findings that ceramide triggers photoreceptor apoptosis suggested sphingolipids might also control photoreceptor development. The present study investigated whether sphingosine-1-phophate (S1P), which promotes survival and differentiation in several cell types, regulates photoreceptor proliferation and differentiation and whether it is a mediator in GDNF and DHA effects.. Rat retina neuronal cultures were supplemented at day 0 or 1 with S1P, GDNF, or DHA and were treated with DL-threo-dihydrosphingosine to inhibit S1P synthesis or with brefeldin A (BFA) to block intracellular trafficking. Proliferation was quantified to determine bromodeoxyuridine uptake and number of mitotic figures. Opsin, peripherin, and sphingosine kinase (SphK), the enzyme required for S1P synthesis, were quantified by immunocytochemistry and Western blot analysis.. S1P increased the proliferation of photoreceptor progenitors. It also stimulated the formation of apical processes, enhanced opsin and peripherin expression, and promoted their localization in these processes; DHA had similar effects. BFA prevented S1P and DHA enhancement of apical process formation without affecting opsin expression. GDNF and DHA enhanced SphK expression in photoreceptors, while inhibiting S1P synthesis blocked GDNF mitogenic effects and DHA effects on differentiation.. The authors propose S1P as a key regulator in photoreceptor development. GDNF and DHA might upregulate SphK levels to promote S1P synthesis, which would initially promote proliferation and then advance photoreceptor differentiation.

Topics: Animals; Blotting, Western; Brefeldin A; Cell Differentiation; Cell Proliferation; Cell Survival; Docosahexaenoic Acids; Enzyme Inhibitors; Glial Cell Line-Derived Neurotrophic Factor; Immunohistochemistry; Intermediate Filament Proteins; Lysophospholipids; Membrane Glycoproteins; Nerve Tissue Proteins; Opsins; Peripherins; Phosphotransferases (Alcohol Group Acceptor); Photoreceptor Cells, Vertebrate; Rats; Rats, Wistar; Sphingosine

Studies in skeletal muscle demonstrate that elevation of plasma FFAs increases the sphingolipid ceramide. We aimed to determine the impact of FFA oversupply on total sphingolipid profiles in a skeletal muscle model. C2C12 myotubes were treated with palmitate (PAL). Lipidomics analysis revealed pleiotropic effects of PAL on cell sphingolipids not limited to ceramides. (13)C labeling demonstrated that PAL activated several branches of sphingolipid synthesis by distinct mechanisms. Intriguingly, PAL increased sphingosine-1-phosphate independently of de novo synthesis. Quantitative real-time PCR demonstrated that PAL increased sphingosine kinase 1 (SK1) mRNA by approximately 4-fold. This was accompanied by a 2.3-fold increase in sphingosine kinase enzyme activity. This upregulation did not occur upon treatment with oleate, suggesting some level of specificity for PAL. These findings were recapitulated in the diet-induced obesity mouse model, in which high-fat feeding increased SK1 message in skeletal muscle over 2.3-fold. These data suggest that the impact of elevated FFA on sphingolipids reaches beyond ceramides and de novo sphingolipid synthesis. Moreover, these findings identify PAL as a novel regulatory stimulus for SK1.

Topics: Animals; Cell Line; Ceramides; Diet; Enzyme Activation; Humans; Isotope Labeling; Lysophospholipids; Mice; Muscle Fibers, Skeletal; Obesity; Oleic Acid; Palmitates; Phosphotransferases (Alcohol Group Acceptor); Rats; Serine C-Palmitoyltransferase; Signal Transduction; Sphingosine; Substrate Specificity; Up-Regulation

Sphingosine 1-phosphate (S1P) is an important factor for the regulation of cell motility acting both inside and outside the cells. The precise role of S1P in the control of cell motility, however, remains unclear. Here we describe the roles of S1P in the regulation of cell motility by dissecting them into intracellular and extracellular actions using a liposomal S1P transfer technique. In a Boyden chamber assay free S1P enhanced directional cell movement, whereas liposomal S1P induced nondirectional cell movement. Furthermore, inhibition of sphingosine kinase (SphK) 1 by several inhibitors or knockdown of the enzyme expression by siRNA caused reduced wound-faced cell polarity formation as assessed by wound-healing assay. Moreover, S1P-induced cell migration was strongly inhibited by SphK inhibitors. These results indicate that extracellular S1P acting through S1P receptors facilitates the formation of cell polarity, whereas S1P generated inside the cells functions as an intracellular mediator per se to enhance nondirectional cell movement, thus S1P enhances directional cell movement in a coordinated fashion.

Topics: Animals; Cell Movement; Cell Polarity; Cells, Cultured; Enzyme Inhibitors; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptors, Lysosphingolipid; RNA, Small Interfering; Sphingosine

Defeating pancreatic cancer resistance to the chemotherapeutic drug gemcitabine remains a challenge to treat this deadly cancer. Targeting the sphingolipid metabolism for improving tumor chemosensitivity has recently emerged as a promising strategy. The fine balance between intracellular levels of the prosurvival sphingosine-1-phosphate (S1P) and the proapoptotic ceramide sphingolipids determines cell fate. Among enzymes that control this metabolism, sphingosine kinase-1 (SphK1), a tumor-associated protein overexpressed in many cancers, favors survival through S1P production, and inhibitors of SphK1 are used in ongoing clinical trials to sensitize epithelial ovarian and prostate cancer cells to various chemotherapeutic drugs. We here report that the cellular ceramide/S1P ratio is a critical biosensor for predicting pancreatic cancer cell sensitivity to gemcitabine. A low level of the ceramide/S1P ratio, associated with a high SphK1 activity, correlates with a robust intrinsic pancreatic cancer cell chemoresistance toward gemcitabine. Strikingly, increasing the ceramide/S1P ratio, by using pharmacologic (SphK1 inhibitor or ceramide analogue) or small interfering RNA-based approaches to up-regulate intracellular ceramide levels or reduce SphK1 activity, sensitized pancreatic cancer cells to gemcitabine. Conversely, decreasing the ceramide/S1P ratio, by up-regulating SphK1 activity, promoted gemcitabine resistance in these cells. Development of novel pharmacologic strategies targeting the sphingolipid metabolism might therefore represent an interesting promising approach, when combined with gemcitabine, to defeat pancreatic cancer chemoresistance to this drug.

Topics: Blotting, Western; Cell Proliferation; Cell Survival; Ceramides; Deoxycytidine; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gemcitabine; Humans; Lysophospholipids; Pancreatic Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Reverse Transcriptase Polymerase Chain Reaction; Ribonucleotide Reductases; RNA, Messenger; Sphingosine; Tumor Cells, Cultured

Myofibroblasts play a central role in the pathogenesis of liver fibrosis. Myofibroblasts of bone marrow (BM) origin have recently been identified in fibrotic liver. However, little is known about the mechanism that controls their mobilization in vivo. Here we confirmed that BM mesenchymal stem cells (BMSCs) can migrate to the damaged liver and differentiate into myofibroblasts. We also investigated the mechanism underlying the homing of BMSCs after liver injury.. ICR mice were lethally irradiated and received BM transplants from enhanced green fluorescent protein transgenic mice. Carbon tetrachloride or bile duct ligation was used to induce liver fibrosis. The fibrotic liver tissue was examined by immunofluorescent staining to identify BM-derived myofibroblasts.. BMSCs contributed significantly to myofibroblast population in fibrotic liver. Moreover, analysis in vivo and in vitro suggested that homing of BMSCs to the damaged liver was in response to sphingosine 1-phosphate (S1P) gradient between liver and BM. Furthermore, S1P receptor type 3 (S1P3) was required for migration of BMSCs triggered by S1P.. S1P mediates liver fibrogenesis through homing of BMSCs via S1P3 receptor, which may represent a novel therapeutic target in liver fibrosis through inhibiting S1P formation and/or receptor activation.

Topics: Animals; Base Sequence; Bone Marrow Cells; Bone Marrow Transplantation; Cell Differentiation; Cell Movement; DNA Primers; Green Fluorescent Proteins; In Vitro Techniques; Liver Cirrhosis; Lysophospholipids; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred ICR; Mice, Transgenic; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Recombinant Proteins; Sphingosine; Suramin; Transforming Growth Factor beta1

A challenging task of the immune system is to fight cancer cells. However, a variety of human cancers educate immune cells to become tumor supportive. This is exemplified for tumor-associated macrophages (TAMs), which are polarized towards an anti-inflammatory and cancer promoting phenotype. Mechanistic explanations, how cancer cells influence the macrophage phenotype are urgently needed to address potential anti-cancer strategies along this line. One potential immune modulating compound, sphingosine-1-phosphate (S1P), was recently highlighted in both tumor growth and immune modulation. Using a xenograft model in nude mice, we demonstrate a supportive role of sphingosine kinase 2 (SphK2), one of the S1P-producing enzymes for tumor progression. The growth of SphK2-deficient MCF-7 breast tumor xenografts was markedly delayed when compared with controls. Infiltration of macrophages in SphK2-deficient and control tumors was comparable. However, TAMs from SphK2-deficient tumors displayed a pronounced anti-tumor phenotype, showing an increased expression of pro-inflammatory markers/mediators such as NO, TNF-alpha, IL-12 and MHCII and a low expression of anti-inflammatory IL-10 and CD206. These data suggest a role for S1P, generated by SphK2, in early tumor development by affecting macrophage polarization.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Polarity; Cell Survival; Female; Humans; Inflammation; Lysophospholipids; Macrophages; Mice; Neoplasm Transplantation; Neoplasms, Experimental; Phenotype; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Transplantation, Heterologous

We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1, but not SphK2, expression. Similarly, IGFR1 phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK1 silencing. SphK1 expression and activity were stimulated by IGFBP-3 approximately 2-fold over 24 h. Silencing of sphingosine 1-phosphate receptor 1 (S1P1) or S1P3, but not S1P2, abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S1P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S1P1 and S1P3. These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-10A cells requires SphK1 activity and S1P1/S1P3, suggesting that S1P, the product of SphK activity and ligand for S1P1 and S1P3, is the "missing link" mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3.

Topics: Breast; Cell Line, Tumor; DNA; Enzyme Activation; Epithelial Cells; ErbB Receptors; Female; Gene Silencing; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Receptor, IGF Type 1; Receptors, Lysosphingolipid; RNA, Small Interfering; Signal Transduction; Sphingosine; Transcriptional Activation

Lung parenchymal strips isolated from ovalbumin-sensitized rats manifest a mast cell-dependent, biphasic contraction when challenged with allergen. The first phase is mediated by the release of preformed 5-HT while the second phase is dependent on de novo synthesis of leukotrienes. Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite which is readily generated in mast cells and has been demonstrated to be an important regulator of allergen-induced mast cell activation. We have used the parenchymal strip to explore the role of sphingosine 1-phosphate and the S1P(2) receptor in the two components of the acute response to allergen. Lung parenchymal strips were prepared from Brown Norway rats actively sensitized to ovalbumin. The strips were set up in organ baths and contractile responses measured isometrically. The inhibitors of sphingosine kinase, D-erythro-NN-dimethylsphingosine (dimethylsphingosine) and 4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol (SKI-II) inhibited concentration-dependently both phases of the contractile response induced by 0.1 microg ml(-1) ovalbumin. The effects were seen at concentrations similar to those which inhibit the purified enzyme and were selective in that neither the contractile response to adenosine nor that to 5-hydroxytryptamine was affected. JTE-013 (a selective S1P(2) receptor antagonist) also blocked the response to ovalbumin (0.1 microg ml(-1)). However, the concentrations of JTE-013 required (microM) were substantially higher than its affinity for the S1P(2) receptors (nM). However, when tested against a lower concentration of ovalbumin (0.03 microg ml(-1)), JTE-013 inhibited the response with nM potency. These data demonstrate the importance of S1P and the S1P(2) receptor as regulators of allergen-induced activation of mast cells in their natural environment in the rat lung.

Topics: Acetates; Adenosine; Allergens; Animals; Bronchoconstriction; Cyclopropanes; Dose-Response Relationship, Drug; Enzyme Inhibitors; Injections, Subcutaneous; Leukotriene Antagonists; Lung; Lysophospholipids; Male; Mast Cells; Methysergide; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Pyrazoles; Pyridines; Quinolines; Rats; Rats, Inbred BN; Receptors, Lysosphingolipid; Serotonin; Serotonin Antagonists; Signal Transduction; Sphingosine; Sulfides; Thiazoles

The importance of bioactive lipid signaling under physiological and pathophysiological conditions is progressively becoming recognized. The disparate distribution of sphingosine kinase (SphK) isoform activity in normal and ischemic brain, particularly the large excess of SphK2 in cerebral microvascular endothelial cells, suggests potentially unique cell- and region-specific signaling by its product sphingosine-1-phosphate. The present study sought to test the isoform-specific role of SphK as a trigger of hypoxic preconditioning (HPC)-induced ischemic tolerance.. Temporal changes in microvascular SphK activity and expression were measured after HPC. The SphK inhibitor dimethylsphingosine or sphingosine analog FTY720 was administered to adult male Swiss-Webster ND4 mice before HPC. Two days later, mice underwent a 60-minute transient middle cerebral artery occlusion and at 24 hours of reperfusion, infarct volume, neurological deficit, and hemispheric edema were measured.. HPC rapidly increased microvascular SphK2 protein expression (1.7+/-0.2-fold) and activity (2.5+/-0.6-fold), peaking at 2 hours, whereas SphK1 was unchanged. SphK inhibition during HPC abrogated reductions in infarct volume, neurological deficit, and ipsilateral edema in HPC-treated mice. FTY720 given 48 hours before stroke also promoted ischemic tolerance; when combined with HPC, even greater (and dimethylsphingosine-reversible) protection was noted.. These findings indicate hypoxia-sensitive increases in SphK2 activity may serve as a proximal trigger that ultimately leads to sphingosine-1-phosphate-mediated alterations in gene expression that promote the ischemia-tolerant phenotype. Thus, components of this bioactive lipid signaling pathway may be suitable therapeutic targets for protecting the neurovascular unit in stroke.

Topics: Animals; Arterioles; Brain Edema; Cerebral Arteries; Cerebrovascular Circulation; Disease Models, Animal; Fingolimod Hydrochloride; Hypoxia-Ischemia, Brain; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Ischemic Preconditioning; Lysophospholipids; Male; Mice; Microcirculation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Reperfusion Injury; RNA, Messenger; Sphingosine

Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.

Topics: Albuminuria; Animals; Cell Line; Connective Tissue Growth Factor; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Down-Regulation; Fibrosis; Gene Expression Regulation, Enzymologic; Humans; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Phosphotransferases (Alcohol Group Acceptor); Podocytes; Promoter Regions, Genetic; Protein Kinase Inhibitors; RNA Interference; RNA, Messenger; Smad4 Protein; Sphingosine; Time Factors; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Up-Regulation

Immunologic processes might contribute to the pathogenesis of pulmonary arterial hypertension (PAH), a fatal condition characterized by progressive pulmonary arterial remodeling, increased pulmonary vascular resistance, and right ventricular failure. Experimental allergen-driven lung inflammation evoked morphologic and functional vascular changes that resembled those observed in patients with PAH. Sphingosine kinase 1 (SphK1) is the main pulmonary contributor to sphingosine-1-phosphate (S1P) synthesis, a modulator of immune and vascular functions.. We sought to investigate the role of SphK1 in allergen-induced lung inflammation.. SphK1-deficient mice and C57Bl/6 littermates (wild-type [WT] animals) were subjected to acute or chronic allergen exposure.. After 4 weeks of systemic ovalbumin sensitization and local airway challenge, airway responsiveness increased less in SphK1(-/-) compared with WT mice, whereas pulmonary vascular responsiveness was greatly increased and did not differ between strains. Acute lung inflammation led to an increase in eosinophils and mRNA expression for S1P phosphatase 2 and S1P lyase in lungs of WT but not SphK1(-/-) mice. After repetitive allergen exposure for 8 weeks, airway responsiveness was not augmented in SphK1(-/-) or WT mice, but pulmonary vascular responsiveness was increased in both strains, with significantly higher vascular responsiveness in SphK1(-/-) mice compared with that seen in WT mice. Increased vascular responsiveness was accompanied by remodeling of the small and intra-acinar arteries.. : The data support a role for SphK1 and S1P in allergen-induced airway inflammation. However, SphK1 deficiency increased pulmonary vascular hyperresponsiveness, which is a component of PAH pathobiology. Moreover, we show for the first time the dissociation between inflammation-induced remodeling of the airways and pulmonary vasculature.

Topics: Acute Disease; Allergens; Animals; Bronchial Hyperreactivity; Chronic Disease; Cytokines; Hypertension, Pulmonary; Lung; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Artery; RNA, Messenger; Sphingosine

Little is known about the contribution of bone marrow-derived progenitor cells (BMPCs) in the regulation endothelial barrier function as defined by microvascular permeability alterations at the level of adherens junctions (AJs).. We investigated the role of BMPCs in annealing AJs and thereby in preventing lung edema formation induced by endotoxin (LPS).. We observed that BMPCs enhanced basal endothelial barrier function and prevented the increase in pulmonary microvascular permeability and edema formation in mice after LPS challenge. Coculture of BMPCs with endothelial cells induced Rac1 and Cdc42 activation and AJ assembly in endothelial cells. However, transplantation of BMPCs isolated from sphingosine kinase-1-null mice (SPHK1(-/-)), having impaired S1P production, failed to activate Rac1 and Cdc42 or protect the endothelial barrier.. These results demonstrate that BMPCs have the ability to reanneal endothelial AJs by paracrine S1P release in the inflammatory milieu and the consequent activation of Rac-1 and Cdc42 in endothelial cells.

Topics: Adherens Junctions; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Capillary Permeability; cdc42 GTP-Binding Protein; Cell Movement; Cell Separation; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Flow Cytometry; Humans; Lipopolysaccharides; Lung; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuropeptides; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Edema; rac GTP-Binding Proteins; rac1 GTP-Binding Protein; Signal Transduction; Sphingosine; Stem Cells; Time Factors

The pleiotropic lipid mediator sphingosine-1-phosphate (S1P) can act intracellularly independently of its cell surface receptors through unknown mechanisms. Sphingosine kinase 2 (SphK2), one of the isoenzymes that generates S1P, was associated with histone H3 and produced S1P that regulated histone acetylation. S1P specifically bound to the histone deacetylases HDAC1 and HDAC2 and inhibited their enzymatic activity, preventing the removal of acetyl groups from lysine residues within histone tails. SphK2 associated with HDAC1 and HDAC2 in repressor complexes and was selectively enriched at the promoters of the genes encoding the cyclin-dependent kinase inhibitor p21 or the transcriptional regulator c-fos, where it enhanced local histone H3 acetylation and transcription. Thus, HDACs are direct intracellular targets of S1P and link nuclear S1P to epigenetic regulation of gene expression.

Topics: Acetylation; Catalytic Domain; Cell Line, Tumor; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p21; Epigenesis, Genetic; Genes, fos; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Lysine; Lysophospholipids; Nucleosomes; Phosphotransferases (Alcohol Group Acceptor); Promoter Regions, Genetic; Repressor Proteins; RNA Interference; Sphingosine; Tetradecanoylphorbol Acetate

Renal fibrosis contributes to glomerulosclerosis and tubulointerstitial damage in chronic kidney disease. A well-established pathway implicated in the progression of fibrosis is the induction of connective tissue growth factor by transforming growth factor-beta, resulting in the accumulation of extracellular matrix proteins. Ren and colleagues demonstrate that sphingosine kinase-1 is involved in the regulation of this pathway in the glomerulus. This raises the possibility of targeting sphingosine kinase-1 to prevent fibrosis in chronic kidney disease patients.

Topics: Animals; Chronic Disease; Connective Tissue Growth Factor; Disease Progression; Fibrosis; Humans; Kidney Diseases; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Podocytes; Protein Kinase Inhibitors; Sphingosine; Transforming Growth Factor beta2

Topics: Adherens Junctions; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Capillary Permeability; cdc42 GTP-Binding Protein; Cell Movement; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Humans; Lung; Lysophospholipids; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Edema; rac GTP-Binding Proteins; Signal Transduction; Sphingosine; Stem Cells; Time Factors

Calcium entry is one of the main regulators of intracellular signaling. Here, we have described the importance of sphingosine, sphingosine kinase 1 (SK1), and sphingosine 1-phosphate (S1P) in regulating calcium entry in thyroid FRTL-5 cells. In cells incubated with the phosphatase inhibitor calyculin A, which evokes calcium entry without mobilizing sequestered intracellular calcium, sphingosine inhibited calcium entry in a concentration-dependent manner. Furthermore, inhibiting SK1 or the ATP-binding cassette ABCC1 multidrug transporter attenuated calcium entry. The addition of exogenous S1P restored calcium entry. Neither sphingosine nor inhibition of SK1 attenuated thapsigargin-evoked calcium entry. Blocking S1P receptor 2 or phospholipase C attenuated calcium entry, whereas blocking S1P receptor 3 did not. Overexpression of wild-type SK1, but not SK2, enhanced calyculin-evoked calcium entry compared with mock-transfected cells, whereas calcium entry was decreased in cells transfected with the dominant-negative G82D SK1 mutant. Exogenous S1P restored calcium entry in G82D cells. Our results suggest that the calcium entry pathway is blocked by sphingosine and that activation of SK1 and the production of S1P, through an autocrine mechanism, facilitate calcium entry through activation of S1P receptor 2. This is a novel mechanism by which the sphingosine-S1P rheostat regulates cellular calcium homeostasis.

Topics: Animals; Autocrine Communication; Calcium; Cell Line; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Thyroid Gland

Sphingosine kinase 1 (SK1) is an important enzyme that regulates the balance between ceramide and sphingosine-1-phosphate (S1P). Potent and novel SK1 inhibitors (6ag, 9ab and 12aa) have been discovered through a series of modifications of sphingosine (1), the substrate of this enzyme.

Topics: Aminobutyrates; Cell Line, Tumor; Drug Design; Enzyme Inhibitors; Homoserine; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Proline; Sphingosine

Sphingosine-1-phosphate, the product of sphingosine kinase (SK) activity, is a sphingolipid metabolite that regulates cell growth, survival and migration. It is also known to affect diuresis, natriuresis and renovascular contraction in rats, although the mechanisms through which it affects these processes are not known. No previous report has addressed the differences among the kidney zones regarding endogenous SK expression and activity. Therefore, we examined SK1 distribution and activity in the various kidney zones: cortex, medulla and papilla. We found that SK1 expression does not correlate with enzyme activity. Study of the expression showed that the enzyme is highly expressed in cortex, followed by medulla and papilla. However, medulla had the highest enzyme activity. In all kidney zones, SK1 expression was mainly cytosolic. Regarding enzyme activity, whereas we found no difference between cytosol, membrane and nucleus in renal medulla, the membrane-bound enzyme presented the highest activity in cortex and papilla. SK1 distribution observed by immunohistochemical staining showed higher expression in cortical proximal convoluted epithelial cells. In medulla, immunostaining was observed as patches of staining, whereas in papilla, positive immunostaining was exclusively restricted to collecting duct cells. We also evaluated the effects of bradykinin and angiotensin II on SK1 activity.

Topics: Angiotensin II; Animals; Bradykinin; Kidney; Kidney Cortex; Kidney Medulla; Lysophospholipids; Male; Organ Specificity; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Sphingosine

In mammalian cells sphingosine-1-phosphate (S1P) is a well-established messenger molecule that participates in a wide range of signalling pathways. The objective of the work reported here was to investigate the extent to which phosphorylated long-chain sphingoid bases, such as sphingosine-1-phosphate and phytosphingosine-1-phosphate (phytoS1P) are used in plant cell signalling. To do this, we manipulated Arabidopsis genes capable of metabolizing these messenger molecules. We show that Sphingosine kinase1 (SPHK1) encodes an enzyme that phosphorylates sphingosine, phytosphingosine and other sphingoid long-chain bases. The stomata of SPHK1-KD Arabidopsis plants were less sensitive, whereas the stomata of SPHK1-OE plants were more sensitive, than wild type to ABA. The rate of germination of SPHK1-KD was enhanced, whereas the converse was true for SPHK1-OE seed. Reducing expression of either the putative Arabidopsis S1P phosphatase (SPPASE) or the DPL1 gene, which encodes an enzyme with S1P lyase activity, individually, had no effect on guard-cell ABA signalling; however, stomatal responses to ABA in SPPASEDPL1 RNAi plants were compromised. Reducing the expression of DPL1 had no effect on germination; however, germination of SPPASE RNAi seeds was more sensitive to applied ABA. We also found evidence that expression of SPHK1 and SPPASE were coordinately regulated, and discuss how this might contribute to robustness in guard-cell signalling. In summary, our data establish SPHK1 as a component in two separate plant signalling systems, opening the possibility that phosphorylated long-chain sphingoid bases such as S1P and phytoS1P are ubiquitous messengers in plants.

Topics: Abscisic Acid; Arabidopsis; Arabidopsis Proteins; Cells, Cultured; Cloning, Molecular; DNA, Bacterial; Gene Expression Regulation, Plant; Germination; Humans; Lysophospholipids; Membrane Proteins; Mutagenesis, Insertional; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Plant Stomata; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Plant; Signal Transduction; Sphingosine; Substrate Specificity

Natural killer (NK) cells play an important role in the innate immune response. Interleukin-18 (IL-18) is a well-known interferon-gamma (IFN-gamma inducing factor, which stimulates immune response in NK and T cells. Sphingosine kinase (SPHK) catalyzes the formation of sphingosine 1-phosphate (S1P), which acts as a second messenger to function as an anti-apoptotic factor and proliferation stimulator of immune cells. In this study, to elucidate whether SPHK is involved in IL-18-induced IFN-gamma production, we measured IL-18-induced IFN-gamma production after pre-treatment with SPHK inhibitor (SKI) in NK-92MI cells. We found that IL-18-induced IFN-gamma expression was blocked by SKI pre-treatment in both mRNA and protein levels. In addition, the increased IFN-gamma production by stimulation with IL-18 is mediated through both SPHK and p38 MAPK. To determine the upstream signals of SKI and p38 MAPK in IL-18-induced IFN-gamma production, phosphorylation levels of p38 MAPK was measured after SKI pre-treatment. As a result, inhibition of SPHK by SKI blocked phosphorylation of p38 MAPK, showing that SPHK activation by IL-18 is an upstream signal of p38 MAPK activation. Inhibition of SPHK by SKI also inhibited IL-18-induced IFN-gamma production in human primary NK cells. In conclusion, SPHK activation is an essential factor for IL-18-induced IFN-gamma production via p38 MAPK.

Topics: Cell Line; Enzyme Activation; Humans; Interferon-gamma; Interleukin-18; Killer Cells, Natural; Lysophospholipids; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Sphingosine

Sphingolipids are present in membranes of all eukaryotic cells. Bioactive sphingolipids also function as signaling molecules that regulate cellular processes such as proliferation, migration, and apoptosis. Human cytomegalovirus (HCMV) exploits a variety of cellular signaling pathways to promote its own replication. However, whether HCMV modulates lipid signaling pathways is an essentially unexplored area of research in virus-host cell interactions. In this study, we examined the accumulation of the bioactive sphingolipids and the enzymes responsible for the biosynthesis and degradation of these lipids. HCMV infection results in increased accumulation and activity of sphingosine kinase (SphK), the enzyme that generates sphingosine 1-phosphate (S1P) and dihydrosphingosine 1-phosphate (dhS1P). We also utilized a mass spectrometry approach to generate a sphingolipidomic profile of HCMV-infected cells. We show that HCMV infection results in increased levels of dhS1P and ceramide at 24 h, suggesting an enhancement of de novo sphingolipid synthesis. Subsequently dihydrosphingosine and dhS1P decrease at 48 h consistent with attenuation of de novo sphingolipid synthesis. Finally, we present evidence that de novo sphingolipid synthesis and sphingosine kinase activity directly impact virus gene expression and virus growth. Together, these findings demonstrate that host cell sphingolipids are dynamically regulated upon infection with a herpes virus in a manner that impacts virus replication.

Topics: Apoptosis; Cell Line, Tumor; Cytomegalovirus; Gene Expression Regulation, Viral; Genes, Immediate-Early; Humans; Lysophospholipids; Mass Spectrometry; Models, Biological; Phosphotransferases (Alcohol Group Acceptor); RNA Interference; Sphingolipids; Sphingosine; Time Factors; Virus Replication

We have previously reported that, in prostate cancer, inhibition of the oncogenic sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) pathway is a key element in chemotherapy-induced apoptosis. Here, we show that selective pharmacologic inhibition of SphK1 triggers apoptosis in LNCaP and PC-3 prostate cancer cells, an effect that is reversed by SphK1 enforced expression. More importantly, we show for the first time that the up-regulation of the SphK1/S1P pathway plays a crucial role in the resistance of prostate cancer cells to chemotherapy. Importantly, pharmacologic SphK1 inhibition with the B-5354c compound sensitizes LNCaP and PC-3 cells to docetaxel and camptothecin, respectively. In vivo, camptothecin and B-5354c alone display a limited effect on tumor growth in PC-3 cells, whereas in combination there is a synergy of effect on tumor size with a significant increase in the ceramide to S1P sphingolipid ratio. To conclude, our study highlights the notion that drugs specifically designed to inhibit SphK1 could provide a means of enhancing the effects of conventional treatment through the prosurvival antiapoptotic SphK1/S1P pathway.

Topics: 4-Aminobenzoic Acid; Animals; Antineoplastic Agents; Apoptosis; Camptothecin; Caspases; Cell Line, Tumor; Cell Survival; Ceramides; Drug Therapy, Combination; Green Fluorescent Proteins; Humans; Irinotecan; Lysophospholipids; Male; Mice; Neoplasm Metastasis; para-Aminobenzoates; Phosphotransferases (Alcohol Group Acceptor); Prostatic Neoplasms; Sphingosine; Treatment Outcome; Xenograft Model Antitumor Assays

Sphingosine-1-phosphate (S1P) is a pleiotropic lysolipid that has recently been implicated in the regulation of tissue fibrosis. However, the fibrogenic potential of S1P in the eye has not previously been investigated. In the current study, we evaluated cells from the anterior and posterior segments of the eye for the presence of S1P and their potential ability to produce and respond to S1P. In addition, we investigated the regulatory role of S1P as a mediator of proliferation, cellular transformation and pro-fibrotic protein expression in human retinal pigmented epithelial cells. Expression of S1P receptors and sphingosine kinases (the enzymes that produce S1P) was examined using RT-PCR, and intracellular localization of S1P was examined using immunoblotting, immunohistochemistry and ELISA in primary human retinal pigmented epithelial (RPE) cells, primary human conjunctival fibroblasts (ConF), and primary human corneal fibroblasts (CF). RPE cell proliferation was determined using an MTT-based cell proliferation assay, and RPE myofibroblast transformation, collagen type I production and profibrotic protein expression were assessed using immunofluorescence, ELISA and immunoblot. S1P(1-3, 5) receptors and sphingosine kinases 1 and 2 were expressed and intracellular pools of S1P were detected in RPE cells, ConF and CF. S1P stimulated RPE cell proliferation in a dose- and time-dependent manner. S1P induced myofibroblast transformation of RPE cells, as indicated by increased alpha-smooth muscle actin (alpha-SMA) expression and its incorporation into prominent stress fibers, and promoted collagen type I production. S1P stimulated the expression of plasminogen activator inhibitor-1 (PAI-1) and heat shock protein 47 (HSP47), two proteins that are linked to increased tissue fibrosis. Combined, these data demonstrate that RPE cells, ConF and CF from the human eye not only have the molecular ability to produce and respond to S1P, but also contain S1P. Furthermore, S1P promotes proliferation, myofibroblast transformation, collagen production and pro-fibrotic protein expression by human RPE cells. These data suggest that S1P is a previously unrecognized mediator of profibrotic cellular function and signaling in the eye.

Topics: Anterior Eye Segment; Cell Proliferation; Cell Transdifferentiation; Cells, Cultured; Collagen Type I; Dose-Response Relationship, Drug; Eye; Fibroblasts; HSP47 Heat-Shock Proteins; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Plasminogen Activator Inhibitor 1; Receptors, Lysosphingolipid; Retinal Pigment Epithelium; Sphingosine

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are lysophospholipid mediators of diverse cellular processes important for cancer progression. S1P is produced by two sphingosine kinases, SphK1 and SphK2. Expression of SphK1 is elevated in many cancers. Here, we report that LPA markedly enhanced SphK1 mRNA and protein in gastric cancer MKN1 cells but had no effect on SphK2. LPA also up-regulated SphK1 expression in other human cancer cells that endogenously express the LPA(1) receptor, such as DLD1 colon cancer cells and MDA-MB-231 breast cancer cells, but not in HT29 colon cancer cells or MDA-MB-453 breast cancer cells, which do not express the LPA(1) receptor. An LPA(1) receptor antagonist or down-regulation of its expression prevented SphK1 and S1P(3) receptor up-regulation by LPA. LPA transactivated the epidermal growth factor receptor (EGFR) in these cells, and the EGFR inhibitor AG1478 attenuated the increased SphK1 and S1P(3) expression induced by LPA. Moreover, down-regulation of SphK1 attenuated LPA-stimulated migration and invasion of MNK1 cells yet had no effect on expression of neovascularizing factors, such as interleukin (IL)-8, IL-6, urokinase-type plasminogen activator (uPA), or uPA receptor induced by LPA. Finally, down-regulation of S1P(3), but not S1P(1), also reduced LPA-stimulated migration and invasion of MKN1 cells. Collectively, our results suggest that SphK1 is a convergence point of multiple cell surface receptors for three different ligands, LPA, EGF, and S1P, which have all been implicated in regulation of motility and invasiveness of cancer cells.

Topics: Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; Chemotaxis; Colonic Neoplasms; ErbB Receptors; Humans; Interleukin-6; Interleukin-8; Lysophospholipids; Neoplasm Invasiveness; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysophosphatidic Acid; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine; Stomach Neoplasms; Transcriptional Activation; Tumor Cells, Cultured; Up-Regulation; Urokinase-Type Plasminogen Activator

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. In order to investigate the role of sphingosine kinase-1 (SphK-1)/sphingosine 1-phosphate (S1P) signal pathway in the expression of CML cells, and to explore whether P210(bcr/abl) involved is activating SphK-1/S1P signal pathwey, the expressions of SphK-1 and S1P receptor mRNA in bcr/abl positive K562 cells and bcr/abl positive primary CML cells were detected by RT-PCR, the imatinib mesylate, the specific inhibitor of P210(bcr/abl) was employed to inhibit the P210(bcr/abl) tyrosine kinases of K562 cells and CML primary cells, and then the intracellular SphK-1 activity was assayed. The results indicated that after being cultured with 2.5 micromol/L imatinib mesylate for 0.5, 2, 6, 24 and 48 hours, the intensions of inhibiting SphK-1 activity were 0.007%, 38.9%, 34.6%, 28.1% and 76.1% resepectively. SphK-1 activity in CML cells also was reduced by 2.5 micromol/L imatinib mesylate (16.8% - 41.9% decrease). It is concluded that the CML cells express SphK-1 and different S1P receptor, and P210(bcr/abl) fusion protein in CML cells can activate SphK-1.

Topics: Benzamides; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Piperazines; Pyrimidines; RNA, Messenger; Signal Transduction; Sphingosine

We investigated the regulation of the sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) axis and its role during pregnancy in the rat myometrium. SphK1 and SphK2 were coexpressed in myometrium during gestation. The levels and activity of SphK1/2 were modest at midgestation (d 12), increased at d 19 and progressively declined to low at postpartum. Similar patterns were observed for the phosphorylation of ERK and protein kinase C (PKC). Inhibition of PKC and ERK reduced SphK1/2 activity. In late pregnancy, levels of cyclooxygenase 2 (COX2) increased in parallel to SphK levels. Using a pharmacological approach, we demonstrated that in primary cultures of myometrial cells from d-19 pregnant rats, induction of COX2 was mediated by 4beta-phorbol 12,13-dibutyrate and IL-1beta through sequential activation of PKC, ERK1/2, and SphK1. S1P produced by SphK1 was released in the medium. Addition of S1P, IL-1beta or 4beta-phorbol 12,13-dibutyrate enhanced COX2 levels via Gi protein. Interestingly, S1P was also released by myometrial tissues at late gestation. This event was dependent on PKC/ERK/SphK1. By contrast, in d-12 myometrial tissues, the release of S1P was markedly reduced in association with low levels of SphK1 and COX2. However, prolonged incubation of myometrium from midgestation led to the induction of COX2. This effect was blocked by SphK inhibitors, providing evidence of the close relationship between SphK activity and COX2 induction in rat myometrium. Overall, our findings provided insight into the physiological relevance of the SphK activation and S1P release in uterine smooth muscle during gestation.

Topics: Animals; Cells, Cultured; Cyclooxygenase 2; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Female; Gestational Age; Lysophospholipids; Models, Biological; Myometrium; Phosphotransferases (Alcohol Group Acceptor); Postpartum Period; Pregnancy; Protein Kinase C; Rats; Rats, Wistar; Signal Transduction; Sphingosine

Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.

Topics: Animals; Behavior, Animal; Cyclic AMP; Fluorescence Resonance Energy Transfer; Lysophospholipids; Mice; Mice, Knockout; Neurons; Pain; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Signal Transduction; Sphingosine; Spine

Glioblastoma multiforme is an invasive primary brain tumor, which evades the current standard treatments. The invasion of glioblastoma cells into healthy brain tissue partly depends on the proteolytic and nonproteolytic activities of the plasminogen activator system proteins, including the urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), and a receptor for uPA (uPAR). Here we show that sphingosine-1-phosphate (S1P) and the inflammatory mediator interleukin-1 (IL-1) increase the mRNA and protein expression of PAI-1 and uPAR and enhance the invasion of U373 glioblastoma cells. Although IL-1 enhanced the expression of sphingosine kinase 1 (SphK1), the enzyme that produces S1P, down-regulation of SphK1 had no effect on the IL-1-induced uPAR or PAI-1 mRNA expression, suggesting that these actions of IL-1 are independent of S1P production. Indeed, the S1P-induced mRNA expression of uPAR and PAI-1 was blocked by the S1P(2) receptor antagonist JTE013 and by the down-regulation of S1P(2) using siRNA. Accordingly, the inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 and Rho-kinase, two downstream signaling cascades activated by S1P(2), blocked the activation of PAI-1 and uPAR mRNA expression by S1P. More importantly, the attachment of glioblastoma cells was inhibited by the addition of exogenous PAI-1 or siRNA to uPAR, whereas the invasion of glioblastoma cells induced by S1P or IL-1 correlated with their ability to enhance the expression of PAI-1 and uPAR. Collectively, these results indicate that S1P and IL-1 activate distinct pathways leading to the mRNA and protein expression of PAI-1 and uPAR, which are important for glioblastoma invasiveness.

Topics: Blotting, Northern; Blotting, Western; Brain Neoplasms; Cell Adhesion; Glioblastoma; Humans; Interleukin-1; Lysophospholipids; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Phosphotransferases (Alcohol Group Acceptor); Plasminogen Activator Inhibitor 1; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sphingosine; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The lipid mediator sphingosine-1-phosphate (S1P), the product of sphingosine kinase (SPHK)-induced phosphorylation of sphingosine, is known to stabilize interendothelial junctions and prevent microvessel leakiness. Here, we investigated the role of SPHK1 activation in regulating the increase in pulmonary microvessel permeability induced by challenge of mice with lipopolysaccharide or thrombin ligation of protease-activating receptor (PAR)-1. Both lipopolysaccharide and thrombin increased mouse lung microvascular permeability and resulted in a delayed activation of SPHK1 that was coupled to the onset of restoration of permeability. In contrast to wild-type mice, Sphk1(-/-) mice showed markedly enhanced pulmonary edema formation in response to lipopolysaccharide and PAR-1 activation. Using endothelial cells challenged with thrombin concentration (50 nmol/L) that elicited a transient but reversible increase in endothelial permeability, we observed that increased SPHK1 activity and decreased intracellular S1P concentration preceded the onset of barrier recovery. Thus, we tested the hypothesis that released S1P in a paracrine manner activates its receptor S1P1 to restore the endothelial barrier. Knockdown of SPHK1 decreased basal S1P production and Rac1 activity but increased basal endothelial permeability. In SPHK1-depleted cells, PAR-1 activation failed to induce Rac1 activation but augmented RhoA activation and endothelial hyperpermeability response. Knockdown of S1P1 receptor in endothelial cells also enhanced the increase in endothelial permeability following PAR-1 activation. S1P treatment of Sphk1(-/-) lungs or SPHK1-deficient endothelial cells restored endothelial barrier function. Our results suggest the crucial role of activation of the SPHK1-->S1P-->S1P1 signaling pathway in response to inflammatory mediators in endothelial cells in regulating endothelial barrier homeostasis.

Topics: Animals; Capillary Permeability; Cells, Cultured; Enzyme Activation; Hemostatics; Humans; Inflammation Mediators; Intercellular Junctions; Lipopolysaccharides; Lung; Lysophospholipids; Mice; Mice, Knockout; Neuropeptides; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Edema; rac GTP-Binding Proteins; rac1 GTP-Binding Protein; Receptor, PAR-1; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Thrombin

Because nerve growth factor (NGF) is elevated during inflammation, plays a causal role in the initiation of hyperalgesia, and is known to activate the sphingomyelin signalling pathway, we examined whether NGF and its putative second messenger, ceramide, could modulate the excitability of capsaicin-sensitive adult sensory neurons. Using the whole-cell patch-clamp recording technique, exposure of isolated sensory neurons to either 100 ng/mL NGF or 1 mmol/L N-acetyl sphingosine (C2-ceramide) produced a 3-4 fold increase in the number of action potentials (APs) evoked by a ramp of depolarizing current in a time-dependent manner. Intracellular perfusion with bacterial sphingomyelinase (SMase) also increased the number of APs suggesting that the release of native ceramide enhanced neuronal excitability. Glutathione, an inhibitor of neutral SMase, completely blocked the NGF-induced augmentation of AP firing, whereas dithiothreitol, an inhibitor of acidic SMase, was without effect. In the presence of glutathione and NGF, exogenous ceramide still enhanced the number of evoked APs, indicating that the sensitizing action of ceramide was downstream of NGF. To investigate the mechanisms of actions for NGF and ceramide, isolated membrane currents were examined. Both NGF and ceramide facilitated the peak amplitude of the TTX-resistant sodium current (TTX-R I(Na)) by approximately 1.5-fold and shifted the activation to more hyperpolarized voltages. In addition, NGF and ceramide suppressed an outward potassium current (I(K)) by ~35%. The inflammatory prostaglandin, PGE2, produced an additional suppression of I(K) after exposure to ceramide (~35%), suggesting that these agents might act on different targets. Based on the existing literature, it is not clear whether this NGF-induced sensitization is mediated by the high-affinity TrkA receptor or the low-affinity p75 neurotrophin receptor. Pretreatment with the p75 blocking antibody completely prevents the NGF-induced increase in the number of APs evoked by the current ramp. Although the sensitization by NGF was blocked, the antibody had no effect on the capacity of ceramide, a putative downstream signalling molecule, to enhance the excitability. Ceramide can be metabolized by ceramidase to sphingosine (Sph) and Sph to sphingosine 1-phosphate (S1P) by sphingosine kinase. It is well established that each of these products of sphingomyelin metabolism can act as intracellular signalling molecules. This raises the question as to

Topics: Action Potentials; Animals; Cells, Cultured; Ceramides; Lysophospholipids; Nerve Growth Factor; Patch-Clamp Techniques; Phosphotransferases (Alcohol Group Acceptor); Sensory Receptor Cells; Signal Transduction; Sphingomyelins; Sphingosine

Sphingosine kinase (SphK) is a key enzyme in the sphingolipid metabolic pathway responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P). SphK/S1P play a critical role in angiogenesis, inflammation, and various pathologic conditions. Recently, S1P(1) receptor was found to be expressed in rheumatoid arthritis (RA) synovium, and S1P signaling via S1P(1) enhances synoviocyte proliferation, COX-2 expression, and prostaglandin E(2) production. Here, we examined the role of SphK/S1P in RA using a potent SphK inhibitor, N,N-dimethylsphingosine (DMS), and a molecular approach against one of its isoenzymes, SphK1. We observed that levels of S1P in the synovial fluid of RA patients were significantly higher than those of osteoarthritis patients. Additionally, DMS significantly reduced the levels of TNF-alpha, IL-6, IL-1beta, MCP-1, and MMP-9 in cell-contact assays using both Jurkat-U937 cells and RA PBMCs. In a murine collagen-induced arthritis model, i.p. administration of DMS significantly inhibited disease severity and reduced articular inflammation and joint destruction. Treatment of DMS also down-regulated serum levels IL-6, TNF-alpha, IFN-gamma, S1P, and IgG1 and IgG2a anti-collagen Ab. Furthermore, DMS-treated mice also displayed suppressed proinflammatory cytokine production in response to type II collagen in vitro. Moreover, similar reduction in incidence and disease activity was observed in mice treated with SphK1 knock-down via small interfering RNA approach. Together, these results demonstrate SphK modulation may provide a novel approach in treating chronic autoimmune conditions such as RA by inhibiting the release of pro-inflammatory cytokines.

Topics: Animals; Arthritis, Rheumatoid; Cell Proliferation; Collagen Type II; Cyclooxygenase 2; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Humans; Inflammation; Inflammation Mediators; Jurkat Cells; Leukocytes, Mononuclear; Lysophospholipids; Matrix Metalloproteinase 9; Mice; Mice, Inbred DBA; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Small Interfering; Signal Transduction; Sphingosine; Synovial Fluid; U937 Cells

Under most conditions, resorbed bone is nearly precisely replaced in location and amount by new bone. Thus, it has long been recognized that bone loss through osteoclast-mediated bone resorption and bone replacement through osteoblast-mediated bone formation are tightly coupled processes. Abundant data conclusively demonstrate that osteoblasts direct osteoclast differentiation. Key questions remain, however, as to how osteoblasts are recruited to the resorption site and how the amount of bone produced is so precisely controlled. We hypothesized that osteoclasts play a crucial role in the promotion of bone formation. We found that osteoclast conditioned medium stimulates human mesenchymal stem (hMS) cell migration and differentiation toward the osteoblast lineage as measured by mineralized nodule formation in vitro. We identified candidate osteoclast-derived coupling factors using the Affymetrix microarray. We observed significant induction of sphingosine kinase 1 (SPHK1), which catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), in mature multinucleated osteoclasts as compared with preosteoclasts. S1P induces osteoblast precursor recruitment and promotes mature cell survival. Wnt10b and BMP6 also were significantly increased in mature osteoclasts, whereas sclerostin levels decreased during differentiation. Stimulation of hMS cell nodule formation by osteoclast conditioned media was attenuated by the Wnt antagonist Dkk1, a BMP6-neutralizing antibody, and by a S1P antagonist. BMP6 antibodies and the S1P antagonist, but not Dkk1, reduced osteoclast conditioned media-induced hMS chemokinesis. In summary, our findings indicate that osteoclasts may recruit osteoprogenitors to the site of bone remodeling through SIP and BMP6 and stimulate bone formation through increased activation of Wnt/BMP pathways.

Topics: Animals; Bone Morphogenetic Protein 6; Calcification, Physiologic; Cell Differentiation; Cell Line; Cell Movement; Chemokines; Culture Media, Conditioned; Humans; Intercellular Signaling Peptides and Proteins; Lysophospholipids; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Osteoclasts; Osteogenesis; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins; Signal Transduction; Sphingosine; Wnt Proteins

Sphingosine kinase (SphK) is a conserved lipid kinase that catalyzes the formation of sphingosine 1-phosphate (S1P), an important lipid mediator, which regulates fundamental biological processes. Here, we provide evidence that SphK is required for the achievement of cell growth arrest as well as myogenic differentiation of C2C12 myoblasts. Indeed, SphK activity, SphK1 protein content and S1P formation were found to be enhanced in myoblasts that became confluent as well as in differentiating cells. Enforced expression of SphK1 reduced the myoblast proliferation rate, enhanced the expression of myogenic differentiation markers and anticipated the onset of differentiated muscle phenotype. Conversely, down-regulation of SphK1 by specific silencing by RNA interference or overexpression of the catalytically inactive SphK1, significantly increased cell growth and delayed the beginning of myogenesis; noticeably, exogenous addition of S1P rescued the biological processes. Importantly, stimulation of myogenesis in SphK1-overexpressing myoblasts was abrogated by treatment with short interfering RNA specific for S1P(2) receptor. This is the first report of the role of endogenous SphK1 in myoblast growth arrest and stimulation of myogenesis through the formation of S1P that acts as morphogenic factor via the engagement of S1P(2).

Topics: Animals; Cell Differentiation; Cell Line; Cell Proliferation; Culture Media, Conditioned; Down-Regulation; Gene Expression Regulation, Enzymologic; Isoenzymes; Lysophospholipids; Mice; Muscle, Skeletal; Myoblasts; Oligonucleotides, Antisense; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sphingosine; Transfection

Sphingosine 1-phosphate (S1P) mediates a number of cellular responses, including growth and proliferation. Skeletal muscle possesses the full enzymatic machinery to generate S1P and expresses the transcripts of S1P receptors. The aim of this work was to localize S1P receptors in rat skeletal muscle and to investigate whether S1P exerts a trophic action on muscle fibers. RT-PCR and Western blot analyses demonstrated the expression of S1P(1) and S1P(3) receptors by soleus muscle. Immunofluorescence revealed that S1P(1) and S1P(3) receptors are localized at the cell membrane of muscle fibers and in the T-tubule membranes. The receptors also decorate the nuclear membrane. S1P(1) receptors were also present at the neuromuscular junction. The possible trophic action of S1P was investigated by utilizing the denervation atrophy model. Rat soleus muscle was analyzed 7 and 14 days after motor nerve cut. During denervation, S1P was continuously delivered to the muscle through a mini osmotic pump. S1P and its precursor, sphingosine (Sph), significantly attenuated the progress of denervation-induced muscle atrophy. The trophic effect of Sph was prevented by N,N-dimethylsphingosine, an inhibitor of Sph kinase, the enzyme that converts Sph into S1P. Neutralization of circulating S1P by a specific antibody further demonstrated that S1P was responsible for the trophic effects of S1P during denervation atrophy. Denervation produced the down regulation of S1P(1) and S1P(3) receptors, regardless of the presence of the receptor agonist. In conclusion, the results suggest that S1P acts as a trophic factor of skeletal muscle.

Topics: Animals; Antibodies; Cell Enlargement; Cell Membrane; Disease Models, Animal; Enzyme Inhibitors; Hypertrophy; Infusion Pumps, Implantable; Lysophospholipids; Male; Muscle Denervation; Muscle, Skeletal; Muscular Atrophy; MyoD Protein; Myogenin; Myosin Heavy Chains; Neuromuscular Junction; Nuclear Envelope; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Receptors, Lysosphingolipid; RNA, Messenger; Sciatic Nerve; Sphingosine; Time Factors

Beside their structural role for the cell membrane the family of sphingolipids act as effector molecules in signal transduction with links to various aspects of cancer initiation, progression and treatment response. The "sphingolipid rheostat" balances between apoptosis inducing ceramid and growth promoting sphingosine-1-phosphate. We analyzed gene expression of 43 proteins from this pathway in different subtypes of breast cancer using microarray data of 1,269 tumor samples (test set n=171; validation sets n=1098) and observed significant differences for several genes. Sphingosine kinase 1 (SPHK1), ceramide galactosyltransferase (UGT8), and Ganglioside GD3-Synthase (ST8SIA1) displayed higher expression among ER negative tumors. In contrast, glucosylceramidsynthase (GCS), dihydroceramidsynthases (LASS4, LASS 6) and acid ceramidase (ASAH1) were higher expressed in ER positive samples. Survival analysis revealed a worse outcome of patients with high SPHK1 expression. To avoid a confounding effect of the ER status we also restricted the analysis to 750 patients with ER positive tumors. Again a worse outcome was observed for tumors displaying high SPHK1 expression. While 75.8+/-1.9% of the patients with tumors low in SPHK1 expression were free of metastasis at 5 years, this was the case for only 64.9+/-3.6% of patients with tumors displaying high SPHK1 expression (P=0.008). Immunohistochemistry identified the carcinoma cells as the major source of SPHK1 expression in the tumor. The correlation of SPHK1 with a poor prognosis as well as its high expression among ER negative tumors are in line with the antiapoptotic and proliferative properties of its product sphingosine-1-phosphate. Targeting of the sphingolipid rheostat may thus open new treatment options.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Female; Gene Expression Profiling; Humans; Immunoenzyme Techniques; Lysophospholipids; Middle Aged; Oligonucleotide Array Sequence Analysis; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Sphingosine; Survival Rate

Sphingosine 1-phosphate (S1P), a lysophospholipid, plays an important chemotactic role in the migration of lymphocytes and germ cells, and is known to regulate aspects of central nervous system development such as neurogenesis and neuronal migration. Its role in axon guidance, however, has not been examined. We show that sphingosine kinase 1, an enzyme that generates S1P, is expressed in areas surrounding the Xenopus retinal axon pathway, and that gain or loss of S1P function in vivo causes errors in axon navigation. Chemotropic assays reveal that S1P elicits fast repulsive responses in retinal growth cones. These responses require heparan sulfate, are sensitive to inhibitors of proteasomal degradation, and involve RhoA and LIM kinase activation. Together, the data identify downstream components that mediate S1P-induced growth cone responses and implicate S1P signalling in axon guidance.

Topics: Animals; Axons; Embryo, Nonmammalian; Enzyme Activation; Gene Expression Regulation, Developmental; Growth Cones; Heparitin Sulfate; Humans; Lim Kinases; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Proteasome Inhibitors; Receptors, Lysosphingolipid; Retina; rhoA GTP-Binding Protein; Signal Transduction; Sphingosine; Visual Pathways; Xenopus laevis

Sphingosine kinase 1 (SK1) and its product sphingosine-1-phosphate (S1P) have been implicated in the regulation of many cellular processes including growth regulation, protection from apoptosis, stimulation of angiogenesis, and most recently as mediators of the TNF-alpha inflammatory response. In this study we set out to examine the role of SK1/S1P in the RAW macrophage response to the potent inflammatory stimulus lipopolysaccharide (LPS). We show that LPS increases cellular levels of SK1 message and protein. This increase is at the transcriptional level and is accompanied by increased SK activity and generation of S1P. S1P is able to cause increases in COX-2 and PGE2 levels in RAW cells. Knockdown of SK1 using siRNA is able to inhibit the TNF but not the LPS inflammatory response. Moreover, knockdown of SK1 enhances both TNF- and LPS-induced apoptosis. These data indicate that there is a dual and distinct role for SK1 and S1P in the TNF and the LPS inflammatory pathways.

Topics: Animals; Apoptosis; Cell Line, Tumor; Inflammation; Lipopolysaccharides; Lysophospholipids; Macrophages; Mice; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tumor Necrosis Factor-alpha; Up-Regulation

Removal of apoptotic cells by phagocytes is considered a pivotal immune regulatory process. Although considerable knowledge has been obtained on the postphagocytic macrophage phenotype, there is little information on molecular mechanisms, which provoke macrophage polarization. In this study, we show that human apoptotic Jurkat cells (AC) or AC-conditioned medium (CM) rapidly induces cyclooxygenase-2 (COX-2) expression in mouse RAW264.7 macrophages via sphingosine-1-phosphate (S1P). Pharmacological inhibition of S1P release from AC or using CM from cells with a knockdown of sphingosine kinase 2 in human MCF-7 cells abrogates this effect. Expression of COX-2 resulted from an increase in mRNA stability via its 3'-untranslated region (UTR), shown by COX-2-3'-UTR and AU-rich element-driven reporter assays. Western analysis corroborated increased nucleocytoplasmic shuttling of the RNA-binding protein HuR after CM treatment. RNA EMSA analysis revealed an S1P- and CM-mediated increase in HuR-RNA binding to a COX-2-specific UTR, whereas HuR knockdown pointed to its importance for S1P in CM-induced COX-2 expression. Immunofluorescence microscopy of phospholipase A2 (PLA2) and ELISA analysis of PGE2 revealed activation of PLA2 and production of PGE2 in response to CM but not S1P. S1P, released from AC, uses HuR to stabilize COX-2 mRNA and thus to increase COX-2 protein expression. However, only CM also activates PLA2 to provide the substrate for COX-2. Our data underscore the importance of S1P in AC-mediated immune regulation, by stabilizing COX-2 mRNA in macrophages, a prerequisite for PGE2 formation.

Topics: Animals; Antigens, Surface; Apoptosis; Cell Communication; Culture Media, Conditioned; Cyclooxygenase 2; ELAV Proteins; ELAV-Like Protein 1; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Enzymologic; Humans; Jurkat Cells; Lysophospholipids; Macrophages; Mice; Phosphotransferases (Alcohol Group Acceptor); Protein Biosynthesis; RNA Stability; RNA-Binding Proteins; RNA, Messenger; Sphingosine

Sphingosine-1-phosphate (S1P) is now emerging as a potent lipid mediator produced by mast cells that contributes to inflammatory and allergic responses. In contrast to its weak effect on degranulation of murine mast cells, S1P potently induced degranulation of the human LAD2 mast-cell line and cord blood-derived human mast cells (hMCs). S1P also stimulated production and secretion of cytokines, TNF-alpha and IL-6, and markedly enhanced secretion of a chemokine, CCL2/MCP-1, important modulators of inflammation. S1P is produced in mast cells by the 2 sphingosine kinases, SphK1 and SphK2. SphK1 but not SphK2 plays a critical role in IgE/Ag-induced degranulation, migration toward antigen, and CCL2 secretion from hMCs, as determined by specifically down-regulating their expression. However, both isoenzymes were required for efficient TNF-alpha secretion. Taken together, our data suggest that differential formation of S1P by SphK1 and SphK2 has distinct and important actions in hMCs.

Topics: Antigens; Cell Degranulation; Cell Line; Chemokine CCL2; Chemotaxis; Down-Regulation; Humans; Lysophospholipids; Mast Cells; Pertussis Toxin; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tumor Necrosis Factor-alpha

The lysophospholipid mediator sphingosine-1-phosphate (S1P) acts on vascular endothelial cells to stimulate migration, proliferation, and capillary-like tube formation in vitro. It is unknown whether S1P stimulates in vivo angiogenesis induced under tissue ischaemia. We investigated the effects of both exogenously and endogenously overproduced S1P on post-ischaemic angiogenesis in murine hindlimbs.. The effects of locally injected S1P on blood flow recovery, angiogenesis, and vascular permeability in mouse ischaemic hindlimbs that underwent femoral arteriectomy were assessed by a laser Doppler blood flow (LDBF) analysis, anti-CD31 immunohistochemistry, and Miles assay, respectively, and compared with those induced by fibroblast growth factor (FGF)-2. Blood flow recovery and angiogenesis in sphingosine kinase 1-transgenic mice that overproduce S1P endogenously were also assessed and compared with wild-type mice. The LDBF analysis showed that daily intramuscular administration of S1P dose-dependently stimulated blood flow recovery, resulting in up to twice as much blood flow when compared with vehicle control, which was accompanied by 1.7-fold increase in the capillary density. The optimal S1P effects were comparable with those obtained with FGF-2. S1P injection did not increase vascular permeability. The post-ischaemic blood flow recovery and angiogenesis were accelerated in sphingosine kinase 1-transgenic mice, which showed 40-fold higher sphingosine kinase activity and 1.8-fold higher S1P content in skeletal muscle than in wild-type (WT) mice, without an increase in the vascular permeability when compared with WT mice.. These results indicate that either local exogenous S1P administration or endogenous S1P overproduction promotes post-ischaemic angiogenesis and blood flow recovery. These observations suggest potential therapeutic usefulness of S1P for tissue ischaemia.

Topics: Angiogenesis Inducing Agents; Animals; Blood Flow Velocity; Capillaries; Capillary Permeability; Disease Models, Animal; Fibroblast Growth Factor 2; Hindlimb; Immunohistochemistry; Ischemia; Laser-Doppler Flowmetry; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Skeletal; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Platelet Endothelial Cell Adhesion Molecule-1; Regional Blood Flow; Sphingosine; Time Factors

Recent evidence suggests that the epoxyeicosatrienoic acids (EETs), which are products of cytochrome P450 (CYP) epoxygenases, possess mitogenic and angiogenic effects in vascular endothelial cells. However, the mechanisms underlying these effects are not fully elucidated. Because sphingosine kinase (SK) and its product S1P play essential roles in cell growth, survival and migration, we hypothesized that SK activation by EETs may mediate some of its angiogenic effects.. We studied the effects of EETs on SK activity in human umbilical vein endothelial cells (HUVECs). Treatment with EETs, particularly 11,12-EET, markedly augmented SK activity in HUVECs. At the concentration of 1 micromol/L, 11,12-EET increased SK activity by 110% and the maximal effect on SK activation was observed at 20 min after 11,12-EET addition. Furthermore, inhibition of SK by a specific inhibitor, SKI-II, markedly attenuated 11,12-EET-induced EC proliferation. Importantly, 11,12-EET-induced activation of Akt kinase and transactivation of the epidermal growth factor (EGF) receptor was also inhibited by SKI-II. To investigate the isoform-specific role of SK in EET-induced angiogenesis, inhibition of SK1 by expression of dominant-negative SK1(G82D) substantially attenuated 11,12-EET-induced EC proliferation, migration, and tube formation in vitro and Matrigel plug angiogenesis in vivo. Furthermore, knockdown of SK1 expression by specific siRNA also inhibited 11,12-EET-induced EC proliferation and migration, whereas SK2 siRNA knockdown was without effect.. These results suggest that SK1 is an important mediator of the 11,12-EET-induced angiogenic effects in human ECs. Thus, SK1 may represent a novel therapeutic modality for the treatment of angiogenesis-related diseases such as cancer and ischaemia.

Topics: 8,11,14-Eicosatrienoic Acid; Cell Movement; Cell Proliferation; Cells, Cultured; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Humans; Lysophospholipids; Neovascularization, Physiologic; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Signal Transduction; Sphingosine; Thiazoles

Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate. The assay was demonstrated to be capable of determining the in vitro activity of both kinase and phosphatase using purified enzymes. The KM of sphingosine kinase for its fluorescently labeled substrate was 38 +/- 18 microM with a Vmax of 0.4 +/- 0.2 microM/min and a kcat of 3900 s-1. Pharmacologic inhibition of sphingosine kinase in a concentration-dependent manner was also demonstrated. Moreover, the fluorescent substrate was shown to be readily taken up by mammalian cells making it possible to study the endogenous activity of sphingosine kinase activity in living cells. The method was readily adaptable to the use of either bulk cell lysates or very small numbers of intact cells. This new methodology provides enhancements over standard methods in sensitivity, quantification, and manpower for both in vitro and cell-based assays.

Topics: Animals; Cell Line, Tumor; Fluorescein; Lysophospholipids; Mice; Molecular Structure; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Reproducibility of Results; Signal Transduction; Sphingosine; Staining and Labeling; Time Factors

Endothelial cells (ECs) regulate the barrier function of blood vessels. Here we show that basal and angiopoietin-1 (Ang-1)-regulated control of EC permeability is mediated by 2 different functional states of sphingosine kinase-1 (SK-1). Mice depleted of SK-1 have increased vascular leakiness, whereas mice transgenic for SK-1 in ECs show attenuation of leakiness. Furthermore, Ang-1 rapidly and transiently stimulates SK-1 activity and phosphorylation, and induces an increase in intracellular sphingosine-1-phosphate (S1P) concentration. Overexpression of SK-1 resulted in inhibition of permeability similar to that seen for Ang-1, whereas knockdown of SK-1 by small interfering RNA blocked Ang-1-mediated inhibition of permeability. Transfection with SKS225A, a nonphosphorylatable mutant of SK-1, inhibited basal leakiness, and both SKS225A and a dominant-negative SK-1 mutant removed the capacity of Ang-1 to inhibit permeability. These effects were independent of extracellular S1P as knockdown or inhibition of S1P1, S1P2, or S1P3, did not affect the Ang-1 response. Thus, SK-1 levels in ECs powerfully regulate basal permeability in vitro and in vivo. In addition, the Ang-1-induced inhibition of leakiness is mediated through activation of SK-1, defining a new signaling pathway in the Ang-1 regulation of permeability.

Topics: Amino Acid Substitution; Angiopoietin-1; Animals; Capillary Permeability; Cells, Cultured; Endothelial Cells; Enzyme Activation; Humans; Lysophospholipids; Mice; Mice, Knockout; Mutation, Missense; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Small Interfering; Sphingosine; Transfection

Sphingosine 1-phosphate (S1P), an abundant lipid mediator in plasma, regulates vascular and immune cells by activating S1P receptors. In this report, we investigated the mechanisms by which high plasma S1P levels are maintained in mice. We found that plasma S1P turns over rapidly with a half-life of approximately 15 minutes, suggesting the existence of a high-capacity biosynthetic source(s). Transplantation of bone marrow from wild-type to Sphk1(-/-)Sphk2(+/-) mice restored plasma S1P levels, suggesting that hematopoietic cells are capable of secreting S1P into plasma. However, plasma S1P levels were not appreciably altered in mice that were thrombocytopenic, anemic, or leukopenic. Surprisingly, reconstitution of Sphk1(-/-)Sphk2(+/-) bone marrow cells into wild-type hosts failed to reduce plasma S1P, suggesting the existence of an additional, nonhematopoietic source for plasma S1P. Adenoviral expression of Sphk1 in the liver of Sphk1(-/-) mice restored plasma S1P levels. In vitro, vascular endothelial cells, but not hepatocytes, secreted S1P in a constitutive manner. Interestingly, laminar shear stress downregulated the expression of S1P lyase (Sgpl) and S1P phosphatase-1 (Sgpp1) while concomitantly stimulating S1P release from endothelial cells in vitro. Modulation of expression of endothelial S1P lyase with small interfering RNA and adenoviral expression altered S1P secretion, suggesting an important role played by this enzyme. These data suggest that the vascular endothelium, in addition to the hematopoietic system, is a major contributor of plasma S1P.

Topics: Adenoviridae; Aldehyde-Lyases; Anemia; Animals; Antibodies, Monoclonal; Bone Marrow Cells; Bone Marrow Transplantation; Cell Line; Cell Line, Tumor; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Genetic Vectors; Half-Life; Humans; Leukopenia; Liver; Lysophospholipids; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenylhydrazines; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Platelet Glycoprotein GPIb-IX Complex; RNA Interference; RNA, Small Interfering; Sphingosine; Stress, Mechanical; Thrombocytopenia; Time Factors; Transduction, Genetic; Whole-Body Irradiation

Sphingosine 1-phosphate (S1P) has many important roles in mammalian cells, including contributing to the control of cell survival and proliferation. S1P is generated by sphingosine kinases (SKs), of which two mammalian isoforms have been identified (SK1 and SK2). To gain a better understanding of SK regulation, we have used a yeast two-hybrid screen to identify SK1-interacting proteins and established elongation factor 1A (eEF1A) as one such protein that associates with both SK1 and SK2. We show the direct interaction of eEF1A with the SKs in vitro, whereas the physiological relevance of this association was demonstrated by co-immunoprecipitation of the endogenous proteins from cell lysates. Although the canonical role of eEF1A resides in protein synthesis, it has also been implicated in other roles, including regulating the activity of some signaling enzymes. Thus, we examined the potential role of eEF1A in regulation of the SKs and show that eEF1A is able to directly increase the activity of SK1 and SK2 approximately 3-fold in vitro. Substrate kinetics demonstrated that eEF1A increased the catalytic rate of both SKs, while having no observable effect on substrate affinities of these enzymes for either ATP or sphingosine. Overexpression of eEF1A in quiescent Chinese hamster ovary cells increased cellular SK activity, whereas a small interfering RNA-mediated decrease in eEF1A levels in MCF7 cells substantially reduced cellular SK activity and S1P levels, supporting the in vivo physiological relevance of this interaction. Thus, this study has established a novel mechanism of regulation of both SK1 and SK2 that is mediated by their interaction with eEF1A.

Topics: Animals; Catalysis; Cell Proliferation; Cell Survival; CHO Cells; Cricetinae; Cricetulus; Enzyme Activation; Humans; Isoenzymes; Lysophospholipids; Peptide Elongation Factor 1; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; RNA, Small Interfering; Signal Transduction; Sphingosine

We recently reported increased sphingosine kinase 1 (SPHK1) and decreased neutral sphingomyelinase 2 (NSMase2) gene expression in myelodysplastic syndromes and acute leukemia. This alteration is supposed to change the cellular sphingolipid metabolites; however, positive correlations were observed between daunorubicin (DA)-IC50 and the SPHK1 message but not between DA-IC50 and NSMase2 messages, when 16 different leukemia cell lines were used to analyze the relationship between gene expressions and chemosensitivity against DA. Using two cell lines with either the highest or lowest SPHK1 expression, cellular ceramides and sphingosine 1-phosphate (S1P) were quantified by liquid chromatography/mass spectrometry. Increased ceramide was observed in DA-sensitive, but not in DA-resistant cell lines treated with low doses of DA. Upon DA treatment, S1P decreased more in the sensitive cell lines than in resistant cell lines. A SPHK inhibitor recovered the DA sensitivity of DA-resistant cells. The modulation of SPHK1 gene expression by either overexpression or using siRNA affected the DA sensitivity of representative cell lines. Results clearly show that SPHK1 is both a good marker to predict the DA sensitivity of leukemia cells and a potential therapeutic target for leukemia with high SPHK1 expression, and suggest that the sphingolipid rheostat plays a significant role in DA-induced cytotoxicity.

Topics: Antibiotics, Antineoplastic; Biomarkers; Cell Line, Tumor; Daunorubicin; Drug Resistance, Neoplasm; Gene Expression Profiling; Humans; Leukemia; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

In the present study, we studied N,N-dimethyl-D-erythro-sphingosine (DMS)-induced cell death and its signaling mechanism in U937 human monocytes. We found that DMS induced cell death in a concentration-dependent manner, while sphingosine 1-phosphate did not. DMS also induced DNA fragmentation, nuclear disruption, and cytochrome c release from mitochondria in a concentration- and time-dependent manner, implying apoptotic cell death. DMS was found to increase mitochondrial membrane potential (MMP) immediately after addition of DMS and to decrease MMP at 2h after addition. However, sphingosine kinase inhibitors and PKC inhibitors did not induce cell death in U937 cells, a result that appears to exclude sphingosine kinase and PKC as target molecules of DMS in the cell death induction process. Furthermore, DMS modulated the activity of several signaling molecules. DMS induced activation of JNK and p38 MAP kinase, while it decreased the activity of ERK and Akt kinase. However, decrease of MMP, inhibition of JNK, p38 MAP kinase, ERK, or Akt with specific inhibitors could not mimic the DMS-induced cell death, implying multiple concerted processes are involved in DMS-induced cell death. In summary, DMS induced apoptotic cell death via modulation of MMP, JNK, p38 MAP kinase, ERK, and Akt kinase, but not through inhibition of sphingosine kinase or PKC in U937 cells.

Topics: Apoptosis; Blotting, Western; Cytochromes c; DNA Fragmentation; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; Membrane Potential, Mitochondrial; p38 Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Proto-Oncogene Proteins c-akt; Signal Transduction; Sphingosine; U937 Cells

Sphingosine-1-phosphate (S1P), which is formed by phosphorylation of sphingosine through a process catalysed by sphingosine kinase (SK), is a multifunctional mediator of a variety of cellular responses including proliferation, differentiation, motility, and survival. K6PC-5, which was recently synthesized as a novel SK activator, is expected to increase S1P levels. Indeed studies have already demonstrated that K6PC-5 exhibits anti-aging effects on intrinsic aged murine skin by increasing fibroblasts, collagen synthesis, dermal thickness, and epidermal differentiation. However, photoaging and intrinsic aging have highly different clinical and histopathological properties. In this study, we developed a photoaged murine model by exposing mice that were 56 weeks old to ultraviolet (UV)B and UVA radiation for 8 weeks. We then investigated whether K6PC-5, as an SK activator, had anti-aging effects on photoaged murine skin in addition to its effects on intrinsic aged murine skin and determined the mechanism. K6PC-5 increased dermal collagen density in photoaged skin through increases in fibroblasts and collagen production. Photoaged murine skin treated with K6PC-5 showed an increase in stratum corneum (SC) integrity with increased corneodesmosome density and an improvement in barrier recovery rate. Matrix metalloproteinase 13 remained unchanged. These results indicate that topical application of K6PC-5 improves photoaged skin by improving skin barrier and increasing fibroblast count and function. In conclusion, K6PC-5, as an S1P activator, improves long-term UV-exposed aged skin as well as intrinsic aged skin.

Topics: Amides; Animals; Biomarkers; Cell Division; Collagen; Dermis; Enzyme Activation; Epidermal Cells; Epidermis; Female; Fibroblasts; Lysophospholipids; Mice; Mice, Hairless; Phosphotransferases (Alcohol Group Acceptor); Skin Aging; Sphingosine; Ultraviolet Rays

Sphingosine 1-phosphate (S1P) produced by sphingosine kinase (SPHK) is implicated in acute immunoresponses, however, mechanisms of SPHK/S1P signaling in the pathogenesis of bronchial asthma are poorly understood. In this study, we hypothesized that SPHK inhibition could ameliorate lung inflammation in ovalbumin (OVA)-challenged mouse lungs. Six- to eight-week-old C57BL/6J mice were sensitized and exposed to OVA for 3 consecutive days. Twenty-four hours later, mice lungs and bronchoalveolar lavage (BAL) fluid were analyzed. For an inhibitory effect, either of the two different SPHK inhibitors, N,N-dimethylsphingosine (DMS) or SPHK inhibitor [SK-I; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole], was nebulized for 30 min before OVA inhalation. OVA inhalation caused S1P release into BAL fluid and high expression of SPHK1 around bronchial epithelial walls and inflammatory areas. DMS or SK-I inhalation resulted in a decrease in S1P amounts in BAL fluid to basal levels, accompanied by decreased eosinophil infiltration and peroxidase activity. The extent of inhibition caused by DMS inhalation was higher than that caused by SK-I. Like T helper 2 (Th2) cytokine release, OVA inhalation-induced increase in eotaxin expression was significantly suppressed by DMS pretreatment both at protein level in BAL fluid and at mRNA level in lung homogenates. Moreover, bronchial hyperresponsiveness to inhaled methacholine and goblet cell hyperplasia were improved by SPHK inhibitors. These data suggest that the inhibition of SPHK affected acute eosinophilic inflammation induced in antigen-challenged mouse model and that targeting SPHK may provide a novel therapeutic tool to treat bronchial asthma.

Topics: Administration, Inhalation; Aniline Compounds; Animals; Asthma; Bronchoalveolar Lavage Fluid; Cells, Cultured; Chemokines, CC; Disease Models, Animal; Enzyme Inhibitors; Goblet Cells; Humans; Hyperplasia; Interleukins; Lysophospholipids; Mice; Mice, Inbred C57BL; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Respiratory Mucosa; Sphingosine; Thiazoles

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates myriad important cellular processes, including growth, survival, cytoskeleton rearrangements, motility, and immunity. Here we report that treatment of Jurkat and U937 leukemia cells with the pan-sphingosine kinase (SphK) inhibitor N,N-dimethylsphingosine to block S1P formation surprisingly caused a large increase in expression of SphK1 concomitant with induction of apoptosis. Another SphK inhibitor, D,L-threo-dihydrosphingosine, also induced apoptosis and produced dramatic increases in SphK1 expression. However, up-regulation of SphK1 was not a specific effect of its inhibition but rather was a consequence of apoptotic stress. The chemotherapeutic drug doxorubicin, a potent inducer of apoptosis in these cells, also stimulated SphK1 expression and activity and promoted S1P secretion. The caspase inhibitor ZVAD reduced not only doxorubicin-induced lethality but also the increased expression of SphK1 and secretion of S1P. Apoptotic cells secrete chemotactic factors to attract phagocytic cells, and we found that S1P potently stimulated chemotaxis of monocytic THP-1 and U937 cells and primary monocytes and macrophages. Collectively, our data suggest that apoptotic cells may up-regulate SphK1 to produce and secrete S1P that serves as a "come-and-get-me" signal for scavenger cells to engulf them in order to prevent necrosis.

Topics: Antibiotics, Antineoplastic; Apoptosis; Chemotaxis, Leukocyte; Doxorubicin; Enzyme Inhibitors; Humans; In Vitro Techniques; Jurkat Cells; Lysophospholipids; Monocytes; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; U937 Cells; Up-Regulation

Topics: Aldehyde-Lyases; Anemia; Animals; Antibodies, Monoclonal; Bone Marrow Cells; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Half-Life; Humans; Leukopenia; Liver; Lysophospholipids; Membrane Proteins; Phenylhydrazines; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Platelet Glycoprotein GPIb-IX Complex; Research Design; Signal Transduction; Sphingosine; Stress, Mechanical; Thrombocytopenia; Time Factors

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(2+) signaling, growth, survival, and differentiation. Because sphingosine kinase (SphK) is the enzyme directly responsible for production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. Here we synthesized a previously unidentified SphK activator, K6PC-5, and have studied its effects on intracellular Ca(2+) signaling in HaCaT cells and epidermal differentiation in murine skin. K6PC-5, a hydrophobic compound chemically named N-(1,3-dihydroxyisopropyl)-2-hexyl-3-oxo-decanamide, activated SphK (obtained from C57BL/6 murine blood and F9-12 cell lysates) in a dose-dependent manner. K6PC-5 induced both intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in HaCaT cells and Ca(2+) mobilization in hairless mouse epidermis. Both dimethylsphingosine (DMS) and dihydroxysphingosine (DHS), SphK inhibitors, and transfection of SphK1-siRNA blocked K6PC-5-induced increases in [Ca(2+)](i). The K6PC-5-induced [Ca(2+)](i) oscillations were dependent on thapsigargin-sensitive Ca(2+) stores and Ca(2+) entry, but independent of the classical phospholipase C-mediated pathway. In addition, K6PC-5 enhanced the expression of involucrin and filaggrin, specific differentiation-associated marker proteins in HaCaT cells, whereas transfection of SphK1-siRNA blocked the increase of involucrin. Topical K6PC-5 also enhanced the expression of involucrin, loricrin, filaggrin, and keratin 5 in intact murine epidermis. Finally, topical K6PC-5 inhibited epidermal hyperplasia by exerting antiproliferative effects on keratinocytes in murine epidermis. These results suggest that K6PC-5 acts to regulate both differentiation and proliferation of keratinocytes via [Ca(2+)](i) responses through S1P production. Thus, regulation of S1P levels may represent a novel approach for treatment of skin disorders characterized by abnormal differentiation and proliferation, such as atopic dermatitis and psoriasis.

Topics: Amides; Animals; Calcium; Calcium Signaling; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Proliferation; Enzyme Activators; Epidermis; Female; Filaggrin Proteins; Humans; Hyperplasia; Intermediate Filament Proteins; Keratin-5; Keratinocytes; Lysophospholipids; Membrane Proteins; Mice; Mice, Hairless; Mice, Inbred C57BL; Neoplasms, Germ Cell and Embryonal; Phosphotransferases (Alcohol Group Acceptor); Protein Precursors; Sphingosine; Teratocarcinoma

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(2+) signaling, growth, survival, and differentiation. Because sphingosine kinase (SK) is the enzyme directly responsible for the production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. To date, there are no reports to demonstrate a chemically induced, direct activation of SK.. Here we have studied the effects of K6PC-5 as a newly synthesized SK activator on fibroblast proliferation in both human fibroblasts and aged mouse skin. To demonstrate that K6PC-5 has S1P-mediated action mechanism in fibroblasts, we have measured SK-dependent intracellular Ca(2+) signaling.. Fibroblasts were cultured primarily from human foreskin and were used to study the effect of K6PC-5 and S1P on intracellular Ca(2+) signaling and fibroblast proliferation. Changes in intracellular Ca(2+) were detected by fluorescence with fura-2/AM. To study skin anti-aging effects of K6PC-5, we used intrinsically aged hairless mice (56 weeks old).. K6PC-5 promoted fibroblast proliferation and procollagen production in human fibroblasts significantly. K6PC-5 induced intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in human fibroblasts. Both dimethylsphingosine and dihydroxysphingosine, SK inhibitors, and the transfection of SK1-siRNA blocked the K6PC-5-induced increases in [Ca(2+)](i), an effect independent of the classical PLC/IP(3)-mediated pathway. The K6PC-5-induced [Ca(2+)](i) oscillations were dependent on thapsigargin-sensitive Ca(2+) stores and Ca(2+) entry. Topical application of K6PC-5 for 2 weeks to intrinsically aged hairless mice enhanced fibroblast proliferation, collagen production, and eventually increased dermal thickness (10%). K6PC-5 also promoted specific epidermal differentiation marker proteins, including involucrin, loricrin, filaggrin, and keratin 5, without any alterations on epidermal barrier function.. These results suggest that K6PC-5 acts to regulate fibroblast proliferation through intracellular S1P production, and can further promote keratinocyte differentiation. We anticipate that the regulation of S1P levels may represent a novel approach for the treatment of skin disorders, including skin aging.

Topics: Aging; Amides; Animals; Calcium; Calcium Signaling; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Fibroblasts; Filaggrin Proteins; Humans; Lysophospholipids; Male; Mice; Mice, Hairless; Models, Animal; Phosphotransferases (Alcohol Group Acceptor); Procollagen; Skin; Sphingosine

Sphingosine-1-phosphate (S1P) is a lipid-signaling molecule produced by sphingosine kinase in response to a wide number of stimuli. By acting through a family of widely expressed G protein-coupled receptors, S1P regulates diverse physiological processes. Here we examined the role of S1P signaling in neurodegeneration using a mouse model of Sandhoff disease, a prototypical neuronopathic lysosomal storage disorder. When sphingosine kinase 1 (Sphk1) was deleted in Sandhoff disease mice, a milder disease course occurred, with decreased proliferation of glial cells and less-pronounced astrogliosis. A similar result of milder disease course and reduced astroglial proliferation was obtained by deletion of the gene for the S1P(3) receptor, a G protein-coupled receptor enriched in astrocytes. Our studies demonstrate a functional role of S1P synthesis and receptor expression in astrocyte proliferation leading to astrogliosis during the terminal stages of neurodegeneration in Sandhoff disease mice. Because astrocyte responses are involved in many types of neurodegeneration, the Sphk1/S1P receptor signaling axis may be generally important during the pathogenesis of neurodegenerative diseases.

Topics: Animals; Astrocytes; Cell Proliferation; Disease Models, Animal; Gene Deletion; Gliosis; Lysophospholipids; Male; Mice; Mice, Mutant Strains; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sandhoff Disease; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Spinal Cord

Sphingolipids are constituents of biological membranes. Ceramide and sphingosine 1-phosphate (S1P) also act as second messengers and are part of a rheostat system, in which ceramide promotes cell death and growth arrest, and S1P induces proliferation and maintains cell survival. As macroautophagy is a lysosomal catabolic mechanism involved in determining the duration of the lifetime of cells, we raised the question of its regulation by sphingolipid messengers. Using chemical and genetic methods, we have shown by GFP-LC3 staining and analysis of the degradation of long-lived proteins that both ceramide and S1P stimulate autophagy.

Topics: Autophagy; Cell Line, Tumor; Ceramides; Chromatography, Thin Layer; Diacylglycerol Kinase; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingolipids; Sphingosine

Transforming growth factor beta (TGFbeta) plays a dual role in oncogenesis, acting as both a tumor suppressor and a tumor promoter. These disparate processes of suppression and promotion are mediated primarily by Smad and non-Smad signaling, respectively. A central issue in understanding the role of TGFbeta in the progression of epithelial cancers is the elucidation of the mechanisms underlying activation of non-Smad signaling cascades. Because the potent lipid mediator sphingosine-1-phosphate (S1P) has been shown to transactivate the TGFbeta receptor and activate Smad3, we examined its role in TGFbeta activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and promotion of migration and invasion of esophageal cancer cells. Both S1P and TGFbeta activate ERK1/2, but only TGFbeta activates Smad3. Both ligands promoted ERK1/2-dependent migration and invasion. Furthermore, TGFbeta rapidly increased S1P, which was required for TGFbeta-induced ERK1/2 activation, as well as migration and invasion, since downregulation of sphingosine kinases, the enzymes that produce S1P, inhibited these responses. Finally, our data demonstrate that TGFbeta activation of ERK1/2, as well as induction of migration and invasion, is mediated at least in part by ligation of the S1P receptor, S1PR2. Thus, these studies provide the first evidence that TGFbeta activation of sphingosine kinases and formation of S1P contribute to non-Smad signaling and could be important for progression of esophageal cancer.

Topics: Cell Line, Tumor; Cell Movement; Chemotaxis; Disease Progression; Enzyme Activation; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Biological; Neoplasm Invasiveness; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Transforming Growth Factor beta

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) activation is a regulatory step in the control of arachidonic acid (AA) liberation for eicosanoid formation. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator involved in the regulation of many important proinflammatory processes and has been found in the airways of asthmatic subjects. We investigated the mechanism of S1P-induced AA release and determined the involvement of cPLA(2)alpha in these events in A549 human lung epithelial cells. S1P induced AA release rapidly within 5 min in a dose- and time-dependent manner. S1P-induced AA release was inhibited by the cPLA(2)alpha inhibitors methyl arachidonyl fluorophosphonate (MAFP) and pyrrolidine derivative, by small interfering RNA-mediated downregulation of cPLA(2)alpha, and by inhibition of S1P-induced calcium flux, suggesting a significant role of cPLA(2)alpha in S1P-mediated AA release. Knockdown of the S1P3 receptor, the major S1P receptor expressed on A549 cells, inhibited S1P-induced calcium flux and AA release. The S1P-induced calcium flux and AA release was associated with sphingosine kinase 1 (Sphk1) expression and activity. Furthermore, Rho-associated kinase, downstream of S1P3, was crucial for S1P-induced cPLA(2)alpha activation. Our data suggest that S1P acting through S1P3, calcium flux, and Rho kinase activates cPLA(2)alpha and releases AA in lung epithelial cells. An understanding of S1P-induced cPLA(2)alpha activation mechanisms in epithelial cells may provide potential targets to control inflammatory processes in the lung.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Asthma; Calcium; Calcium Signaling; Cell Line; Eicosanoids; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Group IV Phospholipases A2; Humans; Lung; Lysophospholipids; Organophosphonates; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Respiratory Mucosa; rho-Associated Kinases; RNA, Small Interfering; Sphingosine

Sphingosine 1-phosphate (S1P), a bioactive sphingolipid involved in diverse biological processes, is generated by sphingosine kinase (SphK) and acts via intracellular and/or extracellular mechanisms. We used biochemical, pharmacological, and physiological approaches to investigate in rat myometrium the contractile effect of exogenous S1P and the possible contribution of SphK in endothelin-1 (ET-1)-mediated contraction. S1P stimulated uterine contractility (EC(50) = 1 microM and maximal response = 5 microM) by a pertussis toxin-insensitive and a phospholipse C (PLC)-independent pathway. Phosphorylated FTY720, which interacts with all S1P receptors, except S1P(2) receptors, failed to mimic S1P contractile response, indicating that the effects of S1P involved S1P(2) receptors that are expressed in myometrium. Contraction mediated by S1P and ET-1 required extracellular calcium and Rho kinase activation. Inhibition of SphK reduced ET-1-mediated contraction. ET-1, via ET(A) receptors coupled to pertussis toxin-insensitive G proteins, stimulated SphK1 activity and induced its translocation to the membranes. Myometrial contraction triggered by ET-1 is consecutive to the sequential activation of PLC, protein kinase C, SphK1 and Rho kinase. Prolonged exposure of the myometrium to S1P downregulated S1P(2) receptors and abolished the contraction induced by exogenous S1P. However, in these conditions, the tension triggered by ET-1 was not reduced, indicating that SphK activated by ET-1 contributed to its contractile effect via a S1P(2) receptor-independent process. Our findings demonstrated that exogenous S1P and SphK activity regulated myometrial contraction and may be of physiological relevance in the regulation of uterine motility during gestation and parturition.

Topics: Animals; Biological Transport; Calcium; Cytosol; Endothelin-1; Enzyme Activation; Extracellular Fluid; Female; GTP-Binding Proteins; Intracellular Signaling Peptides and Proteins; Lysophospholipids; Myometrium; Pertussis Toxin; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Protein Serine-Threonine Kinases; Rats; Rats, Wistar; Receptors, Cell Surface; Receptors, Lysosphingolipid; rho GTP-Binding Proteins; rho-Associated Kinases; Sphingosine; Type C Phospholipases; Uterine Contraction

Fumonisin B(1), a mycotoxin, is an inhibitor of ceramide synthase causing marked dysregulation of sphingolipid metabolism in cells. This mycotoxin causes accumulation of free sphingoid bases (sphingosine and dihydrosphingosine or sphinganine) and their metabolites, important messengers involved in signal transduction leading to either cell survival or death. Free sphingoid bases are known apoptotic molecules whereas sphingosine 1-phosphate is protective. We previously reported that fumonisin B(1) caused sphingosine kinase (SPHK) induction along with the increase of serine palmitoyltransferase (SPT). Fumonisin B(1) also increased inducible nitric oxide synthase (iNOS) expression. In the current study we employed a mouse strain with the targeted deletion of iNOS gene (Nos-KO) to evaluate the role of nitric oxide (NO) on fumonisin B(1)-induced hepatotoxicity. The Nos-KO mice exhibited increased hepatotoxicity after subacute fumonisin B(1) exposure compared to their wild type counterparts, the liver regeneration was lower in Nos-KO compared to that in the WT mice. Increased hepatotoxicity in Nos-KO was not related to the extent of free sphingoid base accumulation after fumonisin B(1) treatment; however, it was accompanied by a lack of fumonisin B(1)-induced SPHK induction. The fumonisin B(1)-induced SPT was unaffected by lack of iNOS gene. Deletion of iNOS gene did not prevent fumonisin B(1)-dependent induction of inflammatory cytokines, namely tumor necrosis factor alpha, interferon gamma and interleukin-12. The lack of fumonisin B(1)-induced SPHK induction in Nos-KO was supported by a similar effect on phosphorylated metabolites of sphingoid bases; the equilibrium between sphingoid bases and their phosphates is maintained by SPHK. We therefore conclude that iNOS induction produced by fumonisin B(1) modulates SPHK activity; the lack of iNOS prevents generation of sphingosine 1-phosphate and deprives cells from its protective effects.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Carcinogens, Environmental; Cell Proliferation; Chemical and Drug Induced Liver Injury; Fumonisins; Hepatocytes; In Situ Nick-End Labeling; Interferon-gamma; Interleukin-12; Liver Diseases; Liver Regeneration; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Sphingosine; Tumor Necrosis Factor-alpha; Weight Loss

Sphingosine 1-phosphate (S1P) levels in cells and, consequently, its bioactivity as a signalling molecule are controlled by the action of enzymes responsible for its synthesis and degradation. In the present report, we examined alterations in expression patterns of enzymes involved in S1P-metabolism (sphingosine kinases including their splice variants, sphingosine 1-phosphate phosphatases, and sphingosine 1-phosphate lyase) under certain inflammatory conditions. We found that sphingosine kinase type 1 (SPHK1) mRNA could be triggered in a cell type-specific manner; individual SPHK1 splice variants were induced with similar kinetics. Remarkably, expression and activity of S1P phosphatase 2 (SPP2) was found to be highly upregulated by inflammatory stimuli in a variety of cells (e.g., neutrophils, endothelial cells). Bandshift analysis using oligonucleotides spanning predicted NFkappaB sites within the SPP2 promoter and silencing of NFkappaB/RelA via RelA-directed siRNA demonstrated that SPP2 is an NFkappaB-dependent gene. Silencing of SPP2 expression in endothelial cells, in turn, led to a marked reduction of TNF-alpha-induced IL-1beta mRNA and protein and to a partial reduction of induced IL-8, suggesting a pro-inflammatory role of SPP2. Notably, up-regulation of SPP2 was detected in samples of lesional skin of patients with psoriasis, an inflammatory skin disease. This study provides detailed insights into the regulation of SPP2 gene expression and suggests that SPP2 might be a novel player in pro-inflammatory signalling.

Topics: Binding Sites; Cells, Cultured; Endothelial Cells; Enzyme Induction; Gene Expression Profiling; Gene Silencing; Humans; Inflammation; Interleukin-1beta; Lipopolysaccharides; Lysophospholipids; Membrane Proteins; Neutrophils; NF-kappa B; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Promoter Regions, Genetic; Psoriasis; RNA, Messenger; RNA, Small Interfering; Skin; Sphingosine; Transcription, Genetic; Tumor Necrosis Factor-alpha; Up-Regulation

Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive lipid that transmits potent signals through a family of G protein coupled receptors with resultant anti-apoptotic and pro-angiogenic effects. We have recently reported that lymphoblastoid B cell lines (LCLs) from rheumatoid arthritis (RA) patients are resistant to Fas-mediated cell death due to over-production of S1P, secondary to over-activity of sphingosine kinase-1 (SphK1). Here we investigated the signaling events that S1P triggers in those cells. Our results show that RA-derived LCLs display increased constitutive enzymatic activity of phosphatidylinositol 3-kinase (PI3K). Incubation of LCLs with a PI3K inhibitor wortmannin reversed PI3K over-activity and the resistance to Fas-mediated cell death. Incubation of RA LCLs with nanomolar concentration of S1P triggered exaggerated activation of both SphK and PI3K in RA LCLs compared to control cells. PI3K was mapped upstream of SphK, since wortmannin could block SphK activation by S1P. S1P signaling effect could be blocked by the Gi/G0 protein inhibitor, pertussis toxin and by an inhibitor of S1P-receptor interaction, suramin. S1P receptor expression levels did not appear to be the cause of disparate S1P-triggered signaling, since LCLs from RA patients and their healthy twin controls did not show statistically significant differences in the expression levels of the five known S1P receptors, as determined by quantitative real time reverse transcription-polymerase chain reaction analyses. Thus, we conclude that Fas death signaling aberration in RA LCLs is caused by extracellular S1P, which triggers PI3K-dependent SphK over-activity through a Gi protein-coupled receptor-mediated signaling cascade.

Topics: Apoptosis; Arthritis, Rheumatoid; B-Lymphocytes; Cell Line; Cell Survival; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Lysophospholipids; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

The mitochondrial enzyme monoamine oxidase (MAO), its isoform MAO-A, plays a major role in reactive oxygen species-dependent cardiomyocyte apoptosis and postischemic cardiac damage. In the current study, we investigated whether sphingolipid metabolism can account for mediating MAO-A- and reactive oxygen species-dependent cardiomyocyte apoptosis. In H9c2 cardiomyoblasts, MAO-A-dependent reactive oxygen species generation led to mitochondria-mediated apoptosis, along with sphingosine kinase-1 (SphK1) inhibition. These phenomena were associated with generation of proapoptotic ceramide and decrease in prosurvival sphingosine 1-phosphate. These events were mimicked by inhibition of SphK1 with either pharmacological inhibitor or small interfering RNA, as well as by extracellular addition of C(2)-ceramide or H(2)O(2). In contrast, enforced expression of SphK1 protected H9c2 cells from serotonin- or H(2)O(2)-induced apoptosis. Analysis of cardiac tissues from wild-type mice subjected to ischemia/reperfusion revealed significant upregulation of ceramide and inhibition of SphK1. It is noteworthy that SphK1 inhibition, ceramide accumulation, and concomitantly infarct size and cardiomyocyte apoptosis were significantly decreased in MAO-A-deficient animals. In conclusion, we show for the first time that the upregulation of ceramide/sphingosine 1-phosphate ratio is a critical event in MAO-A-mediated cardiac cell apoptosis. In addition, we provide the first evidence linking generation of reactive oxygen species with SphK1 inhibition. Finally, we propose sphingolipid metabolites as key mediators of postischemic/reperfusion cardiac injury.

Topics: Animals; Apoptosis; Cells, Cultured; Ceramides; Down-Regulation; Drug Resistance; Hydrogen Peroxide; Lysophospholipids; Mice; Mice, Knockout; Mitochondria, Heart; Monoamine Oxidase; Myocardial Reperfusion Injury; Myocytes, Cardiac; Oxidants; Oxidative Stress; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Serotonin; Sphingolipids; Sphingosine; Up-Regulation

Sphingosine 1-phosphate (Sph-1-P), a product of sphingomyelin metabolism, can act via a family of cognate G protein-coupled receptors or as an intracellular second messenger for agonists acting through their membrane receptors. In view of the general growth promoting and developmental effects of Sph-1-P on target cells, we hypothesized that it plays a role in adaptation of the uterus to pregnancy. We analyzed its potential role and that of the related lysophospholipid lysophosphatidic acid in the pregnant rat uterus by examining changes in mRNA levels of cognate receptors and enzymes involved in their turnover. Of these, only sphingosine kinase-1 (SphK1) was markedly changed ( approximately 30-fold increase), being localized in the glandular epithelium, vasculature, and the myometrium. Uterine SphK1 mRNA and protein levels paralleled those of serum progesterone, and treatment with progesterone or an antagonist elevated or reduced SphK1 mRNA expression, respectively. Progesterone also increased SphK1 mRNA steady-state levels in a rat myometrial/leiomyoma cell line (ELT3). Overexpressing human SphK1 in these cells resulted in increased levels of the cell cycle regulator cyclin D1 and increased myosin light-chain phosphorylation. Ectopic expression of SphK1 also resulted in increased proliferation rates, possibly in conjunction with increased cyclin D1 expression. These studies suggest that the uterine expression of SphK1 mediates processes involved in growth and differentiation of uterine tissues during pregnancy.

Topics: Animals; bcl-X Protein; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzymes; Female; Genes, Dominant; Humans; Immunologic Techniques; Leiomyoma; Lysophospholipids; Mutation; Myometrium; Myosin Light Chains; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pregnancy; Pregnancy, Animal; Progesterone; Progestins; Rats; Rats, Sprague-Dawley; Receptors, Lysophospholipid; RNA, Messenger; Signal Transduction; Sphingosine; Uterus

Sphingosine kinase (Sphk) has been shown to be activated by growth factor and survival factors, and one of its products, sphingosine-1-phosphate, plays an important role in the regulation of various cellular responses. However, the effect of Sphk on the maturation and immunostimulatory function of dendritic cells (DCs) still remains largely unknown. In this study, we examined whether sphingosine kinase inhibitor (SKI) can influence co-stimulatory molecules (CD40, CD80, CD86 and MHC class II) and cytokine production (IL-12 and IL-10) in murine bone marrow-derived DCs. SKI significantly inhibited co-stimulatory molecules in DCs. SKI suppressed IL-12 production by DCs and IFN-gamma production by T cells. In addition, SKI-inhibited LPS induced the translocation of nuclear factor-kappaB, whereas it did not affect the degradation of IL-1 receptor-associated kinase-1 by LPS. These novel findings provide new insight into the immunopharmacological role of SKI in terms of its effects on DCs. These findings open a possibility for further understanding of the immunopharmacological functions of SKI, as well as therapeutic adjuvants for the treatment of DC-related acute and chronic diseases.

Topics: Animals; Antigens, CD; Apoptosis; Bone Marrow Cells; Cell Aggregation; Cell Differentiation; Cells, Cultured; Coculture Techniques; Dendritic Cells; Enzyme Inhibitors; Flow Cytometry; Interleukin-10; Interleukin-12; Lipopolysaccharides; Lysophospholipids; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mutation; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Small Interfering; Sphingosine; Th1 Cells; Transcription Factor RelA; Transfection

P-glycoprotein (P-gp), an ABC-transporter highly expressed in brain capillaries, protects the brain by extruding xenobiotics. However, its overexpression has also been associated with the multidrug resistance phenotype in tumors. Here, we have investigated the regulation of P-gp transport activity by sphingosine kinase 1 (SphK-1) in brain endothelial cells. We first demonstrated that SphK-1 is overexpressed in endothelial cells (EC) isolated from rat brain tumors compared with EC from normal brain. We also provide evidence that the overexpression of SphK-1 in the cerebral EC line RBE4 leads to the up-regulation of P-gp, both at the gene and protein levels, and that this modulation depends on the catalytic activity of SphK-1. Moreover, we determined the effect of sphingosine-1-phosphate (S1P), the product of SphK-1, on P-gp function. S1P strongly stimulates P-gp transport activity, without modulating its expression. Finally, we found that the S1P-mediated stimulation of P-gp activity is mediated by S1P-1 and S1P-3 receptors at the RBE4 cell surface. Altogether, these results indicate that SphK-1 and its product S1P are involved in the control of P-gp activity in RBE4 cells. Since SphK-1 is overexpressed in EC from brain tumors, these data also suggest that this kinase and its product could contribute to the acquisition and the maintenance of the multidrug resistance phenotype in brain tumor-derived endothelial cells.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Brain; Cells, Cultured; Endothelial Cells; Endothelium, Vascular; Green Fluorescent Proteins; Lysophospholipids; Male; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Inbred Lew; Receptors, Lysosphingolipid; Recombinant Fusion Proteins; Sphingosine; Tumor Cells, Cultured; Up-Regulation

Activation of sphingosine kinase (SphK), which has two known isoforms, is responsible for the synthesis of sphingosine 1-phosphate (S1P), a cell survival factor. We tested the following hypotheses: 1] cardiac myocytes null for the SphK1 gene are more vulnerable to the stress of hypoxia+glucose deprivation; 2] the monoganglioside GM-1, which activates SphK via protein kinase C epsilon, is ineffective in SphK1-null myocytes; 3] S1P generated by SphK activation requires cellular export to be cardioprotective.. We cultured adult mouse cardiac myocytes from wildtype and SphK1-null mice (deletion of exons 3-6) and measured cell viability by trypan blue exclusion.. In wildtype adult mouse cardiomyocytes subjected to 4 h of hypoxic stress+glucose deprivation, cell viability was significantly higher than in SphK1-null cardiomyocytes. SphK1-null cells also displayed more mitochondrial cytochrome C release. Cell death induced by hypoxia+glucose deprivation was substantially prevented by pretreatment with exogenous S1P in both wildtype and SphK1-null myocytes, but S1P was effective at a lower concentration in wildtype cells. Hence, the absence of the Sphk1 gene did not affect receptor coupling or downstream signal transduction. Pretreatment for 1 h with 1 microM of the monoganglioside GM-1 increased survival in wildtype cells, but not in SphK1-null myocytes. Thus, activation of SphK1 by GM-1 leads to cell survival. In wildtype cells, enhanced survival produced by GM-1 was abrogated by pretreatment either with 300 nM of the S1P(1) receptor-selective antagonist VPC23019 or with 100 ng/ml of pertussis toxin for 16 h before exposure to hypoxia+glucose deprivation.. As the effect of GM-1 is blocked both at the receptor and the G-protein (Gi) levels, we conclude that S1P generated by GM-1 treatment must be exported from the cell and acts in a paracrine or autocrine manner to couple with its cognate receptor.

Topics: Animals; Apoptosis; Biomarkers; Blotting, Western; Cell Hypoxia; Cell Survival; Cells, Cultured; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Ginsenosides; Glucose; Hypoglycemic Agents; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Heart; Myocardial Ischemia; Myocytes, Cardiac; Pertussis Toxin; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine-1-phosphate (S1P) has been shown to regulate numerous and diverse cell functions, including smooth muscle contraction. Here we assessed the role of S1P/Sphingosine kinase (SPK) pathway in the regulation of bronchial tone. Our objective was to determine, using an integrated pharmacologic and molecular approach, (1) the role of S1P as endogenous modulator of the bronchial tone, and (2) the linkage between S1P pathway and bronchial hyperresponsiveness. We evaluated S1P effects on isolated bronchi and whole lungs, harvested from Balb/c mice sensitized to ovalbumin (OVA) versus vehicle-treated mice, by measuring bronchial reactivity and lung resistance. We found that S1P administration on nonsensitized mouse bronchi does not cause any direct effect on bronchial tone, while a significant increase in Ach-induced contraction occurs after S1P challenge. Conversely, in OVA-sensitized mice S1P/SPK pathway triggers airway hyperesponsiveness. Indeed, S1P causes a dose-dependent contraction of isolated bronchi. Similarly, in the whole lung system S1P increased airway resistance only in OVA-sensitized mice. The action on bronchi of S1P is coupled to an enhanced expression of SPK(1) and SPK(2) as well as of S1P(2) and S1P(3) receptors. In these experiments the key role for S1P/SPK in hyperreactivity has been confirmed by pharmacologic modulation of SPKs. S1P/SPK pathway does not seem to play a major role in physiologic conditions, while it may become critical in pathologic conditions. These results open new windows for therapeutic strategies in diseases like asthma.

Topics: Acetylcholine; Animals; Bronchi; Bronchial Hyperreactivity; Cholinergic Agents; Dose-Response Relationship, Drug; Lysophospholipids; Mice; Mice, Inbred BALB C; Muscle Tonus; Muscle, Smooth; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid produced by sphingosine kinase (SphK1 and 2). We previously showed that S1P receptors (S1P1, S1P2, and S1P3) are expressed in hepatic myofibroblasts (hMF), a population of cells that triggers matrix remodeling during liver injury. Here we investigated the function of these receptors in the wound healing response to acute liver injury elicited by carbon tetrachloride, a process that associates hepatocyte proliferation and matrix remodeling. Acute liver injury was associated with the induction of S1P2, S1P3, SphK1, and SphK2 mRNAs and increased SphK activity, with no change in S1P1 expression. Necrosis, inflammation, and hepatocyte regeneration were similar in S1P2-/- and wild-type (WT) mice. However, compared with WT mice, S1P2-/- mice displayed reduced accumulation of hMF, as shown by lower induction of smooth muscle alpha-actin mRNA and lower induction of TIMP-1, TGF-beta1, and PDGF-BB mRNAs, overall reflecting reduced activation of remodeling in response to liver injury. The wound healing response was similar in S1P3-/- and WT mice. In vitro, S1P enhanced proliferation of cultured WT hMF, and PDGF-BB further enhanced the mitogenic effect of S1P. In keeping with these findings, PDGF-BB up-regulated S1P2 and SphK1 mRNAs, increased SphK activity, and S1P2 induced PDGF-BB mRNA. These effects were blunted in S1P2-/- cells, and S1P2-/- hMF exhibited reduced mitogenic and comitogenic responses to S1P. These results unravel a novel major role of S1P2 in the wound healing response to acute liver injury by a mechanism involving enhanced proliferation of hMF.

Topics: Acute Disease; Animals; Becaplermin; Carbon Tetrachloride Poisoning; Cell Division; Cells, Cultured; Chemical and Drug Induced Liver Injury; DNA Replication; Enzyme Induction; Extracellular Matrix; Fibroblasts; Gene Expression Regulation; Liver Regeneration; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Myoblasts, Smooth Muscle; Necrosis; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-sis; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta1

Sphingosine-1-phosphate, a key mediator in immune cell trafficking, is elevated in the lungs of asthmatic patients and regulates pulmonary epithelium permeability. Stimulation of mast cells by allergens induces two mammalian sphingosine kinases (Sphk1 and Sphk2) to produce sphingosine-1-phosphate (S1P). Little is known about the individual role of these kinases in regulating immune cell function. Here we show that in mast cells, Sphk2 is required for production of S1P, for calcium influx, for activation of protein kinase C, and for cytokine production and degranulation. However, susceptibility to in vivo anaphylaxis is determined both by S1P within the mast cell compartment and by circulating S1P generated by Sphk1 predominantly from a non-mast cell source(s). Thus, sphingosine kinases are determinants of mast cell responsiveness, demonstrating a previously unrecognized relationship with anaphylaxis.

Topics: Anaphylaxis; Animals; Biomarkers; Lysophospholipids; Mast Cells; Mice; Mice, Mutant Strains; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Lymphocytes require sphingosine-1-phosphate (S1P) receptor-1 to exit lymphoid organs, but the source(s) of extracellular S1P and whether S1P directly promotes egress are unknown. By using mice in which the two kinases that generate S1P were conditionally ablated, we find that plasma S1P is mainly hematopoietic in origin, with erythrocytes a major contributor, whereas lymph S1P is from a distinct radiation-resistant source. Lymphocyte egress from thymus and secondary lymphoid organs was markedly reduced in kinase-deficient mice. Restoration of S1P to plasma rescued egress to blood but not lymph, and the rescue required lymphocyte expression of S1P-receptor-1. Thus, separate sources provide S1P to plasma and lymph to help lymphocytes exit the low-S1P environment of lymphoid organs. Disruption of compartmentalized S1P signaling is a plausible mechanism by which S1P-receptor-1 agonists function as immunosuppressives.

Topics: Animals; Bone Marrow; Chemotaxis, Leukocyte; Chromatography, Liquid; Endothelium, Vascular; Female; Hematopoietic Stem Cells; Lymphocytes; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine; Tandem Mass Spectrometry

Sphingosine 1-phosphate (S1P) regulates diverse cellular functions through extracellular ligation to S1P receptors, and it also functions as an intracellular second messenger. Human pulmonary artery endothelial cells (HPAECs) effectively utilized exogenous S1P to generate intracellular S1P. We, therefore, examined the role of lipid phosphate phosphatase (LPP)-1 and sphingosine kinase1 (SphK1) in converting exogenous S1P to intracellular S1P. Exposure of (32)P-labeled HPAECs to S1P or sphingosine (Sph) increased the intracellular accumulation of [(32)P]S1P in a dose- and time-dependent manner. The S1P formed in the cells was not released into the medium. The exogenously added S1P did not stimulate the sphingomyelinase pathway; however, added [(3)H]S1P was hydrolyzed to [(3)H]Sph in HPAECs, and this was blocked by XY-14, an inhibitor of LPPs. HPAECs expressed LPP1-3, and overexpression of LPP-1 enhanced the hydrolysis of exogenous [(3)H]S1P to [(3)H]Sph and increased intracellular S1P production by 2-3-fold compared with vector control cells. Down-regulation of LPP-1 by siRNA decreased intracellular S1P production from extracellular S1P but had no effect on the phosphorylation of Sph to S1P. Knockdown of SphK1, but not SphK2, by siRNA attenuated the intracellular generation of S1P. Overexpression of wild type SphK1, but not SphK2 wild type, increased the accumulation of intracellular S1P after exposure to extracellular S1P. These studies provide the first direct evidence for a novel pathway of intracellular S1P generation. This involves the conversion of extracellular S1P to Sph by LPP-1, which facilitates Sph uptake, followed by the intracellular conversion of Sph to S1P by SphK1.

Topics: Adenoviridae; Biotin; Blotting, Western; Cells, Cultured; Ceramides; Chromatography, Liquid; Cytoplasm; Endothelium, Vascular; Humans; Kidney; Lysophospholipids; Mass Spectrometry; Phosphatidate Phosphatase; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Artery; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Sphingosine

Topics: Animals; Endothelium, Vascular; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Lymphocytes; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine

Sphingosine kinases (SPHKs) catalyze the formation of the bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P), which plays important roles in a wide variety of intra- and extracellular functions. Conventionally, SPHK activity has been determined using radioisotope thin layer chromatography (TLC) and autoradiography to detect the product S1P. Here we describe the development of a simple and robust in vitro SPHK assay in 384-well format with no requirement for any separation steps such as extraction and TLC. The assay is based on (33)P-phosphate transfer from [gamma-(33)P]ATP to sphingosine and subsequent detection of the [(33)P]S1P using AquaBind plates (Asahi Techno Glass, Tokyo, Japan). Enzymatic and inhibition characteristics determined with this assay are in good agreement with previously reported values determined in the conventional TLC assay. K(m) values for D-erythro-sphingosine and ATP were determined to be 17.5 microM and 19.2 microM, respectively. The kinase reaction could be inhibited by ADP and N,N-dimethylsphingosine with a 50% inhibitory concentration of 410 microM and 450 microM, respectively. The established assay format was easily adapted to an automated screening platform and is characterized by a high signal-to-background ratio, small variation, and excellent Z factors.

Topics: Adenosine Triphosphate; Automation; Drug Evaluation, Preclinical; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Escherichia coli; Kinetics; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Recombinant Proteins; Sphingosine

The chicken embryo has been well used in studies of the developmental process, and during development sphingosine and sphingosine 1-phosphate (So1P) are considered critical mediators of cell death and survival. In this study, we compared the sphingolipid contents of chicken embryos during the early embryonic development period from day 3 to day 6. HPLC analyses of sphingosine and So1P in chicken embryos revealed that sphingosine levels were greatly reduced on day 4 whereas So1P levels were not significantly changed. Sphingosine kinase (Sphk) activities, which require sphingosine as substrate to produce So1P, were also greatly reduced on day 4. Collectively, we found sphingosine levels and Sphk activities, but not So1P levels are changed in early stage of chicken embryos development.

Topics: Animals; Chick Embryo; Embryonic Development; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine kinase (SphK) is a key enzyme in the synthesis of sphingosine 1-phosphate (S1P), a bioactive sphingolipid. SphK is involved in ischemic preconditioning (IPC). To date no studies in genetically altered animals have examined the role of SphK1 in myocardial ischemia/reperfusion (IR) injury and IPC.. Wild-type and SphK1 null mouse hearts were subjected to IR (50 min global ischemia and 40 min reperfusion) in a Langendorff apparatus. IPC consisted of 2 min of global ischemia and 2 min of reperfusion for two cycles. At baseline, there were no differences in left ventricular developed pressure (LVDP), +/-dP/dtmax, and LV end-diastolic pressure (EDP) between SphK1 mutant and wild-type (WT) mouse hearts. In the mutants, total SphK enzyme activity was reduced by 44% and S1P levels were decreased by 41%. SphK1 null hearts subjected to IR exhibited more cardiac damage compared with WT: LVDP and +/-dP/dtmax decreased, LVEDP increased, and infarct size increased (n=6, P<0.05). Apoptosis was markedly enhanced in SphK1 mutant IR mouse hearts. IPC was cardioprotective in WT hearts, but this protection appeared to be ineffective in SphK1 null hearts. There was no change in infarct size in the IPC+IR group compared to the IR group in the null hearts (50.1+/-5.0% vs 45.0+/-3.8%, n=6, P=NS). IPC remained ineffective in the null hearts even when the index ischemia time was shortened by 10 min.. Deletion of the SphK1 gene sensitizes the myocardium to IR injury and appears to impair the protective effect of IPC. These data provide the first genetic evidence that the SphK1-S1P pathway is a critical mediator of IPC and cell survival.

Topics: Animals; Apoptosis; Blotting, Western; Creatine Kinase; Disease Susceptibility; In Situ Nick-End Labeling; Ischemic Preconditioning, Myocardial; Lysophospholipids; Mice; Mice, Knockout; Mutation; Myocardium; Perfusion; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Sphingosine

Sphingosine-1-phosphate (S1P) is a lipid mediator that exerts multiple cellular functions through activation of a subfamily of G-protein-coupled receptors. Although there is evidence that S1P plays a role in the developing and adult CNS, little is known about the ability of brain parenchyma to synthesize this lipid. We have therefore analyzed the brain distribution of the enzymatic activity of the S1P synthesizing enzyme, sphingosine kinase (SPHK) [EC:2.7.1.91], as well as mRNA distribution for one of the two isoforms of this enzyme, sphingosine kinase 2. SPHK activity, measured by the conversion of [(3)H]sphingosine to [(3)H]S1P, is highest in cerebellum, followed by cortex and brainstem. Lowest activities were found in striatum and hippocampus. Sensitivity to 0.1% Triton-X suggests that this activity is accounted for by SPHK2. RT-PCR and in situ hybridization studies show that mRNA for this isoform has a distribution similar to that of SPHK activity. In vivo and in vitro ischemia increase SPHK activity and SPHK2 mRNA levels. These results indicate that SPHK2 is the predominant S1P-synthesizing isoform in normal brain parenchyma. Its heterogeneous distribution, in particular laminar distribution in cortex, suggests a neuronal localization and a possible role in cortical and cerebellar functions, in normal as well as ischemic brain.

Topics: Animals; Blotting, Northern; Blotting, Western; Brain; Brain Chemistry; Cells, Cultured; Female; Glucose; Hypoxia, Brain; In Situ Hybridization; Infarction, Middle Cerebral Artery; Lysophospholipids; Male; Mice; Mice, Inbred ICR; Neuroglia; Neurons; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine

Ca2+ signaling plays an important role in endothelial cell (EC) functions including the regulation of barrier integrity. Recently, the endogenous lipid derivative, sphingosine-1-phosphate (S1P), has emerged as an important modulator of EC barrier function. We investigated the role of endogenously generated S1P in Ca2+ metabolism and barrier function in human umbilical endothelial cells (HUVECs) stimulated by thrombin, histamine, or other agonists. Barrier function was assessed by dextran diffusion through HUVEC monolayers, and Ca2+ transients were measured using a fluoroprobe. Thrombin or histamine increased Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ entry through store-operated channels (SOCs) that was accompanied by increased EC permeability. Inhibition of S1P synthesis by a specific sphingosine kinase inhibitor (SKI) decreased thrombin or histamine-induced increased permeability and decreased Ca2+ entry via SOC in a concentration-dependent fashion. SKI had minuscule effects on thrombin or histamine-induced Ca2+ release from ER. SKI also inhibited thapsigargin or ionomycin-induced Ca2+ entry via SOC without affecting Ca2+ release from the ER. In contrast to the effects of endogenously generated S1P, when S1P was administered externally, it initiated Ca2+ release from ER similar to thrombin and histamine while decreasing EC permeability. These observations indicate that after agonist-induced conditions, endogenously generated S1P functions as a positive modulator of Ca2+ entry via SOC and a mediator of increased cell permeability. In contrast, extracellular exposure to S1P has different signaling mechanisms and effects. Thus, the potential dual roles of endogenous and exogenous S1P on EC function need to be considered in pharmacological studies targeting sphingosine metabolism.

Topics: Calcium; Calcium Signaling; Cells, Cultured; Endothelial Cells; Enzyme Inhibitors; Histamine; Humans; Lysophospholipids; Permeability; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Thrombin

Macrophage polarization contributes to a number of human pathologies. This is exemplified for tumor-associated macrophages (TAMs), which display a polarized M2 phenotype, closely associated with promotion of angiogenesis and suppression of innate immune responses. We present evidence that induction of apoptosis in tumor cells and subsequent recognition of apoptotic debris by macrophages participates in the macrophage phenotype shift. During coculture of human primary macrophages with human breast cancer carcinoma cells (MCF-7) the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-alpha and interleukin (IL) 12-p70 production, but increased formation of IL-8 and -10. Alternative macrophage activation required tumor cell death because a coculture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. Interestingly, phenotype alterations were achieved with conditioned media from apoptotic tumor cells, arguing for a soluble factor. Knockdown of sphingosine kinase (Sphk) 2, but not Sphk1, to attenuate S1P formation in MCF-7 cells, restored classical macrophage responses during coculture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P suppressed nuclear factor (NF)-kappaB signaling. These findings suggest that tumor cell apoptosis-derived S1P contributes to macrophage polarization.

Topics: Anti-Inflammatory Agents; Apoptosis; Cell Line, Tumor; Cell Polarity; Cell Survival; Coculture Techniques; Cytokines; Humans; Lysophospholipids; Macrophage Activation; Macrophages; Neoplasms; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Staurosporine

Resistance to chemotherapeutic drugs often limits their clinical efficacy. Previous studies have implicated the bioactive sphingolipid sphingosine-1-phosphate (S-1-P) in regulating sensitivity to cisplatin [cis-diamminedichloroplatinum(II)] and showed that modulating the S-1-P lyase can alter cisplatin sensitivity. Here, we show that the members of the sphingosine kinase (SphK1 and SphK2) and dihydroceramide synthase (LASS1/CerS1, LASS4/CerS4, and LASS5/CerS5) enzyme families each have a unique role in regulating sensitivity to cisplatin and other drugs. Thus, expression of SphK1 decreases sensitivity to cisplatin, carboplatin, doxorubicin, and vincristine, whereas expression of SphK2 increases sensitivity. Expression of LASS1/CerS1 increases the sensitivity to all the drugs tested, whereas LASS5/CerS5 only increases sensitivity to doxorubicin and vincristine. LASS4/CerS4 expression has no effect on the sensitivity to any drug tested. Reflecting this, we show that the activation of the p38 mitogen-activated protein (MAP) kinase is increased only by LASS1/CerS1, and not by LASS4/CerS4 or LASS5/CerS5. Cisplatin was shown to cause a specific translocation of LASS1/CerS1, but not LASS4/CerS4 or LASS5/CerS5, from the endoplasmic reticulum (ER) to the Golgi apparatus. Supporting the hypothesis that this translocation is mechanistically involved in the response to cisplatin, we showed that expression of SphK1, but not SphK2, abrogates both the increased cisplatin sensitivity in cells stably expressing LASS1/CerS and the translocation of the LASS1/CerS1. The data suggest that the enzymes of the sphingolipid metabolic pathway can be manipulated to improve sensitivity to the widely used drug cisplatin.

Topics: Antineoplastic Agents; Blotting, Western; Cell Survival; Ceramides; Cisplatin; Drug Resistance, Neoplasm; Enzyme Activation; Fluorescent Antibody Technique; Golgi Apparatus; Humans; Immunoprecipitation; Lysophospholipids; Membrane Proteins; Oxidoreductases; p38 Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Sphingosine N-Acyltransferase

Fumonisins are causative agents of diseases in mice and rats, including liver and renal toxicities, as well as cancer, and are specific inhibitors of ceramide synthase in the metabolism of sphingolipid. The purpose of this study was to determine whether an elevated level of sphingoid base 1-phosphate was related to the expressions of metabolism enzymes in the liver of fumonisin B1 (FB1)-treated mice and acted as a contributing factor to hepatotoxicity. In our previous study, FB1 was confirmed to be toxic to both liver and kidneys, coupled with simultaneous elevation of sphinganine 1-phosphate. ICR mice were treated intraperitoneally with 10 mg/kg/day FB1 for 5 days, with the concentrations of sphingolipid metabolites in the serum and liver measured using HPLC following Bligh-Dyer extraction. The levels of sphingoid bases and their 1-phosphates in the serum and liver were markedly elevated in response to treatment with FB1. In the liver, FB1 increased the expression of sphingosine kinase and inhibited the expression of sphingosine 1-phosphate lyase. The cleaved form of caspase-3 was detected in the liver of FB1-treated mice, indicating the occurrence of apoptosis in the liver following exposure to FB1. The expressions of proapoptotic signaling molecules, such as phosphorylated forms of c-Jun N-terminus kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), were increased in the liver of FB1-treated mice. In conclusion, these results suggest the elevation of sphingoid base 1-phosphate, as a result of the activation of sphingosine kinase and the inhibition of sphingosine 1-phosphate lyase, may be a major target for FB1-induced hepatotoxicity via the activation of an apoptotic signaling pathway.

Topics: Aldehyde-Lyases; Animals; Caspase 3; Chemical and Drug Induced Liver Injury; Fumonisins; Liver; Lysophospholipids; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinases; Mycotoxins; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine kinase (SK) is a signaling enzyme that phosphorylates sphingosine to produce sphingosine 1-phosphate. Sphingosine and sphingosine 1-phosphate (S1P) belong to a class of bioactive sphingolipid metabolites that are critical in a number of cellular processes, yet often have opposing biological functions. The intracellular localization of sphingosine kinase has been demonstrated in multiple studies to be a critical aspect of its signaling function. To date, assays of sphingosine kinase activity have been developed for measuring activity in lysates, where the effects of localization are lost. Here we outline a system in which the rate of production of S1P can be measured in intact cells using exogenously added radiolabeled ATP instead of tritiated sphingosine. The surprising ability of ATP to enter unpermeabilized monolayers is one aspect that makes this assay simple, efficient, and inexpensive, yet sensitive enough to measure endogenous enzyme activity. The assay is well behaved in terms of kinetics and substrate dependence. Overall, this assay is ideal for future studies to identify changes in S1P production in intact cells such as those that result from the differential intracellular targeting of sphingosine kinase.

Topics: Adenosine Triphosphate; Cells, Cultured; Chromatography, Thin Layer; HeLa Cells; Humans; Kinetics; Lysophospholipids; Phosphorus Radioisotopes; Phosphotransferases (Alcohol Group Acceptor); Sensitivity and Specificity; Sphingosine; Unilamellar Liposomes

The inhalational anesthetic isoflurane has been shown to protect against renal ischemia-reperfusion (IR) injury. Previous studies demonstrated that isoflurane modulates sphingolipid metabolism in renal proximal tubule cells. We sought to determine whether isoflurane stimulates sphingosine kinase (SK) activity and synthesis of sphingosine-1-phosphate (S1P) in renal proximal tubule cells to mediate renal protection via the S1P signaling pathway. Isoflurane anesthesia reduced the degree of renal failure and necrosis in a murine model of renal IR injury. This protection with isoflurane was reversed by SK inhibitors (DMS and SKI-II) as well as an S1P(1) receptor antagonist (VPC23019). In addition, mice deficient in SK1 enzyme were not protected from IR injury with isoflurane. SK activity as well as SK1 mRNA expression increased in both cultured human proximal tubule cells (HK-2) and mouse kidneys after exposure to isoflurane. Finally, isoflurane increased the generation of S1P in HK-2 cells. Taken together, our findings indicate that isoflurane activates SK in renal tubule cells and initiates S1P-->S1P(1) receptor signaling to mediate the renal protective effects. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated renal protection and lead to new therapeutic applications of inhalational anesthetics during the perioperative period.

Topics: Anesthetics, Inhalation; Animals; Cell Line; Creatinine; Enzyme Inhibitors; Humans; Isoflurane; Kidney Diseases; Kidney Tubules; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sphingosine

Sphingosine kinase (Sphk) phosphorylates sphingosine into sphingosine-1-phosphate (S1P), but its recently identified isoform Sphk2 has been suggested to have distinct subcellular localization and substrate specificity. We demonstrate here that, surprisingly, Sphk2(-/-) CD4(+) T cells exhibit a hyperactivated phenotype with significantly enhanced proliferation and cytokine secretion in response to IL-2 as well as reduced sensitivity to regulatory T cell-mediated suppression in vitro, apparently independent of effects upon S1P. Such findings appear to reflect a requirement for Sphk2 to suppress IL-2 signaling because, in Sphk2(-/-) CD4(+) T cells, IL-2 induced abnormally accentuated STAT5 phosphorylation and small interfering RNA knockdown of STAT5 abrogated their hyperactive phenotype. This pathway physiologically modulates autoinflammatory responses, because Sphk2(-/-) T cells induced more rapid and robust inflammatory bowel disease in scid recipients. Thus, Sphk2 regulates IL-2 pathways in T cells, and the modulation of Sphk2 activity may be of therapeutic utility in inflammatory and/or infectious diseases.

Topics: Animals; Autoimmunity; Inflammatory Bowel Diseases; Interleukin-2; Intestinal Mucosa; Intestines; Lysophospholipids; Mice; Mice, Knockout; Phenotype; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; T-Lymphocytes

Sphingosine 1-phosphate (S1P), a lysophospholipid mediator that signals through G protein-coupled receptors, regulates a wide plethora of biological responses such as angiogenesis and immune cell trafficking. Detection and quantification of S1P in biological samples is challenging due to its unique physicochemical nature and occurrence in trace quantities. In this report, we describe a new method to selectively enrich S1P and dihydro-S1P from biological samples by the Fe(3+) gel immobilized metal affinity chromatography (IMAC). The eluted S1P from IMAC was dephosphorylated, derivatized with o-phthalaldehyde (OPA), and detected by high-performance liquid chromatography (HPLC) coupled to a fluorescence detector. IMAC purification of S1P was linear for a wide range of S1P concentration. Using this assay, secretion of endogenous S1P from endothelial cells, fibroblasts and colon cancer cells was demonstrated. We also show that dihydro-S1P was the major sphingoid base phosphate secreted from HUVEC over expressed with Sphk1 cDNA. Pharmcological antagonists of ABC transporters, glyburide and MK-571 attenuated endogenous S1P release. This assay was also used to demonstrate that plasma S1P levels were not altered in mice deficient for ABC transporters, Abca1, Abca7 and Abcc1/Mrp1. IMAC-based affinity-enrichment coupled with a HPLC-based separation and detection system is a rapid and sensitive method to accurately quantify S1P.

Topics: Adenoviridae; Animals; ATP-Binding Cassette Transporters; Chromatography, Affinity; Chromatography, High Pressure Liquid; Endothelial Cells; Ferric Compounds; Humans; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Plasma; Sphingosine; Transduction, Genetic

Sphingosine kinase (SphK) is a conserved lipid kinase that catalyzes formation of important regulators of inter- and intracellular signaling, sphingosine-1 phosphate (S1P), and dihydrosphingosine 1-phosphate (dhS1P). In this study, we investigated the role of SphK1 in the regulation of expression of matrix metalloproteinase 1 (MMP1) in dermal fibroblasts, a key event in regulation of extra cellular matrix. We show that overexpression of SphK1 up-regulated MMP1 protein, MMP1 mRNA, and MMP1 promoter activity, and this action of SphK1 required activation of the ERK1/2-Ets1 and NF-kappaB pathways. Furthermore, experiments using SphK1 specific siRNA demonstrated that SphK1 is required for the TNF-alpha stimulation of MMP1. Additional data revealed a specific role of dhS1P, and not S1P, as a mediator of SphK1-dependent activation of ERK1/2 and up-regulation of MMP1. The stimulatory effect of dhS1P was sensitive to pertussis toxin, suggesting a possible involvement of a G-protein-coupled receptor. In contrast, S1P, but not dhS1P, stimulated the induction of COX-2, which demonstrated selective actions of these two closely related bioactive lipids. In conclusion, this study describes a novel mode of SphK1 signaling through generation of dhS1P with a key role in mediating transcriptional responses to TNF-alpha. This is the first report of selective function of dhS1P as compared with the better studied S1P.

Topics: Ceramides; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gene Expression Regulation, Enzymologic; Humans; Lysophospholipids; Matrix Metalloproteinase 1; NF-kappa B; Pertussis Toxin; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Protein c-ets-1; Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha; Up-Regulation

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells results in the production and secretion of sphingosine 1-phosphate (S1P), a lipid metabolite present in the lungs of allergen-challenged asthmatics. Herein we report that two isoforms of sphingosine kinase (SphK1 and SphK2) are expressed and activated upon FcepsilonRI engagement of bone marrow-derived mast cells (BMMC). Fyn kinase is required for FcepsilonRI coupling to SphK1 and -2 and for subsequent S1P production. Normal activation of SphK1 and -2 was restored by expression of wild type Fyn but only partly with a kinase-defective Fyn, indicating that induction of SphK1 and SphK2 depended on both catalytic and noncatalytic properties of Fyn. Downstream of Fyn, the requirements for SphK1 activation differed from that of SphK2. Whereas SphK1 was considerably dependent on the adapter Grb2-associated binder 2 and phosphatidylinositol 3-OH kinase, SphK2 showed minimal dependence on these molecules. Fyn-deficient BMMC were defective in chemotaxis and, as previously reported, in degranulation. These functional responses were partly reconstituted by the addition of exogenous S1P to FcepsilonRI-stimulated cells. Taken together with our previous study, which demonstrated delayed SphK activation in Lyn-deficient BMMC, we propose a cooperative role between Fyn and Lyn kinases in the activation of SphKs, which contributes to mast cell responses.

Topics: Animals; Cell Degranulation; Cell Line; Chemotaxis; Enzyme Activation; Humans; Immunoglobulin E; Lysophospholipids; Mast Cells; Mice; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-fyn; Receptors, IgE; Sphingosine; src-Family Kinases; Transfection

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to form sphingosine 1-phosphate (S1P), which has the ability to promote cell proliferation and survival and stimulate angiogenesis. The SK1/S1P pathway also plays a critical role in regulation of cyclooxygenase-2 (COX-2), a well-established pathogenic factor in colon carcinogenesis. Therefore, we examined the expression of SK1 and COX-2 in rat colon tumors induced by azoxymethane (AOM) and the relationship of these two proteins in normal and malignant intestinal epithelial cells. Strongly positive SK1 staining was found in 21/28 (75%) of rat colon adenocarcinomas induced by AOM, whereas no positive SK1 staining was observed in normal mucosa. The increase in SK1 and COX-2 expression in AOM-induced rat colon adenocarcinoma was confirmed at the level of mRNA by real-time RT-PCR. In addition, it was found that 1) down-regulation of SK1 in HT-29 human colon cancer cells by small interfering RNA (siRNA) decreases COX-2 expression and PGE2 production; 2) overexpression of SK1 in RIE-1 rat intestinal epithelial cells induces COX-2 expression; and 3) S1P stimulates COX-2 expression and PGE2 production in HT-29 cells. These results suggest that the SK1/S1P pathway may play an important role in colon carcinogenesis, in part, by regulating COX-2 expression and PGE2 production.

Topics: Animals; Azoxymethane; Cell Line, Tumor; Colon; Colonic Neoplasms; Cyclooxygenase 2; Dinoprostone; Epithelial Cells; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Intestinal Mucosa; Lysophospholipids; Male; Mice; Mice, Inbred BALB C; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Inbred F344; Sphingosine; Up-Regulation

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts both as an extracellular ligand for endothelial differentiation gene receptor family and as an intracellular second messenger. Cellular levels of S1P are low and tightly regulated by sphingosine kinase (SPK). Recent studies have suggested that eNOS pathway may function as a downstream target for the biological effects of receptor-mediated S1P. Here we have studied the possible interplay between intracellular SIP generation and the eNOS activation pathway. S1P causes an endothelium-dependent vasorelaxation in rat aorta that is PTX sensitive, inhibited by L-NAME that involves eNOS phosphorylation, and mainly dependent on hsp90. When rat aorta rings were incubated with the SPK inhibitor DL-threo-dihydrosphingosine (DTD), there was a concentration-dependent reduction of Ach-induced vasorelaxation, implying a consistent contribution of sphingolipid pathway through intracellular sphingosine release and phosphorylation. Co-immunoprecipitation experiments consistently showed increased association of hsp90 with eNOS after exposure of cells to S1P as well to BK or calcium ionophore A-23187. Interestingly, as opposite to A-23187, BK and S1P effect were significantly inhibited by pretreatment with the SPK inhibitor DTD. In conclusion, our data demonstrate that an interplay exists among eNOS, hsp90, and intracellularly generated S1P where eNOS coupling to hsp90 is a major determinant for NO release as confirmed by our functional and molecular studies.

Topics: Animals; Aorta; Calcium; Cattle; Cells, Cultured; Endothelial Cells; HSP90 Heat-Shock Proteins; Lysophospholipids; Male; Nitric Oxide; Nitric Oxide Synthase; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Sphingosine; Vasodilation

(R)-4-(3,4-Dihydro-8,8-dimethyl)-2H,8H-benzo[1,2-b:3,4-b'] dipyran-3yl)-1,3-benzenediol (glabridin) is known to have anti-inflammatory, antimicrobial, and cardiovascular protective activities. In the present study, we report the inhibitory effect of glabridin on intercellular adhesion molecule-1 (ICAM-1) expression in tumor necrosis factor-alpha (TNF-alpha)-stimulated human umbilical vein endothelial cells (HUVECs). Glabridin inhibited THP-1 cell adhesion to HUVECs stimulated by TNF-alpha and cell surface expression of ICAM-1 in TNF-alpha-stimulated HUVECs. The mRNA expression of adhesion molecules, including ICAM-1, vascular cell adhesion molecule-1, and E-selectin, was also suppressed by glabridin. Further study demonstrated the inhibitory effect of glabridin on nuclear factor (NF)-kappaB/Rel DNA binding, inhibitory factor-kappaB alpha (IkappaB alpha), and IkappaB beta degradation, IkappaB kinase activation, and p65 nuclear translocation in TNF-alpha-stimulated HUVECs. Treatment of a variety of cell lines with glabridin revealed that inhibitory effect of glabridin on NF-kappaB/Rel activation is not cell type-specific, and both inducible and constitutive NF-kappaB/Rel activation was suppressed by glabridin treatment. Moreover, TNF-alpha-induced phosphorylation of Akt and extracellular signal-regulated kinase (ERK) was blocked by glabridin treatment in HUVECs. Glabridin also suppressed sphingosine-1-phosphate (S1P)-induced cell surface expression and mRNA expression of ICAM-1. Further study demonstrated that TNF-alpha-induced sphingosine kinase activity was inhibited by glabridin, and the inhibitory effect of glabridin on TNF-alpha-induced ICAM-1 expression was reversed by addition of exogenous S1P. Together, our results indicate that the inhibitory effect of glabridin on ICAM-1 expression might be mediated, at least in part, by inhibiting sphingosine kinase pathway and subsequent inhibition of signaling pathways, including Akt, ERK, and NF-kappaB/Rel signaling pathway.

Topics: DNA; Down-Regulation; Endothelium, Vascular; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; I-kappa B Kinase; I-kappa B Proteins; Intercellular Adhesion Molecule-1; Isoflavones; Lysophospholipids; Phenols; Phosphotransferases (Alcohol Group Acceptor); Protein Transport; Proto-Oncogene Proteins c-akt; Signal Transduction; Sphingosine; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Umbilical Veins

Complex sphingolipids are abundant as eukaryotic cell membrane components, whereas their metabolites, in particular ceramide, sphingosine, and sphingosine 1-phosphate, are involved in diverse cell signaling processes. In mammals, degradation of ceramide by ceramidase yields sphingosine, which is phosphorylated by the action of sphingosine kinase to generate sphingosine 1-phosphate. Therefore, ceramidases are key enzymes in the regulation of the cellular levels of ceramide, sphingosine, and sphingosine 1-phosphate. To explore the physiological functions of a neutral ceramidase with diverse cellular locations, we disrupted the Asah2 gene in mice. Asah2 null mice have a normal life span and do not show obvious abnormalities or major alterations in total ceramide levels in tissues. The Asah2-encoded neutral ceramidase is highly expressed in the small intestine along the brush border, suggesting that the neutral ceramidase may be involved in a pathway for the digestion of dietary sphingolipids. Indeed, Asah2 null mice were deficient in the intestinal degradation of ceramide. Thus, the results indicate that the Asah2-encoded neutral ceramidase is a key enzyme for the catabolism of dietary sphingolipids and regulates the levels of bioactive sphingolipid metabolites in the intestinal tract.

Topics: Alleles; Amidohydrolases; Animals; Blotting, Northern; Blotting, Western; Cell Membrane; Ceramidases; Ceramides; Chromatography, Liquid; Genetic Vectors; Heterozygote; Immunohistochemistry; Intestinal Mucosa; Intestine, Small; Lysophospholipids; Mass Spectrometry; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Biological; Models, Genetic; Neutral Ceramidase; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Spectrometry, Mass, Electrospray Ionization; Sphingolipids; Sphingomyelins; Sphingosine; Time Factors; Tissue Distribution

The mitogenic role of sphingosine-1-phosphate (S1P) and its involvement in basic fibroblast growth factor (bFGF)-induced proliferation were examined in primary cultures of cerebellar astrocytes. Exposure to bFGF resulted in a rapid increase of extracellular S1P formation, bFGF inducing astrocytes to release S1P, but not sphingosine kinase, in the extracellular milieu. The SK inhibitor N,N-dimethylsphingosine inhibited S1P release as well as bFGF-induced growth stimulation. S1P application in quiescent astrocytes caused a dose-dependent increase in DNA synthesis. This gliotrophic effect was induced by a brief exposure to low nanomolar S1P, mimicked by the S1P receptor agonist dihydro-S1P, and inhibited by pertussis toxin (PTX), an inactivator of G(i)/G(o)-proteins. S1P also induced activation of extracellular signal-regulated kinase that was inhibited again by PTX. Moreover, the S1P lyase inhibitor 4-deoxypyridoxine induced the cellular accumulation of S1P but did not affect DNA synthesis. These results support the view that S1P exerted a mitogenic effect on cerebellar astrocytes extracellularly, most likely through cell surface S1P receptors. In agreement, mRNAs for S1P1, S1P2, and S1P3 receptors are expressed in cerebellar astrocytes (Anelli et al., 2005. J Neurochem 92:1204-1215). Ceramide, a negative regulator of astrocyte proliferation and down-regulated by bFGF (Riboni et al., 2002. Cerebellum 1:129-135), efficiently inhibited S1P-induced proliferation. The S1P action appears to be part of an autocrine/paracrine cascade stimulated by bFGF and, together with ceramide down-regulation, essential for astrocytes to respond to bFGF. The results suggest that S1P and bFGF/S1P may play an important role in physiopathological glial proliferation, such as brain development, reactive gliosis and brain tumor formation.

Topics: Animals; Animals, Newborn; Astrocytes; Astrocytoma; Brain Neoplasms; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Ceramides; Cerebellum; DNA Replication; Enzyme Inhibitors; Extracellular Fluid; Extracellular Signal-Regulated MAP Kinases; Fibroblast Growth Factor 2; Gliosis; GTP-Binding Protein alpha Subunits, Gi-Go; Lysophospholipids; Mitosis; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Myogenic vasoconstriction, an intrinsic response to elevated transmural pressure (TMP), requires the activation of sphingosine kinase (Sk1) and the generation of reactive oxygen species (ROS). We hypothesized that pressure-induced Sk1 signaling and ROS generation are functionally linked. Using a model of cannulated resistance arteries isolated from the hamster gracilis muscle, we monitored vessel diameter and smooth muscle cell (SMC) Ca2+i (Fura-2) or ROS production (dichlorodihydrofluorescein). Elevation of TMP stimulated the translocation of a GFP-tagged Sk1 fusion protein from the cytosol to the plasma membrane, indicative of enzymatic activation. Concurrently, elevation of TMP initiated a rapid and transient production of ROS, which was enhanced by expression of wild-type Sk1 (hSk(wt)) and inhibited by its dominant-negative mutant (hSk(G82D)). Exogenous sphingosine-1-phosphate (S1P) also stimulated ROS generation is isolated vessels. Chemical (1 micromol/L DPI), peptide (gp91ds-tat/gp91ds), and genetic (N17Rac) inhibition strategies indicated that NADPH oxidase was the source of the pressure-induced ROS. NADPH oxidase inhibition attenuated myogenic vasoconstriction and reduced the apparent Ca2+ sensitivity of the SMC contractile apparatus, without affecting Ca2+-independent, RhoA-mediated vasoconstriction in response to exogenous S1P. Our results indicate a mandatory role for Sk1/S1P in mediating pressure-induced, NADPH oxidase-derived ROS formation. In turn, ROS generation appears to increase Ca2+ sensitivity, necessary for full myogenic vasoconstriction.

Topics: Animals; Arteries; Calcium; Gene Expression Regulation, Enzymologic; Lysophospholipids; Mice; Muscle, Skeletal; NADPH Oxidases; Phosphotransferases (Alcohol Group Acceptor); Pressure; Reactive Oxygen Species; Sphingosine; Vasoconstriction

Sphingosine kinases catalyze the formation of sphingosine 1-phosphate, a bioactive lipid involved in many aspects of cellular regulation, including the fundamental biological processes of cell growth and survival. A diverse range of cell agonists induce activation of human sphingosine kinase 1 (hSK1) and, commonly, its translocation to the plasma membrane. Although the activation of hSK1 in response to at least some agonists occurs directly via its phosphorylation at Ser225 by ERK1/2, many aspects governing the regulation of this phosphorylation and subsequent translocation remain unknown. Here, in an attempt to understand some of these processes, we have examined the known interaction of hSK1 with calmodulin (CaM). By using a combination of limited proteolysis, peptide interaction analysis, and site-directed mutagenesis, we have identified that the CaM-binding site of hSK1 resides in the region spanned by residues 191-206. Specifically, Phe197 and Leu198 are critically involved in the interaction because a version of hSK1 incorporating mutations of both Phe197 --> Ala and Leu198 --> Gln failed to bind CaM. We have also shown for the first time that human sphingosine kinase 2 (hSK2) binds CaM, and does so via a CaM binding region that is conserved with hSK1 because comparable mutations in hSK2 also ablate CaM binding to this protein. By using the CaM-binding-deficient version of hSK1, we have begun to elucidate the role of CaM in hSK1 regulation by demonstrating that disruption of the CaM-binding site ablates agonist-induced translocation of hSK1 from the cytoplasm to the plasma membrane, while having no effect on hSK1 phosphorylation and catalytic activation.

Topics: Amino Acid Sequence; Binding Sites; Calmodulin; Carcinogens; Cell Membrane; Cells, Cultured; Humans; Kidney; Lysophospholipids; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Peptide Fragments; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Protein Transport; Sphingosine; Tetradecanoylphorbol Acetate

Sphingosine kinase 1 (SK1) is one of the two known kinases, which generates sphingosine-1-phosphate (S1P), a potent endogenous lipid mediator involved in cell survival, proliferation, and cell-cell interactions. Activation of SK1 and intracellular generation of S1P were suggested to be part of the growth and survival factor-induced signaling, and overexpression of SK1 provoked cell tumorigenic transformation. Using a highly selective and sensitive LC-MS/MS approach, here we show that SK1 overexpression, but not SK2, in different primary cells and cultured cell lines results in predominant upregulation of the synthesis of dihydrosphingosine-1-phosphate (DHS1P) compared to S1P. Stable isotope pulse-labeling experiments in conjunction with LC-MS/MS quantitation of different sphingolipids demonstrated strong interference of overexpressed SK1 with the de novo sphingolipid biosynthesis by deviating metabolic flow of newly formed sphingoid bases from ceramide formation toward the synthesis of DHS1P. On the contrary, S1P biosynthesis was not directly linked to the de novo sphingoid bases transformations and was dependent on catabolic generation of sphingosine from complex sphingolipids. As a result of SK1 overexpression, migration and Ca2+-response of human pulmonary artery endothelial cells (HPAEC) to stimulation with external S1P, but not thrombin, was strongly impaired. In contrast, selective increase in intracellular content of DHS1P or S1P through the uptake and phosphorylation of corresponding sphingoid bases had no effect on S1P-induced signaling or facilitation of wound healing. Furthermore, infection of human bronchial epithelial cells (HBEpC) with RSV A-2 virus increased SK1-mediated synthesis of DHS1P and S1P, whereas TNF-alpha enhanced only S1P production in HPAEC. These findings uncover a new functional role for SK1, which can control survival/death (DHS1P-S1P/ceramides) balance by targeting sphingolipid de novo biosynthesis and selectively generating DHS1P at a metabolic step preceding ceramide formation.

Topics: Avian Sarcoma Viruses; Calcium; Cell Movement; Cells, Cultured; Endothelial Cells; Gene Expression; Humans; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); RNA, Small Interfering; Serine C-Palmitoyltransferase; Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha; Up-Regulation

The mechanisms regulating spiral modiolar artery (SMA) tone are not known, yet their characterization is pivotal for understanding inner ear blood flow regulation. Sphingosine-1-phosphate (S1P), known to stimulate vasoconstriction in several vascular beds, is a candidate regulator of SMA tone with potential pathophysiological relevance.. Gerbil SMAs were isolated, cannulated and pressurized (30 mm Hg transmural) for experimentation under near-in vivo conditions. For functional experiments, vascular diameter and intracellular Ca2+ were simultaneously measured. Standard RT-PCR and immunohistochemical techniques were also employed.. mRNA transcripts encoding sphingosine kinase, S1P phosphohydrolase and three S1P receptors (S1P(1-3)) were detected in the SMA. S1P induced dose-dependent vasoconstriction of the SMA (EC50 = 115 nmol/L), and enhanced the apparent Ca2+-sensitivity of the contractile apparatus. Noradrenaline did not elicit vasoconstriction. The Rho kinase inhibitor Y27632 (1 micromol/L) reversed S1P-induced vasoconstriction and the S1P-mediated enhancement of Ca2+-sensitivity. RhoA was observed to translocate to the plasma membrane in response to stimulation with 30 micromol/L S1P.. We conclude that all key signalling pathway constituents are present at the mRNA level for S1P to act as an endogenous regulator of SMA tone. S1P stimulates potent, RhoA/Rho kinase-dependent SMA vasoconstriction and Ca2+ sensitization. The high sensitivity to S1P suggests that SMA vasoconstriction is likely to occur under pathological conditions that increase intramural S1P concentrations (i.e., inflammation). From a clinical perspective, the present study identifies new potential therapeutic targets for the treatment of vascular-based, "stroke-like" inner ear pathologies: the enzymes responsible for S1P bioavailability and the S1P receptors.

Topics: Amides; Animals; Arteries; Biological Transport; Calcium; Dose-Response Relationship, Drug; Ear, Inner; Endothelium, Vascular; Gerbillinae; Immunohistochemistry; In Vitro Techniques; Lysophospholipids; Muscle, Smooth, Vascular; Phosphotransferases (Alcohol Group Acceptor); Pyridines; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; RNA, Messenger; Sphingosine; Vasoconstrictor Agents

Monocytes play an important role in inflammation, angiogenesis, and atherosclerosis. During these processes monocytes release pre-formed proinflammatory mediators from granules, and synthesize de novo cytokines and chemokines important in the amplification of the inflammatory response. One of the most prominent triggers of inflammatory responses is the cytokine TNFalpha. However, the intracellular signaling cascades triggered by TNFalpha are not fully understood. In this study we investigated the roles of SPHK on the TNFalpha-triggered responses on human primary monocytes. We show that TNFalpha rapidly triggers S1P generation and activation of SPHK. Moreover, our data shows that SPHK1 is the isoform activated by TNFalpha, and plays an essential role on the TNFalpha-triggered intracellular Ca2+ signals, degranulation, cytokine production, and activation of NFkappaB, thus suggesting a pivotal role for SPHK1 on the proinflammatory responses triggered by TNFalpha.

Topics: Blotting, Western; Calcium; Cells, Cultured; Cytokines; Enzyme Activation; Gene Expression Regulation, Enzymologic; Humans; Immune System; Inflammation; Isoenzymes; Lysophospholipids; Monocytes; NF-kappaB-Inducing Kinase; Phosphotransferases (Alcohol Group Acceptor); Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha

Sphingosine 1-phosphate (S1P), produced by Sphks (sphingosine kinases), is a multifunctional lipid mediator that regulates immune cell trafficking and vascular development. Mammals maintain a large concentration gradient of S1P between vascular and extravascular compartments. Mechanisms by which S1P is released from cells and concentrated in the plasma are poorly understood. We recently demonstrated [Ancellin, Colmont, Su, Li, Mittereder, Chae, Stefansson, Liau and Hla (2002) J. Biol. Chem. 277, 6667-6675] that Sphk1 activity is constitutively secreted by vascular endothelial cells. In the present study, we show that among the five Sphk isoforms expressed in endothelial cells, the Sphk-1a isoform is selectively secreted in HEK-293 cells (human embryonic kidney cells) and human umbilical-vein endothelial cells. In sharp contrast, Sphk2 is not secreted. The exported Sphk-1a isoform is enzymatically active and produced sufficient S1P to induce S1P receptor internalization. Wild-type mouse plasma contains significant Sphk activity (179 pmol x min(-1) x g(-1)). In contrast, Sphk1-/- mouse plasma has undetectable Sphk activity and approx. 65% reduction in S1P levels. Moreover, human plasma contains enzymatically active Sphk1 (46 pmol x min(-1) x g(-1)). These results suggest that export of Sphk-1a occurs under physiological conditions and may contribute to the establishment of the vascular S1P gradient.

Topics: Animals; Cells, Cultured; Culture Media, Conditioned; Endothelium, Vascular; Extracellular Space; Humans; Intracellular Space; Isoenzymes; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Protein Transport; Receptors, Lysosphingolipid; Sphingosine

Sphingosine kinase (SK) is an oncogenic sphingolipid-metabolizing enzyme that catalyzes the formation of the mitogenic second messenger sphingosine-1-phosphate (S1P) at the expense of proapoptotic ceramide. Thus, SK is an attractive target for cancer therapy because blockage of S1P formation leads to inhibition of proliferation, as well as the induction of apoptosis in cancer cells. We have recently identified novel SK inhibitors with nanomolar to low micromolar potencies toward recombinant human SK. This study describes the continuing analysis of these inhibitors through in vitro and in vivo experiments. All three structurally diverse SK inhibitors tested showed antitumor activity in mice without exhibiting toxicity. Blood and tumor inhibitor concentrations exceeded in vitro potency levels. Cell signaling analyses in vitro revealed mixed inhibition of mitogen-activated protein kinase kinase and Akt phosphorylation by the SK inhibitors. Importantly, 4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol (SKI-II) is orally bioavailable, detected in the blood for at least 8 h, and showed a significant inhibition of tumor growth in mice. These compounds are the first examples of nonlipid selective inhibitors of SK with in vivo antitumor activity and provide leads for further development of inhibitors of this important molecular target.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Availability; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Female; Half-Life; Lysophospholipids; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Multidrug Resistance-Associated Proteins; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Sphingosine; Thiazoles

Both acute and chronic hypoxia had no effect on S1P(1), S1P(3) or LPA(1) receptor transcript expression in human pulmonary smooth muscle cells. However, acute hypoxia increased sphingosine kinase SK1/2 and LPP1 mRNA transcript levels, while chronic hypoxia increased SK1 mRNA transcript alone. Acute hypoxia had no effect on S1P-, PDGF- or phorbol ester (PMA)-stimulated activation of ERK-1/2, but increased the ability of S1P to activate p38 MAPK. Chronic hypoxia increased the ability of S1P to stimulate the phosphorylation of ERK-1/2. Therefore, we have demonstrated for the first time that hypoxia can lead to marked changes in the expression of genes involved in S1P production and may modify post S1P receptor signal transduction pathways.

Topics: Cell Hypoxia; Cells, Cultured; Gene Expression Regulation; Humans; Lung; Lysophospholipids; MAP Kinase Signaling System; Myocytes, Smooth Muscle; Phosphatidate Phosphatase; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysophosphatidic Acid; Receptors, Lysosphingolipid; Sphingosine

Dendritic cells (DC) are inducers of primary immune responses and represent an attractive vector for cancer immunotherapy. Sphingosine kinase (SphK) and its product sphingosine-1-phosphate (S1P) play an important role in the regulation of immune cells and cancer, affecting processes such as differentiation, growth or migration. We studied the role of SphK and S1P on migration of DC. RT-PCR showed mRNA expression of SphK in DC, declining from immature (iDC) to mature DC (mDC) to antigen-loaded mDC. Expression of S1P receptors was S1P(1) > S1P(2) = S1P(3), unrelated to maturation or antigen uptake. In transwell assays, iDC migrated towards SDF-1, MIP-1alpha, MCP and S1P, whereby S1P combined with a chemokine had a synergistic effect. mDC migrated towards 6Ckine and MIP-3beta, but not towards S1P. The SphK-inhibitor dihydro-sphingosine (DHS) reduced migration of iDC but not of mDC. In addition S1P(3)-inhibitor suramin inhibited DC migration in response to S1P. DHS had a reverse effect on endocytosis, enhancing the uptake of FITC dextran. We also observed an anti-apoptotic effect of S1P on mDC for the first time. This indicates that SphK/S1P may play a role in accumulation of peripheral iDC at the location of antigen and subsequent antigen-uptake. These findings may help to optimise DC-based cancer immunotherapy by modulation of SphK/S1P.

Topics: Apoptosis; Cell Movement; Cell Survival; Chemokines; Dendritic Cells; Endocytosis; Humans; Immunotherapy, Adoptive; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Messenger; Sphingosine; Statistics, Nonparametric

Our previous studies found that nerve growth factor (NGF), via ceramide, enhanced the number of action potentials (APs) evoked by a ramp of depolarizing current in capsaicin-sensitive sensory neurons. Ceramide can be metabolized by ceramidase to sphingosine (Sph), and Sph to sphingosine 1-phosphate (S1P) by sphingosine kinase. It is well established that each of these products of sphingomyelin metabolism can act as intracellular signalling molecules. This raises the question as to whether the enhanced excitability produced by NGF was mediated directly by ceramide or required additional metabolism to Sph and/or S1P. Sph applied externally did not affect the neuronal excitability, whereas internally perfused Sph augmented the number of APs evoked by the depolarizing ramp. Furthermore, internally perfused S1P enhanced the number of evoked APs. This sensitizing action of NGF, ceramide and internally perfused Sph was abolished by dimethylsphingosine (DMS), an inhibitor of sphingosine kinase. In contrast, internally perfused S1P enhanced the number of evoked APs in the presence of DMS. These observations support the idea that the metabolism of ceramide/Sph to S1P is critical for the sphingolipid-induced modulation of excitability. Both internally perfused Sph and S1P inhibited the outward K+ current by 25-35% for the step to +60 mV. The Sph- and S1P-sensitive currents had very similar current-voltage relations, suggesting that they were likely to be the same. In addition, the Sph-induced suppression of the K+ current was blocked by pretreatment with DMS. These findings demonstrate that intracellular S1P derived from ceramide acts as an internal second messenger to regulate membrane excitability; however, the effector system whereby S1P modulates excitability remains undetermined.

Topics: Action Potentials; Animals; Cells, Cultured; Enzyme Inhibitors; Ganglia, Spinal; Lysophospholipids; Male; Nerve Growth Factor; Neurons, Afferent; Patch-Clamp Techniques; Perfusion; Phosphotransferases (Alcohol Group Acceptor); Potassium Channels; Rats; Rats, Sprague-Dawley; Signal Transduction; Sphingosine; Time Factors

Although several studies have shown that a subset of insulin-like growth factor (IGF) signals require the activation of heterotrimeric G proteins, the molecular mechanisms underlying IGF-stimulated G protein signaling remain poorly understood. Here, we have studied the mechanism by which endogenous IGF receptors activate the ERK1/2 mitogen-activated protein kinase cascade in HEK293 cells. In these cells, treatment with pertussis toxin and expression of a Galpha(q/11)-(305-359) peptide that inhibits G(q/11) signaling additively inhibited IGF-stimulated ERK1/2 activation, indicating that the signal was almost completely G protein-dependent. Treatment with IGF-1 or IGF-2 promoted translocation of green fluorescent protein (GFP)-tagged sphingosine kinase (SK) 1 from the cytosol to the plasma membrane, increased endogenous SK activity within 30 s of stimulation, and caused a statistically significant increase in intracellular and extracellular sphingosine 1-phosphate (S1P) concentration. Using a GFP-tagged S1P1 receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that IGF-1 and IGF-2 induced GFP-S1P receptor internalization and that the effect was blocked by pretreatment with the SK inhibitor, dimethylsphingosine. Treating cells with dimethylsphingosine, silencing SK1 expression by RNA interference, and blocking endogenous S1P receptors with the competitive antagonist VPC23019 all significantly inhibited IGF-stimulated ERK1/2 activation, suggesting that IGFs elicit G protein-dependent ERK1/2 activation by stimulating SK1-dependent transactivation of S1P receptors. Given the ubiquity of SK and S1P receptor expression, S1P receptor transactivation may represent a general mechanism for G protein-dependent signaling by non-G protein-coupled receptors.

Topics: Animals; Enzyme Activation; Green Fluorescent Proteins; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphotransferases (Alcohol Group Acceptor); Protein Structure, Tertiary; Signal Transduction; Somatomedins; Sphingosine; Transcriptional Activation

Sphingosine kinase (Sphk) enzymes are important in intracellular sphingolipid metabolism as well as in the biosynthesis of sphingosine 1-phosphate (S1P), an extracellular lipid mediator. Here, we show that Sphk1 is expressed and is required for small intestinal tumor cell proliferation in Apc Min/+ mice. Adenoma size but not incidence was dramatically reduced in Apc Min/+ Sphk(-/-) mice. Concomitantly, epithelial cell proliferation in the polyps was significantly attenuated, suggesting that Sphk1 regulates adenoma progression. Although the S1P receptors (S1P1R, S1P2R, and S1P3R) are expressed, polyp incidence or size was unaltered in Apc Min/+ S1p2r(-/-), Apc Min/+ S1p3r(-/-), and Apc Min/+ S1p1r(+/-) bigenic mice. These data suggest that extracellular S1P signaling via its receptors is not involved in adenoma cell proliferation. Interestingly, tissue sphingosine content was elevated in the adenomas of Apc Min/+ Sphk1(-/-) mice, whereas S1P levels were not significantly altered. Concomitantly, epithelial cell proliferation and the expression of the G1/S cell cycle regulator CDK4 and c-myc were diminished in the polyps of Apc Min/+ Sphk1(-/-) mice. In rat intestinal epithelial (RIE) cells in vitro, Sphk1 overexpression enhanced cell cycle traverse at the G1/S boundary. In addition, RIE cells treated with sphingosine but not C6-ceramide exhibited reduced cell proliferation, reduced retinoblastoma protein phosphorylation, and cyclin-dependent kinase 4 (Cdk4) expression. Our findings suggest that Sphk1 plays a critical role in intestinal tumor cell proliferation and that inhibitors of Sphk1 may be useful in the control of intestinal cancer.

Topics: Adenomatous Polyps; Animals; Apoptosis; Cell Cycle Proteins; Cell Differentiation; Cell Proliferation; Down-Regulation; G1 Phase; Gastrointestinal Neoplasms; Gene Deletion; Gene Expression Regulation, Neoplastic; Humans; Intestinal Mucosa; Lysophospholipids; Mice; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptors, Lysosphingolipid; RNA, Messenger; S Phase; Sphingosine

Sphingosine 1-phosphate (S1P), produced by sphingosine kinase (SPHK), acts both by intracellular and extracellular modes. We evaluated the role of SPHK1 and S1P in osteoclastogenesis using bone marrow-derived macrophage (BMM) single and BMM/osteoblast coculture systems. In BMM single cultures, the osteoclastogenic factor receptor activator of NF-kappaB ligand (RANKL) upregulated SPHK1 and increased S1P production and secretion. SPHK1 siRNA enhanced and SPHK1 overexpression attenuated osteoclastogenesis via modulation of p38 and ERK activities, and NFATc1 and c-Fos levels. Extracellular S1P had no effect in these cultures. These data suggest that intracellular S1P produced in response to RANKL forms a negative feedback loop in BMM single cultures. In contrast, S1P addition to BMM/osteoblast cocultures greatly increased osteoclastogenesis by increasing RANKL in osteoblasts via cyclooxygenase-2 and PGE(2) regulation. S1P also stimulated osteoblast migration and survival. The RANKL elevation and chemotactic effects were also observed with T cells. These results indicate that secreted S1P attracts and acts on osteoblasts and T cells to augment osteoclastogenesis. Taken together, S1P plays an important role in osteoclastogenesis regulation and in communication between osteoclasts and osteoblasts or T cells.

Topics: Animals; Cell Differentiation; Cell Survival; Chemotaxis; Coculture Techniques; Cyclooxygenase 2; Dinoprostone; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Enzymologic; HeLa Cells; Humans; Lymphocyte Activation; Lysophospholipids; Mice; NFATC Transcription Factors; Osteoblasts; Osteoclasts; Osteogenesis; p38 Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-fos; RANK Ligand; RNA, Messenger; Sphingosine; T-Lymphocytes

Activation of Sphingosine kinase (Sphk) increases a bioactive lipid, sphingosine 1-phosphate (S1P) and has been observed in a variety of cancer cells. Therefore, inhibition of Sphk activity was an important target for the development of anticancer drugs. As a searching tool for Sphk inhibitor, we developed fluorescent Sphk activity assay combined with high performance liquid chromatography (HPLC). Previously we established murine teraticarcinoma mutant F9-12 cells which lack S1P lyase and stably express Sphk1. By using F9-12 cells, optimal assay conditions were established as follows; 100 microM of C17-Sph and 30 microg protein of F9-12 cells lysate in 20 min. Sphingosine analog C17-Sph was efficiently phosphorylated by Sphk activity (Km:67.08 microM, Vmax :1507.5 pmol/min/mg). New product C17,S1P was separated from S1P in reversed-phase HPLC. In optimized conditions, 300 nM of phorbol 12-myristate 13-acetate (PMA) increased Sphk activity approximately twice while 20 microM of N,N-dimethylsphingosine (DMS) reduced 70% of Sphk activity in F9-12 cells lysate. In conclusion, we established non-radioactive but convenient Sphk assay system by using HPLC and F9-12 cells.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Cell Line, Tumor; Chromatography, High Pressure Liquid; Indicators and Reagents; Lysophospholipids; Mice; o-Phthalaldehyde; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Spectrometry, Fluorescence; Sphingosine; Tetradecanoylphorbol Acetate

In recent years sphingolipids have emerged as important signaling molecules regulating fundamental cell responses such as cell death and differentiation, proliferation and aspects of inflammation. Especially ceramide has been a main focus of research since it possesses pro-apoptotic capacity in many cell types. A counterplayer of ceramide was found in sphingosine-1-phosphate (S1P), which is generated from ceramide by the consecutive actions of ceramidase and sphingosine kinase. S1P can potently induce cell proliferation via binding to and activation of the Edg family of receptors which have now been renamed as S1P receptors. Obviously, a delicate balance between ceramide and sphingosine-1-phosphate determines whether cells undergo apoptosis or proliferate, two cell responses that are critically involved in tumor development. Directing the balance in favor of ceramide, i.e. by inhibiting ceramidase or sphingosine kinase activities may support the pro-apoptotic action of ceramide and thus may have beneficial effects in cancer therapy. This review will summarize novel insights into the regulation of sphingolipid formation and their potential involvement in tumor development. Finally, we will pinpoint potential new targets for tumor therapy.

Topics: Amidohydrolases; Animals; Antineoplastic Agents; Apoptosis; Cell Movement; Cell Proliferation; Ceramidases; Ceramides; Enzyme Inhibitors; Fingolimod Hydrochloride; Humans; Lipid Metabolism; Lysophospholipids; Neoplasms; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

To establish the methods for determining the activity of sphingosine kinase(SPK) and the content of sphingosine 1-phosphate (S1P) in biological samples.. The ECV304 cells were transfected with pcDNA3 vector encoding Flag-labeled SPK gene. The expression of SPK was measured by Western blot assay and the activity of SPK was determined by enzymatic reaction, isotope incorporation and thin-layer chromatography methods. The S1P in biological samples was extracted, digested by alkaline phosphatase and then catalyzed by SPK. The S1P contents were determined according to the amounts of products.. SPK gene transfection could enhance the expression and activity of SPK in cells markedly, and the cellular S1P was also increased obviously. HGF stimulation could increase the activity of SPK and cellular S1P in ECV304 cells.. Methods for determining the activity of SPK and the content of SPK in biological samples were established.

Topics: Cell Line; Cytophotometry; Humans; Isotope Labeling; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Lysophosphatidic acid (LPA) is a bioactive lipid, which is structurally similar to sphingosine 1-phosphate (S1P) and which can mobilize Ca2+ in multiple cell types. We recently showed that S1P induces Ca2+ entry directly through store-operated Ca2+ entry (SOCE) channels in human polymorphonuclear neutrophils (PMN). We therefore examined the mechanisms by which LPA induces intracellular Ca2+ mobilization in PMN. External application of low micromolar LPA caused dose-dependent Ca2+ influx without releasing Ca2+ stores, whereas G-protein-coupled (GPC) LPA receptors respond to nanomolar LPA. Additive Ca2+ influx by LPA compared with 100 nM ionomycin-induced Ca2+ influx suggests that LPA-induced Ca2+ influx does not pass through SOCE channels. Ca2+ influx was resistant to inhibition of Gi/o by pertussis toxin, of phospholipase C by U73122, and of G12/13/Rho by Y27632, all demonstrating GPC receptor independence. This Ca2+ influx was inhibited by Gd3+, La3+, Zn2+, or MRS1845 but not by Ni2+ or the sphingosine kinase inhibitor dimethylsphingosine. In addition, we found that LPA has no effect on neutrophil chemotaxis; however, it has stimulatory effects on neutrophil respiratory burst in a dose-response manner. These findings suggest that LPA-induced Ca2+ influx in PMN occurs through a mechanism other than SOCE channels, independent of Ca2+ store-depletion and S1P synthesis, and that the characteristics of LPA-induced Ca2+ influx are similar to those of S1P-induced influx in terms of sensitivity to inorganic inhibitors. Unlike S1P, LPA has stimulatory effects on neutrophil respiratory burst.

Topics: Calcium; Calcium Signaling; Chemotaxis; Estrenes; HL-60 Cells; Humans; Ionomycin; Lysophospholipids; Models, Biological; Neutrophils; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidinones; Receptors, G-Protein-Coupled; Respiratory Burst; Signal Transduction; Sphingosine

A solvent-extraction-based radioassay for measuring sphingosine kinase (SKase) activity has been developed. The assay utilizes [3H]sphingosine substrate and differentially extracts the [3H]sphingosine-1-phosphate product. The extracted radioactivity is demonstrated to be primarily [3H]sphingosine-1-phosphate with less than 1% contamination by [3H]sphingosine. When assaying SKase activity in the soluble cell fraction, the extraction efficiency of the labeled sphingosine-1-phosphate product is a reproducible 78%, which allows for a simple back calculation to correct for the 22% extraction loss. With minor modification, the assay is also a reproducible procedure for determining SKase activity in subcellular membrane fractions. The assay is far more rapid than thin-layer chromatography and high-performance liquid chromatography methods, which makes it possible to do a large number of assays in a short period of time. The utility of the assay is demonstrated by using it to conduct a complete bisubstrate kinetic analysis of rat heart SKase.

Topics: Animals; Kinetics; Lysophospholipids; Myocardium; Phosphotransferases (Alcohol Group Acceptor); Radioligand Assay; Rats; Scintillation Counting; Sphingosine; Tritium

Sphingomyelin derivatives exert various second messenger actions in numerous tissues. Sphingosine (SPH) and sphingosine 1-phosphate (S1P) are two major sphingomyelin derivatives present at high levels in blood. The aim of the present work was to investigate whether S1P and SPH exert relevant actions in mouse skeletal muscle contractility and fatigue. Exogenous S1P and SPH administration caused a significant reduction of tension decline during fatigue of extensor digitorum longus muscle. Final tension after the fatiguing protocol was 40% higher than in untreated muscle. Interestingly, N,N-dimethylsphingosine, an inhibitor of SPH kinase (SK), abolished the effect of supplemented SPH but not that of S1P, suggesting that SPH acts through its conversion to S1P. Moreover, SPH was not effective in Ca(2+)-free solutions, in agreement with the hypothesis that SPH action is dependent on its conversion to S1P by the Ca(2+)-requiring enzyme SK. In contrast to SPH, S1P produced its positive effects on fatigue in Ca(2+)-free conditions, indicating that S1P action does not require Ca(2+) entry and most likely is receptor mediated. The effects of S1P could be ascribed in part to its ability to prevent the reduction (-20 mV) of action potential amplitude caused by fatigue. In conclusion, these results indicate that extracellular S1P has protective effects during the development of muscle fatigue and that the extracellular conversion of SPH to S1P may represent a rheostat mechanism to protect skeletal muscle from possible cytotoxic actions of SPH.

Topics: Animals; Calcium; Dose-Response Relationship, Drug; Indoles; Lysophospholipids; Maleimides; Mice; Mice, Inbred Strains; Muscle Contraction; Muscle Fatigue; Muscle, Skeletal; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

In this study, the effects of short-term diabetes (4 days) on rat renal glomerular cells proliferation and the potential involvement of sphingolipids in this process were investigated. Immunohistochemical analysis showed that streptozotocin (STZ)-induced diabetes promoted increased intra-glomerular hyperplasia, particularly marked for mesangial cells. This was associated with a concomitant increase in neutral ceramidase and sphingosine-kinase activities and the accumulation of the pro-proliferative sphingolipid sphingosine-1-phosphate, in glomeruli isolated from kidney cortex of STZ-treated rats. These results suggest a possible involvement of sphingolipid metabolites in the glomerular proliferative response during the early stages of diabetic nephropathy.

Topics: Amidohydrolases; Animals; Cell Proliferation; Ceramidases; Diabetic Nephropathies; Kidney Glomerulus; Lysophospholipids; Male; Neutral Ceramidase; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Sphingosine; Streptozocin; Time Factors

Sphingosine-1-phosphate (S1P) is a potent biomediator that can act as either an intracellular or an intercellular messenger. In the nervous system it exerts a wide range of actions, and specific membrane receptors for it have been identified in various regions. However, the physiological origin of extracellular S1P in the nervous system is largely unknown. We investigated cerebellar granule cells at different stages of differentiation and astrocytes in primary cultures as possible origins of extracellular S1P. Although these cells show marked differences in S1P metabolism, we found that they can all release S1P and express mRNAs for S1P specific receptors. Extracellular S1P derives from the export of newly synthesized intracellular S1P, and not from the action of a released sphingosine kinase. S1P release is rapid, efficient, and can be regulated by exogenous stimuli. Phorbol ester treatment resulted in an increase in sphingosine kinase 1 activity in the membranes, accompanied by a significant increase in extracellular S1P. S1P release in cells from the cerebellum emerges as a regulated mechanism, possibly related to a specific pool of newly synthesized S1P. To our knowledge, this is the first evidence of the extracellular release of S1P by primary cells from the CNS, which supports a role of S1P as autocrine/paracrine physiological messenger in the cerebellum.

Topics: Animals; Animals, Newborn; Astrocytes; Autoradiography; Cell Differentiation; Cell Membrane; Cells, Cultured; Cerebellum; Chromatography, Thin Layer; Culture Media, Conditioned; Dose-Response Relationship, Drug; Electrophoresis, Gel, Pulsed-Field; Extracellular Space; Gene Expression Regulation; Lysophospholipids; Neurons; Phosphotransferases (Alcohol Group Acceptor); Potassium Chloride; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine; Time Factors; Tritium

We analyzed the intracellular action of sphingosine 1-phosphate (Sph-1-P), formed from sphingosine (Sph) by sphingosine kinase (SPHK), in platelets. When sphingosine kinase activity was inhibited by N,N-dimethylsphingosine (DMS), Ca2+ mobilization induced by convulxin, an agonist of the collagen receptor glycoprotein VI (GPVI), was moderately but specifically abolished; that induced via G protein-coupled receptors was not affected. Under the same conditions, however, tyrosine phosphorylation of Syk and phospholipase Cgamma2, which is essential for the GPVI-mediated signaling, was not inhibited. Sphingosine kinase activity of the platelet membrane fraction increased specifically upon stimulation with convulxin or collagen. Our results suggest that intracellular sphingosine 1-phosphate is related to Ca2+ mobilization in GPVI-mediated signaling pathways.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Blood Platelets; Calcium; Calcium Signaling; Collagen; Crotalid Venoms; Humans; Lectins, C-Type; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Platelet Membrane Glycoproteins; Receptors, Thrombin; Sphingosine; Tyrosine

Sphingosine 1-phosphate (S1P), a product of sphingosine kinases (SphK), mediates diverse biological processes such as cell differentiation, proliferation, motility, and apoptosis. In an effort to search and identify specific inhibitors of human SphK, the inhibitory effects of synthetic sphingoid analogs on kinase activity were examined. Among the analogs tested, we found two, SG12 and SG14, that have specific inhibitory effects on hSphK2. N,N-Dimethylsphingosine (DMS), a well-known SphK inhibitor, displayed inhibitory effects for both SphK1 and SphK2, as well as protein kinase C. In contrast, SG12 and SG14 exhibited selective inhibitory effects on hSphK2. Furthermore, SG14 did not affect PKC. In isolated platelets, SG14 blocked the conversion of sphingosine into sphingosine 1-phosphate significantly. This is the first report on the identification of a hSphK2-specific inhibitor, which may provide a useful tool for studying the biological functions of hSphK2.

Topics: Animals; Blood Platelets; Cell Survival; CHO Cells; Cricetinae; Enzyme Inhibitors; Evaluation Studies as Topic; Humans; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Sphingosine; Transfection

Sphingosine-1 phosphate (S1P) is a bioactive sphingolipid with the potential to mobilize Ca2+, to inhibit apoptosis, and to promote mitogenesis. Sphingosine kinase (SPHK) and S1P were characterized in INS-1 insulinoma cells and isolated rat islets of Langerhans. SPHK activity increased in INS-1 cell homogenates treated with interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha), and responses were additive. IL-1beta or TNF-alpha increased islet SPHK activity within 15 min to 1 h; activity remained elevated after 8 h. SPHK2 was the predominant active isoform in INS-1 cells; little or no SPHK1 activity was detected. Cytokines increased endogenous S1P biosynthesis in 32P(i)-prelabeled INS-1 cells, and cycloheximide inhibited the response after 8 h, suggesting that protein synthesis mediated the response. There was no [32P]S1P release from cells. Compared with basal values, IL-1beta and TNF-alpha induced increases in SPHK1a mRNA levels relative to 18S ribosomal RNA in INS-1 cells within 1 h; relative SPHK2 mRNA levels were unchanged after cytokine treatment. IL-1beta, but not TNF-alpha, induced relative SPHK1a mRNA expression levels within 1 h in islets, whereas SPHK2 mRNA levels were unchanged. Thus, IL-1beta and TNF-alpha induced an early and sustained increase in SPHK activity in INS-1 cells and isolated islets, suggesting that S1P plays a role in the pathological response of pancreatic beta-cells to cytokines.

Topics: Animals; Base Sequence; Cell Line; Cell Line, Tumor; Cytokines; DNA Primers; Interleukin-1; Islets of Langerhans; Kinetics; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Rats; RNA, Messenger; Sphingosine; Substrate Specificity; Tumor Necrosis Factor-alpha

Protein kinase A (PKA) has long been recognized as playing a major role in many regulatory processes in cells through its activation by the ubiquitous second messenger cAMP. We show here a novel mode of activation of PKA type II that is independent of cAMP and is, instead, dependent on sphingosine. PKA type II is specifically activated by sphingosine and its analog, dimethylsphingosine, but not by sphingosine-1-phosphate or other lipids. Like cAMP, sphingosine activates PKA holoenzyme but not the catalytic subunit alone, suggesting that the activation is mediated by the regulatory subunits. However, sphingosine-activated PKA, but not cAMP-activated PKA, is inhibited by phosphatidylserine, suggesting a distinct mechanism of activation. Furthermore, unlike cAMP, sphingosine does not induce the dissociation of PKA holoenzyme into catalytic and regulatory subunits. Modulation of sphingosine levels in vivo results in alteration in basal membrane-associated PKA activity consistent with a direct effect of membrane sphingosine on PKA type II. Importantly, sphingosine-dependent but not cAMP-dependent activation of PKA specifically phosphorylates Ser58 of the multifunctional adapter protein 14-3-3zeta, promoting the conversion of dimeric 14-3-3 to a monomeric state, thus potentially modulating several biological functions. These results define a new mode of PKA activation that is sphingosine-dependent and mechanistically different from the classical cAMP-dependent activation of PKA. Furthermore, they suggest that stimuli that induce sphingosine accumulation and modulate phospholipid content at the cell membrane have the potential to activate PKA, thereby inducing the phosphorylation of distinct substrates and biological activities.

Topics: 14-3-3 Proteins; Animals; Catalytic Domain; Cattle; Cell Membrane; Chromatography, Ion Exchange; COS Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Dimerization; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Ethanolamines; Fibroblasts; Gene Expression Regulation, Enzymologic; Immunoblotting; Kinetics; Lipid Metabolism; Lysophospholipids; Mice; Mice, Inbred BALB C; Myocardium; NIH 3T3 Cells; Phosphatidylserines; Phospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Structure, Tertiary; Serine; Signal Transduction; Sphingosine; Substrate Specificity

In peripheral airways, acetylcholine induces contraction via activation of muscarinic M2-and M3-receptor subtypes (M2R and M3R). Cholinergic hypersensitivity is associated with chronic obstructive pulmonary disease and asthma, and therefore the identification of muscarinic signaling pathways are of great therapeutic interest. A pathway that has been shown to be activated via MR and to increase [Ca2+]i includes the activation of sphingosine kinases (SPHK) and the generation of the bioactive sphingolipid sphingosine 1-phosphate (S1P). Whether the SPHK/S1P signaling pathway is integrated in the muscarinic control of peripheral airways is not known.. To address this issue, we studied precision cut lung slices derived from FVB and M2R-KO and M3R-KO mice.. In peripheral airways of FVB, wild-type, and MR-deficient mice, SPHK1 was mainly localized to smooth muscle. Muscarine induced a constriction in all investigated mouse strains which was reduced by inhibition of SPHK using D, L-threo-dihydrosphingosine (DHS) and N, N-dimethyl-sphingosine (DMS) but not by N-acetylsphingosine (N-AcS), a structurally related agent that does not affect SPHK function. The initial phase of constriction was nearly absent in peripheral airways of M3R-KO mice when SPHK was inhibited by DHS and DMS but was unaffected in M2R-KO mice. Quantitative RT-PCR revealed that the disruption of the M2R and M3R genes had no significant effect on the expression levels of the SPHK1-isoform in peripheral airways.. These results demonstrate that the SPHK/S1P signaling pathway contributes to cholinergic constriction of murine peripheral airways. In addition, our data strongly suggest that SPHK is activated via the M2R. Given the important role of muscarinic mechanisms in pulmonary disease, these findings should be of considerable therapeutic relevance.

Topics: Animals; Cells, Cultured; Gene Expression Regulation, Enzymologic; Lung; Lysophospholipids; Mice; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Receptor, Muscarinic M2; Receptor, Muscarinic M3; Signal Transduction; Sphingosine; Tissue Distribution

Immunotherapeutic drugs that mimic sphingosine 1-phosphate (S1P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-1,3-propanediol (FTY720), stimulates S1P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated sphingosine kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S1P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional sphingosine kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.

Topics: Animals; Fingolimod Hydrochloride; Flow Cytometry; Homozygote; Immunosuppressive Agents; Lymphocytes; Lymphopenia; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prodrugs; Propylene Glycols; Reverse Transcriptase Polymerase Chain Reaction; Sphingosine

Neutral ceramidase is a type II integral membrane protein, which is occasionally secreted into the extracellular milieu after the processing of its N-terminal anchor. We found that when overexpressed in CHOP cells, neutral ceramidase hydrolyzed cell surface ceramide, which increased in amount after the treatment of cells with bacterial sphingomyelinase, leading to an increase in the cellular level of sphingosine and sphingosine 1-phosphate. On the other hand, knockdown of the endogenous enzyme by siRNA decreased the cellular level of both sphingolipid metabolites. The treatment of cells with bovine serum albumin significantly reduced the cellular level of sphingosine, but not sphingosine 1-phosphate, generated by overexpression of the enzyme. The cellular level of sphingosine 1-phosphate increased with overexpression of the cytosolic sphingosine kinase. These results suggest that sphingosine 1-phosphate is mainly produced inside of the cell after the incorporation of sphingosine generated on the plasma membranes. The enzyme also seems to participate in the hydrolysis of serum-derived ceramide in the vascular system. Significant amounts of sphingosine as well as sphingosine 1-phosphate were generated in the cell-free conditioned medium of ceramidase transfectants, compared with mock transfectants. No increase in these metabolites was observed if serum or bacterial sphingomyelinase was omitted from the conditioned medium, suggesting that the major source of ceramide is the serum-derived sphingomyelin. A sphingosine 1-phosphate receptor, S1P(1), was internalized much faster by the treatment of S1P(1)-overexpressing cells with conditioned medium of ceramidase transfectants than that of mock transfectants. Collectively, these results clearly indicate that the enzyme is involved in the metabolism of ceramide at the plasma membrane and in the extracellular milieu, which could regulate sphingosine 1-phosphate-mediated signaling through the generation of sphingosine.

Topics: Amidohydrolases; Animals; Blotting, Western; Cattle; Cell Membrane; Cells, Cultured; Ceramidases; Ceramides; Chromatography, High Pressure Liquid; Culture Media, Conditioned; Extracellular Matrix; Immunoenzyme Techniques; Lysophospholipids; Microscopy, Fluorescence; Phosphotransferases (Alcohol Group Acceptor); Plasmids; Receptors, Lysosphingolipid; RNA, Small Interfering; Serum Albumin, Bovine; Signal Transduction; Sphingomyelin Phosphodiesterase; Sphingomyelins; Sphingosine; Transfection

Sphingosine 1-phosphate is a bioactive sphingolipid that regulates cell growth and suppresses programmed cell death. The biosynthesis of sphingosine 1-phosphate is catalyzed by sphingosine kinase (SK) but the mechanism by which the subcellular localization and activity of SK is regulated in response to various stimuli is not fully understood. To elucidate the origin and structural determinant of the specific subcellular localization of SK, we performed biophysical and cell studies of human SK1 (hSK1) and selected mutants. In vitro measurements showed that hSK1 selectively bound phosphatidylserine over other anionic phospholipids and strongly preferred the plasma membrane-mimicking membrane to other cellular membrane mimetics. Mutational analysis indicates that conserved Thr54 and Asn89 in the putative membrane-binding surface are essential for lipid selectivity and membrane targeting both in vitro and in the cell. Also, phosphorylation of Ser225 enhances the membrane affinity and plasma membrane selectivity of hSK1, presumably by modulating the interaction of Thr54 and Asn89 with the membrane. Collectively, these studies suggest that the specific plasma membrane localization and activation of SK1 is mediated largely by specific lipid-protein interactions.

Topics: Amino Acid Sequence; Asparagine; Cell Line; Cell Membrane; DNA Mutational Analysis; Green Fluorescent Proteins; Humans; Kinetics; Lipids; Lysophospholipids; Mass Spectrometry; Microscopy, Confocal; Microscopy, Fluorescence; Models, Biological; Molecular Sequence Data; Mutation; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pressure; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Serine; Sphingosine; Surface Plasmon Resonance; Threonine; Time Factors; Transfection

We had found previously that neurotrophin-3 (NT-3) is a potent stimulator of cAMP-response element binding protein (CREB) phosphorylation in cultured oligodendrocyte progenitors. Here, we show that CREB phosphorylation in these cells is also highly stimulated by sphingosine-1-phosphate (S1P), a sphingolipid metabolite that is known to be a potent mediator of numerous biological processes. Moreover, CREB phosphorylation in response to NT-3 involves sphingosine kinase 1 (SphK1), the enzyme that synthesizes S1P. Immunocytochemistry and confocal microscopy indicated that NT-3 induces translocation of SphK1 from the cytoplasm to the plasma membrane of oligodendrocytes, a process accompanied by increased SphK1 activity in the membrane fraction where its substrate sphingosine resides. To examine the involvement of SphK1 in NT-3 function, SphK1 expression was down-regulated by treatment with SphK1 sequence-specific small interfering RNA. Remarkably, the capacity of NT-3 to protect oligodendrocyte progenitors from apoptotic cell death induced by growth factor deprivation was abolished by down-regulating the expression of SphK1, as assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Altogether, these results suggest that SphK1 plays a crucial role in the stimulation of oligodendrocyte progenitor survival by NT-3, and demonstrate a functional link between NT-3 and S1P signaling, adding to the complexity of mechanisms that modulate neurotrophin function and oligodendrocyte development.

Topics: Animals; Animals, Newborn; Blotting, Western; Cell Membrane; Cells, Cultured; Cyclic AMP Response Element-Binding Protein; Drug Interactions; Gene Expression Regulation; In Situ Nick-End Labeling; Lysophospholipids; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Models, Biological; Neurotrophin 3; Oligodendroglia; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Sphingosine; Stem Cells; Thiazoles; Thiazolidinediones; Thiones; Time Factors

Sphingosine-1-phosphate (S1P), an important sphingolipid metabolite, regulates diverse cellular processes, including cell survival, growth, and differentiation. Here we show that S1P signaling is critical for neural and vascular development. Sphingosine kinase-null mice exhibited a deficiency of S1P which severely disturbed neurogenesis, including neural tube closure, and angiogenesis and caused embryonic lethality. A dramatic increase in apoptosis and a decrease in mitosis were seen in the developing nervous system. S1P(1) receptor-null mice also showed severe defects in neurogenesis, indicating that the mechanism by which S1P promotes neurogenesis is, in part, signaling from the S1P(1) receptor. Thus, S1P joins a growing list of signaling molecules, such as vascular endothelial growth factor, which regulate the functionally intertwined pathways of angiogenesis and neurogenesis. Our findings also suggest that exploitation of this potent neuronal survival pathway could lead to the development of novel therapeutic approaches for neurological diseases.

Topics: Animals; Apoptosis; Blood Vessels; Lysophospholipids; Mice; Mice, Knockout; Mitosis; Neovascularization, Physiologic; Nervous System; Neural Tube Defects; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Systemic chemotherapy was considered of modest efficacy in prostate cancer until the recent introduction of taxanes. We took advantage of the known differential effect of camptothecin and docetaxel on human PC-3 and LNCaP prostate cancer cells to determine their effect on sphingosine kinase-1 (SphK1) activity and subsequent ceramide/sphingosine 1-phosphate (S1P) balance in relation with cell survival. In vitro, docetaxel and camptothecin induced strong inhibition of SphK1 and elevation of the ceramide/S1P ratio only in cell lines sensitive to these drugs. SphK1 overexpression in both cell lines impaired the efficacy of chemotherapy by decreasing the ceramide/S1P ratio. Alternatively, silencing SphK1 by RNA interference or pharmacologic inhibition induced apoptosis coupled with ceramide elevation and loss of S1P. The differential effect of both chemotherapeutics was confirmed in an orthotopic PC-3/green fluorescent protein model established in nude mice. Docetaxel induced a stronger SphK1 inhibition and ceramide/S1P ratio elevation than camptothecin. This was accompanied by a smaller tumor volume and the reduced occurrence and number of metastases. SphK1-overexpressing PC-3 cells implanted in animals developed remarkably larger tumors and resistance to docetaxel treatment. These results provide the first in vivo demonstration of SphK1 as a sensor of chemotherapy.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Camptothecin; Ceramides; Disease Models, Animal; Docetaxel; Flow Cytometry; Green Fluorescent Proteins; Humans; Lysophospholipids; Male; Mice; Mice, Nude; Microscopy, Fluorescence; Neoplasm Recurrence, Local; Neoplasms, Hormone-Dependent; Phosphotransferases (Alcohol Group Acceptor); Prostatic Neoplasms; RNA Interference; Sphingosine; Taxoids; Tumor Cells, Cultured

To elucidate possible biochemical links between growth arrest from antiproliferative chemotherapeutic agents and apoptosis, our work has focused on agents (EGCg, capsaicin, cis platinum, adriamycin, anti-tumor sulfonylureas, phenoxodiol) that target tNOX. tNOX is a cancer-specific cell surface NADH oxidase (ECTO-NOX protein), that functions in cancer cells as the terminal oxidase for plasma membrane electron transport. When tNOX is active, coenzyme Q(10) (ubiquinone) of the plasma membrane is oxidized and NADH is oxidized at the cytosolic surface of the plasma membrane. However, when tNOX is inhibited and plasma membrane electron transport is diminished, both reduced coenzyme Q(10) (ubiquinol) and NADH would be expected to accumulate. To relate inhibition of plasma membrane redox to increased ceramide levels and arrest of cell proliferation in G(1) and apoptosis, we show that neutral sphingomyelinase, a major contributor to plasma membrane ceramide, is inhibited by reduced glutathione and ubiquinone. Ubiquinol is without effect or stimulates. In contrast, sphingosine kinase, which generates anti-apoptotic sphingosine-1-phosphate, is stimulated by ubiquinone but inhibited by ubiquinol and NADH. Thus, the quinone and pyridine nucleotide products of plasma membrane redox, ubiquinone and ubiquinol, as well as NAD(+) and NADH, may directly modulate in a reciprocal manner two key plasma membrane enzymes, sphingomyelinase and sphingosine kinase, potentially leading to G(1) arrest (increase in ceramide) and apoptosis (loss of sphingosine-1-phosphate). As such, the findings provide potential links between coenzyme Q(10)-mediated plasma membrane electron transport and the anticancer action of several clinically-relevant anticancer agents.

Topics: Alkyl and Aryl Transferases; Apoptosis; Cell Membrane; Ceramides; Electron Transport; G1 Phase; HeLa Cells; Humans; Isoflavones; Lysophospholipids; NAD; NADH, NADPH Oxidoreductases; Phosphotransferases (Alcohol Group Acceptor); Sphingomyelin Phosphodiesterase; Sphingosine; Ubiquinone

Clostridium perfringens alpha-toxin induces hemolysis of rabbit erythrocytes through the activation of glycerophospholipid metabolism. Sheep erythrocytes contain large amounts of sphingomyelin (SM) but not phosphatidylcholine. We investigated the relationship between the toxin-induced hemolysis and SM metabolic system in sheep erythrocytes. Alpha-toxin simultaneously induced hemolysis and a reduction in the levels of SM and formation of ceramide and sphingosine 1-phosphate (S1P). N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells. Furthermore, dl-threo-dihydrosphingosine and B-5354c, isolated from a novel marine bacterium, both sphingosine kinase inhibitors, blocked the toxin-induced hemolysis and production of S1P and caused sphingosine to accumulate. These observations suggest that the toxin-induced activation of the SM metabolic system is closely related to hemolysis. S1P potentiated the toxin-induced hemolysis of saponin-permeabilized erythrocytes but had no effect on that of intact cells. Preincubation of lysated sheep erythrocytes with pertussis toxin blocked the alpha-toxin-induced formation of ceramide from SM. In addition, incubation of C. botulinum C3 exoenzyme-treated lysates of sheep erythrocytes with alpha-toxin caused an accumulation of sphingosine and inhibition of the formation of S1P. These observations suggest that the alpha-toxin-induced hemolysis of sheep erythrocytes is dependent on the activation of the SM metabolic system through GTP-binding proteins, especially the formation of S1P.

Topics: 4-Aminobenzoic Acid; ADP Ribose Transferases; Amidohydrolases; Animals; Bacterial Toxins; Botulinum Toxins; Calcium-Binding Proteins; Ceramidases; Chromatography, Thin Layer; Diglycerides; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Erythrocytes; Ethanolamines; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hemolysis; Inositol 1,4,5-Trisphosphate; Lysophospholipids; Oleic Acids; para-Aminobenzoates; Pertussis Toxin; Phosphatidylcholines; Phosphorylcholine; Phosphotransferases (Alcohol Group Acceptor); Rabbits; Sheep; Sphingomyelins; Sphingosine; Time Factors; Toxins, Biological; Type C Phospholipases

Respiratory syncytial virus (RSV) preferentially infects lung epithelial cells. Infected cells remain viable well into the infection. This prolonged survival results from RSV-induced activation of pro-survival pathways, including Akt and extracellular signal-related kinase (ERK). Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite with demonstrated links to cell survival. It is enzymatically generated by sequential activation of ceramidase (generation of sphingosine) and sphingosine kinase (generation of S1P). In these studies, we found that RSV stimulated neutral ceramidase and sphingosine kinase activities in lung epithelial cells. The combined effect of activation of these two enzymes would decrease proapoptotic ceramide and increase antiapoptotic S1P. S1P activated Akt and ERK within minutes, and inhibition of sphingosine kinase blocked RSV-induced ERK and Akt activation, leading to accelerated cell death after viral infection. RSV infection does eventually kill infected cells but activation of cell survival pathways significantly delays cell death. The studies are the first evidence linking sphingolipid metabolites to cell survival mechanisms in the context of a viral infection.

Topics: Amidohydrolases; Animals; Cell Line; Cell Survival; Ceramidases; Enzyme Activation; Epithelial Cells; Humans; Lysophospholipids; Mitogen-Activated Protein Kinases; Neutral Ceramidase; Phosphotransferases (Alcohol Group Acceptor); Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Respiratory Mucosa; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Signal Transduction; Sphingosine

Sphingosine kinase enzymatic activity is commonly measured using radiolabeled substrates, with thin-layer chromatography and/or solvent extraction needed to detect the reaction product sphingosine-1-phosphate. We developed a fluorescence-based assay, using a sphingosine derivative labeled with a 7-nitrobenz-2-oxa-1,3-diazole moiety (15-NBD-Sph). Separation of substrate (15-NBD-Sph) from product (the corresponding phosphate) is achieved by extraction with chloroform/methanol at pH 8.5. The phosphate derivative is recovered by >98% in the aqueous phase and is directly detected and quantified by its fluorescence. 15-NBD-Sph is readily phosphorylated by human and murine sphingosine kinases 1 and 2. The suitability of the assay for measuring the activity of the kinases, both in the purified state and when contained in lysates of mammalian cells, was demonstrated. The present method is a convenient alternative to the radiometric assays and is particularly suited to the search for inhibitors of sphingosine kinases.

Topics: Animals; Cell Extracts; Cell Line; Enzyme Inhibitors; Fluorescence; Humans; Hydrogen-Ion Concentration; Lysophospholipids; Mice; Molecular Structure; Phosphotransferases (Alcohol Group Acceptor); Spectrometry, Fluorescence; Sphingosine

Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator generated from sphingosine by sphingosine kinase (SPHK). S1P acts both extracellularly and intracellularly as a signaling molecule, although its intracellular targets are still undefined. Intracellular level of S1P is under strict regulatory control of SPHK regulation, S1P degradation, and S1P dephosphorylation. Therefore, clarifying the mechanisms regulating SPHK activity may help us understand when and where S1P is generated. In this study, we performed yeast two-hybrid screening to search for SPHK1a-binding molecules that may be involved in the regulation of the kinase localization or activity. Platelet endothelial cell adhesion molecule-1 (PECAM-1) was identified as a protein potentially associating with SPHK1a. Their association was confirmed by co-immunoprecipitation analysis using HEK293 cells overexpressing PECAM-1 and SPHK1a. Moreover, the kinase activity appeared to be reduced in stable PECAM-1-expressing cells. PECAM-1 is expressed on the cell surface of vascular cells, and several stimuli are known to induce phosphorylation of its tyrosine residues. We found that such phosphorylation attenuated its association with SPHK1a. This association/dissociation of SPHK with PECAM-1, regulated by the phosphorylated state of the membrane protein, may be involved in the control of localized kinase activity in certain cell types.

Topics: Binding Sites; Cell Line; Humans; Lysophospholipids; Mutagenesis, Site-Directed; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Plasmids; Platelet Endothelial Cell Adhesion Molecule-1; Protein Binding; Sphingosine; Two-Hybrid System Techniques; Wheat Germ Agglutinins

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC(20)) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P(1) and S1P(2) receptors. S1P activated G(q), G(13), and all G(i) isoforms and stimulated PLC-beta1, PLC-beta3, and Rho kinase activities. PLC-beta activity was partially inhibited by pertussis toxin (PTX), Gbeta or Galpha(q) antibody, PLC-beta1 or PLC-beta3 antibody, and by expression of Galpha(q) or Galpha(i) minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of Galpha(13) or Galpha(q) minigene and abolished by expression of both. S1P stimulated Ca(2+) release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC(50) 1 nM). Initial contraction and MLC(20) phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and Galpha(q) or Gbeta antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC(20) phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P(2) and S1P(1) involving concurrent activation of PLC-beta1 and PLC-beta3 via Galpha(q) and Gbetagamma(i), respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca(2+) release and MLCK-mediated MLC(20) phosphorylation, and 2) sustained contraction exclusively mediated by S1P(2) involving activation of RhoA via Galpha(q) and Galpha(13), resulting in Rho kinase- and PKC-dependent MLC(20) phosphorylation.

Topics: Adenylyl Cyclase Inhibitors; Animals; Calcium; Cells, Cultured; Enzyme Activation; GTP-Binding Proteins; Intracellular Signaling Peptides and Proteins; Isoenzymes; Lysophospholipids; Molecular Weight; Muscle Contraction; Muscle, Smooth; Myosin Light Chains; Phospholipase C beta; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Serine-Threonine Kinases; Rabbits; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; rho-Associated Kinases; Signal Transduction; Sphingosine; Stomach; Type C Phospholipases

Vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) have been implicated as important stimulatory factors for retinal neovascularization. In this study, we used intraocular gene transfer with gutless adenoviral (AGV) vectors to determine the effect of increased intraocular expression of VEGF, IGF-1, or sphingosine kinase (SPK), which produces sphingosine-1-phosphate, another angiogenic factor. Retinal neovascularization did not occur from intravitreous AGV-vectored VEGF, IGF-1, SPK, or combined VEGF and IGF-1, except occasionally adjacent to the retinal penetration site from the injection. However, corneal and iris neovascularization occurred after 2 weeks in all eyes injected with AGV.VEGF, but not those injected with only AGV.IGF-1 or AGV.SPK. These data suggest that the superficial capillary bed of the retina is relatively insensitive to VEGF, IGF-1, or SPK in adult mice, except when combined with retinal trauma. However, AGV-vectored VEGF is sufficient to consistently cause severe corneal and iris neovascularization. This provides a model for anterior segment neovascularization, which unlike previous models is relatively inexpensive and is not plagued by spontaneous regression, and therefore, may be useful for identification of new treatments.

Topics: Adenoviridae; Animals; DNA Primers; Eye; Genetic Vectors; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Insulin-Like Growth Factor I; Lac Operon; Lysophospholipids; Mice; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Retinal Neovascularization; Reverse Transcriptase Polymerase Chain Reaction; Sphingosine; Transduction, Genetic; Vascular Endothelial Growth Factor A

Sphingosine (Sph) has been implicated as a modulator of membrane signal transduction systems and as a regulatory element of cardiac and skeletal muscle physiology, but little information is presently available on its precise mechanism of action. Recent studies have shown that sphingosine 1-phosphate (S1P), generated by the action of sphingosine kinase (SphK) on Sph, also possesses biological activity, acting as an intracellular messenger, as well as an extracellular ligand for specific membrane receptors. At present, however, it is not clear whether the biological effects elicited by Sph are attributable to its conversion into S1P. In the present study, we show that Sph significantly stimulated phospholipase D (PLD) activity in mouse C2C12 myoblasts via a previously unrecognized mechanism that requires the conversion of Sph into S1P and its subsequent action as extracellular ligand. Indeed, Sph-induced activation of PLD was inhibited by N,N-dimethyl-D-erythro-sphingosine (DMS), at concentrations capable of specifically inhibiting SphK. Moreover, the crucial role of SphK-derived S1P in the activation of PLD by Sph was confirmed by the observed potentiated effect of Sph in myoblasts where SphK1 was overexpressed, and the attenuated response in cells transfected with the dominant negative form of SphK1. Notably, the measurement of S1P formation in vivo by employing labelled ATP revealed that cell-associated SphK activity in the extracellular compartment largely contributed to the transformation of Sph into S1P, with the amount of SphK released into the medium being negligible. It will be important to establish whether the mechanism of action identified in the present study is implicated in the multiple biological effects elicited by Sph in muscle cells.

Topics: Animals; Cell Line; Enzyme Activation; Lysophospholipids; Mice; Myoblasts, Skeletal; Phospholipase D; Phosphotransferases (Alcohol Group Acceptor); Second Messenger Systems; Sphingosine

Sphingosine 1-phosphate (S1P) is a pleiotropic lysophospholipid mediator involved in many cellular responses, including transient calcium mobilization, activation of MAP kinase signaling, inhibition of adenylyl cyclase and increased cell migration. S1P has been shown to be an effective activator of vascular endothelial cells via the interaction with cell surface G protein-coupled receptors (GPCRs), namely S1P-R (formerly EDG-R). The potent immunomodulator, FTY720, is phosphorylated by sphingosine kinase (SK) to FTY720-P. Recently it was shown that FTY720-P, not FTY720, can bind to four out of five of the S1P-R. In the present study, we evaluated the effects of FTY720, FTY720-P, and analogues of FTY720-P: an active (R)-enantiomer [AFD(R)] and an inactive (S)-enantiomer [AFD(S)], on endothelial cell functions. Treatment of HUVEC with FTY720-P, but not FTY720, lead to a robust transient increase in calcium mobilization, detected using the fluorometric imaging plate reader (FLIPR) assay. Additionally, only the phosphorylated derivative (FTY720-P) stimulated MAPK activation. We also observed complementary activities of S1P and FTY720-P in an established in vitro endothelial morphogenesis (Matrigel tube formation) assay and an in vitro endothelial cell migration assay. Using a potent inhibitor of sphingosine kinase, N,N-dimethylsphingosine (DMS), FTY720's effects were inhibited in the migration assay, suggesting that FTY720-P is the active mediator. The effects of FTY720-P in these assays were inhibited by pre-treatment with PTx (pertussis toxin), indicating the requirement of a Gi-coupled S1P receptor. These findings suggest that agonist of S1P-R are able to regulate important endothelial cell properties, which may lead to a greater insight into vascular functions.

Topics: Calcium Signaling; Cells, Cultured; Endothelium, Vascular; Fingolimod Hydrochloride; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Immunosuppressive Agents; Isomerism; Lysophospholipids; Mitogen-Activated Protein Kinases; Molecular Structure; Organogenesis; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine; Structure-Activity Relationship; Umbilical Veins

Advanced glycation end-products (AGE) are generated by chronic hyperglycaemia and may cause diabetic microvascular complications such as diabetic nephropathy. Many factors influence the development of diabetic nephropathy; however, dysregulation of mesangial cell (MC) proliferation appears to play an early and crucial role. In this study, we investigated the effects of AGE on rat MC proliferation and the involvement of sphingolipids in the AGE response. Results show a bimodal effect of AGE on MC proliferation. Thus, low AGE concentrations (<1 microm) induced a significant increase (+26%) of MC proliferation, whereas higher concentrations (10 microm) markedly reduced it (-24%). In parallel, AGE exerted biphasic effects on neutral ceramidase expression and activity. Low AGE concentrations increased neutral ceramidase activity and expression, whereas high AGE concentrations showed opposite effects. Surprisingly, neutral ceramidase modulation did not result in changes of ceramide levels. However, the AGE (10 microm)-inhibitory effect on MC proliferation was associated with accumulation of sphingosine and was specifically prevented by blocking glucosylceramide synthesis, suggesting that the high AGE concentration effects are mediated by sphingosine and/or glycolipids. On the other hand, treatment of cells with low AGE concentrations led to an increase of sphingosine kinase activity and sphingosine-1-phosphate production that drove the increase of MC proliferation. Interestingly, in glomeruli isolated from streptozotocin-diabetic rats, a time-dependent modulation of ceramidase activity was observed as compared with controls. These results suggest that AGE regulate MC growth by modulating neutral ceramidase and endogenous sphingolipids.

Topics: Amidohydrolases; Animals; Blotting, Western; Cell Division; Cells, Cultured; Ceramidases; Chromatography, High Pressure Liquid; Diabetes Mellitus, Experimental; Diacylglycerol Kinase; Dose-Response Relationship, Drug; Gene Expression Regulation; Glomerular Mesangium; Glucosylceramides; Glycation End Products, Advanced; Lysophospholipids; Male; Models, Biological; Neutral Ceramidase; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Sphingolipids; Sphingosine; Thymidine; Time Factors

Plasminogen activators (tPA and uPA) are serine proteases that convert the circulating zymogen plasminogen to active plasmin and mediate fibrin degradation. These multifunctional proteins trigger various biological events such as extracellular matrix degradation, cell adhesion, migration, and proliferation, through not yet fully characterized mechanisms. We report that, in smooth muscle cells and ECV-304 carcinoma cells, tPA and ATF (the N-terminal catalytically inactive fragment of tPA) elicited DNA synthesis that requires activation of the sphingomyelin/ceramide/sphingosine-1-phosphate (Spm/Cer/S1P), signaling pathway and was blocked by D-erythro-2-(N-myristoylamino)-1-phenyl-propanol (D-MAPP) and N-N'-dimethyl sphingosine (DMS), two classical inhibitors of sphingosine-1-phosphate biosynthesis. Binding of tPA to its receptor uPAR triggered the coordinated activation of two key enzymes of the Spm/Cer/S1P pathway, the neutral sphingomyelinase and the sphingosine kinase-1 that was mediated by a common pertussis toxin (PTX)-sensitive mechanism. The tPA-induced sphingosine kinase-1 activation was mediated by Src, since it was inhibited by herbimycin A and in SrcK- cells (overexpressing a dominant negative kinase defective form of Src) and by ERK1/2 (early phase peaking at 15 min). Sphingosine kinase-1 activation was followed by a second phase of ERK1/2 phosphorylation (peaking at 120 min) and subsequent DNA synthesis, which were inhibited by D-MAPP and DMS, by anti-EGD-1 antibodies and in SrcK- cells (in which the mitogenic signaling was rescued by sphingosine-1-phosphate). Altogether, these data underline a pivotal role for the Spm/Cer/S1P pathway in the tPA-induced mitogenic signaling.

Topics: Benzoquinones; Cell Line; Cell Proliferation; Ceramides; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Lactams, Macrocyclic; Lysophospholipids; MAP Kinase Signaling System; Mitogens; Myocytes, Smooth Muscle; Peptide Fragments; Pertussis Toxin; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Quinones; Rifabutin; Signal Transduction; Sphingomyelins; Sphingosine; Tissue Plasminogen Activator; Up-Regulation; Urokinase-Type Plasminogen Activator

We report that prosaposin binds to U937 and is active as a protective factor on tumor necrosis factor alpha (TNFalpha)-induced cell death. The prosaposin-derived saposin C binds to U937 cells in a concentration-dependent manner, suggesting that prosaposin behaves similarly. Prosaposin binding induces U937 cell death prevention, reducing both necrosis and apoptosis. This effect was inhibited by mitogen-activated protein ERK kinase (MEK) and sphingosine kinase (SK) inhibitors, indicating that prosaposin prevents cell apoptosis by activation of extracellular signal-regulated kinases (ERKs) and sphingosine kinase. Prosaposin led to rapid ERK phosphorylation in U937 cells as detected by anti-phospho-p44/42 mitogen-activated protein (MAP) kinase and anti-phosphotyrosine reactivity on ERK immunoprecipitates. It was partially prevented by apo B-100 and pertussis toxin (PT), suggesting that both lipoprotein receptor-related protein (LRP) receptor and Go-coupled receptor may play a role in the prosaposin-triggered pathway. Moreover, sphingosine kinase activity was increased by prosaposin treatment as demonstrated by the enhanced intracellular formation of sphingosine-1-phosphate (S-1-P). The observation that the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the prosaposin effect on cell apoptosis suggests that sphingosine kinase exerts its anti-apoptotic activity by the PI3K-Akt pathway. Thus, cell apoptosis prevention by prosaposin occurs through ERK phosphorylation and sphingosine kinase. The biological effect triggered by prosaposin might be extended to primary cells because it triggers Erk phosphorylation in peripheral blood mononuclear cells (PBMCs). This is the first evidence of a biological effect consequent to a signal transduction pathway triggered by prosaposin in cells of non-neurological origin.

Topics: Apoptosis; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glycoproteins; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Lysophospholipids; Mitogen-Activated Protein Kinases; Monocytes; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Receptors, LDL; Saposins; Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha; U937 Cells

Hepatocyte growth factor (HGF)-induced migration of endothelial cells is critical for angiogenesis. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms. The aim of this study was to investigate whether activation of SPK is involved in the migration of endothelial cells induced by HGF. The biological functions of HGF are mediated through the activation of its high-affinity tyrosine kinase receptor, c-met protooncogene. In the present study, Treatment of ECV304 endothelial cells with HGF resulted in tyrosine phosphorylation of c-Met and activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitor of the PI3K and ERK/MAPK pathways, respectively, blocked the HGF-induced activation of SPK. HGF stimulation significantly increased intracellular S1P level, but no detectable secretion of S1P into the cell culture medium was observed. Treatment of ECV304 cells with pertussis toxin (PTX) has no effect on the HGF-induced migration, indicating extracellular S1P is dispensable for this process. Overexpression of wild-type SPK gene in ECV 304 cells increased the intracellular S1P and enhanced the HGF-induced migration, whereas inhibition of cellular SPK activity by N,N-dimethylsphingosine (DMS), a potent inhibitor of SPK, or by expression of a dominant-negative SPK (DN-SK) blocked the HGF-induced migration of ECV 304 cells. It is suggested that PI3K and ERK/MAPK mediated the activation of SPK and would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which intracellularly generated S1P mediates signaling from HGF/c-Met to the endothelial cell migration.

Topics: Cell Line; Cell Movement; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Inhibitors; Hepatocyte Growth Factor; Humans; Lysophospholipids; Mitogen-Activated Protein Kinases; Neovascularization, Physiologic; Pertussis Toxin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-met; Signal Transduction; Sphingosine; Up-Regulation

The anaphylatoxin C5a is produced following the activation of the complement system and is associated with a variety of pathologies, including septic shock and adult respiratory distress syndrome, and with immune complex-dependent diseases such as rheumatoid arthritis. C5a has been shown to regulate inflammatory functions by interacting with its receptor, C5aR, which belong to the rhodopsin family of seven-transmembrane GPCRs. However, the intracellular signaling pathways triggered by C5aR on immune-effector cells are not well understood. In this report we present data showing that, in human monocyte-derived macrophages, C5aR uses the intracellular signaling molecule sphingosine kinase (SPHK)1 to trigger various physiological responses. Our data show that C5a rapidly stimulates the generation of sphingosine-1-phosphate, SPHK activity, and membrane translocation of SPHK1. Using an antisense oligonucleotide against SPHK1, we show that knockdown of SPHK1 abolishes the C5a-triggered intracellular Ca(2+) signals, degranulation, cytokine generation, and chemotaxis. Our study shows for the first time that SPHK1 not only plays a key role in the generation and release of proinflammatory mediators triggered by anaphylatoxins from human macrophages but is also involved in the process of immune cell motility, thus pointing out SPHK1 as a potential therapeutic target for the treatment of inflammatory and autoimmune diseases.

Topics: beta-N-Acetylhexosaminidases; Calcium Signaling; Cells, Cultured; Chemotaxis; Complement C5a; Cytokines; Cytoplasmic Granules; Humans; Interleukin-6; Interleukin-8; Lysophospholipids; Macrophages; Oligodeoxyribonucleotides, Antisense; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Receptor, Anaphylatoxin C5a; Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha; Type C Phospholipases

Here we have investigated the role of sphingosine-1-phosphate (S1P) signaling in the process of vasculogenesis in the mouse embryo. At stages preceding the formation of blood vessels (7.5-8 dpc) in the embryo proper, yolk sac, and allantois, the S1P receptor S1P(2) is expressed in conjunction with S1P(1) and/or S1P(3). Additionally, sphingosine kinase-2 (SK2), an enzyme that catalyzes the formation of S1P, is expressed in these tissues throughout periods of vasculogenesis. Using the cultured mouse allantois explant model of blood vessel formation, we found that vasculogenesis was dependent on S1P signaling. We showed that S1P could replace the ability of serum to promote vasculogenesis in cultured allantois explants. Instead of small poorly reticulated clusters of rounded endothelial cells that formed under serum-free conditions, S1P promoted the formation of elongated endothelial cells that arranged into expansive branched networks of capillary-like vessels. These effects could not be reproduced by vascular endothelial growth factor or basic fibroblast growth factor administration. The ability of S1P to promote blood vessel formation was not due to effects on cell survival or on changes in numbers of endothelial cells (Flk1(+)/PECAM(+)), angioblasts (Flk1(+)/PECAM(-)), or undifferentiated mesodermal cells (Flk1(-)/PECAM(-)). The S1P effect on blood vessel formation was attributed to it promoting migratory activities of angioblasts and early endothelial cells required for the expansion of vascular networks. Together, our findings suggest that migratory events critical to the de novo formation of blood vessels are under the influence of S1P, possibly synthesized via the action of SK2, with signaling mediated by S1P receptors that include S1P(1), S1P(2), and S1P(3).

Topics: Allantois; Animals; Cell Movement; Lysophospholipids; Mice; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Phosphorylation of sphingosine by sphingosine kinase (SK) is the rate-limiting step in the cellular synthesis of sphingosine 1-phosphate (S1P). The monoganglioside GM1, which stimulates SK, is cardioprotective in part through increased generation of S1P that protects myocytes by diverse mechanisms. Because protein kinase C (PKC)epsilon activation is necessary for myocardial ischemic preconditioning (IPC) and PKC activators increase SK activity, we tested the hypothesis that SK may be a central mediator of IPC.. In adult murine hearts, IPC sufficient to reduce infarct size significantly increased cardiac SK activity, induced translocation of SK protein from the cytosol to membranes, and enhanced cardiac myocyte survival. IPC did not increase SK activity in PKCepsilon-null mice. The SK antagonist N,N-dimethylsphingosine inhibited PKCepsilon activation and directly abolished the protective effects of IPC and the enhanced SK activity induced by IPC.. These findings demonstrate that PKCepsilon is thus recruited by IPC and induces activation of SK that then mediates IPC-induced cardioprotection in murine heart.

Topics: Animals; Cardiotonic Agents; Enzyme Activation; G(M1) Ganglioside; Ischemic Preconditioning, Myocardial; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Cardiovascular; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Protein Kinase C-epsilon; Protein Transport; Signal Transduction; Sphingosine

Sphingosine-1-phosphate (S1P), a lipid signaling molecule that regulates many cellular functions, is synthesized from sphingosine and ATP by the action of sphingosine kinase. Two such kinases have been identified, SPHK1 and SPHK2. To begin to investigate the physiological functions of sphingosine kinase and S1P signaling, we generated mice deficient in SPHK1. Sphk1 null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in most Sphk1-/-tissues was substantially, but not completely, reduced indicating the presence of multiple sphingosine kinases. S1P levels in most tissues from the Sphk1-/- mice were not markedly decreased. In serum, however, there was a significant decrease in the S1P level. Although S1P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs of Sphk1-/- mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in the Sphk1 null mice showing that SPHK1 is not required for the functional activation of this sphingosine analogue prodrug. The results with these Sphk1 null mice reveal that some key physiologic processes that require S1P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK1.

Topics: Animals; Base Sequence; DNA; Fingolimod Hydrochloride; Immunosuppressive Agents; Lymphocytes; Lymphopenia; Lysophospholipids; Mice; Mice, Knockout; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prodrugs; Propylene Glycols; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Transforming growth factor-beta (TGF-beta) signaling plays a pivotal role in extracellular matrix deposition by stimulating collagen production and other extracellular matrix proteins and by inhibiting matrix degradation. The present study was undertaken to define the role of sphingosine kinase (SphK) in TGF-beta signaling. TGF-beta markedly up-regulated SphK1 mRNA and protein amounts and caused a prolonged increase in SphK activity in dermal fibroblasts. Concomitantly, TGF-beta reduced sphingosine-1-phosphate phosphatase activity. Consistent with the changes in enzyme activity, corresponding changes in sphingolipid levels were observed such that sphingosine 1-phosphate (S1P) was increased (approximately 2-fold), whereas sphingosine and ceramide were reduced after 24 h of TGF-beta treatment. Given the relatively early induction of SphK gene expression in response to TGF-beta, we examined whether SphK1 may be involved in the regulation of TGF-beta-inducible genes that exhibit compatible kinetics, e.g. tissue inhibitor of metalloproteinase-1 (TIMP-1). We demonstrate that decreasing SphK1 expression by small interfering RNA (siRNA) blocked TGF-beta-mediated up-regulation of TIMP-1 protein suggesting that up-regulation of SphK1 contributes to the induction of TIMP-1 in response to TGF-beta. The role of SphK1 as a positive regulator of TIMP-1 gene expression was further corroborated by using ectopically expressed SphK1 in the absence of TGF-beta. Adenovirally expressed SphK1 led to a 2-fold increase of endogenous S1P and to increased TIMP-1 mRNA and protein production. In addition, ectopic SphK1 and TGF-beta cooperated in TIMP-1 up-regulation. Mechanistically, experiments utilizing TIMP-1 promoter constructs demonstrated that the action of SphK1 on the TIMP-1 promoter is through the AP1-response element, consistent with the SphK1-mediated up-regulation of phospho-c-Jun levels, a key component of AP1. Together, these experiments demonstrate that SphK/S1P are important components of the TGF-beta signaling pathway involved in up-regulation of the TIMP-1 gene.

Topics: Adenoviridae; Ceramides; Enzyme Induction; Fibroblasts; Gene Expression Regulation; Genetic Vectors; Humans; Kinetics; Lysophospholipids; Membrane Proteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Sphingosine; Tissue Inhibitor of Metalloproteinase-1; Transfection; Transforming Growth Factor beta

Prolonged elevations of cytosolic calcium concentrations ([Ca2+]i) are required for optimal neutrophil (PMN) activation responses to G-Protein coupled chemoattractants. We recently showed that the coupling of endosomal Ca2+ store depletion to more prolonged entry of external Ca2+ depends on cellular conversion of sphingosine to sphingosine 1-phosphate (S1P) by sphingosine kinase (SK). We therefore hypothesized that inhibition of SK might inhibit PMN activation and thus ameliorate lung injury after trauma and hemorrhagic shock (T/HS).. Chemotaxis (CTX) of human PMN was studied using modified Boyden chambers in the presence or absence of the selective SK inhibitor, SKI-2. After determining the concentration of SKI-2 that inhibited human PMN CTX by 50% (IC50) we subjected rats to T/HS (laparotomy, hemorrhage to 30-40 mm Hg x 90 minutes, 3 hours resuscitation). We then studied rat PMN CD11b expression using flow cytometry and lung injury using the Evans Blue dye technique in the presence of IC50 doses of SKI-2 or vehicle given in pretreatment at laparotomy.. Human PMN CTX was suppressed slightly more than 50% by 40 micromol/L SKI-2 (233 +/- 20 vs 103 +/- 12 x 10(3) cells/well, p < 0.001). Rat PMN expression of CD11b after T/HS was decreased from 352 +/- 30 to 232 +/- 7 MFU (p < 0.001) in the presence 30 micromol/L SKI-2. Lung permeability to Evans Blue was decreased from 9.5 +/- 2 to 4.1 +/- 0.7% (p = 0.036.). SKI-2 did not cause hemodynamic instability or alter resuscitation requirements.. Modulation of PMN Ca entry via SK inhibition inhibits PMN CTX in vitro, and inhibits CD11b expression in vivo without major effects on hemodynamics. These cellular changes were associated with amelioration of lung injury in vivo in a rat model of T/HS. These findings suggest that SK inhibition allows modulation of inflammation via control of [Ca2+]i without the cardiovascular compromise expected with Ca2+ channel blockade. SK inhibition therefore appears to be an important novel candidate therapy for inflammatory organ injury after shock.

Topics: Animals; Calcium; Chemotaxis, Leukocyte; Disease Models, Animal; Humans; Inflammation; Lysophospholipids; Neutrophils; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Respiratory Distress Syndrome; Shock, Hemorrhagic; Shock, Traumatic; Sphingosine

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts both extracellularly and intracellularly. The SPL gene encodes a mammalian S1P lyase that degrades S1P. Here, we have disrupted the SPL gene in mouse F9 embryonal carcinoma cells by gene targeting. This is the first report of gene disruption of mammalian S1P lyase. The SPL-null cells exhibited no S1P lyase activity, and intracellular S1P was increased approximately 2-fold, compared with wild-type cells. Treatment of F9 embryonal carcinoma cells with retinoic acid induces differentiation to primitive endoderm (PrE). An acceleration in this PrE differentiation was observed in the SPL-null cells. This effect was apparently caused by the accumulated S1P, since N,N-dimethylsphingosine, a S1P synthesis inhibitor, had an inhibitory effect on the PrE differentiation. Moreover, F9 cells stably expressing sphingosine kinase also exhibited an acceleration in the differentiation. Exogenous S1P had no effect on differentiation, indicating that intracellular but not extracellular S1P is involved. Moreover, we determined that expression of the SPL protein is up-regulated during the progression to PrE. We also showed that sphingosine kinase activity is increased in PrE-differentiated cells. These results suggest that intracellular S1P has a role in the PrE differentiation and that SPL may be involved in the regulation of intracellular S1P levels during this differentiation.

Topics: Aldehyde-Lyases; Animals; Cell Differentiation; Cell Membrane; Cyclic AMP; DNA, Complementary; Endoderm; Exons; Immunoblotting; Kinetics; Lysophospholipids; Mice; Mice, Knockout; Models, Biological; Models, Genetic; Phosphotransferases (Alcohol Group Acceptor); Reverse Transcriptase Polymerase Chain Reaction; Sphingosine; Tretinoin; Tumor Cells, Cultured; Up-Regulation

One-third of the world's population is infected with Mycobacterium tuberculosis (Mtb), and three million people die of tuberculosis each year. Following its ingestion by macrophages (MPs), Mtb inhibits the maturation of its phagosome, preventing progression to a bactericidal phagolysosome. Phagocytosis of Mtb is uncoupled from the elevation in MP cytosolic Ca(2+) that normally accompanies microbial ingestion, resulting in inhibition of phagosome-lysosome fusion and increased intracellular viability. This study demonstrates that the mechanism responsible for this failure of Ca(2+)-dependent phagosome maturation involves mycobacterial inhibition of MP sphingosine kinase. Thus, inhibition of sphingosine kinase directly contributes to survival of Mtb within human MPs and represents a novel molecular mechanism of pathogenesis.

Topics: Animals; Calcium; Calcium Signaling; Cell Fractionation; CHO Cells; Cricetinae; Enzyme Activation; Humans; Lysophospholipids; Macrophage-1 Antigen; Macrophages; Mycobacterium tuberculosis; Phagocytosis; Phagosomes; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tuberculosis Vaccines; Vaccines, Inactivated

In animals, the sphingolipid metabolite sphingosine-1-phosphate (S1P) functions as both an intracellular messenger and an extracellular ligand for G-protein-coupled receptors of the S1P receptor family, regulating diverse biological processes ranging from cell proliferation to apoptosis. Recently, it was discovered in plants that S1P is a signalling molecule involved in abscisic acid (ABA) regulation of guard cell turgor. Here we report that the enzyme responsible for S1P production, sphingosine kinase (SphK), is activated by ABA in Arabidopsis thaliana, and is involved in both ABA inhibition of stomatal opening and promotion of stomatal closure. Consistent with this observation, inhibition of SphK attenuates ABA regulation of guard cell inward K(+) channels and slow anion channels, which are involved in the regulation of stomatal pore size. Surprisingly, S1P regulates stomatal apertures and guard cell ion channel activities in wild-type plants, but not in knockout lines of the sole prototypical heterotrimeric G-protein alpha-subunit gene, GPA1 (refs 5, 6, 7-8). Our results implicate heterotrimeric G proteins as downstream elements in the S1P signalling pathway that mediates ABA regulation of stomatal function, and suggest that the interplay between S1P and heterotrimeric G proteins represents an evolutionarily conserved signalling mechanism.

Topics: Abscisic Acid; Anions; Arabidopsis; Enzyme Activation; Heterotrimeric GTP-Binding Proteins; Ion Channel Gating; Ion Channels; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Potassium; Signal Transduction; Sphingolipids; Sphingosine

In this study we addressed the role of sphingolipid metabolism in the inflammatory response. In a L929 fibroblast model, tumor necrosis factor-alpha (TNF) induced prostaglandin E2 (PGE2) production by 4 h and cyclooxygenase-2 (COX-2) induction as early as 2 h. This TNF-induced PGE2 production was inhibited by NS398, a COX-2 selective inhibitor. GC-MS analysis revealed that only COX-2-generated prostanoids were produced in response to TNF, thus providing further evidence of COX-2 selectivity. As sphingolipids have been implicated in mediating several actions of TNF, their role in COX-2 induction and PGE2 production was evaluated. Sphingosine-1-phosphate (S1P) induced both COX-2 and PGE2 in a dose-responsive manner with an apparent ED50 of 100-300 nM. The related sphingolipid sphingosine also induced PGE2, though with much less efficacy. TNF induced a 3.5-fold increase in sphingosine-1-phosphate levels at 10 min that rapidly returned to baseline by 40 min. Small interfering RNAs (siRNAs) directed against mouse SK1 decreased (typically by 80%) SK1 protein and inhibited TNF-induced SK activity. Treatment of cells with RNAi to SK1 but not SK2 almost completely abolished the ability of TNF to induce COX-2 or generate PGE2. By contrast, cells treated with RNAi to S1P lyase or S1P phosphatase enhanced COX-2 induction leading to enhanced generation of PGE2. Treatment with SK1 RNAi also abolished the effects of exogenous sphingosine and ceramide on PGE2, revealing that the action of sphingosine and ceramide are due to intracellular metabolism into S1P. Collectively, these results provide novel evidence that SK1 and S1P are necessary for TNF to induce COX-2 and PGE2 production. Based on these findings, this study indicates that SK1 and S1P could be implicated in pathological inflammatory disorders and cancer.

Topics: Animals; Cell Line; Cyclooxygenase 2; Dinoprostone; Enzyme Induction; Humans; Isoenzymes; Lysophospholipids; Membrane Proteins; Mice; Phosphotransferases (Alcohol Group Acceptor); Prostaglandin-Endoperoxide Synthases; RNA Interference; Signal Transduction; Sphingosine; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Sphingosine 1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility. However, whether it also has an intracellular function is still a matter of great debate. Overexpression of sphingosine kinase type 1, which generated S1P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion. We have dissected biological responses dependent on intracellular S1P from those that are receptor-mediated by specifically blocking signaling of Galphaq, Galphai, Galpha12/13, and Gbetagamma subunits, the G proteins that S1P receptors (S1PRs) couple to and signal through. We found that intracellular S1P signaled "inside out" through its cell-surface receptors linked to G12/13-mediated stress fiber formation, important for cell motility. Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S1P were independent of GPCRs and inside-out signaling. Using fibroblasts from embryonic mice devoid of functional S1PRs, we also demonstrated that, in contrast to exogenous S1P, intracellular S1P formed by overexpression of sphingosine kinase type 1 promoted growth and survival independent of its GPCRs. Hence, exogenous and intracellularly generated S1Ps affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent intracellular function of S1P, reminiscent of its action in yeast cells that lack S1PRs.

Topics: Animals; Cell Division; Cell Movement; Cells, Cultured; Embryo, Mammalian; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; GTP-Binding Protein alpha Subunits, G12-G13; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Protein-Tyrosine Kinases; Receptors, G-Protein-Coupled; Signal Transduction; Sphingosine; Stress Fibers; Transfection

G-protein-coupled bombesin receptors are capable of signaling through the G(i) protein even when receptor-coupling to G(q) is blocked by [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P (SpD), a neurokinin-1 receptor antagonist and "biased" agonist to bombesin receptors. As bombesin is a monocyte and tumor cell attractant, we were interested in the effects of SpD on cell migration. Chemotaxis of monocytes was tested in micropore filter assays. SpD was a dose-dependent agonist in monocyte migration and was not inhibited by antagonists to neurokinin-1 or -2 receptors. SpD failed to inhibit chemotaxis toward bombesin, suggesting that inhibition of bombesin receptor coupling to G(q) with SpD does not impair migratory responses elicited by bombesin. As pertussis toxin inhibited migration, coupling of receptors to G(i) may signal migration. Chemotaxis toward SpD was inhibited by bombesin receptor antagonists as well as by blocking signaling enzymes downstream of G(q) (phospholipase-3 and protein kinase C with wortmannin and bisindolylmaleimide, respectively), suggesting transactivation of G(q)-mediated chemotaxis signaling by SpD via bombesin receptors. Protein kinase C that induces sphingosine kinase activation and production of sphingosine-1-phosphate, which may lead to G(q)-dependent chemoattraction, was involved in SpD-dependent migration. Inhibition of sphingosine-1-phosphate production with dimethylsphingosine inhibited monocyte migration toward SpD. Data suggest that SpD induces migration in monocytes and signaling events involving activation of sphingosine kinase in a G(i) protein- and protein kinase C-dependent fashion. "Biased" agonism of SpD at bombesin receptors may affect normal and tumor cell migration.

Topics: Androstadienes; Bombesin; Cell Movement; Cells, Cultured; Chemotaxis; Enzyme Activation; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Indoles; Leukocytes, Mononuclear; Lipopolysaccharide Receptors; Lysophospholipids; Maleimides; Neurokinin-1 Receptor Antagonists; Pertussis Toxin; Phospholipases; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Receptors, Bombesin; Sphingosine; Substance P; Wortmannin

We have studied sphingomyelin metabolism in the papillae of neonatal (10-day-old) and adult (70-day-old) kidneys of male Wistar rats because sphingolipid second messengers generated by sphingomyelin metabolism are involved in cellular processes such as proliferation, differentiation and apoptosis. We showed that sphingomyelin and ceramide concentrations in homogenized rat papilla tissue increase with the time whereas sphingosine-1-phosphate content decreases. This is consistent with the finding of a higher biosynthesis of the latter sphingolipid in neonatal than in adult rat papillae. De novo synthetized ceramide was, however, higher in adult than in neonatal papilla homogenates probably accounting for the high ceramide content of the adult rat papilla. These results suggest an active de novo pathway not ending in sphingomyelin but instead arresting at ceramide in adult rat papilla tissue, partially metabolized into sphingosine-1-phosphate in neonatal rat papillae. The activity and the expression of sphingosine kinase, one of the enzymes involved in ceramide metabolism, was found to be higher in neonatal than adult rat kidney tissue. The intracellular distribution of sphingosine kinase was also different; in neonatal rat tissue the enzyme was predominantly associated with plasma membranes but it was cytosolic in adult rat papilla tissue. These findings seem to indicate that, in rat renal papillae, the developmental regulation of sphingosine kinase expression and activity addresses the sphingolipid metabolism to the formation of the proliferative metabolite sphingosine-1-phosphate in the neonatal period, and ceramide, which is associated with cell arrest and differentiation in the adult tissue. These data are consistent with the proliferative state necessary for tubular elongation during the neonatal period and the maintenance of the differentiated state in the adult tissue.

Topics: Aging; Animals; Animals, Newborn; Kidney Medulla; Lysophospholipids; Male; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Sphingolipids; Sphingosine; Tissue Distribution

Lysophosphatidic acid (1-acyl-2-lyso-sn-glycero-3-phosphate; LPA) and sphingosine-1-phosphate (S1P) are bioactive phospholipids which respectively act as agonists for the G-protein-coupled lpA receptors (LPA1, LPA2, and LPA3) and s1p receptors (S1P1, S1P2, S1P3, S1P4, and S1P5), collectively referred to as lysophospholipid receptors (lpR). Since astrocytes are responsive to LPA and S1P, we examined mechanisms of lpR signaling in rat cortical secondary astrocytes. Rat cortical astrocyte mRNA expression by quantitative TaqMan polymerase chain reaction (PCR) analysis revealed the following order of relative expression of lpR mRNAs: s1p3>s1p1>lpa1>s1p2=lpa3>>s1p5. Activation of lpRs by LPA or S1P led to multiple pharmacological effects, including the influx of calcium, phosphoinositide (PI) hydrolysis, phosphorylation of extracellular receptor regulated kinase (ERK) and release of [3H]-arachidonic acid (AA). These signalling events downstream of lpR activation were inhibited to varying degrees by pertussis toxin (PTX) pretreatment or by the inhibition of sphingosine kinase (SK), a rate-limiting enzyme in the biosynthesis of S1P from sphingosine. These results suggest that astrocyte lpR signalling mechanisms likely involve both Gi- and Gq-coupled GPCRs and that receptor-mediated activation of SK leads to intracellular generation of S1P, which in turn amplifies the lpR signalling in a paracrine/autocrine manner.

Topics: Actins; Animals; Arachidonic Acid; Astrocytes; Blotting, Western; Calcium Signaling; Cells, Cultured; Cerebral Cortex; Cloning, Molecular; Enzyme Activation; Inositol Phosphates; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Receptor Cross-Talk; Receptors, Lipoprotein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sphingosine

We identified and cloned the mouse orthologue of human GPR6 as a new member of the lysophospholipid-receptor family. Sphingosine-1-phosphate (S1P) activated GPR6, transiently expressed in frog oocytes or in Chinese hamster ovary (CHO) cells, with high specificity and nanomolar affinity. The GPR6 gene was found to be located on chromosome 10B1 and a single exon coded for the entire open-reading frame. Signal transduction of S1P was inhibited by pertussis toxin, suggesting a coupling of GPR6 to an inhibitory G protein. In CHO cells transfected with GPR6, the sphingosine-kinase pathway mediated Ca(2+) mobilization from internal stores. Apoptotic cell death was induced by serum deprivation or H(2)O(2) treatment and was prevented by S1P in GPR6-, but not in vector-transfected CHO cells. The antiapoptotic effect of S1P required activation of sphingosine kinase and was accompanied by an increase in MAP-kinase phosphorylation.

Topics: Amino Acid Sequence; Animals; Apoptosis; Calcium; Cells, Cultured; CHO Cells; Cricetinae; Ligands; Lysophospholipids; Mice; Molecular Sequence Data; Oocytes; Phosphotransferases (Alcohol Group Acceptor); Receptors, G-Protein-Coupled; Recombinant Proteins; Signal Transduction; Sphingosine; Xenopus laevis

Sphingosine-1-phosphate (S1P) and phosphatidylinositol-4 phosphate [PtdIns(4)P] are important second messengers in various cellular processes. Here, we show that S1P and PtdIns(4)P are formed in purified basolateral membranes (BLM) derived from kidney proximal tubules, indicating the presence of a plasma membrane associated SPK (BLM-SPK) and phosphatidylinositol-4 kinase (PI-4K). We observed that S1P synthesis is linear with time, dependent on protein concentration, and saturable in the presence of increasing concentrations of sphingosine. Different from the observations on cytosolic SPKs, the formation of S1P by BLM-SPK is Mg(2+)-independent and insensitive to the classical inhibitor of the cytosolic SPKs, DL-threo-dihydrosphingosine. With sphingosine as substrate, the enzyme shows cooperative kinetics (n = 3.4) with a K(0.5) value of 0.12 mM, suggesting that BLM-SPK is different from the previously characterized cytosolic SPK. The formation of PtdIns(4)P markedly inhibits BLM-SPK activity. Conversely, a strong activation of PtdIns(4)P synthesis by the formation of S1P is observed. Taken together, these results indicate that (i) basolateral membranes from kidney cells harbor a SPK activity that potentially regulates renal epithelium function, and (ii) the formation of S1P mediated by SPK enhances PI-4K activity, while PtdIns(4)P in turn inhibits SPK, suggesting an interplay between these lipid signaling molecules. These findings suggest the possibility of crosstalk between sphingolipids and glycerolipids, which might be involved in the regulation of transepithelial fluxes across the BLM of kidney cells.

Topics: 1-Phosphatidylinositol 4-Kinase; Animals; Cell Membrane; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cytosol; Dose-Response Relationship, Drug; Edetic Acid; Kidney; Kinetics; Lysophospholipids; Magnesium; Phospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats; Signal Transduction; Sphingolipids; Sphingosine; Swine; Time Factors

Proof for the role of triacylglycerol-rich lipoproteins (TRLs) in the development of cardiovascular events is accumulating. We recently reported that postprandial TRLs bind to and internalize into human aortic vascular smooth muscle cells (HA-VSMCs) by a lipid-dependent mechanism. We now show that postprandial TRLs triggered hydrolysis of sphingomyelin and stimulation of the sphingosine kinase producing sphingosine 1-phosphate (S1P). In addition, postprandial TRLs exhibited survival and mitogenic effects. Interestingly, the signals were modulated by the nature of the fatty acids located at the sn-2 position in the triacylglycerol molecules of TRL. This lipid-stereospecific regulation of S1P cellular levels in HA-VSMCs provides a novel insight into the intrinsic role of dietary fatty acids and the mechanism mediated by triacylglycerol-containing postprandial lipoproteins in the pathogenesis of atherosclerosis.

Topics: Antibiotics, Antineoplastic; Apoptosis; Doxorubicin; Humans; Lipoproteins; Lysophospholipids; Mitosis; Myocytes, Smooth Muscle; Phosphotransferases (Alcohol Group Acceptor); Receptors, G-Protein-Coupled; Signal Transduction; Sphingomyelins; Sphingosine; Time Factors; Triglycerides

Sphingosine-1-phosphate (S1P) is a highly bioactive sphingolipid involved in diverse biological processes leading to changes in cell growth, differentiation, motility, and survival. S1P generation is regulated via sphingosine kinase (SK), and many of its effects are mediated through extracelluar action on G-protein-coupled receptors. In this study, we have investigated the mechanisms regulating SK, where this occurs in the cell, and whether this leads to release of S1P extracellularly. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced early activation of SK in HEK 293 cells, and this activation was more specific to the membrane-associated SK. Therefore, we next investigated whether PMA induced translocation of SK to the plasma membrane. PMA induced translocation of both endogenous and green fluorescent protein (GFP)-tagged human SK1 (hSK1) to the plasma membrane. PMA also induced phosphorylation of GFP-hSK1. The PMA-induced translocation was abrogated by preincubation with known PKC inhibitors (bisindoylmaleimide and calphostin-c) as well as by the indirect inhibitor of PKC, C(6)-ceramide, supporting a role for PKC in mediating translocation of SK to the plasma membrane. SK activity was not necessary for translocation, because a dominant negative G82D mutation also translocated in response to PMA. Importantly, PKC regulation of SK was accompanied by a 4-fold increase in S1P in the media. These results demonstrate a novel mechanism by which PKC regulates SK and increases secretion of S1P, allowing for autocrine/paracrine signaling.

Topics: Amino Acid Sequence; Base Sequence; Cell Line; Cell Membrane; Cloning, Molecular; DNA Primers; Enzyme Activation; Green Fluorescent Proteins; Humans; Luminescent Proteins; Lysophospholipids; Molecular Sequence Data; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Protein Transport; Recombinant Fusion Proteins; Sphingosine; Tetradecanoylphorbol Acetate

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), formed by phosphorylation of sphingosine, has been implicated in cell growth, suppression of apoptosis, and angiogenesis. In this study, we have examined the contribution of intracellular S1P to tumorigenesis of breast adenocarcinoma MCF-7 cells. Enforced expression of sphingosine kinase type 1 (SPHK1) increased S1P levels and blocked MCF-7 cell death induced by anti-cancer drugs, sphingosine, and TNF-alpha. SPHK1 also conferred a growth advantage, as determined by proliferation and growth in soft agar, which was estrogen dependent. While both ERK and Akt have been implicated in MCF-7 cell growth, SPHK1 stimulated ERK1/2 but had no effect on Akt. Surprisingly, parental growth of MCF-7 cells was only weakly stimulated by S1P or dihydro-S1P, ligands for the S1P receptors which usually mediate growth effects. When injected into mammary fat pads of ovariectomized nude mice implanted with estrogen pellets, MCF-7/SPHK1 cells formed more and larger tumors than vector transfectants with higher microvessel density in their periphery. Collectively, our results suggest that SPHK1 may play an important role in breast cancer progression by regulating tumor cell growth and survival.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Breast Neoplasms; Estrogens; Humans; Lysophospholipids; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasms, Hormone-Dependent; Phosphotransferases (Alcohol Group Acceptor); Receptors, Cell Surface; Receptors, Estrogen; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Sphingosine; Tumor Cells, Cultured

Lipids, in addition to being structural components of cell membranes, can act as signaling molecules. Bioactive lipids, such as sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA), may act intracellularly as second messengers or be secreted and act as intercellular signaling molecules. Such molecules can affect a variety of cellular processes including apoptosis, proliferation, differentiation and motility. To investigate possible sources of bioactive lipids during development we have searched the Drosophila genome for homologs of genes involved in mammalian S1P and LPA metabolism. Here we report the developmental expression of 31 such genes by in situ hybridization to Drosophila embryos. Most show expression in specific tissues, with expression in the gut and nervous system being recurring patterns.

Topics: Aldehyde-Lyases; Amidohydrolases; Animals; Ceramidases; Diacylglycerol Kinase; Drosophila; Gene Expression Profiling; In Situ Hybridization; Lysophospholipids; Phospholipase D; Phospholipases A; Phosphotransferases (Alcohol Group Acceptor); Sphingomyelin Phosphodiesterase; Sphingosine

The enzyme sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a bioactive lipid that acts extracellularly on G protein-coupled receptors of the S1P(1)/EDG-1 subfamily. Although S1P is formed in the cytosol of various cells, S1P release is not understood and is controversial because this lipid mediator is also regarded as a second messenger. In this report, we describe the existence of an extracellular S1P-generating system in vascular endothelial cells. Endothelial cells release SK constitutively and form S1P in the range of receptor stimulation. Levels of sphingosine but not ATP in the extracellular environment are rate-limiting. Treatment of endothelial cells with small interfering RNA for SK-1 transcript specifically inhibited SK export, and SK-1-transfected human embryonic kidney 293 cells exhibited enhanced release of SK-1. The export of SK-1 is constitutive and is inhibited by cytochalasin D and treatment at 4 degrees C but not by brefeldin A or nocodazole, suggesting that a nonclassical secretory pathway that requires the actin cytoskeleton dynamics is involved. Because S1P regulates angiogenesis and vascular maturation, we overexpressed SK-1 using an adenoviral vector in vivo in the Matrigel system of angiogenesis. Overexpression of SK-1 resulted in enhanced release of SK activity and induced angiogenesis and vascular maturation. These findings suggest that S1P is made in the extracellular milieu and that extracellular export of SK contributes to the action of S1P in the vascular system.

Topics: Animals; Calcium; Cell Line; Culture Media, Conditioned; Cytosol; Endothelium, Vascular; Female; Gene Expression Regulation, Enzymologic; Gene Silencing; Homeodomain Proteins; Humans; Immediate-Early Proteins; Kidney; Kinetics; Lysophospholipids; Neovascularization, Physiologic; Oocytes; Phosphotransferases (Alcohol Group Acceptor); Protein Transport; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Repressor Proteins; RNA, Small Interfering; RNA, Untranslated; Sphingosine; Transfection; Xenopus; Xenopus Proteins; Zinc Finger E-box Binding Homeobox 2

EDG-1 is a heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) for sphingosine-1-phosphate (SPP). Cell migration toward platelet-derived growth factor (PDGF), which stimulates sphingosine kinase and increases intracellular SPP, was dependent on expression of EDG-1. Deletion of edg-1 or inhibition of sphingosine kinase suppressed chemotaxis toward PDGF and also activation of the small guanosine triphosphatase Rac, which is essential for protrusion of lamellipodia and forward movement. Moreover, PDGF activated EDG-1, as measured by translocation of beta-arrestin and phosphorylation of EDG-1. Our results reveal a role for receptor cross-communication in which activation of a GPCR by a receptor tyrosine kinase is critical for cell motility.

Topics: Animals; Arrestins; Becaplermin; beta-Arrestins; Cell Line; Cell Membrane; Cells, Cultured; Chemotaxis; Gene Deletion; Humans; Immediate-Early Proteins; Lysophospholipids; Mice; Muscle, Smooth, Vascular; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Receptor Cross-Talk; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Receptors, Platelet-Derived Growth Factor; Recombinant Fusion Proteins; Signal Transduction; Sphingosine; Transfection

Recently, a family of G-protein-coupled receptors named endothelial differentiation gene (Edg) receptor family has been identified, which are specifically activated by the two serum lipids, sphingosine-1-phosphate and lysophosphatidic acid. Sphingosine-1-phosphate can also act intracellularly to release Ca2+ from intracellular stores. Since in several cell types, G-protein-coupled lysophosphatidic acid or sphingosine-1-phosphate receptors mobilize Ca2+ in the absence of a measurable phospholipase C stimulation, it was analysed here whether intracellular sphingosine-1-phosphate production was the signalling mechanism used by extracellular sphingosine-1-phosphate for mobilization of stored Ca2+. Sphingosine-1-phosphate and the low affinity sphingosine-1-phosphate receptor agonist, sphingosylphosphorylcholine, induced a rapid, transient and nearly complete pertussis toxin-sensitive Ca2+ mobilization in human embryonic kidney (HEK-293) cells. The G-protein-coupled sphingosine-1-phosphate receptors, Edg-1, Edg-3 and Edg-5, were found to be endogenously expressed in these cells. Most interestingly, sphingosine-1-phosphate and sphingosylphosphorylcholine did not induce a measurable production of inositol-1,4,5-trisphosphate or accumulation of inositol phosphates. Instead, sphingosine-1-phosphate and sphingosylphosphorylcholine induced a rapid and transient increase in production of intracellular sphingosine-1-phosphate with a maximum of about 1.4-fold at 30 s. Stimulation of sphingosine-1-phosphate formation by sphingosine-1-phosphate and sphingosylphosphorylcholine was fully blocked by pertussis toxin, indicating that extracellular sphingosine-1-phosphate via endogenously expressed G(i)-coupled receptors induces a stimulation of intracellular sphingosine-1-phosphate production. As sphingosine-1-phosphate- and sphingosylphosphorylcholine-induced increases in intracellular Ca2+ were blunted by sphingosine kinase inhibitors, this sphingosine-1-phosphate production appears to mediate Ca2+ signalling by extracellular sphingosine-1-phosphate and sphingosylphosphorylcholine in HEK-293 cells.

Topics: Calcium Signaling; Carbachol; Cell Line; Cholinergic Agonists; DNA-Binding Proteins; GTP-Binding Proteins; Humans; I-kappa B Proteins; Immediate-Early Proteins; Lysophospholipids; NF-KappaB Inhibitor alpha; Phosphorylcholine; Phosphotransferases (Alcohol Group Acceptor); Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Sphingosine; Type C Phospholipases

Sphingosine kinase phosphorylates sphingosine to generate sphingosine 1-phosphate, a phospholipid that has been implicated in signaling by a number of transmembrane receptors and was recently shown to act as a ligand for a specific class of G protein-coupled receptors. Here we show that the G protein-coupled bradykinin B2 receptor activates sphingosine kinase leading to a time- and dose-dependent elevation of cellular sphingosine 1-phosphate levels that was blocked by the sphingosine kinase inhibitor dihydrosphingosine. Furthermore, increasing doses of this inhibitor partially affected the bradykinin-mediated ERK/MAP kinase activation and fully blocked the protein kinase C-independent component of the signaling pathway from the B2 receptor to the ERK/MAP kinase cascade. Overexpression of sphingosine kinase did not additionally increase the bradykinin-induced ERK/MAP kinase activity, indicating a permissive rather than activating role of sphingosine 1-phosphate in B2 receptor-mediated mitogenic signaling.

Topics: Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Humans; Lysophospholipids; Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Receptor, Bradykinin B2; Receptors, Bradykinin; Signal Transduction; Sphingosine

Human hepatocytes usually are resistant to TNF-alpha cytotoxicity. In mouse or rat hepatocytes, repression of NF-kappaB activation is sufficient to induce TNF-alpha-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IkappaBalpha (Ad5IkappaB), which almost completely blocks NF-kappaB activation, >80% of the cells survived 24 h after TNF-alpha stimulation. Here, we report that TNF-alpha activates other antiapoptotic factors, such as sphingosine kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N,N-dimethylsphingosine (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-alpha in Ad5IkappaB-infected Huh-7 and Hc cells. TNF-alpha-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous sphingosine 1-phosphate, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-alpha-induced apoptosis. Although Akt has been reported to activate NF-kappaB, DMS and LY 294002 failed to prevent TNF-alpha-induced NF-kappaB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-kappaB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. Sphingosine 1-phosphate administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-kappaB but also SphK and PI3K/Akt are involved in the signaling pathway(s) for protection of human hepatocytes from the apoptotic action of TNF-alpha and probably FasL.

Topics: Adenoviridae; Adjuvants, Immunologic; Apoptosis; Caspases; Cell Line; DNA Fragmentation; Enzyme Activation; Fas Ligand Protein; fas Receptor; Hepatocytes; Humans; I-kappa B Proteins; Ligands; Lysophospholipids; Membrane Glycoproteins; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphotransferases (Alcohol Group Acceptor); Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Sphingosine; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Topics: Chemistry Techniques, Analytical; Chloroform; Chromatography, Thin Layer; Ethanol; Lysophospholipids; Micelles; Phosphotransferases (Alcohol Group Acceptor); Solvents; Sphingosine

In hepatoma Huh-7 cells, inhibition of sphingosine kinase (SphK) activity by N,N-dimethylsphingosine (DMS) resulted in up-regulated production of liver-specific serum proteins including albumin and alpha-fetoprotein (AFP). The changes in these protein levels coincided well with those of two liver-enriched transcription factors, hepatocyte nuclear factor (HNF)-1 and -4, which regulate a number of liver-specific genes at the transcriptional level. Moreover, DMS induced the expression of retinoic acid receptor-alpha and retinoid X receptor-alpha. In DMS-treated cells, 9-cis retinoic acid (RA) further enhanced HNF-4alpha and albumin expression but it inhibited AFP accumulation. These results suggest that activation of SphK disengages cells from their liver-specific phenotype, and that 9-cis RA further induces differentiation of hepatoma cells when SphK activity is inhibited.

Topics: Alitretinoin; alpha-Fetoproteins; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Carcinoma, Hepatocellular; Cell Differentiation; DNA-Binding Proteins; Enzyme Inhibitors; Hepatocyte Nuclear Factor 1; Hepatocyte Nuclear Factor 1-alpha; Hepatocyte Nuclear Factor 1-beta; Hepatocyte Nuclear Factor 4; Hepatocytes; Humans; Liver; Lysophospholipids; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nuclear Proteins; Phosphoproteins; Phosphotransferases (Alcohol Group Acceptor); Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Serum Albumin; Sphingosine; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) stimulate cellular proliferation and affect numerous cellular functions by signaling through G protein-coupled endothelial differentiation gene-encoded (Edg) receptors. S1P and LPA also act as survival factors in many cell types, but have not previously been studied in cardiac myocytes. We incubated neonatal rat cardiac myocytes either in room air/1% CO2 (normoxia) or in an atmosphere of 99% N2/1%CO2 (hypoxia) at 37 degrees C for 18-20 h in the absence of glucose. Cell viability was measured using a calcein ester green fluorescence assay. Under normoxic conditions 88.7+/-1.0% of the cells were viable after 18-20 h. Severe hypoxia reduced viability to 61.3+/-4.3% (n=6, P<0.05). In myocytes preincubated with either 10 microM S1P or 1 microM LPA for 2 h, the effects of severe hypoxia on cell viability were prevented resulting in survival equivalent to normoxia. Neither the protein kinase C inhibitor chelethyrine (1 microM) nor the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoic acid, (5-HD, 100 microM) had any effect on myocyte survival during severe hypoxia, but both agents completely abolished the ability of S1P to rescue cardiac myocytes from hypoxic cell death. We also tested the effects of dimethylsphingosine (DMS), which inhibits sphingosine kinase synthesis of S1P. Incubation of neonatal rat cardiac myocytes with 10 microM DMS for 2 h in the presence of serum resulted in 25-30% cell death during 18-20 h of normoxia. DMS-induced cell death was prevented by concurrent preincubation with either S1P or GM-1, a ganglioside that activates sphingosine kinase to increase intracellular levels of S1P. We conclude that both S1P and LPA are cardioprotective for hypoxic neonatal rat ventricular myocytes. S1P acts through cellular membrane receptors by signaling mechanisms involving protein kinase C and mitochondrial K(ATP) channels. Both endogenous and exogenously applied S1P are effective in preventing cell death induced by inhibition of sphingosine kinase.

Topics: Alkaloids; Animals; Animals, Newborn; Anti-Arrhythmia Agents; Benzophenanthridines; Cardiotonic Agents; Cell Hypoxia; Cell Survival; Cells, Cultured; Culture Media, Serum-Free; Decanoic Acids; Enzyme Inhibitors; Gelsolin; Heart; Hydroxy Acids; Lysophospholipids; Myocardium; Phenanthridines; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Sphingosine

Owing to its ability to induce growth arrest and differentiation of keratinocytes, 1alpha,25-dihydroxyvitamin D3 and its analogs are useful for the treatment of hyperproliferative skin diseases, such as psoriasis vulgaris. It has been implicated that the 1alpha,25-dihydroxyvitamin D3-induced differentiation of keratinocytes is mediated, at least in part, by the formation of ceramides; however, ceramides have also been identified to induce apoptosis in many cells, including keratinocytes. Therefore, it was of interest to investigate the influence of 1alpha,25-dihydroxyvitamin D3 on apoptosis in keratinocytes. Most interestingly, physiological concentrations of 1alpha,25-dihydroxyvitamin D3 did not induce apoptosis in keratinocytes, despite the formation of ceramides. Moreover, 1alpha,25-dihydroxyvitamin D3 appeared cytoprotective and made keratinocytes resistant to apoptosis induced by ceramides, ultraviolet irradiation, or tumor necrosis factor-alpha. The cytoprotective effect was accompanied by the formation of the sphingolipid breakdown product sphingosine-1-phosphate, which prevented apoptosis in analogy to 1alpha,25-dihydroxyvitamin D3. The effect of 1alpha,25-dihydroxyvitamin D3 was specific as the almost inactive precursor cholecalciferol neither induced sphingosine-1-phosphate formation nor prevented cells from apoptosis. Besides this, the cytoprotective aptitude of 1alpha,25-dihydroxyvitamin D3 was completely abolished by the sphingosine kinase inhibitor N,N-dimethylsphingosine, which blocked sphingosine-1-phosphate formation. Moreover, sphingosine-1-phosphate was able to restore the cytoprotective effect of 1alpha,25-dihydroxyvitamin D3 in the presence of N,N-dimethylsphingosine. Taken together, here we report for the first time that 1alpha,25-dihydroxyvitamin D3 protects keratinocytes from apoptosis and additionally this cytoprotection is mediated via the formation of sphingosine-1-phosphate.

Topics: Apoptosis; Calcitriol; Cell Division; Cell Survival; Cells, Cultured; Ceramides; Cytoprotection; Humans; Hydroxycholecalciferols; Keratinocytes; Lysophospholipids; Necrosis; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-bcl-2; Sphingosine; Tumor Necrosis Factor-alpha; Ultraviolet Rays

Sphingosine-1-phosphate (SPP), formed by sphingosine kinase, is the ligand for EDG-1, a GPCR important for cell migration and vascular maturation. Here we show that cytoskeletal rearrangements, lamellipodia extensions, and cell motility induced by platelet-derived growth factor (PDGF) are abrogated in EDG-1 null fibroblasts. However, EDG-1 appears to be dispensable for mitogenicity and survival effects, even those induced by its ligand SPP and by PDGF. Furthermore, PDGF induced focal adhesion formation and activation of FAK, Src, and stress-activated protein kinase 2, p38, were dysregulated in the absence of EDG-1. In contrast, tyrosine phosphorylation of the PDGFR and activation of extracellular signal regulated kinase (ERK1/2), important for growth and survival, were unaltered. Our results suggest that EDG-1 functions as an integrator linking the PDGFR to lamellipodia extension and cell migration. PDGF, which stimulates sphingosine kinase, leading to increased SPP levels in many cell types, also induces translocation of sphingosine kinase to membrane ruffles. Hence, recruitment of sphingosine kinase to the cell's leading edge and localized formation of SPP may spatially and temporally stimulate EDG-1, resulting in activation and integration of downstream signals important for directional movement toward chemoattractants, such as PDGF. These results may also shed light on the vital role of EDG-1 in vascular maturation.

Topics: 3T3 Cells; Animals; Apoptosis; Biological Transport; Cell Division; Cell Movement; Cells, Cultured; Chemotaxis; Cytoskeleton; DNA; Embryo, Mammalian; Enzyme Activation; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Genotype; Green Fluorescent Proteins; Immediate-Early Proteins; Luminescent Proteins; Lysophospholipids; Mice; Mice, Knockout; Microscopy, Confocal; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Protein-Tyrosine Kinases; Pseudopodia; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Receptors, Platelet-Derived Growth Factor; Recombinant Fusion Proteins; Signal Transduction; Sphingosine; src-Family Kinases; Time Factors

Formation of sphingosine-1-phosphate (SPP) by sphingosine kinase serves as a signalling pathway for various membrane receptors. Here, we show that membrane depolarisation is another mechanism by which this pathway can be activated. Formation of [(3)H]SPP as well as levels of endogenous SPP were rapidly and transiently increased in PC12 pheochromocytoma cells depolarised with high KCl. Time course and maximum were similar to those induced by bradykinin. Depolarisation-induced SPP production was also observed in RINm5F insulinoma cells, dependent on extracellular Ca(2+) and fully suppressed by verapamil, thus apparently caused by Ca(2+) influx via voltage-gated Ca(2+) channels. Studies with sphingosine kinase inhibitors and overexpression of sphingosine kinase revealed a partial contribution of this pathway to depolarisation-induced noradrenaline release and Ca(2+) increase.

Topics: Animals; Bradykinin; Calcium Channels; Calcium Signaling; Cell Membrane; Lysophospholipids; Norepinephrine; PC12 Cells; Phosphotransferases (Alcohol Group Acceptor); Potassium Chloride; Rats; Recombinant Proteins; Sphingosine; Verapamil

A common intracellular signal activating polymorphonuclear leukocytes (PMN) in inflammation is a change in cytosolic calcium concentration. Previously, we have shown that interferon-gamma (IFN-gamma) induces transient calcium signals in PMN, but only after intracellular calcium store depletion. Using a digital imaging system, we show that adhesion of PMN is critical for IFN-gamma-induced calcium signals, and with PMN attached to the optimal coating, the calcium signals are evoked even in presence of extracellular calcium, that is, non-depleted calcium stores. Adhesion to fibronectin, pure or extracted from plasma by gelatin, improved the IFN-gamma responses compared with serum, plasma, or vitronectin coats. In accordance with previous observations, IFN-gamma-induced calcium signals in fibronectin adherent cells were totally abolished by the G-protein inhibitor pertussis toxin and were also inhibited by the sphingosine kinase inhibitors dimethylsphingosine (DMS) and N-acetylsphingosine (N-Ac-Sp). PMN contact with fibronectin alone, measured in cells sedimenting onto a fibronectin-coated surface or by addition of fibronectin to glass-adherent cells, evoked transient calcium signals. However, PMN in suspension did not respond to the addition of fibronectin or arginine-glycine-aspartate (RGD). The fibronectin-induced calcium signals were also clearly depressed by pertussis toxin and by the sphingosine kinase inhibitors DMS, dihydrosphingosine (DHS), and N-Ac-Sp. When the product of sphingosine kinase activity, sphingosine 1-phosphate (S1-P), was added to the cells, similar calcium signals were induced, which were dependent on a pertussis toxin-sensitive G-protein activity. Finally, addition of S1-P to the cells prior to stimulation with IFN-gamma partly mimicked the priming effect of fibronectin. In conclusion, fibronectin contact evokes by itself a calcium signal in PMN and further promotes calcium signaling by IFN-gamma. We suggest that fibronectin might activate sphingosine kinase, and that the sphingosine 1-phosphate thereby generated induces a calcium signal via a G-protein-dependent mechanism. Apparently, sphingosine kinase activity is also involved in IFN-gamma induced calcium signals.

Topics: Calcium; Calcium Signaling; Cell Adhesion; Cytosol; Fibronectins; Gelatin; Heterotrimeric GTP-Binding Proteins; Humans; Interferon-gamma; Lysophospholipids; Neutrophils; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Topics: Lysophospholipids; Phosphorus Radioisotopes; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine kinase catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (SPP), a novel lipid mediator with both intra- and extracellular functions. Based on sequence identity to murine sphingosine kinase (mSPHK1a), we cloned and characterized the first human sphingosine kinase (hSPHK1). The open reading frame of hSPHK1 encodes a 384 amino acid protein with 85% identity and 92% similarity to mSPHK1a at the amino acid level. Similar to mSPHK1a, when HEK293 cells were transfected with hSPHK1, there were marked increases in sphingosine kinase activity resulting in elevated SPP levels. hSPHK1 also specifically phosphorylated D-erythro-sphingosine and to a lesser extent sphinganine, but not other lipids, such as D,L-threo-dihydrosphingosine, N, N-dimethylsphingosine, diacylglycerol, ceramide, or phosphatidylinositol. Northern analysis revealed that hSPHK1 was widely expressed with highest levels in adult liver, kidney, heart and skeletal muscle. Thus, hSPHK1 belongs to a highly conserved unique lipid kinase family that regulates diverse biological functions.

Topics: Amino Acid Sequence; Animals; Cell Line; Cloning, Molecular; Cytosol; Gene Expression Profiling; Humans; Intracellular Membranes; Kinetics; Lysophospholipids; Mice; Molecular Sequence Data; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Sequence Alignment; Sequence Homology, Amino Acid; Sphingolipids; Sphingosine; Substrate Specificity; Transfection

Topics: Cell Division; Cyclooxygenase Inhibitors; Endothelin-1; Enzyme Activation; Humans; Liver; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine 1-phosphate is an intermediate of sphingosine catabolism as well as a potent signaling compound. Conditions were established for the extraction and analysis of sphingosine 1-phosphate and other sphingoid base 1-phosphates from in vitro sphingosine kinase assays and other biological samples. The sphingoid base 1-phosphates were extracted in high yield (85%) using small C-18 reverse-phase columns (LiChroprep RP-18). After the extracts were treated with 0.1 N KOH to remove glycerolipids, the sphingoid base 1-phosphates were converted to fluorescent o-phthalaldehyde derivatives that were separated by HPLC using C-18 columns with a mobile phase of methanol:10 mM potassium phosphate (pH 7.2):1 M tetrabutylammonium dihydrogen phosphate (in water) (83:16:1, v/v/v). The o-phthalaldehyde derivative of sphingosine 1-phosphate was reasonably stable (t(1/2) > or = 18 h) when EDTA was present and could be detected in picomole amounts. The HPLC retention time of the sphingoid base 1-phosphates could be shifted by adjusting the mobile phase to pH 5.5, which is useful in separating overlapping compounds (such as sphingosine 1-phosphate and 4-D-hydroxysphinganine) and in confirming the identity of sphingoid base 1-phosphates in biological samples. The extraction procedure and HPLC method facilitated assays of sphingosine kinase with different sphingoid bases as substrates and/or inhibitors and enabled the quantitation of sphingoid base 1-phosphates in human plasma, serum, and platelets as well as in strains of Saccharomyces cerevisae with mutations in sphingolipid metabolism.

Topics: Blood Platelets; Chromatography, High Pressure Liquid; Humans; Lysophospholipids; o-Phthalaldehyde; Phosphates; Phosphotransferases (Alcohol Group Acceptor); Saccharomyces cerevisiae; Spectrometry, Fluorescence; Sphingosine

Sphingosine induces a biphasic increase in cytosolic-free Ca(2+)([Ca(2+)](i)) with an initial peak followed by a sustained increase in HL-60 cells differentiated into neutrophil-like cells. The initial peak is not affected by the presence of ethylene glycol bis (beta-aminoethyl ether) N, N, N', N-tetraacetic acid (EGTA) in the buffer and appears to be dependent on conversion of sphingosine to sphingosine -1-phosphate (S1P) by sphingosine kinase, since it is blocked by the presence of N, N-dimethylsphingosine (DMS), which, like sphingosine, causes a sustained increase itself. The sustained increase that is elicited by sphingosine or DMS is abolished by the presence of EGTA in the buffer. The sustained sphingosine-induced Ca(2+)influx does not appear due to Ca(2+)influx through store-operated Ca(2+)(SOC) channels, since the influx is not inhibited by SKF 96365, nor is it augmented by loperamide. In addition, sphingosine and DMS attenuate the Ca(2+)influx through SOC channels that occurs after depletion of intracellular stores by ATP or thapsigargin. Both the initial peak and the sustained increase in [Ca(2+)](i)elicited by sphingosine can be blocked by phorbol 12-myristate 13-acetate (PMA)-elicited activation of protein kinase C. Thus, in HL-60 cells sphingosine causes a mobilization of Ca(2+)from intracellular Ca(2+)stores, which requires conversion to S1P, while both sphingosine and DMS elicit a Ca(2+)influx through an undefined Ca(2+)channel and cause a blockade of SOC channels.

Topics: Calcium; Calcium Channels; Calcium Signaling; HL-60 Cells; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a lipid messenger that plays an important role in a variety of mammalian cell processes, including inhibition of apoptosis and stimulation of cell proliferation. Basal levels of S1P in cells are generally low but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity. To date, elucidation of the exact signaling pathways affected by these elevated S1P levels has relied on the use of SK inhibitors that are known to have direct effects on other enzymes in the cell. Furthermore, these inhibitors block basal SK activity, which is thought to have a housekeeping function in the cell. To produce a specific inhibitor of SK activation we sought to generate a catalytically inactive, dominant-negative SK. This was accomplished by site-directed mutagenesis of Gly(82) to Asp of the human SK, a residue identified through sequence similarity to the putative catalytic domain of diacylglycerol kinase. This mutant had no detectable SK activity when expressed at high levels in HEK293T cells. Activation of endogenous SK activity by tumor necrosis factor-alpha (TNFalpha), interleukin-1beta, and phorbol esters in HEK293T cells was blocked by expression of this inactive sphingosine kinase (hSK(G82D)). Basal SK activity was unaffected by expression of hSK(G82D). Expression of hSK(G82D) had no effect on TNFalpha-induced activation of protein kinase C and sphingomyelinase activities. Thus, hSK(G82D) acts as a specific dominant-negative SK to block SK activation. This discovery provides a powerful tool for the elucidation of the exact signaling pathways affected by elevated S1P levels following SK activation. To this end we have employed the dominant-negative SK to demonstrate that TNFalpha activation of extracellular signal-regulated kinases 1 and 2 (ERK1,2) is dependent on SK activation.

Topics: Amino Acid Sequence; Catalysis; Cells, Cultured; Enzyme Activation; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tumor Necrosis Factor-alpha

Topics: 3T3 Cells; Adenosine Triphosphate; Animals; Cell Line; HL-60 Cells; Humans; Kidney; Lysophospholipids; Mice; PC12 Cells; Phospholipids; Phosphorus Radioisotopes; Phosphotransferases (Alcohol Group Acceptor); Radioisotope Dilution Technique; Rats; Recombinant Proteins; Sphingosine; Transfection; Tumor Cells, Cultured

Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.

Topics: 3T3 Cells; Animals; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Genes, ras; Humans; Lysophospholipids; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasms, Experimental; Oncogenes; Phosphotransferases (Alcohol Group Acceptor); ras Proteins; Signal Transduction; Sphingosine; Transfection

Sphingosine-1-phosphate (SPP) produced from sphingosine by sphingosine kinase has recently been reported to act as intracellular second messenger for a number of plasma membrane receptors. In the present study, we investigated whether the sphingosine kinase/SPP pathway is involved in cellular signaling of the Gi protein-coupled formyl peptide receptor in myeloid differentiated human leukemia (HL-60) cells. Receptor activation resulted in rapid and transient production of SPP by sphingosine kinase, which was abolished after pertussis toxin treatment. Direct activation of heterotrimeric G proteins by AlF4- also rapidly increased SPP formation in intact HL-60 cells. In cytosolic preparations of HL-60 cells, sphingosine kinase activity was stimulated by the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate). Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine did not affect phospholipase C stimulation and superoxide production but markedly inhibited receptor-stimulated Ca2+ mobilization and enzyme release. We conclude that the formyl peptide receptor stimulates through Gi-type G proteins SPP production by sphingosine kinase, that the enzyme is also stimulated by direct G protein activation, and that the sphingosine kinase/SPP pathway apparently plays an important role in chemoattractant signaling in myeloid differentiated HL-60 cells.

Topics: Calcium; Enzyme Inhibitors; GTP-Binding Proteins; HL-60 Cells; Humans; Lysophospholipids; N-Formylmethionine Leucyl-Phenylalanine; Phosphotransferases (Alcohol Group Acceptor); Receptors, Formyl Peptide; Receptors, Immunologic; Receptors, Peptide; Second Messenger Systems; Signal Transduction; Sphingosine; Superoxides; Type C Phospholipases

We have investigated the extracellular and intracellular actions of sphingosine 1-phosphate (S1P) by using cultured airway smooth muscle cells. We have demonstrated that exogenous S1P elicited an activation of mitogen-activated protein kinase (p42/p44 MAPK) that was abolished by pertussis toxin (0.1 microg/mL, 24 h), which was used to inactivate Gi. The effect of exogenous S1P might therefore be attributed to an action at a putative Gi-coupled receptor. The regulation of the p42/p44 MAPK cascade by S1P was also shown to include a protein kinase C (PKC)-dependent intermediate step. Platelet-derived growth factor (PDGF) stimulates intracellular S1P formation and was therefore used to evaluate the intracellular action of S1P. This has previously been investigated by others using the sphingosine kinase inhibitors D,L-threo-dihydrosphingosine and N,N-dimethylsphingosine. We have demonstrated here that both inhibitors block the PDGF-dependent activation of p42/p44 MAPK. However, both are also PKC inhibitors, which might account for their effect because PDGF utilises PKC as an intermediate in the regulation of the p42/p44 MAPK cascade. Significantly, sphingosine, which is the substrate of sphingosine kinase and a PKC inhibitor, blocked the activation of p42/p44 MAPK by PDGF with an almost identical concentration dependence compared with D,L-threo-dihydrosphingosine and N,N-dimethylsphingosine. Therefore, the use of so-called sphingosine kinase inhibitors might lead to misleading interpretations because of their additional effect on PKC. Other approaches, such as oligodeoxynucleotide anti-sense against sphingosine kinase, are required to address the intracellular role of S1P.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Enzyme Activation; Extracellular Space; Guinea Pigs; Intracellular Fluid; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Models, Biological; Muscle, Smooth, Vascular; Pertussis Toxin; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Sphingosine; Virulence Factors, Bordetella

Sphingosine 1-phosphate (Sph-1-P) is considered to play a dual role in cellular signaling, acting intercellularly as well as intracellularly. In this study, we examined the role of Sph-1-P as a signaling molecule in human platelets, using DL-threo-dihydrosphingosine (DHS) and N,N-dimethylsphingosine (DMS), inhibitors of Sph kinase and protein kinase C. Both DMS and DL-threo-DHS were confirmed to be competitive inhibitors of Sph kinase obtained from platelet cytoplasmic fractions. In intact platelets labeled with [3H]Sph, stimulation with 12-O-tetradecanoylphorbol 13-acetate or thrombin did not affect [3H]-Sph-1-P formation. While both DMS and DL-threo-DHS inhibited not only [3H]Sph-1-P formation but also protein kinase C-dependent platelet aggregation, staurosporine, a potent protein kinase inhibitor, only inhibited the protein kinase C-dependent reaction. Hence, it is unlikely that Sph kinase activation and the resultant Sph-1-P formation are mediated by protein kinase C in platelets. Furthermore, Ca2+ mobilization induced by platelet agonists that act on G protein-coupled receptor was not affected by DMS or DL-threo-DHS. Our results suggest that Sph-1-P does not mediate intracellular signaling, including Ca2+ mobilization, in platelets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Blood Platelets; Calcium Signaling; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Signal Transduction; Sphingosine; Thrombin

Sphingosine 1-phosphate (SPP) is a sphingolipid metabolite which has novel dual actions acting as both an intracellular second messenger and a ligand for a family of G protein-coupled receptors. This paper describes a rapid enzymatic method to quantify mass levels of SPP in serum, mammalian tissues, and cultured cells. The assay utilizes an alkaline lipid extraction to selectively separate SPP from other phospholipids and sphingolipids, including sphingosine. Extracted SPP is efficiently converted to sphingosine by alkaline phosphatase treatment. Sphingosine thus formed is then quantitatively phosphorylated to [(32)P]SPP using recombinant sphingosine kinase and [gamma-(32)P]ATP. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.25 pmol to 2.5 nmol. In various rat tissues, levels of SPP varied between 0. 5 and 6 pmol/mg wet wt. The lowest levels were found in heart and testes, while brain contained the highest levels. The method was adapted easily to measure minute amounts of SPP present in various cultured cell types. The amount of SPP in cell extracts was proportional to the cell number and varied between 0.04 and 2 pmol/10(6) cells. Concurrent measurements of sphingosine levels revealed that its concentration was significantly higher than SPP in most cells and tissues. Furthermore, with this assay we were able to measure increases in intracellular SPP levels in rat pheochromocytoma PC12 cells after treatment with exogenous sphingosine or with nerve growth factor which stimulates sphingosine kinase activity.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Humans; In Vitro Techniques; Lysophospholipids; Male; Nerve Growth Factors; PC12 Cells; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats; Recombinant Proteins; Sphingosine; Tissue Distribution; Tumor Cells, Cultured

Oxidized LDL (oxLDL) have been implicated in diverse biological events leading to the development of atherosclerotic lesions. We previously demonstrated that the proliferation of cultured vascular smooth muscle cells (SMC) induced by oxLDL is preceded by an increase in neutral sphingomyelinase activity, sphingomyelin turnover to ceramide, and stimulation of mitogen-activated protein kinases (Augé, N., Escargueil-Blanc, I., Lajoie-Mazenc, I., Suc, I., Andrieu-Abadie, N., Pieraggi, M. T., Chatelut, M., Thiers, J. C., Jaffrézou, J. P., Laurent, G., Levade, T., Nègre-Salvayre, A., and Salvayre, R. (1998) J. Biol. Chem. 273, 12893-12900). Since ceramide can be converted to other bioactive metabolites, such as the well established mitogen sphingosine 1-phosphate (S1P), we investigated whether additional ceramide metabolites are involved in the oxLDL-induced SMC proliferation. We report here that incubation of SMC with oxLDL increased the activities of both acidic and alkaline ceramidases as well as sphingosine kinase, and elevated cellular sphingosine and S1P. Furthermore, the mitogenic effect of oxLDL was inhibited by D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and N,N-dimethylsphingosine which are inhibitors of ceramidase and sphingosine kinase, respectively. These findings suggest that S1P is a key mediator of the mitogenic effect of oxLDL. In agreement with this conclusion, exogenous addition of sphingosine stimulated the proliferation of cultured SMC, and this effect was abrogated by dimethylsphingosine but not by fumonisin B1, an inhibitor of the acylation of sphingosine to ceramide. Exogenous S1P also promoted SMC proliferation. Altogether, these results strongly suggest that the mitogenic effect of oxLDL in SMC involves the combined activation of sphingomyelinase(s), ceramidase(s), and sphingosine kinase, resulting in the turnover of sphingomyelin to a number of sphingolipid metabolites, of which at least S1P is critical for mitogenesis.

Topics: Amidohydrolases; Animals; Cell Division; Cells, Cultured; Ceramidases; Enzyme Activation; Femoral Artery; Humans; Kinetics; Lipoproteins, LDL; Lysophospholipids; Muscle, Smooth, Vascular; Oxidation-Reduction; Phosphotransferases (Alcohol Group Acceptor); Rabbits; Sphingomyelin Phosphodiesterase; Sphingosine

Ceramide is involved as a mediator of apoptosis induced by a variety of signaling molecules or stressful events. Ceramide-derived sphingosine 1-phosphate behaves as an antiapoptotic agent. The ganglioside GM1 is known to protect neuronal cell lines from apoptosis induced by serum/growth factor withdrawal and its effect is mediated in part by the direct activation of the trkA NGF receptor [G. Ferrari et al. (1995) J. Biol. Chem. 270, 3074-3080]. We show that GM1, similarly to sphingosine 1-phosphate, protects rat heart fibroblasts from apoptosis induced by the protein kinase C inhibitor staurosporine and by C2-ceramide. Furthermore, we show that GM1 induces the synthesis of sphingosine 1-phosphate and that this effect is partially prevented by the sphingosine kinase inhibitor N,N-dimethylsphingosine. We conclude that the antiapoptotic action of GM1 is largely to be ascribed to an increased sphingosine kinase activity.

Topics: Animals; Apoptosis; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; G(M1) Ganglioside; Heart; Lysophospholipids; Myocardium; Phosphotransferases (Alcohol Group Acceptor); Rats; Second Messenger Systems; Sphingosine

Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein-coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229-240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G(1)/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of cells. Hence, SPP belongs to a novel class of lipid mediators that can function inside and outside cells.

Topics: Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cell Division; Cell Line; Cell Survival; Cytosol; DNA; Enzyme Activation; fas Receptor; Humans; Lysophospholipids; Mice; Peptide Hydrolases; Pertussis Toxin; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Recombinant Fusion Proteins; Second Messenger Systems; Sphingosine; Transfection; Virulence Factors, Bordetella

The ability of high density lipoproteins (HDL) to inhibit cytokine-induced adhesion molecule expression has been demonstrated in their protective function against the development of atherosclerosis and associated coronary heart disease. A key event in atherogenesis is endothelial activation induced by a variety of stimuli such as tumor necrosis factor-alpha (TNF), resulting in the expression of various adhesion proteins. We have recently reported that sphingosine 1-phosphate, generated by sphingosine kinase activation, is a key molecule in mediating TNF-induced adhesion protein expression. We now show that HDL profoundly inhibit TNF-stimulated sphingosine kinase activity in endothelial cells resulting in a decrease in sphingosine 1-phosphate production and adhesion protein expression. HDL also reduced TNF-mediated activation of extracellular signal-regulated kinases and NF-kappaB signaling cascades. Furthermore, HDL enhanced the cellular levels of ceramide which in turn inhibits endothelial activation. Thus, the regulation of sphingolipid signaling in endothelial cells by HDL provides a novel insight into the mechanism of protection against atherosclerosis.

Topics: Arteriosclerosis; Cell Adhesion Molecules; Cells, Cultured; Ceramides; E-Selectin; Endothelium, Vascular; Enzyme Activation; Humans; Lipoproteins, HDL; Lysophospholipids; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingomyelins; Sphingosine; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Short-chain sphinganine analogues 8, 9, 18, and 19, as well as 3-fluoro-sphingosine analogues 25 and 26 were synthesized. Their potential as sphingosine kinase inhibitors was investigated, in combination with previously synthesized sphingosine and fluorinated sphinganine analogues.

Topics: Cell Line; Enzyme Inhibitors; Fluorine Compounds; Humans; Lysophospholipids; Molecular Structure; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Stereoisomerism; Structure-Activity Relationship

Human umbilical vein endothelial cells (HUVEC), like most normal cells, are resistant to tumor necrosis factor-alpha (TNF)-induced apoptosis in spite of TNF activating sphingomyelinase and generating ceramide, a known inducer of apoptosis. Here we report that TNF activates another key enzyme, sphingosine kinase (SphK), in the sphingomyelin metabolic pathway resulting in production of sphingosine-1-phosphate (S1P) and that S1P is a potent antagonist of TNF-mediated apoptosis. The TNF-induced SphK activation is independent of sphingomyelinase and ceramidase activities, suggesting that TNF affects this enzyme directly other than through a mass effect on sphingomyelin degradation. In contrast to normal HUVEC, in a spontaneously transformed endothelial cell line (C11) TNF stimulation failed to activate SphK and induced apoptosis as characterized by morphological and biochemical criteria. Addition of exogenous S1P or increasing endogenous S1P by phorbol ester markedly protected C11 cell line from TNF-induced apoptosis. Conversely, N, N-dimethylsphingosine, an inhibitor of SphK, profoundly sensitized normal HUVEC to killing by TNF. Thus, we demonstrate that the activation of SphK by TNF is an important signaling for protection from the apoptotic effect of TNF in endothelial cells.

Topics: Amidohydrolases; Apoptosis; Cell Line, Transformed; Cell Survival; Cells, Cultured; Ceramidases; Ceramides; DNA Fragmentation; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Humans; In Situ Nick-End Labeling; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingomyelin Phosphodiesterase; Sphingosine; Tumor Necrosis Factor-alpha

Contribution of sphingosine kinase (SPK)-catalyzed production of sphingosine-1-phosphate (SPP), in comparison to phospholipase C (PLC), to Ca(2+) signalling by epidermal growth factor (EGF) was studied in two HEK-293 cell clones (HEK2 and HEK3), expressing functional EGF receptors and exhibiting release of stored Ca(2+) by intracellular SPP. In HEK3 cells, EGF increased [Ca(2+)](i) and stimulated both, SPK and PLC. [Ca(2+)](i) increase, but not PLC stimulation, was strongly reduced by SPK inhibition. In HEK2 cells, EGF similarly stimulated PLC, but did not increase [Ca(2+)](i) or stimulate SPK, suggesting that intracellular SPP production plays a major role for Ca(2+) signalling by EGF in HEK-293 cells.

Topics: Calcium Signaling; Cell Line; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Inositol 1,4,5-Trisphosphate; Kidney; Lysophospholipids; MAP Kinase Signaling System; Phosphatidylinositol Diacylglycerol-Lyase; Phosphotransferases (Alcohol Group Acceptor); Receptors, Muscarinic; Sphingosine; Type C Phospholipases; Virulence Factors, Bordetella

Exogenous sphingosine-1-phosphate (SPP) inhibits chemotactic motility of several transformed cell lines. We have found that SPP at high micromolar concentrations decreased chemotaxis of estrogen-independent (MDA-MB-231 and BT 549) and estrogen-dependent (MCF-7 and ZR-75-1) human breast cancer cells. Because SPP has been implicated as a lipid-signaling molecule with novel dual intra- and intercellular actions, it was of interest to determine whether the effect of SPP on chemotactic motility of human breast cancer cells is mediated intracellularly or through the recently identified endothelial differentiation gene (EDG) family of G protein-coupled SPP receptors. There was no detectable specific binding of [32P]SPP to MDA-MB-231 or MCF-7 cells; however, reverse transcription-PCR analysis revealed that both MDA-MB-231 and MCF-7 cells expressed moderate levels of EDG-3, neither expressed EDG-1, and EDG-5 mRNA was expressed in MCF-7 but not in MDA-MB-231 cells. In contrast to SPP, sphinganine-1-phosphate, which binds to and signals through SPP receptors EDG-1, EDG-3, and EDG-5, had no effect on chemotactic motility of MDA-MB-231 or MCF-7 cells. To further discriminate between intracellular and receptor-mediated actions of SPP, we used caged SPP, a photolyzable derivative of SPP that elevates intracellular levels of SPP after illumination. Caged SPP inhibited chemotactic motility of MDA-MB-231 cells only upon UV irradiation. In addition, in MCF-7 cells, overexpression of sphingosine kinase, the enzyme that produces SPP, inhibited chemotactic motility compared with vector-transfected cells and markedly increased cellular SPP levels in the absence of detectable secretion. Our results suggest that the inhibitory effect of SPP on chemotactic motility of human breast cancer cells is likely mediated through intracellular actions of SPP rather than through cell surface receptors.

Topics: Breast Neoplasms; Chemotaxis; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Photolysis; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Sphingosine; Tumor Cells, Cultured

Aggregation of receptors specific for the constant region of immunoglobulin G activates a repertoire of monocyte responses that can lead ultimately to targeted cell killing via antibody-directed cellular cytotoxicity. The high affinity receptor, FcgammaRI, contains no recognized signaling motif in its cytoplasmic tail but rather utilizes the gamma-chain of FcepsilonRI as an accessory molecule to recruit tyrosine kinases for signal transduction. We show here that, in a human monocytic cell line primed with interferon-gamma, FcgammaRI mobilizes intracellular calcium stores using a novel pathway that involves tyrosine kinase coupling to phospholipase D and resultant downstream activation of sphingosine kinase. Moreover, FcgammaRI is not coupled to phospholipase C; hence, calcium release from intracellular stores occurred in the absence of any measurable rise in inositol triphosphate. Finally, as this novel activation pathway is also shown to be responsible for mediating the vesicular trafficking of internalized immune complexes for degradation, it is likely to play a key role in controlling intracellular events triggered by FcgammaRI.

Topics: Antibodies, Monoclonal; Calcium; Cell Compartmentation; Cell Line; Cross-Linking Reagents; Diglycerides; Endocytosis; Enzyme Activation; Genistein; Humans; Inositol 1,4,5-Trisphosphate; Interferon-gamma; Kinetics; Lysophospholipids; Organelles; Phospholipase D; Phosphotransferases (Alcohol Group Acceptor); Protein-Tyrosine Kinases; Receptors, IgG; Signal Transduction; Sphingosine; Type C Phospholipases

Sphingolipid breakdown products [ceramide, sphingosine, and sphingosine-1-phosphate (SPP)] are emerging as a new class of bioactive molecules. In agreement with previous studies, treatment of human promyelocytic leukemia HL-60 cells with 1-alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induced a transient increase of ceramide levels within 2 h, which then returned to basal levels within 8 h. In contrast, sphingosine kinase activity increased more slowly and reached maximal levels only after 20 h of exposure, leading to a concomitant increase in SPP level. Unlike treatments with cell-permeable ceramide analogues or sphingomyelinase, which induce apoptosis, 1,25-(OH)2D3 did not induce apoptosis, despite the early formation of ceramide. Moreover, prolonged treatment of HL-60 cells with 1,25-(OH)2D3 suppressed ceramide-induced apoptosis. There was a correlation between the time course and dose response of the activation of sphingosine kinase by 1,25-(OH)2D3 and the protection against apoptosis. In contrast, treatment with all-trans-retinoic acid neither stimulated sphingosine kinase activity nor protected cells from ceramide-induced apoptosis. Treatment with SPP protected HL-60 cells from ceramide-induced apoptosis, and N,N-dimethylsphingosine (DMS), a competitive inhibitor of sphingosine kinase, prevented the survival effect of 1,25-(OH)2D3. The effect of DMS was counteracted by SPP, suggesting that SPP is a critical component of the cytoprotective effect of 1,25-(OH)2D3. Chelerythrine chloride, an inhibitor of protein kinase C, markedly reduced sphingosine kinase activity and the apoptosis-sparing effect of 1,25-(OH)2D3, and conversely, the tumor promoter 12-O-tetradecanoylphorhol-13-acetate not only suppressed ceramide-induced apoptosis but also stimulated sphingosine kinase activity. Moreover, the protective effect of 12-O-tetradecanoylphorbol-13-acetate was blocked by DMS. Collectively, our observations indicate that the cytoprotective effect of 1,25-(OH)2D3 is mediated by SPP, which is formed as a consequence of activation of sphingosine kinase.

Topics: Alkaloids; Apoptosis; Benzophenanthridines; Calcitriol; Carcinogens; Cell Survival; Ceramides; Dose-Response Relationship, Drug; Enzyme Inhibitors; HL-60 Cells; Humans; Lysophospholipids; Phenanthridines; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tetradecanoylphorbol Acetate; Time Factors

Formation of inositol 1,4,5-trisphosphate (IP3) by phospholipase C (PLC) with subsequent release of Ca2+ from intracellular stores, is one of the major Ca2+ signalling pathways triggered by G-protein-coupled receptors (GPCRs). However, in a large number of cellular systems, Ca2+ mobilization by GPCRs apparently occurs independently of the PLC-IP3 pathway, mediated by an as yet unknown mechanism. The present study investigated whether sphingosine kinase activation, leading to production of sphingosine-1-phosphate (SPP), is involved in GPCR-mediated Ca2+ signalling as proposed for platelet-derived growth factor and FcepsilonRI antigen receptors. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by m2 and m3 muscarinic acetylcholine receptors (mAChRs) expressed in HEK-293 cells without affecting mAChR-induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulated production of SPP in HEK-293 cells. Finally, intracellular injection of SPP induced a rapid and transient Ca2+ mobilization in HEK-293 cells which was not antagonized by heparin. We conclude that mAChRs utilize the sphingosine kinase-SPP pathway in addition to PLC-IP3 to mediate Ca2+ mobilization. As Ca2+ signalling by various, but not all, GPCRs in different cell types was likewise attenuated by the sphingosine kinase inhibitors, we suggest a general role for sphingosine kinase, besides PLC, in mediation of GPCR-induced Ca2+ signalling.

Topics: Animals; Calcium; Cattle; Cell Line; Enzyme Activation; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Lysophospholipids; Microinjections; Phosphotransferases (Alcohol Group Acceptor); Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Bradykinin; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Receptors, Muscarinic; Signal Transduction; Sphingosine; Tumor Cells, Cultured

Sphingosine kinase (SK) catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, which is a second messenger involved in the proliferative responses of mammalian cells. Although the yeast Saccharomyces cerevisiae has similar phosphorylated sphingoid bases which appear to be involved in growth regulation and the response to stress, SK activity had not been previously demonstrated in yeast. In this study, an in vitro system was set up to characterize yeast SK activity. Activity was detected in the cytosol at neutral pH and 37 degreesC. Yeast SK phosphorylated the sphingoid bases sphingosine, dihydrosphingosine and phytosphingosine. (d,l)-threo-dihydrosphingosine, an inhibitor of mammalian SK, did not inhibit the yeast enzyme. Unique properties of yeast SK were an optimal temperature of 43 degreesC, and in vivo activation during nutrient deprivation. Spontaneous mutants with diminished SK activity were isolated utilizing a screen for resistance to sphingosine in a sphingosine-phosphate-lyase deletion background. Abnormal growth and heat sensitivity were observed in these mutants. These findings suggest that SK may function as a stress-response protein in yeast.

Topics: Cell Division; Cell Survival; Enzyme Activation; Enzyme Inhibitors; Heat-Shock Proteins; Hydrogen-Ion Concentration; Kinetics; Lysophospholipids; Mutation; Phenotype; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Saccharomyces cerevisiae; Sphingosine; Spores; Temperature

During the exposure of human myelocytic leukemia HL-60 cells to phorbol diester, nonadherent cells die by apoptosis, but adherent cells survive and growth-arrest at G1 phase of the cell cycle. Here we have shown that the adherent cells rapidly died by apoptosis after forced detachment (anoikis), indicating that phorbol diester induced apoptosis by default. Dimethylsphingosine induced apoptosis in the adherent cells, and sphingosine-1-phosphate rescued the detached cells from apoptosis. Sphingosine kinase activity in adherent cells was higher than that in nonadherent cells and was decreased by forced detachment. It is likely that the phorbol diester-induced apoptosis and the adhesion-mediated survival are modulated by sphingosine and sphingosine-1-phosphate, respectively. The adherent cells were reverted and reproliferated when allowed to spontaneously detach from plastic surfaces by removal of phorbol diester. This result suggests that after removal of phorbol diester, the commitment signal of apoptosis by default is lost faster than the survival signal by adherence.

Topics: Apoptosis; Cell Adhesion; HL-60 Cells; Humans; Indoles; Lysophospholipids; Nuclear Proteins; Phorbol 12,13-Dibutyrate; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Transcription Factors

The phosphorylated derivative of sphingosine, sphingosine-1-phosphate, is a short-living metabolite of ultimate ceramide degradation and was shown to act as an intracellular signaling molecule, stimulating cell proliferation in quiescent Swiss 3T3 fibroblasts and inducing the release of calcium from intracellular stores (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell. Biol. 114, 155-167). In the present study, 24-h treatment of Swiss 3T3 fibroblasts with the synthetic sphingosine analogue cis-4-methylsphingosine resulted in proliferation of quiescent Swiss 3T3 fibroblasts that was 3-fold stronger than that of equimolar sphingosine-1-phosphate. The phosphorylated derivative of cis-4-methylsphingosine accumulated drastically in the cells. Simultaneous treatment with the sphingosine kinase inhibitor L-threo-sphinganine reduced both the amount of phosphorylated cis-4-methylsphingosine and cell proliferation induced by this compound by about 50%, indicating that the phosphorylated derivative mediated the proliferative stimulus. The mitogenic effect of cis-4-methylsphingosine was associated with a mobilization of intracellular calcium in Swiss 3T3 fibroblasts that was similar to that induced by sphingosine-1-phosphate. The results demonstrate that the phosphorylated derivative of cis-4-methylsphingosine mimics the previously reported mitogenic action of sphingosine-1-phosphate in Swiss 3T3 cells, and the stronger effect most likely corresponds to the unusual accumulation of this compound.

Topics: 3T3 Cells; Animals; Apoptosis; Calcium; Cell Division; Fibroblasts; Isomerism; Lysophospholipids; Mice; Mitogens; Molecular Mimicry; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Sphingosine 1-phosphate (SPP), a lipid second messenger formed by the action of sphingosine kinase, has been implicated in regulating diverse biological processes, including growth, survival, and differentiation. N,N-Dimethylsphingosine (DMS) inhibits sphingosine kinase and has been used to investigate the biological roles of SPP; however, little is known of the mechanism of inhibition of sphingosine kinase by DMS. In addition, DMS has been shown to inhibit protein kinase C in vitro. Here we report that DMS is a competitive inhibitor of sphingosine kinase from U937 monoblastic leukemia cells, Swiss 3T3 fibroblasts, and PC12 pheochromocytoma cells. DMS decreases basal levels of SPP and prevents increases in SPP in response to physiological stimuli known to activate sphingosine kinase. DMS also effectively increases cellular levels of ceramide in a variety of cell types, and resetting of the ceramide/SPP rheostat may account for the pro-apoptotic effects of DMS. Moreover, DMS, at concentrations which effectively inhibit sphingosine kinase, has no effect on protein kinase C activity or its membrane translocation. Thus, DMS acts as a specific competitive inhibitor of sphingosine kinase in diverse cell types and is a useful tool to elucidate the role of SPP as an intracellular second messenger.

Topics: 3T3 Cells; Animals; Ceramides; Enzyme Activation; Enzyme Inhibitors; Humans; Lysophospholipids; Mice; PC12 Cells; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Rats; Sphingosine; Tumor Cells, Cultured

The signaling pathways that couple tumor necrosis factor-alpha (TNFalpha) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFalpha induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFalpha resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFalpha on endothelial cells leading to extracellular signal-regulated kinases and NF-kappaB activation, whereas ceramide or sphingosine was not. Furthermore, N, N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFalpha-induced extracellular signal-regulated kinases and NF-kappaB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFalpha-induced endothelial cell activation.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; E-Selectin; Endothelium, Vascular; Enzyme Activation; Gene Expression Regulation; Humans; JNK Mitogen-Activated Protein Kinases; Kinetics; Lysophospholipids; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingomyelins; Sphingosine; Tumor Necrosis Factor-alpha; Umbilical Veins; Vascular Cell Adhesion Molecule-1

Platelet-derived growth factor (PDGF) and serum, but not epidermal growth factor (EGF), stimulated sphingosine kinase activity in Swiss 3T3 fibroblasts and increased intracellular concentrations of sphingosine 1-phosphate (SPP), a sphingolipid second messenger (Olivera, A., and Spiegel, S. (1993) Nature 365, 557-560). We report herein that DL-threo-dihydrosphingosine (DHS), a competitive inhibitor of sphingosine kinase that prevents PDGF-induced SPP formation, specifically inhibited the activation of two cyclin-dependent kinases (p34(cdc2) kinase and Cdk2 kinase) induced by PDGF, but not by EGF. SPP reversed the inhibitory effects of DHS on PDGF-stimulated cyclin-dependent kinases and DNA synthesis, demonstrating that the DHS effects were mediated via inhibition of sphingosine kinase. DHS also markedly reduced PDGF-stimulated but not EGF-stimulated mitogen-activated protein kinase activity and DNA binding activity of activator protein-1. Examination of the early signaling events of PDGF action revealed that DHS did not affect PDGF-induced autophosphorylation of the growth factor receptor or phosphorylation of the SH2/SH3 adaptor protein Shc and its association with Grb2. This sphingosine kinase inhibitor did not abrogate activation of phosphatidylinositol 3-kinase by PDGF. In agreement, treatment with SPP had no effect on these responses but did, however, potently stimulate phosphorylation of Crk, another SH2/SH3 adaptor protein. Moreover, DHS inhibited PDGF-stimulated, but not EGF-stimulated, Crk phosphorylation. Thus, regulation of sphingosine kinase activity defines divergence in signal transduction pathways of PDGF and EGF receptors leading to mitogen-activated protein kinase activation.

Topics: 3T3 Cells; Animals; Calcium-Calmodulin-Dependent Protein Kinases; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; DNA; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Kinetics; Lysophospholipids; Mice; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; Receptors, Platelet-Derived Growth Factor; Second Messenger Systems; Signal Transduction; Sphingosine; Transcription Factor AP-1

Sphingolipid metabolites, such as ceramide and sphingosine-1-phosphate (SPP), are emerging as a new class of second messengers involved in cellular proliferation, differentiation, and apoptosis. Nerve growth factor (NGF), a neurotrophic factor for pheochromocytoma PC12 cells, induced a biphasic increase in the activity of sphingosine kinase, the enzyme that catalyzes the formation of SPP. This activation was blocked by K252a, an inhibitor of tyrosine kinase A (trkA). A rapid 1.7-fold increase was followed by a marked prolonged increase reaching a maximum of fourfold to fivefold stimulation with a concomitant increase in SPP levels and a corresponding decrease in endogenous sphingosine levels. Levels of ceramide, the precursor of sphingosine, were only slightly decreased by NGF in serum-containing medium. However, NGF decreased the elevation of ceramide induced by serum withdrawal. Treatment of PC12 cells with SPP did not induce neurite outgrowth or neurofilament expression, yet it enhanced neurofilament expression elicited by suboptimal doses of NGF. Moreover, SPP also protected PC12 cells from apoptosis induced by serum withdrawal. To further substantiate a role for SPP in the cytoprotective actions of NGF, we found that N, N-dimethylsphingosine, a competitive inhibitor of sphingosine kinase, also induced apoptosis and interfered with the survival effect of NGF. These effects were counteracted by exogenous SPP. Moreover, other structurally related compounds, such as dihydrosphingosine 1-phosphate and lysophosphatidic acid, had no significant protective effects. Our results suggest that activation of sphingosine kinase and subsequent formation of SPP may play an important role in the differentiation and survival effects induced by NGF.

Topics: Animals; Apoptosis; Carrier Proteins; Cell Differentiation; Cell Survival; Culture Media, Serum-Free; Lysophospholipids; Membrane Proteins; Nerve Growth Factors; Neurons; PC12 Cells; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Rats; Receptor, trkA; Signal Transduction; Sphingosine

Exogenous sphingosine 1-phosphate (S1P), like lysophosphatidic acid (LPA), induced neurite retraction or cell rounding in differentiated PC12 cells. The lysosphingolipid-induced shape change was detected at as low as 1 nM; however, a significant accumulation of intracellular S1P was not detected until 1 microM S1P was applied. Moreover, although exogenous sphingosine caused a significant increase in intracellular S1P by sphingosine kinase-catalyzed phosphorylation, the effect on the shape change was marginal. Exposure of the cells to the immobilized S1P in which the lipid was covalently linked to a glass carrier also resulted in the shape change. These results suggest that the exogenous S1P-induced shape change does not require uptake of the lipid into the cells but possibly requires interaction with a cell surface receptor in the neuronal cells.

Topics: Animals; Cell Differentiation; Cell Size; Lysophospholipids; Neurites; PC12 Cells; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptors, Cell Surface; Sphingosine; Tetradecanoylphorbol Acetate

Potential sphingosine (Sph) metabolites include phosphorylated, N-acylated, and N-methylated derivatives. Phosphorylated Sph, i.e., sphingosine 1-phosphate (Sph-1-P), may act as an autocrine stimulator of blood platelets, as it is abundantly stored in platelets and released extracellularly and its exogenous addition induces platelet activation. In this study, we evaluated Sph-1-P formation and its effects in human platelets in the presence of other Sph metabolites. On addition of [3H]Sph to intact platelets, the label was rapidly converted to Sph-1-P. This conversion into [3H]Sph-1-P was inhibited by N,N-dimethylsphingosine (DMS) in a dose-dependent manner, but not by other structurally related Sph derivatives, including ceramide. The inhibition of Sph-1-P formation by DMS was reproduced using a cell-free system (Sph kinase obtained from platelet cytosolic fractions) and much stronger than that by DL-threo-dihydrosphingosine, which had been considered to be the strongest inhibitor of Sph kinase. Administration of DMS to intact platelets resulted in a decrease in Sph-1-P mass and an increase in Sph mass. Furthermore, DMS inhibited the release of Sph-1-P from platelets stimulated with 12-O-tetradecanoylphorbol 13-acetate and inhibited platelet aggregation induced by exogenous addition of Sph-1-P. Collectively, our results indicate that DMS is useful as a Sph kinase inhibitor and that Sph-1-P actions as an autocrine stimulator of platelets are inhibited by DMS.

Topics: Blood Platelets; Cell-Free System; Ceramides; Enzyme Inhibitors; Humans; In Vitro Techniques; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Platelet Aggregation; Sphingosine; Tetradecanoylphorbol Acetate

Calcium mobilization through antigen receptors, including high-affinity IgE receptors (Fc epsilon RI), is thought to be mediated by inositol-1,4,5-trisphosphate production (InsP3). Here we show that antigen clustering of Fc epsilon RI on the rat mast-cell line (RBL-2H3) activates a sphingosine kinase (SK) and produces sphingosine-1-phosphate (S1P), and alternative second messenger for intracellular calcium mobilization. The sphingosine analogue, D-L-threo-dihydrosphingosine (DHS), inhibits the SK enzyme competitively with a dissociation constant, K1, of 5 to 18 microM. This inhibition substantially suppresses the Fc epsilon RI-mediated calcium signal, but leaves intact the syk tyrosine kinase activation and the small InsP3 production. The entire InsP3-dependent pathway activated by a transfected G-protein coupled receptor, used here as a positive control, also remained intact. Thus Fc epsilon RI principally utilizes a SK pathway to mobilize calcium.

Topics: 3T3 Cells; Animals; Calcium; Cell Line; Cytosol; Enzyme Activation; Enzyme Inhibitors; Genistein; Humans; Inositol 1,4,5-Trisphosphate; Isoflavones; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Protein-Tyrosine Kinases; Rats; Receptors, IgE; Receptors, Muscarinic; Signal Transduction; Sphingosine

Ceramide is an important regulatory participant of programmed cell death (apoptosis) induced by tumour-necrosis factor (TNF)-alpha and Fas ligand, members of the TNF superfamily. Conversely, sphingosine and sphingosine-1-phosphate, which are metabolites of ceramide, induce mitogenesis and have been implicated as second messengers in cellular proliferation induced by platelet-derived growth factor and serum. Here we report that sphingosine-1-phosphate prevents the appearance of the key features of apoptosis, namely intranucleosomal DNA fragmentation and morphological changes, which result from increased concentrations of ceramide. Furthermore, inhibition of ceramide-mediated apoptosis by activation of protein kinase C results from stimulation of sphingosine kinase and the concomitant increase in intracellular sphingosine-1-phosphate. Finally sphingosine-1-phosphate not only stimulates the extracellular signal-regulated kinase (ERK) pathway, it counteracts the ceramide-induced activation of stress-activated protein kinase (SAPK/JNK). Thus, the balance between the intracellular levels of ceramide and sphingosine-1-phosphate and their regulatory effects on different family members of mitogen-activated protein kinases determines the fate of the cell.

Topics: 3T3 Cells; Animals; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Ceramides; DNA Damage; Enzyme Activation; fas Receptor; HL-60 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mitosis; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, is mitogenic for quiescent Swiss 3T3 fibroblasts. Recently, sphingolipids metabolites, ceramide, sphingosine and sphingosine-1-phosphate, have been implicated as second messengers in cell growth regulation and differentiation. In this paper, we examined the possibility that interaction of the B subunit with membrane GM1 leads to alterations in metabolism of glycosphingolipids and that increased levels of sphingolipids metabolites may mediate the biological effects of the B subunit. While the B subunit did not induce a change in the level of ceramide or sphingosine, the level of sphingosine-1-phosphate was rapidly and transiently increased. The B subunit also transiently activated cytosolic sphingosine kinase activity, which catalyzes the phosphorylation of the primary hydroxyl group of sphingosine to produce sphingosine-1-phosphate. To determine whether the increase in sphingosine-1-phosphate level plays a role in B subunit-induced mitogenicity, we used a competitive inhibitor of sphingosine kinase, D,L-threo-dihydrosphingosine. D,L-thereo-Dihydrosphingosine not only inhibited B subunit-induced DNA synthesis by 26%, it also reduced its ability to stimulate DNA-binding activity of the transcription factor AP-1. This sphingosine kinase inhibitor also inhibited B subunit-induced increases in the activity of cell cycle-regulated, cyclin-dependent serine/threonine kinases, cdk2 and p34cdc2. These findings suggest that sphingosine-1-phosphate may play a role in the signal transduction pathways activated by binding of the B subunit to endogenous ganglioside GM1.

Topics: 3T3 Cells; Animals; Antibodies; Binding, Competitive; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Division; Ceramides; Cholera Toxin; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; DNA; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; G(M1) Ganglioside; Lysophospholipids; Mice; Mitogens; Peptide Fragments; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; Sphingolipids; Sphingosine; Tetradecanoylphorbol Acetate; Transcription Factor AP-1

Sphingosine-1-phosphate (Sph-1-P) has been implicated as a second messenger in control of cell motility and proliferation (e.g., Sadahira Y, et al., PNAS 89:9686, 1992; Olivera A & Spiegel S, Nature 365:557, 1993). The control mechanism for its synthesis, as catalyzed by sphingosine kinase, is crucial in signal transduction. Synthesis of Sph-1-P in Balb/c 3T3 fibroblasts (A31 variant) is strongly up-regulated by brief treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA). Level of Sph-1-P in PKC-depleted cells is 10-fold higher than in undepleted cells, and a further 5-fold increase occurs after treatment with TPA. In Swiss 3T3 and B16 melanoma cells, Sph-1-P level was unaffected by TPA treatment. Thus, the effect of TPA on Sph-1-P synthesis appears to be cell type-specific.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3T3 Cells; Alkaloids; Animals; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Isoenzymes; Isoquinolines; Kinetics; Lysophospholipids; Mice; Mice, Inbred BALB C; Phosphotransferases (Alcohol Group Acceptor); Piperazines; Protein Kinase C; Sphingosine; Staurosporine; Tetradecanoylphorbol Acetate

Growth signalling networks that use glycerophospholipid metabolites as second messengers have been well characterized, but less is known of the second messengers derived from sphingolipids, another major class of membrane lipids. A tantalizing link between sphingolipids and cellular proliferation has emerged from the discovery that the sphingolipid metabolites sphingosine and sphingosine-1-phosphate stimulate growth of quiescent Swiss 3T3 fibroblasts by a pathway that is independent of protein kinase C. Sphingosine-1-phosphate is rapidly produced from sphingosine and may mediate its biological effects. Furthermore, sphingosine-1-phosphate triggers the dual signal transduction pathways of calcium mobilization and activation of phospholipase D, prominent events in the control of cellular proliferation. Here we report that activation of sphingosine kinase and the formation of sphingosine-1-phosphate are important in the signal transduction pathways activated by the potent mitogens platelet-derived growth factor (PDGF) and fetal calf serum (FCS).

Topics: 3T3 Cells; Animals; Cattle; Cell Division; DNA; Enzyme Activation; Epidermal Growth Factor; Fetal Blood; Humans; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Second Messenger Systems; Sphingosine