jte-013 and sphingosine-1-phosphate

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

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

Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator generated when a cell membrane or its components are damaged by various factors. S1P regulates diverse cell activities via S1P receptors (S1PRs). Keratinocytes express S1PR1-5. Although it is known that S1PRs control keratinocyte differentiation, apoptosis, and wound healing, S1PR functions in keratinocyte infections have not been fully elucidated. We propose that the S1P-S1PR axis in keratinocytes works as a biosensor for bacterial invasion. Indeed, in human impetigo infection, we found high epidermal expression of S1PR1 and S1PR2 in the skin. Furthermore, in normal human epidermal keratinocytes in vitro, treatment with Staphylococcus aureus bacterial supernatant not only induced S1P production but also increased the transcription of S1PR2, confirming our in vivo observation, as well as increased the levels of TNFA, IL36G, IL6, and IL8 mRNAs. However, direct treatment of normal human epidermal keratinocytes with S1P increased the expressions of IL36G, TNFA, and IL8, but not IL6. In both S1P- and S. aureus bacterial supernatant-treated normal human epidermal keratinocytes, S1PR1 knockdown reduced IL36G, TNFA, and IL8 transcription, and the S1PR2 antagonist JTE013 blocked the secretion of these cytokines. Overall, we have proven that during infections, keratinocytes communicate damage by using S1P release and tight control of S1PR1 and 2.

Topics: Cells, Cultured; Cytokines; Gene Knockdown Techniques; Host-Pathogen Interactions; Humans; Impetigo; Keratinocytes; Lysophospholipids; Primary Cell Culture; Pyrazoles; Pyridines; Signal Transduction; Skin; Sphingosine; Sphingosine-1-Phosphate Receptors; Staphylococcus aureus

Epicardial progenitor cells (EpiCs) which are derived from the proepicardium have the potential to differentiate into coronary vascular smooth muscle cells during development. Whether sphingosine 1-phosphate (S1P), a highly hydrophobic zwitterionic lysophospholipid in signal transduction, induces the differentiation of EpiCs is unknown. In the present study, we demonstrated that S1P significantly induced the expression of smooth muscle cell specific markers α-smooth muscle actin and myosin heavy chain 11 in the EpiCs. And the smooth muscle cells differentiated from the EpiCs stimulated by S1P were further evaluated by gel contraction assay. To further confirm the major subtype of sphingosine 1-phosphate receptors (S1PRs) involved in the differentiation of EpiCs, we used the agonists and antagonists of different S1PRs. The results showed that the S1P1/S1P3 antagonist VPC23019 and the S1P2 antagonist JTE013 significantly attenuated EpiCs differentiation, while the S1P1 agonist SEW2871 and antagonist W146 did not affect EpiCs differentiation. These results collectively suggested that S1P, principally through its receptor S1P3, increases EpiCs differentiation into VSMCs and thus indicated the importance of S1P signaling in the embryonic coronary vasculature, while S1P2 plays a secondary role.

Topics: Actins; Animals; Cell Differentiation; Cells, Cultured; Gene Expression Regulation; Lysophospholipids; Mice; Mice, Inbred C57BL; Mouse Embryonic Stem Cells; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Pericardium; Phosphoserine; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Sphingosine

We conducted this experimental study to analyze the relationship between sphingosine-1-phosphate (S1P)-induced mitogen-activated protein (MAP) kinase pathways and keloid formation. We collected samples of the normal tissue and the keloid tissue from 10 normal healthy individuals and 12 patients with keloid scars, respectively. Then, we compared the level of sphingosine-1-phosphate receptor (S1PR1/S1PR2) mRNA/protein expression between the normal tissue and the keloid tissue. Moreover, we also compared the level of S1PR protein expression, that of S1P-induced COL1A1 (collagen Type I, α-1 chain) expression, that of S1P-induced JNK/ERK phosphorylation, that of S1P-induced COL1A1 expression following the treatment with 30 μM PD98059 (ERK inhibitor) or 30 μM SP600125 (JNK inhibitor) and that of S1P-induced COL1A1 expression following the treatment with W146 (S1PR1 inhibitor) or JTE013 (S1PR2 inhibitor) between the normal fibroblasts and the keloid fibroblasts. We found that the level of S1PR1/S1PR2 mRNA/protein expression was significantly higher in the keloid tissue as compared with the normal tissue. Our results also showed that the level of S1P-induced COL1A1 expression and that of S1P-induced JNK/ERK phosphorylation were significantly higher in the keloid fibroblasts as compared with the normal ones (P < 0.05). Furthermore, there were significant decreases in the level of S1P-induced COL1A1 expression when the keloid fibroblasts were treated with 30 μM SP600125 or 30 μM PD98059 and that of S1P-induced COL1A1 expression when the treated with 100 nM W146 or 100 nM JTE013 (P < 0.05). Our results indicate that S1P-induced signal transduction is associated with increased collagen synthesis via S1PR-mediated signaling pathways in the keloid tissue.

Topics: Adult; Anilides; Anthracenes; Cell Line; Collagen Type I; Collagen Type I, alpha 1 Chain; Female; Fibroblasts; Flavonoids; Humans; Keloid; Lysophospholipids; Male; MAP Kinase Signaling System; Middle Aged; Mitogen-Activated Protein Kinases; Organophosphonates; Phosphorylation; Pyrazoles; Pyridines; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Young Adult

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

Multilineage-differentiating stress enduring (Muse) cells, pluripotent marker stage-specific embryonic antigen-3. The main objective of this study is to clarify the efficiency of intravenously infused rabbit autograft, allograft, and xenograft (human) bone marrow-Muse cells in a rabbit acute myocardial infarction model and their mechanisms of tissue repair.. In vivo dynamics of Nano-lantern-labeled Muse cells showed preferential homing of the cells to the postinfarct heart at 3 days and 2 weeks, with ≈14.5% of injected GFP (green fluorescent protein)-Muse cells estimated to be engrafted into the heart at 3 days. The migration and homing of the Muse cells was confirmed pharmacologically (S1PR2 [sphingosine monophosphate receptor 2]-specific antagonist JTE-013 coinjection) and genetically (S1PR2-siRNA [small interfering ribonucleic acid]-introduced Muse cells) to be mediated through the S1P (sphingosine monophosphate)-S1PR2 axis. They spontaneously differentiated into cells positive for cardiac markers, such as cardiac troponin-I, sarcomeric α-actinin, and connexin-43, and vascular markers. GCaMP3 (GFP-based Ca calmodulin probe)-labeled Muse cells that engrafted into the ischemic region exhibited increased GCaMP3 fluorescence during systole and decreased fluorescence during diastole. Infarct size was reduced by ≈52%, and the ejection fraction was increased by ≈38% compared with vehicle injection at 2 months, ≈2.5 and ≈2.1 times higher, respectively, than that induced by mesenchymal stem cells. These effects were partially attenuated by the administration of. Muse cells may provide reparative effects and robust functional recovery and may, thus, provide a novel strategy for the treatment of acute myocardial infarction.

Topics: Allografts; Animals; Autografts; Cell Differentiation; Cell Movement; GATA4 Transcription Factor; Graft Survival; Green Fluorescent Proteins; Heterografts; Humans; Luciferases; Luminescent Proteins; Lysophospholipids; Male; Myocardial Infarction; Pluripotent Stem Cells; Pyrazoles; Pyridines; Rabbits; Receptors, Lysosphingolipid; Recombinant Fusion Proteins; RNA Interference; RNA, Small Interfering; Species Specificity; Sphingosine; Sphingosine-1-Phosphate Receptors

Macrophages, the most plastic cells in the haematopoietic system, are found in all tissues and show great functional heterogeneity. Sphingosine 1-phosphate (S1P)/ S1P receptors (S1PRs) system is widely involved in the process of inflammatory disease, whereas little evidence concerning its role in functional macrophage polarization is available. Thus, the present study was designed to evaluate the effects of S1P/S1PRs on functional polarization of macrophage in mouse bone marrow (BM)-derived monocyte/macrophages (BMMs).. For the detection of M1 macrophage markers, such as CD86, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1/ chemokine (C-C motif) ligand (CCL) 2, nitric oxide synthase (NOS) 2, and macrophage inflammatory protein (MIP)-1β, RT-qPCR and cytometric bead array (CBA) were performed in cultured primary BMMs after the treatment with selective S1PR2/3 antagonists or specific S1PRs siRNA. Western blotting and immunofluorescence were used for the detection of phosphorylation of JNK1/2.. BMMs expressed S1PR1-3 and interestingly, S1PR2/3, but not S1PR1, mediates S1P-induced M1 macrophage polarization of BMMs as their siRNA or antagonists reduced M1 genes' expression. We found that PTX (inhibitor of G(α)i/o), LY294002 (inhibitor of PI3K) or SP600125 (inhibitor of JNK1/2) prevented up-regulation of M1 genes expression mediated by S1P/S1PR2/3 signal, and S1P-induced JNK phosphorylation was inhibited by antagonists of S1PR2/3, PTX or LY294002.. Collectively, our results demonstrate that S1P/S1PR2/3 plays a key role in regulating M1 type polarization of BMMs and acts by activating G(α)i/o/PI3K/JNK signaling pathway, with potential implications for new approaches to inflammatory liver disease therapy.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Arginase; Cell Movement; Chemokines; Chromones; Cytokines; GTP-Binding Protein alpha Subunits, Gi-Go; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; Macrophages; Mice; Mice, Inbred ICR; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Pyrazoles; Pyridines; Receptors, Cell Surface; Receptors, Lysosphingolipid; RNA Interference; RNA, Small Interfering; Signal Transduction; Sphingosine

At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)-S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells.

Topics: Animals; Carcinogenesis; Cell Movement; Dogs; Enzyme Activation; Epithelial Cells; Filamins; Humans; Lysophospholipids; Madin Darby Canine Kidney Cells; Mutation, Missense; Neoplasms; Proto-Oncogene Proteins p21(ras); Pyrazoles; Pyridines; Receptors, Lysosphingolipid; rho-Associated Kinases; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Diabetes strongly associates with microvascular complications that ultimately promote multiorgan failure. Altered myogenic responsiveness compromises tissue perfusion, aggravates hypertension, and sets the stage for later permanent structural changes to the microcirculation. We demonstrate that skeletal muscle resistance arteries isolated from patients with diabetes have augmented myogenic tone, despite reasonable blood glucose control. To understand the mechanisms, we titrated a standard diabetes mouse model (high-fat diet plus streptozotocin [HFD/STZ]) to induce a mild increase in blood glucose levels. HFD/STZ treatment induced a progressive myogenic tone augmentation in mesenteric and olfactory cerebral arteries; neither HFD nor STZ alone had an effect on blood glucose or resistance artery myogenic tone. Using gene deletion models that eliminate tumor necrosis factor (TNF) or sphingosine kinase 1, we demonstrate that vascular smooth muscle cell TNF drives the elevation of myogenic tone via enhanced sphingosine-1-phosphate (S1P) signaling. Therapeutically antagonizing TNF (etanercept) or S1P (JTE013) signaling corrects this defect. Our investigation concludes that vascular smooth muscle cell TNF augments resistance artery myogenic vasoconstriction in a diabetes model that induces a small elevation of blood glucose. Our data demonstrate that microvascular reactivity is an early disease marker and advocate establishing therapies that strategically target the microcirculation.

Topics: Animals; Blood Glucose; Cerebral Arteries; Diabetes Mellitus, Experimental; Etanercept; Humans; Lysophospholipids; Mice; Muscle, Smooth, Vascular; Myography; Pyrazoles; Pyridines; Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha; Vascular Resistance

Sphingosine-1-phosphate (S1P) is a bioactive phospholipid that acts as a signal transducer by binding to S1P receptors (S1PR) 1 to 5. The S1P/S1PRs pathway has been associated with remodeling and allergic inflammation in asthma, but the expression pattern of S1PR and its effects on non-immune cells have not been completely clarified. The aim of this study was to examine the contribution of the signaling of S1P and S1PRs expressed in airway epithelial cells (ECs) to asthma responses in mice.. Bronchial asthma was experimentally induced in BALB/c mice by ovalbumin (OVA) sensitization followed by an OVA inhalation challenge. The effects of S1PR antagonists on the development of asthma were analyzed 24 h after the OVA challenge.. Immunohistological analysis revealed S1PR1-3 expression on mouse airway ECs. Quantitative real-time polymerase chain reaction demonstrated that S1P greatly stimulated the induction of CCL3 and TIMP2 mRNA in human airway ECs, i.e., BEAS-2B cells, in a dose-dependent manner. Pretreatment with the S1PR2 antagonist JTE013 inhibited the CCL3 gene expression in BEAS-2B cells. Immunohistological analysis also showed that the expression level of CCL3 was attenuated by JTE013 in asthmatic mice. Furthermore, JTE013 as well as anti-CCL3 antibody attenuated allergic responses. Intratracheal administration of JTE013 also attenuated eosinophilic reactions in bronchoalveolar lavage fluids. S1P induced transcription factor NFκB activation, while JTE013 greatly reduced the NFκB activation.. JTE013 attenuated allergic airway reactions by regulating CCL3 production from bronchial ECs. The intratracheal administration of JTE013 may be a promising therapeutic strategy for bronchial asthma.

Topics: Animals; Anti-Asthmatic Agents; Anti-Inflammatory Agents; Asthma; Bronchi; Chemokine CCL3; Cytokines; Disease Models, Animal; Epithelial Cells; Female; Inflammation Mediators; Lysophospholipids; Mice, Inbred BALB C; NF-kappa B; Ovalbumin; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; STAT3 Transcription Factor; Tissue Inhibitor of Metalloproteinase-2

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid produced by mast cells (MCs) on cross-linking of their high-affinity receptors for IgE by antigen that can amplify MC responses by binding to its S1P receptors. An acute MC-dependent allergic reaction can lead to systemic shock, but the early events of its development in lung tissues have not been investigated, and S1P functions in the onset of allergic processes remain to be examined.. We used a highly specific neutralizing anti-S1P antibody (mAb) and the sphingosine-1-phosphate receptor 2 (S1PR2) antagonist JTE-013 to study the signaling contributions of S1P and S1PR2 to MC- and IgE-dependent airway allergic responses in mice within minutes after antigen challenge.. Allergic reaction was triggered by a single intraperitoneal dose of antigen in sensitized mice pretreated intraperitoneally with anti-S1P, isotype control mAb, JTE-013, or vehicle before antigen challenge.. Kinetics experiments revealed early pulmonary infiltration of mostly T cells around blood vessels of sensitized mice 20 minutes after antigen exposure. Pretreatment with anti-S1P mAb inhibited in vitro MC activation, as well as in vivo development of airway infiltration and MC activation, reducing serum levels of histamine, cytokines, and the chemokines monocyte chemoattractant protein 1/CCL2, macrophage inflammatory protein 1α/CCL3, and RANTES/CCL5. S1PR2 antagonism or deficiency or MC deficiency recapitulated these results. Both in vitro and in vivo experiments demonstrated MC S1PR2 dependency for chemokine release and the necessity for signal transducer and activator of transcription 3 activation.. Activation of S1PR2 by S1P and downstream signal transducer and activator of transcription 3 signaling in MCs regulate early T-cell recruitment to antigen-challenged lungs through chemokine production.

Topics: Adoptive Transfer; Animals; Antigens; Cell Degranulation; Chemokines; Cytokines; Disease Models, Animal; Female; Humans; Hypersensitivity; Lung; Lysophospholipids; Macrophage Activation; Macrophages; Mast Cells; Mice; Mice, Transgenic; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; STAT3 Transcription Factor; T-Lymphocytes

Glucolipotoxic stress has been identified as a key player in the progression of pancreatic β-cell dysfunction contributing to insulin resistance and the development of type 2 diabetes mellitus (T2D). It has been suggested that bioactive lipid intermediates, formed under lipotoxic conditions, are involved in these processes. Here, we show that sphingosine 1-phosphate (S1P) levels are not only increased in palmitate-stimulated pancreatic β-cells but also regulate β-cell homeostasis in a divergent manner. Although S1P possesses a prosurvival effect in β-cells, an enhanced level of the sphingolipid antagonizes insulin-mediated cell growth and survival via the sphingosine 1-phosphate receptor subtype 2 (S1P2) followed by an inhibition of Akt-signaling. In an attempt to investigate the role of the S1P/S1P2 axis in vivo, the New Zealand obese (NZO) diabetic mouse model, characterized by β-cell loss under high-fat diet (HFD) conditions, was used. The occurrence of T2D was accompanied by an increase of plasma S1P levels. To examine whether S1P contributes to the morphologic changes of islets via S1P2, the receptor antagonist JTE-013 was administered. Most interestingly, JTE-013 rescued β-cell damage clearly indicating an important role of the S1P2 in β-cell homeostasis. Therefore, the present study provides a new therapeutic strategy to diminish β-cell dysfunction and the development of T2D.

Topics: Animals; Diabetes Mellitus, Type 2; Diet, High-Fat; Disease Models, Animal; Insulin; Insulin Resistance; Insulin-Secreting Cells; Lysophospholipids; Male; Mice; Mice, Obese; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Cholangiocarcinoma (CCA) is characterized by a uniquely aggressive behavior and lack of effective targeted therapies. After analyzing the gene expression profiles of seven paired intrahepatic CCA microarrays, a novel sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) pathway and a novel target gene, SPHK1, were identified. We hypothesized that therapeutic targeting of this pathway can be used to kill intrahepatic cholangiocarcinoma (CCA) cells. High levels of SPHK1 protein expression, which was evaluated by immunohistochemical staining of samples from 96 patients with intrahepatic CCA, correlated with poor overall survival. The SPHK1 inhibitor SK1-I demonstrated potent antiproliferative activity in vitro and in vivo. SK1-I modulated the balance of ceramide-sphinogosine-S1P and induced CCA apoptosis. Furthermore, SK1-I combined with JTE013, an antagonist of the predominant S1P receptor S1PR2, inhibited the AKT and ERK signaling pathways in CCA cells. Our preclinical data suggest SPHK1/S1P pathway targeting may be an effective treatment option for patients with CCA.

Topics: Adaptor Proteins, Signal Transducing; Amino Alcohols; Animals; Apoptosis; Bile Duct Neoplasms; Bile Ducts; Cell Line, Tumor; Cell Proliferation; Cell Survival; Ceramides; Cholangiocarcinoma; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Profiling; Humans; Lysophospholipids; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Middle Aged; Prognosis; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Xenograft Model Antitumor Assays

Sphingosine 1-phosphate (S1P)/S1P receptor (S1PR) system has been implicated in the pathological process of liver injury. This study was designed to evaluate the effects of S1P/S1PR on bone marrow-derived monocyte/macrophage (BMM) migration in mouse models of cholestatic liver injury, and identify the signaling pathway underlying this process. S1PR1-3 expression in BMM was characterized by immunofluorescence, RT-PCR and Western blot. Cell migration was determined in Boyden chambers. In vivo, the chimera mice, which received BM transplants from EGFP-transgenic mice, received an operation of bile duct ligation (BDL) to induce liver injury with the administration of S1PR2/3 antagonists. The results showed that S1PR1-3 were all expressed in BMMs. S1P exerted a powerful migratory action on BMMs via S1PR2 and S1PR3. Furthermore, PTX and LY-294002 (PI3K inhibitor) prevented S1PR2/3-mediated BMM migration, and Rac1 activation by S1P was inhibited by JTE-013, CAY-10444 or LY294002. Administration of S1PR2/3 antagonists in vivo significantly reduced BMM recruitment in BDL-treated mice, and attenuated hepatic inflammation and fibrosis. In conclusion, S1P/S1PR2/3 system mediates BMM motility by PTX-PI3K-Rac1 signaling pathway, which provides new compelling information on the role of S1P/S1PR in liver injury and opens new perspectives for the pharmacological treatment of hepatic fibrosis.

Topics: Animals; Bone Marrow Cells; Cell Movement; Cells, Cultured; Chemokines; Chromones; Cytokines; Enzyme-Linked Immunosorbent Assay; Fatty Liver; Fibrosis; Liver; Lysophospholipids; Macrophages; Mice; Mice, Inbred ICR; Mice, Transgenic; Microscopy, Fluorescence; Morpholines; Neuropeptides; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pyrazoles; Pyridines; rac1 GTP-Binding Protein; Real-Time Polymerase Chain Reaction; Receptors, Lysosphingolipid; RNA Interference; RNA, Small Interfering; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Sphingosine-1-phosphate (S1P) modulates many cell functions such as lymphocyte trafficking and signaling as well as keratinocyte proliferation. However, less is known about the specific effects of S1P on cytokine production, particularly on the interaction between dendritic cells (DCs) and keratinocytes, cell types which are crucial for the initiation and maintenance of chronic inflammatory skin diseases like atopic dermatitis or psoriasis. Especially the cytokines of the IL-12 family play a dominant role in many inflammatory diseases as they have a significant impact on T-helper cell function. In the present study we show that S1P decreased the production of the pro-inflammatory cytokines IL-12 and IL-23 in LPS-stimulated DCs via the common subunit p40 as well as in the crosstalk with activated keratinocytes. By using specific S1P receptor agonists (SEW2871, FTY720-P) and antagonist (JTE013) we identified an important role for S1P receptor 1 in the modulation of the cytokine profile. While diminishing IL-12 and IL-23 secretion, S1P enhanced IL-27 production in DCs. To elucidate the mechanism of the different impact on the IL-12 family cytokine production, we investigated the mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase (PI3K) pathways in DCs. By using specific MAPK-Inhibitors (U0126, SB202190, SP600125) we demonstrated that ERK, p38 and JNK differently regulate each pathway of each cytokine. While p38 and JNK did not seem to play a role in the modulation properties of S1P on cytokine production, ERK is at least partially involved in the S1P mediated modulation of IL-12 and IL-27. The PI3K-Inhibitor abrogated the S1P-induced decrease of IL-12 and IL-23 secretion, while it had no influence on the S1P-induced increase of IL-27 production. These data implicate, that S1P has an anti-inflammatory impact on the production of IL-12 family cytokines, indicating therapeutic potential for S1P treatment of several inflammatory diseases like psoriasis.

Topics: Androstadienes; Animals; Bone Marrow Cells; Cells, Cultured; Dendritic Cells; Enzyme Inhibitors; Gene Expression; Immunoblotting; Interleukin-12; Interleukin-23; Interleukin-27; Lipopolysaccharides; Lysophospholipids; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Oxadiazoles; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sphingosine; Thiophenes; Wortmannin

Although sphingosine 1-phosphate (S1P), a bioactive lipid derived from activated platelets, has a variety of physiologic effects on vessels, no reports have described the effect of S1P on the retinal circulation. We examined the effect and underlying mechanism of the vasomotor action of S1P on porcine retinal arterioles. The porcine retinal arterioles were isolated, cannulated, and pressurized without flow for in vitro study. S1P-induced diameter changes were recorded using videomicroscopic techniques. S1P elicited concentration-dependent (1 nM-10 μM) vasoconstriction of the retinal arterioles that was abolished by the S1P receptor 2 (S1PR2) antagonist JTE-013. S1P-induced vasoconstriction was abolished by the Rho kinase (ROCK) inhibitor H-1152 and was inhibited partly by the protein kinase C (PKC) inhibitor Gö-6983. The inhibition of phospholipase C by U73122 and L-type voltage-operated calcium channels (L-VOCCs) by nifedipine inhibited S1P-induced vasoconstriction; a combination of both inhibitors abolished S1P-induced vasoconstriction. Furthermore, inhibition of myosin light chain kinase (MLCK) by ML-9 significantly blocked S1P-induced vasoconstriction; further coadministration of ML-9 with H-1152 or Gö-6983 abolished S1P-induced vasoconstriction. The current data suggest that S1P elicits vasoconstriction of the retinal arterioles via S1PR2 in vascular smooth muscle cells and this vasoconstriction may be mediated by the Ca2+ -sensitive pathway via activation of PKC leading to activation of ROCK and the Ca2+ -dependent pathway via activation of L-VOCCs resulting in activation of MLCK.

Topics: Actins; Animals; Arterioles; Calcium; Calcium Channels, L-Type; Constriction, Pathologic; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; Fluorescent Antibody Technique, Indirect; Indoles; Lysophospholipids; Male; Maleimides; Muscle, Smooth, Vascular; Myosin Light Chains; Nitric Oxide Synthase Type III; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Retinal Artery; rho-Associated Kinases; Sphingosine; Swine

Various cytokines derived from placental cells are essential for normal placenta development and successful pregnancy. Interleukin-6 (IL-6) is a multifunctional cytokine produced by extravillous and cytotrophoblasts regulating the functions of these cells, e.g. migration, invasion, trophoblast differentiation and proliferation. In macrophages, newly synthesized IL-6 accumulates in the Golgi complex and exits in tubulovesicular carriers fused with recycling endosomes and secreted as a soluble protein. Sphingosine-1-phosphate (S1P) induces various cytokine secretions including IL-6 in different cell types. The signaling mechanisms regulating the IL-6 secretion are unknown. In this study, we found that S1PR2 was the major S1P receptor being expressed in BeWo cells. S1P regulated IL-6 protein secretion in early phase (6 h) and gene expression in later phase (24 h). IL-6 secretion was completely inhibited via inhibitor of transcription (Actinomycin D) or protein synthesis (Cycloheximide) confirming that IL-6 releases constitutively from BeWo cells. By using specific S1PR2 inhibitor JTE-013 and S1PR2 gene silencing, we found that S1PR2 was the main receptor that regulates IL-6 secretion. Furthermore, S1P induced RhoGTPases-dependent pathways that are required for IL-6 secretion. Pretreatment of cells with specific Rho-kinase inhibitor (Y27632) and Rac1 inhibitor (NSC23766) drastically inhibited S1P-induced IL-6 secretion. By using a specific Phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), we found that basal activity of PI3K was required for secretion but was independent of S1P/S1PR2 axis activation. In summary, we report first time that binding of S1P to S1PR2 activates multiple RhoGTPases-dependent pathways that coordinate with PI3K pathway for secretion of IL-6 in BeWo cells.

Topics: Amides; Aminoquinolines; Cell Line; Chromones; Cycloheximide; Dactinomycin; Female; Humans; Interleukin-6; Lysophospholipids; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Pregnancy; Pyrazoles; Pyridines; Pyrimidines; rac1 GTP-Binding Protein; Receptors, Lysosphingolipid; rho GTP-Binding Proteins; rho-Associated Kinases; RNA Interference; RNA, Small Interfering; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Trophoblasts

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

We studied the effects of S1P on the diameter and spontaneous contraction of murine iliac collecting lymph vessels.. The isolated lymph vessel was cannulated with two glass micropipettes and then pressurized to 4 cmH(2) O at the intraluminal pressure. The changes in lymph vessel diameter were measured using a custom-made diameter-detection device. Immunohistochemical studies were also performed to confirm S1P receptors on the lymph vessels.. S1P (10(-7) M) had no significant effect on the frequency or amplitude of the lymph vessels' spontaneous contractions. In contrast, S1P (10(-8) -10(-6) M) produced a concentration-related reduction in lymph vessel diameter (tonic contraction). Pretreatment with 10(-4) M l-NAME or 10(-5) M aspirin had no significant effect on the S1P-induced tonic contraction of the lymph vessels. To evaluate the intracellular signal transduction pathway responsible for the S1P-induced tonic contractions and their Ca(2+) -dependence, we investigated the effects of JTE013, VPC23019, U-73122, xestospongin C, and nifedipine on the S1P-induced tonic contractions. All of these inhibitors except VPC23019 and nifedipine significantly reduced the S1P-induced tonic contractions. S1P (5x10(-7) M) also induced significant tonic contractions in the lymph vessels that had been superfused with high K(+) Krebs-bicarbonate solution or Ca(2+) -free high K(+) Krebs solution containing 1 mM EGTA. S1P2 receptors were immunohistochemically detected in the lymph vessels.. These findings suggest that neither endogenous NO nor prostaglandins are involved in the S1P-induced tonic contraction of lymph vessels, which is mainly caused by Ca(2+) release from intracellular Ca(2+) stores through the activation of S1P2 and 1,4,5 IP(3) receptors.

Topics: Animals; Aspirin; Biomechanical Phenomena; Calcium Signaling; Endothelium, Lymphatic; Estrenes; Lymphatic Vessels; Lysophospholipids; Macrocyclic Compounds; Male; Mice; Muscle Contraction; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nifedipine; Nitric Oxide; Oxazoles; Pressure; Pyrazoles; Pyridines; Pyrrolidinones; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Interstitial cells of Cajal (ICC) are the pacemaker cells that generate the rhythmic oscillation responsible for the production of slow waves in gastrointestinal smooth muscle. Spingolipids are known to present in digestive system and are responsible for multiple important physiological and pathological processes. In this study, we are interested in the action of sphingosine 1-phosphate (S1P) on ICC. S1P depolarized the membrane and increased tonic inward pacemaker currents. FTY720 phosphate (FTY720P, an S1P(1,3,4,5) agonist) and SEW 2871 (an S1P(1) agonist) had no effects on pacemaker activity. Suramin (an S1P(3) antagonist) did not block the S1P-induced action on pacemaker currents. However, JTE-013 (an S1P(2) antagonist) blocked the S1P-induced action. RT-PCR revealed the presence of the S1P(2) in ICC. Calphostin C (a protein kinase C inhibitor), NS-398 (a cyclooxygenase-2 inhibitor), PD 98059 (a p42/44 inhibitor), or SB 203580 (a p38 inhibitor) had no effects on S1P-induced action. However, c-jun NH(2)-terminal kinase (JNK) inhibitor II suppressed S1P-induced action. External Ca(2+)-free solution or thapsigargin (a Ca(2+)-ATPase inhibitor of endoplasmic reticulum) suppressed action of S1P on ICC. In recording of intracellular Ca(2+) ([Ca(2+)](i)) concentration using fluo-4/AM S1P increased intensity of spontaneous [Ca(2+)](i) oscillations in ICC. These results suggest that S1P can modulate pacemaker activity of ICC through S1P(2) via regulation of external and internal Ca(2+) and mitogenactivated protein kinase activation.

Topics: Animals; Antinematodal Agents; Calcium; Cells, Cultured; Enzyme Inhibitors; Female; Interstitial Cells of Cajal; Intestine, Small; Ion Channels; Lysophospholipids; Male; Membrane Potentials; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Naphthalenes; Organophosphates; Patch-Clamp Techniques; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Suramin

Accumulation of extracellular matrix including fibronectin in mesangium is one of the major pathologic characteristics in diabetic nephropathy. In the current study, we explored role of sphingosine-1-phosphate (S1P) receptor in fibronectin expression and underlying molecular mechanism. Among five S1P receptors the mRNA level of S1P2 receptor was the most abundant in kidney of diabetic rats and mesangial cells under high glucose condition. S1P augmentation of fibronectin was significantly inhibited by S1P2 receptor antagonist JTE-013 and S1P2-siRNA. S1P-stimulated fibronectin expression was remarkably blocked by ERK1/2 inhibitor PD98059 and p38MAPK inhibitor SB203580. Phospho-ERK1/2 and phospho-p38MAPK level induced by S1P were markedly abrogated by JTE-013 and S1P2-siRNA. In conclusion, S1P2 receptor was significantly up-regulated under diabetic condition. S1P2 receptor mediated fibronectin expression through the activation of S1P-S1P2-MAPK (ERK1/2 and p38MAPK) axis in mesangial cells under high glucose condition, suggesting that it might be a potential therapeutic target for diabetic nephropathy treatment.

Topics: Animals; Diabetic Nephropathies; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Fibronectins; Flavonoids; Glucose; Hyperglycemia; Imidazoles; Kidney; Lysophospholipids; MAP Kinase Signaling System; Mesangial Cells; p38 Mitogen-Activated Protein Kinases; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; RNA Interference; RNA, Messenger; RNA, Small Interfering; Sphingosine; Sphingosine-1-Phosphate Receptors

Previously we demonstrated that sphingosine 1-phosphate receptor 1 (S1PR(1)) played a prominent, but not exclusive, role in enhancing the excitability of small-diameter sensory neurons, suggesting that other S1PRs can modulate neuronal excitability. To examine the potential role of S1PR(2) in regulating neuronal excitability we used the established selective antagonist of S1PR(2), JTE-013. Here we report that exposure to JTE-013 alone produced a significant increase in excitability in a time- and concentration-dependent manner in 70-80% of recorded neurons. Internal perfusion of sensory neurons with guanosine 5'-O-(2-thiodiphosphate) (GDP-β-S) via the recording pipette inhibited the sensitization produced by JTE-013 as well as prostaglandin E(2). Pretreatment with pertussis toxin or the selective S1PR(1) antagonist W146 blocked the sensitization produced by JTE-013. These results indicate that JTE-013 might act as an agonist at other G protein-coupled receptors. In neurons that were sensitized by JTE-013, single-cell RT-PCR studies demonstrated that these neurons did not express the mRNA for S1PR(2). In behavioral studies, injection of JTE-013 into the rat's hindpaw produced a significant increase in the mechanical sensitivity in the ipsilateral, but not contralateral, paw. Injection of JTE-013 did not affect the withdrawal latency to thermal stimulation. Thus JTE-013 augments neuronal excitability independently of S1PR(2) by unknown mechanisms that may involve activation of other G protein-coupled receptors such as S1PR(1). Clearly, further studies are warranted to establish the causal nature of this increased sensitivity, and future studies of neuronal function using JTE-013 should be interpreted with caution.

Topics: Action Potentials; Analysis of Variance; Anilides; Animals; Capsaicin; Cell Line, Tumor; Cell Movement; Dinoprostone; Dose-Response Relationship, Drug; Drug Interactions; Ganglia, Spinal; Guanosine Diphosphate; Hyperalgesia; Lysophospholipids; Male; Melanoma; Mice; Organophosphonates; Pain Threshold; Patch-Clamp Techniques; Pertussis Toxin; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sensory Receptor Cells; Sensory System Agents; Sphingosine; Thionucleotides; Time Factors; Wound Healing

Elevated intraocular pressure is the main risk factor in primary open-angle glaucoma, involving an increased resistance to aqueous humor outflow in the juxtacanalicular region of the conventional outflow pathway which includes the trabecular meshwork (TM) and the inner wall of Schlemm's canal (SC). Previously, sphingosine-1-phosphate (S1P) was shown to decrease outflow facility in porcine and human eyes, thus increasing outflow resistance and intraocular pressure. Owing to S1P's known effect of increasing barrier function in endothelial cells and the robust expression of the S1P₁ receptor on the inner wall of SC, we hypothesized that S1P₁ receptor activation promotes junction formation and decreases outflow facility. The effects of subtype-specific S1P receptor compounds were tested in human and porcine whole-eye perfusions and human primary cultures of SC and TM cells to determine the receptor responsible for S1P effects on outflow resistance. The S1P₁-specific agonist SEW2871 failed to both mimic S1P effects in paired human eye perfusions, as well as increase myosin light chain (MLC) phosphorylation in cell culture, a prominent outcome in S1P-treated SC and TM cells. In contrast, the S1P₂ antagonist JTE-013, but not the S1P₁ or S1P₁,₃ antagonists, blocked the S1P-promoted increase in MLC phosphorylation. Moreover, JTE-013 prevented S1P-induced decrease in outflow facility in perfused human eyes (P < 0.05, n = 6 pairs). Similarly, porcine eyes perfused with JTE-013 + S1P did not differ from eyes with JTE-013 alone (P = 0.53, n = 3). These results demonstrate that S1P₂ , and not S1P₁ or S1P₃, receptor activation increases conventional outflow resistance and is a potential target to regulate intraocular pressure.

Topics: Animals; Aqueous Humor; Cells, Cultured; Glaucoma, Open-Angle; Humans; Intraocular Pressure; Lysophospholipids; Myosin Light Chains; Oxadiazoles; Phosphorylation; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Sphingosine; Swine; Thiophenes; Trabecular Meshwork

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

To determine whether or not sphingosine-1-phosphate (S1P) is involved in the augmented bronchial smooth muscle (BSM) contractility, one of the causes of airway hyperresponsiveness in asthmatics, the effects of S1P on BSM tone were investigated in control and repeatedly antigen-challenged mice. Both in the control and antigen-challenged animals, S1P had no effect on basal tone of the isolated BSM tissues. However, in the BSMs pre-depolarized by 60mM K(+), S1P caused a significant increase in tension in the control mice. The S1P-mediated contraction was abolished by JTE-013, a selective S1P receptor 2 (S1PR2) antagonist, but not by W123, a selective S1PR1 antagonist, and BML-241, a selective S1PR3 antagonist. The S1P-mediated contraction observed in BSMs of the control mice was also inhibited by Y-27632, a Rho-kinase inhibitor, suggesting that the contraction is mediated via activations of S1PR2 and probably its downstream Rho-kinase. On the other hand, interestingly, the S1P-mediated contraction was not observed at all in BSMs of the repeatedly antigen-challenged mice. A marked and significant downregulation of mRNA for S1PR2 was also observed in BSM tissues of the diseased animals. In conclusion, S1P could augment the BSM contraction via activations of its JTE-013-sensitive receptor, probably S1PR2, and the RhoA/Rho-kinase signaling in normal mice. In BSMs of the repeatedly antigen-challenged mice, the expression level of S1PR2 was much decreased, resulting in a loss of the S1P-mediated contraction.

Topics: Animals; Asthma; Bronchi; Down-Regulation; Lysophospholipids; Male; Mice; Mice, Inbred BALB C; Muscle Contraction; Muscle, Smooth; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; rho-Associated Kinases; RNA, Messenger; Sphingosine; Sphingosine-1-Phosphate Receptors; Thiazolidines

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates a multitude of cellular functions, including cell proliferation, survival, migration and angiogenesis. S1P mediates its effects either by signaling through G protein-coupled receptors (GPCRs) or through an intracellular mode of action. In this study, we have investigated the mechanism behind S1P-induced survival signalling.. We found that S1P protected cells from FasL-induced cell death in an NF-kappaB dependent manner. NF-kappaB was activated by extracellular S1P via S1P2 receptors and Gi protein signaling. Our study also demonstrates that extracellular S1P stimulates cells to rapidly produce and secrete additional S1P, which can further amplify the NF-kappaB activation.. We propose a self-amplifying loop of autocrine S1P with capacity to enhance cell survival. The mechanism provides increased understanding of the multifaceted roles of S1P in regulating cell fate during normal development and carcinogenesis.

Topics: Adaptor Proteins, Signal Transducing; Autocrine Communication; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Tumor; Cell Survival; Enzyme Activation; Fas Ligand Protein; Feedback, Physiological; Flavonoids; Humans; Lysophospholipids; NF-kappa B; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; RNA, Small Interfering; Signal Transduction; Sphingosine; Transgenes

Sphingosine-1-phosphate (S1P) has been demonstrated to enhance endothelial barrier function in vivo and in vitro. However, different S1P receptor subtypes have been indicated to play different or even opposing roles in the regulation of vascular barrier function. This study aims to differentiate the roles of endogenous endothelial S1P subtype receptors in the regulation of permeability in intact microvessels using specific receptor agonist and antagonists. Microvessel permeability was measured with hydraulic conductivity (L(p)) in individually perfused rat mesenteric venules. S1P-mediated changes in endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in fura-2-loaded venules. Confocal images of fluorescent immunostaining illustrated the spatial expressions of three S1P subtype receptors (S1P(R1-3)) in rat venules. The application of S1P (1 μM) in the presence of S1P(R1-3) inhibited platelet-activating factor- or bradykinin-induced permeability increase. This S1P effect was reversed only with a selective S1P(R1) antagonist, W-146, and was not affected by S1P(R2) or S1P(R3) antagonists JTE-013 and CAY-10444, respectively. S1P(R1) was also identified as the sole receptor responsible for S1P-mediated increases in endothelial [Ca(2+)](i). S1P(R2) or S1P(R3) antagonist alone affected neither basal L(p) nor platelet-activating factor-induced permeability increase. The selective S1P(R1) agonist, SEW-2871, showed similar [Ca(2+)](i) and permeability effect to that of S1P. These results indicate that, despite the presence of S1P(R1-3) in the intact venules, only the activation of endothelial S1P(R1) is responsible for the protective action of S1P on microvessel permeability and that endogenous S1P(R2) or S1P(R3) did not exhibit functional roles in the regulation of permeability under basal or acutely stimulated conditions.

Topics: Anilides; Animals; Calcium; Capillary Permeability; Endothelium, Vascular; Female; Lysophospholipids; Mesentery; Models, Animal; Organophosphonates; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sphingosine; Venules

We demonstrate here that the bioactive lipid sphingosine 1-phosphate (S1P) uses sphingosine 1-phosphate receptor 4 (S1P(4)) and human epidermal growth factor receptor 2 (HER2) to stimulate the extracellular signal regulated protein kinase 1/2 (ERK-1/2) pathway in MDA-MB-453 cells. This was based on several lines of evidence. First, the S1P stimulation of ERK-1/2 was abolished by JTE013, which we show here is an S1P(2/4) antagonist and reduced by siRNA knockdown of S1P(4). Second, the S1P-stimulated activation of ERK-1/2 was almost completely abolished by a HER2 inhibitor (ErbB2 inhibitor II) and reduced by siRNA knockdown of HER2 expression. Third, phyto-S1P, which is an S1P(4) agonist, stimulated ERK-1/2 activation in an S1P(4)- and HER2-dependent manner. Fourth, FTY720 phosphate, which is an agonist at S1P(1,3,4,5) but not S1P(2) stimulated activation of ERK-1/2. Fifth, S1P stimulated the tyrosine phosphorylation of HER2, which was reduced by JTE013. HER2 which is an orphan receptor tyrosine kinase is the preferred dimerization partner of the EGF receptor. However, EGF-stimulated activation of ERK-1/2 was not affected by siRNA knockdown of HER2 or by ErbB2 (epidermal growth factor receptor 2 (or HER2)) inhibitor II in MDA-MB-453 cells. Moreover, S1P-stimulated activation of ERK-1/2 does not require an EGF receptor. Thus, S1P and EGF function in a mutually exclusive manner. In conclusion, the magnitude of the signaling gain on the ERK-1/2 pathway produced in response to S1P can be increased by HER2 in MDA-MB-453 cells. The linkage of S1P with an oncogene suggests that S1P and specifically S1P(4) may have an important role in breast cancer progression.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Activation; Epidermal Growth Factor; Female; Fingolimod Hydrochloride; HEK293 Cells; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Propylene Glycols; Pyrazoles; Pyridines; Receptor, ErbB-2; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Sphingosine

Sphingosine-1-phosphate (S1P), a lipid mediator enriched in blood, controls the dynamic migration of osteoclast (OC) precursors (OPs) between the blood and bone, in part via the S1P receptor 1 (S1PR1) which directs positive chemotaxis toward S1P. We show that OPs also express S1PR2, an S1P receptor which mediates negative chemotaxis (or chemorepulsion). OP-positive chemotaxis is prominent in gradients with low maximal concentrations of S1P, whereas such behavior is minimal in fields with high maximal S1P concentrations. This reverse-directional behavior is caused by S1PR2-mediated chemorepulsion acting to override S1PR1 upgradient motion. S1PR2-deficient mice exhibit moderate osteopetrosis as a result of a decrease in osteoclastic bone resorption, suggesting that S1PR2 contributes to OP localization on the bones mediated by chemorepulsion away from the blood where S1P levels are high. Inhibition of S1PR2 function by the antagonist JTE013 changed the migratory behavior of monocytoid cells, including OPs, and relieved osteoporosis in a mouse model by limiting OP localization and reducing the number of mature OCs attached to the bone surface. Thus, reciprocal regulation of S1P-dependent chemotaxis controls bone remodeling by finely regulating OP localization. This regulatory axis may be promising as a therapeutic target in diseases affecting OC-dependent bone remodeling.

Topics: Animals; Bone and Bones; Bone Remodeling; Cell Line; Cell Movement; Cells, Cultured; Chemotaxis; CX3C Chemokine Receptor 1; Flow Cytometry; Glutathione Transferase; Green Fluorescent Proteins; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microscopy, Fluorescence; Osteoclasts; Pyrazoles; Pyridines; RANK Ligand; Receptors, Chemokine; Receptors, Lysosphingolipid; Recombinant Fusion Proteins; RNA Interference; Sphingosine; Sphingosine-1-Phosphate Receptors; Stem Cells

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

Phylogenetic analysis of transmembrane regions of GPCRs using PHYLIP indicated that the orphan receptor P2Y(10) receptor was classified into the cluster consisting nucleotide and lipid receptors. Based on the results, we studied the abilities of nucleotides and lipids to activate the P2Y(10) receptors. As a result, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) evoked intracellular Ca(2+) increases in the CHO cells stably expressing the P2Y(10) fused with a G(16alpha) protein. These Ca(2+) responses were inhibited by S1P receptor and LPA receptor antagonists. The introduction of siRNA designed for P2Y(10) receptor into the P2Y(10)-CHO cells effectively blocked both S1P- and LPA-induced Ca(2+) increases. RT-PCR analysis showed that the mouse P2Y(10) was expressed in reproductive organs, brain, lung and skeletal muscle, suggesting the receptor plays physiological roles throughout the whole body. In conclusion, the P2Y(10) receptor is the first receptor identified as a dual lysophospholipid receptor.

Topics: Amino Acid Sequence; Animals; Calcium; CHO Cells; Cricetinae; Cricetulus; Humans; Ligands; Lysophospholipids; Mice; Molecular Sequence Data; Phylogeny; Protein Conformation; Receptors, Lysophosphatidic Acid; Receptors, Lysosphingolipid; Receptors, Purinergic P2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sequence Analysis, Protein; Sphingosine; Tissue Distribution

We have previously shown that the sphingosine 1-phosphate (S1P)/S1P receptor-1 (S1P(1)R) axis contributes to the migration of transplanted neural progenitor cells (NPCs) toward areas of spinal cord injury. In the current study, we examined a strategy to increase endogenous NPC migration toward the injured central nervous system to modify S1PR.. S1P concentration in the ischemic brain was measured in a mouse thrombosis model of the middle cerebral artery. NPC migration in vitro was assessed by a Boyden chamber assay. Endogenous NPC migration toward the insult was evaluated after ventricular administration of the S1P(2)R antagonist JTE-013.. The concentration of S1P in the brain was increased after ischemia and was maximal 14 days after the insult. The increase in S1P in the infarcted brain was primarily caused by accumulation of microglia at the insult. Mouse NPCs mainly expressed S1P(1)R and S1P(2)R as S1PRs, and S1P significantly induced the migration of NPCs in vitro through activation of S1P(1)R. However, an S1P(1)R agonist failed to have any synergistic effect on S1P-mediated NPC migration, whereas pharmacologic or genetic inhibition of S1P(2)R by JTE-013 or short hairpin RNA expression enhanced S1P-mediated NPC migration but did not affect proliferation and differentiation. Interestingly, administration of JTE-013 into a brain ventricle significantly enhanced endogenous NPC migration toward the area of ischemia.. Our findings suggest that S1P is a chemoattractant for NPCs released from an infarcted area and regulation of S1P(2)R function further enhances the migration of NPCs toward a brain infarction.

Topics: Animals; Brain; Brain Ischemia; Cell Differentiation; Cell Division; Cell Movement; Cells, Cultured; Cerebral Infarction; Chemotaxis; Drug Evaluation, Preclinical; Embryonic Stem Cells; Female; Injections, Intraventricular; Lymphocyte Subsets; Lysophospholipids; Mice; Mice, Inbred C57BL; Microglia; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; RNA Interference; RNA, Small Interfering; Sphingosine; Sphingosine-1-Phosphate Receptors

Sphingosine 1-phosphate (S1P) selectively and potently constricts isolated cerebral arteries, but this response has not been pharmacologically characterized.. The receptor subtype(s) involved in S1P-induced cerebrovascular constriction were characterized using genetic (S1P(2) and S1P(3) receptor null mice) and pharmacological tools (phospho-FTY720, a S1P(1/3/4/5) receptor agonist; SEW2871, a S1P(1) receptor agonist, JTE-013, a S1P(2) receptor antagonist, VPC23019, a S1P(1/3) receptor antagonist). Isolated basilar or peripheral (femoral, mesenteric resistance) arteries, from either rat or mouse, were studied in a wire myograph.. S1P concentration-dependently constricted basilar artery in rat, wild-type (WT) and S1P(2) null mice, but barely affected vascular tone in S1P(3) null mice. Vasoconstriction to U46619 (a thromboxane analogue) or to endothelin-1 did not differ between WT, S1P(2) and S1P(3) null mice. JTE-013 inhibited not only S1P-induced vasoconstriction, but also KCl-, U46619- and endothelin-1-induced constriction. This effect was observed in WT as well as in S1P(2) null mice. VPC23019 increased the concentration-dependent vasoconstriction to S1P in both rat and mouse basilar arteries with intact endothelium, but not in rat basilar artery without endothelium. Phospho-FTY720 concentration-dependently constricted rat basilar arteries, but not femoral or mesenteric resistance arteries, while SEW2871 did not induce any response in the same arteries.. S1P constricts cerebral arteries through S1P(3) receptors. The purported S1P(2) receptor antagonist JTE-013 does not appear to be selective, at least in rodents. Enhancement of S1P-induced contraction by VPC23019 might be related to blockade of S1P(1) receptors and NO generation.

Topics: Animals; Cerebral Arteries; Dose-Response Relationship, Drug; Fingolimod Hydrochloride; In Vitro Techniques; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Oxadiazoles; Propylene Glycols; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sphingosine; Thiophenes; Vasoconstriction

The lysophospholipid mediator sphingosine-1-phosphate (S1P) activates G protein-coupled receptors (GPCRs) to induce potent inhibition of platelet-derived growth factor (PDGF)-induced Rac activation and, thereby, chemotaxis in rat vascular smooth muscle cells (VSMCs). We explored the heterotrimeric G protein and the downstream mechanism that mediated S1P inhibition of Rac and cell migration in VSMCs.. S1P inhibition of PDGF-induced cell migration and Rac activation in VSMCs was abolished by the selective S1P(2) receptor antagonist JTE-013. The C-terminal peptides of Galpha subunits (Galpha-CTs) act as specific inhibitors of respective G protein-GPCR coupling. Adenovirus-mediated expression of Galpha(12)-CT, Galpha(13)-CT, and Galpha(q)-CT, but not that of Galpha(s)-CT or LacZ or pertussis toxin treatment, abrogated S1P inhibition of PDGF-induced Rac activation and migration, indicating that both G(12/13) and G(q) classes are necessary for the S1P inhibition. The expression of Galpha(q)-CT as well as Galpha(12)-CT and Galpha(13)-CT also abolished S1P-induced Rho stimulation. C3 toxin, but not a Rho kinase inhibitor or a dominant negative form of Rho kinase, abolished S1P inhibition of PDGF-induced Rac activation and cell migration. The angiotensin II receptor AT(1), which robustly couples to G(q), did not mediate either Rho activation or inhibition of PDGF-induced Rac activation or migration, suggesting that activation of G(q) alone was not sufficient for Rho activation and resultant Rac inhibition. However, the AT(1) receptor fused to Galpha(12) was able to induce not only Rho stimulation but also inhibition of PDGF-induced Rac activation and migration. Phospholipase C inhibition did not affect S1P-induced Rho activation, and protein kinase C activation by a phorbol ester did not mimic S1P action, suggesting that S1P inhibition of migration or Rac was not dependent on the phospholipase C pathway.. These observations together suggest that S1P(2) mediates inhibition of Rac and migration through the coordinated action of G(12/13) and G(q) for Rho activation in VSMCs.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; ADP Ribose Transferases; Animals; Botulinum Toxins; Calcium; Cell Movement; Cells, Cultured; Dose-Response Relationship, Drug; GTP-Binding Protein alpha Subunits, G12-G13; GTP-Binding Protein alpha Subunits, Gq-G11; Lysophospholipids; Male; Muscle, Smooth, Vascular; Protein Kinase C; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-sis; Pyrazoles; Pyridines; rac GTP-Binding Proteins; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Receptors, Lysosphingolipid; Recombinant Fusion Proteins; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction; Sphingosine; Transfection

S1P acts via the S1PR family of G protein-coupled receptors to regulate a variety of physiological responses. Whereas S1P1R activates G(i)- and PI-3-kinase-dependent signals to inhibit vascular permeability, the related S1P2R inhibits the PI-3-kinase pathway by coupling to the Rho-dependent activation of the PTEN phosphatase. However, cellular consequences of S1P2R signaling in the vascular cells are not well understood.. Selective signaling of the S1P2R was achieved by adenoviral-mediated expression in endothelial cells. Secondly, endogenously expressed S1P2R was blocked by the specific pharmacological antagonist JTE013. Activation of S1P2R in endothelial cells resulted in Rho-ROCK- and PTEN-dependent disruption of adherens junctions, stimulation of stress fibers, and increased paracellular permeability. JTE013 treatment of naive endothelial cells potentiated the S1P1R-dependent effects such as formation of cortical actin, blockade of stress fibers, stimulation of adherens junction assembly, and improved barrier integrity. This observation was extended to the in vivo model of vascular permeability in the rat lung: the S1P2R antagonist JTE013 significantly inhibited H2O2-induced permeability in the rat lung perfused model.. S1P2R activation in endothelial cells increases vascular permeability. The balance of S1P1 and S1P2 receptors in the endothelium may determine the regulation of vascular permeability by S1P.

Topics: Adherens Junctions; Animals; Antigens, CD; Cadherins; Capillary Permeability; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Humans; Hydrogen Peroxide; Intracellular Signaling Peptides and Proteins; Lysophospholipids; Phosphorylation; Protein Serine-Threonine Kinases; PTEN Phosphohydrolase; Pulmonary Edema; Pyrazoles; Pyridines; rac GTP-Binding Proteins; Rats; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Stress Fibers; Time Factors; Transfection

Blood platelets store sphingosine 1-phosphate (S1P) abundantly and release this bioactive lipid extracellularly. S1P acts as an intercellular mediator through interaction with the endothelial differentiation gene (EDG)/S1P family of G protein-coupled receptors. Of the EDG family S1P receptors, EDG-5 (S1P2) is inhibited in migration induced by S1P. Diabetes impairs numerous aspects of tissue repair. Failure of wound angiogenesis is known to delay diabetic wound healing.. We examined whether S1P subcutaneous injection could improve the healing of full-thickness skin wounds in healthy and diabetic mice. We further determine if the combined S1P and EDG-5 (S1P2) antagonist injection in diabetic mice could affect wound healing. Finally, we examined the histopathological findings of the wound following S1P injection in diabetic mice.. Eight- to 10-week-old BALA/c mice, diabetic db/db mice and Wister rats were used for the studies. A full-thickness wound was made on the dorsal skin of the healthy and diabetic mice. Either 10 microM or 100 microM of S1P or vehicle control (BSA/PBS) was injected into the wound bed every day. We calculated the wound area after each injection. EDG-5 (S1P2) antagonist (JTE-013) or vehicle (DMSO) was then injected in addition to the S1P around the dorsal wound of diabetic mice and the wound diameter was measured. Wound tissue samples were excised following injection for histopathological examination.. Wound area in normal BALA/c mice did not significantly decrease upon S1P injection compared to S1P-untreated controls. S1P injection alone showed significant promotion of wound healing in diabetic mice compared to no S1P treatment. The combination of S1P and EDG-5 (S1P2) receptor antagonist administration induced maximal wound healing in diabetic mice. Histopathological examination revealed that S1P induces neo-vascularization potential in rats and diabetic mice wound.. S1P injection in diabetic mice significantly accelerated cutaneous wound healing in the neo-vascularization process. The results demonstrate that S1P affects and sustains all key cellular processes responsible for wound repair and point to a unique potential for this molecule in the therapy of diabetic wounds, particularly as an angiogenic agent in treatment of diabetic wounds.

Topics: Animals; Diabetes Mellitus, Experimental; Disease Models, Animal; Drug Therapy, Combination; Injections, Subcutaneous; Lysophospholipids; Male; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Neovascularization, Physiologic; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptors, Lysosphingolipid; Skin; Sphingosine; Wound Healing

Topics: Animals; Blood Platelets; Coronary Vessels; Humans; In Vitro Techniques; Lysophospholipids; Muscle Contraction; Muscle, Smooth, Vascular; Pyrazoles; Pyridines; Sphingosine; Vasoconstriction

To analyze the involvement in allergic reactions of platelets and sphingosine 1-phosphate (Sph-1-P), a lysophospholipid mediator released from activated platelets, the effects of Sph-1-P and a supernatant prepared from activated platelets on mast cell line RBL-2H3 were examined. Sph-1-P strongly inhibited the migration of both non-stimulated and fibronectin-stimulated RBL-2H3 cells, which was reversed by JTE-013, a specific antagonist of G protein-coupled Sph-1-P receptor S1P(2); S1P(2) was confirmed to be expressed in these cells. A similar anti-motility effect of Sph-1-P was observed in a phagokinetic assay. Consistent with these results, treatment of RBL-2H3 cells with Sph-1-P resulted in a rounded cell morphology, which was blocked by JTE-013. Under the present conditions, Sph-1-P failed to induce intracellular Ca(2+) mobilization or histamine degranulation, responses postulated to be elicited by intracellular Sph-1-P. Importantly, the Sph-1-P effect, i.e., the regulation of RBL-2H3 cell motility, was mimicked by the supernatant (both with and without boiling) prepared from activated platelets, and this effect of the supernatant was also blocked by JTE-013. Our results suggest that the motility of mast cells can be regulated by Sph-1-P and also platelets (which release Sph-1-P), via cell surface receptor S1P(2) (not through intracellular Sph-1-P actions, postulated previously in the same cells).

Topics: Animals; Blood Platelets; Cell Line; Cell Movement; Cell Shape; Cytoskeleton; Lysophospholipids; Mast Cells; Platelet Activation; Pyrazoles; Pyridines; Rats; Receptors, Lysosphingolipid; Sphingosine

Although structural changes are most important to determine vascular resistance in portal hypertension, vasoactive mediators also contribute to its regulation. Hepatic stellate cells (HSCs) are assumed to play a role in modulating intrahepatic vascular resistance based on their residence in the space of Disse and capacity to contract. Because sphingosine 1-phosphate (S1P) has been shown to stimulate HSC contractility, we wondered if S1P could regulate portal pressure. S1P at 0.5-5 microM increased portal pressure in isolated rat perfused liver. This effect was abrogated in the presence of a binding antagonist for S1P2, JTE-013. Perfusion of isolated rat liver with 5 microM S1P increased Rho activity in the liver, and co-perfusion with JTE-013 cancelled S1P-induced Rho activation. Because S1P is present in human plasma at approximately 0.2 microM, S1P might readily regulate portal vascular tone in physiological and pathological status. The antagonist for S1P2 merits consideration for treatment of portal hypertension.

Topics: Animals; Cells, Cultured; Culture Techniques; Dose-Response Relationship, Drug; Hepatocytes; Liver; Lysophospholipids; Male; Metabolic Clearance Rate; Portal Pressure; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; rho GTP-Binding Proteins; Sphingosine; Sphingosine-1-Phosphate Receptors

Sphingosine 1-phosphate (S1P), a ligand for G protein-coupled endothelial differentiation gene-1 (Edg-1), Edg-3, Edg-5, Edg-6, and Edg-8, elicits a variety of responses by cells. Prominent among these is cell proliferation. S1P is abundantly stored in platelets and released upon their activation, suggesting that S1P plays a pathophysiologic role in vivo. Because the major part of injected S1P was distributed into the liver in mice, we wondered whether the liver would be one of its targets. The effects of S1P on hepatocytes, the major constituent cells in the liver, were examined.. Northern blot analysis revealed the expression of Edg-1 and Edg-5 messenger RNA (mRNA) in cultured rat hepatocytes, in which S1P decreased DNA synthesis induced by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) without affecting total protein synthesis. This inhibitory effect was attenuated by inactivation of small GTPase Rho with C3 exotoxin but not by inactivation of G(i) with pertussis toxin. Moreover, in the presence of JTE-013, a newly developed and specific binding antagonist for Edg-5, the inhibitory effect was also cancelled. Finally, the administration of S1P after 70% partial hepatectomy in rats reduced the peak of DNA synthesis in hepatocytes with increased Rho activity. Furthermore, Edg-5 but not Edg-1 mRNA expression was enhanced in hepatocytes 24-72 hours after partial hepatectomy, which coincides with decreasing hepatocyte proliferation.. S1P has an antiproliferative property in rat hepatocytes by activating Rho via Edg-5. Our results raise the possibility that S1P is a negative regulator in liver regeneration.

Topics: ADP Ribose Transferases; Animals; Botulinum Toxins; Cell Division; Cells, Cultured; CHO Cells; Cricetinae; DNA; Hepatocytes; Liver; Liver Regeneration; Lysophospholipids; Male; Pertussis Toxin; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; rho GTP-Binding Proteins; RNA, Messenger; Sphingosine

Sphingosine 1-phosphate (Sph-1-P), a bioactive lipid derived from activated platelets, may play an important role in coronary artery spasm and hence the pathogenesis of ischemic heart diseases, since we reported that a decrease in coronary blood flow was induced by this lysophospholipid in an in vivo canine heart model [Cardiovasc. Res. 46 (2000) 119]. In this study, metabolism related to and cellular responses elicited by Sph-1-P were examined in human coronary artery smooth muscle cells (CASMCs).. [3H]Sphingosine (Sph), incorporated into CASMCs, was converted to [3H]Sph-1-P intracellularly, but its stimulation-dependent formation and extracellular release were not observed. Furthermore, the cell surface Sph-1-P receptors of S1P family (previously called EDG) were found to be expressed in CASMCs. Accordingly, Sph-1-P seems to act as an extracellular mediator in CASMCs. Consistent with Sph-1-P-elicited coronary vasoconstriction in vivo, Sph-1-P strongly induced CASMC contraction, which was inhibited by JTE-013, a newly-developed specific antagonist of S1P(2) (EDG-5). Furthermore, C3 exoenzyme or Y-27632 inhibited the CASMC contraction induced by Sph-1-P, indicating Rho involvement. Finally, exogenously-added [3H]Sph-1-P underwent a rapid degradation. Since lipid phosphate phosphatases, ectoenzymes capable of dephosphorylating Sph-1-P, were expressed in CASMCs, Sph-1-P may be dephosphorylated by the ectophosphatases.. Sph-1-P, derived from platelets and dephosphorylated on the cell surface, may induce the contraction of coronary artery smooth muscle cells through the S1P(2)/Rho signaling.

Topics: Acute-Phase Proteins; Amides; Blood Platelets; Cells, Cultured; Coronary Vessels; Humans; In Vitro Techniques; Lysophospholipids; Muscle, Smooth, Vascular; Pyrazoles; Pyridines; Signal Transduction; Sphingosine; Vasoconstriction

We investigated mechanisms for inhibition of B16 melanoma cell migration and invasion by sphingosine-1-phosphate (S1P), which is the ligand for the Edg family G protein-coupled receptors and also implicated as an intracellular second messenger. S1P, dihydro-S1P, and sphingosylphosphorylcholine inhibited B16 cell migration and invasion with the relative potencies expected as S1P2 receptor agonists. The S1P2-selective antagonist JTE013 completely abolished the responses to these agonists. In addition, JTE013 abrogated the inhibition by sphingosine, which is the S1P precursor but not an agonist for S1P receptors, indicating that the sphingosine effects were mediated via S1P2 stimulation, most likely by S1P that was converted from sphingosine. S1P induced inhibition and activation, respectively, of Rac and RhoA in B16 cells, which were abrogated by JTE013. Adenovirus-mediated expression of N17Rac mimicked S1P inhibition of migration, whereas C3 toxin pretreatment, but not Rho kinase inhibitors, reversed the S1P inhibition. Overexpression of S1P2 sensitized, and that of either S1P1 or S1P3 desensitized, B16 cells to S1P inhibition of Rac and migration. In JTE013-pretreated, S1P3-overexpressing B16 cells, S1P stimulated cellular RhoA but failed to inhibit either Rac or migration, indicating that RhoA stimulation itself is not sufficient for inhibition of migration. These results provide compelling evidence that endogenously expressed S1P2 negatively regulates cell motility and invasion through ligand-dependent reciprocal regulation of cellular Rac and RhoA activities. In the presence of JTE013, S1P instead stimulated Rac and migration in B16 cells that overexpress either S1P1 or S1P3, unveiling counteractions between S1P2 and S1P1 or S1P3 chemotactic receptor.

Topics: Adenoviridae; Animals; Blotting, Northern; Blotting, Western; Calcium; Cell Movement; CHO Cells; Cricetinae; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genetic Vectors; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Ligands; Lysophospholipids; Melanoma, Experimental; Mice; Neoplasm Invasiveness; Phosphorylcholine; Plasmids; Protein Isoforms; Pyrazoles; Pyridines; rac GTP-Binding Proteins; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; rhoA GTP-Binding Protein; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Temperature; Time Factors

Sphingosine 1-phosphate (Sph-1-P), a bioactive lysophospholipid capable of inducing a wide spectrum of biological responses, acts as an intercellular mediator, through interaction with the endothelial differentiation gene (EDG)/S1P family of G protein-coupled receptors. In this study, the effects of JTE-013, a specific antagonist of the migration-inhibitory receptor EDG-5, on Sph-1-P-elicited responses were examined in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (SMCs), which expressed EDG-5 protein weakly and abundantly, respectively. This pyrazolopyridine compound reversed the inhibitory effect of Sph-1-P on SMC migration and further enhanced Sph-1-P-stimulated HUVEC migration. In contrast, its effect on Sph-1-P-induced intracellular Ca(2+) mobilization was marginal. Our results indicate that specific regulation of Sph-1-P-modulated migration responses in vascular cells can be achieved by EDG-5 antagonists and that manipulation of Sph-1-P biological activities by each EDG antagonist may lead to a therapeutical application to control vascular diseases.

Topics: Calcium Signaling; Cell Movement; Cells, Cultured; Endothelium, Vascular; Fluorescent Dyes; Fura-2; Humans; Immediate-Early Proteins; Lysophospholipids; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Pyrazoles; Pyridines; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Sphingosine