bml-241 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

Hyperglycemia aggravates hepatic ischemia/reperfusion injury (IRI), but the underlying mechanism for the aggravation remains elusive. Sphingosine-1-phosphate (S1P) and sphingosine-1-phosphate receptors (S1PRs) have been implicated in metabolic and inflammatory diseases. Here, we discuss whether and how S1P/S1PRs are involved in hyperglycemia-related liver IRI. For our in vivo experiment, we enrolled diabetic patients with benign hepatic disease who had liver resection, and we used streptozotocin (STZ)-induced hyperglycemic mice or normal mice to establish a liver IRI model. In vitro bone marrow-derived macrophages (BMDMs) were differentiated in high-glucose (HG; 30 mM) or low-glucose (LG; 5 mM) conditions for 7 days. The expression of S1P/S1PRs was analyzed in the liver and BMDMs. We investigated the functional and molecular mechanisms by which S1P/S1PRs may influence hyperglycemia-related liver IRI. S1P levels were higher in liver tissues from patients with diabetes mellitus and mice with STZ-induced diabetes. S1PR3, but not S1PR1 or S1PR2, was activated in liver tissues and Kupffer cells under hyperglycemic conditions. The S1PR3 antagonist CAY10444 attenuated hyperglycemia-related liver IRI based on hepatic biochemistry, histology, and inflammatory responses. Diabetic livers expressed higher levels of M1 markers but lower levels of M2 markers at baseline and after ischemia/reperfusion. Dual-immunofluorescence staining showed that hyperglycemia promoted M1 (CD68/CD86) differentiation and inhibited M2 (CD68/CD206) differentiation. Importantly, CAY10444 reversed hyperglycemia-modulated M1/M2 polarization. HG concentrations in vitro also triggered S1P/S1PR3 signaling, promoted M1 polarization, inhibited M2 polarization, and enhanced inflammatory responses compared with LG concentrations in BMDMs. In contrast, S1PR3 knockdown significantly retrieved hyperglycemia-modulated M1/M2 polarization and attenuated inflammation. In conclusion, our study reveals that hyperglycemia specifically triggers S1P/S1PR3 signaling and exacerbates liver IRI by facilitating M1 polarization and inhibiting M2 polarization, which may represent an effective therapeutic strategy for liver IRI in diabetes.

Topics: Aged; Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Female; Humans; Hyperglycemia; Liver; Liver Diseases; Liver Transplantation; Lysophospholipids; Macrophages; Male; Mice; Middle Aged; Reperfusion Injury; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Streptozocin; Thiazolidines

Pericytes have long been regarded merely to maintain structural and functional integrity of blood-brain barrier (BBB). Nevertheless, it has also been identified as a component of scar-forming stromal cells after spinal cord injury (SCI). In process of enlargement of spinal cavity after SCI, the number of pericytes increased and outnumbered astrocytes. However, the mechanism of proliferation of pericytes remains unclear. Sphingosine-1-phosphate (S1P) has been reported to play important roles in the formation of glia scar, but previous studies had paid more attention to the astrocytes. The present study aimed to observe the effects of S1P and S1P receptors (S1PRs) on proliferation of pericytes and investigate the underlying mechanism. By double immunostaining, we found that the number of PDGFRβ-positive pericytes was gradually increased and sealed the cavity, which surrounded by reactive astrocytes. Moreover, the subtype of S1PR3 was found to be induced by SCI and mainly expressed on pericytes. Further, by use of CAY10444, an inhibitor of S1PR3, we showed that S1P/S1PR3 mediated the proliferation of pericytes through Ras/pERK pathway. Moreover, CAY10444 was found to have the effects of enhancing neuronal survival, alleviating glial scar formation, and improving locomotion recovery after SCI. The results suggested that S1P/S1PR3 might be a promising target for clinical therapy for SCI.

Topics: Animals; Cell Proliferation; Locomotion; Lysophospholipids; MAP Kinase Signaling System; Pericytes; ras Proteins; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Recovery of Function; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Spinal Cord Injuries; Thiazolidines

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts through a family of five G-protein-coupled receptors (S1PR1-5) and plays a key role in regulating the inflammatory response. Our previous studies demonstrated that rat sensory neurons express the mRNAs for all five S1PRs and that S1P increases neuronal excitability primarily, but not exclusively, through S1PR1. This raises the question as to which other S1PRs mediate the enhanced excitability.. Isolated sensory neurons were treated with either short-interfering RNAs (siRNAs) or a variety of pharmacological agents targeted to S1PR1/R2/R3 to determine the role(s) of these receptors in regulating neuronal excitability. The excitability of isolated sensory neurons was assessed by using whole-cell patch-clamp recording to measure the capacity of these cells to fire action potentials (APs).. After siRNA treatment, exposure to S1P failed to augment the excitability. Pooled siRNA targeted to S1PR1 and R3 also blocked the enhanced excitability produced by S1P. Consistent with the siRNA results, pretreatment with W146 and CAY10444, selective antagonists for S1PR1 and S1PR3, respectively, prevented the S1P-induced increase in neuronal excitability. Similarly, S1P failed to augment excitability after pretreatment with either VPC 23019, which is a S1PR1 and R3 antagonist, or VPC 44116, the phosphonate analog of VPC 23019. Acute exposure (10 to 15 min) to either of the well-established functional antagonists, FTY720 or CYM-5442, produced a significant increase in the excitability. Moreover, after a 1-h pretreatment with FTY720 (an agonist for S1PR1/R3/R4/R5), neither SEW2871 (S1PR1 selective agonist) nor S1P augmented the excitability. However, after pretreatment with CYM-5442 (selective for S1PR1), SEW2871 was ineffective, but S1P increased the excitability of some, but not all, sensory neurons.. These results demonstrate that the enhanced excitability produced by S1P is mediated by activation of S1PR1 and/or S1PR3.

Topics: Action Potentials; Anilides; Animals; Cells, Cultured; Dinoprostone; Enzyme Inhibitors; Fingolimod Hydrochloride; Ganglia, Spinal; Gene Expression Regulation; Immunosuppressive Agents; Lysophospholipids; Mice; Mice, Inbred C57BL; Organophosphonates; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; RNA, Small Interfering; Sensory Receptor Cells; Sphingosine; Sphingosine-1-Phosphate Receptors; Thiazolidines

Endothelial progenitor cells (EPCs) play a fundamental role in neoangiogenesis and tumor angiogenesis. Through the sphingosine-1-phosphate receptor 3 (S1PR3), sphingosine-1-phosphate (S1P) can stimulate the functional capacity of EPCs. Platelet-derived growth factor receptor-beta (PDGFR-β) contributes to the migration and angiogenesis of EPCs. This study aimed to investigate whether S1P induces the migration and angiogenesis of EPCs through the S1PR3/PDGFR-β/Akt signaling pathway. We used the Transwell system and the Chemicon In Vitro Angiogenesis Assay Kit with CAY10444 (an S1PR3 antagonist), AG1295 (a PDGFR kinase inhibitor) and sc-221226 (an Akt inhibitor) to examine the role of the S1PR3/PDGFR-β/Akt pathway in the S1Pinduced migration and angiogenesis of EPCs.

Topics: Animals; Cell Movement; Cells, Cultured; Endothelial Progenitor Cells; Lysophospholipids; Male; Mice, Inbred C57BL; Neovascularization, Physiologic; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptor, Platelet-Derived Growth Factor beta; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Spleen; Thiazolidines; Tyrphostins; Vascular Endothelial Growth Factor A

Emerging evidence suggests a role for sphingosine-1-phosphate (S1P) in various aspects of rheumatoid arthritis (RA) pathogenesis. In this study we compared the effect of chemical hypoxia induced by cobalt chloride (CoCl2) on the expression of S1P metabolic enzymes and cytokine/chemokine secretion in normal fibroblast-like synoviocytes (FLS) and RAFLS. RAFLS incubated with CoCl2, but not S1P, produced less IL-8 and MCP-1 than normal FLS. Furthermore, incubation with the S1P2 and S1P3 receptor antagonists, JTE-013 and CAY10444, reduced CoCl2-mediated chemokine production in normal FLS but not in RAFLS. RAFLS showed lower levels of intracellular S1P and enhanced mRNA expression of S1P phosphatase 1 (SGPP1) and S1P lyase (SPL), the enzymes that are involved in intracellular S1P degradation, when compared to normal FLS. Incubation with CoCl2 decreased SGPP1 mRNA and protein and SPL mRNA as well. Inhibition of SPL enhanced CoCl2-mediated cytokine/chemokine release and restored autocrine activation of S1P2 and S1P3 receptors in RAFLS. The results suggest that the sphingolipid pathway regulating the intracellular levels of S1P is dysregulated in RAFLS and has a significant impact on cell autocrine activation by S1P. Altered sphingolipid metabolism in FLS from patients with advanced RA raises the issue of synovial cell burnout due to chronic inflammation.

Topics: Arthritis, Rheumatoid; Cell Hypoxia; Chemokines; Cobalt; Fibroblasts; Humans; Lysophospholipids; Membrane Proteins; Phosphoric Monoester Hydrolases; Signal Transduction; Sphingosine; Stress, Physiological; Synovial Membrane; Thiazolidines

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

We examined the ability of sphingosine-1-phosphate (S1P) to desensitize extracellular signal-related kinase (ERK), a mitogen-activated protein kinase linked to antiapoptotic responses in the heart. In isolated adult mouse cardiomyocytes, S1P (10 nM-5 microM) induced ERK phosphorylation in a time- and dose-dependent manner. S1P stimulation of ERK was completely inhibited by an S1P1/3 subtype receptor antagonist (VPC23019), by a Gi protein inhibitor (pertussis toxin) and by a mitogen-activated protein kinase/ERK kinase inhibitor (PD98059). A selective S1P3 receptor antagonist (CAY10444) had no effect on S1P-induced ERK activation. The selective S1P1 agonist SEW2871 also induced ERK phosphorylation. Activation of ERK by restimulation with 100 nM S1P was suppressed after 1 hour of preincubation with 100 nM S1P but recovered fully the next day, suggesting receptor recycling. Similar results were obtained in protein kinase C epsilon-null cardiomyocytes. Treatment with the nonselective S1P receptor agonist FTY720 for 1 hour also reduced phospho-ERK expression in response to subsequent S1P stimulation. In contrast to S1P, some desensitization to FTY720 persisted after overnight exposure. Cell death induced by hypoxia/reoxygenation was reduced by pretreatment with exogenous S1P. This enhanced survival was abrogated by pretreatment with PD98059, VPC23019, or pertussis toxin. Thus, exogenous S1P induces rapid and reversible S1P1-mediated ERK phosphorylation. S1P-induced adult mouse cardiomyocyte survival requires ERK activation mediated via an S1P1-Gi pathway.

Topics: Animals; Cell Death; Cell Hypoxia; Cell Survival; Cells, Cultured; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; In Vitro Techniques; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Cardiac; Phosphorylation; Protein Kinase C; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Thiazolidines

The lysophospholipids, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), stimulate chemotaxis and induce differentiation of human keratinocytes. As Ca(2+) plays an important role in keratinocyte differentiation, we studied Ca(2+) signaling by S1P and LPA in these cells, known to express mRNA transcripts of the S1P(1-5) and LPA(1-3) receptors, and the receptor subtypes involved in this process. S1P and LPA caused transient increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)), with pEC(50) values of 8.5+/-0.11 and 7.5+/-0.23, respectively. The [Ca(2+)](i) increases are apparently mediated by stimulation of phospholipase C and involve Ca(2+) mobilization from thapsigargin-sensitive stores and subsequent Ca(2+) influx. The LPA-induced [Ca(2+)](i) increases were not inhibited by the LPA(1/3) receptor antagonist, dioctanoylglycerol pyrophosphate. The S1P-induced [Ca(2+)](i) increases were largely inhibited by the putative S1P(3) antagonist, BML-241, and the S1P(1/3) antagonist, VPC23019. The S1P(1)-specific agonist, SEW2871, did not increase [Ca(2+)](i) but stimulated chemotaxis of keratinocytes, which was fully blocked by S1P(1) antisense oligonucleotides. The data indicate that LPA and S1P potently increase [Ca(2+)](i) in human keratinocytes and that the effect of LPA is mediated by LPA(2), whereas that of S1P is mediated at least to a large part by S1P(3). The S1P(1) receptor, without stimulating [Ca(2+)](i) increases, mediates chemotaxis of keratinocytes.

Topics: Calcium; Calcium Signaling; Cell Movement; Chemotaxis; Green Fluorescent Proteins; Humans; Keratinocytes; Lysophospholipids; Models, Biological; Receptors, Lysophospholipid; Receptors, Lysosphingolipid; Sphingosine; Thapsigargin; Thiazolidines; Type C Phospholipases

Cytokines mediate pancreatic islet beta-cell apoptosis and necrosis, leading to loss of insulin secretory capacity and type 1 diabetes mellitus. The cytokines, IL-1beta and interferon-gamma, induced terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining of rat islet cells within 48 h by about 25-30%, indicative of apoptosis and/or necrosis. Sphingosine 1-phosphate (S1P) at nanomolar concentrations significantly reduced islet cell cytokine-induced TUNEL staining. Similar effects were observed in INS-1 cells. The dihydro analog of S1P also reduced the percentage of TUNEL stained islet and INS-1 cells, whereas the S1P receptor antagonist BML-241 blocked the protective effects. Pertussis toxin did not affect the S1P protective response. In the presence of a phospholipase C antagonist, U73122, there was significant inhibition of the S1P protective effects against apoptosis/necrosis. S1P stimulated INS-1 cell protein kinase C activity. Carbamylcholine chloride acting through muscarinic receptors also inhibited cytokine-induced TUNEL staining in pancreatic islet cells. S1P and/or dihydro-S1P also antagonized cytokine-induced increases in cytochrome c release from mitochondria and caspase-3 activity in INS-1 cells, which are indicative of cell apoptosis vs. necrosis. S1P failed to affect nitric oxide synthase activity after 48 h. Thus, the evidence suggests that S1P acting on S1P receptors coupled to G(q) mediates protective effects on islet beta-cells against cytokine-induced apoptosis.

Topics: Animals; Apoptosis; Carbachol; Caspase 3; Caspases; Cell Survival; Cytochromes c; Cytokines; Enzyme Inhibitors; In Situ Nick-End Labeling; In Vitro Techniques; Indoles; Insulin-Secreting Cells; Lysophospholipids; Male; Maleimides; Nicotinic Agonists; Nitric Oxide Synthase Type II; Protein Kinase C; Rats; Rats, Sprague-Dawley; Sphingosine; Thiazolidines; Type C Phospholipases