sew2871 and sphingosine-kinase

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

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

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

Directed migration of hepatic myofibroblasts (hMFs) contributes to the development of liver fibrosis. However, the signals regulating the motility of these cells are incompletely understood. We have recently shown that sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) are involved in mouse liver fibrogenesis. Here, we investigated the role of S1P/S1PRs signals in human liver fibrosis involving motility of human hMFs.. S1P level in the liver was examined by high-performance liquid chromatography. Expression of S1PRs was characterized, in biopsy specimens of human liver and cultured hMFs, by immunofluorescence and real-time RT-PCR or Western blot analysis. Cell migration was determined in Boyden chambers, by using the selective S1P receptor agonist or antagonist and silencing of S1PRs expression with small interfering RNA.. S1P level in the human fibrotic liver was increased through up-regulation of sphingosine kinase (SphK), irrespective of the etiology of fibrosis. S1P receptors type 1, 2, and 3 (S1P(1,2,3)) were expressed in human hMFs in vivo and in vitro. Interestingly, S1P(1,3) were strongly induced in human fibrotic samples, whereas expression of S1P(2) was massively decreased. S1P exerted a powerful migratory action on human hMFs. Furthermore, the effect of S1P was mimicked by SEW2871 (an S1P(1) agonist), and blocked by suramin (an S1P(3) antagonist) and by silencing S1P(1,3) expression. In contrast, JTE-013 (an S1P(2) antagonist) and silencing of S1P(2) expression enhanced S1P-induced migration.. SphK/S1P/S1PRs signaling axis plays an important role in human liver fibrosis and is involved in the directed migration of human hMFs into the damaged areas.

Topics: Base Sequence; Cell Movement; Cells, Cultured; Humans; Liver Cirrhosis; Lysophospholipids; Myofibroblasts; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Suramin; Thiophenes

Sphingosine 1-phosphate (S1P), a pleiotropic lipid mediator, binds to five related G-protein-coupled receptors to exert its effects. As S1P1 receptor (S1P1R) activation blocks kidney inflammation in acute renal injury, we tested whether activation of S1P1Rs ameliorates renal injury in early-stage diabetic nephropathy (DN) in rats. Urinary albumin excretion increased in vehicle-treated diabetic rats (single injection of streptozotocin), compared with controls, and was associated with tubule injury and increased urinary tumor necrosis factor-α (TNF-α) at 9 weeks. These effects were significantly reduced by FTY720, a non-selective, or SEW2871, a selective S1P1R agonist. Interestingly, only FTY720 was associated with reduced total lymphocyte levels. Albuminuria was reduced by SEW2871 in both Rag-1 (T- and B-cell deficient) and wild-type diabetic mice after 6 weeks, suggesting that the effect was independent of lymphocytes. Another receptor, S1P3R, did not contribute to the FTY720-mediated protection, as albuminuria was also reduced in diabetic S1P3R knockout mice. Further, both agonists restored WT-1 staining along with podocin and nephrin mRNA expression, suggesting podocyte protection. This was corroborated in vitro, as SEW2871 reduced TNF-α and vascular endothelial growth factor mRNA expression in immortalized podocytes grown in media containing high glucose. Whether targeting kidney S1P1Rs will be a useful therapeutic measure in DN will need direct testing.

Topics: Animals; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fingolimod Hydrochloride; Kidney; Lymphocytes; Mice; Mice, Inbred C57BL; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); Podocytes; Propylene Glycols; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sphingosine; Thiophenes; Tumor Necrosis Factor-alpha

Sphingosine-1-phosphate (S1P) has been implicated in angiogenesis, inflammation, cancerogenesis, neurological excitability and immune regulation and is synthesized by two different sphingosine kinases (SphK). It was suggested that mice lacking the gene for SphK1 exhibit no obvious phenotype, because SphK2 compensates for its absence. However, recent investigations revealed that under challenge SphK1 contributed to pro-inflammatory processes favoring Th2 and Th17 rather than Th1-type reactions. To investigate the immune modulatory role of SphK1 as opposed to SphK2 specifically for the Th1 propagating IL-12p70 we compared WT and SphK1(-/-) splenocytes and Flt3-ligand differentiated BMCs of WT and SphK1(-/-), representing dendritic cells as major producers of IL-12p70, incubated with LPS. We determined the impact on IL-12p70 in comparison to other inflammatory cytokines, and on DC and macrophage surface marker expression, SphK mRNA, protein expression and enzymatic activity in splenocytes. Our data demonstrated that SphK1 deficiency enhanced LPS-induced IL-12p70 production although SphK2 was present. To further characterize SphK1-dependent IL-12p70 regulation we exogenously applied S1P, SEW2871 and the new potent S1P1 agonist CYM5442. Both S1P and S1P1-specific analogs fully compensated the increase of IL-12p70 production in SphK1-deficient splenocytes. The use of pertussis toxin, to block G(i)-coupled signaling downstream of S1P1, again increased IL-12p70 and neglected the compensation achieved by addition of S1P and S1P1 agonists pointing on the importance of this specific S1P-receptor. Given that, in parallel to a prominent IL-12p35 increase following LPS stimulation, LPS also enhanced SphK expression and total SphK activity, we concluded that SphK1-derived S1P acting via S1P1 is a major mechanism of this negative IL-12p70 feedback loop, which did not affect other cytokines. Moreover, our data showed that SphK2 activity failed to compensate for SphK1 deficiency. These findings clearly point to a divergent and cytokine-specific impact of immune cell SphK1 and SphK2 in chronic inflammation and cancer.

Topics: Animals; Bone Marrow Cells; CD8 Antigens; Cell Differentiation; Dendritic Cells; Enzyme Assays; Flow Cytometry; Gene Deletion; Gene Expression Regulation; Indans; Interleukin-12; Interleukin-12 Receptor beta 2 Subunit; Interleukin-12 Subunit p40; Lipopolysaccharides; Lymphocyte Activation; Lysophospholipids; Membrane Proteins; Mice; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Sphingosine; Spleen; Thiophenes; Toll-Like Receptors

The sphingosine kinase (SphK)/sphingosine 1-phosphate (S1P) pathway, known to determine the fate and growth of various cell types, can enhance cardiac myocyte survival in vitro and provide cardioprotection in acute ex vivo heart preparations. However, the relevance of these findings to chronic cardiac pathology has never been demonstrated. We hypothesized that S1P signaling is impaired during chronic remodeling of the uninfarcted ventricle during the evolution of post-myocardial infarction (MI) cardiomyopathy and that a therapeutic enhancement of S1P signaling would ameliorate ventricular dysfunction. SphK expression and activity were measured in the remote, uninfarcted myocardium (RM) of C57Bl/6 mice subjected to coronary artery ligation. The mRNA expression of S1P receptor isoforms was also measured, as was the activation of the downstream S1P receptor mediators. A cardioprotective role for S1P(1) receptor agonism was tested via the administration of the S1P(1)-selective agonist SEW2871 during and after MI. As a result, the expression data suggested that a dramatic reduction in SphK activity in the RM early after MI may reflect a combination of posttranscriptional and posttranslational modulation. SphK activity continued to decline gradually during chronic post-MI remodeling, when S1P(1) receptor mRNA also fell below baseline. The S1P(1)-specific agonism with oral SEW2871 during the first 2-wk after MI reduced apoptosis in the RM and resulted in improved myocardial function, as reflected in the echocardiographic measurement of fractional shortening. In conclusion, these results provide the first documentation of alterations in S1P-mediated signaling during the in situ development of cardiomyopathy and suggest a possible therapeutic role for the pharmacological S1P receptor agonism in the post-MI heart.

Topics: Animals; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocytes, Cardiac; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Messenger; Signal Transduction; Sphingolipids; Thiophenes; Ventricular Remodeling