jte-013 and dihydrosphingosine-1-phosphate

Prostacyclin (PGI2) is an important regulator of vascular homeostasis. Our goal was to analyze the role of sphingosine 1-phosphate (S1P) and its receptors in the up-regulation of cyclooxygenase-2 (Cox-2) induced by HDL in human vascular smooth muscle cells (VSMC). S1P induces Cox-2 expression in a time-and dose-dependent manner at concentrations (0.02-1 microM) compatible with those present in physiological HDL levels. The effect was mimicked by dihydro-S1P (DhS1P), a S1P derivative that only acts through cell surface S1P receptors. Desensitization of S1P receptors with S1P (or DhS1P) abolished HDL-induced Cox-2 up-regulation and PGI2 release. Inhibition of S1P receptors by suramin (inhibitor of S1P3), JTE013 (inhibitor of S1P2) or VPC23019 (inhibitor of S1P1 and S1P3) reduced the up-regulation of Cox-2 induced by HDL and S1P. The combination of suramin and JTE013 increased the inhibitory effect compared to that observed in cells treated with each inhibitor alone. siRNA against S1P2 or S1P3 significantly reduced the ability of HDL and S1P to up-regulate Cox-2. Simvastatin induced over-expression of S1P3 and potentiated the induction of Cox-2 expression produced by HDL (or S1P). Finally, suramin, JTE013 and VPC23019 inhibited p38 MAPK and ERK1/2 signaling pathways activated by HDL (or S1P) and the downstream activation of CREB, a key transcription factor involved in Cox-2 transcriptional up-regulation. These results indicate that S1P receptors, in particular S1P2 and S1P3, are involved in the Cox-2-dependent effects of HDL on vascular cells. Strategies aimed to therapeutically modulate S1P or S1P receptors could be useful to improve cardiovascular protection.

Topics: Cells, Cultured; Cyclic AMP Response Element-Binding Protein; Cyclooxygenase 2; Dose-Response Relationship, Drug; Enzyme Induction; Epoprostenol; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lipoproteins, HDL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; p38 Mitogen-Activated Protein Kinases; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; RNA Interference; RNA, Messenger; RNA, Small Interfering; Simvastatin; Sphingosine; Sphingosine-1-Phosphate Receptors; Suramin; Time Factors; Up-Regulation

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