Sphingosine-1-phosphate induces G(alphai)-coupled, PI3K/ras-dependent smooth muscle cell migration.
BACKGROUND
Sphingolipids such as sphingosine-1-phosphate (S-1-P) are potent extracellular mediators released in response to vessel injury. S-1-P binds to G-protein-coupled receptors, which can be either G(alphai) or G(alphaq) linked. This study examines the signaling pathways involved in vascular smooth muscle cell migration after stimulation by S-1-P. We hypothesized that S-1-P stimulates migration of smooth muscle cells that is dependent upon a G(alphai)-coupled receptor, ras, phosphoinositol 3-kinase (PI3-K), and ERK 1/2.


METHODS
Vascular smooth muscle cells were cultured in vitro. A linear wound assay and Boyden chamber assay of migration were employed in the presence of S-1-P and inhibitors of G(alphai) [pertussis toxin (PTx), 100 ng/ml], G(alphaq) (GP-2A, 10 microM), ras [manumycin A (MA), 10 microM], PI3-K [Wortmannin (Wn), 10 microM], and MEK1 [PD98059 (PD), 25 microM]. Western blotting was performed separately to examine p42/p44 MAP kinase (ERK 1/2) activation in response to S-1-P with these inhibitors.


RESULTS
S-1-P induced vascular smooth muscle cell migration. This response was decreased by preincubation with PTx, suggesting a receptor linked, G(alphai)-mediated response. Application of a G(alphaq) inhibitor did not affect this response. S-1-P induced ERK 1/2 phosphorylation in a time-dependent manner. This S-1-P-induced cell migration was PD-sensitive in the Boyden chamber assay, confirming that it is MEK1- and ERK1/2-dependent. Inhibition of ras with MA and PI3-K with Wn also reduced ERK phosphorylation and smooth muscle cell migration in response to S-1-P.


CONCLUSIONS
S-1-P induces smooth muscle cell migration through a G(alphai)-linked, ras- and PI3-K-coupled, ERK 1/2-dependent process. Understanding signal transduction will allow targeted molecular interventions to treat the response of a vessel to injury.
Tanski
William
W
Vascular Biology and Therapeutics Program, Department of Surgery, University of Rochester, New York 14642, USA.
Roztocil
Elisa
E
Davies
Mark G
MG
D000818:Animals; D001011:Aorta / Q000166:cytology; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D015194:Diffusion Chambers, Culture; D004305:Dose-Response Relationship, Drug; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D008246:Lysophospholipids; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000502:physiology; D008297:Male; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D018631:ras Proteins / Q000378:metabolism
D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D008246:Lysophospholipids; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D013110:Sphingosine; D018631:ras Proteins; C060506:sphingosine 1-phosphate; C482127:Map2k1 protein, mouse