Sphingosine-1-phosphate induces G(alphai)-coupled, PI3K/ras-dependent smooth muscle cell migration

Abstract

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.