Mechanisms of sphingosine-1-phosphate-induced akt-dependent smooth muscle cell migration.

Abstract

Sphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid released from activated platelets that stimulates migration of vascular smooth muscle cells (VSMC) in vitro. S-1-P will activate akt, which can regulate multiple cellular functions including cell migration. Akt activation is downstream of phosphatidylinositol 3'-kinase (PI3-K) and phosphoinositide-dependent protein kinase-1 (PDK1). The objective of this study was to examine the regulation of akt signaling during smooth muscle cell (SMC) migration in response to S-1-P. Murine arterial SMC were cultured in vitro. Linear wound and microchemotaxis assays of migration in Boyden chambers were performed in the presence of S-1-P with and without an akt inhibitor (aktI). Assays were performed for PI3-K, PDK1, akt, and GSK3beta in the presence of various inhibitors and after transfection with the G beta gamma inhibitor beta ARK(CT). S-1-P induced time-dependent PI3-K, PDK1, and akt activation. The migratory responses in both assays to S-1-P were blocked by aktI. Activation of akt and dephosphorylation of its downstream kinase, GSK3 beta, were inhibited by aktI. Inhibition of PI3-K with LY294002 significantly decreased activation of both PI3-K and akt. Inhibition of G beta gamma inhibited akt activation through a decrease in activation of both PI3-K and PDK1. Although inhibition of the ras with manumycin A had no effect, inhibition of rho with C3 limited activation of both PI3K and akt. PDK1 responses were unchanged by inhibition of GTPases. Inhibiting the generation of reactive oxygen species with N-acetylcysteine and of epidermal growth factor receptor with AG1478 inhibited PDK1 activation in response to S-1-P. S-1-P-mediated migration is akt-dependent. S-1-P-mediated akt phosphorylation is controlled by a G beta gamma-dependent PI3-K activation, which requires the GTPase rho and G beta gamma. PDK1 activation requires G beta gamma-dependent generation of reactive oxygen species and epidermal growth factor receptor activation.