SPHINGOSINE‐1‐PHOSPHATE INDUCED SMOOTH MUSCLE CELL MIGRATION REQUIRES RHO DEPENDENT AKT ACTIVATION

Abstract

Background: S-1-P stimulates migration of smooth muscle cells in vitro through Gαi G-proteins and MAPK activation and has been shown to utilize the small GTPase, rho. S-1-P will activate akt, which can regulates multiple cellular functions including migration. akt activation is downstream of phosphatidyl - inositol 3 kinase (PI3 - K). The control of akt signaling during migration in response to S-1-P is poorly understood. Methods: Murine arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of S-1-P with and without an akt inhibitor (aktI). Western blotting was performed for PI3-K, akt and GSK3β activation in the presence of the PI3-K inhibitors, Wortmannin (WN) and LY294002 (LY), aktI, the rho inhibitor, C3 and the rho kinase inhibitor, Y27632. Additional experiments were performed with transfections of siRNA to rho to decrease rho expression in SMC. Results: S-1-P produced a dose-dependent cell migration in both migration assays and induced PI3-K and akt activation. The migratory responses in both assays to S-1-P were blocked by aktI. Inhibition of PI3-K significantly reduced akt activation. Activation of akt and dephosphorylation of its downstream kinase, GSK3β, were inhibited by aktI. S-1-P activated rho and this activation was inhibited by C3, but not by Y27632. In the presence of C3, the rho inhibitor, PI3-K activation and akt phosphorylation were significantly reduced in response to S-1-P. Inhibition of rho kinase had no effect. siRNA to rho decreased rho expression by >80% without affecting cell viability and resulted in significantly decreased PI3-K activation and akt phosphorylation in response to S-1-P. Conclusion: S-1-P mediated migration is akt dependent and is is controlled by rho dependent PI3-K activation.