Sphingosine-1-phosphate induced smooth muscle cell migration requires src activation

Abstract

Abstract Introduction: The object of this study is to examine the role of src signaling axis during smooth muscle cell (SMC) migration in response to sphingosine-1-phosphate (S-1-P). S-1-P is a bioactive sphingolipid released from activated platelets stimulates migration of SMC in vitro through Gi G-proteins and both MAPK and p70S6 kinase activation. Activation of the src signaling axis regulates multiple cellular functions (transcription and translation) during cell migration. Methods: Rat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of S-1-P in the presence and absence of the src inhibitor (PP2, 10μM). Western blotting was performed for MEK1/2 and ERK 1/2, MKK3/6 and p38(MAPK), and PI3K/p70S6 kinase phosphorylation after stimulation with S-1-P in the presence and absence of the src inhibitor. Results: S-1-P produced concentration-dependent cell migration in both assays and induced time dependent MEK1/2 and ERK1/2, MKK3/6 and p38(MAPK), and PI3K and p70S6 kinase phosphorylation. Cell migraton was blocked in both assays by src inhibition. PP2 inhibited MKK3/6 and p38(MAPK) but not MEK1/2, ERK1/2 or p70S6kinase activation. S-1-P mediated activation of PI3K was marginally reduced by src inhibition. Conclusions: src mediated p38(MAPK) activation is important in S-1-P induced SMC migrationand ocurs in part in part through PI3K activation. While ERK/2 and p70S6 kinase are involved in migration due to S-1-P, src is not a crucial upstream kinase in the activation of these pathways.