Abstract: Background: S-1-P is a bioactive sphingolipid released from activated platelets, which 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 phosphatidyl-inositol 3’ kinase (PI3-K) and Phosphoinositide-dependent protein kinase-1 (PDK1) Objective: To examine the role of akt signaling during smooth muscle cell migration in response to sphingosine-1-phosphate (S-1-P) 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). Assays were performed for PI3-K, PDK1, akt and GSK3β activation in the presence of various inhibitors and after transfection with the Gβγ inhibitor. βARKCT. Results: S-1-P produced a dose-dependent cell migration and induced time dependent PI3-K, PDK1 and akt activation. The migratory responses in both assays to S-1-P were blocked by akt inhibitor (aktI). Activation of akt and dephosphorylation of its downstream kinase, GSK3β, were inhibited by aktI. Inhibition of PI3-K with LY294002 significantly reduced both PI3-K and akt activation. Inhibition of Gβγ inhibited akt activation through a reduction in both PI3-K and PDK1 activation. While inhibition of the ras with manumycin A had no effect, inhibition of rho with C3 limited both PI3-K and akt activation. PDK1 responses were unchanged by inhibition of GTPases. Inhibition of oxygen free radical generation with N-acetylcysteine and of EGFR with AG1478 inhibited PDK1 activation in response to S-1-P. Conclusion: S-1-P mediated migration is akt dependent. S-1-P mediated akt phosphorylation is controlled by Gβγ dependent, PI3-K activation, which requires the GTPase rho and Gβγ dependent PDK1 activation which requires oxygen free radical generation and EGFR activation.
Publication Year: 2008
Publication Date: 2008-02-01
Language: en
Type: article
Indexed In: ['crossref']
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