Sphingosine-1-phosphate-induced oxygen free radical generation in smooth muscle cell migration requires Gα12/13 protein-mediated phospholipase C activation

Background: Sphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid that stimulates the migration of vascular smooth muscle cell (VSMC) through G-protein coupled receptors; it has been shown to activate reduced nicotinamide dinucleotide phosphate hydrogen (NAD[P]H) oxidase. The role of phospholipase C (PLC) in oxygen free radical generation, and the regulation of VSMC migration in response to S-1-P, are poorly understood. Methods: Rat arterial VSMC were cultured in vitro. Oxygen free radical generation was measured by fluorescent redox indicator assays in response to S-1-P (0.1μM) in the presence and absence of the active PLC inhibitor (U73122; U7, 10nM) or its inactive analog U73343 (InactiveU7, 10nM). Activation of PLC was assessed by immunoprecipitation and Western blotting for the phosphorylated isozymes (β and γ). Small interfering (si) RNA to the G-proteins Gαi, Gαq, and Gα12/13 was used to downregulate specific proteins. Statistics were by one-way analysis of variance (n = 6). Results: S-1-P induced time-dependent activation of PLC-β and PLC-γ; PLC-β but not PLC-γ activation was blocked by U7 but not by InactiveU7. PLC-β activation was Gαi-independent (not blocked by pertussis toxin, a Gαi inhibitor, or Gαi2 and Gαi3 siRNA) and Gαq-independent (not blocked by glycoprotein [GP] 2A, a Gαq inhibitor, or Gαq siRNA). PLC-β activation and cell migration was blocked by siRNA to Gα12/13. Oxygen free radical generation induced by S-1-P, as measured by dihydroethidium staining, was significantly inhibited by U7 but not by InactiveU7. Inhibition of oxygen free radicals with the inhibitor diphenyleneiodonium resulted in decreased cell migration to S-1-P. VSMC mitogen-activated protein kinase activation and VSMC migration in response to S-1-P was inhibited by PLC- inhibition. Conclusion: S-1-P induces oxygen free radical generation through a Gα12/13, PLC-β-mediated mechanism that facilitates VSMC migration. To our knowledge, this is the first description of PLC-mediated oxygen free radical generation as a mediator of S-1-P VSMC migration and illustrates the need for the definition of cell signaling to allow targeted strategies in molecular therapeutics for restenosis.