Sphingosine-1-phosphate stimulates smooth muscle cell migration through Gα_i- and PI3-kinase-dependent P38^MAPK activation

Background. Sphingosine-1-phosphate (S-1-P) is an extracellular mediator released in response to vessel injury. S-1-P binds to G-protein-coupled receptors, which can be Gα_i-, Gα_q-, or G _12/13-linked. This study examines the role of p38 mitogen-activated protein kinase (p38^MAPK) in vascular smooth muscle cell migration after stimulation with S-1-P, and pathways leading to p38^MAPK activation. S-1-P has previously been shown to stimulate migration of vascular smooth muscle cells (VSMCs) in vitro through ERK1/2 and G_i. We hypothesized that S-1-P-induced VSMC migration is also dependent on p38 ^MAPK activation through a G_i-coupled extracellular receptor and phosphoinositide 3-kinase (PI3-K). Methods. VSMCs were cultured in vitro. A linear wound assay was performed in the presence of S-1-P and inhibitors of p38^MAPK (SB203580) or epidermal growth factor (EGF) receptor kinase (AG1478). Chemotaxis stimulated by S-1-P was also assayed in a modified Boyden chamber with and without SB203580 pretreatment. Western blotting was performed to examine p38^MAPK activation in response to S-1-P with and without SB203580, AG1478, or inhibitors of G_i (pertussis toxin), PI3-K (Wortmannin and LY294002), or MEK1 (PD98059). Western blotting and immunoprecipitation for targets of p38^MAPK (MAPKAP kinase-2) and PI3-K (Akt) were also performed. Results. S-1-P stimulated migration of VSMCs in both wound and Boyden transwell assays. This migration was inhibited by SB203580 to the level of control, whereas AG478 had no effect. S-1-P stimulated activation of p38^MAPK that peaked at 10 min, as well as activation of MAPKAP kinase-2. Activation of p38^MAPK was significantly inhibited by SB203580, pertussis toxin, Wortmannin, and LY294002, but not by PD98059 or AG1478; MAPKAP kinase-2 activation was inhibited by SB203580. Akt was activated by S-1-P at 3 to 5 min; this response was inhibited by Wortmannin and LY294002, but not by SB203580 or pertussis toxin. Conclusions. S-1-P induced VSMC migration through a G_i-linked and a PI3-K coupled, p38^MAPK- dependent process. PI3-K appears to function upstream of p38^MAPK, but was not G_i-dependent. S-1-P-stimulated activation of p38^MAPK does not signal via transactivation of the EGF receptor. Understanding signal transduction will allow targeted molecular interventions to treat the response of a vessel to injury.