Sphingosine-1-phosphate stimulates smooth muscle cell migration through Gαi- and PI3-kinase-dependent P38MAPK activation

Allison J. Fegley, William J. Tanski, Elisa Roztocil, Mark G. Davies

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

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 Gαi-, Gαq-, or G 12/13-linked. This study examines the role of p38 mitogen-activated protein kinase (p38MAPK) in vascular smooth muscle cell migration after stimulation with S-1-P, and pathways leading to p38MAPK activation. S-1-P has previously been shown to stimulate migration of vascular smooth muscle cells (VSMCs) in vitro through ERK1/2 and Gi. We hypothesized that S-1-P-induced VSMC migration is also dependent on p38 MAPK activation through a Gi-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 p38MAPK (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 p38MAPK activation in response to S-1-P with and without SB203580, AG1478, or inhibitors of Gi (pertussis toxin), PI3-K (Wortmannin and LY294002), or MEK1 (PD98059). Western blotting and immunoprecipitation for targets of p38MAPK (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 p38MAPK that peaked at 10 min, as well as activation of MAPKAP kinase-2. Activation of p38MAPK 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 Gi-linked and a PI3-K coupled, p38MAPK- dependent process. PI3-K appears to function upstream of p38MAPK, but was not Gi-dependent. S-1-P-stimulated activation of p38MAPK 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.

Original languageEnglish (US)
Pages (from-to)32-41
Number of pages10
JournalJournal of Surgical Research
Volume113
Issue number1
DOIs
StatePublished - Jul 2003
Externally publishedYes

Keywords

  • G-protein signaling
  • Migration
  • P38 MAP kinase
  • PI3-kinase
  • Signal transduction
  • Smooth muscle cells
  • Sphingosine-1-phosphate

ASJC Scopus subject areas

  • Surgery

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