Objective: To determine the role of rhosignaling in sphingosine-1-phosphate (S-1-P)-induced smooth muscle cell migration.
Background: S-1-P is a bioactive sphingolipid released from activated platelets stimulating migration of smooth muscle cells (SMC) in vitro through Galphai G-proteins and MAPK activation. Rho is one of the key small GTPases required for cytoskeletal reorganization and MAPK activation during migration. We hypothesized that S-1-P-stimulated migration is regulated by the rho-signaling pathway.
Methods: Rat arterial SMCs were cultured in vitro. Linear wound assays of migration were performed in the presence of S-1-P with and without C3 (a rho antagonist) and Y (Y27632, a Rho kinase inhibitor). Western blotting was performed for MEK1-ERK1/2 and MMK3/MKK6-p38(MAPK) phosphorylation after stimulation with S-1-P with and without pre-incubation with the inhibitors. Statistics were analyzed by one-way ANOVA.
Results: S-1-P stimulated migration of SMCs in a wound assay (2-fold over control; P < 0.01), which was blocked by Rho inhibition (P < 0.05). S-1-P activated rho and induced a time-dependent increase in ERK1/2 and p38(MAPK) activation. In the presence of C3, MEK1 and ERK1/2 phosphorylation were significantly decreased, while MKK3/6 and p38(MAPK) phosphorylation were unchanged. In contrast, when rho kinase was inhibited, there was an increase in ERK1/2 and a decrease in p38(MAPK) phosphorylation. Rho kinase inhibition resulted in a decrease in MEK1/2 and MKK3/6 phosphorylation.
Conclusions: S-1-P differentially regulates the MAPK pathway through components of the rho pathway. Rho regulates ERK1/2 activation through MEK1/2, while Rho kinase negatively modulates ERK1/2 in a MEK1/2-independent manner and regulates p38(MAPK) through MKK3/6. This is the first description of differential MAPK regulation by a G-protein-coupled receptor through the rho pathway. Understanding signal transduction in SMCs will contribute to the development of molecular therapeutics for intimal hyperplasia.