Sphingosine-1-phosphate receptor-2 regulates expression of smooth muscle alpha-actin after arterial injury.

OBJECTIVE This study tests the hypothesis that S1P2R regulates expression of SMC differentiation genes after arterial injury. METHODS AND RESULTS Carotid ligation injury was performed in wild-type and S1P2R-null mice. At various time points after injury, expression of multiple SMC differentiation genes, myocardin, and S1P receptors (S1P1R, S1P2R, and S1P3R) was measured by quantitative PCR. These experiments demonstrate that at day 7 after injury, S1P2R specifically regulates expression of smooth muscle alpha-actin (SMA) and that this is not mediated by changes in expression of myocardin or any of the S1PRs. In vitro studies using carotid SMCs prepared from wild-type and S1P2R-null mice show that S1P stimulates expression of all SMC-differentiation genes tested, but S1P2R significantly regulates expression of SMA and SM22 alpha only. Chromatin immunoprecipitation assays suggest that S1P-induced recruitment of serum response factor to the SMA promoter and enhancer largely depends on S1P2R. S1P-stimulated SMA expression requires S1P2R-dependent activation of RhoA and mobilization of calcium from intracellular stores. Chelation of calcium does not affect the activation of RhoA by S1P, whereas blockade of Rho by C3 exotoxin partially inhibits the mobilization of calcium by S1P. CONCLUSIONS The results of this study support the hypothesis that S1P2R regulates expression of SMA after injury. We further conclude that transcriptional regulation of SMA by S1P in vitro requires S1P2R-dependent activation of RhoA and mobilization of calcium from intracellular calcium stores.