The role of protease‐activated receptor‐4 in vascular smooth muscle cell migration and proliferation

Abstract

Recent studies have shown that thrombin stimulates the activation of extracellular regulated kinase (ERK1/2) and Rho/Rho kinase, which are associated with cellular proliferation and migration in vascular diseases such as hypertrophy and atherosclerosis. Thrombin activates these kinases through protease‐activated receptors (PARs), which are a family of G‐protein coupled receptors activated by the enzymatic cleavage of the extracellular NH2 terminus. After verifying that PAR‐4 was expressed in vascular smooth muscle cells (VSMCs), we used a PAR‐4 activating peptide (AYPGKF) to stimulate the cells in a time‐ and concentration‐dependent manner. Using Western blot analysis, we observed that 25uM of the PAR‐4 agonist maximally phosphorylated ERK1/2 at a time point of 15 minutes. To determine if PAR‐4‐induced phosphorylation of ERK1/2 was dependent on Rho kinase, we pretreated VSMCs with 25uM of the Rho kinase inhibitor (Y27632) for one hour and then stimulated the cells with 25uM of AYPGKF for 15 minutes or 5U/ml of thrombin for 5 minutes. ERK1/2 phosphorylation was significantly attenuated in the presence of the Rho kinase inhibitor when stimulated with PAR‐4. In addition, pretreatment with the rho kinase inhibitor significantly decreased thrombin‐induced ERK1/2 phosphorylation. We demonstrated that PAR‐4 signals via Rho kinase in the phosphorylation of ERK1/2. We conclude that PAR‐4 may be a key mediator in the cellular migration and/or proliferation of VSMCs.NIH‐NIGMS‐SCORE