Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of G(i)-linked Ras/mitogen-activated protein kinase pathway

We addressed the mechanisms of restoration of cell surface proteinase-activ ated receptor-1 (PAR-1) by investigating thrombin-activated signaling pathw ays involved in PAR-1 re-expression in endothelial cells. Exposure of endot helial cells transfected with PAR-I promoter-luciferase reporter construct to either thrombin or PAR-I activating peptide increased the steady-state P AR-1 mRNA and reporter activity, respectively. Pretreatment of reporter-tra nsfected endothelial cells with pertussis toxin or co-expression of a minig ene encoding Il-amino acid sequence of COOH-terminal Ga; prevented the thro mbin-induced increase in reporter activity. Pertussis toxin treatment also prevented thrombin-induced MAPK phosphorylation, indicating a role of G alp ha(i) in activating the downstream MAPK pathway. Expression of constitutive ly active G alpha(i2) mutant or G beta 1 gamma 2 subunits increased reporte r activity 3-4-fold in the absence of thrombin stimulation. Go-expression o f dominant negative mutants of either Ras or MEK1 with the reporter constru ct inhibited the thrombin-induced PAR-1 expression, whereas constitutively active forms of either Ras or MEK1 activated PAR-1 expression in the absenc e of thrombin stimulation. Expression of dominant negative Src kinase or in hibitors of phosphoinositide 3-kinase also prevented the MAPK activation an d PAR-1 expression. We conclude that thrombin-induced activation of PAR-1 m ediates PAR-1 expression by signaling through G(i1/2) coupled to Src and ph osphoinositide 3-kinase, and thereby activating the downstream Ras/MAPK cas cade.