Protease-activated receptors and endothelial prostanoid production

Abstract

The principal mechanism through which serine proteases regulate cell behaviour is by activation of a unique family of G-protein coupled receptors, referred to as protease-activated receptors (PARs 1–4). The functional significance of PARs in endothelial cells is largely undefined and the intracellular consequences of their activation are poorly understood. We have shown that the serine protease thrombin, as well as PAR-1 and -2-selective peptides induces cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of PGI2, and this was inhibited by the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38mapk and pharmacological or molecular blockade of MEK or p38mapk strongly inhibited thrombin- and PAR-2-induced COX-2 expression and PGI2 formation. Thrombin, and peptide agonists of PAR-1 and PAR-2, increased luciferase activity in HUVEC infected with an NF-κB-dependent luciferase reporter adenovirus and this, as well as PAR-induced PGI2 synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IκBα. Thrombin- and PAR-2-induced COX-2 expression and 6-keto-PGF1α generation were markedly attenuated by the NF-κB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. PAR-1 or PAR-2 stimulation caused nuclear translocation and phosphorylation of p65-NF-κB, and thrombin-, but not PAR-2-induced p65-NF-κB phosphorylation was reduced by inhibition of MEK or p38mapk. Activation of PAR-4 increased phosphorylation of ERK1/2 and p38mapk without modifying NF-κB activation or COX-2 induction. These findings provide new insights into the molecular mechanisms used by proteases and PARs to regulate generation of the cytoprotective molecule PGI2 through changes in endothelial COX-2 expression.