Cyclooxygenase-2 Induction and Prostacyclin Release by Protease-activated Receptors in Endothelial Cells Require Cooperation between Mitogen-activated Protein Kinase and NF-κB Pathways

The functional significance of protease-activated receptors (PARs) in endothelial cells is largely undefined, and the intracellular consequences of their activation are poorly understood. Here, we show that the serine protease thrombin, a PAR-1-selective peptide (TFLLRN), and SLIGKV (PAR-2-selective peptide) induce 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 6-keto-PGF 1α , the stable hydrolysis product of prostacyclin, and this was inhibited by indomethacin and the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38 MAPK , and pharmacological blockade of MEK with either PD98059 or U0126 or of p38 MAPK by SB203580 or SB202190 strongly inhibited thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF 1α formation. Thrombin and peptide agonists of PAR-1 and PAR-2 increased luciferase activity in human umbilical vein endothelial cells infected with an NF-κB-dependent luciferase reporter adenovirus, and this, as well as PAR-induced 6-keto-PGF 1α synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IκBα. Thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF 1α generation were markedly attenuated by the NF-κB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. Activation of PAR-1 or PAR-2 promoted nuclear translocation and phosphorylation of p65-NF-κB, and thrombin-induced but not PAR-2-induced p65-NF-κB phosphorylation was reduced by inhibition of MEK or p38 MAPK . Activation of PAR-4 by AYPGKF increased phosphorylation of ERK1/2 and p38 MAPK without modifying NF-κB activation or COX-2 induction. Our data show that PAR-1 and PAR-2, but not PAR-4, are coupled with COX-2 expression and sustained endothelial production of vasculoprotective prostacyclin by mechanisms that depend on ERK1/2, p38 MAPK , and IκBα-dependent NF-κB activation.