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

Farisa Syeda,Jennifer Grosjean,Rebecca A Houliston,Rosemary J Keogh,Tom D Carter,Ewa Paleolog,Caroline P.D Wheeler-Jones

doi:10.1074/jbc.m509292200

Abstract

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-PGF1alpha, 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 p38MAPK, and pharmacological blockade of MEK with either PD98059 or U0126 or of p38MAPK by SB203580 or SB202190 strongly inhibited thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha formation. Thrombin and peptide agonists of PAR-1 and PAR-2 increased luciferase activity in human umbilical vein endothelial cells infected with an NF-kappaB-dependent luciferase reporter adenovirus, and this, as well as PAR-induced 6-keto-PGF1alpha synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IkappaBalpha. Thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha generation were markedly attenuated by the NF-kappaB 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-kappaB, and thrombin-induced but not PAR-2-induced p65-NF-kappaB phosphorylation was reduced by inhibition of MEK or p38MAPK. Activation of PAR-4 by AYPGKF increased phosphorylation of ERK1/2 and p38MAPK without modifying NF-kappaB 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, p38MAPK, and IkappaBalpha-dependent NF-kappaB activation.

Highlights

The principal mechanism through which serine proteases regulate cell behavior is by activation of a unique family of G-protein-coupled receptors, referred to as protease-activated receptors (PARs)2 [1, 2]
PAR Agonists Differentially Induce COX-2 Expression and PGI2 Synthesis in HUVEC—Peptide agonists for PARs have been extensively characterized for PAR selectivity and are currently in wide use to interrogate the functional significance of PARs in a number of systems
We have recently shown that thrombin enhances expression of COX-2 protein and mRNA in HUVEC and that this is accompanied by prolonged PGI2 synthesis [12]

Summary

Introduction

The principal mechanism through which serine proteases regulate cell behavior is by activation of a unique family of G-protein-coupled receptors, referred to as protease-activated receptors (PARs)2 [1, 2]. We provided evidence that the human PAR-2-selective agonist peptide SLIGKV increased COX-2 protein in HUVEC and enhanced COX-2 mRNA and PGI2 release after 2 and 6 h, respectively [12].

Results

Conclusion