Cyclooxygenase-2 induction and prostacyclin release by protease-activated receptors in endothelial cells require cooperation between mitogen-activated protein kinase and NF-kappaB 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-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.
Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, United Kingdom.
Farisa
F
Syeda
Jennifer
J
Grosjean
Rebecca A
RA
Houliston
Rosemary J
RJ
Keogh
Tom D
TD
Carter
Ewa
E
Paleolog
Caroline P D
CP
Wheeler-Jones
D015121:6-Ketoprostaglandin F1 alpha / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000494:pharmacology; D002478:Cells, Cultured; D051545:Cyclooxygenase 1 / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D011464:Epoprostenol / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000378:metabolism; D044462:Receptors, Proteinase-Activated / Q000378:metabolism*; D015398:Signal Transduction; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects / Q000502:physiology
D051017:Apoptosis Regulatory Proteins; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D010446:Peptide Fragments; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D044462:Receptors, Proteinase-Activated; C101296:prostate apoptosis response-4 protein; D015121:6-Ketoprostaglandin F1 alpha; D011464:Epoprostenol; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D051550:I-kappa B Kinase; D020928:Mitogen-Activated Protein Kinases