Proteinase-activated receptor-2 mediated inhibition of TNFalpha-stimulated JNK activation - A novel paradigm for G(q/11) linked GPCRs.
In this study we examined the potential for PAR(2) and TNFalpha to synergise at the level of MAP kinase signalling in PAR(2) expressing NCTC2544 cells. However, to our surprise we found that activation of PAR(2) by trypsin or the specific activating peptide SLIGKV-OH strongly inhibited both the phosphorylation and activity of JNK. In contrast neither p38 MAP kinase nor ERK activation was affected although TNFalpha stimulated IkappaBalpha loss was partially reversed. The inhibitory effect was not observed in parental cells nor in cells expressing PAR(4), however inhibition was reversed by pre-incubation with the novel PAR(2) antagonist K14585, suggesting that the effect is specific for PAR(2) activation. SLIGKV-OH was found to be more potent in inhibiting TNFalpha-induced JNK activation than in stimulating JNK alone, suggesting agonist-directed signalling. The PKC activator PMA, also mimicked the inhibitory effect of SLIGKV-OH, and the effects of both agents were reversed by pre-treatment with the PKC inhibitor, GF109203X. Furthermore, incubation with the novel G(q/11) inhibitor YM25480 also reversed PAR(2) mediated inhibition. Activation of PAR(2) was found to disrupt TNFR1 binding to RIP and TRADD and this was reversed by both GF109203X and YM25480. A similar mode of inhibition observed in HUVECs through PAR(2) or P2Y2 receptors demonstrates the potential of a novel paradigm for GPCRs linked to G(q/11), in mediating inhibition of TNFalpha-stimulated JNK activation. This has important implications in assessing the role of GPCRs in inflammation and other conditions.
Department of Physiology and Pharmacology, University of Strathclyde, Strathclyde Institute for Pharmacy and Biomedical Sciences, Glasgow, Scotland, UK.
Kathryn
K
McIntosh
Margaret R
MR
Cunningham
Laurence
L
Cadalbert
John
J
Lockhart
Gary
G
Boyd
W R
WR
Ferrell
Robin
R
Plevin
D002478:Cells, Cultured; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008301:Maleimides / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000378:metabolism; D015398:Signal Transduction; D014357:Trypsin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D007211:Indoles; D008301:Maleimides; D009842:Oligopeptides; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; D014409:Tumor Necrosis Factor-alpha; C109968:seryl-leucyl-isoleucyl--glycyl-lysyl-valine; D051550:I-kappa B Kinase; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases; D014357:Trypsin; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C112908:protease-activated receptor 4; C070515:bisindolylmaleimide I