Dual effect of the novel peptide antagonist K-14585 on proteinase-activated receptor-2-mediated signaling

Abstract

Background and purpose: Here we have examined the effects of the novel peptide antagonist N-[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H- indol-5-yl]aminocarbonyl}-glycinyl-L-lysinyl-L-phenylalanyl-N-benzhydrylamide (K-14585) on proteinase-activated receptor (PAR) 2-mediated intracellular signalling events. Experimental approach: Using NCTC2544 cells expressing PAR 2, we assessed the effects of K-14585 on PAR 2-mediated [ 3H] inositol phosphate accumulation, MAP kinase activation, p65 NFκB phosphorylation and DNA binding and IL-8 production. Key results: Pretreatment with K-14585 (5 μM) inhibited [ 3H] inositol phosphate levels stimulated by PAR 2- activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) in PAR 2-expressing NCTC2544 cells. K-14585 pretreatment did not influence PAR 2-mediated extracellular regulated kinase activation but inhibited p38 MAP kinase phosphorylation. At a higher concentration (30 μM), K-14585 alone stimulated p38 MAP kinase activation. These effects were replicated in EAhy926 cells, endogenously expressing PAR 2, but not in parental or PAR 4-expressing NCTC2544 cells, suggesting these effects were PAR 2-dependent. SLIGKV-mediated stimulation of p38 MAP kinase phosphorylation was substantially reduced by the G q/11 inhibitor YM-254890, without affecting K-14585-mediated phosphorylation. Pretreatment with K-14585 inhibited PAR 2-mediated p65 NFκB phosphorylation and NFκB-DNA binding. K-14585 (30 μM) alone stimulated comparable NFκB reporter activity to SLIGKV-OH. K-14585 inhibited SLIGKV-stimulated IL-8 production, but given alone increased IL-8. While SLIGKV-induced IL-8 formation was reduced by both SB203580 and YM-254890, the response to K-14585 was sensitive to SB203580 but not YM-254890. Conclusions and implications: These data reveal that K-14585 has a duality of action functioning both as an antagonist and agonist due to either partial agonist actions or possible agonist-directed signalling. The data also suggest two modes of p38 MAP kinase activation emanating from PAR 2, one G q/11-dependent and the other G q/11-independent. © 2009 The British Pharmacological Society.