Dual effect of the novel peptide antagonist K‐14585 on proteinase‐activated receptor‐2‐mediated signalling

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 PAR₂, we assessed the effects of K‐14585 on PAR₂‐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 PAR₂‐activating peptide Ser‐Leu‐Ile‐Gly‐Lys‐Val (SLIGKV‐OH) in PAR₂‐expressing NCTC2544 cells. K‐14585 pretreatment did not influence PAR₂‐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 PAR₂, but not in parental or PAR₄‐expressing NCTC2544 cells, suggesting these effects were PAR₂‐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 PAR₂‐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 PAR₂, one G_q/11‐dependent and the other G_q/11‐independent.

DOI: 10.1111/j.1476-5381.2009.00415.x

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