Proteinase-activated receptor-2( PAR-2) and tumour necrosis factor-alpha ( TNFα) signalling in inflammation

Proteinase activated receptor-2 (PAR-2) is a novel G-protein coupled receptor, that is activated by means of proteolytic cleavage (Macfarlane et al, 2001) and has both pro and anti-inflammatory actions depending upon the system examined. PAR-2 have been linked to the stress-activated protein kinases (SAPKs), JNK and p38 MAP kinase and NFK13 signalling (Kanke et al, 2001; Sabri et al, 2000), pathways known to be involved in proinflammatory responses in several cell types. TNFa has been demonstrated to up-regulate PAR-2 expression in a variety of cell types (Nystedt et al, 1996; Ritchie et al, 2007). Since both TNFa and PAR-2 are implicated in inflammation, we examined the possibility of altered PAR-2 trafficking under inflammatory conditions and possible crosstalk between PAR-2 and TNFa at the level of intracellular signalling. Previous studies have characterised P AR-2 trafficking in transfected cell lines, however the effects of inflammatory stimuli on the kinetics of PAR-2 trafficking has not been investigated. The study sought to re-characterise PAR-2 trafficking in the presence of inflammatory stimuli. NCTC2544 cells transfected with YFP epitope tagged PAR-2, demonstrated clear PAR-2 expression and trafficking of the receptor was successfully characterised, however no significant differences in the kinetics of P AR-2 trafficking under inflammatory conditions compared to control was observed. The latter part of the study examined PAR-2 and TNFa mediated activation of the MAPK and NFK13 pathways. In a keratinocyte cell line stably expressing PAR-2 (CloneG), trypsin, SLIGKV-OH, and TNFa, caused a time and concentration-dependent increase in p38 MAPK and JNK phosphorylation however, preliminary results failed to show evidence of synergy between the receptors. Surprisingly however, pre-activation of P AR-2 substantially reduced the ability of TNFa to activate JNK. The inhibitory effect of P AR-2 was mimicked by the protein kinase C activator PMA, partially reversed by the PKC inhibitor GF109203X, and completely reversed by the novel Gaqlll inhibitor YM-254890, consistent with a role for both Ca2+ -dependent and independent PKC isoforms and for P AR-2 coupling to Gaq/11 to mediate this agonist driven inhibitory response. These results indicate a potential mechanistic explanation for both the anti and pro-inflammatory actions of P AR- 2, and highlight a possible novel therapeutic avenue for the development ofPAR-2 agonists as anti-inflammatory drugs.