PAR-2 mediated inhibition of TNFα stimulated JNK activity

Kathryn McIntosh1, Robin Plevin1, John C Lockhart2, William R Ferrell3, Laurence Cadalbert1
1Strathclyde University, Glasgow, United Kingdom, 2Paisley University, Paisley, United Kingdom, 3Glasgow University, Glasgow, United Kingdom

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 has been linked to the stress-activated protein (SAP) (SAP), JNK and p38 MAP kinase and also NFκB signalling (Kanke et al, 2001; Sabri et al, 2001), pathways known to be involved in pro-inflammatory responses in several cell types. The cytokine TNF-α is also an important mediator of pro-inflammatory responses, and indeed signals through both the SAPK and NFκB pathways. Since both TNF-α and PAR-2 activators are released during inflammation, we examined the possibility of crosstalk between PAR-2 and TNFα at the level of the SAP kinases.

The NCTC2544 stably expressing PAR-2 (Clone G) was used for this study. Phosphorylation of p38 and JNK was examined using Western blotting, using phospho-specific antibodies, and JNK activity was measured using solid phase in vitro kinase activity assays using GST-c-Jun as a substrate.

In Clone G cells trypsin (50nM), SLIGKV-OH (200μM) and TNF-α (10ng/ml) caused a time and concentration-dependent- increase in p38 and JNK phosphorylation however, preliminary results failed to show evidence of synergy; PAR-2 stimulation failed to potentiate either phospho-p38 or phospho-JNK activation mediated by TNF-α and vice versa. However, surprisingly activation of PAR-2 using low concentrations of AP (1-20μm) substantially reduced the ability of TNF-α to stimulate JNK-mediated phosphorylation of c-Jun although phosphorylation of JNK itself was not effected (TNF = 107.21 fold +/- 1.64 plus peptide = 6.09 fold +/- 1.45 p<0.01, n=3). This inhibitory effect was not apparent in non transfected cells, nor replicated in PAR-4 expressing cells. The inhibitory effect of PAR-2 was mimicked by the protein kinase C activator PMA (100nM), completely reversed by the PKC inhibitor (GF109203X) (TNF & peptide & GFX = 78.98 +/- 14.02 p<0.01, n=3), and partially reversed by the novel Gq/11 inhibitor YM-254890 (TNF& peptide = 5.5 fold +/- 0.20 p<0.05, plus YM = 12.20 fold +/- 1.20 p<0.01, n=3), consistent with a role for both Ca2+ -dependent and independent PKC isoforms in the inhibitory response.

These results indicate a potential mechanistic explanation for both the anti and pro-inflammatory actions of PAR-2, dependant upon the occupancy of the receptor and highlight a possible novel therapeutic avenue for the development of PAR-2 agonists as anti-inflammatory drugs.

MacFarlane SR et al. (2001) Pharmacol Rev 53:245-82
Sabri, Muske et al. (2000) Circulation Research 86(10): 1054-1061.
Kanke, MacFarlane et al. (2001) Journal Biological Chemistry 34(276):31657-66.

Kathryn McIntosh is funded by a University of Paisley Studentship. This work was funded by The Wellcome Trust.