Proteinase-activated receptor-2 (PAR-2) is one of 4 PARs activated by enzymatic cleavage of the N-terminus (Macfarlane et al., 2001), and is implicated in a number of inflammatory processes such as arthritis and skin hypersensitivity (Ferrell et al., 2003, Kawagoe et al., 2002). PAR-2 is believed to couple to a number of intracellular signalling pathways relevant to its pro-inflammatory actions, including the MAP kinases and nuclear factor kappa B (NFB). As PAR-2 is also coupled to intracellular Ca²⁺ signalling (Seater et al., 2004), we sought to determine the influence of intracellular Ca²⁺ increases upon the activation of the NFB pathway and the upstream kinases intermediates, the inhibitory kappa B kinases (IKKs).

NCTC2544 cells expressing hPAR-2 (clone G) were used in this study. NFB transcriptional activity was assessed by luciferase reporter assay. NFB-DNA binding was assayed by EMSA, whilst IKK activity measured by in vitro kinase assay.

Trypsin (30nM) stimulated an approximate 10-15 fold increase in NFB DNA transcriptional activity over a 6hr time period (trypsin, 6hr =12.2 ± 3.2 fold, n=4). This value was significant reduced by pretreatment of cells with BAPTA-AM (50 µM) but not BAPTA (1mM); (trypsin + BAPTA-AM =1.3 ± 0.1 fold, p<0.05). Trypsin-stimulated NFB DNA-binding and activation of IKK and IKKß was effected in a similar manner. (IKK , fold stim; trypsin = 3.31 ± 0.37, trypsin +BAPTA-AM = 1.1 ± 0.1, p<.0.05, n=4). Trypsin also stimulated the phosphorylation of p42/44 MAP kinase and p38 MAP kinase. Neither BAPTA-AM nor BAPTA pre-treatment significantly reduced these stimulations. Pre-treatment of cells with the CaM kinase II inhibitor, KN-62 (10 µM) substantially increased NFB activation whilst the calcineurin inhibitor FK506 was without effect. However, the PKC and PLC inhibitors, GF109203X (10 µM) and U73122 (10 µM) significantly decreased both IKK and NFB-DNA binding. Transient transfection of parental NCTC2544 cells with a C-terminal mutant PAR-2 ( d34-43 C-PAR), shown previously to be unable to couple to Ca²⁺ signalling (Seatter et al., 2004), exhibited a substantially reduced ability to increase activation of either IKK nor NF B relative to levels stimulated through the wild type receptor.

Taken together, these data indicate the importance of intracellular Ca²⁺ in the regulation of NFB in response to PAR-2 stimulation in clone G cells. It also indicates that the NFB and MAP kinase cascades are differentially regulated via Ca²⁺-dependent and independent mechanisms. These findings may be relevant in conditions such as psoriasis where PAR-2 is implicated and an impairment in Ca²⁺ signalling is observed.

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Seatter MJ et al. (2003) Cell Signal 16: 21-29.