The role of intracellular Ca2+ in the regulation of proteinase‐activated receptor‐2 mediated nuclear factor kappa B signalling in keratinocytes

1 In this study, we examined the role of Ca2+ in linking proteinase‐activated receptor‐2 (PAR2) to the nuclear factor kappa B (NFκB) pathway in a skin epithelial cell line NCTC2544 stably expressing PAR2 (clone G). 2 In clone G, PAR2‐mediated NFκB luciferase reporter activity and NFκB DNA‐binding activity was reduced by preincubation with BAPTA‐AM but not BAPTA. Trypsin stimulation of inhibitory kappa B kinases, IKKα and IKKβ, was also inhibited following pretreatment with BAPTA‐AM. 3 BAPTA/AM also prevented PAR2‐mediated IKKα activation in cultured primary human keratinocytes. 4 The effect of BAPTA‐AM was also selective for the IKK/NFκB signalling axis; PAR2 coupling to ERK, or p38 MAP kinase was unaffected. 5 Pharmacological inhibition of the Ca2+‐dependent regulatory protein calcineurin did not inhibit trypsin‐stimulated IKK activity or NFκB‐DNA binding; however, inhibition of Ca2+‐dependent protein kinase C isoforms or InsP3 formation using GF109203X or the phospholipase C inhibitor U73122, respectively, reduced both IKK activity and NFκB‐DNA binding. 6 Mutation of PAR2 within the C‐terminal to produce a mutant receptor, which does not couple to Ca2+ signalling, but is able to activate ERK, abrogated NFκB‐DNA binding and IKK activity stimulated by trypsin. 7 These results suggest a predominant role for the InsP3/Ca2+ axis in the regulation of IKK signalling and NFκB transcriptional activation. British Journal of Pharmacology (2005) 145, 535–544. doi:10.1038/sj.bjp.0706204