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 (NF kappa B) pathway in a skin epithelial cell line NCTC2544 stably expressing PAR2 (clone G). 2 In clone G, PAR2-mediated NF kappa B luciferase reporter activity and NF kappa B DNA-binding activity was reduced by preincubation with BAPTA-AM but not BAPTA. Trypsin stimulation of inhibitory kappa B kinases, IKK alpha and IKK beta, was also inhibited following pretreatment with BAPTA-AM. 3 BAPTA/AM also prevented PAR2-mediated IKK alpha activation in cultured primary human keratinocytes. 4 The effect of BAPTA-AM was also selective for the IKK/NF kappa B 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 NF kappa B-DNA binding; however, inhibition of Ca2+-dependent protein kinase C isoforms or InsP(3) formation using GF109203X or the phospholipase C inhibitor U73122, respectively, reduced both IKK activity and NFkB-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 NF kappa B-DNA binding and IKK activity stimulated by trypsin. 7 These results suggest a predominant role for the InsP(3)/Ca2+ axis in the regulation of IKK signalling and NFkB transcriptional activation.