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

In this study, we examined the role of Ca ²⁺ 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). 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. BAPTA/AM also prevented PAR2-mediated IKKα activation in cultured primary human keratinocytes. The effect of BAPTA-AM was also selective for the IKK/NFκB signalling axis; PAR2 coupling to ERK, or p38 MAP kinase was unaffected. Pharmacological inhibition of the Ca ²⁺-dependent regulatory protein calcineurin did not inhibit trypsin-stimulated IKK activity or NFκB-DNA binding; however, inhibition of Ca ²⁺-dependent protein kinase C isoforms or InsP ₃ formation using GF109203X or the phospholipase C inhibitor U73122, respectively, reduced both IKK activity and NFκB-DNA binding. Mutation of PAR2 within the C-terminal to produce a mutant receptor, which does not couple to Ca ²⁺ signalling, but is able to activate ERK, abrogated NFκB-DNA binding and IKK activity stimulated by trypsin. These results suggest a predominant role for the InsP ₃/Ca ₂₊ axis in the regulation of IKK signalling and NFκB transcriptional activation.