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.
- inhibitory kappa B kinase
- nuclear factor kappa B
- proteinase-activated receptor-2