TY - JOUR
T1 - The role of intracellular Ca 2+ in the regulation of proteinase-activated receptor-2 mediated nuclear factor kappa B signalling in keratinocytes
AU - Macfarlane, Scott R.
AU - Sloss, Callum M.
AU - Cameron, Pamela
AU - Kanke, Toru
AU - McKenzie, Roderick C.
AU - Plevin, Robin
PY - 2005/6/1
Y1 - 2005/6/1
N2 - 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.
AB - 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.
KW - inhibitory kappa B kinase
KW - keratinocytes
KW - nuclear factor kappa B
KW - proteinase-activated receptor-2
UR - http://www.scopus.com/inward/record.url?scp=22044453675&partnerID=8YFLogxK
U2 - 10.1038/sj.bjp.0706204
DO - 10.1038/sj.bjp.0706204
M3 - Article
C2 - 15821758
AN - SCOPUS:22044453675
VL - 145
SP - 535
EP - 544
JO - British Journal of Pharmacology
JF - British Journal of Pharmacology
SN - 0007-1188
IS - 4
ER -