Article date: June 2005
By: Scott R Macfarlane, Callum M Sloss, Pamela Cameron, Toru Kanke, Roderick C McKenzie, Robin Plevin in Volume 145, Issue 4, pages 535-544
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).
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 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.
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.
These results suggest a predominant role for the InsP3/Ca2+ axis in the regulation of IKK signalling and NFκB transcriptional activation.
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).
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 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.
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.
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
DOI: 10.1038/sj.bjp.0706204
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