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 (NFkappaB) pathway in a skin epithelial cell line NCTC2544 stably expressing PAR2 (clone G). 2 In clone G, PAR2-mediated NFkappaB luciferase reporter activity and NFkappaB DNA-binding activity was reduced by preincubation with BAPTA-AM but not BAPTA. Trypsin stimulation of inhibitory kappa B kinases, IKKalpha and IKKbeta, was also inhibited following pretreatment with BAPTA-AM. 3 BAPTA/AM also prevented PAR2-mediated IKKalpha activation in cultured primary human keratinocytes. 4 The effect of BAPTA-AM was also selective for the IKK/NFkappaB 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 NFkappaB-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 NFkappaB-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 NFkappaB-DNA binding and IKK activity stimulated by trypsin. 7 These results suggest a predominant role for the InsP3/Ca2+ axis in the regulation of IKK signalling and NFkappaB transcriptional activation.
Macfarlane
Scott R
SR
Department of Physiology & Pharmacology, University of Strathclyde, Strathclyde Institute for Biomedical Sciences, 27 Taylor Street, Glasgow G4 0NR, Scotland.
Sloss
Callum M
CM
Cameron
Pamela
P
Kanke
Toru
T
McKenzie
Roderick C
RC
Plevin
Robin
R
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D006801:Humans; D051550:I-kappa B Kinase; D007211:Indoles / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008301:Maleimides / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009841:Oligonucleotides / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D044464:Receptor, PAR-2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D016559:Tacrolimus / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D004963:Estrenes; D007211:Indoles; D008301:Maleimides; D016328:NF-kappa B; D009841:Oligonucleotides; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D044464:Receptor, PAR-2; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D014357:Trypsin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016559:Tacrolimus