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Proteinase-activated receptor-2 mediated phosphorylation of p65 NFκB is regulated by an inhibitory kappa b kinase and PKC - dependent pathway in keratinocytes Proteinase-activated receptors (PARs) are a unique family of GPCRs activated by serine protease cleavage. To date 4 members exist, PARs 1-4 (Macfarlane et al., 2001). PAR-2 has been found to mediate pro-inflammatory cellular responses (Ferrell et al., 2003), in part due to activation of the nuclear factor kappa B (NFκB) pathway. The PAR-2 agonist trypsin stimulates NFκB-DNA binding, an event regulated by protein kinase C (PKC)-mediated activation of the inhibitory kappa B kinases (IKKs) (Kanke et al., 2001). An additional important point of regulation within the NFκB pathway is serine-526 phosphorylation of p65 NFκB which allows transcriptional activation following initial DNA binding. In this study we sought to examine the mechanisms which regulate the phosphorylation of NFκB in response to PAR-2 activation. PAR-2 expressing NCTC2544 cells (clone G) and cultured human primary keratinocytes were used. Infection with adenovirus encoding dominant IKKs (Adv. IKK+/-) and transfection with iRNA for PKCα was for 48-72hr. Cellular levels of IκBα , p-p65NFκB and p65NFκB were assessed by Western blotting using specific antibodies. NFκB reporter activity was assayed in clone G expressing a 3 x NF k B luciferase plasmid. In clone G cells trypsin (50nM) and PAR-2 activating peptide (AP) 2-f-LIGKV-OH (50μM) stimulated a 6-12 fold increases in NFκB phosphorylation over a sixty minute period. Total p65 levels were not affected. A similar increase was observed in human primary keratinocytes. Infection with 300pfu of Adv. IKK2+/- but not IKK-1+/- reduced trypsin-stimulated phosphorylation of p65NFκB (Fold stim; trypsin = 11.54 ± 0.67, +Adv. IKK2+/- = 3.3±0.9 n= 4, p<0.05 +). A similar effect was observed in primary keratinocytes. Infection with Adv. IKK2 or the IKK2 inhibitor SC514 also reduced trypsin-stimulated NFκB reporter activity. In clone G pre-treatment with the PKC inhibitors GF109203X or rottlerin (1-10 μM) also reduced p-pNFκB levels (Fold stim; trypsil = 6.86 ± 0.76, +GF109203X = 3.1±0.8 n=4, p<0.05), but did not the effect activation of the MAP kinases. Transfection with iRNA for PKCα reduced the expression of PKC a by approximately 60% and reduced trypsin-induced phosphorylation of NFκB by a similar amount. Taken together the studies show that PAR-2-mediated phosphorylation of p65 NFκB is also strongly regulated by IKK2 which is likely to be in turn regulated by PKC isoforms.
Ferrell WRet al., (2003) J Clin Invest 111, 35-41. |
