G-protein-dependent and -independent pathways regulate proteinase-activated receptor-2 mediated p65 NFκB serine 536 phosphorylation in human keratinocytes
G-protein-dependent and -independent pathways regulate proteinase-activated receptor-2 mediated p65 NFκB serine 536 phosphorylation in human keratinocytes
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DOI：
10.1016/j.cellsig.2008.02.015
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发表时间：
2008-07-01
影响因子：
4.8
通讯作者：
Plevin, Robin
中科院分区：
文献类型：
--
作者：
Goh, Fui Goon;Sloss, Callum M.;Plevin, Robin
GPCRs的偶联机制，如PAR-2，与p65核因子kappaB的磷酸化之间的偶联机制尚未被研究。在目前的研究中，我们发现胰酶和选择性的PAR-2激活肽2f-LIGKV-OH可以刺激p65/relA在转染性皮肤上皮细胞系和原代角质形成细胞中丝氨酸536磷酸化水平的大幅和持续增加。平行实验表明，在两种类型的细胞中，p65核因子kappaB的磷酸化是通过选择性激活IKK2来介导的。用PKC抑制剂GF109203X或PKCαsiRNA处理可降低15min的磷酸化水平，但不能降低30min的磷酸化水平，而选择性PKC Delta抑制剂rotlerin和PKC Delta siRNA可降低这两个时间点的反应。用新型GQ/11抑制剂YM-254890和GQ/11siRNA预处理细胞，可引起类似的抑制模式，并降低PAR-2介导的NF-kappa B转录活性。此外，一种新的PAR-2突变体PAR-234-43刺激细胞，延迟了p65的磷酸化，但不影响ERK的激活动力学。百日咳毒素对Gi或G12/13通路的抑制或RGS突变体LSC的过度表达也不影响NF-kappa B的磷酸化。综上所述，这些数据表明在p65核因子kappaB的早期磷酸化过程中依赖于Gq/11，这随后影响了最初依赖于nfkappaB的基因转录活性，但后来对p65的调控没有受到影响。总体而言，这些新的数据表明，在角质形成细胞中，一条依赖IKK2的、主要不依赖于C蛋白的通路参与了PAR-2对核因子kappa B磷酸化的调节。(C)2008 Elsevier Inc.保留所有权利。
The mechanisms underpinning the coupling of GPCRs, such as PAR-2, to the phosphorylation of p65 NF kappa B have not been investigated. In the current study we found that trypsin and the selective PAR-2 activating peptide, 2f-LIGKV-OH, stimulated large and sustained increases in the serine 536 phosphorylation of p65/RelA in a transfected skin epithelial cell line and primary keratinocytes. Parallel experiments showed that in both cell types, p65 NF kappa B phosphorylation is mediated through the selective activation of IKK2. Treatment with PKC inhibitor GF109203X or PKC alpha siRNA reduced phosphorylation at 15 min but not 30 min, whilst rottlerin, a selective PKC delta inhibitor and PKC delta siRNA reduced the response at both time points. Pre-treatment of cells with the novel Gq/11 inhibitor YM-254890 and Gq/11 siRNA caused a similar pattern of inhibition and also reduced PAR-2-mediated NF kappa B transcriptional activity. Furthermore, stimulation of cells through a novel PAR-2 mutant PAR-234-43, delayed p65 phosphorylation but was without effect on the kinetics of ERK activation. Inhibition of Gi or G12/13 pathways by pertussis toxin pre-treatment or over-expression of the RGS mutant Lsc, also did not effect NF kappa B phosphorylation. Taken together these data indicate dependency for Gq/11 in early phosphorylation of p65 NF kappa B and this subsequently affects initial NF kappa B-dependent gene transcriptional activity, however later regulation of p65 is unaffected. Overall these novel data demonstrate an IKK2-dependent, predominantly C-protein-independent pathway involved in PAR-2 regulation of NF kappa B phosphorylation in keratinocytes. (c) 2008 Elsevier Inc. All rights reserved.