Ritchie E, Saka M, Mackenzie C, Drummond R, Wheeler-Jones C, Kanke T, Plevin R
The Department of Physiology and Pharmacology, The University of Strathclyde, Strathclyde Institute for Biomedical Sciences, Glasgow, UK.
Br J Pharmacol. 2007 Apr;150(8):1044-54. doi: 10.1038/sj.bjp.0707150. Epub 2007 Mar 5.
Up-regulation of proteinase-activated receptor-2 (PAR2) is a factor in a number of disease states and we have therefore examined the signalling pathways involved in the expression of the receptor.
We investigated the effects of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), trypsin and the PAR2 activating peptide, 2-furoyl(2f)-LIGKV-OH on both mRNA and functional expression of PAR2 in human umbilical vein endothelial cells (HUVECs). The effect of specific chemical inhibitors and dominant negative adenovirus constructs of the mitogen-activated protein kinase (MAPK) cascade and the nuclear factor kappa B (NF-kappaB) signalling pathway was assessed. Methods included semi-quantitative and quantitative RT-PCR, [(3)H]inositol phosphate (IP) accumulation and Ca(2+)-dependent fluorescence.
The above agonists induced both mRNA and functional expression of PAR2; PAR4 mRNA, but not that for PAR1 or PAR-3, also increased following TNFalpha treatment. Inhibition of p38 MAP kinase reduced PAR2 and PAR4 expression, whilst inhibition of MEK1/ERK/JNK was without effect. A similar dependency upon p38 MAP kinase was observed for the expression of PAR4. TNFalpha -induced enhancement of PAR2 stimulated [(3)H]-inositol phosphate accumulation (IP) and Ca(2+) signalling was abolished following SB203580 pre-treatment. Infection with adenovirus encoding dominant-negative IKKbeta (Ad.IKKbeta(+/-)) and to a lesser extent dominant-negative IKKalpha (Ad.IKKalpha(+/-)), substantially reduced both control and IL-1beta- induced expression of both PAR2 and PAR4 mRNA and enhancement of PAR2-stimulated IP accumulation and Ca(2+) mobilisation.
These data reveal for the first time the signalling events involved in the upregulation of both PAR2 and PAR4 during pro-inflammatory challenge.
蛋白酶激活受体-2（PAR2）的上调是多种疾病状态中的一个因素，因此我们研究了参与该受体表达的信号通路。
我们研究了肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、胰蛋白酶以及PAR2激活肽2-呋喃甲酰（2f）-LIGKV-OH对人脐静脉内皮细胞（HUVECs）中PAR2的mRNA和功能表达的影响。评估了丝裂原活化蛋白激酶（MAPK）级联反应和核因子κB（NF-κB）信号通路的特异性化学抑制剂和显性负性腺病毒构建体的作用。方法包括半定量和定量逆转录-聚合酶链反应（RT-PCR）、[³H]肌醇磷酸（IP）积累和钙依赖性荧光检测。
上述激动剂诱导了PAR2的mRNA和功能表达；TNFα处理后，PAR4的mRNA增加，但PAR1或PAR-3的mRNA未增加。抑制p38 MAP激酶可降低PAR2和PAR4的表达，而抑制MEK1/ERK/JNK则无作用。PAR4的表达对p38 MAP激酶也有类似的依赖性。SB203580预处理后，TNFα诱导的PAR2刺激的[³H] - 肌醇磷酸积累（IP）和钙信号增强被消除。用编码显性负性IKKβ（Ad.IKKβ(+/-)）的腺病毒感染，以及在较小程度上用编码显性负性IKKα（Ad.IKKα(+/-)）的腺病毒感染，均可显著降低对照和IL-1β诱导的PAR2和PAR4 mRNA表达，以及PAR2刺激的IP积累和钙动员增强。
这些数据首次揭示了促炎刺激期间PAR2和PAR4上调所涉及的信号事件。
