Lin Chih-Chung, Tseng Hsiao-Wei, Hsieh Hsi-Lung, Lee Chiang-Wen, Wu Cheng-Ying, Cheng Ching-Yi, Yang Chuen-Mao
Department of Anesthetics, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
Toxicol Appl Pharmacol. 2008 Jun 15;229(3):386-98. doi: 10.1016/j.taap.2008.01.032. Epub 2008 Feb 9.
Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-alpha (TNF-alpha) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-alpha in human A549 cells remain unclear. Here, we showed that TNF-alpha induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-kappaB (helenalin), and transfection with dominant negative mutants of ERK2 (DeltaERK) and JNK (DeltaJNK), and siRNAs for MEK1, p42 and JNK2. TNF-alpha-stimulated phosphorylation of p42/p44 MAPK and JNK were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of DeltaERK and DeltaJNK. Furthermore, the involvement of NF-kappaB in TNF-alpha-induced MMP-9 production was consistent with that TNF-alpha-stimulated degradation of IkappaB-alpha and translocation of NF-kappaB into the nucleus which were blocked by helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-kappaB was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-alpha in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by helenalin, U0126, or SP600125. In contrast, TNF-alpha-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-kappaB MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-alpha-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-kappaB are essential for TNF-alpha-induced MMP-9 gene expression.
基质金属蛋白酶（MMPs），尤其是MMP-9，已被证明可由包括肿瘤坏死因子-α（TNF-α）在内的细胞因子诱导产生，并参与气道炎症反应。然而，TNF-α诱导人A549细胞中MMP-9表达的机制仍不清楚。在此，我们通过酶谱分析、蛋白质印迹法、逆转录-聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）表明，TNF-α诱导的MMP-9蛋白和mRNA的产生可被MEK1/2抑制剂（U0126）、JNK抑制剂（SP600125）和NF-κB抑制剂（海兔毒素）以及用显性负性突变体ERK2（ΔERK）和JNK（ΔJNK）转染，以及针对MEK1、p42和JNK2的小干扰RNA（siRNAs）所减弱。用抑制剂U0126和SP600125预处理或用显性负性突变体ΔERK和ΔJNK转染可减弱TNF-α刺激的p42/p44丝裂原活化蛋白激酶（MAPK）和JNK的磷酸化。此外，免疫荧光染色显示，NF-κB参与TNF-α诱导的MMP-9产生与TNF-α刺激的IκB-α降解和NF-κB易位至细胞核一致，这被海兔毒素阻断，但未被U0126和SP600125阻断。基因荧光素酶活性测定进一步证实了MAPKs和NF-κB对MMP-9基因转录的调控。在用野生型MMP-9-Luc转染的A549细胞中，TNF-α增强了MMP-9启动子活性，这被海兔毒素、U0126或SP600125抑制。相反，在用突变型-NF-κB MMP-9-luc转染的细胞中，TNF-α刺激的MMP-9荧光素酶活性完全丧失。此外，用放线菌素D和环己酰亚胺预处理可减弱TNF-α诱导的MMP-9表达。这些结果表明，在A549细胞中，p42/p44 MAPK、JNK的磷酸化以及NF-κB的反式激活对于TNF-α诱导的MMP-9基因表达至关重要。
