TNF-α induces MMP-9 expression via activation of Src/EGFR, PDGFR/PI3K/Akt cascade and promotion of NF-κB/p300 binding in human tracheal smooth muscle cells
TNF-α induces MMP-9 expression via activation of Src/EGFR, PDGFR/PI3K/Akt cascade and promotion of NF-κB/p300 binding in human tracheal smooth muscle cells
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DOI：
10.1152/ajplung.00311.2006
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发表时间：
2007-03-01
影响因子：
4.9
通讯作者：
Yang, Chuen-Mao
中科院分区：
文献类型：
--
作者：
Lee, Chiang-Wen;Lin, Chih-Chung;Yang, Chuen-Mao
tnf - α已被证明可诱导基质金属蛋白酶-9 （MMP-9）表达，进而在炎症反应中降解细胞外基质。然而，tnf - α诱导MMP-9的机制尚不清楚。在人气管平滑肌细胞中，tnf - α以一种时间依赖性的方式诱导MMP-9的表达和Akt的磷酸化，而Src （PP1）、表皮生长因子受体（AG1478）、PDGFR （AG1296）和PI3K （LY294002）的抑制剂分别能减弱MMP-9的表达和Akt的磷酸化，这一结果通过报告基因检测、RT-PCR、酶谱分析和Western blot分析显示。转染c-Src （KM， K295M[激酶失活突变体]）、p85和Akt （KA, K179A）的显性阴性突变体也降低了MMP-9的表达。这些结果表明，MMP-9的表达受到PI3K/Akt通过反激活生长因子受体的调控。此外，LY294002或wortmannin抑制Akt磷酸化，但对被helenalin阻断的NF-kappa B易位没有影响。MMP-9启动子和helenalin中突变的NF-kappa B DNA结合元件也能减弱MMP-9的表达，提示PI3K/Akt和NF-kappa B独立调控MMP-9的表达。为了支持这一观点，应用免疫荧光染色和免疫沉淀来表征参与这些反应的转录因子。结果表明，LY294002和姜黄素阻断Akt转位至细胞核。相比之下，p300、乙酰组蛋白（H3）和NF-kappa B p65被发现与磷酸化的Akt共免疫沉淀，表明这些与MMP-9启动子相关的成分通过染色质免疫沉淀试验被发现。因此，我们的研究为通过Src和生长因子受体的反激活介导的tnf - α刺激Akt磷酸化可能刺激p300的募集、组装转录因子（p65），进而导致MMP-9表达的分子机制提供了新的视角。
TNF-alpha has been shown to induce matrix metalloproteinase-9 (MMP-9) expression, which, in turn, degrades extracellular matrix in the inflammatory responses. However, the inductive mechanisms of the MMP-9 by TNF-alpha remain unclear. In human tracheal smooth muscle cells, TNF-alpha induced MMP-9 expression and Akt phosphorylation in a time-dependent manner, which was attenuated by the inhibitors of Src (PP1), epidermal growth factor receptor (AG1478), PDGFR (AG1296), and PI3K (LY294002), respectively, revealed by reporter gene assay, RT-PCR, zymographic, and Western blot analyses. Transfection with the dominant negative mutants of c-Src (KM, K295M [kinase inactive mutant]), p85, and Akt (KA, K179A) also reduced MMP-9 expression. These findings indicated that MMP-9 expression was regulated by PI3K/Akt via the transactivation of growth factor receptors. Furthermore, LY294002 or wortmannin inhibited Akt phosphorylation but had no effect on NF-kappa B translocation, which was blocked by helenalin. Mutated NF-kappa B DNA binding element in the MMP-9 promoter and helenalin also attenuated MMP-9 expression, suggesting that PI3K/Akt and NF-kappa B independently regulated MMP-9 expression. To support this notion, immunofluorescence staining and immunoprecipitation were applied to characterize the transcription factors involved in these responses. The results showed that LY294002 and curcumin blocked Akt translocation into nucleus. In contrast, p300, acetyl-histone (H3), and NF-kappa B p65 were found to be coimmunoprecipitated with the phosphorylated Akt, indicating that these components associated with the MMP-9 promoter are revealed by chromatin immunoprecipitation assay. Thus, our study provides a new insight into the molecular mechanisms that TNF-alpha-stimulated Akt phosphorylation mediated through transactivation of Src and growth factor receptors may stimulate the recruitment of p300, assemble transcription factor (p65), and then lead to MMP-9 expression.