Molecular mechanism of transforming growth factor (TGF)-β1-induced glutathione depletion in alveolar epithelial cells -: Involvement of AP-1/ARE and Fra-1
Molecular mechanism of transforming growth factor (TGF)-β1-induced glutathione depletion in alveolar epithelial cells -: Involvement of AP-1/ARE and Fra-1
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DOI：
10.1074/jbc.m112145200
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发表时间：
2002-06-14
影响因子：
4.8
通讯作者：
Rahman, I
中科院分区：
文献类型：
--
作者：
Jardine, H;MacNee, W;Rahman, I
谷胱甘肽（GSH）是肺上皮细胞和肺衬里液中普遍存在的抗氧化剂。转化生长因子β（1）（TGF-β（1））是一种参与细胞增殖和分化的多效性细胞因子。TGF-β（1）的水平在许多与氧化剂/抗氧化剂失衡相关的慢性炎性肺病中升高。在这项研究中，我们发现TGF-β（1）通过下调负责其形成的酶γ-谷氨酰半胱氨酸合成酶（γ-GCS）的表达来消耗GSH，并诱导II型肺泡上皮细胞（A549）中活性氧的产生。为了研究谷胱甘肽合成抑制的分子机制，我们采用了含有γ-GCS重亚基（h）启动子区片段的报告基因，该基因编码γ-GCS的催化亚基。我们发现TGF-β 1减少了长γ-GCSh结构（-3802/GCSh 5 '-Luc）的表达，这表明抗氧化反应元件（ARE）可能负责介导TGF-β 1的作用。有趣的是，电泳迁移率变动分析显示，在TGF-β（1）处理的上皮细胞中，激活蛋白-1（A-P-1）和ARE的DNA结合活性均增加。γ-GCSh ARE包含一个嵌入其中的完美AP-1位点，该内部AP-1序列的突变（而不是周围的ARE）阻止了DNA结合。进一步的研究表明，c-Jun和Fra-1二聚体，AP-1家族的成员，以前显示对II期基因表达产生负面影响，结合到ARE序列。我们提出了TGF-β下调γ-GCSh的新机制（1），涉及c-Jun和Fra-1二聚体与远端启动子的结合。本研究结果为炎症反应中谷胱甘肽生物合成的调控提供了重要信息。
Glutathione (GSH) is a ubiquitous antioxidant in lung epithelial cells and lung lining fluid. Transforming growth factor beta(1) (TGF-beta(1)) is a pleiotropic cytokine involved in cellular proliferation and differentiation. The level of TGF-beta(1), is elevated in many chronic inflammatory lung disorders associated with oxidant/antioxidant imbalance. In this study, we show that TGF-beta(1), depletes GSH by down-regulating expression of the enzyme responsible for its formation, gamma-glutamylcysteine synthetase (gamma-GCS) and induces reactive oxygen species production in type II alveolar epithelial cells (A549). To investigate the molecular mechanisms of inhibition of glutathione synthesis, we employed reporters containing fragments from the promoter region of the gamma-GCS heavy subunit (h), the gene that encodes the catalytic subunit of gamma-GCS. We found that TGF-beta(1), reduced the expression of the long gamma-GCSh construct (-3802/GCSh5'-Luc), suggesting that an antioxidant response element (ARE) may be responsible for mediating the TGF-beta(1) effect. Interestingly, the electrophoretic mobility shift assay revealed that the DNA binding activity of both activator protein-1 (A-P-1) and ARE was increased in TGF-beta(1)-treated epithelial cells. The gamma-GCSh ARE contains a perfect AP-1 site embedded within it, and mutation of this internal AP-1 sequence, but not the surrounding ARE, prevented DNA binding. Further studies revealed that c-Jun and Fra-1 dimers, members of the AP-1 family previously shown to exert a negative effect on phase II gene expression, bound to the ARE sequence. We propose a novel mechanism of gamma-GCSh down-regulation by TGF-beta(1), that involves the binding of c-Jun and Fra-1 dimers to the distal promoter. The findings of this study provide important information, which may e used for the modulation of glutathione biosynthesis in inflammation.