Takamura Yoshihiro, Fatma Nigar, Kubo Eri, Singh Dhirendra P
Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198-5840, USA.
Am J Physiol Cell Physiol. 2006 Feb;290(2):C554-66. doi: 10.1152/ajpcell.00398.2005.
TNF-alpha induces oxidative stress by generating reactive oxygen species (ROS). This molecule elevates the expression of gamma-glutamylcysteine synthetase heavy subunit (gamma-GCS-HS). Lens epithelium-derived growth factor (LEDGF)/p75, a transcriptional protein, is inducible by oxidative stress and protects cells from various stresses by upregulating stress-responsive genes. This paper presents evidence that TNF-alpha elevates the expression of LEDGF and that LEDGF is one of the transactivators of gamma-GCS-HS gene. An analysis of the gamma-GCS-HS promoter sequence (-819 to +518 nt) revealed the presence of putative sites for LEDGF binding. Gel mobility assay confirmed the binding of LEDGF to the heat shock element (nGAAn) and the stress response element (A/TGGGGA/T) present in gamma-GCS-HS promoter. Transactivation experiments showed activation of gamma-GCS-HS promoter in cells overexpressing LEDGF or treated with a sublethal dose of TNF-alpha (20 ng/ml). Downregulation of gamma-GCS-HS promoter activity in cells transfected with LEDGF small interfering RNA validated the finding. Notably, cells treated with TNF-alpha (20 ng/ml) for 24 h had an increased abundance of LEDGF and gamma-GCS-HS mRNA and protein. In contrast, cells treated with TNF-alpha for longer periods or with higher concentrations of TNF-alpha showed reduced expression of LEDGF and gamma-GCS-HS and increased cellular death with higher ROS levels. Cells overexpressing LEDGF revealed elevated GSH levels (10-15%), a condition that may potentially eliminate the insult to cells induced by TNF-alpha. Thus TNF-alpha regulation of LEDGF may be physiologically important, as elevated expression of LEDGF increases the expression of endogenous gamma-GCS-HS gene, the catalytic subunit of the regulating enzyme in GSH biosynthesis that may constitute a protective mechanism in limiting oxidative stress induced by inflammatory cytokines.
肿瘤坏死因子-α（TNF-α）通过产生活性氧（ROS）诱导氧化应激。该分子可提高γ-谷氨酰半胱氨酸合成酶重亚基（γ-GCS-HS）的表达。晶状体上皮衍生生长因子（LEDGF）/p75是一种转录蛋白，可被氧化应激诱导，并通过上调应激反应基因来保护细胞免受各种应激。本文提供的证据表明，TNF-α可提高LEDGF的表达，且LEDGF是γ-GCS-HS基因的反式激活因子之一。对γ-GCS-HS启动子序列（-819至+518 nt）的分析揭示了存在LEDGF结合的推定位点。凝胶迁移试验证实了LEDGF与γ-GCS-HS启动子中存在的热休克元件（nGAAn）和应激反应元件（A/TGGGGA/T）的结合。反式激活实验表明，在过表达LEDGF或用亚致死剂量的TNF-α（20 ng/ml）处理的细胞中，γ-GCS-HS启动子被激活。用LEDGF小干扰RNA转染的细胞中γ-GCS-HS启动子活性的下调证实了这一发现。值得注意的是，用TNF-α（20 ng/ml）处理24小时的细胞中LEDGF和γ-GCS-HS mRNA及蛋白的丰度增加。相反，用TNF-α处理更长时间或更高浓度TNF-α的细胞显示LEDGF和γ-GCS-HS的表达降低，且细胞死亡增加，ROS水平更高。过表达LEDGF的细胞显示谷胱甘肽（GSH）水平升高（10 - 15%），这种情况可能潜在地消除TNF-α对细胞的损伤。因此，TNF-α对LEDGF的调节可能具有重要的生理意义，因为LEDGF表达的升高会增加内源性γ-GCS-HS基因的表达，γ-GCS-HS是谷胱甘肽生物合成调节酶的催化亚基，可能构成一种保护机制来限制炎性细胞因子诱导的氧化应激。
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