Harvey C J, Thimmulappa R K, Singh A, Blake D J, Ling G, Wakabayashi N, Fujii J, Myers A, Biswal S
Department of Environmental Health Science, Bloomberg School of Public Health, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.
Free Radic Biol Med. 2009 Feb 15;46(4):443-53. doi: 10.1016/j.freeradbiomed.2008.10.040. Epub 2008 Nov 5.
Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is the primary transcription factor protecting cells from oxidative stress by regulating cytoprotective genes, including the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and death. GSH homeostasis is regulated by de novo synthesis as well as GSH redox state; previous studies have demonstrated that Nrf2 regulates GSH homeostasis by affecting de novo synthesis. We report that Nrf2 modulates the GSH redox state by regulating glutathione reductase (GSR). In response to oxidants, lungs and embryonic fibroblasts (MEFs) from Nrf2-deficient (Nrf2(-/-)) mice showed lower levels of GSR mRNA, protein, and enzyme activity relative to wild type (Nrf2(+/+)). Nrf2(-/-) MEFs exhibited greater accumulation of glutathione disulfide and cytotoxicity compared to Nrf2(+/+) MEFs in response to t-butylhydroquinone, which was rescued by restoring GSR. Microinjection of glutathione disulfide induced greater apoptosis in Nrf2(-/-) MEFs compared to Nrf2(+/+) MEFs. In silico promoter analysis of the GSR gene revealed three putative antioxidant-response elements (ARE1, -44; ARE2, -813; ARE3, -1041). Reporter analysis, site-directed mutagenesis, and chromatin immunoprecipitation assays demonstrated binding of Nrf2 to two AREs distal to the transcription start site. Overall, Nrf2 is critical for maintaining the GSH redox state via transcriptional regulation of GSR and protecting cells against oxidative stress.
核因子红细胞2 p45相关因子2（Nrf2）是通过调节细胞保护基因（包括抗氧化剂谷胱甘肽（GSH）途径）来保护细胞免受氧化应激的主要转录因子。GSH维持细胞的氧化还原状态，并影响氧化还原信号传导、细胞增殖和死亡。GSH的稳态由从头合成以及GSH氧化还原状态调节；先前的研究表明，Nrf2通过影响从头合成来调节GSH稳态。我们报告Nrf2通过调节谷胱甘肽还原酶（GSR）来调节GSH氧化还原状态。响应氧化剂时，与野生型（Nrf2(+/+)）相比，Nrf2基因缺陷（Nrf2(-/-)）小鼠的肺和胚胎成纤维细胞（MEF）中GSR mRNA、蛋白质和酶活性水平较低。与Nrf2(+/+) MEF相比，Nrf2(-/-) MEF在响应叔丁基对苯二酚时表现出更高的谷胱甘肽二硫化物积累和细胞毒性，通过恢复GSR可挽救这种情况。与Nrf2(+/+) MEF相比，显微注射谷胱甘肽二硫化物在Nrf2(-/-) MEF中诱导了更大程度的细胞凋亡。对GSR基因的计算机启动子分析揭示了三个假定的抗氧化反应元件（ARE1，-44；ARE2，-813；ARE3，-1041）。报告基因分析、定点诱变和染色质免疫沉淀试验表明Nrf2与转录起始位点远端的两个ARE结合。总体而言，Nrf2对于通过对GSR的转录调节维持GSH氧化还原状态以及保护细胞免受氧化应激至关重要。
