Nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) is a transcription factor that binds to the antioxidant response element (ARE) in the upstream promoter region of various antioxidant-responsive genes. Hence, at least in nonruminants, the NFE2L2-ARE signaling pathway plays an important role in the cellular antioxidant defense system. Whether oxidative stress in bovine mammary epithelial cells alters NFE2L2 or the NFE2L2-ARE pathway is unclear. Therefore, the objective of this study was to examine the response in NFE2L2- and NFE2L2-ARE-related components in bovine mammary epithelial cells (BMEC) under oxidative stress. An in silico analysis to identify potential phosphorylation sites on NFE2L2 and the protein kinases was performed with Netphos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) and Scansite (http://scansite.mit.edu) software. Isolated BMEC were exposed to H₂O₂ (600 μM) for 6 h to induce oxidative stress. In silico analysis revealed ataxia telangiectasia-mutated (ATM) serine/threonine kinase as a key kinase responsible for the phosphorylation of NFE2L2. Thus, after the 6 h incubation with H₂O₂, BMEC were transiently transfected with ATM-small interfering RNA (siRNA) 1, 2, or 3. Compared with the control, transfection with ATM-siRNA3 resulted in proliferation rates that were 60.7 and 36.2% lower with or without H₂O₂. In addition, production of reactive oxygen species and malondialdehyde increased markedly, but activities of superoxide dismutase, glutathione peroxidase, catalase, and glutathione-S-transferase decreased markedly in transfected cells without or with H₂O₂ compared with the control. Transfected cells had markedly lower protein and mRNA expression of NFE2L2 without or with H₂O₂ compared with the control. In addition, fluorescent activity of the ARE in transfected BMEC indicated that NFE2L2-driven transcriptional activation decreased under oxidative stress. Overall, results indicate that ATM is a physiologically relevant NFE2L2 kinase. Furthermore, inhibition of ATM activity can cause marked alterations in oxidative stress leading to cell death as a result of diminished capacity of BMEC to cope with H₂O₂-induced cytotoxicity. The relevance of this kinase in vivo merits further study.

核因子（类胡萝卜素衍生的2）样因子2（NFE2L2，以前称为Nrf2）是一种转录因子，可与各种抗氧化剂响应基因的上游启动子区域中的抗氧化剂响应元件（ARE）结合。因此，至少在非反刍动物中，NFE2L2-ARE信号传导途径在细胞抗氧化剂防御系统中起着重要作用。牛乳腺上皮细胞中的氧化应激是否会改变NFE2L2或NFE2L2-ARE途径尚不清楚。因此，本研究的目的是研究牛乳腺上皮细胞（BMEC）在氧化应激下NFE2L2-和NFE2L2-ARE相关成分的反应。使用Netphos 3.1（http://www.cbs.dtu.dk/services/NetPhos/）和Scansite（http://scansite.mit.edu）软件。分离BMEC暴露至H ₂ ö ₂（600μ中号）6小时以诱导氧化应激。在计算机分析中发现，共济失调的毛细血管扩张突变（ATM）丝氨酸/苏氨酸激酶是负责NFE2L2磷酸化的关键激酶。因此，在与H ₂ O ₂孵育6小时后，BMEC被ATM-小干扰RNA（siRNA）1、2或3瞬时转染。与对照相比，ATM-siRNA3的转染导致增殖率为60.7。有或没有H ₂ O _2时降低了36.2％。另外，与对照相比，在没有或有H ₂ O _2的转染细胞中，活性氧和丙二醛的产生显着增加，但是超氧化物歧化酶，谷胱甘肽过氧化物酶，过氧化氢酶和谷胱甘肽-S-转移酶的活性显着降低。无论是否加入H ₂ O _2，转染的细胞均明显降低NFE2L2的蛋白质和mRNA表达。与对照相比。此外，转染的BMEC中ARE的荧光活性表明NFE2L2驱动的转录激活在氧化应激下降低。总体而言，结果表明ATM是生理相关的NFE2L2激酶。此外，由于BMEC应对H ₂ O ₂诱导的细胞毒性的能力降低，对ATM活性的抑制可引起氧化应激的显着改变，从而导致细胞死亡。该激酶在体内的相关性值得进一步研究。