Department of Molecular Biology, University of Occupational and Environmental Health, Japan, Fukuoka, Japan.
Invest Ophthalmol Vis Sci. 2011 Mar 2;52(3):1226-34. doi: 10.1167/iovs.10-5775.
PURPOSE: Oxidative stress plays an important role in the pathogenesis of various ocular diseases such as retinopathy, glaucoma, and age-related macular degeneration. Activating transcription factor 4 (ATF4) is induced by various stressors, including endoplasmic reticulum (ER) and oxidative stress, and ATF4 expression is regulated translationally through the PERK pathway of eIF2α phosphorylation. Transcriptional regulation of the ATF4 gene under oxidative stress was investigated in human papillomavirus 16 (HPV-16)-transformed retinal pigment epithelial ARPE-19/HPV-16 cells. METHODS: Retinal pigment epithelial cells, trabecular meshwork cells, and corneal endothelial cells were treated with anoxia and thapsigargin (TG). Gene expression of ATF4 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and transcription factors was investigated by Western blot analysis, reporter assays, chromatin immunoprecipitation (ChIP) assays, and small interfering (si)RNA strategies. Cellular sensitivity to oxidative stress was determined. RESULTS: The expression of two transcriptional factors, ATF4 and Nrf2, was significantly induced by anoxia and TG. The Nrf2 regulator Keap1 was downregulated by anoxia. Downregulation of Nrf2 abolished ATF4 expression. On the other hand, downregulation of Keap1 enhanced the expression of both Nrf2 and ATF4. The promoter activity of ATF4 was transactivated by the co-transfection of Nrf2 expression plasmids and reduced by the transfection of Nrf2-specific siRNA. The ChIP assays demonstrated that Nrf2 bound to the promoter of the ATF4 gene. Nrf2 downregulation nearly abolished the ATF4 induction by anoxia and TG. Consistent with these findings, the promoter activity of ATF4 was augmented by treatment with TG, HCA, H(2)O(2), and anoxia. However, stress induction of ATF4 promoter activity was observed, even when a mutation was introduced into the antioxidant-responsive elements site. Furthermore, stress induction of the ATF4 promoter was completely abolished when the 5' untranslated region of the ATF4 gene was deleted. Downregulation of ATF4 rendered ARPE-19/HPV-16 cells sensitive to oxidative stress. CONCLUSIONS: These results suggest that the stress induction of ATF4 is significantly regulated transcriptionally through a Nrf2-dependent mechanism and may be a double-edged sword in the pathogenesis of various retinopathies.
目的：氧化应激在多种眼部疾病的发病机制中起着重要作用，如视网膜病变、青光眼和年龄相关性黄斑变性。激活转录因子 4（ATF4）可被多种应激源诱导，包括内质网（ER）和氧化应激，ATF4 的表达通过 PERK 通路的 eIF2α 磷酸化进行翻译调控。本研究探讨了人乳头瘤病毒 16（HPV-16）转化的视网膜色素上皮 ARPE-19/HPV-16 细胞中氧化应激下 ATF4 基因的转录调控。
方法：用缺氧和他普西苷（TG）处理视网膜色素上皮细胞、小梁网细胞和角膜内皮细胞。通过 Western blot 分析、报告基因检测、染色质免疫沉淀（ChIP）检测和小干扰（si）RNA 策略研究 ATF4 和核因子（红细胞衍生 2）样 2（Nrf2）以及转录因子的基因表达。测定细胞对氧化应激的敏感性。
结果：缺氧和 TG 显著诱导两种转录因子 ATF4 和 Nrf2 的表达。Nrf2 调节剂 Keap1 被缺氧下调。Nrf2 的下调消除了 ATF4 的表达。另一方面，Keap1 的下调增强了 Nrf2 和 ATF4 的表达。Nrf2 表达质粒的共转染可使 ATF4 启动子活性转激活，而 Nrf2 特异性 siRNA 的转染则降低其活性。ChIP 检测表明 Nrf2 结合于 ATF4 基因的启动子。Nrf2 的下调几乎消除了缺氧和 TG 对 ATF4 的诱导。与这些发现一致，用 TG、HCA、H2O2 和缺氧处理可增强 ATF4 启动子的活性。然而，即使在引入抗氧化反应元件位点突变的情况下，仍观察到 ATF4 启动子对应激的诱导。此外，当删除 ATF4 基因的 5'非翻译区时，ATF4 启动子的应激诱导完全被消除。ATF4 的下调使 ARPE-19/HPV-16 细胞对氧化应激敏感。
结论：这些结果表明，ATF4 的应激诱导通过 Nrf2 依赖性机制显著进行转录调控，在多种视网膜病变的发病机制中可能是一把双刃剑。
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