Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA.
Invest Ophthalmol Vis Sci. 2012 Aug 24;53(9):5806-13. doi: 10.1167/iovs.12-10119.
This study sought to determine factors involved in nuclear factor erythroid 2-related factor 2 (Nrf2) regulation and their response to oxidative stress in Fuchs endothelial corneal dystrophy (FECD) and normal corneal endothelial cells (CECs).
FECD corneal buttons were obtained from transplantations and normal human corneas from tissue banks. Oxidative stress was induced by tert-butyl hydroperoxide (tBHP). Protein and mRNA levels of Nrf2, DJ-1, p53, and Kelch-like ECH-associated protein1 (Keap1) were investigated using Western blotting and real-time PCR. Immunoprecipitation was used to detect levels of oxidized DJ-1 protein and Cullin 3- (Cul3)-regulated degradation of DJ-1 in immortalized FECD (FECDi) and normal CEC (HCECi) cell lines. Nrf2 subcellular localization was assessed by immunocytochemistry.
Nrf2 protein stabilizer, DJ-1, decreased significantly in FECD CECs compared with normal, whereas Nrf2 protein repressor, Keap1, was unchanged at baseline but increased under oxidative stress. Under oxidative stress, normal CECs upregulated DJ-1 protein synthesis, whereas FECD CECs did not. DJ-1 decline correlated with increased DJ-1 oxidative modification and carbonylation in FECDi as compared with HCECi. Increased labeling of immunoprecipitated DJ-1 protein with anti-Cul3 antibody indicated enhanced DJ-1 degradation in FECDi as compared with HCECi. Following tBHP treatment, Nrf2 translocated from cytoplasm to nuclei in normal CECs, whereas Nrf2 nuclear localization was not observed in FECD.
Decreased levels of DJ-1 in FECD at baseline and under oxidative stress correlate with impaired Nrf2 nuclear translocation and heightened cell susceptibility to apoptosis. Targeting the DJ-1/Nrf2 axis could yield a mechanism to slow CEC degeneration in FECD.
本研究旨在确定核因子红细胞 2 相关因子 2（Nrf2）调节的相关因素及其在 Fuchs 内皮角膜营养不良（FECD）和正常角膜内皮细胞（CEC）中对氧化应激的反应。
从移植中获得 FECD 角膜瓣，从组织库中获得正常人类角膜。使用叔丁基过氧化物（tBHP）诱导氧化应激。使用 Western blot 和实时 PCR 研究 Nrf2、DJ-1、p53 和 Kelch 样 ECH 相关蛋白 1（Keap1）的蛋白和 mRNA 水平。使用免疫沉淀检测 DJ-1 氧化蛋白水平和 Cullin 3-（Cul3）调控的 DJ-1 降解。通过免疫细胞化学评估 Nrf2 亚细胞定位。
与正常 CEC 相比，FECD CEC 中 Nrf2 蛋白稳定剂 DJ-1 显著下降，而 Nrf2 蛋白抑制剂 Keap1 基础水平不变，但在氧化应激下增加。在氧化应激下，正常 CEC 上调 DJ-1 蛋白合成，而 FECD CEC 则没有。与 HCECi 相比，FECDi 中 DJ-1 下降与 DJ-1 氧化修饰和羰基化增加相关。与 HCECi 相比，用抗 Cul3 抗体标记的免疫沉淀 DJ-1 蛋白增加表明 FECDi 中 DJ-1 降解增强。tBHP 处理后，Nrf2 从正常 CEC 的细胞质转移到细胞核，而在 FECD 中未观察到 Nrf2 核定位。
在基线和氧化应激下，FECD 中 DJ-1 水平降低与 Nrf2 核易位受损和细胞对凋亡的敏感性增加相关。靶向 DJ-1/Nrf2 轴可能为减缓 FECD 中 CEC 变性提供一种机制。
