Decline in DJ-1 and Decreased Nuclear Translocation of Nrf2 in Fuchs Endothelial Corneal Dystrophy
Decline in DJ-1 and Decreased Nuclear Translocation of Nrf2 in Fuchs Endothelial Corneal Dystrophy
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DOI：
10.1167/iovs.12-10119
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发表时间：
2012-08-01
影响因子：
4.4
通讯作者：
Jurkunas, Ula V.
中科院分区：
文献类型：
--
作者：
Bitar, Maya S.;Liu, Cailing;Jurkunas, Ula V.
目的.本研究旨在确定Fuchs角膜内皮营养不良（FECD）和正常角膜内皮细胞（CEC）中参与核因子红细胞2相关因子2（Nrf 2）调节的因子及其对氧化应激的反应。FECD角膜扣获自移植物和来自组织库的正常人角膜。氧化应激由叔丁基过氧化氢（tBHP）诱导。采用Western blotting和real-time PCR检测Nrf 2、DJ-1、p53和Kelch样ECH相关蛋白1（Keap 1）的蛋白和mRNA水平。使用免疫沉淀法检测永生化FECD（FECDi）和正常CEC（HCECi）细胞系中氧化DJ-1蛋白和Cullin 3-（Cul 3）调节的DJ-1降解的水平。通过免疫细胞化学法评估Nrf 2亚细胞定位。与正常相比，Nrf 2蛋白稳定剂DJ-1在FECD CEC中显著降低，而Nrf 2蛋白阻遏物Keap 1在基线时不变，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没有。与HCECi相比，DJ-1下降与FECDi中增加的DJ-1氧化修饰和羰基化相关。用抗Cul 3抗体标记免疫沉淀的DJ-1蛋白的增加表明与HCECi相比，FECDi中的DJ-1降解增强。tBHP处理后，Nrf 2在正常CEC中由胞浆转位至胞核，而在FECD中未观察到Nrf 2核定位。基线和氧化应激下FECD中DJ-1水平降低与Nrf 2核转位受损和细胞对凋亡的敏感性升高相关。靶向DJ-1/Nrf 2轴可以产生减缓FECD中CEC变性的机制。（Invest Ophthalmol维斯科学。2012; 53：5806-5813）DOI：10.1167/iovs.12-10119
PURPOSE. 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).METHODS. 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.RESULTS. 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.CONCLUSIONS. 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. (Invest Ophthalmol Vis Sci. 2012; 53:5806-5813) DOI:10.1167/iovs.12-10119