Essential roles of the PI3 kinase/Akt pathway in regulating Nrf2-dependent antioxidant functions in the RPE
Essential roles of the PI3 kinase/Akt pathway in regulating Nrf2-dependent antioxidant functions in the RPE
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DOI：
10.1167/iovs.07-1099
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发表时间：
2008-04-01
影响因子：
4.4
通讯作者：
Cai, Jiyang
中科院分区：
文献类型：
--
作者：
Wang, Ling;Chen, Yan;Cai, Jiyang
目的。目的 研究培养的人视网膜色素上皮 (RPE) 细胞中磷脂酰肌醇 3 激酶 (PI3K)/Akt 通路与核因子红细胞 2 相关因子 2 (Nrf2) 依赖性抗氧化系统之间的功能相互作用。用不同浓度的 PI3K 抑制剂处理培养的 ARPE-19 细胞，然后暴露于萝卜硫素（一种 Nrf2 诱导剂）。通过蛋白质印迹分析检测 Akt 磷酸化。通过 HPLC 测定细胞内谷胱甘肽 (GSH) 含量。通过定量 RTPCR 测量 Nrf2 下游基因的表达，包括谷氨酸半胱氨酸连接酶 (GCL) 和谷胱甘肽 S-转移酶。在转染含有抗氧化反应元件 (ARE) 的报告质粒后，通过双荧光素酶测定来测量 Nrf2 活性。小干扰RNA方法被用来敲低RPE中的Nrf2。通过亚细胞分级分离和蛋白质印迹分析确定 Nrf2 定位。结果。 PI3K 抑制剂渥曼青霉素和 LY294002 在培养的 RPE 细胞中引起剂量依赖性细胞和线粒体 GSH 消耗以及 GCL 调节亚基的下调。渥曼青霉素和 LY294002 抑制基础 Nrf2 活性和诱导的 Nrf2 活性。 Akt 组成型活性形式的过度表达增强了 Nrf2 的激活，而 Akt 的作用被敲低 Nrf2 的 siRNA 所阻断。 LY294002 还抑制萝卜硫素诱导的 Nrf2 核转位。结论。 PI3K/Akt 通路在调节 Nrf2-ARE 依赖性 RPE 氧化应激保护中发挥着关键作用。
PURPOSE. To investigate functional interactions between the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant system in cultured human retinal pigment epithelium (RPE) cells.METHODS. Cultured ARPE-19 cells were treated with different concentrations of PI3K inhibitors, followed by exposure to sulforaphane, an Nrf2 inducer. Akt phosphorylation was detected by Western blot analysis. Intracellular glutathione (GSH) content was measured by HPLC. Expression of genes downstream of Nrf2, including glutamate-cysteine ligase (GCL) and glutathione S-transferase, was measured by quantitative RTPCR. Nrf2 activity was measured by a dual luciferase assay after transfection of a reporter plasmid containing the antioxidant response element ( ARE). The small interference RNA approach was used to knock down Nrf2 in the RPE. Nrf2 localization was determined by subcellular fractionation and Western blot analyses.RESULTS. PI3K inhibitors wortmannin and LY294002 caused dose-dependent cellular and mitochondrial GSH depletion and downregulation of the modulatory subunit of GCL in cultured RPE cells. Both the basal and the induced Nrf2 activities were inhibited by wortmannin and LY294002. Overexpression of a constitutively active form of Akt potentiated Nrf2 activation, and the effect of Akt was blocked by siRNA that knocked down Nrf2. LY294002 also inhibited sulforaphane-induced Nrf2 nuclear translocation.CONCLUSIONS. The PI3K/Akt pathway plays key roles in regulating Nrf2-ARE-dependent protection against oxidative stress in the RPE.