Oxidant-mediated akt activation in human RPE cells
Oxidant-mediated akt activation in human RPE cells
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DOI：
10.1167/iovs.06-0140
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发表时间：
2006-10-01
影响因子：
4.4
通讯作者：
Jaffe, Glenn J.
中科院分区：
文献类型：
--
作者：
Yang, Ping;Peairs, James J.;Jaffe, Glenn J.
目的.为了确定模型氧化剂过氧化氢（H2 O2）是否影响Akt活化，如果是，Akt活化是否促进视网膜色素上皮（RPE）细胞存活。用单独的培养基、磷脂酰肌醇-3激酶（PI 3 K）及其下游效应物Akt的抑制剂LY 294002（LY）或Akt/蛋白激酶B信号传导抑制剂（API）-2（Akt特异性抑制剂）预处理培养的人RPE细胞，然后用不同剂量的H2 O2刺激不同时间。使用针对磷酸化Akt（Ser 473）的抗体通过Western印迹评估Akt磷酸化。通过四唑盐（WST-1）测定和乳酸脱氢酶（LDH）释放测定来评估Akt阻断对RPE细胞活力的影响。半胱天冬酶介导的细胞角蛋白裂解，一个早期凋亡标志物，通过M30抗体染色进行评估。凋亡诱导因子（AIF）核转位法检测Caspase非依赖性凋亡。电镜观察RPE细胞形态。使用针对横纹肌肉瘤中磷酸化叉头（FKHR）和磷酸化糖原合酶激酶（GSK）-3 β-Akt的抗体，通过蛋白质印迹检测H2 O2对下游Akt靶的作用。H2 O2以剂量依赖性方式诱导Akt磷酸化，并且还诱导下游效应物FKHR和GSK-3 β的磷酸化。LY可显著抑制H2 O2介导的Akt磷酸化，并显著增强caspase相关性和caspase非依赖性RPE细胞死亡。模型氧化剂H2 O2诱导PI 3 K，从而激活Akt。Akt活化增强RPE细胞存活，因此可以在正常情况下和在疾病状态如年龄相关性黄斑变性（AMD）中保护RPE细胞免于氧化剂诱导的细胞死亡。
PURPOSE. To determine whether a model oxidant, hydrogen peroxide (H2O2), influences Akt activation and, if so, whether Akt activation promotes retinal pigment epithelial (RPE) cell survival.METHODS. Cultured human RPE cells were pretreated with medium alone, with LY294002 (LY), an inhibitor of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt, or with Akt/protein kinase B signaling inhibitor (API)-2, a specific Akt inhibitor, and then were stimulated with H2O2 at different doses for various times. Akt phosphorylation was evaluated by Western blot using antibody against phosphorylated Akt (Ser473). The effect of Akt blockade on RPE cell viability was assessed by tetrazolium salt (WST-1) assay and a lactate dehydrognease (LDH) release assay. Caspase-mediated cytokeratin cleavage, an early apoptosis marker, was assessed by M30 antibody staining. Caspase-independent apoptosis was determined by nuclear translocation of apoptosis-inducing factor (AIF). RPE cell morphology was evaluated by electron microscopy. The effect of H2O2 on downstream Akt targets was examined by Western blot using antibody against phosphorylated forkhead in rhabdomyosarcoma (FKHR) and phosphorylated glycogen synthase kinase (GSK)-3 beta.RESULTS. H2O2 induced Akt phosphorylation in a dose-dependent manner and also induced the phosphorylation of downstream effectors FKHR and GSK-3 beta. LY markedly inhibited H2O2-mediated Akt phosphorylation and significantly enhanced caspase-associated and caspase-independent RPE cell death.CONCLUSIONS. A model oxidant, H2O2, induces PI3K and thereby activates Akt. Akt activation enhances RPE cell survival and thus may protect RPE cells from oxidant-induced cell death under normal circumstances and in disease states such as age-related macular degeneration (AMD).