The protective effect of functional connexin43 channels on a human epithelial cell line exposed to oxidative stress
The protective effect of functional connexin43 channels on a human epithelial cell line exposed to oxidative stress
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DOI：
10.1167/iovs.07-0717
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发表时间：
2008-02-01
影响因子：
4.4
通讯作者：
Shao, Qing
中科院分区：
文献类型：
--
作者：
Hutnik, Cindy M. L.;Pocrnich, Cady E.;Shao, Qing
目的.目的探讨缝隙连接蛋白43（Cx43）和缝隙连接细胞间通讯（GJIC）在人视网膜色素上皮细胞株ARPE-19对氧化应激反应中的作用。用化学氧化剂叔丁基过氧化氢（t-BOOH）处理ARPE-19细胞，并通过MTT [3-（4，5-二甲基噻唑-2-基）-2，5-二苯基溴化四唑]测定评估细胞活力。采用刮片/染料转移法和显微注射法检测GJIC，Western blot和免疫荧光染色结合共聚焦显微镜分析检测Cx43的表达。使用靶向Cx43的shRNA载体或编码Cx43、Cx 26和疾病相关显性阴性Cx43突变体（G21 R）的载体逆转录病毒感染ARPE-19细胞，并评估对细胞活力的影响。t-BOOH诱导的ARPE-19细胞死亡与GJIC和Cx43蛋白表达总水平的降低相关。Cx 26和Cx43的过表达增加了氧化剂处理的ARPE-19细胞的活力。相反，Cx43的shRNA敲除、疾病相关显性负性Cx43突变体的表达以及用18 β-大黄酸和氟灭酸阻断GJIC均增加t-BOOH诱导的ARPE-19细胞死亡。Cx43介导的保护ARPE-19细胞免受氧化应激诱导的死亡依赖于功能性Cx43通道。
PURPOSE. To determine the role of connexin43 (Cx43) and gap junctional intercellular communication (GJIC) in the response of the human retinal pigment epithelial cell line ARPE-19 to oxidative stress.METHODS. ARPE-19 cells were treated with the chemical oxidant tert-butyl hydroperoxide (t-BOOH), and cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. GJIC was evaluated by scrape loading/dye transfer and microinjection assays, and Cx43 expression was detected by Western blot and immunofluorescent staining combined with confocal microscopy analysis. Retroviral infection of ARPE-19 cells with shRNA vectors targeting Cx43 or vectors encoding Cx43, Cx26, and a disease-linked dominant negative Cx43 mutant (G21R) were used, and the effect on cell viability was assessed.RESULTS. t-BOOH-induced ARPE-19 cell death was correlated with reductions in GJIC and in the total level of Cx43 protein expression. Overexpression of Cx26 and Cx43 increased the viability of oxidant-treated ARPE-19 cells. Conversely, shRNA knockdown of Cx43, expression of a disease-linked dominant negative Cx43 mutant, and blocking GJIC with 18 beta-glycyrrhetinic acid and flufenamic acid all increased t-BOOH-induced ARPE-19 cell death.CONCLUSIONS. Cx43-mediated protection of ARPE-19 cells from oxidative stress-induced death is dependent on functional Cx43 channels.