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Cindy M. L. Hutnik, Cady E. Pocrnich, Hong Liu, Dale W. Laird, Qing Shao; The Protective Effect of Functional Connexin43 Channels on a Human Epithelial Cell Line Exposed to Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2008;49(2):800-806. doi: https://doi.org/10.1167/iovs.07-0717.
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© ARVO (1962-2015); The Authors (2016-present)
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 18β-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.
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