Gap Junctional Intercellular Communication Protects Human Retinal Pigment Epithelial Cells From Oxidative Stress-Induced Injury

Abstract

Purpose:: To determine the roles of connexin 43 (Cx43) and gap junctional intercellular communication (GJIC) in oxidant-stressed cultured human retinal pigment epithelial (hRPE) cells.

Methods:: Cultured hRPE cells were treated with the chemical oxidant tert-butyl hydroperoxide (t-BOOH). Cell viability was assessed by the MTT assay. GJIC was evaluated by scrape loading/dye transfer and microinjection assays. Cx43 expression was detected by Western blot and immunofluorescent staining combined with confocal microscopy analysis. Retroviral infection of hRPE cells with disease-linked dominant negative Cx43(G21R), and dominant active mutants of both Cx43 and Cx26 was performed and the effect on cell viability was assessed.

Results:: t-BOOH induced hRPE cell death by necrosis. GJIC was hindered by t-BOOH. Phosphorylation of Cx43 and a decrease in overall expression of Cx43 was observed following t-BOOH treatment. Retroviral infection with disease-linked dominant negative Cx43 increased t-BOOH-induced cell death, as did treatment with gap junction blockers 18b-glycyrrhetinic acid (GZA) and flufenamic acid (FFA). Conversely, over-expression of Cx26 and Cx43 increased the viability of oxidant-treated hRPE cells from 45.2% to 74.8% (p<0.01) and to 83.2% (p<0.01), respectively.

Conclusions:: Connexin-mediated protection of hRPE cells from oxidative injury is GJIC-dependent.

Keywords: retinal pigment epithelium • gap junctions/coupling • oxidation/oxidative or free radical damage