The protective effect of functional connexin43 channels on a human epithelial cell line exposed to oxidative stress.
OBJECTIVE
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 18beta-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.
Hutnik
Cindy M L
CM
Department of Ophthalmology, University of Western Ontario, London, ON, Canada. cindyh@sjhc.london.on.ca
Pocrnich
Cady E
CE
Liu
Hong
H
Laird
Dale W
DW
Shao
Qing
Q
D015153:Blotting, Western; D002450:Cell Communication / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival; D000072259:Connexin 26; D018031:Connexin 43 / Q000502:physiology*; D017630:Connexins / Q000378:metabolism; D019610:Cytoprotection; D019084:Fluorescent Antibody Technique, Indirect; D017629:Gap Junctions / Q000502:physiology*; D020868:Gene Silencing; D006801:Humans; D018613:Microscopy, Confocal; D018384:Oxidative Stress; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000187:drug effects; D034622:RNA Interference; D013778:Tetrazolium Salts; D013844:Thiazoles; D020122:tert-Butylhydroperoxide / Q000633:toxicity
D018031:Connexin 43; D017630:Connexins; C000607042:GJB2 protein, human; D013778:Tetrazolium Salts; D013844:Thiazoles; D000072259:Connexin 26; D020122:tert-Butylhydroperoxide; C022616:thiazolyl blue