title: The protective effect of functional connexin43 channels on a human epithelial cell line exposed to oxidative stress.
abstract: 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.
authors.affiliation: Department of Ophthalmology, University of Western Ontario, London, ON, Canada. cindyh@sjhc.london.on.ca
authors.forename: Cindy M L
authors.initials: CM
authors.lastname: Hutnik
authors.forename: Cady E
authors.initials: CE
authors.lastname: Pocrnich
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.forename: Dale W
authors.initials: DW
authors.lastname: Laird
authors.forename: Qing
authors.initials: Q
authors.lastname: Shao
mesh_terms: 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
chemical_list: D018031:Connexin 43; D017630:Connexins; C000607042:GJB2 protein, human; D013778:Tetrazolium Salts; D013844:Thiazoles; D000072259:Connexin 26; D020122:tert-Butylhydroperoxide; C022616:thiazolyl blue