title: 17-β estradiol protects ARPE-19 cells from oxidative stress through estrogen receptor-β.
abstract: OBJECTIVE
  To elucidate the mechanism of 17-β estradiol (17β-E(2))-mediated protection of retinal pigment epithelium (RPE) from oxidative stress.
  
  
  METHODS
  Cultured ARPE-19 cells were subjected to oxidative stress with t-butyl hydroxide or hydrogen peroxide in the presence or absence of 17β-E(2). Reactive oxygen species (ROS) were measured using H(2)DCFDA fluorescence. Apoptosis was evaluated by cell-death ELISA kit and Hoechst-3486 staining. Mitochondrial membrane potential was measured using the JC-1 assay. Cellular localization of estrogen receptor (ER) was evaluated by confocal microscopy. Gene expression and protein expression was quantified using qRT-PCR and western blotting. Superoxide dismutase and ATP levels were measured using commercial kits.
  
  
  RESULTS
  ARPE-19 cells expressed significant amounts of ERα and ERβ. Pretreatment with 17β-E2 protected ARPE-19 cells from oxidative stress and apoptosis. 17β-E(2) reduced the ROS levels and mitochondrial depolarization. The 17β-E(2)-mediated cytoprotection was inhibited by ER antagonists ICI (ERα and ERβ) and THC (ERβ) but not by tamoxifen (ERα). Knockdown of ERβ expression by siRNA abolished the protective effects of 17β-E(2). Further, qRT-PCR analysis revealed that 17β-E(2) pretreatment upregulated the expression of ERβ and phase II cellular antioxidant genes.
  
  
  CONCLUSIONS
  These results indicate that 17β-E(2) protects ARPE-19 cells from oxidative stress through an ERβ-dependent mechanism. 17β-E(2)-mediated cytoprotection occurred through the preservation of mitochondrial function, reduction of ROS production, and induction of cellular antioxidant genes.
authors.affiliation: Preclinical Research and Development, GTx Inc., Memphis, Tennessee 38163, USA.
authors.forename: Anand
authors.initials: A
authors.lastname: Giddabasappa
authors.forename: Matthew
authors.initials: M
authors.lastname: Bauler
authors.forename: Muralimohan
authors.initials: M
authors.lastname: Yepuru
authors.forename: Edward
authors.initials: E
authors.lastname: Chaum
authors.forename: James T
authors.initials: JT
authors.lastname: Dalton
authors.forename: Jeetendra
authors.initials: J
authors.lastname: Eswaraka
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D017209:Apoptosis; D015153:Blotting, Western; D002478:Cells, Cultured; D019610:Cytoprotection; D004797:Enzyme-Linked Immunosorbent Assay; D004958:Estradiol / Q000494:pharmacology*; D047629:Estrogen Receptor beta / Q000235:genetics / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D053078:Membrane Potential, Mitochondrial; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects*; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000378:metabolism; D020122:tert-Butylhydroperoxide / Q000633:toxicity
chemical_list: D047629:Estrogen Receptor beta; D004967:Estrogens; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D004958:Estradiol; D000255:Adenosine Triphosphate; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase