title: Role of Glutathione Peroxidase 4 in Corneal Endothelial Cells.
abstract: To investigate the role of glutathione peroxidase 4 (GPx4) in corneal endothelial cells.
  
  
  
  An immortalized human corneal endothelial cell line was used. Cells were transfected with either siRNA specifically silencing GPx4 or scrambled control siRNA. Knockdown was confirmed by Real-time RT-PCR and immunoblotting. Lipid peroxidation was evaluated by 4-hydroxy-2-nonenal immunostaining. Cytotoxicity, cell death, and cell proliferation were evaluated using a lactate dehydrogenase (LDH) activity assay, Annexin V staining, and WST-8, respectively. Furthermore, cells transfected with GPx4 siRNA or control siRNA were treated with hydrogen peroxide or ferrous sulfate, and cytotoxicity was evaluated using the LDH activity assay.
  
  
  
  The treatment of siRNA decreased the expression of GPx4 at both mRNA and protein levels. The knockdown of GPx4 significantly increased the levels of lipid oxidation and LDH activity. Annexin V-positive cells increased in GPx4 siRNA-treated cells. The proliferation of GPx4 siRNA-treated cells was downregulated compared with that of control siRNA-treated cells. GPx4 knockdown enhanced hydrogen peroxide- and ferrous sulfate-induced cytotoxicity.
  
  
  
  These results suggest that GPx4 is an important antioxidant enzyme for maintaining redox status and protecting corneal endothelial cells from oxidative stress.
authors.affiliation: a Department of Ophthalmology, Graduate School of Medicine and Faculty of Medicine , The University of Tokyo , Tokyo , Japan.|b Research Laboratories of Drug Development , Senju Pharmaceutical Co., Ltd ., Kobe , Japan.
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Uchida
authors.affiliation: b Research Laboratories of Drug Development , Senju Pharmaceutical Co., Ltd ., Kobe , Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Sakai
authors.affiliation: c School of Pharmaceutical Sciences , Kitasato University , Tokyo , Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Imai
authors.affiliation: a Department of Ophthalmology, Graduate School of Medicine and Faculty of Medicine , The University of Tokyo , Tokyo , Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueta
mesh_terms: D015153:Blotting, Western; D016923:Cell Death; D002460:Cell Line; D049109:Cell Proliferation; D003316:Corneal Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004728:Endothelium, Corneal / Q000201:enzymology* / Q000473:pathology; D005786:Gene Expression Regulation; D005979:Glutathione Peroxidase / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D015227:Lipid Peroxidation; D018384:Oxidative Stress; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D012313:RNA; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase
keywords: Antioxidant; corneal endothelial cells; glutathione peroxidase; oxidative stress; reactive oxygen species