title: Role of glutathione peroxidase 4 in conjunctival epithelial cells.
abstract: OBJECTIVE
  The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in conjunctival epithelial cells.
  
  
  METHODS
  An immortalized human conjunctival epithelial cell line was used. Cells were transfected with catalase, GPx1, GPx4, SOD1, SOD2, or control siRNA. Knockdown was confirmed by RT-PCR and immunoblotting. The cytotoxicity induced by knockdown of these antioxidant enzymes was examined by assay of LDH activity. Furthermore, evaluations of lipid peroxidation, cellular levels of reactive oxygen species, cell proliferation, and apoptosis were conducted in cells treated with GPx4 or control siRNA. In oxidative stress study, cells treated with GPx4 or control siRNA were applied with hydrogen peroxide or ferric sulfide, and their cytotoxicity was evaluated by assay of LDH activity.
  
  
  RESULTS
  Small interfering RNA of catalase, GPx1, GPx4, SOD1, and SOD2 siRNA remarkably inhibited the mRNA and protein expression of each gene. Knockdown of GPx4 and SOD1 but not catalase, GPx1, and SOD2 significantly induced cytotoxicity. Glutathione peroxidase 4 knockdown increased lipid oxidation and reactive oxygen species. The proliferation of GPx4 siRNA-treated cells was reduced compared with control siRNA-treated cells. Moreover, cell death in GPx4 siRNA-treated cells was characterized by positive staining for annexin V. In an oxidation stress study, GPx4 siRNA knockdown enhanced the cytotoxicity induced by hydrogen peroxide or ferric sulfide.
  
  
  CONCLUSIONS
  These results suggest that GPx4 is essential for maintaining oxidative homeostasis and keeping defense against oxidative stress in conjunctival epithelial cells.
authors.affiliation: Department of Ophthalmology, University of Tokyo, Tokyo, Japan Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Kobe, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Sakai
authors.affiliation: Department of Ophthalmology, University of Tokyo, Tokyo, Japan Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Kobe, Japan.
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Uchida
authors.affiliation: School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Imai
authors.affiliation: Department of Ophthalmology, University of Tokyo, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueta
authors.affiliation: Inouye Eye Hospital, Tokyo, Japan Miyata Eye Hospital, Miyazaki, Japan.
authors.forename: Shiro
authors.initials: S
authors.lastname: Amano
mesh_terms: D017209:Apoptosis; D049109:Cell Proliferation; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000201:enzymology*; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology*; D005786:Gene Expression Regulation; D005979:Glutathione Peroxidase / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D018384:Oxidative Stress / Q000235:genetics*; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D012333:RNA, Messenger; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase
keywords: antioxidants; conjunctiva; epithelial cells; glutathione peroxidase