title: Glutaredoxin 1 (Grx1) Protects Human Retinal Pigment Epithelial Cells From Oxidative Damage by Preventing AKT Glutathionylation.
abstract: OBJECTIVE
  Glutaredoxin 1 (Grx1) belongs to the oxidoreductase family and is a component of the endogenous antioxidant defense system. However, its physiological function remains largely unknown. In this study, we investigated whether and how Grx1 overexpression protects the retinal pigment epithelial (RPE) cells against H2O2-induced apoptosis.
  
  
  METHODS
  Human retinal pigment epithelial (ARPE-19) cells were transfected with either a Grx1-containing plasmid or an empty vector. Primary human RPE cells were transfected with Grx1 small interfering RNA (siRNA) or scrambled siRNA. Cell viability was measured with the WST8 assay. Apoptosis was quantitatively measured by annexin V/propidium iodide (PI) double staining. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Protein kinase B (AKT) activation was examined by Western blot. Protein kinase B glutathionylation was detected by immunoprecipitation followed by immunoblotting with anti-PSSG antibody.
  
  
  RESULTS
  Glutaredoxin 1 overexpression protected ARPE-19 cells from H2O2-induced cell viability loss. Conversely, Grx1 gene knockdown sensitized primary human RPE cells to H2O2. Assessment of apoptosis indicated that cells transfected with the Grx1-containing plasmid were more resistant to H2O2 with fewer cells undergoing apoptosis as compared to empty vector-transfected cells. Hydrogen peroxide-induced PSSG accumulation was also attenuated by Grx1 enrichment. Furthermore, Grx1 overexpression prevented H2O2-induced AKT glutathionylation, resulting in a sustained phospho-AKT elevation in RPE cells.
  
  
  CONCLUSIONS
  Glutaredoxin 1 can protect RPE cells against oxidative stress-induced apoptosis. The mechanism of this protection is associated with its ability to stimulate the phosphorylation of AKT by preventing AKT glutathionylation. Considering Grx1's protective abilities in RPE cells, Grx1 could be a potential pharmacological target for retinal degenerative diseases.
authors: Pharmaceutical Sciences University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors: Xiaobin
authors: X
authors: Liu
authors: Pharmaceutical Sciences University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors: Jamieson
authors: J
authors: Jann
authors: Pharmaceutical Sciences University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors: Christy
authors: C
authors: Xavier
authors: Pharmaceutical Sciences University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, United States 2North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Wort.
authors: Hongli
authors: H
authors: Wu
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019610:Cytoprotection / Q000502:physiology; D055785:Gene Knockdown Techniques; D054477:Glutaredoxins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism* / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis
chemical_list: C494826:BAX protein, human; C516005:GLRX protein, human; D054477:Glutaredoxins; D026901:Membrane Transport Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C086860:glutathione transporter; D006861:Hydrogen Peroxide; D051057:Proto-Oncogene Proteins c-akt; D005978:Glutathione; D019803:Glutathione Disulfide