title: Hepatocyte growth factor protects RPE cells from apoptosis induced by glutathione depletion.
abstract: OBJECTIVE
  To study the mechanism of the protective effect of hepatocyte growth factor (HGF) in oxidative injury to RPE cells induced by glutathione (GSH) depletion.
  
  
  METHODS
  RPE cells were treated with HGF for 24 hours (20 ng/mL) and then were treated with DL-buthionine-(S,R)-sulfoximine (BSO) for an additional 24 hours. Cell death, apoptosis, and GSH levels were measured. Levels of intracellular reactive oxygen species (ROS) and their cellular localization were assessed by confocal microscopy. Expression of Bcl-2 and release of cytochrome c from mitochondria were quantified. The effect of BSO on caspase-3 activation and expression was determined. Gene expression of key enzymes of GSH metabolism by real-time PCR and regulation and translocation of the transcription factor NF-E2-related factor (Nrf2) by BSO were examined.
  
  
  RESULTS
  Treatment with BSO-induced apoptosis in RPE caused a significant decrease in intracellular GSH and in GSH/GSSG ratios. Marked increases in lipid peroxidase (LPO), ROS, and mitochondrial cytochrome c release and a decrease in Bcl-2 expression were observed. Elevated GSH/GSSG ratio (especially in mitochondria), decreased LPO and ROS, attenuation of apoptosis, and partial restoration of Bcl-2 expression were found in the HGF-pretreated cells. BSO activated caspase-3, and this effect was significantly blocked by HGF. Both HGF and BSO induced anti-oxidant gene expression. Nrf2 translocated to the nuclear region after treatment with BSO, whereas HGF did not induce such translocation.
  
  
  CONCLUSIONS
  The protective effect of HGF may be attributed in part to the elevation of mitochondrial GSH. BSO and HGF act in concert to enhance GSH-related gene expression in stressed RPE cells.
authors.affiliation: Arnold and Mabel Beckman Macular Research Center, Los Angeles, California, USA.
authors.forename: Manlin
authors.initials: M
authors.lastname: Jin
authors.forename: Jennifer
authors.initials: J
authors.lastname: Yaung
authors.forename: Ram
authors.initials: R
authors.lastname: Kannan
authors.forename: Shikun
authors.initials: S
authors.lastname: He
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Ryan
authors.forename: David R
authors.initials: DR
authors.lastname: Hinton
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D019328:Buthionine Sulfoximine / Q000633:toxicity; D053148:Caspase 3; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D019610:Cytoprotection / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000633:toxicity; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000502:physiology; D005978:Glutathione / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D006801:Humans; D008054:Lipid Peroxides / Q000378:metabolism; D018613:Microscopy, Confocal; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D004791:Enzyme Inhibitors; D008054:Lipid Peroxides; D051267:NF-E2-Related Factor 2; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D019328:Buthionine Sulfoximine; D017228:Hepatocyte Growth Factor; D045304:Cytochromes c; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione