title: Vitreous induces heme oxygenase-1 expression mediated by transforming growth factor-beta and reactive oxygen species generation in human retinal pigment epithelial cells.
abstract: OBJECTIVE
  When human retinal pigment epithelial (RPE) cells come in contact with vitreous, they undergo changes in gene expression that include inflammatory and anti-oxidant responses. The effects of vitreous on expression of heme oxygenase-1 (HO-1), metallothionein (MT) -1a and -2a, and c-fos were investigated. Activator protein-1 (AP-1) binding sites are located in the promoter region of HO-1 and MT genes and the effects of vitreous on c-fos activity were investigated.
  
  
  METHODS
  Low passage cultures of human RPE cells were grown in the presence or absence of vitreous or transforming growth factor-beta (TGF-beta). The expression of HO-1 and MTs was measured by real time PCR and, in the case of HO-1, by immunoblotting and immunofluorescence microscopy. Specific inhibitors were used to investigate possible signaling pathways. The effect of vitreous on activation of AP-1 transcription factor was determined by immunoblotting, electrophoretic mobility shift assays, or immunofluorescence microscopy.
  
  
  RESULTS
  Incubation of RPE cells with vitreous resulted in increased expression of HO-1, MT-1a and MT-2a. TGF-beta caused an increase in HO-1 expression, although not to the extent mediated by vitreous, but had little effect on MT expression. Addition of inhibitors of TGF-beta signaling (SB431542 or TGF-beta-neutralizing antibodies) decreased the vitreous induction of HO-1. Several reactive oxygen species (ROS) quenchers inhibited the TGF-beta-induced or vitreous-induced elevation of HO-1 mRNA but had no effect on vitreous-mediated induction of MT expression. Inhibitors of the mitogen-activated protein kinase (p38MAPK; SB203580) and Jun N-terminal kinase (JNK; SP600125) pathways inhibited vitreous-induction of HO-1. C-fos, a component of AP-1 transcription factor complexes, exhibited increased expression and activation in the presence of vitreous.
  
  
  CONCLUSIONS
  TGF-beta, a known component of vitreous, can account for some but not all of the regulation of the anti-oxidant, anti-inflammatory HO-1 gene in human RPE cells, but it does not participate in the vitreous-mediated upregulation of MTs. Both vitreous and TGF-beta signals increased HO-1 expression via ROS but the latter were not involved in vitreous-mediated MT expression. Increased p38, JNK, and c-fos activation may be implicated in vitreous modulation of HO-1.
authors.affiliation: Department of Pathology and Microbiology, University of South Carolina School of Medicine, Columbia, SC 29208, USA. rhartung@med.sc.edu <rhartung@med.sc.edu>
authors.forename: Ren
authors.initials: R
authors.lastname: Hartung
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Parapuram
authors.forename: Ramapriya
authors.initials: R
authors.lastname: Ganti
authors.forename: D Margaret
authors.initials: DM
authors.lastname: Hunt
authors.forename: Kakarla V
authors.initials: KV
authors.lastname: Chalam
authors.forename: Richard C
authors.initials: RC
authors.lastname: Hunt
mesh_terms: D030201:Activin Receptors, Type I / Q000037:antagonists & inhibitors; D001549:Benzamides / Q000494:pharmacology; D001692:Biological Transport / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004149:Dioxoles / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology; D051547:Heme Oxygenase-1 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000502:physiology*; D014822:Vitreous Body / Q000166:cytology / Q000502:physiology*
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D004149:Dioxoles; D020033:Protein Isoforms; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D018125:Receptors, Transforming Growth Factor beta; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D008668:Metallothionein; D051547:Heme Oxygenase-1; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I