title: Transcriptional regulation of activating transcription factor 4 under oxidative stress in retinal pigment epithelial ARPE-19/HPV-16 cells.
abstract: OBJECTIVE
  Oxidative stress plays an important role in the pathogenesis of various ocular diseases such as retinopathy, glaucoma, and age-related macular degeneration. Activating transcription factor 4 (ATF4) is induced by various stressors, including endoplasmic reticulum (ER) and oxidative stress, and ATF4 expression is regulated translationally through the PERK pathway of eIF2α phosphorylation. Transcriptional regulation of the ATF4 gene under oxidative stress was investigated in human papillomavirus 16 (HPV-16)-transformed retinal pigment epithelial ARPE-19/HPV-16 cells.
  
  
  METHODS
  Retinal pigment epithelial cells, trabecular meshwork cells, and corneal endothelial cells were treated with anoxia and thapsigargin (TG). Gene expression of ATF4 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and transcription factors was investigated by Western blot analysis, reporter assays, chromatin immunoprecipitation (ChIP) assays, and small interfering (si)RNA strategies. Cellular sensitivity to oxidative stress was determined.
  
  
  RESULTS
  The expression of two transcriptional factors, ATF4 and Nrf2, was significantly induced by anoxia and TG. The Nrf2 regulator Keap1 was downregulated by anoxia. Downregulation of Nrf2 abolished ATF4 expression. On the other hand, downregulation of Keap1 enhanced the expression of both Nrf2 and ATF4. The promoter activity of ATF4 was transactivated by the co-transfection of Nrf2 expression plasmids and reduced by the transfection of Nrf2-specific siRNA. The ChIP assays demonstrated that Nrf2 bound to the promoter of the ATF4 gene. Nrf2 downregulation nearly abolished the ATF4 induction by anoxia and TG. Consistent with these findings, the promoter activity of ATF4 was augmented by treatment with TG, HCA, H(2)O(2), and anoxia. However, stress induction of ATF4 promoter activity was observed, even when a mutation was introduced into the antioxidant-responsive elements site. Furthermore, stress induction of the ATF4 promoter was completely abolished when the 5' untranslated region of the ATF4 gene was deleted. Downregulation of ATF4 rendered ARPE-19/HPV-16 cells sensitive to oxidative stress.
  
  
  CONCLUSIONS
  These results suggest that the stress induction of ATF4 is significantly regulated transcriptionally through a Nrf2-dependent mechanism and may be a double-edged sword in the pathogenesis of various retinopathies.
authors.affiliation: Department of Molecular Biology, University of Occupational and Environmental Health, Japan, Fukuoka, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Miyamoto
authors.forename: Hiroto
authors.initials: H
authors.lastname: Izumi
authors.forename: Rie
authors.initials: R
authors.lastname: Miyamoto
authors.forename: Han
authors.initials: H
authors.lastname: Bin
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kondo
authors.forename: Akihiko
authors.initials: A
authors.lastname: Tawara
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Sasaguri
authors.forename: Kimitoshi
authors.initials: K
authors.lastname: Kohno
mesh_terms: D051701:Activating Transcription Factor 4 / Q000235:genetics*; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral / Q000502:physiology*; D047369:Chromatin Immunoprecipitation; D004728:Endothelium, Corneal / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology*; D052162:Human papillomavirus 16 / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D000860:Hypoxia; D051267:NF-E2-Related Factor 2 / Q000502:physiology*; D018384:Oxidative Stress; D010957:Plasmids; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D014129:Trabecular Meshwork / Q000378:metabolism
chemical_list: C497109:ATF4 protein, human; D004791:Enzyme Inhibitors; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D051701:Activating Transcription Factor 4; D019284:Thapsigargin; D006861:Hydrogen Peroxide