title: Oxidative stress increases HO-1 expression in ARPE-19 cells, but melanosomes suppress the increase when light is the stressor.
abstract: OBJECTIVE
  Phagocytized melanosomes in ARPE-19 cells were previously shown to decrease susceptibility to oxidative stress induced by hydrogen peroxide treatment and increase stress due to light irradiation relative to cells containing control black latex beads. Here we asked whether differential expression of antioxidant enzymes in cells containing pigment granules could explain the outcomes.
  
  
  METHODS
  ARPE-19 cells were loaded by phagocytosis with porcine RPE melanosomes or black latex beads (control particles). Heme oxygenase-1 (HO-1), HO-2, glutathione peroxidase (GPx), and catalase were quantified by Western blot analysis before and after treatment with sublethal hydrogen peroxide or blue light (400-450 nm). The stress was confirmed as sublethal by cell survival analysis using real-time quantification of propidium iodide fluorescence.
  
  
  RESULTS
  Phagocytosis itself produced transient changes in protein levels of some antioxidant enzymes, but steady-state levels (7 days after phagocytosis) did not differ in cells containing melanosomes versus beads. Sublethal stress, induced by either hydrogen peroxide or light, had no effect on catalase or HO-2 in either particle-free or particle-loaded cells. In contrast, HO-1 protein was upregulated by treatment with both hydrogen peroxide and light. Particle content did not affect the HO-1 increase induced by hydrogen peroxide, but the increase induced by blue light irradiation was partially blocked in cells containing black beads and blocked even more in cells containing melanosomes.
  
  
  CONCLUSIONS
  The results do not implicate differential antioxidant enzyme levels in stress protection by melanosomes against hydrogen peroxide, but they suggest a multifaceted role for melanosomes in regulating light stress susceptibility in RPE cells.
authors.affiliation: Department of Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Krakow, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Pilat
authors.forename: Anja M
authors.initials: AM
authors.lastname: Herrnreiter
authors.forename: Christine M B
authors.initials: CM
authors.lastname: Skumatz
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Sarna
authors.forename: Janice M
authors.initials: JM
authors.lastname: Burke
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D005979:Glutathione Peroxidase / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008027:Light; D020460:Melanosomes / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects*; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects*; D010587:Phagocytosis / Q000502:physiology; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D013552:Swine; D000094765:Glutathione Peroxidase GPX1
chemical_list: D000975:Antioxidants; D016877:Oxidants; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; D000094765:Glutathione Peroxidase GPX1