June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Iron Loaded Melanosomes Change Antioxidant Enzyme Levels in ARPE-19 Cells
Author Affiliations & Notes
  • Anna Pilat
    Biophysics, Jagiellonian University, Krakow, Poland
  • Christine Skumatz
    Ophthalmology, Medical College of Wisconsin, Milwaukee, WI
  • Magdalena Olchawa
    Biophysics, Jagiellonian University, Krakow, Poland
  • Mariusz Zareba
    Ophthalmology, Medical College of Wisconsin, Milwaukee, WI
  • Janice Burke
    Ophthalmology, Medical College of Wisconsin, Milwaukee, WI
  • Tadeusz Sarna
    Biophysics, Jagiellonian University, Krakow, Poland
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1190. doi:
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      Anna Pilat, Christine Skumatz, Magdalena Olchawa, Mariusz Zareba, Janice Burke, Tadeusz Sarna; Iron Loaded Melanosomes Change Antioxidant Enzyme Levels in ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1190.

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Abstract

Purpose: ARPE-19 cells containing phagocytized photobleached or non-photobleached melanosomes were previously shown to exhibit differential ability to bind metal ions by the containing melanin, which could affect the cell susceptibility to oxidative stress. Here we asked whether the protective mechanism(s) involves differential expression of antioxidant enzymes in cells containing pigment granules by quantifying enzyme levels after enrichment cells with iron-preloaded particles.

Methods: ARPE-19 cells were loaded by phagocytosis with porcine RPE melanosomes, non-treated or photo-bleached, or with control particles - black latex beads. Prior phagocytosis, granules were exposed to 10µM iron donor to increase their iron content. Heme oxygenase-1 (HO-1), ferritin, HO-2, glutathione peroxidase (GPx), and catalase were quantified by western blot analysis after selected time post-phagocytosis. ARPE-19 cells containing iron-loaded particles were examined by cell survival analysis using real-time quantification of propidium iodide fluorescence or MTT assay.

Results: Phagocytosis itself produced transient changes in protein levels of some antioxidant enzymes, but their steady state levels (7 days post phagocytosis) did not differ in cells containing melanosomes versus beads. Phagocytosis of iron-loaded porcine RPE melanosomes induced increase in ferritin, HO-1 expression, but had no effect on HO-2, catalase or GPx protein level 24hrs post-phagocytosis. ARPE-19 cells containing phagocytized photobleached RPE melanosomes induced even higher level of HO-1 and GPx but did not increase ferritin, catalase or HO-2 protein level.

Conclusions: Although melanin is known for its ability to bind metal ions, this is a reversible process and under certain conditions bound to melanin metal ions can be released. Indeed, it was recently demonstrated that iron-loaded melanosomes induced increased levels of the iron storage protein ferritin, which was used as a reporter for iron release into the cytosol. This observation not only implies a broader role for pigment granules in regulating cellular iron homeostasis, but it also raises the possibility that cells containing pigment granules may differ in expression levels of other iron sensitive proteins aside from ferritin. Taken together the results indicate a role for HO-1 and ferritin in the RPE iron homeostasis.

Keywords: 634 oxidation/oxidative or free radical damage • 424 antioxidants • 701 retinal pigment epithelium  
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