April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
The Oxidative Stress-resistance Of Arpe-19 Cells May Be A Function Of High Basal Levels Of Iron-binding Proteins
Author Affiliations & Notes
  • Markus Karlsson
    Dept of Ophthalmology,
    University of Linkoping, Linkoping, Sweden
  • Christina I. Frennesson
    Dept of Ophthalmology,
    University of Linkoping, Linkoping, Sweden
  • Sven Erik G. Nilsson
    Dept of Ophthalmology,
    University of Linkoping, Linkoping, Sweden
  • Tino Kurz
    Dept of Pharmacology,
    University of Linkoping, Linkoping, Sweden
  • Footnotes
    Commercial Relationships  Markus Karlsson, None; Christina I. Frennesson, None; Sven Erik G. Nilsson, None; Tino Kurz, None
  • Footnotes
    Support  Linkoping University Hospital Research Fund (ALF) and Crown Princess Margaretha's Foundation for the Visually Handicapped (KMA).
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 4435. doi:
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      Markus Karlsson, Christina I. Frennesson, Sven Erik G. Nilsson, Tino Kurz; The Oxidative Stress-resistance Of Arpe-19 Cells May Be A Function Of High Basal Levels Of Iron-binding Proteins. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4435.

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Abstract

Purpose: : Oxidative stress has emerged as a tentative factor in the pathogenesis of lipofuscin formation and subsequent development of age-related macular degeneration (AMD). Lysosomal redox-active iron plays an important role as catalyst in the generation of hydrogen peroxide-derived hydroxyl radicals. We showed previously that ARPE-19 cells can tolerate about 150 times higher hydrogen peroxide concentrations than J774 cells, which also are professional scavengers, implying that ARPE-19 cells either have less iron within their lysosomes or are able to keep it in a non redox-active state. Since previous studies showed an iron-chelating capacity of metallothionein (MT), the aim of the present work was to investigate whether basal levels of this phase-II protein differ between ARPE-19 and J774 cells. If so, this might indicate that autophagy of Fe-binding proteins would keep lysosomal redox-active Fe down.

Methods: : 24 h after seeding, immortalized human ARPE-19 cells and murine J774 cells were exposed to 100 µM ZnSO4 for 24 h. Cells were then lysed and protein samples subjected to immunodetection of MT. In additional experiments, the level of total intracellular iron was measured using atomic absorption spectroscopy (AAS). The amount of loosely bound iron was detected by the sulfide-silver method (SSM).

Results: : Both ARPE-19 and J774 cells seem to contain comparable amounts of total iron as measured by AAS. Using the SSM, it was shown that ARPE-19 cells did not contain less iron inside their lysosomes compared to J774. Western blot analyses showed that the basal level of MT in ARPE-19 cells was ≥37% higher than in J774 cells. Pretreatment with 100 µM ZnSO4 increased the MT level in J774, but not in ARPE-19 cells, indicating an already high expression of this protein that could not be further upregulated.

Conclusions: : Despite having equal amounts of total as well as intra-lysosomal iron, ARPE-19 cells have been proven to withstand oxidative stress better than most other cell types. Considering that the formation of hydroxyl radicals from hydrogen peroxide is dependent on iron-catalysed Fenton-reaction, ARPE-19 cells therefore seem to have a capacity to keep their intra-lysosomal iron in a non-redox-active form. ARPE-19 cells contain a high basal level of the iron-binding protein MT that, following normal autophagy, relocates to the lysosomal compartment. This could be part of the explanation why ARPE-19 cells are resistant to oxidative stress and, hence, why lipofuscin accumulates slowly and AMD usually develops only at old age.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • age-related macular degeneration 
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