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Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuroscience. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
2016 (English)In: Acta Ophthalmologica, ISSN 1755-375X, E-ISSN 1755-3768, Vol. 94, no 6, 556-565 p.Article in journal (Refereed) Published
Abstract [en]


Oxidative stress-related damage to retinal pigment epithelial (RPE) cells is an important feature in the development of age-related macular degeneration. Iron-catalysed intralysosomal production of hydroxyl radicals is considered a major pathogenic factor, leading to lipofuscin formation with ensuing depressed cellular autophagic capacity, lysosomal membrane permeabilization and apoptosis. Previously, we have shown that cultured immortalized human RPE (ARPE-19) cells are extremely resistant to exposure to bolus doses of hydrogen peroxide and contain considerable amounts of the iron-binding proteins metallothionein (MT), heat-shock protein 70 (HSP70) and ferritin (FT). According to previous findings, autophagy of these proteins depresses lysosomal redox-active iron. The aim of this study was to investigate whether up- or downregulation of these proteins would affect the resistance of ARPE-19 cells to oxidative stress.


The sensitivity of ARPE-19 cells to H2O2 exposure was tested following upregulation of MT, HSP70 and/or FT by pretreatment with ZnSO4, heat shock or FeCl3, as well as siRNA-mediated downregulation of the same proteins.


Upregulation of MT, HSP70 and FT did not improve survival following exposure to H2O2. This was interpreted as existence of an already maximal protection. Combined siRNA-mediated attenuation of both FT chains (H and L), or simultaneous downregulation of all three proteins, made the cells significantly more susceptible to oxidative stress confirming the importance of iron-binding proteins.


The findings support our hypothesis that the oxidative stress resistance exhibited by RPE cells may be explained by a high autophagic influx of iron-binding proteins that would keep levels of redox-active lysosomal iron low.

Place, publisher, year, edition, pages
Wiley-Blackwell Publishing Inc., 2016. Vol. 94, no 6, 556-565 p.
Keyword [en]
age-related macular degeneration, ARPE-19, ferritin, HSP70, iron metallothionein, oxidative stress, retinal pigment epithelium
National Category
Clinical Medicine
URN: urn:nbn:se:liu:diva-111557DOI: 10.1111/aos.13076ISI: 000383520800034OAI: diva2:757979

At the time for thesis presentation publication was in status: Manuscript

Funding agencies:  Crown Princess Margaretas Foundation for the Visually Handicapped; Edvin Jordan Foundation for Ophthalmological Research; Linkoping University Hospital Research Fund (ALF)

Available from: 2014-10-24 Created: 2014-10-24 Last updated: 2016-10-18Bibliographically approved
In thesis
1. Oxidative stress-related damage of retinal pigment epithelial cells: possible protective properties of autophagocytosed iron-binding proteins
Open this publication in new window or tab >>Oxidative stress-related damage of retinal pigment epithelial cells: possible protective properties of autophagocytosed iron-binding proteins
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Oxidative stress is a major pathogenic factor in the development of age-related macular degeneration (AMD), which is the most common cause of severe central visual impairment in the elderly population in the western world.

It is believed that the degenerative process starts in the retinal pigment epithelium (RPE). The post-mitotic RPE is a single layer of pigmented cells located behind the photoreceptors – rods and cones – of the retina. Daily, the RPE cells phagocytose and recycle the expended tips of the photoreceptor outer segments. This heavy phagocytic burden leads to substantial oxidative stress in the cells, which is further enhanced by intense illumination and a high oxygen tension. A hallmark of early AMD is a progressive build-up of the non-degradable age pigment lipofuscin (LF) in lysosomes of the RPE. LF accumulation hampers phagocytosis and autophagy in the RPE, resulting in increased amounts of cellular debris in and around the cells. This decreases the function and viability of both RPE cells and photoreceptors.

Iron is known to accumulate in the retina with increasing age, particularly in AMDaffected eyes, and amplifies oxidative stress by acting as a potent catalyst in the generation of hydroxyl radicals. These highly reactive radicals contribute to LF formation and may, if abundantly present, also directly damage lysosomal membranes. The subsequent leakage of degrading enzymes to the cytosol initiates cell death via apoptosis or necrosis.

In this thesis, we have investigated the oxidative stress response of human RPE (ARPE-19) cells compared to murine J774 cells, another type of lysosome-rich cells with a high phagocytic capacity. The ARPE-19 cells were found to be extremely resistant to oxidative stress and tolerated exposure to single doses of H2O2 in concentrations up to 150 times higher than the J774 cells before lysosomal rupture and ensuing cell death occurred. This resistance was increased even further when the cells were protected with a potent iron chelator that prevents redox-active iron to participate in hydroxyl radical generation. Both cell lines were shown to be equally effective in degrading H2O2 and seem to contain comparable amounts of total as well as intralysosomal iron. Therefore, we reasoned that the insensitivity of ARPE-19 cells to H2O2 exposure might be related to a mechanism which keeps their intralysosomal iron bound in a non redox-active form. This theory was supported by our finding of very high basal expression levels of metallothionein (MT), heat shock-protein 70 (HSP70) and ferritin (FT) in ARPE-19 cells compared to J774 cells. All of these proteins have previously been shown to possess potent iron-binding properties. The ARPE-19 cells were also shown to have a higher basal rate of autophagy. SiRNA-mediated attenuation of MT, HSP70 and FT levels in the ARPE-19 cells resulted, to some degree, in an increased sensitivity to H2O2 treatment. Furthermore, a human cell stress array showed several other stress-related proteins to be up-regulated in ARPE-19 cells.

Additionally, we evaluated the commonly used, but frequently misinterpreted, H2DCF test for oxidative stress. It was demonstrated that oxidation of H2DCF into fluorescent DCF mainly reflects relocation to the cytosol of lysosomal iron and mitochondrial cytochrome c, rather than being the result of some poorly defined “general” oxidative stress.

In conclusion, our results indicate that the extreme resistance to oxidative stress exhibited by the ARPE-19 cells might be related to a high continuous autophagic influx of iron-binding proteins into the lysosomal compartment. Before being degraded, such proteins will temporarily keep intralysosomal iron bound in a non redox-active form, thereby inhibiting hydroxyl radical formation. This may partly explain why RPE cells, in spite of their exposed location and heavy burden of phagocytosis, usually manage to survive and evade significant LF accumulation until late in life.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2014. 93 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1426
National Category
Clinical Medicine Biochemistry and Molecular Biology
urn:nbn:se:liu:diva-111558 (URN)10.3384/diss.diva-111558 (DOI)978-91-7519-209-3 (print) (ISBN)
Public defence
2014-11-28, Nils-Holgersalen, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Available from: 2014-10-24 Created: 2014-10-24 Last updated: 2015-09-29Bibliographically approved

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