Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress.
OBJECTIVE
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.


METHODS
The sensitivity of ARPE-19 cells to H2 O2 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.


RESULTS
Upregulation of MT, HSP70 and FT did not improve survival following exposure to H2 O2 . 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.


CONCLUSIONS
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.
Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
Markus
M
Karlsson
Department of Medical and Health Sciences, Linköping University, Linköping, Sweden.
Tino
T
Kurz
D015153:Blotting, Western; D002452:Cell Count; D002460:Cell Line; D002470:Cell Survival; D004351:Drug Resistance; D005293:Ferritins / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D034741:RNA, Small Interfering / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D015854:Up-Regulation
D018840:HSP70 Heat-Shock Proteins; D034741:RNA, Small Interfering; D005293:Ferritins; D008668:Metallothionein; D006861:Hydrogen Peroxide
ARPE-19; HSP70; age-related macular degeneration; ferritin; iron; metallothionein; oxidative stress; retinal pigment epithelium