The Remarkable Resistance Of ARPE-19 Cells To Oxidative Stress May Result From High Basal Levels Of Iron-binding Proteins And Their Autophagy

Abstract

Purpose: : Iron-mediated oxidative stress has been suggested as a possible pathogenic factor in the development of age-related macular degeneration (AMD). Free redox-active iron mainly exists in lysosomes where it can catalyze hydroxyl radical formation with ensuing formation of lipofuscin and/or lysosomal burst resulting in cell death. Although the post-mitotic retinal pigment epithelial (RPE) cells reside in an unfavorable environment (high in oxygen and light flux) and have a high phagocytic activity, AMD usually develops remarkably late in life. This suggests that RPE cell lysosomes contain only small amounts of redox-active iron. In earlier studies we showed cultured ARPE-19 cells to be up to 150 times more resistant to oxidative stress (H₂O₂) than J774 cells, which also are professional scavengers. Since both cell types seem to contain comparable amounts of total as well as intralysosomal iron, ARPE-19 cells therefore should be able to keep their iron in a non-redox-active state. The aim of this study was to investigate whether the intracellular basal levels of iron-binding proteins - such as heat shock-protein 70 (HSP70), metallothionein (MT) and ferritin (FT) - differ between the two cell types.

Methods: : Immortalized human ARPE-19 and murine J774 cells were grown for 24 h at standard culture conditions and then exposed to either heat shock (43°C; 30 min), an iron-phosphate complex (via 200 or 500 µM FeCl₃) or 100 µM ZnSO₄. Protein samples taken 24 h after the different treatments were then subjected to immunoblotting.

Results: : ARPE-19 cells were shown to contain high basal levels of FT, HSP70 and MT, whereas these proteins seemed to be almost non-detectable (HSP70) or present to a lesser extent (MT and FT) in control J774 cells. As expected, increased expression of HSP70, MT and FT was found in J774 cells after exposure to heat shock, Zn or Fe. With the exception of FT, their levels were only moderately increased in ARPE-19 cells following the same treatment.

Conclusions: : Our results suggest that the extreme resistance of ARPE-19 cells to oxidative stress may depend on high basal levels of several iron-binding proteins and a high capacity for up-regulation of FT. Pronounced autophagy of iron-binding proteins by RPE cells may explain the normally late development of AMD.

Keywords: oxidation/oxidative or free radical damage • retinal pigment epithelium • protective mechanisms