Purchase this article with an account.
Jill Harned, Jenny Ferrell, Marilyn M. Lall, Lloyd N. Fleisher, Steven Nagar, Małgorzata Goralska, M. Christine McGahan; Altered Ferritin Subunit Composition: Change in Iron Metabolism in Lens Epithelial Cells and Downstream Effects on Glutathione Levels and VEGF Secretion. Invest. Ophthalmol. Vis. Sci. 2010;51(9):4437-4446. doi: 10.1167/iovs.09-3861.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The iron storage protein ferritin is necessary for the safe storage of iron and for protection against the production of iron-catalyzed oxidative damage. Ferritin is composed of 24 subunits of two types: heavy (H) and light (L). The ratio of these subunits is tissue specific, and alteration of this ratio can have profound effects on iron storage and availability. In the present study, siRNA for each of the chains was used to alter the ferritin H:L chain ratio and to determine the effect of these changes on ferritin synthesis, iron metabolism, and downstream effects on iron-responsive pathways in canine lens epithelial cells.
Primary cultures of canine lens epithelial cells were used. The cells were transfected with custom-made siRNA for canine ferritin H- and L-chains. De novo ferritin synthesis was determined by labeling newly synthesized ferritin chains with 35S-methionine, immunoprecipitation, and separation by SDS-PAGE. Iron uptake into cells and incorporation into ferritin was measured by incubating the cells with 59Fe-labeled transferrin. Western blot analysis was used to determine the presence of transferrin receptor, and ELISA was used to determine total ferritin concentration. Ferritin localization in the cells was determined by immunofluorescence labeling. VEGF, glutathione secretion levels, and cystine uptake were measured.
FHsiRNA decreased ferritin H-chain synthesis, but doubled ferritin L-chain synthesis. FLsiRNA decreased both ferritin H- and L-chain synthesis. The degradation of ferritin H-chain was blocked by both siRNAs, whereas only FHsiRNA blocked the degradation of ferritin L-chain, which caused significant accumulation of ferritin L-chain in the cells. This excess ferritin L-chain was found in inclusion bodies, some of which were co-localized with lysosomes. Iron storage in ferritin was greatly reduced by FHsiRNA, resulting in increased iron availability, as noted by a decrease in transferrin receptor levels and iron uptake from transferrin. Increased iron availability also increased cystine uptake and glutathione concentration and decreased nuclear translocation of hypoxia-inducible factor 1-α and vascular endothelial growth factor (VEGF) accumulation in the cell-conditioned medium.
Most of the effects of altering the ferritin H:L ratio with the specific siRNAs were due to changes in the availability of iron in a labile pool. They caused significant changes in iron uptake and storage, the rate of ferritin synthesis and degradation, the secretion of VEGF, and the levels of glutathione in cultured lens epithelial cells. These profound effects clearly demonstrate that maintenance of a specific H:L ratio is part of a basic cellular homeostatic mechanism.
This PDF is available to Subscribers Only