Altered ferritin subunit composition: change in iron metabolism in lens epithelial cells and downstream effects on glutathione levels and VEGF secretion.

PURPOSE: 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. METHODS: 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. RESULTS: 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-alpha and vascular endothelial growth factor (VEGF) accumulation in the cell-conditioned medium. CONCLUSIONS: 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.

Iron regulates L-cystine uptake and glutathione levels in lens epithelial and retinal pigment epithelial cells by its effect on cytosolic aconitase.

PURPOSE: The authors previously published the novel finding that iron regulates L-glutamate synthesis and accumulation in the cell-conditioned medium (CCM) by increasing cytosolic aconitase activity in cultured lens epithelial cells (LECs), retinal pigment epithelial (RPE) cells, and neurons. The present study was designed to determine whether iron-induced L-glutamate accumulation in the CCM regulates L-cystine uptake and glutathione (GSH) levels through the aconitase pathway in LECs and RPE cells. METHODS: The presence of xCT, the light chain of X(c)(-), a glutamate/cystine antiporter, was analyzed by RT-PCR, immunoblotting, and immunocytochemistry. Uptake of L-[(35)S]cystine and L-[(3)H]glutamate was measured in the presence or absence of transporter inhibitors. L-cystine uptake and intracellular GSH concentration were measured in the presence or absence of iron-saturated transferrin, the iron chelator dipyridyl (DP), or oxalomalic acid (OMA), an aconitase inhibitor. RESULTS: LECs and RPE cells express xCT, as evidenced by RT-PCR analysis and immunoblotting. xCT was localized by immunocytochemistry. The authors found that the iron-induced increase in L-glutamate availability increased L-cystine uptake, with subsequent increases in GSH levels. In addition, L-glutamate production, L-cystine uptake, and GSH concentration were inhibited by OMA and DP, indicating a central role for iron-regulated aconitase activity in GSH synthesis in LECs and RPE cells. CONCLUSIONS: These results demonstrate for the first time that iron regulates L-cystine uptake and the downstream production of GSH in two mammalian cell types. It is possible that the increase in intracellular antioxidant concentration induced by iron serves as a protective mechanism against the well-established capacity of iron to induce oxidative damage.

Iron alters glutamate secretion by regulating cytosolic aconitase activity.

Glutamate has many important physiological functions, including its role as a neurotransmitter in the retina and the central nervous system. We have made the novel observations that retinal pigment epithelial cells underlying and intimately interacting with the retina secrete glutamate and that this secretion is significantly affected by iron. In addition, iron increased secretion of glutamate in cultured lens and neuronal cells, indicating that this may be a common mechanism for the regulation of glutamate production in many cell types. The activity of the iron-dependent enzyme cytosolic aconitase (c-aconitase) is increased by iron. The conversion of citrate to isocitrate by c-aconitase is the first step in a three-step process leading to glutamate formation. In the present study, iron increased c-aconitase activity, and this increase was associated with an increase in glutamate secretion. Inhibition of c-aconitase by oxalomalate decreased glutamate secretion and completely inhibited the iron-induced increase in glutamate secretion. Derangements in both glutamate secretion and iron metabolism have been noted in neurological diseases and retinal degeneration. Our results are the first to provide a functional link between these two physiologically important substances by demonstrating a significant role for iron in the regulation of glutamate production and secretion in mammalian cells resulting from iron regulation of aconitase activity. Glutamatergic systems are found in many nonneuronal tissues. We provide the first evidence that, in addition to secreting glutamate, retinal pigment epithelial cells express the vesicular glutamate transporter VGLUT1 and that regulated vesicular release of glutamate from these cells can be inhibited by riluzole.