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

Ferritin is a multimeric iron-storage protein consisting of 24 subunits of two types: heavy (H) and light (L). The ratio of these two chains is tissue specific and controls iron storage and availability.1 Ferritin H- and L-chain expression is regulated at both the transcriptional2 and translational levels.3 In addition, cells can regulate the H:L ratio through differential rates of degradation and secretion for each chain.4,5 The role of ferritin H- and L-chains and the effects of alteration of the H:L ratio on iron storage and physiological and pathologic processes, have been studied by inducing the overexpression and underexpression (siRNA knockdown) of each chain. Overexpression of H-chain ferritin decreases the cytosolic labile iron pool (LIP; considered to be a pool of available iron in the cytosol), increases iron incorporation into ferritin, and increases cell resistance to oxidative stress and UV irradiation in cultured erythroid cells and in lens epithelial cells (LECs).6–8 In addition, altering the H:L ratio with siRNA has significant effects on iron availability in HeLa cells. Ferritin L-chain siRNA (FLsiRNA) decreases L-chain levels by 80%, but has no effect on H-chain levels or any parameters indicative of iron availability. On the other hand, ferritin H-chain siRNA (FHsiRNA) decreases H-chain levels by 75% and increases L-chain levels threefold. FHsiRNA also increases iron availability and decreases resistance to oxidative stress induced by hydrogen peroxide.9 We have recently shown that ferritin chains are significantly modified in canine and human lens fiber cells and that these modifications increase with age.10 Although it is unclear how these modifications affect the assembly of the whole ferritin molecule, they most likely affect the iron storage capability of ferritin. Such changes could contribute to cataractogenesis, either by the aggregation of abnormal chains or by increasing the availability of reactive iron that cannot be safely stored. Ferritin chains have other physiologic roles beyond safely storing potentially reactive iron. For example, siRNA knockdown showed that H-chain ferritin contributes to malignant mesothelioma cell resistance to apoptosis11 and that L-chain ferritin plays a role in iron-independent cellular proliferation.9 Indeed, the physiological roles of iron are numerous, and new activities have recently been uncovered (Fig. 1), such as the regulation of the levels of hypoxia inducible factor (HIF)-1,12,13 a transcription factor that regulates many genes, including vascular endothelial growth factor (VEGF),14,15 which is made by the lens.16 We have recently shown that iron controls VEGF secretion in both LECs (McGahan MC, et al. IOVS 2007;48:ARVO E-Abstract 2436) and retinal pigmented epithelial (RPE) cells (Ferrell J, Harned J, unpublished data, 2008). Furthermore, iron controls the synthesis and secretion of glutamate in LECs, RPE cells, and neurons.17 The iron-regulated secretion of glutamate had downstream effects on cystine uptake by the cystine/glutamate antiporter and the levels of glutathione (GSH) in these cells.18 Figure 1. The role of iron in HIF-1 activation and GSH production. The goal of the present study was to alter the ferritin H:L ratio with siRNA for each ferritin chain and to determine how these changes affect iron storage and availability, ferritin levels (including the kinetics of its synthesis and degradation), cystine uptake, GSH levels, HIF-1α translocation to the nucleus, and VEGF production in LECs.