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M. McGahan, J. Ferrell, M. Mukunnemkeril, L. Fleisher, S. Nagar, M. Goralska, J. Harned; Alteration of Ferritin Subunit Composition Changes Iron Metabolism in Lens Epithelial Cells With Downstream Effects on Glutathione Levels and Vascular Endothelial Growth Factor Secretion. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2436.
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Ferritin is a multimeric iron storage protein consisting of 24 subunits of two types, heavy (H) and light (L). The ratio of these subunits is tissue specific and is thought to control iron storage ability. We have recently demonstrated that in addition to catalyzing free radical damage, iron increases glutathione (GSH) levels and decreases VEGF secretion from cultured lens epithelial cells (LEC). The purpose of this study was to alter the H:L ratio using siRNA for each chain and to determine the effects of these changes on iron storage and downstream effects on intracellular GSH levels in and VEGF secretion by cultured canine LEC.
Custom siRNAs for canine ferritin H- and L-chains were obtained from Dharmacon. A non-targeting siRNA served as a control. De novo synthesis of H- and L-chain ferritins was determined by labeling with 35S-methionine. The amount of GSH in the LEC was determined fluorometrically and the amount of VEGF in the bathing media was determined using ELISA.
Transfection with siRNA for H-chain ferritin resulted in a 54% decrease in H-chain synthesis. Iron incorporation into ferritin was decreased by 53%. Four/five days after transfection GSH levels were increased while VEGF secretion was reduced by 54%. In contrast, transfection with siRNA for L-chain caused a 37% decrease in L-chain synthesis. There was a concomitant increase in iron incorporation into ferritin (53%). This treatment did not alter GSH levels in these cells and had only a minor effect on VEGF secretion.
Reducing synthesis of H-chain ferritin decreased iron incorporation into ferritin. This should increase iron availability and could account for the observed increase in GSH levels and reduction in VEGF secretion. Conversely, decreasing L-chain synthesis increased iron incorporation into ferritin, which should decrease iron availablility. However, the siRNA-induced decrease in L-chain synthesis did not have significant effects on either GSH levels within, or VEGF secretion by LEC. These results may reflect differential effects of these two treatments on discrete intracellular iron pools resulting in differential downstream effects, and could provide important clues about how intracellular iron availability affects normal physiological functions.
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