Abstract
Abstract: :
Purpose: Ferritin is an Fe storage protein, which consists of 24 subunits of two types, heavy (H) and light (L). Changes in ferritin content and its tissue specific ratio of H/L chains modify the Fe storage capacity of cells. In a previous study we overexpressed ferritin chains in lens epithelial cells (LEC) transfected with H and L chain clones and discovered that transfected cells secrete overexpressed, assembled ferritin into the media. The purpose of this study was to determine if secreted ferritin plays a role in maintaining cellular Fe homeostasis and how an altered ferritin subunit ratio affects this process. Methods: LEC were transfected with pTargeT mammalian vector containing cloned sequences of H- and L-chain ferritin cDNAs for 24h and then labeled with 59-Fe transferrin for 20h. After labeling, the media were replaced with fresh, label free DMEM for additional 24h. Fe content of cellular and extracellular ferritin was determined after analyzing ferritin bands on PAGE gel using electronic radioactivity detector. Results: Cellular ferritin of H-transfectants contained twice as much 59-Fe than ferritin of control, plasmid transfectants. L-transfectants showed two pools of Fe containing ferritin, one comigrating with 59-Fe-ferritin present in plasmid- and H-transfectants and the other of slightly lower mobility. Combined radioactivity of both 59-Fe containing ferritins was 60% higher than that of plasmid ferritin. Analysis of media from transfected cells, 24h after labeling, showed presence of ferritin which in all experimental groups contained 30-40% of 59-Fe found in bands of corresponding cell lysates. Extracellular ferritin of L-transfectants had the same pattern observed in their cell lysates, however secretion of the lower mobility band was lower (21% Fe) than the higher mobility band (33%). We used ferritin containing media from transfected cells to treat nontransfected cells in the presence of 59-Fe-transferrin. The treatment did not change total 59-Fe uptake and 59-Fe incorporation into ferritin of nontransfected cells. Conclusions: Overexpression of both chains increased long term storage of Fe in ferritin. H-chain was most likely incorporated into existing pool of endogenous ferritin while the L-chain overexpression resulted in formation of separate L-rich pool of ferritin of lower mobility. A significant amount of Fe can be removed from the cells by secreted ferritin however the secreted ferritin does not facilitate Fe uptake and may have some other role in Fe metabolism.
Keywords: gene/expression • protein structure/function • metabolism