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Abstract
The primary cultures of canine lens epithelial cells were transiently transfected with cDNAs for dog ferritin H- or L-chains in order to study differential expression of these chains. By using chain-specific antibodies, we determined that at 48 h after transfection overexpression of L-chain was much higher (9-fold over control) than that of H-chain (1.7-fold). We discovered that differentially transfected cells secrete overexpressed chains as homopolymeric ferritin into the media. Forty-eight hours after transfection accumulation of H-ferritin in the media was much higher (3-fold) than that of L-ferritin. This resulted in lowering of the concentration of H-chain in the cytosol. Co-transfection of cells with both H- and L-chain cDNAs increased the intracellular levels of H-chain and eliminated secretion of H-ferritin to the media. We concluded that lens epithelial cells differentially regulate concentration of both ferritin chains in the cytosol. The overexpressed L-chain accumulated in the cytosol as predominantly homopolymeric L-ferritin. This is in contrast to H-chain, which is removed to the media unless there is an L-chain available to form heteropolymeric ferritin. These data indicate that the inability of cells to more strictly control cytosolic levels of L-chain may augment its accumulation in lenses of humans with hereditary hyperferritinemia cataract syndrome, which is caused by overexpression of L-chain due to mutation in the regulatory element in the untranslated region of the mRNA of the chain.
Highlights
Ble for uptake and oxidation of Fe2ϩ into ferric ions
We used chain-specific antibodies to determine whether the discrepancy in overexpression of both chains by transfected LEC resulted from a difference in the ability to detect each ferritin chain
The time was selected based on the transfection efficiency determined with pEGFP vector containing green fluorescent protein cDNA and confirmed by dramatic overexpression of L-chain in Ltransfectants detected at that time
Summary
Ble for uptake and oxidation of Fe2ϩ into ferric ions. Translocation of these ions into the core of the ferritin shell and their mineralization for long term storage is facilitated by the L subunit. The H and L subunit ratio of mammalian ferritins varies and is highly tissue-specific; mechanisms by which cells maintain the chain-specific ratio are not fully understood Both chains complement each other in the process of sequestering and storing iron, H-chain has a broader range of functions. In our previous studies on iron metabolism and resistance to UV irradiation of dog lens epithelial cells, we used a speciesand tissue-specific model to change ferritin chain ratio by transiently transfecting primary cell culture with canine H- or L-chain ferritin cDNA [13]. These studies revealed a discrepancy between expression of H- and L-ferritin chains under the
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