Repression of the heavy ferritin chain increases the labile iron pool of human K562 cells (original) (raw)
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Blood, 2001
The role of ferritin expression on the labile iron pool of cells and its implications for the control of cell proliferation were assessed. Antisense oligodeoxynucleotides were used as tools for modulating the expression of heavy and light ferritin subunits of K562 cells. mRNA and protein levels of each subunit were markedly reduced by 2-day treatment with antisense probes against the respective subunit. Although the combined action of antisense probes against both subunits reduced their protein expression, antisense repression of one subunit led to an increased protein expression of the other. Antisense treatment led to a rise in the steady-state labile iron pool, a rise in the production of reactive oxygen species after pro-oxidative challenges and in protein oxidation, and the down-regulation of transferrin receptors. When compared to the repression of individual subunits, co-repression of each subunit evoked a more than additive increase in the labile iron pool and the extent of ...
Role of Ferritin in the Control of the Labile Iron Pool in Murine Erythroleukemia Cells
Journal of Biological Chemistry, 1998
In vitro studies have shown that ferritin iron incorporation is mediated by a ferroxidase activity associated with ferritin H subunits (H-Ft) and a nucleation center associated with ferritin L subunits (L-Ft). To assess the role played by the ferritin subunits in regulating intracellular iron distribution, we transfected mouse erythroleukemia cells with the H-Ft subunit gene mutated in the iron-responsive element. Stable transfectants displayed high H-Ft levels and reduced endogenous L-Ft levels, resulting in a marked change in the H:L subunit ratio from 1:1 in control cells to as high as 20:1 in some transfected clones. The effects of H-Ft overexpression on the labile iron pool were determined in intact cells by a novel method based on the fluorescent metallosensor calcein. H-Ft overexpression resulted in a significant reduction in the iron pool, from 1.3 M in control cells to 0.56 M in H-Ft transfectants, and in higher buffering capacity following iron loads. A fraction of the H-Ftassociated iron was labile, available to cell-permeant, but not cell-impermeant, chelators. The results of this study provide the first in vivo direct demonstration of the capacity of H-Ft to sequester cell iron and to regulate the levels of the labile iron pool.
The Cellular Labile Iron Pool and Intracellular Ferritin in K562 Cells
2010
http://bloodjournal.hematologylibrary.org/content/94/6/2128.full.html Updated information and services can be found at: (1174 articles) Red Cells Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub\_requests
The Role of Ferritin in Health and Disease: Recent Advances and Understandings
Metabolites
Systemic iron homeostasis needs to be tightly controlled, as both deficiency and excess iron cause major global health concerns, such as iron deficiency anemia, hemochromatosis, etc. In mammals, sufficient dietary acquisition is critical for fulfilling the systemic iron requirement. New questions are emerging about whether and how cellular iron transport pathways integrate with the iron storage mechanism. Ferritin is the intracellular iron storage protein that stores surplus iron after all the cellular needs are fulfilled and releases it in the face of an acute demand. Currently, there is a surge in interest in ferritin research after the discovery of novel pathways like ferritinophagy and ferroptosis. This review emphasizes the most recent ferritin-related discoveries and their impact on systemic iron regulation.
1999
Human erythroid precursors grown in culture possess membrane receptors that bind and internalize acid isoferritin. These receptors are regulated by the iron status of the cell, implying that ferritin iron uptake may represent a normal physiologic pathway. The present studies describe the fate of internalized ferritin, the mechanisms involved in the release of its iron, and the recognition of this iron by the cell. Normal human erythroid precursors were grown in a 2-phase liquid culture that supports the proliferation, differentiation, and maturation of erythroid precursors. At the stage of polychromatic normoblasts, cells were briefly incubated with 59 Fe-and/or 125 I-labeled acid isoferritin and chased. The 125 I-labeled ferritin protein was rapidly degraded and only 50% of the label remained in intact ferritin protein after 3 to 4 hours. In parallel, 59 Fe decreased in ferritin and increased in hemoglobin. Extracellular holoferritin uptake elevated the cellular labile iron pool (LIP) and reduced iron regulatory protein (IRP) activity; this was inhibited by leupeptin or chloroquine. Extracellular apoferritin taken up by the cell functioned as an iron scavenger: it decreased the level of cellular LIP and increased IRP activity. We suggest that the iron from extracellular is metabolized in a similar fashion by developing erythroid cells as is intracellular ferritin. Following its uptake, extracellular ferritin iron is released by proteolytic degradation of the protein shell in an acid compartment. The released iron induces an increase in the cellular LIP and participates in heme synthesis and in intracellular iron regulatory pathways.
Archives of Biochemistry and Biophysics, 1997
Heme (ferroprotoporphyrin IX) is the well known Hemin (ferriprotoporphyrin IX), the oxidized pros-prosthetic group of hemoglobin and other hemoproteins. thetic group of hemoglobin, is a source of potentially When oxidized to the ferric state, heme (now designated cytotoxic iron, but in chronic low doses can induce cytoas hemin) is bound less tightly by hemoglobin and can protection against iron-stimulated oxidative stress. The interact more readily with acceptors such as other prolatter property of hemin has been examined, using muteins or lipid membranes in cells (1-3). Hemin's amphirine L1210 cells and three different oxidant generating philic properties tend to favor such interactions. This, systems: (i) glucose/glucose oxidase, (ii) near-ultraviolet combined with the ability of ligated iron to undergo reirradiation, and (iii) dye-mediated photodynamic action. dox cycling and, in the process, catalyze damaging oxi-Cells treated with the lipophilic iron donor ferric-8-hydative reactions makes hemin a potentially cytotoxic droxyquinoline, Fe(HQ) 2 (1 mM, 30 min) were found to compound. Numerous examples of hemin prooxidant acbe more sensitive to oxidative killing than nontreated tivity have been reported (3-5). Of special interest are controls. However, cells challenged after long-term (20recent studies showing that acute in vitro exposure of 24 h) exposure to hemin (10 mM) were substantially more endothelial cells (4, 6) or breast tumor cells (7) to hemin resistant than controls and were sensitized far less by itself or methemoglobin as a source of hemin enhanced Fe(HQ) 2. Immunoblot analyses of 24-h hemin-treated their sensitivity to lethal injury by H 2 O 2 or activated cells indicated that the ferritin heavy (H) subunit was neutrophils. Whereas this response was observed when elevated 12-to 15-fold, whereas the light (L) subunit was oxidative stress was applied shortly (Ç1 h) after hemin, essentially unchanged. Experiments carried out with a striking hyperresistance was evident when stress was 55 Fe(HQ) 2 showed that iron uptake capacity of cells was delayed for 16 h. The latter cells were more resistant greatly enhanced after hemin treatment. More specifithan controls, even when challenged in the presence of a cally, hemin-stimulated cells were found to contain Ç9 second dose of hemin. Using photodynamically imposed times more immunoprecipitable ferritin iron after incuoxidative stress, we have described similar behavior in bation with saturating levels (4-5 mM) of 55 Fe(HQ) 2 and Ç3 times more iron per ferritin molecule compared with L1210 leukemia cells that had been chronically exposed nonstimulated controls. The nonferritin iron content of to synthetic iron chelates or to hemin itself (8, 9). In the latter was estimated to be Ç40 times greater than agreement with others (4, 6, 7), we found that ironthat of the former following low-level (0.5 mM) 55 Fe(HQ) 2 treated cells were enriched in immunodetectable ferritin treatment. These results are consistent with the idea (predominantly the heavy chain), induction of which corthat induced ferritin, enriched in H-chain, sequesters related strongly with the development of hyperresisredox active iron rapidly and copiously, thereby enhanctance. In the present study we confirm that hemin-stiming cellular resistance to oxidants. ᭧ 1997 Academic Press ulated L1210 cells are upregulated in ferritin and show Key Words: hemin; iron; ferritin; oxidative stress; leuthat these cells are more resistant to several different kemia cells. 1 This work was supported by USPHS Grants 2-PO1 CA49089 and CA70823 from the National Cancer Institute. hydroxyquinoline as a donor. Our results indicate that 2 This paper is based on work described in a dissertation submitted although stimulated cells have a greater capacity for by F. Lin in partial fulfillment of the requirements for a Ph.D. degree iron binding, most of the iron is directed to ferritin, in Biochemistry at the Medical College of Wisconsin. where it is shielded from damaging redox reactions.
Journal of Neurochemistry, 2009
Insertional mutations in exon 4 of the ferritin light chain (FTL) gene are associated with hereditary ferritinopathy (HF) or neuroferritinopathy, an autosomal dominant neurodegenerative disease characterized by progressive impairment of motor and cognitive functions. To determine the pathogenic mechanisms by which mutations in FTL lead to neurodegeneration, we investigated iron metabolism and markers of oxidative stress in the brain of transgenic (Tg) mice that express the mutant human FTL498-499InsTC cDNA. Compared with wild-type mice, brain extracts from Tg (FTL-Tg) mice showed an increase in the cytoplasmic levels of both FTL and ferritin heavy chain polypeptides, a decrease in the protein and mRNA levels of transferrin receptor-1, and a significant increase in iron levels. Transgenic mice also showed the presence of markers for lipid peroxidation, protein carbonyls, and nitrone-protein adducts in the brain. However, gene expression analysis of iron management proteins in the liver of Tg mice indicates that the FTL-Tg mouse liver is iron deficient. Our data suggest that disruption of iron metabolism in the brain has a primary role in the process of neurodegeneration in HF and that the pathogenesis of HF is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates in the brain. Keywords animal model; hereditary ferritinopathy; neuroferritinopathy Ferritin, the main iron storage protein, plays a central role in the maintenance of cellular iron balance (Theil 1990; Harrison and Arosio 1996; Chasteen 1998). The protein is composed of 24 subunits of both ferritin heavy chain (FTH1) and ferritin light chain (FTL). The FTH1 subunit is thought to play a role in the rapid detoxification of iron, whereas the FTL subunit facilitates iron nucleation, mineralization, and long-term iron storage (Rucker et al. 1996). Because of its ability to participate directly as a donor or acceptor in electron transfer reactions,
Biochemical Pharmacology, 2000
Toxic and carcinogenic free radical processes induced by drugs and other chemicals are probably modulated by the participation of available iron. To see whether endogenous iron was genetically variable in normal mice, the common strains C57BL/10ScSn, C57BL/6J, BALB/c, DBA/2, and SWR were examined for major differences in their hepatic non-heme iron contents. Levels in SWR mice were 3-to 5-fold higher than in the two C57BL strains, with intermediate levels in DBA/2 and BALB/c mice. Concentrations in kidney, lung, and especially spleen of SWR mice were also greater than those in C57BL mice. Non-denaturing PAGE of hepatic ferritin from all strains showed a major holoferritin band at approximately 600 kDa, with SWR mice having Ͼ3-fold higher levels than C57BL strains. SDS PAGE showed a band of 22 kDa, mainly representing L-ferritin subunits. A trace of a subunit at 18 kDa was also detected in ferritin from SWR mice. The 18 kDa subunit and a 500 kDa holoferritin from which it originates were observed in all strains after parenteral iron overload, and there was no major variation in ferritin patterns. Although iron uptake studies showed no evidence for differential duodenal absorption between strains to explain the variation in basal iron levels, acquisition of absorbed iron by the liver was significantly higher in SWR mice than C57BL/6J. As with iron and ferritin contents, total iron regulatory protein (IRP-1) binding capacity for mRNA iron responsive element (IRE) and actual IRE/IRP binding in the liver were significantly greater in SWR than C57BL/6J mice. Cytosolic aconitase activity, representing unbound IRP-1, tended to be lower in the former strain. SWR mice were more susceptible than C57BL/10ScSn mice to the toxic action of diquat, which is thought to involve iron catalysis. If extrapolated to humans, the findings could suggest that some people might have the propensity for greater basal hepatic iron stores than others, which might make them more susceptible to iron-catalysed toxicity caused by oxidants.