Transient increase of the labile iron pool in HepG2 cells by intravenous iron preparations.

Intravenous iron, used for the treatment of anemia in chronic renal failure and other diseases, represents a possible source of free iron in tissue cells, particularly in the liver. In this study we examined the effect of different sources of intravenous iron (IVI) on the labile iron pool (LIP) which represents the nonferritin-bound, redox-active iron that is implicated in oxidative stress and cell injury. Furthermore, we examined the role of the LIP for the synthesis of ferritin. We used HepG2 cells as a well known model for hepatoma cells and monitored the LIP with the metal-sensitive fluorescent probe, calcein-AM, the fluorescence of which is quenched on binding to iron. We showed that steady state LIP levels in HepG2 cells were increased transiently, up to three-fold compared to control cells, as an adaptive response to long-term IVI exposure. In relation to the amount of iron in the LIP, the ferritin levels increased and the iron content of ferritin decreased. As any fluctuation in the LIP, even when it is only transient (e.g. after exposure to intravenous iron in this study), may result either in impairment of synthesis of iron containing proteins or in cell injury by pro-oxidants. Such findings in nonreticuloendothelial cells may have important implications in the generation of the adverse effects of chronic iron exposure reported in dialysis patients.     

Red blood cells upregulate cytoprotective proteins and the labile iron pool in dividing human T cells despite a reduction in oxidative stress

We have recently reported that red blood cells (RBC) promote T cell growth and survival by inhibiting activation-induced T cell death. In the present study, we have examined parameters of oxidative stress and intracellular iron in activated T cells and correlated these data with the expression of ferritin, heme oxygenase-1 (HO-1), and the transferrin receptor CD71. T cells growing in the presence of RBC had reduced levels of reactive oxygen species (ROS) and oxidatively modified proteins, suggesting that RBC efficiently counteracted ROS production on the activated T cells. Flow cytometry and immunodetection demonstrated that T cells dividing in the presence of RBC had increased levels of intracellular ferritin rich in L-subunits and HO-1 along with a downmodulation in CD71 expression. Finally, using the fluorescent iron indicator calcein and flow cytometry analysis, we were able to show that a relative amount of the labile iron pool (LIP) was upregulated in T cells growing in the presence of RBC. These findings are consistent with a typical response to iron overload. However, neither heme compounds nor ferric iron reproduced the levels of expansion and survival of T cells induced by intact RBC. Altogether, these data suggest that RBC inhibit apoptosis of activated T cells by a combination of ROS scavenging and upregulation of cytoprotective proteins such as ferritin and HO-1, which may counteract a possible toxic effect of the increased intracellular free iron.     

Sulfide increases labile iron pool in RD4 cells

A linkage between sulfur and iron metabolism has been suggested since sulfide has the ability to release iron from ferritin in the presence of iron acceptors in vitro. Nevertheless, this linkage is still lacking evidence in vivo as well as in cellular models. In this study we have treated human RD4 skeletal muscle cells with sodium sulfide and measured the level of the labile iron pool (LIP) as well as the intracellular sulfide concentration. We have also detected the amounts of L-ferritin protein as well as the iron regulatory protein 2 (IRP2). The sulfide treatment resulted in a 100% increase in the amount of LIP after 1 and 2 h. We also found that the raise of the LIP levels was coupled to an elevation of the amounts of intracellular sulfide that increased by 60%. The bioavailability of the released iron was confirmed by a 100% increase in L-ferritin protein as well as a 60% decrease of the IRP2 protein levels. These results suggest that there is a linkage between sulfur metabolism and intracellular iron regulation in mammalian cells.     

Tumor necrosis factor-alpha-induced reactive oxygen species formation is mediated by JNK1-dependent ferritin degradation and elevation of labile iron pool

Tumor necrosis factor alpha induces increased reactive oxygen species (ROS) generation in different experimental models. However, the nature of this phenomenon is still unknown. We hypothesized that TNF-induced ROS formation is due to JNK-regulated ferritin degradation and an increase in labile iron pool (LIP). We used as a model human prostate cancer cells, DU145. TNF treatment induced ROS formation, which was reduced to the control level in cells pretreated with desferrioxamine, an iron chelator. TNF induced a drop in light chain of the ferritin level, as judged by immunoblotting and an increase in LIP, evaluated by calcein fluorescence. Moreover, we observed that the JNK inhibitor SP600125 abolished TNF-induced changes in LIP, which suggests that JNK kinases are involved in this process. To explore which one of the JNK kinases is responsible for these effects, DU145 cells were transiently transfected with plasmids encoding inactive mutants of JNK1 or JNK2. The cells expressing inactive JNK1 mutant, but not cells expressing JNK2 mutant or possessing an empty vector, were completely resistant to TNF-induced ROS generation, ferritin degradation, and an increase in LIP. These data suggest that TNF-induced ROS formation is mediated by JNK1, which regulates ferritin degradation and thus the level of highly reactive iron.     

Distinct roles of basal steady-state and induced H-ferritin in tumor necrosis factor-induced death in L929 cells

Tumor necrosis factor (TNF) alpha is a cytokine capable of inducing caspase-dependent (apoptotic) cell death in some cells and caspase-independent (necrosis-like) cell death in others. Here, using a mutagenesis screen for genes critical in TNF-induced death in L929 cells, we have found that H-ferritin deficiency is responsible for TNF resistance in a mutant line and that, upon treatment with TNF, this line fails to elevate levels of labile iron pool (LIP), critical for TNF-induced reactive oxygen species (ROS) production and ROS-dependent cell death. Since we found that TNF-induced LIP in L929 cells is primarily furnished by intracellular storage iron, the lesser induction of LIP in H-ferritin-deficient cells results from a reduction of intracellular iron storage caused by less H-ferritin. Different from some other cell lines, the H-ferritin gene in L929 cells is not TNF inducible; however, when H-ferritin is expressed in L929 cells under a TNF-inducible system, the TNF-induced LIP and subsequent ROS production and cell death were all prevented. Thus, LIP is a common denominator of ferritin both in the enhancement of cell death by basal steady-state H-ferritin and in protection against cell death by induced H-ferritin, thereby acting as a key determinant of TNF-induced cell death.     

P66Shc mediated ferritin degradation--a novel mechanism of ROS formation

Diallyl trisulfide (DATS) has been shown to induce the formation of reactive oxygen species (ROS) in prostate cancer cells, which was accompanied by a decrease in the ferritin protein level and an increase in the labile iron pool (LIP). However, the mechanism of the ferritin degradation has not been fully elucidated. In this paper we demonstrate that DATS-induced ROS formation depends on p66Shc. In cells stably expressing a dominant negative mutant of p66Shc (p66ShcS36A), DATS did not induce ROS formation. In addition, in cells expressing p66ShcS36A neither an increase in ferritin H degradation nor an increase in LIP were observed. Cells stably expressing p66ShcS36A also possess higher levels of ferritin H compared to PC-3 cells transfected with an empty vector. Moreover, DATS-induced G2/M arrest is completely abrogated in cells expressing p66ShcS36A. Mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) or p66Shc knockout mouse have been used to evaluate if p66Shc involvement in DATS-induced signaling is cell specific. DATS induced G2/M arrest in WT MEFs but had no effect in the p66Shc^−/− cell line. Moreover, increases in LIP and ROS formation were significantly attenuated in p66Shc^−/− MEFs treated with DATS.     

Repression of ferritin expression modulates cell responsiveness to H-ras-induced growth

We assessed the role of the cell labile iron pool in mediating oncogene-induced cell proliferation via repression of ferritin expression. When HEK-293 cells, engineered to inducibly express either active (+) or dominant-negative (-) forms of the H-ras oncogene, were treated with antisense nucleotides to ferritin subunits they displayed (a) decreased ferritin levels, (b) increased labile iron pool and either (c) faster growth in cells induced to express H-Ras (+) or (d) recovery from growth retardation in dominant-negative H-Ras-induced cells. Our studies support the view that the role of down-modulation of ferritin expression by some oncogene-evoked proliferation proceeds via expansion of the cellular labile iron pool.     

Does the calcein-AM method assay the total cellular 'labile iron pool' or only a fraction of it?

The calcein-AM (calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular 'labile iron pool' (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting calcein does not significantly chelate iron at 相似文献   

The level of 8-oxo-7,8-dihydro-2′-deoxyguanosine is positively correlated with the size of the labile iron pool in human lymphocytes

It appears that the labile iron pool (LIP, low molecular weight iron) presence in cells can result in the production of reactive oxygen species (ROS). ROS may be responsible for the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in cellular DNA. In the present study we report on the relationship between LIP and the endogenous level of 8-oxodGuo in human lymphocytes. Good correlation has been determined between LIP and the oxidatively modified nucleoside. This in turn points out the possibility that under physiological condition there is the availability of LIP for catalyzing Fenton-type reactions in close proximity to cellular DNA. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0335-x.     

Detection of iron-containing proteins contributing to the cellular labile iron pool by a native electrophoresis metal blotting technique

The labile iron pool (LIP) plays a role in generation of free radicals and is thus the target of chelators used for the treatment of iron overload. We have previously shown that the LIP is bound mostly to high molecular weight carriers (MW>5000). However, the iron does not remain associated with these proteins during native gel electrophoresis. In this study we describe a new method to reconstruct the interaction of iron with iron-binding proteins. Proteins were separated by native gradient polyacrylamide gel electrophoresis and transfered to polyvinilidene difluoride membrane under native conditions. The immobilized iron-binding proteins are then labeled by 59Fe using a 'titrational blotting' technique and visualized by storage phosphorimaging. At least six proteins, in addition to ferritin and transferrin, are specifically labeled in cellular lysates of human erythroleukemic cells. This technique enables separation and detection of iron-binding proteins or other metal-protein complexes under near-physiological conditions and facilitates identification of weak iron-protein complexes. Using a new native metal blotting method, we have confirmed that specific high molecular weight proteins bind the labile iron pool.     

Glutathione depletion in immortalized midbrain-derived dopaminergic neurons results in increases in the labile iron pool: Implications for Parkinson's disease

Glutathione depletion is one of the earliest detectable events in the Parkinsonian substantia nigra (SN), but whether it is causative for ensuing molecular events associated with the disease is unknown. Here we report that reduction in levels of glutathione in immortalized midbrain-derived dopaminergic neurons results in increases in the cellular labile iron pool (LIP). This increase is independent of either iron regulatory protein/iron regulatory element (IRP/IRE) or hypoxia inducible factor (HIF) induction but is both H₂0₂ and protein synthesis-dependent. Our findings suggest a novel mechanistic link between dopaminergic glutathione depletion and increased iron levels based on translational activation of TfR1. This may have important implications for neurodegeneration associated with Parkinson's disease in which both glutathione reduction and iron elevation have been implicated.     

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Converging evidence leaves little doubt that a change in the conformation of prion protein (PrP^C) from a mainly α-helical to a β-sheet rich PrP-scrapie (PrP^Sc) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrP^Sc, nor the normal function of PrP^C is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrP^C mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrP^C increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrP^C on ferritin iron content is enhanced by stimulating PrP^C endocytosis, and reversed by cross-linking PrP^C on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP^102L decreases ferritin iron content significantly relative to PrP^C expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrP^C nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrP^C in cellular iron uptake and transport to ferritin, and dysfunction of PrP^C as a significant contributing factor of brain iron imbalance in prion disorders.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Overexpression of the ferritin iron-responsive element decreases the labile iron pool and abolishes the regulation of iron absorption by intestinal epithelial (Caco-2) cells

Mammalian cells regulate iron levels tightly through the activity of iron-regulatory proteins (IRPs) that bind to RNA motifs called iron-responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Likewise, intestinal epithelial cells regulate iron absorption by a process that also depends on the intracellular levels of iron. Although intestinal epithelial cells have an active IRE/IRP system, it has not been proven that this system is involved in the regulation of iron absorption in these cells. In this study, we characterized the effect of overexpression of the ferritin IRE on iron absorption by Caco-2 cells, a model of intestinal epithelial cells. Cells overexpressing ferritin IRE had increased levels of ferritin, whereas the levels of the transferrin receptor were decreased. Iron absorption in IRE-transfected cells was deregulated: iron uptake from the apical medium was increased, but the capacity to retain this newly incorporated iron diminished. Cells overexpressing IRE were not able to control iron absorption as a function of intracellular iron, because both iron-deficient cells as well as iron-loaded cells absorbed similarly high levels of iron. The labile iron pool of IRE-transfected cell was extremely low. Likewise, the reduction of the labile iron pool in control cells resulted in cells having increased iron absorption. These results indicate that cells overexpressing IRE do not regulate iron absorption, an effect associated with decreased levels of the regulatory iron pool.     

Role of intracellular labile iron,ferritin, and antioxidant defence in resistance of chronically adapted Jurkat T cells to hydrogen peroxide

To examine the role of intracellular labile iron pool (LIP), ferritin (Ft), and antioxidant defence in cellular resistance to oxidative stress on chronic adaptation, a new H₂O₂-resistant Jurkat T cell line “HJ16” was developed by gradual adaptation of parental “J16” cells to high concentrations of H₂O₂. Compared to J16 cells, HJ16 cells exhibited much higher resistance to H₂O₂-induced oxidative damage and necrotic cell death (up to 3 mM) and had enhanced antioxidant defence in the form of significantly higher intracellular glutathione and mitochondrial ferritin (FtMt) levels as well as higher glutathione-peroxidase (GPx) activity. In contrast, the level of the Ft H-subunit (FtH) in the H₂O₂-adapted cell line was found to be 7-fold lower than in the parental J16 cell line. While H₂O₂ concentrations higher than 0.1 mM fully depleted the glutathione content of J16 cells, in HJ16 cells the same treatments decreased the cellular glutathione content to only half of the original value. In HJ16 cells, H₂O₂ concentrations higher than 0.1 mM increased the level of FtMt up to 4-fold of their control values but had no effect on the FtMt levels in J16 cells. Furthermore, while the basal cytosolic level of LIP was similar in both cell lines, H₂O₂ treatment substantially increased the cytosolic LIP levels in J16 but not in HJ16 cells. H₂O₂ treatment also substantially decreased the FtH levels in J16 cells (up to 70% of the control value). In contrast in HJ16 cells, FtH levels were not affected by H₂O₂ treatment. These results indicate that chronic adaptation of J16 cells to high concentrations of H₂O₂ has provoked a series of novel and specific cellular adaptive responses that contribute to higher resistance of HJ16 cells to oxidative damage and cell death. These include increased cellular antioxidant defence in the form of higher glutathione and FtMt levels, higher GPx activity, and lower FtH levels. Further adaptive responses include the significantly reduced cellular response to oxidant-mediated glutathione depletion, FtH modulation, and labile iron release and a significant increase in FtMt levels following H₂O₂ treatment.     

Detection and quantitation of iron in ferritin,transferrin and labile iron pool (LIP) in cardiomyocytes using 55Fe and storage phosphorimaging

Dysregulated iron metabolism has a detrimental effect on cardiac function. The importance of iron homeostasis in cardiac health and disease warrants detailed studies of cardiomyocyte iron uptake, utilization and recycling at the molecular level. In this study, we have performed metabolic labeling of primary cultures of neonatal rat cardiomyocytes with radioactive iron coupled with separation of labeled iron-containing molecules by native electrophoresis followed by detection and quantification of incorporated radioiron by storage phosphorimaging. For the radiolabeling we used a safe and convenient beta emitter ⁵⁵Fe which enabled sensitive and simultaneous detection and quantitation of iron in cardiomyocyte ferritin, transferrin and the labile iron pool (LIP). The LIP is believed to represent potentially dangerous redox–active iron bound to uncharacterized molecules. Using size-exclusion chromatography spin micro columns, we demonstrate that iron in the LIP is bound to high molecular weight molecule(s) (≥5000?Da) in the neonatal cardiomyocytes.     

Induction of iron regulatory protein 1 RNA-binding activity by nitric oxide is associated with a concomitant increase in the labile iron pool: implications for DNA damage

Iron regulatory protein 1 (IRP1) is a bifunctional [4Fe-4S] protein that controls iron homeostasis. Switching off its function from an aconitase to an apo-IRP1 interacting with iron-responsive element-containing mRNAs depends on the reduced availability of iron in labile iron pool (LIP). Although the modulation of IRP1 by nitric oxide has been characterized, its impact on LIP remains unknown. Here, we show that inhibition of IRP1 aconitase activity and induction of its IRE-binding activity during exposure of L5178Y mouse lymphoma cells to NO are associated with an increase in LIP levels. Removal of NO resulted in a reverse regulation of IRP1 activities accompanied by a decrease of LIP. The increased iron burden in LIP caused by NO exacerbated hydrogen peroxide-induced genotoxicity in L5178Y cells. We demonstrate that the increase in LIP levels in response to chronic but not burst exposure of L5178Y cells to NO is associated with alterations in the expression of proteins involved in iron metabolism.