HFE Downregulates Iron Uptake From Transferrin and Induces Iron-Regulatory Protein Activity in Stably Transfected Cells

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3915-3921 ◽  
Author(s):  
H.D. Riedel ◽  
M.U. Muckenthaler ◽  
S.G. Gehrke ◽  
I. Mohr ◽  
K. Brennan ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Iron Uptake ◽  
Iron Homeostasis ◽  
Hereditary Hemochromatosis ◽  
Regulatory Protein ◽  
Intracellular Iron ◽  
Iron Storage ◽  
Iron Regulatory Proteins ◽  
Cellular Iron

Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular “labile iron pool.” The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.

HFE Downregulates Iron Uptake From Transferrin and Induces Iron-Regulatory Protein Activity in Stably Transfected Cells

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3915-3921 ◽  
Author(s):  
H.D. Riedel ◽  
M.U. Muckenthaler ◽  
S.G. Gehrke ◽  
I. Mohr ◽  
K. Brennan ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Iron Uptake ◽  
Iron Homeostasis ◽  
Hereditary Hemochromatosis ◽  
Regulatory Protein ◽  
Intracellular Iron ◽  
Iron Storage ◽  
Iron Regulatory Proteins ◽  
Cellular Iron

Abstract Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular “labile iron pool.” The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.

scholarly journals Nitric Oxide–Mediated Induction of Ferritin Synthesis in J774 Macrophages by Inflammatory Cytokines: Role of Selective Iron Regulatory Protein-2 Downregulation

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 1059-1066 ◽  
Author(s):  
Stefania Recalcati ◽  
Donatella Taramelli ◽  
Dario Conte ◽  
Gaetano Cairo
Keyword(s):  
Nitric Oxide ◽  
Posttranscriptional Regulation ◽  
Iron Homeostasis ◽  
Regulatory Protein ◽  
Intracellular Iron ◽  
Iron Regulatory Proteins ◽  
Ferritin Synthesis ◽  
Direct Demonstration ◽  
J774 Cells

Cytokine-treated macrophages represent a useful model to unravel the molecular basis of reticuloendothelial (RE) iron retention in inflammatory conditions. In the present study, we showed that stimulation of murine macrophage J774 cells with interferon (IFN)-γ/lipopolysaccharide (LPS) resulted in a nitric oxide-dependent modulation of the activity of iron regulatory proteins (IRP)-1 and 2, cytoplasmic proteins which, binding to RNA motifs called iron responsive elements (IRE), control ferritin translation. Stimulation with cytokines caused a small increase of IRP-1 activity and a strong reduction of IRP-2 activity accompanied by increased ferritin synthesis and accumulation. Cytokines induced only a minor increase of H chain ferritin mRNA, thus indicating that IRP-2–mediated posttranscriptional regulation plays a major role in the control of ferritin expression. This was confirmed by direct demonstration that the translational repression function of IRP was impaired in stimulated cells. In fact, translation in cell-free extracts of a reporter transcript under the control of an IRE sequence was repressed less efficiently by IRP-containing lysates from cytokine-treated cells than by lysates from control cells. Our findings throw light on the role of IRP-2 showing that: (1) this protein responds to a stimulus in opposite fashion to IRP-1; (2) when abundantly expressed, as in J774 cells, IRP-2 is sufficient to regulate intracellular iron metabolism in living cells; and (3) by allowing increased ferritin synthesis, IRP-2 may play a role in the regulation of iron homeostasis in RE cells during inflammation.

scholarly journals Crucial function of vertebrate glutaredoxin 3 (PICOT) in iron homeostasis and hemoglobin maturation

2013 ◽  
Vol 24 (12) ◽  
pp. 1895-1903 ◽  
Author(s):  
Petra Haunhorst ◽  
Eva-Maria Hanschmann ◽  
Lars Bräutigam ◽  
Oliver Stehling ◽  
Bastian Hoffmann ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Iron Homeostasis ◽  
Regulatory Protein ◽  
Iron Regulation ◽  
Iron Starvation ◽  
Intracellular Iron ◽  
Cellular Iron ◽  
Cellular Iron Uptake ◽  
S Proteins

The mechanisms by which eukaryotic cells handle and distribute the essential micronutrient iron within the cytosol and other cellular compartments are only beginning to emerge. The yeast monothiol multidomain glutaredoxins (Grx) 3 and 4 are essential for both transcriptional iron regulation and intracellular iron distribution. Despite the fact that the mechanisms of iron metabolism differ drastically in fungi and higher eukaryotes, the glutaredoxins are conserved, yet their precise function in vertebrates has remained elusive. Here we demonstrate a crucial role of the vertebrate-specific monothiol multidomain Grx3 (PICOT) in cellular iron homeostasis. During zebrafish embryonic development, depletion of Grx3 severely impairs the maturation of hemoglobin, the major iron-consuming process. Silencing of human Grx3 expression in HeLa cells decreases the activities of several cytosolic Fe/S proteins, for example, iron-regulatory protein 1, a major component of posttranscriptional iron regulation. As a consequence, Grx3-depleted cells show decreased levels of ferritin and increased levels of transferrin receptor, features characteristic of cellular iron starvation. Apparently, Grx3-deficient cells are unable to efficiently use iron, despite unimpaired cellular iron uptake. These data suggest an evolutionarily conserved role of cytosolic monothiol multidomain glutaredoxins in cellular iron metabolism pathways, including the biogenesis of Fe/S proteins and hemoglobin maturation.

The hereditary hemochromatosis protein, HFE, lowers intracellular iron levels independently of transferrin receptor 1 in TRVb cells

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2564-2570 ◽  
Author(s):  
Hanqian Carlson ◽  
An-Sheng Zhang ◽  
William H. Fleming ◽  
Caroline A. Enns
Keyword(s):  
Transferrin Receptor ◽  
Iron Homeostasis ◽  
Hereditary Hemochromatosis ◽  
Cell Types ◽  
Single Amino Acid ◽  
Intracellular Iron ◽  
Iron Storage ◽  
Transferrin Receptor 1 ◽  
A Cell

AbstractHereditary hemochromatosis (HH) is an autosomal recessive disease that leads to parenchymal iron accumulation. The most common form of HH is caused by a single amino acid substitution in the HH protein, HFE, but the mechanism by which HFE regulates iron homeostasis is not known. In the absence of transferrin (Tf), HFE interacts with transferrin receptor 1 (TfR1) and the 2 proteins co-internalize, and in vitro studies have shown that HFE and Tf compete for TfR1 binding. Using a cell line lacking endogenous transferrin receptors (TRVb cells) transfected with different forms of HFE and TfR1, we demonstrate that even at low concentrations Tf competes effectively with HFE for binding to TfR1 on living cells. Transfection of TRVb cells or the derivative line TRVb1 (which stably expresses human TfR1) with HFE resulted in lower ferritin levels and decreased Fe2+ uptake. These data indicate that HFE can regulate intracellular iron storage independently of its interaction with TfR1. Earlier studies found that in HeLa cells, HFE expression lowers Tf-mediated iron uptake; here we show that HFE lowers non–Tf-bound iron in TRVb cells and add to a growing body of evidence that HFE may play different roles in different cell types.

scholarly journals Heterotypic interactions between transferrin receptor and transferrin receptor 2

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 2008-2014 ◽  
Author(s):  
Todd M. Vogt ◽  
Aaron D. Blackwell ◽  
Anthony M. Giannetti ◽  
Pamela J. Bjorkman ◽  
Caroline A. Enns
Keyword(s):  
Transferrin Receptor ◽  
Iron Uptake ◽  
Iron Homeostasis ◽  
Critical Role ◽  
Hereditary Hemochromatosis ◽  
Metabolic Labeling ◽  
Western Blots ◽  
Cellular Iron ◽  
Transferrin Receptor 2

Cellular iron uptake in most tissues occurs via endocytosis of diferric transferrin (Tf) bound to the transferrin receptor (TfR). Recently, a second transferrin receptor, transferrin receptor 2 (TfR2), has been identified and shown to play a critical role in iron metabolism. TfR2 is capable of Tf-mediated iron uptake and mutations in this gene result in a rare form of hereditary hemochromatosis unrelated to the hereditary hemochromatosis protein, HFE. Unlike TfR, TfR2 expression is not controlled by cellular iron concentrations and little information is currently available regarding the role of TfR2 in cellular iron homeostasis. To investigate the relationship between TfR and TfR2, we performed a series of in vivo and in vitro experiments using antibodies generated to each receptor. Western blots demonstrate that TfR2 protein is expressed strongest in erythroid/myeloid cell lines. Metabolic labeling studies indicate that TfR2 protein levels are approximately 20-fold lower than TfR in these cells. TfR and TfR2 have similar cellular localizations in K562 cells and coimmunoprecipitate to only a very limited extent. Western analysis of the receptors under nonreducing conditions reveals that they can form heterodimers.

scholarly journals Nitric Oxide–Mediated Induction of Ferritin Synthesis in J774 Macrophages by Inflammatory Cytokines: Role of Selective Iron Regulatory Protein-2 Downregulation

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 1059-1066 ◽  
Author(s):  
Stefania Recalcati ◽  
Donatella Taramelli ◽  
Dario Conte ◽  
Gaetano Cairo
Keyword(s):  
Nitric Oxide ◽  
Posttranscriptional Regulation ◽  
Iron Homeostasis ◽  
Regulatory Protein ◽  
Intracellular Iron ◽  
Iron Regulatory Proteins ◽  
Ferritin Synthesis ◽  
Direct Demonstration ◽  
J774 Cells

Abstract Cytokine-treated macrophages represent a useful model to unravel the molecular basis of reticuloendothelial (RE) iron retention in inflammatory conditions. In the present study, we showed that stimulation of murine macrophage J774 cells with interferon (IFN)-γ/lipopolysaccharide (LPS) resulted in a nitric oxide-dependent modulation of the activity of iron regulatory proteins (IRP)-1 and 2, cytoplasmic proteins which, binding to RNA motifs called iron responsive elements (IRE), control ferritin translation. Stimulation with cytokines caused a small increase of IRP-1 activity and a strong reduction of IRP-2 activity accompanied by increased ferritin synthesis and accumulation. Cytokines induced only a minor increase of H chain ferritin mRNA, thus indicating that IRP-2–mediated posttranscriptional regulation plays a major role in the control of ferritin expression. This was confirmed by direct demonstration that the translational repression function of IRP was impaired in stimulated cells. In fact, translation in cell-free extracts of a reporter transcript under the control of an IRE sequence was repressed less efficiently by IRP-containing lysates from cytokine-treated cells than by lysates from control cells. Our findings throw light on the role of IRP-2 showing that: (1) this protein responds to a stimulus in opposite fashion to IRP-1; (2) when abundantly expressed, as in J774 cells, IRP-2 is sufficient to regulate intracellular iron metabolism in living cells; and (3) by allowing increased ferritin synthesis, IRP-2 may play a role in the regulation of iron homeostasis in RE cells during inflammation.

scholarly journals Effect of nitric oxide on expression of transferrin receptor and ferritin and on cellular iron metabolism in K562 human erythroleukemia cells

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2962-2966 ◽  
Author(s):  
R Oria ◽  
L Sanchez ◽  
T Houston ◽  
MW Hentze ◽  
FY Liew ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Iron Metabolism ◽  
Transferrin Receptor ◽  
Regulatory Protein ◽  
Inhibitory Effect ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Intracellular Iron ◽  
Cellular Iron ◽  
Erythroleukemia Cells

Nitric oxide (NO) is known to increase the affinity of the intracellular iron-regulatory protein (IRP) for iron-response elements (IREs) in transferrin receptor and ferritin mRNAs, suggesting that it may act as a regulator of cellular iron metabolism. In this study, exogenous NO produced by adding the NO-generator S-nitroso-N-acetyl penicillamine gave a dose-dependent upregulation of transferrin receptor expression by K562 erythroleukemia cells and increased levels of transferrin receptor mRNA. NO did not affect the affinity of transferrin binding by the transferrin receptor. NO alone did not alter intracellular ferritin levels, but it did abrogate the inhibitory effect of the iron chelator desferrioxamine and potentiated the stimulatory effect of additional iron. NO also caused some increase in ferritin mRNA levels, which might mask any IRP-/IRE-mediated inhibitory effect of NO on ferritin translation. Although NO did not affect net iron uptake, it increased release of iron from K562 cells pulsed previously with 59Fe, and subcellular fractionation showed that it also increased the proportion of intracellular iron bound to ferritin. These findings provide direct evidence that NO can affect cellular iron metabolism and suggest that NO produced in vivo by activated bone marrow macrophages might affect erythropoiesis.

Overexpression of mitochondrial ferritin causes cytosolic iron depletion and changes cellular iron homeostasis

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 2161-2167 ◽  
Author(s):  
Guangjun Nie ◽  
Alex D. Sheftel ◽  
Sangwon F. Kim ◽  
Prem Ponka
Keyword(s):  
Iron Homeostasis ◽  
Rna Binding ◽  
Binding Activity ◽  
Intracellular Iron ◽  
Free Radical Damage ◽  
Iron Regulatory Proteins ◽  
Cellular Iron ◽  
Cellular Iron Uptake ◽  
A Cell ◽  
Cellular Iron Homeostasis

AbstractCytosolic ferritin sequesters and stores iron and, consequently, protects cells against iron-mediated free radical damage. However, the function of the newly discovered mitochondrial ferritin (MtFt) is unknown. To examine the role of MtFt in cellular iron metabolism, we established a cell line that stably overexpresses mouse MtFt under the control of a tetracycline-responsive promoter. The overexpression of MtFt caused a dose-dependent iron deficiency in the cytosol that was revealed by increased RNA-binding activity of iron regulatory proteins (IRPs) along with an increase in transferrin receptor levels and decrease in cytosolic ferritin. Consequently, the induction of MtFt resulted in a dramatic increase in cellular iron uptake from transferrin, most of which was incorporated into MtFt. The induction of MtFt caused a shift of iron from cytosolic ferritin to MtFt. In addition, iron inserted into MtFt was less available for chelation than that in cytosolic ferritin and the expression of MtFt was associated with decreased mitochondrial and cytosolic aconitase activities, the latter being consistent with the increase in IRP-binding activity. In conclusion, our results indicate that overexpression of MtFt causes a dramatic change in intracellular iron homeostasis and that shunting iron to MtFt likely limits its availability for active iron proteins.

Iron homeostasis: new tales from the crypt

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4020-4027 ◽  
Author(s):  
Cindy N. Roy ◽  
Caroline A. Enns
Keyword(s):  
Iron Uptake ◽  
Iron Transport ◽  
Iron Homeostasis ◽  
Molecular Mechanisms ◽  
Hereditary Hemochromatosis ◽  
The Body ◽  
Regulatory Proteins ◽  
Iron Regulatory Proteins ◽  
Duodenal Epithelium ◽  
Related Proteins

Abstract The enterocyte is a highly specialized cell of the duodenal epithelium that coordinates iron uptake and transport into the body. Until recently, the molecular mechanisms underlying iron absorption and iron homeostasis have remained a mystery. This review focuses on the proteins and regulatory mechanisms known to be present in the enterocyte precursor cell and in the mature enterocyte. The recent cloning of a basolateral iron transporter and investigations into its regulation provide new insights into possible mechanisms for iron transport and homeostasis. The roles of proteins such as iron regulatory proteins, the hereditary hemochromatosis protein (HFE)–transferrin receptor complex, and hephaestin in regulating this transporter and in regulating iron transport across the intestinal epithelium are discussed. A speculative, but testable, model for the maintenance of iron homeostasis, which incorporates the changes in the iron-related proteins associated with the life cycle of the enterocyte as it journeys from the crypt to the tip of the villous is proposed.

The Zebrafish Hypochromic Mutant [iItalic]Shiraz[/iItalic] Encodes a Novel Mitochondrial Glutaredoxin That Establishes a Link between Heme and Fe/S Production.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 51-51
Author(s):  
Rebecca A. Wingert ◽  
Bruce Barut ◽  
Helen Foott ◽  
Paula Fraenkel ◽  
Kimberly Dooley ◽  
...  
Keyword(s):  
Positional Cloning ◽  
Iron Homeostasis ◽  
Rna Binding ◽  
Fetal Liver ◽  
Regulatory Protein ◽  
Cluster Assembly ◽  
Hemoglobin Synthesis ◽  
Cellular Iron ◽  
Hemoglobin Production

Abstract Iron is required in the mitochondria both to produce heme, which is used for hemoglobin synthesis, and to make iron-sulfur (Fe/S) clusters, which confer electron transfer or catalytic functions to proteins. Cellular iron utilization and Fe/S cluster production are thought to occur independently, yet the processes are coordinated through currently uncharacterized pathways. The shiraz (sir) zebrafish mutant manifests a hypochromic, microcytic anemia. Positional cloning of sir discovered a deletion at the locus that included the zebrafish orthologue to glutaredoxin 5 (grx5), a gene required in yeast for Fe/S cluster assembly. We found that grx5 is highly expressed in the developing blood and fetal liver of both zebrafish and mouse embryos. Antisense-mediated morpholino knockdown of grx5 prevented hemoglobin production, and overexpression of zebrafish, yeast, mouse, or human grx5 RNA in sir embryos completely rescued hemoglobin production, indicating that grx5 is the gene responsible for the sir phenotype. Expression of zebrafish grx5 was found to rescue Fe/S protein production in the yeast Δgrx5 strain, demonstrating that the role of grx5 in Fe/S cluster assembly is conserved among eukaryotes. The surprising finding that mutating a gene necessary for Fe/S cluster assembly caused a lack of hemoglobin synthesis suggested that we had discovered a connection between these pathways. In vertebrates, iron regulatory protein 1 (IRP1) acts as a sensor of intracellular iron levels and controls cellular iron homeostasis via posttranscriptional regulation of iron uptake, storage, and utilization genes. For instance, IRP1 binds to the 5′ iron response element (IRE) in the aminolevulinate synthase 2 (ALAS2) mRNA, blocking translation when cellular iron is low. However, when cellular iron is replete, IRP1 binds a Fe/S cluster and its RNA-binding activity is abolished. We hypothesized that the loss of Fe/S cluster assembly in sir would activate IRP1 and block ALAS2 synthesis, resulting in hypochromia. In support of this model, overexpression of ALAS2 RNA without the 5′ IRE rescued sir hypochromia, while overexpression of ALAS2 including the IRE did not facilitate rescue. Furthermore, antisense morpholino knockdowns of IRP1 caused rescue of hemoglobin synthesis in sir embryos. The combination of these data indicate that hemoglobin production in the differentiating red cell is monitored through Fe-S cluster assembly as a mechanism to gauge iron levels and accordingly direct heme synthesis. This finding illustrates a crucial role for the mitochondrial Fe/S cluster assembly machinery during hemoglobin production, and has broad implications for the role of such genes in human hypochromic anemias.

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