Physiology of iron transport and the hemochromatosis gene

Iron is essential for fundamental cell functions but is also a catalyst for chemical reactions involving free radical formation, potentially leading to oxidative stress and cell damage. Cellular iron levels are therefore carefully regulated to maintain an adequate substrate while also minimizing the pool of potentially toxic “free iron.” The main control of body iron homeostasis in higher organisms is placed in the duodenum, where dietary iron is absorbed, whereas no controlled means of eliminating unwanted iron have evolved in mammals. Hereditary hemochromatosis, the prototype of deregulated iron homeostasis in humans, is due to inappropriately increased iron absorption and is commonly associated to a mutated HFE gene. The HFE protein is homologous to major histocompatibility complex class I proteins but is not an iron carrier, whereas biochemical and cell biological studies have shown that the transferrin receptor, the main protein devoted to cellular uptake of transferrin iron, interacts with HFE. This review focuses on recent advances in iron research and presents a model of HFE function in iron metabolism.

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CBIO-10. REDUCED IRON EXPORT FUNCTIONS IN A CELL INTRINSIC MANNER TO DRIVE GLIOBLASTOMA GROWTH

Neuro-Oncology ◽

10.1093/neuonc/noaa215.070 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii17-ii17

Author(s):

Katie Troike ◽

Erin Mulkearns-Hubert ◽

Daniel Silver ◽

James Connor ◽

Justin Lathia

Keyword(s):

Tumor Cells ◽

Iron Uptake ◽

Iron Homeostasis ◽

Iron Status ◽

Tumor Grade ◽

Hfe Gene ◽

Iron Release ◽

Female Patients ◽

Cellular Iron

Abstract Glioblastoma (GBM), the most common primary malignant brain tumor in adults, is characterized by invasive growth and poor prognosis. Iron is a critical regulator of many cellular processes, and GBM tumor cells have been shown to modulate expression of iron-associated proteins to enhance iron uptake from the surrounding microenvironment, driving tumor initiation and growth. While iron uptake has been the central focus of previous investigations, additional mechanisms of iron regulation, such as compensatory iron efflux, have not been explored in the context of GBM. The hemochromatosis (HFE) gene encodes a transmembrane glycoprotein that aids in iron homeostasis by limiting cellular iron release, resulting in a sequestration phenotype. We find that HFE is upregulated in GBM tumors compared to non-tumor brain and that expression of HFE increases with tumor grade. Furthermore, HFE mRNA expression is associated with significantly reduced survival specifically in female patients with GBM. Based on these findings, we hypothesize that GBM tumor cells upregulate HFE expression to augment cellular iron loading and drive proliferation, ultimately leading to reduced survival of female patients. To test this hypothesis, we generated Hfe knockdown and overexpressing mouse glioma cell lines. We observed significant alterations in the expression of several iron handling genes with Hfe knockdown or overexpression, suggesting global disruption of iron homeostasis. Additionally, we show that knockdown of Hfe in these cells increases apoptosis and leads to a significant impairment of tumor growth in vivo. These findings support the hypothesis that Hfe is a critical regulator of cellular iron status and contributes to tumor aggression. Future work will include further exploration of the mechanisms that contribute to these phenotypes as well as interactions with the tumor microenvironment. Elucidating the mechanisms by which iron effulx contributes to GBM may inform the development of next-generation targeted therapies.

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Physiologic systemic iron metabolism in mice deficient for duodenal Hfe

Blood ◽

10.1182/blood-2006-07-036186 ◽

2007 ◽

Vol 109 (10) ◽

pp. 4511-4517 ◽

Cited By ~ 57

Author(s):

Maja Vujic Spasic ◽

Judit Kiss ◽

Thomas Herrmann ◽

Regina Kessler ◽

Jens Stolte ◽

...

Keyword(s):

Iron Metabolism ◽

Iron Homeostasis ◽

Iron Absorption ◽

Binding Capacity ◽

Hereditary Hemochromatosis ◽

Direct Interaction ◽

Hfe Gene ◽

Hepatic Iron ◽

Primary Role ◽

Genes Encoding

Abstract Mutations in the Hfe gene result in hereditary hemochromatosis (HH), a disorder characterized by increased duodenal iron absorption and tissue iron overload. Identification of a direct interaction between Hfe and transferrin receptor 1 in duodenal cells led to the hypothesis that the lack of functional Hfe in the duodenum affects TfR1-mediated serosal uptake of iron and misprogramming of the iron absorptive cells. Contrasting this view, Hfe deficiency causes inappropriately low expression of the hepatic iron hormone hepcidin, which causes increased duodenal iron absorption. We specifically ablated Hfe expression in mouse enterocytes using Cre/LoxP technology. Mice with efficient deletion of Hfe in crypt- and villi-enterocytes maintain physiologic iron metabolism with wild-type unsaturated iron binding capacity, hepatic iron levels, and hepcidin mRNA expression. Furthermore, the expression of genes encoding the major intestinal iron transporters is unchanged in duodenal Hfe-deficient mice. Our data demonstrate that intestinal Hfe is dispensable for the physiologic control of systemic iron homeostasis under steady state conditions. These findings exclude a primary role for duodenal Hfe in the pathogenesis of HH and support the model according to which Hfe is required for appropriate expression of the “iron hormone” hepcidin which then controls intestinal iron absorption.

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An improved structural model of the human iron exporter ferroportin. Insight into the role of pathogenic mutations in hereditary hemochromatosis type 4

Bio-Algorithms and Med-Systems ◽

10.1515/bams-2017-0029 ◽

2017 ◽

Vol 13 (4) ◽

Author(s):

Valentina Tortosa ◽

Maria Carmela Bonaccorsi di Patti ◽

Valentina Brandi ◽

Giovanni Musci ◽

Fabio Polticelli

Keyword(s):

Membrane Protein ◽

Crystal Structures ◽

Iron Homeostasis ◽

Structural Model ◽

Hereditary Hemochromatosis ◽

Structural Models ◽

Pivotal Role ◽

Cellular Iron ◽

Insight Into

AbstractFerroportin (Fpn) is a membrane protein representing the major cellular iron exporter, essential for metal translocation from cells into plasma. Despite its pivotal role in human iron homeostasis, many questions on Fpn structure and biology remain unanswered. In this work, we present two novel and more reliable structural models of human Fpn (hFpn; inward-facing and outward-facing conformations) obtained using as templates the recently solved crystal structures of a bacterial homologue of hFpn,

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

Blood ◽

10.1182/blood.v94.11.3915.423k27_3915_3921 ◽

1999 ◽

Vol 94 (11) ◽

pp. 3915-3921 ◽

Cited By ~ 2

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.

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Enhanced erythropoiesis in Hfe-KO mice indicates a role for Hfe in the modulation of erythroid iron homeostasis

Blood ◽

10.1182/blood-2010-09-307462 ◽

2011 ◽

Vol 117 (4) ◽

pp. 1379-1389 ◽

Cited By ~ 31

Author(s):

Pedro Ramos ◽

Ella Guy ◽

Nan Chen ◽

Catia C. Proenca ◽

Sara Gardenghi ◽

...

Keyword(s):

Iron Overload ◽

Iron Uptake ◽

Iron Homeostasis ◽

Hereditary Hemochromatosis ◽

Dietary Iron ◽

Erythroid Cells ◽

Hepcidin Expression ◽

Stress Erythropoiesis ◽

Cellular Iron ◽

Cellular Iron Uptake

Abstract In hereditary hemochromatosis, mutations in HFE lead to iron overload through abnormally low levels of hepcidin. In addition, HFE potentially modulates cellular iron uptake by interacting with transferrin receptor, a crucial protein during erythropoiesis. However, the role of HFE in this process was never explored. We hypothesize that HFE modulates erythropoiesis by affecting dietary iron absorption and erythroid iron intake. To investigate this, we used Hfe-KO mice in conditions of altered dietary iron and erythropoiesis. We show that Hfe-KO mice can overcome phlebotomy-induced anemia more rapidly than wild-type mice (even when iron loaded). Second, we evaluated mice combining the hemochromatosis and β-thalassemia phenotypes. Our results suggest that lack of Hfe is advantageous in conditions of increased erythropoietic activity because of augmented iron mobilization driven by deficient hepcidin response. Lastly, we demonstrate that Hfe is expressed in erythroid cells and impairs iron uptake, whereas its absence exclusively from the hematopoietic compartment is sufficient to accelerate recovery from phlebotomy. In summary, we demonstrate that Hfe influences erythropoiesis by 2 distinct mechanisms: limiting hepcidin expression under conditions of simultaneous iron overload and stress erythropoiesis, and impairing transferrin-bound iron uptake by erythroid cells. Moreover, our results provide novel suggestions to improve the treatment of hemochromatosis.

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Complete Scanning of the Hereditary Hemochromatosis Gene (HFE) by Use of Denaturing HPLC

Clinical Chemistry ◽

10.1093/clinchem/47.9.1633 ◽

2001 ◽

Vol 47 (9) ◽

pp. 1633-1640 ◽

Cited By ~ 25

Author(s):

Gerald Le Gac ◽

Catherine Mura ◽

Claude Férec

Keyword(s):

Hereditary Hemochromatosis ◽

Hfe Gene ◽

Test Accuracy ◽

Melting Profile ◽

Coding Region ◽

Hemochromatosis Gene ◽

Hfe Mutations ◽

Methods Analytical ◽

Positive Controls ◽

Experimental Melting

Abstract Background: Between 4% and 35% of hereditary hemochromatosis (HC) probands are C282Y or H63D heterozygotes or lack both of these two common HFE mutations, and 15 novel HFE mutations have been described recently. We evaluated denaturing HPLC (DHPLC) for screening of the whole HFE coding region and further defined whether HC probands with an incomplete HFE genotype carry uncommon mutations. Methods: Analytical conditions for each coding exon were determined by a combination of computer melting profile predictions and experimental melting curves. To test accuracy for scanning the complete HFE coding region and optimize DHPLC running conditions, each melting domain was investigated with at least one mutation or one polymorphism as reference. We tested 100 DNA samples harboring the C282Y, H63D, or S65C mutations and 17 artificially created positive controls that carried either 1 of the 14 other known HFE mutations or 3 selected polymorphisms. Results: Investigations on each of the coding exons 1, 2, 4, 5, and 6 could be performed at one analysis temperature. Coding exon 3 displayed a more complex melting profile and required two analysis temperatures. DHPLC detected all known HFE mutations as well as the three selected polymorphisms. Conclusions: DHPLC can be used to scan the HFE gene in HC probands in whom at least one chromosome lacks an assigned mutation.

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Expression of hepcidin is down-regulated in TfR2 mutant mice manifesting a phenotype of hereditary hemochromatosis

Blood ◽

10.1182/blood-2004-04-1416 ◽

2005 ◽

Vol 105 (1) ◽

pp. 376-381 ◽

Cited By ~ 155

Author(s):

Hiroshi Kawabata ◽

Robert E. Fleming ◽

Dorina Gui ◽

Seo Y. Moon ◽

Takayuki Saitoh ◽

...

Keyword(s):

Iron Homeostasis ◽

Hereditary Hemochromatosis ◽

Isolated Hepatocytes ◽

Mutant Mice ◽

Wild Type ◽

Hepcidin Expression ◽

Cellular Iron ◽

Inflammatory Stimuli ◽

Cellular Iron Uptake ◽

Hepcidin Mrna

Abstract Transferrin receptor 2 (TfR2) is a membrane glycoprotein that mediates cellular iron uptake from holotransferrin. Homozygous mutations of this gene cause one form of hereditary hemochromatosis in humans. We recently reported that homozygous TfR2(Y245X) mutant mice, which correspond to the TfR2(Y250X) mutation in humans, showed a phenotype similar to hereditary hemochromatosis. In this study, we further analyzed the phenotype as well as iron-related gene expression in these mice by comparing the TfR2-mutant and wild-type siblings. Northern blot analyses showed that the levels of expression of hepcidin mRNA in the liver were generally lower, whereas those of duodenal DMT1, the main transporter for uptake of dietary iron, were higher in the TfR2-mutant mice as compared to the wild-type siblings. Expression of hepcidin mRNA in the TfR2 mutant mice remained low even after intraperitoneal iron loading. In isolated hepatocytes from both wild-type and TfR2 mutant mice, interleukin-6 and lipopolysaccharide each induced expression of hepcidin mRNA. These results suggest that up-regulation of hepcidin expression by inflammatory stimuli is independent of TfR2 and that TfR2 is upstream of hepcidin in the regulatory pathway of body iron homeostasis. (Blood. 2005;105:376-381)

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Co-localization of the Mammalian Hemochromatosis Gene Product (HFE) and a Newly Identified Transferrin Receptor (TfR2) in Intestinal Tissue and Cells

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540305100507 ◽

2003 ◽

Vol 51 (5) ◽

pp. 613-623 ◽

Cited By ~ 36

Author(s):

William J.H. Griffiths ◽

Timothy M. Cox

Keyword(s):

Transferrin Receptor ◽

Iron Homeostasis ◽

Receptor Gene ◽

Hfe Gene ◽

Transport Pathway ◽

Intestinal Crypt ◽

Hemochromatosis Gene ◽

Small Intestinal ◽

Potential Interactions ◽

Crypt Cells

Mutations in the HFE gene and a newly identified second transferrin receptor gene, TfR2, cause hemochromatosis. The cognate proteins, HFE and TfR2, are therefore of key importance in human iron homeostasis. HFE is expressed in small intestinal crypt cells where transferrin-iron entry may determine subsequent iron absorption by mature enterocytes, but the physiological function of TfR2 is unknown. Using specific peptide antisera, we examined the duodenal localization of HFE and TfR2 in humans and mice, with and without HFE deficiency, by confocal microscopy. We also investigated potential interactions of these proteins in human intestinal cells in situ. Duodenal expression of HFE and TfR2 (but not TfR1) in wild-type mice and humans was restricted to crypt cells, in which they co-localized. HFE deficiency disrupted this interaction, altering the cellular distribution of TfR2 in human crypts. In human Caco-2 cells, HFE and TfR2 co-localized to a distinct CD63-negative vesicular compartment showing marked signal enhancement on exposure to iron-saturated transferrin ligand, indicating that HFE preferentially interacts with TfR2 in a specialized early endosomal transport pathway for transferrin-iron. This interaction occurs specifically in small intestinal crypt cells that differentiate to become iron-absorbing enterocytes. Our immunohistochemical findings provide evidence for a novel mechanism for the regulation of iron balance in mammals.

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Heterotypic interactions between transferrin receptor and transferrin receptor 2

Blood ◽

10.1182/blood-2002-09-2742 ◽

2003 ◽

Vol 101 (5) ◽

pp. 2008-2014 ◽

Cited By ~ 31

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.

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Frequency of the Hemochromatosis Gene (HFE) Variants in a Jordanian Arab Population and in Diabetics from the Same Region

Disease Markers ◽

10.1155/2009/329603 ◽

2009 ◽

Vol 27 (1) ◽

pp. 17-22 ◽

Cited By ~ 6

Author(s):

Asem Alkhateeb ◽

Amal Uzrail ◽

Khaldon Bodoor

Keyword(s):

Blood Donor ◽

Hereditary Hemochromatosis ◽

University Hospital ◽

European Ancestry ◽

Hfe Gene ◽

Arab Population ◽

Population Type ◽

Significant Difference ◽

Hemochromatosis Gene

Hereditary HFE-linked hemochromatosis is a frequent recessive disorder among individuals of northern European ancestry. The clinical characteristic of this disease is the gradual accumulation of iron in internal organs, which ultimately may lead to organ damage and death. Three allelic variants of HFE gene have been correlated with hereditary hemochromatosis: C282Y is significantly associated with hereditary hemochromatosis in populations of Celtic origin, H63D and S65C are associated with milder form of iron overload. In this study we performed mutation analysis to identify allele frequency of the three variants of HFE gene in Jordanian Arab population, to assess deviations of these frequencies from those detected elsewhere, and to determine if there is an increased frequency of these variants in a diabetic population (Type 2 diabetes) from the same area. DNA was extracted from blood samples of 440 individuals attending King Abdullah University Hospital for ambulatory services. We used polymerase chain reaction (PCR) to amplify exons 2 and 4 of the HFE gene then restriction fragment length polymorphism (RFLP) method to detect the variants. There were neither homozygous nor heterozygous for C282Y variant. For the H63D variant, 0.68% were homozygous and 21.1% were heterozygous. For the S65C variant, there were no homozygous and 0.23% were heterozygous. Allelic frequencies were, 0%, 11.25%, and 0.11% for C282Y, H63D, and S65C, respectively. Our samples were subdivided into two categories of type 2 diabetic (89 cases) and controls (blood donors, 204 cases) and compared with regard to the H63D variant. Both groups did not have homozygous H63D variant. H63D heterozygous in diabetics were 23.60% and in blood donor controls 22.55%. Allelic frequency of the mutant H63D allele was 11.80% in diabetics and 11.27% for the blood donor controls. This is the first study to show the frequency of the three hemochromatosis gene variants in Jordan with the interesting finding of no C282Y allele detected in 440 samples. Additionally, no significant difference was observed in H63D variant frequency in type 2 diabetics as compared to controls.

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