Hepcidin in iron overload disorders

Abstract Hepcidin is the principal regulator of iron absorption in humans. The peptide inhibits cellular iron efflux by binding to the iron export channel ferroportin and inducing its internalization and degradation. Either hepcidin deficiency or alterations in its target, ferroportin, would be expected to result in dysregulated iron absorption, tissue maldistribution of iron, and iron overload. Indeed, hepcidin deficiency has been reported in hereditary hemochromatosis and attributed to mutations in HFE, transferrin receptor 2, hemojuvelin, and the hepcidin gene itself. We measured urinary hepcidin in patients with other genetic causes of iron overload. Hepcidin was found to be suppressed in patients with thalassemia syndromes and congenital dyserythropoietic anemia type 1 and was undetectable in patients with juvenile hemochromatosis with HAMP mutations. Of interest, urine hepcidin levels were significantly elevated in 2 patients with hemochromatosis type 4. These findings extend the spectrum of iron disorders with hepcidin deficiency and underscore the critical importance of the hepcidin–ferroportin interaction in iron homeostasis.

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An Essential Role For Transferrin Receptor 2 In Erythropoiesis During Iron Restriction

Blood ◽

10.1182/blood.v122.21.429.429 ◽

2013 ◽

Vol 122 (21) ◽

pp. 429-429

Author(s):

Daniel F Wallace ◽

Cameron J McDonald ◽

Eriza S Secondes ◽

Lesa Ostini ◽

Gautam Rishi ◽

...

Keyword(s):

Iron Deficiency ◽

Iron Overload ◽

Iron Deficiency Anemia ◽

Transferrin Receptor ◽

Iron Homeostasis ◽

Hereditary Hemochromatosis ◽

Deficiency Anemia ◽

Erythroid Cells ◽

Iron Restriction ◽

Extramedullary Erythropoiesis

Abstract Iron deficiency and iron overload are common clinical conditions that impact on the health and wellbeing of up to 30% of the world’s population. Understanding mechanisms regulating iron homeostasis will provide improved strategies for treating these disorders. The liver-expressed proteins matriptase-2 (encoded by TMPRSS6), HFE and transferrin receptor 2 (TFR2) play important and opposing roles in systemic iron homeostasis by regulating expression of the iron regulatory hormone hepcidin. Mutations in TMPRSS6 lead to iron refractory iron deficiency anemia, whereas mutations in HFE and TFR2 lead to the iron overload disorder hereditary hemochromatosis. To elucidate the competing roles of these hepcidin regulators, we created mice lacking matriptase-2, Hfe and Tfr2. Tmprss6 -/-/Hfe-/-/Tfr2-/- mice had iron deficiency anemia resulting from hepatic hepcidin over-expression and activation of Smad1/5/8, indicating that matriptase-2 predominates over Hfe and Tfr2 in hepcidin regulation. Surprisingly, this anemia was more severe than in the Tmprss6-/- mice, demonstrated by more extensive alopecia, lower hematocrit and significant extramedullary erythropoiesis in the spleen. There was increased expression of erythroid-specific genes in the spleens of Tmprss6-/-/Hfe-/-/Tfr2-/- mice, consistent with the extramedullary erythropoiesis. Expression of Tfr2 but not Hfe in the spleen was increased in the Tmprss6-/- mice compared to wild type and correlated with the expression of erythroid genes, suggesting that Tfr2 is expressed in erythroid cells. Further analysis of gene expression in the bone marrow suggests that the loss of Tfr2 in the erythroid cells of Tmprss6-/-/Hfe-/-/Tfr2-/- mice causes a delay in the differentiation process leading to a more severe phenotype. In conclusion, our results indicate that Hfe and Tfr2 act upstream of matriptase-2 in hepcidin regulation or in a way that is overridden when matriptase-2 is deleted. These results indicate that inhibition of matriptase-2 would be useful in the treatment of iron overload conditions such as hereditary hemochromatosis. We have also identified a novel role for Tfr2 in erythroid differentiation that is separate from its canonical role as a regulator of iron homeostasis in the liver. This important role of Tfr2 in erythropoiesis only becomes apparent during conditions of iron restriction. Our results provide novel insights into mechanisms regulating and linking iron homeostasis and erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

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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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The Effect of the Flavonol Rutin on Serum and Liver Iron Content in a Genetic Mouse Model of Iron Overload

Bioscience Reports ◽

10.1042/bsr20210720 ◽

2021 ◽

Author(s):

Zachary Hawula ◽

Eriza Secondes ◽

Daniel Wallace ◽

Gautam Rishi ◽

V. Nathan Subramaniam

Keyword(s):

Mouse Model ◽

Iron Overload ◽

Transferrin Receptor ◽

Iron Homeostasis ◽

Binding Capacity ◽

Hereditary Hemochromatosis ◽

Transferrin Saturation ◽

Iron Loading ◽

Genetic Mouse Model ◽

Transferrin Receptor 2

The flavonol rutin has been shown to possess antioxidant and iron chelating properties in vitro and in vivo. These dual properties are beneficial as therapeutic options to reduce iron accumulation and the generation of reactive oxygen species resultant from excess free iron. The effect of rutin on iron metabolism has been limited to studies performed in wild type mice either injected or fed high iron diets. The effect of rutin on iron overload caused by genetic dysregulation of iron homeostasis has not yet been investigated. In this study we examined the effect of rutin treatment on tissue iron loading in a genetic mouse model of iron overload, which mirrors the iron loading associated with Type 3 hereditary hemochromatosis patients who have a defect in Transferrin Receptor 2. Male Transferrin Receptor 2 knockout mice were administered rutin via oral gavage for 21 continuous days. Following treatment, iron levels in serum, liver, duodenum, and spleen were assessed. In addition, hepatic ferritin protein levels were determined by western blotting, and expression of iron homeostasis genes by quantitative real-time PCR. Rutin treatment resulted in a significant reduction in hepatic ferritin protein expression and serum transferrin saturation. In addition, trends towards decreased iron levels in the liver and serum, and increased serum unsaturated iron binding capacity were observed. This is the first study to explore the utility of rutin as a potential iron chelator and therapeutic in an animal model of genetic iron overload.

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Defective palmitoylation of transferrin receptor triggers iron overload in Friedreich's ataxia fibroblasts

Blood ◽

10.1182/blood.2020006987 ◽

2021 ◽

Author(s):

Floriane Petit ◽

Anthony Drecourt ◽

Michaël Dussiot ◽

Coralie Zangarelli ◽

Olivier Hermine ◽

...

Keyword(s):

Iron Overload ◽

Transferrin Receptor ◽

Iron Homeostasis ◽

Mitochondrial Protein ◽

Friedreich’S Ataxia ◽

Friedreich's Ataxia ◽

Gene Encoding ◽

Iron Sulfur Cluster ◽

The Road ◽

Cellular Iron

Friedreich's ataxia (FRDA) is a frequent autosomal recessive disease caused by a GAA repeat expansion in the FXN gene encoding frataxin, a mitochondrial protein involved in iron-sulfur cluster (ISC) biogenesis. Resulting frataxin deficiency affects ISC-containing proteins and causes iron to accumulate in the brain and heart of FRDA patients. Here we report on abnormal cellular iron homeostasis in FRDA fibroblasts inducing a massive iron overload in the cytosol and mitochondria. We observe membrane transferrin receptor 1 (TfR1) accumulation, increased TfR1 endocytosis, and delayed transferrin recycling, ascribing this to impaired TfR1 palmitoylation. Frataxin deficiency is shown to reduce coenzyme A (CoA) availability for TfR1 palmitoylation. Finally, we demonstrate that artesunate, CoA, and dichloroacetate improve TfR1 palmitoylation and decrease iron overload, paving the road for evidence-based therapeutic strategies at the actionable level of TfR1 palmitoylation in FRDA.

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

Blood ◽

10.1182/blood.v94.11.3915 ◽

1999 ◽

Vol 94 (11) ◽

pp. 3915-3921 ◽

Cited By ~ 66

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.

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DHPLC Scan of Iron Genes in Consecutive Patients with Suspected Iron Overload.

Blood ◽

10.1182/blood.v104.11.3206.3206 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3206-3206

Author(s):

Domenico Girelli ◽

Giorgio Biasiotto ◽

Barbara Foglieni ◽

Claudia Bozzini ◽

Anna Polotti ◽

...

Keyword(s):

Iron Overload ◽

Functional Role ◽

Iron Homeostasis ◽

Clinical Phenotype ◽

Tertiary Care ◽

Hereditary Hemochromatosis ◽

Rapid Identification ◽

Hfe Gene ◽

Heterozygous Male ◽

Juvenile Hemochromatosis

Abstract Background: Hereditary Hemochromatosis, once considered a monogenic disorder, is now seen as a polygenic disease, with clinical phenotype also influenced by several environmental factors. Besides the classic HFE gene, other genes involved in modulation of iron homeostasis and clinical phenotype include those coding for hemojuvelin and hepcidin (both responsible for Juvenile Hemochromatosis), ferroportin, and, possibly, H-ferritin. Methods: we used DHPLC to scan mutations in the above mentioned genes in 55 consecutive patients recently referred to our tertiary care unit for iron overload disorders. Many of them had at least biochemical signs of iron overload not explained, or not completely explained, by classic or rare HFE mutations. Main results: the −72 C→T variation in the promoter of hepcidin gene, near the putative TATA box, was found in a H63D heterozygous male with unexplained biochemical signs (serum ferritin 660 μg/L, TS 45%), who, rather, should have been protected by several previous blood donations. We recently found this new hepcidin mutation in a family from another cohort (Biasiotto et al., in press): genotype/phenotype correlation data were also consisting with a functional role of this mutation. Two other hepcidin variations were found in this series, the 108 G→A (new), and the 212 G→A. While the first appeared silent, familial study suggests that the second may be functional in association with C282Y. No new or functional variations were found in hemojuvelin or H-ferritin genes. Several polymorphisms were detected in the ferroportin gene, including two new sequence variations (−116 T→C in the promoter; 691+20C C→T in intron 4), that occurred in two unrelated subjects with wild-type HFE genotype. Their functional role is currently under investigation by extensive family studies and/or quantitative evaluation of hepatic iron by MRI/liver biopsy. Conclusions: DHPLC scan of iron genes appears as a helpful tool for integrating clinical data in selected patients referring for suspected iron overload, as well as for rapid identification of new mutations.

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