The Human-Specific BOLA2 Duplication Modifies Iron Homeostasis and Anemia Predisposition in Chromosome 16p11.2 Autism Individuals.

Human-specific duplications at chromosome 16p11.2 mediate recurrent pathogenic 600 kbp BP4-BP5 copy-number variations, which are among the most common genetic causes of autism. These copy-number polymorphic duplications are under positive selection and include three to eight copies of BOLA2, a gene involved in the maturation of cytosolic iron-sulfur proteins. To investigate the potential advantage provided by the rapid expansion of BOLA2, we assessed hematological traits and anemia prevalence in 379,385 controls and individuals who have lost or gained copies of BOLA2: 89 chromosome 16p11.2 BP4-BP5 deletion carriers and 56 reciprocal duplication carriers in the UK Biobank. We found that the 16p11.2 deletion is associated with anemia (18/89 carriers, 20%, p = 4e-7, OR = 5), particularly iron-deficiency anemia. We observed similar enrichments in two clinical 16p11.2 deletion cohorts, which included 6/63 (10%) and 7/20 (35%) unrelated individuals with anemia, microcytosis, low serum iron, or low blood hemoglobin. Upon stratification by BOLA2 copy number, our data showed an association between low BOLA2 dosage and the above phenotypes (8/15 individuals with three copies, 53%, p = 1e-4). In parallel, we analyzed hematological traits in mice carrying the 16p11.2 orthologous deletion or duplication, as well as Bola2+/- and Bola2-/- animals. The Bola2-deficient mice and the mice carrying the deletion showed early evidence of iron deficiency, including a mild decrease in hemoglobin, lower plasma iron, microcytosis, and an increased red blood cell zinc-protoporphyrin-to-heme ratio. Our results indicate that BOLA2 participates in iron homeostasis in vivo, and its expansion has a potential adaptive role in protecting against iron deficiency.

The role of iron in mediating testosterone's effects on erythropoiesis in mice.

Testosterone stimulates iron-dependent erythropoiesis and suppresses hepcidin. To clarify the role of iron in mediating testosterone's effects on erythropoiesis, we induced iron deficiency in mice by feeding low iron diet. Iron-replete and iron-deficient mice were treated weekly with testosterone propionate or vehicle for 3 weeks. Testosterone treatment increased red cell count in iron-replete mice, but, surprisingly, testosterone reduced red cell count in iron-deficient mice. Splenic stress erythropoiesis was stimulated in iron-deficient mice relative to iron-replete mice, and further increased by testosterone treatment, as indicated by the increase in red pulp area, the number of nucleated erythroblasts, and expression levels of TfR1, GATA1, and other erythroid genes. Testosterone treatment of iron-deficient mice increased the ratio of early-to-late erythroblasts in the spleen and bone marrow, and serum LDH level, consistent with ineffective erythropoiesis. In iron-deficient mice, erythropoietin levels were higher but erythropoietin-regulated genes were generally downregulated relative to iron-replete mice, suggesting erythropoietin resistance. Conclusion: Testosterone treatment stimulates splenic stress erythropoiesis in iron-replete as well as iron-deficient mice. However, testosterone worsens anemia in iron-deficient mice because of ineffective erythropoiesis possibly due to erythropoietin resistance associated with iron deficiency. Iron plays an important role in mediating testosterone's effects on erythropoiesis.

Global loss of Tfr2 with concomitant induced iron deficiency greatly ameliorates the phenotype of a murine thalassemia intermedia model.

ß-thalassemias result from mutations in ß-globin, causing ineffective erythropoiesis and secondary iron overload due to inappropriately low levels of the iron regulatory hormone hepcidin. Mutations in transferrin receptor 2 (TFR2) lead to hereditary hemochromatosis (HH) as a result of inappropriately increased iron uptake from the diet, also due to improperly regulated hepcidin. TFR2 is also thought to be required for efficient erythropoiesis through its interaction with the erythropoietin receptor in erythroid progenitors. Transmembrane serine protease 6 (TMPRSS6), a membrane serine protease expressed selectively in the liver, participates in regulating hepcidin production in response to iron stores by cleaving hemojuvelin (HJV). We have previously demonstrated that inhibiting TMPRSS6 expression with a hepatocyte-specific siRNA formulation, induces hepcidin, mitigates anemia, and reduces iron overload in murine models of ß-thalassemia intermedia and HH. Here, we demonstrate that Tmprss6 siRNA treatment of double mutant Tfr2Y245X/Y245X HH Hbbth3/+ thalassemic mice induces hepcidin and diminishes tissue and serum iron levels. Importantly, treated double mutant animals produce more mature red blood cells and have a nearly 50% increase in hemoglobin compared to untreated ß-thalassemic mice. Furthermore, we also show that treatment of Tfr2Y245X/Y245X HH mice leads to increased hepcidin expression and reduced total body iron burden. These data indicate that siRNA suppression of Tmprss6, in conjunction with the targeting of TFR2, may be superior to inhibiting Tmprss6 alone in the treatment of the anemia and secondary iron loading in ß-thalassemia intermedia and may be useful as a method of suppressing the primary iron overload in TFR2-related (type 3) hereditary hemochromatosis.

Mild iron deficiency does not ameliorate the phenotype of a murine erythropoietic protoporphyria model.

Reduced ferrochelatase activity in erythropoietic protoporphyria (EPP) causes the accumulation of protoporphyrin IX (PPIX) leading to acute cutaneous photosensitivity and liver injury. Many EPP patients also have a mild hypochromic, microcytic anemia and iron deficiency. Iron deficiency can lead to decreased PPIX accumulation in another erythropoietic porphyria, congenital erythropoietic porphyria (CEP). Expression of the iron regulatory peptide hepcidin is negatively regulated by the serine protease TMPRSS6. Hepcidin induction by siRNA-mediated inhibition of TMPRSS6 expression reduces iron availability and induces iron deficiency. To interrogate the therapeutic potential of iron deficiency to modify EPP, we treated an ethylnitrosourea-induced mouse model of EPP, Fech m1Pas , with a GalNAc-conjugated Tmprss6 siRNA and PPIX levels, anemia and iron parameters were monitored. The GalNAc-RNAi therapeutic reduces Tmprss6 expression and induces mild iron deficiency in Fech m1Pas animals. However, decreases in erythrocyte PPIX levels and liver PPIX accumulation were not seen. These results indicate short-term induction of iron deficiency, at least in a murine model of EPP, does not lead to decreased PPIX production.

NCOA4 maintains murine erythropoiesis via cell autonomous and non-autonomous mechanisms.

Ncoa4 mediates autophagic degradation of ferritin, the cytosolic iron storage complex, to maintain intracellular iron homeostasis. Recent evidence also supports a role for Ncoa4 in systemic iron homeostasis and erythropoiesis. However, the specific contribution and temporal importance of Ncoa4-mediated ferritinophagy in regulating systemic iron homeostasis and erythropoiesis is unclear. Here, we show that Ncoa4 has a critical role in basal systemic iron homeostasis and both cell autonomous and non-autonomous roles in murine erythropoiesis. Using an inducible murine model of Ncoa4 knockout, acute systemic disruption of Ncoa4 impaired systemic iron homeostasis leading to tissue ferritin and iron accumulation, a decrease in serum iron, and anemia. Mice acutely depleted of Ncoa4 engaged the Hif2a-erythropoietin system to compensate for anemia. Mice with targeted deletion of Ncoa4 specifically in the erythroid compartment developed a pronounced anemia in the immediate postnatal stage, a mild hypochromic microcytic anemia at adult stages, and were more sensitive to hemolysis with higher requirements for the Hif2a-erythropoietin axis and extramedullary erythropoiesis during recovery. These studies demonstrate the importance of Ncoa4-mediated ferritinophagy as a regulator of systemic iron homeostasis and define the relative cell autonomous and non-autonomous contributions of Ncoa4 in supporting erythropoiesis in vivo.

A recurring mutation in the respiratory complex 1 protein NDUFB11 is responsible for a novel form of X-linked sideroblastic anemia.

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited blood disorders characterized by pathological mitochondrial iron deposition in erythroid precursors. Each known cause has been attributed to a mutation in a protein associated with heme biosynthesis, iron-sulfur cluster biogenesis, mitochondrial translation, or a component of the mitochondrial respiratory chain. Here, we describe a recurring mutation, c.276_278del, p.F93del, in NDUFB11, a mitochondrial respiratory complex I-associated protein encoded on the X chromosome, in 5 males with a variably syndromic, normocytic CSA. The p.F93del mutation results in respiratory insufficiency and loss of complex I stability and activity in patient-derived fibroblasts. Targeted introduction of this allele into K562 erythroleukemia cells results in a proliferation defect with minimal effect on erythroid differentiation potential, suggesting the mechanism of anemia in this disorder.

RNAi-mediated reduction of hepatic Tmprss6 diminishes anemia and secondary iron overload in a splenectomized mouse model of ß-thalassemia intermedia.

Diminished ß-globin synthesis in ß-thalassemia is associated with ineffective erythropoiesis, leading to secondary iron overload caused by inappropriately low levels of hepcidin and to splenomegaly in the symptomatic thalassemias. Splenectomy is often employed in patients with ß-thalassemia to reduce hemolysis. Expression of the iron regulatory peptide hormone hepcidin is repressed by the serine protease TMPRSS6. Hepcidin induction by RNAi-mediated inhibition of TMPRSS6 expression reduces iron overload and mitigates anemia in murine models of ß-thalassemia intermedia. To interrogate the efficacy of RNAi-mediated reduction of Tmprss6 in splenectomized ß-thalassemia, splenectomized ß-thalassemic Hbbth3/+ animals were treated with a GalNAc-conjugated siRNA targeting Tmprss6 (GalNAc-Tmprss6) and their hematological and iron parameters monitored. We demonstrate that treatment with GalNAc-Tmprss6 significantly diminishes Tmprss6 expression and appropriately elevates hepcidin expression in splenectomized Hbbth3/+ animals. Similar to unsplenectomized animals, treated animals have markedly improved anemia due to diminished ineffective erythropoiesis and reduced iron loading in both serum and tissue. These results suggest that RNAi-mediated reduction of Tmprss6 may have positive outcomes even in splenectomized ß-thalassemia patients.

Differing impact of the deletion of hemochromatosis-associated molecules HFE and transferrin receptor-2 on the iron phenotype of mice lacking bone morphogenetic protein 6 or hemojuvelin.

UNLABELLED: Hereditary hemochromatosis, which is characterized by inappropriately low levels of hepcidin, increased dietary iron uptake, and systemic iron accumulation, has been associated with mutations in the HFE, transferrin receptor-2 (TfR2), and hemojuvelin (HJV) genes. However, it is still not clear whether these molecules intersect in vivo with bone morphogenetic protein 6 (BMP6)/mothers against decapentaplegic (SMAD) homolog signaling, the main pathway up-regulating hepcidin expression in response to elevated hepatic iron. To answer this question, we produced double knockout mice for Bmp6 and ß2-microglobulin (a surrogate for the loss of Hfe) and for Bmp6 and Tfr2, and we compared their phenotype (hepcidin expression, Bmp/Smad signaling, hepatic and extrahepatic tissue iron accumulation) with that of single Bmp6-deficient mice and that of mice deficient for Hjv, alone or in combination with Hfe or Tfr2. Whereas the phenotype of Hjv-deficient females was not affected by loss of Hfe or Tfr2, that of Bmp6-deficient females was considerably worsened, with decreased Smad5 phosphorylation, compared with single Bmp6-deficient mice, further repression of hepcidin gene expression, undetectable serum hepcidin, and massive iron accumulation not only in the liver but also in the pancreas, the heart, and the kidneys. CONCLUSION: These results show that (1) BMP6 does not require HJV to transduce signal to hepcidin in response to intracellular iron, even if the loss of HJV partly reduces this signal, (2) another BMP ligand can replace BMP6 and significantly induce hepcidin expression in response to extracellular iron, and (3) BMP6 alone is as efficient at inducing hepcidin as the other BMPs in association with the HJV/HFE/TfR2 complex; they provide an explanation for the compensatory effect of BMP6 treatment on the molecular defect underlying Hfe hemochromatosis in mice.

Congenital sideroblastic anemia due to mutations in the mitochondrial HSP70 homologue HSPA9.

The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases characterized by defects in mitochondrial heme synthesis, iron-sulfur (Fe-S) cluster biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homolog located in the chromosome 5q deletion syndrome 5q33 critical deletion interval and involved in mitochondrial Fe-S biogenesis, result in CSA inherited as an autosomal recessive trait. In a fraction of patients with just 1 severe loss-of-function allele, expression of the clinical phenotype is associated with a common coding single nucleotide polymorphism in trans that correlates with reduced messenger RNA expression and results in a pseudodominant pattern of inheritance.

Regulation of Iron Metabolism by Hepcidin under Conditions of Inflammation.

Iron is a redox-active metal required as a cofactor in multiple metalloproteins essential for a host of life processes. The metal is highly toxic when present in excess and must be strictly regulated to prevent tissue and organ damage. Hepcidin, a molecule first characterized as an antimicrobial peptide, plays a critical role in the regulation of iron homeostasis. Multiple stimuli positively influence the expression of hepcidin, including iron, inflammation, and infection by pathogens. In this Minireview, I will discuss how inflammation regulates hepcidin transcription, allowing for sufficient concentrations of iron for organismal needs while sequestering the metal from infectious pathogens.

HFE interacts with the BMP type I receptor ALK3 to regulate hepcidin expression.

Mutations in HFE are the most common cause of hereditary hemochromatosis (HH). HFE mutations result in reduced expression of hepcidin, a hepatic hormone, which negatively regulates iron absorption from the duodenum and iron release from macrophages. However, the mechanism by which HFE regulates hepcidin expression in hepatocytes is not well understood. It is known that the bone morphogenetic protein (BMP) pathway plays a central role in controlling hepcidin expression in the liver. Here we show that HFE overexpression increased Smad1/5/8 phosphorylation and hepcidin expression, whereas inhibition of BMP signaling abolished HFE-induced hepcidin expression in Hep3B cells. HFE was found to associate with ALK3, inhibiting ALK3 ubiquitination and proteasomal degradation and increasing ALK3 protein expression and accumulation on the cell surface. The 2 HFE mutants associated with HH, HFE C282Y and HFE H63D, regulated ALK3 protein ubiquitination and trafficking differently, but both failed to increase ALK3 cell-surface expression. Deletion of Hfe in mice resulted in a decrease in hepatic ALK3 protein expression. Our results provide evidence that HFE induces hepcidin expression via the BMP pathway: HFE interacts with ALK3 to stabilize ALK3 protein and increase ALK3 expression at the cell surface.

An RNAi therapeutic targeting Tmprss6 decreases iron overload in Hfe(-/-) mice and ameliorates anemia and iron overload in murine ß-thalassemia intermedia.

Mutations in HFE lead to hereditary hemochromatosis (HH) because of inappropriately high iron uptake from the diet resulting from decreased hepatic expression of the iron-regulatory hormone hepcidin. -thalassemia is a congenital anemia caused by partial or complete loss of -globin synthesis causing ineffective erythropoiesis, anemia, decreased hepcidin production, and secondary iron overload. Tmprss6 is postulated to regulate hepcidin production by cleaving Hemojuvelin (Hjv), a key modulator of hepcidin expression, from the hepatocyte surface. On this basis, we hypothesized that treatment of mouse models of HH (Hfe(-/-)) and -thalassemia intermedia (Hbb(th3/+)) with Tmprss6 siRNA formulated in lipid nanoparticles (LNPs) that are preferentially taken up by the liver would increase hepcidin expression and lessen the iron loading in both models. In the present study, we demonstrate that LNP-Tmprss6 siRNA treatment of Hfe(-/-) and Hbb(th3/+) mice induces hepcidin and diminishes tissue and serum iron levels. Furthermore, LNP-Tmprss6 siRNA treatment of Hbb(th3/+) mice substantially improved the anemia by altering RBC survival and ineffective erythropoiesis. Our results indicate that pharmacologic manipulation of Tmprss6 with RNAi therapeutics isa practical approach to treating iron overload diseases associated with diminished hepcidin expression and may have efficacy in modifying disease-associated morbidities of -thalassemia intermedia.

A competitive enzyme-linked immunosorbent assay specific for murine hepcidin-1: correlation with hepatic mRNA expression in established and novel models of dysregulated iron homeostasis.

Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including ß-thalassemia intermedia (Hbb(th3/+)), hereditary hemochromatosis (Hfe(-/-), Hjv(-/-), and Tfr2(Y245X/Y245X)), hypotransferrinemia (Trf(hpx/hpx)), heterozygous transferrin receptor 1 deficiency (Tfrc(+/-)) and iron refractory iron deficiency anemia (Tmprss6(-/-) and Tmprss6(hem8/hem8)). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups.

Combination therapy with a Tmprss6 RNAi-therapeutic and the oral iron chelator deferiprone additively diminishes secondary iron overload in a mouse model of ß-thalassemia intermedia.

ß-thalassemias result from diminished ß-globin synthesis and are associated with ineffective erythropoiesis and secondary iron overload caused by inappropriately low levels of the iron regulatory hormone hepcidin. The serine protease TMPRSS6 attenuates hepcidin production in response to iron stores. Hepcidin induction reduces iron overload and mitigates anemia in murine models of ß-thalassemia intermedia. To further interrogate the efficacy of an RNAi-therapeutic downregulating Tmprss6, ß-thalassemic Hbb(th3/+) animals on an iron replete, an iron deficient, or an iron replete diet also containing the iron chelator deferiprone were treated with Tmprss6 siRNA. We demonstrate that the total body iron burden is markedly improved in Hbb(th3/+) animals treated with siRNA and chelated with oral deferiprone, representing a significant improvement compared to either compound alone. These data indicate that siRNA suppression of Tmprss6, in conjunction with oral iron chelation therapy, may prove superior for treatment of anemia and secondary iron loading seen in ß-thalassemia intermedia.

X-linked sideroblastic anemia due to ALAS2 intron 1 enhancer element GATA-binding site mutations.

X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia. In affected males, it is uniformly associated with partial loss-of-function missense mutations in the erythroid-specific heme biosynthesis protein 5-aminolevulinate synthase 2 (ALAS2). Here, we report five families with XLSA owing to mutations in a GATA transcription factor binding site located in a transcriptional enhancer element in intron 1 of the ALAS2 gene. As such, this study defines a new class of mutations that should be evaluated in patients undergoing genetic testing for a suspected diagnosis of XLSA.

The transferrin receptor modulates Hfe-dependent regulation of hepcidin expression.

Hemochromatosis is caused by mutations in HFE, a protein that competes with transferrin (TF) for binding to transferrin receptor 1 (TFR1). We developed mutant mouse strains to gain insight into the role of the Hfe/Tfr1 complex in regulating iron homeostasis. We introduced mutations into a ubiquitously expressed Tfr1 transgene or the endogenous Tfr1 locus to promote or prevent the Hfe/Tfr1 interaction. Under conditions favoring a constitutive Hfe/Tfr1 interaction, mice developed iron overload attributable to inappropriately low expression of the hormone hepcidin. In contrast, mice carrying a mutation that interferes with the Hfe/Tfr1 interaction developed iron deficiency associated with inappropriately high hepcidin expression. High-level expression of a liver-specific Hfe transgene in Hfe-/- mice was also associated with increased hepcidin production and iron deficiency. Together, these models suggest that Hfe induces hepcidin expression when it is not in complex with Tfr1.

Murine mutants in the study of systemic iron metabolism and its disorders: an update on recent advances.

Many past and recent advances in the field of iron metabolism have relied upon the use of mouse models of disease. These models have arisen spontaneously in breeder colonies or have been engineered for global or conditional ablation or overexpression of select genes. Full phenotypic characterization of these models typically involves maintenance on iron-loaded or -deficient diets, treatment with oxidative or hemolytic agents, breeding to other mutant lines or other stresses. In this review, we focus on systemic iron biology and the contributions that mouse model-based studies have made to the field. We have divided the field into three broad areas of research: dietary iron absorption, regulation of hepcidin expression and cellular iron metabolism. For each area, we begin with an overview of the current understanding of key molecular and cellular determinants then discuss recent advances. Finally, we conclude with brief comments on prospects for future study. This article is part of a Special Issue entitled: Cell Biology of Metals.

Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever arenaviruses.

At least five arenaviruses cause viral haemorrhagic fevers in humans. Lassa virus, an Old World arenavirus, uses the cellular receptor alpha-dystroglycan to infect cells. Machupo, Guanarito, Junin and Sabia viruses are New World haemorrhagic fever viruses that do not use alpha-dystroglycan. Here we show a specific, high-affinity association between transferrin receptor 1 (TfR1) and the entry glycoprotein (GP) of Machupo virus. Expression of human TfR1, but not human transferrin receptor 2, in hamster cell lines markedly enhanced the infection of viruses pseudotyped with the GP of Machupo, Guanarito and Junin viruses, but not with those of Lassa or lymphocytic choriomeningitis viruses. An anti-TfR1 antibody efficiently inhibited the replication of Machupo, Guanarito, Junin and Sabia viruses, but not that of Lassa virus. Iron depletion of culture medium enhanced, and iron supplementation decreased, the efficiency of infection by Junin and Machupo but not Lassa pseudoviruses. These data indicate that TfR1 is a cellular receptor for New World haemorrhagic fever arenaviruses.

Hepcidin induction by transgenic overexpression of Hfe does not require the Hfe cytoplasmic tail, but does require hemojuvelin.

Mutations in HFE cause the most common form of hereditary hemochromatosis (HH). We previously showed that liver-specific, transgenic overexpression of murine Hfe stimulates production of the iron regulatory hormone hepcidin. Here, we developed several additional transgenic mouse strains to further interrogate the structural basis of HFE function in the pathophysiology of HH. We hypothesized that the small, cytoplasmic domain of HFE might be necessary for HFE-mediated induction of hepcidin. We demonstrate that, like the full-length protein, overexpression of Hfe proteins lacking the cytoplasmic domain leads to hepcidin induction, iron deficiency and a hypochromic, microcytic anemia. However, high-level expression of a liver-specific Hfe transgene carrying the mouse equivalent of the common HFE C282Y human disease-causing mutation (murine C294Y) did not cause iron deficiency. Furthermore, hepcidin induction by transgenes encoding both WT Hfe and Hfe lacking its cytoplasmic domain is greatly attenuated in the absence of hemojuvelin (Hjv). Our observations indicate that the extracellular and transmembrane domains of Hfe are sufficient, and Hjv is essential, for Hfe-mediated induction of hepcidin expression.

Transgenic HFE-dependent induction of hepcidin in mice does not require transferrin receptor-2.

Hereditary hemochomatosis (HH) is caused by mutations in several genes, including HFE and transferrin receptor-2 (TFR2). Loss of either protein decreases expression of the iron regulatory hormone hepcidin by the liver, leading to inappropriately high iron uptake from the diet, and resulting in systemic iron overload. In tissue culture, overexpressed HFE and TFR2 physically interact. Hepatocellular overexpression of Hfe in vivo increases hepcidin expression, despite an associated decrease in Tfr2. On this basis, we hypothesized that Tfr2 would not be required for Hfe-dependent up-regulation of hepcidin. We show that hepatocellular overexpression of Hfe in Tfr2(Y245X/Y245X) mice leads to hepcidin induction eventuating in iron deficiency and a hypochromic, microcytic anemia. Furthermore, coimmunoprecipitation studies using liver lysates did not provide evidence for physical interaction between Hfe and Tfr2 in vivo. In conclusion, we demonstrate that Tfr2 is not essential for Hfe-mediated induction of hepcidin expression, supporting the possibility that TFR2 may regulate iron metabolism in an HFE-independent manner.