Recovery from blood loss requires a greatly enhanced supply of iron to support expanded erythropoiesis. After hemorrhage, suppression of the iron-regulatory hormone hepcidin allows increased iron absorption and mobilization from stores. We identified a new hormone, erythroferrone (ERFE), which mediates hepcidin suppression during stress erythropoiesis. ERFE is produced by erythroblasts in response to erythropoietin. ERFE-deficient mice fail to suppress hepcidin rapidly after hemorrhage and exhibit a delay in recovery from blood loss. ERFE expression is greatly increased in murine HbbTh3/+ thalassemia intermedia where it contributes to the suppression of hepcidin and systemic iron overload characteristic of this disease.

Expression of hepcidin, a key regulator of intestinal iron absorption, can be induced in vitro by several bone morphogenetic proteins (BMPs), including BMP2, BMP4 and BMP9 (refs. 1,2). However, in contrast to BMP6, expression of other BMPs is not regulated at the mRNA level by iron in vivo, and their relevance to iron homeostasis is unclear. We show here that targeted disruption of Bmp6 in mice causes a rapid and massive accumulation of iron in the liver, the acinar cells of the exocrine pancreas, the heart and the renal convoluted tubules. Despite their severe iron overload, the livers of Bmp6-deficient mice have low levels of phosphorylated Smad1, Smad5 and Smad8, and these Smads are not significantly translocated to the nucleus. In addition, hepcidin synthesis is markedly reduced. This indicates that Bmp6 is critical for iron homeostasis and that it is functionally nonredundant with other members of the Bmp subfamily. Notably, Bmp6-deficient mice retain their capacity to induce hepcidin in response to inflammation. The iron burden in Bmp6 mutant mice is significantly greater than that in mice deficient in the gene associated with classical hemochromatosis (Hfe), suggesting that mutations in BMP6 might cause iron overload in humans with severe juvenile hemochromatosis for which the genetic basis has not yet been characterized.

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Inherited anemias with ineffective erythropoiesis, such as β-thalassemia, manifest inappropriately low hepcidin production and consequent excessive absorption of dietary iron, leading to iron overload. Erythroferrone (ERFE) is an erythroid regulator of hepcidin synthesis and iron homeostasis. Erfe expression was highly increased in the marrow and spleen of HbbTh3/+ mice (Th3/+), a mouse model of thalassemia intermedia. Ablation of Erfe in Th3/+ mice restored normal levels of circulating hepcidin at 6 weeks of age, suggesting ERFE could be a factor suppressing hepcidin production in β-thalassemia. We examined the expression of Erfe and the consequences of its ablation in thalassemic mice from 3 to 12 weeks of age. The loss of ERFE in thalassemic mice led to full restoration of hepcidin mRNA expression at 3 and 6 weeks of age, and significant reduction in liver and spleen iron content at 6 and 12 weeks of age. Ablation of Erfe slightly ameliorated ineffective erythropoiesis, as indicated by reduced spleen index, red cell distribution width, and mean corpuscular volume, but did not improve the anemia. Thus, ERFE mediates hepcidin suppression and contributes to iron overload in a mouse model of β-thalassemia.

In response to iron loading, hepcidin synthesis is homeostatically increased to limit further absorption of dietary iron and its release from stores. Mutations in HFE, transferrin receptor 2 (Tfr2), hemojuvelin (HJV), or bone morphogenetic protein 6 (BMP6) prevent appropriate hepcidin response to iron, allowing increased absorption of dietary iron, and eventually iron overload. To understand the role each of these proteins plays in hepcidin regulation by iron, we analyzed hepcidin messenger RNA (mRNA) responsiveness to short and long-term iron challenge in iron-depleted Hfe, Tfr2, Hjv, and Bmp6 mutant mice. After 1-day (acute) iron challenge, Hfe 2/2 mice showed a smaller hepcidin increase than their wild-type strain-matched controls, Bmp6 2/2 mice showed nearly no increase, and Tfr2 and Hjv mutant mice showed no increase in hepcidin expression, indicating that all four proteins participate in hepcidin regulation by acute iron changes. After a 21-day (chronic) iron challenge, Hfe and Tfr2 mutant mice increased hepcidin expression to nearly wildtype levels, but a blunted increase of hepcidin was seen in Bmp6 2/2 and Hjv 2/2 mice. BMP6, whose expression is also regulated by iron, may mediate hepcidin regulation by iron stores. None of the mutant strains (except Bmp6 2/2 mice) had impaired BMP6 mRNA response to chronic iron loading. Conclusion: TfR2, HJV, BMP6, and, to a lesser extent, HFE are required for the hepcidin response to acute iron loading, but are partially redundant for hepcidin regulation during chronic iron loading and are not involved in the regulation of BMP6 expression. Our findings support a model in which acute increases in holotransferrin concentrations transmitted through HFE, TfR2, and HJV augment BMP receptor sensitivity to BMPs. A distinct regulatory mechanism that senses hepatic iron may modulate hepcidin response to chronic iron loading. (HEPATOLOGY 2011;53:1333-1341 S ystemic iron homeostasis is dependent on the hepatic peptide hormone hepcidin, its receptor/iron channel ferroportin, and the feedback regulation of the two molecules by iron. Hepcidin controls the delivery of dietary and recycled iron to plasma by binding to the iron exporter ferroportin and inducing its endocytosis and degradation. 1 Increases in body iron levels stimulate hepcidin production in the liver, limiting further absorption of iron. The mechanism of hepcidin regulation by iron is not completely understood. Human studies have shown that blood hepcidin concentrations rapidly increase in response to oral iron challenge, are proportional to increases in diferric transferrin (holotransferrin) concentrations, 2,3 and strongly correlate with iron stores as reflected by serum ferritin. 3 In mice, hepcidin messenger RNA (mRNA) increase within 24 hours after the switch from a lowiron diet to a standard diet 4 and in vivo imaging of hepcidin promoter-reporter constructs in mice 5 confirmed high responsiveness of hepcidin promoter Abbreviations: ANOVA, analysis of variance; apo-Tf, apotransferrin; BMP6, bone morphogenetic pr...

The deficiency of hepcidin, the hormone that controls iron absorption and its tissue distribution, is the cause of iron overload in nearly all forms of hereditary hemochromatosis and in untransfused iron-loading anemias. In a recent study, we reported the development of minihepcidins, small drug-like hepcidin agonists. Here we explore the feasibility of using minihepcidins for the prevention and treatment of iron overload in hepcidindeficient mice. An optimized minihepcidin (PR65) was developed that had superior potency and duration of action compared with natural hepcidin or other minihepcidins, and favorable cost of synthesis. PR65 was administered by subcutaneous injection daily for 2 weeks to iron-depleted or iron-loaded hepcidin knockout mice. PR65 administration to iron-depleted mice prevented liver iron loading, decreased heart iron levels, and caused the expected iron retention in the spleen and duodenum. At high doses, PR65 treatment also caused anemia because of profound iron restriction. PR65 administration to hepcidin knockout mice with pre-existing iron overload had a more moderate effect and caused partial redistribution of iron from the liver to the spleen. Our study demonstrates that minihepcidins could be beneficial in iron overload disorders either used alone for prevention or possibly as adjunctive therapy with phlebotomy or chelation. IntroductionProduced by the liver, hepcidin is a 25 amino acid peptide hormone that circulates in plasma and homeostatically controls body iron balance. 1 Iron levels in turn regulate hepcidin production: in healthy individuals, hepcidin production increases when plasma or tissue iron concentrations rise and decreases after iron depletion. The hormone binds to its receptor ferroportin, the sole exporter of cellular iron into plasma. Ferroportin is prominently expressed in enterocytes, iron-recycling macrophages and hepatocytes. Hepcidin binding initiates the endocytosis and proteolysis of ferroportin and thereby decreases iron flow into plasma. 2 In hereditary hemochromatoses (HH) types I-III, mutations in genes encoding hepcidin regulators, or hepcidin itself lead to diminished production of hepcidin thus decreasing the inhibitory effect of hepcidin on duodenal iron absorption and causing clinical iron overload. 3 Hepcidin deficiency and hyperabsorption of dietary iron are major factors not only in HH but also in iron overload associated with hereditary anemias caused by ineffective erythropoiesis. 4 Hepcidin replacement therapy with pharmacologically optimized agonists would provide a rational treatment for these disorders. In HH or ␤-thalassemia intermedia, early diagnosis may allow preventive treatment with hepcidin agonists to normalize iron regulation, and reduce the potential for iron toxicity and the need for phlebotomy or chelation. Aside from inhibiting dietary iron absorption, hepcidin or hepcidin agonists may also have a protective effect on the liver, heart, and other organs by causing redistribution of iron into macrophages of the liver and spleen...

Anemia is very common in patients suffering from infections or chronic inflammation and can add substantially to the morbidity of the underlying disease. It is mediated by excessive production of the iron-regulatory peptide hepcidin, but the signaling pathway responsible for hepcidin up-regulation in the inflammatory context is still not understood completely. In the present study, we show that activin B has an unexpected but crucial role in the induction of hepcidin by inflammation. There is a dramatic induction of Inhbb mRNA, encoding the activin ␤ B -subunit, in the livers of mice challenged with lipopolysaccharide, slightly preceding an increase in Smad1/5/8 phosphorylation and Hamp mRNA. Activin B also induces Smad1/5/8 phosphorylation in human hepatoma-derived cells and, synergistically with IL-6 and STAT-3 signaling, upregulates hepcidin expression markedly, an observation confirmed in mouse primary hepatocytes. Pretreatment with a bone morphogenic protein type I receptor inhibitor showed that the effect of activin B on hepcidin expression is entirely attributable to its effect on bone morphogenetic protein signaling, most likely via activin receptor-like kinase 3. Activin B is therefore a novel and specific target for the treatment of anemia of inflammation. (Blood. 2012;120(2):431-439) IntroductionAnemia of inflammation develops as a complication of an acute or chronic activation of the immune response. It is particularly common in hospitalized patients and in the elderly and has a negative impact on recovery and survival. 1,2 Most chronic bacterial, fungal, viral, or parasitic infections with systemic manifestations can cause anemia of inflammation, as do rheumatologic disorders, systemic autoimmune disorders, inflammatory bowel disease, chronic kidney diseases, and some malignancies. 3 The limitation of iron supply to erythropoiesis is a major factor in the development of this anemia. Treatment includes administration of erythropoiesis-stimulating agents and intravenous iron. However, iron is rapidly trapped in the macrophage compartment, 4,5 rendering it unavailable for erythropoiesis once the initial iron bolus is incorporated into RBCs and body stores.Anemia of inflammation appears to be caused, at least in part, by the induction of the iron-regulatory hormone hepcidin. [6][7][8] Hepcidin acts by binding to the sole known iron-export channel, ferroportin, which is found on duodenal enterocytes, macrophages, and hepatocytes, the cell types that export iron into plasma. Binding of hepcidin to ferroportin induces its internalization and degradation, 9 which progressively inhibit iron efflux from these cells, leading to hypoferremia. The fundamental mechanisms causing increased hepcidin production by the liver during inflammation are still incompletely understood, but a cross-talk with the pathway through which hepcidin is regulated by iron is not excluded.Iron overload induces the expression of bone morphogenetic protein 6 (BMP6), a member of the TGF-␤ superfamily. Binding of BMP6 to paired serine/thr...

Erythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in response to stimulation by erythropoietin (EPO). We previously demonstrated that ERFE messenger RNA expression and serum protein concentration increase in mice subjected to hemorrhage or EPO therapy, that ERFE acts on hepatocytes to suppress hepcidin, and that the resulting decrease in hepcidin augments iron delivery for intensified erythropoiesis. We also showed that ERFE contributes to pathological hepcidin suppression and iron overload in mice with nontransfused β-thalassemia. We now report the development and technical validation of a rabbit monoclonal antibody-based sandwich immunoassay for human ERFE. We use this assay to show that blood loss or EPO administration increases serum ERFE concentrations in humans, and that patients with both nontransfused and transfused β-thalassemia have very high serum ERFE levels, which decrease after blood transfusion. The assay should be useful for human studies of normal and disordered erythropoiesis and its effect on iron homeostasis.