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Meynard D, Kautz L, Darnaud V, Canonne-Hergaux F, Coppin H, Roth M-P. Lack of the bone morphogenetic protein BMP6 induces massive iron overload. Nat Genet 2008;41:478–481. (Reprinted with permission.)

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 vivo3, 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.

Andriopoulos Jr B, Corradini E, Xia Y, Faasse SA, Chen S, Grgurevic L, et al. BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism. Nat Genet 2009;41:482–487. www.nature.com(Reprinted with permission.)

Juvenile hemochromatosis is an iron-overload disorder caused by mutations in the genes encoding the major iron regulatory hormone hepcidin (HAMP) and hemojuvelin (HFE2). We have previously shown that hemojuvelin is a co-receptor for bone morphogenetic proteins (BMPs) and that BMP signals regulate hepcidin expression and iron metabolism. However, the endogenous BMP regulator(s) of hepcidin in vivo is unknown. Here we show that compared with soluble hemojuvelin (HJV.Fc), the homologous DRAGON.Fc is a more potent inhibitor of BMP2 or BMP4 but a less potent inhibitor of BMP6 in vitro. In vivo, HJV.Fc or a neutralizing antibody to BMP6 inhibits hepcidin expression and increases serum iron, whereas DRAGON.Fc has no effect. Notably, Bmp6-null mice have a phenotype resembling hereditary hemochromatosis, with reduced hepcidin expression and tissue iron overload. Finally, we demonstrate a physical interaction between HJV.Fc and BMP6, and we show that BMP6 increases hepcidin expression and reduces serum iron in mice. These data support a key role for BMP6 as a ligand for hemojuvelin and an endogenous regulator of hepcidin expression and iron metabolism in vivo.

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Our understanding of how the body controls the intake and distribution of iron is essential for understanding human disorders where iron homeostasis is perturbed. These range from iron loading diseases, such as the hemochromatosis syndromes and thalassemias, to disorders of erythropoiesis and the anemia of chronic disease. A variety of primarily genetic studies over the last 10-15 years have led to the realization that the liver plays a central role in controlling iron metabolism through its synthesis of the bioactive iron-regulatory peptide hepcidin. How hepcidin itself is regulated has been the subject of intense investigation in recent years. Two recent articles in Nature Genetics1, 2 provide compelling evidence that bone morphogenetic protein 6 (BMP6) is a key endogenous regulator of hepcidin (Fig. 1).

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Figure 1. Regulation of hepcidin production by BMP6. BMP6 responds to increased iron and binds to the BMP receptors on the hepatocyte surface in conjunction with the coreceptor HJV. This stimulates phosphorylation of SMAD1, SMAD5, and SMAD8, which in turn bind to the co-SMAD (SMAD4). The complex translocates to the nucleus where it stimulates transcription of HAMP, the gene encoding hepcidin. Hepcidin is secreted by hepatocytes and acts on the iron exporter ferroportin (FPN) on various target cells to limit iron release. Iron status may also influence HAMP expression via TfR2 and the HFE/TfR1 complex, but whether these proteins signal through the BMP-SMAD pathway or independently has not been resolved.

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Iron is essential for basic cellular processes but it is also toxic when present in excess, so the levels of iron in the body must be maintained in the optimal physiological range. Because humans do not possess an active mechanism for iron excretion, this regulation is brought about largely by modulating the intake of iron from the diet across the wall of the proximal small intestine. The same factors that regulate iron absorption also regulate iron donation to the plasma from macrophages and other body cells, and this in turn is necessary to supply the iron that is critical for erythropoiesis and other metabolic activities.

In 2001, two studies identified hepcidin as the dominant factor regulating iron entry into the plasma.3, 4 Hepcidin acts to repress iron efflux from intestinal enterocytes, macrophages, and likely most body cells by binding to the only known iron export protein (ferroportin) and facilitating its internalization and degradation.5 Thus, when hepcidin levels are high, iron absorption is decreased and iron accumulates in macrophages. However, when hepcidin levels drop, iron absorption is increased and macrophages divest themselves of iron. Primary iron loading disorders due to mutations in the genes encoding hereditary hemochromatosis (HFE), transferrin receptor 2 (TfR2), hemojuvelin (HJV), or hepcidin (all of which are strongly expressed in the liver) are characterized by low or absent hepcidin expression, leading to inappropriately high iron absorption and body iron loading.6

How is hepcidin itself regulated in response to such stimuli as changes in body iron stores, altered erythropoiesis, hypoxia, and inflammation? We don't have the full story yet, but because hepcidin levels are reduced when HFE, TfR2, or HJV are mutated, these are likely to be upstream regulators of the pathway. The signal transduction pathways through which HFE and TfR2 influence hepcidin expression have yet to be defined, but the analysis of how HJV affects hepcidin expression has been particularly revealing. HJV is one of a family of proteins known as repulsive guidance molecules (RGMs) and has the alternative name of RGMc. RGMa and RGMb are involved in development of the central nervous system (a characteristic not shared by HJV) and have been shown to act as cofactors for the signaling of the BMPs. We now know that HJV is also a BMP coreceptor and signals through this pathway to alter hepcidin expression.7 The BMPs are members of the transforming growth factor-β superfamily of ligands and act through BMP receptors to activate the multifunctional SMAD (mothers against decapentaplegic homologue) signaling pathway. Mice in which the key signaling component SMAD4 is deleted develop severe iron overload, indicating the importance of this pathway in iron homeostasis.8

A range of BMPs have been shown to stimulate hepcidin gene transcription both in vitro and in vivo,9, 10 but these have varying potencies and it has been unclear what the true endogenous BMP ligand is for the regulation of hepcidin. The recent studies by Meynard et al. and Andriopoulos et al. provide some answers.

Meynard and coworkers1 hypothesized that BMP6 may be particularly important in hepcidin regulation, because they had previously found that BMP6 expression was positively correlated with body iron levels, the only BMP they found to show such regulation.11 Therefore, they investigated an existing Bmp6-deficient mouse line12 and found that these animals did indeed have substantially reduced hepcidin levels and showed an iron-loading phenotype similar to animals lacking the gene encoding Hjv, a BMP coreceptor. Iron accumulated in liver, heart, pancreas, and kidney. The periportal, hepatocyte-dominant distribution of iron in the liver is consistent with elevated iron absorption and, correspondingly, genes encoding proteins involved in iron absorption were up-regulated in the proximal small intestine of Bmp6−/− mice. The BMP receptors signal through SMAD1, SMAD5, and SMAD8, and the phosphorylation of each of these was decreased in Bmp6 null mice, as was their translocation to the nucleus. Taken together, these data provide very strong evidence that BMP6 plays a critical role in the regulation of hepcidin, and particularly its response to iron.

In the second study, Andriopoulos et al.2 took a different approach by first examining the effectiveness of soluble forms of HJV (sHJV) and its homolog RGMb (sRGMb) in blocking BMP-mediated hepcidin stimulation. Their previous studies had shown that sHJV could interfere with this signaling process.13 In vitro experiments showed that both sHJV and sRGMb were able to inhibit BMP stimulation of hepcidin transcription, and that sRGMb was less effective at inhibiting the actions of certain BMPs (such as BMP6) than sHJV. However, when these reagents were administered to mice, only sHJV was able to block hepcidin expression and increase plasma iron levels. In view of this result and the demonstration that sHJV was a more potent BMP6 antagonist, the investigators reasoned that BMP6 may be a key player in hepcidin regulation and examined the effects of altering BMP6 levels in mice. Consistent with their prediction, the administration of neutralizing antibodies to BMP6 reduced hepcidin expression and increased plasma iron levels, whereas administration of BMP6 itself had the opposite effect. These studies confirmed that BMP6 does indeed play an important role in maintaining hepcidin expression. This group also examined Bmp6 null mice and confirmed the iron loading phenotype that Meynard et al. described. Finally, they provided evidence that HJV was able to physically interact with BMP6 in biochemical studies.

Taken together, these two studies provide compelling evidence that BMP6 is a major regulator of hepcidin gene expression and that it represents an important part of the hepcidin regulatory network. Interestingly, Bmp6−/− mice have quite a mild phenotype, with only minor skeletal abnormalities being observed in embryos and no clear pathologies in other organ systems (other than the iron-related changes).12 In contrast, deletion of a number of other BMPs, such as Bmp1, Bmp2, Bmp4, and Bmp7, leads to far more widespread and severe defects. This strengthens the case that BMP6 plays a specific role in iron homeostasis.

Hepcidin can be strongly induced by inflammatory stimuli, and proinflammatory cytokines such as interleukin-6 are known to alter hepcidin transcription by signaling through the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway.14 However, this response is abrogated in Smad4 knockout mice,8 indicating that there is crosstalk between the JAK-STAT and BMP-SMAD pathways and that signaling through the SMADs is necessary for the hepcidin gene to respond to an inflammatory event. Meynard and colleagues1 found that the usual robust response of hepcidin to inflammation remained strong in Bmp6−/− mice, indicating that the necessary SMAD signal must be originating from a BMP receptor ligand other than BMP6. This is an area that requires further investigation.

The fact that BMP6 is regulated by iron suggests that it may play a pivotal role in monitoring body iron requirements. Previous studies have suggested that HFE and TfR2 are also involved in monitoring iron levels, so defining the links between BMP6 and these hepatocyte proteins should be a priority for future research. Further investigations are also required on the expression pattern of BMP6 in relation to other proteins involved in hepcidin regulation. For hepcidin itself, as well as HFE and TfR2, the hepatocyte is the primary site of expression. HJV is expressed most strongly in skeletal and cardiac muscle, but it is also expressed in hepatocytes at reasonably high levels. Less is known about BMP6 expression, but one study15 reported relatively low expression in the liver and much of that was in nonparenchymal cells. Cell-specific knockout of the BMP6 gene may be required to fully define its role.

Could knowledge of the role played by BMP6 be used in the therapy of iron-related disorders? The answer is maybe. Supplying BMP6 or a BMP6 agonist could certainly increase hepcidin levels, and this could be used to treat at least some classes of patients with iron overload. BMP6 antagonists could also play a role in treating patients with anemia arising from chronic disease. However, we really need to know much more about the biology of BMP6 and its interactions with the other proteins involved in hepcidin regulation before these possibilities can be entertained seriously. On the basis of our current knowledge, it may be more appropriate to target hepcidin itself in these conditions rather than an upstream regulator to reduce the possibility of off-target effects. Nevertheless, the delineation of the role played by BMP6 in hepcidin regulation represents an important advance in our understanding of an essential regulatory pathway in the body.

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