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Meeting Report

The first presentation ‘Hepcidin regulation by the BMP pathway’ was by Jodie L Babitt, Division of Nephrology, Massachusetts General Hospital, Boston, MA, USA.

Babitt discussed the role of the bone morpho-genetic protein (BMP) pathway in the regula-tion of hepcidin expression and iron metabo-lism. Liver-specific Smad-4-knockout mice first highlighted the potential importance of the BMP pathway as the mice exhibited hepcidin deficiency and developed severe iron overload [1]. Babitt et al. then demonstrated that hemojuvelin, a glycosylphosphatidyl-inositol-linked membrane protein known to regulate hepcidin expression, acts as a BMP coreceptor [2]. Hemojuvelin muta-tions in humans or mice cause the loss of hepcidin expression and severe iron overload, presumably owing to decreased BMP signaling [3].

Babitt has demonstrated that several BMPs can increase hepcidin production in vitro and in vivo, but that BMP6 may be the principal endogenous BMP regulating hepcidin. BMP6-null mice had almost undetectable hepcidin mRNA, and devel-oped severe iron overload of the liver and other organs but no other significant abnormalities [4,5]. BMP6 may also be important for the appropriate increase of hepcidin in response to iron loading. When mice were fed an iron-rich diet, Bmp6 expression in hepatocytes increased, suggesting

that BMP6 concentration may be a signal reflect-ing iron stores [6]. However, the mechanism by which iron affects BMP6 expression remains to be determined.

Based on the essential role of BMP6 in iron homeostasis in the mouse, BMP6 mutations and variants could play a role in human dis-ease. BMP6 deficiency could be the cause of some rare cases of hereditary hemochromatosis, which resemble the juvenile hemochromatosis caused by hemojuvelin or hepcidin mutations. Furthermore, BMP6 variants may be modifiers of iron disorders, such as the human hemochro-matosis protein (HFE), which has variable pen-etrance. Babitt also presented that HFE itself affects the pathway downstream of BMP6, as HFE-knockout mice exhibited impaired BMP signaling, but not BMP6 mRNA expression [7]. It remains to be determined if BMP6 is also involved in hepcidin regulation by inflammation and erythropoiesis.

Babitt proposed that interfering with BMP6 pathway may be effective for treating diseases of hepcidin excess. Considering that Bmp6-null mice did not have obvious problems other than iron dysregulation, selective BMP6 blockers may have relatively few off-target effects. Likewise, BMP6 agonists may be helpful for managing iron overload in patients with hepcidin deficiency.

Elizabeta NemethDavid Geffen School of Medicine, UCLA, 10833 LeConte Ave, CHS 37-131, Los Angeles, CA 90095, USA Tel.: +1 310 825 7499 Fax: +1 310 206 8766 enemeth@mednet.ucla.edu

Scientific Program on Iron and Heme, 51st Annual Meeting of the American Society of HematologyErnest N Morial Convention Center, New Orleans, LA, USA December 5–8, 2009

The hepatic peptide hormone hepcidin regulates plasma iron concentrations and tissue iron distribution by inhibiting dietary iron absorption and mobilization of iron from stores in macrophages and hepatocytes. Dysregulation of hepcidin production underlies many iron disorders. Deficient production of hepcidin causes systemic iron overload in hereditary hemochromatosis and iron-loading anemias, such as b-thalassemia, whereas hepcidin excess contributes to the development of anemia in inflammatory disorders and chronic kidney disease, and may cause erythropoietin resistance. The Scientific Program on Iron and Heme session at the 51st ASH annual meeting discussed recent advances in understanding hepcidin biology and explored the potential for hepcidin therapeutic applications. The session included three 30-min presentations.

Hepcidin biology and therapeutic applicationsExpert Rev. Hematol. 3(2), 153–155 (2010)

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Nemeth

The second presentation ‘Matriptase-2, a novel suppressor of hepcidin’ was by Clara Camaschella, Antonella Nai, Alessia Pagani and Laura Silvestri from Università Vita-Salute and San Raffaele Scientific Institute, Milan, Italy.

Matriptase-2 (also known as TMPRSS6) is a membrane ser-ine protease that was recently demonstrated to have a role in iron metabolism. Using N-ethyl-N-nitrosourea mutagenesis, Du et al. generated mice that had a mutation in matriptase-2 and exhibited iron-deficiency anemia and associated loss of body hair (‘mask mouse’) [8]. A similar phenotype was observed in the matriptase-2-knockout mouse [9]. The apparent cause of anemia in these mice was increased hepcidin expression, high-lighting the essential role of matriptase-2 in the regulation of hepcidin and iron. Within several months, a number of studies were published demonstrating that matriptase-2 mutations in humans cause iron-refractory iron-deficiency anemia. Clinical and laboratory features of iron-refractory iron-deficiency anemia include autosomal recessive inheritance, microcytic hypochro-mic anemia, extremely low iron and transferrin saturation from infancy, and inappropriately high serum/urinary hepcidin levels. It appears that the disease-causing mutations all lead to the loss of function of matriptase-2, either by affecting proteolytic activ-ity, autocatalytic activation of the protease, or its cell surface localization [10].

Camaschella’s group recently identified the likely mechanism by which matriptase-2 affects iron metabolism [11]. They demon-strated in vitro that matriptase-2 binds to and cleaves membrane hemojuvelin, a BMP coreceptor and essential regulator of hepci-din. With loss of matriptase-2 activity, increased concentration of membrane hemojuvelin would lead to higher expression of hepcidin, blocking dietary iron absorption and the release of recycled iron from macrophages. Iron-refractory iron-deficiency anemia, thus, appears to be a phenotypic opposite of juvenile hemochromatosis caused by hemojuvelin mutations.

Further support for hemojuvelin as the substrate of matrip-tase-2 came from mice that were mutated in both hemojuvelin and matriptase-2 [12]. These mice exhibited characteristics of hemojuvelin-deficient mice, including high plasma iron and liver iron content, and low hepcidin mRNA levels, an oppo-site phenotype of that exhibited by matriptase-2-mutant mice. Hemojuvelin, however, may not be the only biologically relevant substrate of matriptase-2. The protease is not only expressed in the liver, the site of hemojuvelin expression, but also in the kidney and olfactory epithelium. Whether matriptase-2 has a discernible role in those tissues remains to be determined.

Most patients are homozygous or compound heterozygous for TMPRSS6 mutations but, in a few patients, a single muta-tion was identified. Camaschella raised a possibility that even heterozygote carriers may be susceptible to iron deficiency or that TMPRSS6 variants may affect iron status and erythro-poiesis in the general population. Indeed, common polymorphic changes in the TMPRSS6 sequence have been reported, and genome-wide studies identified an association between certain TMPRSS6 variants and serum iron, transferrin saturation, MCV and hemoglobin levels [13,14].

Camaschella highlighted some questions that arose from the recent findings. It is unknown how TMPRSS6 is regulated by iron or other stimuli, or what its relationship is with other hepci-din activators/inhibitors. It also remains to be determined which TMPRSS6 variants are associated with susceptibility to iron deficiency and whether they can act as modifiers of HFE hemo-chromatosis. Finally, Camaschella pointed out that matriptase-2 may provide additional pathways for therapeutic manipulation of iron disorders.

Presentation three was titled ‘Development of Hepcidin Agonists and Antagonists’ and was given by Elizabeta Nemeth, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.

Nemeth described the role of hepcidin in the pathogenesis of iron disorders and proposed that hepcidin-targeted therapies may improve treatment options for patients suffering from those diseases. Although no hepcidin therapies are yet available, several candidates are currently under development as hepcidin agonists or antagonists.

Hepcidin agonists should be useful for preventing or amelio-rating iron overload in hereditary hemochromatosis, b-thalasse-mias and other iron-loading anemias, and possibly chronic liver diseases. Detailed structure–function studies of hepcidin and its receptor allowed identification of the components of both molecules critical for formation of the ligand–receptor complex. Nemeth et al. demonstrated that the N-terminus of hepcidin was essential for its function, and that synthetic small peptides based on hepcidin N-terminal region were active in vitro. Candidate peptides were modified to improve peptide activity, resistance to proteolysis and oral bioavailability and were tested in mice. Similar to injections of wild-type hepcidin, which cause a rapid drop in serum iron, intraperitoneal injections of several candidate agonists caused a comparable effect. Furthermore, some modi-fied peptides were able to decrease serum iron in mice after oral administration by gavage [Nemeth E, Unpublished Data]. The small size of the peptides may allow the eventual development of orally available agonists for humans.

Hepcidin antagonists would be expected to benefit patients with diseases of hepcidin excess manifested as iron-restricted anemia and when very prolonged, as with systemic iron deficiency. Elevated hep-cidin is the cause of iron-refractory iron-deficiency anemia, and may contribute to anemia of chronic kidney disease and erythropoietin resistance. Inappropriately high hepcidin has also been observed in anemias associated with inflammatory disorders, such as different infections, autoimmune diseases, critical illness and obesity. Several approaches have been undertaken to develop hepcidin antagonists. To directly interfere with hepcidin activity, Sasu et al. generated hepcidin-neutralizing antibodies [101]. In a mouse model of anemia of inflammation, they demonstrated that, while administration of erythropoietin by itself could not ameliorate anemia, a combination treatment using erythropoietin and hepcidin-neutralizing antibody was effective in preventing anemia.

Another approach to antagonize hepcidin is to target path-ways regulating hepcidin production. Two molecules that tar-get the BMP pathway have been described. Dorsomorphin, a

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small -molecule inhibitor of BMP signaling, was observed to block hepcidin induction by iron in vivo [15]. Soluble hemojuv-elin, another antagonist of BMP signaling, decreased hepcidin baseline expression in mice and concurrently increased liver iron content [16].

Some existing therapies may also be effective in lowering hepcidin and, thus, increasing iron supply for erythropoiesis. Anticytokine therapies, such as anti-IL-6 receptor antibody, were demonstrated to suppress hepcidin production and improve anemia in humans [17,18]. Similarly, some erythropoiesis-stimu-lating agents, such as prolyl hydroxylase inhibitors, effectively

suppressed hepcidin in animal model of inflammation [19]. Hepcidin-targeted treatment approaches are being developed, but their risks and relative clinical benefits remain to be evaluated.

Financial & competing interests disclosureElizabeta Nemeth is a cofounder and an officer of Intrinsic LifeSciences, LLC, a biotech company developing iron-related diagnostics. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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SMAD4 in iron metabolism through the positive regulation of hepcidin expression. Cell. Metab. 2, 399–409 (2005).

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3 Niederkofler V, Salie R, Arber S. Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload. J. Clin. Invest. 115, 2180–2186 (2005).

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5 Meynard D, Kautz L, Darnaud V et al. Lack of the bone morphogenetic protein BMP6 induces massive iron overload. Nat. Genet. 41(4), 478–481 (2009).

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10 Silvestri L, Guillem F, Pagani A et al. Molecular mechanisms of the defective hepcidin inhibition in TMPRSS6 mutations associated with iron-refractory iron deficiency anemia. Blood 113, 5605–5608 (2009).

11 Silvestri L, Pagani A, Nai A et al. The serine protease matriptase-2 (TMPRSS6) inhibits hepcidin activation by cleaving membrane hemojuvelin. Cell Metab. 8, 502–511 (2008).

12 Truksa J, Gelbart T, Peng H et al. Suppression of the hepcidin-encoding gene Hamp permits iron overload in mice lacking both hemojuvelin and matriptase-2/TMPRSS6. Br. J. Haematol. 147, 571–581 (2009).

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15 Yu PB, Hong CC, Sachidanandan C et al. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat. Chem. Biol. 4, 33–41 (2008).

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18 Kawabata H, Tomosugi N, Kanda J et al. Anti-interleukin 6 receptor antibody tocilizumab reduces the level of serum hepcidin in patients with multicentric Castleman’s disease. Haematologica 92, 857–858 (2007).

19 Klaus S, Arend M, Fourney P et al. Induction of erythropoiesis and iron utilization by the HIF prolyl hydroxylase inhibitor FG-4592. J. Am. Soc. Nephrol. 16 (2005).

Patent

101 Sasu BJ, Hainu M, Boone TC et al. Hepcidin, hepcidin antagonists and methods of use. Amgen Inc. Thousand Oaks, CA, USA 12/022515. 9 April 2008.

Affiliation • Elizabeta Nemeth, PhD

David Geffen School of Medicine, UCLA, 10833 LeConte Ave, CHS 37-131, Los Angeles, CA 90095, USA Tel.: +1 310 825 7499 Fax: +1 310 206 8766 enemeth@mednet.ucla.edu