A human anti-matriptase-2 antibody limits iron overload, α-globin aggregates, and splenomegaly in ß-thalassemic mice.

ABSTRACT: Iron plays a major role in the deterioration of ß-thalassemia. Indeed, the high levels of transferrin saturation and iron delivered to erythroid progenitors are associated with production of α-globin precipitates that negatively affect erythropoiesis. Matriptase-2/TMPRSS6, a membrane-bound serine protease expressed in hepatocytes, negatively modulates hepcidin production and thus is a key target to prevent iron overload in ß-thalassemia. To address safety concerns raised by the suppression of Tmprss6 by antisense oligonucleotides or small interfering RNA, we tested a fully human anti-matriptase-2 antibody, RLYB331, which blocks the protease activity of matriptase-2. When administered weekly to Hbbth3/+ mice, RLYB331 induced hepcidin expression, reduced iron loading, prevented the formation of toxic α-chain/heme aggregates, reduced ros oxygen species formation, and improved reticulocytosis and splenomegaly. To increase the effectiveness of RLYB331 in ß-thalassemia treatment even further, we administered RLYB331 in combination with RAP-536L, a ligand-trapping protein that contains the extracellular domain of activin receptor type IIB and alleviates anemia by promoting differentiation of late-stage erythroid precursors. RAP-536L alone did not prevent iron overload but significantly reduced apoptosis in the erythroid populations of the bone marrow, normalized red blood cell counts, and improved hemoglobin and hematocrit levels. Interestingly, the association of RLYB331 with RAP-536L entirely reversed the ß-thalassemia phenotype in Hbbth3/+ mice and simultaneously corrected iron overload, ineffective erythropoiesis, splenomegaly, and hematological parameters, suggesting that a multifunctional molecule consisting of the fusion of RLYB331 with luspatercept (human version of RAP-536L) would allow administration of a single medication addressing simultaneously the different pathophysiological aspects of ß-thalassemia.

ATGL-dependent white adipose tissue lipolysis controls hepatocyte PPARα activity.

In hepatocytes, peroxisome proliferator-activated receptor α (PPARα) orchestrates a genomic and metabolic response required for homeostasis during fasting. This includes the biosynthesis of ketone bodies and of fibroblast growth factor 21 (FGF21). Here we show that in the absence of adipose triglyceride lipase (ATGL) in adipocytes, ketone body and FGF21 production is impaired upon fasting. Liver gene expression analysis highlights a set of fasting-induced genes sensitive to both ATGL deletion in adipocytes and PPARα deletion in hepatocytes. Adipose tissue lipolysis induced by activation of the ß3-adrenergic receptor also triggers such PPARα-dependent responses not only in the liver but also in brown adipose tissue (BAT). Intact PPARα activity in hepatocytes is required for the cross-talk between adipose tissues and the liver during fat mobilization.

Regulators of hepcidin expression.

Iron, an essential nutrient, is required for many biological processes but is also toxic in excess. The lack of a mechanism to excrete excess iron makes it crucial for the body to regulate the amount of iron absorbed from the diet. This regulation is mediated by the hepatic hormone hepcidin. Hepcidin also controls iron release from macrophages that recycle iron and from hepatocytes that store iron. Hepcidin binds to the only known iron export protein, ferroportin, inducing its internalization and degradation and thus limiting the amount of iron released into the plasma. Important regulators of hepcidin, and therefore of systemic iron homeostasis, include plasma iron concentrations, body iron stores, infection and inflammation, hypoxia and erythropoiesis, and, to a lesser extent, testosterone. Dysregulation of hepcidin production contributes to the pathogenesis of many iron disorders: hepcidin deficiency causes iron overload in hereditary hemochromatosis and non-transfused ß-thalassemia, whereas overproduction of hepcidin is associated with iron-restricted anemias seen in patients with chronic inflammatory diseases and inherited iron-refractory iron-deficiency anemia. The present review summarizes our current understanding of the molecular mechanisms and signaling pathways contributing to hepcidin regulation by these factors and highlights the issues that still need clarification.

Deletion of BMP6 worsens the phenotype of HJV-deficient mice and attenuates hepcidin levels reached after LPS challenge.

Lack of either bone morphogenetic protein 6 (BMP6) or the BMP coreceptor hemojuvelin (HJV) in mice leads to a similar phenotype with hepcidin insufficiency, hepatic iron loading, and extrahepatic iron accumulation in males. This is consistent with the current views that HJV is a coreceptor for BMP6 in hepatocytes. To determine whether BMP6 and HJV may also signal to hepcidin independently of each other, we intercrossed Hjv-/- and Bmp6-/- mice and compared the phenotype of animals of the F2 progeny. Loss of Bmp6 further repressed Smad signaling and hepcidin expression in the liver of Hjv-/- mice of both sexes, and led to iron accumulation in the pancreas and the heart of females. These data suggest that, in Hjv-/- females, Bmp6 can provide a signal adequate to maintain hepcidin to a level sufficient to avoid extrahepatic iron loading. We also examined the impact of Bmp6 and/or Hjv deletion on the regulation of hepcidin by inflammation. Our data show that lack of 1 or both molecules does not prevent induction of hepcidin by lipopolysaccharide (LPS). However, BMP/Smad signaling in unchallenged animals is determinant for the level of hepcidin reached after stimulation, which is consistent with a synergy between interleukin 6/STAT3 and BMP/SMAD signaling in regulating hepcidin during inflammation.

Limiting hepatic Bmp-Smad signaling by matriptase-2 is required for erythropoietin-mediated hepcidin suppression in mice.

Hepcidin, the main regulator of iron homeostasis, is repressed when erythropoiesis is acutely stimulated by erythropoietin (EPO) to favor iron supply to maturing erythroblasts. Erythroferrone (ERFE) has been identified as the erythroid regulator that inhibits hepcidin in stress erythropoiesis. A powerful hepcidin inhibitor is the serine protease matriptase-2, encoded by TMPRSS6, whose mutations cause iron refractory iron deficiency anemia. Because this condition has inappropriately elevated hepcidin in the presence of high EPO levels, a role is suggested for matriptase-2 in EPO-mediated hepcidin repression. To investigate the relationship between EPO/ERFE and matriptase-2, we show that EPO injection induces Erfe messenger RNA expression but does not suppress hepcidin in Tmprss6 knockout (KO) mice. Similarly, wild-type (WT) animals, in which the bone morphogenetic protein-mothers against decapentaplegic homolog (Bmp-Smad) pathway is upregulated by iron treatment, fail to suppress hepcidin in response to EPO. To further investigate whether the high level of Bmp-Smad signaling of Tmprss6 KO mice counteracts hepcidin suppression by EPO, we generated double KO Bmp6-Tmprss6 KO mice. Despite having Bmp-Smad signaling and hepcidin levels that are similar to WT mice under basal conditions, double KO mice do not suppress hepcidin in response to EPO. However, pharmacologic downstream inhibition of the Bmp-Smad pathway by dorsomorphin, which targets the BMP receptors, improves the hepcidin responsiveness to EPO in Tmprss6 KO mice. We concluded that the function of matriptase-2 is dominant over that of ERFE and is essential in facilitating hepcidin suppression by attenuating the BMP-SMAD signaling.

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.

MicroRNA-130a is up-regulated in mouse liver by iron deficiency and targets the bone morphogenetic protein (BMP) receptor ALK2 to attenuate BMP signaling and hepcidin transcription.

Systemic iron balance is controlled by the liver peptide hormone hepcidin, which is transcriptionally regulated by the bone morphogenetic protein (BMP)-SMAD pathway. In iron deficiency, liver BMP-SMAD signaling and hepcidin are suppressed as a compensatory mechanism to increase iron availability. MicroRNAs are small regulatory RNAs that have an increasingly recognized role in many biologic processes but are only recently implicated in iron homeostasis regulation. Here, we demonstrate that liver expression of the microRNA miR-130a is up-regulated by iron deficiency in mice. We identify the BMP6-SMAD signaling pathway as a functional target of miR-130a in hepatoma-derived Hep3B cells. Although the TGF-ß/BMP common mediator SMAD4 was previously reported to be an miR-130a target to inhibit TGF-ß signaling, we do not confirm SMAD4 as an miR-130a target in our biologic system. Instead, we determine that the BMP type I receptor ALK2 is a novel target of miR-130a and that miR-130a binds to two specific sites in the 3'-untranslated region to reduce ALK2 mRNA stability. Moreover, we show in mice that the increased liver miR-130a during iron deficiency is associated with reduced liver Alk2 mRNA levels. Finally, we demonstrate that down-regulation of ALK2 by miR-130a has a functional effect to inhibit BMP6-induced hepcidin transcription in Hep3B cells. Our data suggest that iron deficiency increases liver miR-130a, which, by targeting ALK2, may contribute to reduce BMP-SMAD signaling, suppress hepcidin synthesis, and thereby promote iron availability.

The role of TMPRSS6/matriptase-2 in iron regulation and anemia.

Matriptase-2, encoded by the TMPRSS6 gene, is a member of the type II transmembrane serine protease family. Matriptase-2 has structural and enzymatic similarities to matriptase-1, which has been implicated in cancer progression. Matriptase-2 was later established to be essential in iron homeostasis based on the phenotypes of iron-refractory iron deficiency anemia identified in mouse models as well as in human patients with TMPRSS6 mutations. TMPRSS6 is expressed mainly in the liver and negatively regulates the production of hepcidin, the systemic iron regulatory hormone. This review focuses on the current understanding of matriptase-2 biochemistry, and its role in iron metabolism and cancer progression. In light of recent investigations, the function of matriptase-2 in hepcidin regulation, how it is being regulated, as well as the therapeutic potential of matriptase-2 are also discussed.

The liver: conductor of systemic iron balance.

Iron is a micronutrient essential for almost all organisms: bacteria, plants, and animals. It is a metal that exists in multiple redox states, including the divalent ferrous (Fe(2+)) and the trivalent ferric (Fe(3+)) species. The multiple oxidation states of iron make it excellent for electron transfer, allowing iron to be selected during evolution as a cofactor for many proteins involved in central cellular processes including oxygen transport, mitochondrial respiration, and DNA synthesis. However, the redox cycling of ferrous and ferric iron in the presence of H2O2, which is physiologically present in the cells, also leads to the production of free radicals (Fenton reaction) that can attack and damage lipids, proteins, DNA, and other cellular components. To meet the physiological needs of the body, but to prevent cellular damage by iron, the amount of iron in the body must be tightly regulated. Here we review how the liver is the central conductor of systemic iron balance and show that this central role is related to the secretion of a peptide hormone hepcidin by hepatocytes. We then review how the liver receives and integrates the many signals that report the body's iron needs to orchestrate hepcidin production and maintain systemic iron homeostasis.

Inflammation regulates TMPRSS6 expression via STAT5.

TMPRSS6 is a regulated gene, with a crucial role in the regulation of iron homeostasis by inhibiting hepcidin expression. The main regulator of iron homeostasis, the antimicrobial peptide hepcidin, which also has a role in immunity, is directly upregulated by inflammation. In this study, we analyzed whether inflammation is also a modulator of TMPRSS6 expression in vitro and in vivo and we determined the mechanism of this regulation A Human Hepatoma cell line was treated with interleukin-6 and mice were injected with lipopolysaccharide and TMPRSS6 expression and the regulatory mechanism were addressed. In this study, we demonstrate that inflammation downregulates TMPRSS6 expression in vitro and in vivo. The downregulation of Tmprss6 by inflammation in mice is not dependent on the Bmp-Smad pathway but occurs through a decrease in Stat5 phosphorylation. Moreover, Stat5 positively regulates Tmprss6 expression directly by binding to a Stat5 element located on the Tmprss6 promoter. Importantly, our results highlight the functional role of inflammatory modulation of TMPRSS6 expression in the regulation of hepcidin. TMPRSS6 inhibition via decreased STAT5 phosphorylation may be an additional mechanism by which inflammation stimulates hepcidin expression to regulate iron homeostasis and immunity.

A novel validated enzyme-linked immunosorbent assay to quantify soluble hemojuvelin in mouse serum.

Hemojuvelin is a critical regulator of hepcidin expression and can be cleaved by proteases to form soluble hemojuvelin. Soluble hemojuvelin has been recently identified in human serum but the presence and quantity of soluble hemojuvelin in mouse serum is unknown. We developed a two-site enzyme-linked immunosorbent assay using a monoclonal anti-hemojuvelin as the capture antibody and a biotinylated polyclonal anti-hemojuvelin as the detection antibody to quantify the levels of soluble hemojuvelin in mouse serum. We validated this assay using cell-conditioned media and serum from Hemojuvelin-null and Bone morphogenetic protein 6-null mice. We also used this validated assay to measure serum soluble hemojuvelin concentrations in mice receiving an acute low iron or high iron treatment. This two-site enzyme-linked immunosorbent assay was highly specific for mouse hemojuvelin, with a lower limit of detection at 13.2-26.8 ng/mL of soluble hemojuvelin in mouse serum. The median serum soluble hemojuvelin concentration in wild-type C57BL/6J mice was 57.9 ± 22 ng/mL, which is 4- to 20-fold less than that reported in healthy human volunteers. After acute low iron diet treatment in these mice, serum soluble hemojuvelin levels were increased and correlated with lowered serum iron levels and decreased hepatic hepcidin expression. An acute high iron diet in wild-type mice or chronically iron-overloaded Bone morphogenetic protein 6-null mice did not significantly lower serum soluble hemojuvelin concentrations. Here we report reliable quantitation of mouse serum soluble hemojuvelin using a novel and validated enzyme-linked immunosorbent assay. This assay may provide a useful tool to elucidate the source and physiological role of serum soluble hemojuvelin in hepcidin regulation and iron metabolism using well-established mouse models of iron-related disorders.

Iron regulation of hepcidin despite attenuated Smad1,5,8 signaling in mice without transferrin receptor 2 or Hfe.

BACKGROUND & AIMS: HFE and transferrin receptor 2 (TFR2) are each necessary for the normal relationship between body iron status and liver hepcidin expression. In murine Hfe and Tfr2 knockout models of hereditary hemochromatosis (HH), signal transduction to hepcidin via the bone morphogenetic protein 6 (Bmp6)/Smad1,5,8 pathway is attenuated. We examined the effect of dietary iron on regulation of hepcidin expression via the Bmp6/Smad1,5,8 pathway using mice with targeted disruption of Tfr2, Hfe, or both genes. METHODS: Hepatic iron concentrations and messenger RNA expression of Bmp6 and hepcidin were compared with wild-type mice in each of the HH models on standard or iron-loading diets. Liver phospho-Smad (P-Smad)1,5,8 and Id1 messenger RNA levels were measured as markers of Bmp/Smad signaling. RESULTS: Whereas Bmp6 expression was increased, liver hepcidin and Id1 expression were decreased in each of the HH models compared with wild-type mice. Each of the HH models also showed attenuated P-Smad1,5,8 levels relative to liver iron status. Mice with combined Hfe/Tfr2 disruption were most affected. Dietary iron loading increased hepcidin and Id1 expression in each of the HH models. Compared with wild-type mice, HH mice demonstrated attenuated (Hfe knockout) or no increases in P-Smad1,5,8 levels in response to dietary iron loading. CONCLUSIONS: These observations show that Tfr2 and Hfe are each required for normal signaling of iron status to hepcidin via the Bmp6/Smad1,5,8 pathway. Mice with combined loss of Hfe and Tfr2 up-regulate hepcidin in response to dietary iron loading without increases in liver Bmp6 messenger RNA or steady-state P-Smad1,5,8 levels.

Regulation of TMPRSS6 by BMP6 and iron in human cells and mice.

Mutations in transmembrane protease, serine 6 (TMPRSS6), encoding matriptase-2, are responsible for the familial anemia disorder iron-refractory iron deficiency anemia (IRIDA). Patients with IRIDA have inappropriately elevated levels of the iron regulatory hormone hepcidin, suggesting that TMPRSS6 is involved in negatively regulating hepcidin expression. Hepcidin is positively regulated by iron via the bone morphogenetic protein (BMP)-SMAD signaling pathway. In this study, we investigated whether BMP6 and iron also regulate TMPRSS6 expression. Here we demonstrate that, in vitro, treatment with BMP6 stimulates TMPRSS6 expression at the mRNA and protein levels and leads to an increase in matriptase-2 activity. Moreover, we identify that inhibitor of DNA binding 1 is the key element of the BMP-SMAD pathway to regulate TMPRSS6 expression in response to BMP6 treatment. Finally, we show that, in mice, Tmprss6 mRNA expression is stimulated by chronic iron treatment or BMP6 injection and is blocked by injection of neutralizing antibody against BMP6. Our results indicate that BMP6 and iron not only induce hepcidin expression but also induce TMPRSS6, a negative regulator of hepcidin expression. Modulation of TMPRSS6 expression could serve as a negative feedback inhibitor to avoid excessive hepcidin increases by iron to help maintain tight homeostatic balance of systemic iron levels.

Serum and liver iron differently regulate the bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway in mice.

UNLABELLED: The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is a central regulator of hepcidin expression and systemic iron balance. However, the molecular mechanisms by which iron is sensed to regulate BMP6-SMAD signaling and hepcidin expression are unknown. Here we examined the effects of circulating and tissue iron on Bmp6-Smad pathway activation and hepcidin expression in vivo after acute and chronic enteral iron administration in mice. We demonstrated that both transferrin saturation and liver iron content independently influence hepcidin expression. Although liver iron content is independently positively correlated with hepatic Bmp6 messenger RNA (mRNA) expression and overall activation of the Smad1/5/8 signaling pathway, transferrin saturation activates the downstream Smad1/5/8 signaling cascade, but does not induce Bmp6 mRNA expression in the liver. Hepatic inhibitory Smad7 mRNA expression is increased by both acute and chronic iron administration and mirrors overall activation of the Smad1/5/8 signaling cascade. In contrast to the Smad pathway, the extracellular signal-regulated kinase 1 and 2 (Erk1/2) mitogen-activated protein kinase (Mapk) signaling pathway in the liver is not activated by acute or chronic iron administration in mice. CONCLUSION: Our data demonstrate that the hepatic Bmp6-Smad signaling pathway is differentially activated by circulating and tissue iron to induce hepcidin expression, whereas the hepatic Erk1/2 signaling pathway is not activated by iron in vivo.

Iron overload induces BMP6 expression in the liver but not in the duodenum.

BACKGROUND: The bone morphogenetic protein BMP6 regulates hepcidin production by the liver. However, it is not yet known whether BMP6 derives from the liver itself or from other sources such as the small intestine, as has been recently suggested. This study was aimed at investigating the source of BMP6 further. DESIGN AND METHODS: We used three different strains of mice (C57BL/6, DBA/2, and 129/Sv) with iron overload induced either by an iron-enriched diet or by inactivation of the Hfe gene. We examined Bmp6 expression at both the mRNA (by quantitative PCR) and protein (by immunohistochemistry and Western blotting analyses) levels. RESULTS: We showed that iron overload induces Bmp6 mRNA expression in the liver but not in the duodenum of these mice. Bmp6 is also detected by immunohistochemistry in liver tissue sections of mice with iron overload induced either by an iron-enriched diet or by inactivation of the Hfe gene, but not in liver tissue sections from iron-loaded Bmp6-deficient mice. Bmp6 in the duodenum was below immunodetection threshold, thus confirming quantitative PCR data. Lack of specificity of available antibodies together with slight heterogeneity between 129 substrains may account for the differences with previously published data. CONCLUSIONS: Our data strongly support the importance of liver BMP6 for regulation of iron metabolism. Indeed, they demonstrate that intestinal Bmp6 expression is modulated by iron neither at the mRNA nor at the protein level.

BMP6 treatment compensates for the molecular defect and ameliorates hemochromatosis in Hfe knockout mice.

BACKGROUND & AIMS: Abnormal hepcidin regulation is central to the pathogenesis of HFE hemochromatosis. Hepatic bone morphogenetic protein 6 (BMP6)-SMAD signaling is a main regulatory mechanism controlling hepcidin expression, and this pathway was recently shown to be impaired in Hfe knockout (Hfe(-/-)) mice. To more definitively determine whether HFE regulates hepcidin expression through an interaction with the BMP6-SMAD signaling pathway, we investigated whether hepatic Hfe overexpression activates the BMP6-SMAD pathway to induce hepcidin expression. We then investigated whether excess exogenous BMP6 administration overcomes the BMP6-SMAD signaling impairment and ameliorates hemochromatosis in Hfe(-/-) mice. METHODS: The BMP6-SMAD pathway and the effects of neutralizing BMP6 antibody were examined in Hfe transgenic mice (Hfe Tg) compared with wild-type (WT) mice. Hfe(-/-) and WT mice were treated with exogenous BMP6 and analyzed for hepcidin expression and iron parameters. RESULTS: Hfe Tg mice exhibited hepcidin excess and iron deficiency anemia. Hfe Tg mice also exhibited increased hepatic BMP6-SMAD target gene expression compared with WT mice, whereas anti-BMP6 antibody administration to Hfe Tg mice improved the hepcidin excess and iron deficiency. In Hfe(-/-) mice, supraphysiologic doses of exogenous BMP6 improved hepcidin deficiency, reduced serum iron, and redistributed tissue iron to appropriate storage sites. CONCLUSIONS: HFE interacts with the BMP6-SMAD signaling pathway to regulate hepcidin expression, but HFE is not necessary for hepcidin induction by BMP6. Exogenous BMP6 treatment in mice compensates for the molecular defect underlying Hfe hemochromatosis, and BMP6-like agonists may have a role as an alternative therapeutic strategy for this disease.

BMP/Smad signaling is not enhanced in Hfe-deficient mice despite increased Bmp6 expression.

Impaired regulation of hepcidin expression in response to iron loading appears to be the pathogenic mechanism for hereditary hemochromatosis. Iron normally induces expression of the BMP6 ligand, which, in turn, activates the BMP/Smad signaling cascade directing hepcidin expression. The molecular function of the HFE protein, involved in the most common form of hereditary hemochromatosis, is still unknown. We have used Hfe-deficient mice of different genetic backgrounds to test whether HFE has a role in the signaling cascade induced by BMP6. At 7 weeks of age, these mice have accumulated iron in their liver and have increased Bmp6 mRNA and protein. However, in contrast to mice with secondary iron overload, levels of phosphorylated Smads 1/5/8 and of Id1 mRNA, both indicators of BMP signaling, are not significantly higher in the liver of these mice than in wild-type livers. As a consequence, hepcidin mRNA levels in Hfe-deficient mice are similar or marginally reduced, compared with 7-week-old wild-type mice. The inappropriately low levels of Id1 and hepcidin mRNA observed at weaning further suggest that Hfe deficiency triggers iron overload by impairing hepatic Bmp/Smad signaling. HFE therefore appears to facilitate signal transduction induced by the BMP6 ligand.