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

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.     

Tmprss6 is a genetic modifier of the Hfe-hemochromatosis phenotype in mice

The hereditary hemochromatosis protein HFE promotes the expression of hepcidin, a circulating hormone produced by the liver that inhibits dietary iron absorption and macrophage iron release. HFE mutations are associated with impaired hepatic bone morphogenetic protein (BMP)/SMAD signaling for hepcidin production. TMPRSS6, a transmembrane serine protease mutated in iron-refractory iron deficiency anemia, inhibits hepcidin expression by dampening BMP/SMAD signaling. In the present study, we used genetic approaches in mice to examine the relationship between Hfe and Tmprss6 in the regulation of systemic iron homeostasis. Heterozygous loss of Tmprss6 in Hfe(-/-) mice reduced systemic iron overload, whereas homozygous loss caused systemic iron deficiency and elevated hepatic expression of hepcidin and other Bmp/Smad target genes. In contrast, neither genetic loss of Hfe nor hepatic Hfe overexpression modulated the hepcidin elevation and systemic iron deficiency of Tmprss6(-/-) mice. These results indicate that genetic loss of Tmprss6 increases Bmp/Smad signaling in an Hfe-independent manner that can restore Bmp/Smad signaling in Hfe(-/-) mice. Furthermore, these results suggest that natural genetic variation in the human ortholog TMPRSS6 might modify the clinical penetrance of HFE-associated hereditary hemochromatosis, raising the possibility that pharmacologic inhibition of TMPRSS6 could attenuate iron loading in this disorder.     

Induction of activin B by inflammatory stimuli up-regulates expression of the iron-regulatory peptide hepcidin through Smad1/5/8 signaling

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 β(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, up-regulates 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.     

Iron metabolism in hepcidin1 knockout mice in response to phenylhydrazine-induced hemolysis

Hepcidin, an iron regulatory peptide, plays a central role in the maintenance of systemic iron homeostasis by inducing the internalization and degradation of the iron exporter, ferroportin. Hepcidin expression in the liver is regulated in response to several stimuli including iron status, erythropoietic activity, hypoxia and inflammation. Hepcidin expression has been shown to be reduced in phenylhydrazine-treated mice, a mouse model of acute hemolysis. In this mouse model, hepcidin suppression was associated with increased expression of molecules involved in iron transport and recycling. The present study aims to explore whether the response to phenylhydrazine treatment is affected by hepcidin deficiency and/or the subsequently altered iron metabolism. Hepcidin1 knockout (Hamp(-/-)) and wild type mice were treated with phenylhydrazine or saline and parameters of iron homeostasis were determined 3 days after the treatment. In wild type mice, phenylhydrazine administration resulted in significantly reduced serum iron, increased tissue non-heme iron levels and suppressed hepcidin expression. The treatment was also associated with increases in membrane ferroportin protein levels and spleen heme oxygenase 1 mRNA expression. In addition, trends toward increased mRNA expression of duodenal iron transporters were also observed. In contrast, serum iron and tissue non-heme iron levels in Hamp(-/-) mice were unaffected by the treatment. Moreover, the effects of phenylhydrazine on the expression of ferroportin and duodenal iron transporters were not observed in Hamp(-/-) mice. Interestingly, mRNA levels of molecules involved in splenic heme uptake and degradation were significantly induced by Hamp disruption. In summary, our study demonstrates that the response to phenylhydrazine-induced hemolysis differs between wild type and Hamp(-/-) mice. This observation may be caused by the absence of hepcidin per se or the altered iron homeostasis induced by the lack of hepcidin in these mice.     

Stimulated erythropoiesis with secondary iron loading leads to a decrease in hepcidin despite an increase in bone morphogenetic protein 6 expression

The BMP/SMAD signalling pathway plays an important role in iron homeostasis, regulating hepcidin expression in response to body iron levels. However, the role of this pathway in the reduction in hepcidin associated with increased erythropoiesis (and secondary iron loading) is unclear. To investigate this, we established a mouse model of chronic stimulated erythropoiesis with secondary iron loading using the haemolytic agent phenylhydrazine. We then examined the expression of components of the BMP6/SMAD signalling pathway in these animals. We also examined this pathway in the Hbb(th3/+) mouse, a model of the iron loading anaemia β-thalassaemia intermedia. Increasing doses of phenylhydrazine led to a progressive increase in both liver iron levels and Bmp6 mRNA expression, but, in contrast, hepatic Hamp expression declined. The increase in Bmp6 expression was not associated with a corresponding change in the phosphorylation of hepatic SMAD1/5/8, indicating that stimulated erythropoiesis decreases the ability of BMP6 to alter SMAD phosphorylation. Increased erythropoiesis also reduces the capacity of phosphorylated SMAD (pSMAD) to induce hepcidin, as Hamp levels declined despite no changes in pSMAD1/5/8. Similar results were seen in Hbb(th3/+) mice. Thus the erythroid signal probably affects some components of BMP/SMAD signalling, but also may exert some independent effects.     

Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation

Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb^th1/th1 (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.     

Iron-deficiency anemia from matriptase-2 inactivation is dependent on the presence of functional Bmp6

Hepcidin is the master regulator of iron homeostasis. In the liver, iron-dependent hepcidin activation is regulated through Bmp6 and its membrane receptor hemojuvelin (Hjv), whereas, in response to iron deficiency, hepcidin repression seems to be controlled by a pathway involving the serine protease matriptase-2 (encoded by Tmprss6). To determine the relationship between Bmp6 and matriptase-2 pathways, Tmprss6(-/-) mice (characterized by increased hepcidin levels and anemia) and Bmp6(-/-) mice (exhibiting severe iron overload because of hepcidin deficiency) were intercrossed. We showed that loss of Bmp6 decreased hepcidin levels; increased hepatic iron; and, importantly, corrected hematologic abnormalities in Tmprss6(-/-) mice. This finding suggests that elevated hepcidin levels in patients with familial iron-refractory, iron-deficiency anemia are the result of excess signaling through the Bmp6/Hjv pathway.     

Effect of phlebotomy on hepcidin expression in hemojuvelin-mutant mice

Hemojuvelin (Hjv) is an essential component of the pathway regulating hepcidin (Hamp1) gene expression. Mice with targeted disruption of the Hjv gene (Hjv-/- mice) fail to upregulate hepatic Hamp1 expression following iron overload. The main aim of the study was to determine whether the Hjv protein is also necessary for Hamp1 downregulation. In addition, sex differences in Hamp1 expression in Hjv-/- mice were also examined. Male and female Hjv-/- mice (129SvJ background) were used for the experiments, tissue Hamp1 and Hamp2 mRNA content was determined by real-time PCR. Hepatic Hamp1 mRNA content in male Hjv-/- mice was low (0.6% of Hjv+/+ males), however, female Hjv-/- mice displayed only moderately reduced (to 17%) Hamp1 mRNA levels. Hepatic non-heme iron concentration was similar in Hjv-/- mice of both sexes. Disruption of the Hjv gene did not affect Hamp1 mRNA content in the myocardium or Hamp2 mRNA content in the pancreas. Single phlebotomy resulted in significant reduction of Hamp1 mRNA in both male and female Hjv+/+ mice (to 17% and 27% of controls respectively), measured 20 h after treatment. In Hjv-/- mice, phlebotomy decreased Hamp1 mRNA content to 46% in males and to 11% in females. Bleeding also significantly decreased (to 16%) hepatic Hamp2 mRNA levels in Hjv-/- females. The obtained results indicate that the pathway mediating hepcidin downregulation by phlebotomy does not require functional hemojuvelin protein. In addition, they confirm a significant effect of sex on hepcidin gene expression.     

Expression of Rgmc, the murine ortholog of hemojuvelin gene, is modulated by development and inflammation, but not by iron status or erythropoietin

Mutations of hepcidin (HAMP) and hemo-juvelin (HJV) genes have been recently demonstrated to result in juvenile hemochromatosis. Expression of HAMP is regulated by iron status or infection, whereas regulation of HJV is yet unknown. Using quantitative real-time polymerase chain reaction, we compared expression of Hamp and Rgmc (the murine ortholog of HJV) in livers of mice treated with iron, erythropoietin, or lipopolysaccharide (LPS), as well as during fetal and postnatal development. Iron overload increased Hamp expression without effect on Rgmc mRNA. Erythropoietin decreased Hamp mRNA, but Rgmc expression was unchanged. Hamp mRNA level decreased after birth by 4 orders of magnitude, without significant changes in Rgmc expression. Administration of LPS elevated Hamp mRNA levels, while markedly decreasing hepatic Rgmc mRNA levels (to approximately 5% after 6 hours). The responses of Hamp and Rgmc were quite different and suggested that human HJV expression could be modulated by inflammation.     

Different expression pattern of hepcidin genes in the liver and pancreas of C57BL/6N and DBA/2N mice

BACKGROUND/AIMS: Male C57BL/6 and DBA/2 mice differ in their liver iron content. The aim of this study was to examine possible differences in the expression of hepcidin genes (Hamp and Hamp2) between the two strains. METHODS: Hepatic mRNAs were quantified by real-time PCR. RESULTS: Ferroportin1, transferrin receptor 2 and HAMP mRNA levels displayed no significant strain differences. However, HAMP2 mRNA levels were higher in DBA/2N mice. In both strains, HAMP2 mRNA content was sex-dependent, with higher values in female animals. Both hepatic HAMP and HAMP2 mRNA levels were elevated by iron overload, but treatment with lipopolysaccharide increased only HAMP mRNA. Lipopolysaccharide also elevated the amount of HAMP mRNA in the pancreas, while pancreatic HAMP2 mRNA levels were decreased. Sequence analysis of hepcidin amplicons from DBA/2N mice predicted an Asn-->Lys substitution at position 73 of the HAMP peptide and a Ser-->Phe substitution at position 76 of the HAMP2 peptide. CONCLUSIONS: Hepatic Hamp2 expression displays considerable strain- and sex-dependent variation. Lipopolysaccharide increases expression of Hamp both in the liver and pancreas, but Hamp2 does not respond to lipopolysaccharide treatment. The significance of the amino acid substitutions in hepcidin peptides in DBA/2N mice is at present unknown.     

Hepcidin Regulation in Wild-Type and Hfe Knockout Mice in Response to Alcohol Consumption: Evidence for an Alcohol-Induced Hypoxic Response

Background /Aims:  Expression of Hamp1 , the gene encoding the iron regulatory peptide hepcidin, is inappropriately low in HFE-associated hereditary hemochromatosis and Hfe knockout mice ( Hfe ^−/− ). Since chronic alcohol consumption is also associated with disturbances in iron metabolism, we investigated the effects of alcohol consumption on hepcidin mRNA expression in Hfe ^−/− mice.
Methods:  Hfe ^−/− and C57BL/6 (wild-type) mice were pair-fed either an alcohol liquid diet or control diet for up to 8 weeks. The mRNA levels of hepcidin and ferroportin were measured at the mRNA level by RT-PCR and protein expression of hypoxia inducible factor-1 alpha (HIF-1α) was measured by western blot.
Results:  Hamp1 mRNA expression was significantly decreased and duodenal ferroportin expression was increased in alcohol-fed wild-type mice at 8 weeks. Time course experiments showed that the decrease in hepcidin mRNA was not immediate, but was significant by 4 weeks. Consistent with the genetic defect, Hamp1 mRNA was decreased and duodenal ferroportin mRNA expression was increased in Hfe ^−/− mice fed on the control diet compared with wild-type animals and alcohol further exacerbated these effects. HIF-1α protein levels were elevated in alcohol-fed wild-type animals compared with controls.
Conclusion:  Alcohol may decrease Hamp1 gene expression independently of the HFE pathway possibly via alcohol-induced hypoxia.     

Deletion of TMPRSS6 attenuates the phenotype in a mouse model of β-thalassemia

Inappropriately low expression of the key iron regulator hepcidin (HAMP) causes iron overload in untransfused patients affected by β-thalassemia intermedia and Hamp modulation provides improvement of the thalassemic phenotype of the Hbb(th3/+) mouse. HAMP expression is activated by iron through the bone morphogenetic protein (BMP)-son of mothers against decapentaplegic signaling pathway and inhibited by ineffective erythropoiesis through an unknown "erythroid regulator." The BMP pathway is inactivated by the serine protease TMPRSS6 that cleaves the BMP coreceptor hemojuvelin. Here, we show that homozygous loss of Tmprss6 in Hbb(th3/+) mice improves anemia and reduces ineffective erythropoiesis, splenomegaly, and iron loading. All these effects are mediated by Hamp up-regulation, which inhibits iron absorption and recycling. Because Hbb(th3/+) mice lacking Tmprss6 show residual ineffective erythropoiesis, our results indicate that Tmprss6 is essential for Hamp inhibition by the erythroid regulator. We also obtained partial correction of the phenotype in Tmprss6 haploinsufficient Hbb(th3/+) male but not female mice and showed that the observed sex difference reflects an unequal balance between iron and erythropoiesis-mediated Hamp regulation. Our study indicates that preventing iron overload improves β-thalassemia and strengthens the essential role of Tmprss6 for Hamp suppression, providing a proof of concept that Tmprss6 manipulation can offer a novel therapeutic option in this condition.     

Evidence for distinct pathways of hepcidin regulation by acute and chronic iron loading in mice

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(-/-) mice showed a smaller hepcidin increase than their wild-type strain-matched controls, Bmp6(-/-) 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 wild-type levels, but a blunted increase of hepcidin was seen in Bmp6(-/-) and Hjv(-/-) mice. BMP6, whose expression is also regulated by iron, may mediate hepcidin regulation by iron stores. None of the mutant strains (except Bmp6(-/-) 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.     

Ineffective erythropoiesis in beta-thalassemia is characterized by increased iron absorption mediated by down-regulation of hepcidin and up-regulation of ferroportin

Progressive iron overload is the most salient and ultimately fatal complication of beta-thalassemia. However, little is known about the relationship among ineffective erythropoiesis (IE), the role of iron-regulatory genes, and tissue iron distribution in beta-thalassemia. We analyzed tissue iron content and iron-regulatory gene expression in the liver, duodenum, spleen, bone marrow, kidney, and heart of mice up to 1 year old that exhibit levels of iron overload and anemia consistent with both beta-thalassemia intermedia (th3/+) and major (th3/th3). Here we show, for the first time, that tissue and cellular iron distribution are abnormal and different in th3/+ and th3/th3 mice, and that transfusion therapy can rescue mice affected by beta-thalassemia major and modify both the absorption and distribution of iron. Our study reveals that the degree of IE dictates tissue iron distribution and that IE and iron content regulate hepcidin (Hamp1) and other iron-regulatory genes such as Hfe and Cebpa. In young th3/+ and th3/th3 mice, low Hamp1 levels are responsible for increased iron absorption. However, in 1-year-old th3/+ animals, Hamp1 levels rise and it is rather the increase of ferroportin (Fpn1) that sustains iron accumulation, thus revealing a fundamental role of this iron transporter in the iron overload of beta-thalassemia.     

Suppression of the hepcidin-encoding gene Hamp permits iron overload in mice lacking both hemojuvelin and matriptase-2/TMPRSS6

Hepcidin, the master regulator of enteric iron absorption, is controlled by the opposing effects of pathways activated in response to iron excess or iron attenuation. Iron excess is regulated through a pathway involving the cell surface receptor hemojuvelin ( HFE2 ) that stimulates expression of the hepcidin encoding gene ( HAMP ). Iron attenuation is countered through a pathway involving the hepatocyte-specific plasma membrane protease matriptase-2 encoded by TMPRSS6 , leading to suppression of HAMP expression. The non-redundant function of hemojuvelin and matriptase-2 has been deduced from the phenotype imparted by mutations of HFE2 and TMPRSS6 , which cause iron excess and iron deficiency respectively. Hemojuvelin is positioned to be the ideal substrate for matriptase-2. To examine the relationship between hemojuvelin and matriptase-2 in vivo , we crossed mice lacking the protease domain of matriptase-2 with mice lacking hemojuvelin. Mice lacking functional matriptase-2 and hemojuvelin exhibited low Hamp ( Hamp1 ) expression, high serum and liver iron, and high transferrin saturation. Surprisingly, the double mutant mice also exhibited lower levels of iron in the heart compared to hemojuvelin-deficient mice, demonstrating a possible cardioprotective effect resulting from the loss of matriptase-2. This phenotype is consistent with hemojuvelin being a major substrate for matriptase-2/TMPRSS6 protease activity.