Hfe acts in hepatocytes to prevent hemochromatosis

Hereditary hemochromatosis (HH) is a prevalent, potentially fatal disorder of iron metabolism hallmarked by intestinal hyperabsorption of iron, hyperferremia, and hepatic iron overload. In both humans and mice, type I HH is associated with mutations in the broadly expressed HFE/Hfe gene. To identify where Hfe acts to prevent HH, we generated mice with tissue-specific Hfe ablations. This work demonstrates that local Hfe expression in hepatocytes serves to maintain physiological iron homeostasis, answering a long-standing question in medicine and explaining earlier clinical observations.     

Increased glucose disposal and AMP-dependent kinase signaling in a mouse model of hemochromatosis

Hereditary hemochromatosis is an inherited disorder of increased iron absorption that can result in cirrhosis, diabetes, and other morbidities. We have investigated the mechanisms underlying supranormal glucose tolerance despite decreased insulin secretion in a mouse model of hemochromatosis with deletion of the hemochromatosis gene (Hfe(-/-)). Hfe(-/-) mice on 129Sv or C57BL/6J backgrounds have decreased glucose excursions after challenge compared with controls. In the C57BL/6J/ Hfe(-/-), for example, incremental area under the glucose curve is reduced 52% (p < 0.001) despite decreased serum insulin, and homeostasis model assessment insulin resistance is decreased 50% (p < 0.05). When studied by the euglycemic clamp technique 129Sv/Hfe(-/-) mice exhibit a 20% increase in glucose disposal (p < 0.05) at submaximal insulin but no increase at maximal insulin compared with wild types. [1,2-(13)C]D-glucose clearance from plasma is significantly increased in Hfe(-/-) mice (19%, p < 0.05), and lactate derived from glycolysis is elevated 5.1-fold in Hfe(-/-) mice (p < 0.0001). Basal but not insulin-stimulated glucose uptake is elevated in isolated soleus muscle from Hfe(-/-) mice (p < 0.03). Compared with controls Hfe(-/-) mice exhibit no differences in serum lipid, insulin, glucagon, or thyroid hormone levels; adiponectin levels are elevated 41% (p < 0.05), and the adiponectin message in adipocytes is increased 83% (p = 0.04). Insulin action measured by phosphorylation of Akt is not enhanced in muscle, but phosphorylation of AMP-dependent kinase is increased. We conclude that supranormal glucose tolerance in iron overload is characterized by increased glucose disposal that does not result from increased insulin action. Instead, the Hfe(-/-) mice demonstrate increased adiponectin levels and activation of AMP-dependent kinase.     

Iron-mediated inhibition of mitochondrial manganese uptake mediates mitochondrial dysfunction in a mouse model of hemochromatosis

Previous phenotyping of glucose homeostasis and insulin secretion in a mouse model of hereditary hemochromatosis (Hfe(-/-)) and iron overload suggested mitochondrial dysfunction. Mitochondria from Hfe(-/-) mouse liver exhibited decreased respiratory capacity and increased lipid peroxidation. Although the cytosol contained excess iron, Hfe(-/-) mitochondria contained normal iron but decreased copper, manganese, and zinc, associated with reduced activities of copper-dependent cytochrome c oxidase and manganese-dependent superoxide dismutase (MnSOD). The attenuation in MnSOD activity was due to substantial levels of unmetallated apoprotein. The oxidative damage in Hfe(-/-) mitochondria is due to diminished MnSOD activity, as manganese supplementation of Hfe(-/-) mice led to enhancement of MnSOD activity and suppressed lipid peroxidation. Manganese supplementation also resulted in improved insulin secretion and glucose tolerance associated with increased MnSOD activity and decreased lipid peroxidation in islets. These data suggest a novel mechanism of iron-induced cellular dysfunction, namely altered mitochondrial uptake of other metal ions.     

Hfe deficiency impairs pulmonary neutrophil recruitment in response to inflammation

Regulation of iron homeostasis and the inflammatory response are tightly linked to protect the host from infection. Here we investigate how imbalanced systemic iron homeostasis in a murine disease model of hereditary hemochromatosis (Hfe(-/-) mice) affects the inflammatory responses of the lung. We induced acute pulmonary inflammation in Hfe(-/-) and wild-type mice by intratracheal instillation of 20 μg of lipopolysaccharide (LPS) and analyzed local and systemic inflammatory responses and iron-related parameters. We show that in Hfe(-/-) mice neutrophil recruitment to the bronchoalveolar space is attenuated compared to wild-type mice although circulating neutrophil numbers in the bloodstream were elevated to similar levels in Hfe(-/-) and wild-type mice. The underlying molecular mechanisms are likely multifactorial and include elevated systemic iron levels, alveolar macrophage iron deficiency and/or hitherto unexplored functions of Hfe in resident pulmonary cell types. As a consequence, pulmonary cytokine expression is out of balance and neutrophils fail to be recruited efficiently to the bronchoalveolar compartment, a process required to protect the host from infections. In conclusion, our findings suggest a novel role for Hfe and/or imbalanced iron homeostasis in the regulation of the inflammatory response in the lung and hereditary hemochromatosis.     

Quantitative evaluation of expression of iron-metabolism genes in ceruloplasmin-deficient mice

Aceruloplasminemia is an autosomal recessive disorder caused by mutations in the ceruloplasmin (CP) gene, and is characterized by a unique combination of neurovisceral iron overload and iron deficiency anemia. We generated CP-deficient (CP(-/-)) mice to investigate the functional involvement of CP in iron metabolism. The mice showed a marked iron overload in the liver and mild iron deficiency anemia. We examined the expression of iron-metabolism genes in the duodenum and liver using TaqMan RT-PCR. The divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), and hephaestin (HEPH) genes were not up-regulated in the duodenum from CP(-/-) mice. These data suggest that the mechanism of hepatic iron overload in aceruloplasminemia is quite different from that in hemochromatoses and atransferrinemia. In the liver, CP(-/-) mice showed no increase of gene expression for DMT1 and transferrin receptors (TFR and TFR2), indicating that none of the known pathways of iron uptake is activated in hepatocytes of CP(-/-) mice. This result supports the hypothesis that CP mainly acts to release iron from cells in the liver.     

Distinct requirements for Hfe in basal and induced hepcidin levels in iron overload and inflammation

Hepcidin is a negative regulator of iron absorption produced mainly by the liver in response to changes in iron stores and inflammation, and its levels have been shown to regulate the intestinal basolateral iron transporter ferroportin1 (Fp1). Hereditary hemochromatosis patients and Hfe-deficient mice show inappropriate expression of hepcidin but, in apparent contradiction, still retain the ability to regulate iron absorption in response to alterations of iron metabolism. To further understand the molecular relationships among Hfe, hepcidin, and Fp1, we investigated hepcidin and Fp1 regulation in Hfe-deficient mice (Hfe-/- and beta2m-/-) in response to iron deprivation, iron loading, and acute inflammation. We found that whereas basal hepcidin levels were manifestly dependent on the presence of Hfe and on the mouse background, all Hfe-deficient mice were still able to regulate hepcidin in situations of altered iron homeostasis. In the liver, Fp1 was modulated in opposite directions by iron and LPS, and its regulation in Hfe-deficient mice was similar to that observed in wild-type mice. In addition, we found that iron-deprived mice were able to mount a robust response after LPS challenge and that Toll-like receptor 4 (TLR-4)-deficient mice fail to regulate hepcidin expression in response to LPS. In conclusion, these results suggest that although Hfe is necessary for the establishment of hepcidin basal levels, it is dispensable for hepcidin regulation through both the iron-sensing and inflammatory pathways, and hepatic Fp1 regulation is largely independent of hepcidin and Hfe. The inflammatory pathway overrides the iron-sensing pathway and is TLR-4 dependent.     

Accelerated CCl4-induced liver fibrosis in Hjv-/- mice, associated with an oxidative burst and precocious profibrogenic gene expression

Hereditary hemochromatosis is commonly associated with liver fibrosis. Likewise, hepatic iron overload secondary to chronic liver diseases aggravates liver injury. To uncover underlying molecular mechanisms, hemochromatotic hemojuvelin knockout (Hjv-/-) mice and wild type (wt) controls were intoxicated with CCl₄. Hjv-/- mice developed earlier (by 2-4 weeks) and more acute liver damage, reflected in dramatic levels of serum transaminases and ferritin and the development of severe coagulative necrosis and fibrosis. These responses were associated with an oxidative burst and early upregulation of mRNAs encoding α1-(I)-collagen, the profibrogenic cytokines TGF-β1, endothelin-1 and PDGF and, notably, the iron-regulatory hormone hepcidin. Hence, CCl4-induced liver fibrogenesis was exacerbated and progressed precociously in Hjv−/− animals. Even though livers of naïve Hjv−/− mice were devoid of apparent pathology, they exhibited oxidative stress and immunoreactivity towards α-SMA antibodies, a marker of hepatic stellate cells activation. Furthermore, they expressed significantly higher (2–3 fold vs. wt, p<0.05) levels of α1-(I)-collagen, TGF-β1, endothelin-1 and PDGF mRNAs, indicative of early fibrogenesis. Our data suggest that hepatic iron overload in parenchymal cells promotes oxidative stress and triggers premature profibrogenic gene expression, contributing to accelerated onset and precipitous progression of liver fibrogenesis.     

Regulatory networks for the control of body iron homeostasis and their dysregulation in HFE mediated hemochromatosis

Although the recent identification of several genes has extended our knowledge on the maintenance of body iron homeostasis, their tissue specific expression patterns and the underlying regulatory networks are poorly understood. We studied C57black/Sv129 mice and HFE knockout (HFE -/-) variants thereof as a model for hemochromatosis, and investigated the expression of iron metabolism genes in the duodenum, liver, and kidney as a function of dietary iron challenge. In HFE +/+ mice dietary iron supplementation increased hepatic expression of hepcidin which was paralleled by decreased iron regulatory protein (IRP) activity, and reduced expression of divalent metal transporter-1 (DMT-1) and duodenal cytochrome b (Dcytb) in the enterocyte. In HFE -/- mice hepcidin formation was diminished upon iron challenge which was associated with decreased hepatic transferrin receptor (TfR)-2 levels. Accordingly, HFE -/- mice presented with high duodenal Dcytb and DMT-1 levels, and increased IRP and TfR expression, suggesting iron deficiency in the enterocyte and increased iron absorption. In parallel, HFE -/- resulted in reduced renal expression of Dcytb and DMT-1. Our data suggest that the feed back regulation of duodenal iron absorption by hepcidin is impaired in HFE -/- mice, a model for genetic hemochromatosis. This change may be linked to inappropriate iron sensing by the liver based on decreased TfR-2 expression, resulting in reduced circulating hepcidin levels and an inappropriate up-regulation of Dcytb and DMT-1 driven iron absorption. In addition, iron excretion/reabsorption by the kidneys may be altered, which may aggravate progressive iron overload.     

The transferrin receptor modulates Hfe-dependent regulation of hepcidin expression

Hemochromatosis is caused by mutations in HFE, a protein that competes with transferrin (TF) for binding to transferrin receptor 1 (TFR1). We developed mutant mouse strains to gain insight into the role of the Hfe/Tfr1 complex in regulating iron homeostasis. We introduced mutations into a ubiquitously expressed Tfr1 transgene or the endogenous Tfr1 locus to promote or prevent the Hfe/Tfr1 interaction. Under conditions favoring a constitutive Hfe/Tfr1 interaction, mice developed iron overload attributable to inappropriately low expression of the hormone hepcidin. In contrast, mice carrying a mutation that interferes with the Hfe/Tfr1 interaction developed iron deficiency associated with inappropriately high hepcidin expression. High-level expression of a liver-specific Hfe transgene in Hfe-/- mice was also associated with increased hepcidin production and iron deficiency. Together, these models suggest that Hfe induces hepcidin expression when it is not in complex with Tfr1.     

Genetic modification of iron metabolism in mice affects the gut microbiota

The composition of the gut microbiota is affected by environmental factors as well as host genetics. Iron is one of the important elements essential for bacterial growth, thus we hypothesized that changes in host iron homeostasis, may affect the luminal iron content of the gut and thereby the composition of intestinal bacteria. The iron regulatory protein 2 (Irp2) and one of the genes mutated in hereditary hemochromatosis Hfe , are both proteins involved in the regulation of systemic iron homeostasis. To test our hypothesis, fecal metal content and a selected spectrum of the fecal microbiota were analyzed from Hfe-/-, Irp2-/- and their wild type control mice. Elevated levels of iron as well as other minerals in feces of Irp2-/- mice compared to wild type and Hfe-/- mice were observed. Interestingly significant variation in the general fecal-bacterial population-patterns was observed between Irp2-/- and Hfe-/- mice. Furthermore the relative abundance of five species, mainly lactic acid bacteria, was significantly different among the mouse lines. Lactobacillus (L.) murinus and L. intestinalis were highly abundant in Irp2-/- mice, Enterococcus faecium species cluster and a species most similar to Olsenella were highly abundant in Hfe-/- mice and L. johnsonii was highly abundant in the wild type mice. These results suggest that deletion of iron metabolism genes in the mouse host affects the composition of its intestinal bacteria. Further studying the relationship between gut microbiota and genetic mutations affecting systemic iron metabolism in human should lead to clinical implications.     

Ca2+ channel blockers reverse iron overload by a new mechanism via divalent metal transporter-1

Hereditary hemochromatosis and transfusional iron overload are frequent clinical conditions associated with progressive iron accumulation in parenchymal tissues, leading to eventual organ failure. We have discovered a new mechanism to reverse iron overload-pharmacological modulation of the divalent metal transporter-1 (DMT-1). DMT-1 mediates intracellular iron transport during the transferrin cycle and apical iron absorption in the duodenum. Its additional functions in iron handling in the kidney and liver are less well understood. We show that the L-type calcium channel blocker nifedipine increases DMT-1-mediated cellular iron transport 10- to 100-fold at concentrations between 1 and 100 microM. Mechanistically, nifedipine causes this effect by prolonging the iron-transporting activity of DMT-1. We show that nifedipine mobilizes iron from the liver of mice with primary and secondary iron overload and enhances urinary iron excretion. Modulation of DMT-1 function by L-type calcium channel blockers emerges as a new pharmacological therapy for the treatment of iron overload disorders.     

Circulating gastrin is increased in hemochromatosis

Gastric acid production is important in intestinal iron absorption. The peptide hormone gastrin exists in both amidated and non-amidated forms, which stimulate and potentiate gastric acid secretion, respectively. Since non-amidated gastrins require ferric ions for biological activity in vitro, this study investigated the connection between iron status and gastrin by measurement of circulating gastrin concentrations in mice and humans with hemochromatosis. Gastrin concentrations are increased in the plasma and gastric mucosa of Hfe(-/-) mice, and in the sera of humans with HFE-related hemochromatosis. The discovery of a relationship between iron status and circulating gastrin concentrations opens a new perspective on the mechanisms of iron homeostasis.     

Smad6 and Smad7 are co-regulated with hepcidin in mouse models of iron overload

The inhibitory Smad7 acts as a critical suppressor of hepcidin, the major regulator of systemic iron homeostasis. In this study we define the mRNA expression of the two functionally related Smad proteins, Smad6 and Smad7, within pathways known to regulate hepcidin levels. Using mouse models for hereditary hemochromatosis (Hfe-, TfR2-, Hfe/TfR2-, Hjv- and hepcidin1-deficient mice) we show that hepcidin, Smad6 and Smad7 mRNA expression is coordinated in such a way that it correlates with the activity of the Bmp/Smad signaling pathway rather than with liver iron levels. This regulatory circuitry is disconnected by iron treatment of Hfe ?/? and Hfe/TfR2 mice that significantly increases hepatic iron levels as well as hepcidin, Smad6 and Smad7 mRNA expression but fails to augment pSmad1/5/8 levels. This suggests that additional pathways contribute to the regulation of hepcidin, Smad6 and Smad7 under these conditions which do not require Hfe.     

Disorders associated with systemic or local iron overload: from pathophysiology to clinical practice

In healthy subjects, the rate of dietary iron absorption, as well as the amount and distribution of body iron are tightly controlled by hepcidin, the iron regulatory hormone. Disruption of systemic iron homeostasis leads to pathological conditions, ranging from anemias caused by iron deficiency or defective iron traffic, to iron overload (hemochromatosis). Other iron-related disorders are caused by misregulation of cellular iron metabolism, which results in local accumulation of the metal in mitochondria. Brain iron overload is observed in neurodegenerative disorders. Secondary hemochromatosis develops as a complication of another disease. For example, repeated blood transfusions, a standard treatment of various anemias characterized by ineffective erythropoiesis, promote transfusional siderosis, while chronic liver diseases are often associated with mild to moderate secondary iron overload. In this critical review, we discuss pathophysiological and clinical aspects of all types of iron metabolism disorders (265 references).     

小鼠模型在铁代谢研究中的应用

铁代谢在维持生命活动中至关重要,机体铁代谢紊乱会导致贫血和人类遗传性血色病等诸多疾病,对人体健康造成危害。在铁代谢研究领域,小鼠模型具有人群及细胞模型所不具备的优势,可以最准确的表现相应基因及通路在铁代谢调控中的生理作用。利用基因敲除及转基因小鼠模型,许多铁代谢相关的基因及调控通路被发现,有助于深入了解铁稳态调控的分子机制。这些小鼠模型为治疗铁代谢紊乱相关疾病潜在药物的开发和评估提供了理想的平台。