Molecular mechanisms of iron homeostasis

Iron metabolism in mammals requires a complex and tightly regulated molecular network. The classical view of iron metabolism has been challenged over the past ten years by the discovery of several new proteins, mostly Fe (II) iron transporters, enzymes with ferro-oxydase (hephaestin or ceruloplasmin) or ferri-reductase (Dcytb) activity or regulatory proteins like HFE and hepcidin. Furthermore, a new transferrin receptor has been identified, mostly expressed in the liver, and the ability of the megalin-cubilin complex to internalise the urinary Fe (III)-transferrin complex in renal tubular cells has been highlighted. Intestinal iron absorption by mature duodenal enterocytes requires Fe (III) iron reduction by Dcytb and Fe (II) iron transport through apical membranes by the iron transporter Nramp2/DMT1. This is followed by iron transfer to the baso-lateral side, export by ferroportin and oxidation into Fe (III) by hephaestin prior to binding to plasma transferrin. Macrophages play also an important role in iron delivery to plasma transferrin through phagocytosis of senescent red blood cell, heme catabolism and recycling of iron. Iron egress from macrophages is probably also mediated by ferroportin and patients with heterozygous ferroportin mutations develop progressive iron overload in liver macrophages. Iron homeostasis at the level of the organism is based on a tight control of intestinal iron absorption and efficient recycling of iron by macrophages. Signalling between iron stores in the liver and both duodenal enterocytes and macrophages is mediated by hepcidin, a circulating peptide synthesized by the liver and secreted into the plasma. Hepcidin expression is stimulated in response to iron overload or inflammation, and down regulated by anemia and hypoxia. Hepcidin deficiency leads to iron overload and hepcidin overexpression to anemia. Hepcidin synthesis in response to iron overload seems to be controlled by the HFE molecule. Patients with hereditary hemochromatosis due to HFE mutation have impaired hepcidin synthesis and forced expression of an hepcidin transgene in HFE deficient mice prevents iron overload. These results open new therapeutic perspectives, especially with the possibility to use hepcidin or antagonists for the treatment of iron overload disorders.     

小肠铁释放机制及相关疾病研究进展

铁是生物体必需的微量元素。铁缺乏和铁过载均会导致铁代谢紊乱相关疾病,因此有关机体铁水平稳态的调节机制已成为了目前铁代谢领域的研究热点。小肠吸收细胞是调节肠铁吸收、肠铁释放,以及维持机体铁稳态的重要部位。最新的研究表明,铁从小肠吸收细胞基底端释放入血液循环,主要是由膜铁转运蛋白（ferroportin1,Fp1）介导,并在膜铁转运辅助蛋白（haphaestin,Hp）和铜蓝蛋白（ceruloplasmin,Cp）的参与下完成。其中Fp1在小肠铁释放过程中起着至关重要的作用。本文重点阐述铁释放相关蛋白Fp1的作用机制及其调节机制,并详细介绍Fp1基因突变导致的铁代谢相关疾病方面的最新研究讲展。     

Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination

Ferroportin exports iron into plasma from absorptive enterocytes, erythrophagocytosing macrophages, and hepatic stores. The hormone hepcidin controls cellular iron export and plasma iron concentrations by binding to ferroportin and causing its internalization and degradation. We explored the mechanism of hepcidin-induced endocytosis of ferroportin, the key molecular event in systemic iron homeostasis. Hepcidin binding caused rapid ubiquitination of ferroportin in cell lines overexpressing ferroportin and in murine bone marrow-derived macrophages. No hepcidin-dependent ubiquitination was observed in C326S ferroportin mutant which does not bind hepcidin. Substitutions of lysines between residues 229 and 269 in the third cytoplasmic loop of ferroportin prevented hepcidin-dependent ubiquitination and endocytosis of ferroportin, and promoted cellular iron export even in the presence of hepcidin. The human ferroportin mutation K240E, previously associated with clinical iron overload, caused hepcidin resistance in vitro by interfering with ferroportin ubiquitination. Our study demonstrates that ubiquitination is the functionally relevant signal for hepcidin-induced ferroportin endocytosis.     

Hepcidin induction limits mobilisation of splenic iron in a mouse model of secondary iron overload

Venesection has been proposed as a treatment for hepatic iron overload in a number of chronic liver disorders that are not primarily linked to mutations in iron metabolism genes. Our aim was to analyse the impact of venesection on iron mobilisation in a mouse model of secondary iron overload. C57Bl/6 mice were given oral iron supplementation with or without phlebotomy between day 0 (D0) and D22, and the results were compared to controls without iron overload. We studied serum and tissue iron parameters, mRNA levels of hepcidin1, ferroportin, and transferrin receptor 1, and protein levels of ferroportin in the liver and spleen. On D0, animals with iron overload displayed elevations in iron parameters and hepatic hepcidin1 mRNA. By D22, in the absence of phlebotomies, splenic iron had increased, but transferrin saturation had decreased. This was associated with high hepatic hepcidin1 mRNA, suggesting that iron bioavailability decreased due to splenic iron sequestration through ferroportin protein downregulation. After 22 days with phlebotomy treatments, control mice displayed splenic iron mobilisation that compensated for the iron lost due to phlebotomy. In contrast, phlebotomy treatments in mice with iron overload caused anaemia due to inadequate iron mobilisation. In conclusion, our model of secondary iron overload led to decreased plasma iron associated with an increase in hepcidin expression and subsequent restriction of iron export from the spleen. Our data support the importance of managing hepcidin levels before starting venesection therapy in patients with secondary iron overload that are eligible for phlebotomy.     

The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis

Ferroportin (SLC40A1) is an iron transporter postulated to play roles in intestinal iron absorption and cellular iron release. Hepcidin, a regulatory peptide, binds to ferroportin and causes it to be internalized and degraded. If ferroportin is the major cellular iron exporter, ineffective hepcidin function could explain manifestations of human hemochromatosis disorders. To investigate this, we inactivated the murine ferroportin (Fpn) gene globally and selectively. Embryonic lethality of Fpn(null/null) animals indicated that ferroportin is essential early in development. Rescue of embryonic lethality through selective inactivation of ferroportin in the embryo proper suggested that ferroportin has an important function in the extraembryonic visceral endoderm. Ferroportin-deficient animals accumulated iron in enterocytes, macrophages, and hepatocytes, consistent with a key role for ferroportin in those cell types. Intestine-specific inactivation of ferroportin confirmed that it is critical for intestinal iron absorption. These observations define the major sites of ferroportin activity and give insight into hemochromatosis.     

Iron disorders of genetic origin: a changing world

Iron disorders of genetic origin are mainly composed of iron overload diseases, the most frequent being HFE-related hemochromatosis. Hepcidin deficiency underlies iron overload in HFE-hemochromatosis as well as in several other genetic iron excess disorders, such as hemojuvelin or hepcidin-related hemochromatosis and transferrin receptor 2-related hemochromatosis. Deficiency of ferroportin, the only known cellular protein iron exporter, produces iron overload in the typical form of ferroportin disease. By contrast, genetically enhanced hepcidin production, as observed in matriptase-2 deficiency, generates iron-refractory iron deficiency anemia. Diagnosis of these iron storage disorders is usually established noninvasively through combined biochemical, imaging and genetic approaches. Moreover, improved knowledge of the molecular mechanisms accounting for the variations of iron stores opens the way of novel therapeutic approaches aiming to restore normal iron homeostasis. In this review, we will summarize recent findings about these various genetic entities that have been identified owing to an exemplary interplay between clinicians and basic scientists.     

Impact of D181V and A69T on the function of ferroportin as an iron export pump and hepcidin receptor

Mutations in the only known mammalian iron exporter ferroportin cause a rare iron overload disorder termed ferroportin disease. Two distinct clinical phenotypes are caused by different disease mechanisms: mutations in ferroportin either cause loss of iron export function or gain of function due to resistance to hepcidin, the peptide hormone that normally downregulates ferroportin. The aim of the present study was to examine the disease mechanisms of the thus far unclassified A69T and D181V ferroportin mutations. We overexpressed wild-type and mutant ferroportin fused to green fluorescent protein in human embryonic kidney cells and used a ⁵⁹Fe-assay, intracellular ferritin concentrations, confocal microscopy and flow cytometry to study iron export function, subcellular localization and the responsiveness to hepcidin. While the A69T ferroportin mutation seems not to affect the iron export function it causes dose-dependent hepcidin resistance. We further found that D181V mutated ferroportin is iron export defective and hepcidin resistant, similar to the loss of function mutations A77D and C367X. This indicates that intact iron export might be necessary for hepcidin-induced downregulation of ferroportin. This hypothesis was investigated by studying the hepcidin response under modulation of iron availability. Incubation of wild-type ferroportin overexpressing cells with holo-transferrin increases the hepcidin effect whereas chelating extracellular ferrous iron causes hepcidin resistance. In this study we present data that postulates to classify the D181V ferroportin mutation as loss of function and the A69T mutation as dose-dependent hepcidin resistant and outline a possible causal link between iron export function and the hepcidin effect.     

Hepcidin非铁调控因子研究进展

铁调素（Hepcidin）是由肝细胞分泌的维持人体系统性铁平衡的核心因子,其通过改变细胞膜铁转运蛋白（ferroportin,Fpn）的表达量以调控肠黏膜细胞和巨噬细胞内铁的转出水平,从而决定机体循环铁水平并影响肝脏等主要储铁脏器的铁负荷程度。根据近年来的研究发现,影响Hepcidin表达的主要因素可以归纳为两个方面：一是机体本身对铁的需求,而由于铁本身又是Hb（hemoglobin,血红蛋白）的合成原料以及携氧成份,因此还应包括机体对Hb合成和缺氧的反应,介导因子主要包括携铁转铁蛋白（holo—transferrin,holo—Tf）、促红细胞生成素（erythropoietin,EPO）和缺氧诱导因子-1（hypoxia．inducible factor1,HIF．1）;另一则是源于疾病病理过程中相关致病因素、细胞因子、激素等非铁调控因子的改变对其表达调控机制产生的影响,并通过扰乱机体铁稳态加速疾病的发展或加重病情。随着研究资料的积累,糖尿病、部分心血管疾病、酒精性或非酒精性脂肪肝等慢性疾病存在铁过负荷已是不争的事实,多种hepcidin非铁调控因子在代谢紊乱型铁过负荷综合征（sysmetabolic iron overload syndrome）发生过程中的作用受到了广泛重视。对一些常见疾病中引起hepcidin表达变化异常和铁代谢紊乱的非铁因子及其作用机制的研究进展进行综述。     

Iron imports. IV. Hepcidin and regulation of body iron metabolism

Hepcidin, a small peptide synthesized in the liver, controls extracellular iron by regulating its intestinal absorption, placental transport, recycling by macrophages, and release from stores. Hepcidin inhibits the cellular efflux of iron by binding to and inducing the degradation of ferroportin, the sole iron exporter in iron-transporting cells. In turn, hepcidin synthesis is increased by iron loading and decreased by anemia and hypoxia. Hepcidin is markedly induced during inflammation, trapping iron in macrophages, decreasing plasma iron concentrations, and contributing to the anemia of inflammation. Hepcidin deficiency due to the dysregulation of its synthesis causes most known forms of hemochromatosis.     

Comparative studies of duodenal and macrophage ferroportin proteins

Intestinal epithelial cells and reticuloendothelial macrophages are, respectively, involved in diet iron absorption and heme iron recycling from senescent erythrocytes, two critical processes of iron homeostasis. These cells appear to use the same transporter, ferroportin (Slc40a1), to export iron. The aim of this study was to compare the localization, expression, and regulation of ferroportin in both duodenal and macrophage cells. Using a high-affinity purified polyclonal antibody, we analyzed the localization and expression of ferroportin protein in the spleen, liver, and duodenum isolated from normal mice as well as from well-characterized mouse models of altered iron homeostasis. Ferroportin was found to be predominantly expressed in enterocytes of the duodenum, in splenic macrophages, and in liver Kupffer cells. Interestingly, the protein species detected in these cells migrated differently on SDS-PAGE. These differences in apparent molecular masses were partly explained by posttranslational complex N-linked glycosylations. In addition, in enterocytes, the transporter was mostly expressed at the basolateral membrane, whereas in bone marrow-derived macrophages, ferroportin was found predominantly localized in the intracellular vesicular compartment. However, some microdomains positive for ferroportin were also detected at the plasma membrane of macrophages. Despite these differences, we observed a parallel upregulation of ferroportin expression in tissue macrophages and enterocytes in response to iron-restricted erythropoiesis, suggesting that iron homeostasis is likely maintained through coordinate expression of the iron exporter in both intestinal and phagocytic cells. Our data also confirm a predominant regulation of ferroportin through systemic regulator(s) likely including hepcidin.     

Iron and the heart: A paradigm shift from systemic to cardiomyocyte abnormalities

Iron is a key micronutrient for the human body and participates in biological processes, such as oxygen transport, storage, and utilization. Iron homeostasis plays a crucial role in the function of the heart and both iron deficiency and iron overload are harmful to the heart, which is partly mediated by increased oxidative stress. Iron enters the cardiomyocyte through the classic pathway, by binding to the transferrin 1 receptor (TfR1), but also through other routes: T-type calcium channel (TTCC), divalent metal transporter 1 (DMT1), L-type calcium channel (LTCC), Zrt-, Irt-like Proteins (ZIP) 8 and 14. Only one protein, ferroportin (FPN), extrudes iron from cardiomyocytes. Intracellular iron is utilized, stored bound to cytoplasmic ferritin or imported by mitochondria. This cardiomyocyte iron homeostasis is controlled by iron regulatory proteins (IRP). When the cellular iron level is low, expression of IRPs increases and they reduce expression of FPN, inhibiting iron efflux, reduce ferritin expression, inhibiting iron storage and augment expression of TfR1, increasing cellular iron availability. Such cellular iron homeostasis explains why the heart is very susceptible to iron overload: while cardiomyocytes possess redundant iron importing mechanisms, they are equipped with only one iron exporting protein, ferroportin. Furthermore, abnormalities of iron homeostasis have been found in heart failure and coronary artery disease, however, no clear picture is emerging yet in this area. If we better understand iron homeostasis in the cardiomyocyte, we may be able to develop better therapies for a variety of heart diseases to which abnormalities of iron homeostasis may contribute.     

Duodenal Ferroportin Is Up-Regulated in Patients with Chronic Hepatitis C

Hepatitis C virus (HCV) infection is a leading cause of liver-related mortality. Chronic hepatitis C (CHC) is frequently associated with disturbances in iron homeostasis, with serum iron and hepatic iron stores being elevated. Accumulating evidence indicates that chronic HCV infection suppresses expression of hepatic hepcidin, a key mediator of iron homeostasis, leading to iron overload conditions. Since hepcidin mediates degradation of ferroportin, a basolateral transporter involved in the release of iron from cells, diminished hepcidin expression probably leads to up-regulation of ferroportin-1 (Fpn1) in patients with CHC. In this study, we determined the protein levels of duodenal Fpn1, and found that its expression was significantly up-regulated in patients with CHC. The expression of duodenal Fpn1 is negatively correlated with mRNA levels of hepcidin, and positively correlated with serum iron parameters. Although iron is a critical factor for growth of a variety of pathogenic bacteria, our results suggest that iron overload in blood does not increase the infection rate of bacteria in patients with CHC.     

Hepcidin regulation of ferroportin 1 expression in the liver and intestine of the rat

Hepcidin has been implicated as the iron stores regulator: a hepatic signaling molecule that regulates intestinal iron absorption by undefined mechanisms. The possibility that hepcidin regulates the expression of ferroportin 1 (FPT1), the basolateral iron transporter, was examined in rats after administration of LPS, an iron chelator, or His-tagged recombinant hepcidin (His-rHepc). In the liver, LPS stimulated a biphasic increase of hepcidin mRNA with peaks of mRNA at 6 and 36 h. Concurrently, hepatic FPT1 mRNA expression decreased to minimal level at 6 h and then increased with a peak at 24-36 h. LPS also induced biphasic changes in intestinal FPT1 mRNA expression, with decreased levels at 6 h and increased expression at 48 h. Whereas the initial decrease of FPT1 coincides with an LPS-induced decrease in serum iron, both intestinal and hepatic FPT1 expression recovered, whereas serum iron concentration continued to decrease for at least 24 h. Dietary iron ingestion increased intestinal ferritin protein production but did not reduce intestinal FPT1 mRNA expression. The iron chelator pyrrolidinedithiocarbamate (PDTC) stimulated hepatic hepcidin without suppressing intestinal FPT1 expression. In PDTC-treated rats, LPS stimulated no additional hepatic hepcidin expression but did increase intestinal FPT1 expression. Administration of HisrHepc induced significant reduction of intestinal FPT1 expression. Taken together, these data suggest that hepcidin mediates LPS-induced downregulation of intestinal FPT1 expression and that the hepcidin signaling pathway involves a PDTC-sensitive step.     

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.     

Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone

Hepcidin is a liver produced cysteine-rich peptide hormone that acts as the central regulator of body iron metabolism. Hepcidin is synthesized under the form of a precursor, prohepcidin, which is processed to produce the biologically active mature 25 amino acid peptide. This peptide is secreted and acts by controlling the concentration of the membrane iron exporter ferroportin on intestinal enterocytes and macrophages. Hepcidin binds to ferroportin, inducing its internalization and degradation, thus regulating the export of iron from cells to plasma. The aim of the present study was to develop a novel method to produce human and mouse recombinant hepcidins, and to compare their biological activity towards their natural receptor ferroportin. Hepcidins were expressed in Escherichia coli as thioredoxin fusion proteins. The corresponding peptides, purified after cleavage from thioredoxin, were properly folded and contained the expected four-disulfide bridges without the need of any renaturation or oxidation steps. Human and mouse hepcidins were found to be biologically active, promoting ferroportin degradation in macrophages. Importantly, biologically inactive aggregated forms of hepcidin were observed depending on purification and storage conditions, but such forms were unrelated to disulfide bridge formation.     

膜铁转运蛋白1，铁调素的靶分子？

膜铁转运蛋白1是重要的跨膜铁输出分子,主要分布于十二指肠和单核巨噬系统的细胞膜上,参与机体的肠铁吸收和巨噬细胞对铁的再循环等过程。铁调素是调节机体铁代谢平衡的激素,机体通过肝脏分泌的铁调素对铁转运相关蛋白的表达进行调控,从而实现机体自身的铁稳态。最新研究显示,铁调素的靶分子可能是膜铁转运蛋白1,它通过直接的作用引起膜铁转运蛋白1的内化(internalization)、降解,从而调节其在细胞膜上的表达量,进而控制肠铁吸收和巨噬细胞对铁的再循环过程,以维持机体的铁稳态。