Hepcidin regulation: ironing out the details

Hepcidin is a peptide hormone secreted by the liver that plays a central role in the regulation of iron homeostasis. Increased hepcidin levels result in anemia while decreased expression is the causative feature in most primary iron overload diseases. Mutations in hemochromatosis type 2 (HFE2), which encodes the protein hemojuvelin (HJV), result in the absence of hepcidin and an early-onset form of iron overload disease. HJV is a bone morphogenetic protein (BMP) coreceptor and HJV mutants have impaired BMP signaling. In this issue of the JCI, Babitt and colleagues show that BMPs are autocrine hormones that induce hepcidin expression (see the related article beginning on page 1933). Administration of a recombinant, soluble form of HJV decreased hepcidin expression and increased serum iron levels by mobilizing iron from splenic stores. These results demonstrate that recombinant HJV may be a useful therapeutic agent for treatment of the anemia of chronic disease, a disorder resulting from high levels of hepcidin expression.     

Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload

Iron homeostasis plays a critical role in many physiological processes, notably synthesis of heme proteins. Dietary iron sensing and inflammation converge in the control of iron absorption and retention by regulating the expression of hepcidin, a regulator of the iron exporter ferroportin. Human mutations in the glycosylphosphatidylinositol-anchored protein hemojuvelin (HJV; also known as RGMc and HFE2) cause juvenile hemochromatosis, a severe iron overload disease, but the way in which HJV intersects with the iron regulatory network has been unclear. Here we show that, within the liver, mouse Hjv is selectively expressed by periportal hepatocytes and also that Hjv-mutant mice exhibit iron overload as well as a dramatic decrease in hepcidin expression. Our findings define a key role for Hjv in dietary iron sensing and also reveal that cytokine-induced inflammation regulates hepcidin expression through an Hjv-independent pathway.     

Iron regulation by hepcidin

Hepcidin is a key hormone that is involved in the control of iron homeostasis in the body. Physiologically, hepcidin is controlled by iron stores, inflammation, hypoxia, and erythropoiesis. The regulation of hepcidin expression by iron is a complex process that requires the coordination of multiple proteins, including hemojuvelin, bone morphogenetic protein 6 (BMP6), hereditary hemochromatosis protein, transferrin receptor 2, matriptase-2, neogenin, BMP receptors, and transferrin. Misregulation of hepcidin is found in many disease states, such as the anemia of chronic disease, iron refractory iron deficiency anemia, cancer, hereditary hemochromatosis, and ineffective erythropoiesis, such as β-thalassemia. Thus, the regulation of hepcidin is the subject of interest for the amelioration of the detrimental effects of either iron deficiency or overload.     

A mouse model of juvenile hemochromatosis

Hereditary hemochromatosis is an iron-overload disorder resulting from mutations in proteins presumed to be involved in the maintenance of iron homeostasis. Mutations in hemojuvelin (HJV) cause severe, early-onset juvenile hemochromatosis. The normal function of HJV is unknown. Juvenile hemochromatosis patients have decreased urinary levels of hepcidin, a peptide hormone that binds to the cellular iron exporter ferroportin, causing its internalization and degradation. We have disrupted the murine Hjv gene and shown that Hjv-/- mice have markedly increased iron deposition in liver, pancreas, and heart but decreased iron levels in tissue macrophages. Hepcidin mRNA expression was decreased in Hjv-/- mice. Accordingly, ferroportin expression detected by immunohistochemistry was markedly increased in both intestinal epithelial cells and macrophages. We propose that excess, unregulated ferroportin activity in these cell types leads to the increased intestinal iron absorption and plasma iron levels characteristic of the juvenile hemochromatosis phenotype.     

Hepcidin as a therapeutic tool to limit iron overload and improve anemia in β-thalassemic mice

Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.     

Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload

Iron overload is the hallmark of hereditary hemochromatosis and a complication of iron-loading anemias such as β-thalassemia. Treatment can be burdensome and have significant side effects, and new therapeutic options are needed. Iron overload in hereditary hemochromatosis and β-thalassemia intermedia is caused by hepcidin deficiency. Although transgenic hepcidin replacement in mouse models of these diseases prevents iron overload or decreases its potential toxicity, natural hepcidin is prohibitively expensive for human application and has unfavorable pharmacologic properties. Here, we report the rational design of hepcidin agonists based on the mutagenesis of hepcidin and the hepcidin-binding region of ferroportin and computer modeling of their docking. We identified specific hydrophobic/aromatic residues required for hepcidin-ferroportin binding and obtained evidence in vitro that a thiol-disulfide interaction between ferroportin C326 and the hepcidin disulfide cage may stabilize binding. Guided by this model, we showed that 7–9 N-terminal amino acids of hepcidin, including a single thiol cysteine, comprised the minimal structure that retained hepcidin activity, as shown by the induction of ferroportin degradation in reporter cells. Further modifications to increase resistance to proteolysis and oral bioavailability yielded minihepcidins that, after parenteral or oral administration to mice, lowered serum iron levels comparably to those after parenteral native hepcidin. Moreover, liver iron concentrations were lower in mice chronically treated with minihepcidins than those in mice treated with solvent alone. Minihepcidins may be useful for the treatment of iron overload disorders.     

Progesterone receptor membrane component-1 regulates hepcidin biosynthesis

Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism.     

Hereditary hemochromatosis: genetic complexity and new diagnostic approaches

Since the discovery of the hemochromatosis gene (HFE) in 1996, several novel gene defects have been detected, explaining the mechanism and diversity of iron-overload diseases. At least 4 main types of hereditary hemochromatosis (HH) have been identified. Surprisingly, genes involved in HH encode for proteins that all affect pathways centered around liver hepcidin synthesis and its interaction with ferroportin, an iron exporter in enterocytes and macrophages. Hepcidin concentrations in urine negatively correlate with the severity of HH. Cytokine-mediated increases in hepcidin appear to be an important causative factor in anemia of inflammation, which is characterized by sequestration of iron in the macrophage system. For clinicians, the challenge is now to diagnose HH before irreversible damage develops and, at the same time, to distinguish progressive iron overload from increasingly common diseases with only moderately increased body iron stores, such as the metabolic syndrome. Understanding the molecular regulation of iron homeostasis may be helpful in designing innovative and reliable DNA and protein tests for diagnosis. Subsequently, evidence-based diagnostic strategies must be developed, using both conventional and innovative laboratory tests, to differentiate between the various causes of distortions of iron metabolism. This review describes new insights in mechanisms of iron overload, which are needed to understand new developments in diagnostic medicine.     

The gene encoding the iron regulatory peptide hepcidin is regulated by anemia,hypoxia, and inflammation

The present study was aimed at determining whether hepcidin, a recently identified peptide involved in iron metabolism, plays a role in conditions associated with both iron overload and iron deficiency. Hepcidin mRNA levels were assessed in two models of anemia, acute hemolysis provoked by phenylhydrazine and bleeding provoked by repeated phlebotomies. Hepcidin response to hypoxia was also studied, both ex vivo, in human hepatoma cells, and in vivo. Anemia and hypoxia were associated with a dramatic decrease in liver hepcidin gene expression, which may account for the increase in iron release from reticuloendothelial cells and increase in iron absorption frequently observed in these situations. A single injection of turpentine for 16 hours induced a sixfold increase in liver hepcidin mRNA levels and a twofold decrease in serum iron. The hyposideremic effect of turpentine was completely blunted in hepcidin-deficient mice, revealing hepcidin participation in anemia of inflammatory states. These modifications of hepcidin gene expression further suggest a key role for hepcidin in iron homeostasis under various pathophysiological conditions, which may support the pharmaceutical use of hepcidin agonists and antagonists in various iron homeostasis disorders.     

Hepcidin and iron metabolism: From laboratory to clinical implications

Hepcidin is a liver-synthesized hormone that inhibits the cellular efflux of iron by binding to ferroportin and its subsequent degradation. The main role of hepcidin is regulation of ferroportin expression and cell membrane function. Recent studies implicate hepcidin in a variety of iron disorders in addition to its primary role in the regulation of systemic iron homeostasis. Hepcidin excess has a key pathologic role in anemia of inflammation, chronic kidney disease, and iron-refractory iron deficiency anemia, while hepcidin deficiency is responsible for most cases of familial hemochromatosis and iron-loading anemia. The most important advances on the role of hepcidin in normal and abnormal iron metabolism and the main clinical and diagnostic implications are summarized in this review.     

慢性病贫血铁调节蛋白研究新进展

慢性病贫血是一种正细胞正色素贫血,其发病机制之一是铁代谢紊乱。肝脏分泌的铁调节蛋白（hepcidin）被认为是调节铁代谢的激素,它作用于铁转运蛋白（ferroporlin）,使后者内化,减少细胞内铁向细胞外转运,从而降低血清铁。慢性疾病时的高炎症因子状态会通过JAK2／STAT3途径促进肝细胞高表达hepcidin,导致慢性病贫血时的铁代谢紊乱。本文就铁调节蛋白及其与铁代谢及慢性贫血的关系进行综述。     

Iron homeostasis in the Metabolic Syndrome

The metabolic syndrome (MetS) affects iron homeostasis in a many‐faceted fashion. On the one side, hyperferritinaemia with normal or mildly elevated transferrin saturation is observed in approximately one‐third of patients with non‐alcoholic fatty liver disease (NAFLD) or the MetS. This constellation has been named the ‘dysmetabolic iron overload syndrome (DIOS)’. Current evidence suggests that elevated body iron stores exert a detrimental effect on the clinical course of obesity‐related conditions and that iron removal improves insulin sensitivity and delays the onset of T2DM. On the other side, iron deficiency is a frequent finding in more progressed stages of obesity. The mechanisms underlying the DIOS and obesity‐related iron deficiency appear strikingly similar as elevated hepcidin concentrations, low expression of duodenal ferroportin (FPN) and impaired iron absorption are found in both conditions. This review summarizes the current knowledge about the dysregulation of iron homeostasis in the MetS and particularly in its hepatic manifestation NAFLD.     

Hypoxia-inducible factor regulates hepcidin via erythropoietin-induced erythropoiesis

Iron demand in bone marrow increases when erythropoiesis is stimulated by hypoxia via increased erythropoietin (EPO) synthesis in kidney and liver. Hepcidin, a small polypeptide produced by hepatocytes, plays a central role in regulating iron uptake by promoting internalization and degradation of ferroportin, the only known cellular iron exporter. Hypoxia suppresses hepcidin, thereby enhancing intestinal iron uptake and release from internal stores. While HIF, a central mediator of cellular adaptation to hypoxia, directly regulates renal and hepatic EPO synthesis under hypoxia, the molecular basis of hypoxia/HIF-mediated hepcidin suppression in the liver remains unclear. Here, we used a genetic approach to disengage HIF activation from EPO synthesis and found that HIF-mediated suppression of the hepcidin gene (Hamp1) required EPO induction. EPO induction was associated with increased erythropoietic activity and elevated serum levels of growth differentiation factor 15. When erythropoiesis was inhibited pharmacologically, Hamp1 was no longer suppressed despite profound elevations in serum EPO, indicating that EPO by itself is not directly involved in Hamp1 regulation. Taken together, we provide in vivo evidence that Hamp1 suppression by the HIF pathway occurs indirectly through stimulation of EPO-induced erythropoiesis.     

A tincture of hepcidin cures all: the potential for hepcidin therapeutics

Iron overload as a result of blood transfusions and excessive intestinal iron absorption can be a complication of chronic anemias such as β-thalassemia. Inappropriately low levels of hepcidin, a negative regulator of iron absorption and recycling, underlie the pathophysiology of the intestinal hyperabsorption. In this issue of the JCI, Gardenghi et al. demonstrate that increasing hepcidin expression to induce iron deficiency in murine β-thalassemia not only mitigates the iron overload, but also the severity of the anemia. These data illustrate the therapeutic potential of modulating hepcidin expression in diseases associated with altered iron metabolism.     

Iron regulation by hepatocytes and free radicals

Iron is an essential metallic microelement for life. However, iron overload is toxic. The liver serves an important role as a storehouse for iron in the body. About 20–25 mg of iron is required each day for hemoglobin synthesis. To maintain iron homeostasis, transferrin and transferrin receptors are primarily involved in the uptake of iron into hepatocytes, ferritin in its storage, and ferroportin in its export. Moreover, hepcidin controls ferroportin and plays a central role in the iron metabolism. Excess “free” reactive iron produces damaging free radicals via Fenton or Harber-Weiss reactions. Produced free radicals attack cellular proteins, lipids and nucleic acid. Several detoxification system and anti-oxidant defense mechanisms exist to prevent cellular damage by free radicals. Excessive free radicals can lead to hepatocellular damage, liver fibrosis, and hepatocarcinogenesis.     

The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice

Systemic iron homeostasis is mainly controlled by the liver through synthesis of the peptide hormone hepcidin (encoded by Hamp), the key regulator of duodenal iron absorption and macrophage iron release. Here we show that the liver-specific microRNA miR-122 is important for regulating Hamp mRNA expression and tissue iron levels. Efficient and specific depletion of miR-122 by injection of a locked-nucleic-acid-modified (LNA-modified) anti-miR into WT mice caused systemic iron deficiency, characterized by reduced plasma and liver iron levels, mildly impaired hematopoiesis, and increased extramedullary erythropoiesis in the spleen. Moreover, miR-122 inhibition increased the amount of mRNA transcribed by genes that control systemic iron levels, such as hemochromatosis (Hfe), hemojuvelin (Hjv), bone morphogenetic protein receptor type 1A (Bmpr1a), and Hamp. Importantly, miR-122 directly targeted the 3′ untranslated region of 2 mRNAs that encode activators of hepcidin expression, Hfe and Hjv. These data help to explain the increased Hamp mRNA levels and subsequent iron deficiency in mice with reduced miR-122 levels and establish a direct mechanistic link between miR-122 and the regulation of systemic iron metabolism.