Maintaining physiologic iron concentrations in tissues is critical for metabolism
and host defense. Iron absorption in the duodenum, recycling of iron from senescent
erythrocytes, and iron mobilization from storage in macrophages and hepatocytes constitute
the major iron flows into plasma for distribution to tissues, predominantly for
erythropoiesis. All iron transfer to plasma occurs through the iron exporter ferroportin.
The concentration of functional membrane-associated ferroportin is controlled by its
ligand, the iron-regulatory hormone hepcidin, and fine-tuned by regulatory mechanisms
serving iron homeostasis, oxygen utilization, host defense and erythropoiesis. Fundamental
questions about the structure and biology of ferroportin remain to be answered.
Minihepcidins are in vitro and in vivo active mimetics of iron-regulatory hormone hepcidin. They contain various unusual amino acids including: N-substituted, β-homo-, and D-amino acids with their combination depending on particular minihepcidin. In the current study, we sought to limit the use of unusual/more expensive amino acids derivatives by peptide cyclisation. Novel cyclic mimetics of hepcidin were synthesized and tested in vitro and showed activity at low nanomolar concentration. Nonetheless, the most active cyclic compound (mHS17) is approximately ten times less active than the parental minihepcidin PR73. Collectively, our findings suggest that cyclisation is viable approach in the synthesis of hepcidin mimetics.
Minihepcidins; Peptides; S-Alkylation of peptides; Iron; Cyclisation
Iron is an essential trace element for multicellular organisms and nearly all microorganisms. Although iron is abundant in the environment, common forms of iron are minimally soluble and therefore poorly accessible to biological organisms. Microorganisms entering a mammalian host face multiple mechanisms that further restrict their ability to obtain iron and thereby limit their pathogenicity. Iron levels also modulate host defence, as iron content in macrophages regulates their cytokine production. Here, we review recent advances that highlight the role of systemic and cellular iron-regulating mechanisms in protecting hosts from infection, emphasizing aspects that are applicable to human health and disease.
Hepcidin is the key regulator of iron absorption and recycling, and its expression is suppressed by red blood cell production. When erythropoiesis is expanded, hepcidin expression decreases. To gain insight into the stage of erythroid differentiation at which the regulation might originate, we measured serum hepcidin levels in archived pure red cell aplasia samples from patients whose block in erythroid differentiation was well defined by hematopoietic colony assays and marrow morphologic review. Hepcidin values are high or high normal in pure red cell aplasia patients in whom erythropoiesis is inhibited prior to the proerythroblast stage, but are suppressed in patients with excess proerythroblasts and few later erythroid cells. These data suggest that the suppressive effect of erythropoietic activity on hepcidin expression can arise from proerythroblasts, the stage at which transferrin receptor 1 expression peaks, prompting the hypothesis that transferrin receptor 1 expression on erythroid precursors is a proximal mediator of the erythroid regulator of hepcidin expression. Our characterization of erythropoiesis, iron status, and hepcidin expression in mice with global or hematopoietic cell-specific haploinsufficiency of transferrin receptor 1 provides initial supporting data for this model. The regulation appears independent of erythroferrone and growth differentiation factor 15, supporting the concept that several mechanisms signal iron need in response to an expanded erythron.
Minihepcidins are small peptides that mimic biological activity of the iron-regulatory hormone hepcidin. Structurally, they contain thiol-free-cysteine residue in position 7 which is crucial for their bioactivity. Nonetheless, free sulfhydryl group is not desirable in pharmaceutical entities as it may lead to dermatological side effects. Moreover free thiol moiety is quite reactive and depending on conditions/reagents may be alkylated and/or oxidized giving various Cys-derivatives: S-alkyl cysteines, sulfoxides, sulfones, disulfides, cysteinesulfinic and cysteic acids. To limit such reactivity and maintain bioactivity of minihepcidin(s) we used thiol-protection strategy based on activated vinyl thioethers. Novel S-protected analogs of physiologically active minihepcidin PR73 were synthesized and tested in vitro showing activity comparable to parental molecule. The most active compound, PR73SH was also tested in vivo showing activity profile analogous to PR73. Collectively, our findings suggest that S-vinyl-derivatization of minihepcidin(s) may be a suitable approach in the development of physiologically active agonists of hepcidin.
Minihepcidins; Peptides; S-Alkylation of peptides; Iron
Hereditary hemochromatosis, an iron overload disease caused by a deficiency in the iron-regulatory hormone hepcidin, is associated with lethal infections by siderophilic bacteria. To elucidate the mechanisms of this susceptibility, we infected wild-type and hepcidin-deficient mice with the siderophilic bacterium Vibrio vulnificus, and found that hepcidin deficiency results in increased bacteremia and decreased survival of infected mice, which can be partially ameliorated by dietary iron depletion. Additionally, timely administration of hepcidin agonists to hepcidin-deficient mice induces hypoferremia that decreases bacterial loads and rescues these mice from death, regardless of initial iron levels. Studies of Vibrio vulnificus growth ex vivo show that high iron sera from hepcidin-deficient mice support extraordinarily rapid bacterial growth, and that this is inhibited in hypoferremic sera. Our findings demonstrate that hepcidin-mediated hypoferremia is a host defense mechanism against siderophilic pathogens and suggest that hepcidin agonists may improve infection outcomes in patients with hereditary hemochromatosis or thalassemia.
Hepcidin is the master regulator of iron homeostasis in vertebrates. The synthesis of hepcidin is induced by systemic iron levels and by inflammatory stimuli. While the role of hepcidin in iron regulation is well established, its contribution to host defense is emerging as complex and multifaceted. In this review, we summarize the literature on the role of hepcidin as a mediator of antimicrobial immunity. Hepcidin induction during infection causes depletion of extracellular iron, which is thought to be a general defense mechanism against many infections by withholding iron from invading pathogens. Conversely, by promoting iron sequestration in macrophages, hepcidin may be detrimental to cellular defense against certain intracellular infections, although critical in vivo studies are needed to confirm this concept. It is not yet clear whether hepcidin exerts any iron-independent effects on host defenses.
Anemia of inflammation (AI, also called anemia of chronic disease) is a common, typically normocytic normochromic anemia that is caused by an underlying inflammatory disease. It is diagnosed when serum iron concentrations are low despite adequate iron stores, as evidenced by serum ferritin that is not low. In the setting of inflammation, AI may be difficult to differentiate from iron deficiency anemia, and the two conditions may coexist. In AI, erythropoiesis is iron-restricted by hepcidin-mediated hypoferremia and erythrocyte production is suppressed by cytokines acting on erythroid progenitors. Decreased erythropoiesis is unable to compensate for shortened erythrocyte lifespan caused by enhanced erythrophagocytosis by cytokine-activated macrophages. Treatment should focus on the underlying disease. If this is not feasible and the anemia limits the quality of life or the performance of daily activities, a combination of erythropoiesis-stimulating agents and intravenous iron may be effective but should be attempted only after careful consideration of risk and benefit. Recent advances in molecular understanding of AI are stimulating the development of new pathophysiologically targeted experimental therapies.
anemia of chronic disease; hepcidin; ferroportin; cytokines; erythropoiesis-stimulating agents
Recovery from blood loss requires a greatly enhanced supply of iron to support expanded erythropoiesis. After hemorrhage, suppression of the iron-regulatory hormone hepcidin allows increased iron absorption and mobilization from stores. We identified a new hormone, erythroferrone (ERFE), which mediates hepcidin suppression during stress erythropoiesis. ERFE is produced by erythroblasts in response to erythropoietin. ERFE-deficient mice fail to suppress hepcidin rapidly after hemorrhage and exhibit a delay in recovery from blood loss. ERFE expression is greatly increased in murine HbbTh3/+ thalassemia intermedia where it contributes to the suppression of hepcidin and systemic iron overload characteristic of this disease.
Hepcidin, the iron-regulatory hormone and acute phase reactant, is proposed to contribute to the pathogenesis of atherosclerosis by promoting iron accumulation in plaque macrophages, leading to increased oxidative stress and inflammation in the plaque (the “iron hypothesis”). Hepcidin and iron may thus represent modifiable risk factors in atherosclerosis. We measured hepcidin expression in Apoe−/− mice with varying diets and ages. To assess the role of macrophage iron in atherosclerosis, we generated Apoe−/− mice with macrophage-specific iron accumulation by introducing the ferroportin ffe mutation. Macrophage iron loading was also enhanced by intravenous iron injection. Contrary to the iron hypothesis, we found that hepatic hepcidin expression was not increased at any stage of the atherosclerosis progression in Apoe−/− or Apoe/ffe mice and the atherosclerotic plaque size was not increased in mice with elevated macrophage iron. Our results strongly argue against any significant role of macrophage iron in atherosclerosis progression in mice.
Hepcidin, the iron-regulatory hormone, is increased during infection or inflammation, causing hypoferremia. This response is thought to be a host defense mechanism that restricts iron availability to invading pathogens. It is not known if hepcidin is differentially induced by bacterial versus viral infections, whether the stimulation of pattern recognition receptors directly regulates hepcidin transcription, or which of the proposed signaling pathways are essential for hepcidin increase during infection. We analyzed hepcidin induction and its dependence on interleukin-6 (IL-6) in response to common bacterial or viral infections in mice or in response to a panel of pathogen-derived molecules (PAMPs) in mice and human primary hepatocytes. In wild-type (WT) mice, hepcidin mRNA was induced several hundred-fold both by a bacterial (Streptococcus pneumoniae) and a viral infection (influenza virus PR8) within 2 to 5 days. Treatment of mice and human primary hepatocytes with most Toll-like receptor ligands increased hepcidin mRNA within 6 h. Hepcidin induction by microbial stimuli was IL-6 dependent. IL-6 knockout mice failed to increase hepcidin in response to S. pneumoniae or influenza infection and had greatly diminished hepcidin response to PAMPs. In vitro, hepcidin induction by PAMPs in primary human hepatocytes was abolished by the addition of neutralizing IL-6 antibodies. Our results support the key role of IL-6 in hepcidin regulation in response to a variety of infectious and inflammatory stimuli.
The pathogenesis of dysmetabolic iron overload syndrome (DIOS) is still unclear. Hepcidin is the key regulator of iron homeostasis controlling iron absorption and macrophage release.
To investigate hepcidin regulation by iron in DIOS.
We analysed urinary hepcidin at baseline and 24 h after a 65 mg oral iron dose in 24 patients at diagnosis and after iron depletion (n=13) and compared data with those previously observed in 23 healthy controls. Serum iron indices, liver histology and metabolic data were available for all patients.
At diagnosis, hepcidin values were significantly higher than in controls (P<0.001). After iron depletion, hepcidin levels decreased to normal values in all patients. At baseline, a significant response of hepcidin to iron challenge was observed only in the subgroup with lower basal hepcidin concentration (P=0.007). In iron-depleted patients, urinary hepcidin significantly increased after oral iron test (P=0.006).
Ours findings suggest that in DIOS, the progression of iron accumulation is counteracted by the increase in hepcidin production and progressive reduction of iron absorption, explaining why these patients develop a mild–moderate iron overload that tends to a plateau.
Despite fluctuations in dietary iron intake and intermittent losses through bleeding, the plasma iron concentrations in humans remain stable at 10–30 μM. While most of the iron entering blood plasma comes from recycling, appropriate amount of iron is absorbed from the diet to compensate for losses and maintain nontoxic amounts in stores. Plasma iron concentration and iron distribution are similarly regulated in laboratory rodents. The hepatic peptide hepcidin was identified as the systemic iron-regulatory hormone. In the efferent arc, hepcidin regulates intestinal iron absorption, plasma iron concentrations, and tissue iron distribution by inducing degradation of its receptor, the cellular iron exporter ferroportin. Ferroportin exports iron into plasma from absorptive enterocytes, from macrophages that recycle the iron of senescent erythrocytes, and from hepatocytes that store iron. In the more complex and less well understood afferent arc, hepatic hepcidin synthesis is transcriptionally regulated by extracellular and intracellular iron concentrations through a molecular complex of bone morphogenetic protein receptors and their iron-specific ligands, modulators and iron sensors. Through as yet undefined pathways, hepcidin is also homeostatically regulated by the iron requirements of erythroid precursors for hemoglobin synthesis. In accordance with the role of hepcidin-mediated iron redistribution in host defense, hepcidin production is regulated by inflammation as well. Increased hepcidin concentrations in plasma are pathogenic in iron-restrictive anemias including anemias associated with inflammation, chronic kidney disease and some cancers. Hepcidin deficiency causes iron overload in hereditary hemochromatosis and ineffective erythropoiesis. Hepcidin, ferroportin and their regulators represent potential targets for the diagnosis and treatment of iron disorders and anemias.
Iron overload; iron deficiency; anemia
The review summarizes the current understanding of the role of hepcidin and ferroportin in normal iron homeostasis and its disorders. It further discusses the various approaches to therapeutic targeting of hepcidin and ferroportin in iron overload diseases (mainly hereditary hemochromatosis and β-thalassemia) and iron-restrictive anemias (anemias associated with infections, inflammatory disorders and certain malignancies, anemia of chronic kidney diseases, iron-refractory iron deficiency anemia).
Anemia of cancer (AC) may contribute to cancer-related fatigue and impair quality of life. Improved understanding of the pathogenesis of AC could facilitate better treatment, but animal models to study AC are lacking. We characterized four syngeneic C57BL/6 mouse cancers that cause AC. Mice with two different rapidly-growing metastatic lung cancers developed the characteristic findings of anemia of inflammation (AI), with dramatically different degrees of anemia. Mice with rapidly-growing metastatic melanoma also developed a severe anemia by 14 days, with hematologic and inflammatory parameters similar to AI. Mice with a slow-growing peritoneal ovarian cancer developed an iron-deficiency anemia, likely secondary to chronically impaired nutrition and bleeding into the peritoneal cavity. Of the four models, hepcidin mRNA levels were increased only in the milder lung cancer model. Unlike in our model of systemic inflammation induced by heat-killed Brucella abortus, ablation of hepcidin in the ovarian cancer and the milder lung cancer mouse models did not affect the severity of anemia. Hepcidin-independent mechanisms play an important role in these murine models of AC.
Mutations in ferroportin (Fpn) result in iron overload. We correlate the behavior of three Fpn mutants in vitro with patients’ phenotypes. Patients with Fpn mutations A77D or N174I showed macrophage iron loading. In cultured cells, FpnA77D did not reach the cell surface and cells did not export iron. Fpn mutant N174I showed plasma membrane and intracellular localization, and did not transport iron. Fpn mutation G80S was targeted to the cell surface and was transport competent, however patients showed macrophage iron. We suggest that FpnG80S represents a class of Fpn mutants whose behavior in vitro does not explain the patients’ phenotype.
ferroportin; hemochromatosis; hepcidin; iron transport
The hepatic peptide hormone hepcidin controls the duodenal absorption of iron, its storage and its systemic distribution. Hepcidin production is often insufficient in chronic hepatitis C and alcoholic liver disease, leading to hyperabsorption of iron and its accumulation in the liver. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) mediate the hepatic regeneration after liver injury. We examined the effect of the growth factors on hepcidin synthesis by hepatocytes. Results: HGF and EGF treatment of primary mouse hepatocytes, as well as EGF administration in mice, suppressed hepcidin mRNA synthesis. The suppression of hepcidin by these growth factors was transcriptional, and was mediated by a direct effect of HGF and EGF on the BMP pathway regulating hepcidin synthesis. We further showed that growth factors interfered with nuclear localization of activated Smads and increased the nuclear pool of the BMP transcriptional co-repressor TG-interacting factor (TGIF). In a kinase screen with small-molecule kinase inhibitors, inhibitors in the PI3 kinase pathway and in the MEK/ERK pathway prevented HGF suppression of hepcidin in primary mouse hepatocytes. Conclusion: HGF and EGF suppress hepatic hepcidin synthesis, in part through PI3 kinase MEK/ERK kinase pathways which may be modulating the nuclear localization of BMP pathway transcriptional regulators including activated Smads1/5/8 and the co-repressor TGIF. EGF, HGF and possibly other growth factors that activate similar pathways may contribute to hepcidin suppression in chronic liver diseases, promote iron accumulation in the liver and exacerbate the destructive disease processes.
Chronic liver disease; iron overload; hepatocyte growth factor; epidermal growth factor; bone morphogenetic protein pathway
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, a 25-amino acid peptide hormone, is the principal regulator of plasma iron concentrations. Hepcidin binding to its receptor, the iron exporter ferroportin, induces ferroportin internalization and degradation, thus blocking iron efflux from cells into plasma. The aim of this study was to characterize the fate of hepcidin after binding to ferroportin. We show that hepcidin is taken up by ferroportin-expressing cells in a temperature- and pH-dependent manner, and degraded together with its receptor. When Texas red-labeled hepcidin (TR-Hep) was added to ferroportin-GFP (Fpn-GFP) expressing cells, confocal microscopy showed co-localization of TR-Hep with Fpn-GFP. Using flow cytometry, we showed that the peptide was almost completely degraded by 24 h after its addition, but that lysosomal inhibitors completely prevented degradation of both ferroportin and hepcidin. In addition, using radio-labeled hepcidin and HPLC analysis we show that hepcidin is not recycled, and that only degradation products are released from the cells. Together these results show that the hormone hepcidin and its receptor ferroportin are internalized together and trafficked to lysosomes where both are degraded.
Hepcidin is a regulatory hormone that plays a major role in controlling body iron homeostasis. Circulating factors (holotransferrin, cytokines, erythroid regulators) might variably contribute to hepcidin modulation in different pathological conditions. There are few studies analysing the relationship between hepcidin transcript and related protein expression profiles in humans. Our aims were: a. to measure hepcidin expression at either hepatic, serum and urinary level in three paradigmatic iron overload conditions (hemochromatosis, thalassemia and dysmetabolic iron overload syndrome) and in controls; b. to measure mRNA hepcidin expression in two different hepatic cell lines (HepG2 and Huh-7) exposed to patients and controls sera to assess whether circulating factors could influence hepcidin transcription in different pathological conditions. Our findings suggest that hepcidin assays reflect hepatic hepcidin production, but also indicate that correlation is not ideal, likely due to methodological limits and to several post-trascriptional events. In vitro study showed that THAL sera down-regulated, HFE-HH and C-NAFLD sera up-regulated hepcidin synthesis. HAMP mRNA expression in Huh-7 cells exposed to sera form C-Donors, HFE-HH and THAL reproduced, at lower level, the results observed in HepG2, suggesting the important but not critical role of HFE in hepcidin regulation.
Hemoglobinopathies and other disorders of erythroid cells are often associated with abnormal iron homeostasis. We review the molecular physiology of intracellular and systemic iron regulation, and the interactions between erythropoiesis and iron homeostasis. Finally, we discuss iron disorders that affect erythropoiesis as well as erythroid disorders that cause iron dysregulation.
Homeostatic mechanisms maintain plasma iron concentrations at 10–30 μm. Chronically low or high concentrations lead to ineffective erythropoiesis, anemia, generation of nontransferrin-bound iron, and tissue damage.
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 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−/− showed a smaller hepcidin increase than their wild-type strain-matched controls, Bmp6−/− nearly no increase, and Tfr2 and Hjv mutants 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 mutants 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 (excepting Bmp6−/− mice) had impaired BMP6 mRNA response to chronic iron loading. Conclusion: TfR2, HJV and 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.
Hereditary hemochromatosis; bone morphogenetic protein 6; hemojuvelin; HFE; transferrin receptor 2
The peptide hormone hepcidin is a key homeostatic regulator of iron metabolism and involved in pathological regulation of iron in response to infection, inflammation, hypoxia and anaemia. It acts by binding to the iron exporter ferroportin, causing it to be internalised and degraded; however, little is known about the structure/activity relationships of the interaction of hepcidin with ferroportin. Here we show that there are key residues within the N-terminal region of hepcidin that influence its interaction with ferroportin, and we explore the structure/function relationships at these positions. We found that the interaction is primarily hydrophobic with critical stereochemical requirements at positions 4 and 6. In addition, a series of hepcidin mutants in which disulfide bonds had been replaced with diselenide bonds showed no change in biological activity compared to native hepcidin. The results provide mechanistic insight into the interaction between hepcidin and ferroportin and identify important constraints for the development of hepcidin congeners for the treatment of hereditary 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.