3-Amidinophenylalanine-derived matriptase inhibitors can modulate hepcidin production in vitro.

Matriptase-2 (MT-2) is a type II transmembrane serine protease and predominantly attached to the surface of hepatocytes. MT-2 decreases the production of hepcidin, a key regulator of iron homeostasis. In this study, the effects of four 3-amidinophenylalanine-derived combined matriptase-1/matriptase-2 (MT-1/2) inhibitors (MI-432, MI-441, MI-460, and MI-461) on hepcidin production were investigated in hepatocyte mono- and hepatocyte-Kupffer cell co-cultures. In MI-461-treated cell cultures, the extracellular hydrogen peroxide contents and the interleukin-6 and -8 (IL-6 and IL-8) levels were determined and compared to controls. Hepcidin overproduction was observed in hepatocytes upon treatment with MI-432, MI-441 and MI-461 at 50 µM. In contrast, extracellular hydrogen peroxide levels were not elevated significantly after matriptase inhibition with MI-461. Furthermore, MI-461 did not induce increases in IL-6 and IL-8 levels in these hepatic models. A model of the binding mode of inhibitor MI-461 in complex with MT-2 revealed numerous polar contacts contributing to the nanomolar potency of this compound. Based on the in vitro data on hepcidin regulation, treatment with MI-461 might be valuable in pathological states of iron metabolism without causing excessive oxidative stress.

Hepcidin.

Dysregulation of metabolism and utilization of iron can lead to the development and maintenance of anemia of CKD. Anemia is prevalent among patients with CKD. The markers of iron sufficiency or availability of iron are far from perfect which results in inaccurate diagnosis and treatment of anemia with poor outcomes. Hepcidin, a 25 amino acid peptide produced by the hepatocytes, has emerged as the key regulator of uptake and release of iron in the tissues to maintain a steady supply of iron to erythron and other tissues while avoiding higher levels of iron that could be detrimental to the organs. Hepcidin itself is regulated by the supply of iron, the need for erythropoiesis, and the state of inflammation. Alterations in hepcidin levels are associated with restricted erythropoiesis, anemia, and iron overload. Discovery of hepcidin and elucidation of its mechanism of action and consequences of its upregulation and suppression have unraveled important insight into many hematologic disorders including anemia of CKD. This knowledge has also unlocked unique opportunities to modulate hepcidin via agonists and antagonists of hepcidin and its feedback pathways to treat clinical conditions. Many such agents are being developed and have potential therapeutic utility in future.

New thiazolidinones reduce iron overload in mouse models of hereditary hemochromatosis and ß-thalassemia.

Genetic iron-overload disorders, mainly hereditary hemochromatosis and untransfused ß-thalassemia, affect a large population worldwide. The primary etiology of iron overload in these diseases is insufficient production of hepcidin by the liver, leading to excessive intestinal iron absorption and iron efflux from macrophages. Hepcidin agonists would therefore be expected to ameliorate iron overload in hereditary hemochromatosis and ß-thalassemia. In the current study, we screened our synthetic library of 210 thiazolidinone compounds and identified three thiazolidinone compounds, 93, 156 and 165, which stimulated hepatic hepcidin production. In a hemochromatosis mouse model with hemochromatosis deficiency, the three compounds prevented the development of iron overload and elicited iron redistribution from the liver to the spleen. Moreover, these compounds also greatly ameliorated iron overload and mitigated ineffective erythropoiesis in ß-thalassemic mice. Compounds 93, 156 and 165 acted by promoting SMAD1/5/8 signaling through differentially repressing ERK1/2 phosphorylation and decreasing transmembrane protease serine 6 activity. Additionally, compounds 93, 156 and 165 targeted erythroid regulators to strengthen hepcidin expression. Therefore, our hepcidin agonists induced hepcidin expression synergistically through a direct action on hepatocytes via SMAD1/5/8 signaling and an indirect action via eythroid cells. By increasing hepcidin production, thiazolidinone compounds may provide a useful alternative for the treatment of iron-overload disorders.

Hepcidin Protects against Lethal Escherichia coli Sepsis in Mice Inoculated with Isolates from Septic Patients.

Iron is an essential micronutrient for most microbes and their hosts. Mammalian hosts respond to infection by inducing the iron-regulatory hormone hepcidin, which causes iron sequestration and a rapid decrease in the plasma and extracellular iron concentration (hypoferremia). Previous studies showed that hepcidin regulation of iron is essential for protection from infection-associated mortality with the siderophilic pathogens Yersinia enterocolitica and Vibrio vulnificus However, the evolutionary conservation of the hypoferremic response to infection suggests that not only rare siderophilic bacteria but also common pathogens may be targeted by this mechanism. We tested 10 clinical isolates of Escherichia coli from children with sepsis and found that both genetic iron overload (by hepcidin-1 knockout [HKO]) and iatrogenic iron overload (by intravenous iron) potentiated infection with 8 out of the 10 studied isolates: after peritoneal injection of E. coli, iron-loaded mice developed sepsis with 60% to 100% mortality within 24 h, while control wild-type mice suffered 0% mortality. Using one strain for more detailed study, we show that iron overload allows rapid bacterial multiplication and dissemination. We further found that the presence of non-transferrin-bound iron (NTBI) in the circulation is more important than total plasma or tissue iron in rendering mice susceptible to infection and mortality. Postinfection treatment of HKO mice with just two doses of the hepcidin agonist PR73 abolished NTBI and completely prevented sepsis-associated mortality. We demonstrate that the siderophilic phenotype extends to clinically common pathogens. The use of hepcidin agonists promises to be an effective early intervention in patients with infections and dysregulated iron metabolism.

Hepcidin agonists as therapeutic tools.

Hepcidin agonists are a new class of compounds that regulate blood iron levels, limit iron absorption, and could improve the treatment of hemochromatosis, ß-thalassemia, polycythemia vera, and other disorders in which disrupted iron homeostasis causes or contributes to disease. Hepcidin agonists also have the potential to prevent severe complications of siderophilic infections in patients with iron overload or chronic liver disease. This review highlights the preclinical studies that support the development of hepcidin agonists for the treatment of these disorders.

Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin.

Nonclassical ferroportin disease (FD) is a form of hereditary hemochromatosis caused by mutations in the iron transporter ferroportin (Fpn), resulting in parenchymal iron overload. Fpn is regulated by the hormone hepcidin, which induces Fpn endocytosis and cellular iron retention. We characterized 11 clinically relevant and 5 nonclinical Fpn mutations using stably transfected, inducible isogenic cell lines. All clinical mutants were functionally resistant to hepcidin as a consequence of either impaired hepcidin binding or impaired hepcidin-dependent ubiquitination despite intact hepcidin binding. Mapping the residues onto 2 computational models of the human Fpn structure indicated that (1) mutations that caused ubiquitination-resistance were positioned at helix-helix interfaces, likely preventing the hepcidin-induced conformational change, (2) hepcidin binding occurred within the central cavity of Fpn, (3) hepcidin interacted with up to 4 helices, and (4) hepcidin binding should occlude Fpn and interfere with iron export independently of endocytosis. We experimentally confirmed hepcidin-mediated occlusion of Fpn in the absence of endocytosis in multiple cellular systems: HEK293 cells expressing an endocytosis-defective Fpn mutant (K8R), Xenopus oocytes expressing wild-type or K8R Fpn, and mature human red blood cells. We conclude that nonclassical FD is caused by Fpn mutations that decrease hepcidin binding or hinder conformational changes required for ubiquitination and endocytosis of Fpn. The newly documented ability of hepcidin and its agonists to occlude iron transport may facilitate the development of broadly effective treatments for hereditary iron overload disorders.

Matriptase-2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway.

Systemic iron homeostasis is maintained by regulation of iron absorption in the duodenum, iron recycling from erythrocytes, and iron mobilization from the liver and is controlled by the hepatic hormone hepcidin. Hepcidin expression is induced via the bone morphogenetic protein (BMP) signaling pathway that preferentially uses two type I (ALK2 and ALK3) and two type II (ActRIIA and BMPR2) BMP receptors. Hemojuvelin (HJV), HFE, and transferrin receptor-2 (TfR2) facilitate this process presumably by forming a plasma membrane complex with BMP receptors. Matriptase-2 (MT2) is a protease and key suppressor of hepatic hepcidin expression and cleaves HJV. Previous studies have therefore suggested that MT2 exerts its inhibitory effect by inactivating HJV. Here, we report that MT2 suppresses hepcidin expression independently of HJV. In Hjv-/- mice, increased expression of exogenous MT2 in the liver significantly reduced hepcidin expression similarly as observed in wild-type mice. Exogenous MT2 could fully correct abnormally high hepcidin expression and iron deficiency in MT2-/- mice. In contrast to MT2, increased Hjv expression caused no significant changes in wild-type mice, suggesting that Hjv is not a limiting factor for hepcidin expression. Further studies revealed that MT2 cleaves ALK2, ALK3, ActRIIA, Bmpr2, Hfe, and, to a lesser extent, Hjv and Tfr2. MT2-mediated Tfr2 cleavage was also observed in HepG2 cells endogenously expressing MT2 and TfR2. Moreover, iron-loaded transferrin blocked MT2-mediated Tfr2 cleavage, providing further insights into the mechanism of Tfr2's regulation by transferrin. Together, these observations indicate that MT2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway.

Endogenous hepcidin and its agonist mediate resistance to selected infections by clearing non-transferrin-bound iron.

The iron-regulatory hormone hepcidin is induced early in infection, causing iron sequestration in macrophages and decreased plasma iron; this is proposed to limit the replication of extracellular microbes, but could also promote infection with macrophage-tropic pathogens. The mechanisms by which hepcidin and hypoferremia modulate host defense, and the spectrum of microbes affected, are poorly understood. Using mouse models, we show that hepcidin was selectively protective against siderophilic extracellular pathogens (Yersinia enterocolitica O9) by controlling non-transferrin-bound iron (NTBI) rather than iron-transferrin concentration. NTBI promoted the rapid growth of siderophilic but not nonsiderophilic bacteria in mice with either genetic or iatrogenic iron overload and in human plasma. Hepcidin or iron loading did not affect other key components of innate immunity, did not indiscriminately promote intracellular infections (Mycobacterium tuberculosis), and had no effect on extracellular nonsiderophilic Y enterocolitica O8 or Staphylococcus aureus Hepcidin analogs may be useful for treatment of siderophilic infections.

Interleukin-1ß (IL-1ß) transcriptionally activates hepcidin by inducing CCAAT enhancer-binding protein δ (C/EBPδ) expression in hepatocytes.

Hepcidin is a liver-derived hormone that negatively regulates serum iron levels and is mainly regulated at the transcriptional level. Previous studies have clarified that in addition to hepatic iron levels, inflammation also efficiently increases hepatic hepcidin expression. The principle regions responsible for efficient hepcidin transcription are bone morphogenetic protein-responsive elements (BMP-REs) 1 and 2 as well as the signal transducer and activator of transcription 3-binding site (STAT-BS). Here, we show that the proinflammatory cytokine interleukin-1ß (IL-1ß) efficiently increases hepcidin expression in human HepG2 liver-derived cells and primary mouse hepatocytes. The primary region responsible for IL-1ß-mediated hepcidin transcription was the putative CCAAT enhancer-binding protein (C/EBP)-binding site (C/EBP-BS) at the hepcidin promoter spanning nucleotides -329 to -320. IL-1ß induces the expression of C/EBPδ but neither C/EBPα nor C/EBPß in hepatocytes, and C/EBPδ bound to the C/EBP-BS in an IL-1ß-dependent manner. Lipopolysaccharide (LPS) induced the expression of IL-1ß in Kupffer cells and hepatocytes in the mouse liver; furthermore, the culture supernatants from the macrophage-like cell line RAW264.7 treated with LPS potentiated the stimulation of hepcidin expression in hepatocytes. The present study reveals that: 1) inflammation induces IL-1ß production in Kupffer cells and hepatocytes; 2) IL-1ß increases C/EBPδ expression in hepatocytes; and 3) induction of C/EBPδ activates hepcidin transcription via the C/EBP-BS that has been uncharacterized yet. In cooperation with the other pathways activated by inflammation, IL-1ß pathway stimulation leads to excess production of hepcidin, which could be causative to anemia of inflammation.

Estado actual del metabolismo del hierro: implicaciones clínicas y terapéuticas / Current status of iron metabolism: clinical and therapeutic implications

La hepcidina es el principal regulador del metabolismo del hierro y el factor patogénico más importante en sus trastornos. La deficiencia de hepcidina provoca sobrecarga de hierro, mientras que su exceso da lugar o contribuye al desarrollo de anemias por déficit o restricción de hierro en las enfermedades crónicas. Conocemos los mecanismos implicados en la síntesis de hepcidina y, en condiciones fisiológicas, hay un equilibrio entre las señales activadoras e inhibidoras que regulan su síntesis. Las primeras incluyen las relacionadas con la concentración plasmática de hierro y con las enfermedades inflamatorias. Las señales inhibidoras más importantes están relacionadas con la eritropoyesis activa y con la matriptasa-2. Conocer cómo se sintetiza la hepcidina ha servido para diseñar nuevos tratamientos farmacológicos cuya diana principal es la hepcidina. En un futuro próximo, se dispondrá de tratamientos eficaces dirigidos a corregir el defecto de muchos de los trastornos del metabolismo del hierro (AU)

Hepcidin is the main regulator of iron metabolism and a pathogenic factor in iron disorders. Hepcidin deficiency causes iron overload, whereas hepcidin excess causes or contributes to the development of iron-restricted anaemia in chronic inflammatory diseases. We know the mechanisms involved in the synthesis of hepcidin and, under physiological conditions, there is a balance between activating signals and inhibitory signals that regulate its synthesis. The former include those related to plasmatic iron level and also those related to chronic inflammatory diseases. The most important inhibitory signals are related to active erythropoiesis and to matriptase-2. Knowing how hepcidin is synthesised has helped design new pharmacological treatments whose main target is the hepcidin. In the near future, there will be effective treatments aimed at correcting the defect of many of these iron metabolism disorders (AU)

Regulation of Hepcidin by Erythropoiesis: The Story So Far.

Hepcidin is the master regulator of systemic iron homeostasis, facilitating iron balance by controlling intestinal iron absorption and recycling. Hepcidin levels are suppressed when erythropoiesis is stimulated, for example following acute blood loss, appropriately enhancing cellular iron export to the plasma to support production of new red blood cells. However, persistent increased and ineffective erythropoiesis, for example in thalassemia, results in sustained elevations in iron absorption, which cause iron overload with associated organ toxicities. The ligands, receptors, and canonical pathways by which iron loading and inflammation upregulate hepcidin expression have been largely established. However, although several mechanisms have been proposed, the means by which erythropoiesis causes hepcidin suppression have been unclear. The erythroid-derived hormone erythroferrone appears to be a convincing candidate for the link between increased erythropoiesis and hepcidin suppression. If confirmed to be clinically and physiologically relevant in humans, potentiation or inhibition of erythroferrone activity could be a crucial pharmaceutical strategy.

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 ß-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in ß-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.

Modulation of hepcidin to treat iron deregulation: potential clinical applications.

The secreted peptide hormone hepcidin regulates systemic and local iron homeostasis through degradation of the iron exporter ferroportin. Dysregulation of hepcidin leads to altered iron homeostasis and development of pathological disorders including hemochromatosis, and iron loading and iron restrictive anemias. Therapeutic modulation of hepcidin is a promising method to ameliorate these conditions. Several approaches have been taken to enhance or reduce the effects of hepcidin in vitro and in vivo. Based on these approaches, hepcidin modulating drugs have been developed and are undergoing clinical evaluation. In this article we review the rationale for development of these drugs, the data concerning their safety and efficacy, their therapeutic uses, and potential future prospects.

Small cyclic agonists of iron regulatory hormone hepcidin.

Minihepcidins are in vitro and in vivo active mimetics of iron-regulatory hormone hepcidin. They contain various unusual amino acids including: N-substituted, ß-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 cyclization. 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 cyclization is viable approach in the synthesis of hepcidin mimetics.

[Current management of thalassemia intermedia]. / Prise en charge actuelle des thalassémies intermédiaires.

Thalassemia intermedia is a clinical entity where anemia is mild or moderate, requiring no or occasional transfusion. Non-transfusion-dependent thalassemia encompasses 3 main clinical forms: beta-thalassemia intermedia, hemoglobin E/beta-thalassemia and alpha-thalassemia intermedia (HbH disease). Clinical severity of thalassemia intermedia increases with age, with more severe anemia and more frequent complications such as extramedullary hematopoiesis and iron overload mainly related to increased intestinal absorption. Numerous adverse events including pulmonary hypertension and hypercoagulability have been associated with splenectomy, often performed in thalassemia intermedia patients. The potential preventive benefit of transfusion and chelation therapies on the occurrence of numerous complications supports the strategy of an earlier therapeutic intervention. Increasing knowledge about pathophysiological mechanisms involved in thalassemia erythropoiesis and related iron overload is currently translating in novel therapeutic approaches.

A chemical screen identifies small molecules that regulate hepcidin expression.

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Bone morphogenic protein and Stat3 signaling regulate Hepcidin's transcription. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 2.7kb of the human Hepcidin promoter upstream of a firefly reporter gene. We used high throughput methods to screen 10,169 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1 standard deviation below the mean of the other chemicals (z-score >3 or <1), while not adversely affecting cell viability, quantified by fluorescence assay. Following validation assays, we identified 16 chemicals in a broad range of functional classes that promote Hepcidin expression. All of the chemicals identified increased expression of bone morphogenic protein-dependent and/or Stat3-dependent genes, however none of them strongly increased phosphorylation of Smad1,5,8 or Stat3.

Quercetin prevents ethanol-induced iron overload by regulating hepcidin through the BMP6/SMAD4 signaling pathway.

Emerging evidence has demonstrated that chronic ethanol exposure induces iron overload, enhancing ethanol-mediated liver damage. The purpose of this study was to explore the effects of the naturally occurring compound quercetin on ethanol-induced iron overload and liver damage, focusing on the signaling pathway of the iron regulatory hormone hepcidin. Adult male C57BL/6J mice were pair-fed with isocaloric-Lieber De Carli diets containing ethanol (accounting for 30% of total calories) and/or carbonyl iron (0.2%) and treated with quecertin (100 mg/kg body weight) for 15 weeks. Mouse primary hepatocytes were incubated with ethanol (100 mM) and quercetin (100 µM) for 24 h. Mice exposed to either ethanol or iron presented significant fatty infiltration and iron deposition in the liver; these symptoms were exacerbated in mice cotreated with ethanol and iron. Quercetin attenuated the abnormity induced by ethanol and/or iron. Ethanol suppressed BMP6 and intranuclear SMAD4 as well as decreased hepcidin expression. These effects were partially alleviated by quercetin supplementation in mice and hepatocytes. Importantly, ethanol caused suppression of SMAD4 binding to the HAMP promoter and of hepcidin messenger RNA expression. These effects were exacerbated by anti-BMP6 antibody and partially alleviated by quercetin or human recombinant BMP6 in cultured hepatocytes. In contrast, co-treatment with iron and ethanol, especially exposure of iron alone, activated BMP6/SMAD4 pathway and up-regulated hepcidin expression, which was also normalized by quercetin in vivo. Quercetin prevented ethanol-induced hepatic iron overload different from what carbonyl iron diet elicited in the mechanism, by regulating hepcidin expression via the BMP6/SMAD4 signaling pathway.

The pathophysiology and pharmacology of hepcidin.

Inappropriate production of the iron-regulatory hormone hepcidin contributes to the pathogenesis of common iron disorders. Absolute or relative deficiency of hepcidin causes iron overload in hereditary hemochromatosis and iron-loading anemias. Elevated hepcidin causes iron restriction in inflammatory conditions including autoimmune disease, critical illness, some cancers, and chronic kidney disease. Multiple agents targeting hepcidin and its regulators are under development as novel therapeutics for iron disorders. This review summarizes hepcidin biology and discusses the current landscape for hepcidin-targeting therapeutic strategies.

Friend of GATA and GATA-6 modulate the transcriptional up-regulation of hepcidin in hepatocytes during inflammation.

Hepcidin is an antimicrobial peptide hormone that plays a central role in the metabolism of iron and its expression in the liver can be induced through two major pathways: the inflammatory pathway, mainly via IL-6; and the iron-sensing pathway, mediated by BMP-6. GATA-proteins are group of evolutionary conserved transcriptional regulators that bind to the consensus motif-WGATAR-in the promoter region. In hepatoma cells, GATA-proteins 4 and 6 in conjunction with the co-factor friend of GATA (FOG) were shown to modulate the transcription of HAMP. However, it is unclear as to which of the GATA-proteins drive the expression of HAMP in vivo. In this study, using in vitro and in vivo approaches, we investigated the relevance of GATA and FOG proteins in the expression of hepcidin following treatment with IL-6 and BMP-6. We found that treatment of Huh7 cells with either IL-6 or BMP-6 increased the HAMP promoter activity. The HAMP promoter activity following treatment with IL-6 or BMP-6 was further increased by co-transfection of the promoter with GATA proteins 4 and 6. However, co-transfection of the HAMP promoter with FOG proteins 1 or 2 repressed the promoter response to treatments with either IL-6 or BMP-6. The effects of both GATA and FOG proteins on the promoter activity in response to IL-6 or BMP-6 treatment were abrogated by mutation of the GATA response element-TTATCT-in the HAMP promoter region -103/-98. In vivo, treatment of mice with lipopolysaccharide led to a transient increase of Gata-6 expression in the liver that was positively correlated with the expression of hepcidin. Our results indicate that during inflammation GATA-6 is up-regulated in concert with hepcidin while GATA-4 and FOG (1 and 2) are repressed.