Decreased Hepcidin Expression Research Articles

Erythroferrone contributes to iron mobilization for embryo erythropoiesis in iron-deficient mouse pregnancies.

Erythroferrone (ERFE) is an erythroblast-secreted regulator of iron metabolism. The production of ERFE increases during stress erythropoiesis, leading to decreased hepcidin expression and mobilization of iron. Pregnancy requires a substantial increase in iron availability to sustain maternal erythropoietic expansion and fetal development and is commonly affected by iron deficiency. To define the role of ERFE during iron-replete or iron-deficient pregnancy, we utilized mouse models expressing a range of ERFE levels: transgenic (TG) mice overexpressing ERFE, wild-type (WT), and ERFE knockout (KO) mice. We altered maternal iron status using diets with low or standard iron content and performed the analysis at E18.5. Iron deficiency increased maternal ERFE in WT pregnancy. Comparing different maternal genotypes, ERFE TG dams had lower hepcidin relative to their liver iron load but similar hematological parameters to WT dams on either diet. In ERFE KO dams, most hematologic and iron parameters were comparable to WT, but mean corpuscular volume (MCV) was decreased under both iron conditions. Similar to dams, TG embryos had lower hepcidin on both diets, but their hematologic parameters did not differ from those of WT embryos. ERFE KO embryos had lower MCV than WT embryos on both diets. The effect was exacerbated under iron-deficient conditions where ERFE KO embryos had higher hepcidin, lower Hb and Hct, and lower brain iron concentration compared to WT embryos, indicative of iron restriction. Thus, under iron-deficient conditions, maternal and embryo ERFE facilitate iron mobilization for embryonic erythropoiesis.

Caffeine Decreases Hepcidin Expression to Alleviate Aberrant Iron Metabolism under Inflammation by Regulating the IL-6/STAT3 Pathway.

Caffeine is well-known as a psychostimulant, and it can also be beneficial in numerous diseases such as diabetes and different types of cancer. Previous studies have shown that caffeine can have a protective role in bacterial infection-induced inflammation and hyperoxia-mediated pulmonary inflammation. Hepcidin, which is regulated by the IL-6/STAT3 inflammation pathway, is a peptide hormone that maintains systemic iron homeostasis. We hypothesized that caffeine’s effects on inflammation may also influence hepcidin production and therefore systemic iron metabolism. To this end, we treated 2-month-old mice with caffeine by daily intragastric administration for 7 days, administering intraperitoneal LPS after the final caffeine treatment. Twelve hours after LPS treatment the mice were euthanized, and tissues were collected. We found that caffeine decreased hepatic hepcidin expression and attenuated LPS-induced hepatic hepcidin overexpression. IL-6 expression and STAT3 phosphorylation were also reduced upon caffeine administration. Additionally, hepatic and splenic FPN1 levels increased after caffeine treatment, leading to lower iron levels in liver and spleen tissues and higher iron levels in serum. Caffeine also prevented the increase in spleen weight and decrease in body weight after LPS treatment. Together, our findings suggest that caffeine decreases hepcidin expression via inhibiting inflammation and the activation of the IL-6/STAT3 pathway, thus presenting an attractive, potential therapeutic for the treatment of anemia of inflammation.

Alcohol exposure increases manganese accumulation in the brain and exacerbates manganese-induced neurotoxicity in mice.

Environmental and occupational exposure to heavy metals remains one of the major concerns in public health. Increased levels of manganese (Mn) pollution are associated with profound neurotoxic effects, including neurobehavioral deficits and disturbances resembling Parkinson's disease. While Mn absorption is in part mediated by iron transporters, recent studies have shown that the levels of iron transporters are modified by alcohol and that chronic alcohol consumption increases body iron stores. However, it is largely unexplored whether alcohol exposure influences the transport and neurotoxicity of Mn. To address this question, we exposed mice to ethanol (10%; v/v) by drinking water for 4weeks, during which period MnCl2 (5mg/kg) or saline solutions were administered daily by intranasal instillation. Ethanol consumption in mice increased brain Mn levels in a dose-dependent manner after Mn instillation, determined by inductively-coupled plasma mass spectrometry, which was accompanied by up-regulation of iron transporters, as assessed by western blotting and qPCR. In addition, alcohol drinking increased hypoxic response and decreased hepcidin expression, providing the molecular mechanism of increased iron transporters and Mn uptake upon alcohol consumption. Moreover, brain dopamine levels, analyzed by HPLC, were decreased after intranasal Mn instillation, which was worsened by alcohol. Likewise, alcohol-Mn co-exposure synergistically altered dopaminergic protein expression. Finally, alcohol binge-drinking, which resembles alcohol drinking manner in humans, increased brain Mn content along with upregulation of iron transporters. Our study suggests that individuals who consume alcohol may have a higher risk of Mn neurotoxicity upon Mn exposure.

Hepatocyte neogenin is required for hemojuvelin-mediated hepcidin expression and iron homeostasis in mice

AbstractNeogenin (NEO1) is a ubiquitously expressed multifunctional transmembrane protein. It interacts with hemojuvelin (HJV), a BMP coreceptor that plays a pivotal role in hepatic hepcidin expression. Earlier studies suggest that the function of HJV relies on its interaction with NEO1. However, the role of NEO1 in iron homeostasis remains controversial because of the lack of an appropriate animal model. Here, we generated a hepatocyte-specific Neo1 knockout (Neo1fl/fl;Alb-Cre+) mouse model that circumvented the developmental and lethality issues of the global Neo1 mutant. Results show that ablation of hepatocyte Neo1 decreased hepcidin expression and caused iron overload. This iron overload did not result from altered iron utilization by erythropoiesis. Replacement studies revealed that expression of the Neo1L1046E mutant that does not interact with Hjv, was unable to correct the decreased hepcidin expression and high serum iron in Neo1fl/fl;Alb-Cre+ mice. In Hjv−/− mice, expression of HjvA183R mutant that has reduced interaction with Neo1, also displayed a blunted induction of hepcidin expression. These observations indicate that Neo1-Hjv interaction is essential for hepcidin expression. Further analyses suggest that the Hjv binding triggered the cleavage of the Neo1 cytoplasmic domain by a protease, which resulted in accumulation of truncated Neo1 on the plasma membrane. Additional studies did not support that Neo1 functions by inhibiting Hjv shedding as previously proposed. Together, our data favor a model in which Neo1 interaction with Hjv leads to accumulation of cleaved Neo1 on the plasma membrane, where Neo1 acts as a scaffold to induce the Bmp signaling and hepcidin expression.

Abstract 4028: Nanocyclix® ALK2 Inhibitors to overcome cancer-induced anemia

Abstract Introduction: Anemia is a condition characterized by low Hemoglobin levels. Anemia is observed during tumor development through the release of inflammatory cytokines, which will affect iron availability, a key component of red blood cells. In recent years, consequences of anemia of cancer on patients is getting growing recognition from a quality of life perspective, as well as its impact on the treatment scheme and hospitalization duration. Cancer-induced anemia has been identified in 30% of treatment-naive cancers (ECAS study, 2011 - WHO methodology). The proportion of anaemic patients reaches 70% following treatments, such as chemotherapies. Current approaches consider erythropoietin derivatives, formulated iron or transfusions, which does not address underlying chronic inflammation. Moreover, their use is limited as a consequence of toxicities (EPO, formulated iron) or availability (transfusion). Increased expression of Hepcidin, a peptidic hormone regulating the storage of bioavailable iron, has been demonstrated as downstream effector of key inflammatory cytokines activation of ALK2/Smad axis. ALK2 (Activin-like receptor kinase 2) plays a critical role in the Smad signaling pathway and the production haemoglobin, through the regulation Hepcidin production in the liver. As a consequence, the selective inhibition of ALK2 as emerged as a valuable target for the treatment of Hepcidin-driven anemia of cancer to provide an alternative to current treatments. Results: OD52, a potent and selective macrocyclic ALK2 inhibitor identified from Nanocyclix® technology platform, was used as in vitro pharmacological tool to investigate the impact of ALK2 inhibition on Hepcidin expression level in HepG2 cells, as well as in mice hepatocytes. BMP6-induced expression of Hepcidin mRNA was completely inhibited by LDN-193189 to low detectable levels, whereas OD52 normalized its expression. Further validation was performed in mice hepatocytes, where OD52 decreased Hepcidin expression to a lower extend as compared with LDN-193189. These experiments confirmed the importance of selectivity within BMP-kinase receptor family, where ALK3 component represents an undesired off-target. In vivo evaluation of the compound in the acute model of turpentine-induced anemia demonstrated a normalization of haemoglobin level. In contrast, LDN-193189 increased haemoglobin above vehicle group to a level mimicking hemochromatosis, likely due to its ALK3 component. Through further medicinal chemistry optimization, OD66 was identified as suitable in vivo pharmacological tool and was evaluated in a cancer-induced anemia mouse model. The compound demonstrated a normalization of haemoglobin levels to comparable levels with the tumor-free group without displaying toxicity. Conclusion: The identification of potent and selective Nanocyclix® ALK2 inhibitors and their evaluation in relevant in vitro and in vivo models demonstrated a normalization of haemoglobin levels through the modulation of Hepcidin expression. This collaborative work warrants further optimization towards the identification of a preclinical candidate. Citation Format: Pascal Benderitter, Josselin Caradec, Edwige Nicodeme, Anne Bouillot, Sophie Vaulont, Benoit Viollet. Nanocyclix® ALK2 Inhibitors to overcome cancer-induced anemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4028.

Hepatocyte-specific deletion of peroxisomal protein PEX13 results in disrupted iron homeostasis

Peroxisomes are organelles, abundant in the liver, involved in a variety of cellular functions, including fatty acid metabolism, plasmalogen synthesis and metabolism of reactive oxygen species. Several inherited disorders are associated with peroxisomal dysfunction; increasingly many are associated with hepatic pathologies. The liver plays a principal role in regulation of iron metabolism. In this study we examined the possibility of a relationship between iron homeostasis and peroxisomal integrity.We examined the effect of deleting Pex13 in mouse liver on systemic iron homeostasis. We also used siRNA-mediated knock-down of PEX13 in a human hepatoma cell line (HepG2/C3A) to elucidate the mechanisms of PEX13-mediated regulation of hepcidin.We demonstrate that transgenic mice lacking hepatocyte Pex13 have defects in systemic iron homeostasis. The ablation of Pex13 expression in hepatocytes leads to a significant reduction in hepatic hepcidin levels. Our results also demonstrate that a deficiency of PEX13 gene expression in HepG2/C3A cells leads to decreased hepcidin expression, which is mediated through an increase in the signalling protein SMAD7, and endoplasmic reticulum (ER) stress.This study identifies a novel role for a protein involved in maintaining peroxisomal integrity and function in iron homeostasis. Loss of Pex13, a protein important for peroxisomal function, in hepatocytes leads to a significant increase in ER stress, which if unresolved, can affect liver function. The results from this study have implications for the management of patients with peroxisomal disorders and the liver-related complications they may develop.

Hepcidin as a key iron regulator mediates glucotoxicity-induced pancreatic β-cell dysfunction.

It has been well established that glucotoxicity induces pancreatic β-cells dysfunction; however, the precise mechanism remains unclear. Our previous studies demonstrated that high glucose concentrations are associated with decreased hepcidin expression, which inhibits insulin synthesis. In this study, we focused on the role of low hepcidin level-induced increased iron deposition in β-cells and the relationship between abnormal iron metabolism and β-cell dysfunction. Decreased hepcidin expression increased iron absorption by upregulating transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) expression, resulting in iron accumulation within cells. Prussia blue stain and calcein-AM assays revealed greater iron accumulation in the cytoplasm of pancreatic tissue isolated from db/db mice, cultured islets and Min6 cells in response to high glucose stimulation. Increased cytosolic iron deposition was associated with greater Fe2+ influx into the mitochondria, which depolarized the mitochondria membrane potential, inhibited ATP synthesis, generated excessive ROS and induced oxidative stress. The toxic effect of excessive iron on mitochondrial function eventually resulted in impaired insulin secretion. The restricted iron content in db/db mice via reduced iron intake or accelerated iron clearance improved blood glucose levels with decreased fasting blood glucose (FBG), fasting blood insulin (FIns), HbA1c level, as well as improved intraperitoneal glucose tolerance test (IPGTT) results. Thus, our study may reveal the mechanism involved in the role of hepcidin in the glucotoxcity impaired pancreatic β cell function pathway.

Novel Insights Into Systemic Iron Regulation

Iron, an essential element in mammals, is absorbed by duodenal enterocytes, enters the circulation through the iron exporter ferroportin, (FPN), circulates bound to transferrin and is uptaken through Transferrin Receptor 1. If in excess, iron is stored in macrophages and hepatocytes and released when needed. To maintain systemic iron homeostasis and to avoid the formation of “non transferrin bound iron” (NTBI), a highly reactive form which causes organ damage, the liver synthetizes hepcidin that, binding FPN, blocks iron export to the circulation. Hepcidin integrates signals from body iron, erythropoiesis and inflammatory cytokines. Defective hepcidin production causes iron overload and organ failure in Hereditary Hemochromatosis and Thalassemia; hepcidin excess leads to anemia in Iron Refractory iron Deficiency Anemia (IRIDA) and Anemia of Inflammation (AI).In hepatocytes hepcidin is under the control of the BMP-SMAD pathway, which is activated in a paracrine manner by BMP2 and BMP6 produced by liver sinusoidal endothelial cells. BMP2 maintains hepcidin basal levels, while BMP6 controls its expression in response to iron. The two ligands have different affinity for BMP type I receptors ALK2 and ALK3, suggesting two distinct branches of the hepcidin activation pathway. This possibility is consistent with the non-redundant function of BMP2 and BMP6, the different iron phenotype of hepatocyte-conditional ALK2 and ALK3 KO mice and the residual ability of BMP6 to activate hepcidin in hemochromatosis mice. Moreover ALK2, but not ALK3, is inhibited by the immunophilin FKBP12 in the absence of ligands. The BMP pathway activation depends upon the coreceptor hemojuvelin (HJV), the MHC class I protein HFE and the second transferrin receptor (TFR2). Mutations of all these proteins lead to decreased hepcidin expression in hemochromatosis.Hepcidin expression is inhibited in iron deficiency, hypoxia and when erythropoiesis is increased. Inhibitors are the liver transmembrane serine protease TMPRSS6, whose genetic inactivation causes IRIDA, and the erythroid hormone erythroferrone (ERFE), which is released by erythropoietin-stimulated erythroblasts. The mechanism of hepcidin inhibition by ERFE is unclear; still to allow ERFE function the BMP-SMAD pathway has not to be hyperactive. Intriguingly, both iron deficiency and erythropoiesis require epigenetic modifications at the hepcidin locus with HDAC3-dependent reversible loss of H3K9ac and H3K4me3.Hepcidin also acts as an antimicrobial peptide since its expression, increased by proinflammatory cytokines, such as IL6 through JAK2-STAT3 signaling, restricts iron availability for microbial growth. This first-line of defense against infections negatively influences erythropoiesis since chronic hepcidin activation causes AI. Despite persistent JAK2-STAT3 activation, inhibition of the BMP-SMAD pathway reduces hepcidin activation in AI experimental rodent models, suggesting that hepcidin activation in inflammation requires a functional BMP-SMAD pathway. Independently from hepcidin, inflammation also reduces FPN mRNA levels, favoring macrophage iron sequestration.The identification of hepcidin-ferroportin axis molecular players has translational implications. In primary and secondary iron overload hepcidin agonists (hepcidin peptides or mimics, agents that inhibit the hepcidin inhibitor TMPRSS6 and likely the ALK2-inhibitor FKBP12) and ferroportin inhibitors are potentially useful to prevent iron overload and/or to favor iron redistribution to macrophages. In case of AI, hepcidin antagonists (including anti-hepcidin, anti-HJV and anti-BMP6 monoclonal antibodies, L-enantiomeric oligonucleotides targeting hepcidin, siRNA against hepcidin, non-anticoagulant heparins, the ALK2 inhibitor momelotinib) might improve erythropoiesis increasing iron availability.The effect of some agents that have now entered the clinical phase will become apparent in the coming years. DisclosuresCamaschella:vifor Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Target identification of hepcidin production inhibitors by a combination of chemical proteomics and radioactive compound binding assay

Hepcidin is a peptide hormone and has emerged as the central molecule regulating systemic iron homeostasis. Hepcidin inhibition could be a strategy for treating anemia of chronic disease. We previously reported the discovery of DS79182026, a new inhibitor of hepcidin production, from phenotypic screening using the human hepatocyte HepG2 cell line. In this study, we utilized a combination of affinity purification-based chemical proteomics and radioactive compound binding assay, and identified several candidate proteins. Purified recombinant proteins were subjected to radioactive compound binding assays for validation, and ALK2 and ALK3 demonstrated specific binding to the compound. Since ALK2 is known to be related to hepcidin production, we focused on ALK2 and found that its knockdown decreased hepcidin expression; we also found a strong correlation (R = 0.920) between pharmacological activity and compound affinity to ALK2. These results indicate that ALK2 is the primary target protein of our new hepcidin production inhibitors.

The C19S Substitution Enhances the Stability of Hepcidin While Conserving Its Biological Activity.

Hepcidin, the key hormone of iron homeostasis is responsible for lowering the serum iron level through its interaction with iron exporter ferroportin. Thus, hepcidin agonists provide a promising opportunity in the treatment of iron disorders caused by lacking or decreased hepcidin expression. We investigated the importance of each of the eight highly conserved cysteines for the biological activity of hepcidin. Eight cysteine mutants were created with site directed mutagenesis. The binding ability of these hepcidin mutants to the hepcidin receptor ferroportin was determined using bacterial two-hybrid system and WRL68 human hepatic cells. The biological activity of hepcidin mutants was determined by western blot analysis of ferroportin internalization and ferroportin ubiquitination. To investigate the effect of mutant hepcidins on the iron metabolism of the WRL68 cells, total intracellular iron content was measured with a colorimetric assay. The stability of M6 hepcidin mutant was determined using ELISA technique. Our data revealed that serine substitution of the sixth cysteine (M6) yielded a biologically active but significantly more stable peptide than the original hormone. This result may provide a promising hepcidin agonist worth testing in animal models.

The Effects of Angelica Sinensis Polysaccharide on Tumor Growth and Iron Metabolism by Regulating Hepcidin in Tumor-Bearing Mice

Background/Aims: Iron plays a fundamental role in cell biology and its concentration must be precisely regulated. It is well documented that excess iron burden contributes to the occurrence and progression of cancer. Hepcidin secreted by liver plays an essential role in orchestrating iron metabolism. In the present study, we aimed to investigate the ability of angelica sinensis polysaccharide (ASP) to decrease iron burden in tumor-bearing mice and the mechanism of ASP regulation hepcidin expression. Methods: Western blot, RT-PCR, immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) were used to detect the regulation of hepcidin and related cytokines by ASP. The role of ASP in tumor proliferation was investigated using in vivo assays. Iron depositions and iron concentrations in organs were determined by hematoxylin-eosin (H&E) staining and atomic absorption spectrophotometer. Results: We found that ASP could inhibit tumor growth in mice xenografted with 4T1 and H22 cancer cells. In vivo experiments also showed that ASP could potently regulate hepcidin expression in liver and serum and decrease iron burden in liver, spleen and grafted tumors in mouse model. Treatment with ASP in hepatic cell lines reproduced comparable results in decreasing hepcidin as in mouse liver. Furthermore, we found that ASP markedly suppressed the expression of interleukin-6 (IL-6), JAK2, p-STAT3, and p-SMAD1/5/8 in liver, suggesting that JAK/STAT and BMP-SMAD pathways were involved in the regulation of hepcidin expression by ASP. We also found down-regulation of iron-related cytokines in ASP treated mice. Conclusion: The present study provides new evidence that ASP decreases hepcidin expression, which can reduce iron burden and inhibit tumor proliferation. These findings might aid ASP developed as a potential candidate for cancer treatment in patients with iron overload.

Effect of erythropoietin administration on proteins participating in iron homeostasis in Tmprss6-mutated mask mice.

Tmprss6-mutated mask mice display iron deficiency anemia and high expression of hepcidin. The aim of the study was to determine the effect of erythropoietin administration on proteins participating in the control of iron homeostasis in the liver and spleen in C57BL/6 and mask mice. Administration of erythropoietin for four days at 50 IU/mouse/day increased hemoglobin and hematocrit in C57BL/6 mice, no such increase was seen in mask mice. Erythropoietin administration decreased hepcidin expression in C57BL/6 mice, but not in mask mice. Erythropoietin treatment significantly increased the spleen size in both C57BL/6 and mask mice. Furthermore, erythropoietin administration increased splenic Fam132b, Fam132a and Tfr2 mRNA content. At the protein level, erythropoietin increased the amount of splenic erythroferrone and transferrin receptor 2 both in C57BL/6 and mask mice. Splenic ferroportin content was decreased in erythropoietin-treated mask mice in comparison with erythropoietin-treated C57BL/6 mice. In mask mice, the amount of liver hemojuvelin was decreased in comparison with C57BL/6 mice. The pattern of hemojuvelin cleavage was different between C57BL/6 and mask mice: In both groups, a main hemojuvelin band was detected at approximately 52 kDa; in C57BL/6 mice, a minor cleaved band was seen at 47 kDa. In mask mice, the 47 kDa band was absent, but additional minor bands were detected at approximately 45 kDa and 48 kDa. The results provide support for the interaction between TMPRSS6 and hemojuvelin in vivo; they also suggest that hemojuvelin could be cleaved by another as yet unknown protease in the absence of functional TMPRSS6. The lack of effect of erythropoietin on hepcidin expression in mask mice can not be explained by changes in erythroferrone synthesis, as splenic erythroferrone content increased after erythropoietin administration in both C57BL/6 and mask mice.

Hepcidin links gluco-toxicity to pancreatic beta cell dysfunction by inhibiting Pdx-1 expression.

ObjectiveGluco-toxicity is a term used to convey the detrimental effect of hyperglycemia on β-cell function through impaired insulin synthesis. Although it is known that the expression and activity of several key insulin transcription regulators is inhibited, other molecular mechanisms that mediate gluco-toxicity are poorly defined. Our objective was to explore the role of hepcidin in β-cell gluco-toxicity.DesignWe first confirmed that high glucose levels inhibited hepcidin expression in the mouse insulinoma cell line, MIN6. The downregulation of hepcidin decreased Pdx-1 expression, which reduced insulin synthesis.MethodsMIN6 cells were exposed to high glucose concentrations (33.3 mmol/L). Glucose-stimulated insulin secretion (GSIS) and serum hepcidin levels were measured by ELISA. The mRNA levels of insulin1, insulin2, Pdx-1 and hepcidin were measured by real-time polymerase chain reaction. Western blot analysis was used to detect the changes in PDX-1 expression. Transient overexpression with hepcidin was used to reverse the downregulation of Pdx-1 and insulin synthesis induced by gluco-toxicity.ResultsExposure of MIN6 cells to high glucose significantly decreased GSIS and inhibited insulin synthesis as well as Pdx-1 transcriptional activity and expression at both the mRNA and protein levels. High glucose also decreased hepcidin expression and secretion. Hepcidin overexpression in MIN6 cells partially reversed the gluco-toxicity-induced downregulation of Pdx-1 and insulin expression and improved GSIS. The restoration of insulin synthesis by transfection of a hepcidin overexpression plasmid confirmed the role of hepcidin in mediating the gluco-toxic inhibition of insulin synthesis.ConclusionsOur observations suggest that hepcidin is associated with gluco-toxicity-reduced pancreatic β-cell insulin synthesis in type 2 diabetes by inhibiting Pdx-1 expression.

Roles of transferrin receptors in erythropoiesis.

Erythropoiesis requires large amounts of iron for hemoglobin synthesis, which is mainly provided by macrophages and the intestines in a transferrin (Tf)-bound form. Bone marrow erythroblasts incorporate Tf through endocytosis, which is mediated by transferrin receptor 1 (TFR1). Recently, human TFR1, aside from its role as a Tf receptor, was also found to be a receptor for the H-subunit of ferritin (FTH). In humans, hematopoietic erythroid precursor cells express high levels of TFR1 and specifically take up the FTH homopolymer (H-ferritin). H-ferritin inhibits the formation of burst forming unit-erythroid colonies in vitro. TFR2, which is also a Tf receptor, is predominantly expressed in hepatocytes and erythroid precursor cells. In the liver, TFR2 forms a complex with HFE, a hereditary hemochromatosis-associated protein, and acts as an iron sensor. In mice, hepatocyte-specific knockout of the TFR2 gene has been shown to cause systemic iron-overload with decreased expression of hepcidin, the central regulator of iron homeostasis. In erythroid cells, TFR2 forms a complex with the erythropoietin receptor and facilitates its trafficking to the cell membrane. Moreover, hematopoietic cell-specific knockout of the TFR2 gene causes microcytic erythrocytosis in mice. This review focuses on the molecular evolution and functions of these TFRs and their ligands.

CD81 Promotes Both the Degradation of Transferrin Receptor 2 (TfR2) and the Tfr2-mediated Maintenance of Hepcidin Expression

Mutations in transferrin receptor 2 (TfR2) cause a rare form of the hereditary hemochromatosis, resulting in iron overload predominantly in the liver. TfR2 is primarily expressed in hepatocytes and is hypothesized to sense iron levels in the blood to positively regulate the expression of hepcidin through activation of the BMP signaling pathway. Hepcidin is a peptide hormone that negatively regulates iron egress from cells and thus limits intestinal iron uptake. In this study, a yeast two-hybrid approach using the cytoplasmic domain of TfR2 identified CD81 as an interacting protein. CD81 is an abundant tetraspanin in the liver. Co-precipitations of CD81 with different TfR2 constructs demonstrated that both the cytoplasmic and ecto-transmembrane domains of TfR2 interact with CD81. Knockdown of CD81 using siRNA significantly increased TfR2 levels by increasing the half-life of TfR2, indicating that CD81 promotes degradation of TfR2. Previous studies showed that CD81 is targeted for degradation by GRAIL, an ubiquitin E3 ligase. Knockdown of GRAIL in Hep3B-TfR2 cells increased TfR2 levels, consistent with inhibition of CD81 ubiquitination. These results suggest that down-regulation of CD81 by GRAIL targets TfR2 for degradation. Surprisingly, knockdown of CD81 decreased hepcidin expression, implying that the TfR2/CD81 complex is involved in the maintenance of hepcidin mRNA. Moreover, knockdown of CD81 did not affect the stimulation of hepcidin expression by BMP6 but increased both the expression of ID1 and SMAD7, direct targets of BMP signaling pathway, and the phosphorylation of ERK1/2, indicating that the CD81 regulates hepcidin expression differently from the BMP and ERK1/2 signaling pathways.

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 action of JAK, SMAD and ERK signal pathways on hepcidin suppression by polysaccharides from Angelica sinensis in rats with iron deficiency anemia.

A crude polysaccharide was obtained by water extraction and ethanol precipitation from the root of Angelica sinensis (AS) to investigate its suppressive effect on hepcidin expression in rats with iron deficiency anemia (IDA). In this study, we showed that the Angelica sinensis polysaccharide (ASP) was mainly composed of arabinose, glucose and galactose in a molar ratio of 1 : 2.5 : 7.5, with the molecular weight of 72 900 Da and the sugar content of 95.1%. ASP markedly decreased hepcidin expression by suppressing the expression of JAK1/2, phospho-JAK1/2, phospho-SMAD1/5/8, phospho-ERK1/2, and promoting the expression of SMAD7 in the liver. These findings suggest that ASP can prevent the janus-kinase (JAK), son of mother against decapentaplegic (SMAD) and extracellular signal-regulated kinase (ERK) pathways to down-regulate hepcidin expression in IDA rats, and may be useful for the treatments of diseases induced by hepcidin over-expression.

Hepatitis C virus core+1/ARF protein decreases hepcidin transcription through an AP1 binding site

Chronic viral hepatitis C is characterized by iron accumulation in the liver, and hepcidin regulates iron absorption. Hepatitis C virus (HCV) core+1/ARFP is a novel protein produced by a second functional ORF within the core gene. Here, using reporter assays and HCV bicistronic replicons, we show that, similarly to core, core+1/ARFP decreases hepcidin expression in hepatoma cells. The activator protein 1 (AP1) binding site of the human hepcidin promoter, shown here to be relevant to basal promoter activity and to the repression by core, is essential for the downregulation by core+1/ARFP while the previously described C/EBP (CCAAT/enhancer binding protein) and STAT (signal transducer and activator of transcription) sites are not. Consistently, expression of the AP1 components c-jun and c-fos obliterated the repressive effect of core and core+1/ARFP. In conclusion, we provide evidence that core+1/ARFP downregulates AP1-mediated transcription, providing new insights into the biological role of core+1/ARFP, as well as the transcriptional modulation of hepcidin, the main regulator of iron metabolism.

The Liver as a Conductor of Systemic Iron Regulation: the Central Role of Hepcidin and BMP Signaling

AbstractAbstract SCI-22Iron is a necessary trace element found in nearly all living organisms and is critical to many cellular processes, including electron transport in the mitochondrial membrane, DNA synthesis, and hemoglobin synthesis. Iron deficiency leads to anemia, and excess iron can promote free radical formation and result in cellular injury (1). Thus, iron balance is tightly regulated to provide sufficient amounts of iron for critical cellular processes, while avoiding the detrimental effects of excess iron. Iron homeostasis is disrupted in many hematologic diseases, including hemochromatosis, the anemia of chronic disease, and β-thalassemia, affecting hundreds of millions of people worldwide. Hepcidin is a peptide hormone secreted by the liver that plays a central role in iron homeostasis. Hepcidin binding to ferroportin, the only known mammalian cellular iron exporter, leads to internalization and degradation of ferroportin in lysosomes, which decreases iron absorption from the diet and iron release from macrophages that recycle iron from senescent erythrocytes. As a central regulator of body iron metabolism, hepcidin is modulated by the same factors that alter iron homeostasis, including changes in body iron stores, inflammation, rate of erythropoiesis, anemia, and hypoxia. Hepcidin levels are increased in response to oral and parenteral iron loading and decreased under iron deficient conditions (2). Inflammation stimulates hepcidin expression by increasing IL-6 levels, which activates the JAK/STAT signaling cascade and binding of the transcriptional activator STAT3 to the hepcidin promoter (3, 4). In contrast, anemia and hypoxia are associated with decreased hepcidin levels (5). Studies of patients with autosomal recessive hemochromatosis, an iron overload disorder, have revealed that several hepatocyte proteins are required for appropriate hepcidin regulation, including those coded by HFE (6), TFR2 (7), and HJV (also called HFE2 and RGMc) (8). Mutations in either the hepcidin gene (HAMP) or HJV cause a more severe form of hemochromatosis that presents in the first or second decade of life. In juvenile hemochromatosis, patients who carry HJV mutations, urinary hepcidin is undetectable, suggesting that HJV is a key regulator of hepcidin (8). Disruption of Hjv in the mouse results in decreased hepcidin expression and increased iron deposition in the liver, pancreas, and heart but decreased iron levels in tissue macrophages (9, 10). HJV (RGMc) encodes hemojuvelin, a member of the repulsive guidance molecule family, which resides on the cell membrane as a glycosylphosphatidylinositol (GPI)-linked protein. It functions as a BMP6 coreceptor to activate hepcidin expression through a BMP/SMAD signaling pathway (11, 12, 13). Accordingly, disease-associated mutations in HJV result in decreased BMP/SMAD signaling and decreased hepcidin expression (11, 14). Hemojuvelin, acting through the BMP signaling pathway, thus plays an essential role in the regulation of hepcidin expression. BMP signaling also appears to be central in order for iron and inflammation to regulate hepcidin expression. Disclosures:Lin:Ferrumax Pharmaceuticals: Equity Ownership.

The Red Cell: How Erythropoiesis Modulates Hepcidin Expression

Abstract SCI-24Erythroid precursors in the bone marrow require transferrin-bound iron for hemoglobin synthesis. Therefore, it is not surprising that the regulation of erythropoiesis and iron metabolism is interlinked. Iron demand for erythropoiesis is communicated to the iron-regulatory machinery through incompletely understood mechanisms. At the core of systemic iron homeostasis is the peptide hormone hepcidin, restricting cellular iron export to plasma by inducing the endocytosis and proteolysis of ferroportin. Hepcidin, a liver-synthesized peptide hormone, is released in response to increased iron load, and there is early evidence that circulating hepcidin concentrations affect the distribution of iron between the macrophage storage compartment (favored by higher hepcidin concentrations) and parenchymal cells, including cardiac myocytes and hepatocytes (favored by low hepcidin). Furthermore, ferroportin has recently been identified on erythroid precursors. Its purpose in this cell type and its function in the interface between erythropoiesis and iron metabolism are unclear. Additionally, in response to bleeding or the administration of erythropoietin, expansion of erythroid precursors suppresses hepcidin, most likely through one or more mediators released by the bone marrow and acting on hepatocytes. Iron-loading anemias with ineffective erythropoiesis, in particular β-thalassemia, demonstrate the effects of pathological “erythroid regulators” of hepcidin. Although erythrocyte transfusions are the main cause of iron loading in patients who receive them (β-thalassemia major), lethal iron overload is seen also in patients who are rarely or never transfused (β-thalassemia intermedia). Here, iron hyperabsorption is the cause of iron overload and, as in hereditary hemochromatosis, is caused by low hepcidin. Decreased hepcidin expression in β-thalassemia, with concurrent ineffective erythropoiesis and iron overload, indicates that the “erythroid regulator” may play an even more substantial role in iron metabolism than the “stores regulator.” Two members of the bone morphogenetic protein (BMP) family, growth differentiation factor (GDF) 15 and Twisted Gastrulation (TWSG1), have been implicated as candidate bone marrow-derived hepcidin suppressors in β-thalassemia. Neither factor is responsible for the physiologic hepcidin suppression in response to hemorrhage-induced stress erythropoiesis, and the physiologic suppressor is not known. We focus here on the current state of knowledge regarding the regulation of iron metabolism and attempt to elucidate the interface between iron regulation and erythropoiesis using evidence in part derived from animal models of β-thalassemia. A more complete understanding of the coregulation of erythropoiesis and iron metabolism may lay the foundation for improving diagnosis, increasing treatment options, and ultimately impacting the well-being of patients afflicted with different anemias and/or iron overload related-disorders. Disclosures:No relevant conflicts of interest to declare.