Regulation Of Hepcidin Expression Research Articles

HFE interacts with the BMP type I receptor ALK3 to regulate hepcidin expression

AbstractMutations in HFE are the most common cause of hereditary hemochromatosis (HH). HFE mutations result in reduced expression of hepcidin, a hepatic hormone, which negatively regulates iron absorption from the duodenum and iron release from macrophages. However, the mechanism by which HFE regulates hepcidin expression in hepatocytes is not well understood. It is known that the bone morphogenetic protein (BMP) pathway plays a central role in controlling hepcidin expression in the liver. Here we show that HFE overexpression increased Smad1/5/8 phosphorylation and hepcidin expression, whereas inhibition of BMP signaling abolished HFE-induced hepcidin expression in Hep3B cells. HFE was found to associate with ALK3, inhibiting ALK3 ubiquitination and proteasomal degradation and increasing ALK3 protein expression and accumulation on the cell surface. The 2 HFE mutants associated with HH, HFE C282Y and HFE H63D, regulated ALK3 protein ubiquitination and trafficking differently, but both failed to increase ALK3 cell-surface expression. Deletion of Hfe in mice resulted in a decrease in hepatic ALK3 protein expression. Our results provide evidence that HFE induces hepcidin expression via the BMP pathway: HFE interacts with ALK3 to stabilize ALK3 protein and increase ALK3 expression at the cell surface.

Intestinal inflammation modulates expression of the iron-regulating hormone hepcidin depending on erythropoietic activity and the commensal microbiota.

States of chronic inflammation such as inflammatory bowel disease are often associated with dysregulated iron metabolism and the consequent development of an anemia that is caused by maldistribution of iron. Abnormally elevated expression of the hormone hepcidin, the central regulator of systemic iron homeostasis, has been implicated in these abnormalities. However, the mechanisms that regulate hepcidin expression in conditions such as inflammatory bowel disease are not completely understood. To clarify this issue, we studied hepcidin expression in mouse models of colitis. We found that dextran sulfate sodium-induced colitis inhibited hepcidin expression in wild-type mice but upregulated it in IL-10-deficient animals. We identified two mechanisms contributing to this difference. Firstly, erythropoietic activity, as indicated by serum erythropoietin concentrations and splenic erythropoiesis, was higher in the wild-type mice, and pharmacologic inhibition of erythropoiesis prevented colitis-associated hepcidin downregulation in these animals. Secondly, the IL-10 knockout mice had higher expression of multiple inflammatory genes in the liver, including several controlled by STAT3, a key regulator of hepcidin. The results of cohousing and fecal transplantation experiments indicated that the microbiota was involved in modulating the expression of hepcidin and other STAT3-dependent hepatic genes in the context of intestinal inflammation. Our observations thus demonstrate the importance of erythropoietic activity and the microbiota in influencing hepcidin expression during colitis and provide insight into the dysregulated iron homeostasis seen in inflammatory diseases.

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.

Hepcidin expression in liver cells: evaluation of mRNA levels and transcriptional regulation.

Hepcidin produced in the liver negatively regulates intestinal iron absorption, and the bone morphogenetic protein (BMP) pathway is well-known to stimulate hepcidin expression. However, the regulation of hepcidin expression has not been fully elucidated. In this study, we evaluate different systems that can be used to determine how hepcidin expression is regulated. The basal expression of hepcidin in liver cell lines, such as HepG2 cells and Hepa1-6 cells, was lower than that in the liver and primary hepatocytes; the expression levels of hepcidin in the cell lines were near the limit of detection for RT-PCR and RT-qPCR analyses. Treatment with trichostatin A, RNAlater, or MG-132 enhanced the expression of hepcidin in HepG2 cells, suggesting that histone deacetylation, instability of mRNA, or proteosomal degradation of the protein(s) that positively regulate hepcidin expression may be responsible for the decreased expression of hepcidin in HepG2 cells. In luciferase-based reporter assays, BMP induced the transcription of a reporter, hepcidin(-2018)-luc, that contains nt -2018 through nt -35 of the hepcidin promoter in HepG2 cells and Hepa1-6 cells. However, BRE-luc, a representative reporter used to evaluate BMP signaling, was unresponsive to BMP in HepG2 cells. These results suggest that hepcidin transcription can be best evaluated in liver cell lines and that the hepcidin promoter senses BMP signaling with high sensitivity. The present study demonstrates that studies regarding the regulation of hepcidin expression at the mRNA level should be evaluated in primary hepatocytes, and liver cell lines are well-suited for studies examining the transcriptional regulation of hepcidin.

Hemojuvelin and bone morphogenetic protein (BMP) signaling in iron homeostasis.

Mutations in hemojuvelin (HJV) are the most common cause of the juvenile-onset form of the iron overload disorder hereditary hemochromatosis. The discovery that HJV functions as a co-receptor for the bone morphogenetic protein (BMP) family of signaling molecules helped to identify this signaling pathway as a central regulator of the key iron hormone hepcidin in the control of systemic iron homeostasis. This review highlights recent work uncovering the mechanism of action of HJV and the BMP-SMAD signaling pathway in regulating hepcidin expression in the liver, as well as additional studies investigating possible extra-hepatic functions of HJV. This review also explores the interaction between HJV, the BMP-SMAD signaling pathway and other regulators of hepcidin expression in systemic iron balance.

Ferristatin II induces hepatic hepcidin expression and Stat3 phosphorylation (907.3)

Administration of Ferristatin II (FS2) to rats is associated with decreased serum iron, reduced transferrin saturation and increased hepatic hepcidin expression without changes in liver non‐heme iron levels. To better understand the mechanisms underlying hepcidin induction by FS2, its effects on ER stress were examined. XBP1 splicing, a marker of ER stress, was not altered in livers from treated rats. Likewise, levels of the ER stress protein Grp94 were unchanged in FS2‐treated Hep3B cells. SMAD signaling, a second regulatory pathway, did not appear to be activated since transcript levels of downstream targets SMAD7, BMP6 and Id1 were all reduced in livers of FS2‐treated rats. However, mRNA levels of alpha‐2‐microglobulin, alpha‐1‐acid glycoprotein and C‐reactive protein were up‐regulated. Western blot experiments confirmed that FS2 treatment induced phosphorylation of hepatic Stat3, a known mediator of the acute phase response and a regulator of hepcidin expression. Accordingly, FS2 induced Stat3 phosphorylation in HepG2 cells in vitro. These results suggest that FS2 induces hepatic hepcidin expression in a Stat3‐dependent manner. The pharmacological induction of hepcidin by ferristatin II holds clinical potential to decrease iron absorption in iron overload diseases.Grant Funding Source: Supported by NIH NIDDK (DK064750)

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 role of vitamin D in regulating the iron-hepcidin-ferroportin axis in monocytes

Chronic kidney disease affects 40% of adults aged 65 and older. Anemia of CKD is present in 30% of patients with CKD and is associated with increased cardiovascular risk, decreased quality of life, and increased mortality. Hepcidin-25 (hepcidin), the key iron regulating hormone, prevents iron egress from macrophages and thus prevents normal recycling of the iron needed to support erythropoiesis. Hepcidin levels are increased in adults and children with CKD. Vitamin D insufficiency is highly prevalent in CKD and is associated with erythropoietin hyporesponsiveness. Recently, hepcidin levels were found to be inversely correlated with vitamin D status in CKD. The aim of this study was to investigate the role of vitamin D in the regulation of hepcidin expression in vitro and in vivo. This study reports that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D, is associated with decreased hepcidin and increased ferroportin expression in lipopolysaccharide (LPS) stimulated THP-1 cells. 1,25(OH)2D3 also resulted in a dose-dependent decrease in pro-hepcidin cytokines, IL-6 and IL-1β, release in vitro. Further, we show that high-dose vitamin D therapy impacts systemic hepcidin levels in subjects with early stage CKD. These data suggest that improvement in vitamin D status is associated with lower systemic concentrations of hepcidin in subjects with CKD. In conclusion, vitamin D regulates the hepcidin-ferroportin axis in macrophages which may facilitate iron egress. Improvement in vitamin D status in patients with CKD may reduce systemic hepcidin levels and may ameliorate anemia of CKD.

Altered Hepcidin Expression is Part of the Choroid Plexus Response to IL-6/Stat3 Signaling Pathway in Normal Aging Rats

Accumulating evidence has revealed that brain iron concentrations increase with aging, and the choroid plexus may be at the basis of iron-mediated toxicity and the increase in inflammation and oxidative stress that occurs with aging. However, nothing is known concerning the correlation between the IL-6/Stat3 signaling pathway and the levels of hepcidin expression at the choroid plexus (CP) in normal aging. The morphological modifications as a function of aging were investigated and the present study used quantitative real time PCR (qPCR) and western blotting (WB) to determine the alterations in specific mRNA and corresponding protein changes at the CP on aging at ages 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33 and 36 m Brown-Norway/Fischer (B-N/F) rats. Results showed that a striking deterioration of the CP epithelial cells, and results also firstly demonstrated that hepcidin expression at the choroid plexus increased with aging at the mRNA level and might cause corresponding changes in protein expression. These alterations in normal aging were in accordance with the expression and secretion of IL-6 and Stat3. Our data suggest that IL-6 regulate hepcidin expression at the choroid plexus, upon interaction with the cognate cellular receptor, and through the Stat3 signaling transduction pathway. The enhanced Stat3 signaling responsiveness to proinflammatory factors may impact on mechanisms of Alzheimer’s disease.

The Role Of Liver Endothelial Sinusoidal Cells In Iron-Mediated Bmp6 Regulation

IntroductionIron-dependent regulation of hepcidin responds to circulating (holo-Tf) and hepatic iron. Although the signals that modulate hepcidin according to holo-Tf are still unknown, hepatic iron regulates hepcidin expression through transcriptional modulation of Bmp6. Bmp6 is controlled by iron only in the liver. Heterogeneous cell types with different functions in the liver include hepatocytes (HC) and non-parenchymal cells as Kupffer cells (KC), sinusoidal endothelial cells (LSEC), stellate cells. It has been reported that LSEC and KC, but not HC, upregulate Bmp6 independently of intracellular iron (Enns et al., Plos One 2013). Here we extend this observation analyzing Bmp6 regulation in isolated liver cells after acute and chronic changes of the iron status in wild type mice. In addition we studied pathogenic models of iron deregulation due to low (iron loaded Hjv KO mice) and high (iron deficient Tmprss6 KO mice) hepcidin. MethodsMice were maintained in accordance with the European Union guidelines. The study was approved by the IACUC of San Raffaele Scientific Institute, Milan, Italy. For chronic changes of the iron status, four weeks-old male mice were maintained an iron-replete (IB, 200 mg/kg iron), iron-deficient diet (ID, <3 mg /kg iron) or iron-loaded (IL, 8.3 g/kg iron) diet for 3 weeks. To induce acute iron changes, we used two approaches: iron dextran injection (1 g/kg body weight i.p.) or 2 weeks ID diet followed by 1, 3 and 6 days of IB, ID or IL diet. Hjv and Tmprss6 KO mice were studied in basal conditions at 7-8 weeks. Liver cells were isolated according to Liu et al. (Proteomics 2011) and cell purity validated by mRNA expression of specific genes. RNA was extracted using RNeasy Mini kit. Total RNA was retro-transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem). Gene expression levels were measured by quantitative real-time PCR using TaqMan Gene Expression Master Mix (Applied Biosystem). Transferrin saturation (TS) and liver iron content (LIC) was measured as previously described (Pagani et al. Blood 2011). ResultsIn wild type animals hepcidin in basal condition is expressed exclusively in HC, whereas Bmp6 is expressed in non-parenchymal cells, mainly LSEC. In all cell types, Bmp6 levels inversely correlate with Tfr1 expression, suggesting that cellular iron influences Bmp6. In mice chronically maintained an IB, ID and IL diet HC, LSEC and KC modulate Bmp6 according to both cellular iron and TS. To investigate which cell type first regulates Bmp6, we injected wild type mice with a single dose of iron dextran and separated cells 3 and 6 hrs post injection. At 6 hrs, when TS and LIC are increased and Hamp is activated in HC, only LSEC upregulate Bmp6. As for the pathogenic models, Bmp6 is upregulated in HC, LSEC and KC cells in Hjv KO mice and downregulated in Tmprss6 KO animals. However, at difference with HC, regulation of Bmp6 in LSEC and KC is independent on cellular iron (as shown by Tfr1 mRNA), and related to TS in both animal models. To distinguish between the contribution of cellular and circulating iron in the regulation of Bmp6, we fed mice previously treated with an ID diet, with an ID, IB and IL diet for 1-3-6 days. At day 1, TS increases but LIC does not change in IB and ID mice while total liver Bmp6 is modulated according to TS. These results will be analyzed in separated cells. In vitro, exogenous BMP6 transcriptionally activates BMP6 expression in hepatoma-derived cells, even at short time point, suggesting that a paracrine mechanism contributes to BMP6 regulation. ConclusionsIn our hands Bmp6 expression is modulated not only in LSEC and KC but also in HC in animals fed a variable iron content diet. LSEC are the first iron “sensor” that, through Bmp6, activates hepcidin expression in HC. Bmp6 in LSEC and KC responds mainly to TS changes. We also suggest that an autocrine/paracrine mechanism activates liver Bmp6 to amplify signaling for hepcidin increase. Disclosures:No relevant conflicts of interest to declare.

Serum-induced up-regulation of hepcidin expression involves the bone morphogenetic protein signaling pathway

Hepcidin is a peptide hormone that is secreted by the liver and that functions as the central regulator of systemic iron metabolism in mammals. Its expression is regulated at the transcriptional level by changes in iron status and iron requirements, and by inflammatory cues. There is considerable interest in understanding the mechanisms that influence hepcidin expression because dysregulation of hepcidin production is associated with a number of disease states and can lead to iron overload or iron-restricted anemia. In order to shed light on the factors that alter hepcidin expression, we carried out experiments with HepG2 and HuH7, human hepatoma cell lines that are widely used for this purpose. We found that the addition of heat-inactivated fetal calf serum to these cells resulted in a significant dose- and time-dependent up-regulation of hepcidin expression. Serum also activated signaling events known to be downstream of bone morphogenetic proteins (BMPs), a group of molecules that have been implicated previously in hepcidin regulation. Inhibition of these signals with dorsomorphin significantly suppressed serum-induced hepcidin up-regulation. Our results indicate that a BMP or BMP-like molecule present in serum may play an important role in regulating hepcidin expression.

Liver iron modulates hepcidin expression during chronically elevated erythropoiesis in mice

The liver-derived peptide hepcidin controls the balance between iron demand and iron supply. By inhibiting the iron export activity of ferroportin, hepcidin modulates iron absorption and delivery from the body's stores. The regulation of hepcidin, however, is not completely understood and includes a variety of different signals. We studied iron metabolism and hepcidin expression in mice constitutively overexpressing erythropoietin (Epo) (Tg6 mice), which leads to excessive erythropoiesis. We observed a very strong down-regulation of hepcidin in Tg6 mice that was accompanied by a strong increase in duodenal expression of ferroportin and divalent metal tranporter-1, as well as enhanced duodenal iron absorption. Despite these compensatory mechanisms, Tg6 mice displayed marked circulating iron deficiency and low levels of iron in liver, spleen, and muscle. To elucidate the primary signal affecting hepcidin expression during chronically elevated erythropoiesis, we increased iron availability by either providing iron (thus further increasing the hematocrit) or reducing erythropoiesis-dependent iron consumption by means of splenectomy. Both treatments increased liver iron and up-regulated hepcidin expression and the BMP6/SMAD pathway despite continuously high plasma Epo levels and sustained erythropoiesis. This suggests that hepcidin expression is not controlled by erythropoietic signals directly in this setting. Rather, these results indicate that iron consumption for erythropoiesis modulates liver iron content, and ultimately BMP6 and hepcidin. Analysis of the BMP6/SMAD pathway targets showed that inhibitor of DNA binding 1 (ID1) and SMAD7, but not transmembrane serine protease 6 (TMPRSS6), were up-regulated by increased iron availability and thus may be involved in setting the upper limit of hepcidin. We provide evidence that under conditions of excessive and effective erythropoiesis, liver iron regulates hepcidin expression through the BMP6/SMAD pathway.

The Evaluation of iron overload through hepcidin level and its related factors in myelodysplastic syndromes

We chose hepcidin and its related factors as evaluating indicators to determine the degrees of iron overload in myelodysplastic syndromes (MDS) patients. A total of 73 patients and 28 healthy volunteers were enrolled in this study. We performed enzyme-linked immunosorbent assay to measure both bone marrow and peripheral blood serum hepcidin. Real-time quantitative polymerase chain reaction was used to determine the gene expression of growth differentiation factor 15 and twisted gastrulation 1. Serum ferritin (SF), C-reactive protein (CRP), and erythropoietin were measured by routine standard laboratory assays. CD4+ and CD19+ lymphocytes and Th polarization were detected by flow cytometry. Twenty-four MDS patients were measured their cardiac and liver iron deposition levels through magnetic resonance imaging (MRI) T2* examination. No significant difference was found between the bone marrow hepcidin levels and peripheral blood hepcidin levels (P = 0.134). Stratified according to different World Health Organization subtypes, refractory anemia with ringed sideroblasts patients had the lowest hepcidin levels (105.40 ± 5.13 ng/ml), while refractory anemia with excess blasts-1 had the highest levels (335.71 ± 25.16 ng/ml). Stratified according to International Prognostic Scoring System and WHO Classification-based Prognostic Scoring System, there was a significant difference of hepcidin levels between low-risk group and high-risk group in two systems, respectively (P = 0.033 and 0.009). The hepcidin levels of CD4+ high-expression group were demonstrated higher than the normal expression groups (P = 0.02), but the CD19+ high-expression group did not show the same result (P = 0.206). Meanwhile, patients with a Th1 polarization trend had a high level of hepcidin versus normal group (P < 0.001). Liver iron concentration (LIC) measured by MRI T2* had a closer correlation (r = 0.582, P < 0.001) to hepcidin than serum ferritin, by stepwise regression. C-reactive protein and LIC seemed to be the key determinants of hepcidin, by multivariate regression. Inflammation plays an important role in the regulation of hepcidin expression. T-lymphocyte activation and Th polarization trend might participate in the regulatory mechanism partly. The capability of organ iron load assessment of MRI T2* seems better than that of SF. It seems that hepcidin with CRP and LIC measured by MRI T2* are potential indicators of iron overload in MDS patients.

Iron regulation by hepcidin

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

A Chemical Screen Reveals New Modulators of Hepcidin Expression

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing the human Hepcidin promoter upstream of a firefly reporter gene. We then used high throughput methods to screen 10,360 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change &gt;;3 standard deviations above or &gt;;1.5 standard deviations below the mean of the other chemicals, while not adversely affecting cell viability. Using these criteria, we identified 32 small molecules that upregulated and 3 that downregulated Hepcidin expression. On retesting assays, we confirmed 22 of the initial positives (69%) and 1 of the initial negatives as regulators of Hepcidin expression. Functional classification of the positive regulators indicated: 4 anti‐inflammatory agents, 4 antimicrobials, 6 antineoplastic drugs, 4 kinase inhibitors, and 4 with other or unknown functions. We are now evaluating the mechanisms of action in human hepatocytes and in a zebrafish model. The best candidates will subsequently be tested in mouse models of iron overload syndromes. Funding: NIDDK R01 DK085250–01A1.

Hemojuvelin and Iron Metabolism in Kidney and Heart Allograft Recipients

IntroductionHemojuvelin plays an essential role in the regulation of hepcidin expression, specifically in the iron-sensing pathway. Dietary iron sensing and inflammatory pathways converge in the regulation of the key regulator hepcidin. Hepcidin is a small defensin-like peptide whose production by hepatocytes is modulated in response to anemia, hypoxia, or inflammation. We studied correlations of hemojuvelin with markers of iron status and of inflammation among 61 prevalent kidney allograft recipients and 136 prevalent heart transplant recipients. MethodsComplete blood count, urea, serum lipids, fasting glucose, and creatinine were measured using standard laboratory methods in the central laboratory of the hospital. Hepcidin, soluble transferin receptor (sTFR), interleukin-6 (IL-6) and hemojuvelin were assayed by enzyme immunosorbent assay using commercially available kits. ResultsAmong heart transplant recipients hemojuvelin correlated strongly with kidney function, transferrin saturation and white blood cell count; moderately with red blood cell count, hepcidin, IL-6, high-sensitivity C-reactive protein (hsCRP) and weakly with sTfR. Multiple regression analysis revealed the predictors of hemojuvelin to be kidney function and TSAT, explaining 79% of the variations among hemojuvelin values in heart transplant recipients. Among kidney transplant recipients hemojuvelin correlated with kidney function, TSAT, hepcidin and hsCRP, and tended to correlate with IL-6. Predictors of hemojuvelin on multiple regression analysis were TSAT and creatinine. ConclusionsElevated hemojuvelin as well as hepcidin levels in kidney or heart transplant recipients may be due to the impaired kidney function and disturbed iron status frequently encountered among this population.

Matriptase-2, a Regulatory Protease of Iron Homeostasis: Possible Substrates, Cleavage Sites and Inhibitors

Matriptase-2 is a cell surface serine protease with a modular structure. The exploration of its function in iron homeostasis was of significance for the understanding of the regulation of hepcidin expression, the master protein in iron control. Mutations in matriptase- 2 cause iron-refractory iron deficiency anemia (IRIDA), an iron deficiency disorder where the level of hepcidin is inappropriately high. Matriptase-2 controls hepcidin expression through the suppression of bone morphogenetic protein (BMP)/sons of mothers against decapentaplegic homologue protein (SMAD) signaling, probably by cleaving the BMP co-receptor hemojuvelin. Since prospective studies revealed that genetic inactivation of matriptase-2 reduces iron loading in different mouse models, matriptase-2 becomes highly attractive as a novel target for the design of low-molecular weight inhibitors. The first synthetic peptidomimetic matriptase-2 inhibitors have been reported. A computational model of the active site of matriptase-2 based on the X-ray crystal structure of the close homologue matriptase was generated and mutational studies were performed in order to identify critical amino acids specifying the preferred recognition site of matriptase-2. So far, the only known putative natural substrates of matriptase-2 are hemojuvelin and matriptase-2 itself, as this protease undergoes complex auto-processing during zymogen activation. Cleavage sites within both natural substrates were identified.

Acidic milieu augments the expression of hepcidin, the central regulator of iron homeostasis

Hepcidin is the central regulator of body iron homeostasis, and dysregulation of hepcidin expression causes various clinical disorders, such as anemia and hemochromatosis. Various stimuli, including iron load and interleukin-6, are involved in the regulation of hepcidin expression. We previously reported that serum hepcidin levels were high in patients with end-stage renal disease, compared with healthy subjects. Since metabolic acidosis is commonly observed in these patients, we hypothesized that acidic milieu might augment hepcidin expression. In this study, we investigated the effect of changes in the pH of the microenvironment on hepcidin expression in human hepatoma and leukemia cell lines. We found that hepcidin expression in these cells was augmented by the acidic milieu created with lactic acid, hydrochloric acid and excess carbon dioxide. Acidic milieu did not clearly enhance hepcidin promoter activity, but rather stabilized hepcidin transcript in the hepatoma cells. We speculate that metabolic acidosis may contribute in part to the elevation of serum hepcidin levels in patients with end-stage chronic kidney disease. Further studies are needed to elucidate the association between acidosis and hepcidin expression in various clinical settings.

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.

A Screen for Small Molecules That Regulate Hepcidin Expression

Abstract 611Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. 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 3 kilobases of the human Hepcidin promoter upstream of a firefly reporter gene. We then used high throughput methods to screen 10,360 chemicals in duplicate from the Harvard Institute of Chemistry and Cell Biology library for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1.5 standard deviations below the mean of the other chemicals (z-score >3 or <-1.5), while not adversely affecting cell viability, quantified by a nonlytic fluorescence assay. Using these criteria, we identified 32 small molecules that upregulated and 3 that downregulated Hepcidin expression. Functional classification of the positive regulators indicated: 4 anti-inflammatory agents, 4 antimicrobials, 6 antineoplastic drugs, 6 kinase inhibitors, and 12 with other or unknown function. Of the positive modulators, two were flavones, consistent with our prior discovery that the isoflavone genistein upregulates Hepcidin expression. Of the negative regulators, one was a kinase inhibitor and two were of unknown function. Experiments are underway to characterize the mechanism of action of these regulators. The best candidates will subsequently be tested in mouse models of iron overload syndromes with the intention of developing new therapies for diseases in which Hepcidin is inappropriately regulated. Disclosures:No relevant conflicts of interest to declare.