Activation and inactivation of the iron hormone hepcidin: Biochemical characterization of prohepcidin cleavage and sequential degradation to N-terminally truncated hepcidin isoforms

The hormone hepcidin is produced mainly in the liver in response to iron loading and inflammation and secreted into the circulation as a 25-amino acid peptide. The 84-amino acid prohormone undergoes limited proteolytic cleavage at a conserved proprotein convertase (PC) recognition site. In addition to the 25-amino acid hepcidin, N-terminally truncated isoforms of lower biological activity are found in plasma and urine.Here we show that a redundant system of proprotein convertases cleaves prohepcidin at the predicted site releasing active hepcidin-25 from the proprotein. In addition to furin mediated cleavage of prohepcidin, we found prohepcidin peptidase activity of proprotein convertases PC5/6, PC7/LPC, PC1/3 and PC2 which was specific for the release of hepcidin-25 from prohepcidin as shown by mass spectrometry. In native tissue extracts, a calcium-dependent prohepcidin peptidase activity is present specifically releasing the 25-mer hepcidin isoform from the recombinant prohormone. In contrast, the 20-mer isoform of hepcidin is generated by a calcium-independent tissue activity which cleaves the 25-mer peptide but has no activity on the entire prohormone. This finding demonstrates the presence of an additional peptidase in this inactivation mechanism for hepcidin. An inhibitor of prohepcidin cleavage was designed and synthesized from d-amino acids (QRRRRR). Biochemical studies indicated that this is a potent and generic inhibitor of prohepcidin cleavage.Biochemical and inhibitor studies of endogenous tissue peptidase activities support the implication of proprotein convertases in the activation of hepcidin. Inactivation of the peptide hormone by N-terminal truncation is mediated by other distinct peptidases, which appear to act sequentially to initial release of hepcidin-25 from the proprotein.     

Regulatory failure of serum prohepcidin levels in patients with hepatitis C

BACKGROUND/AIMS: Elevated serum ferritin and hepatic iron concentrations are frequently observed in chronic hepatitis C (CHC), which may be related to hepcidin. Because the role of hepcidin in CHC patients remains unknown, we aimed in this study to generate some information about hepcidin in CHC. METHODS: To determine whether serum hepcidin correlates with markers of iron status in patients with viral hepatitis, we measured serum prohepcidin levels in patients with hepatitis C virus (HCV) and hepatitis B virus (HBV) infection and in healthy controls. RESULTS: Serum prohepcidin and ferritin levels were negatively correlated (r=-0.182, P=0.037) in HCV patients and positively correlated in HBV patients and in healthy controls. The total iron scores in liver specimens from HCV patients were also negatively correlated (r=-0.403, P=0.013). Serum prohepcidin levels in patients with liver cirrhosis (LC) were significantly lower than in patients with chronic hepatitis (CH). In both CH and LC patients, serum prohepcidin levels were significantly lower in HCV patients than in HBV patients. CONCLUSION: Failure of homeostatic regulation of serum prohepcidin concentrations may be induced by HCV infection, resulting in elevation of serum ferritin levels, which leads to the progression of liver injury by iron overload in CHC patients.     

Up-regulation of Transferrin Receptor Expression in Hepatocytes by Habitual Alcohol Drinking Is Implicated in Hepatic Iron Overload in Alcoholic Liver Disease

Background Hepatic iron overload is often seen in alcoholic liver disease (ALD). We previously reported that the expression of 4-hydroxy-2-nonenal-protein adducts, which is a lipid peroxidative product and can be used as a marker of radical-mediated cellular damage, was increased in iron-overloaded hepatocytes with ALD. However, the mechanism of hepatic iron overload in ALD has not been clarified. In this study, to elucidate the mechanism of hepatic iron overload in ALD, we immunohistochemically investigated the expression of transferrin receptor (TfR), which mainly acts for cellular iron uptake.
Methods Hepatic tissues were obtained from 31 patients with ALD and 5 normal livers by percutaneous needle biopsy under laparoscopy or ultrasound guidance. Chemical detection of hepatic iron accumulation was performed by Perls' Prussian blue stain. Immunohistochemical detection of TfR expression was done using human monoclonal anti-TfR antibody (TR104) according to the avidin-biotin complex method with alkaline phosphatase.
Results Excess iron accumulation was found in 22 hepatic tissues with ALD but not in any normal hepatic tissues. TfR expression was increased in hepatocytes of 18 hepatic tissues with ALD but was not detected in any normal hepatic tissues. The mean duration of abstinence of patients who demonstrated positive TfR expression in hepatocytes was significantly shorter than that of patients who demonstrated negative TfR expression (positive: 14 days; negative: 30 days). However, total ethanol consumption, daily ethanol intake, and serum aspartate aminotransferase and γ-glutamyl transpeptidase values on admission were not significantly correlated with TfR expression in hepatocytes.
Conclusions The up-regulation of TfR expression in hepatocytes is implicated in hepatic iron overload in ALD, and habitual alcohol drinking is an important factor for the induction of TfR expression.     

Prohepcidin localises to the Golgi compartment and secretory pathway in hepatocytes

BACKGROUND/AIMS: Hepcidin is a liver-expressed peptide which plays an important role in the regulation of iron metabolism. It is a negative regulator of iron absorption and release of iron from cells. The aims of this study were to analyse the expression and localisation of prohepcidin in liver and cell lines. METHODS: We generated antibodies against recombinant mouse prohepcidin and studied its expression in cell lines, primary hepatocytes and livers of normal mice and mice with abnormalities in iron metabolism. RESULTS: Prohepcidin localised to the secretory pathway, primarily the Golgi apparatus in liver cells and tissues. Hfe and beta2-microglobulin knockout mice have similar levels of prohepcidin protein expression as compared to wild-type mice despite increased iron stores. Sex-linked anaemia mice have iron deficiency and no prohepcidin expression in the liver. CONCLUSIONS: Prohepcidin protein is present in the secretory pathway of liver cells. Despite iron loading, mouse models of haemochromatosis have comparatively normal levels of prohepcidin expression whereas mice with iron deficiency have no prohepcidin expression.     

Iron and steatohepatitis

As the main iron storage site in the body and the main source of the iron-regulatory hormone, hepcidin, the liver plays a pivotal role in iron homeostasis. A variable degree of hepatic iron accumulation has long been recognized in a number of chronic liver diseases. Both alcoholic and non-alcoholic steatohepatitis display increased iron deposits in the liver, with an hepatocellular, mesenchymal, or mixed pattern, and recent reports have documented a concomitant aberrant hepcidin expression that could be linked to different coincidental pathogenic events (e.g. the etiological agent itself, necroinflammation, metabolic derangements, genetic predisposition). The present study reviews the pathogenic mechanisms of iron accumulation in steatohepatitis during alcoholic and non-alcoholic liver disease and the role of excess iron in chronic disease progression.     

Contribution of Hfe expression in macrophages to the regulation of hepatic hepcidin levels and iron loading

Hereditary hemochromatosis (HH), an iron overload disease associated with mutations in the HFE gene, is characterized by increased intestinal iron absorption and consequent deposition of excess iron, primarily in the liver. Patients with HH and Hfe-deficient (Hfe-/-) mice manifest inappropriate expression of the iron absorption regulator hepcidin, a peptide hormone produced by the liver in response to iron loading. In this study, we investigated the contribution of Hfe expression in macrophages to the regulation of liver hepcidin levels and iron loading. We used bone marrow transplantation to generate wild-type (wt) and Hfe-/- mice chimeric for macrophage Hfe gene expression. Reconstitution of Hfe-deficient mice with wt bone marrow resulted in augmented capacity of the spleen to store iron and in significantly decreased liver iron loading, accompanied by a significant increase of hepatic hepcidin mRNA levels. Conversely, wt mice reconstituted with Hfe-deficient bone marrow had a diminished capacity to store iron in the spleen but no significant alterations of liver iron stores or hepcidin mRNA levels. Our results suggest that macrophage Hfe participates in the regulation of splenic and liver iron concentrations and liver hepcidin expression.     

mRNA expression of iron regulatory genes in beta-thalassemia intermedia and beta-thalassemia major mouse models

beta-Thalassemia is an inherited anemia in which synthesis of the hemoglobin beta-chain is decreased. The excess unmatched alpha-globin chains accumulate in the growing erythroid precursors, causing their premature death (ineffective erythropoiesis). Clinical features of beta-thalassemia include variably severe anemia and iron accumulation due to increased intestinal iron absorption. The most anemic patients require regular blood transfusions, which exacerbate their iron overload and result in damage to vital organs. The hepatic peptide hepcidin, a key regulator of iron metabolism in mammals, was recently found to be low in the urine of beta-thalassemia patients, compared with healthy controls, despite their iron overload. In our work, we measured by RQ-PCR the liver mRNA expression of hepcidin and other iron regulatory genes in beta-thalassemia major mouse model (C57Bl/6 Hbb(th3/th3)), and compared it with beta-thalassemia intermedia mouse model (C57Bl/6 Hbb(th3/+)) and control mice. We found decreased expression of hepcidin and TfR2 and increased expression of TfR1 and NGAL in the beta-thalassemia mouse models, compared with the control mice. Significant down-regulation of hepcidin expression in beta-thalassemia major, despite iron overload, might explain the increased iron absorption typically observed in thalassemia.     

Reversal of hemochromatosis by apotransferrin in non-transfused and transfused Hbbth3/+ (heterozygous b1/b2 globin gene deletion) mice

Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb^th3/+ mice. Mice with the Hbb^th3/+ phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb^th3/+ mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice.     

Effects of alcohol consumption on iron metabolism in mice with hemochromatosis mutations

Background: Alcoholic liver disease is associated with increased hepatic iron accumulation. The liver-derived peptide hepcidin is the central regulator of iron homeostasis and recent animal studies have demonstrated that exposure to alcohol reduces hepcidin expression. This down-regulation of hepcidin in vivo implies that disturbed iron sensing may contribute to the hepatosiderosis seen in alcoholic liver disease. Alcohol intake is also a major factor in expression of the hemochromatosis phenotype in patients homozygous for the C282Y mutation of the HFE gene. Methods: To assess the effect of alcohol in mice with iron overload, alcohol was administered to mice with disrupted Hfe and IL-6 genes and Tfr2 mutant mice and their respective 129x1/SvJ, C57BL/6J, and AKR/J wild-type congenic strains. Iron absorption, serum iron levels, and hepcidin expression levels were then measured in these mice compared with water-treated control mice. Results: Alcohol was shown to have a strain-specific effect in 129x1/SvJ mice, with treated 129x1/SvJ mice showing a significant increase in iron absorption, serum iron levels, and a corresponding decrease in hepcidin expression. C57BL/6J and AKR/J strain mice showed no effect from alcohol treatment. 129x1/SvJ mice heterozygous or homozygous for the Hfe knockout had a diminished response to alcohol. All 3 strains were shown to have high blood alcohol levels. Conclusions: The effect of alcohol on iron homeostasis is dependent on the genetic background in mice. In an alcohol-susceptible strain, mutation of the Hfe gene diminished the response of the measured iron indices to alcohol treatment. This indicates that either maximal suppression of hepcidin levels had already occurred as a result of the Hfe mutation or that Hfe was a component of the pathway utilized by EtOH in suppressing hepcidin production and increasing iron absorption.     

Lower serum prohepcidin levels associated with lower iron and erythropoietn requirements in hemodialysis patients with chronic hepatitis C

ABSTRACT: BACKGROUND: Patients with chronic HCV infection have increased liver iron. Recently identified protein hepcidin synthesized in the liver, is thought to be a key regulator for iron homeostasis and is induced by infection and inflammation. It was previously reported lower erythropoietin and iron supplementation requirement in HD patients with HCV infection. We investigated the association of prohepcidin with inflammation and iron parameters in HD patients with and without chronic HCV infection. METHODS: Sixty patients (27 male, 33 female, mean age 50 +/-15 years) on chronic HD were included. Parameters related to iron metabolism (ferritin, serum iron and total iron binding capacity (TIBC)), inflammation (hs-CRP, TNF-alpha and IL-6) and prohepcidin levels were measured. The response to treatment (erythropoiesis-stimulating agent (ESA) resistance index) was assessed from the ratio of the weekly erythropoietin (rhuEPO) dose to hemoglobin (Hb) per unit weight. RESULTS: Serum prohepcidin levels of HCV positive patients (135+/-25 ng/mL) were significantly lower than HCV negative patients [148+/-18 ng/mL, (p=0.025)]. Serum IL-6 levels of HCV positive patients were also significantly lower than HCV negative patients (p=0.016). Serum prohepcidin levels was positively correlated with ferritin (r=0.405, p=0.001) and IL-6 (r=0.271, p=0.050) levels in HD patients. In the HCV positive group, serum prohepcidin levels significantly correlated with ferritin levels (r=0.514 p=0.004). In the HCV negative group, serum prohepcidin levels significantly correlated with serum IL-6 levels (r=0.418, p=0.027). In multiple regression analysis performed to predict prohepcidin in HCV positive patients, serum ferritin was found to be an independent variable (r=0.28, p=0.008). CONCLUSIONS: HCV positive HD patients have low levels of serum prohepcidin and IL-6 which might account for iron accumulation and/or lower iron and rhuEPO requirement in these patients.     

Long-Term Ethanol Feeding Enhances Susceptibility of the Liver to Orally Administered Lipopolysaccharides in Rats

Background Endotoxin has been implicated in the pathogenesis and progression of alcoholic liver disease. However, it is still unclear how long-term ethanol feeding affects absorption of endotoxin from the intestine and susceptibility of the liver to gut-derived endotoxin. The object of this study was to determine the effect of long-term ethanol feeding on hepatic susceptibility to orally administered endotoxin.
Methods Male Wistar rats that weighed approximately 150 g were pair-fed with an ethanol-containing liquid diet or a control diet for 35 days. In some experiments, 0, 10, or 20 mg/kg of lipopolysaccharides (LPS) was added to the liquid diet for 7 days beginning on day 29. On day 36, the animals were killed for blood biochemistry and histologic examination of the liver. We also determined plasma endotoxin levels after 20 mg/kg of LPS administration using a gastric tube. In another set of experiments, we determined intestinal permeability using FD4 (fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4000 D).
Results With 10 mg/kg of LPS, serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels were significantly increased in the ethanol-fed rats but not in controls. After 20 mg/kg of LPS administration, more substantial increases in serum ALT and ALP levels were observed in ethanol-fed rats as compared with control diet-fed rats. Plasma endotoxin levels in long-term ethanol-fed rats were higher than those in control rats after intragastric administration of high-dose endotoxin (20 mg/kg). Furthermore, intestinal permeability to FD4 was increased by long-term ethanol administration.
Conclusions Long-term ethanol feeding increases intestinal permeability to and absorption of endotoxin, which can sequentially enhance hepatic susceptibility to orally administered endotoxin. This model has potential as a subclinical experimental model for the study of alcoholic liver disease.     

A competitive enzyme-linked immunosorbent assay specific for murine hepcidin-1: correlation with hepatic mRNA expression in established and novel models of dysregulated iron homeostasis

Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbb^th3/+), hereditary hemochromatosis (Hfe^−/−, Hjv^−/−, and Tfr2^Y245X/Y245X), hypotransferrinemia (Trf^hpx/hpx), heterozygous transferrin receptor 1 deficiency (Tfrc^+/−) and iron refractory iron deficiency anemia (Tmprss6^−/− and Tmprss6^hem8/hem8). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups.     

Decreased liver hepcidin expression in the Hfe knockout mouse

Hepcidin is a circulating antimicrobial peptide which has been proposed to regulate the uptake of dietary iron and its storage in reticuloendothelial macrophages. Transgenic mice lacking hepcidin expression demonstrate abnormalities of iron homeostasis similar to Hfe knockout mice and to patients with HFE-associated hereditary hemochromatosis (HH). To identify any association between liver hepcidin expression and the iron homeostasis abnormalities observed in HH, we compared liver hepcidin mRNA content in wild type and Hfe knockout mice. Because the iron homeostasis abnormalities in the Hfe knockout mice are greatest early in life, we analyzed mice at different ages. At four weeks of age, Hfe knockout mice had significantly decreased liver hepcidin mRNA expression compared to wild type mice. The decreased hepcidin expression was associated with hepatic iron deposition, elevated transferrin saturations, and decreased splenic iron concentrations. At 10 weeks of age, despite marked hepatic iron loading, Hfe knockout mice demonstrated liver hepcidin mRNA expression similar to that observed in wild type mice. Placing 8 week-old wild type and Hfe knockout mice on a 2% carbonyl iron diet for 2 weeks led to a similar degree of hepatic iron loading in each group. However, while the wild type mice demonstrated a mean five-fold increase in liver hepcidin mRNA, no change was observed in the Hfe knockout mice. The lack of an increase in liver hepcidin expression in these iron-loaded Hfe knockout mice was associated with sparing of iron deposition into the spleen. These data indicate that the normal relationship between body iron stores and liver hepcidin mRNA levels is altered in Hfe knockout mice, such that liver hepcidin expression is relatively decreased. We speculate that decreased hepcidin expression relative to body iron stores contributes to the iron homeostasis abnormalities characteristic of HH.     

Role of Kupffer Cell-Derived Reactive Oxygen Intermediates in Alcoholic Liver Disease in Rats In Vivo

The pathophysiology of alcoholic liver disease (ALD) remains largely unknown. In this work, we have developed an experimental rat model to elucidate the mechanism of liver injury, including ALD, in which Kupffer cell-derived reactive oxygen intermediates (ROIs) might be involved. Groups of male Wistar rats were pair-fed on a liquid high-fat diet containing ethanol (36% of total calories) or isocaloric carbohydrate with or without dietary carbonyl iron (0.5% w/v) for 3 weeks. In this rat model, we investigated Kupffer cell-derived ROI generation, which affected hepatocellular injury and hepatic fibrosis in ALD. The production of ROIs in Kupffer cells isolated from the iron-fed, the ethanol-fed, and the ethanol plus iron-fed rats were significantly increased, compared with that in Kupffer cells isolated from control rats (iron > ethanol + iron > ethanol ≫ control). However, hepatic vitamin E content in the ethanol plus iron-fed rats was decreased rather than that in the iron-fed rats. Then, lipid peroxidation of isolated microsomes was assessed as malondialdehyde equivalents determined by thiobarbituric acid assay. Compared with controls, the malondialdehyde equivalents were elevated in experimental groups (ethanol + iron > ethanol > iron > control). Serum ALT levels were greatly elevated in rats fed a diet containing both ethanol and iron (ethanol + iron > iron > ethanol > control). Hepatic content of hydroxyproline was significantly increased in ethanol plus iron-fed rats, compared with rats other than the ethanol plus iron-fed group (ethanol + iron > iron > ethanol > control). These results suggested that the enhanced Kupffer cell-derived ROI generation could itself contribute to the increased susceptibility to lipid peroxidation, which might cause hepatocellular injury and lead to hepatic fibrosis in ALD.