Purpose of review
Growth differentiation factor 15 (GDF15) was identified as a hepcidin-suppression factor that is expressed at high levels in patients with ineffective erythropoiesis. This review addresses the regulation, expression and potential functions of GDF15 in the context of erythroid biology.
GDF15 expression during late erythroid differentiation was discovered as part of an erythroblast transcriptome project. Since GDF15 expression is associated with cellular stress or apoptosis, further investigation of the cytokine was focused upon its involvement in ineffective erythropoiesis. Remarkably high serum levels were detected in patients with thalassemia syndromes, congenital dyserythropoiesis and some acquired sideroblastic anemias. Similarly high-level GDF15 expression is not a feature of normal erythropoiesis, or erythroid recovery after bone marrow transplantation. Since GDF15 is a TGF-β superfamily member, it was investigated as an effector of ineffective erythropoiesis that suppresses hepcidin expression despite iron overloading.
In contrast to the low-levels of GDF15 expressed during normal erythropoiesis, ineffective erythropoiesis causes high-level expression of GDF15. In patients with thalassemia and related anemias, GDF15 expression may contribute to iron overloading or other features of the disease phenotype.
GDF15; ineffective erythropoiesis; iron regulation
Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera.
Methods and Findings
An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10–1500 µg/L and a detection limit of 5.4 µg/L. The intra- and interassay coefficients of variance ranged from 8–15% and 5–16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 µg/L) and 10 patients with iron deficiency anemia (15.7 µg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 µg/L) compared to 32 age-matched healthy controls (42.7 µg/L).
We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.
The TGF-β family member GDF-15 promotes lesion formation and plaque instability in atherosclerosis-prone LDLr-deficient mice.
Growth differentiation factor (GDF) 15 is a member of the transforming growth factor β (TGF-β) superfamily, which operates in acute phase responses through a currently unknown receptor. Elevated GDF-15 serum levels were recently identified as a risk factor for acute coronary syndromes. We show that GDF-15 expression is up-regulated as disease progresses in murine atherosclerosis and primarily colocalizes with plaque macrophages. Hematopoietic GDF-15 deficiency in low density lipoprotein receptor−/− mice led to impaired initial lesion formation and increased collagen in later lesions. Although lesion burden in GDF-15−/− chimeras was unaltered, plaques had reduced macrophage infiltrates and decreased necrotic core formation, all features of improved plaque stability. In vitro studies pointed to a TGFβRII-dependent regulatory role of GDF-15 in cell death regulation. Importantly, GDF-15−/− macrophages displayed reduced CCR2 expression, whereas GDF-15 promoted macrophage chemotaxis in a strictly CCR2- and TGFβRII-dependent manner, a phenomenon which was not observed in G protein–coupled receptor kinase 2+/− macrophages. In conclusion, GDF-15 deletion has a beneficial effect both in early and later atherosclerosis by inhibition of CCR2-mediated chemotaxis and by modulating cell death. Our study is the first to identify GDF-15 as an acute phase modifier of CCR2/TGFβRII-dependent inflammatory responses to vascular injury.
Hepcidin is a regulatory hormone that plays a major role in controlling body iron homeostasis. Circulating factors (holotransferrin, cytokines, erythroid regulators) might variably contribute to hepcidin modulation in different pathological conditions. There are few studies analysing the relationship between hepcidin transcript and related protein expression profiles in humans. Our aims were: a. to measure hepcidin expression at either hepatic, serum and urinary level in three paradigmatic iron overload conditions (hemochromatosis, thalassemia and dysmetabolic iron overload syndrome) and in controls; b. to measure mRNA hepcidin expression in two different hepatic cell lines (HepG2 and Huh-7) exposed to patients and controls sera to assess whether circulating factors could influence hepcidin transcription in different pathological conditions. Our findings suggest that hepcidin assays reflect hepatic hepcidin production, but also indicate that correlation is not ideal, likely due to methodological limits and to several post-trascriptional events. In vitro study showed that THAL sera down-regulated, HFE-HH and C-NAFLD sera up-regulated hepcidin synthesis. HAMP mRNA expression in Huh-7 cells exposed to sera form C-Donors, HFE-HH and THAL reproduced, at lower level, the results observed in HepG2, suggesting the important but not critical role of HFE in hepcidin regulation.
Iron overload is the principal cause of morbidity and mortality in β-thalassemia with or without transfusion dependence. Iron homeostasis is regulated by the hepatic peptide hormone hepcidin. Hepcidin controls dietary iron absorption, plasma iron concentrations, and tissue iron distribution. Hepcidin deficiency is the main or contributing factor of iron overload in iron-loading anemias such as β-thalassemia. Hepcidin deficiency results from a strong suppressive effect of the high erythropoietic activity on hepcidin expression. Although in thalassemia major patients iron absorption contributes less to the total iron load than transfusions, in non-transfused thalassemia, low hepcidin and the consequent hyperabsorption of dietary iron is the major cause of systemic iron overload. Hepcidin diagnostics and future therapeutic agonists may help in management of patients with β-thalassemia.
hepcidin; β-thalassemia; iron overload
The aim of the study was to establish an optimized protocol of iron dextran administration to pig neonates, which better meets the iron demand for erythropoiesis. Here, we monitored development of red blood cell indices, plasma iron parameters during a 28-day period after birth (till the weaning), following intramuscular administration of different concentrations of iron dextran to suckling piglets. To better assess the iron status we developed a novel mass spectrometry assay to quantify pig plasma levels of the iron-regulatory peptide hormone hepcidin-25. This hormone is predominantly secreted by the liver and acts as a negative regulator of iron absorption and reutilization. The routinely used protocol with high amount of iron resulted in the recovery of piglets from iron deficiency but also in strongly elevated plasma hepcidin-25 levels. A similar protocol with reduced amounts of iron improved hematological status of piglets to the same level while plasma hepcidin-25 levels remained low. These data show that plasma hepcidin-25 levels can guide optimal dosing of iron treatment and pave the way for mixed supplementation of piglets starting with intramuscular injection of iron dextran followed by dietary supplementation, which could be efficient under condition of very low plasma hepcidin-25 level.
Iron deficiency is a common cause of anemia. In end-stage renal disease (ESRD), iron deficiency impairs the therapeutic efficacy of recombinant erythropoietin. Oral or parental iron supplements usually are effective in treating iron deficiency anemia (IDA). Some patients, however, respond poorly to iron supplements and are diagnosed as having iron-refractory iron deficiency anemia (IRIDA). The disease represents a medical challenge but its underlying mechanism was unclear. Hepcidin is a central player in iron homeostasis. It down-regulates the iron exporter ferroportin, thereby inhibiting iron absorption, release and recycling. In ESRD, plasma hepcidin levels are elevated, which contributes to iron deficiency in patients. Matriptase-2, a liver transmembrane serine protease, has been found to have a major role in controlling hepcidin gene expression. In mice, defects in the Tmprss6 gene encoding matriptase-2 result in high hepcidin expression and cause severe microcytic anemia. Similarly, mutations in the human TMPRSS6 gene have been identified in patients with IRIDA. Thus, matriptase-2 is critical for iron homeostasis and may play a role in renal disease.
matriptase-2; TMPRSS6; hepcidin; end-stage renal disease; EPO resistance
Introduction. Anemia is a frequent problem in hospitalized geriatric patients, and the anemia of chronic disease (ACD) and iron deficiency anemia (IDA) are the 2 most prevalent causes. The aim of the study was to assess the possible role of serum hepcidin in the differential diagnosis between ACD and IDA. Methods. We investigated serum hepcidin, iron status, anemia, and C-reactive protein in 39 consecutive geriatric patients with ACD and IDA. Serum hepcidin levels were determined using a commercial ELISA kit (DRG Instruments, Marburg, Germany). We also measured hepcidin in 26 healthy controls. Results. The serum hepcidin levels were not significantly higher in the 28 patients with ACD as compared to the 11 patients with IDA. Conclusions. The serum hepcidin levels measured using the commercial ELISA kit (DRG) do not appear to increase in older patients with ACD. It should be noted that an assay-specific problem could explain our results.
The recent discovery of hepcidin, the key iron regulatory hormone, has changed our view of iron metabolism, which in turn is long known to be linked with insulin resistant states, including type 2 diabetes mellitus and the Metabolic Syndrome (MetS). Serum ferritin levels are often elevated in MetS (Dysmetabolic hyperferritinemia - DHF), and are sometimes associated with a true mild-to-moderate hepatic iron overload (dysmetabolic iron overload syndrome - DIOS). However, the pathophysiological link between iron and MetS remains unclear. This study was aimed to investigate, for the first time, the relationship between MetS and hepcidin at population level. We measured serum hepcidin levels by Mass Spectrometry in 1,391 subjects from the Val Borbera population, and evaluated their relationship with classical MetS features. Hepcidin levels increased significantly and linearly with increasing number of MetS features, paralleling the trend of serum ferritin. In multivariate models adjusted for relevant variables including age, C-Reactive Protein, and the HFE C282Y mutation, ferritin was the only significant independent predictor of hepcidin in males, while in females MetS was also independently associated with hepcidin. Overall, these data indicate that the fundamental iron regulatory feedback is preserved in MetS, i.e. that hepcidin tends to progressively increase in response to the increase of iron stores. Due to recently discovered pleiotropic effects of hepcidin, this may worsen insulin resistance and contribute to the cardiovascular complications of MetS.
Systemic iron balance is regulated by hepcidin, a peptide hormone secreted by the liver. By decreasing cell surface expression of the iron exporter ferroportin, hepcidin decreases iron absorption from the intestine and iron release from reticuloendothelial stores. Hepcidin excess has been implicated in the pathogenesis of anemia of chronic disease, while hepcidin deficiency has a key role in the pathogenesis of the iron overload disorder hemochromatosis. We have recently shown that hemojuvelin is a coreceptor for bone morphogenetic protein (BMP) signaling and that BMP signaling positively regulates hepcidin expression in liver cells in vitro. Here we show that BMP-2 administration increases hepcidin expression and decreases serum iron levels in vivo. We also show that soluble hemojuvelin (HJV.Fc) selectively inhibits BMP induction of hepcidin expression in vitro and that administration of HJV.Fc decreases hepcidin expression, increases ferroportin expression, mobilizes splenic iron stores, and increases serum iron levels in vivo. These data support a role for modulators of the BMP signaling pathway in treating diseases of iron overload and anemia of chronic disease.
Growth differentiation factor (GDF)‐15 is a distant and divergent member of the transforming growth factor‐β superfamily (TGF‐β) . There is growing evidence indicating the involvement of GDF‐15 in various pathologies. Expression of GDF‐15 is induced under conditions of inflammation and increased GDF‐15 serum levels are suggested as a risk factor for cardiovascular diseases.
Methods and Results
We show here that GDF‐15 and proinflammatory cytokine interleukin (IL)‐6 levels are highly increased (5‐fold) in cultured oxidized low‐density lipoproteins–stimulated peritoneal macrophages derived from GDF‐15+/+/apolipoprotein (apo) E−/−, mice. Notably, IL‐6 induction on oxidized low‐density lipoproteins stimulation is completely abolished in the absence of GDF‐15. Consistent with our in vitro data GDF‐15 mRNA expression and protein levels are upregulated (2.5‐ to 6‐fold) in the atherosclerotic vessel wall of GDF‐15+/+/apoE−/− mice after a cholesterol‐enriched diet. GDF‐15 deficiency inhibits lumen stenosis (52%) and 18FDG uptake (34%) in the aortic arch despite increased serum triglyceride/cholesterol levels and elevated body weight. Immunohistomorphometric investigations of atherosclerotic lesions reveal a decreased percentage of inflammatory CD11b+ (57%) or IL‐6+, leukocytes, and apoptotic cells (74%) after 20 weeks. However, the total number of macrophages and cell density in atherosclerotic lesions of the innominate artery are increased in GDF‐15−/−/apoE−/− mice.
Our data suggest that GDF‐15 is involved in orchestrating atherosclerotic lesion progression by regulating apoptotic cell death and IL‐6–dependent inflammatory responses to vascular injury.
atherosclerosis; GDF‐15; inflammation; interleukins
Growth-differentiation factor-15 (GDF-15) is a stress-responsive, transforming growth factor-β-related cytokine, which has recently been reported to be elevated in serum of patients with idiopathic pulmonary arterial hypertension (IPAH). The aim of the study was to examine the expression and biological roles of GDF-15 in the lung of patients with pulmonary arterial hypertension (PAH).
GDF-15 expression in normal lungs and lung specimens of PAH patients were studied by real-time RT-PCR and immunohistochemistry. Using laser-assisted micro-dissection, GDF-15 expression was further analyzed within vascular compartments of PAH lungs. To elucidate the role of GDF-15 on endothelial cells, human pulmonary microvascular endothelial cells (HPMEC) were exposed to hypoxia and laminar shear stress. The effects of GDF-15 on the proliferation and cell death of HPMEC were studied using recombinant GDF-15 protein.
GDF-15 expression was found to be increased in lung specimens from PAH patients, com-pared to normal lungs. GDF-15 was abundantly expressed in pulmonary vascular endothelial cells with a strong signal in the core of plexiform lesions. HPMEC responded with marked upregulation of GDF-15 to hypoxia and laminar shear stress. Apoptotic cell death of HPMEC was diminished, whereas HPMEC proliferation was either increased or decreased depending of the concentration of recombinant GDF-15 protein.
GDF-15 expression is increased in PAH lungs and appears predominantly located in vascular endothelial cells. The expression pattern as well as the observed effects on proliferation and apoptosis of pulmonary endothelial cells suggest a role of GDF-15 in the homeostasis of endothelial cells in PAH patients.
Hepcidin is upregulated by inflammation and iron. Inherited (HFE genotype) and treatment-related factors (blood units (BU), Iron overload) affecting hepcidin (measured by C-ELISA) were studied in 42 consecutive patients with AML prior to and after allogeneic hematopoietic cell transplantation (HCT). Results. Elevated serum ferritin pre- and post-HCT was present in all patients. Median hepcidin pre- and post-HCT of 358 and 398 ng/mL, respectively, were elevated compared to controls (median 52 ng/mL) (P < .0001). Liver and renal function, prior chemotherapies, and conditioning had no impact on hepcidin. Despite higher total BU after HCT compared to pretransplantation (P < .0005), pre- and posttransplant ferritin and hepcidin were similar. BU influenced ferritin (P = .001) and hepcidin (P = .001). No correlation of pre- or posttransplant hepcidin with pretransplant ferritin was found. HFE genotype did not influence hepcidin. Conclusions. Hepcidin is elevated in AML patients pre- and post-HCT due to transfusional iron-loading suggesting that hepcidin synthesis remains intact despite chemotherapy and HCT.
The present study was aimed at determining whether hepcidin, a recently identified peptide involved in iron metabolism, plays a role in conditions associated with both iron overload and iron deficiency. Hepcidin mRNA levels were assessed in two models of anemia, acute hemolysis provoked by phenylhydrazine and bleeding provoked by repeated phlebotomies. Hepcidin response to hypoxia was also studied, both ex vivo, in human hepatoma cells, and in vivo. Anemia and hypoxia were associated with a dramatic decrease in liver hepcidin gene expression, which may account for the increase in iron release from reticuloendothelial cells and increase in iron absorption frequently observed in these situations. A single injection of turpentine for 16 hours induced a sixfold increase in liver hepcidin mRNA levels and a twofold decrease in serum iron. The hyposideremic effect of turpentine was completely blunted in hepcidin-deficient mice, revealing hepcidin participation in anemia of inflammatory states. These modifications of hepcidin gene expression further suggest a key role for hepcidin in iron homeostasis under various pathophysiological conditions, which may support the pharmaceutical use of hepcidin agonists and antagonists in various iron homeostasis disorders.
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.
Aim of this study was to evaluate whether the A736V TMPRSS6 polymorphism, a major genetic determinant of iron metabolism in healthy subjects, influences serum levels of hepcidin, the hormone regulating iron metabolism, and erythropoiesis in chronic hemodialysis (CHD).
To this end, we considered 199 CHD patients from Northern Italy (157 with hepcidin evaluation), and 188 healthy controls without iron deficiency, matched for age and gender. Genetic polymorphisms were evaluated by allele specific polymerase chain reaction assays, and hepcidin quantified by mass spectrometry.
Serum hepcidin levels were not different between the whole CHD population and controls (median 7.1, interquartile range (IQR) 0.55-17.1 vs. 7.4, 4.5-17.9 nM, respectively), but were higher in the CHD subgroup after exclusion of subjects with relative iron deficiency (p = 0.04). In CHD patients, the A736V TMPRSS6 polymorphism influenced serum hepcidin levels in individuals positive for mutations in the HFE gene of hereditary hemochromatosis (p < 0.0001). In particular, the TMPRSS6 736 V variant was associated with higher hepcidin levels (p = 0.017). At multivariate analysis, HFE and A736V TMPRSS6 genotypes predicted serum hepcidin independently of ferritin and C reactive protein (p = 0.048). In patients without acute inflammation and overt iron deficiency (C reactive protein <1 mg/dl and ferritin >30 ng/ml; n = 86), hepcidin was associated with lower mean corpuscular volume (p = 0.002), suggesting that it contributed to iron-restricted erythropoiesis. In line with previous results, in patients without acute inflammation and severe iron deficiency the “high hepcidin” 736 V TMPRSS6 variant was associated with higher erythropoietin maintenance dose (p = 0.016), independently of subclinical inflammation (p = 0.02).
The A736V TMPRSS6 genotype influences hepcidin levels, erythropoiesis, and anemia management in CHD patients. Evaluation of the effect of TMPRSS6 genotype on clinical outcomes in prospective studies in CHD may be useful to predict the outcomes of hepcidin manipulation, and to guide treatment personalization by optimizing anemia management.
Anemia; Chronic kidney disease; Erythropoietin; Genetics; Inflammation; Iron; Hemodialysis; Hepcidin; Hfe gene; Matriptase-2; Tmprss6
Hemojuvelin (HJV) is highly expressed in the liver, skeletal muscles, and heart, seems to play a role in iron absorption and release from cells, and has anti-inflammatory properties. Moreover, HJV plays an essential role in the regulation of hepcidin expression, specifically in the iron-sensing pathway. Hepcidin has emerged as a key regulator of iron homeostasis. In this study we tested for the first time the hypothesis that HJV is related to iron metabolism in hemodialysis (HD) patients.
Iron status, complete blood count, and serum creatinine, albumin, and lipids were assessed, using standard laboratory methods. Serum levels of soluble transferrin receptor (sTFR), high-sensitivity CRP, IL-6, hepcidin, and HJV were measured using commercially available kits.
Serum HJV, hepcidin, ferritin, IL-6, hsCRP, and serum creatinine were significantly higher (all P < 0.001), whereas serum iron, sTFR, transferrin, hemoglobin, and erythrocyte count were significantly lower in HD patients, compared to healthy volunteers (all P < 0.001). In univariate analysis, HJV was strongly correlated (P < 0.001) with ferritin, transferrin saturation, and TIBC, as well as with hsCRP, hepcidin, Kt/V (P < 0.01) and residual renal function, the presence of diabetes, APKD, and coronary heart disease. Predictors of HJV level in multiple regression analysis were ferritin (beta value was 0.50, P = 0.00004) and transferrin saturation (beta value was 0.47, P = 0.0002), explaining 81% of the HJV variations.
Serum HJV is elevated in HD patients and related predominantly to kidney function and iron metabolism. However, HJV is probably not correlated to inflammation. HJV appears to be a new player in iron metabolism in these patients.
Iron metabolism; Hemodialysis; Inflammation; Hepcidin; Hemojuvelin
Hepcidin is a central regulator of iron metabolism. Serum hepcidin levels are increased in patients with renal insufficiency, which may contribute to anemia. Urine hepcidin was found to be increased in some patients after cardiac surgery, and these patients were less likely to develop acute kidney injury. It has been suggested that urine hepcidin may protect by attenuating heme-mediated injury, but processes involved in urine hepcidin excretion are unknown.
To assess the role of tubular reabsorption we compared fractional excretion (FE) of hepcidin-25 with FE of β2-microglobulin (β2m) in 30 patients with various degrees of tubular impairment due to chronic renal disease. To prove that hepcidin is reabsorbed by the tubules in a megalin-dependent manner, we measured urine hepcidin-1 in wild-type and kidney specific megalin-deficient mice. Lastly, we evaluated FE of hepcidin-25 and β2m in 19 patients who underwent cardiopulmonary bypass surgery. Hepcidin was measured by a mass spectrometry assay (MS), whereas β2m was measured by ELISA.
In patients with chronic renal disease, FE of hepcidin-25 was strongly correlated with FE of β2m (r = 0.93, P <0.01). In megalin-deficient mice, urine hepcidin-1 was 7-fold increased compared to wild-type mice (p < 0.01) indicating that proximal tubular reabsorption occurs in a megalin- dependent manner. Following cardiac surgery, FE of hepcidin-25 increased despite a decline in FE of β2m, potentially indicating local production at 12–24 hours.
Hepcidin-25 is reabsorbed by the renal tubules and increased urine hepcidin-25 levels may reflect a reduction in tubular uptake. Uncoupling of FE of hepcidin-25 and β2m in cardiac surgery patients suggests local production.
AKI; β2-microglobulin; Hepcidin; Megalin; Kidney tubules
Iron is an essential micronutrient, as it is required for adequate erythropoietic function, oxidative metabolism and cellular immune responses. Although the absorption of dietary iron (1-2 mg/d) is regulated tightly, it is just balanced with losses. Therefore, internal turnover of iron is essential to meet the requirements for erythropoiesis (20-30 mg/d). Increased iron requirements, limited external supply, and increased blood loss may lead to iron deficiency (ID) and iron-deficiency anemia. Hepcidin, which is made primarily in hepatocytes in response to liver iron levels, inflammation, hypoxia and anemia, is the main iron regulatory hormone. Once secreted into the circulation, hepcidin binds ferroportin on enterocytes and macrophages, which triggers its internalization and lysosomal degradation. Thus, in chronic inflammation, the excess of hepcidin decreases iron absorption and prevents iron recycling, which results in hypoferremia and iron-restricted erythropoiesis, despite normal iron stores (functional ID), and anemia of chronic disease (ACD), which can evolve to ACD plus true ID (ACD + ID). In contrast, low hepcidin expression may lead to iron overload, and vice versa. Laboratory tests provide evidence of iron depletion in the body, or reflect iron-deficient red cell production. The appropriate combination of these laboratory tests help to establish a correct diagnosis of ID status and anemia.
Iron metabolism; Iron deficiency; Functional iron deficiency; Hepcidin; Anemia of chronic disease
Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. Recent in vivo studies have shown that hepcidin is down-regulated by erythropoiesis, anemia, and hypoxia, which meets the need of iron input for erythrocyte production. Erythropoietin (EPO) is the primary signal that triggers erythropoiesis in anemic and hypoxic conditions. Therefore, a direct involvement of EPO in hepcidin regulation can be hypothesized. We report here the regulation of hepcidin expression by EPO, in a dose-dependent manner, in freshly isolated mouse hepatocytes and in the HepG2 human hepatocyte cell model. The effect is mediated through EPOR signaling, since hepcidin mRNA levels are restored by pretreatment with an EPOR-blocking antibody. The transcription factor C/EBPα showed a pattern of expression similar to hepcidin, at the mRNA and protein levels, following EPO and anti-EPOR treatments. Chromatin immunoprecipitation experiments showed a significant decrease of C/EBPα binding to the hepcidin promoter after EPO supplementation, suggesting the involvement of this transcription factor in the transcriptional response of hepcidin to EPO.
Hepcidin is an acute-phase response antimicrobial peptide that has emerged as a central regulator of iron absorption. Circulating hepcidin levels have been shown to affect iron uptake, release and storage. Hepcidin is mainly liver-derived and regulated, at least in part, transcriptionally. Hypoxia, erythroid demand, iron content and inflammation each have been shown to influence hepcidin mRNA expression in intact animals. In vitro, regulation of hepcidin by cytokines and by hypoxia is readily demonstrated in primary hepatocytes or in hepatocyte lines, but incubating the same cell lines with iron does not increase transcription of hepcidin. Thus, how iron excess stimulates hepcidin production in hepatocytes remains unknown. In addition, there is no current technique available that can investigate how iron induces hepcidin expression. To provide a better understanding of hepcidin gene expression in response to these regulatory stimuli, we have established a whole animal in vivo bioluminescence imaging assay to measure the activity of hepcidin promoter constructs in the animals liver after hydrodynamic transfection of hepcidin promoter/luciferase constructs into mice. Transfected hepcidin promoter constructs were shown to respond to both inflammatory and increasing iron stimuli in vivo. This work highlights the ability of this new imaging technique to investigate the key regions of the hepcidin promoter involved in iron induction of hepcidin expression.
Hepcidin; IVIS; Luciferase; Iron Stimulation
Hepcidin is the master regulator of iron homeostasis. In the liver, iron-dependent hepcidin activation is regulated through Bmp6 and its membrane receptor hemojuvelin (Hjv) whereas, in response to iron deficiency, hepcidin repression seems to be controlled by a pathway involving the serine protease matriptase-2 (encoded by Tmprss6). To determine the relationship between Bmp6 and matriptase-2 pathways, Tmprss6−/− mice (characterized by increased hepcidin levels and anemia) and Bmp6−/− mice (exhibiting severe iron overload due to hepcidin deficiency) were intercrossed. We showed that loss of Bmp6 decreased hepcidin levels, increased hepatic iron and, importantly, corrected hematological abnormalities in Tmprss6−/− mice. This suggests that elevated hepcidin levels in patients with familial iron-refractory iron deficiency anemia are due to excess signaling through the Bmp6/Hjv pathway.
Anemia, Iron-Deficiency; metabolism; Animals; Antimicrobial Cationic Peptides; metabolism; Bone Morphogenetic Protein 6; metabolism; Female; Iron; metabolism; Iron, Dietary; metabolism; Liver; metabolism; Membrane Proteins; metabolism; Mice; Mice, Knockout; Serine Endopeptidases; metabolism; Signal Transduction; physiology; hepcidin; hemojuvelin; bmp6; matriptase2; tmprss6
Systemic iron homeostasis involves a negative feedback circuit in which the expression level of the peptide hormone hepcidin depends on and controls the iron blood levels. Hepcidin expression is regulated by the BMP6/SMAD and IL6/STAT signaling cascades. Deregulation of either pathway causes iron-related diseases such as hemochromatosis or anemia of inflammation. We quantitatively analyzed how BMP6 and IL6 control hepcidin expression. Transcription factor (TF) phosphorylation and reporter gene expression were measured under co-stimulation conditions, and the promoter was perturbed by mutagenesis. Using mathematical modeling, we systematically analyzed potential mechanisms of cooperative and competitive promoter regulation by the transcription factors, and experimentally validated the model predictions. Our results reveal that hepcidin cross-regulation primarily occurs by combinatorial transcription factor binding to the promoter, whereas signaling crosstalk is insignificant. We find that the presence of two BMP-responsive elements enhances the steepness of the promoter response towards the iron-sensing BMP signaling axis, which promotes iron homeostasis in vivo. IL6 co-stimulation reduces the promoter sensitivity towards the BMP signal, because the SMAD and STAT transcription factors compete for recruiting RNA polymerase to the transcription start site. This may explain why inflammatory signals disturb iron homeostasis in anemia of inflammation. Taken together, our results reveal why the iron homeostasis circuit is sensitive to perturbations implicated in disease.
The nutritional iron uptake is tightly regulated because the body has limited capacity of iron excretion. Mammals maintain iron homeostasis by a negative feedback loop, in which the peptide hepcidin senses the iron blood level and controls iron resorption. Molecular perturbations in the homeostasis loop lead to iron-related diseases such as hemochromatosis or anemia of inflammation. Quantitative studies are required to understand the dynamics of the iron homeostasis circuitry in health and disease. We investigated how the biological activity of hepcidin is regulated by combining experiments with mathematical modeling. We present a multi-scale model that describes the signaling network and the gene promoter controlling hepcidin expression. Possible scenarios of hepcidin regulation were systematically tested against experimental data, and interpreted using a network model of iron metabolism in vivo. The analysis showed that the presence of multiple redundant regulatory elements in the hepcidin gene promoter facilitates homeostasis, because changes in iron blood levels are sensed with high sensitivity. We further suggest that inflammatory signals establish molecular competition at the hepcidin promoter, thereby reducing its iron sensitivity and leading to a loss of homeostasis in anemia of inflammation. We conclude that quantitative insights into hepcidin expression regulation explain features of systemic iron homeostasis.
Hepcidin, a peptide that is released into the blood in response to inflammation, prevents cellular iron export and results in declines in iron status. Elevated serum and urinary levels of hepcidin have been observed in athletes following exercise, and declines in iron status have been reported following prolonged periods of training. The objective of this observational study was to characterize the effects of an occupational task, military training, on iron status, inflammation, and serum hepcidin.
Volunteers (n = 21 males) included Norwegian Soldiers participating in a 7-day winter training exercise that culminated in a 3-day, 54 km ski march. Fasted blood samples were collected at baseline, on day 4 (PRE, prior to the ski march), and again on day 7 (POST, following the ski march). Samples were analyzed for hemoglobin, serum ferritin, soluble transferrin receptor (sTfR), interleukin-6 (IL-6), and serum hepcidin. Military training affected inflammation and serum hepcidin levels, as IL-6 and hepcidin concentrations increased (P < 0.05) from the baseline to POST (mean ± SD, 9.1 ± 4.9 vs. 14.5 ± 8.4 pg/mL and 6.5 ± 3.5 vs. 10.2 ± 6.9 ng/mL, respectively). Iron status was not affected by the training exercise, as sTfR levels did not change over the course of the 7-day study.
Military training resulted in significant elevations in IL-6 and serum hepcidin. Future studies should strive to identify the role of hepcidin in the adaptive response to exercise, as well as countermeasures for the prevention of chronic or repeated elevations in serum hepcidin due to exercise or sustained occupational tasks which may result in longer term decrements in iron status.
Physical activity; Operational stress; Military; Ferritin; Inflammation; Iron absorption; Soluble transferrin receptor
Growth differentiation factor 11 (GDF11) is one of the significant genes that control skeletal formation. Knockout of GDF11 function causes abnormal patterning of the anterior/posterior axial skeleton. The mRNA of GDF11 is initially translated to a precursor protein that undergoes a proteolytic cleavage to generate the C-terminal peptide or mature GDF11, and the N-terminal peptide named GDF11 propeptide. The propeptide can antagonize GDF11 activity in vitro. To investigate the effects of GDF11 propeptide on GDF11 function in vivo, we generated transgenic mice that over-express the propeptide cDNA in skeletal tissue. The transgenic mice showed formation of extra ribs on the seventh cervical vertebra (C7) as a result of transformation of the C7 vertebra into a thoracic vertebra. The GDF11 propeptide transgene mRNA was detected in tail tissue in embryos and was highly expressed in tail and calvaria bones after birth. A high frequency of C7 rib formation was noticed in the transgenic mouse line with a high level of transgene expression. The anterior boundaries of Hoxa-4 and Hoxa-5 mRNA in situ expressions showed cranial shifts from their normal prevertebra locations in transgenic embryos. These results demonstrated significant effects of GDF11 propeptide transgene on vertebral formation, which are likely occurring through depressing GDF11 function and altered locations of Hoxa-4 and Hoxa-5 expression.