Mammalian Iron Metabolism Research Articles

СОВРЕМЕННЫЕ ПРЕДСТАВЛЕНИЯ О ГОМЕОСТАЗЕ ЖЕЛЕЗА В ОРГАНИЗМЕ ЧЕЛОВЕКА

Iron is a necessary element for all living organisms, since it is part of the functional groups of oxygentransporting proteins and enzymes that catalyze energy generation reactions and metabolic processes. New research in the fi eld of iron homeostasis has allowed to learn in more detail the molecular mechanisms of absorption, export, transport, storage, recycling of iron, the mechanisms of regulation of iron metabolism in mammals at the cellular and systemic level are disclosed in detail. This article summarizes modern scientifi c data on iron homeostasis at all stages, from absorption to recycling

ГЕПСИДИН, ТРАНСФЕРИН, ФЕРИТИН: ФІЗІОЛОГІЧНА РОЛЬ ЯК ЦЕНТРАЛЬНИХ РЕГУЛЯТОРІВ ОБМІНУ ЗАЛІЗА В ОРГАНІЗМІ

The knowledge about mammalian iron metabolism has advanced dramatically over the past decades. Studies of genetics, biochemistry and molecular biology allowed us the identification and characterization of many of the molecules involved in regulation of iron homeostasis. Important progresses were made after the discovery in 2000 of a small peptide – hepsidin – that has been proved to play a central role in orchestration on iron metabolism also providing a link between iron metabolism and inflammation and innate immunity. Hepsidin directly interacts with ferroportin, the only known mammalian iron exporter, which is expressed by enterocytes, macrophages and hepatocytes. The direct hepsidin- ferroportin interaction allows an adaptative response from the body in situations that alter normal iron homeostasis (hypoxia, anemia, iron deficiency, iron overload, and inflammation). In clause the items of information on transport protein of iron - transferrin are stated. Its physiological role and clinical importance is shown. Dynamics of the contents of the hepsidin, transferrin, ferritin in persons with latent deficiency of iron. The conclusion about importance of the given parameter for laboratory diagnostics of iron deficiency condition is made. In the article the items of information about the ferritin - protein - depot of iron in body are given. Its physiological role and clinical importance is displayed. Dynamics of changes of the contents ferritin during treatment of the patients with iron deficiency anemia and persons with latent deficiency of iron is shown. The conclusion about the level of the ferritin in serum of blood is the important dynamic parameter for laboratory diagnostics iron deficiency of condition is made.

Lipogenic SREBP-1a/c transcription factors activate expression of the iron regulator hepcidin, revealing cross-talk between lipid and iron metabolisms

The sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors best known for stimulating the expression of genes encoding key lipogenic enzymes. However, SREBP functions beyond lipid metabolism are less understood. Here, we show that hepcidin antimicrobial peptide (Hamp), encoding the hormone hepcidin essential for iron homeostasis and regulated by dietary iron and inflammation, is a target gene of the two SREBP isoforms SREBP-1a/c. We found that in tissue culture, mature, active, and nuclear forms of the SREBP-1a/c proteins induce endogenous Hamp gene expression and increase the Hamp promoter activity primarily via three regulatory sequences, including an E-box. Moreover, ChIP experiments revealed that SREBP-1a binds to the Hamp gene promoter. Overexpression of nuclear SREBP-1a under the control of the phosphoenolpyruvate carboxylase-1 (Pck1) promoter in mice increased hepatic Hamp mRNA and blood hepcidin levels, and as expected, caused fatty liver. Consistent with the known effects of Hamp up-regulation, SREBP-1a-overexpressing mice displayed signs of dysregulation in iron metabolism, including reduced serum iron and increased hepatic and splenic iron storage. Conversely, liver-specific depletion of the nuclear forms of SREBPs, as in SREBP cleavage-activating protein knockout mice, impaired lipopolysaccharide-induced up-regulation of hepatic Hamp Together, these results indicate that the SREBP-1a/c transcription regulators activate hepcidin expression and thereby contribute to the control of mammalian iron metabolism.

Ironing out the Brain.

Our understanding of the broad principles of cellular and systemic iron homeostasis in man are well established with the exception of the brain. Most of the proteins involved in mammalian iron metabolism are present in the brain, although their distribution and precise roles in iron uptake, intracellular metabolism and export are still uncertain, as is the way in which systemic iron is transferred across the blood-brain barrier. We briefly review current concepts concerning the uptake and distribution of iron in the brain, before turning to the ways in which brain iron homeostasis might be regulated. The distribution of iron between different brain regions is then discussed as is the increase in brain iron with normal aging, and the different forms in which iron is present. The increased levels of iron found in specific brain regions and their potential contribution to neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease and other polyglutamine expansion diseases, amyotrophic lateral sclerosis, Friedreich's ataxia, as well as a number of neurodegenerative diseases with iron accumulation, are discussed. The interactions between neuroinflammation and iron are presented, and the chapter concludes with a review of current clinical studies and discussion of the potential and efficacy of iron chelation therapy in the treatment of neurodegenerative diseases.

Large scale expression and purification of secreted mouse hephaestin.

Hephaestin is a large membrane-anchored multicopper ferroxidase involved in mammalian iron metabolism. Newly absorbed dietary iron is exported across the enterocyte basolateral membrane by the ferrous iron transporter ferroportin, but hephaestin increases the efficiency of this process by oxidizing the transported iron to its ferric form and promoting its release from ferroportin. Deletion or mutation of the hephaestin gene leads to systemic anemia with iron accumulation in the intestinal epithelium. The crystal structure of human ceruloplasmin, another multicopper ferroxidase with 50% sequence identity to hephaestin, has provided a framework for comparative analysis and modelling. However, detailed structural information for hephaestin is still absent, leaving questions relating to metal coordination and binding sites unanswered. To obtain structural information for hephaestin, a reliable protocol for large-scale purification is required. Here, we present an expression and purification protocol of soluble mouse hephaestin, yielding milligram amounts of enzymatically active, purified protein using the baculovirus/insect cell system.

The Interface Between Iron Metabolism and Gene-Based Iron Contrast for MRI.

Using a gene-based approach to track cellular and molecular activity with magnetic resonance imaging (MRI) has many advantages. The strong correlation between transverse relaxation rates and total cellular iron content provides a basis for developing sensitive and quantitative detection of MRI reporter gene expression. In addition to biophysical concepts, general features of mammalian iron regulation add valuable context for interpreting molecular MRI predicated on gene-based iron labeling. With particular reference to the potential of magnetotactic bacterial gene expression as a magnetic resonance (MR) contrast agent for mammalian cell tracking, studies in different cell culture models highlight the influence of intrinsic iron regulation on the MRI signal. The interplay between dynamic regulation of mammalian iron metabolism and expression systems designed to sequester iron biominerals for MRI is presented from the perspective of their potential influence on MR image interpretation.

Hepcidin and its potential clinical utility.

A number of pathophysiological conditions are related to iron metabolism disturbances. Some of them are well known, others are newly discovered or special. Hepcidin is a newly identified iron metabolism regulating hormone, which could be a promising biomarker for many disorders. In this review, we provide background information about mammalian iron metabolism, cellular iron trafficking, and the regulation of expression of hepcidin. Beside these molecular biological processes, we summarize the methods that have been used to determine blood and urine hepcidin levels and present those pathological conditions (cancer, inflammation, neurological disorders) when hepcidin measurement may have clinical relevance.

Liver congestion in heart failure contributes to inappropriately increased serum hepcidin despite anemia.

Hepcidin is a key regulator of mammalian iron metabolism and mainly produced by the liver. Hepcidin excess causes iron deficiency and anemia by inhibiting iron absorption from the intestine and iron release from macrophage stores. Anemia is frequently complicated with heart failure. In heart failure patients, the most frequent histologic appearance of liver is congestion. However, it remains unclear whether liver congestion associated with heart failure influences hepcidin production, thereby contributing to anemia and functional iron deficiency. In this study, we investigated this relationship in clinical and basic studies. In clinical studies of consecutive heart failure patients (n = 320), anemia was a common comorbidity (41%). In heart failure patients without active infection and ongoing cancer (n = 30), log-serum hepcidin concentration of patients with liver congestion was higher than those without liver congestion (p = 0.0316). Moreover, in heart failure patients with liver congestion (n = 19), the anemia was associated with the higher serum hepcidin concentrations, which is a type of anemia characterized by induction of hepcidin. Subsequently, we produced a rat model of heart failure with liver congestion by injecting monocrotaline that causes pulmonary hypertension. The monocrotaline-treated rats displayed liver congestion with increase of hepcidin expression at 4 weeks after monocrotaline injection, followed by anemia and functional iron deficiency observed at 5 weeks. We conclude that liver congestion induces hepcidin production, which may result in anemia and functional iron deficiency in some patients with heart failure.

HERC2 Targets the Iron Regulator FBXL5 for Degradation and Modulates Iron Metabolism

FBXL5 (F-box and leucine-rich repeat protein 5) is the F-box protein subunit of, and therefore responsible for substrate recognition by, the SCF(FBXL5) ubiquitin-ligase complex, which targets iron regulatory protein 2 (IRP2) for proteasomal degradation. IRP2 plays a central role in the maintenance of cellular iron homeostasis in mammals through posttranscriptional regulation of proteins that contribute to control of the intracellular iron concentration. The FBXL5-IRP2 axis is integral to control of iron metabolism in vivo, given that mice lacking FBXL5 die during early embryogenesis as a result of unrestrained IRP2 activity and oxidative stress attributable to excessive iron accumulation. Despite its pivotal role in the control of iron homeostasis, however, little is known of the upstream regulation of FBXL5 activity. We now show that FBXL5 undergoes constitutive ubiquitin-dependent degradation at the steady state. With the use of a proteomics approach to the discovery of proteins that regulate the stability of FBXL5, we identified the large HECT-type ubiquitin ligase HERC2 (HECT and RLD domain containing E3 ubiquitin protein ligase 2) as an FBXL5-associated protein. Inhibition of the HERC2-FBXL5 interaction or depletion of endogenous HERC2 by RNA interference resulted in the stabilization of FBXL5 and a consequent increase in its abundance. Such accumulation of FBXL5 in turn led to a decrease in the intracellular content of ferrous iron. Our results thus suggest that HERC2 regulates the basal turnover of FBXL5, and that this ubiquitin-dependent degradation pathway contributes to the control of mammalian iron metabolism.

Mechanisms of iron metabolism in Caenorhabditis elegans.

Iron is involved in many biological processes essential for sustaining life. In excess, iron is toxic due to its ability to catalyze the formation of free radicals that damage macromolecules. Organisms have developed specialized mechanisms to tightly regulate iron uptake, storage and efflux. Over the past decades, vertebrate model organisms have led to the identification of key genes and pathways that regulate systemic and cellular iron metabolism. This review provides an overview of iron metabolism in the roundworm Caenorhabditis elegans and highlights recent studies on the role of hypoxia and insulin signaling in the regulation of iron metabolism. Given that iron, hypoxia and insulin signaling pathways are evolutionarily conserved, C. elegans provides a genetic model organism that promises to provide new insights into mechanisms regulating mammalian iron metabolism.

Investigating the role of transferrin in the distribution of iron, manganese, copper, and zinc.

The essential role of transferrin in mammalian iron metabolism is firmly established. Integral to our understanding of transferrin, studies in hypotransferrinemic mice, a model of inherited transferrin deficiency, have demonstrated that transferrin is essential for iron delivery for erythropoiesis and in the regulation of expression of hepcidin, a hormone that inhibits macrophage and enterocyte iron efflux. Here we investigate a potential role for transferrin in the distribution of three other physiologic metals, manganese, copper, and zinc. We first assessed metal content in transferrin-rich fractions of wild-type mouse sera and demonstrate that although both iron and manganese cofractionated predominantly with transferrin, the absolute levels of manganese are several orders of magnitude lower than those of iron. We next measured metal content in multiple tissues in wild-type and hypotransferrinemic mice of various ages. Tissue metal imbalances were severe for iron and minimal to moderate for some metals in some tissues in hypotransferrinemic mice. Metal levels measured in a transferrin-replete yet hepcidin-deficient and iron-loaded mouse strain suggested that the observed imbalances in tissue copper, zinc, and manganese levels were not all specific to hypotransferrinemic mice or caused directly by transferrin deficiency. Overall, our results suggest that transferrin does not have a primary role in the distribution of manganese, copper, or zinc to tissues and that the abnormalities observed in tissue manganese levels are not attributable to a direct role for transferrin in manganese metabolism but rather are attributable to an indirect effect of transferrin deficiency on hepcidin expression and/or iron metabolism.

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.

Hepcidin, show some self-control! How the hormone of iron metabolism regulates its own expression

Does the hormone of iron metabolism, hepcidin, exhibit 'self-control'? Hepcidin is a small, disulfide-rich peptide synthesized by the liver, which plays a keystone role in regulating systemic iron metabolism in mammals. Hepcidin acts by binding and triggering the lysosomal degradation of the cellular iron exporter ferroportin. Ultimately, decreased ferroportin leads to decreased plasma iron levels. Although various modulators of HAMP (the hepcidin antimicrobial peptide gene) expression are known, no auto-regulatory pathway has been described. In their paper published in the Biochemical Journal in April 2013, Pandur et al. identify an auto-regulatory pathway in which prohepcidin regulates HAMP expression. The authors observe that prohepcidin can bind to the inflammation-regulated STAT3 (signal transducer and activator of transcription 3)-binding site in the HAMP promoter to negatively regulate HAMP expression. Furthermore, the authors find that the prohepcidin-binding partner, α-1 antitrypsin, inhibits prohepcidin's ability to decrease HAMP activity. This is significant as α-1 antitrypsin, similar to hepcidin, is an acute-phase reactant that is up-regulated by inflammation. In conclusion, the discovery of a hepcidin auto-regulatory pathway, first, supports the emerging notion that hepcidin regulation is exquisitely fine-tuned through a process of combinatorial control; and secondly, suggests that hepcidin may play a hand in its own deregulation in diseases of iron metabolism that involve aberrant cytokine signalling (e.g. the anaemia of inflammation).

Proteomic analysis and molecular modelling characterize the iron-regulatory protein haemojuvelin/repulsive guidance molecule c.

HJV (haemojuvelin) plays a key role in iron metabolism in mammals by regulating expression of the liver-derived hormone hepcidin, which controls systemic iron uptake and release. Mutations in HJV cause juvenile haemochromatosis, a rapidly progressing iron overload disorder in humans. HJV, also known as RGMc (repulsive guidance molecule c), is a member of the three-protein RGM family. RGMs are GPI (glycosylphosphatidylinositol)-linked glycoproteins that share ~50% amino acid identity and several structural motifs, including the presence of 14 cysteine residues in analogous locations. Unlike RGMa and RGMb, HJV/RGMc is composed of both single-chain and two-chain isoforms. To date there is no structural information for any member of the RGM family. In the present study we have mapped the disulfide bonds in mouse HJV/RGMc using a proteomics strategy combining sequential MS steps composed of ETD (electron transfer dissociation) and CID (collision-induced dissociation), in which ETD induces cleavage of disulfide linkages, and CID establishes disulfide bond assignments between liberated peptides. The results of the present study identified an HJV/RGMc molecular species containing four disulfide linkages. We predict using ab initio modelling that this molecule is a single-chain HJV/RGMc isoform. Our observations outline a general approach using tandem MS and ab initio molecular modelling to define unknown structural features in proteins.

Iron Regulatory Proteins Control a Mucosal Block to Intestinal Iron Absorption

Mammalian iron metabolism is regulated systemically by the hormone hepcidin and cellularly by iron regulatory proteins (IRPs) that orchestrate a posttranscriptional regulatory network. Through ligand-inducible genetic ablation of both IRPs in the gut epithelium of adult mice, we demonstrate that IRP deficiency impairs iron absorption and promotes mucosal iron retention via a ferritin-mediated "mucosal block." We show that IRP deficiency does not interfere with intestinal sensing of body iron loading and erythropoietic iron need, but rather alters the basal expression of the iron-absorption machinery. IRPs thus secure sufficient iron transport across absorptive enterocytes by restricting the ferritin "mucosal block" and define a basal set point for iron absorption upon which IRP-independent systemic regulatory inputs are overlaid.

HRG1 Is Essential for Heme Transport from the Phagolysosome of Macrophages during Erythrophagocytosis

Adult humans have about 25 trillion red blood cells (RBCs), and each second we recycle about 5 million RBCs by erythrophagocytosis (EP) in macrophages of the reticuloendothelial system. Despite the central role for EP in mammalian iron metabolism, the molecules and pathways responsible for heme trafficking during EP remain unknown. Here, we show that the mammalian homolog of HRG1, a transmembrane heme permease in C.elegans, is essential for macrophage iron homeostasis and transports heme from the phagolysosome to the cytoplasm during EP. HRG1 is strongly expressed in macrophages of thereticuloendothelial system and specifically localizes to the phagolysosomal membranes during EP. Depletion of Hrg1 in mouse macrophages causes attenuation of heme transport from the phagolysosomal compartment. Importantly, missense polymorphisms in human HRG1 are defective in heme transport. Our results reveal HRG1 as the long-sought heme transporter for heme-iron recycling in macrophages and suggest that genetic variations in HRG1 could be modifiers of human iron metabolism.

Iron and intestinal immunity

Recent advances in the study of iron metabolism have led to a better understanding of the molecular basis for the interactions between iron and the inflammatory response. We will review this new information in the context of the gastrointestinal tract. The effects of iron on microbial enteropathogens are well known. Recent work has demonstrated that iron also has potentially important effects on the intestinal microbiota. On the host side, hepcidin, a key regulator of mammalian iron metabolism, has emerged as an important mediator of the cross-talk between iron homeostasis and inflammation. Hepcidin-dependent changes in iron flux can influence the expression of inflammatory cytokines, and conversely, inflammatory cytokines can induce hepcidin expression and alter iron homeostasis. Hepcidin levels have been found to be elevated in some studies of inflammatory bowel disease, while manipulating hepcidin expression in animal models of this condition has beneficial effects on both inflammation and dysregulated iron metabolism. The information on iron metabolism that has become available in recent years has shed new light on the pathogenesis of inflammatory diseases of the gastrointestinal tract, and is also starting to suggest new approaches to treating such diseases.

The role of iron regulatory proteins in the control of iron metabolism in mammals

The iron regulatory proteins (IRP1 and IRP2) are two cytoplasmic RNA-binding proteins involved in the mechanisms that control iron metabolism in mammalian cells. They modulate the expression of iron-related proteins at a post-transcriptional level by binding to specific iron regulatory elements (IREs) on their mRNAs. IRP-IRE interaction can block protein synthesis or stabilize the mRNA. At low intracellular iron concentration, IRPs bind to the IRE of ferritin or ferroportin mRNAs and block their translation. Direct interactions between IRPs and several IRE motifs stabilize transferrin receptor mRNA. The converse regulation of ferritin and TfR synthesis, being a consequence of the lack of binding of IRPs to IRE, occurs in cells with high iron level. Thus, IRP-mediated regulation rapidly restores the physiological level of iron during its deficiency as well as excess. The role of IRPs in maintaining the intracelluar iron balance has been relatively well characterized in numerous types of mammalian cells. However, the importance of IRPs in the regulation of systemic iron metabolism in mammals, particularly, in signaling between the cells which play major roles in body iron metabolism, such as duodenal enterocytes, reticuloendothelial macrophages, hepatocytes, and bone marrow precursors of red blood cells, is only beginning to be investigated. Several studies have shown that IRP2 is a predominant regulator of iron homeostasis in mice housed under standard conditions, thus limiting the impact of IRP1 on this metabolic pathway. Although IRP1-deficient mice do not display a strong pathological phenotype, a deletion of both IRPs is embryonic lethal. In addition, in vitro and in vivo studies have reported that nitric oxide (NO) and hydrogen peroxide (H2O2), which are produced during inflammation, are potent IRP1 regulators that mediate the disassembly of Fe-S cluster of IRP1. There is also an increasing evidence that NO and superoxide anion (O2 ) may induce a strong down-regulation of IRP1 at the protein level and thus have an impact on the binding of IRP1 to IREs. All these data suggest a predominant role of IRP1 in the regulation of iron homeostasis under specific physiopathological conditions.

Iron and Immunity: Immunological Consequences of Iron Deficiency and Overload

The influence of iron on immune function has been long appreciated. However, the molecular basis for this interaction is less well understood. Recently, there have been several important advances that have shed light on the mechanisms that regulate mammalian iron metabolism. The new insights provide a conceptual framework for understanding and manipulating the cross-talk between iron homeostasis and the immune system. This article will review what is currently known about how disturbances of iron metabolism can affect immunity and how activation of the immune system can lead to alterations in iron balance.

Quantitation of hepcidin in serum using ultra‐high‐pressure liquid chromatography and a linear ion trap mass spectrometer

Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Biological levels are increased in end-stage renal disease and during inflammation but suppressed in hemochromatosis. Thus hepcidin levels have diagnostic importance. This study describes the development of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. The fragmentation of hepcidin was investigated using triple quadrupole and linear ion trap mass spectrometers. A standard quantity of a stable isotopically labelled hepcidin internal standard was added to serum samples. Extraction was performed by protein precipitation and weak cation-exchange magnetic nanoparticles. Chromatography was carried out on sub 2 microm particle stationary phase, using ultra-high-pressure liquid chromatography and a linear ion trap for quantitation. The lower limit of quantitation was 0.4 nmol/L with less than 20% accuracy and precision. The mean hepcidin concentration in sera for controls was 4.6 +/- 2.7 nmol/L, in patients with sickle cell disease, 7.0 +/- 8.9 nmol/L; in patients with end-stage renal disease, 30.5 +/- 15.7 nmol/L; and patients with penetrant hereditary hemochromatosis, 1.4 +/- 0.8 nmol/L.