The roles of iron in health and disease

Iron is vital for almost all living organisms by participating in a wide variety of metabolic processes, including oxygen transport, DNA synthesis, and electron transport. However, iron concentrations in body tissues must be tightly regulated because excessive iron leads to tissue damage, as a result of formation of free radicals. Disorders of iron metabolism are among the most common diseases of humans and encompass a broad spectrum of diseases with diverse clinical manifestations, ranging from anemia to iron overload and, possibly, to neurodegenerative diseases. The molecular understanding of iron regulation in the body is critical in identifying the underlying causes for each disease and in providing proper diagnosis and treatments. Recent advances in genetics, molecular biology and biochemistry of iron metabolism have assisted in elucidating the molecular mechanisms of iron homeostasis. The coordinate control of iron uptake and storage is tightly regulated by the feedback system of iron responsive element-containing gene products and iron regulatory proteins that modulate the expression levels of the genes involved in iron metabolism. Recent identification and characterization of the hemochromatosis protein HFE, the iron importer Nramp2, the iron exporter ferroportin1, and the second transferrin-binding and -transport protein transferrin receptor 2, have demonstrated their important roles in maintaining body's iron homeostasis. Functional studies of these gene products have expanded our knowledge at the molecular level about the pathways of iron metabolism and have provided valuable insight into the defects of iron metabolism disorders. In addition, a variety of animal models have implemented the identification of many genetic defects that lead to abnormal iron homeostasis and have provided crucial clinical information about the pathophysiology of iron disorders. In this review, we discuss the latest progress in studies of iron metabolism and our current understanding of the molecular mechanisms of iron absorption, transport, utilization, and storage. Finally, we will discuss the clinical presentations of iron metabolism disorders, including secondary iron disorders that are either associated with or the result of abnormal iron accumulation.     

Chemistry and biology of eukaryotic iron metabolism

With rare exceptions, virtually all studied organisms from Archaea to man are dependent on iron for survival. Despite the ubiquitous distribution and abundance of iron in the biosphere, iron-dependent life must contend with the paradoxical hazards of iron deficiency and iron overload, each with its serious or fatal consequences. Homeostatic mechanisms regulating the absorption, transport, storage and mobilization of cellular iron are therefore of critical importance in iron metabolism, and a rich biology and chemistry underlie all of these mechanisms. A coherent understanding of that biology and chemistry is now rapidly emerging. In this review we will emphasize discoveries of the past decade, which have brought a revolution to the understanding of the molecular events in iron metabolism. Of central importance has been the discovery of new proteins carrying out functions previously suspected but not understood or, more interestingly, unsuspected and surprising. Parallel discoveries have delineated regulatory mechanisms controlling the expression of proteins long known--the transferrin receptor and ferritin--as well as proteins new to the scene of iron metabolism and its homeostatic control. These proteins include the iron regulatory proteins (IRPs 1 and 2), a variety of ferrireductases in yeast an mammalian cells, membrane transporters (DMT1 and ferroportin 1), a multicopper ferroxidase involved in iron export from cells (hephaestin), and regulators of mitochondrial iron balance (frataxin and MFT). Experimental models, making use of organisms from yeast through the zebrafish to rodents have asserted their power in elucidating normal iron metabolism, as well as its genetic disorders and their underlying molecular defects. Iron absorption, previously poorly understood, is now a fruitful subject for research and well on its way to detailed elucidation. The long-sought hemochromatosis gene has been found, and active research is underway to determine how its aberrant functioning results in disease that is easily controlled but lethal when untreated. A surprising connection between iron metabolism and Friedreich's ataxia has been uncovered. It is no exaggeration to say that the new understanding of iron metabolism in health and disease has been explosive, and that what is past is likely to be prologue to what is ahead.     

Iron uptake and metabolism in the new millennium

Iron is an essential element for metabolic processes intrinsic to life, and yet the properties that make iron a necessity also make it potentially deleterious. To avoid harm, iron homeostasis is achieved through iron transport, storage and regulatory proteins. The functions of some of these molecules are well described, for example transferrin and transferrin receptor-1, whereas the roles of others, such as the transferrin homolog melanotransferrin, remain unclear. The past decade has seen the identification of new molecules involved in iron metabolism, such as divalent metal transporter-1, ferroportin-1, hepcidin, hemojuvelin and heme carrier protein-1. Here, we focus on these intriguing new molecules and the insights gained from them into cellular iron uptake and the regulation of iron metabolism.     

Characterization of transferrin receptors on plasma membranes of lactating rat mammary tissue

Little is known about the transport of iron into the mammary secretory cell and the process of milk iron secretion. The concentration of iron in milk is remarkably unaffected by maternal iron status, suggesting that the uptake of iron into the mammary gland is regulated. It is known that iron enters other cells via transferrin receptor-mediated endocytosis. This study was designed to isolate and characterize the mammary gland transferrin receptor in lactating rat mammary tissue using immunochemical techniques. The existence of functional mammary gland transferrin receptors in lactating rodents was demonstrated using radiolabel-binding techniques. Isolation of mammary transferrin receptors by affinity chromatography was confirmed using immunoelectrophoresis and slot blot analysis. The intact transferrin receptor was found to have a molecular weight of 176 kd as determined by Western blotting followed by scanning densitometry. Reduction of the receptor with beta-mercaptoethanol gave a molecular weight of 98 kd. An additional immunoreactive band of 135 kd was observed. The presence of transferrin receptors in normal lactating rat mammary tissue is likely to explain iron transport into mammary tissue for both cellular metabolism and milk iron secretion.     

The ferroportin-ceruloplasmin system and the mammalian iron homeostasis machine: regulatory pathways and the role of lactoferrin

In the last 20 years, several new genes and proteins involved in iron metabolism in eukaryotes, particularly related to pathological states both in animal models and in humans have been identified, and we are now starting to unveil at the molecular level the mechanisms of iron absorption, the regulation of iron transport and the homeostatic balancing processes. In this review, we will briefly outline the general scheme of iron metabolism in humans and then focus our attention on the cellular iron export system formed by the permease ferroportin and the ferroxidase ceruloplasmin. We will finally summarize data on the role of the iron binding protein lactoferrin on the regulation of the ferroportin/ceruloplasmin couple and of other proteins involved in iron homeostasis in inflamed human macrophages.     

Hypoxic preconditioning increases iron transport rate in astrocytes

The mechanisms involved in the neuroprotection induced by hypoxic preconditioning (HP) have not been fully elucidated. The involvement of hypoxia-inducible factor-1 alpha (HIF-1alpha) in such neuroprotection has been confirmed. There is also evidence showing that a series of genes with important functions in iron metabolism, including transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1), are regulated by HIF-1alpha in response to hypoxia in extra-neural organs or cells. We therefore hypothesized that HP is able to affect the expression of iron metabolism proteins in the brain and that changes in these proteins induced by HP might be associated with the HP-induced neuroprotection. We herein demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (ferroportin1) proteins, and thus increase tansferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release in astrocytes. Moreover, HP could lead to a progressive increase in cellular iron content. We concluded that HP has the ability to increase iron transport speed in astrocytes. Based on our findings and the importance of astrocytes in neuronal survival in hypoxic/ischemic preconditioning, we proposed that the increase in iron transport rate and cellular iron in astocytes might be one of the mechanisms associated with the HP-induced neuroprotection. We also demonstrated that ferroportin1 expression was significantly affected by HIF-1alpha in astrocytes, implying that the gene encoding this iron efflux protein might be a hypoxia-inducible one.     

Hypoxic preconditioning increases iron transport rate in astrocytes

The mechanisms involved in the neuroprotection induced by hypoxic preconditioning (HP) have not been fully elucidated. The involvement of hypoxia-inducible factor-1 alpha (HIF-1alpha) in such neuroprotection has been confirmed. There is also evidence showing that a series of genes with important functions in iron metabolism, including transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1), are regulated by HIF-1alpha in response to hypoxia in extra-neural organs or cells. We therefore hypothesized that HP is able to affect the expression of iron metabolism proteins in the brain and that changes in these proteins induced by HP might be associated with the HP-induced neuroprotection. We herein demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (ferroportin1) proteins, and thus increase tansferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release in astrocytes. Moreover, HP could lead to a progressive increase in cellular iron content. We concluded that HP has the ability to increase iron transport speed in astrocytes. Based on our findings and the importance of astrocytes in neuronal survival in hypoxic/ischemic preconditioning, we proposed that the increase in iron transport rate and cellular iron in astocytes might be one of the mechanisms associated with the HP-induced neuroprotection. We also demonstrated that ferroportin1 expression was significantly affected by HIF-1alpha in astrocytes, implying that the gene encoding this iron efflux protein might be a hypoxia-inducible one.     

低氧与铁代谢相关蛋白

氧和铁这两种元素对生命活动十分重要. 低氧诱导因子(hypoxia-inducible factors, HIFs)作为转录因子,参与一系列靶基因的表达调控以适应低氧. 铁参与 DNA合成、氧气运输、代谢反应等多种细胞活动,过量游离铁会通过Haber-Weiss或 Fenton反应产生毒性自由基. 细胞通过与铁吸收、存储和利用有关的多种铁代谢相 关蛋白之间的协同作用来维持铁稳态. 与铁稳态相关的一些基因是HIFs的靶基因或 者间接受低氧调控,包括转铁蛋白、转铁蛋白受体、二价金属转运体1、铁调素、膜 铁转运蛋白、血浆铜蓝蛋白、铁蛋白等,而胞内铁浓度的改变能影响HIFs的表达. 本文就低氧与铁代谢相关蛋白的关系,尤其是低氧对铁代谢相关蛋白的调节作一综 述.     

Angiotensin II Inhibits Iron Uptake and Release in Cultured Neurons

Based on the well-confirmed roles of angiotensin II (ANGII) in iron transport of peripheral organs and cells, the causative link of excess brain iron with and the involvement of ANGII in neurodegenerative disorders, we speculated that ANGII might also have an effect on expression of iron transport proteins in the brain. In the present study, we investigated effects of ANGII on iron uptake and release using the radio-isotope methods as well as expression of cell iron transport proteins by Western blot analysis in cultured neurons. Our findings demonstrated for the first time that ANGII significantly reduced transferrin-bound iron and non-transferrin bound iron uptake and iron release as well as expression of two major iron uptake proteins transferrin receptor 1 and divalent metal transporter 1 and the key iron exporter ferroportin 1 in cultured neurons. The findings suggested that endogenous ANGII might have a physiological significance in brain iron metabolism.     

Increased IRP1 and IRP2 RNA binding activity accompanies a reduction of the labile iron pool in HFE-expressing cells.

Iron regulatory proteins (IRPs), the cytosolic proteins involved in the maintenance of cellular iron homeostasis, bind to stem loop structures found in the mRNA of key proteins involved iron uptake, storage, and metabolism and regulate the expression of these proteins in response to changes in cellular iron needs. We have shown previously that HFE-expressing fWTHFE/tTA HeLa cells have slightly increased transferrin receptor levels and dramatically reduced ferritin levels when compared to the same clonal cell line without HFE (Gross et al., 1998, J Biol Chem 273:22068-22074). While HFE does not alter transferrin receptor trafficking or non-transferrin mediated iron uptake, it does specifically reduce (55)Fe uptake from transferrin (Roy et al., 1999, J Biol Chem 274:9022-9028). In this report, we show that IRP RNA binding activity is increased by up to 5-fold in HFE-expressing cells through the activation of both IRP isoforms. Calcein measurements show a 45% decrease in the intracellular labile iron pool in HFE-expressing cells, which is in keeping with the IRP activation. These results all point to the direct effect of the interaction of HFE with transferrin receptor in lowering the intracellular labile iron pool and establishing a new set point for iron regulation within the cell.     

Iron metabolism genes in Antarctic notothenioids: A review

Iron is an essential element for metabolic processes intrinsic to life, but the properties that make iron a necessity also make it potentially deleterious. To avoid harm, iron homeostasis is achieved through iron transport, storage and regulatory proteins. The functions of some of these molecules are well described, for example transferrin and ferritin, whereas the roles of others remain unclear. The past decade has seen the identification of new molecules involved in iron metabolism, such as divalent metal transporter-1, and hepcidin. The present review aims at surveying the studies carried out on some of the most important genes involved in transport and storage of iron in Antarctic Notothenioidei, a dominating fish group endowed of a number of striking adaptive characters, including reduced (or absence of) hematocrit. This unique peculiarity among vertebrates makes this fish group a suitable system to studying the relationship between hemoglobin and iron metabolism and to understanding the adaptive changes occurred in Antarctic fish metabolism during their evolution to avoid the deleterious effects of iron overload in the absence of hemoglobin. The results summarised here indicate that the loss of hemoglobin in the most specialized group of Antarctic notothenioids, belonging to the Channychthyidae family, is accompanied by remodulation of the iron metabolism.     

Molecular mechanisms of iron homeostasis

Iron metabolism in mammals requires a complex and tightly regulated molecular network. The classical view of iron metabolism has been challenged over the past ten years by the discovery of several new proteins, mostly Fe (II) iron transporters, enzymes with ferro-oxydase (hephaestin or ceruloplasmin) or ferri-reductase (Dcytb) activity or regulatory proteins like HFE and hepcidin. Furthermore, a new transferrin receptor has been identified, mostly expressed in the liver, and the ability of the megalin-cubilin complex to internalise the urinary Fe (III)-transferrin complex in renal tubular cells has been highlighted. Intestinal iron absorption by mature duodenal enterocytes requires Fe (III) iron reduction by Dcytb and Fe (II) iron transport through apical membranes by the iron transporter Nramp2/DMT1. This is followed by iron transfer to the baso-lateral side, export by ferroportin and oxidation into Fe (III) by hephaestin prior to binding to plasma transferrin. Macrophages play also an important role in iron delivery to plasma transferrin through phagocytosis of senescent red blood cell, heme catabolism and recycling of iron. Iron egress from macrophages is probably also mediated by ferroportin and patients with heterozygous ferroportin mutations develop progressive iron overload in liver macrophages. Iron homeostasis at the level of the organism is based on a tight control of intestinal iron absorption and efficient recycling of iron by macrophages. Signalling between iron stores in the liver and both duodenal enterocytes and macrophages is mediated by hepcidin, a circulating peptide synthesized by the liver and secreted into the plasma. Hepcidin expression is stimulated in response to iron overload or inflammation, and down regulated by anemia and hypoxia. Hepcidin deficiency leads to iron overload and hepcidin overexpression to anemia. Hepcidin synthesis in response to iron overload seems to be controlled by the HFE molecule. Patients with hereditary hemochromatosis due to HFE mutation have impaired hepcidin synthesis and forced expression of an hepcidin transgene in HFE deficient mice prevents iron overload. These results open new therapeutic perspectives, especially with the possibility to use hepcidin or antagonists for the treatment of iron overload disorders.     

Lactoferrin: Role in iron homeostasis and host defense against microbial infection

The transferrin family of non-heme iron binding glycoproteins are believed to play a central role in iron metabolism and have been implicated in iron transport, cellular iron delivery and control of the level of free iron in external secretions. Lactoferrin (LF) is a member of this family that is widely localized in external fluids including milk and mucosal secretions, in addition to being a prominent component of the secondary granules of neutrophils. Although structurally related to transferrin, LF appears to have a broader functional role mediated by both iron dependent and iron independent mechanisms. In this review, we will focus on our current understanding on the role of LF in regulating iron homeostasis and its role in host protection against microbial infection at the mucosal surface. In addition, recent insights obtained from analyzing the phenotypic consequences of LF ablation in lactoferrin knockout mice (LFKO), which challenge the long held dogma that LF is required for intestinal iron absorption in the neonate, are summarized.     

乳铁蛋白对铁代谢的影响及其在神经退行性疾病中的应用

乳铁蛋白是一种具有多种生理功能的铁结合性糖蛋白,是铁在机体内代谢及转运关键载体。目前,有关乳铁蛋白对神经退行性疾病的防治作用及应用研究已成为该领域的新热点。本文主要介绍了铁在机体内的代谢;铁转运蛋白-乳铁蛋白转运系统：铁转运主要是由转铁蛋白受体和乳铁蛋白受体介导的,铁转运入脑的途径主要是转铁蛋白-转铁蛋白受体途径,还有乳铁蛋白-乳铁蛋白受体途径及其他途径;铁对脑损伤的作用机制,其中铁参与的氧化应激反应以及铁代谢和铁转运相关基因的突变或缺失可能都是引起脑损伤的原因;最后简述乳铁蛋白在防治神经退行性疾病中最新的进展,乳铁蛋白修饰的纳米粒子可能是目前最有效治疗神经退行性疾病的方法之一。     

A structural comparison of human serum transferrin and human lactoferrin

The transferrins are a family of proteins that bind free iron in the blood and bodily fluids. Serum transferrins function to deliver iron to cells via a receptor-mediated endocytotic process as well as to remove toxic free iron from the blood and to provide an anti-bacterial, low-iron environment. Lactoferrins (found in bodily secretions such as milk) are only known to have an anti-bacterial function, via their ability to tightly bind free iron even at low pH, and have no known transport function. Though these proteins keep the level of free iron low, pathogenic bacteria are able to thrive by obtaining iron from their host via expression of outer membrane proteins that can bind to and remove iron from host proteins, including both serum transferrin and lactoferrin. Furthermore, even though human serum transferrin and lactoferrin are quite similar in sequence and structure, and coordinate iron in the same manner, they differ in their affinities for iron as well as their receptor binding properties: the human transferrin receptor only binds serum transferrin, and two distinct bacterial transport systems are used to capture iron from serum transferrin and lactoferrin. Comparison of the recently solved crystal structure of iron-free human serum transferrin to that of human lactoferrin provides insight into these differences.     

Iron gathering by zoopathogenic fungi

Iron is a metal required by most microorganisms and is prominently used in the transfer of electrons during metabolism. The gathering of iron is, then, an essential process and its fulfillment becomes a crucial pathogenetic event for zoopathogenic fungi. Iron is rather unavailable because it occurs on the earth's surface in its insoluble ferric form in oxides and hydroxides. In the infected host iron is bound to proteins such as transferrin and ferritin. Solubilization of ferric iron is the major problem confronting microorganisms. This process is achieved by two major mechanisms: ferric reduction and siderophore utilization. Ferric reductase is frequently accompanied by a copper oxidase transport system. There is one example of direct ferric iron transport apparently without prior reduction. Ferric reduction may also be accomplished by low molecular mass compounds. Some fungi have evolved a process of iron acquisition involving the synthesis of iron-gathering compounds called siderophores. Even those fungi that do not synthesize siderophores have developed permeases for transport of such compounds formed by other organisms. Fungi can also reductively release iron from siderophores and transport the ferrous iron often by the copper oxidase transport system. There is a great diversity of iron-gathering mechanisms expressed by pathogenic fungi and such diversity may be found even in a single species.     

Brain iron homeostasis.

The anatomical and cellular distribution of non-haem iron, ferritin, transferrin, and the transferrin receptor have been studied in postmortem human brain and these studies, together with data on the uptake and transport of labeled iron, by the rat brain, have been used to elucidate the role of iron and other metal ions in certain neurological disorders. High levels of non-haem iron, mainly in the form of ferritin, are found in the extrapyramidal system, associated predominantly with glial cells. In contrast to non-haem iron, the density of transferrin receptors is highest in cortical and brainstem structures and appears to relate to the iron requirement of neurones for mitochondrial respiratory activity. Transferrin is synthesized within the brain by oligodendrocytes and the choroid plexus, and is present in neurones, consistent with receptor mediated uptake. The uptake of iron into the brain appears to be by a two-stage process involving initial deposition of iron in the brain capillary endothelium by serum transferrin, and subsequent transfer of iron to brain-derived transferrin and transport within the brain to sites with a high transferrin receptor density. A second, as yet unidentified mechanism, may be involved in the transfer of iron from neurones possessing transferrin receptors to sites of storage in glial cells in the extrapyramidal system. The distribution of iron and the transferrin receptor may be of relevance to iron-induced free radical formation and selective neuronal vulnerability in neurodegenerative disorders.     

Transferrin receptor 2 is emerging as a major player in the control of iron metabolism

Our knowledge of mammalian iron metabolism has advanced dramatically over recent years. Iron is an essential element for virtually all living organisms. Its intestinal absorption and accurate cellular regulation is strictly required to ensure the coordinated synthesis of the numerous iron-containing proteins involved in key metabolic processes, while avoiding the uptake of excess iron that can lead to organ damage. A range of different proteins exist to ensure this fine control within the various tissues of the body. Among these proteins, transferrin receptor (TFR2) seems to play a key role in the regulation of iron homeostasis. Disabling mutations in TFR2 are responsible for type 3 hereditary hemochromatosis (Type 3 HH). This review describes the biological properties of this membrane receptor, with a particular emphasis paid to the structure, function and cellular localization. Although much information has been garnered on TFR2, further efforts are needed to elucidate its function in the context of the iron regulatory network.     

Iron metabolism in insect disease vectors: mining the Anopheles gambiae translated protein database

All animals require iron for survival. This requirement reflects the role of this mineral as a cofactor of numerous proteins. However, under physiological conditions, Fe(2+) oxidizes to Fe(3+) encouraging the formation of toxic free radicals. In mammals, the potential for oxidative damage from iron is minimized by binding iron to proteins. Mammalian iron metabolism is complex and numerous proteins are involved in iron absorption, transport, uptake and utilization. We have analyzed the Anopheles gambiae translated protein database for candidates that show identity to proteins involved in mammalian iron metabolism (Holt et al., 2002. The genome sequence of the malaria mosquito Anopheles gambiae. Science 298, 129-149). Our results indicate that proteins involved in iron absorption and intracellular iron utilization are, for the most part, conserved in A. gambiae. In contrast, proteins involved in the pathways of iron export from the gut, transport in hemolymph and uptake at peripheral tissues in mosquitos differ from those for mammals.     

Immunolocalization of transferrin and transferrin receptor in mouse small intestinal absorptive cells

The mechanisms by which the duodenal mucosa absorbs iron are unknown. Insorption into absorptive cells of luminal iron bound to transferrin via receptor-mediated endocytosis has been hypothesized, but transferrin and transferrin receptor are absent in apical microvillous brush borders of small bowel biopsies taken from fasted patients and normal volunteers. We hypothesized that a normal iron-containing diet might induce the transient appearance of transferrin and transferrin receptor in apical brush borders of small intestinal absorptive cells in a normal mouse that was provided iron-containing chow until the moment of sacrifice. Light and electron microscopic immunolocalization of transferrin and transferrin receptor in proximal small intestinal absorptive cells was limited to basolateral membranes and coated pits of cells predominantly in the crypts and basal regions of the villi. Transferrin and transferrin receptor were not detected in apical microvillous brush border membranes of these enterocytes. In parallel immunolocalization protocols designed to show the ability to immunodetect other antigens at these locations, maltase and proteoglycan were demonstrated in apical microvillous brush border membranes and in basolateral membranes, respectively, in absorptive cells of small intestinal villous tip, base, and crypt regions. Furthermore, transferrin and transferrin receptor were immunolocalized in hepatocyte sinusoidal microvillus membranes. We conclude that food does not induce the appearance of immunodetectable transferrin and transferrin receptor in the apical microvilli of small intestinal absorptive cells and, therefore, that these iron transport proteins are not involved in the apical microvillous membrane transport of luminal dietary iron.