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Molecular control of iron metabolism
Best Practice & Research Clinical Haematology Vol. 18, No. 2, pp. 159–169, 2005 doi:10.1016/j.beha.2004.10.004 available online at http://www.sciencedirect.com
1 Molecular control of iron metabolism Nancy C. Andrews*
MD, PhD
Professor of Pediatrics; Dean for Basic Sciences and Graduate Studies, Investigator Children’s Hospital Boston, Karp Family Research Laboratories 8-125, Howard Hughes Medical Institute, Harvard Medical School and Dana-Farber Cancer Institute, 300 Longwood Ave, Boston, MA 02115-5737, USA
As described in this volume, our knowledge of mammalian iron metabolism has advanced dramatically over the past decade. While basic physiological concepts were described through elegant experiments in the 1950s and 1960s, many molecular details remained unknown. Modern techniques in genetics, biochemistry and molecular biology have allowed for the identification and characterization of most, but probably not all, of the key molecules that move iron into, out of, and between cells. Insight into the complex regulation of these iron transport proteins has been accelerated through the identification of hemochromatosis disease genes. Although much of the information has been gleaned from experiments in animal models, it is likely that it can be directly extrapolated to humans. Key words: iron; homeostasis; anemia; hemochromatosis; metal transport; transferrin; ferritin.
Iron is required by all mammalian cells. Its major roles include oxygen delivery (as a heme cofactor in hemoglobin and myoglobin) and enzymatic transfer of electrons (in cytochromes, ribonucleotide reductase, and enzymes that deal with oxygen radicals). However, excess free iron promotes the formation of damaging oxygen radicals that attack cellular lipids, proteins and nucleic acids. Normal iron homeostasis allows cells to benefit from the usefulness of iron while avoiding its deleterious effects. At a systemic level, iron balance averts both iron deficiency and iron overload. Systemic iron homeostasis is essentially a closed system. There appears to be no regulated mechanism for iron excretion through the liver or kidneys; iron losses occur primarily through bleeding and sloughing of mucosal and skin cells. To maintain iron balance, intestinal iron uptake is meticulously controlled; only a fraction of the iron in a normal diet is absorbed. Compared to the total iron endowment, the amounts of iron entering and leaving the body are insignificant when measured over hours to days, though important in the long term. Iron deficiency is most often a consequence of
* Tel.: C1 617 919 2116; Fax: C1 617 730 0934. E-mail address: [email protected] 1521-6926/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
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increased losses (e.g. gastrointestinal blood loss), excessive requirements (e.g. during rapid growth in childhood) or inadequate dietary supply. In contrast, iron overload usually results from a genetic error that interferes with normal regulation and leads to chronically increased dietary absorption. Iron overload may also result from excessive iron therapy, most commonly through hypertransfusion.
MECHANISMS OF IRON TRANSPORT Iron cannot passively enter cells because it cannot negotiate the lipid bilayer without the aid of specific carrier proteins. All eukaryotic cells probably require mechanisms to take up (import) iron. Some specialized cells—particularly intestinal epithelial cells, hepatocytes and macrophages—also require mechanisms to release (export) it. In addition, the toxicity of free iron requires specialized proteins to bind iron in extracellular fluids and in intracellular storage sites. More than half of the body’s iron in normal individuals is found in hemoglobin in erythrocytes and their precursors. About 20–25 mg iron is required on a daily basis to support the hemoglobinization of new erythrocytes. Very little of this is supplied by the 0.5–2.0 mg acquired through intestinal absorption. Most of it must come from recycling of iron already in the body. The primary source is a specialized population of tissue macrophages, which phagocytose damaged erythrocytes to scavenge their iron from hemoglobin and return it to the circulation. The amount of iron passing through this macrophage recycling system every day approximates the amount needed for erythropoiesis. Additional iron may be obtained through mobilization of cellular iron stores, particularly from hepatocytes. Iron in the circulation Iron circulates bound to transferrin (TF), a very abundant plasma protein that binds two iron atoms with extremely high affinity. TF keeps iron soluble, preventing the Fe3C ion from precipitating out of aqueous plasma. It also prevents iron from reacting with other molecules by attenuating its redox activity. It aids in delivery of iron to cells by binding to a specific cell-surface transferrin receptor (TFR1). Each TFR1 homodimer binds two molecules of TF. TFR1 is present in low abundance on most if not all mammalian cell types, but it is expressed at high levels by developing erythroid precursors, activated lymphocytes, placental syncytiotrophoblasts and tumor cells. Each of these cell types has an unusually large need for iron for hemoglobin production (in the case of erythroid precursors), iron transfer (placental syncytiotrophoblasts) or cell proliferation. Iron uptake through the TF cycle In many tissues, cellular iron uptake occurs through a process known as the ‘TF cycle’ (reviewed in Ref. [1]). When iron-loaded TF attaches to TFR1, the ligand-receptor interaction triggers clathrin-mediated invagination of the cell membrane and formation of intracellular TF-TFR1-containing endosomes. The pH inside the endosomes is rapidly lowered through the action of proton pumps, resulting in conformational changes of both the TF and TFR1 proteins. Iron is released from the complex and reduced to its Fe2C form. Fe2C is transported across the endosomal membrane into the cytoplasm by divalent metal transporter 1 (DMT1, previously known as Nramp2,
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DCT1), a proton-dependent iron carrier with 12 membrane-spanning segments.2 In the cytoplasm, iron is either incorporated into protoporphyrin to produce heme or retained in storage forms (discussed below). Meanwhile, the endosomes containing TF and TFR1 return the proteins to the cell surface for re-use. The TF cycle is clearly very important for erythropoiesis and lymphopoiesis. Knockout mice lacking TFR1 die during mid-gestation from severe anemia.3 Chimeric mice containing a mixture of wild-type and TFR-null cells generate their entire erythroid and lymphoid cell populations from wild-type cells, although other tissues have mixed contributions from both cell sources.4 Mice and human patients deficient in TF also have severe anemia5,6 and lymphoid abnormalities.7 The TF cycle also appears to be important early in the development of the central nervous system, although its involvement is not well understood.3,8 As discussed later, it is clear that some cells can take iron up directly, without the TF cycle. Why has this complex mechanism evolved? There are at least two likely answers. First, the avidity with which TF binds iron is advantageous while the metal is circulating, but problematic forcellular uptake. The TF cycle allows for pH-dependent release of iron from TF in a controlled intracellular compartment. Second, receptor-mediated uptake of iron bound to TF probably allows for much higher local iron concentrations within endosomes containing transmembrane iron transporters than could be achieved by passive interactions between circulating ions and the cell surface. This interpretation is consistent with the fact that the TF cycle functions primarily in cells with excessive iron needs. Intestinal iron absorption The absorptive epithelial cells of the intestine (enterocytes) are responsible for bringing new iron into the body. They are organized in finger-like villous structures to increase the absorptive surface area. Undifferentiated cells at the base of the villi divide to give rise to several cell types, including enterocytes. Over the course of several days the cells differentiate and migrate up the villous. They carry out their absorptive function during this migration, but when they reach the top, they senesce and slough into the intestinal lumen. The apical (luminal) membrane of the enterocyte is organized into a microvillus brush border. The first step in iron absorption is transfer across this surface into the cell. We now know that most of the transfer of non-heme iron is carried out by DMT1, the same protein that performs transmembrane iron transport as part of the TF cycle in erythroid cells.9,10 DMT1 is capable of transporting avariety of divalent metal cations, but iron is probably the most important. As most dietary non-heme iron is in the ferric (Fe3C) form, it must first be reduced to ferrous (Fe2C) iron. A cytochrome b-like brush border ferrireductase, DCYTB, is probably involved in enzymatic reduction of iron11, though there is no direct evidence that DCYTB and DMT1 form a physical complex. The amounts of both DMT1 and DCYTB in enterocytes increase dramatically in iron deficiency.10,11 Once inside the enterocyte, iron has two possible fates. Some remains stored within the cell; that iron is ultimately lost from the body at the end of the enterocyte lifespan. The remainder is transferred across the basolateral surface through a distinct transport mechanism. The major molecule that carries iron across the membrane is ferroportin (also known as IREG1, MTP1).12–14 It is similar to DMT1 in several respects: it also is a multi-transmembrane segment protein, and it appears to prefer ferrous iron. However, there is no sequence homology between these two iron transporters. A multicopper ferroxidase, hephaestin, acts in some way to facilitate basolateral iron transport15,
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although its mechanism of action is not yet known. It may serve to oxidize iron to allow binding to the plasma iron carrier, TF. Macrophage iron recycling Relatively little is known about macrophage iron recycling, in spite of its great physiological importance. Tissue macrophages recognize old and damaged erythrocytes16 and phagocytose them to remove them from circulation. Within the macrophages the erythrocytes are lysed and hemoglobin is degraded. Heme oxygenase catalyzes the liberation of iron from heme. Although, the handling of iron within the cell is poorly understood, it appears likely that macrophages release iron through the action of ferroportin17 with the aid of the ferroxidase ceruloplasmin.18 The amount of iron that is recycled from effete red cells and returned to plasma TF approximates the amount needed for the synthesis of new erythrocytes. Placental iron transfer During pregnancy, the mother transfers iron to the developing fetus to establish generous newborn iron stores. However, in spite of its importance, very little is known about placental iron transfer. It does not appear to depend on DMT1 (unpublished observation) but it does involve TFR1, ferroportin, hephaestin and hepcidin.15,19,20 Hepatocyte iron transport Hepatocytes serve as the major storage depot for iron in excess of tissue needs. The details of hepatocyte iron uptake have not been worked out, but several mechanisms are likely to be involved. Hepatocytes express both TFR1 and a homolog, transferrin receptor-2 (TFR2)21, but neither appears to be essential for iron uptake. Expression of TFR1 is not necessary for the formation of normal liver.4 While the amount of TFR2 is much greater than the amount of TFR1, mutations in TFR2 result in hepatocyte iron overload, not iron deficiency.22,23 Finally, animals and patients lacking TF develop marked liver iron overload.24,25 There is abundant evidence for at least one non-TF iron uptake pathway in the liver.26,27 While not yet understood on a molecular level, it is likely that this pathway is important in hepatic iron accumulation in hemochromatosis, when serum iron levels exceed the binding capacity of TF. Recently, L-type calcium channels were shown to be involved in cardiomyocyte uptake of non-TF-bound iron;28 it is possible that they (or similar molecules) also mediate hepatic uptake. Hepatocytes must also export iron to mobilize their stores for use as needed. As ferroportin is the only described cellular iron exporter, and it is expressed by hepatocytes, it seems likely that ferroportin is involved. It probably functions with ferroxidase assistance from ceruloplasmin, similarly to iron export from macrophages. However, this has not yet been proven experimentally.
SITES OF IRON STORAGE In general, two cell types are considered important for systemic storage of iron that is not needed for immediate use. As discussed above, macrophages recover iron from
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effete erythrocytes; they also internalize iron administered by injection of the medicinal forms iron dextran, iron sucrose and ferrous gluconate. Their large capacity for storage is obvious when tissues from patients with transfusional iron overload are stained for iron: large amounts of iron accumulate in macrophages before deposition in parenchymal cells is apparent. Hepatocytes acquire both TF-bound and non-TF-bound iron from the plasma. They, too, have a large capacity for iron storage. Patients with hereditary hemochromatosis may have tenfold more hepatic iron than normal individuals without any evidence of hepatocellular damage. Although enterocytes can store iron taken up from the diet (and probably also from the plasma), they are not considered part of the iron storage system. The iron that they retain leaves the body when the cells senesce and are sloughed into the gut lumen. At a molecular level, iron storage occurs primarily in ferritin (reviewed in Ref. [29]). Light (L) and heavy (H) ferritin polypeptides are similar proteins of approximately 20 kDa. They assemble in varying proportions into 24-subunit heteromultimers that sequester mineralized iron in a central core. Each ferritin molecule can accommodate up to 4500 iron atoms. Iron entry is facilitated by an oxidase activity unique to the H-ferritin polypeptide. Iron release occurs when intracellular pools become depleted. Ferritin can frequently be found within lysosomes; degradation of ferritin leads to the formation of hemosiderin, a non-homogeneous conglomerate of iron, protein and membrane breakdown products. Hemosiderin iron is mobilized poorly, if at all. Both ferritin and hemosiderin react with the Perls Prussian blue iron stain, allowing detection in tissue sections.30
REGULATION OF IRON HOMEOSTASIS Because iron is important for many biological processes, yet is highly reactive, its availability must be controlled not only at a cellular level but also systemically. At least four situations lead to measurable changes in intestinal iron absorption and tissue iron distribution: abnormal iron availability (iron overload or deficiency), accelerated erythropoiesis, hypoxia, and inflammation. Iron absorption and plasma availability must be decreased in the settings of iron overload and inflammation, and increased in response to iron deficiency, accelerated erythropoiesis and hypoxia. The responses to these stimuli are coordinated. In general, situations that require decreased iron availability are associated with interruption of intestinal absorption and retention of iron by recycling macrophages. In contrast, situations requiring increased iron availability are associated with increased intestinal absorption and enhanced macrophage iron release. A circulating peptide was recently discovered which probably explains how iron release from absorptive enterocytes and macrophages is regulated in concert. Hepcidin (also called HAMP) is a 25-amino-acid molecule that is structurally similar to the defensin antimicrobial peptides.31–33 It is primarily produced by the liver, secreted into plasma, and cleared by the kidneys. Increased levels of hepcidin are produced in the setting of non-genetic iron overload34,35 and inflammation.36–38 Decreased hepcidin expression is seen in iron deficiency, accelerated erythropoiesis and hypoxia.36,37 Conversely, forced constitutive expression of hepcidin in transgenic mice leads to iron deficiency and anemia20, whereas genetic loss of hepcidin results in hemochromatosis.32,39 Taken together, these findings indicate that hepcidin is a negative regulator of
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intestinal iron absorption. Studies of cellular iron distribution in mutant animals suggest that it is also a negative regulator of macrophage iron release. The regulation of hepcidin expression has not yet been worked out in detail. An inflammatory cytokine, interleukin-6, stimulates hepcidin production in vivo and in vitro,38,40 presumably by binding to its cell-surface receptor and initiating a signaling cascade. This accounts for induction of hepcidin in inflammation, but it is not yet known how the cells producing hepcidin register cues about iron status, erythropoiesis and hypoxia. The iron occupancy of circulating TF (TF saturation) may provide hepatocytes with information about iron stores and erythropoietic rate. The mechanism of action of hepcidin has not yet been reported. However, it probably involves inhibition of cellular iron export, either directly through interaction with ferroportin or indirectly through a signaling cascade that regulates ferroportin or an associated protein. It will be important to understand this pathway in some detail, because pharmacological manipulation may be useful in iron disorders, including hereditary hemochromatosis, the anemia of chronic disease, and perhaps even iron deficiency.
GENES MUTATED IN HUMAN IRON DISORDERS Iron is the predominant metal in the earth’s crust, making it abundant in our environment. Most genetic disorders of iron homeostasis result in iron overload (hemochromatosis) rather than iron deficiency, suggesting that human evolution has been biased to keep iron out of the body rather than to facilitate its uptake. However, this does not appear to be the case for all mammalian species. For example, the black rhinoceros is such an avid iron absorber that it develops a hemochromatosis-like illness from eating the plant-based diet provided in captivity.41 The difference in our regulation of absorption may be attributable to the fact that we are carnivorous. HFE hemochromatosis Mutations in four genes (described below) are known to result in similar patterns of pathological iron overload, involving the parenchymal cells of the liver, heart and endocrine tissues and inherited in an autosomal recessive pattern. The most common form of hemochromatosis is genetically linked to the HLA complex on human chromosome 6p. It results from homozygous mutations in an HLA class I-like gene, HFE.42 This disorder has been modeled in mice through mutations that inactivate the murine Hfe gene or introduce a common disease-causing missense mutation (C282Y) into it.43–45 The molecular function of HFE has remained obscure, but there are several important observations that provide clues. HFE forms a tight protein-protein complex with TFR.46 The binding sites for HFE and TF overlap, preventing both proteins from binding to the same TFR molecule simultaneously.47,48 There is controversy in the literature about the consequences of HFE/TFR complex formation; some investigators have reported that it leads to decreased iron uptake,49,50 whereas others have found that it leads to increased iron uptake.51 Because those studies were carried out in transfected cells, rather than in a native system, it is possible that both sets of findings are correct, but not necessarily informative about the normal situation. Using a genetic approach, we and others have implicated HFE in the regulation of hepcidin expression. Patients and mice homozygous for HFE mutations express
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hepcidin at levels that are inappropriately low for their iron overload state.35,52–54 We have reported that mice lacking HFE do not mount an appropriate hepcidin response to inflammation55, though others disagree with our conclusions.56,57 TFR2 hemochromatosis A clinically indistinguishable disorder is found in patients who have normal HFE genes but are homozygous for mutations in the gene encoding TFR2. TFR2 knockout mice show similar findings.23 TFR2 is expressed at enormous levels in hepatocytes, but is not found in most other cell types. The major function of TFR2 has not yet been established, but it can mediate endocytosis of diferric TF, albeit with lower efficiency than TFR1.58 It does not form a complex with HFE59 and its role in regulating iron homeostasis is unknown. However, it was recently shown that increased saturation of TF with iron leads to increased hepatocellular TFR2 protein, suggesting that it may, in some way, help to ‘interpret’ iron stores signals.60,61 To date, no link between TFR2 and hepcidin has been reported in the literature, but it seems likely that they participate in the same overall regulatory pathway. Juvenile hemochromatosis More severe, early-onset iron overload is seen in patients with ‘juvenile’ hemochromatosis due to mutations either in the gene encoding hepcidin39 or in a novel gene, hemojuvelin (HJV).62 In both instances, homozygosity for deleterious mutations leads to very similar iron-overload phenotypes: cardiomyopathy, endocrinopathies and liver dysfunction. The striking similarity between these disorders suggests that hepcidin and HJV are components of the same regulatory system. This is further supported by the fact that, similar to patients with HFE hemochromatosis, patients with HJV mutations have decreased urinary hepcidin levels.62 The likely function of hepcidin has already been discussed, but the function of HJV remains unknown. At the time of its discovery last year, there was only one known protein that had a similar structure: repulsive guidance molecule (RGM) from chickens.63 It is now clear that mammals have three RGM-like molecules; HJV is also known as RGM-C.64 Hemojuvelin has several sequence motifs in common with other proteins, including a signal peptide, an arginine-glycine-aspartic acid (RGD) motif, a von Willebrand factor type D domain, and a putative GPI anchor site. HJV is expressed at high levels in muscle and heart, and at lower levels in the liver. However, none of this information gives real insight into how hemojuvelin participates in the regulation of iron homeostasis. Much more is likely to be learned over the coming years. While each of these four hemochromatosis disease genes was initially discovered through the study of patients homozygous for mutations in a single gene, it is now clear that some patients with severe hemochromatosis carry mutations in more than one gene. A small subset of individuals with HFE hemochromatosis also carries one mutated hepcidin (HAMP) gene,65,66 apparently resulting in a more rapid onset of iron overload. As homozygous HFE mutations are relatively common in individuals of Northern European descent, it is likely that patients with HFE mutations accompanied by mutations in the TFR2 or HJV genes will be identified in the future.
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Ferroportin-associated iron overload Mutations in the ferroportin gene cause an iron overload disorder with a distinct pathology. Although sometimes called ‘hemochromatosis’, the ferroportin-associated disease shows autosomal dominant inheritance and macrophage-predominant iron loading.67,68 Many patients have biochemical evidence of aberrant iron homeostasis (increased serum ferritin) in the absence of clinical signs or symptoms. Only a fraction appear to develop classical iron-related toxicity in the liver and endocrine tissues. It seems counterintuitive that mutations in the iron exporter, ferroportin, should cause iron overload, particularly because the mutations probably interfere with, rather than enhance, ferroportin function. The explanation for this paradox probably lies in the fact that ferroportin is important not only for intestinal iron absorption but also for macrophage iron recycling. At present, the best guess is that partial loss of ferroportin function in macrophages leads to iron-restricted erythropoiesis. This, in turn, stimulates increased intestinal iron absorption in spite of a ferroportin deficit. Consistent with this hypothesis, several patients heterozygous for ferroportin mutations were reported to be anemic early in their course.69 However, the hypothesis is not yet proven, and it is quite possible that the details of the pathophysiology will be more complex.
FUTURE DIRECTIONS Although enormous progress has been made in understanding mammalian iron metabolism, many unanswered questions remain. I will mention a few of the more pressing questions here. Quantitatively, macrophage recycling of hemoglobin iron makes the most important contribution to the iron economy, yet very little is understood of the intracellular processes involved. We need better knowledge of the subcellular compartments that iron enters and leaves within the macrophage. We need to understand the controls that determine whether liberated iron will remain in storage or be exported by ferroportin. It is likely that the export apparatus involves other as yet unknown components in addition to ferroportin; some of these might distinguish macrophage iron export from enterocyte iron export, and might be differentially affected by mutations in patients with autosomal dominant, ferroportin-associated iron overload. It will be important to determine whether there are other physiologically significant transmembrane iron carriers in addition to DMT1 and ferroportin. Does non-TFbound iron enter hepatocytes through the action of L-type calcium channels, or through some other mechanism? Is ferroportin the only molecule that can mediate export of iron? Finally, much remains to be learned about the proteins identified as hemochromatosis disease genes. How does HFE normally function, and what is the significance of its association with TFR? What purpose does TFR2 serve? How can HJV, a protein that is highly expressed in muscle and heart, be directly involved in the regulation of iron homeostasis? Over the next decade, many of these questions will be answered. New proteins important for iron metabolism are likely to be discovered. Ultimately, it should be possible to develop a robust, comprehensive and quantitative model of systemic iron homeostasis. With such a model, it should be possible to predict how perturbation of
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one part of the system—e.g. by genetic mutations or treatment with a drug—affects the entire organism. When that is accomplished, we will truly understand hemochromatosis and other iron disorders.
REFERENCES *1. Hentze MW, Muckenthaler MU & Andrews NC. Balancing acts; molecular control of mammalian iron metabolism. Cell 2004; 117: 285–297. 2. Fleming MD, Romano MA, Su MA et al. Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport. Proc Natl Acad Sci USA 1998; 95: 1148–1153. 3. Levy JE, Jin O, Fujiwara Y et al. Transferrin receptor is necessary for development of erythrocytes and the nervous system. Nat Genet 1999; 21: 396–399. 4. Ned RM, Swat W & Andrews NC. Transferrin receptor 1 is differentially required in lymphocyte development. Blood 2003; 102: 3711–3718. 5. Beutler E, Gelbart T, Lee P et al. Molecular characterization of a case of atransferrinemia. Blood 2000; 96: 4071–4074. 6. Bernstein SE. Hereditary hypotransferrinemia with hemosiderosis, a murine disorder resembling human atransferrinemia. J Lab Clin Med 1987; 110: 690–705. 7. Macedo MF, de Sousa M, Ned RM et al. Transferrin is required for early T-cell differentiation. Immunology 2004; 112: 543–549. 8. Dickinson T & Connor JR. Histological analysis of selected brain regions of hypotransferrinemic mice. Brain Res 1994; 635: 169–178. *9. Fleming MD, Trenor CC, Su MA et al. Microcytic anemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet 1997; 16: 383–386. *10. Gunshin H, Mackenzie B, Berger UV et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 1997; 388: 482–488. 11. McKie AT, Barrow D, Latunde-Dada GO et al. An iron-regulated ferric reductase associated with the absorption of dietary iron. Science 2001; 291: 1755–1759. 12. Abboud S & Haile DJ. A novel mammalian iron-regulated protein involved in intracellular iron metabolism. J Biol Chem 2000; 275: 19906–19912. *13. Donovan A, Brownlie A, Zhou Y et al. Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature 2000; 403: 776–781. *14. McKie AT, Marciani P, Rolfs A et al. A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol Cell 2000; 5: 299–309. 15. Vulpe CD, Kuo YM, Murphy TL et al. Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse. Nat Genet 1999; 21: 195–199. 16. Bratosin D, Mazurier J, Tissier JP et al. Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. A review. Biochimie 1998; 80: 173–195. 17. Knutson MD, Vafa MR, Haile DJ & Wessling-Resnick M. Iron loading and erythrophagocytosis increase ferroportin 1 (FPN1) expression in J774 macrophages. Blood 2003; 102: 4191–4197. 18. Harris ZL, Durley AP, Man TK & Gitlin JD. Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux. Proc Natl Acad Sci USA 1999; 96: 10812–10817. 19. Bradley J, Leibold EA, Harris ZL et al. The influence of gestational age and fetal iron status on iron regulatory protein activity and iron transporter protein expression in third trimester human placenta. Am J Physiol Regul Integr Comp Physiol 2004. 20. Nicolas G, Bennoun M, Porteu A et al. Severe iron deficiency anemia in transgenic mice expressing liver hepcidin. Proc Natl Acad Sci USA 2002; 99: 4596–4601. 21. Kawabata H, Yang R, Hirama T et al. Molecular cloning of transferrin receptor 2. A new member of the transferrin receptor-like family. J Biol Chem 1999; 274: 20826–20832. 22. Camaschella C, Roetto A, Cali A et al. The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22. Nat Genet 2000; 25: 14–15.
168 N. C. Andrews 23. Fleming RE, Ahmann JR, Migas MC et al. Targeted mutagenesis of the murine transferrin receptor-2 gene produces hemochromatosis. Proc Natl Acad Sci USA 2002; 99: 10653–10658. 24. Craven CM, Alexander J, Eldridge M et al. Tissue distribution and clearance kinetics of non-transferrinbound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis. Proc Natl Acad Sci USA 1987; 84: 3457–3461. 25. Hamill RL, Woods JC & Cook BA. Congenital atransferrinemia: a case report and review of the literature. Am J Clin Pathol 1991; 96: 215–218. 26. Wright TL, Brissot P, Ma WL & Weisiger RA. Characterization of non-transferrin-bound iron clearance by rat liver. J Biol Chem 1986; 261: 10909–10914. 27. Brissot P, Wright TL, Ma WL & Weisiger RA. Efficient clearance of non-transferrin-bound iron by rat liver. Implications for hepatic iron loading in iron overload states. J Clin Invest 1985; 76: 1463–1470. 28. Oudit GY, Sun H, Trivieri MG et al. L-type Ca(2C) channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy. Nat Med 2003; 9: 1187–1194. 29. Theil EC. Ferritin: at the crossroads of iron and oxygen metabolism. J Nutr 2003; 133: 1549S–1553S. 30. Klavins JV, Pickett JP & Wessely Z. Staining of minerals and solubility of iron in tissues. Ann Clin Lab Sci 1976; 6: 214–222. 31. Krause A, Neitz S, Magert HJ et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett 2000; 480: 147–150. *32. Nicolas G, Bennoun M, Devaux I et al. Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice. Proc Natl Acad Sci USA 2001; 98: 8780–8785. 33. Park CH, Valore EV, Waring AJ & Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 2001; 276: 7806–7810. 34. Pigeon C, Ilyin G, Courselaud B et al. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 2001; 276: 7811–7819. *35. Muckenthaler M, Roy CN, Custodio AO et al. Regulatory defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis. Nat Genet 2003; 34: 102–107. 36. Weinstein DA, Roy CN, Fleming MD et al. Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease. Blood 2002; 100: 3776–3781. *37. Nicolas G, Chauvet C, Viatte L et al. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest 2002; 110: 1037–1044. 38. Nemeth E, Valore EV, Territo M et al. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood 2003; 101: 2461–2463. *39. Roetto A, Papanikolaou G, Politou M et al. Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis. Nat Genet 2003; 33: 21–22. 40. Nemeth E, Rivera S, Gabayan Vet al. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Invest 2004; 113: 1271–1276. 41. D.E. Paglia, P. Dennis. Role of chronic iron overload in multiple disorders of captive black rhinoceroses (Diceros bicornis). In: Proceedings of the American Association of Zoo Veterinarians; 1999; Columbus, Ohio; p. 163–171. *42. Feder JN, Gnirke A, Thomas W et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996; 13: 399–408. 43. Zhou XY, Tomatsu S, Fleming RE et al. HFE gene knockout produces mouse model of hereditary hemochromatosis. Proc Natl Acad Sci USA 1998; 95: 2492–2497. 44. Levy JE, Montross LK, Cohen DE et al. The C282Y mutation causing hereditary hemochromatosis does not produce a null allele. Blood 1999; 94: 9–11. 45. Bahram S, Gilfillan S, Kuhn LC et al. Experimental hemochromatosis due to MHC class I HFE deficiency: immune status and iron metabolism. Proc Natl Acad Sci USA 1999; 96: 13312–13317. 46. Bennett MJ, Lebron JA & Bjorkman PJ. Crystal structure of the hereditary haemochromatosis protein HFE complexed with transferrin receptor. Nature 2000; 403: 46–53. 47. West Jr. AP, Giannetti AM, Herr AB et al. Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites. J Mol Biol 2001; 313: 385–397. 48. Giannetti AM & Bjorkman PJ. HFE and transferrin directly compete for transferrin receptor in solution and at the cell surface. J Biol Chem 2004; 279: 25866–25875.
Molecular control of iron metabolism 169 49. Roy CN, Penny DM, Feder JN & Enns CA. The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells. J Biol Chem 1999; 274: 9022–9028. 50. Salter-Cid L, Brunmark A, Li Yet al. Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis. Proc Natl Acad Sci USA 1999; 96: 5434–5439. 51. Waheed A, Grubb JH, Zhou XY et al. Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis. Proc Natl Acad Sci USA 2002; 99: 3117–3122. 52. Bridle KR, Frazer DM, Wilkins SJ et al. Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis. Lancet 2003; 361: 669–673. 53. Ahmad KA, Ahmann JR, Migas MC et al. Decreased liver hepcidin expression in the hfe knockout mouse. Blood Cells Mol Dis 2002; 29: 361–366. 54. Nicolas G, Viatte L, Lou DQ et al. Constitutive hepcidin expression prevents iron overload in a mouse model of hemochromatosis. Nat Genet 2003; 34: 97–101. 55. Roy CN, Custodio AO, De Graaf J et al. An Hfe-dependent pathway mediates hyposideremia in response to lipopolysaccharide-induced inflammation in mice. Nat Genet 2004; 36: 481–485. 56. Frazer DM, Wilkins SJ, Millard KN et al. Increased hepcidin expression and hypoferraemia associated with an acute phase response are not affected by inactivation of HFE. Br J Haematol 2004; 126: 434–436. 57. Lee P, Peng H, Gelbart T & Beutler E. The IL-6- and lipopolysaccharide-induced transcription of hepcidin in HFE-, transferrin receptor 2-, and beta 2-microglobulin-deficient hepatocytes. Proc Natl Acad Sci USA 2004; 101: 9263–9265. 58. Kawabata H, Germain RS, Vuong PT et al. Transferrin receptor 2-{alpha} supports cell growth both in iron-chelated cultured cells and in vivo. J Biol Chem 2000; 275: 16618–16625. 59. West Jr. AP, Bennett MJ, Sellers VM et al. Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE. J Biol Chem 2000; 275: 38135–38138. 60. Johnson MB & Enns CA. Regulation of transferrin receptor 2 by transferrin: diferric transferrin regulates transferrin receptor 2 protein stability. Blood 2004. 61. Robb A & Wessling-Resnick M. Regulation of transferrin receptor 2 protein levels by transferrin. Blood 2004;. *62. Papanikolaou G, Samuels ME, Ludwig EH et al. Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis. Nat Genet 2004; 36: 77–82. 63. Monnier PP, Sierra A, Macchi P et al. RGM is a repulsive guidance molecule for retinal axons. Nature 2002; 419: 392–395. 64. Schmidtmer J & Engelkamp D. Isolation and expression pattern of three mouse homologues of chick Rgm. Gene Expr Patterns 2004; 4: 105–110. 65. Jacolot S, Le Gac G, Scotet V et al. HAMP as a modifier gene that increases the phenotypic expression of the HFE pC282Y homozygous genotype. Blood 2004; 103: 2835–2840. 66. Merryweather-Clarke AT, Cadet E, Bomford A et al. Digenic inheritance of mutations in HAMP and HFE results in different types of haemochromatosis. Hum Mol Genet 2003; 12: 2241–2247. 67. Njajou OT, Vaessen N, Joosse M et al. A mutation in SLC11A3 is associated with autosomal dominant hemochromatosis. Nat Genet 2001; 28: 213–214. 68. Montosi G, Donovan A, Totaro A et al. Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene. J Clin Invest 2001; 108: 619–623. 69. Pietrangelo A. The ferroportin disease. Blood Cells Mol Dis 2004; 32: 131–138.
1 Molecular control of iron metabolism Nancy C. Andrews*
MD, PhD
Professor of Pediatrics; Dean for Basic Sciences and Graduate Studies, Investigator Children’s Hospital Boston, Karp Family Research Laboratories 8-125, Howard Hughes Medical Institute, Harvard Medical School and Dana-Farber Cancer Institute, 300 Longwood Ave, Boston, MA 02115-5737, USA
As described in this volume, our knowledge of mammalian iron metabolism has advanced dramatically over the past decade. While basic physiological concepts were described through elegant experiments in the 1950s and 1960s, many molecular details remained unknown. Modern techniques in genetics, biochemistry and molecular biology have allowed for the identification and characterization of most, but probably not all, of the key molecules that move iron into, out of, and between cells. Insight into the complex regulation of these iron transport proteins has been accelerated through the identification of hemochromatosis disease genes. Although much of the information has been gleaned from experiments in animal models, it is likely that it can be directly extrapolated to humans. Key words: iron; homeostasis; anemia; hemochromatosis; metal transport; transferrin; ferritin.
Iron is required by all mammalian cells. Its major roles include oxygen delivery (as a heme cofactor in hemoglobin and myoglobin) and enzymatic transfer of electrons (in cytochromes, ribonucleotide reductase, and enzymes that deal with oxygen radicals). However, excess free iron promotes the formation of damaging oxygen radicals that attack cellular lipids, proteins and nucleic acids. Normal iron homeostasis allows cells to benefit from the usefulness of iron while avoiding its deleterious effects. At a systemic level, iron balance averts both iron deficiency and iron overload. Systemic iron homeostasis is essentially a closed system. There appears to be no regulated mechanism for iron excretion through the liver or kidneys; iron losses occur primarily through bleeding and sloughing of mucosal and skin cells. To maintain iron balance, intestinal iron uptake is meticulously controlled; only a fraction of the iron in a normal diet is absorbed. Compared to the total iron endowment, the amounts of iron entering and leaving the body are insignificant when measured over hours to days, though important in the long term. Iron deficiency is most often a consequence of
* Tel.: C1 617 919 2116; Fax: C1 617 730 0934. E-mail address: [email protected] 1521-6926/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
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increased losses (e.g. gastrointestinal blood loss), excessive requirements (e.g. during rapid growth in childhood) or inadequate dietary supply. In contrast, iron overload usually results from a genetic error that interferes with normal regulation and leads to chronically increased dietary absorption. Iron overload may also result from excessive iron therapy, most commonly through hypertransfusion.
MECHANISMS OF IRON TRANSPORT Iron cannot passively enter cells because it cannot negotiate the lipid bilayer without the aid of specific carrier proteins. All eukaryotic cells probably require mechanisms to take up (import) iron. Some specialized cells—particularly intestinal epithelial cells, hepatocytes and macrophages—also require mechanisms to release (export) it. In addition, the toxicity of free iron requires specialized proteins to bind iron in extracellular fluids and in intracellular storage sites. More than half of the body’s iron in normal individuals is found in hemoglobin in erythrocytes and their precursors. About 20–25 mg iron is required on a daily basis to support the hemoglobinization of new erythrocytes. Very little of this is supplied by the 0.5–2.0 mg acquired through intestinal absorption. Most of it must come from recycling of iron already in the body. The primary source is a specialized population of tissue macrophages, which phagocytose damaged erythrocytes to scavenge their iron from hemoglobin and return it to the circulation. The amount of iron passing through this macrophage recycling system every day approximates the amount needed for erythropoiesis. Additional iron may be obtained through mobilization of cellular iron stores, particularly from hepatocytes. Iron in the circulation Iron circulates bound to transferrin (TF), a very abundant plasma protein that binds two iron atoms with extremely high affinity. TF keeps iron soluble, preventing the Fe3C ion from precipitating out of aqueous plasma. It also prevents iron from reacting with other molecules by attenuating its redox activity. It aids in delivery of iron to cells by binding to a specific cell-surface transferrin receptor (TFR1). Each TFR1 homodimer binds two molecules of TF. TFR1 is present in low abundance on most if not all mammalian cell types, but it is expressed at high levels by developing erythroid precursors, activated lymphocytes, placental syncytiotrophoblasts and tumor cells. Each of these cell types has an unusually large need for iron for hemoglobin production (in the case of erythroid precursors), iron transfer (placental syncytiotrophoblasts) or cell proliferation. Iron uptake through the TF cycle In many tissues, cellular iron uptake occurs through a process known as the ‘TF cycle’ (reviewed in Ref. [1]). When iron-loaded TF attaches to TFR1, the ligand-receptor interaction triggers clathrin-mediated invagination of the cell membrane and formation of intracellular TF-TFR1-containing endosomes. The pH inside the endosomes is rapidly lowered through the action of proton pumps, resulting in conformational changes of both the TF and TFR1 proteins. Iron is released from the complex and reduced to its Fe2C form. Fe2C is transported across the endosomal membrane into the cytoplasm by divalent metal transporter 1 (DMT1, previously known as Nramp2,
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DCT1), a proton-dependent iron carrier with 12 membrane-spanning segments.2 In the cytoplasm, iron is either incorporated into protoporphyrin to produce heme or retained in storage forms (discussed below). Meanwhile, the endosomes containing TF and TFR1 return the proteins to the cell surface for re-use. The TF cycle is clearly very important for erythropoiesis and lymphopoiesis. Knockout mice lacking TFR1 die during mid-gestation from severe anemia.3 Chimeric mice containing a mixture of wild-type and TFR-null cells generate their entire erythroid and lymphoid cell populations from wild-type cells, although other tissues have mixed contributions from both cell sources.4 Mice and human patients deficient in TF also have severe anemia5,6 and lymphoid abnormalities.7 The TF cycle also appears to be important early in the development of the central nervous system, although its involvement is not well understood.3,8 As discussed later, it is clear that some cells can take iron up directly, without the TF cycle. Why has this complex mechanism evolved? There are at least two likely answers. First, the avidity with which TF binds iron is advantageous while the metal is circulating, but problematic forcellular uptake. The TF cycle allows for pH-dependent release of iron from TF in a controlled intracellular compartment. Second, receptor-mediated uptake of iron bound to TF probably allows for much higher local iron concentrations within endosomes containing transmembrane iron transporters than could be achieved by passive interactions between circulating ions and the cell surface. This interpretation is consistent with the fact that the TF cycle functions primarily in cells with excessive iron needs. Intestinal iron absorption The absorptive epithelial cells of the intestine (enterocytes) are responsible for bringing new iron into the body. They are organized in finger-like villous structures to increase the absorptive surface area. Undifferentiated cells at the base of the villi divide to give rise to several cell types, including enterocytes. Over the course of several days the cells differentiate and migrate up the villous. They carry out their absorptive function during this migration, but when they reach the top, they senesce and slough into the intestinal lumen. The apical (luminal) membrane of the enterocyte is organized into a microvillus brush border. The first step in iron absorption is transfer across this surface into the cell. We now know that most of the transfer of non-heme iron is carried out by DMT1, the same protein that performs transmembrane iron transport as part of the TF cycle in erythroid cells.9,10 DMT1 is capable of transporting avariety of divalent metal cations, but iron is probably the most important. As most dietary non-heme iron is in the ferric (Fe3C) form, it must first be reduced to ferrous (Fe2C) iron. A cytochrome b-like brush border ferrireductase, DCYTB, is probably involved in enzymatic reduction of iron11, though there is no direct evidence that DCYTB and DMT1 form a physical complex. The amounts of both DMT1 and DCYTB in enterocytes increase dramatically in iron deficiency.10,11 Once inside the enterocyte, iron has two possible fates. Some remains stored within the cell; that iron is ultimately lost from the body at the end of the enterocyte lifespan. The remainder is transferred across the basolateral surface through a distinct transport mechanism. The major molecule that carries iron across the membrane is ferroportin (also known as IREG1, MTP1).12–14 It is similar to DMT1 in several respects: it also is a multi-transmembrane segment protein, and it appears to prefer ferrous iron. However, there is no sequence homology between these two iron transporters. A multicopper ferroxidase, hephaestin, acts in some way to facilitate basolateral iron transport15,
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although its mechanism of action is not yet known. It may serve to oxidize iron to allow binding to the plasma iron carrier, TF. Macrophage iron recycling Relatively little is known about macrophage iron recycling, in spite of its great physiological importance. Tissue macrophages recognize old and damaged erythrocytes16 and phagocytose them to remove them from circulation. Within the macrophages the erythrocytes are lysed and hemoglobin is degraded. Heme oxygenase catalyzes the liberation of iron from heme. Although, the handling of iron within the cell is poorly understood, it appears likely that macrophages release iron through the action of ferroportin17 with the aid of the ferroxidase ceruloplasmin.18 The amount of iron that is recycled from effete red cells and returned to plasma TF approximates the amount needed for the synthesis of new erythrocytes. Placental iron transfer During pregnancy, the mother transfers iron to the developing fetus to establish generous newborn iron stores. However, in spite of its importance, very little is known about placental iron transfer. It does not appear to depend on DMT1 (unpublished observation) but it does involve TFR1, ferroportin, hephaestin and hepcidin.15,19,20 Hepatocyte iron transport Hepatocytes serve as the major storage depot for iron in excess of tissue needs. The details of hepatocyte iron uptake have not been worked out, but several mechanisms are likely to be involved. Hepatocytes express both TFR1 and a homolog, transferrin receptor-2 (TFR2)21, but neither appears to be essential for iron uptake. Expression of TFR1 is not necessary for the formation of normal liver.4 While the amount of TFR2 is much greater than the amount of TFR1, mutations in TFR2 result in hepatocyte iron overload, not iron deficiency.22,23 Finally, animals and patients lacking TF develop marked liver iron overload.24,25 There is abundant evidence for at least one non-TF iron uptake pathway in the liver.26,27 While not yet understood on a molecular level, it is likely that this pathway is important in hepatic iron accumulation in hemochromatosis, when serum iron levels exceed the binding capacity of TF. Recently, L-type calcium channels were shown to be involved in cardiomyocyte uptake of non-TF-bound iron;28 it is possible that they (or similar molecules) also mediate hepatic uptake. Hepatocytes must also export iron to mobilize their stores for use as needed. As ferroportin is the only described cellular iron exporter, and it is expressed by hepatocytes, it seems likely that ferroportin is involved. It probably functions with ferroxidase assistance from ceruloplasmin, similarly to iron export from macrophages. However, this has not yet been proven experimentally.
SITES OF IRON STORAGE In general, two cell types are considered important for systemic storage of iron that is not needed for immediate use. As discussed above, macrophages recover iron from
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effete erythrocytes; they also internalize iron administered by injection of the medicinal forms iron dextran, iron sucrose and ferrous gluconate. Their large capacity for storage is obvious when tissues from patients with transfusional iron overload are stained for iron: large amounts of iron accumulate in macrophages before deposition in parenchymal cells is apparent. Hepatocytes acquire both TF-bound and non-TF-bound iron from the plasma. They, too, have a large capacity for iron storage. Patients with hereditary hemochromatosis may have tenfold more hepatic iron than normal individuals without any evidence of hepatocellular damage. Although enterocytes can store iron taken up from the diet (and probably also from the plasma), they are not considered part of the iron storage system. The iron that they retain leaves the body when the cells senesce and are sloughed into the gut lumen. At a molecular level, iron storage occurs primarily in ferritin (reviewed in Ref. [29]). Light (L) and heavy (H) ferritin polypeptides are similar proteins of approximately 20 kDa. They assemble in varying proportions into 24-subunit heteromultimers that sequester mineralized iron in a central core. Each ferritin molecule can accommodate up to 4500 iron atoms. Iron entry is facilitated by an oxidase activity unique to the H-ferritin polypeptide. Iron release occurs when intracellular pools become depleted. Ferritin can frequently be found within lysosomes; degradation of ferritin leads to the formation of hemosiderin, a non-homogeneous conglomerate of iron, protein and membrane breakdown products. Hemosiderin iron is mobilized poorly, if at all. Both ferritin and hemosiderin react with the Perls Prussian blue iron stain, allowing detection in tissue sections.30
REGULATION OF IRON HOMEOSTASIS Because iron is important for many biological processes, yet is highly reactive, its availability must be controlled not only at a cellular level but also systemically. At least four situations lead to measurable changes in intestinal iron absorption and tissue iron distribution: abnormal iron availability (iron overload or deficiency), accelerated erythropoiesis, hypoxia, and inflammation. Iron absorption and plasma availability must be decreased in the settings of iron overload and inflammation, and increased in response to iron deficiency, accelerated erythropoiesis and hypoxia. The responses to these stimuli are coordinated. In general, situations that require decreased iron availability are associated with interruption of intestinal absorption and retention of iron by recycling macrophages. In contrast, situations requiring increased iron availability are associated with increased intestinal absorption and enhanced macrophage iron release. A circulating peptide was recently discovered which probably explains how iron release from absorptive enterocytes and macrophages is regulated in concert. Hepcidin (also called HAMP) is a 25-amino-acid molecule that is structurally similar to the defensin antimicrobial peptides.31–33 It is primarily produced by the liver, secreted into plasma, and cleared by the kidneys. Increased levels of hepcidin are produced in the setting of non-genetic iron overload34,35 and inflammation.36–38 Decreased hepcidin expression is seen in iron deficiency, accelerated erythropoiesis and hypoxia.36,37 Conversely, forced constitutive expression of hepcidin in transgenic mice leads to iron deficiency and anemia20, whereas genetic loss of hepcidin results in hemochromatosis.32,39 Taken together, these findings indicate that hepcidin is a negative regulator of
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intestinal iron absorption. Studies of cellular iron distribution in mutant animals suggest that it is also a negative regulator of macrophage iron release. The regulation of hepcidin expression has not yet been worked out in detail. An inflammatory cytokine, interleukin-6, stimulates hepcidin production in vivo and in vitro,38,40 presumably by binding to its cell-surface receptor and initiating a signaling cascade. This accounts for induction of hepcidin in inflammation, but it is not yet known how the cells producing hepcidin register cues about iron status, erythropoiesis and hypoxia. The iron occupancy of circulating TF (TF saturation) may provide hepatocytes with information about iron stores and erythropoietic rate. The mechanism of action of hepcidin has not yet been reported. However, it probably involves inhibition of cellular iron export, either directly through interaction with ferroportin or indirectly through a signaling cascade that regulates ferroportin or an associated protein. It will be important to understand this pathway in some detail, because pharmacological manipulation may be useful in iron disorders, including hereditary hemochromatosis, the anemia of chronic disease, and perhaps even iron deficiency.
GENES MUTATED IN HUMAN IRON DISORDERS Iron is the predominant metal in the earth’s crust, making it abundant in our environment. Most genetic disorders of iron homeostasis result in iron overload (hemochromatosis) rather than iron deficiency, suggesting that human evolution has been biased to keep iron out of the body rather than to facilitate its uptake. However, this does not appear to be the case for all mammalian species. For example, the black rhinoceros is such an avid iron absorber that it develops a hemochromatosis-like illness from eating the plant-based diet provided in captivity.41 The difference in our regulation of absorption may be attributable to the fact that we are carnivorous. HFE hemochromatosis Mutations in four genes (described below) are known to result in similar patterns of pathological iron overload, involving the parenchymal cells of the liver, heart and endocrine tissues and inherited in an autosomal recessive pattern. The most common form of hemochromatosis is genetically linked to the HLA complex on human chromosome 6p. It results from homozygous mutations in an HLA class I-like gene, HFE.42 This disorder has been modeled in mice through mutations that inactivate the murine Hfe gene or introduce a common disease-causing missense mutation (C282Y) into it.43–45 The molecular function of HFE has remained obscure, but there are several important observations that provide clues. HFE forms a tight protein-protein complex with TFR.46 The binding sites for HFE and TF overlap, preventing both proteins from binding to the same TFR molecule simultaneously.47,48 There is controversy in the literature about the consequences of HFE/TFR complex formation; some investigators have reported that it leads to decreased iron uptake,49,50 whereas others have found that it leads to increased iron uptake.51 Because those studies were carried out in transfected cells, rather than in a native system, it is possible that both sets of findings are correct, but not necessarily informative about the normal situation. Using a genetic approach, we and others have implicated HFE in the regulation of hepcidin expression. Patients and mice homozygous for HFE mutations express
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hepcidin at levels that are inappropriately low for their iron overload state.35,52–54 We have reported that mice lacking HFE do not mount an appropriate hepcidin response to inflammation55, though others disagree with our conclusions.56,57 TFR2 hemochromatosis A clinically indistinguishable disorder is found in patients who have normal HFE genes but are homozygous for mutations in the gene encoding TFR2. TFR2 knockout mice show similar findings.23 TFR2 is expressed at enormous levels in hepatocytes, but is not found in most other cell types. The major function of TFR2 has not yet been established, but it can mediate endocytosis of diferric TF, albeit with lower efficiency than TFR1.58 It does not form a complex with HFE59 and its role in regulating iron homeostasis is unknown. However, it was recently shown that increased saturation of TF with iron leads to increased hepatocellular TFR2 protein, suggesting that it may, in some way, help to ‘interpret’ iron stores signals.60,61 To date, no link between TFR2 and hepcidin has been reported in the literature, but it seems likely that they participate in the same overall regulatory pathway. Juvenile hemochromatosis More severe, early-onset iron overload is seen in patients with ‘juvenile’ hemochromatosis due to mutations either in the gene encoding hepcidin39 or in a novel gene, hemojuvelin (HJV).62 In both instances, homozygosity for deleterious mutations leads to very similar iron-overload phenotypes: cardiomyopathy, endocrinopathies and liver dysfunction. The striking similarity between these disorders suggests that hepcidin and HJV are components of the same regulatory system. This is further supported by the fact that, similar to patients with HFE hemochromatosis, patients with HJV mutations have decreased urinary hepcidin levels.62 The likely function of hepcidin has already been discussed, but the function of HJV remains unknown. At the time of its discovery last year, there was only one known protein that had a similar structure: repulsive guidance molecule (RGM) from chickens.63 It is now clear that mammals have three RGM-like molecules; HJV is also known as RGM-C.64 Hemojuvelin has several sequence motifs in common with other proteins, including a signal peptide, an arginine-glycine-aspartic acid (RGD) motif, a von Willebrand factor type D domain, and a putative GPI anchor site. HJV is expressed at high levels in muscle and heart, and at lower levels in the liver. However, none of this information gives real insight into how hemojuvelin participates in the regulation of iron homeostasis. Much more is likely to be learned over the coming years. While each of these four hemochromatosis disease genes was initially discovered through the study of patients homozygous for mutations in a single gene, it is now clear that some patients with severe hemochromatosis carry mutations in more than one gene. A small subset of individuals with HFE hemochromatosis also carries one mutated hepcidin (HAMP) gene,65,66 apparently resulting in a more rapid onset of iron overload. As homozygous HFE mutations are relatively common in individuals of Northern European descent, it is likely that patients with HFE mutations accompanied by mutations in the TFR2 or HJV genes will be identified in the future.
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Ferroportin-associated iron overload Mutations in the ferroportin gene cause an iron overload disorder with a distinct pathology. Although sometimes called ‘hemochromatosis’, the ferroportin-associated disease shows autosomal dominant inheritance and macrophage-predominant iron loading.67,68 Many patients have biochemical evidence of aberrant iron homeostasis (increased serum ferritin) in the absence of clinical signs or symptoms. Only a fraction appear to develop classical iron-related toxicity in the liver and endocrine tissues. It seems counterintuitive that mutations in the iron exporter, ferroportin, should cause iron overload, particularly because the mutations probably interfere with, rather than enhance, ferroportin function. The explanation for this paradox probably lies in the fact that ferroportin is important not only for intestinal iron absorption but also for macrophage iron recycling. At present, the best guess is that partial loss of ferroportin function in macrophages leads to iron-restricted erythropoiesis. This, in turn, stimulates increased intestinal iron absorption in spite of a ferroportin deficit. Consistent with this hypothesis, several patients heterozygous for ferroportin mutations were reported to be anemic early in their course.69 However, the hypothesis is not yet proven, and it is quite possible that the details of the pathophysiology will be more complex.
FUTURE DIRECTIONS Although enormous progress has been made in understanding mammalian iron metabolism, many unanswered questions remain. I will mention a few of the more pressing questions here. Quantitatively, macrophage recycling of hemoglobin iron makes the most important contribution to the iron economy, yet very little is understood of the intracellular processes involved. We need better knowledge of the subcellular compartments that iron enters and leaves within the macrophage. We need to understand the controls that determine whether liberated iron will remain in storage or be exported by ferroportin. It is likely that the export apparatus involves other as yet unknown components in addition to ferroportin; some of these might distinguish macrophage iron export from enterocyte iron export, and might be differentially affected by mutations in patients with autosomal dominant, ferroportin-associated iron overload. It will be important to determine whether there are other physiologically significant transmembrane iron carriers in addition to DMT1 and ferroportin. Does non-TFbound iron enter hepatocytes through the action of L-type calcium channels, or through some other mechanism? Is ferroportin the only molecule that can mediate export of iron? Finally, much remains to be learned about the proteins identified as hemochromatosis disease genes. How does HFE normally function, and what is the significance of its association with TFR? What purpose does TFR2 serve? How can HJV, a protein that is highly expressed in muscle and heart, be directly involved in the regulation of iron homeostasis? Over the next decade, many of these questions will be answered. New proteins important for iron metabolism are likely to be discovered. Ultimately, it should be possible to develop a robust, comprehensive and quantitative model of systemic iron homeostasis. With such a model, it should be possible to predict how perturbation of
Molecular control of iron metabolism 167
one part of the system—e.g. by genetic mutations or treatment with a drug—affects the entire organism. When that is accomplished, we will truly understand hemochromatosis and other iron disorders.
REFERENCES *1. Hentze MW, Muckenthaler MU & Andrews NC. Balancing acts; molecular control of mammalian iron metabolism. Cell 2004; 117: 285–297. 2. Fleming MD, Romano MA, Su MA et al. Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport. Proc Natl Acad Sci USA 1998; 95: 1148–1153. 3. Levy JE, Jin O, Fujiwara Y et al. Transferrin receptor is necessary for development of erythrocytes and the nervous system. Nat Genet 1999; 21: 396–399. 4. Ned RM, Swat W & Andrews NC. Transferrin receptor 1 is differentially required in lymphocyte development. Blood 2003; 102: 3711–3718. 5. Beutler E, Gelbart T, Lee P et al. Molecular characterization of a case of atransferrinemia. Blood 2000; 96: 4071–4074. 6. Bernstein SE. Hereditary hypotransferrinemia with hemosiderosis, a murine disorder resembling human atransferrinemia. J Lab Clin Med 1987; 110: 690–705. 7. Macedo MF, de Sousa M, Ned RM et al. Transferrin is required for early T-cell differentiation. Immunology 2004; 112: 543–549. 8. Dickinson T & Connor JR. Histological analysis of selected brain regions of hypotransferrinemic mice. Brain Res 1994; 635: 169–178. *9. Fleming MD, Trenor CC, Su MA et al. Microcytic anemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet 1997; 16: 383–386. *10. Gunshin H, Mackenzie B, Berger UV et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 1997; 388: 482–488. 11. McKie AT, Barrow D, Latunde-Dada GO et al. An iron-regulated ferric reductase associated with the absorption of dietary iron. Science 2001; 291: 1755–1759. 12. Abboud S & Haile DJ. A novel mammalian iron-regulated protein involved in intracellular iron metabolism. J Biol Chem 2000; 275: 19906–19912. *13. Donovan A, Brownlie A, Zhou Y et al. Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature 2000; 403: 776–781. *14. McKie AT, Marciani P, Rolfs A et al. A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol Cell 2000; 5: 299–309. 15. Vulpe CD, Kuo YM, Murphy TL et al. Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse. Nat Genet 1999; 21: 195–199. 16. Bratosin D, Mazurier J, Tissier JP et al. Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. A review. Biochimie 1998; 80: 173–195. 17. Knutson MD, Vafa MR, Haile DJ & Wessling-Resnick M. Iron loading and erythrophagocytosis increase ferroportin 1 (FPN1) expression in J774 macrophages. Blood 2003; 102: 4191–4197. 18. Harris ZL, Durley AP, Man TK & Gitlin JD. Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux. Proc Natl Acad Sci USA 1999; 96: 10812–10817. 19. Bradley J, Leibold EA, Harris ZL et al. The influence of gestational age and fetal iron status on iron regulatory protein activity and iron transporter protein expression in third trimester human placenta. Am J Physiol Regul Integr Comp Physiol 2004. 20. Nicolas G, Bennoun M, Porteu A et al. Severe iron deficiency anemia in transgenic mice expressing liver hepcidin. Proc Natl Acad Sci USA 2002; 99: 4596–4601. 21. Kawabata H, Yang R, Hirama T et al. Molecular cloning of transferrin receptor 2. A new member of the transferrin receptor-like family. J Biol Chem 1999; 274: 20826–20832. 22. Camaschella C, Roetto A, Cali A et al. The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22. Nat Genet 2000; 25: 14–15.
168 N. C. Andrews 23. Fleming RE, Ahmann JR, Migas MC et al. Targeted mutagenesis of the murine transferrin receptor-2 gene produces hemochromatosis. Proc Natl Acad Sci USA 2002; 99: 10653–10658. 24. Craven CM, Alexander J, Eldridge M et al. Tissue distribution and clearance kinetics of non-transferrinbound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis. Proc Natl Acad Sci USA 1987; 84: 3457–3461. 25. Hamill RL, Woods JC & Cook BA. Congenital atransferrinemia: a case report and review of the literature. Am J Clin Pathol 1991; 96: 215–218. 26. Wright TL, Brissot P, Ma WL & Weisiger RA. Characterization of non-transferrin-bound iron clearance by rat liver. J Biol Chem 1986; 261: 10909–10914. 27. Brissot P, Wright TL, Ma WL & Weisiger RA. Efficient clearance of non-transferrin-bound iron by rat liver. Implications for hepatic iron loading in iron overload states. J Clin Invest 1985; 76: 1463–1470. 28. Oudit GY, Sun H, Trivieri MG et al. L-type Ca(2C) channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy. Nat Med 2003; 9: 1187–1194. 29. Theil EC. Ferritin: at the crossroads of iron and oxygen metabolism. J Nutr 2003; 133: 1549S–1553S. 30. Klavins JV, Pickett JP & Wessely Z. Staining of minerals and solubility of iron in tissues. Ann Clin Lab Sci 1976; 6: 214–222. 31. Krause A, Neitz S, Magert HJ et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett 2000; 480: 147–150. *32. Nicolas G, Bennoun M, Devaux I et al. Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice. Proc Natl Acad Sci USA 2001; 98: 8780–8785. 33. Park CH, Valore EV, Waring AJ & Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 2001; 276: 7806–7810. 34. Pigeon C, Ilyin G, Courselaud B et al. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 2001; 276: 7811–7819. *35. Muckenthaler M, Roy CN, Custodio AO et al. Regulatory defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis. Nat Genet 2003; 34: 102–107. 36. Weinstein DA, Roy CN, Fleming MD et al. Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease. Blood 2002; 100: 3776–3781. *37. Nicolas G, Chauvet C, Viatte L et al. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest 2002; 110: 1037–1044. 38. Nemeth E, Valore EV, Territo M et al. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood 2003; 101: 2461–2463. *39. Roetto A, Papanikolaou G, Politou M et al. Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis. Nat Genet 2003; 33: 21–22. 40. Nemeth E, Rivera S, Gabayan Vet al. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Invest 2004; 113: 1271–1276. 41. D.E. Paglia, P. Dennis. Role of chronic iron overload in multiple disorders of captive black rhinoceroses (Diceros bicornis). In: Proceedings of the American Association of Zoo Veterinarians; 1999; Columbus, Ohio; p. 163–171. *42. Feder JN, Gnirke A, Thomas W et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996; 13: 399–408. 43. Zhou XY, Tomatsu S, Fleming RE et al. HFE gene knockout produces mouse model of hereditary hemochromatosis. Proc Natl Acad Sci USA 1998; 95: 2492–2497. 44. Levy JE, Montross LK, Cohen DE et al. The C282Y mutation causing hereditary hemochromatosis does not produce a null allele. Blood 1999; 94: 9–11. 45. Bahram S, Gilfillan S, Kuhn LC et al. Experimental hemochromatosis due to MHC class I HFE deficiency: immune status and iron metabolism. Proc Natl Acad Sci USA 1999; 96: 13312–13317. 46. Bennett MJ, Lebron JA & Bjorkman PJ. Crystal structure of the hereditary haemochromatosis protein HFE complexed with transferrin receptor. Nature 2000; 403: 46–53. 47. West Jr. AP, Giannetti AM, Herr AB et al. Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites. J Mol Biol 2001; 313: 385–397. 48. Giannetti AM & Bjorkman PJ. HFE and transferrin directly compete for transferrin receptor in solution and at the cell surface. J Biol Chem 2004; 279: 25866–25875.
Molecular control of iron metabolism 169 49. Roy CN, Penny DM, Feder JN & Enns CA. The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells. J Biol Chem 1999; 274: 9022–9028. 50. Salter-Cid L, Brunmark A, Li Yet al. Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis. Proc Natl Acad Sci USA 1999; 96: 5434–5439. 51. Waheed A, Grubb JH, Zhou XY et al. Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis. Proc Natl Acad Sci USA 2002; 99: 3117–3122. 52. Bridle KR, Frazer DM, Wilkins SJ et al. Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis. Lancet 2003; 361: 669–673. 53. Ahmad KA, Ahmann JR, Migas MC et al. Decreased liver hepcidin expression in the hfe knockout mouse. Blood Cells Mol Dis 2002; 29: 361–366. 54. Nicolas G, Viatte L, Lou DQ et al. Constitutive hepcidin expression prevents iron overload in a mouse model of hemochromatosis. Nat Genet 2003; 34: 97–101. 55. Roy CN, Custodio AO, De Graaf J et al. An Hfe-dependent pathway mediates hyposideremia in response to lipopolysaccharide-induced inflammation in mice. Nat Genet 2004; 36: 481–485. 56. Frazer DM, Wilkins SJ, Millard KN et al. Increased hepcidin expression and hypoferraemia associated with an acute phase response are not affected by inactivation of HFE. Br J Haematol 2004; 126: 434–436. 57. Lee P, Peng H, Gelbart T & Beutler E. The IL-6- and lipopolysaccharide-induced transcription of hepcidin in HFE-, transferrin receptor 2-, and beta 2-microglobulin-deficient hepatocytes. Proc Natl Acad Sci USA 2004; 101: 9263–9265. 58. Kawabata H, Germain RS, Vuong PT et al. Transferrin receptor 2-{alpha} supports cell growth both in iron-chelated cultured cells and in vivo. J Biol Chem 2000; 275: 16618–16625. 59. West Jr. AP, Bennett MJ, Sellers VM et al. Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE. J Biol Chem 2000; 275: 38135–38138. 60. Johnson MB & Enns CA. Regulation of transferrin receptor 2 by transferrin: diferric transferrin regulates transferrin receptor 2 protein stability. Blood 2004. 61. Robb A & Wessling-Resnick M. Regulation of transferrin receptor 2 protein levels by transferrin. Blood 2004;. *62. Papanikolaou G, Samuels ME, Ludwig EH et al. Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis. Nat Genet 2004; 36: 77–82. 63. Monnier PP, Sierra A, Macchi P et al. RGM is a repulsive guidance molecule for retinal axons. Nature 2002; 419: 392–395. 64. Schmidtmer J & Engelkamp D. Isolation and expression pattern of three mouse homologues of chick Rgm. Gene Expr Patterns 2004; 4: 105–110. 65. Jacolot S, Le Gac G, Scotet V et al. HAMP as a modifier gene that increases the phenotypic expression of the HFE pC282Y homozygous genotype. Blood 2004; 103: 2835–2840. 66. Merryweather-Clarke AT, Cadet E, Bomford A et al. Digenic inheritance of mutations in HAMP and HFE results in different types of haemochromatosis. Hum Mol Genet 2003; 12: 2241–2247. 67. Njajou OT, Vaessen N, Joosse M et al. A mutation in SLC11A3 is associated with autosomal dominant hemochromatosis. Nat Genet 2001; 28: 213–214. 68. Montosi G, Donovan A, Totaro A et al. Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene. J Clin Invest 2001; 108: 619–623. 69. Pietrangelo A. The ferroportin disease. Blood Cells Mol Dis 2004; 32: 131–138.