Balancing Acts: Molecular Control of Mammalian Iron Metabolism

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1 Cell, Vol. 117, , April 30, 2004, Copyright 2004 by Cell Press Balancing Acts: Molecular Control of Mammalian Iron Metabolism Review Matthias W. Hentze, 1 Martina U. Muckenthaler, 2 and Nancy C. Andrews 3, * 1 European Molecular Biology Laboratory Meyerhofstrasse 1 D Heidelberg Germany 2 University of Heidelberg Im Neuenheimer Feld 153 D Heidelberg Germany 3 Children s Hospital Harvard Medical School and Howard Hughes Medical Institute Children s Hospital sequences of systemic iron overload result from chronic iron accumulation in tissues. Systemic iron imbalance is not a sine qua non for iron-related pathology. Local mismanagement of iron homeostasis contributes, e.g., to the pathogenesis of neurodegenerative disorders such as Parkinson s disease. The dual challenge of avoiding iron deficiency and iron overload requires distinct mechanisms to achieve iron homeostasis in individual cells, in tissues, and systemically. Cellular Iron Homeostasis Cellular iron homeostasis necessitates tight control of iron uptake, storage, and export and management of intra- 300 Longwood Avenue cellular iron distribution. Although unicellular organisms Boston, Massachusetts face similar challenges, vertebrates have evolved distinct, highly specialized mechanisms for this purpose. Open access under CC BY-NC-ND license. Cellular Iron Uptake In normal individuals, extracellular iron circulates in the Iron is ubiquitous in the environment and in biology. The study of iron biology focuses on physiology and plasma bound to transferrin (Tf), an abundant protein homeostasis understanding how cells and organiron with extraordinarily high affinity for iron. In this form, isms regulate their iron content, how diverse tissues is nonreactive, but also difficult to extract. To deal orchestrate iron allocation, and how dysregulated iron with this, there are several Tf-dependent iron uptake homeostasis leads to common hematological, metaprotein mechanisms that mediate internalization of the entire iron- bolic, and neurodegenerative diseases. This has protransferrin complex (Figure 1). The uptake of diferric-tf via vided novel insights into gene regulation and unveiled receptor 1 (TfR1) has been most intensively remarkable links to the immune system. studied (Cheng et al., 2004). Tf/TfR1 complexes localize to clathrin-coated pits, which are endocytosed. Early Tfcontaining endosomes are acidified by a proton pump, The Dual Challenge effecting conformational changes in both Tf and TfR1 Daily production of 200 billion new erythrocytes requires to release iron. An unidentified ferrireductase subse- 20 mg of iron for hemoglobin synthesis, accounting for quently reduces Fe 3 to Fe 2, allowing divalent metal nearly 80% of the iron demand in humans. Not surpris- transporter 1 (DMT-1; also known as DCT1 or Nramp2) ingly, significant iron deficiency leads to anemia, a major to transfer Fe 2 across the endosomal membrane into public health concern affecting up to a billion people the cytoplasm (Fleming et al., 1998). Apo-Tf and TfR1 worldwide. Eukaryotic cells (and most prokaryotic or- are recycled to the cell surface, where each can be used ganisms) also require iron for survival and prolifera- for further cycles of iron binding and uptake. Because tion, as a constituent of other hemoproteins, iron-sulfur TfR1 is ubiquitously expressed, Tf-mediated iron uptake (Fe-S) proteins, and proteins that use iron in other func- is thought to occur in most cell types. However, this tional groups to carry out essential housekeeping func- pathway appears to be most important in developing tions for cellular metabolism. Cellular iron deficiency erythroid precursors because of their enormous need arrests cell growth and leads to cell death. for iron for hemoglobin synthesis. The biological importance of iron is largely attributable A homologous protein, transferrin receptor-2 (TfR2) to its chemical properties as a transition metal. It readily (Kawabata et al., 1999), is restricted to hepatocytes, engages in one-electron oxidation-reduction reactions duodenal crypt cells, and erythroid cells, suggesting a between its ferric (3 ) and ferrous (2 ) states. However, more specialized role. The finding that mutations in the the same chemical property explains why an excess of human TfR2 gene result in hemochromatosis (pathologi- free, reactive iron is toxic. In the cytoplasm, a signifi- cal systemic iron overload) (Camaschella et al., 2000) cant fraction of iron is reduced and can participate in established its importance in iron homeostasis. TfR2 Fenton-type redox chemistry: ferrous iron reacts with binds Tf with approximately 30-fold lower affinity than hydrogen peroxide (H 2 O 2 ) or lipid peroxides to generate TfR1 and, in contrast to TfR1, its expression is not conferric iron, OH, and the highly reactive hydroxyl radical trolled by the IRE/IRP regulatory system (see below). (OH ) or lipid radicals such as LO and LOO. These Polarized epithelial cells of the kidney utilize a third upradicals damage lipid membranes, proteins, and nucleic take mechanism for Tf bound iron, through megalinacids. Since both cellular iron overload and iron defi- dependent, cubilin-mediated endocytosis (Kozyraki et ciency cause cell death, the levels of reactive iron must al., 2001). This may provide the major iron supply for be carefully controlled and limited. Most pathologic con- renal proximal tubules. Although Tf-dependent iron uptake probably predominates *Correspondence: hentze@embl.de under normal circumstances, pathological iron

2 Cell 286 Figure 1. Cellular Iron Metabolism A generic mammalian cell is depicted with an indication of iron import (top) as well as iron export (bottom) pathways. The transferrin receptor-1 (TfR1) is ubiquitously expressed, while transferrin receptor-2 (TfR-2) is restricted to hepatocytes, duodenal crypt cells, and erythroid cells. Polarized epithelial cells of the kidney utilize cubilin for transferrin-mediated iron uptake. DMT-1 is implicated in intestinal iron absorption after Fe 3 from the diet is reduced to Fe 2 by the cytochrome b-like ferrireductase (Dcytb). DMT-1 further functions in iron export from the endosome following uptake via the Tf cycle. The hemoglobin scavenger receptor (CD163) plays a role in haptoglobin-mediated hemoglobin uptake of monocytes and macrophages. The only putative iron exporter identified to date is ferroportin, which has been implicated in iron export from duodenal enterocytes, macrophages, hepatocytes, placenta syncytiotrophoblasts, and cells of the central nervous system (CNS). Ceruloplasmin, and its intestinal homolog hephaestin, oxidize Fe 2 after cellular iron export for loading onto transferrin. Much intracellular iron traffic is directed toward mitochondria, where the iron-dependent step of heme synthesis and critical steps for Fe-S cluster biogenesis are localized. Dietary iron uptake occurs at the apical (luminal) surface of duodenal absorptive cells (enterocytes). Non- heme food iron exists primarily in the bio-unavailable, insoluble Fe 3 form, which must first be reduced to Fe 2 for transport across the intestinal epithelium. The responsible enzyme appears to be a cytochrome b-like ferrireductase (Dcytb), a hemoprotein associated with the apical enterocyte membrane (McKie et al., 2001). Fe 2 then enters the cell through DMT-1. DMT-1 has also been implicated in Fe 2 recapture in the kidney (Ferguson et al., 2001; Hubert and Hentze, 2002) and in iron transport in the brain (Jeong and David, 2003). Iron may also be taken up by cells in other protein bound forms such as acidic isoferritin (Meyron-Holtz et al., 1999) or hemoglobin. The latter is of particular importance in the setting of intravascular hemolysis, observed in disorders such as sickle cell anemia and thalassemia. Under these conditions, plasma hemoglo- bin is captured by the acute phase protein haptoglobin and taken up into the cell by the hemoglobin scavenger receptor (CD163), which is expressed on monocytes and macrophages (Kristiansen et al., 2001). Thus, different means of cellular iron uptake have evolved to accommodate different forms of iron and, probably, to allow differential regulation. Biochemical data suggest that additional iron uptake mechanisms may exist (e.g., through putative receptors for ferritin and hemopexin), but these have not yet been character- ized at a molecular level or in vivo. Finally, substantial iron uptake by tissue macrophages occurs through an indirect route. Old and damaged red blood cells are phagocytosed by a specialized macro- phage population that serves the purpose of recycling iron overload leads to supersaturation of transferrin iron binding sites and circulating free iron. Several lines of evidence indicate that there are distinct transport pathways for non-tf bound iron. First, mice and humans lacking Tf develop massive iron overload in nonhematopoietic tissues, such as liver and pancreas (Trenor et al., 2000). Second, TfR1-deficient mice show normal embryonic organ development before they succumb to severe anemia in mid-gestation (Levy et al., 1999). Third, rapid clearance of plasma Fe is observed in diseases (e.g., hereditary hemochromatosis) where the iron binding capacity of Tf is exceeded. Intestinal absorptive cells take up dietary non-tf bound iron directly, through the action of DMT1, the same metal transporter that mediates iron export from the endosome following uptake via the Tf cycle (Fleming et al., 1997; Gunshin et al., 1997). DMT-1 is a H /divalent metal symporter that also transports other divalent metals (e.g., Mn 2,Cu 2,Zn 2 ). In addition, studies in cultured cells indicate that there is at least one other direct iron uptake system with properties distinct from DMT1 (Sturrock et al., 1990). Calcium channels may, under some circumstances, mediate the uptake of iron (Oudit et al., 2003; Mwanjewe and Grover, 2004). In addition, iron can be complexed by neutrophil gelatinase-associated lipocalin (NGAL/24p3) in a form that is internalized and trafficked to late endosomes with acidic ph (Yang et al., 2002a). Recombinant NGAL/24p3 loaded with iron has been shown to deliver the metal into cultured cells, affecting the expression of iron-responsive genes. This NGAL/24p3 delivery pathway appears to be important in differentiating epithelial cells during early development (Yang et al., 2002a).

3 Review 287 from hemoglobin for re-use (Brittenham, 1994). Engulfed import into tissue culture cells (e.g., Mukhopadhyay et red cells are lysed, hemoglobin is degraded (with help al., 1998), but this has not yet been confirmed. from heme oxygenase), part of the recovered iron is Regulation of Cellular Iron Homeostasis held in storage, and the remainder is exported to reload Proteins involved in iron uptake, storage, utilization, and circulating Tf. This is quantitatively important; only 1 2 export must be regulated in a coordinated fashion. The mg of iron enters the body through the intestine each signals and regulatory mechanisms that orchestrate day, yet 25 mg are needed for erythropoiesis and other their expression involve modulation of transcription, uses. Nearly all of the available iron pool is derived from mrna stability, translation, and posttranslational modi- macrophage recycling (Figure 3). fications. Posttranscriptional regulation is best characterized Cellular Iron Storage (Figure 2). Trans-acting iron regulatory proteins Once iron enters the cell, the portion that is not needed (IRPs) interact with iron-responsive elements (IREs), for immediate use is stored by ferritin, a ubiquitous and which are conserved hairpin structures found in untrans- highly conserved multimeric protein (Figure 1) (Torti and lated portions of mrnas encoding iron-related proteins. Torti, 2002). In vertebrates, ferritin consists of an apoprotein Single IREs are located in the 5 untranslated regions shell of 24 light (L) and heavy (H) chain subunits, (UTRs) of mrnas encoding ferritin H and L chains, ery- surrounding a core of up to 4500 iron atoms. Ferritin throid 5-aminolevulinic acid synthase (the first enzyme sequesters the metal from the intracellular labile iron of heme biosynthesis), mitochondrial aconitase (a citrate pool, locking it up in chemically less reactive ferrihydrite cycle enzyme), and ferroportin (Hentze and Kühn, 1996; to achieve both iron storage and detoxification (Harrison, Donovan et al., 2000; McKie et al., 2000; Eisenstein and 1977). The ratio of H to L subunits varies depending on Ross, 2003). The formation of IRE/IRP complexes on tissue type and physiologic status and changes in re- the 5 UTR serves to inhibit early steps of the translation sponse to inflammation or infection. Ferritin has enzymatic process (Muckenthaler et al., 1998). Proximity of the properties, converting Fe 2 to Fe 3 as iron is IRE to the transcription start site is important for this internalized and packed into the mineral core. This ferroxidase mechanism of translational control, and alteration of the activity is an inherent feature of the H subunit. transcription start site can impart differential responses Degradation of ferritin and concomitant iron release to IRP binding (Paraskeva et al., 1999). helps to mobilize iron for cellular utilization. Little is A second type of regulation occurs when IREs are known about how ferritin is degraded, but both lysosomal located beyond the coding sequence. Multiple IREs in and proteosomal pathways have been implicated. the 3 UTR of TfR1 mrna serve to stabilize the mrna A unique H-type ferritin homopolymer is expressed in upon IRP binding (Hentze and Kühn, 1996). A single IRE mitochondria (mitochondrial ferritin [MtF]) (Levi et al., was identified in the 3 UTR of one isoform of DMT ). Experimental overexpression of MtF results in However, this IRE only marginally mediates iron regulamitochondrial iron accumulation, decreased cytosolic tion in cultured cells (Gunshin et al., 2001), and its mechanism ferritin, and increased TfR1 expression. Interestingly, of action is not well understood. The situation is MtF expression is increased in anemia disorders charac- more complex, as a 5 promoter/exon 1A region acts terized by mitochondrial iron accumulation (sideroblastic with the 3 IRE-containing terminal exon to control anemias), suggesting a protective role of MtF against DMT-1 expression (Hubert and Hentze, 2002). iron-mediated toxicity. Diverse IREs display remarkable structural similarity. Cellular Iron Export Canonical IREs have a six nucleotide apical loop with Duodenal enterocytes, macrophages, hepatocytes, placenta the consensus sequence 5 -CAGUGN-3 on a stem of syncytiotrophoblasts, and cells of the central ner- five paired bases, a small asymmetrical bulge with an vous system (CNS) require mechanisms to release iron unpaired cytosine on the 5 strand, and an additional in a controlled fashion to ensure availability of the metal stem of variable length (Hentze et al., 1988). The nucleotides where it is needed. The only putative iron exporter identified making up the two stem segments may vary conwhere to date is ferroportin (also known as Ireg1, MTP) siderably. (Abboud and Haile, 2000; Donovan et al., 2000; McKie et The cellular labile iron pool regulates binding of IRP1 al., 2000). It is highly expressed in iron-deficient intestine and IRP2 to IREs through distinct mechanisms. When and in tissue macrophages. Ferroportin is located at the iron levels are high, a cubane [4Fe-4S] cluster assembles basolateral membrane of duodenal enterocytes, where in IRP1, inhibiting IRE binding activity and converting it mediates iron export into the bloodstream, apparently IRP1 to an aconitase. When cellular iron stores are low, in concert with hephaestin, a ferroxidase that is homolo- IRP1 binds to IRE targets as an apoprotein. In contrast, gous to the abundant plasma protein ceruloplasmin (Vulpe IRP2 does not contain an Fe-S cluster. It accumulates et al., 1999). in iron-deficient cells and is targeted for degradation Iron export from nonintestinal cells requires ceruloplasmin when iron levels are high (Hentze and Kühn, 1996). Surexported (Cp) (Harris et al., 1999). Cp converts Fe 2, likely prisingly, it was recently reported that mice on an iron- by ferroportin, to Fe 3 that is loaded onto Tf deficient diet activated IRP2 but failed to increase IRP1 for transport in the plasma (Figure 1). In the brain, the activity (Meyron-Holtz et al., 2004). This contrasts with glycosylphosphatidylinositol (GPI)-anchored form of Cp findings from cultured cells and suggests that IRP1 ac- physically interacts with ferroportin to export iron from tivity may not be determined by a simple iron equilibrium astrocytes (Jeong and David, 2003). Patients and mice reaction. with Cp deficiency accumulate iron in macrophages, IRP1 and IRP2 share extensive sequence homology, hepatocytes, and cells of the CNS, resulting in ironrestricted but IRP2 contains a 73 amino acid N-terminal insertion erythropoiesis and neurodegeneration. Some that has been implicated in iron-sensing and iron-medi- reports suggest that Cp may also be involved in iron ated degradation. It can bind heme, triggering oxy-

4 Cell 288 Figure 2. The IRE/IRP Regulatory System Proteins involved in iron storage, erythroid heme synthesis, the TCA cycle, iron export, and iron uptake are coordinately regulated by the interaction of the iron regulatory proteins (IRPs) with conserved RNA secondary structures, the iron-responsive elements (IREs). The binding of IRPs to single IREs in the 5 -untranslated regions (UTRs) of mrnas blocks their translation, while IRP binding to multiple IREs in the 3 UTR stabilizes the TfR-1 mrna. IRPs exist in two isoforms, IRP1 and IRP2. Increased iron levels favor the conversion of IRP1 from its active RNA binding form into an Fe-S cluster containing cytoplasmic aconitase that lacks IRE binding activity as well as the proteasomal degradation of IRP2. Low iron levels or the action of NO promote accumulation of the active apoprotein form of IRP1 and stabilize IRP2. In contrast, H 2 O 2 only activates IRP1, while hypoxia interferes with IRP2 degradation. gen-dependent oxidation, ubiquitination by the E3 ubi- kin-1, interleukin-6, and interferon- (IFN- ) all stimulate quitin-protein ligase HOIL1, and degradation by the H-ferritin expression but reduce TfR1 expression (Torti proteasome (Kang et al., 2003; Yamanaka et al., 2003). and Torti, 2002). IFN- and lipopolysaccharide have However, other reports suggest that this domain is dispensable been reported to induce DMT-1 and inhibit ferroportin for iron-mediated IRP2 degradation and that expression in activated monocytes (Ludwiczek et al., 2-oxoglutarate-dependent oxygenases are involved in 2003; Yang et al., 2002b). The ferroportin response to regulation (Bourdon et al., 2003; Hanson et al., 2003; lipopolysaccharide appears to be mediated by Toll-like Wang et al., 2004). Thus, the mechanisms of IRP1 and receptor 4 and, in part, by IRP-dependent regulation of IRP2 regulation are less clear than they had appeared the IRE-containing ferroportin mrna (Liu et al., 2002). to be. Together, these cytokine-induced changes may contrib- IRPs are also controlled by other effectors including ute to macrophage iron retention observed in inflammatory reactive oxygen species, which lead to disassembly of states, augmenting the host defense against microreactive the Fe-S cluster of IRP1 (Pantopoulos and Hentze, 1998), organisms. and nitric oxide and hypoxia, which affect both IRPs Intracellular Iron Distribution (Hentze and Kühn, 1996; Hanson et al., 1999). Serine Most of the iron within cells is routed to the mitochondria, phosphorylation of IRP1 destabilizes the Fe-S cluster, where substantial amounts are needed for heme suggesting an additional, iron-independent mechanism biosynthesis and maturation of Fe-S clusters (Figure 1). of IRP1 regulation (Brown et al., 1998; Fillebeen et al., In erythroid cells, where nearly all iron is needed for 2003). heme synthesis, iron-loaded endosomes have been proposed It is clear that other translational control mechanisms to make direct contact with mitochondria to facili- will turn out to be important in regulating iron homeosta- tate accumulation of large amounts of iron (Ponka, sis. Exploiting an IRP-independent mechanism, IFN- 1997). This model is intriguing, but not yet proven. silences translation of ceruloplasmin mrna, apparently Mitochondrial iron also accumulates in yeast (Gerber through the interaction of a phosphorylated ribosomal and Lill, 2002) and humans (Allikmets et al., 1999) with protein with a novel element located in the 3 UTR (Ma- defects in proteins involved in Fe-S cluster assembly. zumder et al., 2003). It is not yet known whether this Human ABC7 and its functional yeast ortholog Atm1p mechanism is used for regulation of other mrnas. are thought to function in the export of Fe-S clusters Iron-related genes are also regulated at the level of from mitochondria. Mutations in ABC7 result in anemia transcription. Cytokine responses have been reported and neurological symptoms. Erythroid precursor cells for many. For example, tumor necrosis factor-, interleu- from affected patients contain mitochondrial iron aggre-

5 Review 289 gates. ABC7 may play a role in heme biosynthesis, poiesis, intestinal absorption is enhanced by an erythroid through a direct interaction with ferrochelatase, the final regulator (Finch, 1994). Iron homeostasis is also enzyme in the pathway (Taketani et al., 2003). altered in response to hypoxia, through a humoral hyp- Defects in the frataxin gene cause the neurodegenera- oxia regulator. tive disorder Friedreich ataxia. Friedreich ataxia patients The hierarchy of regulators is discernible in situations and mice with muscle- or neuron-specific frataxin gene where erythropoiesis is iron deficient, yet tissue iron inactivation display progressive deficiency of Fe-S cluswith stores are overfilled. Hypotransferrinemic (Trf hpx ) mice ter containing proteins followed by mitochondrial iron a splice donor mutation that results in near total overload (Rotig et al., 1997). Frataxin is a mitochondrial deficiency of serum Tf are one example (Trenor et al., matrix protein that has postulated roles in mitochondrial 2000). Receptor-mediated endocytosis of Tf is essential iron export (Chen et al., 2002) and iron storage (Adamec for iron uptake by erythroid precursors (Levy et al., 1999) et al., 2000). Recent work in yeast demonstrated that and erythropoiesis is severely impaired in Trf hpx mice frataxin is required for de novo biogenesis of cellular because the supply of iron bound to Tf is inadequate. Fe-S proteins (Muhlenhoff et al., 2003). It binds to the However, as discussed earlier, nonhematopoietic tiscentral Fe-S cluster-assembly complex in an ironpancreas, sues avidly assimilate non-tf bound iron, and the liver, dependent manner where it may support iron loading of and other organs develop iron overload. In the complex (Gerber et al., 2003). Interestingly, frataxin this setting, the erythroid regulator overrides the stores expression is reduced during erythroid differentiation, regulator, resulting in vigorous intestinal absorption of and it is tempting to speculate that this may serve to dietary iron and consequent deposition of excess iron in divert mitochondrial iron toward heme biosynthesis. parenchymal cells. Plasma transferred from Trf hpx mice We still understand little about how iron enters mitoet failed to increase iron absorption in recipients (Buys chondria or how trafficking to mitochondria is regulated. al., 1991), suggesting that, if the erythroid regulator Recently, it was proposed that an Fe-S protein might involves a soluble factor, the factor is short-lived, or the act as a sensor of the mitochondrial iron status. If this signal results from a decrease, rather than increase, in putative sensor lacks its Fe-S cluster, mitochondrial iron its production. A possible caveat to this interpretation would accumulate through increased mitochondrial iron is the fact that Tf levels are unusually low. However, a uptake and/or decreased iron release. Iron sequestraent similar predominance of the erythroid regulator is appar- tion would continue until requirements for Fe-S cluster in patients with thalassemia syndromes. They have assembly were satisfied. Thereafter, the amount of iron increased erythropoietic drive due to destruction of im- remaining in a postmitochondrial, cytoplasmic iron pool mature erythroid cells in the bone marrow, leading to would be sensed, in some fashion, to regulate cellular increased intestinal iron absorption even after other tis- iron absorption (Pandolfo, 2002). However, mutations sues become iron overloaded. in aminolevulinate synthase, the first enzyme in heme Cellular iron retention occurs rapidly in the settings synthesis, also cause mitochondrial iron accumulation. of infection and inflammation, presumably to withhold This suggests a more complicated process for regulatin macrophages that recycle the metal from senescent a nutrient from invading pathogens. Iron accumulates ing mitochondrial iron influx. erythrocytes, and intestinal iron absorption is interrupted. This can be considered an inflammatory regulator. Systemic Iron Homeostasis These different regulators (erythroid, stores, hypoxia, and Systemic Regulators inflammatory) need not be completely independent. Systemic iron homeostasis, the control of iron balance Rather, they might represent quantitative differences in throughout the body, requires mechanisms for regulatresponses mediated by the same molecule(s). ing iron entry into and mobilization from stores, for meet- Serum plasma iron levels are determined both by ining erythropoietic needs, and for scavenging previously testinal absorption and macrophage recycling of iron used iron. There is no efficient pathway for iron excrefrom hemoglobin (Figure 3). Regulatory effectors that tion; hence, intestinal absorption must be modulated to modulate intestinal iron absorption probably also moduprovide enough (but not too much) iron to keep stores late release of iron from tissue macrophages and hepareplete and erythroid demands met. There must be ef- tocytes. The search for these molecules has focused fective communication between cells that consume iron on plasma proteins that might act at multiple sites. Some (primarily erythroid precursors) and cells that acquire plausible candidates, e.g., erythropoietin, have likely and store iron (duodenal enterocytes, hepatocytes, tis- been ruled out (Raja et al., 1986). sue macrophages). Transfer between tissues must be Hepcidin (HAMP, LEAP) appears to be such a regulaorchestrated to maintain homeostasis (Figure 3). tory effector. It is a small, cysteine-rich peptide, cleaved In normal adults, storage iron is deposited in hepato- from a larger precursor (Krause et al., 2000; Park et al., cytes and tissue macrophages and mobilized in re- 2001; Pigeon et al., 2001). Hepcidin is secreted by the sponse to acute need. Intestinal iron absorption is regu- liver and excreted through the kidneys (Krause et al., lated, in part, in response to a signal that communicates 2000; Park et al., 2001). It resembles peptides involved information about the amount of iron in the depot the in innate immunity and has antimicrobial properties stores regulator (Finch, 1994). Erythroid cells are the (Krause et al., 2000; Park et al., 2001). Mice that fail major consumers of iron, and the iron endowment of to express hepcidin have elevated body iron stores, the erythron (bone marrow, erythroid precursors plus presumably due to hyperabsorption, associated with circulating red blood cells) normally exceeds the amount decreased iron in tissue macrophages (Nicolas et al., present in stores. When erythroid demand surpasses 2001, 2002a). Hepcidin expression increases when excess the capacity of storage cells to mobilize iron for erythro- iron is administered (Pigeon et al., 2001; Mucken-

6 Cell 290 Figure 3. Systemic Iron Homeostasis Major pathways of iron traffic between cells and tissues are depicted. Normal (human) values for the iron content of different organs and tissues are stated, and the approximate daily fluxes of iron are also indicated. Note that these values are approximate and subject to significant person-to-person variation. Iron losses result from sloughing of skin and mucosal cells as well as blood loss. Importantly, there exists no regulated excretion pathway to control systemic iron homeostasis. thaler et al., 2003), suggesting a compensatory response transcription. Analysis of the putative promoter revealed to limit intestinal absorption. Transgenic mice constitu- binding sites for CCAAT/enhancer binding protein tively expressing hepcidin have profound iron deficiency (C/EBP) and hepatocyte nuclear factor 4 (HNF4 ) (Nicolas et al., 2002a). These observations make hep- within an 800 bp segment that conferred liver-specific cidin an attractive candidate for a soluble regulator that expression (Courselaud et al., 2002). In reporter assays, acts to attenuate both intestinal iron absorption and C/EBP activated transcription and HNF4 repressed macrophage iron release (Figure 4). it. Furthermore, compared to wild-type animals, C/EBP Increased hepcidin expression in iron-loaded animals knockout mice expressed less hepcidin and developed and hypertransfused human patients suggests that it is mild iron overload, while HNF4 knockout mice exa component of the stores regulator. However, anemic, pressed more hepcidin (Courselaud et al., 2002). Aliron-loaded Trf hpx mice express very little hepcidin though wide ranges in mrna expression have been (Weinstein et al., 2002), suggesting that it is also a com- observed, production may also be regulated at the level ponent of the erythroid regulator. Hepcidin expression of pre-protein cleavage and/or secretion. Owing to its decreases in response to non-anemic hypoxia, implicat- small size, hepcidin is probably cleared quantitatively ing the peptide in the hypoxia regulator (Nicolas et al., by the kidneys, allowing for rapid alteration of circulat- 2002b). Increased hepcidin expression observed in mice ing levels. and human patients with inflammation suggests that it Genetic experiments have suggested that hepcidin contributes to the inflammatory regulator (Weinstein et attenuates iron release from enterocytes, placental synal., 2002; Nemeth et al., 2003; Nicolas et al., 2002b). cytiotrophoblasts, and macrophages. It was recently re- Although correlative changes in expression appear to ported that injection of synthetic hepcidin peptide into link hepcidin to these regulators, the signaling pathways mice inhibited iron uptake in isolated duodenal seghave not been worked out. Interleukin-6 induces hepments (Laftah et al., 2004). Unfortunately, the hepcidin cidin mrna expression in vitro (Nemeth et al., 2003) and peptide mixture was heterogenous, and the amount of in vivo (Nemeth et al., 2004), linking hepcidin production native, active hepcidin was not determined. In contrast to the inflammatory regulator. However, neither ferric to the conclusions of other investigators, these authors iron nor iron-loaded Tf induced hepcidin expression in suggested that hepcidin predominantly acted to dimincultured hepatic cells; in fact, ferric iron treatment led to decreased hepcidin production. No molecular link ish iron transport across the apical membrane, with little has yet been found between hepcidin expression and or no effect on basolateral iron transfer. However, the the erythroid and hypoxia regulators. effects were small, and a time lag between hepcidin Is hepcidin the sole downstream effector for all four injection and effect on absorption makes the experiregulators described above? Although hepcidin producthis issue in future studies. ments difficult to interpret. It will be important to resolve tion is attenuated when erythropoiesis is iron restricted, activation of the erythroid regulator in Trf hpx mice also No hepcidin receptor has been identified to date, but leads to markedly increased expression of Dmt1, ferroprotein. it is assumed that hepcidin interacts with a cell surface portin, and Dcytb mrnas (McKie et al., 2000, 2001; Hepcidin binding might signal the inactivation Canonne-Hergaux et al., 2001). The increased expresexpression of the cellular iron export apparatus through decreased sion of these transport-related proteins may result from or modification of its components. Alterna- an additional signal acting to augment iron absorption, tively, hepcidin might bind directly to ferroportin or an beyond decreased hepcidin production. associated transport molecule. Characterization of the Hepcidin mrna is highly expressed in normal liver, and much regulation appears to occur at the level of mechanism of hepcidin action is a priority for iron biologists.

7 Review 291 Figure 4. Role of Hepcidin, HFE, and Other Molecules in the Regulation of Systemic Iron Homeostasis Hepcidin is an antimicrobial, defensin-like peptide secreted by the liver. It diminishes iron release from reticuloendothelial macrophages and duodenal enterocytes. As a consequence, serum iron levels decrease. Hepcidin expression is regulated by iron levels, inflammatory stimuli, the erythroid iron demand, and hypoxia. Recent data indicate that the former two responses require HFE function. IRE/IRP in Systemic Iron Homeostasis Autosomal dominant hyperferritinemia-cataract syndrome The IRE/IRP regulatory system may also be important is caused by loss-of-function mutations in the in controlling systemic iron balance. IREs are found in IRE of ferritin L chain mrna (Cazzola and Skoda, 2000). mrnas encoding two critical duodenal iron transporters, Multiple, independent mutations have been described ferroportin and DMT1, strongly suggesting that IRPs that impair IRP binding, causing de-repression of ferritin are involved in their posttranscriptional regulation. Direct L chain translation. Remarkably, the loss of this house- experimental evidence for a functional role of the keeping function does not cause severe disturbances of IRE in the 3 -untranslated region of DMT1 mrna is limited, systemic iron metabolism. Bilateral, early-onset cataracts but the IRE in the 5 UTR of ferroportin mrna has and increased serum ferritin levels are the only clini- been shown to mediate iron-dependent translational cal features. control in cell lines (Abboud and Haile, 2000). Iron Overload Disorders Surprisingly, the phenotype of IRP1 / mice is rather A heterogeneous group of inherited iron overload condimild, suggesting that IRP2 can fully compensate for the tions result from increased intestinal absorption and lack of IRP1 under normal circumstances (Meyron-Holtz deposition of iron in vital organs. Mutations in four genes et al., 2004). However, IRP2 / animals misregulate iron (HFE, TFR2, hemojuvelin, and hepcidin) have been found metabolism in the duodenal mucosa, resulting in increased in patients with hemochromatosis, and mutations in expression of DMT1, ferritin, and ferroportin ferroportin have been identified in a pathologically dis- and increased duodenal iron content (LaVaute et al., tinct disorder. 2001). IRP2-deficient mice are also reported to have The prototype, HLA-linked hemochromatosis, is characterized elevated serum ferritin and increased liver iron. As discussed by iron accumulation in the liver, heart, and later, they develop a neurodegenerative pheno- pancreas, with relative sparing of macrophages. Complications, type. Tissue-specific ablation of IRP2 will be necessary resulting from destructive changes, include to elucidate how loss of this protein leads to these pro- liver cirrhosis, heart failure, and diabetes. When identified tean manifestations. before severe damage ensues, hemochromatosis Interestingly, a single point mutation in the IRE of can be treated by phlebotomy (blood-letting) to remove ferritin H chain mrna (A49U) is associated with autosomal iron-rich red blood cells. Although now deleterious, the dominant iron overload affecting three members of hemochromatosis mutation may have been subject to a Japanese family (Kato et al., 2001). The A49U IRE positive selection at times when the diet provided little mutation appears to increase IRP binding, resulting in bioavailable iron. Only a fraction of patients with a decreased H chain ferritin expression. Ferritin L chain hemochromatosis genotype develop iron overload, and levels are increased, possibly due to titration of the IRP environmental factors affect disease penetrance. In addition, repressors by the higher affinity mutant IRE. These observations genetic factors likely determine individual susrepressors suggest that regulation of ferritin (H chain) ceptibility to iron loading. expression by the IRE/IRP system is important for the HFE encodes an atypical major histocompatibility maintenance of systemic iron homeostasis. It will be class I protein that heterodimerizes with -2 microglobulin important to firmly establish the IRE mutation as the but does not bind a small peptide (Feder et al., 1996; genetic cause of the iron overload and to investigate Lebron et al., 1998). Most affected patients are homozygous the underlying mechanism(s). for a missense mutation (C282Y) (Feder et al., 1996)

8 Cell 292 that partially disrupts HFE function. A very common mis- Non-HFE Hemochromatosis sense mutation, H63D, apparently confers a slight prechromatosis Some patients with severe, early onset (juvenile) hemo- disposition to iron loading (Tomatsu et al., 2003) but is are homozygous for mutations in HAMP, rarely clinically significant unless found in patients who the human gene encoding hepcidin (Roetto et al., 2003). also carry a C282Y mutation on their other HFE allele. Furthermore, patients with HFE mutations in combina- Other HFE mutations have been described, but most tion with heterozygous HAMP mutations have a more are rare and their association with clinical disease is not severe phenotype than relatives who only carry HFE always certain. mutations. These observations lend additional support The normal function of HFE is unknown. Importantly, to the idea of an HFE/hepcidin axis. it forms a high-affinity complex with TfR1 (Parkkila et Some non-hfe hemochromatosis patients carry non- al., 1997; Lebron et al., 1998). Co-crystallization revealed sense and missense mutations in the gene encoding an extensive interaction interface between the two molea TfR2 (Camaschella et al., 2000), which does not form complex with HFE. TfR1/TfR2 heterodimers can be cules (Bennett et al., 2000), which overlaps the Tf binding site on TfR1. In vitro, HFE competitively inhibits Tf bindit is not clear that they form in vivo. It is not yet known detected when the two proteins are coexpressed, but ing. At high concentrations of HFE, two HFE molecules bind to the TfR homodimer (Bennett et al., 2000). At how TfR2 relates to HFE and hepcidin. Other patients with autosomal recessive, juvenile lower concentrations, a complex can be formed that hemochromatosis carry mutations in a novel gene, hemincludes HFE, TfR1, and Tf in a 1:2:1 stoichiometry (Leojuvelin (HFE2) (Papanikolaou et al., 2004). While it was bron et al., 1998). It remains unclear what the stoichiomexpected that this second juvenile hemochromatosis etry is in vivo. Fe 2 -Tf binds to TfR1 with higher affinity gene might encode a receptor for hepcidin, it is not and is present in plasma at micromolar concentrations. obvious that hemojuvelin serves that function. First, it However, HFE is tethered to the same membrane suris expressed predominantly in skeletal muscle, heart, face as TfR, favoring a bimolecular interaction. and liver. Second, patients with hemojuvelin mutations Tf-mediated iron uptake was decreased in transfected have decreased urinary hepcidin levels. The only de- HeLa cells expressing HFE (Gross et al., 1998), consisscribed homolog of hemojuvelin is a chicken axonal pattent with the finding that HFE binding precludes binding terning molecule (repulsive guidance molecule, RGM), of Fe 2 -Tf. However, others reported that coexpression which has no known connection to iron metabolism. At of HFE and 2-microglobulin increases iron uptake in present, it is not known whether hemojuvelin is another transfected CHO cells, by enhancing recycling of TfR1 component of the hepcidin regulatory pathway or a key to the cell surface following endosomal iron release molecule in a parallel regulatory pathway. (Waheed et al., 2002). Regardless of the interpretation A distinct iron overload disease with autosomal domiof these conflicting studies, it is important to consider nant inheritance results from heterozygosity for mutathat studies of HFE/TfR1 interactions in transfected cells tions in the gene encoding ferroportin (Montosi et al., may be misleading; the cellular context is inappropriate 2001; Njajou et al., 2001). Affected individuals have iron and the stoichiometry is influenced by the fact that HFE accumulation in macrophages, but relatively normal serum is overexpressed. iron levels. Because there is no robust assay for It is assumed that HFE does not directly modify dietary ferroportin activity, it is not yet certain how these mutairon uptake because the Tf cycle is not operative at the tions affect protein function. The pattern of iron loading apical surface of absorptive enterocytes. Until recently, suggests a defect in the recycling of iron already within it was widely believed that HFE acts in intestinal crypt the body, rather than a primary increase in intestinal cells, helping to program their future absorptive capacity absorption. Ferroportin is highly expressed by the reticas they differentiate into mature enterocytes. However, uloendothelial macrophages that unload iron from he- emerging information about hepcidin suggests that the moglobin scavenged from senescent red blood cells. crypt cell may not be the critical site of HFE function. The flux of iron through these macrophages is much In contrast to mice with induced iron overload, hepcidin greater than the flux through intestinal epithelial cells (Figure 3), suggesting that loss of one ferroportin allele expression was not increased in Hfe / mice (Ahmad et makes recycling less efficient, leading to a misinterpreal., 2002; Muckenthaler et al., 2003; Nicolas et al., 2003) tation of body iron stores (Montosi et al., 2001). Conseor in patients with HFE hemochromatosis (Bridle et al., quently, there may be a signal to increase intestinal 2003). Strikingly, the unique difference in hepatic gene absorption to meet iron demands. While this model is expression between Hfe / mice and wild-type mice attractive, it has not yet been proven. injected with iron dextran was that hepcidin expression Animal Models was not increased in the former (Muckenthaler et al., Historically, animal models have taken advantage of 2003). Constitutive expression of hepcidin on an Hfe / strong parallels between human and rodent iron metabbackground prevented iron overload (Nicolas et al., olism. Recent studies of iron transport have been ex- 2003). Hfe / mice fail to induce hepcidin appropriately tended to other species. In addition, targeted mutagenein response to inflammatory stimuli (Roy et al., 2004). sis has generated new classes of mutant mice (Table Together, these observations suggest that (1) HFE and 1), which serve three general purposes: development of hepcidin are parts of the same regulatory axis and (2) mouse models of human diseases (e.g., hemochro- HFE might function upstream of hepcidin expression matosis, aceruloplasminemia, Friedreich s ataxia), inter- (Figure 4). New models for HFE function must take these rogation of the roles of iron-related proteins in vivo (e.g., facts into account. TfR1, H-ferritin, IRPs), and discovery of previously un-

9 Review 293 Table 1. Animal Models Useful for Understanding Iron Biology and Related Diseases Type of Analogous Strain Gene (Mutation) Mutation Iron Phenotype Human Disease Hfe / and Hfe C282Y/C282Ya Hfe TMM Hepatocellular iron deposition, decreased HFE HC macrophage iron, elevated transferrin saturation 2m / Beta-2 microglobulin TMM Parenchymal iron deposition ND Usf2 / Hepcidin b TMM Hepatocellular iron deposition, decreased Juvenile HC macrophage iron, elevated transferrin saturation Hamp liver spec. transgene Hepcidin TgM Iron deficiency and anemia NA Hfe / and Hamp transgene Hepcidin/HFE CMM Amelioration of hepatic iron loading NA relative to Hfe / mice TfR / Transferrin receptor-1 c TMM Microcytic hypochromic erythrocytes f, ND decreased iron stores TfRr2 245x/245x Transferrin receptor-2 TMM Hepatocellular iron deposition, decreased TfR2 HC macrophage iron, elevated transferrinsaturation hpx Transferrin SMM Microcytic hypochromic anemia, tissue Atransferrinemia iron deposition mk DMT1 (G185R) SMM Systemic iron deficiency; impaired iron ND uptake in the duodenum and in erythroid precursors b (Belgrade rat) DMT1 (G185R) SRM Systemic iron deficiency; impaired iron ND uptake in duodenum and in erythroid precursors cdy (Chardonnay) DMT1 (missense, IZM Hypochromic anemia ND nonsense) weh (Weissherbst) Ferroportin IZM Hypochromic anemia, impaired iron ND transfer from yolk sac to embryo Cp / Ceruloplasmin TMM Iron accumulation in hepatocytes and Aceruloplasminemia macrophages sla Hephaestin (deletion) SMM Microcytic hypochromic anemia, impaired ND intestinal iron transfer Fth / H-Ferritin d TMM Elevated tissue and serum L-ferritin ND Ireb2 / IRE binding protein 2 TMM Iron deposition in enterocytes, neurons ND and oligodendrocytes f (flexed tail) Sideroflexin 1 SMM Transient fetal and neonatal anemia with ND (frameshift) intracellular iron deposits Frda - tissue specific k.o. Frataxin TMM Mitochondrial iron deposits, neurodegen- Friedreich ataxia neuron/heart e eration and cardiomyopathy Frda - tissue specific k.o. Frataxin TMM Mitochondrial iron deposits; Friedreich ataxia muscle e cardiomyopathy Hmox1 / Heme oxygenase 1 TMM Anemia, low serum iron levels, tissue iron Hmox 1 deficiency deposition ND: not described; SMM: spontaneous mouse mutant; TMM: targeted mouse mutant; NA: not applicable; SRM: spontaneous rat mutant; TgM: transgenic mouse; HC: hemochromatosis; IZM: induced zebrafish mutant; CMM: compound mutant mouse a Mutation is in mouse codon 294. b Includes deletion of gene for upstream stimulatory factor-2. c Trfr / mice: embryonic lethal by E d Fth / mice: early embryonic lethality. e Frda / mice: early embryonic lethality; beta-cell ko: loss of beta cells, diabetes mellitus. f Reduction in red cell size and hemoglobin content. suspected links between proteins with other functions There is substantial variation in baseline iron parameters and the regulation of iron balance (e.g., hepcidin). among inbred mouse strains. In the coming years, Breeding and analysis of compound mutants extend quantitative trait locus analysis in mice is likely to yield the power of these genetic studies. Compound mutants information on genes that modify the hemochromatosis have confirmed that iron loading in HFE hemochro- phenotype and/or predispose to iron deficiency in human matosis results from enhanced flux of iron through patients. known transport pathways, rather than through activa- The Iron-Immune System Connection tion of an accessory absorptive pathway (Levy et al., There are remarkable interconnections between iron 2000). As in human patients, HFE hemochromatosis pro- metabolism and the immune system (De Sousa et al., motes a porphyric phenotype in mice heterozygous for 1988). DMT1, the major iron uptake molecule, is a close mutations in the uroporphyrinogen decarboxylase gene homolog of Nramp1, a metal transporter localized to (Phillips et al., 2001). phagolysosomes, which mediates macrophage resis-

10 Cell 294 tance against intracellular pathogens. Tf and TfR1 play oxidative stress without frank neurodegeneration (Thompson essential roles in lymphocyte development and activation. et al., 2003). 2-microglobulin, the heterodimeric partner of Dramatic, localized, iron accumulation is observed in HFE, is a key component of the immune surveillance Hallervorden-Spatz neurodegenerative disease, which mechanism. Lactoferrin and transferrin bind iron to se- is due to mutations in a pantothenate kinase (PANK) quester it from invading pathogens. Hepcidin has intrinsic gene. PANK is essential for the synthesis of coenzyme antimicrobial activity of uncertain importance. Heme A from pantothenate, and its dysfunction results in the oxygenase has immunosuppressive and antiapoptotic accumulation of cysteine in the basal ganglia. As cys- properties. As described earlier, inflammatory cytokines teine efficiently binds iron, it was postulated that this have direct and indirect effects on expression of proteins results in tissue damage by promoting oxidative stress of iron metabolism. Intriguing as it is, we do not (Zhou et al., 2001). An alternative name for this disorder, yet understand the reasons for this complex relationship PKAN (panthothenate kinase-associated neurode- between iron and immunity. generation), has been proposed. HFE is structurally related to major histocompatibility Iron deficiency also has deleterious consequences class I molecules, suggesting an evolutionary and/or for the brain, particularly in early life. Iron deficiency functional link between HFE and the immune system. appears to alter the chemistry of neurotransmission, As described above, HFE is required for appropriate neuronal networks, and myelination. Effects on dopamine hepcidin expression in response to iron overload and receptor and transporter function have been rehepcidin lipopolysaccharide-induced inflammation. Although the ported (Beard, 2003). physiological function of hepcidin as an antimicrobial In spite of these multiple links between iron metabo- effector of the innate immune response warrants further lism, neuronal function, and neurodegeneration, we are investigation, the structural similarities between hepmetabolism still far from understanding many basic aspects of iron cidin and the -defensins suggests another intriguing in the brain. Further convergence between evolutionary link. These connections and parallels can neurobiology and the study of iron metabolism will con- be rationalized to some extent by considering that iron tribute critical insights into normal brain function and is an essential nutrient for invading microbes, and sequestration common neuropathologies. of iron is an effective measure against them. Undoubtedly, however, there are other important links The Challenges Ahead that are yet to be discovered and understood. Although proteins required for heme biosynthesis and Iron in the Brain Fe-S cluster assembly have been identified, we know Deposition of excess iron in the brain is commonly obmitochondria. little about intracellular iron trafficking, particularly to served in neurodegenerative disorders. In some diseases Some of these questions can be ad- (e.g., Parkinson s, Alzheimer s), it is not clear whether dressed using yeast as a model system. Disease models defects in cerebral iron homeostasis cause degenerato such as frataxin- or ABC7-deficient mice will contribute tive processes or whether iron accumulates as a consemals the understanding of Fe-S cluster assembly in mam- quence of another insult. However, accumulating eviidentification and intracellular iron management in general. The dence implicates aberrant neuronal iron metabolism as of other disorders associated with defects a pathogenic factor (Halliwell, 2001; Perry et al., 2002). in Fe-S protein assembly may bring forward new players In Friedreich ataxia, the most common hereditary involved in mammalian mitochondrial iron metabolism. ataxia, a functional deficiency of frataxin affects intracelsystemic On a grander scale, the problem is to understand lular iron distribution as well as Fe-S cluster biogenesis iron homeostasis: how tissues communicate and results in iron accumulation in mitochondria (see information about iron needs and coordinate iron deliv- above). While the molecular pathogenesis is still unclear, ery at a distance. The urgent issue is to understand mitochondrial dysfunction may result from iron-catadetail, the regulation of intestinal iron absorption in molecular lyzed formation of reactive oxygen species, leading to to facilitate the development of new treatments progressive neurodegeneration (Pandolfo, 2002). As disthis for iron deficiency and overload disorders. Efforts to do cussed earlier, aceruloplasminemia leads to defective will likely be facilitated by emerging concepts in cellular iron export and iron accumulation in several systems biology. Large datasets derived from genomic organs, including the brain. Increased iron levels and and proteomic studies may require an integrative, com- consequent lipid peroxidation correlate with the extent putational approach to define the complexities of iron of neurodegeneration in humans and Cp-deficient mice homeostasis at the level of the whole organism inter- (Miyajima et al., 2002). Similarly, a mutation in the car- acting with its environment. boxyl terminus of the ferritin L chain results in a rare Finally, a combination of insights into cellular iron memovement disorder, neuroferritinopathy. Spherical ingrate tabolism and systemic iron homeostasis will help to inte- clusions of ferritin and iron accumulate in the basal gantype-specific our understanding of local, organ-specific or cell glia of patients together with ubiquitin and tau (Curtis management of iron. Iron metabolism cerglia et al., 2001). tainly is not a rusty topic! In mice, global loss of IRP2 results in abnormal iron We apologize to all our colleagues whose work we deposition in the CNS, causing abnormal movements could not cite due to limitations in space. (ataxia, bradykinesia) and tremors. It has been proposed that, in the absence of IRP2, overexpression of H- and References L-ferritin causes axonal degeneration (LaVaute et al., Abboud, S., and Haile, D.J. (2000). 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