Two to Tango: Regulation of Mammalian Iron Metabolism

Disruptions in iron homeostasis from both iron deficiency and overload account for some of the most common human diseases. Iron metabolism is balanced by two regulatory systems, one that functions systemically and relies on the hormone hepcidin and the iron exporter ferroportin, and another that predominantly controls cellular iron metabolism through iron-regulatory proteins that bind iron-responsive elements in regulated messenger RNAs. We describe how the two distinct systems function and how they "tango" together in a coordinated manner. We also highlight some of the current questions in mammalian iron metabolism and discuss therapeutic opportunities arising from a better understanding of the underlying biological principles. Disruptions in iron homeostasis from both iron deficiency and overload account for some of the most common human diseases. Iron metabolism is balanced by two regulatory systems, one that functions systemically and relies on the hormone hepcidin and the iron exporter ferroportin, and another that predominantly controls cellular iron metabolism through iron-regulatory proteins that bind iron-responsive elements in regulated messenger RNAs. We describe how the two distinct systems function and how they "tango" together in a coordinated manner. We also highlight some of the current questions in mammalian iron metabolism and discuss therapeutic opportunities arising from a better understanding of the underlying biological principles. Iron is essential for fundamental metabolic processes in cells and organisms. The key to systemic iron supply and homeostasis lies in the regulation of adequate plasma iron levels. Iron circulates in plasma bound to the glycoprotein transferrin, which has two high-affinity binding sites for Fe(III). Transferrin binding maintains iron in a soluble form, serves as a major vehicle for iron delivery into cells (via the transferrin receptor, TfR1), and limits the generation of toxic radicals. In humans, plasma transferrin is normally about 30% saturated with iron. A transferrin saturation <16% indicates iron deficiency, whereas >45% saturation is a sign of iron overload. When the saturation exceeds 60%, non-transferrin-bound iron begins to accumulate in the circulation and to damage parenchymal cells. The homeostatic system thus has to maintain transferrin saturation at physiological levels, responding to signals from pathways that consume iron (such as erythropoiesis) and sending signals to the cells that supply iron to the blood stream (Figure 1). Iron is released into the circulation from duodenal enterocytes, which absorb 1–2 mg of dietary iron per day, and from macrophages, which internally recycle 20–25 mg of iron from senescent erythrocytes. Hepatocytes play a dual role in systemic iron metabolism: they are the major site of iron storage and they secrete the regulatory hormone hepcidin (Hamp, LEAP1). Hepcidin orchestrates systemic iron fluxes and controls plasma iron levels by binding to the iron exporter ferroportin (SLC40A1, Solute carrier family 40, member 1) on the surface of iron-releasing cells (Figure 1), triggering its degradation and hence reducing iron transfer to transferrin (Nemeth et al., 2004Nemeth E. Tuttle M.S. Powelson J. Vaughn M.B. Donovan A. Ward D.M. Ganz T. Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3043) Google Scholar). Inherited and acquired disorders that perturb hepcidin production consequently cause iron deficiency (high hepcidin levels) or iron overload (hepcidin deficiency). Assessing the concentration of serum ferritin is a clinically useful measure of iron storage. Low serum ferritin levels indicate depleted stores, whereas increased levels may indicate iron overload. Inflammatory conditions (or infections, cancer, and liver disorders) can also increase serum ferritin. Given its clinical utility, it is surprising that the physiological function(s) of serum ferritin and its source (that is, whether it is derived from damaged cells or actively secreted by a regulated mechanism) still remain to be defined. Serum ferritin is predominantly composed of L chain subunits, partially glycosylated, and iron poor. Inorganic dietary iron is absorbed at the brush border of duodenal enterocytes via the divalent metal transporter 1 (DMT1/SLC11A2, solute carrier family 11, member 2) (Gunshin et al., 1997Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Cloning and characterization of a mammalian proton-coupled metal-ion transporter.Nature. 1997; 388: 482-488Crossref PubMed Scopus (2459) Google Scholar). Given that iron largely adopts the oxidized state, it must first be reduced by the membrane-associated ferrireductase DcytB (Cybrd1). DcytB may not be the only ferrireductase of the apical membrane of enterocytes, as knockout mice appear to have normal iron metabolism (see review by McKie, 2008McKie A.T. The role of Dcytb in iron metabolism: an update.Biochem. Soc. Trans. 2008; 36: 1239-1241Crossref PubMed Scopus (66) Google Scholar). Heme iron is absorbed independently by mechanisms that remain uncertain, because the proposed transporter SLC46A1 appears to carry mostly folate (Qiu et al., 2006Qiu A. Jansen M. Sakaris A. Min S.H. Chattopadhyay S. Tsai E. Sandoval C. Zhao R. Akabas M.H. Goldman I.D. Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption.Cell. 2006; 127: 917-928Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). Heme iron is released intracellularly by hemoxygenase, mainly by the inducible hemoxygenase 1 (HOX1) (Ferris et al., 1999Ferris C.D. Jaffrey S.R. Sawa A. Takahashi M. Brady S.D. Barrow R.K. Tysoe S.A. Wolosker H. Barañano D.E. Doré S. et al.Haem oxygenase-1 prevents cell death by regulating cellular iron.Nat. Cell Biol. 1999; 1: 152-157Crossref PubMed Scopus (451) Google Scholar). Cytosolic iron can then be exported into the circulation by the basolateral iron exporter ferroportin (McKie et al., 2000McKie A.T. Marciani P. Rolfs A. Brennan K. Wehr K. Barrow D. Miret S. Bomford A. Peters T.J. Farzaneh F. et al.A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation.Mol. Cell. 2000; 5: 299-309Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar, Donovan et al., 2000Donovan A. Brownlie A. Zhou Y. Shepard J. Pratt S.J. Moynihan J. Paw B.H. Drejer A. Barut B. Zapata A. et al.Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter.Nature. 2000; 403: 776-781Crossref PubMed Scopus (1232) Google Scholar). Enterocytic iron export through ferroportin requires hephaestin, a multicopper oxidase homologous to ceruloplasmin, which oxidases Fe(II) to Fe(III) for loading onto transferrin. Consistent with this function, hephaestin-deficient mice display iron deficiency anemia with mucosal iron retention. Because iron cannot be excreted from the organism in a regulated way, iron absorption represents the critically controlled process. Normally, only 1–2 mg of iron per day are absorbed to compensate for iron losses, for example by sloughing of intestinal epithelial cells, desquamation of skin and urinary cells, blood loss, or sweat. Iron absorption can be enhanced when the needs are higher (for example, because of increased erythropoiesis or pregnancy) and suppressed in iron overload. The lack of an active mechanism for iron excretion accounts for the development of iron overload when the regulation of iron absorption is defective or bypassed (as occurs in blood transfusions). The vast majority of recycled iron (∼25 mg/day) is dedicated to hemoglobin synthesis. TfR1 mediates erythroid iron acquisition, and its expression parallels the maturation of erythroid progenitors. Mouse embryos lacking TfR1 die because of severe anemia (and with neurologic abnormalities), whereas Tfr1 haploinsufficiency or dysfunction of other components of the TfR1 endocytotic cycle (such as DMT1, STEAP3, or EXOC6, see below) cause microcytic anemia (characterized by abnormally small red blood cells) as a result of defective iron utilization in mice; mutations of DMT1 in humans elicit a similar phenotype and cause liver iron accumulation (Table S1 available online) (see review by Iolascon et al., 2009Iolascon A. De Falco L. Beaumont C. Molecular basis of inherited microcytic anemia due to defects in iron acquisition or heme synthesis.Haematologica. 2009; 94: 395-408Crossref PubMed Scopus (100) Google Scholar). A proposed additional route of erythroblast iron acquisition is ferritin released from macrophages in the so-called "erythroblastic islands" (Leimberg et al., 2008Leimberg M.J. Prus E. Konijn A.M. Fibach E. Macrophages function as a ferritin iron source for cultured human erythroid precursors.J. Cell. Biochem. 2008; 151: 88-96Google Scholar). However, the severe iron deficiency anemia of both mice and patients with transferrin deficiency (Table S1) suggests that this process can at best make a minor contribution to erythroid iron acquisition. Erythroblasts not only acquire but also handle large amounts of iron. Potentially, iron may be directly transported from endosomes into mitochondria by a "kiss-and-run mechanism" through a direct contact between both organelles, effectively bypassing the cytosol (Sheftel et al., 2007Sheftel A.D. Zhang A.S. Brown C. Shirihai O.S. Ponka P. Direct interorganellar transfer of iron from endosome to mitochondrion.Blood. 2007; 110: 125-132Crossref PubMed Scopus (178) Google Scholar). Iron is imported into mitochondria by the inner membrane protein mitoferrin 1 (Mfrn1/ SLC25A37, solute carrier family 25, member 37) (Shaw et al., 2006Shaw G.C. Cope J.J. Li L. Corson K. Hersey C. Ackermann G.E. Gwynn B. Lambert A.J. Wingert R.A. Traver D. et al.Mitoferrin is essential for erythroid iron assimilation.Nature. 2006; 440: 96-100Crossref PubMed Scopus (367) Google Scholar). This process is facilitated by the ABCB10 (ATP-binding cassette, subfamily B, member 10) protein, which is thought to stabilize Mfrn1 (Chen et al., 2009Chen W. Paradkar P.N. Li L. Pierce E.L. Langer N.B. Takahashi-Makise N. Hyde B.B. Shirihai O.S. Ward D.M. Kaplan J. Paw B.H. Abcb10 physically interacts with mitoferrin-1 (Slc25a37) to enhance its stability and function in the erythroid mitochondria.Proc. Natl. Acad. Sci. USA. 2009; 106: 16263-16268Crossref PubMed Scopus (139) Google Scholar). Mfrn2/SLC25A28 may represent the housekeeping Mfrn1 homolog. To coordinate the synthesis of the heme precursor protoporphyrin IX with iron availability, δ-aminolevulinic acid synthase 2 (ALAS2), the erythroid-specific first enzyme of protoporphyrin IX synthesis, is posttranscriptionally regulated by iron via the iron-responsive element/iron-regulatory protein (IRE/IRP) system (see below). Genetic defects in ALAS2 cause sideroblastic anemia (Table S1), whereas haploinsufficiency of other enzymes in the pathway cause porphyrias, due to accumulation of toxic heme precursors (see review by Puy et al., 2010Puy H. Gouya L. Deybach J.C. Porphyrias.Lancet. 2010; 375: 924-937Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). How heme is exported from mitochondria remains to be defined. Mitochondrial iron uptake without the ability to use it for heme or Fe/S cluster biogenesis causes iron accumulation, because the excess import is not properly balanced by the export of products. Such mitochondrial iron depositions lead to the appearance of "ringed" sideroblasts (erythroblasts with perinuclear iron accumulations) and occur in conditions such as X-linked sideroblastic anemia due to ALAS2 deficiency or in the autosomal recessive deficiency of SLC25A38 (solute carrier family 25, member 38), a mitochondrial transporter likely involved in the import of ALA substrate glycine (Table S1). Anemia and ringed sideroblasts also appear when proteins involved in Fe/S cluster biogenesis are defective, such as impairment of GLRX5 (glutaredoxin 5) (Ye et al., 2010Ye H. Jeong S.Y. Ghosh M.C. Kovtunovych G. Silvestri L. Ortillo D. Uchida N. Tisdale J. Camaschella C. Rouault T.A. Glutaredoxin 5 deficiency causes sideroblastic anemia by specifically impairing heme biosynthesis and depleting cytosolic iron in human erythroblasts.J. Clin. Invest. 2010; 120: 1749-1761Crossref PubMed Scopus (164) Google Scholar) or the ATP-binding cassette protein ABCB7, which leads to sideroblastic anemia with ataxia (Table S1). Although erythroblasts consume large amounts of iron, they have to maintain safety mechanisms to avoid iron and/or heme excess: iron can be stored in ferritin or exported by ferroportin. Erythroblasts express a ferroportin messenger RNA (mRNA) isoform (1b) that lacks the 5′ IRE and thus evades potential translational repression by IRPs (see below) (Zhang et al., 2009Zhang D.L. Hughes R.M. Ollivierre-Wilson H. Ghosh M.C. Rouault T.A. A ferroportin transcript that lacks an iron-responsive element enables duodenal and erythroid precursor cells to evade translational repression.Cell Metab. 2009; 9: 461-473Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). This isoform is susceptible to hepcidin degradation and may endow erythroid precursors with a mechanism to respond to systemic iron availability. Additionally, erythroblasts have the capacity to export excess heme (for example, when globin synthesis is limiting). The proposed exporter, FLVCR (feline leukemia virus subgroup C cellular receptor), is a multitransmembrane protein, a member of the major facilitator superfamily, and a receptor for a virus that causes severe aplastic anemia in cats. Accumulation of toxic heme at the proerythroblast stage can cause apoptosis, and mice with neonatal FLVCR deficiency develop severe hyperchromic, macrocytic anemia, reticulocytopenia, and a block in erythroid maturation at the proerythroblast stage (Keel et al., 2008Keel S.B. Doty R.T. Yang Z. Quigley J.G. Chen J. Knoblaugh S. Kingsley P.D. De Domenico I. Vaughn M.B. Kaplan J. et al.A heme export protein is required for red blood cell differentiation and iron homeostasis.Science. 2008; 319: 825-828Crossref PubMed Scopus (253) Google Scholar). Macrophages have to shoulder the lion's share of the burden of maintaining adequate levels of plasma iron. Given that less than 10% of the daily iron needs are met by intestinal absorption, the rest is covered by macrophages that recycle iron internally. The amount of plasma iron is just over 10% of the amount used daily, which means that plasma iron is turned over many times each day. Macrophages phagocytose aged or damaged erythrocytes and catabolize heme using hemoxygenase. NRAMP1 (natural resistance-associated macrophages protein 1), a divalent metal transporter homologous to DMT1, is expressed within phagolysosomal membranes and participates in iron export from phagocytic vesicles (Soe-Lin et al., 2009Soe-Lin S. Apte S.S. Andriopoulos Jr., B. Andrews M.C. Schranzhofer M. Kahawita T. Garcia-Santos D. Ponka P. Nramp1 promotes efficient macrophage recycling of iron following erythrophagocytosis in vivo.Proc. Natl. Acad. Sci. USA. 2009; 106: 5960-5965Crossref PubMed Scopus (102) Google Scholar). Export of ferrous iron from macrophages occurs via ferroportin (Figure 1). Reflecting its central role in systemic iron homeostasis, ferroportin expression in macrophages is regulated at multiple levels: ferroportin transcription is induced by erythrophagocytosis and heme iron, its translation is regulated by the IRE/IRP system, and its protein stability by hepcidin (see below). Ferroportin-mediated iron export is coupled to the function of the multicopper oxidase ceruloplasmin, a protein synthesized and secreted by the liver. Ceruloplasmin-deficient mice and humans show hepatocyte and macrophage iron accumulation. Aceruloplasminemia causes anemia (highlighting the critical role of iron release for erythropoiesis), diabetes, a late-onset disorder of the basal ganglia, and retinal degeneration (Table S1). Hepcidin has emerged as the central regulatory molecule of systemic iron homeostasis. It is a defensin family member with strong links to innate immunity. The bioactive, mature 25 amino acid peptide is generated from an 84 amino acid prepropeptide by furin cleavage. Hepcidin is secreted from hepatocytes and circulates in plasma bound to α2-macroglobulin (Peslova et al., 2009Peslova G. Petrak J. Kuzelova K. Hrdy I. Halada P. Kuchel P.W. Soe-Lin S. Ponka P. Sutak R. Becker E. et al.Hepcidin, the hormone of iron metabolism, is bound specifically to alpha-2-macroglobulin in blood.Blood. 2009; 113: 6225-6236Crossref PubMed Scopus (85) Google Scholar). Hepcidin clearance occurs via the kidney or by codegradation with ferroportin. Hepcidin forms a hairpin structure with four intramolecular disulfide bonds (Jordan et al., 2009Jordan J.B. Poppe L. Haniu M. Arvedson T. Syed R. Li V. Kohno H. Kim H. Schnier P.D. Harvey T.S. et al.Hepcidin revisited, disulfide connectivity, dynamics, and structure.J. Biol. Chem. 2009; 284: 24155-24167Crossref PubMed Scopus (144) Google Scholar) and exhibits modest antimicrobial activity in vitro, which has not yet been demonstrated in vivo. Hepcidin binds to ferroportin, triggers its internalization, ubiquitination, and subsequent lysosomal degradation (Nemeth et al., 2004Nemeth E. Tuttle M.S. Powelson J. Vaughn M.B. Donovan A. Ward D.M. Ganz T. Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3043) Google Scholar). Ferroportin binding is mediated by the N terminus of the peptide; Jak2 (Janus kinase 2) has been reported to bind to the hepcidin-ferroportin complex and to phosphorylate ferroportin before internalization (De Domenico et al., 2009De Domenico I. Lo E. Ward D.M. Kaplan J. Hepcidin-induced internalization of ferroportin requires binding and cooperative interaction with Jak2.Proc. Natl. Acad. Sci. USA. 2009; 106: 3800-3805Crossref PubMed Scopus (98) Google Scholar). Research into the molecular mechanisms that underlie hereditary hemochromatosis in patients and murine models has been instrumental to decipher hepcidin regulation in mammals. Hereditary hemochromatosis is an autosomal recessive disease that leads to iron overload of the liver and other organs. Complications, which are preventable by iron depletion therapies, can be fatal and include liver cirrhosis, cancer, diabetes, hypogonadism, heart failure, and arthritis. Family studies implicate four genes in the disorder (Table S1): the most common type, which has a carrier frequency of ∼1:8 in Caucasian populations, is due to a homozygous missense mutation of the HFE gene (C282Y) (Feder et al., 1996Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo Jr., R. Ellis M.C. Fullan A. et al.A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3156) Google Scholar). Less common but clinically more severe forms of hereditary hemochromatosis are caused by mutations of the TfR2, hemojuvelin (HJV), or hepcidin (HAMP) genes. All recessive forms of the disease represent molecular defects of hepatocytes and are caused by inappropriately low hepcidin expression (Figure 2A ): the disease severity and the age of onset roughly correlate with the degree of hepcidin deficiency (see review by Camaschella, 2005Camaschella C. Understanding iron homeostasis through genetic analysis of hemochromatosis and related disorders.Blood. 2005; 106: 3710-3717Crossref PubMed Scopus (157) Google Scholar). HFE encodes a ubiquitously expressed major histocompatibility complex class 1-like molecule. The C282Y mutation abrogates β2-microglobulin binding and HFE surface expression; other HFE mutations are relatively rare. Hepcidin levels in patients may be normal, but are inadequately low for the degree of iron loading (Piperno et al., 2007Piperno A. Girelli D. Nemeth E. Trombini P. Bozzini C. Poggiali E. Phung Y. Ganz T. Camaschella C. Blunted hepcidin response to oral iron challenge in HFE-related hemochromatosis.Blood. 2007; 110: 4096-4100Crossref PubMed Scopus (111) Google Scholar, Ganz et al., 2008Ganz T. Olbina G. Girelli D. Nemeth E. Westerman M. Immunoassay for human serum hepcidin.Blood. 2008; 112: 4292-4297Crossref PubMed Scopus (505) Google Scholar) and display a blunted response to acute oral iron challenges (Piperno et al., 2007Piperno A. Girelli D. Nemeth E. Trombini P. Bozzini C. Poggiali E. Phung Y. Ganz T. Camaschella C. Blunted hepcidin response to oral iron challenge in HFE-related hemochromatosis.Blood. 2007; 110: 4096-4100Crossref PubMed Scopus (111) Google Scholar). The penetrance of HFE mutations is low and clinical manifestations occur most commonly in middle aged males, indicating the importance of environmental and/or additional genetic factors for disease expression (Beutler et al., 2002Beutler E. Felitti V.J. Koziol J.A. Ho N.J. Gelbart T. Penetrance of 845G—> A (C282Y) HFE hereditary haemochromatosis mutation in the USA.Lancet. 2002; 359: 211-218Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar). Juvenile hereditary hemochromatosis due to mutations in the HAMP or HJV genes may lead to irreversible hypogonadism, refractory heart failure, and even death in the second to third decades of life. Patients with HAMP or HJV mutations are phenotypically similar and have virtually undetectable hepcidin levels. HJV is a glycophosphatidlyinositol-linked protein, homologous to repulsive guidance molecules, and mostly expressed in liver, skeletal muscle, and heart. HJV is a bone morphogenetic protein (BMP) coreceptor (Babitt et al., 2006Babitt J.L. Huang F.W. Wrighting D.M. Xia Y. Sidis Y. Samad T.A. Campagna J.A. Chung R.T. Schneyer A.L. Woolf C.J. et al.Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.Nat. Genet. 2006; 38: 531-539Crossref PubMed Scopus (743) Google Scholar) that is required to drive hepcidin transcription via SMAD proteins (Figure 2A) (see below). HAMP mutations are extremely rare. Mice lacking HAMP and HJV recapitulate the organ iron loading observed in humans (Table S1). Hereditary hemochromatosis due to TfR2 mutations may present early, but with a less severe phenotype than the juvenile form (see review by Camaschella, 2005Camaschella C. Understanding iron homeostasis through genetic analysis of hemochromatosis and related disorders.Blood. 2005; 106: 3710-3717Crossref PubMed Scopus (157) Google Scholar). TfR2 is a type II transmembrane protein that binds transferrin with lower affinity than TfR1 (Figure 2A). Targeted Tfr2 gene deletion in mice causes iron overload with low basal hepcidin levels (Table S1); similar observations have been reported in humans with TFR2 mutations. A dominant form of hereditary hemochromatosis is caused by missense mutations in ferroportin (Table S1). Ferroportin is the only known cellular iron exporter and represents the "hepcidin receptor." Mutations that reduce its membrane localization or its ability to export iron cause macrophage iron retention, normal/low plasma iron levels, and in some cases iron-restricted erythropoiesis. A hemochromatosis-like disease with high plasma iron and hepatocyte iron accumulation is caused by hepcidin-resistant ferroportin mutations either because hepcidin fails to bind ferroportin (C326S) or the internalization and degradation of ferroportin following hepcidin binding is impaired (Fernandes et al., 2009Fernandes A. Preza G.C. Phung Y. De Domenico I. Kaplan J. Ganz T. Nemeth E. The molecular basis of hepcidin-resistant hereditary hemochromatosis.Blood. 2009; 114: 437-443Crossref PubMed Scopus (111) Google Scholar). Hepcidin levels are also inappropriately low in "iron-loading anemias" in which erythropoietic signals suppress hepcidin transcription (Figure 2B) even when systemic iron load is high. The prototype of these anemias is β-thalassemia intermedia, characterized by transfusion-independent iron overload and low to absent hepcidin levels. Growth differentiation factor 15 (GDF15) and twisted gastrulation 1 (TWSG1) released by erythroblasts have been proposed to be involved in hepcidin suppression (Tanno et al., 2007Tanno T. Bhanu N.V. Oneal P.A. Goh S.H. Staker P. Lee Y.T. Moroney J.W. Reed C.H. Luban N.L. Wang R.H. et al.High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin.Nat. Med. 2007; 13: 1096-1101Crossref PubMed Scopus (581) Google Scholar, Tanno et al., 2009Tanno T. Porayette P. Sripichai O. Noh S.J. Byrnes C. Bhupatiraju A. Lee Y.T. Goodnough J.B. Harandi O. Ganz T. et al.Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells.Blood. 2009; 114: 181-186Crossref PubMed Scopus (264) Google Scholar). In patients with homozygous β-thalassemia or other anemias with ineffective erythropoiesis, elevated serum GDF15 correlates with diminished hepcidin levels and increased iron absorption. Iron (blood) losses and/or insufficient iron intake/absorption from dietary sources can cause iron deficiency that most commonly manifests as microcytic anemia. Likewise, inappropriately high hepcidin expression lowers plasma iron levels (due to diminished iron release by macrophages and lower iron absorption) and causes anemia. In this context, the common acquired anemia of chronic diseases (ACD) and the genetic iron-refractory iron deficiency anemia (IRIDA) are most interesting. Related to its evolutionary origin, hepcidin transcription is activated by inflammatory cytokines, especially interleukin 6 (Figure 2C). Hypoferremia develops rapidly as a result of decreased macrophage iron release and represents a defense mechanism against (iron-dependent) pathogens. Excessive hepcidin production is also seen in patients with infections, malignancies, chronic kidney diseases, or any type of inflammation. If prolonged, it leads to ACD. In rare cases, hepcidin can be expressed ectopically by hepatic adenomas, which results in microcytic anemia with some features of ACD (Weinstein et al., 2002Weinstein D.A. Roy C.N. Fleming M.D. Loda M.F. Wolfsdorf J.I. Andrews N.C. Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease.Blood. 2002; 100: 3776-3781Crossref PubMed Scopus (508) Google Scholar), but this anemia is fully reversible after removal of the adenoma. Patients with iron deficiency normally have low or undetectable levels of hepcidin. This is not the case in patients with IRIDA, who suffer from a microcytic anemia that is unresponsive to oral and partially refractory to parenteral iron, because of inappropriately high hepcidin levels. IRIDA is caused by mutations in TMPRSS6 (matriptase-2), a gene that encodes a protease that negatively regulates hepcidin expression (Du et al., 2008Du X. She E. Gelbart T. Truksa J. Lee P. Xia Y. Khovananth K. Mudd S. Mann N. Moresco E.M. et al.The serine protease TMPRSS6 is required to sense iron deficiency.Science. 2008; 320: 1088-1092Crossref PubMed Scopus (416) Google Scholar) (see below). Interestingly genetic variants in TMPRSS6, frequent in the general population, may modulate the ability to absorb iron and to synthesize hemoglobin for maturing erythroid cells (Andrews, 2009Andrews N.C. Genes determining blood cell traits.Nat. Genet. 2009; 41: 1161-1162Crossref PubMed Scopus (21) Google Scholar). Whether TMPRSS6 variants may contribute to sporadic iron deficiency by increased hepcidin levels and decreased dietary iron absorption remains to be explored. Hepcidin expression in hepatocytes is regulated by multiple, in part opposing signals, including systemic iron availability (such as diferric transferrin, Tf-Fe2), hepatic iron stores, erythropoietic activity, hypoxia, and inflammatory/infectious states (Figure 2). These different regulatory inputs are integrated transcriptionally. After the discovery of the biological relevance of hepcidin, important progress has been made toward understanding the molecules and pathways that control hepcidin expression in response to iron and the role of the membrane proteins mutated in hereditary hemochromatosis (HFE, HJV, and TfR2) in this process (Figure 2A). HFE has been suggested to act as a bimodal switch between two sensors of the concentration of Tf-Fe2, TfR1, and TfR2, on the plasma membrane of hepatocytes (Goswami and Andrews, 2006Goswami T. Andrews N.C. Hereditary hemochromatosis protein, HFE, interaction with transferrin receptor 2 suggests a molecular mechanism for mammalian iron sensing.J. Biol. Chem. 2006; 281: 28494-28498Crossref PubMed Scopus (271) Google Scholar). This model is supported by the following findings: HFE binds the ubiquitously expressed TfR1 at a site that overlaps the transferrin binding domain, and Tf-Fe2 thus competes with HFE binding to TfR1. By contrast, TfR2 can bind both HFE and Tf-Fe2 simultaneously (Gao et al., 2009Gao J. Chen J. Kramer M. Tsukamoto H. Zhang A.S. Enns C.A. Interaction of the hereditary hemochromatosis protein HFE with transferrin receptor 2 is required for transferrin-induced hepcidin expression.Cell Metab. 2009; 9: 217-227Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Mice bearing an engineered TfR1 mutation with increased HFE binding show low hepcidin expression and systemic iron overload similar to HFE-deficient mice, suggesting that the TfR1 sequesters HFE to prevent its participation in hepcidin activation. Conversely, mutations that abolish the HFE-TfR1 interaction or mice with increased HFE levels display elevated hepcidin expression and succumb to iron deficiency (Schmidt et al., 2008Schmidt P.J. Toran P.T. Giannetti