Erythropoiesis Iron Metabolism: Interactions with Normal And

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Iron Metabolism: Interactions with Normal and Disordered Erythropoiesis

Tomas Ganz and Elizabeta Nemeth

Cold Spring Harb Perspect Med 2012; doi: 10.1101/cshperspect.a011668

Subject Collection Hemoglobin and Its Diseases

Hemoglobin Variants: Biochemical Properties and The Prevention of Thalassemia Clinical Correlates Antonio Cao and Yuet Wai Kan Christopher S. Thom, Claire F. Dickson, David A. Gell, et al. Classification of the Disorders of Hemoglobin The Switch from Fetal to Adult Hemoglobin Bernard G. Forget and H. Franklin Bunn Vijay G. Sankaran and Stuart H. Orkin The Molecular Basis of α-Thalassemia Pathophysiology and Clinical Manifestations of Douglas R. Higgs the β-Thalassemias Arthur W. Nienhuis and David G. Nathan Evolution of Hemoglobin and Its Genes Development of Gene Therapy for Thalassemia Ross C. Hardison Arthur W. Nienhuis and Derek A. Persons The Search for Genetic Modifiers of Disease α-Thalassemia, Mental Retardation, and Severity in the β-Hemoglobinopathies Myelodysplastic Syndrome Guillaume Lettre Richard J. Gibbons World Distribution, Population Genetics, and β-Thalassemia Intermedia: A Clinical Perspective Health Burden of the Hemoglobinopathies Khaled M. Musallam, Ali T. Taher and Eliezer A. Thomas N. Williams and David J. Weatherall Rachmilewitz Iron Metabolism: Interactions with Normal and Hematopoietic Stem Cell Transplantation in Disordered Erythropoiesis Thalassemia and Sickle Cell Anemia Tomas Ganz and Elizabeta Nemeth Guido Lucarelli, Antonella Isgrò, Pietro Sodani, et al. Pluripotent Stem Cells in Research and Treatment of Hemoglobinopathies Natasha Arora and George Q. Daley

For additional articles in this collection, see http://perspectivesinmedicine.cshlp.org/cgi/collection/

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Iron Metabolism: Interactions with Normal and Disordered Erythropoiesis

Tomas Ganz and Elizabeta Nemeth

Department of Medicine and Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095 Correspondence: [email protected]

Hemoglobinopathies and other disorders of erythroid cells are often associated with abnormal iron homeostasis. We review the molecular physiology of intracellular and systemic iron regulation, and the interactions between erythropoiesis and iron homeostasis. Finally, we discuss iron disorders that affect erythropoiesis as well as erythroid disorders that cause iron dysregulation.

ron overload is a common complication of this environment, biological organisms evolved Ihemoglobinopathies treated by erythrocyte to conserve iron. Quantitative analysis of tissue transfusions (1 mL of packed erythrocytes con- iron distribution and ﬂuxes in humans illus- tains about 1 mg of iron) and those associated trates how this is accomplished (Finch 1994). with ineffective erythropoiesis, which stimulates The typical adult human male contains about the hyperabsorption of dietary iron. With the 4 g of iron of which about 2.5 g is in hemoglo- increasing use of transfusion therapy, iron over- bin, 1 g is stored predominantly in hepatocytes load has become a major cause of morbidity and and hepatic and splenic macrophages, and most premature mortality. More recently, the effective of the rest is distributed in myoglobin, cyto- treatment of iron overload by iron chelation has chromes, and other ferroproteins. Only about dramatically improved survival (Cunningham 1–2 mg/d, or ,0.05%/d, is lost from the body 2008; Telfer2009). This work reviews recent ad- predominantly through desquamation and mi- vances in our understanding of the molecular nor blood loss. In the steady state, this amount

www.perspectivesinmedicine.org basis of iron homeostasis and its disorders. is replaced through intestinal iron absorption. Although the loss of iron may increase slightly with increasing iron stores, these changes do not IRON BIOLOGY AND HOMEOSTASIS signiﬁcantly contribute to homeostasis; intestinal iron absorption is by far the predominant Iron Intake determinant of the iron content of the body. A Iron is the most abundant element on Earth by typical Western diet provides about 15 mg of mass and the fourth most abundant in the iron per day and only 10% is absorbed. Re- Earth’s crust but it readily oxidizes into insolu- covery from blood loss causes an increase in iron ble compounds with poor bioavailability. In absorption up to 20-fold, indicating that the

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T. Ganz and E. Nemeth

duodenum where iron absorption takes place so this compartment must turn over every few has a large reserve capacity for iron absorption. hours. Erythrocyte precursors take up iron al- Pathological increase of intestinal iron absorp- most exclusively through transferrin receptors tion is a common cause of iron overload, ac- (TfR1) so the iron supply to erythrocyte precur- counting for the excess iron in hereditary hemo- sors is completely dependent on plasma trans- chromatosis and untransfused b-thalassemia. ferrin. In contrast, hepatocytes and other non- Blood transfusions and parenteral administra- erythroid cells can also take up iron that is not tion of iron compounds bypass the regulatory bound to transferrin (nontransferrin-bound bottleneck of iron absorption and constitute iron or NTBI), a process that becomes impor- the other major cause of iron overload. tant during iron overload when plasma transferrin saturation reaches 100% (Breuer et al. 2000). The predominant cellular storage form Iron Recycling of iron is the hollow spherical protein ferritin Under normal circumstances, the reutilization whose cavity contains iron in ferric form com- of iron recycled from senescent cells accounts plexed with hydroxide and phosphate anions. for most of the iron flux in humans. With the erythrocyte lifespan of 120 d, 20–25 mg of iron Regulation of Plasma Iron Concentrations is required to replace the 20–25 mL of erythrocytes that must be produced every day to main- Despite varying dietary iron intake and changes tain a steady state. Other cell types also turn over in erythropoietic activity owing to occasional but their much lower iron content contributes or periodic blood loss, iron concentrations in relatively little to the iron flux. Macrophages in plasma normally remain in the 10–30 mM range. the liver, spleen, and marrow (formerly called Chronically low concentrations decrease iron the reticuloendothelial system) phagocytose se- supply to erythropoiesis and other processes nescent or damaged erythrocytes, degrade their leading to anemia and dysfunction of other hemoglobin to release heme, extract iron from cell types sensitive to iron deprivation. Chroni- heme using heme oxygenase (Poss and Tonega- cally high iron concentrations lead to intermit- wa 1997), and recycle the iron to the extracellu- tent or steady-state saturation of transferrinwith lar fluid and plasma. Steady-state iron flux from iron and the generation of NTBI with conse- recycling can increase up to 150 mg/d in con- quent deposition of excess iron in the liver, en- ditions with ineffective erythropoiesis in which docrine glands, cardiac myocytes, and other tis- the number of erythroid precursors is increased sues. Excess cellular iron may cause tissue injury and accompanied by the apoptosis of hemoglo- by catalyzing the generation of reactive oxygen www.perspectivesinmedicine.org binized erythrocyte precursors in the marrow species, which can cause DNA damage, lipid and shortened erythrocyte survival (Beguin peroxidation, and oxidation of proteins. et al. 1988). Systemic Iron Homeostasis Iron Distribution and Storage Phenomenological description of systemic iron Free iron is highly reactive and causes cell and homeostasis was developed starting in the 1930s tissue injury through its ability to catalyze the (Finch 1994). Homeostatic mechanisms regu- production of reactive oxygen species. In living late dietary iron absorption and iron deposition organisms, iron is complexed with proteins or into or withdrawal from stores depending on the small organic molecules (citrate, acetate), which amount of stored iron (“stores regulator”) and mitigate its reactivity. Transferrin is the physio- the requirements of erythropoiesis (“erythro- logical carrier of iron in plasma. Normally, only poietic regulator”). The description of the mo- 20%–40% of the available binding sites on lecular processes that underlie iron homeostasis transferrin molecules are occupied by ferric has progressed rapidly in the last two decades iron. The iron content of plasma is only 2–3 mg but is still not complete.

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Iron Metabolism

CELLULAR IRON REGULATION ferrin-TfR1 complex returns to the cell membrane (Fig. 1). The neutral pH at the membrane Cellular Iron causes the apotransferrin to dissociate from Cells require iron predominantly for incorpora- TfR1, whereupon apotransferrin diffuses away tion into various ferroproteins, where iron exists to be loaded with iron again, repeating the cycle. in iron–sulfur clusters, in heme or hemelike From the vesicle, iron is delivered to mitochon- prosthetic moieties, or in other more loosely dria where it is incorporated into protoporphy- associated forms. It now appears that most cell rin IX to form heme, or incorporated into na- types in the body autonomously regulate their scent iron–sulfur clusters. Alternatively, iron iron uptake solely to meet their individual re- can be exported from the vesicle into the cyto- quirements for iron. These cells do not export plasm where it is incorporated into cytoplasmic appreciable amounts of iron and are presumed ferroproteins or stored in cytoplasmic ferritin. to give up their iron only when they undergo cell Nontransferrin-bound iron (NTBI) (Breuer death and are recycled by macrophages. In con- et al. 2000) usually accumulates when the iron- trast, several specialized cell types supplyor store binding capacity of transferrin is exceeded and iron to meet the needs of the entire organism, then circulates complexed mostly with citrate or and are therefore equipped to export iron into acetate. Hemoglobinopathies such as b-thalas- extracellular ﬂuid and plasma. Iron-exporting semia major and intermedia are associated with cells include duodenal enterocytes that absorb particularly high levels of NTBI. Some cells, in- dietary iron, macrophages that recycle iron cluding hepatocytes, cardiomyocytes, and cells from senescent or dead cells, and macrophages of endocrine glands can take up NTBI, although and hepatocytes that store iron and release it to the precise mechanism is not well understood. meet systemic demand. During pregnancy, the Candidate NTBI transporters include L-type placental syncytiotrophoblast must transport voltage-gated calcium channels, DMT-1 and maternal iron into the fetal circulation to meet Zip14. the iron requirements of fetal growth and development. The endothelial cells that form the Intracellular Iron Transport blood–brain barrier must also selectively transport iron as it now appears that the iron con- To undergo transport to the cytoplasm or mi- centrations in the brain are not appreciably in- tochondria, ferric iron must be reduced to its creased in systemic iron overload disorders. ferrous form through the action of ferrireduc- Finally, erythroid precursors need much more tases. Recent studies indicate that Steap (six- iron than any other cell type as each cell synthe- transmembrane epithelial antigen of the pros- www.perspectivesinmedicine.org sizes more than a billion heme molecules, there- tate) proteins 1–4 are among the relevant fer- fore facing greater iron-homeostatic challenges. rireductases, with Steap3 having a particular importance in erythroid precursors (Fig. 1), as- sisted perhaps by Steap2 and Steap4 (Ohgami Cellular Iron Uptake et al. 2006). Toreach the cytoplasm, ferrous iron Transferrin-mediated iron uptake is the best un- must cross the membrane of the vesicle. In many derstood mechanism of cellular iron import. cells, the proton-dependent ferrous iron trans- Although the transferrin receptor (TfR1) is exporter divalent metal transporter-1 (DMT1) pressed in many cell types, erythrocyte precur- appears essential for iron transport from the sors contain most of the TfR1 molecules and vacuole into the cytoplasm but in macrophages take up the great majority of iron-transferrin its homolog natural resistance-associated mac- in the organism. Iron-transferrin is endocytosed rophage protein (Nramp1) may also contribute via the cell membrane TfR1 and internalized (Soe-Lin et al. 2009). Because of its chemical into endosomal recycling vesicles. As the vesicle reactivity, iron is chaperoned in the cytoplasm, acidiﬁes, the low pH releases the transferrin- at least in part by multifunctional poly(RC)- bound ferric iron and the iron-free (apo)trans- binding proteins (PCBPs) (Shi et al. 2008). In

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T. Ganz and E. Nemeth

Tf reloaded with Fe3+ by macrophages and duodenal enterocytes

Tf 3+ Fe Tf TfR1 TfR1

Decreased pH Steap3 Fe3+ Fe3+ 2+ Tf Tf Fe TfR1 TfR1 DMT1

2+ Fe Ch K & R Mfrn1 Hemoglobin

Heme Mfrn1 export

FLVCR1 Fpn Mitochondria

Figure 1. Iron trafﬁc in erythrocyte precursors synthesizing hemoglobin. Iron is taken up as diferric transferrin by the transferrin receptor (TfR1). Acidiﬁcation of the endocytic vesicle releases ferric iron from transferrin, and the membrane ferrireductase Steap3 reduces it to ferrous iron, which is then exported to the cytoplasm by DMT1. The complex of iron-free apotransferrin (Tf ) and TfR1 is returned to the plasma membrane where the neutral pH causes Tf to dissociate from its receptor. The transferrin cycle is completed when Tf is reloaded with ferric iron by duodenal enterocytes or iron-recycling macrophages. Ferrous iron exported by DMT1 may be delivered to mitochondrial mitoferrin-1 (Mfrn1) by direct contact (the kiss-and-run mechanism, K&R) or through intermediate transport by as-yet uncharacterized cytoplasmic chaperones (Fe2þCh). Mitoferrin-1 imports iron into mitochondria where iron is incorporated into newly synthesized heme. Heme is exported via an unknown exporter (Heme export) and incorporated into globin chains to generate hemoglobin. Under some circumstances, iron is exported as ferrous iron via ferroportin (Fpn) or as heme via feline leukemia virus C receptor (FLVCR1). www.perspectivesinmedicine.org

particular, PCBP1 mediates the delivery of iron Mitochondria and Iron to cytoplasmic ferritin and both PCBP1 and 2 are involved in the delivery of iron to cytoplas- Consistent with their autonomous evolutionary mic iron-dependent prolyl and asparaginyl hy- origin, mitochondria are equipped with distinct droxylases that mediate oxygen sensing (Nandal iron transporters. Iron uptake into mitochon- et al. 2011). It is not known how iron is trans- dria depends on the inner mitochondrial mem- ported to mitochondria. In erythroid cells, there brane proteins mitoferrin 1 and 2, with the for- is evidence for a “kiss-and-run” mechanism mer predominantly expressed in erythroid cells (Fig. 1) whereby iron could be transferred and the latter ubiquitously (Paradkar et al. 2009; from endosomal vesicles directly to mitochon- Troadec et al. 2011). In erythroid cells, mitofer- dria (Sheftel et al. 2007) but it is not clear how rin 1 interacts with the ATP-binding transporter much this mechanism contributes to the iron Abcb10 and with ferrochelatase to form a plau- ﬂux into mitochondria and whether it also sible pathway for the delivery of iron for heme functions in nonerythroid cell types. formation (Chen et al. 2010). How heme is

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Iron Metabolism

exported from mitochondria for incorporation and the enzymatic inhibition of aconitase has into hemoglobin and other hemoproteins is not the opposite effect (Bullock et al. 2010; Talbot known. Mitochondria also contain a distinct et al. 2011). Many mRNAs are regulated by the ferritin, mitochondrial ferritin, for local iron IRP/IRE system (Sanchez et al. 2011) and fall storage. into three classes: (1) iron acquisition, generally with IREs in the 30 region resulting in increased protein synthesis during cellular iron depriva- Cellular Iron Homeostasis tion; (2) iron utilization and storage, with IREs Cellular, as opposed to systemic, iron homeosta- in the 50 region, resulting in repressed protein sis assures that sufficient but not excessive synthesis during iron deprivation; and (3) iron amounts of iron are taken up by each cell to export, with IREs also in the 50 region and pro- meet its individual requirement for ferroprotein tein synthesis repressed during iron deprivation. synthesis. The system that has evolved relies Proteins subject to IRE/IRP regulation include on posttranscriptional regulation through the TfR1andDMT1involvedincellularironuptake, interaction of iron-regulatory proteins (IRP1 aminolevulinic acid synthase 2, which catalyzes and IRP2) with iron-regulatory elements (IREs) the first step of the heme synthesis pathway in in messenger RNAs (mRNAs) that encode key erythroid cells, the heavy and light subunits of iron transporters, ferroproteins, and enzymes ferritin involved in iron storage, and ferroportin, involved in iron-utilizing pathways. The IRE/ the iron exporter expressed in tissues and cells IRPsystem effectively regulates iron uptake, pro- involved in iron export to plasma. The net effect vides for the storage of excessiron in ferritin, and of the IRE/IRP response during cellular iron coordinates the synthesis of heme, iron–sulfur deficiency is to increase cellular iron uptake, clusters, and ferroproteins with the availability mobilize iron from cellular storage, decrease of iron. The system in effect acts to decrease iron utilization, and, when iron becomes suffi- wasted expenditure of synthetic energy and sub- cient or excessive to reverse these responses and strates, and to prevent accumulation of toxic direct more iron into cellular storage and ex- forms of iron. Target mRNAs contain IREs that port. Further fine-tuning of iron import and form characteristic stem-loop structures either export is achieved by differential splicing of tar- in the 50 region, where IRP binding represses get mRNAs in different tissues to either include translation and decreases protein synthesis, or or exclude IREs. As an example, systemic adap- in the 30 region where IRP binding prevents en- tation to iron deficiency may be facilitated by donucleases from cleaving sensitive regions of a ferroportin mRNA isoform that lacks IRE, the mRNA, thus stabilizing mRNA and increas- which may allow iron-transporting duodenal www.perspectivesinmedicine.org ing protein synthesis (Casey et al. 1988). IRP1 enterocytes to deliver iron to plasma for system- and IRP2 are structurally related but interact ic needs even if the enterocyte is sensing iron with iron in distinct ways. Both proteins bind deficiency, and may transfer iron from ery- to IREs when cellular iron levels are low. In the throid cells to other tissues more critically de- presence of iron, IRP1 incorporates an iron– pendent on iron (Zhang et al. 2009). sulfur cluster, does not bind IREs, and acts as an aconitase enzyme converting citrate to isoci- Generalized Regulation of Protein Synthesis trate in the Krebs cycle. In contrast, IRP2 is ubiq- by Iron in Erythroid Cells uitinated by a complex iron-dependent process and then degraded in proteasomes (Salahudeen In addition to the regulation of the synthesis of et al. 2009; Vashisht et al. 2009). The dual spe- individual proteins by iron, erythroid cells also cificity of IRP1/aconitase may have a functional contain a mechanism for a generalized adaptive role in the regulation of erythropoiesis by iron response to iron deficiency. This response is af- availability, as the provision of the aconitase fected by the heme-regulated inhibitor kinase product isocitrate reverses some of the suppres- (HRI) belonging to a class of kinases activated sive effect of iron deprivation on erythropoiesis bycellularstress,includingnutrientdeprivation,

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T. Ganz and E. Nemeth

viralinfection,andendoplasmicreticulumstress cellular fluid and blood plasma, and the release (Chen 2007). During iron deficiency as heme of iron from macrophages involved in iron re- concentrations drop, heme dissociates from cycling and from iron-storing hepatocytes. It HRI, causing it to undergo specific autophos- now appears that there is a single systemic reg- phorylation to become a catalytically active ki- ulator of iron, the hepatic peptide hormone nase targeting the a subunit of eukaryotic trans- hepcidin. The hormone inhibits iron delivery lational initiation factor 2 (eIF2a). Activated to plasma and extracellular fluid thereby con- HRIinhibitstranslational initiation by phoshor- trolling the concentration of iron in plasma. ylating eIF2a. Not all protein synthesis is inhib- Hepcidin inhibits the transfer of dietary iron ited however, as activated HRI may promote the from duodenal enterocytes to plasma, the re- synthesis of transcription factors that are protec- lease of recycled iron from macrophages to plas- tive during iron-deficient erythropoiesis (Liu ma, and the release of stored iron from hepato- et al. 2008). A priori, it is not obvious how iron cytes (Fig. 2). Fetal hepcidin inhibits the transfer deficiency results in the production of smaller, of maternal iron across the placenta to the fetal less-hemoglobinized erythrocytes rather than circulation. At the molecular level, hepcidin acts fewer normally sized and hemoglobinized cells. by binding to its receptor, ferroportin, and caus- Studies with HRI-deficient mice showed that ing its endocytosis and proteolysis, which re- HRI protects erythroid precursors from apopto- sults in decreased iron release from cells to plas- sis induced by excessive production of globin ma and extracellular fluid. Ferroportin is found chains and contributes to the microcytosis and at very low concentrations in most cell types but hypochromia seen in iron deficiency, erythro- much higher amounts in professional iron- poietic protoporphyria, and b-thalassemia. transporting tissues, including the duodenal enterocytes and splenic macrophages. Intermedi- ate concentrations of ferroportin are detectable Iron and Hypoxia Sensing in hepatocytes. The hypoxia-sensing pathway may also contribute to cellular iron homeostasis. Prolyl and as- Regulation of Hepcidin Synthesis paraginyl hydroxylases, which inactivate the HIF transcription factors, are not only sensitive to Hepatocytes are the main source of hepcidin, oxygen tension but also to iron concentrations with much lower amounts produced by macro- because they use iron as a catalytic cofactor. In phages, adipocytes, and perhaps other cells. support of the potential role of HIF in iron reg- Hepcidin synthesis is controlled predominantly ulation, tissue-specific deletion of HIF2a in at the transcriptional level and is increased by www.perspectivesinmedicine.org mouse enterocytes decreased intestinal iron ab- plasma iron-transferrin as well as by iron stored sorption as well as the expression of DMT1 in in hepatocytes (the “stores” regulator), is sup- enterocytes (Mastrogiannaki et al. 2009). HIF2a pressed in response to increased iron require- bound to the DMT1 promoter and transacti- ments of erythroid precursors (the “erythroid” vated it. The broader physiologic function of regulator), and is potently stimulated by in- HIF in cellular iron homeostasis still remains to flammation (Fig. 2). be established and may vary in different tissues depending on oxygen tension and other factors. Regulation of Hepcidin Synthesis by Iron Iron regulation of hepcidin is mediated by the SYSTEMIC IRON HOMEOSTASIS canonical bone morphogenetic protein pathway adapted for hepcidin regulation by iron- The Central Role of Hepcidin specific molecular components (Fig. 3). Nor- Systemic iron homeostasis encompasses the reg- mal iron regulation in the murine model ulatory circuitry that controls the absorption of requires the iron-specific ligand bone morpho- dietary iron, the concentration of iron in extra- genetic protein 6 (BMP6), interacting with the

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Iron Metabolism

Inflammation Liver Spleen Hepcidin

Liver Hepcidin Fe Fpn Hepcidin Fpn

Plasma Erythrocytes Fe-Tf Fpn Iron signals

Bone marrow

Duodenum

Erythropoietic signal

Figure 2. Iron homeostasis. Through membrane ferroportin (Fpn), iron flows into plasma (pale blue arrows) from duodenal enterocytes, iron-storing hepatocytes, and iron-recycling macrophages predominantly in the spleen. Iron-transferrin (Fe-Tf ) is mostly delivered to the marrow (pale blue arrow) where iron is incorporated into erythrocyte hemoglobin (red). When the erythrocytes live out their lifespan (normally 120 d in humans), their hemoglobin and heme are degraded in the macrophages, mostly in the spleen, and iron is returned into the plasma iron pool. Hepatocytes secrete hepcidin under the control of stimulatory signals that reflect liver iron stores and plasma iron concentrations (blue), inhibitory signals reflecting erythropoietic activity (red), and inflammatory cytokines (green). Hepcidin causes the degradation of Fpn and thereby inhibits iron delivery to plasma and the erythropoietic bone marrow.

BMP receptor. Another iron-speciﬁc compo- cating that iron-transferrin modulates the sensi- nent required for normal hepcidin regulation tivity of the receptor to its ligands. It is not by iron is the membrane protein hemojuvelin, known with certainty how the concentration of www.perspectivesinmedicine.org which interacts with BMPs as well as with the iron-transferrin is sensed but ablation of HFE or BMP receptor. The BMP receptor is a ligand- TfR2, and especially the combined loss of both activated serine/threonine kinase, which phos- molecules, decreases hepcidin expression and phorylates the cytoplasmic proteins Smad1, interferes with the sensing of transient changes 5, and 8. Together with the common Smad4, in iron-transferrin while preserving the increase the phosphorylated Smads form heterodimers, in BMP6 and the chronic hepcidin response to which reach the nucleus and enhance the tran- iron loading (Wallace et al. 2009; Ramos et al. scription of hepcidin. The synthesis of the BMP6 2011; Corradini et al. 2011). A plausible current ligand appears to be responsive predominantly model postulates that iron-transferrin is sensed to iron stores rather than transferrin saturation, by TfR1 and TfR2, with HFE shuttling between and compared to the large changes in hepcidin the two molecules depending on iron-transfer- expression, BMP6 changes in a relatively narrow rin concentrations. At higher iron-transferrin range. In contrast, changes in extracellular iron- concentrations, the association of TfR2 and transferrin concentration affect signal transduc- HFE somehow potentiates BMP receptor com- tion by the BMP receptor even in the absence of plex signaling. Two other proteins, GPI-linked changes in BMP6 mRNA concentration, indi- hemojuvelin (Papanikolaou et al. 2004) and the

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T. Ganz and E. Nemeth

Inflammation BMP receptor complex Iron sensing

IL-6 HJV HoloTf

BMP6 TfR2 TfR1 MT-2 IL-6R BMP HFE receptor JAk-STAT3 pathway Neogenin

BMP6 Fe Increased BMP6 mRNA Intracellular

Smad pathway Fe sensor

Increased hepcidin mRNA

Hepatocyte Hepcidin

Figure 3. Regulation of hepcidin by iron and inﬂammation. Hepcidin synthesis is transcriptionally regulated by iron through the BMP receptor complex and its Smad pathway (shades of blue) and by inﬂammation predominantly via the IL-6 receptor and its JAK-STAT3 pathway (green). Extracellular iron is sensed by transferrin receptors (TfR1 and TfR2) aided by HFE, which can associate with either TfR but is displaced from TfR1 when TfR1 binds diferric transferrin (HoloTf ). When HoloTf concentrations are high, HFE is associated mostly with TfR2 and stabilizes it. HFE-TfR2 then potentiates BMP receptor signaling through an unknown mechanism. Stored hepatic intracellular iron increases the concentrations of BMP6 mRNA and presumably BMP6 protein in the liver thereby stimulating the BMP receptor, its Smad pathway, and hepcidin transcription.

membrane protease matriptase 2 (MT2, also intheabsenceofanysignificantchangesinserum called transmembrane serine proteinase 6, TM- iron (Ashby et al. 2010). Apparently, stimulated PRSS6) (Du et al. 2008) respectively enhance erythrocyte precursors produce one or more and dampen BMP signaling, with hemojuvelin hepcidin-suppressive factors but the molecular acting as a BMP pathway coreceptor, and MT2 nature of this putative physiological erythroid www.perspectivesinmedicine.org exerting its effect by an inactivating cleavage of regulator of hepcidin is not yet known. The sup- hemojuvelin (Silvestri et al. 2008). pressive effect on hepcidin is even more promi- nent under pathological conditions of expanded but ineffective erythropoiesis, seen in b-thalas- Regulation of Hepcidin by Erythropoiesis semia and congenital dyserythropoietic anemias Hepcidin mRNA is suppressed during anemia (Adamsky et al. 2004; Papanikolaou et al. 2005) or hypoxia (Nicolas et al. 2002) but it now ap- where the large number of apoptosing erythro- pears that this is an indirect effect dependent on cyte precursors could generate additional sup- increased erythropoietin production and the re- pressive factors. Two members of the BMP sulting expansion of erythroid precursors in the family, growth differentiation factor (GDF) 15 marrow (Pak et al. 2006; Vokurka et al. 2006; and twisted gastrulation (TWSG) 1, have been Mastrogiannakietal.2011)andnotadirecteffect proposed to play a role in pathological hepcidin of hypoxia-regulated pathways on the hepcidin suppression during ineffective erythropoiesis promoter.Innormal volunteers,the administra- (Tanno et al. 2007, 2009; Casanovas et al. 2011) tion of erythropoietin was sufficient to lower se- but their specific regulatory role in iron homeo- rum hepcidin profoundly within less than 1 day, stasis or pathology remains to be established.

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Iron Metabolism

Regulation of Hepcidin by Inflammation that restrict the release of recycled iron from macrophages and hepatocyte stores, and inter- During infections and inflammation, the syn- fere with the absorption of dietary iron. Iron thesis and serum concentrations of hepcidin restriction limits hemoglobin synthesis and are greatly increased (Pigeon et al. 2001; Nicolas contributes to anemia although other factors et al. 2002; Nemeth et al. 2003, 2004; Ganz et al. (inadequate erythropoietin production, cyto- 2008). This regulatory circuitry is thought to be kine effects on the marrow, decreased erythro- related to the possible role of hepcidin in host cyte lifespan) may also participate, depending defense whereby hepcidin-mediated iron re- on the underlying disease. Clinically, this disor- striction may limit microbial growth. Multi- der is manifested most often as a normocytic ple cytokines stimulate hepcidin transcription normochromic anemia with hypoferremia, but during inflammation, chief among them IL-6 the anemia can be microcytic, especially in chil- (Nemeth et al. 2003, 2004) and the members dren or very chronic inflammatory disorders. of the BMP family (Maes et al. 2010). Interleu- kin-6 activates the JAK-STAT3 pathway (Fig. 3), with STAT3binding to canonical binding sites in Iron-Refractory Iron Deficiency Anemia the hepcidin promoter, leading to transcription- This relatively rare condition is detected in chil- al stimulation of hepcidin synthesis (Wrighting dren who present with an unexplained hypo- and Andrews 2006; Pietrangelo et al. 2007; Verga ferremia and microcytic anemia resistant to Falzacappa et al. 2007). The BMPand IL-6 path- oral iron administration, and partially resistant ways are synergistic through a mechanism that is even to intravenous iron supplementation. The not yet fully defined (Verga Falzacappa et al. patients have elevated or high normal hepcidin 2008; Maes et al. 2010). Inflammation may con- levels (Finberg et al. 2008) in stark contrast to tribute to elevated serum hepcidin levels seen in common iron deficiency in which serum hepci- many adult patients with sickle cell anemia dinisveryloworundetectable(Ganzetal.2008). (Kroot et al. 2009; Porter 2009).

Iron Overload from Transfusions DISORDERS OF IRON HOMEOSTASIS Blood transfusions deliver 1 mg of iron for Iron Deﬁciency each mL of packed erythrocytes or more than Worldwide, iron deﬁciency is the most common 200 mg per each unit transfused, effectively by- iron disorder (see Miller 2012). Although it is passing the regulatory mechanisms that control thought of as a predominantly acquired prob- iron intake. Excess iron may eventually cause www.perspectivesinmedicine.org lem caused by blood loss and inadequate iron toxicity and organ damage, and can only be re- intake, genetic predisposition may modulate moved by phlebotomy (contraindicated if pa- the susceptibility to this condition as illustrated tient is still anemic) or by treatment with che- by genome-wide association studies (Benyamin lating agents. Extrapolating from clinical data et al. 2009a,b; Chambers et al. 2009; Tanaka for iron-related toxicity in hereditary hemo- et al. 2010). Not surprisingly, associations have chromatosis, chelation therapy is recommended been reported between serum iron concentra- after 10–20 transfusions for those patients who tions and polymorphisms and mutations in need chronic erythrocyte transfusions (Britten- transferrin, HFE, and MT2 (TMPRSS6). ham 2011).

Anemia of Inﬂammation (Anemia of Hereditary Hemochromatosis and Chronic Disease) Related Disorders Infections and inﬂammatory disorders are a Hereditary hemochromatosis is a group of ge- common cause of iron maldistribution, mediat- netic disorders that impede either hepcidin pro- ed by increased plasma hepcidin concentrations duction or its regulation by iron (Nicolas et al.

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T. Ganz and E. Nemeth

2001; Papanikolaou et al. 2005) or, very rarely, et al. 2007) but cause iron overload owing to the cause the resistance of ferroportin to internali- iron content of transfused blood. zation by hepcidin (Fernandes et al. 2009; Sham et al. 2009). In the order of increasing severity of hepcidin deficiency and iron overload, autoso- Modulation of Ineffective Erythropoiesis by Iron Availability mal recessive forms can result from mutations in genes encoding HFE, TfR2, hemojuvelin, and Recent studies in mouse models of thalassemia hepcidin. Ferroportin resistance to hepcidin is suggest that increased concentrations of plasma attributable to autosomal-dominant mutations iron in this condition may further unbalance in ferroportin that either interfere with hepci- heme and globin synthesis and worsen ineffec- din binding or with ferroportin internalization. tive erythropoeisis, and conversely, that re- Additional genes that caused iron overload in stricting theironsupply throughthe administra- transgenic mouse models but have not yet been tion of apotransferrin or hepcidin may improve implicated in humans include BMP6 (Andrio- erythropoiesis (Gardenghi et al. 2010a,b; Li poulos Jr. et al. 2009; Meynard et al. 2009) and et al. 2010). It remains to be seenwhether similar neogenin (Lee et al. 2010). Hepatic iron over- interventions will be helpful in human disease. load can also develop as a part of more complex genetic diseases, including deficiencies of transferrin and ceruloplasmin and loss-of-function CONCLUDING REMARKS mutations in DMT1 (Pietrangelo et al. 2011). In Iron homeostasis is intimately intertwined with these disorders, iron-restrictive anemia devel- erythropoiesis, the main destination of iron in ops because of diminished iron release from humans and other vertebrates. Iron overload is macrophages (ceruloplasmin deficiency), iron a clinically important aspect of various hemo- delivery to erythrocytes (transferrin deficiency), globinopathies because of transfusion-induced or iron utilization by erythrocyte precursors iron overload and because of pathological sup- (DMT1 loss of function). In contrast, simple pression of hepcidin synthesis with resulting hereditary hemochromatosis in humans has hyperabsorption of dietary iron. Advances in only modest effects on erythropoiesis, limited the understanding of iron homeostasis and its to a slight increase in mean corpuscular volume interactions with erythropoiesis should trans- (McLaren et al. 2007). late into improved outcomes for patients with hemoglobinopathies. Iron Overload Associated with Ineffective

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