Critical Review Systemic Iron Homeostasis and Erythropoiesis George Papanikolaou 1 Kostas Pantopoulos 2 *

Critical Review Systemic Iron Homeostasis and Erythropoiesis George Papanikolaou 1 Kostas Pantopoulos 2 * 1 Department of Nutrition and Dietetics, School of Health Science and Education, Harokopion University, Athens, Greece 2 Lady Davis Institute for Medical Research and Department of Medicine, McGill University, Montreal, Quebec, Canada Abstract Iron is an essential nutrient that is potentially toxic due to its redox reactivity. Insufficient iron supply to erythroid cells, the major iron consumers in the body, leads to various forms of anemia. On the other hand, iron overload (hemochromatosis) is associated with tissue damage and diseases of liver, pancreas, and heart. Physiological iron balance is tightly controlled at the cellular and systemic level by iron regulatory proteins (IRP1, IRP2) and the iron regulatory hormone hepcidin, respectively. Underlying mechanisms often intersect to achieve optimal iron utilization, to control immune responses, and to prevent iron toxicity. This review focuses on systemic iron homeostasis in the context of erythropoiesis, a highly iron-demanding process. We discuss the function and regulation of hepcidin by various stimuli, and highlight hepcidin-dependent and -independent mechanisms that link iron utilization with maturation of erythroid progenitor cells. VC 2017 IUBMB Life, 69(6):399 413, 2017 Keywords: iron; hepcidin; ferroportin; transferrin receptor; hypoxia; erythropoietin; erythroferrone; IRP1; IRP2 The Janus Face of Bioiron Iron is integral constituent of several metalloproteins primarily as part of heme or iron-sulfur clusters, and rarely as part of iron-oxo centers. As such, iron is essential for oxygen transport, and for electron transfer and catalytic reactions (1). The biological versatility of iron is based on its capacity to coordinate with proteins and to act as electron donor and acceptor. Thus, iron can readily convert between its two common oxidation states Fe 21 (ferrous) and Fe 31 (ferric), by the loss or gain of one electron. Iron s chemical reactivity has important Abbreviations: IRP1, iron regulatory protein 1; IRP2, iron regulatory protein 2; IRE, iron responsive element; HO-1, heme oxygenase 1; HO-2, heme oxygenase 2; FPN, ferroportin; DMT1, divalent metal transporter 1; Dcytb, duodenal cytochrome b; TfR1, transferrin receptor 1; stfr1, soluble TfR1; TfR2, transferrin receptor 2; NTB1, non-transferrin bound iron; Steap3, Six-transmembrane epithelial antigen of prostate 3; NCOA4, Nuclear receptor coactivator 4; EPO, erythropoietin; EPOR, erythropoietin receptor; BFU-E, burst forming unit - erythroid; CFU-2, colony forming unit - erythroid; ALAS2, erythroid aminolevulinate synthase; FC, ferrochelatase; FBXL5, F-box/LRR-repeat protein 5; HIF2a, hypoxia-inducible factor 2a; HRI, heme regulated inhibitor; STAT3, signal transducer and activator of transcription 3; STAT3-BS, STAT3-binding site; STAT5, signal transducer and activator of transcription 5; GATA-1, GATA-binding protein 1; HH, hereditary hemochromatosis; HFE, high Fe (iron); HJV, hemojuvelin; Zip14, ZRT/IRT-like Protein 14; IRIDA, iron-refractory iron deficiency anemia; AI, anemia of inflammation; ACD, anemia of chronic disease; TMPRSS6, Transmembrane Protease, Serine 6; BMP2, bone-morphogenetic protein 2; BMP6, bone-morphogenetic protein 6; BMP-RE, BMP responsive element; SMAD, 1/5/8, homolog of both the drosophila protein mothers against decapentaplegic and the C. elegans protein SMA 1/5/8; ACVR2A, Activin A Receptor Type 2A; BMPR2, BMP receptor 2; ALK2, activin receptor-like kinase 2; ALK3, activin receptor-like kinase 3; ALK7, activin receptor-like kinase 7; IL-6, interleukin 6; ERK/MAP, extracellular signal regulated kinase / mitogen activated protein; JAK1/2, Janus kinase 1/2; IFNa, interferon-a; IFNc, interferon-c; GDF15, growth differentiation factor 15; TWSG1, twisted gastrulation; ERFE, erythroferrone; TNF, tumor necrosis factor VC 2017 International Union of Biochemistry and Molecular Biology Volume 69, Number 6, June 2017, Pages 399 413 *Address correspondence to: Kostas Pantopoulos, Lady Davis Institute for Medical Research, 3755 C^ote Ste-Catherine Road, Montreal, H3T 1E2, Quebec, Canada. Tel.: (514)-340-8260 ext. 25293. Fax: (514)-340-7502. E-mail: kostas.pantopoulos@mcgill.ca Received 7 February 2017; Accepted 16 March 2017 DOI 10.1002/iub.1629 Published online 6 April 2017 in Wiley Online Library (wileyonlinelibrary.com) IUBMB Life 399

IUBMB LIFE FIG 1 Physiological iron metabolism in humans. Most of body iron is utilized for erythropoiesis. Significant amounts of iron are found in muscles and the liver. Circulating iron is delivered to tissues by transferrin, which contains a small but dynamic amount of iron (turnover rate: 20 25 mg/day). The transferrin iron pool is primarily replenished by iron released from macrophages after phagocytosis of senescent red blood cells. Dietary iron absorbed in the intestine is likewise directed to transferrin, but under physiological conditions this amount is tiny and serves to compensate for non-specific iron losses. implications for its biological properties. First, Fe 21 undergoes spontaneous aerobic oxidation to Fe 31 that is virtually insoluble at physiological ph (KfreeFe 31 5 10217 M). This makes acquisition of iron by cells and organisms challenging, despite its high abundance. Second, free iron acts as catalyst of oxidative stress via Fenton/Haber-Weiss chemistry, which yields hazardous radicals with the capacity to attack cellular macromolecules and cause tissue injury. Consequently, a tight control of iron metabolism is imperative to satisfy metabolic needs for iron, and to prevent accumulation of toxic iron excess. Iron Distribution in the Body The adult human body contains approximately 3 to 5 g of iron, corresponding to 55 mg/kg for males and 44 mg/g for females (2,3). More than 70% of body iron is present as heme within hemoglobin of developing erythroblasts and mature erythrocytes, and is utilized for oxygen binding and transport to tissues (Fig. 1). Except for muscles, which use 2.5% of body iron within myoglobin for oxygenation, all other cell types have much lower iron requirements for metabolic purposes. Significant fractions of body iron are distributed within tissue macrophages (5%) and liver hepatocytes (20%). Macrophages clear senescent erythrocytes, degrade hemoglobinderived heme via heme oxygenases (HO-1 and HO-2), and export the resulting Fe 21 to plasma via ferroportin (Fpn), the sole cellular iron exporter. Hepatocytes store excess of body iron within ferritin, the iron storage protein. Dietary Iron Absorption Dietary iron absorption is high during growth and slows down in adulthood. Because there is no mechanism for iron excretion from the body, healthy adults acquire 1 to 2 mg of iron per day from the diet to compensate for nonspecific losses (mainly due to cell desquamation, menstrual bleeding, or other blood loss). Meat products offer an important nutritional iron source due to their high content of heme, which is considered more bioavailable compared to inorganic iron (4). The pathway for heme iron absorption is incompletely understood and the apical enterocyte heme transporter(s) remain elusive. Nevertheless, it is well established that heme iron assimilation requires catabolism of heme within enterocytes and release of Fe 21, which follows the fate of inorganic dietary iron (Fig. 2). On the other hand, the mechanism for inorganic iron absorption is well characterized (5). This involves reduction of Fe 31 to Fe 21 by ferrireductases (such as Dcytb) or other reducing agents (such as ascorbate) in the duodenal lumen, followed by transport across the apical membrane of enterocytes via the divalent metal transporter 1 (DMT1). Internalized Fe 21 is transferred to the basolateral membrane by an unknown mechanism, and exported to plasma via ferroportin. The efflux 400 Iron and Erythropoiesis

FIG 2 Dietary iron absorption. Ferric iron is reduced in the intestinal lumen by Dcytb or other ferrireductases. Ferrous iron is then transported across the apical membrane of enterocytes by DMT1. Following transport to the basolateral site by unknown mechanisms, iron gets exported to the circulation via ferroportin. This step is coupled with re-oxidation of ferrous to ferric iron by membrane-bound hephaestin or soluble ceruloplasmin. Heme gets internalized by an unknown transporter and following enzymatic degradation, liberated iron follows the fate of absorbed inorganic iron. FIG 3 Iron-dependent heme/globin synthesis in erythroid cells. Iron acquisition involves binding of circulating holo-tf to TfR1 on the plasma membrane, which is followed by receptor-mediated endocytosis. Iron is released from the endosome and delivered to mitochondria, where it gets incorporated to protoporphyrin IX to form heme, in a reaction catalyzed by FC. The first reaction of the heme biosynthetic pathway likewise occurs in the mitochondria and is catalyzed by ALAS2. Heme is directed to the cytosol, where it associates with globin to form hemoglobin. Iron deficiency leads to specific inhibition of ALAS2 synthesis by IRPs. Heme deficiency triggers global inhibition of protein (mainly globin) synthesis by HRI-mediated eif2a phosphorylation. Papanikolaou and Pantopoulos 401

IUBMB LIFE of Fe 21 is associated with its re-oxidation to Fe 31 by the soluble or membrane-bound multicopper ferroxidases ceruloplasmin or hephaestin, respectively (6). Iron Transport in the Bloodstream Exported Fe 31 is captured by transferrin, the plasma iron carrier, and transported to bone marrow erythroblasts and other cells in peripheral tissues (Fig. 1). Circulating transferrin contains a very small (0.1%) but highly dynamic fraction of body iron that turns over >10 times per day to satisfy the daily iron requirement for erythropoiesis (2). The physiological saturation of transferrin with iron is 30%. The buffering capacity of apo-transferrin prevents accumulation of free nontransferrin bound iron (NTBI), which is redox-active and toxic. Transferrin has two iron-binding sites and delivers its Fe 31 cargo upon binding to transferrin receptor 1 (TfR1) on the cell surface via receptor-mediated endocytosis (Fig. 3). Iron is released from transferrin in the acidified endosome, reduced by the ferrireductase Steap3 (six-transmembrane epithelial antigen of prostate 3), and transported across the endosomal membrane to intracellular compartments via DMT1. The cycle is completed by the release of apo-transferrin to the bloodstream, which can recapture iron and engage in further cycles of iron delivery to cells. Apo-transferrin is primarily replenished with iron provided by tissue macrophages following erythrophagocytosis (7). Thus, under physiological conditions, erythropoiesis is sustained by macrophage-mediated recycling of iron from senescent red blood cells. The contribution of dietary iron released by enterocytes to maintenance of the circulating transferrin iron pool is very low. The significance of dietary iron increases in iron deficiency, which stimulates iron absorption, but also mobilization of iron from stores. This involves lysosomal ferritin degradation in hepatocytes following its interaction with the cargo receptor NCOA4 (nuclear receptor coactivator 4) (8). Liberated iron is exported to the bloodstream via ferroportin (9). Iron and Erythropoiesis Bone marrow erythroblasts acquire more than 80% of plasma iron (10). In adult humans, 2 million newly synthesized erythrocytes are released in the circulation every second. Erythropoiesis requires 20 to 30 mg of iron per day, far more than the amount absorbed from diet (2). Erythroid progenitor cells are detected by their ability to form colonies (BFU-E and CFU-E), which further differentiate to proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatic erythroblasts, reticulocytes, and mature erythrocytes (11). Maturation and proliferation of early erythroid progenitor cells depends on erythropoietin (EPO), a cytokine secreted from the kidneys in response to hypoxemia. In cases of acute demands, such as hemorrhage and hemolysis, increased EPO secretion stimulates proliferation of erythroid progenitors (partly through reducing apoptosis of CFU- Es), and accelerates terminal erythrocyte maturation. EPO receptors (EPOR), which appear at a later stage of BFU-E maturation, are highly expressed on the surface of CFU-Es. Iron demand increases during the terminal stages of erythroid cell differentiation where hemoglobin and heme synthesis occur (Fig. 3). Heme is synthesized by a series of enzymatic reactions that take place in the cytosol and mitochondria (12). The first step (condensation of succinyl-coa and glycine) is catalyzed in the cytosol by erythroid aminolevulinate synthase (ALAS2). The final step, iron incorporation into protoporphyrin IX takes place in the mitochondria and is catalyzed by ferrochelatase (FC). The transfer of iron to the mitochondria is extremely efficient and involves the transporter mitoferrin 1 in the inner mitochondrial membrane (13). Experimental evidence suggests an erythroid-specific direct transfer mechanism of iron from the endosomes to the mitochondria via transient interaction of these organelles ( kiss and run ) (14). Iron entry is the limiting step for erythroid heme synthesis. This is mediated by TfR1, which is indispensable for erythropoiesis. Thus, Tfr1 / mice die during embryonic development (15). Expression of TfR1 peaks at the late basophilicpolychromatophilic stage and decreases (in parallel with decreasing heme synthesis) at the orthochromatic stage (16). Erythroid cells shed remaining TfR1 receptors by exocytosis or proteolytic cleavage giving rise to soluble TfR1 (stfr1), whose plasma concentration correlates well with the erythropoietic rate, erythroid mass, and iron need (17). Iron mobilization from ferritin may contribute to erythropoiesis, especially when plasma iron is limiting. Thus, Ncoa4 / mice that exhibit a defect in ferritin degradation, develop a mild hypochromic microcytic anemia, which becomes more severe when fed an iron-deficient diet (18). Ferritinophagy occurs during terminal erythroid differentiation (19) and coincides with NCOA4 expression in orthochromatic erythroblasts (16). Regulation of Cellular Iron Metabolism and Implications on Systemic Iron Balance Cellular iron uptake, storage, efflux and erythroid cell utilization are coordinately regulated by iron regulatory proteins IRP1 and IRP2, the cytosolic iron sensors (20). In iron-starved cells, IRPs bind to iron-responsive elements (IREs) in the untranslated regions of mrnas encoding TfR1, DMT1, ferritin, ferroportin, and ALAS2 (Fig. 4). The IRE/IRP interactions stabilize TfR1 and DMT1 mrnas against degradation, and inhibit translation of ferritin, ferroportin, and ALAS2 mrnas. These responses promote uptake of iron and prevent iron sequestration or efflux, and erythroid heme synthesis. Conversely, IRE/ IRP interactions do not occur in iron-replete cells. This allows TfR1 mrna degradation and synthesis of ferritin, ferroportin and ALAS2. Hence, iron uptake is inhibited and excess iron is 402 Iron and Erythropoiesis

FIG 4 Coordinate regulation of cellular iron metabolism by IRE/IRP interactions. In iron-deficient cells, IRPs bind to mrnas encoding ferritin, ferroportin, ALAS2, HIF2a, TfR1 and DMT1. IRE/IRP interactions in the 5 untranslated region (5 UTR) inhibit mrna translation, and in the 3 UTR prevent mrna degradation. In iron-replete cells IRPs are inactivated for IRE-binding, allowing opposite responses. Thus, IRP1 assembles an iron-sulfur cluster, while IRP2 undergoes degradation. stored intracellularly, exported to the bloodstream and utilized in erythroid cells for heme synthesis. IRP1 and IRP2 are structurally related but are regulated by different mechanisms (20). In iron-replete cells, IRP1 is converted to cytosolic aconitase at the expense of its IRE-binding activity, following insertion of a 4Fe-4S cluster. The 4Fe-4S cluster of IRP1 is sensitive to hyperoxia and is stabilized by hypoxia (21). On the other hand, IRP2 undergoes iron- and oxygen-dependent proteasomal degradation via the ubiquitin ligase FBXL5, which is stabilized in iron-replete oxygenated cells by an Fe-O-Fe bridge. Loss of this sensor in iron deficiency or hypoxia triggers FBXL5 degradation and allows IRP2 accumulation. Another connection between the IRE/IRP system and hypoxia was revealed by the discovery of a functional atypical IRE in the mrna encoding hypoxia-inducible factor 2a (HIF2a), which accounts for its translational regulation by IRPs (22) (Fig. 4). HIF2a transcriptionally induces expression of a battery of genes mediating hypoxic responses, but also iron metabolism and erythropoiesis (Dcytb, DMT1, ferroportin, ceruloplasmin, transferrin, TfR1, ALAS2, EPO). Considering that HIF2a is stabilized in response to iron deficiency and hypoxia, the above findings provide important links between cellular and systemic iron metabolism (23,24). This notion applies particularly to iron-deficient enterocytes, where dietary iron absorption is stimulated by HIF2adependent transcriptional activation of Dcytb, DMT1 and ferroportin, respectively (25 27). The expression of DMT1 is also augmented by IRP-dependent stabilization of an IREcontaining DMT1 transcript. Opposite responses occur in ironloaded enterocytes, where iron efflux to the bloodstream is further limited by its sequestration in ferritin (28). High expression of ferritin imposes a mucosal block, that can be alleviated via its translational repression by IRPs (29). IRPmediated translational inhibition of HIF2a mrna and of an IRE-containing ferroportin transcript may fine-tune dietary iron absorption (23,24). IRP1 and IRP2 are ubiquitously expressed, even though their relative distribution varies in mouse tissues, with IRP1 being more abundant in the kidneys, brown fat and lungs. Targeted disruption of both IRPs is associated with early embryonic lethality, while single Irp1 / or Irp2 / mice are viable but have distinct phenotypes and iron metabolic defects (23). These findings indicate that the functions of IRP1 and IRP2 are only partially redundant. Irp1 / mice develop polycythemia due to misregulation of HIFa mrna (see section Regulation of erythropoiesis by iron ). By contrast, Irp2 / mice exhibit microcytic anemia and erythropoietic protoporphyria due to misregulation of TfR1 and ALAS2 in erythroid precursor cells, which causes erythroid iron deficiency and accumulation of free protoporphyrin IX (30,31). The translational regulation of ALAS2 by the IRE/IRP system in erythroid cell, links the heme biosynthetic pathway to iron supply (23). Moreover, in response to heme deficiency, the heme regulated inhibitor HRI kinase phosphorylates the translation initiation factor eif2a and thereby inhibits globin synthesis (32) (Fig. 3). It should be noted that while IRP2 appears to be essential for efficient TfR1 mrna expression in differentiating erythroblasts (30), TfR1 mrna stability bypasses the negative feedback regulation of IRPs, presumably due to the highly efficient iron transport to mitochondria that does not allow fluctuations in cytosolic iron (33). In this setting, TfR1 expression is predominantly regulated at the transcriptional level by STAT5A (34), GATA-1 (35), and other factors. Papanikolaou and Pantopoulos 403

IUBMB LIFE FIG 5 Hepcidin-mediated regulation of iron efflux to the bloodstream. (a) The iron-regulatory hormone hepcidin is secreted from the liver in response to high iron or inflammatory signals, and binds to ferroportin triggering its degradation; this leads to retention of iron within enterocytes and macrophages. (b) Hepcidin expression is inhibited in response to low iron or high erythropoietic drive, permitting dietary iron absorption by enterocytes and iron release from macrophages. Hormonal Regulation of Systemic Iron Homeostasis by Hepcidin Systemic iron homeostasis is primarily regulated via the hepcidin/ferroportin axis. Hepcidin is a liver-derived peptide hormone that restricts iron fluxes to the bloodstream (Fig. 5). It operates by binding to ferroportin in target cells, mostly macrophages and enterocytes (but also other cell types), which promotes ferroportin internalization and degradation (36). Hepcidin was originally identified as an anti-microbial peptide (37) and as an iron-induced hepatic gene product (38). It is synthesized in hepatocytes as a pre-peptide that undergoes proteolytic processing to a mature hormone consisting of 25 amino acids with eight cysteine residues that form four disulfide bonds. Even though these bonds determine the folding and structure of hepcidin, they appear to be redundant for iron regulation, since a minihepcidin consisting of the N-terminal fragment DTHFPICIF retains ferroportin-binding activity and promotes ferroportin degradation in cells and mice (39). Hepcidin is predominantly produced in the liver by hepatocytes, but is also expressed at low levels in macrophages and in cells from non-hepatic tissues (such as heart, brain, pancreas, stomach, lung, kidney, adipose tissue, retina). Nevertheless, only hepatocyte-derived hepcidin appears to regulate systemic iron trafficking (40), while hepcidin produced by other cells may exert local tissue-specific functions. The expression of hepcidin is regulated transcriptionally mainly in response to iron or inflammation (41). Irondependent induction of hepcidin serves to prevent excessive dietary iron absorption from enterocytes when body iron levels increase. Inflammatory induction of hepcidin contributes to an acute hypoferremic response that is caused by iron retention in macrophages. This is thought to be protective for the host during infection, by depriving bacteria from an essential nutrient (nutritional immunity) (42). The antimicrobial activity of hepcidin may enhance host defense. It should also be noted that hepcidin promotes transcriptional induction of antiinflammatory genes (43) and contributes to the resolution of inflammation in mice (44). Together, these findings highlight hepcidin as an important molecular link between iron metabolism and innate immunity. Hepcidin expression is inhibited by anemia and hypoxia. The ensuing accumulation of ferroportin in enterocytes and macrophages stimulates iron efflux to plasma, to meet the increased iron needs for erythropoiesis. Hepcidin is also regulated by other secondary positive stimuli, such as endoplasmic reticulum stress or gluconeogenesis; or negative signals, such as oxidative stress, gonadal hormones and growth factors (41). Some of these responses may be related to progression of chronic liver diseases (45), but our current understanding of their physiological implications is limited. Disorders of Hepcidin Dysregulation The significance of hepcidin became evident from experiments with mouse models of hepcidin inactivation or overexpression, which develop severe iron overload (46) or iron deficiency anemia (47), respectively. These findings were thereafter extrapolated to human iron-related disorders. A breakthrough was the discovery that hepcidin deficiency is causatively linked to hereditary hemochromatosis (HH), an 404 Iron and Erythropoiesis

FIG 6 Signaling to hepcidin. High serum or body iron (reflected in BMP6) induce hepcidin mrna transcription via the BMP/SMAD pathway. The inflammatory cytokines IL-6 and activin B induce hepcidin mrna transcription via the JAK/STAT and BMP/SMAD pathways, respectively. The erythropoietic regulators ERFE and possibly GDF15 suppress hepcidin transcription by unknown mechanisms. autosomal recessive and genetically heterogeneous endocrine disorder of iron overload (45). Thus, inactivating mutations of the hepcidin gene (HAMP) cause juvenile hemochromatosis, a rare, early onset form of HH (48). A similar clinical phenotype develops in response to inactivation of hemojuvelin (HJV), which leads to severe hepcidin deficiency (49). Moreover, moderate hepcidin deficiency accounts for milder and more prevalent forms of HH, linked to mutations in HFE (50), or transferrin receptor 2 (TfR2) (51), respectively. The hallmarks of HH are hyperabsorption of dietary iron (up to 8 10 mg/day), and failure of tissue macrophages to retain iron recycled during erythrophagocytosis. These responses are caused by aberrant overexpression of ferroportin in enterocytes and macrophages due to inappropriate hepcidin suppression. Unrestricted iron efflux from enterocytes and macrophages leads to hyperferremia, high transferrin saturation and emergence of redox active NTBI. This is readily taken up by hepatocytes and other parenchymal cells, and triggers clinical complications (liver disease, diabetes, cardiomyopathy, arthritis, osteoporosis). Mouse models bearing targeted disruption of HFE, TfR2, HJV or hepcidin recapitulate the pathway of iron overload observed in HH (52). Experiments with Hfe / and Hjv / mice showed that parenchymal iron overload in hepatocytes and pancreatic acinar cells is mediated by the NTBI transporter Slc39a14 (also called Zip14) (53). At the other end of the spectrum, hepcidin overexpression is associated with anemia. The most dramatic phenotype is observed in iron-refractory iron deficiency anemia (IRIDA), a hereditary disease caused by inactivating mutations in the TMPRSS6 gene encoding matriptase-2, a negative regulator of hepcidin (54). IRIDA patients develop microcytic hypochromic anemia that is unresponsive to oral iron supplementation. Under chronic inflammatory conditions, sustained upregulation of hepcidin leads to hypoferremia due to iron sequestration in macrophages. This causes iron-restricted erythropoiesis and contributes to development of the anemia of inflammation (AI), a normocytic normochromic anemia also known as anemia of chronic disease (ACD) (55,56). Hepcidin is not the sole driver of ACD, which is a multifactorial condition that is aggravated by impaired proliferation of erythroid progenitor cells, increased erythrophagocytosis, and reduced EPO expression and responsiveness. In addition, cellular iron retention is favored by cytokine-mediated regulation of iron metabolism genes, such as transcriptional induction of ferritin (57) and suppression of ferroportin (58). Nevertheless, targeting the hepcidin pathway has shown promising therapeutic results in pre-clinical models (59). Regulation of Hepcidin by Iron Increased serum or tissue iron trigger transcriptional induction of hepcidin in hepatocytes (Fig. 6). The exact mechanism for iron sensing is incompletely understood, but it is well established that the iron signal is transmitted to the hepcidin promoter via BMP/SMADs (bone-morphogenetic protein/ Papanikolaou and Pantopoulos 405

IUBMB LIFE homologs of both the drosophila protein mothers against decapentaplegic and the C. elegans protein SMA). The role of the BMP/SMAD pathway was uncovered by the characterization of HJV as a BMP co-receptor (60). HJV binds to BMP ligands in a ph-dependent manner, and may operate by recruiting the BMP ligand on the cell membrane to interact with type II BMP receptors (ACVR2A and BMPR2) (61). According to this model, recruitment of the ligand requires formation of a ternary complex with the HJV-interacting protein neogenin. Following endocytosis and acidification of the endosome, HJV is released, allowing interaction of the complex with endosomal type I BMP receptors (ALK2 and ALK3) to activate signal transduction. This involves phosphorylation of SMAD1, SMAD5 and SMAD8 proteins, interaction of activated p- SMAD1/5/8 with SMAD4, and translocation of the complex to the nucleus for binding to two BMP responsive elements (BMP- RE1 and BMP-RE2) at proximal and distal sites of the HAMP promoter. Even though various BMPs can induce hepcidin in vitro (BMP2, 5, 6, 7 and 9), iron-regulated BMP6 appears to be a major physiologically relevant ligand (62,63). BMP6 is secreted from liver endothelial cells in response to increased hepatic iron stores and acts in a paracrine fashion on hepatocytes (64). BMP6 may also promote termination of iron signaling to hepcidin by a negative feedback mechanism involving induction of matriptase-2 expression (65), a serine protease that cleaves and inactivates HJV (66), and is also induced by iron deficiency (67). Additional BMP ligands appear to have a role in fine tuning of iron signaling, since the combined disruption of Hfe (or Tfr2) and Bmp6 aggravated hepcidin suppression and iron overload in mice (68). In fact, subsequent work revealed that endothelial cell-derived BMP2 is crucial for constitutive hepcidin expression in hepatocytes by a paracrine mechanism (69), further emphasizing the critical role of the liver endothelium in systemic iron homeostasis. HFE and TfR2 are essential for appropriate iron signaling, as their inactivation causes hemochromatosis. Interestingly, hepatocytespecific disruption of either Hfe (70), Tfr2 (71), or Hjv (72) recapitulates the hemochromatotic phenotype, underlying the central role of these proteins in regulation of systemic iron homeostasis via hepcidin. Combined disruption of Hfe did not exacerbate iron overload in Hjv / mice (68,73), suggesting that HFE and HJV operate in the same pathway. Consistently, it has been proposed that HFE stimulates iron signaling by stabilizing the type I BMP receptor ALK3 (74). It remains to be further validated whether this is the principal systemic iron regulatory function of HFE, a major histocompatibility complex (MHC) class I molecule that was discovered as the hemochromatosis protein more than 20 years ago (75). Ensuing biochemical (76) and structural (77) studies demonstrated that HFE physically interacts with TfR1 and thereby inhibits cellular iron uptake. Conversely, TfR1 was shown to inhibit HFE-mediated iron signaling to hepcidin (78). These findings, provided the basis for a model of plasma iron sensing. The model postulates that hepatocellular TfR1 limits iron signaling by sequestering HFE and thereby preventing it from interacting with TfR2 (78). Along these lines, the TfR1/HFE interaction is abrogated by iron-loaded transferrin when plasma iron levels increase, which activates the iron signaling cascade via HFE/TfR2. Even though the model is further supported by the documented stabilization of TfR2 by iron-loaded transferrin (79,80), the capacity of TfR2 to interact with HFE remains controversial (81 83). Furthermore, patients (84) and mice (85) with compound HFE and TfR2 deficiency develop more severe iron overload compared to single HFE or TfR2 inactivation. These genetic data provide evidence that HFE and TfR2 exhibit non-overlapping functions. Experiments in mice suggest that hepcidin is independently regulated by plasma and stores iron (86,87). Thus, a transient increase in transferrin saturation following iron intake can lead to hepcidin induction without having any effect on BMP6 expression. This is associated with increased SMAD1/5/ 8 phosphorylation (87) and is blunted in Hfe /, Tfr2 /, Hjv / and Bmp6 / mice (86). Experiments with primary mouse hepatocytes suggested involvement of the ERK/MAP (extracellular signal regulated kinase/mitogen activated protein) kinase pathway on hepcidin induction by iron-loaded transferrin (88), but these findings were not corroborated in vivo (87). Despite the advances in understanding iron signaling to hepcidin, we still have limited knowledge on upstream molecular events. Thus, a challenge for future studies will be to elucidate the mechanisms by which alterations in plasma or tissue iron levels are sensed and translate into hepcidin responses. Regulation of Hepcidin by Inflammatory Stimuli Early experiments showed that LPS administration promotes a robust increase of liver hepcidin mrna levels in mice (38). Subsequent work established the transcriptional induction of hepcidin via IL-6/STAT3 signaling (Fig. 6). This is initiated upon the binding of IL-6 to gp130 receptor complexes, followed by JAK1/2-mediated phosphorylation of the transcription factor STAT3, which in turn binds to a STAT3-binding site (STAT3-BS) in the proximal HAMP promoter and activates hepcidin transcription (89 91). The JAK/STAT pathway is also used by other cytokines (such as oncostatin M, IL-22 or IFNa) for hepcidin induction (92). The STAT3-BS and BMP-RE1 are located in close proximity to each other within the HAMP promoter, and a crosstalk between the JAK/STAT and BMP/SMAD pathways has been documented (Fig. 6). Thus, IL-6-mediated induction of hepcidin requires the BMP type I receptor ALK3 (93), and can be antagonized by several pharmacological BMP/SMAD inhibitors (59). The BMP/SMAD pathway is also critical for inflammatory induction of hepcidin by activin B, another LPS-inducible cytokine (94) that appears to be secreted by non-parenchymal liver cells (95). Biochemical experiments suggest that hepcidin 406 Iron and Erythropoiesis

FIG 7 Hepcidin suppression in iron-loading anemias. Ineffective erythropoiesis leads to hepcidin suppression via induction of the erythroid regulators ERFE and GDF15. This promotes iron absorption, increased efflux of iron into plasma, buildup of NTBI, and tissue iron overload. induction by activin B involves either SMAD2/3 phosphorylation via canonical activin type I receptor ALK7, or SMAD1/5/ 8 phosphorylation via BMP type I receptors ALK2 and ALK3 (96). However, activin B knockout mice exhibit appropriate induction of hepcidin following LPS administration or Escherichia coli infection, indicating that activin B signaling is not essential for the inflammatory hepcidin response (97). On the other hand, experimental evidence suggests a role of activin B and BMP2 as mediators of hepcidin induction by IL-1b signaling (98). Regulation of Hepcidin by Erythropoiesis Erythropoiesis dominates over iron homeostasis in mammals. In non-anemic individuals, iron stores are the primary regulators of iron absorption and hepcidin levels. In certain anemic conditions such as iron deficiency anemia, hepcidin is suppressed and iron absorption can increase to amounts exceeding the capacity of the stores regulator to absorb iron (20 40 mg/day, if patients receive therapeutic doses of iron) (10,99). Stimulation of erythropoiesis after blood donation, hemorrhage, EPO administration or acute hemolysis, results in a transient imbalance between iron supply and bone marrow needs, which is followed by decreased hepcidin levels as a homeostatic mechanism to restore iron supply (100,101). Furthermore, low hepcidin concentrations are observed in patients with hereditary anemias (thalassemias, congenital dyserythropoietic, or sideroblastic anemias) with bone marrow hyperplasia and ineffective erythropoiesis (102,103). Iron absorption in these patients is increased irrespectively of iron stores, leading to iron overload and severe tissue toxicity (Fig. 7). Finch attributed these effects to an elusive erythroid regulator, presumably a bone marrow-derived cytokine (10). Obvious candidates such as stfr or EPO were excluded through experimental evidence and clinical observations (10,100,104,105). Growth differentiation factor 15 (GDF15) was identified as an erythroid regulator that contributes to hepcidin suppression in patients with b-thalassemia (106) or congenital dyserythropoietic anemias (107,108). Another candidate is Twisted gastrulation (TWSG1), which likewise appears to negatively regulate hepcidin in b-thalassemia (109). Both GDF15 and TWSG1 interfere with the BMP/SMAD signaling pathway and inhibit hepcidin expression in vitro; however, in vivo corroborative data are missing. In fact, wild type and Gdf15 / mice exhibited similar suppression of hepcidin in response to phlebotomy without any significant alterations in Twsg1 mrna (110), suggesting that Gdf15 and Twsg1 are dispensable as erythroid regulators of hepcidin in this setting. More recently, erythroferrone (ERFE) was discovered as an erythroid regulator that mediates hepcidin suppression Papanikolaou and Pantopoulos 407

IUBMB LIFE FIG 8 Mechanisms for regulation of erythropoiesis by iron. When iron is limiting for erythropoiesis, EPO expression and downstream erythropoietic activity are decreased via IRP1-mediated suppression of HIF2a mrna translation in the kidney (a). In addition, erythroid cell differentiation is impaired via protein kinase C a/b hyperactivation followed by overexpression of PU.1 (b), and via TfR2 inactivation (c). during stress erythropoiesis (111) (Fig. 6). ERFE is expressed in erythroblasts but also muscle cells, and appears to operate in a BMP/SMAD-independent manner. Nevertheless, ERFE fails to suppress hepcidin when BMP/SMAD signaling is hyperactive due to matriptase-2 deficiency (112). ERFE was previously described as myonectin, an adiponectin-homologous myokine produced in muscles during exercise and feeding, that enhances fatty acid uptake by myocytes and hepatocytes (113). ERFE is a member of the C1q and TNF related family (C1QTNF) and the ERFE gene (also known as FAM132B, C1QTNF15, CTRP15, FLJ37034) is located on chromosome 2q37. ERFE expression is induced after stimulation of erythropoiesis by EPO via the JAK2/STAT5 signaling pathway (111). Primary mouse hepatocyte cultures exhibited hepcidin suppression after treatment with supernatants of ERFE-transfected cells, while mice treated with recombinant ERFE showed significant reduction in both liver hepcidin mrna and serum peptide levels. Erfe / mice failed to suppress hepcidin in response to phlebotomy (111). When challenged by heat-killed Brucella abortus in a model of ACD, Erfe / mice exhibited more severe anemia and delayed recovery than wild-type controls (114). Furthermore, ablation of Erfe restored normal hepcidin expression and slightly improved indices of ineffective erythropoiesis in Hbb th3/ mice, a model of b-thalassemia intermedia (114). Taken together, these data illustrate a critical physiological role of ERFE in the recovery from anemia due to hemorrhage/hemolysis or inflammation, and a pathological function in the development of iron overload due to ineffective erythropoiesis. The mechanism by which ERFE suppresses hepcidin expression remains to be characterized. In a study comparing the expression of candidate erythroid regulators in different mouse models of anemia, Erfe was the most consistently upregulated (115). Erfe induction was highest in iron deficiency anemia. While these results validate the importance of ERFE as a critical erythroid regulator, they also indicate that ERFE expression does not solely depend on the rate of erythropoiesis, but also on the adequacy of iron supply to the bone marrow. Further work is needed to clarify how the erythroid iron status may interfere with EPO signaling for ERFE expression and secretion. Regulation of Erythropoiesis by Iron Efficient erythropoiesis requires fine tuning between erythrocyte production, iron supply and hemoglobin synthesis. This involves crosstalk between the erythroid bone marrow and the liver, the site of hepcidin production, and coordination of heme and hemoglobin synthesis with EPO signaling and iron supply to maturing erythroblasts. Clinical and experimental evidence shows that EPO and iron are interdependent signals, since bone marrow responsiveness to EPO is diminished in iron-restricted erythropoiesis (116). Iron deficient rats showed a 3.5-fold increase in the CFU-E pool compared to nonanemic rats, but only a 1.7-fold increase in nucleated cells, suggesting enhanced apoptosis at the stage of erythroid progenitors and blockage of terminal 408 Iron and Erythropoiesis

maturation (117). In patients with iron deficiency anemia, erythroblast bone marrow cellularity does not differ from patients with ACD, despite significant increases in serum EPO (118). By contrast, patients with ineffective erythropoiesis due to hemoglobinopathies such as b-thalassemia, display significant erythroid hyperplasia (10). Patients with ACD refractory to EPO treatment might restore responsiveness with intravenous iron administration. In the clinical setting, it is well documented that patients with chronic renal failure and functional iron deficiency are hyporesponsive to EPO, therefore current therapeutic regimens optimize EPO treatment by combining intravenous iron administration (116). In iron deficiency, adaptive homeostatic responses limit erythroid progenitor expansion by modulating EPO signaling and erythroid differentiation. This may prevent iron drainage from other tissues, where iron sufficiency could be more critical. Coordination of iron supply with erythropoiesis can be achieved by various mechanisms (Fig. 8). a. Iron deficiency is known to increase EPO expression via HIF2a stabilization (24). Nevertheless, IRP1-mediated suppression of HIF2a mrna translation in renal interstitial cells (where IRP1 is highly abundant) is expected to antagonize this response and mitigate EPO expression and downstream erythropoietic activity. IRP1 is a bifunctional protein that is regulated by an unusual 4Fe-4S cluster switch. It operates either as IRE-binding protein or as cytosolic aconitase, an enzyme catalyzing the isomerization of citrate to isocitrate (20). Hypoxia and increased iron availability stabilize the 4Fe-4S cluster and maintain IRP1 in the aconitase form, while iron deficiency shifts the equilibrium towards apo-irp1, the IRE-binding protein. The model postulating IRP1-dependent inhibition of EPO expression in iron deficiency is supported by data in Irp1 / mice, which have a normal phenotype when iron replete. However, these animals exhibit polycythemia, splenomegaly, extramedullary hematopoiesis and low hepcidin levels due to unrestricted EPO expression during growth (4 6 weeks of age), or under iron deficiency (119 121). b. Iron deficiency impairs erythroid cell differentiation via protein kinase C a/b hyperactivation and overexpression of PU.1. This transcription factor inhibits erythroid lineage commitment promoting myeloid differentiation, and accounts for IFN-c-induced inhibition of erythropoiesis that contributes to ACD (122). In addition, PU.1 accounts for the sensitization of erythroid progenitors to the inhibitory effects of INFc by iron deprivation (123). Mechanistically, iron deficiency appears to trigger PU.1 induction via aconitase inactivation. The enzymatic activity of both mitochondrial and cytosolic aconitases depends on the 4Fe-4S cluster in their active sites, that is vulnerable to iron limitation. In support of this model, the aconitase inhibitor fluorocitrate inhibited in vitro erythroid differentiation and this effect was reversed with isocitrate supplementation (124). In addition, treatment with isocitrate corrected (at least partly) iron deficiency anemia in mice; yet erythrocytes remained microcytic and hypochromic (124). Moreover, isocitrate normalized hepcidin expression and substantially improved erythropoiesis by suppressing the erythroid iron restriction response in an arthritis-induced rat model of ACD (123). However, isocitrate had negligible effects in a mouse model of more severe ACD triggered by heat-killed Brucella abortus (125). These data emphasize the need for more experimental work to assess the pharmacological potential of isocitrate for the treatment of ACD. c. Iron deficiency inhibits erythroid cell differentiation via TfR2 inactivation. In addition to its role as an iron sensor that regulates hepcidin production in hepatocytes, TfR2 is also expressed in erythroid progenitors as component of the EPOR complex (126). In the absence of iron-loaded transferrin, TfR2 undergoes proteolytic shedding to a soluble inactive form (127). Mice bearing hematopoietic-specific ablation of Tfr2 exhibited normal systemic iron homeostasis, but responded to dietary iron restriction with increased extramedullary erythropoiesis in the liver and spleen compared to controls (128). Furthermore, they accumulated a higher number of immature erythroid cells at the stage of polychromatic erythroblasts, at the expense of mature cells (reticulocytes and erythrocytes). In a different approach, bone marrow transplantation from Tfr2 / mice to isogenic wild type recipients (Tfr2 BMK 8) led to similar impairment in erythroid differentiation under milder dietary iron restriction (129). However, contrary to hematopoietic-specific Tfr2 / mice, the chimeric Tfr2 BMK 8 animals had increased hemoglobin, higher erythrocyte counts, lower hepcidin expression in the liver, and increased Erfe expression in the bone marrow (128,129). These findings are consistent with an inhibitory function of TfR2 on EPO sensitivity. The slight differences in the two models are likely related to variable degrees of anemia and iron loading of Tfr2-deficient erythroid cells. Taken together, the data obtained from both experimental settings suggest that inactivation of erythroid TfR2 in iron deficiency facilitates differentiation of erythroblasts. Another ramification of these findings is that acute expansion of erythropoiesis due to EPO administration or hypoxia, leads to a relative iron deficient state that destabilizes TfR2, increases EPO sensitivity in the bone marrow and promotes hepatic hepcidin downregulation via increased ERFE production. This interpretation positions TfR2 as a key molecule in a signaling pathway connecting erythropoiesis with hepcidin regulation (130,131). Conclusions Iron homeostasis is controlled by elaborate mechanisms at the cellular and systemic level, which aim to optimize utilization and counteract toxicity of iron. Cellular and systemic iron regulatory pathways, mediated by the IRE/IRP and hepcidin/ferroportin networks, respectively, crosstalk to coordinate iron supply to erythroid cells for erythropoiesis. Thus, IRP1 operates as an upstream regulator of EPO, the erythropoietic hormone that Papanikolaou and Pantopoulos 409

IUBMB LIFE also controls hepcidin expression via ERFE and other erythroid regulators. ERFE-mediated control of hepcidin directly couples erythropoietic iron need with iron flux in plasma. Iron and erythropoiesis are further connected by additional pathways. For instance, TfR2 appears to offer a critical link between irondependent hepcidin regulation in the liver, and EPO-dependent erythroid cell maturation in the bone marrow. Better understanding of the underlying mechanisms is expected to pave the way for targeted therapeutic interventions. Acknowledgements This work was supported by a grant from the Canadian Institutes for Health Research (CIHR; MOP-86514). References [1] Papanikolaou, G., and Pantopoulos, K. (2005) Iron metabolism and toxicity. Toxicol. Appl. Pharmacol. 202, 199 211. [2] Gkouvatsos, K., Papanikolaou, G., and Pantopoulos, K. (2012) Regulation of iron transport and the role of transferrin. Biochim. Biophys. Acta 1820, 188 202. [3] Ganz, T. 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