Critical Review
Systemic Iron Homeostasis and Erythropoiesis
George Papanikolaou1
Kostas Pantopoulos2*
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 eryC 2017 IUBMB Life, 69(6):399–413, 2017
throid progenitor cells. V
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 Fe21 (ferrous) and Fe31 (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
C 2017 International Union of Biochemistry and Molecular Biology
V
Volume 69, Number 6, June 2017, Pages 399–413
^ te Ste-Catherine Road, Montreal, H3T 1E2, Que*Address correspondence to: Kostas Pantopoulos, Lady Davis Institute for Medical Research, 3755 Co
bec, 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)
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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, Fe21 undergoes
spontaneous aerobic oxidation to Fe31 that is virtually insolu31
ble at physiological pH (KfreeFe
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 Fe21 to plasma via ferroportin (Fpn), the
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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
Fe21, 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 Fe31
to Fe21 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 Fe21 is
transferred to the basolateral membrane by an unknown
mechanism, and exported to plasma via ferroportin. The efflux
Iron and Erythropoiesis
FIG 2
FIG 3
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.
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.
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of Fe21 is associated with its re-oxidation to Fe31 by the soluble or membrane-bound multicopper ferroxidases ceruloplasmin or hephaestin, respectively (6).
Iron Transport in the Bloodstream
Exported Fe31 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 Fe31 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-
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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
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
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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.
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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
404
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
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
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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/
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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 pSMAD1/5/8 with SMAD4, and translocation of the complex to
the nucleus for binding to two BMP responsive elements (BMPRE1 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
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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
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
Papanikolaou and Pantopoulos
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
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
Hbbth3/– 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
408
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
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
Papanikolaou and Pantopoulos
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 (Tfr2BMK8) led to similar impairment in
erythroid differentiation under milder dietary iron restriction (129). However, contrary to hematopoietic-specific
Tfr2–/– mice, the chimeric Tfr2BMK8 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
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).
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