24 Cell 142, July 9, 2010 ©2010 Elsevier Inc.
Plasma Iron: Key to Iron Overload and Iron Deficiency
Iron is essential for fundamental metabolic processes in cells
and organisms. The key to systemic iron supply and homeo-
stasis lies in the regulation of adequate plasma iron levels.
Iron circulates in plasma bound to the glycoprotein transferrin,
which has two high-affinity binding sites for Fe(III). Transfer-
rin binding maintains iron in a soluble form, serves as a major
vehicle for iron delivery into cells (via the transferrin receptor,
TfR1), and limits the generation of toxic radicals. In humans,
plasma transferrin is normally about 30% saturated with iron. A
transferrin saturation <16% indicates iron deficiency, whereas
>45% saturation is a sign of iron overload. When the saturation
exceeds 60%, non-transferrin-bound iron begins to accumu-
late in the circulation and to damage parenchymal cells.
The homeostatic system thus has to maintain transfer-
rin saturation at physiological levels, responding to signals
from pathways that consume iron (such as erythropoiesis)
and sending signals to the cells that supply iron to the blood
stream (Figure 1). Iron is released into the circulation from
duodenal enterocytes, which absorb 1–2 mg of dietary iron
per day, and from macrophages, which internally recycle
20–25 mg of iron from senescent erythrocytes. Hepatocytes
play a dual role in systemic iron metabolism: they are the
major site of iron storage and they secrete the regulatory hor-
mone hepcidin (Hamp, LEAP1). Hepcidin orchestrates sys-
temic iron fluxes and controls plasma iron levels by binding to
the iron exporter ferroportin (SLC40A1, Solute carrier family
40, member 1) on the surface of iron-releasing cells (Figure 1),
triggering its degradation and hence reducing iron transfer to
transferrin (Nemeth et al., 2004). Inherited and acquired dis-
orders that perturb hepcidin production consequently cause
iron deficiency (high hepcidin levels) or iron overload (hepci-
Assessing the concentration of serum ferritin is a clinically
useful measure of iron storage. Low serum ferritin levels indi-
cate depleted stores, whereas increased levels may indicate
iron overload. Inflammatory conditions (or infections, cancer,
and liver disorders) can also increase serum ferritin. Given its
clinical utility, it is surprising that the physiological function(s)
of serum ferritin and its source (that is, whether it is derived
from damaged cells or actively secreted by a regulated mecha-
nism) still remain to be defined. Serum ferritin is predominantly
composed of L chain subunits, partially glycosylated, and iron
Inorganic dietary iron is absorbed at the brush border of duo-
denal enterocytes via the divalent metal transporter 1 (DMT1/
SLC11A2, solute carrier family 11, member 2) (Gunshin et
al.,1997). Given that iron largely adopts the oxidized state, it
must first be reduced by the membrane-associated ferrire-
ductase DcytB (Cybrd1). DcytB may not be the only ferrire-
ductase of the apical membrane of enterocytes, as knock-
out mice appear to have normal iron metabolism (see review
by McKie, 2008). Heme iron is absorbed independently by
mechanisms that remain uncertain, because the proposed
transporter SLC46A1 appears to carry mostly folate (Qiu et
al., 2006). Heme iron is released intracellularly by hemoxyge-
nase, mainly by the inducible hemoxygenase 1 (HOX1) (Fer-
ris et al., 1999). Cytosolic iron can then be exported into the
circulation by the basolateral iron exporter ferroportin (McKie
et al., 2000; Donovan et al., 2000). Enterocytic iron export
through ferroportin requires hephaestin, a multicopper oxi-
dase homologous to ceruloplasmin, which oxidases Fe(II) to
Fe(III) for loading onto transferrin. Consistent with this func-
tion, hephaestin-deficient mice display iron deficiency ane-
mia with mucosal iron retention.
Two to Tango:
Regulation of Mammalian Iron Metabolism
Matthias W. Hentze,1,2,* Martina U. Muckenthaler,2,3 Bruno Galy,1 and Clara Camaschella4
1European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
2Molecular Medicine Partnership Unit, European Molecular Biology Laboratory, and University of Heidelberg, 69120 Heidelberg, Germany
3Department of Pediatric Oncology, Hematology and Immunology, Im Neuenheimer Feld 153, 69120 Heidelberg, Germany
4Vita-Salute University and Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, Italy
Disruptions in iron homeostasis from both iron deficiency and overload account for some of the
most common human diseases. Iron metabolism is balanced by two regulatory systems, one that
functions systemically and relies on the hormone hepcidin and the iron exporter ferroportin, and
another that predominantly controls cellular iron metabolism through iron-regulatory proteins that
bind iron-responsive elements in regulated messenger RNAs. We describe how the two distinct
systems function and how they “tango” together in a coordinated manner. We also highlight some
of the current questions in mammalian iron metabolism and discuss therapeutic opportunities
arising from a better understanding of the underlying biological principles.
Cell 142, July 9, 2010 ©2010 Elsevier Inc. 25
Because iron cannot be excreted from the organism in a reg-
ulated way, iron absorption represents the critically controlled
process. Normally, only 1–2 mg of iron per day are absorbed to
compensate for iron losses, for example by sloughing of intes-
tinal epithelial cells, desquamation of skin and urinary cells,
blood loss, or sweat. Iron absorption can be enhanced when
the needs are higher (for example, because of increased eryth-
ropoiesis or pregnancy) and suppressed in iron overload. The
lack of an active mechanism for iron excretion accounts for
the development of iron overload when the regulation of iron
absorption is defective or bypassed (as occurs in blood trans-
Iron Utilization: Erythropoiesis
The vast majority of recycled iron (?25 mg/day) is dedicated to
hemoglobin synthesis. TfR1 mediates erythroid iron acquisition,
and its expression parallels the maturation of erythroid pro-
genitors. Mouse embryos lacking TfR1 die because of severe
anemia (and with neurologic abnormalities), whereas Tfr1 hap-
loinsufficiency or dysfunction of other components of the TfR1
endocytotic cycle (such as DMT1, STEAP3, or EXOC6, see
below) cause microcytic anemia (characterized by abnormally
small red blood cells) as a result of defective iron utilization in
mice; mutations of DMT1 in humans elicit a similar phenotype
and cause liver iron accumulation (Table S1 available online)
(see review by Iolascon et al., 2009). A proposed additional
route of erythroblast iron acquisition is ferritin released from
macrophages in the so-called “erythroblastic islands” (Leim-
berg et al., 2008). However, the severe iron deficiency anemia
of both mice and patients with transferrin deficiency (Table S1)
suggests that this process can at best make a minor contribu-
tion to erythroid iron acquisition.
Erythroblasts not only acquire but also handle large
amounts of iron. Potentially, iron may be directly transported
from endosomes into mitochondria by a “kiss-and-run mech-
anism” through a direct contact between both organelles,
effectively bypassing the cytosol (Sheftel et al., 2007). Iron
is imported into mitochondria by the inner membrane pro-
tein mitoferrin 1 (Mfrn1/ SLC25A37, solute carrier family 25,
member 37) (Shaw et al., 2006). This process is facilitated
by the ABCB10 (ATP-binding cassette, subfamily B, member
10) protein, which is thought to stabilize Mfrn1 (Chen et al.,
2009). Mfrn2/SLC25A28 may represent the housekeeping
To coordinate the synthesis of the heme precursor protopor-
phyrin IX with iron availability, δ-aminolevulinic acid synthase 2
(ALAS2), the erythroid-specific first enzyme of protoporphyrin
IX synthesis, is posttranscriptionally regulated by iron via the
iron-responsive element/iron-regulatory protein (IRE/IRP) sys-
tem (see below). Genetic defects in ALAS2 cause sideroblastic
Figure 1. Regulation of Systemic Iron Homeostasis
Cells involved in systemic iron regulation are shown. Divalent metal transporter 1 (DMT1) at the apical membrane of enterocytes takes up iron from the lumen of
the duodenum after DCYTB reduces Fe(III) to Fe(II). Ferroportin at the basolateral membrane cooperates with hephaestin that oxidizes Fe(II) to Fe(III). Iron-loaded
(diferric) transferrin (Tf-Fe2), indicated by red dots, supplies iron to all cells by binding to the transferrin receptor 1 (TfR1) and subsequent endocytosis. TfR1 is
highly expressed on hemoglobin-synthesizing erythroblasts. Hepatocytes sense transferrin saturation/iron stores and release hepcidin accordingly. Red cell iron is
recycled by macrophages via ferroportin and the ferroxidase ceruloplasmin. In iron overload (left), high hepcidin levels inhibit ferroportin-mediated iron export by
triggering internalization and degradation of the complex to reduce transferrin saturation. Hepcidin expression is high. In iron deficiency (right), iron is released by
ferroportin into the circulation. Hemoglobin-derived heme is catabolized in macrophages by hemoxygenase-1 (HOX1). Hepcidin expression is low.
26 Cell 142, July 9, 2010 ©2010 Elsevier Inc.
anemia (Table S1), whereas haploinsufficiency of other enzymes
in the pathway cause porphyrias, due to accumulation of toxic
heme precursors (see review by Puy et al., 2010). How heme is
exported from mitochondria remains to be defined.
Mitochondrial iron uptake without the ability to use it for
heme or Fe/S cluster biogenesis causes iron accumulation,
because the excess import is not properly balanced by the
export of products. Such mitochondrial iron depositions lead
to the appearance of “ringed” sideroblasts (erythroblasts with
perinuclear iron accumulations) and occur in conditions such
as X-linked sideroblastic anemia due to ALAS2 deficiency or in
the autosomal recessive deficiency of SLC25A38 (solute car-
rier family 25, member 38), a mitochondrial transporter likely
involved in the import of ALA substrate glycine (Table S1). Ane-
mia and ringed sideroblasts also appear when proteins involved
in Fe/S cluster biogenesis are defective, such as impairment of
GLRX5 (glutaredoxin 5) (Ye et al., 2010) or the ATP-binding cas-
sette protein ABCB7, which leads to sideroblastic anemia with
ataxia (Table S1).
Although erythroblasts consume large amounts of iron, they
have to maintain safety mechanisms to avoid iron and/or heme
excess: iron can be stored in ferritin or exported by ferroportin.
Erythroblasts express a ferroportin messenger RNA (mRNA)
isoform (1b) that lacks the 5′ IRE and thus evades potential
translational repression by IRPs (see below) (Zhang et al.,
2009). This isoform is susceptible to hepcidin degradation and
may endow erythroid precursors with a mechanism to respond
to systemic iron availability. Additionally, erythroblasts have
the capacity to export excess heme (for example, when globin
synthesis is limiting). The proposed exporter, FLVCR (feline leu-
kemia virus subgroup C cellular receptor), is a multitransmem-
brane protein, a member of the major facilitator superfamily,
and a receptor for a virus that causes severe aplastic anemia
in cats. Accumulation of toxic heme at the proerythroblast
stage can cause apoptosis, and mice with neonatal FLVCR
deficiency develop severe hyperchromic, macrocytic anemia,
reticulocytopenia, and a block in erythroid maturation at the
proerythroblast stage (Keel et al., 2008).
Macrophages have to shoulder the lion’s share of the burden
of maintaining adequate levels of plasma iron. Given that less
than 10% of the daily iron needs are met by intestinal absorp-
tion, the rest is covered by macrophages that recycle iron inter-
nally. The amount of plasma iron is just over 10% of the amount
used daily, which means that plasma iron is turned over many
times each day.
Macrophages phagocytose aged or damaged erythrocytes
and catabolize heme using hemoxygenase. NRAMP1 (natu-
ral resistance-associated macrophages protein 1), a divalent
metal transporter homologous to DMT1, is expressed within
phagolysosomal membranes and participates in iron export
from phagocytic vesicles (Soe-Lin et al., 2009). Export of fer-
rous iron from macrophages occurs via ferroportin (Figure 1).
Reflecting its central role in systemic iron homeostasis, fer-
roportin expression in macrophages is regulated at multiple
levels: ferroportin transcription is induced by erythrophago-
cytosis and heme iron, its translation is regulated by the IRE/
IRP system, and its protein stability by hepcidin (see below).
Ferroportin-mediated iron export is coupled to the function of
the multicopper oxidase ceruloplasmin, a protein synthesized
and secreted by the liver. Ceruloplasmin-deficient mice and
humans show hepatocyte and macrophage iron accumulation.
Aceruloplasminemia causes anemia (highlighting the critical
role of iron release for erythropoiesis), diabetes, a late-onset
disorder of the basal ganglia, and retinal degeneration (Table
Systemic Iron Homeostasis: The Iron Hormone Hepcidin
Hepcidin has emerged as the central regulatory molecule of
systemic iron homeostasis. It is a defensin family member with
strong links to innate immunity. The bioactive, mature 25 amino
acid peptide is generated from an 84 amino acid prepropeptide
by furin cleavage. Hepcidin is secreted from hepatocytes and
circulates in plasma bound to α2-macroglobulin (Peslova et al.,
2009). Hepcidin clearance occurs via the kidney or by codeg-
radation with ferroportin. Hepcidin forms a hairpin structure
with four intramolecular disulfide bonds (Jordan et al., 2009)
and exhibits modest antimicrobial activity in vitro, which has
not yet been demonstrated in vivo.
Hepcidin binds to ferroportin, triggers its internalization,
ubiquitination, and subsequent lysosomal degradation (Nem-
eth et al., 2004). Ferroportin binding is mediated by the N termi-
nus of the peptide; Jak2 (Janus kinase 2) has been reported to
bind to the hepcidin-ferroportin complex and to phosphorylate
ferroportin before internalization (De Domenico et al., 2009).
Research into the molecular mechanisms that underlie heredi-
tary hemochromatosis in patients and murine models has been
instrumental to decipher hepcidin regulation in mammals.
Hereditary hemochromatosis is an autosomal recessive dis-
ease that leads to iron overload of the liver and other organs.
Complications, which are preventable by iron depletion ther-
apies, can be fatal and include liver cirrhosis, cancer, diabe-
tes, hypogonadism, heart failure, and arthritis. Family studies
implicate four genes in the disorder (Table S1): the most com-
mon type, which has a carrier frequency of ?1:8 in Caucasian
populations, is due to a homozygous missense mutation of the
HFE gene (C282Y) (Feder et al., 1996). Less common but clini-
cally more severe forms of hereditary hemochromatosis are
caused by mutations of the TfR2, hemojuvelin (HJV), or hepci-
din (HAMP) genes. All recessive forms of the disease represent
molecular defects of hepatocytes and are caused by inappro-
priately low hepcidin expression (Figure 2A): the disease sever-
ity and the age of onset roughly correlate with the degree of
hepcidin deficiency (see review by Camaschella, 2005).
HFE encodes a ubiquitously expressed major histocom-
patibility complex class 1-like molecule. The C282Y mutation
abrogates β2-microglobulin binding and HFE surface expres-
sion; other HFE mutations are relatively rare. Hepcidin levels
in patients may be normal, but are inadequately low for the
degree of iron loading (Piperno et al., 2007; Ganz et al., 2008)
and display a blunted response to acute oral iron challenges
(Piperno et al., 2007). The penetrance of HFE mutations is low
and clinical manifestations occur most commonly in middle
Cell 142, July 9, 2010 ©2010 Elsevier Inc. 27
aged males, indicating the importance of environmental and/
or additional genetic factors for disease expression (Beutler et
Juvenile hereditary hemochromatosis due to mutations in
the HAMP or HJV genes may lead to irreversible hypogonad-
ism, refractory heart failure, and even death in the second to
third decades of life. Patients with HAMP or HJV mutations are
phenotypically similar and have virtually undetectable hepci-
din levels. HJV is a glycophosphatidlyinositol-linked protein,
homologous to repulsive guidance molecules, and mostly
expressed in liver, skeletal muscle, and heart. HJV is a bone
morphogenetic protein (BMP) coreceptor (Babitt et al., 2006)
that is required to drive hepcidin transcription via SMAD pro-
teins (Figure 2A) (see below). HAMP mutations are extremely
rare. Mice lacking HAMP and HJV recapitulate the organ iron
loading observed in humans (Table S1).
Hereditary hemochromatosis due to TfR2 mutations may
present early, but with a less severe phenotype than the juve-
nile form (see review by Camaschella, 2005). TfR2 is a type II
transmembrane protein that binds transferrin with lower affin-
ity than TfR1 (Figure 2A). Targeted Tfr2 gene deletion in mice
causes iron overload with low basal hepcidin levels (Table S1);
similar observations have been reported in humans with TFR2
A dominant form of hereditary hemochromatosis is caused
by missense mutations in ferroportin (Table S1). Ferropor-
tin is the only known cellular iron exporter and represents
the “hepcidin receptor.” Mutations that reduce its membrane
localization or its ability to export iron cause macrophage iron
retention, normal/low plasma iron levels, and in some cases
iron-restricted erythropoiesis. A hemochromatosis-like dis-
ease with high plasma iron and hepatocyte iron accumulation
is caused by hepcidin-resistant ferroportin mutations either
because hepcidin fails to bind ferroportin (C326S) or the inter-
nalization and degradation of ferroportin following hepcidin
binding is impaired (Fernandes et al., 2009).
Hepcidin levels are also inappropriately low in “iron-loading
anemias” in which erythropoietic signals suppress hepcidin
transcription (Figure 2B) even when systemic iron load is high.
Figure 2. Regulation of Hepcidin Expression
(A) Hepcidin regulation by systemic iron availability. High concentrations of
transferrin-Fe2 (Tf-Fe2) displace HFE from TfR1 to promote its interaction with
transferrin receptor 2 (TfR2). The HFE-TfR2 complex then activates hepcidin
transcription via ERK/MAPK and bone morphogenetic protein (BMP)/SMAD
signaling. The BMP coreceptor hemojuvelin (HJV) interacts with type I and
type II BMP receptors (BMPR) at the plasma membrane to induce phospho-
rylation of receptor-activated SMAD (R-SMAD) proteins, and subsequent
formation of active transcriptional complexes involving the co-SMAD factor
SMAD4. This signaling is inhibited by soluble HJV (sHJV). TMPRSS6 physical-
ly interacts with HJV and causes HJV fragmentation. SMAD7 interferes with
SMAD4-controlled hepcidin activation. Sequence motifs critical for SMAD-
mediated control of the hepcidin promoter are shown.
(B) Hepcidin regulation by erythropoietic signals. GDF15 and TWSG 1 are
released by erythroid precursors to inhibit BMP/SMAD activation of hepcidin.
This situation characterizes iron-loading anemias.
(C) Hepcidin regulation by inflammatory stimuli. Interleukin 6 (IL6) activates
the Janus kinase (JAK)/ signal transducer and activator of transcription (STAT)
signaling pathway and stimulates the hepcidin promoter via a STAT-binding
motif close to the transcription start site. The BMP signaling pathway also
contributes to the inflammatory response via SMAD4.
28 Cell 142, July 9, 2010 ©2010 Elsevier Inc.
The prototype of these anemias is β-thalassemia intermedia,
characterized by transfusion-independent iron overload and
low to absent hepcidin levels. Growth differentiation factor 15
(GDF15) and twisted gastrulation 1 (TWSG1) released by eryth-
roblasts have been proposed to be involved in hepcidin sup-
pression (Tanno et al., 2007, Tanno et al., 2009). In patients with
homozygous β-thalassemia or other anemias with ineffective
erythropoiesis, elevated serum GDF15 correlates with dimin-
ished hepcidin levels and increased iron absorption.
Iron (blood) losses and/or insufficient iron intake/absorption from
dietary sources can cause iron deficiency that most commonly
manifests as microcytic anemia. Likewise, inappropriately high
hepcidin expression lowers plasma iron levels (due to dimin-
ished iron release by macrophages and lower iron absorption)
and causes anemia. In this context, the common acquired ane-
mia of chronic diseases (ACD) and the genetic iron-refractory
iron deficiency anemia (IRIDA) are most interesting.
Related to its evolutionary origin, hepcidin transcription is
activated by inflammatory cytokines, especially interleukin
6 (Figure 2C). Hypoferremia develops rapidly as a result of
decreased macrophage iron release and represents a defense
mechanism against (iron-dependent) pathogens. Excessive
hepcidin production is also seen in patients with infections,
malignancies, chronic kidney diseases, or any type of inflam-
mation. If prolonged, it leads to ACD. In rare cases, hepcidin can
be expressed ectopically by hepatic adenomas, which results
in microcytic anemia with some features of ACD (Weinstein et
al., 2002), but this anemia is fully reversible after removal of the
Patients with iron deficiency normally have low or undetect-
able levels of hepcidin. This is not the case in patients with
IRIDA, who suffer from a microcytic anemia that is unrespon-
sive to oral and partially refractory to parenteral iron, because of
inappropriately high hepcidin levels. IRIDA is caused by muta-
tions in TMPRSS6 (matriptase-2), a gene that encodes a pro-
tease that negatively regulates hepcidin expression (Du et al.,
2008) (see below). Interestingly genetic variants in TMPRSS6,
frequent in the general population, may modulate the ability to
absorb iron and to synthesize hemoglobin for maturing erythroid
cells (Andrews, 2009). Whether TMPRSS6 variants may contrib-
ute to sporadic iron deficiency by increased hepcidin levels and
decreased dietary iron absorption remains to be explored.
Hepcidin expression in hepatocytes is regulated by multiple, in
part opposing signals, including systemic iron availability (such
as diferric transferrin, Tf-Fe2), hepatic iron stores, erythropoietic
activity, hypoxia, and inflammatory/infectious states (Figure 2).
These different regulatory inputs are integrated transcriptionally.
Regulation by Systemic Iron Availability
After the discovery of the biological relevance of hepcidin,
important progress has been made toward understanding
the molecules and pathways that control hepcidin expres-
sion in response to iron and the role of the membrane proteins
mutated in hereditary hemochromatosis (HFE, HJV, and TfR2)
in this process (Figure 2A).
HFE has been suggested to act as a bimodal switch
between two sensors of the concentration of Tf-Fe2, TfR1,
and TfR2, on the plasma membrane of hepatocytes (Gos-
wami and Andrews, 2006). This model is supported by the
following findings: HFE binds the ubiquitously expressed
TfR1 at a site that overlaps the transferrin binding domain,
and Tf-Fe2 thus competes with HFE binding to TfR1. By con-
trast, TfR2 can bind both HFE and Tf-Fe2 simultaneously
(Gao et al., 2009). Mice bearing an engineered TfR1 mutation
with increased HFE binding show low hepcidin expression
and systemic iron overload similar to HFE-deficient mice,
suggesting that the TfR1 sequesters HFE to prevent its par-
ticipation in hepcidin activation. Conversely, mutations that
abolish the HFE-TfR1 interaction or mice with increased HFE
levels display elevated hepcidin expression and succumb
to iron deficiency (Schmidt et al., 2008). Hepcidin activation
by holotransferrin requires both HFE and TfR2 (Gao et al.,
2010). These observations support a model in which high
concentrations of Tf-Fe2 displace HFE from TfR1 to pro-
mote its interaction with TfR2, which is further stabilized by
increased Tf-Fe2 binding to the lower-affinity TfR2. The HFE-
TfR2 complex then activates hepcidin transcription. Future
research is needed to establish the stoichiometry of the pro-
teins involved in this “Tf-Fe2-sensing complex” and to clarify
whether HJV is part of it.
Although HFE and TfR2 clearly contribute to hepcidin acti-
vation, the BMP signaling pathway is quantitatively the most
critical. By as yet only partially understood mechanisms, it
integrates signals from the “Tf-Fe2-sensing complex” and the
hepatocytic iron stores. Central to the latter is BMP6, which
is positively regulated by iron. How BMP6 mRNA expression
is activated by increased iron levels and repressed by iron
deficiency requires further investigation. Bmp6 knockout mice
show hepcidin deficiency and tissue iron overload (Andrio-
poulos et al., 2009; Meynard et al., 2009), although BMP2
and BMP4 can also bind to HJV. BMP6 is thought to act in
an autocrine manner analogous to its role in chondrocyte dif-
ferentiation (Grimsrud et al., 1999) to induce signaling via HJV,
the BMP coreceptor that adapts BMP receptors for iron regula-
tion (Babitt et al., 2006). The BMP/HJV complex joins the type
I (Alk2 and Alk3) and the type II (ACTRIIA) BMP receptors to
induce phosphorylation of receptor activated SMAD (R-SMAD)
proteins and subsequent formation of active transcriptional
complexes involving the co-SMAD factor, SMAD4 (Wang et al.,
2005) (Figure 2A).
Two sequence motifs (the proximal BMP-RE1 and the distal
BMP-RE2) of the hepcidin promoter are critical for transcrip-
tion via HJV, BMP6, and SMAD4 (Casanovas et al., 2009),
and the promoter region that contains BMP-RE2 confers iron
responsiveness to the hepcidin promoter (Truksa et al., 2007).
Multiple lines of evidence highlight the importance of HJV/
BMP/SMAD signaling for hepcidin activation: (1) mice lacking
HJV show attenuated R-SMAD phosphorylation in the liver
(Babitt et al., 2006), (2) administration of BMP2 and BMP6
to mice induces hepcidin mRNA and decreases serum iron
levels, (3) BMP antagonists (such as dorsomorphin) inhibit
hepcidin mRNA expression and increase serum iron levels
(Yu et al., 2008), (4) liver-specific disruption of the co-SMAD4
Cell 142, July 9, 2010 ©2010 Elsevier Inc. 29
causes severe iron overload with diminished hepcidin tran-
scription (Wang et al., 2005), and (5) the inhibitory iSMAD7
potently suppresses hepcidin transcription in cellular models
(Mleczko-Sanecka et al., 2010). Interestingly, R-SMAD phos-
phorylation is also attenuated in mice lacking HFE, suggest-
ing that HJV and HFE act together to activate hepcidin tran-
scription (Corradini et al., 2009; Kautz et al., 2009). Crosstalk
between the BMP/SMAD and p38-MAPK signaling pathways
activates hepcidin mRNA expression in response to Tf-Fe2
in primary hepatocytes. Activation of p38-MAPK and Erk1/2
depends on both HFE and TfR2, as this pathway is attenuated
in mice lacking HFE or TFR2 and in double-knockout mice
(Wallace et al., 2009).
Apart from mutations of the hepcidin gene itself, only HJV
mutations lead to a near absence of hepcidin expression and
the most severe form of hereditary hemochromatosis. Thus,
HJV is central for hepcidin expression, and the point of con-
vergence of multiple regulatory inputs. The membrane-associ-
ated protease TMPRSS6 that is mutated in IRIDA (see above)
physically interacts with HJV and cleaves HJV when both pro-
teins are expressed on the cell surface, suggesting that HJV
is the major TMPRSS6 target for iron regulation (Silvestri et
al., 2008a). Genetically, the combined deficiency of HJV and
TMPRSS6 causes iron overload, suggesting that TMPRSS6
acts upstream of HJV (Truksa et al., 2009, Finberg et al., 2010).
Increased HJV surface expression has however yet to be con-
firmed in Tmprss6-deficient mice or IRIDA patients.
Furin-mediated cleavage releases HJV from cells to gener-
ate soluble HJV (sHJV), which antagonizes BMP-dependent
hepcidin activation. Furin mRNA expression is regulated by
iron and hypoxia, conferring another level of control (Silvestri
et al., 2008b). Because of the high HJV expression in skeletal
muscle, it is tempting to speculate that sHJV is released as a
muscle signal in iron deficiency. Importantly, cleavage of HJV
by other proteases does not seem to be redundant with that
by TMPRSS6, as lack of TMPRSS6 activity causes iron defi-
ciency in humans and mice. Future work needs to address
how TMPRSS6 expression and activity are regulated, and the
relative contributions of TMPRSS6 and furin to the regula-
tion of HJV and systemic iron homeostasis need to be further
Neogenin, a DCC (deleted in colorectal cancer) family mem-
ber, appears to stabilize HJV to enhance BMP signaling and
hepcidin expression. Consistently, mice lacking neogenin
exhibit hepatic iron overload, low hepcidin levels, and reduced
BMP signaling (Lee et al., 2010).
Regulation by Erythropoietic Signals
Erythropoiesis requires considerable quantities of iron, and
the inhibition of hepcidin expression by erythropoietic signals
(Figure 2B) thus is of great physiological importance. None-
theless, the molecular mechanisms and factors responsible
are still poorly understood. Hepcidin suppression in response
to phlebotomy or hemolysis depends on intact erythropoietic
activity in mouse models: irradiation and cytotoxic inhibition of
erythropoiesis prevent hepcidin suppression (Pak et al., 2006).
GDF15 and TWSG1 are both released by erythroid precursors.
High doses of GDF15 are detectable in the serum of patients
with ineffective erythropoiesis such as β-thalassemia (Tanno
et al., 2007). Such pathological concentrations of GDF15 can
suppress hepcidin transcription in cell models, but the under-
lying molecular mechanism has not yet been characterized. By
contrast, lower GDF15 concentrations fail to suppress hepci-
din in cellular models and are apparently ineffective in patients
with sickle cell anemia, myelodysplastic syndrome, and ACD.
TWSG1 expression is increased in thalassemic mice, where it is
produced during early erythroblast maturation. In cellular mod-
els, the BMP-binding protein TWSG1 inhibits BMP-dependent
activation of Smad-mediated signal transduction that leads to
hepcidin activation (Tanno et al., 2009). Correlations between
TWSG1 expression, serum iron parameters, and hepcidin lev-
els have not yet been studied in human anemias.
Regulation by Hypoxia
Liver-specific stabilization of the hypoxia-inducible factor 1
(HIF1) and HIF2 decreases hepcidin expression, and chemi-
cal HIF stabilizers can suppress hepcidin mRNA expression in
hepatoma cells (Peyssonnaux et al., 2007). These findings have
raised the possibility that iron-dependent prolyl hydroxylases
involved in HIF degradation may act as hepatic iron sensors.
Whether or not HIFs directly bind to the hepcidin promoter is
In vivo, hypoxia induces erythropoietin (EPO) synthesis,
which in turn stimulates erythropoiesis. EPO injection into mice
reduces hepcidin levels in a dose-dependent manner and can
override signals that activate hepcidin expression. Even low
dose EPO injections in human volunteers promptly decrease
urinary excretion of hepcidin (Robach et al., 2009). Because
experimental blockade of erythropoietic activity prevents its
effect, EPO likely suppresses hepcidin by stimulation of eryth-
ropoiesis rather than more directly (Pak et al., 2006).
Regulation by Inflammatory and Stress Signals
The inflammatory cytokines IL1 and IL6 are both potent induc-
ers of hepcidin expression, a response whose clinical impor-
tance for ACD has been discussed above. IL6 activates the
Janus kinase (JAK)/signal transducer and activator of tran-
scription (STAT) signaling pathway, which activates the hepci-
din promoter via a STAT-binding motif close to the transcription
start site (Figure 2C) (Fleming 2007). The BMP signaling path-
way also contributes to the inflammatory response via SMAD4
(Casanovas et al., 2009; Wang et al., 2005). Mice injected
with lipopolysaccharide (LPS) augment hepcidin transcription
even in the context of iron overload; likewise, LPS counter-
acts the diminished hepcidin expression in response to iron
deficiency, suggesting that the two signals are integrated at
the hepcidin promoter and that inflammatory and iron stores
regulators operate independently rather than following a strict
hierarchy (Huang, et al., 2009a). Hepcidin expression is also
increased by endoplasmic reticulum (ER) stress. This stress
response can be controlled by the transcription factor cyclic
AMP response element-binding protein H (CREBH) (Vecchi et
al., 2009) or by the stress-inducible transcription factors CHOP
and C/EBPalpha (Oliveira et al., 2009). It has also been sug-
gested that increased hepcidin transcription and iron depriva-
tion may represent defense mechanisms against excessive cell
proliferation and cancer, possibly by binding of the p53 tumor
suppressor protein to a response element in the hepcidin pro-
moter (Weizer-Stern et al., 2007).
30 Cell 142, July 9, 2010 ©2010 Elsevier Inc.
The increasing understanding of the role of hepcidin in iron
overload and deficiency (inclucing ACD) opens new therapeu-
tic avenues in a field that at present is essentially limited to
iron depletion or substitution therapy. Both hepcidin agonists
and antagonists would be useful drug prospects to treat iron-
Current treatment options for diseases hallmarked by
insufficient hepcidin expression would be complemented by
hepcidin agonists. A proof-of-principle study has shown that
transgenic hepcidin expression in mice lacking HFE can pre-
vent iron overload (Nicolas et al., 2003). However, it is not clear
whether hepcidin substitution (similar to insulin treatment of
type 1 diabetes) would complement the current treatment of
hereditary hemochromatosis with phlebotomy (or iron chela-
tors). However, small molecules to augment hepcidin synthesis
(via transcription) or mimic its effects on ferroportin are attrac-
tive candidates for the treatment of thalassemias, other iron-
loading anemias, and the iron overload induced by hepatitis
Hepcidin antagonists would be useful for the treatment of
patients with iron-restricted anemias as a consequence of hep-
cidin excess (e.g., ACD, IRIDA). Hepcidin depletion by neutralizing
antibodies or by hepcidin small interfering RNAs (siRNAs) was
shown to restore normal hemoglobin levels in a mouse model of
anemia of inflammation when applied in combination with EPO
(Sasu et al., 2010). Here, inhibitors of the stimulatory pathways for
hepcidin transcription could offer one class of candidate com-
pounds. Agents like the BMP signaling inhibitor dorsomorphin
(Yu et al., 2008) or sHJV, which decrease baseline expression
of hepcidin in mice, might prevent iron-deficiency anemias due
to excess hepcidin. Alternatively, one can envisage blocking the
effect of hepcidin on its only known target ferroportin, at the level
of binding or the downstream effects that it triggers.
Figure 3. Cell Biology of Iron Metabolism
A generic cell is depicted. Most cells acquire plasma iron via transferrin receptor 1 (TfR1)-mediated endocytosis of transferrin-bound iron. In endosomes, iron
is freed from transferrin and reduced to Fe(II) by STEAP metalloreductases prior its release into cytosol via divalent metal transporter 1 (DMT1); transferrin and
TfR1 return to the plasma membrane to be used for further cycles. DMT1 also functions in the apical absorption of dietary iron after reduction by DCYTB and
possibly other ferrireductases. Other iron acquisition pathways are symbolized (e.g., acquisition of heme iron from red blood cells by macrophages). Iron uptake
systems feed the so-called labile iron pool (LIP). The LIP is utilized for direct incorporation into iron proteins or iron transport to mitochondria via mitoferrin
(Mfrn), where the metal is inserted into heme and Fe/S cluster prosthetic groups. Proteins promoting heme transport into and out of cells have been identified.
The fraction of the LIP that is not utilized for metalation reactions can be exported via ferroportin, which works together with ferroxidases for iron loading onto
transferrin, or stored in a nontoxic form in ferritin shells. Ferritin can be released into the extracellular milieu by unknown mechanisms and interact with specific
receptors on the cell surface. Some cells also express a mitochondrial form of ferritin to protect the organelle against iron-induced toxicity. The size of the LIP
is determined by the rate of iron uptake, utilization, storage, and export; these processes must be coordinately regulated to avoid detrimental iron deficiency
and prevent iron excess.
Cell 142, July 9, 2010 ©2010 Elsevier Inc. 31
Cellular Iron Homeostasis: The IRE/IRP System
The maintenance of iron homeostasis by cells involves tasks
that are very similar to those addressed at the systemic level:
coordination of iron uptake, utilization, and storage to assure
the availability of appropriate supplies and to prevent toxicity.
Remarkably, the machinery and the mechanisms are entirely
different. In contrast to systemic iron metabolism, cellular iron
traffic also involves regulated iron excretion (Figure 3).
Cellular Iron Uptake
Tf-Fe2 is a major iron source for mammalian cells, which they
take up via the high-affinity TfR1. The Tf-Fe2/TfR1 complex is
internalized by clathrin-dependent endocytosis. Subsequent
acidification of early endosomes triggers conformational
changes in both transferrin and its receptor that promote the
release of iron (Dautry-Varsat et al., 1983). The freed iron is
then reduced to Fe(II) by members of the STEAP familly of
metalloreductases (Ohgami et al., 2005, 2006) for transport
into the cytosol via DMT1; thus, DMT1 plays a dual role in iron
metabolism as an apical membrane protein of enterocytes to
mediate systemic iron absorption as well as a ubiquitous endo-
somal protein involved in iron transfer from endosomes to the
cytosol. Apo-transferrin and the TfR1 are then largely recycled
to the cell surface. Optimal kinetics of the transferrin cycle are
important for efficient acquisition of transferrin-bound iron and
require the EXOC6 member of the exocyst, a protein complex
involved in vesicular trafficking (Lim et al., 2005). Although TfR1
is ubiquitously expressed, the transferrin cycle is particularly
important for the massive iron delivery to erythroid precursors.
Humans and mice lacking transferrin expression accumulate
iron in nonhematopoietic tissues such as the liver (Table S1),
and targeted disruption of the Tfr1 locus in the mouse shows
that TfR1 is required for the differentiation of erythroid, lym-
phoid, and neuroepithelial cells, but it is dispensable for the
development of other tissues, at least during fetal life. This
implies that at least some cells can acquire iron independently
of the transferrin cycle.
Biochemical and genetic studies support the existence of
transferrin-independent routes of iron uptake. DMT1 was
once thought to be responsible for non-transferrin-bound iron
uptake by liver cells, but iron loading of DMT1-deficient mouse
hepatocytes indicates that at least one alternative transferrin-
independent uptake pathway must exist (Gunshin et al., 2005);
the metal transporter ZIP14 is a candidate for this (Liuzzi et al.,
2006), but awaits functional validation in vivo.
Under conditions of systemic iron overload, the L-type volt-
age-gated calcium channel mediates transferrin-independent
iron entry into cardiomyocytes (Oudit et al., 2003); calcium
channels may also play a role in iron delivery to neuronal cells
(Gaasch et al., 2007). Interestingly, calcium channel block-
ers such as nifedipine mobilize liver iron and enhance urinary
excretion in iron-loaded mice, probably by increasing DMT1-
mediated iron transport (Ludwiczek et al., 2007). These find-
ings suggest new therapeutic opportunities for the treatment
of systemic iron overload. Receptor-mediated endocytosis
of other forms of protein-bound iron represents an addi-
tional means for specific cell types to take up iron: lipocalin
2-dependent endocytosis of an iron-laden siderophore via the
SLC22A17 lipocalin receptor has been proposed to modulate
the survival of kidney cells in culture (Devireddy et al., 2005),
but the physiological relevance of this iron uptake pathway
remains uncertain as lipocalin 2 knockout mice develop nor-
mally. Serum ferritin can enter cells via the Scara5 (scavenger
receptor class A, member 5) and TIM-2 (T cell immunoglobu-
lin and mucin domain containing 2) ferritin receptors (Li et al.,
2009, Chen et al., 2005).
Finally, specialized cells are able to acquire iron in the form
of heme. The nature of the enterocytic heme importer remains
uncertain; SLC48A1 is the only bona fide heme import mol-
ecule identified so far (Rajagopal et al., 2008), and gene inac-
tivation in mice should help to evaluate its in vivo functions in
Cells also acquire heme indirectly. Macrophages obtain
heme by phagocytosis and processing of dying red blood
cells. In plasma, hemoglobin and free heme arising from intra-
vascular hemolysis are cleared by specific scavenger systems:
hemoglobin forms a complex with haptoglobin that is delivered
to reticuloendothelial cells via CD163-mediated endocytosis.
Among other plasma molecules, free heme binds to hemopexin
and the complex is endocytosed via the CD91 receptor pres-
ent on the surface of macrophages, hepatocytes, and other
cell types. Thus, cells meet their iron needs via different uptake
systems optimized to serve the specific cellular iron demands
Cellular Iron Export
Iron export occurs from many cells including neuronal and
erythroid cells, but it is particularly important in cells that
maintain plasma iron levels. Such cells include macrophages
and duodenal enterocytes, and in fetal develoment iron export
is mediated by cells of the extraembryonic visceral endo-
derm (ExVE) and placental syncythiotrophoblasts. These cells
express relatively high levels of ferroportin (SLC40A1), and the
effects of targeted disruption of the Slc40a1 locus in the mouse
reflects the unique, nonredundant functions of ferroportin in
iron release from these cell types (Donovan et al., 2005). As
mentioned above, ferroportin transports Fe(II) and acts in con-
cert with either of the ferroxidases hephaestin (enterocytes)
or ceruloplasmin (other cell types) that facilitate iron extrac-
tion from the ferroportin channel and subsequent loading onto
plasma transferrin (De Domenico et al., 2007). The fact that both
ceruloplasmin and hephaestin are copper dependent explains
the importance of the copper status for iron metabolism.
In addition to ferroportin-mediated release of elemental iron,
cells appear to be able to export iron in the form of heme; over-
expression and viral interference studies suggest that FLVCR1
could promote heme efflux. The physiological role of heme
export remains unclear, but FLVCR1 is essential in the mouse
and is required for proerythroblast differentiation and mac-
rophage heme iron recycling (Keel et al., 2008). A small fraction
of cellular iron can also exit the cell bound to ferritin, but the
mechanisms and physiological role of ferritin release remain to
be better defined.
Cellular Iron Storage
Iron from the cytoplasmic “labile iron pool” (LIP) that is not uti-
lized for metalation reactions or exported is stored within the
nanocavity of ferritin heteropolymers made of 24 subunits of
heavy (FtH1) and light (FtL) chains (see review by Arosio and
32 Cell 142, July 9, 2010 ©2010 Elsevier Inc.
Levi, 2010). Both ferritin subunits are ubiquitously expressed,
but their expression ratios vary depending on the cell type
and in response to stimuli such as inflammation or infection.
FtH1 carries the ferroxidase activitiy that is necessary for iron
deposition into the nanocage, while FtL facilitates iron nucle-
ation and increases the turnover of the ferroxidase site. Little is
known about how iron is extracted from the LIP and delivered
to ferritin. Poly(rC)-binding protein 1 (PCBP1), an RNA-binding
protein mostly known for its role in posttranscriptional regula-
tion, is required for ferritin iron loading in cultured cells and
can promote ferritin iron loading in vitro (Shi et al., 2008); future
work will address whether PCBP1 is the metallochaperone
responsible for ferritin mineralization in vivo.
Ferritin provides cells with a means to lock up excess iron in
a redox inactive form to prevent iron-mediated cell and tissue
damage; it also constitutes an iron store whose mobilization
involves both proteasomal and lysosomal ferritin degradation.
Ferritin is essential, as shown by the early embryonic lethal-
ity of FtH1 knockout mice (Ferreira et al., 2000). Mutations of
the FtL 5′ IRE (see below) cause the dominant hyperferritine-
mia-cataract syndrome. C-terminal mutations of FtL cause
hereditary ferritinopathy, an adult-onset autosomal dominant
neurodegenerative disease characterized by the presence of
ferritin inclusion bodies and iron deposition in the brain (Table
Homopolymers of a nuclear gene-encoded H-type ferritin
are present in mitochondria (see review by Arosio and Levi,
2010). Akin to its cytosolic counterpart, mitochondrial ferritin
(FtMt) is thought to protect the organelle against iron-mediated
toxicity. In contrast to FtH1 and FtL, FtMt is not ubiquitously
expressed; it has been detected in tissues such as the tes-
tis, heart, endocrine pancreas, and kidney, but not in spleen,
gut, or liver. Another major difference between cytosolic and
mitochondrial ferritins is that FtMt expression is not (directly)
controlled by the IRPs (see below).
Regulation of Cellular Iron Metabolism
While key aspects of systemic iron metabolism are regulated
transcriptionally (hepcidin expression) and posttranslationally
(ferroportin function by hepcidin), cellular iron homeostasis is
coordinately regulated posttranscriptionally by iron regulatory
protein 1 (IRP1) and IRP2 (also known as ACO1 and IREB2,
respectively) (Figure 4). The two orthologous RNA-binding pro-
teins interact with conserved cis-regulatory hairpin structures
known as IREs, which are present in the 5′ or 3′ untranslated
regions (UTRs) of target mRNAs. Either of the two IRPs inhibits
translation initiation when bound to the single 5′ UTR IREs of
ferritin H- or L-chain (iron storage), ferroportin (export), ALAS2
(utilization), mitochondrial aconitase (ACO2), or hypoxia-induc-
ible factor 2α (HIF2α/EPAS1) mRNAs, whereas their binding
to the multiple IRE motifs within the 3′ UTR of TFR1 (uptake)
mRNA prevents its endonucleolytic cleavage and subsequent
degradation (see reviews by Muckenthaler et al., 2008, and
Recalcatti et al., 2010). The IRPs also appear to positively regu-
late DMT1 (uptake) mRNA expression via a single 3′ UTR IRE
motif, but the molecular mechanism is not known. Single IRE-
like structures with restricted phylogenetic conservation have
been identified in the 3′ UTR of the CDC14A (cell division cycle
14 homolog A), HAO1 (hydroxyacid oxidase 1), and CDC42BPA
(CDC42 binding protein kinase α)/MRCKa mRNAs, but their
functional roles are not yet clear.
A canonical IRE is defined by both RNA sequence and struc-
ture and consists of an unpaired cytosine separated from a
CAGUGN loop (where N = U, C, or A) by a 5 base pair upper
stem plus a lower stem of variable length (Muckenthaler et al.,
2008); the IREs of the HIF2α and DMT1 mRNAs have a nonca-
nonical additional bulge on the 3′ strand of the upper stem. The
high specificity and affinity of the IRP1/IRE interaction results
from two spatially distant sites that establish multiple RNA-
protein contacts clustered around the terminal loop and the C
bulge, respectively, of the IRE (Walden et al., 2006); sequence
Figure 4. Regulation of Cellular Iron
In iron-deficient cells (right), iron regulatory protein 1
(IRP1) or IRP2 bind to cis-regulatory hairpin struc-
tures called iron-responsive elements (IREs), present
in the untranslated regions (UTRs) of mRNAs encod-
ing proteins involved in iron transport and storage
(Muckenthaler et al., 2008). The binding of IRPs to
single IREs in the 5′ UTRs of target mRNAs inhibits
their translation, whereas IRP interaction with mul-
tiple 3′ UTR IREs in the transferrin receptor 1 (TfR1)
transcript increases its stability. As a consequence,
TfR1-mediated iron uptake increases whereas iron
storage in ferritin and export via ferroportin decrease,
thereby increasing the LIP. In iron-replete cells (left),
the FBXL5 iron-sensing F-box protein interacts with
IRP1 and IRP2 and recruits the SKP1-CUL1 E3 ligase
complex that promotes IRP ubiquitination and deg-
radation by the proteasome; IRP1 is primarily subject
to regulation via the assembly of a cubane Fe/S clus-
ter that triggers a conformational switch precluding
IRE-binding and conferring aconitase activity to the
holoprotein. IRPs also modulate the translation of
the mRNAs encoding the erythroid-specific ALAS2
heme synthesis enzyme, the mitochondrial aconitase
(ACO2), and the HIF2α hypoxia-inducible transcrip-
tion factor. Single 3′ UTR IRE motifs are present in
the DMT1 and CDC14A mRNAs, but their role and
mechanism of function are not yet fully defined.
Cell 142, July 9, 2010 ©2010 Elsevier Inc. 33
variability of the upper and lower stems also influences IRP1
binding affinity, potentially contributing to the graded response
of IRE-controlled mRNAs to IRP regulation (Goforth et al.,
2010). Approaches based on mutagenesis and systematic
evolution of ligands by exponential enrichment (SELEX) have
yielded high-affinity RNA binders that are substantially differ-
ent from the canonical IRE (Henderson et al., 1994, Butt et al.,
1996), suggesting that the IRP regulon may extend beyond the
current list of IRE-containing mRNAs.
IRP-binding to IREs responds to cellular iron levels (Figure
4). In iron-replete cells, IRP2 (and apo-IRP1, see below) inter-
acts with the FBXL5 (F-box and leucine-rich repeat protein 5)
adaptor protein that recruits a SCF (SKP1-CUL1-F-box) E3
ligase complex, promoting IRP ubiquitination and subsequent
degradation by the proteasome (Salahudeen et al., 2009, Vash-
isht et al., 2009); in iron-deficient cells, the FBXL5-dependent
degradation of IRPs decreases. Iron regulation of IRP turnover
involves a hemerythrin-like domain of FBXL5 that acts as an
iron sensor: direct binding of iron to this domain stabilizes
FBXL5 (hence IRPs are degraded), whereas FBXL5 is other-
IRP1-binding to IREs is subject to an additional layer of
regulation. In iron-replete cells, IRP1 (but not IRP2) ligates a
cubane 4Fe-4S cluster that precludes IRE binding (Walden et
al., 2006; Muckenthaler et al., 2008); remarkably, 4Fe-4S IRP1
functions as a cytosolic aconitase and the protein is hence
bifunctional. In iron-deficient cells, IRP1 loses its Fe/S cluster
and aconitase activity and adopts its IRE-binding (apo-IRP1)
conformation. The molecular details of this iron-regulated Fe/S
cluster assembly/disassembly are not yet known, but disrup-
tion of critical components of Fe/S cluster biogenesis stimulate
the IRE-binding activity of IRP1 (see review by Sheftel and Lill,
2009). It is thus possible that the ratio of holo- to apo-IRP1
depends primarily on mitochondrial iron availability and Fe/S
cluster synthesis, whereas cytosolic iron sensing may primarily
Genetic ablation of both IRPs in the mouse causes embry-
onic lethality (Smith et al., 2006, Galy et al., 2008). By contrast,
animals lacking either protein are viable and fertile. IRP2 knock-
out mice show a mild microcytic anemia, a tendency toward
increased neurodegeneration, and abnormal body iron distri-
bution (Cooperman et al., 2005, Galy et al., 2005). IRP1 knock-
out mice are asymptomatic under laboratory conditions, dem-
onstrating that the cytosolic aconitase activity is not essential
(Meyron-Holtz et al., 2004a). Taken together, IRP expression is
essential, but the two proteins are largely redundant.
Experiments with animals and cultured cells show that the
two IRPs respond differentially to non-iron signals. For exam-
ple, hypoxic conditions inactivate IRP1 by favoring holo-IRP1
formation (Meyron-Holtz et al., 2004b) while stabilizing IRP2
as a result of the oxygen requirement for iron-mediated FBXL5
degradation. Furthermore, reactive oxygen species selectively
activate IRP1 by causing disassembly of Fe/S clusters, likely
via a membrane-initiated signaling pathway (Pantopoulos and
Hentze, 1998). Phosphorylation of IRP1 and IRP2 by specific
kinases can also regulate their activity. Although the exact
physiological and pathophysiological functions of the differ-
ential regulation of IRP1 and IRP2, respectively, remain to be
determined, it could in principle allow cells to finely control iron
metabolism over a wide range of conditions and to alter the set
points of the two regulatory proteins independently from iron
Intracellular Iron Trafficking and Utilization
One of the least well understood problems in iron biology is how
iron moves within cells. In the cytoplasm, iron is present in di-
iron centers directly bound to proteins such as ribonucleotide
reductase, but most iron trafficks to mitochondria, where it is
incorporated into bioactive heme and Fe/S cluster prosthetic
groups. Recent work identifies 2,5 dihydroxybenzoic acid as
the iron-binding moiety of a mammalian siderophore related
to bacterial enterobactin. Disruption of its biosynthesis causes
mitochondrial iron deficiency, implicating its importance for
intracellular iron transport to mitochondria (Devireddy et al.,
2010). In erythroblasts, the main mitochondrial iron importer for
heme and Fe/S cluster biogenesis is Mfrn1. Mfrn1 is required
for primitive and definitive erythropoiesis (Shaw et al., 2006).
Iron management within mitochondria also remains poorly
understood. The metal is inserted into protoporphyrin IX by
ferrochelatase to form heme, or delivered to the Fe/S cluster
biosynthetic machinery possibly by the iron chaperone frataxin
(FXN), a matrix protein that is defective in patients with Frie-
dreich’s ataxia (Table S1) (for details on the biogenesis of Fe/S
cluster proteins, see review by Sheftel and Lill, 2009).
Whether a fraction of elemental iron exits mitochondria is not
known, but heme is exported from the organelle via a yet unde-
fined mechanism and incorporated into proteins throughout the
cell. Likewise, Fe/S clusters are utilized in multiple subcellular
compartments; whether they all originate from mitochondria
is still debated as components of the Fe/S cluster assembly
machinery have been detected in the cytosol and could pro-
mote extramitochondrial Fe/S cluster synthesis or repair (Tong
and Rouault., 2006; Sheftel and Lill, 2009). The central role of
mitochondria in Fe/S cluster metabolism is also underscored
by the inner membrane protein ABCB7 that is required for
the maturation of cytosolic but not mitochondrial Fe/S cluster
proteins; how exactly ABCB7 contributes to the maturation of
extramitochondrial Fe/S cluster proteins is not known, as its
function has not yet been defined.
By making heme and Fe/S clusters, mitochondria represent
the major subcellular site of iron utilization and as such play a
central role in the control of cellular iron metabolism. The IRPs
are essential to secure mitochondrial iron supplies and func-
tion in vivo (Galy et al., 2010). When Fe/S cluster biogenesis is
impaired, iron accumulates in mitochondria, potentially harm-
ing the organelle. A current model of how mitochondria influ-
ence cellular iron metabolism posits that cells sense mitochon-
drial iron insufficiency via an Fe/S cluster-dependent factor
and respond by increasing mitochondrial iron levels; a heme
intermediate could also be involved given that mitochondrial
iron loading also occurs in erythroid cells with heme deficiency
stemming from mutations in ALAS2 or SLC25A38. Diversion of
iron to mitochondria would deplete the cytosol, thereby stim-
ulating IRP binding to IREs; in addition, perturbation of Fe/S
cluster metabolism would activate IRP1. This in turn increases
cellular iron uptake (TfR1, DMT1) and diminishes iron storage
(ferritin) and export (ferroportin), so that more iron becomes
34 Cell 142, July 9, 2010 ©2010 Elsevier Inc.
available; IRPs protect mitochondria against detrimental iron
deficiency (Galy et al., 2010), although additional regulatory
pathways could contribute (Huang et al., 2009b).
In erythroid cells, IRP activation furthermore inhibits ALAS2
translation and heme synthesis to avoid the accumulation of
toxic metabolic intermediates until mitochondrial iron suffi-
ciency is restored. By contrast, impaired Fe/S cluster biogen-
esis with abnormally high IRP activity seems to maintain con-
stitutively high levels of iron, triggering mitochondrial iron (over)
loading and, as observed in erythroblasts with glutaredoxin 5
deficiency, block ALAS2 expression and heme synthesis (Table
S1). Whether and how mitochondria can protect themselves
against iron overload remains to be elucidated.
Crosstalk between Cellular and Systemic Regulators
As described above, systemic and cellular iron homeostasis
are maintained by distinct control systems: hepcidin/ferropor-
tin and IRE/IRP. It seems very likely that there is higher level
coordination between these two systems, and future work will
define in more detail how the two systems tango in harmony
(Figure 5). Three interconnections have already been identi-
First, the ferroportin connection: The expression of ferro-
portin 1, which is critical for plasma iron levels, is subject to
regulation by both systems: the systemic iron status is commu-
nicated posttranslationally via hepcidin, whereas intracellular
iron availability regulates ferroportin synthesis via the 5′ UTR
IRE of the ferroportin mRNA. Mice doubly deficient for IRP1
and IRP2 in enterocytes are unable to limit ferroportin expres-
sion in these iron exporting cells, leading to cellular iron defi-
ciency. This shows that both control mechanisms are required
to assure regulated iron export (Galy et al., 2008). The IRE/
IRP system has to protect the cells exporting iron against det-
rimental iron losses, whereas hepcidin protects the organism
against systemic overload. The creation of ferroportin mouse
mutants in which hepcidin and IRP-dependent regulation are
dissociated may help to more clearly define the relative con-
tribution of the two regulatory systems to the control of cel-
lular iron export under different physiological and pathological
Second, the HIF2α connection: HIF2α mRNA is an IRP tar-
get (Sanchez et al., 2007), and the encoded transcription factor
regulates DMT1 expression at the apical surface of duodenal
enterocytes (Mastrogiannaki et al., 2009). Mice lacking intesti-
nal HIF2α have decreased expression of DMT1 and ferroportin
and thus fail to promote iron absorption even upon lowering
of hepcidin expression. How exactly hepcidin, HIF2α, and IRP
activity depend on each other to assure adequate systemic
iron supplies requires further research. Cross-breeding of
mouse lines with tissue-specific ablations of IRPs, HIF2α, and/
or hepcidin may prove to be informative. It has also been dis-
cussed that hepcidin transcription may be controlled by HIF2α
in response to hypoxia or iron deficiency.
Third, the TfR connection: Hepcidin expression is regulated
by the signaling receptor TfR2 and the “switch factor” HFE
that also binds to TfR1 in competition with plasma Tf-Fe2 (see
above). TfR1 expression is promoted by high IRP activity. The
equilibrium between the amount of plasma iron “sensing” TfR1
and “signaling” TfR2 is thought to be important for hepcidin
activation, and IRP activity may thus indirectly affect hepcidin
expression by regulating TfR1 levels in hepatocytes.
The 1980s and 90s brought us the foundational discoveries
in cellular iron metabolism and its regulation, and we are now
just leaving a decade of research during which systemic iron
metabolism has progressed from enigma to significant under-
standing. What lies ahead?
As discussed above, an urgent issue is to further unravel
how systemic and cellular iron control mechanisms talk to each
other. We should not lose sight of the fact that fundamental
questions of the cell biology of iron remain unanswered. How
does iron traffic inside cells, and what is the importance of the
recently identified 2,5 dihydroxybenzoic acid-containing mam-
malian siderophore? How is iron (and Fe/S clusters and heme)
incorporated into iron containing proteins? We also need to
focus research efforts to a greater degree on iron metabolism
of different organ systems and of cell-cell interactions. For
example, little is known about how the kidney handles iron: iron
undergoes glomerular filtration and reabsorption, which have
important physiological and potentially therapeutic implica-
tions. Similarly, the nervous system poses grand challenges:
For example, why does iron deficiency affect the central ner-
vous system, although hereditary hemochromatosis patients
do not accumulate iron in the brain even in the presence of
extremely high plasma and systemic iron levels? How do local
alterations of iron metabolism contribute to neurodegenera-
tion, and can we devise strategies to prevent or ameliorate
its course? What role do changes in iron metabolism play in
Figure 5. Interplay between Systemic and Cellular Iron-Regulatory
(Left) In hepcidin-producing cells (for example, hepatocytes), iron-regulatory
proteins (IRPs) can influence Hepcidin gene regulation by modulating levels
of transferrin receptor 1 (TfR1) and/or hypoxia-inducible factor 2α (HIF2α);
the iron-responsive element (IRE)/IRP system may also impact on Hepcidin
expression by changing intracellular iron levels (dashed line).
(Right) Ferroportin expression is regulated by both hepcidin and IRPs. Fur-
thermore, IRPs can potentially exert a direct positive effect on iron uptake via
divalent metal transporter 1 (DMT1) or TfR1, or an indirect negative effect via
Cell 142, July 9, 2010 ©2010 Elsevier Inc. 35
interactions between inflamed tissues and cells of the immune
system? Responses to the latter questions will also have impli-
cations for common disorders such as atherosclerosis and
Increasingly, our quest to understand basic cell biological
phenomena will be complemented by exploration of the iron
biology of disease states. Progress in this area will help to
devise new therapeutic concepts. The impressive progress
that has been made during the past few years in unraveling
the regulation of hepcidin expression and its function may well
translate into new drugs to treat iron overload diseases and
some forms of anemia.
Supplemental Information includes one table and can be found with this ar-
ticle online at doi:10.1016/j.cell.2010.06.028.
We are grateful to Petra Riedinger for graphic design. Work in the labora-
tory of M.W.H. was supported by grants from the European Commission (Eu-
roIron) and the BMBF (HepatoSys). M.U.M. acknowledges support from the
Deutsche Forschungsgemeinschaft, BMBF (HepatoSys, Erare), and European
Commission (EuroIron), and C.C. from Telethon Rome, grant GGP08089, and
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HH type 1
Inheritance Gene Protein Hepcidin
B2m -/- mouse
Vujić Spasić et al
HH type 2
Severe systemic iron
Usf2 -/- mouse
Huang et al 2005
Finberg et al 2010
Kawabata et al
Wallace et al 2009
Roetto et al 2010
Zohn et al 2007
Donovan et al 2005
HH type 2
HH type 3
Hepcidin Absent Severe systemic iron
Hfe, Tfr2 double
HH type 4,
Anemia ± ± iron
Matriptase-2 High Iron-refractory
Du et al 2008
Folgueras et al
, Beutler et al 2000
Fraenkel et al 2009
Gunshin et al, 2005
Transferrin Low Microcytic anemia
liver iron overload
liver iron overload
- Hemolytic anemia,
Cp and Heph
Kono & Miyajima
ALAS2 . Microcytic anemia,
liver iron overload
liver iron overload
Camaschella et al,
. N.A. Guernsey et al,
Glutaredoxin 5 - N.A.
Wingert et al, 2005
anemia and ataxia
liver iron overload
Frda -/- tissue
Pondarrè et al,
Huang et al, 2009
Ferrari et al, 2006
Cozzi et al, 2010
Table S1. Genetic disorders of iron proteins
MIM n = Mendelian inheritance in man number at http://www.ncbi.nlm.nih.gov/sites/entrez
HH = hereditary hemochromatosis, AR = autosomal recessive, AD = autosomal dominant, IRIDA = iron-refractory iron deficiency anemia, HHCS =
Hyperferritinemia cataract syndrome, N.A. = not available, β 2m = β 2-microglobulin
°) The spontaneous mutant model have microcytic anemia but not liver iron overload
°°) Mutations of ferroportin may cause two distinct phenotypes (see text for details)
*) Mutations in the 5’UTR IRE of FtL cause HHCS, mutations in C-terminal sequence cause neuroferritinopathy
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