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Cell Metab. Author manuscript; available in PMC 2016 November 03.
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Published in final edited form as:

Cell Metab. 2015 November 3; 22(5): 777–787. doi:10.1016/j.cmet.2015.09.006.

Ironing out ferroportin

Hal Drakesmith1, Elizabeta Nemeth2, and Tomas Ganz2,3
1MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford,
Oxford, United Kingdom
2Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles,
USA
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3Departmentof Pathology, David Geffen School of Medicine, University of California, Los

Angeles, USA

Abstract
Maintaining physiologic iron concentrations in tissues is critical for metabolism and host defense.
Iron absorption in the duodenum, recycling of iron from senescent erythrocytes, and iron
mobilization from storage in macrophages and hepatocytes constitute the major iron flows into
plasma for distribution to tissues, predominantly for erythropoiesis. All iron transfer to plasma
occurs through the iron exporter ferroportin. The concentration of functional membrane-associated
ferroportin is controlled by its ligand, the iron-regulatory hormone hepcidin, and fine-tuned by
regulatory mechanisms serving iron homeostasis, oxygen utilization, host defense and
erythropoiesis. Fundamental questions about the structure and biology of ferroportin remain to be
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answered.

Introduction
A major destination for iron in the body is the heme of hemoglobin in red blood cells.
Humans normally complete the synthesis of at least 2 million erythrocytes per second; each
mature red blood cell contains about 280 million molecules of hemoglobin, and each of the
four globin subunits contains one iron atom in heme, so that the total iron flux required to
maintain erythropoiesis is about 2–3 × 1015 atoms of iron per second in an adult human. The
erythropoietic use of iron governs the lion’s share of iron trafficking, but iron is also
required for mitochondrial function, DNA synthesis and other basic activities in all cells in
the body; and in pregnancy the growing fetus places a further large demand on the maternal
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iron transport system. In humans, >90% of iron demand (or about 20–25 mg/day) is met by
recycling existing iron, with uptake of ‘new’ iron from the diet accounting for about 1 mg of
iron per day. Macrophages in the red pulp of the spleen specialise in the uptake and
digestion of senescent red blood cells, can liberate the iron from heme via the enzyme heme-
oxygenase-1, and release iron back into plasma. Dietary iron uptake is mediated by duodenal

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enterocytes that capture iron from the diet at their apical surface via the ferric reductase
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DcytB and the importer DMT1, and export iron into plasma through their basolateral
membranes (Mackenzie and Garrick, 2005). Dietary heme represents an important source of
iron, especially in meat eaters, but the mechanism of its absorption is not yet understood,
and it is not known to what extent heme is converted to inorganic iron within enterocytes or
transferred intact to plasma. Iron transfer from duodenal enterocytes to plasma (referred to
as “mucosal transfer” in older literature on intestinal iron absorption) has long been known
to be subject to regulation by systemic influences including iron deficiency, anemia and
hypoxia (Finch, 1994). How iron entered plasma from either recycling macrophages or
absorptive enterocytes was unknown until the year 2000, when it became apparent that
despite arising from distinct lineages, and despite their sources of iron being very different,
both macrophages and enterocytes use the same protein to mediate iron export: ferroportin
(SLC40A1, also referred to in the literature as FPN1, MTP1 and IREG1). Moreover,
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ferroportin is also used to export stored iron from hepatocytes and to release maternal iron
from the placenta to the fetal circulation. Three groups (Abboud and Haile, 2000; Donovan
et al., 2000; McKie et al., 2000) independently discovered ferroportin, a
multitransmembrane protein that is well conserved in mammals, appears to be ancient with
orthologs present in plants and worms, and intriguingly is not closely related to any other
known mammalian transporter proteins (although there may be some distant homology to
bacterial transport proteins).

Function and importance in physiology

Early work confirmed that ferroportin is highly expressed by cells and tissues associated
with iron transport: duodenal enterocytes, liver Kupffer cells and splenic red pulp
macrophages, periportal hepatocytes, and the placental syncytiotrophoblast (Figure 1). The
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ability of ferroportin to mediate iron export was shown using radioactive iron isotopes in
Xenopus laevis oocytes injected with ferroportin cRNA and human cell lines transfected
with ferroportin-expressing constructs (Donovan et al., 2000; McKie et al., 2000).

The key role of ferroportin in iron export in vivo was established using a series of transgenic
mice (Donovan et al., 2005). Global Fpnnull/null mice do not complete embryonic
development, likely because ferroportin is required for transfer of iron to the embryo
through epithelial cells of the extraembryonic visceral endoderm (exVE). When ferroportin
was deleted only in embryo proper but spared in exVE and placenta, mice survived to birth
but rapidly became anemic and runted after birth and showed marked iron accumulation in
enterocytes, splenic macrophages and Kupffer cells, and in hepatocytes. Adult mice in
which ferroportin was inducibly and exclusively deleted in intestinal cells also rapidly
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became anemic, demonstrating that ferroportin is essential for intestinal absorption of iron.
Further work inactivating ferroportin in macrophages and in hepatocytes showed that
ferroportin is required in these cells for efficient mobilisation of stored iron; mice lacking
ferroportin in these sites become anemic more rapidly than controls when fed iron-deficient
diets (Zhang et al., 2011b; Zhang et al., 2012). However the same mice on standard diet
maintained intact erythropoiesis, indicating that duodenal ferroportin upregulation and
increased intestinal absorption can compensate for the decreased activity of ferroportin in
recycling and storage compartments, especially in mice which compared to humans derive a

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larger proportion of their iron flows from intestinal absorption. Overall, it appears that
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ferroportin is the only way elemental iron can exit cells to enter plasma.

Multilevel regulation of ferroportin

The regulation of ferroportin expression is complex, with important layers of control at
transcriptional, post-transcriptional, post-translational and cell-lineage levels. These
different phases of control allow multiple and diverse physiological inputs to influence
ferroportin activity at its various sites of expression. Notably, ferroportin regulation varies
among different cell types, allowing added flexibility in controlling systemic iron flow under
different conditions (Figure 2).

As noted above, macrophages are critical to iron homeostasis because they take up senescent
red blood cells (by a process referred to as erythrophagocytosis), liberate heme from
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hemoglobin, liberate iron from heme, and release iron back into plasma via ferroportin.
Although the overall rate of iron release is subject to systemic control, the cellular response
to erythrophagocytosis is cell-autonomously regulated: in the terminal step of this
coordinated sequence ferroportin expression in macrophages is increased by heme and iron
released from digested erythrocytes (Delaby et al., 2008). Ferroportin contains in its
promoter sequence an antioxidant response element (ARE) 7kb upstream of the transcription
start site (Marro et al., 2010); AREs can be bound either by Bach1 (leading to gene
repression) or Nrf2 (leading to gene activation). Heme causes Bach1 degradation, which
may allow Nrf2 to facilitate transcriptional activation of ferroportin (with a similar effect on
heme oxygenase and ferritin, in a coordinated cellular response). Pharmacological activators
of Nrf2 increase Fpn1 mRNA in macrophages in an Nrf2-dependent manner, and can
counteract the inflammation-induced suppression of Fpn1 mRNA caused by LPS (Harada et
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al., 2011). Iron itself also enhances ferroportin expression at the translational level.
Ferroportin mRNA (isoform 1a, which is abundant in macrophages) contains a 5′ iron
response element (IRE), which under conditions of iron deficiency binds IRP proteins,
causing translational repression (Lymboussaki et al., 2003). In erythrophagocytosing
macrophages, iron liberated from heme by heme oxygenase-1 (that is also activated by Nrf2)
inactivates IRPs, resulting in derepression of Fpn1a mRNA translation. Moreover, the 3′
untranslated region of ferroportin is targeted by miR-485-3p microRNA which is induced by
cellular iron deficiency (Sangokoya et al., 2013). As cellular iron levels rise, the
downregulation of this miRNA may also contribute to increased ferroportin expression. The
sequential heme- and iron-mediated induction of ferroportin expression facilitates efficient
recycling or iron from macrophages that have internalised and degraded senescent
erythrocytes (Delaby et al., 2008; Marro et al., 2010) (Figure 2a).
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In duodenal enterocytes, ferroportin production is strongly regulated by iron and hypoxia, all
geared toward increasing absorption of dietary iron during iron deficiency and anemia
(Figure 2b). The molecular basis for these effects is becoming clearer. Hypoxia acts by
stabilisation of hypoxia inducible factors (HIFs) that transcriptionally activate genes in order
to respond to a low cellular oxygen state. Iron deficiency also stabilises HIFs because their
degradation is triggered by iron-dependent hydroxylation of proline residues and therefore
HIFs may also function as sensors of iron scarcity. HIF activation in enterocytes was shown

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to result in the upregulation of both the apical iron import machinery, DcytB and DMT1
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(Shah et al., 2009) and the basolateral iron exporter ferroportin (Taylor et al., 2011). The
promoter of the ferroportin gene contains HIF response elements (HREs) that are targets for
HIF2alpha binding, and mice lacking HIF2alpha, but not HIF1alpha, fail to upregulate
duodenal ferroportin mRNA in response to iron deficiency (Taylor et al., 2011). Thus
HIF2alpha has a demonstrable role in increasing ferroportin transcription during hypoxia
and iron deficiency. Interestingly, HIF2alpha is also the predominant factor in the regulation
of erythropoietin transcription, hinting at its possible role as a coordinator between iron
supply and erythropoiesis during the response to hypoxia.

At first glance, the IRP/IRE system in iron-deficient enterocytes would be expected to

impair ferroportin expression through translational repression by the 5′IRE of ferroportin.
The presence of a 5′ IRE in Hif2alpha mRNA (Sanchez et al., 2007) also raises the question
why the translation of Hif2alpha mRNA is not repressed during enterocyte iron depletion.
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Although such responses would protect enterocytes from cellular iron deficiency, they
would be detrimental to an iron-deficient organism as iron would be retained in enterocytes,
decreasing transfer of iron to plasma. In fact, iron deficiency clearly increases both
ferroportin protein and mRNA expression. The inability of the IRE/IRP system to repress
ferroportin translation in duodenal enterocytes is explained only in part by the presence of a
splicing variant of ferroportin mRNA, termed Fpn1b, which lacks the 5′ IRE but produces
an identical protein (Zhang et al., 2009). It appears that other, perhaps hypoxia-dependent,
mechanisms predominate to limit IRP1-mediated translational repression (Anderson et al.,
2013) and increase the concentrations of HIF2alpha and the transcription and translation of
ferroportin. Therefore under conditions of iron deficiency, increased production of
ferroportin, combined with its decreased hepcidin-mediated endocytosis and proteolysis,
result in higher transfer of iron from the diet into plasma at the expense of iron depletion of
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enterocytes. Enterocytes are short-lived cells which are shed into the intestinal lumen after a
few days, so the organism may tolerate their iron depletion. The relative timing and
amplitude of these complex iron– and hypoxia–sensitive elements in the ferroportin
regulatory circuits has not yet been reported.

Interestingly, ferroportin lacking a 5′ IRE (Fpn1b mRNA isoform) is also expressed by

erythrocyte progenitor cells (Cianetti et al., 2005; Zhang et al., 2009; Zhang et al., 2011a).
Under conditions of iron limitation, Fpn1b would be continually translated, and this may
allow erythropoiesis to be ‘altruistic’ and give up some of its iron in order to increase supply
to other tissues that require it.

In contrast to hypoxia and iron deficiency, inflammation decreases the expression of

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ferroportin, through effects on transcription. This effect was initially observed in vivo for
liver and splenic Fpn1 mRNA as a consequence of inflammation caused by administration
of bacterial lipopolysaccharide (LPS), which is a ligand for the innate immune sensor Toll-
like receptor 4 (TLR4), (Liu et al., 2005a; Yang et al., 2002). Human monocytic cells were
also shown to decrease FPN mRNA in response to LPS and to interferon-gamma
(Ludwiczek et al., 2003). The mechanism of decreased Fpn mRNA caused by inflammation
is only partially understood. Suppression of Fpn1 mRNA by the bacteria Pseudomonas
aeruginosa requires intact TLR4 (Huang et al., 2012; Peyssonnaux et al., 2006), whereas the

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Mycoplasma-derived molecule FSL1 decreases Fpn1 in a TLR2-dependent fashion (Guida

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et al., 2015); however the decrease of Fpn1 mRNA by LPS and the TLR3 ligand poly(I:C)
do not require the intracellular adaptors of TLR signalling, MyD88 or TRIF (Huang et al.,
2012). Even in the absence of hepcidin, these mechanisms are sufficient to cause
downregulation of duodenal ferroportin (and DMT-1) by LPS (Deschemin and Vaulont,
2013) and downregulation of splenic and liver ferroportin by FSL1 (Guida et al., 2015)
leading to acute (3 – 6 hours after injection) hypoferremia in mice. These results suggest that
transcriptional downregulation of ferroportin could contribute to the hypoferremia of
inflammation that can be protective against infection with extracellular pathogens. However,
the degree and duration of hypoferremia caused by this inflammatory suppression of Fpn1
mRNA are relatively minor in the absence of hepcidin, as evidenced by the small effects on
circulating iron after infection with extracellular bacteria in hepcidin knockout mice (Arezes
et al., 2015).
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Another layer of regulation of ferroportin activity arises from macrophage differentiation

and polarization. Macrophages are markedly phenotypically and functionally diverse
depending on their source (yolk sac-derived precursors or circulating bone-marrow derived
monocytes), tissue location (resident in various organs, or recruited from the blood), and
exposure to a variety of signals originating from other cells. Iron-recycling macrophages in
the red pulp of the spleen, which express high levels of ferroportin, depend on the
transcription factor SpiC for their development (Kohyama et al., 2009). SpiC activity is
normally repressed by Bach1; heme causes Bach1 degradation and derepresses SpiC in
monocytes, leading to their differentiation into red pulp macrophages expressing ferroportin
and heme-oxygenase-1 (Haldar et al., 2014). Transcription of ferroportin and heme-
oxygenase-1 increases after heme-mediated Bach1 degradation and Nrf2 activation, as
mentioned before. Thus increased levels of potentially toxic heme, for instance derived from
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hemolytic events, drive both the development of the cell type and the specific molecular
processes that are equipped to detoxify heme and recycle heme iron. However, macrophages
can also be differentiated in multiple other directions, simplified as ranging from the M1
pro-inflammatory phenotype induced by LPS or interferon-gamma to the M2 alternative
specializations characterised by anti-inflammatory and tissue remodelling function (Mosser
and Edwards, 2008). In line with the transcriptional stimulation of hepcidin by inflammatory
signals, ferroportin expression by M1 macrophages is low, and this coupled with increased
ferritin levels confers an iron sequestration phenotype (Recalcati et al., 2010). However,
macrophages exposed to M-CSF, IL-4 or glucocorticoids assume an iron recycling/exporting
phenotype expressing hemoglobin processing machinery, and high levels of ferroportin
(Corna et al., 2010; Recalcati et al., 2010; Sierra-Filardi et al., 2010; Vallelian et al., 2010).
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As described in this section, ferroportin gene transcription and translation is modulated by a

number of multi-layered signals. However, the activity of the functional transporter on cell
membranes is predominantly governed posttranslationally by a systemically acting peptide
hormone, hepcidin (e.g. (Laftah et al., 2004; Rivera et al., 2005). Because hepcidin
expression is itself regulated by a complex integration of many physiological signals
including those arising from iron status, inflammation and erythroid drive (Ganz, 2013),
hepcidin adds a crucial final layer to the control of ferroportin activity (Figure 2c).

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Ferroportin and hepcidin

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Hepcidin is a 25 amino acid peptide secreted into blood plasma predominantly by

hepatocytes. The sole well-documented physiologic activity of hepcidin is its binding to
ferroportin which results in ferroportin endocytosis and proteolysis, predominantly in
lysosomes (Nemeth et al., 2004). Accordingly, low hepcidin states are characterized by high
levels of ferroportin in iron-transporting tissues and brisk iron export to extracellular fluid
and plasma. In contrast, high circulating hepcidin concentrations cause ferroportin to
disappear from cell membranes and iron export to plasma to cease. The central importance
of the hepcidin-ferroportin interaction for iron homeostasis is highlighted by the profound
consequences of hepcidin deficiency or excess. Complete genetic hepcidin deficiency causes
the most severe form of iron overload in mice (Lesbordes-Brion et al., 2006; Nicolas et al.,
2001) and humans (Roetto et al., 2003), and mutations in ferroportin that prevent hepcidin
binding (Altamura et al., 2014) largely phenocopy hepcidin deficiency, with the exception of
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iron-induced pancreatic injury which for unclear reasons causes premature mortality in the
hepcidin-resistant homozygous C326S ferroportin mouse. At the opposite extreme,
transgenic overexpression of hepcidin results in lethal perinatal iron deficiency (Nicolas et
al., 2002) and the administration of synthetic hepcidin or its analogs to mice causes dose-
dependent hypoferremia (Rivera et al., 2005) and inhibits iron absorption (Laftah et al.,
2004), again indicating the dominant effect of hepcidin on the regulation of ferroportin
function.

It has been reported that macrophage ferroportin may be much more sensitive to the effect of
hepcidin than ferroportin on enterocytes (Chaston et al., 2008; Chung et al., 2009). In
principle, such organ-specific ferroportin responses to hepcidin could result from differences
in hepcidin binding, perhaps due to organ-specific differences in ferroportin glycosylation
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(Canonne-Hergaux et al., 2006), possible differences in cellular endocytic machinery (not

specifically analysed) or from differential rates of ferroportin synthesis which strongly
affects the net expression of ferroportin on cell membranes. Definitive determination of
differences in ferroportin responses in the two organs may be difficult. In vivo studies of
radiolabelled hepcidin binding found substantial binding both in the duodenum (proximal
more than distal) and in the spleen where iron-recycling macrophages are abundant (Rivera
et al., 2005) but this technique cannot detect small differences in receptor affinities. Despite
any possible quantitative differences, the phenotypes of major iron disorders and of their
animal models indicate that hepcidin exerts a controlling influence on both duodenal
enterocytes and recycling macrophages: high hepcidin concentrations suppress both
intestinal iron absorption and macrophage iron recycling, and conversely, hepcidin
deficiency leads to both excessive iron absorption and iron depletion of macrophages.
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The structural determinants of the hepcidin-ferroportin interaction are partially understood.

Ferroportin is a 12-transmembrane domain protein (Liu et al., 2005b) (Figure 3) belonging
to the major facilitator superfamily of transporters of small molecules. Although technical
obstacles caused some early disagreement on this point, there is now consensus in the field
that both ferroportin termini are located on the cytoplasmic side of the membrane (Liu et al.,
2005b; Rice et al., 2009). The thiol cysteine C326 is exposed to the extracellular milieu and
is essential for hepcidin binding as demonstrated by the lack of radiolabeled hepcidin

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binding by mutant ferroportins C326S and C326T (Fernandes et al., 2009). The lack of
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hepcidin binding to C326S ferroportin confers a gain of function phenotype in humans and
in mice, resulting in early and severe iron overload as a consequence of hyperabsorption of
dietary iron (Altamura et al., 2014; Sham et al., 2009). Extensive mutagenesis and molecular
modelling identified the N-terminal 9 amino acids of the 25 amino acid mature hepcidin
interacting with the C326-containing exofacial ferroportin segment, mainly through pi-
stacking interactions of aromatic side chains (Nemeth et al., 2006; Preza et al., 2011a). The
confidence in this model was reinforced by the rational design of 9-amino acid
minihepcidins that were as potent as and more drug-like than hepcidin itself (Preza et al.,
2011b; Ramos et al., 2012).

It was reported that an unstructured 19 amino acid peptide based on the C326-containing
ferroportin loop acted as a specific binder of hepcidin and could be used for measurements
of hepcidin in plasma or urine (De Domenico et al., 2008). However, this claim could not be
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replicated in other laboratories, and the publication was retracted as it was found to have
been affected by research misconduct (paper #10 in the report by (McCormack et al., 2013)).

As with other receptors that undergo ligand-induced endocytosis, the interaction of hepcidin
with ferroportin could be expected to trigger a conformational change in ferroportin
followed by covalent modifications of one or more of its cytoplasmic segments to initiate
endocytosis. Mutations that interfere with these mechanisms would confer resistance to
hepcidin. Several ferroportin mutations associated with partial resistance to hepcidin
(Drakesmith et al., 2005) affect amino acids located within the cell membrane and do not
prevent hepcidin binding (Fernandes et al., 2009) but likely interfere with the
conformational change that is required to initiate endocytosis. Although the structural details
have not yet been elucidated, ferroportin endocytosis is now known to depend on the
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hepcidin-induced ubiquitination of multiple cytoplasmic lysines on the extended

cytoplasmic loop connecting the two halves of the ferroportin molecule (Figure 3) (Qiao et
al., 2012; Ross et al., 2012). The specific ubiquitin ligases involved in the endocytosis of
ferroportin remain to be identified.

An earlier proposed mechanism claimed that hepcidin-induced ferroportin endocytosis

depended on JAK2-mediated phosphorylation of ferroportin on a pair of cytoplasmic
tyrosines (Y302 and Y303)(De Domenico et al., 2009; De Domenico et al., 2007b).
However, these studies were subsequently contradicted (Ross et al., 2012) and the articles
were found to be affected by scientific misconduct (papers #2 and #8 in the report by
(McCormack et al., 2013)). Contrary to the original claim that Y302F/Y303F mutations
confer resistance to hepcidin (De Domenico et al., 2007b), these mutations were found to
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prevent ferroportin trafficking to the membrane (Ross et al., 2012). Morover, Y302F/Y303F
homozygosity in mice caused an embryonic lethal phenotype due to inadequate iron transfer
across placenta (Altamura et al., 2011), an opposite phenotype from that expected of
hepcidin resistance. Concerns were also raised about the reported key role of a single lysine
residue K253 and the Nedd4-2 ligase system in hepcidin-dependent and hepcidin-
independent endocytosis of ferroportin (De Domenico et al., 2011). The article was
confounded by misconduct (paper #1 in report by (McCormack et al., 2013)) and was
retracted.

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Compared to most other cell types, macrophages are particularly ferroportin-rich, especially
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in the spleen and the liver where they recycle iron from senescent erythrocytes.
Macrophages also have an important role as orchestrators of innate immunity, in part by
producing cytokines that modify the activity of cells participating in host resistance to
infection. Both functions of macrophages, in iron homeostasis and in host defense, influence
each other. Understanding the effects of iron on host resistance has important implications
for the treatment of iron deficiency in settings where serious infections are endemic.
However, understanding of the effect of iron on macrophage cytokine production has been
complicated by the contradictory findings in different studies. In mouse models, iron
deficiency and the resulting iron depletion of macrophages has been associated with a
heightened inflammatory response to LPS, which could be prevented by hepcidin
administration which promotes macrophage iron retention (Pagani et al., 2011). However, in
HFE−/− mice, where macrophages are also iron-depleted and hepcidin concentrations are
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also low, there was no difference compared to WT mice in TNFα and IL-6 plasma levels
after LPS stimulation (Roy et al., 2004). Furthermore, studies from isolated macrophages
demonstrated an opposite association between macrophage iron and cytokine production:
iron depletion via chelation reduced TNFα and IL-6 secretion (Wang et al., 2008) and iron
accumulation in ferroportin-deficient macrophages potentiated TNFα and IL-6 release
compared to intact macrophages (Zhang et al., 2011b). These apparently conflicting
conclusions from in vivo and in vitro experiments may be caused by the heterogeneity of
macrophage types, their differing exposure to iron sources and varying levels of ferroportin
expression.

In principle, hepcidin could modulate cytokine secretion by macrophages either through

ferroportin-generated signalling or by causing cytoplasmic iron retention and altering iron-
dependent intracellular signalling, including the hypoxia pathways and the iron-regulatory
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protein pathways. One study proposed that hepcidin triggered ferroportin signalling via
JAK2-STAT3 pathway, causing anti-inflammatory transcriptional changes (De Domenico et
al., 2010). The study, however, was affected by research misconduct (paper #3 in the report
by (McCormack et al., 2013)) and the involvement of JAK2-STAT3 was not confirmed in
subsequent studies (Ross et al., 2012). Thus both the pathophysiology of iron effects on
inflammation and the potential mechanisms by which iron, hepcidin and ferroportin
modulate inflammatory function of macrophages remain uncertain (Table 1).

Mechanism of iron transport by ferroportin and the role of ferroxidases

Surprisingly little is known about how ferroportin transports iron. Recent studies of the
efflux of microinjected iron radioisotopes from Xenopus oocytes established that this system
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should prove useful for detailed analysis of iron transport by ferroportin (Mitchell et al.,
2014). Oocytes microinjected with ferroportin-GFP mRNA express ferroportin on their
membranes, export up to 300-times more iron than control oocytes, and iron efflux is
strongly inhibited by the addition of hepcidin. The two common iron forms Fe2+ and Fe3+
are readily interconverted so definitive determination of which species is transported by
ferroportin is challenging. Support for the transport of Fe2+ is provided by the observation
that the very similar cation Co2+ that is more stable to oxidation under the usual
experimental conditions is exported from Xenopus oocytes with a similar rate constant as

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iron. Moreover, multiple experiments with the Xenopus oocyte system found that iron efflux
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was potentiated by apotransferrin (Donovan et al., 2000) (Mitchell et al., 2014). and
ceruloplasmin (McKie et al., 2000; Mitchell et al., 2014)., the latter known as the principal
ferroxidase in blood plasma that catalyzes the conversion of Fe2+ to Fe3+. This potentiating
effect is ascribed to the increased concentration gradient for the release of Fe2+ from
ferroportin facilitating the movement of iron through the transporter.

All three known mammalian multicopper-containing ferroxidases - ceruloplasmin,

hephaestin and zyklopen - oxidize Fe2+ to Fe3+, the form that is loaded onto plasma
transferrin for distribution to tissues. Ceruloplasmin and hephaestin have been shown to
facilitate cellular iron efflux in vivo, especially in situations of increased demand for iron. In
the duodenum, hephaestin is a transmembrane protein on enterocytes with an extracellular
domain that is highly homologous to the plasma protein ceruloplasmin. Ablation of
hephaestin in mice results in mild systemic iron deficiency and mild anemia both of which
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improve with age as iron demand for growth declines (Fuqua et al., 2014). Systemic iron
deficiency was caused by decreased intestinal iron absorption but was associated with
excess iron retention within enterocytes, indicative of impaired basolateral transport of iron
to plasma. The decrement in iron absorption occured despite an increased expression of
ferroportin, demonstrating impaired iron transport by ferroportin. Hephaestin is also found
in the brain where its ablation leads to age-dependent regional iron accumulation (Jiang et
al., 2015).

Ceruloplasmin is known in two forms, a blood plasma protein and a GPI-linked membrane-
associated protein, encoded by splicing variants of the same gene. The GPI-linked
ceruloplasmin has been reported to be particularly abundant in the brain where it has a
different distribution than hephaestin (Jiang et al., 2015). Patients and mouse models that
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lack ceruloplasmin accumulate iron in the liver, the brain and the retina with resulting
damage to these organs. Ceruloplasmin-deficient humans and mice also show signs of
impaired iron mobilization from macrophages and hepatocytes resulting in anemia (Harris et
al., 1999; Osaki and Johnson, 1969). During erythropoietic stress after bleeding,
ceruloplasmin may also facilitate augmented dietary iron transfer to plasma (Cherukuri et
al., 2005). Combined hephaestin-ceruloplasmin deficiency has particularly severe
consequences in the retina where both proteins are expressed, causing a severe form of
macular degeneration (Hadziahmetovic et al., 2008). A third ferroxidase, zyklopen, was
described as a membrane protein highly expressed in the placenta (Chen et al., 2010) but its
physiological function has not yet been reported.

In view of the well-documented ability of the multicopper ferroxidases to influence

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ferroportin function, various modes by which ferroxidases and ferroportin could interact
have been explored. In one model, GPI-linked ceruloplasmin (or an equivalent sink for
Fe2+) is required to remove Fe2+ from ferroportin in astrocytes. In the absence of
ceruloplasmin, Fe2+ becomes “stuck” in ferroportin and this conformational state causes
astrocyte ferroportin to be ubiquitinated on K523 and to undergo endocytosis and
proteolysis (De Domenico et al., 2007a). Although the hypothesis has some attractive
features, including the explanation of why aceruloplasminemia has such severe effects in the
brain where soluble ceruloplasmin concentrations are very low, the evidence for it is again

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 Drakesmith et al. Page 10

confounded by misconduct (paper #7 in the report by (McCormack et al., 2013)) and has not
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yet been independently verified. Others have documented by coimmunoprecipitation that

hephaestin or GPI-ceruloplasmin physically interact with ferroportin (Jeong and David,
2003; Yeh et al., 2009). Physical contact with a ferroxidase would assure very low
concentrations of Fe2+ on the exoface of ferroportin and could facilitate iron efflux.

Not much is known about the mechanism by which intracellular iron is delivered to
ferroportin for export. A recent study (Yanatori et al., 2014) reported that the iron chaperone
protein PCBP2 (but not its paralogue PCBP1) interacts with both DMT1 and ferroportin.
The results suggested that ferrous iron imported into the cytoplasm by DMT1 may be
shuttled to ferroportin via PCBP2.

Fundamental questions about ferroportin, including its structure, the molecular mechanism
of iron transport, the nature of any symported or antiported species and the mechanism by
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which iron is delivered to ferroportin for export, remain open for investigation (Table 1).

Genetic disorders of ferroportin and their effects on systemic iron

homeostasis
In the absence of definitive structural studies, important insights into ferroportin structure
emerged from studies of informative patients with iron disorders. Clinically detectable
ferroportin mutations are very heterogeneous, with a systematic review reporting on 161
patients with 31 distinct mutations (Mayr et al., 2010). They manifest a dominant pattern of
inheritance and fall into two broad phenotypic categories with some overlap. One group of
mutations causes a ferroportin gain-of-function owing to partial or complete resistance to
hepcidin-induced ferroportin endocytosis, as discussed in previous sections. The affected
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patients hyperabsorb iron and present, often at a young age, with high transferrin saturation,
high plasma ferritin, and hepatic and other organ iron overload with evidence of toxic
damage. The phenotype resembles that of patients with classical hemochromatosis from
homozygous or compound heterozygous recessive mutations in hepcidin or its regulators
(HFE, TfR2 or hemojuvelin). Another group of patients have dominant loss-of-function
mutations that cause high plasma ferritin but not high transferrin saturation (a condition
referred to as “ferroportin disease”(Pietrangelo, 2004)). These mutations appear to limit the
rate of iron export from recycling macrophages so that these macrophages retain iron and as
a consequence secrete high amounts of ferritin into plasma (Cohen et al., 2010). Any loss of
transport capacity in the duodenum appears to be sufficiently compensated to prevent
systemic iron deficiency, unless the patients are phlebotomized to remove accumulated iron.
This disorder does not appear to cause clinically important organ damage, presumably
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because macrophages (unlike hepatocytes, cardiac myocytes or endocrine glandular tissue)

are equipped to produce very large amounts of reactive oxygen species during respiratory
burst and are therefore well defended against similar toxins generated when they become
iron overloaded. In principle, some mutations could cause both decreased iron export and
partial resistance to hepcidin and this could help explain the variable iron-loading
phenotypes in patients with the same mutation.

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 Drakesmith et al. Page 11

The ferroportin variant Q248H is common (a few %) in populations of African ancestry and
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has been weakly associated with increased serum ferritin (Gordeuk et al., 2003; Kasvosve et
al., 2015; Rivers et al., 2007). The polymorphism may confer a somewhat decreased
sensitivity of ferroportin to hepcidin, perhaps because of its proximity to cytoplasmic
ubiquitination sites involved in hepcidin-induced ferroportin endocytosis (Nekhai et al.,
2013).

The puzzling feature of ferroportin disease is the structural basis of its mode of inheritance.
Essentially all of the several dozen reported mutations are heterozygous missense mutations
that act dominantly. This suggests that the ferroportin loss-of-function is not due to
haploinsufficiency, otherwise heterozygous nonsense mutations or other gene defects that
completely ablate the production of protein from one of the alleles would also be expected.
At the functional level, the mutations cause either a trafficking defect so that insufficient
ferroportin is displayed on the cell membrane (Liu et al., 2005b; Schimanski et al., 2005), or
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an iron transport defect that reduces the rate of iron efflux per ferroportin molecule on the
cell membrane (Liu et al., 2005b; McGregor et al., 2005). There is evidence that some
mutations known to impair trafficking may also impair iron efflux by membrane-associated
ferroportin (McGregor et al., 2005). Compared to the mouse model of haploinsufficiency
(heterozygous knockout) which had only a very mild form of macrophage iron retention
(Donovan et al., 2005), mice with “ferroportin disease” (flatiron mouse, H32R ferroportin
mutation) showed age-dependent accumulation of iron in hepatic macrophages and
relatively severe iron restriction, manifested by low transferrin saturation and hypochromic
erythrocytes on blood smears (Zohn et al., 2007). Although the comparison was not made
side-by-side under the same conditions and identical strain backgrounds, these findings are
consistent with a dominant negative effect of the mutation. If ferroportin is transported to the
membrane in a multimeric form, a dominant negative effect could result from missense
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mutations that cause mistrafficking of the multimer. However, several dominant mutations
(e.g. G323V, N174I, G80S, R88G,) were reported to cause deficient iron efflux rather than
mistrafficking of ferroportin (Callebaut et al., 2014; De Domenico et al., 2006; Liu et al.,
2005b), and here the dominant negative effect may imply cooperativity between multiple
ferroportin molecules engaged in iron transport. In such a model, a transport-impaired
ferroportin molecule would need to disrupt the function of neighbouring wild-type
molecules.

Although the genetic evidence points to some kind of interaction between ferroportin
molecules that would cause a dominant negative effect, it has not been definitively
determined whether ferroportin is monomeric or multimeric during trafficking or in the cell
membrane. This is in part because the main published evidence supporting the multimeric
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nature of ferroportin (De Domenico et al., 2007c) is clouded by scientific misconduct (paper
#9 in the report by (McCormack et al., 2013)). Several groups reported evidence that
ferroportin is a monomer, as determined by a variety of methods (Goncalves et al., 2006;
Pignatti et al., 2006; Schimanski et al., 2008) but others detected a dimeric form complexed
with hephaestin (Yeh et al., 2009). Detailed physicochemical analysis of recombinant
ferroportin extracted from membranes (Rice et al., 2009) led to the conclusion that the
monomeric form is stable and functional, and binds hepcidin, but that weak, detergent-

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 Drakesmith et al. Page 12

sensitive homophilic interactions favouring multimerization in the cell membrane could not
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definitively be ruled out.

The role of ferroportin in local tissue iron homeostasis and its disorders
In addition to its role as the sole cellular iron exporter in tissues that provide iron for
systemic needs (duodenum, iron-recycling macrophages, hepatocytes and placenta), there is
increasing evidence that ferroportin may also be involved in local iron homeostasis. This
function of ferroportin is best revealed by the consequences of its tissue-specific ablation, as
was done in cardiac myocytes (Lakhal-Littleton et al., 2015), with resulting progressive
impairment of the function of cardiomyocytes and the premature death of the affected mice.
These experiments raise questions about the function of ferroportin in cells that do not
participate in systemic iron homeostasis. Is ferroportin functioning as a safety valve to
release iron generated during subcellular remodelling (e.g. autophagy) or during
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uncontrolled uptake in the course of transient episodes of complete transferrin saturation

with iron? Ferroportin in erythrocyte precursors (Zhang et al., 2011a) may serve as such a
safety valve that also makes erythroblast iron available during periods of severe iron
deficiency to sustain vital organs at the expense of erythropoiesis.

An important role for ferroportin in the brain is presaged by the adverse consequences of
ceruloplasmin deficiency in mice and humans, further exacerbated in mice when both
hephaestin and ceruloplasmin are ablated. It has also been suggested that β-amyloid
precursor protein (APP) may function as a ferroxidase in neurons (Duce et al., 2010),
implicating ferroportin in the pathogenesis of Alzheimer’s disease; however the ferroxidase
activity of APP turned out to be an artefact (Wong et al., 2014). This does not exclude a role
for APP in neuronal iron metabolism as the same authors provided more evidence that APP
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can stabilize ferroportin on the cell membrane and increase iron efflux by an alternative and
as yet undefined mechanism (Wong et al., 2014).

Cancer cells with their high metabolic rates and rapid multiplication have a particularly high
requirement for iron. Recent studies suggest that as cancer cells become more malignant,
they clonally evolve to retain iron by decreasing the expression of ferroportin and increasing
the secretion of hepcidin (Torti and Torti, 2011). These characteristics are accompanied by
increased pool of labile iron in cancer cells. Tumor-associated macrophages appear to have
an iron-exporting phenotype which may provide iron to the cancer cells and further promote
tumor growth (Torti and Torti, 2011).

Ferroportin and infection

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Almost all infectious pathogens need to scavenge iron from the host in order to multiply.
The ferroportin/hepcidin axis controls release of cellular iron from macrophages into
plasma, and therefore may play an important role in infections with microbes that reside
within macrophages or microbes that survive in the bloodstream or other extracellular
spaces (Drakesmith and Prentice, 2012). Iron-dependent extracellular microbes are
exemplified by the pathogen Vibrio vulnificus that causes rapid death in mice lacking
hepcidin. Here unrestricted ferroportin activity provides ample extracellular iron (likely in
the form of non-transferrin-bound iron), potentiating pathogen replication. Blockade of

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 Drakesmith et al. Page 13

ferroportin-mediated iron release into plasma by administration of mini-hepcidin peptides

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restored the ability of these mice to resist Vibrio vulnificus and to survive the infection
(Arezes et al., 2015).

Other work has concentrated on intracellular (macrophage-tropic) pathogens in which

ferroportin plays a different role. In this context, iron release from infected cells into plasma
could be expected to deprive microbes of the iron they need to grow. Consistent with this
notion, increasing ferroportin expression by retroviral gene transfer into cell lines in vitro
decreased multiplication of Salmonella enterica serovar Typhimurium, and this effect could
be reversed by the addition of hepcidin (Chlosta et al., 2006). In vivo, during the first few
days of murine infection with Salmonella enterica serovar Typhimurium, the stimulatory
effect of IL-6 on hepcidin production may lead to loss of ferroportin from cell membranes,
iron retention in macrophages and stimulation of intracellular growth (Kim et al., 2014).
However, countervailing effects may predominate during chronic infection: mechanisms
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that increase ferroportin transcription appear as a part of a natural and protective iron-
modulating innate immune response to Salmonella infection of macrophages, and this
response can be enhanced by interferon-gamma (Nairz et al., 2008). This increased
expression of ferroportin in macrophages is also observed in vivo following infection of
mice, where it results in loss of spleen iron (Brown et al., 2015). The upregulation of
ferroportin in Salmonella-infected macrophages is at least in part driven by nitric-oxide-
mediated Nrf2 activation, which in turn increases transcription of the ferroportin gene (Nairz
et al., 2013).

The interaction of other macrophage-tropic intracellular pathogens with ferroportin activity

has been investigated. Ferroportin over-expression was found to suppress the initial growth
of Mycobacterium tuberculosis in macrophage-like cell lines, although the microbicidal
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activity of these cells was also compromised (Johnson et al., 2010). In the case of
macrophage infection with Listeria monocytogenes, wild-type bacteria that gain access to
the cytosol cause an upregulation of ferroportin (perhaps via nitric oxide formation) which
in turn limits iron availability to the pathogen; in contrast L. monocytogenes lacking
listeriolysin O, which is required for bacteria to escape the phagolysosome and enter the
cytoplasm, do not increase ferroportin expression, and are susceptible to iron-induced, ROS-
mediated microbicidal activity (Haschka et al., 2015).

Some pathogens may have evolved mechanisms for assuring their intracellular iron supply.
In addition to the early hypoferremic and macrophage-iron-sequestering effect of
Salmonella enterica serovar Typhimurium infection (Kim et al., 2014), it has also been
reported that macrophages infected with Leishmania amazonensis decreased ferroportin
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expression in a TLR4- and hepcidin-dependent manner (Ben-Othman et al., 2014).

Furthermore, replication of the parasite was enhanced by iron retention caused by
exogenously added hepcidin or by expression of inactive ferroportin, whereas Leishmania
growth was inhibited by expression of a partially hepcidin-resistant ferroportin mutant
(N144H). In summary, ferroportin-mediated iron release from macrophages can be a critical
determinant for pathogen growth. Enhanced ferroportin expression following Salmonella,
Listeria, Mycobacteria and Leishmania infection is host-protective in each case, but it would
appear that Salmonella (in acute infection) and Leishmania may counteract this effect at

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 Drakesmith et al. Page 14

least temporarily and ensure their own iron supply by downregulating ferroportin. Further
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work is required to elucidate the complex time-dependent iron redistribution during in vivo
infection with various intracellular pathogens. Moreover, the specific role of ferroportin in
other infections that are known to interact with iron homeostasis, for example the liver-stage
of Plasmodium (Portugal et al., 2011), is not clear. Although in cell lines ferroportin-
mediated iron release inhibits replication of HIV-1 (Xu et al., 2010), the interactions of
hepcidin, ferroportin and iron with viral infections remain relatively unexplored.

Conclusions
Ferroportin is a unique iron exporter, essential for dietary iron absorption, recycling of iron
from senescent erythrocytes, mobilization of stored iron, and iron transfer to the developing
fetus. It is the only known receptor for the iron-regulatory hormone hepcidin that binds to
ferroportin, induces its endocytosis and thereby controls its membrane concentration and
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iron-exporting activity. Especially in macrophages, ferroportin synthesis is regulated

transcriptionally and translationally, presumably to meet the rapidly changing requirements
for iron export after erythrophagocytosis and in response to macrophage infection by
intracellular microbes. The role of ferroportin in cellular iron homeostasis, innate immunity
and cancer is becoming increasingly recognized. Despite its importance, the structural basis
of ferroportin function and the mechanism by which ferroportin transports iron are not
understood.

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Figure 1. Systemic flows of elemental iron

The release of iron into blood plasma is mediated by the iron transporter ferroportin which
also serves as the control point for the regulation of iron flows. In plasma, iron is carried by
transferrin (Tf-Fe). Red blood cells (RBCs) contain the majority of the body iron, and iron
fluxes in humans are dominated by the utilization of iron by erythroblasts in the marrow,
and recycling of iron from the hemoglobin of senescent erythrocytes by
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erythrophagocytosing macrophages (iMΦ=iron-recycling macrophages, mostly in the

spleen). Other cells in the body utilize smaller amounts of iron, and this too is recycled by
macrophages after cells die. The liver is the major storage organ for iron from which iron
can be mobilized in times of need, and where it is deposited when there is a surplus of iron
in the body. Duodenal absorption of iron is the sole external source of iron, and compensates
for the losses of iron from the body which are normally relatively small. In pregnant women,
iron is transferred to fetal blood through ferroportin in the placenta.
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Figure 2. Ferroportin regulation

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Cellular iron efflux is proportional to the concentration of ferroportin molecules on the

membrane, and multiple levels of ferroportin regulation exist in different cell types. Panel A:
In iron-recycling macrophages, following erythrophagocytosis both heme and iron increase
ferroportin production. Heme regulates ferroportin transcriptionally via the Bach1/Nrf2
complex (heme causes degradation of repressor Bach1, resulting in the stimulation of Fpn
transcription by Nrf2). Iron regulates ferroportin translationally through modulating the
interaction of IRPs with IREs in the 5′ UTR of ferroportin (iron prevents IRPs from binding
to Fpn mRNA, allowing translation to proceed). During infections with intracellular
pathogens, increased NO production leads to the activation of Nrf2 and increased ferroportin
transcription. Inflammation can also suppress ferroportin transcription through as yet
unknown mechanisms. Ferroportin is post-translationally regulated by hepcidin-mediated
endocytosis and proteolysis. Panel B: In duodenal enterocytes, hypoxia induces ferroportin
transcription via HIF2alpha. Retention of iron in enterocytes during iron deficiency is
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avoided by the presence of ferroportin mRNAs that lack 5′IRE. Hepcidin controls iron
efflux from enterocytes posttranslationallly by inducing the endocytosis and proteolysis of
ferroportin. Panel C: Integration of diverse local and systemic influences on cellular iron
efflux through ferroportin regulation at the transcriptional, translational and posttranslational
level.

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Figure 3. Alternating access model of iron transport by ferroportin

Ferroportin is a member of the major facilitator superfamily of transporters which share a
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structural feature known as the MFS fold: 12 transmembrane segments (TM) organized into
two 6-helix halves, connected by a large cytoplasmic loop between TM-6 and -7. Transport
across the membrane likely occurs via the alternate-access (rocker-switch) mechanism
whereby the two halves of the molecule cycle through inward-facing, occluded and outward-
facing conformations to facilitate substrate transport. We hypothesize that i) ferrous iron
binds to ferroportin in the open-in conformation, ferroportin flips to the open-out
conformation and releases ferrous iron to be oxidized by a ferroxidase (Ferrox) and
delivered to transferrin (Tf) in the ferric form; and ii) that hepcidin binds to the open out
conformation inducing a conformational change that results in ubiquitination of the
connecting loop and endocytosis of ferroportin.
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Table 1

Some unanswered questions about ferroportin

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What is the structure of ferroportin?

What is the molecular mechanism of iron transport?
How does hepcidin cause ferroportin endocytosis?
What is the molecular mechanism of ferroportin disease and its autosomal dominance?
How does ferroportin-mediated iron export affect macrophage immune functions?
What is the role of ferroportin in viral infection?
How does inflammation suppress ferroportin transcription?
What is the role of ferroportin in the heart, the erythroblast, the retina and in the brain?
Does ferroportin have other organ-specific roles?
Is ferroportin activity co-ordinated with fluxes of other nutrients?
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