Med Clin (Barc).

2017;148(5):218–224

www.elsevier.es/medicinaclinica

Review

Current status of iron metabolism: Clinical and therapeutic

implications夽
Susana Conde Diez a , Ricardo de las Cuevas Allende b , Eulogio Conde García c,∗
a
Medicina de Familia, Servicio Cántabro de Salud, Santander (Cantabria), Spain
b
Medicina de Familia, Servicio Cántabro de Salud, Centro de Salud Bajo Asón, Ampuero (Cantabria), Spain
c
Servicio de Hematología, Hospital Universitario Marqués de Valdecilla, Universidad de Cantabria, Santander (Cantabria), Spain

a r t i c l e i n f o a b s t r a c t

Article history: Hepcidin is the main regulator of iron metabolism and a pathogenic factor in iron disorders. Hepcidin
Received 22 September 2016 deficiency causes iron overload, whereas hepcidin excess causes or contributes to the development of
Accepted 26 October 2016 iron-restricted anaemia in chronic inflammatory diseases. We know the mechanisms involved in the
Available online 23 March 2017
synthesis of hepcidin and, under physiological conditions, there is a balance between activating signals
and inhibitory signals that regulate its synthesis. The former include those related to plasmatic iron level
Keywords: and also those related to chronic inflammatory diseases. The most important inhibitory signals are related
Iron metabolism
to active erythropoiesis and to matriptase-2. Knowing how hepcidin is synthesized has helped design
Hepcidin
Ferroportin
new pharmacological treatments whose main target is the hepcidin. In the near future, there will be
Hepcidin agonists effective treatments aimed at correcting the defect of many of these iron metabolism disorders.
Hepcidin antagonists © 2016 Elsevier España, S.L.U. All rights reserved.

Estado actual del metabolismo del hierro: implicaciones clínicas y terapéuticas

r e s u m e n

Palabras clave: La hepcidina es el principal regulador del metabolismo del hierro y el factor patogénico más importante en
Metabolismo del hierro sus trastornos. La deficiencia de hepcidina provoca sobrecarga de hierro, mientras que su exceso da lugar
Hepcidina o contribuye al desarrollo de anemias por déficit o restricción de hierro en las enfermedades crónicas.
Ferroportina
Conocemos los mecanismos implicados en la síntesis de hepcidina y, en condiciones fisiológicas, hay un
Agonistas de la hepcidina
equilibrio entre las señales activadoras e inhibidoras que regulan su síntesis. Las primeras incluyen las
Antagonistas de la hepcidina
relacionadas con la concentración plasmática de hierro y con las enfermedades inflamatorias. Las señales
inhibidoras más importantes están relacionadas con la eritropoyesis activa y con la matriptasa-2. Conocer
cómo se sintetiza la hepcidina ha servido para diseñar nuevos tratamientos farmacológicos cuya diana
principal es la hepcidina. En un futuro próximo, se dispondrá de tratamientos eficaces dirigidos a corregir
el defecto de muchos de los trastornos del metabolismo del hierro.
© 2016 Elsevier España, S.L.U. Todos los derechos reservados.

Introduction
Iron is an essential nutrient in the body which plays a central
role in cellular energy metabolism, anaerobic respiration, synthe-
sis of haemoglobin and nucleotide synthesis, additionally, it is also
involved in many other processes of exudative metabolism and
夽 Please cite this article as: Conde Diez S, de las Cuevas Allende R, Conde García
E. Estado actual del metabolismo del hierro: implicaciones clínicas y terapéuticas.
Med Clin (Barc). 2017;148:218–224.
∗ Corresponding author.
E-mail addresses: euconde@humv.es, condee@unican.es (E. Conde García).
cellular immune response.1 In the adult, the total amount of iron in
the body is 3–4 g, of which 65% is in haemoglobin, 25% in deposit
organs (liver, reticuloendothelial system macrophages and bone
marrow) and the remaining 10% in myoglobin, cytochromes, per-
oxidase and catalases.2 Every day, 1–2 mg/d of iron is absorbed
from the diet, which is the same amount lost daily, but it should
be noted that the organism does not have an active iron excretion
mechanism, so that control of the duodenal absorption plays a vital
role in iron homeostasis.3 Plasma iron circulates bound to trans-
ferrin and it comes from the iron which is absorbed and the iron
that comes from deposit organs, which release it into the plasma
through ferroportin.1–3 Their accumulation leads to iron overload
that is toxic and can damage tissues and cause cell death by free

2387-0206/© 2016 Elsevier España, S.L.U. All rights reserved.

 S. Conde Diez et al. / Med Clin (Barc). 2017;148(5):218–224 219

Hepcidin
1-2 mg/d
FPN Macrophage
Enterocyte FPN
Liver FPN Plasma 20-30
1000mg 3-4mg
mg/d
Fe losses
BM
1-2 mg/d
Fig. 1. Body iron homeostasis. The interaction of hepcidin with ferroportin (FPN)
controls the main efflux of iron into plasma.
radical formation and lipid peroxidation. For this reason, the circu-
lating iron is never found in free form, it is always attached to other
molecules, mainly transferrin, but when concentrations of plasma
iron are high and transferrin is saturated, the excess iron is bound
to other plasma molecules of low molecular weight such as citrate,
acetate and albumin.2
The iron in the body follows a cycle consisting of duodenal
absorption, distribution through the plasma bound to transferrin
and transfer to cells via the transferrin receptor, located in the cyto-
plasmic membrane, for use in different metabolic processes or for
storing it in deposit organs.1–3 When red blood cells age, they are
destroyed in macrophages, mainly in the spleen, and iron is reused
after passing through the plasma (Fig. 1). Foods containing ferric
iron which, after being reduced to the ferrous form, it is absorbed
by duodenal enterocytes and subsequently released into plasma
through ferroportin (Fig. 2).
Regulation of iron homeostasis. Hepcidin-ferroportin axis
Hepcidin is the main hormone that regulates iron metabolism.
It is synthesized in the liver and its main mission is to con-
trol the arrival of iron in plasma from food through enterocytes,
macrophages, which contain the iron coming from recycled senes-
cent red blood cells, and that released from the deposits (Fig. 2).
Ferroportin is responsible for the efflux of iron from enterocytes,
macrophages and hepatocytes into the plasma. Hepcidin binds
to ferroportin for its destruction by endocytosis into the cell’s
lysosomes resulting, on the one hand, in a hyposideremia by low-
ering the iron transferred to the plasma and, on the other hand,
to the accumulation of iron as ferritin enterocytes, macrophages
RBCs
2500 mg and hepatocytes.1–3 The control of iron homeostasis by hepcidin
is a classic endocrine regulation system; in the words of Ganz,
the relationship of hepcidin with iron is similar to that of insulin
with glucose.4 Hepcidin is therefore the principal regulator of iron
and plays a key role in all its abnormalities, whether having to do
with deficiency or excess. Hepcidin deficiency causes iron over-
load, while its excess favours iron sequestration in the liver and
macrophages and contributes to the development of iron deficiency
anaemias or because of its misuse in anaemia of chronic disease.5
In these cases, a functional iron deficiency anaemia occurs because
iron reserves are not available for erythropoiesis.5,6 Increased
hepcidin also occurs in chronic inflammatory processes by an
increase in IL6, which also represents a host defence mechanism
against infection by limiting the availability of extracellular iron
to microorganisms.5 Hepcidin production is negatively regulated
by erythropoiesis through mediators that prevent its production
when iron is required for haemoglobin synthesis.5 Hepcidin is an
acute phase reactant that responds to a variety of inflammatory
mediators and signals that activate transcription through different
signalling pathways.5
Regulation of hepcidin expression
Hepcidin, encoded by the gene HAMP, is the hormone that reg-
ulates iron metabolism. It is a 25-amino acid peptide produced by
hepatocytes interacting with the ferroportin found in the cell mem-
brane of enterocytes, macrophages and hepatocytes.7 Regulation
of hepcidin is a multifactorial process involving different stimu-
latory and inhibitory signals which, in various ways, control its
final transcript.7,8 Hepcidin is regulated by plasma iron through
a feedback mechanism which involves intra and extracellular iron
sensors coupled to one or more signal transduction pathways.

Enterocyte BM: Erythropoiesis

Ferritin
Fe3+
DCYTB Hephaestin
Fe2+
FPN
DMT1

Fer
ritin

Hepcidin
FPN

FPN

Liver Vein Macrophage

DCYTB: ferrireductase; DMT1: Divalent Iron Metal Transporter 1; ferroxidase: hephaestin
Free transferrin and iron bound ; Ferritin ; Hepcidin ; Ferroportin FPN

Fig. 2. Dietary iron is absorbed by duodenal enterocytes. Nonheme iron occurs primarily in the ferric state (Fe 3+) in the intestine and is reduced to ferrous iron (Fe 2+) by the
action of ferrireductases, especially duodenal cytochrome b (Dcytb). Ferrous iron passes through the duodenal enterocytes of the divalent metal transporter-1 (DMT1). Once
in the enterocyte, the ferrous iron can be stored in it as ferritin or can be released into the bloodstream through ferroportin (FPN). Ferrous iron is oxidized by a ferroxidase
identified as hephaestin, which after converting into ferric iron, it binds to transferrin and thus circulates in plasma. Furthermore, senescent erythrocytes are phagocytosed
by macrophages, primarily in the spleen, but also in the liver and the bone marrow (BM). During erythropoiesis, erythroblasts acquire iron for haemoglobin synthesis from
transferrin via transferrin receptors. Excess iron is stored in the liver and in macrophages as ferritin, which is oxidized to hemosiderin. Hepcidin plays a key role in the release
of iron from the deposits depending on the requirements (e.g.: increased erythropoiesis, etc.).
220
tion Iron activation
nhibi
sis i
poie Tf - Fe2
ro
Eryth
Tf - Fe2
Tf - Fe2 BMP6
TfR2 B
e TfR1
HFE
ErF M HJV
P
R
NEO
1
2
SMAD
1/5/8
EPO P SMAD
Hypoxia 1/5/8
SMAD4
STAT3
Fig. 3. Hepcidin synthesis. The main axis that controls the synthesis of hepcidin
is bone morphogenetic protein-hemojuvelin-sons of mothers against decapentaplegic
(BMP-HJV-SMAD). Transferrin bound to iron (Fe2-Tf) is a hepatocyte sensor for
hepcidin transcription control. The binding of Fe2-Tf with transferrin receptors
(TfR1 and TfR2) and human hemochromatosis protein (HFE) is inversely corre-
lated with levels of transferrin saturation by iron, so that, if levels increase, HFE
is displaced from TfR1 to TfR2 to activate BMP. This activation facilitates the bind-
ing of BMP6 with its receptors 1 and 2 (BMPR1 and BMPR2) in the presence of
hemojuvelin (HJV) and neoginin (NEO), and launches SMAD 1/5/8 phosphorylation,
hepcidin antimicrobial peptide (HAMP) gene transcription and the synthesis of hep-
cidin. Inflammation is another important mediator in hepcidin synthesis through IL6
and activin B (Act-B) via janus kinase-signal transducer and activator of transcription
(JAK-STAT3) and BMP. The main hepcidin inhibitory signal comes from the bone
marrow during active erythropoiesis and takes place through different proteins,
highlighting erythroferrone (ErFe), growth differentiation factor (GDF15) and twisted
gastrulation BMP signalling modulator (TWSG1), which inhibit the SMAD pathway.
Matriptase-2 (MT-2), encoded by the gene TMPRSS6, also has an inhibitory effect on
hepcidin transcription through HJV cleavage, preventing BMP complex activation.
Other inhibitory signals of hepcidin synthesis come from situations that generate
tissue hypoxia and from the administration of erythropoiesis stimulating factors
such as erythropoietin (EPO).
Iron-bound transferrin (Fe2-Tf) (holotransferrin) is a hepatocyte
sensor converging in a complex heterotetrameric signalling asso-
ciated with the hepatocyte membrane made up of transferrin
receptors (TfR1 and TfR2), human hemochromatosis protein (HFE),
BMP ligands (bone morphogenetic protein), 2 kinase receptors of
BMP (BMPR1 and BMPR2), a BMP coreceptor (hemojuvelin [HJV])
and a facilitator (neoginin).7–10 There is also an increase in hepcidin
in IL-6-mediated inflammatory processes. Conversely, an effective
erythropoiesis in bone marrow decreases hepcidin levels, ensuring
the supply of iron for erythrocyte production. Therefore, hepcidin
levels reflect the integration of multiple activating and inhibitory
signals involved in iron regulation (Fig. 3).
Activating signals
BMP-HJV-SMAD is the main axis controlling hepcidin synthe-
sis. In situations of iron deficiency, the Fe2-Tf complex binds
to the TfR1 receptor, which is not capable of activating the
BMP-SMAD pathway and therefore hepcidin is not synthesized,
something which facilitates the arrival of iron into plasma. When
iron deficiency is corrected, holotransferrin joins the TfR2 recep-
tor, forming a complex with HFE1,7–11 (Fig. 3). The role of HFE
is to promote the association and stabilization with TfR2, acti-
vate HJV and BMP to bind to its receptors (BMPR1 and BMPR2),
S. Conde Diez et al. / Med Clin (Barc). 2017;148(5):218–224
Inflammation in the presence of neoginin, promoting SMAD1/5/8 phosphoryla-
act tion, HAMP transcription and finally hepcidin synthesis12,13 (Fig. 3).
iva
ti Another important mediator in hepcidin synthesis is the inflamma-
on
tion through IL6 and other cytokines (IL1, IL22) and activin B (Act-B)
which activate the JAK-STAT3 and BMP signalling pathway7,9
B
MT - 2
Act -
B IL6
M
P (Fig. 3).
R
Inhibitory signals
The main hepcidin inhibitory signal comes from erythropoiesis
and is related to different proteins such as erythroferrone, growth
differentiation factor 15 (GDF15) and twisted gastrulation BMP
JAK - STAT3 signalling modulator (TWSG1) that inhibits the SMAD pathway7
(Fig. 3). Matriptase-2 also has an inhibiting effect on hepcidin,
encoded by the TMPRSS6 gene, as it blocks HJV and prevents BMP
complex activation.10 Other inhibitory signals of hepcidin synthe-
sis are tissue hypoxia and erythropoiesis stimulating factors such
as erythropoietin (EPO).1,2
Hepcidin Disorders associated with hepcidin abnormalities
HAMP
Ferroportin and hepcidin abnormalities, whether by interaction
or aberrant expression, cause or contribute to triggering a large
number of iron related disorders ranging from anaemia due to iron
deficiency to iron overload diseases.14,15
Diseases associated with an excess of hepcidin
A high level of hepcidin generates hyposideremia with the
consequent reduction in erythropoiesis due to insufficient sup-
ply of iron, which leads to the development of anaemia in chronic
diseases.15,16
Chronic diseases present with moderate normocytic and nor-
mochromic anaemia but sometimes it may be microcytic and
hypochromic. Anaemia is not only the result of elevated hepcidin,
there is also a direct effect of cytokines on the production and
half-life of red blood cells.5 In chronic kidney disease (CKD), an
elevated hepcidin is the result of a combination of inflammation
and inadequate kidney clearance.10,17 In neoplasms, anaemia is
associated with disease and treatments, but high levels of hep-
cidin and cytokines also play an important role.2 In addition, 10%
of the elderly develop anaemia due to nutritional defects, bleeding,
chronic inflammation, renal failure, MDS and increased hepcidin.1,2
Finally, iron-refractory iron deficiency anaemia must be included in
this section, a genetic anaemia characterized by a mutation in the
TMPRR6 gene encoding matriptase-2.10 The genetic defect is auto-
somal recessive and occurs with elevated hepcidin levels due to the
absence of matriptase-2, resulting in an accumulation of HJV and
activation of BMP pathway.10
Diseases associated with hepcidin decrease or resistance
A hepcidin production deficiency results in iron overload which
is usually genetic and is due to mutations in the HFE or TfR2
gene (adult hemochromatosis) or the HJV or HAMP genes (juve-
nile hemochromatosis).18,19 This decrease of hepcidin contributes
to the normal operation of ferroportin, which results in enterocyte
iron absorption and increased release of iron from macrophages,
which causes hypersideremia with subsequent increase in trans-
ferrin saturation, and the appearance of iron bound to proteins
other than transferrin, called non-transferrin-bound iron (NTBI).7,15
As there is no regulatory mechanism for excreting iron in humans,
excess iron is deposited in tissues that express carriers for NTBI,
mainly the liver, heart, pancreas and other endocrine glands, caus-
ing function failure in the affected organ.
 S. Conde Diez et al. / Med Clin (Barc). 2017;148(5):218–224
Hemochromatosis is the most common cause of genetic iron
overload. There are many mutations that lead to the clinical syn-
drome previously mentioned, but the most common is inherited as
an autosomal recessive trait, affecting the HFE gene, which encodes
the HFE protein (Type 1 Hemochromatosis). The most common sub-
type affects homozygous patients with Cys282Tyr mutation (type
1A). Heterozygous patients may have mixed Cys282Tyr/His63Asp
mutations (type 1B). There are also cases that have mutations in
genotypes other than HFE, for example, Ser65Cys (Type 1C).13,18,19
More penetrating and severe forms of hereditary hemochromato-
sis are rare and are caused by mutations in genes HJV (type 2A),
hepcidin (type 2B), or TfR2 (type 3).9,18 Mutations in the gene that
controls TfR2 prevent this from binding to HFE and consequently,
the BMP-HJV-SMAD complex is not activated and hepcidin is not
synthesized.18 Another rare form of hemochromatosis is due to
mutations in the ferroportin gene (type 4). These mutations, C326S
and SLC11A3, lead to a hepcidin-resistant ferroportin, so that the
iron is exported to the circulation, even in the presence of ele-
vated levels of hepcidin.18,20 Phlebotomy is the treatment of choice
in iron overload and it is estimated that 1 mg of iron is lost with
each millilitre of red cells removed and, therefore, the mobilization
of excess iron accumulated in the tissues is promoted to restore
erythropoiesis.2
ˇ-Thalassemia is another disease that occurs with anaemia,
iron overload and low levels of hepcidin. Iron overload is the
leading cause of morbidity and mortality in these patients,
both in non-transfusion-dependent thalassemia as well as in
the dependent one. Defective ␤-globin chain production during
erythropoiesis results in the precipitation of ␣-chains produced
in excess and many erythroid precursors may die early, which
results in an ineffective erythropoiesis.21,22 To minimize anaemia,
a series of compensatory mechanisms are activated, such as an
increase in erythropoietin which causes a erythroblastic hyperpla-
sia, extramedullary haematopoiesis, splenomegaly and increased
intestinal absorption of iron which, in the absence of transfusion,
is the cause of iron overload.23 The decrease in hepcidin levels
is the result of increased suppressor factors, erythroferrone and
GDF15 during the development of erythroblasts.8 Transfusions par-
tially correct anaemia and hepcidin suppression, but provide very
high amounts of iron with the RBCs transfused. Iron overload in
␤-thalassemia is treated with iron chelators.
Hepcidin in the diagnosis of iron abnormalities
If hepcidin plays a central role in the pathogenesis of many iron
disorders, it seems logical that its quantification should become a
useful tool for the diagnosis and clinical management of associated
diseases. The size of hepcidin in its circulating bioactive form is 25
amino acids and the N-terminal degradation or storage of samples
at room temperature results in smaller isoforms (hepcidin-24, -23,
-22 and -20 amino acids) whose meaning is not known.1 Circulating
hepcidin binds to one alpha-2-macroglobulin and to albumin, it is
excreted by the kidney and reabsorbed in the proximal tubules.24
The development of tests to quantify hepcidin levels in biologi-
cal samples is a not yet resolved challenge. Absolute hepcidin levels
differ widely, up to 10 times in the general population and between
the different clinical trials, so each laboratory should establish its
own reference values. These differences may be due to the fact
that hepcidin is influenced by many physiological and patholog-
ical stimuli that promote or inhibit its synthesis and because it
adheres to plastics.24 Hepcidin determination can be performed
on serum, plasma or urine by immunoassay or by mass spectrom-
etry. The quantification of hepcidin by immunoassay may be more
appropriate for large-scale studies due to its high performance
and relatively low cost. However, immunoassays quantify the total
221
hepcidin, without distinguishing between the complete hepcidin
(hepcidin-25) and the smaller isoforms (hepcidin-20, -22, -23 and
-24), besides, its concentrations are usually higher in CKD due to
a dysfunction in hepcidin clearance.24 Urinary hepcidin measure-
ment correlates with plasma hepcidin, but urinary detection may
be distorted by the high concentration of small isoforms and in
CKD.24
Hepcidin as a therapeutic target
Hepcidin is the main target of diseases related to iron
metabolism disorders and, therefore, new hepcidin agonists or
antagonist drug therapies are being researched.10,11 The interest of
these studies is huge, as evidenced by the fact that in July 2016 there
were 120 registered clinical trials of hepcidin in ClinicalTrials.gov.
Hepcidin agonists
Increase levels of circulating hepcidin may be beneficial in
patients with processes that occur with iron overload, such as
hemochromatosis and thalassemia. This could be achieved with
drugs that have a hepcidin-like activity or stimulating its endoge-
nous production, but the design of a hepcidin similar to that of
oneself does not seem to be the solution, because its half-life is
very short and because of the costs involved in its production.10
Minihepcidins
They have a hepcidin-like action with a better bioavailability
and a longer circulating half-life. Their action is exerted by bind-
ing to ferroportin and blocking the efflux of iron from enterocytes,
macrophages and hepatocytes, leading to decreased plasma iron
levels.11,25–27 Its efficacy has been demonstrated in knockout HAMP
−/− mice, a model of severe hemochromatosis with lack of hep-
cidin, in which a plasma iron reduction and tissue iron overload
normalization is achieved.27 In thalassaemic mice, treatment with
minihepcidins decreases iron overload and improves all haemato-
logical parameters.25–27 This is the result of iron restriction, leading
to a decrease in aggregate formation and intracellular inclusion
bodies and prevents oxidative stress in erythrocytes, damage in
DNA, organelle and cell membrane, with improved erythropoiesis
and anaemia correction.2,20
Hepcidin endogenous production stimulators
Another way to increase hepcidin production is based on the
design of TMPRSS6 antagonists. RNA technologies are used, mainly
antisense oligonucleotides that could silence the mRNA of TMPRSS6
thereby stabilizing HJV, stimulating BMP-HJV-SMAD pathway and
increasing the synthesis of endogenous hepcidin.11,28 These drugs
have also been effective in treating iron overload in knockout mice
and in the treatment of anaemia and iron overload in thalassemia
mouse model (th3/+).29 In addition to increasing the levels of
hepcidin, they improve iron overload, reduce splenomegaly and,
surprisingly, ineffective erythropoiesis is corrected in thalassaemic
mice with homozygous inactivation of TMPRSS6.29,30
Hepcidin antagonists
The high concentration of hepcidin causes iron entrapment
in the cells of the reticuloendothelial system and is the leading
cause of effective iron deficiency in anaemia of chronic disease,
therefore, pharmacological suppression of hepcidin would facil-
itate iron mobilization, promoting erythropoiesis and anaemia
correction. The strategies that are being considered for this pur-
pose are directed to change the BMP-SMAD or IL6/STAT3 axes,
222 S. Conde Diez et al. / Med Clin (Barc). 2017;148(5):218–224

Table 1
Hepcidin inhibitors and their corresponding targets.

Inhibitor Action and target References

BMPs/BMPR/HJV complex
• Anti-HJV MAb (ABT-207, h5F9-AM8) Inhibitor of BMPs/SMAD pathway 33,34

• sHJV-Fc Inhibitor of BMPs/SMAD pathway 11,35

• LDN-193189 BMPR1 phosphorylation inhibitor 36

• Anti-BPM6 MAb BMP6 sequestration 37

• siHep, siHJV, siTfR2 Hepcidin mRNA degradation, HJV or TfR2 36,38

• Modified heparin Inhibitors of BMPs/SMAD pathway 40,41

IL6/SATAT3 pathway
• Anti-IL6 (xiltuximab) IL6 sequestration 42

• Anti-IL6R (tocilizumab) IL6 receptor sequestration 43

• AG490 STAT3 phosphorylation inhibitor 44

Antihepcidin agents
• Antihepcidin MAb (12B9m) Hepcidin protein sequestration 48

• Anticalines (PRS-080) Hepcidin protein sequestration 49

• Aptamers (Spiegelmers: NOX-H94) Hepcidin protein sequestration 50

Hepcidin-ferroportin interaction
• Antiferroportin MAb (LY2928057) Hepcidin-ferroportin binding interference 51

• Fursultiamine Cys326-HS sequestration (FPN-hepcidin binding) 52

Other inhibitors
• TNF-␣ MAb (infliximab, golimumab) Indirect effect of IL6 suppression 53,54

• Vitamin D SMAD 1/5/8 phosphorylation suppressed. Vitamin D recep 55

• 17-Estradiol SMAD 1/5/8 phosphorylation suppressed 56

• Testosterone SMAD 1/5/8 phosphorylation suppressed 57

• Chinese medicinal plant extract: Caulis spatholobi (Jixueteng) SMAD 1/5/8 phosphorylation suppressed 58

MAb: monoclonal antibody; MRNA: messenger ribonucleic acid; BMP: bone morphogenetic protein; BMPR: bone morphogenetic protein receptor; FPN: ferroportin; HJV:
hemojuvelin; sHJV-Fc: fusion protein between the soluble HJV and the Fc fragment of immunoglobulins; IL: interleukin; SMAD: sons of mothers against decapentaplegic;
STAT3: signal transducer and activator of transcription 3; TfR2: transferrin receptor 2; TNF: tumour necrosis factor.
siHep, siHJV, siTfR2: oligonucleotides silencing mRNA expression in genes encoding hepcidin, HJV or TfR2.

interfere in hepcidin-ferroportin binding and enhance their sup- modified heparin analogues have been designed to minimize its
pressive mechanisms10,11,16,31,32 (Table 1). anticoagulant effect, maintaining their inhibitory effect on hepcidin
expression to treat anaemia of inflammatory processes.40,41

Inhibition of bone morphogenetic protein pathway Suppression of the inflammatory pathway

The BMP-HJV-SMAD pathway is the main axis which con-
trols the synthesis of hepcidin and, therefore, inhibiting the BMP
pathway would decrease HAMP expression and blood concentra-
tions of hepcidin. Various molecules have been directed against
HJV as a target. On the one hand, several monoclonal antibodies
(mAbs) targeting the HJV (ABT-207, h5F9-AM8 and h5F9-23) pro-
tein that prevent the binding of BMP with BMPR, block the SMAD
pathway and correct anaemia in rats with chronic inflammatory
processes33,34 have been investigated. A similar effect is achieved
with a fusion protein between the soluble HJV (sHJV) and the Fc
fragment of immunoglobulins (sHJV-Fc) that binds to BMP and pre-
vents BMPR activation.11,35 The same applies to a BMP receptor 1
(BMPR1) inhibitor, the LDN-193189, in rats and human hepatoma
cells36 and with an anti-BPM6 MAb.37 Another way of inhibiting
the BMP pathway is blocking or silencing the genes regulating hep-
cidin synthesis with antisense oligonucleotides that interfere with
RNA and silence the mRNA (siRNA) of hepcidin-encoding genes,
TfR2 and HJV.36,38 The administration of these siRNAs decreases
hepcidin concentrations and corrects anaemia in a chronic inflam-
matory anaemia mouse model.16,39 Furthermore, it is known that
human mutations in the genes encoding hepcidin, the HJV and TfR2,
are associated with iron overload diseases, so in these cases, the
effects on the corresponding targets of these oligonucleotides and
siRNAs may not be effective.10
Heparin is another known hepcidin inhibitor through BMP6
sequestration and the subsequent blocking of the SMAD pathway,
but its anticoagulant activity limits the therapeutic use as an hep-
cidin inhibitor. This action of heparin has been demonstrated in
mice and in patients receiving heparin to prevent deep venous
thrombosis which has achieved an increase in serum iron and trans-
ferrin saturation and a reduction in C-reactive protein.40 Several
Blocking IL6 with xiltuximab42 and IL6 receptor with
tocilizumab43 prevent STAT3 phosphorylation and hepcidin con-
centration decrease and blood iron (level) normalization in
monkeys with arthritis and in patients with Castleman’s disease.
Similar effects were achieved with a chemical inhibitor of JAK2,
the AG490, blocking the IL6-STAT3 pathway.44 The main drawback
of anti-cytokine treatments is that they induce immunosuppres-
sion, with impairment of the host defences and an increased risk of
infections.11
Potentiation of erythropoiesis
Increased erythropoiesis is accompanied by a suppression of
hepcidin, which could represent a new strategy for the treatment of
anaemia of chronic disease. High doses of EPO may be able to over-
come the observed resistance to EPO in these diseases, probably
by partial suppression of hepcidin.45 Similarly, prolylhydroxylase
inhibitors, which stabilize the factors, induce hypoxia and increase
EPO synthesis, decrease hepcidin levels and increase haemoglobin
in patients with CKD.46
Neutralization of circulating hepcidin
The bioactive neutralization of hepcidin may be achieved by
direct binding or sequestration of the circulating hormone with
MAb, anticalines or L-RNA aptamers (called Spiegelmers, in Ger-
man, ‘mirror’). The main advantage of these hepcidin neutralizers
is their high specificity and picomolar affinity to bind to their tar-
gets, but the main challenge they must overcome to demonstrate
their efficacy is the high rate of hepcidin production present in
humans, so that would require massive doses of therapeutic agents
in order to achieve their goal.10 However, it has been shown that the
intermittent or partial neutralization of hepcidin activity may be
 S. Conde Diez et al. / Med Clin (Barc). 2017;148(5):218–224
sufficient to achieve the desired therapeutic effects.10,47 A human
anti-hepcidin antibody, 12B9m, has been evaluated in transgenic
mice with inflammatory anaemia caused by Brucella abortus and
in monkeys. Neutralization of hepcidin by MAb increased ery-
thropoiesis and haemoglobin levels.48 Currently, clinical trials are
being conducted with another anti-hepcidin antibody, LY2787106,
in patients with anaemia associated with cancer.10 Anticalines are
small proteins designed by genetic engineering targeting various
biological ligands.47 One of these compounds is the PRS-080, which
binds to human hepcidin with subnanomolar affinity, achieving an
effective mobilization of iron and hypersideremia in primates.49
Aptamers (Spiegelmers) are l-oligonucleotides designed against the
mirror image of the target, to which they bind with high affin-
ity. They are stable in the circulation and immunologically passive
because their structure contains l-ribose, instead of d-ribose, which
gives them a high degradation resistance due to nucleases.50 NOX-
H94 neutralizes human hepcidin and increases plasma iron and
transferrin saturation.50
Ferroportin as a target. Blocking hepcidin-ferroportin interaction
Hepcidin binding to ferroportin takes place in an extracellu-
lar region of ferroportin containing the sulfhydryl residue Cys326
surrounded by hydrophobic residues.10 Blocking this interaction
prevents endocytosis and ferroportin destruction, which favours
the efflux of iron from cells into plasma. A humanized antifer-
roportin MAb, the LY2928057, is being evaluated. It targets this
region and prevents hepcidin binding the carrier without interfer-
ing with the efflux of iron through ferroportin,51 increasing plasma
iron levels in cynomolgus monkeys. Fursultiamine is a thiol com-
pound that prevents hepcidin binding to ferroportin in vitro and
in vivo in mice.52
Other inhibitors of hepcidin
TNF-␣ inhibitors (golimumab or infliximab) also decrease the
synthesis of hepcidin and improve the anaemia of chronic disease
through an indirect effect on IL653,54 and vitamin D, testosterone
and 17-estradiol, which prevents SMAD55-57 phosphorylation.
Finally, it should be noted that some extracts of Chinese medici-
nal plants, such as Caulis spatholobi, called jixueteng, have a potent
inhibitor effect on HAMP expression through SMAD 1/5/858 phos-
phorylation suppression.
Conclusions
We have reviewed the mechanisms that regulate iron homeo-
stasis and hepcidin expression, as well as diseases related to its
excess or decrease and the advantages of its quantification in the
diagnosis of these diseases. The importance of hepcidin as a thera-
peutic target focusing on different compounds aimed at correcting
iron overload or block/inhibit hepcidin in order to cure the anaemia
of chronic disease has been analyzed. Numerous clinical trials are
being conducted and it is expected that they will be soon applied to
clinical practice, but which of the different therapeutic approaches
will have more chances of success is not yet known.
Authorship/collaboration
SCD, RCA and ECG have contributed to the literature review,
writing and discussion of the content of the work. ECG has made
the final text review.
Conflicts of interest
There are no conflicts of interest.
223
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