2180? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 115? ? ? Number 8? ? ? August 2005
Hemojuvelin is essential for dietary iron
sensing, and its mutation leads
to severe iron overload
Vera Niederkofler, Rishard Salie, and Silvia Arber
Biozentrum, Department of Cell Biology, University of Basel, and Friedrich Miescher Institute, Basel, Switzerland.
Regulation of iron uptake depends on the ability of an organism
to accurately sense systemic iron and adjust its level accordingly.
While iron is an essential physiological cofactor for the production
of many proteins, most notably heme proteins, excess iron can be
harmful to the organism, in part through the generation of oxy-
gen radicals, and is potentially lethal (1, 2). Recent work has estab-
lished the importance of the peptide hormone hepcidin in iron
homeostasis as a negative regulator of iron release into the system
by duodenal enterocytes and reticuloendothelial macrophages.
Hepcidin binds to the iron exporter ferroportin, which results in
ferroportin internalization and degradation (3). How hepcidin
levels are kept in balance through upstream signaling pathways is
still under investigation (4–7).
Multiple pathways are known to regulate expression of hepcidin
and thus indirectly affect iron uptake and retention (7). Hepcidin
expression is induced by an excess of iron and is downregulated
by iron deprivation, consistent with its role as a downstream effec-
tor of iron sensing (8). In addition, hepcidin also responds to acute
inflammation with rapid induction of gene expression. Injection
of LPS, a bacterial endotoxin and potent activator of inflamma-
tory response, induces hepcidin in mice (8), an effect believed to be
dependent on cytokine production (4, 5). During inflammation
induced by pathogenic infection, creation of a hypoferremic envi-
ronment is thought to be a defense mechanism of the host organ-
ism for restricting pathogenic growth, which is partially dependent
on physiological iron levels (9). Thus, both dietary iron sensing and
inflammatory pathways converge in the regulation of the key regu-
lator hepcidin, but how these 2 pathways intersect remains unclear.
An increasing number of genes have been assigned roles in iron
homeostasis through their mutational identification in human
diseases. Some of these mutations cause accumulation of iron and
result in disease states of varying severity (1, 10). Human juvenile
hemochromatosis is an early-onset disorder of iron homeostasis
that results in massive iron overload in various body tissues (1,
10–12). Patients suffer from cardiomyopathy, diabetes, or cirrhosis
attributed to oxidative damage caused by iron loading and often
die before 30 years of age. Recently, a mutation causing juvenile
hemochromatosis, which had been mapped to the human chro-
mosomal position 1q, was identified by positional cloning (13).
The corresponding gene was named hemojuvelin (HJV; also known
as HFE2) and was shown to be a member of a previously identified
glycosylphosphatidylinositol-anchored gene family named after
its founding member, repulsive guidance molecule (RGM), the
function of which has mainly been studied in the nervous system.
The RGM gene family is composed of 3 members: RGMa, RGMb,
and RGMc (14). RGMc only differs from HJV in nomenclature
(13, 14), and its mouse homolog will be referred to as Hjv in this
study. Whereas 2 mouse homologs of this family, mouse RGMa
(mRGMa) and mRGMb, are mainly expressed in the nervous sys-
tem, the expression of Hjv is enriched in skeletal muscle and liver
(13, 14). Functionally, RGMa has been implicated in axon guidance
and neural tube closure (14, 15) whereas the expression of mRGMb
(also known as DRAGON) is regulated by the transcription factor
DRG11 in dorsal root ganglia sensory neurons (16). In contrast,
no function for Hjv has previously been described.
Here we show that Hjv is expressed in periportal hepatocytes and
that disruption of the Hjv gene results in severe iron overload. Mice
with mutations in Hjv fail to express hepcidin in response to dietary
or injected iron; this provides a molecular explanation for the severe
iron accumulation observed in Hjv-mutant mice. In contrast, these
mice retain the ability to upregulate hepcidin expression in response
to acute inflammation induced by either LPS or its downstream
products IL-6 and TNF-α. Moreover, we also show that, upon
induction of inflammation, Hjv expression in wild-type mice is
selectively downregulated in the liver but not in skeletal muscle.
Taken together with previous observations (6), our data suggest
that inflammation, through downregulation of hepatic Hjv, might
induce a temporary elimination of iron sensing. In summary, our
Nonstandard?abbreviations?used: ATG, amino-terminal methionine; eGFP,
enhanced GFP; HJV, hemojuvelin; HNF4α, hepatocyte nuclear factor 4α; RGM,
repulsive guidance molecule; Tfr2, transferrin receptor 2.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 115:2180–2186 (2005).
Related Commentary, page 2079
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
results suggest that Hjv plays an essential role in the regulation of
hepcidin expression, specifically in the iron-sensing pathway.
Generation of Hjv-mutant mice and expression of Hjv in periportal
hepatocytes. The recent genetic linkage of HJV to the iron overload dis-
ease juvenile hemochromatosis in humans (13) has opened the way
to elucidation of the function of Hjv in iron homeostasis under nor-
mal physiological conditions and in disease by use of genetic studies
in mice. We therefore generated Hjv-mutant mice, which coordinate-
ly express lacZ?targeted to the nucleus from the Hjv locus (Figure 1,
A and B). Previous work has shown that the strongest expression of
Hjv in mice is found in skeletal muscles, but a lower level of expres-
sion has also been detected in the liver (14). Mice homozygous for
the mutated Hjv allele showed a complete absence of Hjv mRNA in
all tissues analyzed, including skeletal muscles, providing evidence
for a complete null mutation (Figure 1C and data not shown).
To determine the exact site of expression of Hjv in the liver, we
processed vibratome sections of adult liver from Hjv+/– mice in
order to determine the presence of lacZ activity (Figure 1E). Inter-
estingly, we found a patterned distribution of Hjv expression in the
liver whereas skeletal muscles were stained uniformly (Figure 1E
and data not shown). To determine the identity of these cells, we
analyzed lacZ expression on thin sections and found labeled cells
surrounding portal tracts but not central veins (Figure 1, F and G).
At high magnification, lacZ+ cells were often shown to contain 2
nuclei; this has previously been described as occurring frequently
in hepatocytes (17, 18) (Figure 1G). In addition, double-labeling
immunohistochemistry with an antibody against hepatocyte
nuclear factor 4α (HNF4α), a transcription factor expressed in
hepatocytes (19), confirmed the hepatocytic identity of these lacZ+
cells (Figure 1, H–K). In contrast, lacZ expression was not detected
in any other of the several liver cell types examined, including sinu-
soidal endothelial cells expressing CD31 (20) (data not shown).
Hjv expression in periportal hepatocytes. (A) Targeting strategy used for homologous recombination in ES cells to eliminate Hjv gene function.
The Hjv locus contains 3 coding exons (yellow). A targeting construct containing eGFP (dark gray) followed by IRES-NLS-lacZ-pA (blue) and
thymidine kinase–neomycin (TK-neo) (light gray) cassettes was integrated in frame into the second coding exon of Hjv. The probe used for
genomic Southern blot analysis is indicated in blue. Integrated cassette is not drawn to scale. STOP, carboxyterminal stop codon. (B) Genomic
Southern blot of Hjv+/+, Hjv+/–, and Hjv–/– genomic DNA using the probe indicated in A. (C) Northern blot analysis of total RNA isolated from P21
hindlimb muscles of Hjv+/+ and Hjv–/– mice probed for the expression of Hjv (top) and GAPDH (bottom). (D) Schematic drawing depicting the
territories of liver lobules. Portal tracts (PT) are indicated in blue; central veins (CVs) are shown in red. Note that solid lines in D–G outline the
hexagonally shaped hepatic lobule with PTs at the corners. (E–G) Detection of enzymatic lacZ activity in liver from 3-month-old Hjv+/– mice ana-
lyzed on vibratome (E) or cryostat (F and G) sections. Red circles indicate CV, blue circles indicate PT. Inset in (G) depicts high magnification of
individual binuclear hepatocytes that express lacZ. (H–K) Immunohistochemical detection of HNF4α (H, J, and K: red), lacZ (I, J, and K: green),
and SYTOX green (nuclei; K: blue) in liver from 3-month-old Hjv+/– mice. Arrows point to binuclear Hjv-expressing hepatocytes. Scale bar: 530
µm (E); 260 µm (F); 70 µm (G); 30 µm (inset in G); 40 µm (H–K) .
2182?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
Together, these findings indicate that Hjv expression in the liver is
restricted to hepatocytes surrounding the portal tracts.
Hjv mutation in mice causes severe iron overload. To assess the conse-
quences of Hjv mutation for iron homeostasis in various organs, we
used both histological staining procedures and quantitative deter-
mination of iron content (Figure 2 and Supplemental Figure 1; sup-
plemental material available online with this article; doi:10.1172/
JCI25683DS1). At 2.5 months of age, Hjv-mutant mice showed a
severe increase in iron content in the liver (∼20 fold; iron accumula-
tion in the parenchymal cells of the liver), pancreas (∼25 fold; iron
accumulation in acinar tissue), and heart (∼4.5 fold) (Figure 2, A–D,
I, and J, and Supplemental Figure 1). In contrast, we found a reduc-
tion in iron accumulation in the spleen (∼4.5 fold; Figure 2E–H, I,
and J), probably due to the inability of reticuloendothelial macro-
phages residing in the red pulp to sequester iron. These findings are
consistent with the previously observed distribution of iron content
under conditions of hemochromatosis in both human patients and
other mouse models of this disease (10, 11, 21–24).
A time-course experiment for the determination of?iron content
in various tissues at several postnatal developmental stages of Hjv-
mutant mice showed a rapid and permanent increase in iron accu-
mulation, which reached plateau levels by 4 months of age (Figure
2J and Supplemental Figure
1E). Importantly, the first
signs of hepatic iron over-
load were already detected by
P30 (Figure 2J). These find-
ings reveal that mutation
of Hjv in mice leads to iron
accumulation in multiple
organs with a time course
and tissue distribution com-
parable to that observed in
patients suffering from juve-
nile hemochromatosis (10).
Although we found simi-
larities detected in iron accu-
mulation between human
patients and Hjv-mutant
mice, we did not observe
obvious features of cardio-
myopathy in these mice
as assessed by histology,
analysis of heart weight, and
mRNA expression analysis
of a number of marker genes
known to be altered in car-
Figure 1, F and G, and data
not shown). Moreover, Hjv-
mutant mice did not experi-
ence an increase in mortality
(up to 15 months of age) or
show signs of diabetes (Sup-
plemental Figure 1H and
data not shown). While Hjv-
mutant males were sterile,
they did not show signs of
hypogonadism as assessed by
Scale bar: 270 µm (A and B); 45 µm (C and D); 1.2 mm (E and F); 100 µm (G and H).
determination of testicular size, a phenotype frequently observed in
human sufferers of juvenile hemochromatosis (10). Together, these
findings suggest that Hjv-mutant mice show an iron homeostasis
phenotype highly similar to that of human patients but, surpris-
ingly, do not develop all of the associated pathological conditions.
Lack of hepcidin expression in Hjv-mutant mice. We next began to assess
the molecular mechanism by which absence of Hjv leads to iron accu-
mulation in mice. Hepcidin expression is a well-established indicator
of iron levels and is upregulated by high body iron (25). In the liver of
wild-type rats, hepcidin expression occurs in 2 waves: an early postna-
tal spike (P0–P3) that declines rapidly followed by a second increase
that begins during the fourth postnatal week and continues into
adulthood (26). A very similar time course can be detected in mice
in which adult levels of hepcidin expression in the liver are reached at
P24 (6) (Figure 3A). In contrast to the dynamic expression of hepcidin,
Hjv expression in the liver was already detected at E13.5 and reached
a steady level by late embryonic stages (6) (Figure 3A).
We first determined the level of hepatic hepcidin expression in
adult Hjv-mutant mice by Northern blot analysis and in situ hybrid-
ization experiments. We found that hepcidin mRNA was virtually
undetectable in adult Hjv-mutant mice compared with wild-type
littermates, in which expression was detected broadly throughout
Iron accumulation in Hjv-mutant mice. (A–H) Histological detection of iron content on cryostat sections of liver
(A–D) and spleen (E–H) of wild-type (A, C, E, and G) and Hjv–/– (B, D, F, and H) mice. Note uniform iron accumula-
tion in the liver of 2.5-month-old Hjv-mutant mice and absence thereof in the red pulp of the spleen. (I) Quantitative
determination of iron content (µmol/g dry weight) in various organs of 2.5-month-old wild-type (white), Hjv+/– (gray),
and Hjv–/– (black) mice (n = 5 for each group). Asterisks indicate significant changes (P < 0.05) in Hjv–/– mice as
compared with wild-type littermates. (J) Time course (P12–P300) of iron content (µmol/g dry weight) determined
in Hjv–/– mice (squares) compared with pooled wild-type and Hjv+/– mice (triangles). Liver (green) and spleen (blue)
are depicted in the graph. At least 3 animals per time point and genotype were included in the analysis. Asterisks
indicate significant changes (P < 0.05) in Hjv–/– mice as compared with pooled wild-type and Hjv+/– littermates.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
the liver (≤ 0.3% of wild type; Figure 3, B and D, and Figure 4A).
Moreover, hepatic hepcidin expression in Hjv-mutant mice was
also absent at early postnatal stages, when wild-type mice exhibit
a naturally occurring spike of hepcidin expression (Figure 3B). To
determine whether Hjv-mutant mice exhibit a general block of hep-
cidin regulation in response to iron, we assessed whether artificial
elevation of iron levels in Hjv-mutant mice was capable of inducing
hepcidin expression. We found that subcutaneous injection of iron-
dextran (8) in Hjv-mutant mice did not increase hepcidin expression
significantly whereas the same treatment consistently increased
hepcidin in wild-type mice (Figure 3C).
Together, these findings point to an essential role for Hjv in iron
sensing, that of an upstream regulator of hepcidin expression. More-
over, the massive reduction in hepcidin provides a molecular explana-
tion for the continued iron accumulation and lack of effective regu-
latory mechanisms to decrease iron uptake in Hjv-mutant mice.
Acute inflammation can induce hepcidin expression in Hjv-mutant mice.
Does the lack of hepcidin expression in Hjv-mutant mice represent an
absolute inability to induce hepatic hepcidin expression, or is it possi-
ble to bypass this deficiency by stimulation of the inflammatory path-
way (4, 8)? We found that induction of acute inflammation by lipo-
polysaccharide (LPS) injection led to rapid and robust upregulation
of hepcidin in Hjv-mutant mice compared with sham-injected mutant
animals (∼300 fold; Figure 4A). To determine whether downstream
products of LPS were also sufficient to mimic the effect of LPS on
hepcidin expression in Hjv-mutant mice, we used injections of either
proinflammatory cytokine IL-6 or TNF-α (27). We found that either
IL-6 or TNF-α was sufficient to mimic the effect of LPS, albeit to a
lesser extent (IL-6: ∼130 fold; TNF-α: ∼160 fold) (Figure 4A).
We also assessed whether, in Hjv-mutant mice, inflammation-
mediated upregulation of hepcidin expression was capable of
effectively eliciting appropriate downstream responses. The iron
exporter ferroportin has been shown both to regulate cellular
iron uptake by binding to hepcidin (3) and to be transcriptionally
downregulated by high hepcidin levels (28). Consistent with the
observed lack of hepcidin expression in Hjv-mutant mice, we found
high expression of ferroportin in both untreated and sham-injected
Hjv-mutant mice (Figure 4B and data not shown). In contrast,
upon LPS injection (associated with hepcidin induction), ferropor-
tin mRNA is significantly reduced in Hjv-mutant mice as well as in
wild-type mice (Figure 4B), indicating the presence of intact down-
stream responses to hepcidin in Hjv-mutant mice. These findings
show that the inflammatory pathway can efficiently bypass a
requirement for Hjv in the induction of hepatic hepcidin expres-
sion and assign a specific role to Hjv in the iron-sensing pathway
upstream of hepcidin regulation.
Inflammation induces selective downregulation of Hjv in liver but not
muscle. Normal iron balance is subverted during inflammation
when hepcidin levels are elevated to create a transient hypoferre-
mic environment inhibitory to pathogenic growth (9). This low
serum iron concentration should be perceived as hypoferremia by
the dietary iron-sensing pathway and rapidly counteracted; how-
ever, this does not occur. Interestingly, previous experiments have
shown that Hjv expression in the liver of wild-type mice is strongly
downregulated upon induction of acute inflammation by LPS (6).
These findings raise the question of whether the observed effect
is selective to the liver and whether other genes involved in iron
metabolism (1, 10–12) are regulated in a similar manner.
Interestingly, in contrast to the dramatic downregulation of Hjv
expression observed in the liver of LPS-injected animals (6) (Figure
4C), no decrease in the expression of Hjv in skeletal muscles was
detected under these conditions (Figure 4C). Moreover, we also
found that the expression levels of several hemochromatosis- or iron
metabolism–related genes, such as Hfe, transferrin receptor 2 (Tfr2),
β2-microglobulin, and ceruloplasmin, analyzed in the liver were
unchanged following LPS injection (Supplemental Figure 2). Finally,
a decrease in Hjv expression in the liver was also observed in response
to IL-6 or TNF-α injection (Figure 4D). Together, these findings show
that the inflammatory response induces a transcriptional downregu-
lation of Hjv specifically in the liver and that such a response is not
observed for other genes implicated in iron regulatory pathways.
In this study, we provide evidence that Hjv expression in the liver
is restricted to periportal hepatocytes and that Hjv is an essen-
tial component of the iron-sensing pathway. Our experiments
show that Hjv-mutant mice exhibit an iron overload phenotype
with high similarity to that of human patients suffering from
juvenile hemochromatosis. Despite excessive iron accumulation,
Lack of hepcidin expression in Hjv-mutant mice. (A) Developmental
time course (E13.5–P90) of Hjv, hepcidin, and GAPDH expression lev-
els as determined by Northern blot analysis on total RNA isolated from
liver. (B) Northern blot analysis of total RNA isolated from adult (P90) or
P1.5 liver of wild-type and Hjv–/– mice probed for the expression of Hjv,
hepcidin, and GAPDH. (C) Northern blot analysis of total RNA isolated
from adult (P90) liver of wild-type and Hjv–/– mice sacrificed 7 days after
sham injection (S) or injection with iron-dextran (ID) and probed for the
expression of hepcidin and GAPDH. Scale bar: 100 µm. (D) In situ
hybridization on cryostat sections of liver isolated from adult (P90) wild-
type and Hjv–/– mice probed for the expression of hepcidin.
2184?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
Hjv-mutant mice show a complete lack of hepcidin expression; this
provides a molecular explanation for the observed phenotype. Nev-
ertheless, hepcidin expression can still be induced in Hjv-mutant
mice by activation of the inflammatory pathway, providing evi-
dence for the selective requirement of Hjv in the iron-sensing but
not the inflammatory pathway upstream of hepcidin regulation.
We will discuss our findings with respect to the role of Hjv in iron
homeostasis and potential mechanisms by which Hjv might link
iron sensing and inflammatory pathways in vivo.
The complex regulatory network underlying systemic regulation
of iron homeostasis is tuned to respond to different stimuli by
activation of distinct molecular pathways, all of which funnel into
the regulation of hepatic hepcidin expression (7) (Figure 5, A and
B). In this study, we provide evidence that Hjv is essential in the
iron-sensing pathway. We found that even experimental elevation
of iron levels was not capable of inducing hepcidin expression in
Hjv-mutant mice and that these mice were also devoid of the hepci-
din expression spike that normally occurs during the first postna-
tal week. These findings strongly suggest an essential role for Hjv
in iron metabolism from birth throughout life.
In marked contrast to the defects in hepcidin expression in
response to iron observed in Hjv-mutant mice, regulation of hep-
Selective suppression of Hjv during inflammatory response. (A–C) Northern blot analysis of hepcidin (A), ferroportin (B), and Hjv (C) expres-
sion on total RNA isolated from liver of wild-type or Hjv-mutant mice. Before isolation of total RNA, mice were injected intraperitoneally with PBS
(sham), LPS, IL-6, or TNF-α. At least 3 animals per experimental condition were analyzed, and 1 representative example is shown. Quantifica-
tion of expression levels was performed by normalization of each sample to GAPDH expression probed sequentially on the same blots (data
not shown). Histograms depict wild-type mice in black and Hjv-mutant mice in grey. Asterisks indicate significant changes (P < 0.05) in animals
treated with LPS, IL-6, or TNF-α as compared with sham-injected animals of the same genotype. (D) Northern blot analysis of Hjv expression
on total RNA isolated from skeletal muscle of wild-type mice after sham or LPS injection. Quantification was performed as described in (A–C).
Histogram depicts sham-injected mice in black and LPS-injected mice in white.
An essential role for Hjv in the iron-sensing pathway. (A) Model depicting the dietary iron-sensing pathway in wild-type and Hjv-mutant mice. In
wild-type mice, green arrows indicate responses in the presence of high iron; red arrows show responses in the presence of low iron. Balanced
regulation of this pathway adjusts iron levels to the needs of the healthy organism (follow green or red arrows from left to right). In Hjv-mutant
mice, iron-sensing is defective due to the absence of Hjv (indicated by gray double line). Despite high iron, this leads to an essentially complete
absence of hepcidin expression, iron overload, and hemochromatosis (green arrows in Hjv mutant). Fpn, ferroportin. (B) Model depicting the
impact of acute inflammation on the iron-sensing pathway (blue arrows). Acute-phase cytokines IL-6 and TNF-α act to coordinately downregulate
Hjv expression in the liver while simultaneously inducing hepcidin expression. The reduction of Hjv results in a blockade of the dietary iron-sens-
ing pathway (indicated by blue double line). This mechanism efficiently suppresses the iron-sensing pathway during the inflammatory response,
which results in a low iron serum concentration inhibiting pathogenic growth.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
cidin expression upon inflammation is intact in these mutants.
Interestingly, hepcidin levels in LPS-injected Hjv-mutant animals do
not reach those of wild-type animals. These findings suggest that
the total level of hepcidin expression observed upon inflammation
is additive to the baseline level?and again argue for the existence
of 2 independent pathways that lead to the regulation of hepcidin
expression. Of these 2 pathways, only the iron-sensing pathway
requires functional Hjv (Figure 5).
How do iron sensing and inflammatory pathways interact, and
what possible mechanism could prevent interference between
the 2 pathways? Interestingly, previous experiments have shown
that hepatic Hjv is rapidly downregulated at the transcriptional
level upon induction of inflammation by LPS injection in wild-
type mice (6). Moreover, we now show that Hjv downregulation
upon inflammation is selective to the liver and does not occur in
skeletal muscle, another prominent site of expression of Hjv (Fig-
ure 5B). Together with our observations that Hjv is not required
for hepcidin induction during inflammation, these findings pro-
vide an intriguing potential mechanistic explanation for how the
iron-sensing pathway is switched off during inflammation: by the
rapid and selective extinction of Hjv in the liver (Figure 5B). By
such a mechanism, interference with individual pathways may be
prevented by selective and fast cross-regulatory interactions at the
level of transcriptional gene regulation.
This study also provides evidence that the expression of Hjv in
the liver is restricted to a population of periportal hepatocytes.
In contrast, Hfe, Tfr2, and β2-microglobulin have been described
as expressed broadly throughout the liver (29–31). Periportal
hepatocytes are located close to the portal veins, which deliver
blood to the liver from the gut and are thus in a prime position to
detect the iron content of blood coming directly from the diges-
tive tract. While we cannot exclude a potential role for Hjv in iron-
sensing in skeletal muscles with the currently available mouse
model, our results nevertheless suggest the possibility that the cel-
lular source assigned to iron sensing in the liver may be periportal
hepatocytes, marked by the expression of Hjv. Definitive proof of
the importance of hepatic expression of Hjv, however, awaits either
the generation of tissue-specific Hjv-mutant mice or attempts to
selectively rescue Hjv-mutant phenotypes by tissue-specific expres-
sion. Finally, since hepcidin expression is not restricted to periportal
hepatocytes, this finding excludes a direct molecular link from Hjv
to the intracellular induction of hepcidin expression.
In summary, the findings described in this study reveal a selec-
tive role for Hjv in 1 of 2 pathways, both of which converge on
the downstream expression of the key regulatory peptide hepci-
din. Whereas Hjv is required for hepcidin expression through the
iron-sensing pathway, Hjv is dispensable for induction of hepcidin
through the inflammatory pathway. Our findings also provide
important insights for future therapeutic strategies for treating
diseases affecting iron metabolism.
Generation, maintenance, and analysis of Hjv-mutant mice. A mouse genomic
library was screened using an Hjv-specific probe. The second coding exon
of Hjv was disrupted via homologous recombination in ES cells (129SvJae1
origin; targeting frequency: 1:100) by inserting a cassette containing an
enhanced GFP (eGFP) in frame with the endogenous amino-terminal methi-
onine (ATG), followed by an internal ribosomal entry site–nuclear localization
signal–lacZ–polyA (IRES-NLS-lacZ-pA) and a thymidine kinase–neomycin. ES
cell recombinants were screened by genomic Southern blot: EcoRI digest,
5′ probe (300 bp); oligonucleotides, (a) 5′-CTCAGTGTATTATGTG-
TAGAA-3′?and (b) 5′-AATTCCAGGAACGTTGGTGGC-3′ (Figure 1A). The
identification of Hjv-mutant mice was performed by genomic Southern
blot (Figure 1B) and PCR: (a) 5′-CCAGTGCAAGATCCTCCGCTGC-3′and
(b) 5′-TCCGGATGGTGGTAGCGTTGGC-3′. Hjv mice were maintained in
a 129SvJ genetic background under standard conditions. All experiments
were performed using male mice, and control littermates were processed in
parallel for each experiment. All animal experiments were approved by the
Veterinarian Board Basel-Stadt (Basel, Switzerland).
Northern blot analysis and histology. Northern blot analysis and isolation of
total RNA was performed as previously described (14), using digoxigenin-
labeled probes directed against Hjv (14), hepcidin (BC021587), Tfr2 (BC013654),
β2-microglobulin (BI691504), Hfe (AA255260), ceruloplasmin (AI225600), fer-
roportin (BQ928442), and GAPDH (gift from P. Matthias, Friedrich Miescher
Institute). Expression of mMLP, Marp, ANF (32), BNF (NM_008726), troponin1
(NM_009406), and MLC2a (33) were assessed by Northern blot analysis on
heart total RNA. Signals were quantified using FluoView 500 (Olympus) and
normalized to the expression level of GAPDH. Average values were determined
from at least 3 independent experiments for each data point. Cryostat sections
(16 µm) were processed for immunohistochemistry as previously described
(34), using fluorophore-conjugated secondary antibodies (1:1000; Invitrogen
Corp.). Primary antibodies used in this study were rabbit anti-lacZ (34) and
goat anti-HNF4α (Santa Cruz Biotechnology Inc.). Nuclei were detected using
SYTOX Green (Invitrogen Corp.). Vibratome sections (100 µm) were cut on
a vibratome (Leica). Detection of lacZ enzymatic activity and in situ hybridiza-
tion experiments were performed as previously described (14, 34).
Iron quantification, blood glucose measurement, and statistical analysis. Iron was
detected on cryostat sections using the Accustain Iron Stain Kit (Sigma-
Aldrich). Nonheme iron in dehydrated tissue was quantified according to
a previously described method (35). Animals were fasted for 6 hours before
blood glucose measurement (Glucocard Memory 2, ARKRAY). For statisti-
cal analyses, all P values were calculated with Microsoft Excel XP using a
2-tailed Student’s t test. P values of less than 0.05 were considered statisti-
cally significant. Error bars shown in figures represent SEM.
LPS, cytokine, and iron injection. LPS (1 µg/g body weight; serotype O111:
B4; Sigma-Aldrich), IL-6 (12.5 ng/g body weight; R&D Systems), and TNF-α
(12.5 ng/g body weight; R&D Systems) were injected intraperitoneally, and
organs were isolated for RNA preparation 6 hours after LPS and 4 hours
after IL-6 and TNF-α injections (28). Iron-dextran or PBS/dextran/phenol-
control solution was injected subcutaneously as previously described, and
animals were analyzed 7 days after injection (8).
We thank Jean-Francois Spetz, Patrick Kopp, Bernard Kuche-
mann, and Gisela Niklaus for excellent technical assistance; Nancy
Andrews for sharing unpublished observations; and Caroline Arber,
Pico Caroni, and Thomas Jessell for advice and helpful comments
on the manuscript. S. Arber, V. Niederkofler, and R. Salie were sup-
ported by a grant from the Swiss National Science Foundation, the
Novartis Research Foundation, and by the Kanton of Basel-Stadt.
Received for publication May 18, 2005, and accepted in revised
form June 14, 2005.
Address correspondence to: Silvia Arber, Biozentrum, Department of
Cell Biology, University of Basel, Klingelbergstrasse 70, 4056-Basel,
Switzerland. Phone: 41-61-267-2057; Fax: 41-61-267-2078; E-mail:
Vera Niederkofler and Rishard Salie contributed equally to this work.
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2186? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
1. Hentze, M.W., Muckenthaler, M.U., and Andrews,
N.C. 2004. Balancing acts: molecular control
of mammalian iron metabolism [review]. Cell.
2. Andrews, N.C. 2005. Molecular control of iron
metabolism. Best. Pract. Res. Clin. Haematol.
3. Nemeth, E., et al. 2004. Hepcidin regulates cellular
iron efflux by binding to ferroportin and inducing
its internalization. Science. 306:2090–2093.
4. Nemeth, E., et al. 2004. IL-6 mediates hypoferremia
of inflammation by inducing the synthesis of the
iron regulatory hormone hepcidin. J. Clin. Invest.
5. Lee, P., Peng, H., Gelbart, T., and Beutler, E. 2004.
The IL-6- and lipopolysaccharide-induced tran-
scription of hepcidin in HFE-, transferrin receptor
2-, and beta 2-microglobulin-deficient hepatocytes.
Proc. Natl. Acad. Sci. U. S. A. 101:9263–9265.
6. Krijt, J., Vokurka, M., Chang, K.T., and Necas, E.
2004. Expression of Rgmc, the murine ortholog of
hemojuvelin gene, is modulated by development
and inflammation, but not by iron status or eryth-
ropoietin. Blood. 104:4308–4310.
7. Nicolas, G., et al. 2002. The gene encoding the
iron regulatory peptide hepcidin is regulated by
anemia, hypoxia, and inflammation. J. Clin. Invest.
8. Pigeon, C., et al. 2001. A new mouse liver-specific
gene, encoding a protein homologous to human
antimicrobial peptide hepcidin, is overexpressed
during iron overload. J. Biol. Chem. 276:7811–7819.
9. Luft, F.C. 2004. Hepcidin comes to the rescue.
J. Mol. Med. 82:345–347.
10. Pietrangelo, A. 2004. Hereditary hemochromato-
sis–a new look at an old disease [review]. N. Engl. J.
11. Beutler, E., Hoffbrand, A.V., and Cook, J.D. 2003.
Iron deficiency and overload [review]. Hematology.
(Am. Soc. Hematol. Educ. Program.). 2003:40–61.
12. Brissot, P., Troadec, M.B., and Loreal, O. 2004. The
clinical relevance of new insights in iron transport
and metabolism. Curr. Hematol. Rep. 3:107–115.
13. Papanikolaou, G., et al. 2004. Mutations in HFE2
cause iron overload in chromosome 1q-linked juve-
nile hemochromatosis. Nat. Genet. 36:77–82.
14. Niederkofler, V., Salie, R., Sigrist, M., and Arber, S.
2004. Repulsive guidance molecule (RGM) gene
function is required for neural tube closure but
not retinal topography in the mouse visual system.
J. Neurosci. 24:808–818.
15. Monnier, P.P., et al. 2002. RGM is a repulsive
guidance molecule for retinal axons. Nature.
16. Samad, T.A., et al. 2004. DRAGON: a member of
the repulsive guidance molecule-related family of
neuronal- and muscle-expressed membrane pro-
teins is regulated by DRG11 and has neuronal
adhesive properties. J. Neurosci. 24:2027–2036.
17. Guidotti, J.E., et al. 2003. Liver cell polyploidiza-
tion: a pivotal role for binuclear hepatocytes. J. Biol.
18. Seglen, P.O. 1997. DNA ploidy and autophagic pro-
tein degradation as determinants of hepatocellular
growth and survival. Cell Biol. Toxicol. 13:301–315.
19. Parviz, F., et al. 2003. Hepatocyte nuclear factor
4alpha controls the development of a hepatic
epithelium and liver morphogenesis. Nat. Genet.
20. Benten, D., et al. 2005. Hepatic targeting of trans-
planted liver sinusoidal endothelial cells in intact
mice. Hepatology. doi:10.1002/hep.20746.
21. Kawabata, H., et al. 2005. Expression of hepcidin is
down-regulated in TfR2 mutant mice manifesting
a phenotype of hereditary hemochromatosis. Blood.
22. Fleming, R.E., et al. 2001. Mouse strain differences
determine severity of iron accumulation in Hfe
knockout model of hereditary hemochromatosis.
Proc. Natl. Acad. Sci. U. S. A. 98:2707–2711.
23. Zhou, X.Y., et al. 1998. HFE gene knockout produc-
es mouse model of hereditary hemochromatosis.
Proc. Natl. Acad. Sci. U. S. A. 95:2492–2497.
24. Nicolas, G., et al. 2001. Lack of hepcidin gene
expression and severe tissue iron overload in
upstream stimulatory factor 2 (USF2) knockout
mice. Proc. Natl. Acad. Sci. U. S. A. 98:8780–8785.
25. Andrews, N.C. 2004. Anemia of inflammation: the
cytokine-hepcidin link. J. Clin. Invest. 113:1251–1253.
26. Courselaud, B., et al. 2002. C/EBPalpha regulates
hepatic transcription of hepcidin, an antimicrobial
peptide and regulator of iron metabolism. Cross-
talk between C/EBP pathway and iron metabolism.
J. Biol. Chem. 277:41163–41170.
27. Zetterstrom, M., Sundgren-Andersson, A.K., Ost-
lund, P., and Bartfai, T. 1998. Delineation of the
proinflammatory cytokine cascade in fever induc-
tion. Ann. N. Y. Acad. Sci. 856:48–52.
28. Yeh, K.Y., Yeh, M., and Glass, J. 2004. Hepcidin
regulation of ferroportin 1 expression in the liver
and intestine of the rat. Am. J. Physiol. Gastrointest.
Liver Physiol. 286:G385–G394.
29. Chorney, M.J., Yoshida, Y., Meyer, P.N., Yoshida, M.,
and Gerhard, G.S. 2003. The enigmatic role of the
hemochromatosis protein (HFE) in iron absorp-
tion. Trends Mol. Med. 9:118–125.
30. Zhang, A.S., Xiong, S., Tsukamoto, H., and Enns,
C.A. 2004. Localization of iron metabolism-
related mRNAs in rat liver indicate that HFE is
expressed predominantly in hepatocytes. Blood.
31. Kawabata, H., et al. 2001. Regulation of expression of
murine transferrin receptor 2. Blood. 98:1949–1954.
32. Arber, S., et al. 1997. MLP-deficient mice exhibit a
disruption of cardiac cytoarchitectural organiza-
tion, dilated cardiomyopathy, and heart failure.
33. Gottshall, K.R., et al. 1997. Ras-dependent path-
ways induce obstructive hypertrophy in echo-
selected transgenic mice. Proc. Natl. Acad. Sci. U. S. A.
34. Arber, S., et al. 1999. Requirement for the homeo-
box gene Hb9 in the consolidation of motor neu-
ron identity. Neuron. 23:659–674.
35. Torrance, J.D., and Bothwell, T.H. 1980. Tissue
iron stores. In Methods in hematology. Volume 1. J.D.
Cook, editor. Churchill Livingstone Press. New
York, New York, USA. 90–115.