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Blood First Edition Paper, prepublished online April 14, 2011; DOI 10.1182/blood-2010-12-327957
Skeletal Muscle Hemojuvelin is Dispensable for Systemic Iron Homeostasis
(Hemojuvelin in Skeletal Muscle)
Wenjie Chen1, Franklin W. Huang2, Tomasa Barrientos de Renshaw1, Nancy C.
Andrews1, 3
1
Department of Pharmacology and Cancer Biology, Duke University School of Medicine,
Durham, NC; 2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston,
MA 3Department of Pediatrics, Duke University School of Medicine, Durham, NC
Scientific category:
Red Cells
CORRESPONDING AUTHOR:
Nancy C. Andrews, M.D., Ph.D.
DUMC 2927
Durham, NC 27710
Phone: 919 684 2455
Fax: 919 684 0208
Email: [email protected]
Copyright © 2011 American Society of Hematology
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Abstract
Hepcidin, a hormone produced mainly by the liver, has been shown to inhibit both
intestinal iron absorption and iron release from macrophages. Hemojuvelin, a GPI-linked
membrane protein, acts as a bone morphogenetic protein (BMP) co-receptor to activate
hepcidin expression through a SMAD signaling pathway in hepatocytes. In this report,
we show that loss of hemojuvelin specifically in liver leads to decreased liver hepcidin
production and increased tissue and serum iron levels in mice. Although it does not have
any known function outside of the liver, hemojuvelin is expressed at very high levels in
cardiac and skeletal muscle. To explore possible roles for hemojuvelin in skeletal muscle,
we analyzed conditional knockout mice that lack muscle hemojuvelin. The mutant
animals had no apparent phenotypic abnormalities. We found that systemic iron
homeostasis and liver hepcidin expression were not affected by loss of hemojuvelin in
skeletal muscle, regardless of dietary iron content. We conclude that, in spite of its
expression pattern, hemojuvelin is primarily important in the liver.
2
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Introduction
Hepcidin is a peptide hormone secreted by the liver, which plays a central role in iron
homeostasis. Ferroportin, the only known mammalian cellular iron exporter, is expressed
on the basolateral membrane of intestinal enterocytes and the plasma membrane of
macrophages. Hepcidin binding to ferroportin leads to internalization and degradation of
ferroportin in lysosomes, which decreases iron absorption from the diet and iron release
from macrophages that recycle iron from senescent erythrocytes. High levels of hepcidin
result in iron deficiency and low levels of hepcidin result in iron overload. This
homeostatic mechanism has been confirmed by studies in humans and mice: loss-offunction mutations in the hepcidin gene (HAMP) cause juvenile hemochromatosis with
iron overload;1 inactivation of the hepcidin gene in mice leads to iron overload;2
overexpression of hepcidin in patients with hepatic adenomas causes iron refractory
anemia;3 and transgenic mice that overexpress hepcidin develop severe iron deficiency
anemia before birth.4
As a central regulator of body iron metabolism, hepcidin is modulated by the same
factors that alter iron homeostasis, including changes in body iron stores, the rate of
erythropoiesis, inflammation, anemia and hypoxia. Hepcidin levels are increased in
response to oral and parenteral iron loading and decreased under iron deficient
conditions.5 Inflammation stimulates hepcidin expression by increasing interleukin-6
levels. The interleukin-6 ligand-receptor interaction results in activation of the JAK/STAT
signaling cascade and binding of the transcriptional activator STAT3 to the hepcidin
promoter.6,7 On the other hand, anemia and hypoxia are associated with decreased
hepcidin levels.8 The von Hippel-Lindau/hypoxia-inducible transcription factor (VHL/HIF)
signaling pathway may link hypoxia and hepcidin regulation in vivo. HIF proteins has
been reported to bind to the hepcidin promoter and reduce hepcidin expression in the
3
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liver in response to hypoxia or iron-deficiency,9,10 but a more recent study disputed these
findings.11
Studies of patients with autosomal recessive hemochromatosis, an iron overload
disorder, have revealed that several hepatocyte proteins are required for appropriate
hepcidin regulation, including HFE,12 TFR2,13 and HJV (HFE2)14. Mutations in either the
hepcidin gene (HAMP) or HJV cause a more severe form of hemochromatosis that
presents in the first or second decade of life. In juvenile hemochromatosis patients who
carry HJV mutations, urinary hepcidin is undetectable, suggesting that HJV is a key
regulator of hepcidin.14 To date, about 30 disease-linked mutations have been identified
in the HJV gene, including missense and nonsense mutations. These mutations are
predicted to lead to loss of function of HJV, thus reducing hepcidin expression in
hepatocytes.15 Disruption of Hjv in the mouse results in decreased hepcidin expression
and increased iron deposition in liver, pancreas, and heart but decreased iron levels in
tissue macrophages.16,17
HJV encodes hemojuvelin, a member of the repulsive guidance molecule family, which
resides on the cell membrane as a glycosylphosphatidyl inositol-linked protein. It
functions as a BMP co-receptor to activate hepcidin expression through a BMP/SMAD
signaling pathway.18 Recently, BMP6 was shown to be the key ligand binding to
hemojuvelin in a complex with at least one of the type I BMP receptors.19,20 This ligandreceptor complex initiates a signaling cascade leading to phosphorylation of Smad
proteins that associate with Smad4, resulting in nuclear translocation and activation of
hepcidin transcription. Accordingly, disease-associated mutations in HJV result in
decreased BMP/SMAD signaling and decreased hepcidin expression.18 These findings
are supported by the discovery that loss of Smad4 in hepatocytes leads to a failure of
hepcidin expression and massive iron overload in the mouse.21
4
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In addition to residing on the cell membrane, hemojuvelin can be cleaved and secreted
out of cells in a soluble form. Soluble hemojuvelin can selectively bind to BMP ligands
and inhibit endogenous and BMP-induced hepcidin expression.22,23 Administration of
soluble hemojuvelin decreases hepcidin expression in vivo, leading to increased
ferroportin expression and increased serum iron levels in mice.22 Soluble hemojuvelin is
produced by cleavage by furin, a proprotein convertase, at position 332-335 (RNRR).24,25
Furin is induced by iron deficiency and hypoxia in association with stabilization of HIF-1α,
suggesting that soluble hemojuvelin production may play an important role in iron
deficiency and hypoxia-mediated hepcidin regulation.25 Subsequently, the serine
protease TMPRSS6 was shown to be mutated in patients with iron-refractory iron
deficiency anemia (IRIDA).26 TMPRSS6 cleaves hemojuvelin on the cell membrane and
inhibits hepcidin expression.27 In contrast to the soluble hemojuvelin produced by furin
cleavage, the products of TMPRSS6 cleavage do not inhibit BMP-induced hepcidin
expression.28
Previous studies showed that HJV is expressed in heart and muscle at levels exceeding
its expression in the liver.14 However, hepcidin is absent in skeletal muscle and
expressed at very low levels in the heart.5 These observations suggest that HJV might
have a distinct role in non-hepatic tissues. We hypothesized that soluble hemojuvelin is
produced by skeletal muscle and circulates to the liver to regulate hepcidin expression.
To test our hypothesis, we investigated hepcidin expression and iron homeostasis in
mice lacking hemojuvelin in skeletal muscle.
5
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Methods
Targeting of the murine Hjv locus
We isolated Hjv genomic clones from a strain 129/SvJ mouse library (Stratagene). To
construct a targeting vector with exon 4 of Hjv flanked by LoxP sites (Figure 1A), we first
retrieved a 10 kb fragment containing all 4 exons of Hjv. We then targeted a LoxP-NeoLoxP to intron 3 followed by removal of Neo cassette using Cre recombinase, leaving a
single LoxP site in intron 3. A second FRT-Neo-FRT-LoxP cassette was targeted
downstream of the 3’ end of exon 4. Linearized vector was electroporated into 129 J1
embryonic stem (ES) cells. Correct homologous recombination was confirmed by
Southern blot analysis of flanking sequences from both ends of the targeted region. ES
cell clones with normal karyotypes were injected into C57BL/6 blastocysts. Transmission
of the targeted alleles was confirmed by Southern blot analysis. The animals were
maintained on an inbred 129S6/SvEvTac background. Subsequent genotyping was
carried out by extracting DNA from snipped toe/tail samples and subjecting it to
Southern blot and/or PCR analysis. Mouse lines carrying Cre recombinase transgenes
under the control of tissue-specific promoters were obtained from The Jackson
Laboratory (Bar Harbor, ME). Conditional knockout mice were obtained by intercrossing
male mice with genotype of Hjv fl/+;Cre/+ female mice with genotype of Hjv fl/fl. Hjv
fl/fl;Cre/+ mice were compared to littermate control Hjv fl/fl mice.
Animal care and analysis
All mice were born and housed in the barrier facility at Duke University according to
protocols approved by the Duke University Institutional Animal Care & Use Committee.
Animals were maintained on a standard diet containing 380 parts per million iron (Prolab
Isopro RMH 3000, 5P76). We analyzed mice on three diets: standard diet, iron-deficient
diet and iron-rich diet. For non-standard diet experiments, recently weaned, 3 week old
6
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animals were maintained on a low iron diet (2-5 parts per million iron, Harlan Teklad TD
99397, Harlan Laboratories, Indianapolis, IN) with iron-free water or a high iron diet (380
parts per million iron plus 2% carbonyl iron, Harlan Teklad TD 10066, Harlan
Laboratories, Indianapolis, IN) with regular water for 5 weeks. Food and water were
provided ad libitum. All mice were analyzed at 8 weeks of age.
Iron analysis in blood and tissues
Under Avertin anesthesia (Sigma, St. Louis, MO), whole blood was collected from the
retro-orbital sinus through capillary tubes (Fisher Scientific, Pittsburgh, PA) into serum
separator tubes (Becton, Dickinson, Franklin Lakes, NJ). Serum was prepared and
serum iron levels were determined using a serum iron/UIBC kit (Thermo DMA, Arlington,
TX) according to the manufacturers’ instructions. Non-heme iron concentrations of liver,
spleen, heart, and skeletal muscle (quadriceps femoris) were determined as previously
described.29
RNA extraction, RT-PCR, and quantitative RT-PCR
Total liver RNA was isolated from flash-frozen tissue using the RNeasy mini kit (Qiagen,
Valencia, CA). Total skeletal and cardiac muscle RNA were isolated using the RNeasy
Fibrous Tissue Mini Kit (Qiagen, Valencia, CA). Total RNA was reverse transcribed
using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) according to the
manufacturer’s protocol. Expression of wild-type Hjv transcript was sought by
quantitative PCR using forward primer 5’-TTGGCCCTTGCTAGATAACG-3’, located
within exon 3 and reverse primer 5’-TCAATGCATTCCTGCATGTT-3’, located within
exon 4. Expression of a partial Hjv transcript was sought by quantitative PCR using
forward primer 5’-TCTGACCTGAGTGAGACTGC-3’, located within exon 1, and reverse
primer 5’-GATGATGAGCCTCCTACCTA-3’, located within exon 2. Quantitative PCR to
7
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assess Hamp1, Id1, and Bmp6 transcript abundance was performed as previously
described.20
Statistical analysis
Unpaired, 2-tailed Student’s t-test was performed with Microsoft Excel (PC 2007 version)
software (Redmond, WA). P values less than .05 were considered statistically significant.
8
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Results
Generation of conditional hemojuvelin knockout mice
The mouse genome was modified to generate a Cre recombinase-sensitive conditional
knockout allele of hemojuvelin (Hjv fl) containing one LoxP site in intron 3, a neomycin
(neo) sequence flanked by FRT sites and another LoxP site in untranscribed sequence
downstream of exon 4 (Figure 1A). In the absence of Cre recombinase activity, the Hjv fl
gene retains an intact promoter sequence and encodes full-length wild-type murine
hemojuvelin. The Neo sequence in the Hjv fl allele did not interfere with expression or
function of Hjv; Hjv fl/fl mice had similar iron levels in the liver and spleen when
compared to wild type mice (not shown). Cre-mediated recombination was expected to
lead to a conditional knockout allele (Hjv cko) lacking exon 4, which encodes 208 amino
acids and contains the GPI anchor signal sequence, furin and matriptase-2 cleavage
sites.
We established germline-competent 129S6/SvEvTac mice carrying the Hjv fl allele. Male
mice heterozygous for the Hjv fl allele (Hjv fl/+ mice) were intercrossed to C57BL/6
hemizygous HSA-Cre/+ transgenic female mice (B6.Cg-Tg(ACTA1-cre)79Jme/J strain,
The Jackson Laboratory, ME) that express Cre recombinase restricted to skeletal
muscle to obtain male Hjv fl/+;HSA-Cre/+ mice. Then we intercrossed male Hjv fl/+;HSACre/+ mice to female Hjv fl/fl mice to obtain Hjv fl/fl;HSA-Cre/+ mice (hereafter referred
to as Hjv sk/sk mice). Their littermates with the Hjv fl/fl genotype, but no Cre transgene,
were used as controls in the analyses. All offspring have a mixed
129S6/SvEvTac;C57BL/6 genetic background.
Heterozygous Hjv sk/+ and homozygous Hjv sk/sk mice had no apparent abnormalities.
The numbers of offspring of both genotypes followed a Mendelian inheritance pattern,
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suggesting normal viability during the gestational and neonatal periods. Wild-type Hjv
mRNA was analyzed using RT-PCR primers from exon 3 and 4. Quantitative PCR
results showed that wild-type Hjv mRNA expression was undetectable in skeletal muscle
from Hjv sk/sk mice, but was normal in liver and heart compared to Hjv fl/fl mice (Figure
1B). The predicted, truncated Hjv transcript including the first three exons was assayed
using RT-PCR primers from exon 1 and 2. Quantitative PCR analysis demonstrated the
presence of the truncated Hjv transcript in skeletal muscle of Hjv sk/sk mice was
expressed at approximately 30% of wild type levels. Due to the lack of a reliable
antibody against murine hemojuvelin, we could not test whether a truncated hemojuvelin
protein was produced.
Iron overload phenotype in hemojuvelin liver-specific knockout mice
To confirm that deletion of Hjv exon 4 leads to loss of hemojuvelin function, we
evaluated iron homeostasis in Hjv liver-specific conditional knockout mice by
intercrossing male Hjv fl/+;Alb-Cre/+ transgenic mice to female Hjv fl/fl mice. The AlbCre transgene (B6.Cg-Tg(Alb-cre)21Mgn/J strain, Jackson Laboratory, ME) has been
bred onto a 129S6/SvEvTac background, allowing us to carry out this experiment on a
pure genetic background. Although the truncated Hjv mRNA was expressed at
approximately 50% of wild type levels (data not shown), Hjv full length mRNA expression
was not detected in the liver in Hjv fl/fl;Alb-Cre/+ mice (hereafter referred to as Hjv l/l
mice, Figure 2A). Hjv l/l mice exhibited higher non-heme iron levels in liver, heart and
muscle, but lower non-heme iron levels in spleen (Figure 2B), compared to littermate Hjv
fl/fl mice. Hjv l/l mice also had increased serum iron and transferrin saturation (Figure
2C,D). Hepatic hepcidin and Id1 mRNA expression was decreased in Hjv l/l mice, while
10
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Bmp6 mRNA expression was increased (Figure 2E). While we cannot rule out the
possibility that a truncated protein is produced from the deleted allele, the fact that all
these features recapitulate the phenotype found in Hjv null mice17 strongly suggests that
deletion of Hjv exon 4 leads to loss of function of Hjv.
Iron homeostasis in hemojuvelin skeletal muscle-specific knockout mice
Soluble hemojuvelin has been shown to decrease hepcidin expression both in vitro and
in vivo.22,23 Considering the high expression levels of Hjv mRNA and the lack of hepcidin
in skeletal muscle, we hypothesized that Hjv in skeletal muscle serves as a source of
soluble hemojuvelin, which circulates to liver to regulate hepatic hepcidin expression and
systemic iron homeostasis. We examined parameters of iron homeostasis in
hemojuvelin skeletal muscle-specific knockout mice at 8 weeks of age. Hjv sk/sk mice
did not show significant differences in serum iron (Figure 3A), transferrin saturation
(Figure 3B) or non-heme iron concentration in liver, spleen and skeletal muscle (Figure
3C), compared to Hjv fl/fl mice. Hepatic Hamp1 and Id1 mRNA expression in Hjv sk/sk
mice was similar to Hjv fl/fl mice (Figure 3D,E). Younger Hjv sk/sk mice (aged 4 to 6
weeks), also had tissue iron levels similar to control Hjv fl/fl mice (Figure S1).
We next asked whether Hjv fl/fl and sk/sk mice would respond differently to an irondeficient diet. We weaned 3-week-old pups with Hjv fl/fl and sk/sk genotypes from Hjv
fl/+;ACTA1-Cre/+ and Hjv fl/fl intercross, and fed them an iron-deficient diet for 5 weeks
prior to serum and tissue analysis. Both Hjv fl/fl and sk/sk mice on the iron-deficient diet
showed significant decreases in serum iron (Figure 4A), transferrin saturation, (Figure
4B) and non-heme iron in liver, spleen and skeletal muscle (Figure 4C) compared to
mice on a standard diet (Figure 3). Hepatic Hamp1 mRNA expression was much lower
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in all mice on the iron-deficient diet (Figure 4D). However, hepatic Id1 mRNA expression
did not show a significant change from that observed in mice on a standard diet in either
cohort (Figure 4E). In summary, no differences in parameters of iron homeostasis were
observed comparing Hjv fl/fl and sk/sk mice on an iron-deficient diet.
We also investigated whether an iron-rich diet differentially alters systemic iron
homeostasis in Hjv fl/fl and sk/sk mice. We fed newly weaned, 3 week old Hjv fl/fl and
sk/sk mice an iron-rich diet for 5 weeks. We observed a significant increase in serum
iron (Figure 5A), transferrin saturation (Figure 5B), and non-heme iron concentrations in
liver, spleen (Figure 5C) and skeletal muscle (Figure 5D) compared to mice on a
standard diet (Figure 3). Hepatic Hamp1 and Id1 mRNA expression was also increased
on an iron-rich diet compared to a standard diet (Figure 5E,F). However, there was no
difference between Hjv fl/fl and sk/sk mice in any tested parameters of iron homeostasis.
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Discussion
Hemojuvelin functions as a BMP co-receptor to activate hepcidin expression through a
BMP/SMAD signaling pathway.18 We have demonstrated that loss of hemojuvelin
specifically in the liver leads to an iron overload phenotype similar to that observed in Hjv
null mice. Hepatic hepcidin and Id1 mRNA expression was decreased in these mice,
while Bmp6 mRNA expression was increased, consistent with iron overload. These data
confirm both that hepatic hemojuvelin is critical in maintaining liver hepcidin expression
and systemic iron homeostasis and that the conditional knockout allele does not encode
a functional hemojuvelin protein.
Hemojuvelin is thought to also be cleaved and secreted out of cells in a soluble form.
Soluble hemojuvelin can selectively bind to BMP ligands and inhibit endogenous, BMPinduced hepcidin expression in vitro.22,23 Considering that hemojuvelin and furin are
highly expressed in skeletal muscle, we hypothesized that soluble hemojuvelin produced
by skeletal muscle might regulate hepatic hepcidin expression.
We tested our hypothesis by generating a strain of mice in which the hemojuvelin gene
was specifically inactivated in skeletal muscle. These Hjv conditional knockout mice did
not show any differences in iron homeostasis when compared to control mice and had a
similar response to both iron-deficient and iron-rich diets. These data indicate that
hemojuvelin in skeletal muscle is dispensable for systemic iron homeostasis under the
conditions tested.
There are several limitations to our work and the conclusions we can draw. All mice
used in our experiments have a mixed 129S6/SvEvTac;C57BL/6 genetic background,
and we cannot rule out the possibility that resulting increased variability masks a very
subtle difference between Hjv sk/sk and fl/fl control mice. Although hemojuvelin mRNA is
13
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at high expression levels in skeletal muscle, we cannot confirm that hemojuvelin protein
is also expressed at high levels, due to the lack of a reliable antibody against murine
hemojuvelin. Additionally, production of soluble hemojuvelin has not been definitively
proven in vivo in general, and in muscle in particular. It may be that as yet unidentified
stress conditions are necessary to induce cleavage of soluble hemojuvelin in skeletal
muscle and thus alter systemic iron homeostasis. Finally, hemojuvelin is also expressed
at high levels in cardiac muscle, and cardiac hemojuvelin may compensate for the loss
of hemojuvelin in skeletal muscle. By generating a strain of mice with inactivated
hemojuvelin in both cardiac and skeletal muscle, we can test whether loss of
hemojuvelin in both cardiac and skeletal muscle affects systemic iron homeostasis.
The function of hemojuvelin in skeletal muscle remains unknown. Loss of hemojuvelin in
skeletal muscle did not lead to abnormalities in muscle histology (not shown) and
patients with juvenile hemochromatosis have not been reported to have muscle
abnormalities. Non-heme iron in skeletal muscle from Hjv sk/sk mice was similar to
control mice. Id1 mRNA expression was also similar, suggesting that hemojuvelin does
not regulate an analogous BMP/SMAD signaling pathway in skeletal muscle.
In conclusion, we generated mice lacking hemojuvelin in the liver and replicated iron
overload phenotypes found in hemojuvelin-null mice. We also demonstrated that loss of
hemojuvelin in skeletal muscle did not appreciably affect systemic iron homeostasis
regardless of dietary iron content. At this time, the reason for high hemojuvelin
expression in muscle remains obscure.
14
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Acknowledgements
This work was supported by a Cooley’s Anemia Foundation Research Fellowship (W.C.),
a grant from the Roche Foundation for Anemia Research (N.C.A.) and R01 DK066373
(N.C.A.). We thank Paul Schmidt for assistance with technical aspects of this work and
members of the Andrews laboratory for helpful discussions.
Authorship
Contribution: W.C. designed and performed experiments, analyzed results, and wrote
the manuscript. F.W.H. designed experiments and made the Hjv conditional knockout
vector. T.B.R. performed part of Hjv liver-specific knockout mice experiments. N.C.A.
guided experiments and contributed to writing the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
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hereditary hemochromatosis does not produce a null allele.
Blood.
1999;94(1):9-11.
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FIGURE LEGENDS
Figure 1. Targeted disruption of the Hjv gene. (A) Schematic representation of the
mouse wild-type Hjv genomic sequence (+), LoxP-flanked allele with FRT-flanked neo
sequence (fl) and conditional knock out (cko) alleles. Positions of sequences coding for
features of the protein are indicated: * Tmprss6 cleavage site 28; # Furin cleavage site
24,25
. (B) Hjv mRNA relative expression in liver (L), heart (H) and skeletal muscle (SK) by
quantitative RT-PCR. fl/fl, mice with homozygous fl allele, n=4; sk/sk, mice with
homozygous cko allele in skeletal muscle, n=4. qPCR primers of Hjv: qF, forward primer
in exon 3; qR reverse primer in exon 4. Rpl19 was used as an internal control. Hjv
expression level in liver from fl/fl mice was assigned as 1. Error bars represent standard
deviation (SD).
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Figure 2. Hjv liver-specific knockout mice exhibit iron overload phenotypes. (A)
Relative Hjv mRNA expression in liver determined by quantitative RT-PCR. fl/fl, mice
with homozygous fl allele; l/l, homozygous fl mice expressing the Cre transgene in liver.
Hjv expression level in fl/fl mice liver was assigned a value of 1. (B) Serum iron
concentration. (C) Serum transferrin saturation. (D) Non-heme tissue iron concentrations
(μg/g wet weight) of fl/fl and Hjv liver-specific knockout (l/l) mice. L, liver; S, spleen; H,
heart; SK, skeletal muscle. (E) Hepatic Hamp1, Id1 and Bmp6 relative to Rpl19 mRNA
expression by quantitative RT-PCR. Results are reported as the fold change from fl/fl
mice. (A-E) Mean values from analysis of 8-week-old mice (fl/fl mice: 3 male and 2
female, l/l mice: 4 male and 2 female) are graphed. Error bars represent SD. ‡, # and §
indicate P < .05, P < .005 and P < .0005, respectively, when compared to fl/fl mice.
19
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Figure 3. Iron homeostasis is not altered in Hjv skeletal muscle-specific knockout
mice. (A) Serum iron concentration. (B) Serum transferrin saturation. (C) Non-heme
tissue iron concentrations (μg/g wet weight) of Hjv fl/fl and skeletal muscle-specific
knockout (sk/sk) male mice. L, liver; S, spleen; SK, skeletal muscle. (D) Hepatic Hamp1
relative to Rpl19 mRNA expression. (E) Hepatic Id1 relative to Rpl19 mRNA expression.
Mean values from analysis of 8-week-old male mice (N=8 per genotype) are graphed.
Error bars represent SD. No statistically significant differences were observed.
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Figure 4. Iron homeostasis in Hjv skeletal muscle-specific knockout mice on irondeficient diet. (A) Serum iron concentration. (B) Serum transferrin saturation. (C) Nonheme tissue iron concentrations (μg/g wet weight) of Hjv fl/fl and skeletal muscle-specific
knockout (sk/sk) female mice. L, liver; S, spleen; SK, skeletal muscle. (D) Hepatic
Hamp1 relative to Rpl19 mRNA expression. Note that all values were very low compared
to mice on a standard diet. (E) Hepatic Id1 relative to Rpl19 mRNA expression. Mean
values from analysis of 8-week-old female mice (N=8 per genotype) are graphed. Error
bars represent SD. No statistically significant differences were observed.
21
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Figure 5. Iron homeostasis in Hjv skeletal muscle-specific knockout mice on ironrich diet. (A) Serum iron concentration. (B) Serum transferrin saturation. (C) Non-heme
tissue iron concentrations (μg/g wet weight) of Hjv fl/fl and skeletal muscle-specific
knockout (sk/sk) male mice. L, liver; S, spleen. (D) Non-heme iron concentrations in
skeletal muscle. (E) Hepatic Hamp1 relative to Rpl19 mRNA expression. (F) Hepatic Id1
relative to Rpl19 mRNA expression. Mean values from analysis of 8-week-old male mice
(N=4 for fl/fl and N=6 for sk/sk) are graphed. Error bars represent SD. No statistically
significant differences were observed.
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Prepublished online April 14, 2011;
doi:10.1182/blood-2010-12-327957
Skeletal muscle hemojuvelin is dispensable for systemic iron homeostasis
Wenjie Chen, Franklin W. Huang, Tomasa Barrientos de Renshaw and Nancy C. Andrews
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