altered body iron distribution and microcytosis in mice ......blood first edition paper,...
TRANSCRIPT
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Title
Altered body iron distribution and microcytosis in mice deficient for
Iron Regulatory Protein 2 (IRP2).
Running title: IRP2 in systemic iron metabolism.
Bruno Galy (1), Dunja Ferring (1), Belen Minana (1) +, Oliver Bell (1)†, Heinz G. Janser (2),
Martina Muckenthaler (1)‡, Klaus Schümann (3), and Matthias W. Hentze (1) *.
(1) European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
(2) Institut für Pharmakologie und Toxikologie der Ludwig-Maximilians-Universität, München,
Germany. (3) Lehrstuhl für Ernährungphysiologie, Technical University, München, Germany.
present address: + Centre de Regulació Genòmica, Barcelona, Spain, † Friedrich Miescher
Institute, Basel, Switzerland, ‡ University of Heidelberg, Im Neuenheimer Feld 153, Heidelberg,
Germany.
B.G was the recipient of an EMBO long-term fellowship (ALF199-212). This work was made
possible by funds from the Gottfried Wilhelm Leibniz Prize to M.W.H.
*Corresponding author: Matthias W. Hentze, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg,
Germany. Tel: +496221387501, Fax: +496221387518, e-mail: [email protected]
Word count: Abstract: 191 Text: 5091
Scientific Heading: RED CELLS
Blood First Edition Paper, prepublished online June 14, 2005; DOI 10.1182/blood-2005-04-1365
Copyright © 2005 American Society of Hematology
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Abstract
Iron Regulatory Protein (IRP)-2 deficient mice have been reported to suffer from late
onset neurodegeneration by an unknown mechanism. We report that young adult IRP2-/- mice
display signs of iron mismanagement within the central iron recycling pathway in the
mammalian body, the liver-bone marrow-spleen axis, with altered body iron distribution and
compromised hematopoiesis. In comparison to wild-type littermates, IRP2-/- mice are mildly
microcytic with reduced serum hemoglobin levels and hematocrit. Serum iron and transferrin
saturation are unchanged, and hence microcytosis is not due to an overt decrease in systemic iron
availability. The liver and duodenum are iron loaded, while the spleen is iron-deficient
associated with a reduced expression of the iron exporter ferroportin. A reduction in transferrin
receptor (TfR)1 mRNA levels in the bone marrow of IRP2-/- mice can plausibly explain the
microcytosis by an intrinsic defect in erythropoiesis due to a failure to adequately protect TfR1
mRNA against degradation. This study links a classical regulator of cellular iron metabolism to
systemic iron homeostasis and erythropoietic TfR1 expression. Furthermore, this work uncovers
aspects of mammalian iron metabolism that can or cannot be compensated by the expression of
IRP1.
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Introduction
As both lack and excess of iron are pathological, vertebrates control iron balance at the
cellular and the systemic level by coordinating iron uptake, storage, export, and distribution (for
a recent review, see Hentze et al.1). For systemic iron uptake, dietary iron is transported from the
intestinal lumen into the cytoplasm of duodenal enterocytes via DMT1/Slc11A22. The
ubiquitously expressed transferrin (Tf) receptor 1 (TfR1) is thought to supply body cells with
iron by internalisation of serum Tf. Some specialized macrophages acquire iron indirectly by
breaking down heme following phagocytosis of senescent erythrocytes. Iron that enters cells and
is not used can be sequestered within heteropolymers of ferritin H- and L-chain. The
mechanisms by which cells export iron are less well understood. Ferroportin/Slc40A1, located at
the basolateral membrane of duodenal enterocytes, exports iron into the bloodstream and loads it
onto plasma apo-Tf3, probably in conjunction with the feroxidase hephaestin. Ferroportin is also
expressed by e.g. macrophages and hepatocytes3-5 where it acts in concert with ceruloplasmin3,6.
Systemic iron homeostasis requires communication between cells that need iron (mainly
erythroid precursors) and cells that acquire (duodenal enterocytes), store (hepatocytes and tissue
macrophages) or recycle (tissue macrophages) iron. As no pathway for regulated iron excretion
is known, control of intestinal iron absorption is critical for maintenance of adequate body iron
levels. Duodenal iron absorption responds to changes in dietary iron intake, the status of the iron
stores, and erythropoietic activity7. Hepcidin (Hamp), a soluble β-defensin-like polypeptide
excreted mostly by the liver decreases duodenal iron absorption and iron release from
macrophages by inhibition of ferroportin8. Hamp mRNA levels decrease in response to iron
deficiency and anemia, while dietary iron overload stimulates Hamp expression9.
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Cellular iron metabolism is coordinately controlled by Iron Regulatory Proteins (IRPs) 1 and 2
that bind to Iron-Responsive Elements (IRE), cis-regulatory RNA motifs present in untranslated
regions (UTR) of mRNAs encoding proteins of iron uptake (TfR1, DMT1), storage (ferritin H-
and L-chain), export (ferroportin), or utilization (mitochondrial aconitase, 5-aminolevulinate
synthase). Independently, both IRPs inhibit translation when bound to IREs present in the 5'UTR
(e.g. ferritin, 5-aminolevulinate synthase, mitochondrial aconitase, ferroportin mRNAs), whereas
their association with the IREs present in the 3'UTR of the TfR1 mRNA prevents its
degradation1. The IRE-binding activity from both IRPs is high in iron-deficient cells and low
under conditions of iron load. Failure to coordinate the expression of IRE-containing genes is
associated with pathological conditions as illustrated by the autosomal dominant
hyperferritinemia-cataract syndrome observed in patients carrying mutations in the ferritin L
IRE10,11, by the autosomal dominant iron overload in patients with a mutation in the ferritin H
IRE12, or by a progressive neurodegenerative disorder observed in aged mice lacking IRP213.
The role of the IRP/IRE regulatory network in cellular homeostasis has been extensively
investigated in cell culture1,14-16. The respective roles of IRP1 and IRP2 in mammalian
physiology are only beginning to be investigated13,17-19. To address the role of the IRP/IRE
regulatory network in systemic iron metabolism, we developed mouse lines with targeted
disruptions of the irp1 or irp2 loci18. A detailed analysis of the phenotype of mice lacking IRP2
uncovers a novel role of IRP2 in systemic iron metabolism. We address the mechanisms
underlying the misregulation of systemic iron homeostasis in IRP2-/- mice.
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Methods
Mice.
The generation of the IRP2-deficient mice has been described18. A βGeo gene-trap construct was
inserted into the second intron of the irp2 locus to interrupt the open reading frame near the
amino-terminus, thereby creating a functional null allele. The selection cassette was co-inserted
with LoxP sites flanking exon 3 for excision by the Cre recombinase. Heterozygotes on a mixed
Sv129/Ola / C57BL6/J genetic background were intercrossed to obtain +/+, +/- and -/-
littermates. Animals were kept under a constant light/dark cycle. The iron content of the standard
diet was 200 mg/kg. Mice were made iron-deficient by feeding a low iron diet (< 10 mg/kg) vs. a
control diet (C1038 and C1000, respectively, Altromin, Lage, Germany) for 25 days starting
from weaning age. 10-week old females were injected intraperitoneally with phenylhydrazine
(60 mg/kg of body weight) or NaCl 0.9% on two consecutive days and sacrificed 3 days after the
last injection. Heparinized blood was collected by cardiac puncture. Longitudinal sections of the
proximal duodenum were collected first. Bone marrow cells were flushed out of the femur with
ice-cold PBS and pelleted by centrifugation for RNA and protein extraction. Animal handling
was in accordance with institutional guidelines.
Hematology and iron determination.
Serum iron and unsaturated iron binding capacity (UIBC), respectively, were determined using
the Total iron assay and UIBC assay reagents (Diagnostic Chemicals Limited, Charlottetown,
Canada) together with the calibrators and standards recommended by the manufacturer. Blood
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profiles and hemoglobin content were determined by Laboklin (Laboklin GmbH, Bad-Kissingen,
Germany).
Total non-heme iron was measured using the bathophenantroline chromogen (Sigma,
Taufkirchen, Germany) as described by Torrance and Bothwell20 with the modifications
described by Patel et al.21.
Determination of duodenal iron transport.
Duodenal iron transfer was determined as described previously22. 12-week old mice were fasted
overnight. A tied-off duodenal segment (2-3 cm) was flushed with saline (37°C), filled in situ
with 50-100 µl of physiological medium (125 mM NaCl, 3.5 mM KCl, 10 mM D-glucose, 16
mM Na-HEPES, pH 7.4), containing 100 µM 59Fe3+:nitrilotriacetate 1:2 (NEN, NEZ37,
Dreieich, Germany). The ligated duodenal segment was removed after 15 min, ligating the
mesenteric blood supply to avoid blood losses. The radioactivity associated with the carcass was
measured in a whole body counter (ARMAC 446, Packard, Palo Alto, CA, USA)22.
RNA analyses.
Total RNA was extracted using the Trizol reagent (Invitrogen, Karlsruhe, Germany) and a
polytron homogeniser (Kinematika, Lucerne, Switzerland). Northern-blotting was done
following standard procedures, using a β-actin probe to assess equal loading. Signals were
quantified using a fluorimager (Fujifilm FLA-2000, Amersham Biosciences, Freiburg,
Germany). Microarray experiments were carried out as described previously23,24 using the Mouse
Version 3.0 of the "IronChip".
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Templates for RNase protection assays were generated by PCR from murine cDNAs using the
primers 5'-GATGAATGACTTCCTGAATGTC-3' (sense) and 5'-
TAGTCATCTGGACACCACTG-3' (reverse) (DMT1 3' variants), 5'-
AACACTGTTGTCAGAGAAGTTG-3' (sense) and 5'-ATTCACAGAATAACTTAGTTCTTC-
3' (reverse) (Tfr1), 5'-AGATCTGTGAAGAATAGAGAGCCTAG-3' (sense) and 5'-
GCTGCAGGGGTGTAGAGAGGTC-3' (reverse) (Hepcidin 1 and 2). The PCR products were
cloned into the pCRII Topo vector (Invitrogen). The templates were linearized with BamHI
(DMT1), XhoI (TfRI), or BglII (hepcidin) and antisense probes were generated by in vitro
transcription using the T7 (DMT1, hepcidin) or the SP6 (TfRI) RNA polymerases (Stratagene,
Amsterdam, The Netherlands). 5 µg of total RNA were used for RNase protection assay using
the RPA-III kit (Ambion, Huntingdon, Cambridgeshire, United Kingdom). RNA samples were
co-hybridised with a β-actin probe from the kit. Protected products were resolved on denaturing
acrylamide gels and subjected to autoradiography using a fluorimager (Fujifilm FLA-2000,
Amersham Biosciences).
Protein analyses.
Protein extracts were prepared and subjected to western-blot analysis as described previously18
except that for the detection of ferroportin the samples were not heated prior to loading. Ferritin
H and L subunits were detected using the goat polyclonal antibodies sc-14416 and sc-14420,
respectively (Santa Cruz Biotechnology, Heidelberg, Germany). β-actin (clone AC-15) and TfR1
were detected using mouse monoclonal antibodies, respectively, from Sigma and Zymed (Berlin,
Germany). Ferroportin and IRP2 were detected using immuno-purified rabbit polyclonal
antibodies raised, respectively, against a peptide corresponding to the C-terminus of ferroportin
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(GPDEKEVTDENQPNTS) or against a tandem of the 73 a.a. domain of IRP225 fused to GST.
The specificity of the ferroportin antibody was established using protein extracts from Hela cells
transfected with a mouse ferroportin cDNA as described previously3 (not shown).
Tissues were washed in PBS and incubated in fresh 4% paraformaldehyde solution overnight at
4°C. After extensive washes in PBS, specimens were infused with a PBS/15% sucrose followed
by PBS/30% sucrose. Finally, tissues were washed in PBS and embedded in tissue-Teck OCT
compound (Pelco, Redding, CA, USA). 10 µm tissue sections were prepared on a cryostat
(model CM3050, Leica, Wetzlar, Germany) and attached to SuperFrost®Plus glass slides
(Menzel-Glaser, Braunschweig, Germany). Immunostaining of ferritin H- and L-chain was
performed using the goat polyclonal antibodies sc-14416 and sc-14420 (Santa-Cruz
Biotechnology) and a Vectastain kit (Vector Laboratories, Burlingame, CA, USA). For
immunofluorescent staining, tissue slices were treated with trypsin and incubated in PBS / 0.1%
triton X100 for 20 min, washed in PBS and placed in blocking solution (PBS, BSA 0.2%, normal
goat serum 1/500 (Santa Cruz Biotechnology). Samples were incubated with the anti-ferroportin
antibody described above and a rat monoclonal anti-F4/80 antibody (Serotec, Düsseldorf,
Germany) to detect splenic macrophages. Immune complexes were detected with goat anti-rabbit
(ferroportin) or anti-rat (F4/80) antibodies conjugated to Alexa 488 or Alexa 594 (Molecular
Probes, Invitrogen). Nuclei were stained with Dapi (Molecular Probes, Invitrogen) and samples
were mounted in Fluoromount-G (Southern Biotech., Birmingham, AL, USA).
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Results
Abnormal systemic iron distribution and microcytosis in IRP2-deficient mice.
At 8-10 weeks of age, IRP2-deficient mice present without gross phenotypic abnormalities. Both
males and females are fertile, apparently healthy and display a normal overall posture and
activity pattern. The mean weight is 28.6 ±3.3 g (wild type) vs. 27.8 ±4.7 g (IRP2-/-) for males
and 25.1 ±3.4 g (wild type) vs. 24.3 ±5.2 g (IRP2-/-) for females.
To evaluate the consequences of IRP2 deficiency on systemic iron metabolism, tissue iron was
measured in the liver, a site of iron storage, in the duodenum, the site of iron absorption, and in
the spleen, a major site of iron recycling, as well as in the brain (table 1). Non-heme iron content
is increased in the liver (by ~ 50%) and duodenum (by ~ 80%), in agreement with earlier
observations on older animals of a distinct IRP2 KO mouse line13. By contrast, the spleen of
IRP2-/- mice displays a relative iron deficiency (by ~ 40%) that was not reported before. Non-
heme iron levels in the brain are unchanged.
To assess the impact of the abnormal iron distribution on the major systemic iron utilization
pathway, we determined the hematolgical parameters (table 2). While leucocyte counts are
normal, the hematocrit and serum hemoglobin values are significantly reduced in spite of normal
erythrocyte counts, with lower MCV values (microcytosis) in both male and female IRP2-/-
mice.
Considering the accumulation of iron in the duodenum and the liver, IRP2-/- mice could be
microcytic because of decreased systemic iron availability. However, serum iron levels, the total
iron binding capacity, and the serum transferrin saturation do not significantly differ between
IRP2-/- mice and their wild-type littermates (table 3). Thus, IRP2-deficient mice are mildly
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microcytic and display signs of iron mismanagement with abnormal body iron distribution. The
normal serum iron and transferrin saturation values suggest that the microcytosis is not due to
systemic iron deficiency.
Duodenal iron metabolism in IRP2-/- mice.
To better understand the iron accumulation in the duodenum of IRP2-deficient mice, we
performed Prussian Blue staining (Fig. 1A, upper panels). Confirming the increase in non-heme
iron content detected with the bathophenantroline method (table 1), iron deposits are evident in
the duodenal villi but not crypts of IRP2-/- mice, suggesting that iron deposits do not result from
increased uptake of plasma iron via TfR1 which is preferentially expressed in crypt cells26.
Immunostaining reveals ferritin H- and L-chain expression that mirrors the increase in iron
(Fig.1A, lower panels). By western-blot analysis of protein extracts from four +/+, four +/- and
four -/- mice (Fig. 1B), ferritin H- and L- chain levels are increased 8-fold and 9-fold,
respectively, in IRP2-/- mice. Some interindividual variability could be due to heterogeneity in
the mixed genetic background. Ferritin H- and L-chain mRNAs are unchanged in the same
animals (northern blots Fig. 1B, lower panels), showing that accumulation of ferritin occurs
posttranscriptionally.
A rise in iron and ferritin levels in IRP2-/- enterocytes could be explained by increased iron
influx, decreased efflux, or retention of iron within ferritin that is translationally de-repressed.
Next, we examined the expression of DMT1 and ferroportin, the apical and the basolateral iron
transporters, respectively. Four isoforms of DMT1 that differ in their N- and C- termini are
generated by a combination of alternative splicing and the use of alternative promoters and
polyadenylation sites27. We analysed DMT1 mRNA expression by RNase protection assay,
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focusing on the variants that differ by the absence (noIRE form) or the presence (IRE form) of an
IRE motif in the 3' UTR28 (Fig. 2A). The increased DMT1 mRNA expression, especially of the
IRE form, was ascertained in duodenal samples of iron-deficient mice (Fig. 2A) as a positive
control for regulation29. Analysis of groups of four IRP2+/+, +/- and -/- mice reveals no
significant variation in the expression of the DMT-1 mRNA isoforms. Confirming mRNA data,
DMT1 protein expression was readily detected by western blotting in iron-deficient mice,
whereas basal DMT1 levels remained below the detection limit in both wild-type and IRP2-/-
mice (data not shown).
In the same mice, ferroportin levels are subject to significant interindividual variability (Fig. 3B),
but combining the results obtained from three independent lots of mice shows that ferroportin
expression is not significantly altered in IRP2-/- mice, either at the mRNA or the protein level.
We analysed ferroportin protein expression in total protein extracts, which does not discriminate
cell surface-exposed from intracellular protein. However, internalised ferroportin is targeted for
degradation8 and should therefore contribute little to the total ferroportin signal.
Dcytb, an iron reductase thought to act in conjunction with DMT1, may act as a facilitator of
iron overload in HFE KO mice30. A comparison of duodenal mRNA expression patterns between
IRP2-/- and +/+ mice using the "IronChip"24 revealed unchanged Dcytb mRNA expression
(details of this experiment are included as supplementary information). Similarly, no alteration in
the expression of hephaestin mRNA was observed.
These data show that the accumulation of iron and ferritin in the duodenal mucosa of IRP2-
deficient mice occurs without detectable alteration in the expression of the known iron
transporters. Amongst other possibilities, our data could be explained by partial de-repression of
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ferritin translation as a direct consequence of IRP2 deficiency, and subsequent iron retention
within the increased ferritin stores.
The lack of detectable changes in the levels of duodenal DMT1 and ferroportin in IRP2-/- mice
with diminished hematocrit is intriguing. Duodenal expression of ferroportin and of DMT1 is
modulated by nutritional iron deficiency15,31 and hemolytic anemia32. Therefore, the unchanged
ferroportin and DMT1 expression in IRP2-deficient mice could reflect impaired sensing and/or
signalling of the reduction in hematocrit and serum hemoglobin values. To test whether or not
IRP2-/- mice can respond normally to reduced hematocrit levels, mice were injected with
phenylhydrazine (PHZ) to trigger hemolysis. This treatment similarly diminished the hematocrit
in wild-type and mutant mice (data not shown). Wild-type mice respond to PHZ with the
expected increase in ferroportin protein (Fig. 2D) and DMT1 mRNA, especially the IRE-
containing variant (Fig. 2C) mirrored by the level of the corresponding DMT1 protein (data not
shown). In addition, an IronChip analysis revealed the appropriate upregulation of the Dcytb
mRNA (data not shown). Importantly, the response of IRP2-/- mice to PHZ is identical to that of
wild-type mice (Fig. 2C and 2D). These data show that a mild reduction of the hematocrit and
serum hemoglobin values in IRP2-deficient mice fails to elicit alterations in duodenal expression
of DMT1 and ferroportin, although the regulatory network in IRP2-/- mice is able to respond to
PHZ-induced anemia.
To investigate the functional consequences of increased ferritin levels in the duodenum, we
studied duodenal iron transfer in situ by injecting 59Fe into the lumen of the gut and measuring
the radioactivity recovered in the body after 15 minutes. The rate of iron transport across the
duodenal mucosa is similar in IRP2-/- mice and wild-type littermate controls (Fig. 2E). Thus, the
increase in the ferritin content of enterocytes of IRP2-/- mice does not detectably affect the
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transport of 59Fe measured over a short period of time. It is possible that the increased ferritin
expression favors duodenal iron retention over a more extended time course. Nonetheless, our
data show that IRP2 deficiency has only a minor impact on duodenal iron metabolism and its
ability to respond to hemolysis.
Hepatic iron metabolism in IRP2 deficiency.
As the livers of IRP2-/- mice also display iron loading, ferritin H- and L- chain expression was
analysed by western blotting (Fig. 3, upper panels). L-chain expression is increased 6-fold in
IRP2-/- mice. Ferritin H-chain expression is below the detection limit in the liver of wild-type
mice, but is detectably increased in IRP2-/- animals. Ferritin H- and L- chain mRNA levels are
unchanged (Fig. 3, lower panels), showing that ferritin expression is increased
posttranscriptionally. By contrast, IRP2-/- mice display a faint reduction of TfR1 levels (data not
shown), suggesting that the accumulation of iron and ferritin in the liver of IRP2-deficient mice
occurs without constitutive increase in TfR1-dependent iron acquisition.
Considering both the liver iron accumulation (table 1) and the reduced hematocrit (table 2), we
determined hepcidin expression. Hepcidin mRNA levels show interindividual variability in
mutant mice as well as in wild-type littermates. Combining the results from three independent
lots of mice, we observe a slight increase, although not statistically significant, in hepcidin
mRNA expression in IRP2-/- vs. +/+ mice (Fig. 4A). Unlike humans, mice express two hepcidin
genes. The two hepcidin proteins may play distinct roles in iron homeostasis33. Given their 92%
sequence identity34, the hepcidin 1 and 2 mRNAs cannot be discriminated by Northern blotting.
To check whether IRP2-mutant mice regulate hepcidin 1 and 2 mRNAs selectively, RNase
protection experiments were performed with brain mRNA as a negative control (Fig. 4B). As a
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control for regulation, we analysed hepcidin mRNA expression in females that received an iron-
poor vs. a control diet. In control mice, both hepcidin mRNAs are detected (Fig. 4B). Iron-
deficient female mice display the expected decrease in both hepcidin 1 and hepcidin 2 mRNA
levels, while hepcidin 2 mRNA is barely detectable in IRP2+/+ and -/- males, which express
predominantly hepcidin 1. This difference is likely explained by the sex difference since gender
has been shown to affect the hepcidin1/hepcidin2 ratio35. In agreement with the northern-blot
data, we do not observe significant changes in hepcidin 1 mRNA levels in IRP2-deficient
compared to wild-type mice. This observation is surprising since a reduction in hematocrit and
serum hemoglobin values is expected to elicit hepcidin downregulation. To test whether or not
IRP2-deficient mice can adequately adjust hepcidin expression in response to decreased
hematocrit and serum hemoglobin values, mice were injected with PHZ as described above.
Wild-type mice respond with the expected dramatic reduction in hepcidin mRNA expression
(Fig. 4C). Importantly, the response of IRP2-/- mice to PHZ is identical, showing that IRP2
deficiency does affect the control of hepcidin expression in response to hemolytic anemia.
Altered iron metabolism in the spleen of IRP2-deficient mice.
In agreement with a decrease in non-heme iron content (table 1), sections from the spleens of
IRP2-/- mice show weaker Prussian Blue staining in the red pulp (Fig. 5A, upper panels). This
staining essentially reveals macrophages and is associated with weaker immunostaining for the
ferritin L-chain (Fig. 5A, lower panels), and a reduction in ferritin H (~1.5 fold) and L (~2 fold)
expression in western blots of splenic extracts from IRP2-/- mice (Fig. 5B, upper panels). The
levels of the corresponding mRNAs are unchanged (Fig. 5B, lower panels), suggesting
posttranscriptional downregulation. We detected no signs of erythroid hyperplasia that could
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explain the iron deficiency of splenic macrophages. Indeed, IRP2-/- mice display no
splenomegaly, and the mRNA levels encoding the heme biosynthetic pathway enzymes
ferrochelatase or eALAS are normal as assessed with the IronChip (supplementary
informations). By contrast, the levels of these mRNAs increase when the mice are made strongly
anemic by PHZ injection (data not shown).
The iron deficiency of splenic macrophages could be due to increased iron export and/or
decreased iron acquisition. Several observations indicate that ferroportin is a major mediator of
iron efflux from macrophages36,37. Overexpression (or activity) of ferroportin could hence
explain an iron deficiency of splenic macrophages. Alternatively, if the splenic iron deficiency
was caused by reduced iron acquisition, ferroportin mRNA translation might be repressed via
IRP1. Thus, determination of ferroportin expression could help distinguish between these
scenarios. In the spleen, ferroportin is detected mostly in macrophages expressing the F4/80
antigen4. In F4/80 positive cells of IRP2-/- spleens, total ferroportin protein expression is
reduced, without a concomitant change in ferroportin mRNA levels (Fig. 6). This result directly
supports the second hypothesis according to which the iron deficiency is more readily explained
by reduced iron acquisition than by increased export. A similar decrease in total ferroportin
expression at the posttranscriptional level was observed in the bone marrow of IRP2-/- mice,
another major site of iron recycling (data not shown).
Decreased erythroid TfR1 expression can account for the microcytosis.
Microcytosis in IRP2-/- mice is associated with iron redistribution within the liver-bone marrow-
spleen axis. Nonetheless, serum iron levels and transferrin saturation are not detectably altered,
suggesting that the microcytosis could result from an intrinsic defect in hematopoiesis. To test
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this possibility, IRP target mRNAs were analysed in the bone marrow. Translational
derepression of ferritin due to lack of IRP2 may result in iron sequestration and impair
hematopoiesis. However, western blotting revealed no evidence for increased ferritin expression
in the bone marrow of IRP2-/- versus wild-type mice (data not shown). Similarly, dysregulation
of eALAS expression may affect heme synthesis, although we found eALAS protein
accumulation to be unchanged in IRP2-deficient mice (data not shown). Importantly, TfR1 is
essential for hematopoiesis38. As IRP binding to IREs in the 3' UTR of TfR1 mRNA sustains
TfR1 expression, lack of IRP2 may negatively affect TfR1 expression in erythroid precursors
and diminish their iron acquisition capacity. TfR1 mRNA levels in the bone marrow of IRP2+/+
and -/- mice were measured by RNase protection assay. In the bone marrow, most of the TfR1 is
expressed in erythroid cells39. Indeed, the level of TfR1 mRNA (Fig. 7A) and protein (Fig. 7B) is
significantly reduced in IRP2-/- mice, a result that can plausibly explain the observed phenotype.
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Discussion
Cell culture and in vitro studies demonstrated the importance of the IRP/IRE regulatory
network in cellular iron metabolism1,14-16. We recently generated mice with loss-of-function
alleles for IRP1 and IRP2, respectively18, to extend and complement phenotypic studies on mice
with constitutive deletions of IRP1 and/or IRP213,17. The detailed analyses of mice lacking IRP2
expression reported here have uncovered a previously unrecognised function of IRP2 in securing
physiological iron distribution between the duodenum, the liver and the spleen, and the need for
IRP2 expression for normal erythropoiesis.
Altered iron metabolism in IRP2-deficient mice.
IRP2-deficient mice display mild microcytosis with reduced hematocrit and serum hemoglobin
values, associated with altered iron distribution in the central iron recycling system, the liver-
bone marrow-spleen axis, and iron deposits in the duodenal mucosa. Notably these alterations
occur without detectable change in hepcidin mRNA levels. However, the lack of IRP2 does not
disrupt the physiological regulation of hepcidin in severely anemic mice following PHZ
injection. This result indicates that the reduced hematocrit of IRP2-/- animals may not have
reached the threshold required for hepcidin to respond. Similarly, the iron accumulation in the
liver of IRP2-/- mice may not be sufficient to trigger hepcidin expression, or it may fail to be
sensed as iron overload as a result of its cellular or subcellular distribution. Conceivably, the
antagonistic effects of microcytosis and liver iron loading on hepcidin expression could
neutralize each other.
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The reasons for the altered iron distribution in IRP2-/- mice are not fully resolved. While liver
iron loading and macrophage iron deficiency are reminiscent of type 1 hemochromatosis, the
normal serum Tf saturation and the enterocyte iron loading are not40. Furthermore, the IronChip
analysis revealed unchanged HFE mRNA expression in various organs (supplementary material).
The phenotypic manifestations of IRP2 deficiency have several other interesting implications.
The increased expression of ferritin in enterocytes on the one hand and the unaffected expression
of duodenal iron transport molecules, the normal duodenal transfer of 59Fe and the normal serum
transferrin saturation on the other are striking in terms of the mucosal block hypothesis proposed
by Crosby41. This hypothesis states that duodenal ferritin serves to withhold iron from entering
into circulation and thus as a means to negatively regulate iron transfer. IRP2-/- mice with an 8-
to 9-fold increase in ferritin and seemingly normal iron transfer appear to uncouple the two
events in a way that is not predicted by the mucosal block scenario. One critical issue is whether
IRP2-deficient mice are microcytic because of iron mismanagement within the iron-recycling
pathway. Normal serum iron levels and transferrin saturation values suggest that this is not the
case. As mice with haploinsuficiency for TfR1 suffer from microcytosis38, the ~1.6-fold
reduction of TfR1 mRNA expression accompanied by diminished TfR1 protein expression in
erythroid cells of IRP2-/- mice can plausibly explain the microcytosis. We did not find increased
ferritin expression in total bone marrow samples that could withhold a fraction of the iron needed
for erythropoiesis. However, changes in ferritin levels in erythroid precursors might be masked
by the contribution of ferritin from the other cells of the bone marrow. Indeed, Cooperman et al.
recently reported a small increase in ferritin expression in eryhtroid precursors sorted from the
bone marrow of a distinct IRP2-deficient line42. Although Western blotting detected no changes
in eALAS accumulation in total bone marrow extracts from our IRP2-/- mice (data not shown),
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Cooperman et al. found increased de novo synthesis of eALAS in sorted erythroid precursors
after metabolic labelling42, associated with accumulation of free protoporphyrin IX. Altogether
these data suggest that microcytosis in IRP2-/- mice is likely caused by an intrinsic defect of
intracellular iron availability in erythroid cells.
Using a gene replacement vector to target the irp2 locus non-conditionally, LaVaute et al.13
described the development of a progressive neurodegenerative disease with locomotor
impairment in IRP2-/- mice older than 6 months. We have not observed any overt signs of
neuropathology such as tremor or any obvious postural abnormality in young adults as well as in
8-month old IRP2-/- mice (data not shown). While both IRP2-deficient mouse lines display
duodenal and hepatic accumulation of iron and ferritin, we did not observe increased ferroportin
and DMT1 expression in the duodenum as reported by LaVaute et al.13. Several factors could
explain such discrepancies. While both mouse lines are on similar mixed C57BL6/Sv129 genetic
backgrounds, they were analysed at a different age (8-10 weeks in the present study versus 4
months and older in LaVaute et al.13). However, we tested but did not detect significantly
increased duodenal ferroportin expression in 4 and 8 month-old IRP2-/- mice either (data not
shown). Perhaps more importantly, the targeting strategies are distinct. While LaVaute et al.13
used a gene replacement vector bearing a PGK-Neo cassette, we inserted a promoter-less βGeo
gene-trap construct into an early intron of the irp2 gene18. Artefacts arising from selection
markers are well documented43. Therefore we crossed our IRP2-/- line bearing the βGeo cassette
with a Cre deletor strain to derive an IRP2 null line that lacks any selection marker inserts and
that differs from the wild type only by the absence of exon 3. A preliminary analysis of this IRP2
∆/∆ line also displays a complete loss of IRP2 expression and shows the same phenotype as the
one reported here, including the misdistribution of iron and microcytosis, and excluding
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increased duodenal ferroportin expression or overt symptoms of neuropathology up to 6 months
of age (B.G. et al., unpublished results). This demonstrates that the phenotypic traits reported
here result from IRP2 deficiency per se and are not technical artefacts arising from the presence
of the gene trap construct.
Molecular basis of altered iron metabolism in IRP2-deficient mice.
IRP2 deficiency leads to iron accumulation in the liver and in the duodenum with a concomitant
posttranscriptional increase in ferritin protein expression. The mechanisms underlying iron
deposition in those tissues remain to be elucidated. We did not observe any increase in hepatic
TfR1 expression in mutant animals. A second Tf receptor with about 25-fold lower affinity than
TfR1, TfR2, is expressed on hepatocytes44. However, the IronChip analysis did not reveal any
change in TfR2 mRNA expression either. Hence, a potential increase in liver iron uptake could
potentially involve Tf-independent pathways. A reduction in iron efflux may also account for
iron accumulation, although there is no detectable change in hepatic ferroportin expression (data
not shown). Still unknown ferroportin-independent iron export pathways might be affected, or
ferroportin function could be decreased posttranslationally.
Contrary to what was observed in the liver and in the duodenum, the iron and ferritin content of
splenic macrophages is lower in IRP2-/- animals. Curiously, IRP2-/- macrophages from the
spleen and bone marrow display reduced ferroportin expression, a finding that was not reported
before13,42. This is unexpected, since ferroportin downregulation should result in iron retention.
Hypothetically, an increase in the iron transport activity of ferroportin may overcome the
reduction of its expression, or an as yet unknown iron export pathway may be activated. In that
case, ferroportin downregulation might be secondary to iron deficiency and may result from
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translational repression of the IRE-containing ferroportin mRNA by IRP1 whose IRE-binding
activity would be increased in iron-deficient cells.
A recent study revealed that hepcidin binds ferroportin and augments its turnover in human cell
lines8. This mechanism does not explain the observed reduction in ferroportin expression in
IRP2-/- macrophages, because hepcidin expression in the liver and ferroportin expression in
other tissues are not detectably affected. Splenic macrophages acquire iron mainly from
senescent erythrocytes. As the reduction of the red cell mass (table 2) cannot account for the
decreased iron content of the spleen (table 1), a possible reason for the iron deficiency of
macrophages could be a quantitatively modest impairment of erythrophagocytosis.
Roles of IRP1 and of IRP2 in iron homeostasis.
An intriguing feature of IRP2-deficient mice is the remarkable preservation of mRNA expression
patterns in the brain, duodenum, liver and spleen, assessed with the IronChip (see supplementary
information). These data imply broad functional redundancies between IRP1 and IRP2.
Constitutive inactivation of both irp1 and irp2 genes in the mouse results in embryonic lethality19
(B.G. et al., unpublished data), showing that the IRP/IRE regulatory system is essential for life.
IRP1-deficient animals are almost normal and display none of the phenotypic traits of our IRP2-
deficient mouse line17 (B.G. et al., manuscript in preparation). These findings suggest that the
two IRPs can largely replace each other. Thus, at least under laboratory conditions, IRP2 can
fully compensate for the lack of IRP1, whereas IRP1 can largely but not fully compensate for the
absence of IRP2. The strong redundancy of the two IRPs combined with the embryonic lethality
of doubly-deficient mice poses a challenge to dissect and understand the physiological roles of
the IRP/IRE regulatory system in vivo. The possibility to inactivate one or both of the irp genes
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in a time- and tissue-specific manner using conditional alleles18 provides an experimental system
to further uncover the role of the IRP/IRE regulatory system in mammalian physiology.
Acknowledgements
We are grateful to Patrick Hundsdoerfer and Karen Brennan for helpful discussions and to
Yevhen Vainshtein for help with handling of the microarray data. We thank the EMBL
transgenic mouse service and the staff of the EMBL animal house for their contribution to this
work.
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23
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Table 1: Abnormal iron distribution in IRP2-/- vs. wild-type mice.
+/+ -/-
Liver 195 ±9 (n=27) 300 ±15 (n=24) *
Duodenum 571 ±41 (n=17) 1058 ±113 (n=13)*
Spleen 2191 ±191 (n=15) 1331 ±122 (n=11) *
Brain 157 ±11 (n=18) 148 ±13 (n=14)
Tissue non-heme iron content was determined using the bathophenantroline chromogen20 in 8-10
week-old mice and is given in mg/g of dried tissue. Results are presented as means ±standard
error. The sample size (n) is indicated. (* : P≤0.001, student t-test).
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Table 2: Hematological parameters of IRP2-/- vs. wild-type mice.
males females
+/+ -/- +/+ -/-
RBC (1012/l) 8.43 ±0.25
(n=11)
7.96 ±0.20
(n=7)
7.53 ±0.23
(n=14)
7.27 ±0.15
(n=18)
MCV (fl) 53.04 ±0.57
(n=10)
46.78 ±0.75
(n=7) **
51.11 ±0.58
(n=12)
47.21 ±0.52
(n=17) **
Hematocrits (l/l) 0.45 ±0.01
(n=11)
0.37 ±0.01
(n=7)**
0.39 ±0.01
(n=14)
0.34 ±0.01
(n=18)*
Hemoglobin (g/l) 138 ±3 (n=11) 115 ± 3 (n=7)** 128 ±6 (n=14) 108 ±3 (n=18)*
Reticulocytes (109/l) 148.0 ±12.8
(n=11)
194.2 ±35.6
(n=6)
354.4 ±100.4
(n=7)
347.4 ±121.5
(n=9)
WBC (109/l) 6.318 ±0.643
(n=11)
6.985 ±0.883
(n=7)
5.036 ±0.414
(n=14)
5.322 ±0.396
(n=18)
Neutrophils (109/l) 0.725 ±0.093
(n=7)
0.937 ±0.213
(n=6)
0.716 ±0.228
(n=9)
0.454 ±0.052
(n=9)
Lymphocytes (109/l) 5.601 ±0.834
(n=7)
5.157 ±0.682
(n=6)
4.115 ±0.444
(n=9)
5.038 ±0.461
(n=9)
Monocytes (109/l) 0.121 ±0.024
(n=7)
0.128 ±0.044
(n=6)
0.266 ±0.188
(n=9)
0.252 ±0.195
(n=9)
Thrombocytes
(109/l)
613 ±28 (n=8) 761 ±56 (n=6) 555 ±44 (n=9) 611 ±65 (n=9)
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Results were obtained from 8-10 week-old mice and are given ± standard errors. RBC : red
blood cells, MCV: mean corpuscular volume, WBC : white blood cells. The sample size (n) is
indicated. (* : p≤0.01, ** : p≤0.001, student t-test).
Table 3: Serum iron parameters in IRP2-/- vs. wild-type mice.
+/+ -/-
Serum Fe (µg/dl) 212 ±18 (n=14) 212 ±14 (n=15)
TIBC (µg/dl) 416 ±20 (n=14) 412 ±20 (n=15)
Tf saturation (%) 50.6 ±3.0 (n=14) 51.3 ±1.9 (n=15)
Results were obtained from 8-10 week-old mice and are presented as means ±standard error.
TIBC (total iron binding capacity) = serum Fe + UIBC (unbound iron binding capacity). Tf
saturation = (serum Fe/TIBC)X100. The sample size (n) is indicated.
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Figure Legends
Figure 1: Iron accumulation and increased ferritin expression in the duodenum of IRP-2
deficient mice.
A) The proximal part of the duodenum from 10 week +/+ and -/- males was analysed by Prussian
blue staining (upper panels) or by immunostaining with anti ferritin L-chain (middle panels) or
ferritin H-chain antibodies (lower panels). Prussian blue coloration was counterstained with
Nuclear Fast Red and ferritin immunostaining with Hematoxylin. Pictures were acquired on a
Axiophot microscope (Zeiss) with a 20X objective. B) Expression of ferritin H- and ferritin L-
chains in groups of four +/+, +/- and -/- 10-week-old males. 40 µg of total protein were resolved
by SDS-PAGE and subjected to western-blot analysis (upper panels). Ferritin H- and L-chain
mRNA levels were analysed by northern-blotting using 10 µg of total RNA from the same mice
(lower panels) and β-actin mRNA as a standard. Western-blot and northern-blot signals were
quantified and are presented as a histogram (bottom) after normalization for β-actin expression.
In western blots, the ferritin L-subunit resolves into two bands, the lower one corresponding to a
cleavage product. Both bands were taken into account for quantification. IRP2 expression was
below the detection limit.
Figure 2: Expression of DMT1 and ferroportin, and 59Fe transport in the duodenum of
IRP2-deficient mice.
A) DMT1 expression was analysed by RNase protection assay. The antisense RNA probe
matching a sequence common to the two 3' variants of the DMT1 mRNA isoforms plus a domain
specific for the noIRE form is depicted (top). 5 µg of total RNA were co-hybridised with the
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DMT1 probe together with a β-actin probe as an input control. Arrows indicate the signals
corresponding to full-length probes (DMT1: 500 nt, β-actin: 276 nt), to β-actin (250 nt) and to
the IRE (320 nt)/no-IRE (400 nt) DMT1 isoforms. Total RNA from control and iron-deficient
mice was used as a positive control for regulation of DMT1 expression. The histogram shows the
levels of the DMT1 mRNA isoforms after normalization for β-actin. These figures are
representative of data obtained with 3 independent lots of mice (including four +/+ and four -/-
animals each). B) Ferroportin expression was analysed by western blotting (upper panels) and by
northern-blotting (lower panels). Equal loading was checked by detection of β-actin. The
expression of ferroportin and of the DMT1 mRNA 3' splice variants was determined,
respectively, by western-blotting (D) and RNase protection assay (C) in wild-type and IRP2-/-
mice injected with phenylhydrazine (PHZ) vs. a saline as a control (ctr). β-actin mRNA was used
as a standard. E) Measurement of duodenal 59Fe transfer in vivo. 12 week-old males and females
were fasted overnight and 59Fe transfer analysed as described in materials and methods. The size
(n) of the samples is indicated.
Figure 3: Posttranscriptional increase in ferritin expression in the liver of IRP2-deficient
mice.
Expression of ferritin H- and L-subunits was analysed in groups of four +/+, +/- and -/- mice by
western- (upper panels) and northern-blotting (lower panels) using β-actin as a standard. IRP2
expression is also shown; a non-specific band is indicated with an asterisk. The western-blot and
northern-blot signals were quantified and results are presented in a histogram (bottom) after
normalization for β-actin. Ferritin H-chain was below the detection limit in wild-type mice (n.d.)
and was not quantified in IRP2-/- mice (n.q.) because of background interference. These figures
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are representative of data obtained with 3 independent lots of mice (including four +/+ and four -
/- animals each).
Figure 4: Unchanged hepcidin mRNA expression in the liver of IRP2-deficient mice.
A) Expression of hepcidin was analysed in groups of four +/+, +/- and -/- mice by northern-
blotting using β-actin as a standard. The histogram shows quantification of the hepcidin signals
(normalized for β-actin) from the analysis of 3 independent lots of mice comprising 4 animals in
each group. B) Hepcidin 1 and hepcidin 2 mRNA levels were assayed by RNase protection. 10
µg of total RNA were co-hybridised with the hepcidin probe together with a β-actin probe as an
input control. The signals corresponding to full-length probes (hepcidin: 125 nt, β-actin: 334 nt),
to β-actin (250 nt) and to the hepcidin 1 (40 nt) and hepcidin 2 (54 nt) mRNAs are indicated by
arrows. Total RNA from control vs. iron-deficient mice was used as a positive control for
regulation of hepcidin expression. C) Hepcidin mRNA levels were assayed by northern blotting
in mice injected with phenylhydrazine (PHZ) or a saline as a control (ctr), in wild-type vs. IRP2-
mutant mice. β-actin was used as a standard.
Figure 5: Diminished iron staining and ferritin expression in the spleen of IRP2-deficient
mice.
A) The spleen from 10 week +/+ and -/- males was analysed by Prussian Blue staining (upper
panels) or staining with an anti ferritin L-chain antibody (lower panels). Prussian blue coloration
was counterstained with Nuclear Fast Red and ferritin immunostaining with Hematoxylin.
Pictures were acquired on a Axiophot microscope (Zeiss) with a 20X objective. Ferritin H-chain
expression was below the detection limit and is not shown. B) IRP2 and ferritin H- and L-chain
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35
expression was analysed in groups of four +/+, +/- and -/- mice by western- (upper panels) and
northern-blotting (lower panels) from 40 µg of total protein or 10 µg of total RNA, respectively,
using β-actin as a standard. The histogram represents the level of ferritin expression after
normalization for β-actin.
Figure 6: Reduced ferroportin expression in the spleen of IRP2-deficient mice.
A) Ferroportin expression was analysed by fluorescent immunostaining on spleen sections. The
red pulp (RP) and the white pulp (WP) are indicated. Macrophages of the red pulp were revealed
with an anti-F4/80 antibody. Nuclei were stained with Dapi. Pictures were acquired using a wide
field Axiovert 200M microscope and a 25X immersion objective from Zeiss. B) Ferroportin
expression was analysed in groups of four +/+, +/- and -/- mice by western- (upper panels) and
northern-blotting (lower panels). The histogram depicts the levels of ferroportin expression after
normalization for β-actin. These figures are representative of data obtained with 3 independent
lots of mice (including four +/+ and four -/- animals each).
Figure 7: Reduced TfR1 expression in the bone marrow of IRP2-deficient mice.
A) TfR1 mRNA levels in the bone marrow were assayed by RNase protection in groups of four
wild-type versus four IRP2-/- mice. β-actin was used as a standard. The bands corresponding to
full-length probes (TfR1: 490 nt, β-actin: 276 nt) and to the β-actin (250 nt) and TfR1 (371 nt)
mRNAs are indicated by arrows The signals were quantified using a phosphorimager. The
histogram represents the levels of TfR1 mRNA after normalization for β-actin expression (means
± SD). TfR1 mRNA levels are significantly reduced in mutant mice compared to wild-type mice
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36
(student t-test). B) TfR1 protein levels (middle panel) were analysed in four wild-type versus
four IRP2-/- mice (upper panel) by western blotting using β-actin as a standard (bottom panel).
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37
Galy et al., Fig.1
A +/+ -/-
Fe
Ft-L
B
+/+ +/- -/-
Ft-L
β-actin
RNA
Ft-H
β-actin
Ft-L
Ft-H
β-actin
+/+ +/- -/-
protein
Ft-H
mRNAprotein
Ft-L
arb
itra
ry u
nits
Ft-H
+/+ -/- +/+ -/-0
500
1000
1500
2000
2500
37 kD
26 kD
19 kD
26 kD
19 kD
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38
Galy et al., Fig.2
E
pm
ol/cm
/min
12
10
8
6
4
2
0
+/+ -/-(n=9) (n=10)
D
C
probe
IRE
no IRE
probe
β-actin
DMT1
β-actin
no
RN
An
oR
NA
se
s
+/+ -/-ctr PHZ ctr PHZ
+/+ -/-ctr PHZ ctr PHZ
ferroportin
β-actin
A
B
no
RN
An
oR
NA
se
s
Fe
co
ntr
ol
Fe
de
ficie
nt
probe
IRE
no IRE
probeβ-actin
+/+ +/- -/-
DMT1
β-actin
arb
itra
ry u
nits
20
40
60
80
100
120
140
160
0
IREnoIRE
+/+-/-
ferroportin
β-actin
protein
RNA
ferroportin
β-actin
+/+ +/- -/-
5' pA
5' pADMT1-IRE
DMT1-noIRE
probe5'3'
37 kD
64 kD
82 kD
37 kD
64 kD
82 kD
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39
Galy et al., Fig.3
+/+ +/- -/-
Ft-L
β-actin
RNA
Ft-L
Ft-H
β-actin
+/+ +/- -/-
protein
mRNAprotein
Ft-L
arb
itra
ry u
nits
0
IRP2*
Ft-H
β-actin
Ft-H
1000
800
600
400
200
nd nq
+/+ -/- +/+ -/-
37 kD
19 kD
115 kD
26 kD
82 kD
19 kD
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40
Galy et al., Fig.4
A
Hepcidin
β-actin
+/+ +/- -/-
+/+ -/-
arb
itra
ry u
nits
0
200
80
60
40
100
20
120
140
160
180
Hepcidin
β-actin
+/+ -/-ctr PHZ ctr PHZ
B
no
RN
An
oR
NA
se
s
Fe c
on
tro
lF
e d
eficie
nt
probe
Hepcidin 2
probe
β-actin
+/+ +/- -/-
Hepcidin
β-actin
Hepcidin 1
Bra
in
C
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41
Galy et al., Fig.5
+/+ +/- -/-
IRP2
Ft-L
β-actin
A +/+ -/-
Fe
+/+ +/- -/-
Ft-L
β-actin
protein
RNA
Ft-H
Ft-L
Ft-H
mRNAprotein
Ft-L
arb
itra
ry u
nits
Ft-H
+/+ -/- +/+ -/-0
50
100
150
200
250
B
19 kD
19 kD
37 kD
115 kD
82 kD
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42
Galy et al., Fig.6
ferroportin
β-actin
A
ferroportin
F4/80+
dapi
overlay
WPRP
+/+ +/- -/-
ferroportin
β-actin
+/+ +/- -/-
protein
RNA
120
100
80
60
40
20
0
arb
itra
ry u
nits
+/+ -/-
mRNAprotein
B
37 kD
64 kD
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43
Galy et al., Fig.7
30
25
20
15
10
5
0
arb
itra
ry u
nits
+/+ -/-
probe
TfR1
probe
β-actin
TfR1
β-actinn
oR
NA
no
RN
Ase
s
+/+ -/-
35
p<0.01
+/+ -/-
TfR1
β-actin
A
B
IRP2
37 kD
115 kD
82 kD
115 kD
82 kD
For personal use only.on October 22, 2017. by guest www.bloodjournal.orgFrom
doi:10.1182/blood-2005-04-1365Prepublished online June 14, 2005;
Schumann and Matthias W HentzeBruno Galy, Dunja Ferring, Belen Minana, Oliver Bell, Heinz G Janser, Martina Muckenthaler, Klaus Regulatory Protein 2 (IRP2)Altered body iron distribution and microcytosis in mice deficient for Iron
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