altered body iron distribution and microcytosis in mice ......blood first edition paper,...

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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




13

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




14

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




15

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




16

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.




17

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.




18

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),




19

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




20

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




21

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




22

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.




23

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29

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).




30

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)




31

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.




32

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




33

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




34

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




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




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).




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




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


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




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




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


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




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




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




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




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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