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First published online March 30, 2004
doi: 10.1242/10.1242/dev.01081
1 Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA
2 Department of Medicine, Baylor College of Medicine, Houston, TX 77030,
USA
3 MRC Mammalian Genetics Unit, Medical Research Council, Harwell, Oxfordshire
OX11 0RD, UK
Author for correspondence (e-mail:
armins{at}bcm.tmc.edu)
Accepted 13 January 2004
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Ferroportin 1, Iron homeostasis, Hepcidin
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). Owing to
lack of efficient means of excretion, iron homeostasis is principally
regulated at the level of organismal uptake by the duodenal enterocyte.
Regulation occurs at both the apical and basolateral membranes to effect
transcellular movement of iron from the gut lumen to the portal circulation
(for a review, see Roy and Enns,
2000). At the apical surface, ferrous iron is transported into the
enterocyte via the proton symporter DMT1 (for a review, see
Andrews, 1999). Recently,
ferroportin 1 (Fpn1; also known as MTP1, IREG1, SLC11A3; SLC40A1 – Mouse
Genome Informatics) has been identified as the basolateral iron transporter of
the duodenal enterocyte (Abboud and Haile,
2000; Donovan et al.,
2000; McKie et al.,
2000). Cellular export of iron and loading onto serum transferrin
requires ferroxidase activity, served by the multicopper oxidase hephaestin
(Vulpe et al., 1999) in the
duodenal enterocyte, and by its homologue ceruloplasmin (for a review, see
Hellman and Gitlin, 2002) in
other cells types. From the portal circulation, transferrin-bound iron is
taken up by liver cells via the transferrin (Tf)-transferrin receptor (TfR)
system, the principal mechanism of iron delivery to iron-using cells (for a
review, see Richardson and Ponka,
1997).
Although steady-state iron levels are maintained by enteral absorption of
iron (for a review, see Finch,
1994), the majority of iron used for cellular requirements derives
from iron recycled by the reticuloendothelial macrophage system (for a review,
see Knutson and Wessling-Resnick,
2003). Fpn1 has been implicated in turnover of iron recovered from
scavenged red blood cells in reticuloendothelial macrophages of the splenic
red pulp and hepatic Kupffer cells (Abboud
and Haile, 2000; Yang et al.,
2002). The essential role of Fpn1 in iron homeostasis has been
revealed by mutations in human FPN1, leading to autosomal dominant iron
overload in reticuloendothelial macrophages
(Montosi et al., 2001;
Njajou et al., 2001;
Cazzola et al., 2002;
Devalia et al., 2002;
Arden et al., 2003;
Rivard et al., 2003;
Jouanolle et al., 2003). This
unique pattern of iron accumulation caused by a mutation in FPN1,
comprising type IV hereditary hemochromatosis, contrasts distinctly to the
primarily parenchymal accumulation seen in the more common, recessively
inherited HFE-associated hemochromatosis (type I) (for a review, see
Ajioka and Kushner, 2002). All
known mutations in FPN1 map to the coding region, and both gain- and
loss-of-function mechanisms have been evoked in disease pathogenesis (for a
review, see Fleming and Sly,
2001).
The post-transcriptional regulation of iron-relevant proteins, including
TfR and ferritin, is achieved by the interaction of iron regulatory proteins
(IRPs) with iron responsive elements (IREs) located within the untranslated
regions (UTR) of the mRNA (for a review, see
Aisen et al., 2001). In the
case of ferritin, an IRE sequence in the 5' UTR forms a secondary,
stem-loop structure that is bound by iron regulatory protein 1 (IRP1) in the
absence of intracellular iron, inhibiting protein translation (for a review,
see Hentze and Kuhn, 1996).
Likewise, Fpn1 transcripts contain an IRE sequence in the 5'
UTR, which inhibits translational efficiency of Fpn1 mRNA in the
absence of intracellular iron in cell culture
(Abboud and Haile, 2000;
McKie et al., 2000;
Liu et al., 2002). Here,
positional cloning of the Pcm mutation identified a hypermorphic
allele of Fpn1 which, in the absence of an IRE in the 5'UTR,
affects its translational regulation, leading to increased Fpn1 protein levels
during early postnatal development despite low cellular iron levels.
Dietary absorption of iron responds to body iron levels via a putative
regulator, conveying organismal iron status to remote effectors of iron
balance (for a review, see Finch,
1994). The expression of the hepcidin antimicrobial peptide (Hamp;
also known as Hepc, LEAP-1), a disulfide-bridged oligopeptide produced in the
liver (Park et al., 2001), is
responsive to body iron stores (Pigeon et
al., 2001). Severe dysregulation of iron balance in murine models
of Hamp gain- and loss-of-function
(Nicolas et al., 2001;
Nicolas et al., 2002a), as
well as failure of Hamp induction in a murine model for HFE
hemochromatosis (Ahmad et al.,
2002; Nicolas et al.,
2003) reveal Hamp as the principal hormonal regulator of iron
homeostasis. Furthermore, mutations in human HAMP lead to a severe,
juvenile form of hereditary hemochromatosis (type II)
(Roetto et al., 2003).
In the present study, downregulated expression of Hamp correlated with high levels of Fpn1 protein expression and polycythemia in Pcm mutant animals. Developmental analysis of Hamp expression indicates an interference on Fpn1 regulation that is superimposed on the primary defects caused by the microdeletion in the Fpn1 promoter region, and alleviates the abnormalities in iron homeostasis and erythropoiesis in Pcm mutant animals. Our results provide the most compelling evidence to date implicating Hamp in the systemic regulation of Fpn1 expression in vivo.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). The present
study involved partial congenics on an A/J background; most analyses were
performed on animals from N5 and subsequent backcross generations. A genome
scan to determine the chromosomal location of Pcm by residual
heterozygosity mapping employed N3 and N4 backcross animals at 7 weeks of age.
Twenty-one wild type and 13 mutants showing hematocrit (Hct) greater than 50%
were genotyped by PCR using a simple sequence length polymorphism (SSLP)
marker panel consisting of 81 markers (Research Genetics) polymorphic among
A/J and the parental strains. Markers displayed an average spacing of 15-20
cM. Marker and genome scan information are available from authors upon
request. All animal experiments in this study were approved by the
Institutional Animal Care and Use Committee of Baylor College of Medicine.
Red blood cell analyses
Hematocrit measurements, expressed as a percentage, were obtained by
standard microcapillary determination. Determination of mean corpuscular
volume (MCV), and mean corpuscular hemoglobin content (MCHC) was performed on
the ADVIA 120 Hematology System (Bayer). Blood was collected in EDTA-coated
collection tubes (Sarstedt) at postnatal day 0 (P0) and 12 weeks of age for
analysis.
Serum and tissue iron determination
Quantification of iron concentration was performed based on methodology of
Torrance and Bothwell (Torrance and
Bothwell, 1980). Equal volumes of 0.2 N HCl with 0.05% ascorbic
acid, and 11% trichloroacetic acid were added to serum samples. Tissue samples
were digested in 3 N HCl with 10% trichloroacetic acid at 65°C overnight.
Colorimetric iron determination of supernatants was performed using the Total
Iron Measurement Kit (Sigma).
In vitro bone marrow cultures
Erythroid lineage terminal differentiation as a readout for
erythroid-committed precursor and progenitor cell abundance in the bone marrow
was determined by a fluorescence-activated cell sorting (FACS)-based approach
to quantify erythropoietin (Epo)-dependent expression of erythroid lineage
marker TER-119, expressed in mature erythrocytes. Bone marrow cells were
flushed from dissected femurs, washed and lysed of erythrocytes in buffer
containing 155 mM NH4Cl, 10 mM NH4HCO3 and
0.1 mM EDTA pH 8.0. As determined by Coulter Z2 particle counter,
1x106 cells were plated in Iscove's Modified Dulbecco's
Medium (Invitrogen) containing 30% FBS (Stem Cell) in 1 ml. Parallel cultures
were set up with and without recombinant human Epo (Amgen) at a final
concentration of 3000 mU/ml. Cells were cultured in a humidified incubator at
37°C and 5% CO2. To determine the percentage of
TER-119-expressing cells, uncultured cells (day 0) and cells on day 2 were
harvested, and washed in PBS. Cells were incubated with biotinylated antibody
against mouse TER-119 (BD Pharmingen) for 30 minutes on ice at 1:100 dilution.
After washing, cells were incubated with streptavidin-phycoerythrin (BD
Pharmingen) for 10 minutes at 1:50 dilution, washed and resuspended in PBS.
The percentage of TER-119-expressing cells was determined by flow cytometry
using the Coulter Epics XL-MCL.
Histology
Liver and spleen samples were dissected, fixed in Bouin's reagent,
dehydrated in a graded series of ethanol, embedded in paraffin wax and
sectioned at 5-10 µm. To determine the abundance and localization of iron,
Prussian Blue staining was performed using the Accustain iron staining kit
(Sigma), and photographed using light microscopy. Femurs from mice were
dissected, cleaned and fixed in a 1:1 solution of 37% formaldehyde and B-5
fixative stock solution (Poly Scientific). After light decalcification, femurs
were paraffin wax embedded and sectioned. To assess the relative abundance of
hemoglobinized cells in the bone marrow, paraffin wax-embedded femoral
sections were rehydrated, fixed for 10 minutes in methanol, and subjected to
o-dianidisine (O-D) staining. Slides were incubated for 10 minutes in
O-D working solution containing 5 volumes of 0.2% O-D in methanol, 1 volume of
1% sodium nitroprusside in water and 1 volume of 3% hydrogen peroxide.
RACE and RT-PCR
Total RNA from liver and kidney tissue samples was isolated using TRIzol
Reagent (Invitrogen). The 5' ends of Fpn1 transcripts in
wild-type and homozygous mutant animals were determined by rapid amplification
of cDNA ends (RACE) using the SMART RACE cDNA amplification kit (Clontech) on
1 µg total RNA derived from livers of 3-week-old mice. RACE PCR was
achieved using a gene-specific primer in Fpn1 corresponding to
sequence 5'-ATGACGGACACATTCTGAACCA-3'. RT-PCR to determine
approximate level of transcript abundance for Fpn1 and Hamp
was performed on total RNA derived from livers of 7-week-old animals.
First-stand cDNA was synthesized from 2 µg total RNA using standard
methods. PCR amplification conditions consisted of 25 cycles: denaturation at
94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension
at 72°C for 30 seconds. Primer sequences for Fpn1 are
5'-ACAAACAAGGGGAGAACGC-3' (forward) and
5'-ATGACGGACACATTCTGAACCA-3' (reverse); published primer sequences
for Hamp (Hepc1) and ß-actin were used
(Nicolas et al., 2001).
Quantitative real-time RT-PCR
Epo and Fpn1 mRNA expression were quantified via
real-time RT-PCR on total RNA samples isolated from liver and kidney from
3-week-old animals and kidney from 12-week-old animals. Reactions and signal
detection were performed on an ABI Prism 7000 Sequence Detection System
(Applied Biosystems). Epo primers
[5'-TCAACTTCTATGCTTGGAAAAGAATG-3' (forward),
5'-TGAGAGACAGCGTCAAGATGAGA-3' (reverse)] and TaqMan MGB probe
(5'-CGCTAGCGACCTGGA-3') were labeled with 6-FAM (Applied
Biosystems). Fpn1 primer sequences
[5'-GGGTGGATAAGAATGCCAGACT-3' (forward) and
5'-ATGACGGACACATTCTGAACCA-3' (reverse)] were assayed via SYBR
Green fluorescence detection. 18S rRNA assay was performed using
primers 5'-TCGAGGCCCTGTAATTGGAA-3' (forward),
5'-CCCTCCAATGGATCCTCGTT-3' (reverse); and TaqMan MGB probe
5'-AGTCCACTTTAAATCCTT-3' was labeled with 6-FAM (Applied
Biosystems). Relative amounts of mRNA are expressed as a ratio to 18S
rRNA expression, and normalized to a single wild-type ratio arbitrarily set to
1.
Western analysis of liver and duodenum
For western blot analyses, liver and duodenal samples were collected,
homogenized and lysed in RIPA buffer plus Complete®, EDTA-free protease
inhibitor (Roche). Extract supernatant was collected and protein quantified
using the BioRad DC Protein Assay kit (BioRad). The samples were
mixed with equal volume sample buffer with ß-mercaptoethanol. Unboiled
samples were used for Fpn1 and actin detection, boiled samples for ferritin;
10 µg total protein from liver lysates and 5 µg from duodenum were
loaded per lane. Samples were separated using 8 or 10% SDS-PAGE and
transferred to PVDF membrane. Blocking was achieved by incubation in
TRIS-buffered saline containing 5% BSA and 0.1% Tween 20. Membranes were
incubated overnight at 4°C using primary antibodies against mouse Fpn1
(kindly provided by M. Hentze) at 1:2000, horse ferritin from spleen (Sigma)
at 1:5000, or mouse actin (Santa Cruz) at 1:5000. After washing, membranes
were incubated with horseradish peroxidase-conjugated secondary antibodies
(1:5000) against rabbit (for Fpn1 and ferritin) or goat (for actin), and
signal developed using ECL reagent (Santa Cruz Biotechnology).
Statistical analyses
All data are reported as the mean±s.d. All comparisons were made
versus wild-type cohorts, and analyzed for significant differences using the
Student's unpaired t-test.
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). Mutant
heterozygous animals exhibited an incompletely penetrant increase in Hct up to
75% in young adult animals. Our analysis of the Pcm mutation on a
partially congenic A/J background reiterated the preliminary report on the
increased redness in the ear and footpad vasculature of heterozygous mutant
animals (Cattanach, 1995),
most highly expressed around 7 weeks of age
(Fig. 1A). Consistent with the
external phenotype, Hct analysis of heterozygous mutant animals demonstrated a
highly penetrant polycythemia at 7 weeks of age when compared with wild-type
littermates (Pcm/+ Hct 69.1±12.0% versus +/+ Hct
44.3±3.6%; P<0.001)
(Fig. 1B). The polycythemia in
heterozygous mutants was transient in nature, as the Hct corrected to baseline
by 12 weeks of age. Strikingly, homozygous mutants were remarkable for a
congenital anemia at postnatal day 0 (P0) (Pcm/Pcm Hct
22.4±5.3% versus +/+ Hct 44.8±9.4%; P<0.001). The
homozygous mutants corrected for the significant perinatal anemia by 7 weeks
of age, and were indistinguishable from wild-type and heterozygous Hct levels
by 12 weeks of age (Fig. 1B).
Additionally, there was no evidence for involvement of non-erythroid
hematopoietic cell lineages in polycythemic mutant animals (data not shown).
Wild-type animals were remarkable for a nadir in Hct at 3 weeks of age,
consistent with the physiological anemia observed in young mice
(Bechensteen and Halvorsen,
1996).
|
A 58 bp microdeletion in the Fpn1 promoter represents the Pcm mutation
Severe embryonic anemia has been reported for a loss-of-function allele of
the zebrafish homologue of Fpn1
(Donovan et al., 2000), an
evolutionarily highly conserved iron transporter mapping to within 1 Mb of
D1Mit236 (Fig. 1B). Given the
presence of severe perinatal anemia in Pcm homozygous animals,
Fpn1 represented the strongest candidate gene for Pcm.
However, in contrast to mutant human
(Montosi et al., 2001;
Njajou et al., 2001;
Cazzola et al., 2002;
Devalia et al., 2002;
Arden et al., 2003;
Jouanolle et al., 2003;
Rivard et al., 2003) and
zebrafish (Donovan et al.,
2000) alleles, we detected no coding region mutations in
Fpn1. Instead, a 58 bp microdeletion was detected immediately
upstream to the transcription start site, in close proximity to putative
promoter elements, such as a TATA box (Fig.
2A). The microdeletion, comparable in size to other
radiation-induced lesions (Grosovsky et
al., 1988; Miles and Meuth,
1989), was absent in the parental strains and co-segregated
invariably with the mutant phenotype (Fig.
2B).
|
).
Importantly, mutant RACE products revealed multiple transcription start sites,
both upstream and downstream of the wild-type start site. For example, in a
subset of transcripts, initiation occurred 845 bp upstream of the wild-type
transcription start site, followed by splicing downstream of the IRE, with no
evidence of in-frame, upstream translation initiation sequences
(Fig. 2A). Other transcripts
initiated downstream of the IRE, but upstream of the translation initiation
codon, retaining the wild-type open reading frame. In total, only one of 10
cDNAs retained the IRE present in the Fpn1 wild-type 5'UTR
(Fig. 2A) (Abboud and Haile, 2000;
Donovan et al., 2000;
McKie et al., 2000).
Importantly, mutant transcript abundance was not affected
(Fig. 2D).
Abnormal iron balance in Pcm mice
Several mouse models of defective iron balance are characterized by a
severe, congenital anemia (Bannerman et
al., 1973; Bernstein,
1987; Nicolas et al.,
2002a). Indeed, peripheral blood smears from anemic,
Pcm/Pcm mice were remarkable for hypochromic, microcytic erythrocytes
at P0 (Fig. 3A). Additionally,
quantitative analysis of red cell indices at P0 demonstrated significantly
lowered MCV [Pcm/Pcm MCV 75.2±2.7 fl (n=10), versus
+/+ MCV 104±6.4 fl (n=6); P<0.0001] and MCHC
[Pcm/Pcm MCHC 21.2±4.1 g/dl (n=10), versus +/+ MCHC
29.3±3.3 g/dl (n=6); P<0.01] in homozygous
mutants. Furthermore, whereas P0 wild-type hepatocytes contained significant
iron stores, Pcm/Pcm hepatocytes were devoid of stainable iron
(Fig. 3B). Quantification of
organismal iron levels at P0 showed that mutant pups were iron-deficient, most
severely in homozygous mutants (Fig.
3C). Strikingly, the perinatal iron deficiency progressively
reversed to an iron overload phenotype during postnatal development, resulting
in a predominantly localized hepatic iron accumulation reminiscent of a
reticuloendothelial macrophage distribution pattern at 12 weeks of age
(Fig. 3D)
(Yang et al., 2002). By
contrast, no iron accumulation was observed in livers from wild-type
littermates (Fig. 3D).
Quantification of hepatic iron demonstrated a significant, graded elevation in
iron levels at 12 weeks of age, highest in Pcm homozygous mutant
animals (Fig. 3E). Both at P0
and 12 weeks of age, heterozygous animals exhibited intermediate levels of
hepatic iron staining (data not shown), consistent with tissue iron
quantification (Fig. 3C,E).
Decreased serum iron in Pcm mutant animals at 7 and 12 weeks of age
in the context of hepatic iron accumulation
(Fig. 3D,E) is consistent with
increased tissue iron sequestration and/or use during erythropoiesis
(Fig. 3F).
|
). Recently, similar in vitro culture methods have been shown
to closely approximate in vivo differentiation of erythroid progenitors
(Zhang et al., 2003).
Homozygous mutant bone marrow demonstrated significantly increased erythroid
differentiation in the presence of Epo at 3 weeks of age
(Fig. 4B), consistent with
normalization of the P0 anemia by early adulthood
(Fig. 1B). At 7 weeks of age,
o-dianidisine staining for hemoglobinized cells in heterozygous bone
marrow sections demonstrated hypercellularity and increased abundance of
erythrogenic foci (Fig. 4C).
Additionally, heterozygous bone marrow showed significantly elevated erythroid
differentiation in vitro (Fig.
4D), in agreement with marrow-derived red cell production leading
to polycythemia. By 12 weeks of age, consistent with the resolution of the
transient alterations in hematocrit observed in both heterozygous and
homozygous mutant animals (Fig.
1C), no statistically significant differences in kidney
Epo transcript levels were detected
(Fig. 4E). Additionally, the
microcytosis observed at P0 (Fig.
3A) reversed to moderately elevated MCV at 12 weeks of age in both
heterozygotes [Pcm/+ MCV 44.8±1.2 fl (n=10), versus
+/+ MCV 42.4±0.7 fl (n=4); P<0.01] and homozygotes
[Pcm/Pcm MCV 46.7±2.3 fl (n=6); P<0.01],
whereas no statistically significant differences in MCHC were observed (data
not shown), indicating resolution of hypochromia.
|
; McKie et al.,
2000; Liu et al.,
2002), aberrant Pcm mRNAs lacking an IRE are likely not
to be repressed by low cellular iron states. Indeed, western analysis
demonstrated a graded increase in hepatic Fpn1 expression at P0, with a
several-fold increase in Fpn1 levels in mutant livers, highest in homozygotes
(Fig. 5A). By contrast, the
expression level of ferritin, which directly correlates with intracellular
iron content via IRE-dependent post-transcriptional regulation
(Hentze et al., 1987) (for a
review, see Aisen et al.,
2001), showed the expected graded decrease in mutant livers,
lowest in homozygotes (Fig.
5A). These results were consistent with decreased hepatic iron
staining and organismal iron levels at P0
(Fig. 3B,C). Similarly, a
graded increase in hepatic Fpn1 protein expression was seen in 3-week-old
mutants (Fig. 5B), whereas low
levels of ferritin expression correlated with a lack of hepatic iron staining
in all genotypes at this stage (data not shown). Thus, in the absence of an
IRE, Pcm mutant Fpn1 transcripts are not subject to
iron/IRE-mediated translational regulation. This is manifested as increased
hepatic Fpn1 protein expression during early postnatal life, representing the
direct effect of the Pcm microdeletion in Fpn1. Based on the
increased Fpn1 protein levels and absence of aberrant Fpn1 isoforms by western
analysis (data not shown), Pcm is considered a hypermorphic allele of
Fpn1.
|
).
Although the majority of heterozygous mutant mice were polycythemic at 7 weeks
of age, a subset of animals (12.3%) displayed reduced phenotypic expressivity
with Hcts of less than 50%. Therefore, we determined whether Fpn1 protein
levels correlated with either Hct subgroup. Indeed, in contrast to
polycythemic heterozygotes, non-polycythemic heterozygotes and homozygotes
demonstrated baseline Fpn1 expression (Fig.
5C,D). Thus, elevated duodenal and hepatic Fpn1 levels are likely
to be permissive for the polycythemia at 7 weeks, regulating increased
duodenal iron uptake and decreased cellular iron storage and sequestration. As
Fpn1 transcript abundance was unaffected
(Fig. 5F), the profound
increase in Fpn1 protein levels at 7 weeks of age results from a predominantly
post-transcriptional, rather than transcriptional, mechanism of Fpn1
regulation. Furthermore, this regulatory effect appears to be superimposed on
the primary defect caused by the microdeletion in the Fpn1 promoter
region, i.e. a moderate, graded increase in Fpn1 expression because of the
absence of the IRE (Fig. 5A,B).
By 12 weeks of age, Fpn1 protein levels were downregulated in all genotypes,
whereas increased ferritin expression in mutants reflected increased hepatic
iron localization (Fig. 5E),
confirming hepatic iron accumulation (Fig.
3D,E).
Delayed upregulation of Hamp in polycythemic mutant mice at 7 weeks of age
Recent studies in mice and individuals with juvenile hemochromatosis have
implicated Hamp as the hormonal regulator of iron homeostasis
(Nicolas et al., 2001;
Park et al., 2001;
Pigeon et al., 2001;
Nicolas et al., 2002a;
Ahmad et al., 2002;
Nicolas et al., 2003;
Roetto et al., 2003).
Hamp is expressed at very low levels during most of embryonic and
early postnatal development, and displays significant upregulation by P56
(Nicolas et al., 2002a).
Strikingly, at 7 weeks of age, low levels of Hamp expression were
restricted to polycythemic Pcm mutant mice, whereas upregulation of
Hamp correlated with normal Hct
(Fig. 5F). By 12 weeks of age,
Hamp expression was upregulated in all animals
(Fig. 5G), correlating with
normalization of Hct in both mutant genotypes as well as downregulation of
Fpn1 protein expression (Fig.
5E). Western analysis for Fpn1 and RT-PCR for Hamp were
representative: of the high Hct mutants tested (Hct>60%), 12 of 13 animals
showed high Fpn1 protein levels, while 11 of 13 animals expressed low
Hamp mRNA levels. Conversely, in the low Hct cohort (Hct<50%), all
animals tested (n=8) displayed both downregulated Fpn1 protein levels
and upregulated Hamp expression. All wild-type samples (n=6)
exhibited low Fpn1 protein and upregulated Hamp transcript
levels.
Normalization of hepatic iron accumulation in aged mutant animals
Downregulation of Fpn1 protein levels in Pcm mutant animals at 12
weeks of age (Fig. 5E) would be
predicted to abrogate organismal iron uptake due to Fpn1-mediated duodenal
iron absorption, eventually normalizing iron levels with time. In fact, an
aged cohort of heterozygous mutant animals demonstrated normalization of
hepatic iron levels by 8-10 months of age
(Fig. 3E). This correlated with
greatly decreased hepatic iron staining, characterized by residual
reticuloendothelial localization (data not shown). Interestingly, heterozygous
mutant serum iron levels at this time point were lowered significantly versus
wild type (Fig. 3F), consistent
with tissue iron sequestration resulting from Hamp regulation. Additionally,
Pcm/+ mutants were anemic [Pcm/+ Hct 36.8±2.4%
(n=8), versus +/+ Hct 42.9±2.5% (n=11);
P<0.0001]. Thus, low serum iron levels and sequestration of iron
in reticuloendothelial stores are reminiscent of the anemia of chronic disease
(for a review, see Means,
2003), and appear to be the disease endpoint in Pcm
mutant animals.
|
Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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; McKie et al.,
2000; Liu et al.,
2002), to our knowledge, this is the first report demonstrating
the in vivo role for the Fpn1 IRE. Indeed, there is precedence for
IRE-based mutations causing human disease. The initial mutation identified in
hereditary hyperferritinemia-cataract syndrome involved a point mutation in
the IRE located in the 5'UTR of L-ferritin
(Girelli et al., 1995).
Subsequently, numerous mutations have been identified within the L-ferritin
IRE, including a 29 bp deletion predicted to abolish IRE-mediated regulation
of protein translation (Girelli et al.,
1997). To date, no mutations affecting IRE function have been
identified in the FPN1 locus. Our results suggest that regulatory
mutations may comprise a novel subset of FPN1 alleles, and should be
suspected when features of type IV hemochromatosis are present in the absence
of coding region mutations.
Changes in iron homeostasis induced by acute inflammatory stimuli require
Hamp upregulation (Nicolas et
al., 2002b), which leads to decreased duodenal iron uptake and
increased reticuloendothelial macrophage iron sequestration (for a review, see
Ganz, 2003). Additionally,
downregulation of Fpn1 protein expression in the duodenum and
reticuloendothelial macrophages in response to acute inflammatory stimuli
(Yang et al., 2002) has been
suggested to be mediated by Hamp-regulation of Fpn1 protein expression
(Nicolas et al., 2002a;
Nicolas et al., 2003).
Furthermore, duodenal Fpn1 expression is responsive to systemic regulation,
rather than to local iron levels (Chen et
al., 2003). We provide strong in vivo evidence for this systemic
effect, as high Fpn1 protein levels (Fig.
5C,D) correlate with low Hamp expression
(Fig. 5F) at 7 weeks, whereas,
by contrast, high levels of Hamp expression at 7 and 12 weeks
(Fig. 5G) correlate with low
Fpn1 protein expression (Fig.
5E). Furthermore, as Fpn1 transcript levels appear
unaffected in either 7- or 12-week-old livers
(Fig. 5F,G), our data point to
post-transcriptional regulation of Fpn1 by Hamp, capable of achieving
profound effects on Fpn1 protein levels, as evidenced by polycythemic
heterozygotes at 7 weeks of age (Fig.
5D).
As downregulation of Fpn1 protein expression occurs normally in homozygous
livers, despite the absence of an IRE in mutant Fpn1 transcripts, our
results suggest an IRE-independent mechanism of Fpn1 regulation by
Hamp. It has been suggested that transcriptional, in addition to
post-transcriptional, mechanisms play an important role in regulating Fpn1
expression (Liu et al., 2002;
Yang et al., 2002;
Zoller et al., 2002;
Chen et al., 2003). However,
in the absence of appreciable differences in Fpn1 transcript
abundance (Fig. 5F), it is
possible that a primarily post-transcriptional, IRE-independent mode of
regulation leads to the profound differences in Fpn1 protein levels in
Pcm mutant mice (Fig.
5C). Although the precise nature of this regulation is unknown,
these data may point to a direct interaction between Fpn1 and Hamp proteins as
a potential mechanism.
Based on our results, we propose that persistent Fpn1 expression caused by
delayed postnatal Hamp upregulation forms the basis for the elevated
iron uptake conducive for augmented erythropoiesis in Pcm mutant
animals. As Hamp is a negative regulator of iron balance, and Hamp
expression was upregulated by 12 weeks of age across all genotypes
(Fig. 5G), further organismal
accumulation of iron should cease and iron levels should decrease over time.
Indeed, by 8-10 months of age, Pcm heterozygous mutants exhibited a
significant decrease in hepatic iron content
(Fig. 3E). This contrasts with
the persistent iron accumulation in aged HFE- and Hamp-deficient
animals (Nicolas et al., 2001;
Lebeau et al., 2002;
Nicolas et al., 2003).
Importantly, chronic failure to induce Hamp expression in the context
of iron overload has been shown to be the likely mechanism of dysregulated
iron balance in HFE mutant mice (Ahmad et
al., 2002; Nicolas et al.,
2003). Conversely, return of hepatic iron content to baseline in
aged Pcm heterozygotes (Fig.
3E) is indicative of intact Hamp regulation in Pcm mutant
animals. Additionally, findings of anemia and low serum iron are consistent
with Hamp-mediated, reticuloendothelial iron sequestration, similar to the
anemia of chronic disease, also termed the anemia of inflammation (for a
review, see Jurado, 1997).
Elevation of Epo expression observed in Pcm mutants rules out a
primary polycythemia, such as polycythemia vera (for a review, see
Prchal, 2001). Although the
observed increase in Fpn1 protein expression would appear adequate to supply
sufficient iron required for the augmented erythropoiesis in Pcm
mutant animals, the observed polycythemia in Pcm heterozygous animals
is most likely a secondary, Epo-dependent phenomenon. To explain the increased
Epo expression observed in Pcm mutant mice that leads to the
transient polycythemia, we propose a model by which increased Fpn1 protein
expression and concomitant intracellular iron deficiency leads to Hamp
downregulation. At birth, homozygous mutant animals are severely iron
deficient, leading to hypochromic, microcytic anemia, while heterozygotes have
decreased but sufficient iron stores, permitting near-wild-type Hct. Increased
Fpn1 protein expression in Pcm mutants during early postnatal life,
i.e. the primary effect of the microdeletion in the Pcm promoter
region, causes augmented cellular iron efflux, e.g. in the liver and kidney,
exacerbating further the cellular iron deficiency. This should lead to the
following distinct molecular consequences in cell types involved in hypoxia
and iron sensing:
In the first component of the model, cellular iron deficiency is known to
mimic hypoxia (for a review, see Semenza,
1998), sensing of which requires an iron-dependent prolyl
hydroxylase (Epstein et al.,
2001; Ivan et al.,
2001; Jaakkola et al.,
2001). Thus, increased Fpn1-mediated iron efflux in hypoxia
sensing cells in the liver and kidney leads to inappropriate induction of Epo
expression, as observed at 3 weeks of age
(Fig. 4A). This effect of
increased Epo production is ineffective in homozygotes until the severe
perinatal iron deficiency resolves as a consequence of increased Fpn1-mediated
duodenal uptake during early postnatal development. By contrast, as sufficient
iron for productive erythropoiesis is present, elevated Epo expression in
heterozygotes results in increased erythropoiesis. Although not fully defined
as a clinical entity, mild but significant increases in red cell indices,
including hematocrit, have indeed been observed in the context of iron
deficiency in infants and young children
(Aslan and Altay, 2003).
In the second component of the model, within the iron sensor of the liver
(for a review, see Ganz,
2003), Fpn1-mediated efflux would be sensed as low cellular iron
levels. In a regulatory response, this leads to repression of the negative
hormonal regulator Hamp. Based on the concept that Hamp negatively regulates
Fpn1 protein levels (Nicolas et al.,
2002a; Nicolas et al.,
2003), which is strongly supported by the present data, decreased
Hamp expression would lead to further de-repression of Fpn1 protein levels in
Pcm mutant animals, e.g. as seen at 7 weeks of age. This superimposed
regulatory mechanism further exacerbates cellular iron deficiency in
Fpn1-expressing cells. Concomitantly, in the duodenum, increase in Fpn1
protein expression driven by Hamp downregulation leads to increased iron
absorption during early postnatal life in these mutants, culminating in tissue
iron accumulation by 12 weeks of age (Fig.
3D,E). This represents a paradoxical state in which low iron
levels are sensed cellularly, despite significant organismal iron accumulation
in Pcm mutant animals. Additionally, recent data suggest Epo itself
may downregulate Hamp expression
(Nicolas et al., 2002c). Thus,
Hamp downregulation in polycythemic Pcm mutant animals could
be compounded by elevated Epo levels.
By 12 weeks of age, Hamp is upregulated across all genotypes, including the heterozygotes (Fig. 5G). This leads to decreased duodenal and reticuloendothelial Fpn1 protein expression, and, hence, decreased duodenal iron uptake and sequestration in the reticuloendothelial system. Over time, this would reduce organismal iron load, which is indeed observed in aged heterozygous mutant animal (Fig. 3E). The mechanism of Hamp upregulation in the heterozygous population observed at 12 weeks of age is unknown, yet correlates with return of Epo levels to baseline (Fig. 4E), and is indicative of the disruption of this feed-forward mechanism and reversion of the organismal phenotype over time. Hence, this dynamic model reconciles the molecular findings of Fpn1 and Hamp expression with the changes in iron homeostasis and aberrant erythropoiesis in Pcm mutant animals during postnatal development.
The significant iron deficiency observed at birth in Pcm mutants represents an unexpected finding relative to the augmented Fpn1-mediated organismal iron uptake that is characteristic of early postnatal life. Strikingly, our preliminary data reveal decreased Fpn1 expression in Pcm mutant placenta, consistent with the perinatal iron deficiency, and suggest differential developmental regulation of Fpn1 expression (H.M. and A.S., unpublished).
At present, it is unclear why the transient polycythemia is characteristic of the heterozygous as opposed to the homozygous mutant population. Perhaps in the absence of sufficient iron for productive erythropoiesis during crucial early postnatal timepoints, homozygotes are unable to undergo the significant erythropoiesis observed in heterozygous animals, despite upregulation of Epo expression during early postnatal development (Fig. 4B).
In summary, at birth, Pcm mutant animals are iron deficient. Owing to a gain-of-function mutation causing elevated Fpn1 protein levels in different cellular compartments postnatally, Pcm mutant animals display increased organismal iron uptake during early postnatal life, culminating in significant hepatic iron accumulation by young adulthood. Additionally, heterozygous mutant animals exhibit a transient polycythemia secondary to elevated Epo expression. Hamp upregulation by young adulthood correlates with downregulation of Fpn1 protein expression, preceding the reversion of the iron overload phenotype. Decreased Fpn1-mediated iron uptake in the duodenum abrogates organismal iron accumulation, while decreased Fpn1-mediated iron efflux from reticuloendothelial macrophages leads to sequestration, dynamically explaining the contrasting phenotypes during early postnatal and adult life in Pcm mutant mice.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Aventis Pharmaceuticals, Bridgewater, NJ 08807, USA
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