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RED CELLS
The Nramp2/DMT1 iron transporter is induced in the duodenum of microcytic
anemia mk mice but is not properly targeted to the intestinal brush border
François Canonne-Hergaux, Mark D. Fleming, Joanne E. Levy, Susan Gauthier, Trevor Ralph, Virginie Picard,
Nancy C. Andrews, and Philippe Gros
Microcytic anemia (mk) mice and Belgrade (b) rats are severely iron deficient
because of impaired intestinal iron absorption and defective iron metabolism in
peripheral tissues. Both animals carry a
glycine to arginine substitution at position 185 in the iron transporter known as
Nramp2/DMT1 (divalent metal transporter
1). DMT1 messenger RNA (mRNA) and
protein expression has been examined in
the gastrointestinal tract of mk mice.
Northern blot analysis indicates that, by
comparison to mk/ⴙ heterozygotes,
mk/mk homozygotes show a dramatic
increase in the level of DMT1 mRNA in the
duodenum. This increase in RNA expression is paralleled by a concomitant increase of the 100-kd DMT1 isoform I protein expression in the duodenum.
Immunohistochemical analyses show
that, as for normal mice on a low-iron diet,
DMT1 expression in enterocytes of mk/mk
mice is restricted to the duodenum. However, and in contrast to normal enterocytes, little if any expression of DMT1 is
seen at the apical membrane in mk/mk
mice. These results suggest that the
G185R mutation, which was shown to
impair the transport properties of DMT1,
also affects the membrane targeting of
the protein in mk/mk enterocytes. This
loss of function of DMT1 is paralleled by a
dramatic increase in expression of the
defective protein in mk/mk mice. This is
consistent with a feedback regulation of
DMT1 expression by iron stores. (Blood.
2000;96:3964-3970)
© 2000 by The American Society of Hematology
Introduction
Homeostasis of iron is extremely well balanced, thereby providing
sufficient iron for cellular functions, and yet preventing a toxic
excess of the metal. There is no physiologic pathway for iron
excretion, and absorption into the intestine is assumed to have a
primary role in regulating whole body iron stores.1,2 Intestinal iron
uptake occurs in the proximal portion of the small intestine
(duodenum)3,4 where it involves at least 2 transport steps: one from
the lumen across the apical membrane of absorptive epithelial cells,
termed the mucosal uptake, and another across the basal membrane
of enterocytes into the blood stream, termed mucosal transfer.5
Recently, the characterization of genes and proteins affected in
inherited disorders of iron metabolism in humans and in animal
models has clarified the mechanistic basis of iron transport in
enterocytes.5 The apical iron uptake system (Nramp2/DCT1,
recently renamed DMT1) of enterocytes was identified by several
groups.6-8 DMT1 is a divalent metal transporter identified by
functional cloning in Xenopus laevis oocytes as an electrogenic
metal transporter of broad substrate specificity, transporting Fe2⫹,
Zn2⫹, Mn2⫹, and other ions.6 DMT1-mediated Fe2⫹ transport was
shown to be pH dependent and coupled to proton symport.
Independently, the DMT1 gene was found to be mutated in 2 rodent
models of iron deficiency, the microcytic anemia (mk) mouse and
the Belgrade (b) rat.7,8 Both the mk mouse and the b rat exhibit
severe microcytic, hypochromic anemia because of a defect in iron
uptake in the intestine and utilization in peripheral tissues, includ-
ing red cell precursors.9 The DMT1 gene produces 2 alternatively
spliced transcripts generated by differential use of two 3⬘ exons
encoding distinct C-termini of the protein as well as distinct 3⬘
untranslated regions (UTRs).8,10 Interestingly, one DMT1 messenger RNA (mRNA) contains an iron responsive element (IRE) in its
3⬘ UTR. The second DMT1 splice isoform does not contain an IRE.
It encodes a protein (isoform II) in which the C-terminal 18 amino
acids of the IRE form (isoform I) are replaced by a novel 25 amino
acid segment.8,10 Protein11 and mRNA expression studies6,12 indicate that the IRE-containing isoform I is the one expressed in the
proximal portion of the duodenum, where it is dramatically
up-regulated by dietary iron deprivation. DMT1 expression is
restricted to the distal half of the villi, where it localizes to the brush
border of absorptive epithelial cells.11
DMT1 is an integral membrane protein composed of 12
transmembrane (TM) domains, several of which contain charged
residues. This structural unit defines a protein family highly
conserved from bacteria to man,13,14 including the closely related
macrophage-specific mammalian homologue Nramp1 (78% similarity)15. In macrophages, Nramp1 is recruited to the phagosomal
membrane,16 where it may modulate divalent metal content to
affect microbial replication.16,17 Indeed, naturally occurring
(G169D)18 or experimentally induced mutations19 in Nramp1
abrogate natural resistance to infection with intracellular parasites.
The mk mouse and the b rat carry the same mutation at the
From the Department of Biochemistry, McGill University, Montreal, Canada;
Division of Hematology/Oncology, Children’s Hospital, Boston, MA; Howard
Hughes Medical Institute, Boston, MA; Division of Hematology and Department
of Pathology, Brigham and Women’s Hospital, Boston, MA; and Department of
Pediatrics, Harvard Medical School, Boston, MA.
Research Council of Canada. N.C.A. is an associate investigator of the HHMI.
Submitted January 31, 2000; accepted July 28, 2000.
Supported by National Institutes of Health (NIH) grant AI35237 (P.G.) and
partially supported by NIH grants DK53813 (N.C.A), HL03600 (M.D.F.), and
HL03503 (J.E.L.). P.G. is an International Research Scholar of the Howard
Hughes Medical Institute (HHMI) and a Senior Scientist of the Medical
3964
Reprints: Philippe Gros, Department of Biochemistry, McGill University, 3655
Drummond, Room 907, Montreal, Quebec, Canada, H3G-1Y6; e-mail:
[email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2000 by The American Society of Hematology
BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
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BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
DMT1 locus, a nonconservative glycine to arginine substitution
that introduces a bulky, positively charged residue into predicted
TM4 of the protein.7,8 Transient expression studies in HEK293T
cells have shown that the G185R DMT1 mutant is severely
impaired and cannot significantly stimulate Fe2⫹ uptake.20 Surprisingly, the G185R mutation in DMT1 maps to a residue that is
adjacent to the corresponding Nramp1 residue mutated G169D in
inbred mouse strains susceptible to infections. In macrophages
from mouse strains bearing a Nramp1G169D allele, no mature protein
is detected,21 suggesting that this mutation causes protein instability and degradation. The similarity in nature and position of these
naturally occurring loss-of-function mutations in Nramp1 and
Nramp2/DMT1 suggests that these 2 adjacent glycine residues play
an important structural or functional role. In the present report, we
have analyzed the expression, tissue distribution, and subcellular
localization of the G185R DMT1 variant in the intestine of anemic
mk mice to gain insight into the molecular basis of defect in
intestinal iron absorption in these mice.
Materials and methods
Animals
MK/ReJ-mk/⫹ were originally obtained from the Jackson Laboratory and
subsequently maintained as an inbred stock by breeding male homozygotes
(Nramp2/DMT1G185R/G185R) to obligate female heterozygotes, as previously
described.7 Animals were maintained on a standard rodent diet in the animal
facility at Children’s Hospital (Boston, MA). At the time of the study,
mk/mk homozygotes had significantly reduced liver iron compared with
mk/⫹ animals (49.4 ⫾ 23 ␮g/g versus 88.71 ⫾ 5 ␮g/g). The 129sv mice
were initially purchased from Taconic Farms (Germantown, NY) and
subsequently maintained as a breeding colony in the Animal Care Center at
McGill University (Montreal, QC, Canada). For iron depletion experiments, control inbred 129sv mice (Nramp2/DMT1⫹/⫹) were fed either a
low-iron diet (⫺Fe; ICN, Montreal, QC, Canada) or an identical diet
supplemented with 3% ferric phosphate (⫹Fe; ICN) for 8 weeks before
isolation of tissues.11 Decreased levels of plasma ferritin (128 ⫾ 20 ng/L for
low iron compared to 225 ⫾ 34 ng/L for normal diet) in addition to
decreased expression of ferritin protein in proximal duodenum and liver
(data not shown) suggest that mice kept on the low-iron diet indeed become
iron deficient.
Tissue preparation
For immunoblotting and immunohistochemistry, the first portion of the
small intestine (approximately 4 cm) from mk/⫹ and mk/mk mice was
harvested and dissected in 2 equal segments, I1 and I2, corresponding to the
proximal and distal duodenum, respectively, as previously described.11 A
distal segment (approximately 2 cm) of the small intestine, I3, corresponding to the ileum, as well as a segment (approximately 2 cm) of the colon
were also dissected (see Figure 2 in Canonne-Hergaux et al11). Tissue
samples were snap-frozen in liquid nitrogen and used to prepare crude
membrane fractions or used to isolate RNA. Fresh tissues were also fixed in
Bouin solution (picric acid 9 g/L, acetic acid 4%, methanol 3.6%, and
formaldehyde 25%) for immunohistochemical studies.
Cell culture
LR73 Chinese hamster ovary (CHO) cells22 were grown in ␣-minimal
essential medium supplemented with 10% fetal calf serum, 2 mmol/L
L-glutamine, 50 U/mL penicillin, and 50 ␮g/mL streptomycin. A DMT1
isoform II complementary DNA (cDNA) modified by the insertion of an
antigenic c-Myc epitope at the carboxy terminus23 was introduced in LR73
cells. A cell clone stably expressing the tagged DMT1-cMyc protein was
isolated and propagated as previously described.24
DMT1 EXPRESSION IN THE INTESTINE OF mk MICE
3965
RNA isolation and hybridization studies
Total mouse duodenal RNA was isolated from the proximal 2 cm of
duodenum (I1), using RNA STAT-60 (Leedo Medical Laboratories, Houston, TX). Total RNA (10 ␮g) was electrophoresed on a 1% agarose, 0.7%
formaldehyde gel and blotted onto Hybond N (Amersham, Piscataway, NJ).
DMT1 expression was detected, using a hybridization probe corresponding
to positions 731-1240 of DMT1 (Genbank L33415) incubated in UltraHyb
(Ambion, Austin, TX) and washed according to the manufacturer’s
instructions. The blot was stripped and reprobed with either a probe,
consisting of the noncoding portion (1.6 kilobase [kb]) of a mouse
transferrin receptor cDNA (kindly provided by Dr P. Ponka, Lady Davis
Institute, Montreal, QC, Canada) or with a cDNA probe, consisting of the
entire open reading frame of mouse ␤-actin.
Crude membrane extracts
Crude membrane fractions from cultured CHO cells and from portions of
the gastrointestinal tract were prepared, exactly as we have previously
described.11 Briefly, tissue homogenates and CHO cell lysates were
centrifuged at 6000g for 15 minutes (4°C) to eliminate nuclei and intact
cells, and membrane fractions were recovered from the supernatant by
centrifugation at 80 000g for 60 minutes at 4°C. Final pellets were
resuspended in sucrose histidine buffer (0.25 mol/L sucrose, 0.03 mol/L
histidine, pH 7.2), supplemented with protease inhibitors, and stored frozen at
⫺80°C until use. Protein concentrations of the various membrane fractions
were determined by the Bradford assay (commercially supplied by Biorad,
Hercules, CA).
Production and purification of DMT1 antibodies
For the production of rabbit polyclonal antisera to analyze isoforms I and II
of DMT1 (IRE: isoform I and non-IRE: isoform II), 2 glutathione-Stransferase (GST) fusion proteins were constructed. The first protein
contained a DMT1 peptide segment corresponding to the amino terminus
(residues 1 to 73) that is identical in isoforms I and II, and a second
consisted in GST fusion containing the carboxy terminal region (residues
532-568) of isoform II. The respective polyclonal antiserum generated
against these immunogens were purified against the same DMT1 peptide
segments (NT and CT) fused to dihydrofolate reductase, as previously
described.11 The specificities of the anti–DMT1-NT and anti–DMT1-CT
sera were established by immunoblotting.11
Immunoblot analysis
Crude membrane preparations from tissues (80 ␮g of protein) or control
CHO cells (5 ␮g of protein) were separated by sodium dodecylsulfate–
polyacrylamide gel electrophoresis (SDS-PAGE; 10% polyacrylamide) and
transferred by electroblotting to polyvinylidene fluoride membranes (16
hours, 4°C). For DMT1 detection, samples were incubated for 30 minutes at
room temperature in 1⫻ Laemmli sample buffer (with occasional vortexing) prior to SDS-PAGE because, as for other integral membrane proteins,
heat treatment of DMT1-containing samples was found to cause aggregation of the protein. For transferrin receptor and Biliary glycoprotein 1
(Bgp1) immunodetection, samples in 1⫻ Laemmli sample buffer were
boiled for 5 minutes prior to SDS-PAGE. Similar loading on gel and similar
transfer of proteins to the membrane was verified by staining the blots with
Ponceau S Red (Sigma, St Louis, MO). Immunoblots were pre-incubated
with blocking solution (0.02% Tween20, 7% skim milk in phosphatebuffered saline [PBS]) for 2 hours at 20°C prior to incubation with primary
antibodies for 16 hours at 4°C in blocking solution. Primary antibodies
were used at the following concentrations: rabbit anti–DMT1-NT, 1/200;
rabbit
anti–
DMT1-CT, 1/100; rat monoclonal antimouse transferrin receptor (TfR),
1/500 (Biosource International, Camarillo, CA); rabbit polyclonal antiBgp1 (provided by Dr N. Beauchemin, McGill University), 1/4000.
Membranes were washed (PBS ⫹ 0.2% Tween20) and then incubated with
peroxidase-labeled antirat or antirabbit secondary antibodies (1/10 000;
Amersham, Buckinghamshire, England; 1 hour, 20°C). The signals were
visualized by Enhanced ChemiLuminescence (ECL, Amersham). In certain
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3966
CANONNE-HERGAUX et al
BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
experiments, membranes were stripped (100 mmol/L ␤2-mercaptoethanol,
2% SDS, 62.5 mmol/L Tris-HCl pH 6.8; 50°C, 30 minutes) and then
reprobed with a different primary antibody.
Immunohistochemistry
Portions of the gastrointestinal tract were fixed in Bouin solution (72 hours,
20°C). They were then dehydrated in a series of ethanol (3 ⫻ 20 minutes:
70%, 95%, 100%), ethanol/xylene (1/1), and xylene solutions, followed by
embedding in paraffin. Sections (5 ␮m) were cut and mounted with gelatin
on glass slides. Immunohistochemical staining was performed, using the
peroxidase-antiperoxidase procedure, as previously described.11 Dilutions
of antibodies were as follows: DMT1-NT, 1/40; DMT1-CT, 1/25; nonimmune serum, 1/40; and anti-Bgp1, 1/500. Following incubation with the
primary antibody, 3 washes in PBS, and incubation with the secondary
antibody (swine antirabbit immunoglobulin G, 1:100; DAKO, Carpinteria,
CA), subsequent rabbit peroxidase-antiperoxidase immunostaining (PAP,
1:100; DAKO) was revealed, using 3⬘-diaminobenzidine tetrahydrochloride (DAB) in solution, followed by counterstaining with 0.1% methylene
blue in PBS.
Results
The mRNA and polypeptide corresponding to the IRE-containing
isoform I of DMT1 are normally expressed at low levels throughout the intestine, but their expression is dramatically increased in
the duodenum on dietary iron deprivation.6,11 This finding suggests
a possible feedback regulatory loop of DMT1 isoform I by iron
stores. Thus, the current study aimed at analyzing the effect of
DMT1 G185R loss-of-function mutation on the regulation of
expression of the DMT1 mRNA and protein. In addition, the effect
of the G185R mutation on cellular and subcellular localization of
DMT1 protein was investigated.
Figure 1. DMT1 mRNA expression in intestine from anemic mk/mk mice. The
proximal portion of the small intestine immediately adjacent to the stomach was
dissected from mk/⫹ heterozygotes and mk/mk homozygous anemic mice (2 animals
in each group). Total RNA from each tissue was prepared, separated by electrophoresis through a denaturing agarose gel, and followed by blotting to a hybridization
membrane. The blot was probed with a DMT1 cDNA probe that recognizes both the
IRE (isoform I) and non-IRE (isoform II) containing mRNAs (A) and was washed
under highly stringent conditions. After removal of the probe, the blot was sequentially
rehybridized with probes for TfR (B) and ␤-actin (C).
DMT1 mRNA is overexpressed in duodenum of mk /mk mice
The effect of the G185R mutation on the level of DMT1 mRNA
expression was studied by Northern blot analysis. The duodenum
was dissected from 4- to 5-week-old mk/mk mice and from their
normal mk/⫹ heterozygous littermates. Total RNA was prepared,
and equal amounts of duplicate duodenum RNA samples (2
animals in each group mk/⫹ or mk/mk) were separated in denaturing formaldehyde gels and analyzed by Northern blot analysis,
using a DMT1 probe (nucleotide position 731-1240). Results in
Figure 1A indicate that the DMT1 probe detects low-level expression of a 3- to 3.5-kb mRNA species in the duodenum of mk/⫹
controls. However, DMT1 mRNA expression is dramatically
increased in RNA samples from mk/mk duodenum. The level of
expression of another transcript containing IREs in its 3⬘ untranslated region, the TfR, was also found increased in mk/mk mice
when compared with the heterozygotes mk/⫹ (Figure 1B). Results
from hybridization to a control ␤-actin probe (Figure 1C) indicate
that similar amounts of RNA were loaded on the gel and transferred
to the hybridization membrane. These results indicate that DMT1
and TfR mRNA are up-regulated in the duodenum of anemic
mk/mk mice.
DMT1 protein is overexpressed in duodenum of mk /mk mice
The mouse DMT1 gene produces 2 alternatively spliced mRNAs
that differ at their 3⬘ ends. We have raised an anti-DMT1 rabbit
polyclonal antiserum (DMT1-NT) against the N-terminus of DMT1
(identical in isoforms I and II) and a similar antiserum directed
against the C-terminus of isoform II (the non-IRE isoform). With
the use of these antibodies in combination, we have shown that the
IRE-containing DMT1 isoform I is the predominant isoform
expressed in the intestine.11 The effect of the G185R mutation on
the level of DMT1 protein expression was initially studied by
immunoblotting. For this process, crude microsomal membrane
fractions from intestinal segments of anemic mk/mk homozygous
animals and control mk/⫹ heterozygotes were analyzed by immunoblotting with anti-DMT1 antibody, DMT1-NT (Figure 2A). Two
additional controls were included for comparison with mk/mk and
mk/⫹ tissues. The first consisted of crude microsomal fractions of
proximal duodenum (I1) from normal (⫹/⫹) mice fed either a
normal (lane 1) or a low-iron diet (lane 2). The second control was
membranes from CHO cells that stably expressed DMT1 protein
(CHO-DMT1 isoform II, lane 12) or the parental, untransfected cell
line (CHO, lane 11). The specificity of the DMT1-NT antibody is
shown by the presence of a single immunoreactive species of
approximate molecular mass 90 to 100 kd in the CHO-DMT1
transfectant that is absent in CHO control membranes.
A low level of DMT1 was detected in membrane fractions
from proximal duodenum of normal mice fed a normal diet
(Figure 2, lane 1) and from proximal (I1, lane 3) and distal
duodenum (I2, lane 5) of control heterozygous mk/⫹ animals.
As previously described,11 depletion of dietary iron results in a
dramatic increase in expression of DMT1 in the duodenum (lane
2) of normal mice. Similarly, the level of DMT1 expression was
clearly increased in membrane fractions from the proximal (I1,
lane 4) and to a lesser extent the distal portion of the duodenum
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BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
DMT1 EXPRESSION IN THE INTESTINE OF mk MICE
3967
indicate that DMT1 isoform I expression is strongly increased in
the duodenum of anemic mk/mk mice.
The same microsomal membrane fractions were also analyzed
for expression of the TfR (Figure 2B) and Bgp1 (Figure 2C), a cell
surface adhesion molecule expressed in epithelial cells throughout
the intestinal tract.26 In normal mice (⫹/⫹) and in mk/⫹ heterozygotes, TfR was expressed as an approximately 90-kd protein
species in all intestinal segments tested. Interestingly, in mk/mk
mice, TfR was present at higher levels than in mk/⫹ heterozygotes.
This finding was observed for all intestinal segments tested, but it
was particularly apparent in the duodenum. These observations at
the TfR protein levels are in agreement with results from Northern
blot analysis (Figure 1B). TfR expression level also increased in
duodenum from iron-depleted mice (Figure 2B, compare lane 2
with lane 1). However, Bgp1 was expressed at high levels in all
intestinal sections of ⫹/⫹, mk/⫹, and mk/mk mice (Figure 2C),
indicating that protein degradation, unequal loading, or unequal
transfer to the blot were unlikely to be responsible for differences in
levels of DMT1 and TfR expression detected between wild-type
and mk/mk mice.
Figure 2. Expression of DMT1 protein in the gastrointestinal tract of anemic
mk/mk mice. Proximal (I1) and distal (I2) sections of the duodenum as well as ileum
(I3) and colon (C) were harvested from heterozygous mk/⫹ (lanes 3, 5, 7, and 9) or
homozygous mk/mk mice (lanes 4, 6, 8, and 10). Microsomal fractions were prepared
as previously described11 and 80 ␮g samples of membrane proteins were resolved
on a 10% acrylamide gel and transferred to a polyvinylidene fluoride membrane. For
comparison, microsomal fractions isolated from proximal duodenum (I1) of wild type
mice (⫹/⫹) fed a normal diet (lane 1) or a low-iron diet (lane 2) were included in the
analysis. To demonstrate the specificity of the anti-DMT1 antibody, 5 ␮g of membrane
proteins from CHO cells (lane 11) or CHO cells expressing cMyc-tagged DMT1
isoform II (lane 12) were also included. Immunoblotting was performed with
antibodies raised against the amino terminus of DMT1 (DMT1-NT) (A), the transferrin
receptor (B), and Bgp1 proteins (C). The positions and sizes (in kilodaltons) of
molecular mass markers are indicated on the right.
(I2, lane 6) of homozygous mk/mk mice, when compared with
control mk/⫹ heterozygotes (I1, lane 3; I2, lane 5). Although
strongly induced, the level of expression of DMT1 in mk/mk
duodenum appears lower than that observed in iron-depleted
animals (compare lanes 4 and 2). The electrophoretic mobility
of DMT1 from tissues of iron-deficient mice (lane 2) was
indistinguishable from that of the DMT1 G185R protein seen in
tissues from mk/mk mice (lanes 4 and 6). These results suggest
that the G185R mutation does not grossly impair posttranslational modification of the DMT1 protein, including glycosylation to 90 to 100 kd from a 56-kd precursor.24,25 In agreement
with previous studies in normal mice,11 no DMT1 protein was
detected with our antibody in more distal portions of the small
intestine (I3; lanes 7 and 8) or colon (C; lanes 9 and 10) in either
mk/⫹ or mk/mk samples. In duodenal samples containing
increased amounts of DMT1 (lanes 2, 4, and, to lesser extent,
lane 6), other immunoreactive species of faster electrophoretic
mobility were also detected by the DMT1-NT antiserum (Figure
2A). Other species of low apparent molecular weight (⬍ 45 kd)
were not seen consistently and tended to increase in proportion
to repeated freezing and thawing of the specimens. This finding
suggests that they correspond to degradation products of the
mature 90-kd polypeptide, as previously observed elsewhere.11
In lane 2 of Figure 2, an approximately 60-kd band is seen and
could correspond to the nonglycosylated precursor of DMT1
that is only observed when DMT1 expression is extremely high.
Finally, the DMT1-NT reactive protein detected in tissues from
normal11 or mk/mk mice (data not shown) did not react with the
DMT1-CT antiserum. Together, these immunoblotting results
Cellular and subcellular localization of DMT1 in mk /mk
mouse duodenum
Cellular and subcellular localization of the DMT1 protein was
investigated by immunostaining fixed sections of duodenum from
control heterozygous mk/⫹ (Figure 3D-F) or homozygous mk/mk
mice (Figure 3G-I). As a positive control in these studies, duodenal
sections from wild type (⫹/⫹) animals maintained on a low-iron diet
(⫺Fe) were also examined (Figure 3A-C). Freshly dissected tissues
were fixed in Bouin solution, embedded in paraffin, and immunostained with either rabbit anti–DMT1-NT polyclonal antiserum
(Figure 3A,D,G), rabbit anti–DMT1-CT specific for the non-IRE
isoform II (Figure 3B,E,H), normal pre-immune rabbit serum (not
shown), or a rabbit anti-Bgp1 antiserum (Figure 3C,F,I). Sections
were then counterstained with methylene blue and examined under
400 ⫻ magnification.
As we previously reported,11 intense DMT1 staining in the
duodenum of control mice fed a low-iron diet (Figure 3A) was
limited to villi with no expression in the lamina propria or in
intestinal crypts of Lieberkuhn. In villi, DMT1 was mostly
localized at the luminal surface (Figure 3A, black arrow) and to a
much lesser extent in the apical cytoplasm (Figure 3A, arrowhead)
of villus cells. In heterozygous mk/⫹ mice, no DMT1 staining was
observed in the columnar epithelial cells of the villi (Figure 3D).
This result was anticipated, considering the low level of DMT1
protein expression detected in this tissue by immunoblotting
(Figure 2A, lane 3). However, strong DMT1 staining was detected
in duodenal villi from homozygous mk/mk mice (Figure 3G).
Again, DMT1 staining was restricted to the villi and was absent in
both the vascular and lymphatic cells of the lamina propria and in
crypts cells. In contrast to the staining seen in iron-deficient mice,
DMT1 staining in mk/mk enterocytes was largely intracellular,
mostly in the apical side of epithelial cells (Figure 3G, arrowhead)
with little or no brush border staining (Figure 3G). Incubation with
either normal preimmune serum (not shown) or with the anti–
DMT1-CT antiserum (Figure 3B,E,H) produced no detectable
staining, indicating that the IRE-containing isoform I of DMT1 is
the one up-regulated in iron-deficient mice and in mk/mk homozygotes. Occasionally, staining was detected with the anti–DMT1-NT
antibody in rare crypts cells (Figure 3D, white arrowhead). This
latter staining appeared only after prolonged DAB reaction times
and was independent of iron status of animals.
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3968
BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
CANONNE-HERGAUX et al
homozygotes (Figure 4C) that was restricted to the absorptive
enterocytes with no expression in mucus-secreting goblet cells
(white arrows). In wild type mice (Figure 4A-B), DMT1 was
concentrated at the brush border (black arrows) but also was found
to a lesser extent within the intracellular apical area (arrowheads)
of villus enterocytes. In mk/mk mice (Figure 4C-D), DMT1 was
present in a strong intracellular pattern, predominantly in the apical
half of the epithelial cells (arrowheads). However, little or no
staining of DMT1 was apparent at the luminal surface of mk/mk
enterocytes, suggesting that little of the protein reached the brush
border where it is normally expressed in iron-deficient controls.
Together, these results establish that DMT1 protein is abundantly expressed in the duodenum of mk/mk mice, but it is
not efficiently targeted to the brush border of the epithelial
absorptive cells.
Discussion
Figure 3. Immunohistochemical staining of DMT1 in the intestine of anemic
mk/mk mice. Tissues were fixed in Bouin solution, embedded in paraffin, and
sectioned as described.11 Sections from the proximal duodenum (I1) of wild type mice
on low-iron diet [⫹/⫹ (⫺Fe); A, B, and C], heterozygous mk/⫹ (D, E, and F), and
homozygous mk/mk mice (G, H, and I) were immunostained with rabbit polyclonal
antisera directed against the amino terminus of DMT1 (DMT1-NT; A, D, and G), or the
carboxy terminus of isoform II (non-IRE containing) DMT1 (DMT1-CT; B, E, and H), or
biliary glycoprotein 1 (Bgp1; C, F, and I). Sections were stained with DAB,
counterstained with methylene blue, and photographed at a magnification of 400 ⫻.
Arrows in (A) show intense DMT1 staining at the brush border (arrows) and
intracellularly in the apical region of villus enterocytes (arrowhead). In mk/⫹ mice (D),
DMT1 is not detectable. In mk/mk mice (G), strong DMT1 staining is apparent in the
apical region of villus enterocytes (arrowhead) but not at the brush border. In (C), (F),
and (I), arrows identify Bgp1 staining at the brush border (black arrows), in the
supranuclear, intracellular region (white arrows) of villus enterocytes as well as in
crypts cells (arrowhead). The development time for DAB was generally longer for
mk/⫹ and mk/mk (4-6 minutes) than for low-iron diet (3 minutes), indicating that the
level of DMT1 expression is stronger in normal mice on low-iron diet than in mk/mk
homozygotes.
Anti-Bgp1 antiserum was used as a positive control in these
sections because Bgp1 is known to be highly expressed in the
mouse gastrointestinal tract. Bgp1 protein is localized to the brush
border of crypts and villus epithelial cells.27,28 As expected (Figure
3C,F,I), Bgp1 was detected in all tissue sections at the luminal
surface of the epithelium (black arrows), at the supranuclear, at the
intracellular region of villi epithelial cells (white arrows), and in
the crypts of Lieberkuhn (arrowheads).
Subcellular localization of DMT1 protein in villus epithelial
cells from mk/mk mice was further studied by examination of
additional sections under high magnification (Figure 4C-D). Similar sections from iron-depleted wild type mice were used as
controls [⫹/⫹ (⫺Fe); Figure 4A-B]. Analysis of transverse (Figure
4A,C, ⫻ 600) and longitudinal (Figure 4B,D, ⫻ 1000) sections of
villi show a similar cellular distribution of DMT1 protein expression in normal, iron-depleted mice (Figure 4A) and in mk/mk
In the present study, we have investigated the consequences of loss
of function of DMT1 on the level of expression and on cellular and
subcellular localization of the protein in enterocytes from irondeficient mk/mk mice. We report that mk/mk mice show a dramatic
increase in the expression of DMT1 mRNA in the duodenum
associated with a concomitant increase in isoform I DMT1 protein
expression. In contrast to the situation observed in iron-deficient
wild-type animals, little of the overexpressed DMT1 protein is
found at the brush border of mk/mk enterocytes.
The dramatic induction of DMT1 mRNA and protein observed
in animals deprived of dietary iron6,11 and in mk/mk mice (this
study) suggests a feedback regulatory loop for regulation of DMT1
expression by iron stores. One possibility is that up-regulation of
DMT1 may be through the action of the IRE located in the 3⬘ UTR
of isoform I. IREs mediate changes in protein levels in response to
Figure 4. Subcellular localization of DMT1 in enterocytes from anemic mk/mk
mice. Sections from proximal duodenum (I1) from either wild-type mice on a low-iron
diet [⫹/⫹ (⫺Fe); panels A and B] or mk/mk mice (panels C and D) were analyzed for
DMT1 expression with a rabbit polyclonal antiserum directed against the amino
terminus of DMT1, and examined under high magnification (600 ⫻, panels A and C;
1000⫻, panels B and D). Transverse (A and C) and longitudinal (B and D) sections
are shown. In iron-depleted ⫹/⫹ mice (A and B), DMT1 is concentrated at the brush
border (arrow) and also detectable in the apical region (arrowhead) of villus
enterocytes. In mk/mk mice (C and D), DMT1 staining is intracellular (arrowhead) and
is not (or weakly) detectable at the brush border. In all sections, mucus-secreting
goblet cells remain negative (white arrows).
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BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
DMT1 EXPRESSION IN THE INTESTINE OF mk MICE
iron availability.29,30 When iron is scarce within cells, IRE-binding
proteins (IRP1 and IRP2) are available to bind IREs. It has
previously been established that IRP binding to IREs in the 3⬘ UTR
of the TfR mRNA increases message stability and translation.29 The
observations that in iron-deprived animals, the nonheme iron
content of enterocytes and the IRP activity in the duodenum
decreased and increased, respectively,31,32 and that transferrin
binding activity and both RNA and protein expression of TfR are
increased in wild-type deficient animals31 (this study; Figure 2B)
and in mk/mk mice (this study; Figures 1B and 2B) are consistent
with a regulatory role for IRPs in the up-regulation of DMT1. In
duodenal crypts cells of iron-deprived and of mk mice, the level of
intracellular iron decreases, reflecting the change in body iron
status. We speculate that this results in programming of the
developing enterocyte to absorb more iron when it arrives at the
villus. The mechanism of programming may in part be related to
induction of IRP activity, resulting in stabilization of DMT1
mRNA and consequent up-regulation of apical iron transport
function. However, we do not have direct evidence of such a
regulatory mechanism. Therefore, we cannot exclude the possibility that regulation of DMT1 expression operates without the
participation of the IRE element itself; rather it may reflect changes
at the transcriptional or translational level. Indeed, no absolute
correlation between the level of DMT1 mRNA and protein
expressed could be established in mk/⫹ and mk/mk mice. Interestingly, and despite a dramatic induction of DMT1 expression, the
rate of iron absorption increases modestly (3-fold to 8-fold) in
iron-deficient animals when compared to normal groups.31,32 This
observation also points to possible roles of other proteins in the
regulation of intestinal iron absorption, most likely ferrireductase
or the basal membrane iron transporter,33 that could also be rate
limiting in iron acquisition.
Immunohistochemical localization studies of DMT1 protein in
mk/mk mice indicate that the mutated protein is highly expressed in
absorptive enterocytes of the villi. This pattern of tissue and
cellular expression is similar to that seen for the wild-type protein
in normal mice fed a low-iron diet. However, and in contrast to the
wild-type protein, the G185R variant of DMT1 was not expressed
at the brush border of duodenal enterocytes, but rather was present
intracellularly (Figures 3 and 4). Thus, although the G185R
mutation clearly has a deleterious effect on the transport properties
of the protein measured in vitro,20 it also appears to interfere with
proper targeting to the brush border of the absorptive epithelium in
vivo, indicating that integrity of TM4 is important for proper
targeting of DMT1 to that site. We conclude that replacement of the
glycine residue at position 185 by an arginine affects both the
transport function and the membrane targeting of DMT1. The
mechanism by which the G185R mutation affects targeting of the
protein is unknown, as TM4 does not contain obvious targeting or
sorting signals. Interestingly, transient transfection and expression
of wild type and G185R mutant in human embryonic kidney
HEK293 cells show similar localization of both wild type and
mutant proteins to the plasma membrane and to Tf-positive
recycling endosomes.20 The reason for different results is unclear,
but it may reflect differences in the cell types expressing the mutant
3969
proteins (differentiated mouse enterocytes versus undifferentiated
HEK immortalized human cells20). Moreover, the fact that this
study reflects a physiologic situation as opposed to a transfected
cell clone, and the fact that the effect of the G185R mutation was
assessed on the background of isoform I (this study) as opposed to
isoform II20 may also contribute to this discrepancy.
The Nramp2/DMT1 gene was initially cloned by virtue of its
cross-hybridization to the Nramp1 gene that is involved in macrophage function and host resistance to infection.15,25,34 Interestingly,
a single G169D mutation in predicted TM4 of Nramp1 that
corresponds to the glycine residue immediately adjacent to the
G185R mutation of DMT1 in mk mice impairs resistance of mice to
infection with intracellular parasites. Previous observations21 suggest that the G169D mutation in Nramp1 affects normal processing
of the protein, with possible targeting for degradation. Although
these results with Nramp1 also suggest that integrity of TM4 is
important for proper targeting of Nramp proteins, the G169Dinduced degradation of Nramp1 is different in several respects from
the consequences of the G185R mutation in DMT1 of mk mice.
Indeed, the G185R variant appears quite stable and is readily
detected at high levels in the duodenum of mk mice. Second,
DMT1 is extensively modified by asparagine-linked glycosylation
in primary and transfected cells,24 and this posttranslational
modification is preserved in the G185R mutant, as indicated by the
similar electrophoretic mobility of the wild type and mutant
proteins in duodenal protein extracts (Figure 2). This suggests that
the G185R mutant can pass the quality control check imposed by
the endoplasmic reticulum and Golgi apparatus. Thus, as opposed
to the G169D Nramp1 variant, the G185R mutation in DMT1 does
not markedly affect stability or maturation of the protein. However,
we observed higher level DMT1 protein expression in the normal
mice fed a low-iron diet than in mk/mk homozygotes mice. Thus,
we cannot formally exclude the possibility that the G185R
mutation may indeed have a certain effect on protein stability in
mk/mk mice. The balance between the up-regulation of RNA
expression and the degradation of the mutant protein may lead to a
detection of DMT1 in mk mice that is important but lower than the
one observed in iron-depleted wild type mice.
The present study shows that the G185R mutation in DMT1
affects targeting of the protein to the brush border of the intestinal
epithelium, the normal physiological site for iron uptake. In mutant
mk/mk homozygotes, this loss of function at DMT1 contributes to
the systemic iron deficiency and the severe anemia characteristic of
these mice. In response to their iron deficiency, there is a dramatic
increase in expression of the mutant DMT1 protein in mk/mk
homozygotes, most likely mediated by a feedback regulatory
mechanism sensing the level of body iron stores.
Acknowledgments
The authors are indebted to Dr N. Beauchemin (McGill University, Montreal, QC, Canada) and to Dr P. Ponka (Lady Davis Institute,
Montreal, QC, Canada) for the generous gifts of the anti-Bgp1 antiserum and the transferrin receptor cDNA sequence, respectively.
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2000 96: 3964-3970
The Nramp2/DMT1 iron transporter is induced in the duodenum of
microcytic anemia mk mice but is not properly targeted to the intestinal
brush border
François Canonne-Hergaux, Mark D. Fleming, Joanne E. Levy, Susan Gauthier, Trevor Ralph,
Virginie Picard, Nancy C. Andrews and Philippe Gros
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