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RED CELLS
From the Departments of Biochemistry, Physiology, and
Medicine, McGill University, and the Lady Davis Institute for Medical
Research, Jewish General Hospital, Montreal, QC, Canada.
Divalent metal transporter 1 (DMT1) is the major
transferrin-independent iron uptake system at the apical pole of
intestinal cells, but it may also transport iron across the membrane of
acidified endosomes in peripheral tissues. Iron transport and
expression of the 2 isoforms of DMT1 was studied in erythroid cells
that consume large quantities of iron for biosynthesis of hemoglobin. In mk/mk mice that express a loss-of-function mutant
variant of DMT1, reticulocytes have a decreased cellular iron uptake
and iron incorporation into heme. Interestingly, iron release from transferrin inside the endosome is normal in mk/mk
reticulocytes, suggesting a subsequent defect in
Fe++ transport across the endosomal membrane.
Studies by immunoblotting using membrane fractions from peripheral
blood or spleen from normal mice where reticulocytosis was induced by
erythropoietin (EPO) or phenylhydrazine (PHZ) treatment suggest that
DMT1 is coexpressed with transferrin receptor (TfR) in erythroid cells. Coexpression of DMT1 and TfR in reticulocytes was also detected by
double immunofluorescence and confocal microscopy. Experiments with
isoform-specific anti-DMT1 antiserum strongly suggest that it is
the non-iron-response element containing isoform II of DMT1 that
is predominantly expressed by the erythroid cells. As opposed to
wild-type reticulocytes, mk/mk reticulocytes express little if any DMT1, despite robust expression of TfR, suggesting a possible effect of the mutation on stability and targeting of DMT1 isoform II in
these cells. Together, these results provide further evidence that DMT1
plays a central role in iron acquisition via the transferrin cycle in
erythroid cells.
(Blood. 2001;98:3823-3830) The divalent metal transporter 1 (DMT1), also known
as natural resistance-associated macrophage protein 2 (Nramp2) or
divalent cation transporter 1 (DCT1), is a protein recently
shown to play a pivotal role in iron uptake from both transferrin (Tf)
and non-Tf sources in different anatomic sites.1,2 DMT1 is
an integral membrane protein formed by 12 predicted transmembrane (TM)
domains, several of which contain charged residues. This structural
unit defines a protein family highly conserved from bacteria to
man3,4 and that includes the closely related
phagocyte-specific homologue Nramp1 (78% similarity) involved in
macrophage function and resistance to infections.5
The DMT1 gene encodes 2 messenger RNAs (mRNAs; isoforms I
and II) produced by alternative splicing of two 3' exons, resulting in
transcripts with different 3' untranslated regions (UTRs) and encoding
proteins with distinct C-termini.6,7 One of the DMT1
mRNAs, isoform I, is predicted to contain an iron-responsive element
(IRE). DMT1 isoform II encodes a protein in which the C-terminal 18 amino acids derived from IRE-containing mRNA are replaced by a novel
25-amino acid segment.6,7
Studies in Xenopus laevis oocytes have shown that DMT1
(isoform I) is an electrogenic transporter of divalent cations
including Fe++, Zn++, Mn++, and
others.8 Studies in vitro in cultured mammalian cells have
also demonstrated that both DMT1 isoforms can transport a variety of
divalent cations at the plasma membrane, including Fe++.9-11 In mammalian cells, DMT1-mediated
Fe++ transport was shown to be dependent on pH and coupled
to proton symport.8,11,12 On the other hand,
microfluorescence imaging studies in primary macrophages with a
metal-sensitive fluorescent probe have recently established that Nramp1
also acts as a divalent cation efflux pump at the phagosomal
membrane.13 Genetic studies in rodent models of iron
deficiency and microcytic anemia have shown that DMT1 is
mutated (Gly185Arg) in the mk mouse and in the Belgrade
(b) rat.6,14 Both the mk mouse and
the b rat exhibit severe microcytic, hypochromic anemia due
to a defect in iron uptake in the intestine but also in iron
acquisition and utilization in peripheral tissues, including red blood
cell (RBC) precursors.15,16
In the intestine, expression studies of mRNA8,17 and
protein18 indicate that the DMT1 IRE-containing isoform I
is 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.18
Interestingly, mk/mk mice show a dramatic increase in
expression of the Gly185Arg mutant variant of DMT1 (isoform I) in the
duodenum, but little of the overexpressed protein is detected at the
brush border of mk/mk enterocytes,19 suggesting
that the Gly185Arg mutation impairs not only the transport
properties,9 but also membrane targeting of the protein in
mice. In mk and b animals, the Tf-dependent iron
uptake by erythroid precursors is also impaired.16,20,21 Immature erythroid cells are the most avid consumers of iron in mammals, for use in hemoglobin synthesis. Although non-Tf-bound iron
uptake has been described in vitro,22 most of the iron is
delivered to developing erythroid cells from Tf via receptor-mediated endocytosis.23 This involves binding of diferric Tf to
transferrin receptor (TfR), internalization of Tf within endocytic
vesicles, and the release of iron from the protein following endosomal
acidification.23,24 The metal is then transported through
the endosomal membrane via a Fe++ transporter, a step found
to be impaired in b reticulocytes.22,25-27 In
different cell lines, including murine erythroleukemia (MEL) cells, as
well as in Chinese hamster ovary (CHO), HEK293, and RAW cells
expressing a transfected DMT1 complementary DNA (cDNA), the protein is
detected primarily in Tf-positive compartments, a colocalization that
supports a role for DMT1 in Tf-derived iron uptake within acidified
endosomes.9,28
Therefore, a large body of experimental data support the proposal that,
in addition to its demonstrated role in iron acquisition at the
intestinal brush border, DMT1 also transports iron across the membrane
of acidified endosomes into the cytoplasm. The defect in iron
acquisition noted in peripheral tissues of mk and
b animals, in particular in reticulocytes, suggests an
important role of DMT1 in erythroid cells precursors as well. Here, we
used 2 anti-DMT1 antibodies to examine expression of the 2 DMT1
isoforms in reticulocytes and erythrocytes from either normal mice,
mice treated with phenylhydrazine (PHZ) or erythropoietin (EPO) , and
from anemic mk/mk mice. Our results show abundant expression
of DMT1 isoform II in erythroid cell precursors, suggesting a key role
of this isoform in Fe aquisition and heme biosynthesis. The poor
expression of DMT1 in mk erythroid cells, together with the
demonstrated loss of transport activity associated with the Gly185Arg
mutation,9 may both contribute to the severe impairment of
iron uptake in mk/mk erythroid cells leading to hypochromic
microcytic anemia.
Animal care
Treatment with PHZ and EPO
Cell culture The CHO LR73 cells and MEL cells were cultured as previously described.18,30 The c-Myc-tagged DMT1 (isoform II), and Nramp1 cDNAs inserted in expression plasmid pMT2 were introduced into CHO cells by transfection using calcium phosphate coprecipitation method. The isolation and characterization of cell clones expressing high levels of each protein, including preparation of membrane fractions and immunoblotting were as described.18Salicylaldehyde isonicotinoyl hydrazone and 2,2'-dipyridyl interception experiments Reticulocytes (50 µL packed cells) obtained from mk/mk or EPO-treated mk/+ mice were suspended in 250 µL minimal essential medium (MEM) containing 25 mM HEPES,10 mM NaHCO3, pH 7.4. 59Fe-Tf was added to the final concentration of 5 µM (in terms of Tf concentration). In appropriate groups, salicylaldehyde isonicotinoyl hydrazone (SIH) and 2,2'-dipyridyl (DP; Sigma, St Louis, MO) were added to the final concentration of 100 µM and 1 mM, respectively. Cells were then incubated for 60 minutes at 37°C in a shaking water bath, followed by 3 washes in ice-cold phosphate-buffered saline (PBS) at 4°C. In parallel experiments, 59Fe-Tf was incubated with MEM (without reticulocytes) in the presence of either SIH or DP. Heme-59Fe was extracted by methyl-ethyl ketone,31 and its radioactivity was measured using a gamma counter. For 59Fe-SIH or 59Fe-DP determinations, 200 µL cell mixture was lysed with 600 µL H2O, and proteins were precipitated (ethanol 100%, 20°C, 1 hour). After centrifugation (3000 rpm, 20 minutes),
59Fe-SIH or 59Fe-DP radioactivities in the
supernatant were measured. Reticulocyte-mediated transfer of
59Fe to SIH or DP is expressed as a difference between
radioactivities in the samples containing reticulocytes and those
incubated without the cells. Following ethanol treatment, the
precipitate contains 59Fe associated with protein (Tf,
ferritin, hemoglobin, etc), whereas the supernatant contains
59Fe-SIH or 59Fe-DP.
Preparation of RBC membranes The RBCs (0.4 mL heparinized mouse blood) were washed several times with PBS (5 minutes, 5000-8000g), and the final pellet was resuspended in 2.5 mL of a lysis buffer containing 10 mM Tris-HCl buffer (pH 7.5) and protease inhibitors (PIs; 2 µg/mL leupeptin; 2 µg/mL aprotinin, 1 µg/mL pepstatin; 100 µg/mL phenylmethylsulfonyl fluoride [PMSF] and 2 mM EDTA). The homogenate was then centrifuged at 14 000g for 10 minutes and this was repeated 3 times. The final "ghost" pellet was resuspended in 200 µL of a solubilization buffer (5 mM Tris-HCl, pH 7.5, 0.5% Triton X-100 and PIs) and stored frozen at80°C. All procedures were
carried out at 4°C. Protein concentration of various membrane
fractions was determined by the Bradford assay
(Bio-Rad).
Immunoblotting Crude membrane preparations from tissues (80-120 µg protein), RBCs (80 µg), MEL cells (100 µg), or CHO cells (5 µg protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10% polyacrylamide), and transferred by electroblotting to polyvinylidene fluoride membrane. Because heat treatment of DMT1-containing samples was found to cause aggregation of the protein, samples were incubated for 30 minutes at room temperature in Laemmli buffer (with occasional vortexing) prior to SDS-PAGE. Similar loading and transfer of proteins were verified by staining the blots with Ponceau S (Sigma). Immunoblots were preincubated with blocking solution (0.02% Tween 20, 7% skim milk in PBS) for 3 hours at 20°C, prior to incubation with primary antibodies for16 hours at 4°C in blocking solution. Primary antibodies recognizing either both DMT1 isoforms (anti-DMT1-NT; 1:200), or DMT1 isoform I (anti-DMT1-CT; 1:100) or a rabbit anti-Nramp1-NT (1:200), and rat monoclonal anti-mouse TfR (1:500) were used for immunoblotting exactly as we have previously described.18,28Immunofluorescence For confocal microscopy, CD1 mice were injected with EPO (100 U/d for 3 days), and blood was taken 48 hours later. Following centrifugation of blood, buffy coat was removed. In the RBC fraction, no white blood cells (mononuclear and polymorphonuclear cells) were found in smears by Giemsa staining. Cells were resuspended in MEM supplemented with 10 mM NaHCO3 and 25 mM HEPES (pH 7.4) at 15 × 106 reticulocytes/mL followed by 30 minutes of incubation at 37°C to eliminate endogenous Tf. Cells were washed once with PBS and resuspended in MEM containing Tf (Alexa-Tf; 500 nM; Molecular Probes, Eugene, OR). Cells were incubated 30 minutes on ice, followed by 20 minutes at 37°C and 3 washes with PBS at 4°C. Cells were fixed with paraformaldehyde (30 minutes at 4°C), washed 3 times in PBS, and permeabilized with 0.05% NP-40 (Sigma) in PBS containing 1% bovine serum albumin (BSA) (Sigma) and 5% normal goat serum (Life Technologies, Montreal, QC, Canada) at 4°C for 30 minutes. Cells were blocked for 1 hour at room temperature in PBS containing 1% BSA, 10% goat serum, 10% CD1 mouse serum in PBS (1 hour, 20°C). Cells were washed and incubated with anti-DMT1-NT antibody (1:100, 1 hour, 20°C) followed by washing and incubation with the secondary rhodamine-coupled donkey anti-rabbit antiserum (1:200). Colocalization studies were performed using a Bio-Rad scanning confocal fluorescence microscope and digitizing equipment, as we have previously described.28
DMT1 expression in spleens and reticulocytes from EPO-treated, PHZ-treated, and mk/mk mice Erythroid precursors are avid consumers of iron during heme biosynthesis, and animals bearing loss-of-function mutations in the DMT1 gene show a profound defect in iron acquisition by peripheral tissues, including RBC precursors.16,20,21,32 Therefore, the aims of the present study were to identify the cellular and subcellular site of DMT1 expression in mature or precursors RBCs and analyze its expression in genetically anemic mice (mk/mk), and in response to experimental treatments known to induce erythropoiesis.The 129sv mice were treated with EPO or PHZ, 2 agents known to
induce reticulocytosis. EPO directly stimulates erythropoiesis, whereas
PHZ causes profound anemia, which triggers vigorous EPO-mediated erythropoiesis both in the spleen and bone marrow. Erythropoiesis response was monitored after EPO and PHZ treatments (see "Materials and methods"), by recording splenomegaly (spleen index [SI]), and
enumerating reticulocytes in blood samples (percent of total RBCs;
Figure 1). Compared to saline-injected
control mice, both EPO and PHZ treatments induced strong
reticulocytosis (Figure 1A) consistent with splenomegaly (Figure 1B).
Both responses were stronger in PHZ-treated (35% reticulocytes;
SI = 1.3) than in EPO-treated mice (15% reticulocytes;
SI = 0.9). The microcytic anemia induced in mk/mk mice by
loss of DMT1 function is concomitant to enhanced reticulocytosis.
Indeed, homozygous mk/mk mice show increased splenomegaly
(SI = 1) and increased reticulocytosis (11.4%, 35.8%, and 8.3%)
when compared with normal mk/+ heterozygotes 0.4,% 0.8%,
and 0.8%) or 129sv controls (SI
Although impaired iron assimilation by erythroid cells of
mk/mk mice has been reported,20,21 defects in
Tf-mediated iron transport are poorly characterized in these animals.
To clarify mechanisms underlying impaired utilization of Tf-bound iron
by mk/mk erythroid cells, reticulocytes from
mk/mk and mk/+ (following EPO treatment) mice
were incubated with 59Fe-labeled Tf following which
59Fe uptake by the cells and its incorporation into heme
were measured. Two permeable Fe chelators, SIH (binds Fe+++
and Fe++) and DP (binds Fe++ only) were
exploited to estimate relative levels of Fe+++ and
Fe++ present in endosomes of reticulocytes from
mk/+ and mk/mk mice. In these experiments, no
significant difference was seen in total cellular iron uptake between
wild-type +/+ (9.7 ± 0.1 pmoles/106 cells per hour) and
mk/+ (9.6 ± 0.3 pmoles/106 cells per hour).
On the other hand, as compared to mk/+ cells, mk/mk reticulocytes had decreased cellular 59Fe
uptake (4.4 ± 0.5 versus 9.6 ± 0.3 pmoles/106 cells
per hour) and exhibited a significant inhibition of 59Fe
incorporation into heme (3 ± 0.2 versus 8.3 ± 0.1
pmoles/106 cells per hour; Figure
2). The reduced rate of heme synthesis in
mk/mk mice was not due to a defect in heme synthesis pathway because the heme synthesis rate can be brought to the control levels by
means of non-Tf Fe donor (Fe-SIH, not shown). Neither SIH nor DP
affected Tf cycle (data not shown) but these chelators completely
inhibited heme synthesis in both mk/+ and mk/mk
reticulocytes. Importantly, the rates of 59Fe transfer from
59Fe-Tf to DP or SIH were not decreased in mk/mk
reticulocytes, suggesting that in mk/mk reticulocytes the
endosomal Fe metabolism (Fe release from Tf and its reduction by a
putative Fe+++ reductase) are functionally normal. These
results indicate that the basic defect in mk/mk
reticulocytes, as in those from b
rats,22,25-27 resides in an impaired Fe++
transport efficiency across the endosomal membrane.
Thus far, the expression of DMT1 protein in erythroid cells including
cellular and subcellular localization, has not been studied. Membrane
fractions were prepared from spleens of normal, PHZ-treated (Figure
3A), or EPO-treated animals (Figure 3B),
as well as from mk/+ and mk/mk mutants (Figure
3C), separated by SDS-PAGE and analyzed by immunoblotting.
Immunodetection was done with an affinity-purified polyclonal rabbit
anti-DMT1 antiserum directed against the amino terminus of the protein
(DMT1-NT) (Figure 3Ai,Bi,Ci). Membrane fractions from CHO cells
transfected with either Nramp1 (CHO-Nramp1) or DMT1 isoform II
(CHO-DMT1) were included as negative and positive controls,
respectively. Low-level expression of immunoreactive DMT1 of apparent
molecular mass 70 kd was detected in spleen membranes from control mice
(
DMT1 isoform II is expressed in the spleen of PHZ-treated mice By alternative splicing, DMT1 produces 2 products that can be distinguished by the presence (IRE, isoform I) or absence (non-IRE, isoform II) of an IRE in the 3' UTR, as well as distinct C-terminal peptide sequences (Figure 4A). To gain information into which of the 2 DMT1 isoforms is expressed during erythropoiesis in the spleen, an immunoblot containing membrane fractions from spleens of control and PHZ-treated animals was analyzed with an isoform II-specific rabbit anti-DMT1 antiserum (anti-DMT1-CT) (Figure 4A,B). This anti-DMT1-CT antiserum detected a specific immunoreactive product in the PHZ-treated spleen (Figure 4B; open arrowhead), with apparent electrophoretic mobility ( 70 kd) similar to the DMT1 species detected by the
anti-DMT1-NT antiserum (Figure 3). The DMT1 isoform II observed in the
spleen migrated considerably faster than the same protein overexpressed
in CHO transfectants (Figure 4B; filled arrowhead).
DMT1 isoform II protein is expressed in normal RBC membrane Expression of DMT1 protein was then measured in membrane fractions (erythrocyte ghosts) prepared directly from heparinized blood of normal mice, treated or not with EPO (Figure 5A) or PHZ (Figure 5B). DMT1 expression was also monitored in Friend erythroleukemia virus-transformed MEL erythroblastoid cells (Figure 5C). Immunoblots were analyzed using either the anti-DMT1-NT (Figure 5Ai,Bi,Ci; recognizes isoforms I and II), or the anti-DMT1-CT (Figure 5 Aii,Bii,Cii; isoform II specific) or anti-TfR antisera (Figure 5Aiii,Biii,Ciii). The DMT1-NT-reactive 70-kd protein (Figure 5Ai,Bi) was expressed at low levels in circulating RBCs from normal mice, and its expression was dramatically increased in similar samples from PHZ- and EPO-treated animals. The stronger DMT1 expression seen in blood from PHZ-treated than EPO-treated animals parallels both immunoblotting results with spleen membranes (Figure 3), and the extent of reticulocytosis in these groups (Figure 1). The 70-kd DMT1 species was also detected with our anti-DMT1-CT antibody in the RBC membranes after EPO and PHZ treatment (Figure 5Aii,Bii, open arrowheads). An additional prominent cross-reactive species of slower mobility was also detected by this antiserum in membranes from both untreated and treated mice (Figure 5Aii,Bii; asterisks). The identity of this cross-reactive species is currently unknown; however, the size of this protein (> 100 kd), its restricted expression in RBCs, together with its lack of reactivity with the non-isoform-specific DMT1-NT antiserum strongly suggest that it corresponds to a protein unrelated to DMT1 but expressed at high levels in RBCs. The expression of TfR in those samples paralleled that of DMT1 and was strongly enhanced during EPO-induced (Figure 5Aiii) or PHZ-induced (Figure 5Biii) reticulocytosis. Finally, results in Figure 5C show that MEL erythroid cells express abundant levels of DMT1 (70 kd) that is immunoreactive with both the anti-DMT1-NT and -CT antisera indicating high-level expression of isoform II in these cells. Together, these results combined with those obtained with splenic membranes (Figures 3 and 4) identify the erythroid compartment as a normal physiologic site of expression of non-IRE DMT1 isoform II, as opposed to the intestine, which is the primary site of the IRE-containing isoform I.18
Expression of DMT1 protein in mk/mk RBC membranes Expression of DMT1 was analyzed in membrane fractions prepared from heparinized blood of mk/+, mk/mk, and PHZ-treated mk/+ mice (anti-DMT1-NT; Figure 6Ai,Bi). For comparison, DMT1 detection was monitored in parallel using RBC membranes from normal and EPO-treated wild-type mice (Figure 6Ai). The degree of reticulocytosis associated with these treatments (Figure 6Aiii,Biii), and the level of TfR expression in the corresponding samples (Figure 6Aii,Bii) were also measured and are shown. Blood specimens from mk/+ controls show low reticulocyte counts in the normal range (Figure 6Aiii, bar 3; Figure 6Biii, bar 2), and membrane fractions from these samples show low levels of DMT1 (Figure 6Ai,Bi) and TfR expression (Figure 6Aii,Bii). PHZ treatment of mk/+ mice resulted in robust reticulocytosis (Figure 6Biii, bar 1), which was similar to that seen for normal +/+ controls treated with EPO (Figure 6Aiii, bar 2), and was concomitant to a strong increase in DMT1 (Figure 6Bi) and TfR (Figure 6Bii) in RBC membrane fractions from these mice. On the other hand, membrane fractions prepared from heparinized blood of mk/mk mice showed little if any DMT1 expression when compared to normal mk/+ controls (Figure 6Ai,Bi), and this despite constitutive reticulocytosis (Figure 6Aiii, bar 4; Figure 6Biii, bar 3) associated with increased TfR expression in these membrane fractions (Figure 6Aii,Bii). After longer exposures (not shown), DMT1 was poorly detected in membranes prepared from mk/mk RBCs, suggesting only residual expression of the mutated protein. These results indicate that the loss-of-function mutation at DMT1 in mk mice affects not only protein function but may also affect stability or targeting of the protein in reticulocytes.
Subcellular localization of DMT1 in the RBCs The subcellular localization of DMT1 was then investigated in erythroid cells by immunofluorescence, using heparinized blood from normal mice treated with EPO. RBCs were prepared and labeled with Alexa-conjugated Tf (Alexa-Tf) before fixation and immunostaining with the anti-DMT1-NT antiserum (see "Materials and methods"). Analysis by confocal microscopy (Figure 7) indicated that in a small fraction of RBCs (approximately 10%), Tf (green) produced a punctate intracellular staining consistent with a localization in recycling endosomes (Figure 7B,F). It is very likely that these Tf-positive cells correspond to immature erythroid cells (about 10% by methylene blue staining). A similar staining was observed in the same percentage of cells with the anti-DMT1-NT antiserum (red, Figure 7C,G; arrowhead). Superimposition of the 2 images (Figure 7D,H) clearly shows overlapping staining (yellow) of the 2 signals. A large number of cells corresponding to mature erythrocytes did not stain for either DMT1 or Tf (Figure 7B-C, arrows). Together, these results confirm that DMT1 is expressed in erythroid cell precursors and indicate that DMT1 partially colocalizes with Tf in the early recycling endosomal compartment of these cells.
The present studies show that erythroid cell precursors are a major site of expression of DMT1 protein. Western analyses of membrane fractions from spleen, a major site of erythropoiesis in mice, have shown that the level of DMT1 isoform II and TfR expression positively correlate with reticulocytosis induced by EPO and PHZ treatment. Similar results were obtained using membrane fractions prepared from heparinized blood of animals undergoing reticulocytosis. These results suggest concomitant expression of the DMT1 isoform II and TfR in immature erythroid cells, in agreement with the known requirement for iron of these cells. Additional evidence for this was provided by immunofluorescence studies and confocal microscopy that identified DMT1 staining in immature erythroid cells, sharing overlapping distribution with Tf. Expression of DMT1 isoform II in young RBCs and during reticulocytosis suggests that it may transport iron across the membrane of acidified endosomes and into the cytoplasm of erythroid precursor cells. This proposal is in agreement with (1) the overlapping intracellular staining of Alexa-Tf and DMT1 reported here for reticulocytes; (2) the observation that the depressed 59Fe-heme synthesis noted in mk/mk reticulocytes expressing a mutant DMT1 protein does not rest with impaired iron release from Tf inside the endosome, nor reduction of Fe+++ to Fe++ (Figure 2); (3) the recent demonstration that isoform II of DMT1 expressed in CHO cells can transport Fe++ into the calcein-accessible labile iron pool.11 DMT1 plays a key role in iron acquisition at the brush border of the duodenum.14,18,19 As opposed to absorptive cells of intestinal villi that express exclusively the IRE-containing isoform I of DMT1, analysis of erythroid cell (blood and MEL cells) expression with antibodies directed against either the amino terminus (isoform I + II) or the carboxy terminus of DMT1 (isoform II specific) suggest that the non-IRE-containing isoform II is the major DMT1 isoform expressed in erythroid cell precursors. However, because we do not have available an isoform-specific anti-isoform I antibody, the possibility that this isoform is also present at low levels in reticulocyte membranes cannot be excluded. The isoform II of DMT1 expressed in erythroid cells showed an apparent molecular mass of approximately 70 kd, in agreement with the mass predicted from cDNA sequence (62.3 kd). The observed 70-kd mass is smaller than the 80 to 100 kd measured for DMT1 isoform II expressed in transfected CHO cells. Because up to 50% of the molecular mass of the DMT1 isoform II expressed in CHO cells is contributed by N-linked glycosylation,28 it is likely that the differences in electrophoretic mobility of isoform II measured in erythroid versus CHO cells result from distinct posttranslational modifications of the protein. In addition, isoform I of DMT1 expressed at the intestinal brush border of epithelial cells shows a molecular mass of 80 to 90 kd, when analyzed by immunoblotting with the same antibody and the same gel,18,19,34 suggesting likely modification by glycosylation. Such a modification may provide protection against degradative attacks by the intestinal content, or may be important for proper maturation and targeting of the protein to the apical pole of intestinal enterocytes,35 requirements that may not be relevant for DMT1 isoform II expression in the Tf-positive compartment of erythroid cells. Therefore, our results suggest considerable heterogeneity in posttranslational modification of isoforms I and II in different cell types and tissues, perhaps contributing to variability of molecular mass reported for DMT1 by different groups.9,12 Finally, we have also noted large differences in the apparent molecular mass of the close DMT1 homologue Nramp1, as estimated by SDS-PAGE either in primary macrophages,33 or in transfected CHO and RAW.2647,18,36 or in membrane fractions from total spleen (this study), varying between 110 kd, 90 kd, and 65 kd, respectively. The role of posttranslational modifications of DMT1 and Nramp1 by glycosylation, phosphorylation, or others on the activity and transport properties of these proteins remains to be characterized. The DMT1 mRNA coding isoform II does not contain in its 3' UTR the IRE characteristic of isoform I.6,7 IREs are bound by a set of regulatory proteins (IRP, or IRE- binding proteins) that together ultimately regulate stability and translation of mRNAs such as TfR and ferritin in response to iron availability.37-39 Furthermore, it has been proposed that in erythroid cells additional mechanisms such as transcriptional control may play an important role in regulation of TfR expression.23 The absence of IRE in the DMT1 isoform II expressed in reticulocytes suggests that DMT1 may not be regulated by iron status in erythroid cells. The high level of expression of DMT1 and TfR noted in membrane fractions from spleen and circulating RBCs from normal mice undergoing reticulocytosis in response to PHZ and EPO treatment may simply reflect expansion of the TfR/DMT1-positive reticulocyte pool without any modulatory activity at the mRNA or protein levels in response to iron. This proposition is supported by the observation that DMT1 isoform II protein levels in MEL cells are insensitive to exposure of these cells to high concentrations of iron or iron chelators (unpublished results, 2001). This is in sharp contrast to the DMT1 isoform I expressed in duodenum, which is clearly regulated by iron stores possibly via the IRE/IRP system.18,19 The precise characterization of transcriptional or posttranscriptional regulation of DMT1 expression in erythroid cells (if any) remains to be clarified. Loss of function in mk/mk mice is associated with constitutive reticulocytosis, a compensatory response by these mice to the chronic defect in the synthesis of functional heme due to lack of available Fe++. Despite robust reticulocytosis and strong expression of TfR, mk/mk reticulocytes show very little, if any, DMT1 protein. This is in contrast with EPO- and PHZ-treated mice, where expansion of the erythroid pool is concomitant to increased expression of both DMT1 and TfR proteins. Although, we cannot rule out the possibility that the Gly185Arg mutation of mk/mk mice may have an unknown effect on expression of the protein (mRNA stability, translation, or other), these findings suggest that the Gly185Arg mutation may affect either stability or targeting of DMT1 to a functional site in erythroid cells. We have previously reported up-regulation of the Gly185Arg variant (isoform I) expression in mk/mk enterocytes, but the protein was not properly targeted to the brush border, a defect that may contribute to the systemic iron deficiency and the severe anemia in mice.19 Although the DMT1 protein (isoform I) could be readily detected in membrane fractions from mk/mk intestine, and thus is not rapidly degraded, it is possible that the targeting defect associated with the Gly185Arg mutation may have more severe consequences on the stability of the protein in reticulocytes (mostly isoform II) than enterocytes in mk/mk mice. Interestingly, a recent study by immunofluorescence suggested that b/b reticulocytes express similar quantities of DMT1 when compared to normal or b/+ reticulocytes.40 The possible cause of differences between mk mice and b rat remains to be clarified. The Nramp2/DMT1 gene was initially cloned by virtue of its cross-hybridization to the Nramp1 gene.41 In Nramp1, a single Gly169Asp mutation in predicted TM4, impairs macrophage function and causes susceptibility to infections by intracellular pathogens. Interestingly, Gly169Asp in Nramp1 is immediately adjacent to the Gly185Arg mutation in the TM4 of DMT1 in mk mice. The Gly169Asp mutation in Nramp1 results in absence of mature polypeptide, as a result altered targeting and stability of the protein.33 Therefore, there is considerable similarity between the genotype and phenotype of the 2 known loss-of-function missense mutations in these 2 members of the Nramp family. A residual expression of Gly185Arg DMT1 isoform II detected in mk/mk erythroid precursors, in addition to the residual activity of the mutated DMT1 demonstrated by Su and coworkers,9 likely contributes to the survival of homozygotes mk/mk mice. However, mk/mk reticulocytes have about 45% of normal Fe uptake (Figure 2) suggesting that an alternative iron transporter may be active in these cells.
Submitted April 6, 2001; accepted August 6, 2001.
Supported by National Institutes of Health (NIH) grant AI355237 (to P.G.), Canadian Institutes of Health Research (CIHR) grant MT-14100 (to P.P.), and Milestone Medica Corporation Award grant (to F.C-H.) P.G. is an International Research Scholar of the Howard Hughes Medical Institute (HHMI) and a Senior Scientist of the CIHR.
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.
Reprints: Philippe Gros, Department of Biochemistry, McGill University, 3655 William Osler Promenade, Rm 907, Montreal, QC, Canada, H3G-1Y; e-mail: gros{at}med.mcgill.ca.
1. Andrews NC. Iron homeostasis: insights from genetics and animal models. Nat Rev Genet. 2000;1:208-217[Medline] [Order article via Infotrieve]. 2. Wessling-Resnick M. Iron transport. Annu Rev Nutr. 2000;20:129-151[CrossRef][Medline] [Order article via Infotrieve].
3.
Cellier M, Prive G, Belouchi A, et al.
Nramp defines a family of membrane proteins.
Proc Natl Acad Sci U S A.
1995;92:10089-10093
4.
Agranoff D, Monahan IM, Mangan JA, Butcher PD, Krishna S.
Mycobacterium tuberculosis expresses a novel pH-dependent divalent cation transporter belonging to the Nramp family.
J Exp Med.
1999;190:717-724 5. Canonne-Hergaux F, Gruenheid S, Govoni G, Gros P. The Nramp1 protein and its role in resistance to infection and macrophage function. Proc Assoc Am Physicians. 1999;111:283-289[CrossRef][Medline] [Order article via Infotrieve].
6.
Fleming MD, Romano MA, Su MA, Garrick LM, Garrick MD, Andrews NC.
Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport.
Proc Natl Acad Sci U S A.
1998;95:1148-1153 7. Lee PL, Gelbart T, West C, Halloran C, Beutler E. The human Nramp2 gene: characterization of the gene structure, alternative splicing, promoter region and polymorphisms. Blood Cells Mol Dis. 1998;24:199-215[CrossRef][Medline] [Order article via Infotrieve]. 8. Gunshin H, Mackenzie B, Berger UV, et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature. 1997;388:482-488[CrossRef][Medline] [Order article via Infotrieve].
9.
Su MA, Trenor CC, Fleming JC, Fleming MD, Andrews NC.
The G185R mutation disrupts function of the iron transporter Nramp2.
Blood.
1998;92:2157-2163 10. Zhang L, Lee T, Wang Y, Soong TW. Heterologous expression, functional characterization and localization of two isoforms of the monkey iron transporter Nramp2. Biochem J. 2000;349:289-297[CrossRef][Medline] [Order article via Infotrieve].
11.
Picard V, Govoni G, Jabado N, Gros P.
Nramp2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein-accessible cytoplasmic pool.
J Biol Chem.
2000;275:35738-35745
12.
Tandy S, Williams M, Leggett A, et al.
Nramp2 expression is associated with pH-dependent iron uptake across the apical membrane of human intestinal Caco-2 cells.
J Biol Chem.
2000;275:1023-1029
13.
Jabado N, Jankowski A, Dougaparsad S, Picard V, Grinstein S, Gros P.
Natural resistance to intracellular infections: natural resistance-associated macrophage protein1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane.
J Exp Med.
2000;192:1237-1248 14. Fleming MD, Trenor CC 3rd, Su MA, et al. Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet. 1997;16:383-386[CrossRef][Medline] [Order article via Infotrieve].
15.
Edwards JA, Hoke JE.
Red cell iron uptake in hereditary microcytic anemia.
Blood.
1975;46:381-388
16.
Oates PS, Morgan EH.
Defective iron uptake by the duodenum of Belgrade rats fed diets of different iron contents.
Am J Physiol.
1996;270:G826-832
17.
Fleming RE, Migas MC, Zhou X, et al.
Mechanism of increased iron absorption in murine model of hereditary hemochromatosis: increased duodenal expression of the iron transporter DMT1.
Proc Natl Acad Sci U S A.
1999;96:3143-3148
18.
Canonne-Hergaux F, Gruenheid S, Ponka P, Gros P.
Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron.
Blood.
1999;93:4406-4417
19.
Canonne-Hergaux F, Fleming MD, Levy JE, et al.
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.
Blood.
2000;96:3964-3970
20.
Harrison DE.
Marrow transplantation and iron therapy in mouse hereditary microcytic anemia.
Blood.
1972;40:893-901 21. Bannerman RM, Edwards JA, Kreimer-Birnbaum M, McFarland E, Russell ES. Hereditary microcytic anaemia in the mouse; studies in iron distribution and metabolism. Br J Haematol. 1972;23:235-245[Medline] [Order article via Infotrieve]. 22. Garrick LM, Dolan KG, Romano MA, Garrick MD. Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells. J Cell Physiol. 1999;178:349-358[CrossRef][Medline] [Order article via Infotrieve]. 23. Ponka P, Lok CN. The transferrin receptor: role in health and disease. Int J Biochem Cell Biol. 1999;31:1111-1137[CrossRef][Medline] [Order article via Infotrieve]. 24. Richardson DR, Ponka P. The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochim Biophys Acta. 1997;1331:1-40[Medline] [Order article via Infotrieve].
25.
Savigni DL, Morgan EH.
Transport mechanisms for iron and other transition metals in rat and rabbit erythroid cells.
J Physiol (Lond).
1998;508:837-850
26.
Garrick MD, Gniecko K, Liu Y, Cohan DS, Garrick LM.
Transferrin and the transferrin cycle in Belgrade rat reticulocytes.
J Biol Chem.
1993;268:14867-14874 27. Farcich EA, Morgan EH. Uptake of transferrin-bound and nontransferrin-bound iron by reticulocytes from the Belgrade laboratory rat: comparison with Wistar rat transferrin and reticulocytes. Am J Hematol. 1992;39:9-14[Medline] [Order article via Infotrieve].
28.
Gruenheid S, Canonne-Hergaux F, Gauthier S, Hackam DJ, Grinstein S, Gros P.
The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes.
J Exp Med.
1999;189:831-841
29.
Devault A, Gros P.
Two members of the mouse mdr gene family confer multidrug resistance with overlapping but distinct drug specificities.
Mol Cell Biol.
1990;10:1652-1663
30.
Lok CN, Ponka P.
Identification of a hypoxia response element in the transferrin receptor gene.
J Biol Chem.
1999;274:24147-24152 31. Teale FW. Cleavage of haem protein link by acid methyl-ethylketones. Biochim Biophys Acta. 1959;35:543[Medline] [Order article via Infotrieve]. 32. Sladic-Simic D, Zivkovic N, Pavic D. Iron metabolism in Belgrade laboratory (b-b) rats. Rev Eur Etud Clin Biol. 1972;17:197-200[Medline] [Order article via Infotrieve]. 33. Vidal SM, Pinner E, Lepage P, Gauthier S, Gros P. Natural resistance to intracellular infections: Nramp1 encodes a membrane phosphoglycoprotein absent in macrophages from susceptible (Nramp1 D169) mouse strains. J Immunol. 1996;157:3559-3568[Abstract].
34.
Canonne-Hergaux F, Levy JE, Fleming MD, Montross LK, Andrews NC, Gros P.
Expression of the DMT1 (NRAMP2/DCT1) iron transporter in mice with genetic iron overload disorders.
Blood.
2001;97:1138-1140 35. Gut A, Kappeler F, Hyka N, Balda MS, Hauri HP, Matter K. Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins. EMBO J. 1998;17:1919-1929[CrossRef][Medline] [Order article via Infotrieve].
36.
Gruenheid S, Pinner E, Desjardins M, Gros P.
Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome.
J Exp Med.
1997;185:717-730 37. Eisenstein RS. Iron regulatory proteins and the molecular control of mammalian iron metabolism. Annu Rev Nutr. 2000;20:627-662[CrossRef][Medline] [Order article via Infotrieve].
38.
Hentze MW, Kuhn LC.
Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress.
Proc Natl Acad Sci U S A.
1996;93:8175-8182
39.
Ponka P.
Tissue-specific regulation of iron metabolism and heme synthesis: distinct control mechanisms in erythroid cells [see comments].
Blood.
1997;89:1-25 40. Garrick MD, Fletcher RJ, Dolan KG, et al. The iron transporter DMT1 (or Nramp2 or DCT1) is located mostly in endosomes in normal and belgrade rat reticulocytes. [abstract]. Blood. 1999;94:403a. 41. Gruenheid S, Cellier M, Vidal S, Gros P. Identification and characterization of a second mouse Nramp gene. Genomics. 1995;25:514-525[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
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  S. Soe-Lin, S. S. Apte, B. Andriopoulos Jr., M. C. Andrews, M. Schranzhofer, T. Kahawita, D. Garcia-Santos, and P. Ponka Nramp1 promotes efficient macrophage recycling of iron following erythrophagocytosis in vivo PNAS, April 7, 2009; 106(14): 5960 - 5965. [Abstract] [Full Text] [PDF]
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 N. J. Foot, H. E. Dalton, L. M. Shearwin-Whyatt, L. Dorstyn, S.-S. Tan, B. Yang, and S. Kumar Regulation of the divalent metal ion transporter DMT1 and iron homeostasis by a ubiquitin-dependent mechanism involving Ndfips and WWP2 Blood, November 15, 2008; 112(10): 4268 - 4275. [Abstract] [Full Text] [PDF]
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M. Schranzhofer, M. Schifrer, J. A. Cabrera, S. Kopp, P. Chiba, H. Beug, and E. W. Mullner Remodeling the regulation of iron metabolism during erythroid differentiation to ensure efficient heme biosynthesis Blood, May 15, 2006; 107(10): 4159 - 4167. [Abstract] [Full Text] [PDF]
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C. Beaumont, J. Delaunay, G. Hetet, B. Grandchamp, M. de Montalembert, and G. Tchernia Two new human DMT1 gene mutations in a patient with microcytic anemia, low ferritinemia, and liver iron overload Blood, May 15, 2006; 107(10): 4168 - 4170. [Abstract] [Full Text] [PDF]
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 Y.-Z. Chang, Y. Ke, J.-R. Du, G. M. Halpern, K.-P. Ho, L. Zhu, X.-S. Gu, Y.-J. Xu, Q. Wang, L.-Z. Li, et al. Increased Divalent Metal Transporter 1 Expression Might Be Associated with the Neurotoxicity of L-DOPA Mol. Pharmacol., March 1, 2006; 69(3): 968 - 974. [Abstract] [Full Text] [PDF]
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 F. Canonne-Hergaux, A. Donovan, C. Delaby, H.-j. Wang, and P. Gros Comparative studies of duodenal and macrophage ferroportin proteins Am J Physiol Gastrointest Liver Physiol, January 1, 2006; 290(1): G156 - G163. [Abstract] [Full Text] [PDF]
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 S. A. B. Knight, G. Vilaire, E. Lesuisse, and A. Dancis Iron Acquisition from Transferrin by Candida albicans Depends on the Reductive Pathway Infect. Immun., September 1, 2005; 73(9): 5482 - 5492. [Abstract] [Full Text] [PDF]
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 A. Fortier, G. Min-Oo, J. Forbes, S. Lam-Yuk-Tseung, and P. Gros Single gene effects in mouse models of host: pathogen interactions J. Leukoc. Biol., June 1, 2005; 77(6): 868 - 877. [Abstract] [Full Text] [PDF]
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M. P. Mims, Y. Guan, D. Pospisilova, M. Priwitzerova, K. Indrak, P. Ponka, V. Divoky, and J. T. Prchal Identification of a human mutation of DMT1 in a patient with microcytic anemia and iron overload Blood, February 1, 2005; 105(3): 1337 - 1342. [Abstract] [Full Text] [PDF]
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 B. Zhou, X. Kong, and D. I. H. Linzer Enhanced Recovery from Thrombocytopenia and Neutropenia in Mice Constitutively Expressing a Placental Hematopoietic Cytokine Endocrinology, January 1, 2005; 146(1): 64 - 70. [Abstract] [Full Text] [PDF]
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A. C. G. Chua, J. K. Olynyk, P. J. Leedman, and D. Trinder Nontransferrin-bound iron uptake by hepatocytes is increased in the Hfe knockout mouse model of hereditary hemochromatosis Blood, September 1, 2004; 104(5): 1519 - 1525. [Abstract] [Full Text] [PDF]
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J. R. Forbes and P. Gros Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and Nramp2 (Slc11a2) expressed at the plasma membrane Blood, September 1, 2003; 102(5): 1884 - 1892. [Abstract] [Full Text] [PDF]
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N. Touret, W. Furuya, J. Forbes, P. Gros, and S. Grinstein Dynamic Traffic through the Recycling Compartment Couples the Metal Transporter Nramp2 (DMT1) with the Transferrin Receptor J. Biol. Chem., July 3, 2003; 278(28): 25548 - 25557. [Abstract] [Full Text] [PDF]
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N. Jabado, P. Cuellar-Mata, S. Grinstein, and P. Gros Iron chelators modulate the fusogenic properties of Salmonella-containing phagosomes PNAS, May 13, 2003; 100(10): 6127 - 6132. [Abstract] [Full Text] [PDF]
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S. Lam-Yuk-Tseung, G. Govoni, J. Forbes, and P. Gros Iron transport by Nramp2/DMT1: pH regulation of transport by 2 histidines in transmembrane domain 6 Blood, May 1, 2003; 101(9): 3699 - 3707. [Abstract] [Full Text] [PDF]
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 M. Tabuchi, N. Tanaka, J. Nishida-Kitayama, H. Ohno, and F. Kishi Alternative Splicing Regulates the Subcellular Localization of Divalent Metal Transporter 1 Isoforms Mol. Biol. Cell, December 1, 2002; 13(12): 4371 - 4387. [Abstract] [Full Text] [PDF]
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N. Jabado, F. Canonne-Hergaux, S. Gruenheid, V. Picard, and P. Gros Iron transporter Nramp2/DMT-1 is associated with the membrane of phagosomes in macrophages and Sertoli cells Blood, September 18, 2002; 100(7): 2617 - 2622. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
0.5; < 2%
reticulocytes). As observed for wild-type mice, PHZ treatment of
mk/+ heterozygotes caused a splenomegaly response
(SI = 1.24) and blood reticulocytosis (21.7% ± 0.6%), which were
similar to those seen in normal mice similarly treated. These results
indicate robust splenic erythropoietic response occurring either
naturally in mk/mk mutants lacking DMT1 function or induced
by treatment with EPO or during recovery from PHZ-induced hemolytic
anemia.
