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RED CELLS
Cellular and Subcellular Localization of the Nramp2 Iron Transporter
in the Intestinal Brush Border and Regulation by Dietary Iron
By F. Canonne-Hergaux, S. Gruenheid, P. Ponka, and P. Gros
Genetic studies in animal models of microcytic anemia and
biochemical studies of transport have implicated the Nramp2
gene in iron transport. Nramp2 generates two alternatively
spliced mRNAs that differ at their 38 untranslated region by
the presence or absence of an iron-response element (IRE)
and that encode two proteins with distinct carboxy termini.
Antisera raised against Nramp2 fusion proteins containing
either the carboxy or amino termini of Nramp2 and that can
help distinguish between the two Nramp2 protein isoforms
(IRE: isoform I; non-IRE: isoform II) were generated. These
antibodies were used to identify the cellular and subcellular
localization of Nramp2 in normal tissues and to study
possible regulation by dietary iron deprivation. Immunoblotting experiments with membrane fractions from intact organs show that Nramp2 is expressed at low levels throughout the small intestine and to a higher extent in kidney.
Dietary iron starvation results in a dramatic upregulation of
the Nramp2 isoform I in the proximal portion of the duode-
num only, whereas expression in the rest of the small
intestine and in kidney remains largely unchanged in response to the lack of dietary iron. In proximal duodenum,
immunostaining studies of tissue sections show that Nramp2
protein expression is abundant under iron deplete condition
and limited to the villi and is absent in the crypts. In the villi,
staining is limited to the columnar absorptive epithelium of
the mucosa (enterocytes), with no expression in mucussecreting goblet cells or in the lamina propria. Nramp2
expression is strongest in the apical two thirds of the villi
and is very intense at the brush border of the apical pole of
the enterocytes, whereas the basolateral membrane of these
cells is negative for Nramp2. These results strongly suggest
that Nramp2 is indeed responsible for transferrin-independent iron uptake in the duodenum. These findings are
discussed in the context of overall mechanisms of iron
acquisition by the body.
r 1999 by The American Society of Hematology.
I
uptake by the enterocytes from the lumen across the apical
membrane, (2) intracellular transport, and (3) transfer across the
basolateral membrane into plasma. It has been suggested that
the rate-limiting step for absorption of inorganic iron is uptake
of the ingested metal across the brush border membrane of the
enterocyte.12 Uptake of ferrous iron by the enterocyte is an
energy-dependent and carrier-mediated process (saturation kinetics, temperature dependence, and sensitivity to proteases),13,14 and a large number of candidates for iron binding
proteins and/or transport activities have been suggested by
labeling, cross-linking, or direct transport measurements in
brush border membrane vesicles.15 The recent identification of
genes mutated in animal models of deficit in iron metabolism
has shed considerable light on the proteins possibly involved in
iron transport in the intestine as well as peripheral tissues. The
mk mutant mouse strain16,17 and the Belgrade (b) rat18 are two
rodent models of iron deficiency, both exhibiting a severe
microcytic hypochromic anemia marked by a severe defect in
iron absorption by intestinal cells and in erythroid iron use.
Moreover, studies in vivo have shown that this anemia cannot
be corrected by increased dietary iron19,20 or by direct iron
injection,21 suggesting also a second block in iron uptake by red
blood cell precursors and other peripheral tissues. It has recently
been demonstrated that the Nramp2 (natural resistanceassociated macrophage protein 2) gene is mutated in both the
mk and b animal models.22,23 Indeed, both the mk mouse and the
b rat have been shown to carry the same mutation at Nramp2, ie,
a glycine to arginine (Gly185Arg) substitution in one of the
predicted transmembrane domains (TM4) of the protein.22,23 In
addition, studies in transiently transfected HEK293T cells have
shown that overexpression of the wild-type but not the mk/b
mutant variant of Nramp2 stimulates cellular iron uptake.23,24
Finally, studies in Xenopus laevis oocytes have suggested that
Nramp2 may act as a pH-dependent divalent cation transporter,
functioning by a proton symport mechanism.25
The Nramp2 gene was initially identified as encoding a
RON IS AN ESSENTIAL element for all forms of life, and
living organisms have developed sophisticated means to
recycle, scavenge, and recruit iron from their environment.1,2
Excessive accumulation of iron can be potentially harmful
through the generation of free radicals and other toxic substances.3 In mammals, there is no physiological excretion
system for iron, which is normally lost from the organism by
nonspecific mechanisms such as cell desquamation. Therefore,
body iron levels appear to be primarily regulated at the level of
absorption and through efficient storage.4,5 In mammals, nutritional iron absorption (both heme and nonheme iron) occurs
primarily in the intestine.6,7 Heme iron constitutes only a small
fraction of the available dietary iron, but it is highly available
for absorption.8,9 On the other hand, the absorption of nonheme
iron is low and markedly regulated in the first part of the
duodenum,6,7 in which the acidic pH promotes solubilization of
iron transformed to Fe2⫹ by ferrireductase10 and ascorbate.11
Inorganic iron absorption occurs as a three-step process: (1)
From the Departments of Biochemistry and Physiology, McGill
University, Montreal, Quebec, Canada; and the Lady Davis Institute for
Medical Research, Jewish General Hospital, Montreal, Quebec, Canada.
Submitted December 29, 1998; accepted February 16, 1999.
Supported by a research grant to P.G. from NIAID (Grant No.
AI35237). S.G. is supported by a studentship from the Medical Research
Council of Canada (MRC). P.G. is an International Research Scholar of
the Howard Hughes Medical Institute and is a senior scientist of the
MRC.
Address reprint requests to P. Gros, PhD, 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. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1999 by The American Society of Hematology.
0006-4971/99/9312-0021$3.00/0
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Blood, Vol 93, No 12 (June 15), 1999: pp 4406-4417
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NRAMP2 PROTEIN EXPRESSION IN INTESTINE
protein highly similar to the natural resistance-associated macrophage protein 1 (Nramp1).26,27 Naturally occurring28,29 and
experimentally induced mutations30 at Nramp1 abrogate natural
resistance to infection with unrelated intracellular microorganisms such as Salmonella, Leishmania, and Mycobacteria
in mice.31,32 Likewise, polymorphic variants at the human
NRAMP1 locus are associated with differential susceptibility of
humans to tuberculosis and leprosy in endemic areas of
disease.33,34 Nramp1 and Nramp2 are highly hydrophobic
integral membrane glycoproteins composed of 12 transmembrane (TM) domains that possess several structural characteristics of ion channels and transporters.35,36 As opposed to
Nramp1, which is expressed exclusively in mononuclear phagocytes such as tissue macrophages,37,38 Nramp2 mRNA expression is more ubiquitous and has been detected in most tissues
and cell types analyzed27,39; however, it is higher in brain,
thymus, proximal intestine, kidney, and bone marrow.25 cDNA
cloning and sequencing experiments have indicated that Nramp2
produces two alternatively spliced transcripts generated by
alternative use of two 38 exons encoding distinct C-termini of
the protein as well as distinct 38 untranslated regions (UTRs).40
Interestingly, one Nramp2 mRNA contains an iron-responsive
element (IRE) in its 38UTR. The IRE is an RNA secondary
structure present in the 58- or the 38-UTR of animal mRNAs
encoding proteins involved in iron metabolism.41,42 In ironreplete cells, iron-regulatory proteins interact with IREs to
either enhance the stability or inhibit translation of the tagged
RNAs, thus regulating the amount of protein expressed.43,44 The
second Nramp2 splice isoform (non-IRE) 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 acids
segment and codes for a distinct 38 UTR lacking the IRE.
Gunshin et al25 have observed that Nramp2 mRNA expression
is regulated by dietary iron.
Immunolocalization studies with protein-specific antibodies
have shown that Nramp1 is expressed in macrophages in the
late endosomal/lysosomal compartment.45 Upon phagocytosis,
Nramp1 is recruited to the membrane of the phagosome,45
where it may act to modulate the intravesicular cation content to
affect intracellular microbial replication.46 On the other hand,
the absence of isoform-specific anti-Nramp2 antibodies has so
far precluded the identification of the organ and cell types
expressing the protein. Likewise, the cellular and subcellular
localization in normal tissues of the two Nramp2 protein
isoforms as well as their possible regulation by iron remain
unknown. These aspects of Nramp2 need to be better characterized to understand the role of this protein in dietary iron uptake,
possible trans-epithelial transport of iron, and iron metabolism
in peripheral tissues. In the current study, we have generated
affinity-purified, isoform-specific anti-Nramp2 antibodies and
have determined the organ-specific, cell-specific, as well as
subcellular distribution of the Nramp2 isoforms in normal
tissues. We have also analyzed the regulation of these two
isoforms by dietary iron.
MATERIALS AND METHODS
Animals. Normal inbred mouse strain 129sv were initially obtained
from Taconic Farms (Germantown, NY) and subsequently bred and
handled in our animal care facility according to the rules and regulations
4407
of the Canadian Council on Animal Care. For iron depletion studies,
animals were fed a low iron diet (ICN, Costa Mesa, CA) for a period of
8 weeks, whereas control animals were fed an identical diet supplemented with 3% ferric phosphate (ICN).
Cell culture and transfections. Chinese hamster ovary (CHO) cells
LR7347 were grown in ␣-minimum essential medium (␣-MEM) supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine, 50 U/mL
penicillin, and 50 µg/mL streptomycin. All media and media supplements were purchased from GIBCO/BRL (Grand Island, NY). An
expression plasmid was constructed45 using the mammalian expression
vector pMT2 containing the entire coding sequence of mouse Nramp1
cDNA modified at its carboxy terminus by the in-frame addition of four
consecutive antigenic peptide epitopes (EQKLISEEDL) derived from
the human c-Myc protein. In a similar manner, a c-Myc–tagged Nramp2
expression plasmid was constructed by introducing the c-Myc–tagged
Nramp2 cDNA from plasmid pBluescript48 in mammalian expression
vector pMT2. pMT2 uses SV40 and adenovirus regulatory sequences to
overexpress exogenous cDNAs and is introduced into mammalian cells
by cotransfection with pSV2neo to allow selection of cotransfectants in
geneticin (G418). Plasmids were introduced in CHO LR73 cells by
transfection, using the calcium phosphate coprecipitation method.49
Individual G418R cell clones were isolated, expended in culture, and
analyzed for recombinant Nramp1 and Nramp2 protein expression by
immunofluorescence and immunoblotting.
Microsomal membrane fractions preparation. Crude membrane
fractions from homogenized tissues and various CHO-transfected cell
clones were prepared according to a previously described procedure.50
Kidney, liver, spleen, and portions of the gastrointestinal system
including colon were removed immediately after death, were frozen in
liquid nitrogen, and were ground to a fine powder using mortar and
pestle precooled on carbonic ice. CHO cells were grown to 70%
confluency and harvested in cold phosphate-buffered saline (PBS)citrate. Tissue powder or CHO cell pellets were then resuspended in 10
mL/g of tissue of a solution consisting in 0.25 mol/L sucrose/0.03 mol/L
histidine (pH 7.2) supplemented with 2 mmol/L EDTA, 0.1 mg/mL
phenylmethylsulfonyl fluoride, 2 µg/mL leupeptin, 1 µg/mL pepstatin,
and 2 µg/mL aprotinin. Tissues and cells were homogenized using a
glass potter with a tight fitting teflon pestle rotated at 1,300 revolutions/
min. The homogenate was then centrifuged at 6,000g for 15 minutes.
The sediment was resuspended several times in the original volume of
sucrose-histidine solution and centrifuged again at 6,000g for 15
minutes. The combined supernatants were then centrifuged at 80,000g
for 30 minutes and the pellet corresponding to the microsomal fraction
was resuspended in sucrose/histidine buffer and stored frozen at ⫺80°C
until use. Protein concentration of the various membrane fractions was
determined by the Bradford assay (Bio-Rad, Hercules, CA).
Preparation of Nramp immunogens. The fusion protein containing
the amino terminal domain (region 1-54) of Nramp1 fused in-frame to
glutathione-S-transferase (GST) was constructed and used to generate
anti-Nramp1 (mN1-NT) antibodies, as previously described.51 For
producing polyclonal antisera against the two protein isoforms of
Nramp2 (IRE: isoform I; non-IRE: isoform II), a fusion protein was
produced containing GST fused to a peptide segment corresponding to
the amino terminus (residues 1-73) region of Nramp2. To generate a
specific antibody against the isoform II, a GST fusion protein containing
the carboxy terminal coding region (residues 532-568) of the Nramp2
non-IRE mRNA was constructed. Nucleotide sequences of final constructs were verified before production of the proteins in Escherichia
coli.52 GST-chimeras were purified over glutathione-agarose, analyzed
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), and excised from the gel after light staining with Coomassie
blue. Antisera were raised in New Zealand White rabbits using purified
protein (0.1 mg per rabbit per injection) emulsified in Freund’s
complete or incomplete adjuvant. Rabbits were boosted at 6-week
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4408
intervals, and blood (10 mL) was obtained by arterial puncture 11 days
after each boost. Serum was isolated and kept frozen at ⫺20°C.
Affınity purification of anti-Nramp antisera. The same strategy was
used for affinity purification of anti-Nramp1 and anti-Nramp2 antibodies and consisted in using the same Nramp peptide segments fused to
dihydrofolate reductase (DHFR). Briefly, each pGEX-Nramp construct
(see above) was digested with EcoRI, and the resulting overhangs were
repaired using the Klenow fragment of DNA polymerase I (Pharmacia,
Uppsala, Sweden) before digestion with the BamHI and ligation into
Bgl II- and Sma I-digested pQE40 bacterial expression vector (Qiagen,
Mississauga, Ontario, Canada). The in-frame (His)6-DHFR-Nramp1 or
Nramp2 fusion protein constructs were transformed into E coli strain
M15(pREP4) for expression (Qiagen). Purification of DHFR chimeras
was performed on Ni-NTA agarose according to experimental conditions suggested by the manufacturer (Qiagen). The polyclonal antiserum fractions were then purified by a preparative immunoblot procedure.53 The fusion proteins were loaded onto a 12% SDS-polyacrylamide
gel using a preparative comb and transferred to a polyvinylidene
fluoride membrane (PVDF; westran; Schleicher & Schuell, Keene,
NH). The band corresponding to a fusion protein was localized on the
membrane by Ponceau S staining (Sigma, St Louis, MO), excised, cut in
small pieces, and incubated for 2 hours at room temperature with 5%
skim milk powder in PBS/0.2 % Tween 20 to block nonspecific sites.
PVDF membrane fragments were further incubated 16 hours at 4°C
with the immune serum diluted 2:3 in PBS. After 5 washes with PBS,
bound antibodies were eluted with 0.2 mol/L glycine, pH 2.2, for 3
minutes. The pH of the eluate was quickly neutralized by adding 1
mol/L Tris base to pH ⬃7.5, and bovine serum albumin (BSA) and
glycerol were added to a final concentration of 0.1% and 50%,
respectively. Aliquots of the purified sera were kept at ⫺20°C.
Immunoblotting. Crude membrane preparations from tissues (100
µg) or CHO cells (25 µg) were dissolved in Laemmli buffer and
incubated for 30 minutes at room temperature before SDS-PAGE (10%
polyacrylamide) and transfer to PVDF membranes. Similar loading and
transfer of proteins was verified by staining the blots with Ponceau S
(Sigma). PVDF membranes were preincubated with blocking solution
(0.02% Tween20, 5% skim milk in PBS) for 2 hours at room
temperature, followed by incubation with primary antibodies for 16
hours at 4°C in blocking solution. For immunoblotting of tissue
membrane extracts, primary antibodies were used at the following
dilutions: rabbit anti-Nramp1 (1/200), rabbit anti-Nramp2 NT (1/100),
rabbit anti-Nramp2 CT (1/75), rat monoclonal anti-mouse transferrin
receptor (1/250; Biosource International, Camarillo, CA), and rabbit
polyclonal anti-biliary glycoprotein 1 (Bgp1; 1/4,000; provided by Dr
N. Beauchemin, McGill University, Montreal, Quebec, Canada). For
immunoblotting of CHO membrane extracts, purified anti-Nramp 1 and
anti-Nramp 2 as well as mouse monoclonal anti-cMyc (9E10) were used
at the 1/100 dilution. After incubation with the primary antibody and
washing with PBS ⫹ 0.2% Tween20, membranes were incubated with
peroxidase labeled anti-rat, anti-rabbit, or anti-mouse secondary antibodies (1/10,000; Amersham, Arlington Heights, IL) for 1 hour at room
temperature, and the signal was visualized by ECL (Amersham). After
ECL detection, some membranes were incubated at 50°C for 30 minutes
in stripping solution (100 mmol/L ␤2-mercaptoethanol, 2% SDS, 62.5
mmol/L Tris-HCl, pH 6.8) and then reprobed with a different primary
antibody.
Immunohistochemistry. Tissues were fixed in Bouin’s solution for
72 hours at room temperature. They were then dehydrated in a series of
ethanol (3 times for 20 minutes: 70%, 95%, and 100%), ethanol/xylene
(1/1), and xylene solutions, followed by embedding in paraffin.
Five-micrometer sections were cut and mounted with gelatin on glass
slides. Before labeling, sections were deparaffinized in xylene and
rehydrated in a graded series of ethanol baths (100%, 95%, 70%, 50%,
and 30%). Endogenous peroxidase activity in the deparaffinized sections was blocked with ethanol 70%-peroxide 1% solution. Sections
CANONNE-HERGAUX ET AL
were then incubated in PBS/100 mmol/L glycine, rinsed in PBS, and
blocked for at least 1 hour at room temperature in PBS containing 1%
BSA (Fraction V; fatty acid free; Boehringer Mannheim, Indianapolis,
IN) and 10% normal goat serum (GIBCO). Incubation with the primary
antibody diluted in blocking solution was performed in a wet chamber
overnight at 4°C. Immunohistochemical staining of fixed paraffinembedded sections was performed using the peroxidase/antiperoxidase
procedure. After incubation with the primary antibody, three washes in
PBS, and incubation with the secondary antibody (swine antirabbit IgG,
1:100; DAKO), subsequent rabbit peroxidase-antiperoxidase immunostaining (PAP; 1:100; DAKO) was shown using 38-diaminobenzidine
tetrahydrochloride (DAB) in solution. Sections were then counterstained with 0.1% methylene blue in PBS. Finally, sections were
dehydrated again in ethanol and xylene and mounted in Permount. For
immunofluorescence, after incubation with the primary antibodies,
sections were further incubated with an anti-rabbit secondary antibody
conjugated to Cy3/Rhodamine (Jackson Immunochemicals, West Grove,
PA). Sections were then washed with PBS and directly mounted in
ImmuMount (Shandon, Pittsburgh, PA). Immunofluorescence was
analyzed with a Nikon microscope using the 100⫻ oil immersion
objective.
RESULTS
Production and characterization of isoform-specific antiNramp2 antisera. To further study localization and regulation
of the Nramp2 protein in normal tissues, we aimed at generating
specific anti-Nramp2 antisera that would not react with Nramp1.
In addition, it has been previously observed that the Nramp2
gene can generate by alternative splicing two Nramp2 mRNAs
and two corresponding polypeptides that differ markedly at
their 38 untranslated region (presence or absence of ironresponse element [IRE]) and at their carboxyl termini, respectively (Fig 1A). For clarity, we have designated proteins
encoded by the IRE-containing and non-IRE containing Nramp2
mRNAs, Nramp2 isoform I and isoform II, respectively. Thus,
we attempted to raise antisera that could also distinguish the two
Nramp2 protein variants. For this, we have used two fusion
proteins as immunogens in rabbits. The first one comprised the
first 73 amino terminal residues of Nramp2 (NT, identical in
both proteins), and antibodies raised against this protein should
recognize Nramp2 isoforms I and II. The second immunogen
comprised residues 532-568 from the carboxy terminus of
Nramp2 encoded by the non-IRE containing mRNA (CT), and
antibodies against this sequence should be specific for isoform
II (Fig 1A). Using both antibodies in parallel should allow one
to monitor the expression of either isoform in test cells and
tissue samples.
Hyperimmune antisera were generated in rabbits and the
anti-Nramp2 Ig fraction was purified by affinity chromatography (see Materials and Methods). The specificity of the two
antisera was tested by immunoblotting using membrane fractions from CHO cells and from CHO cells that have been
transfected and overexpress either Nramp1 or isoform II of
Nramp2. In addition, these transfected proteins contain a cMyc
epitope tag fused in-frame at their C-terminus that allows one to
verify the specificity of the signal obtained by immunoblotting
using an anti-cMyc monoclonal antibody (9E10). Results from
these analyses are shown in Fig 1B. The 9E10 antibody
specifically detects a single protein species of 80 to 90 kD in the
Nramp1-transfected cells (Fig 1B, lane 16) and a species of 90
to 100 kD in the Nramp2-transfected cells (Fig 1B, lane 8).
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NRAMP2 PROTEIN EXPRESSION IN INTESTINE
4409
Fig 1. Detection of the Nramp1 and Nramp2 cMyctagged proteins in CHO cell membranes. (A) Schematic representation of the two mRNAs corresponding to the IRE-containing form and to the non-IRE
form of Nramp2 generated by alternative splicing of
a 38 end exon (see Lee et al40). The two mRNAs
harbor distinct 38 untranslated region (38UTR) and
encode two polypeptides with distinct carboxy termini (hatched boxes). The mN2 (NT) antibody was
raised against an amino terminal segment of Nramp2
that is common to the two Nramp2 protein isoforms
(solid boxes). The mN2 (CT) antibody was raised
against the carboxy terminal extremity of the protein
encoded by the Nramp2 non-IRE mRNA (Nramp2
isoform II). (B) Crude membrane extracts from untransfected CHO cells (lanes 1, 3, 5, 7, 9, 11, 13, and
15), from Nramp1 transfectants (lanes 10, 12, 14, and
16), and from Nramp2 isoform II transfectants (lanes
2, 4, 6, and 8) were separated by SDS-PAGE on a 10%
acrylamide gel followed by transfer to PVDF membranes. Immunodetection was with affinity-purified
mNramp1 [mN1 (NT); lanes 1, 2, 9, and 10], mN2 (NT;
lanes 3, 4, 11, and 12), and mN2 (CT; lanes 5, 6, 13, and
14) antibodies and monoclonal anti-cMyc antibody
9E10 (lanes 7, 8, 15, and 16), as described in Materials
and Methods. The positions and sizes (in kilodaltons)
of molecular weight markers are shown.
These immunoreactive bands are absent in membrane extracts
from untransfected CHO controls (Fig 1B, lanes 1, 3, 5, 7, 9, 11,
13, and 15). In extracts from Nramp1 transfectants, the affinitypurified Nramp1 antiserum [mN1 (NT)] recognizes a single
protein of apparent molecular mass 80 to 90 kD (Fig 1B, lane
10). Both affinity-purified anti-Nramp2 antibodies raised against
the N terminus [mN2 (NT)] or the C terminus [mN2 (CT)]
fusions detect a band of apparent mobility 90 to 100 kD in
membranes from Nramp2-transfected cells (Fig 1B, lanes 4 and
6, respectively). The electrophoretic mobility characteristics of
Nramp proteins detected with affinity-purified anti-Nramp antisera were very similar to that of the species detected by the
anti-cMyc antibody in the same cell extracts. Finally, no
cross-reactivity was seen between the anti-Nramp1 and the
anti-Nramp2 antibodies (compare lanes 2, 4, and 6 with lanes
10, 12, and 14, respectively). Together, these results clearly
establish that our affinity-purified anti-Nramp1 and antiNramp2 antisera are protein specific.
Tissue expression of Nramp2 and regulation by dietary iron.
In normal mouse tissues, Nramp2 mRNA is present in low
abundance in most tissues and cell types analyzed.27 Northern
blot analysis of RNA from rat tissues also showed broad
expression of Nramp2 (DCT1), but also identified upregulation
of Nramp2 in response to depletion of dietary iron.25 However,
the organ- and cell-specific expression of the two Nramp2
protein isoforms as well as their subcellular distribution remain
unknown. This issue was investigated using the two antiNramp2 antisera. Considering both the previous mRNA expression data and the known gastrointestinal site of iron absorption,
we restricted the analysis to a few organs (small intestine [I],
colon [C], kidney [K], spleen [S], and liver [L]; Fig 2). We also
investigated the possible role of dietary iron depletion on
Nramp2 protein expression. For these studies, mice were fed
either a low iron diet (⫺Fe) or the same diet supplemented with
iron (⫹Fe; Fig 3). After 8 weeks, mice were killed and organs
were collected. Two-centimeter sections corresponding to the
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4410
CANONNE-HERGAUX ET AL
Fig 2. Dissection scheme for the gastrointestinal
tract. Different segments of the gastrointestinal tract
were dissected from mice fed a low iron diet or fed a
normal diet. The first part of the small intestine was
divided into two pieces, I1 and I2, corresponding to
the proximal and distal parts of the duodenum,
respectively. The distal part of the small intestine,
the ileum (I3), as well as the colon (C) were also
dissected.
proximal duodenum (I1), distal duodenum (I2), distal ileum
(I3), and colon (C) were dissected (Fig 2), and microsomal
membrane fractions were isolated by centrifugation after tissue
homogenization. These fractions were then separated by gel
A
B
Fig 3. Effect of dietary iron
depletion on Nramp protein expression in normal tissues. Organs were harvested from mice
maintained on low iron diet (ⴚFe)
or on the same diet but supplemented with iron (ⴙFe). One hundred micrograms of microsomal
membrane fractions isolated
from proximal duodenum (I1),
distal duodenum (I2), ileum (I3),
colon (C), kidney (K), spleen (S),
and liver (L) were resolved on a
10% acrylamide gel, transferred
to a PVDF membrane, and analyzed by immunoblotting. To control the specificity of the antiNramp antibodies, 25 ␮g of
membrane proteins from control
CHO cells (CHO) or CHO transfectants expressing either a cMyctagged Nramp1 (CHOmN1) or a
cMyc-tagged Nramp2 isoform II
(CHOmN2) were included. Immunoblotting was performed with
antibodies raised against Nramp2
N-terminus (A; mNramp2 NT),
Nramp2 C-terminus (B; mNramp2
CT), Nramp1 (C; mNramp1 NT),
TfR (D), and Bgp1 proteins (E). The
positions and sizes (in kilodaltons) of molecular weight markers are shown. The exposure
times of (A) and (B) were adjusted to produce a similar reference signal by the two antisera
against the protein expressed in
transfected CHO cells.
C
D
E
electrophoresis and analyzed with the two anti-Nramp2 antisera
(NT and CT), as well as with the anti-Nramp1 antiserum (Fig
3A, B, and C, respectively). In these experiments, membrane
fractions from CHO cells and CHO transfectants expressing
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NRAMP2 PROTEIN EXPRESSION IN INTESTINE
either Nramp1 or Nramp2 isoform II were included as controls
(Fig 3, lanes 15, 16, and 17).
In mice fed on a normal diet (⫹ Fe), the anti-Nramp2
antiserum directed against the N-terminus [mN2 (NT)] and
recognizing both Nramp2 isoforms detected a low level of
Nramp2 protein expression (80 to 90 kD) in the two duodenum
fractions (Fig 3A, lanes 1 and 3), as well as in the ileum fraction
(Fig 3A, lane 5) and to a higher extent in the kidney (Fig 3A,
lane 9). No Nramp2 protein expression was detected in colon
(Fig 3A, lane 7), spleen, and liver (Fig 3A, lanes 11 and 13).
After prolonged exposure, a low level of Nramp2 expression
was also detected in colon, spleen, and liver (data not shown).
The specificity of the antiserum was confirmed by the presence
of the immunoreactive band seen only in the Nramp2 CHO
transfectant (Fig 3A, compare lanes 15 and 16 with lane 17).
After diet-induced iron depletion (⫺Fe), Nramp2 protein expression was dramatically enhanced (by a factor of 50- to 100-fold)
in the first part of the small intestine (I1) corresponding to the
proximal duodenum (Fig 3A, lane 2). This strong upregulation
of Nramp2 protein expression appeared only in the first 2 cm of
the duodenum and was not seen in more distal portions of the
small intestine (Fig 3A, lanes 4 and 6) or in the colon (Fig 3A,
lane 8), spleen (Fig 3A, lane 12), and liver (Fig 3A, lane 14).
Two additional immunoreactive species of faster electrophoretic mobility were also detected in the duodenum sample from
iron-deprived animals. Because these protein species are not
detected in any of the other tissue samples analyzed and because
they are of lower apparent molecular mass than that of the
predicted peptide backbone of Nramp2, they most likely
represent partial degradation products from the mature protein.
Nramp2 protein expression was also increased by iron depletion
in the kidney (2 independent experiments), but only by a factor
of twofold (Fig 3A, lanes 9 v 10). Together, these results
identify strong upregulation of Nramp2 protein expression in
proximal duodenum in response to dietary-induced iron depletion.
To gain information into which of the two Nramp2 isoforms
is upregulated by dietary iron, an immunoblot containing the
same membrane fractions was probed with the anti-Nramp2
[mN2 (CT)] antiserum directed against isoform II (Fig 3B). The
anti-Nramp2 (CT) antiserum did not detect specific immunoreactive products in any of the tissues analyzed, either under
normal or low iron diet conditions. The absence of immunoreactive bands did not appear to result from lack of reactivity of the
anti-Nramp2 (CT) antiserum, because a strong signal was seen
in membrane fractions from CHO transfectants expressing
Nramp2 isoform II (Fig 3B, lane 17). The signal obtained with
the anti-Nramp2 CT antiserum in Nramp2 CHO transfectants
was adjusted to that produced by the anti-Nramp2 NT antiserum
in the same sample (compare lanes 17 in Fig 3A v B) to facilitate
comparison between other lanes of the two panels. The strong
iron-induced upregulation of Nramp2 in the proximal duodenum, together with the apparent absence of Nramp2 isoform II
expression in this tissue, strongly suggest that it is the isoform I
of Nramp2 that is expressed and regulated in the proximal
intestine. The observed constitutive and iron-inducible expression of Nramp2 in duodenum is consistent with the known site
of iron and other divalent cations uptake in the gut.11,13
Additional controls were included (Fig 3C, D, and E). First,
4411
expression of Nramp1 was investigated in the same tissue panel,
using a protein specific anti-Nramp1 antiserum.51 Nramp1
expression was detected only in spleen microsomal fractions
(Fig 3C, lanes 11 and 12), in agreement with the known
expression of Nramp1 mRNA in reticuolendothelial organs28
and the known phagocyte-specific expression of the Nramp1
protein.45 Nramp1 protein expression in spleen was not influenced by the dietary iron status of the mice (Fig 3C, lanes 11
and 12).
Expression of the transferrin receptor (TfR) was studied in
the same samples (Fig 3D). TfR was expressed as an approximately 90-kD species present in the three intestinal segments
analyzed, in the colon, in the kidney, and also in the spleen.
However, TfR was expressed only at very low levels in liver
(Fig 3D, lane 13). Upon iron depletion, a small increase in TfR
expression was noted in colon (lanes 7 v 8) and kidney (lanes 9 v
10), whereas clear induction was seen in the liver (lanes 13 v
14).
Finally, a control was included to ascertain that protein
degradation during sample preparation did not contribute to
differences in Nramp2 protein expression in different tissues
and in response to iron depletion. Biliary glycoproteins (Bgps)
are a group of surface glycoproteins abundantly expressed in
epithelial cells of the gastrointestinal tract, including small
intestine, colon, and also liver.54 An anti-Bgp1 antiserum was
used to monitor Bgp in the various membrane fractions (Fig
3E). Abundant Bgp expression was seen in all intestinal
segments (lanes 1 through 8) as well as in the liver (lanes 13 and
14), whereas low level expression was seen in kidney (lanes 9
and 10) and spleen (lanes 11 and 12). The levels of Bgp in these
tissues were not influenced by the dietary status of the mice.
This Bgp expression pattern is in agreement with that previously published55-57 and indicates that protein degradation did
not contribute significantly to the differential expression of
Nramp2 isoforms observed in normal tissues and in response to
dietary iron.
Thus, (1) the robust induction of Nramp2 seen in duodenum
under low iron diet and (2) the noted increase in TfR expression
in liver in response to low iron diet, together with (3) decreased
plasma ferritin levels measured in low iron diet animals (128 ⫾
20 µg/L, mean ⫾ SE of 3 mice), compared with levels measured
in control animals (225 ⫾ 34 µg/L, mean ⫾ SE of 3 mice),
indicate that the diet used indeed resulted in iron depletion in
these mice.
Cellular and subcellular localization of the Nramp2 protein
in epithelial cells of the intestinal villi. The cellular and
subcellular sites of expression of Nramp2 in the proximal
portion of the duodenum were investigated by immunostaining
of histological sections obtained from mice fed normal or
iron-depleted diets. Freshly dissected tissues were fixed in
Bouin’s solution and embedded in parafilm, and sections were
analyzed with either normal preimmune serum, anti-Nramp2
(NT) antiserum, or anti-Bgp1 antiserum, followed by counterstaining with methylene blue (Fig 4). In mice fed a normal diet,
we were unable to detect a positive Nramp2 staining in any of
the structures examined (Fig 4A). This was somehow anticipated, considering the relatively low level of Nramp2 noted in
these tissues by Western blot analyses (Fig 3, lane 1). However,
in tissue sections from mice fed a low iron diet, a very intense
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
4412
CANONNE-HERGAUX ET AL
Fig 4. Immunohistochemical
staining for Nramp2 in the intestine. Tissue sections were prepared as described in Materials
and Methods and were immunostained with polyclonal rabbit antimouse Nramp2 (NT) antibody
(A and D), preimmune serum (B
and E), and polyclonal rabbit antimouse Bgp1 antibody (C and F).
Sections were counterstained
with methylene blue, before microscopic examination and photography. Low magnification
(400ⴛ) of histological sections of
proximal duodenum (I1; Fig 2)
from mice fed with a control (A,
B, and C) or low iron (D, F, and E)
diet. Position of villi and crypts
are identified. Arrows in (D) identify Nramp2 brush border staining (arrow), intracellular staining
(arrowhead), or negative goblet
cells (white arrow). In (C) and (F),
arrows identify Bgp1 staining in
the brush border and in the
crypts (arrow), in the supranuclear intracellular region (arrowhead), and in the lumen
(white arrow).
Nramp2 immunostaining was observed (Fig 4D). This Nramp2
staining was detected in villi only and was absent from the
crypts of Lieberkuhn located more proximal and near the
muscularis mucosae (Fig 4D). In addition, in several individual
villi analyzed, Nramp2 staining seemed to be most intense in
the most luminal half of the villi. In villi, staining was restricted
to columnar epithelial cells (enterocytes) and was absent from
mucus-secreting goblet cells (Fig 4D, white arrow). No Nramp2
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NRAMP2 PROTEIN EXPRESSION IN INTESTINE
staining was evident in the different cell types and structures of
the central internal portion of the villus (lamina propria). In
epithelial absorptive cells, staining was most intense at the
luminal surface of the epithelium, possibly associated with the
brush border (microvilli; Fig 4D, arrow). In several villi
examined, Nramp2 staining was reduced in a region at the tip of
the villi, possibly corresponding to the zone of exfoliating
epithelial cells. The Nramp2 staining observed in these sections
was specific and was not seen in control sections incubated with
preimmune serum (Fig 4E). Staining of additional sections with
the anti-Nramp2 (CT) antiserum specific for isoform II failed to
detect a signal in intestinal villi (data not shown), in agreement
with immunoblotting results (Fig 3B) and strongly suggesting
that it is the Nramp2 isoform I that is prominently expressed at
that site. Anti-Bgp 1 antibody was used as a positive control on
these sections (Fig 4C and F), because it is known that biliary
glycoproteins are expressed at the surface of the brush border of
epithelial cells of the villi.57 This antibody indeed produced
strong immunostaining at the brush border of epithelial cells
(Fig 4C and F; villi, arrows) which was similar to that seen for
Nramp2 (Fig 4D). Bgp1 was also detected in the supranuclear
intracellular region of the epithelial cells (Fig 4C and F,
arrowheads) in the luminal space itself (Fig 4F, white arrow), as
well as in the crypts of Lieberkuhn (Fig 4C and F, arrows) and in
goblet cells of the villi. The latter four staining patterns are
clearly distinct from that observed for Nramp2. Finally, Bgp1
staining was independent of iron dietary status of the animals.
Subcellular localization of Nramp2 was examined further by
immunohistochemical staining and by immunofluorescence
under higher (1,000⫻) magnification (Fig 5). Results in Fig 5
confirm that Nramp2 expression is restricted to the villi portion
of the intestinal epithelium (Fig 5B and D), whereas both the
internal lamina propria of the villi and the crypts of Lieberkuhn
are negative for Nramp2 (Fig 5A and C). These high magnification pictures also confirm that only absorptive epithelial cells
and not goblet cells (Fig 5B and D, white arrows) express
Nramp2. They also show that, although Nramp2 is most
concentrated at the brush border of the epithelial cells (Fig 5B
and D; arrow), staining can also be observed as a punctate
intracellular pattern in the apical half portion of the cells (Fig
5B and D; arrowhead). The latter staining pattern suggests that
Nramp2 may also be expressed in a subcellular membranous
compartment as well. This staining was not an artifact of the
Cy3 fluorophore used and was also observed with a rhodaminecoupled secondary antibody (data not shown) and by immunochemical staining (Fig 5B). In addition, this staining was not
seen in the crypts (Fig 5C) or in animals fed normal diet (data
not shown).
Together, these results establish that, under iron-deplete
conditions, Nramp2 is abundantly expressed in the brush border
of absorptive epithelial cells of the duodenum.
DISCUSSION
Nramp2 was cloned originally by cross-hybridization to the
Nramp1 gene.27,28 Nramp1 is expressed in macrophages and
mutations in this gene cause susceptibility to infection with
intracellular parasites.30,51 The Nramp1 protein is an integral
membrane phosphoglycoprotein expressed in the lysosomal
compartment of macrophages. It is targeted to the membrane of
4413
Fig 5. Subcellular localization of Nramp2 in enterocytes. Tissue
sections from the proximal duodenum (I1; Fig 2) from mice maintained on a low iron diet were analyzed for Nramp2 expression.
Immunochemistry (A and B) or immunofluorescence (C and D) were
performed on sections using the anti-Nramp2 NT antibody. Crypts of
Lieberkuhn (A and C) and villi (B and C) are shown. Nramp2 staining
was observed in the brush border (arrow) and intracellularly (arrowhead), whereas goblet cells (white arrow) and lamina propria remain
negative. (Original magnification ⴛ 1,000.)
the phagosome after phagocytosis, where it is believed to
modify the intravesicular milieu of the phagosome to affect in
situ microbial replication.45,46 Nramp1 and Nramp2 share 78%
sequence similarity with highly conserved primary sequence
motifs and secondary structures (12 TM domains) that define a
large protein family conserved throughout the eukaryotic and
prokaryotic kingdoms.36 Recent studies have indicated that
Nramp2 plays an important role in divalent cations transport,
including iron. (1) Nramp2 is homologous to the SMF yeast
genes family of Mn2⫹ transporters,58 and Nramp2 can functionally complement a SMF1/SMF2 null mutant.48 (2) Nramp2 has
also been identified in expression cloning studies in Xenopus
laevis oocytes as DCT1, a pH-dependent divalent cation proton
cotransporter.25 (3) Nramp2 is mutated in two rodent models of
hypochromic, microcytic anemia, the mk mouse,22 and the b
rat.23 The inability to correct the microcytic anemia in mk and b
animals by dietary iron19,20 or iron injections21 has suggested
that Nramp2 may be involved not only in intestinal iron uptake
but also in iron metabolism in peripheral tissues.
Nramp2 mRNA is ubiquitously expressed,27 but is abundant
in kidney and intestine25 and is dramatically upregulated by iron
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4414
starvation in the latter tissue.25 The Nramp2 gene encodes two
mRNAs generated by alternative splicing of two 38 terminal
exons.40 The two mRNAs code for two proteins with distinct
carboxy termini and also have different 38 untranslated regions
in their respective mRNAs, with one showing an IRE. However,
the tissue, cellular and subcellular localization of the Nramp2
protein isoforms I (IRE) and II (non-IRE) have not yet been
determined and need to be elucidated to better understand the
role of these two proteins in iron metabolism in the intestine and
in peripheral tissues. In the current study, we have generated an
antiserum against the N-terminus of Nramp2 that is identical in
both Nramp2 isoforms and another one against the C-terminus
of isoform II (Fig 1A). We show that these antisera are specific
and do not cross-react with the closely related Nramp1 protein
(Fig 1B). Furthermore, the simultaneous use of these two
antisera facilitated the analysis of both isoforms in immunoblotting, immunochemistry, and immunofluorescence studies.
The expression of the two Nramp2 protein isoforms was
monitored in a subset of organs and tissues that are important
for iron uptake and storage or that were previously known to
express Nramp2 mRNA. In tissues and in CHO transfectants
controls, Nramp2 was expressed as a broad immunoreactive
species of molecular mass 80 to 100 kD. This is larger than that
predicted from cDNA sequencing (60 kD). We have recently
shown that this is caused by extensive N-linked glycosylation of
the protein.59 In general, the isoform II of Nramp2 could not be
detected by our antibody in these tissues by immunoblotting
(Fig 3) and immunostaining (data not shown). The lack of
immunoreactive isoform II expression in gut tissues was
somewhat surprising and could be explained by one of three
possibilities: (1) isoform II is not expressed in these tissues; (2)
it is expressed but only at low levels and our antiserum is not
reactive enough to detect it; and (3) it is expressed but
posttranslationally modified in such a way that it is no longer
recognized by the antiserum. Although the antiserum directed
against isoform II is indeed less reactive than that directed
against the amino terminus, the following observations suggest
that it is clearly not isoform II that is upregulated in the
duodenum. First, the anti-isoform II antiserum detects the
protein expressed in CHO cells (Figs 1 and 3). Secondly, the
antibody recognizes isoform II expressed in other cell types
such as macrophages (J774, RAW) and erythroleukemia cells
(MEL; F.C.-H., unpublished data). On the other hand, isoform I
was detected by our antiserum in membrane fractions from
duodenum, proximal intestine, and from kidneys. In addition,
expression of isoform I was dramatically upregulated by dietary
iron starvation. This regulation was specific for the duodenum
and was not seen in other tissues positive for expression (Fig 3).
In the duodenum, Nramp2 isoform I is expressed in the mucosa,
is localized to the villi, and is absent in the crypts. In villi, it is
expressed in the apical two thirds part and is restricted to
absorptive epithelial cells (absent in goblet cells), where it is
concentrated at the brush border (Figs 4 and 5). Moreover,
Nramp2 protein expression was highest in the proximal duodenum and decreased more distally (data not shown). In the rat, it
has been shown that DCT1 (Nramp2) mRNA expression is
highest in the duodenum and also decreases more distally.25
This proximal to distal gradient of Nramp2 expression in the
duodenum and the localization of Nramp2 at the brush border of
CANONNE-HERGAUX ET AL
the enterocytes are in agreement with the known physiological
site of ferrous iron uptake in the gut, which is mostly limited to
the brush border of the proximal intestine.13,14 In addition, the
location of the Nramp2 protein restricted to the proximal part of
the duodenum is compatible with the demonstrated pHdependence divalent cation transport by Nramp2,25 in which a
relatively acidic pH of the gastric juice60 could provide the
gating mechanism for Nramp2 activation. Finally, the localization of Nramp2 to the brush border of the villi is in agreement
with a carrier-mediated process for ferrous iron uptake and with
the large body of published data documenting the presence of
specific iron-binding proteins15,61 and iron transport activities in
brush border membrane preparations.11,62
Iron homeostasis is maintained primarily by controlling
intestinal iron absorption.4,5 This is a complicated process
performed by the enterocyte that involves conversion of iron to
a transportable form, transport across the apical membrane of
the epithelium, intracellular movement to the basal pole, and
transport across the basolateral membrane and ultimately across
endothelial cells into the blood stream.63 Thus, iron has to
traverse several biological barriers in the form of cell membranes to which it is fairly impermeant, and the carrier-mediated
process seems to play a key role in this journey. For example, in
the unicellular eukaryote Saccharomyces cerevisiae (yeast), up
to 6 distinct transport activities for iron have been functionally
identified,64 and there is no reason to believe that this number
would be lower in mammals. In the duodenal lumen, ferric iron
is converted to ferrous iron by the action of ferrireductase.11
Conversion of Fe(III) to Fe(II) has been shown to be a
prerequisite for intestinal iron absorption by mice and humans,10 and inhibition of ferrireductase in Caco-2 cells reduces
iron transport.11,65 The transport of iron across the apical pole of
the enterocyte has generally been believed to be a carriermediated process.61,66 Teichmann and Stremmel67 have described a saturable, temperature-dependent, iron transport activity (FeIII) in brush border membrane vesicles. These
investigators were able to isolate a putative iron transporter as a
membrane glycoprotein of 160 kD, and composed of a trimer of
a 54-kD monomeric unit.67 More recently, Ikeda-Moore et al15
described low- and high-affinity binding sites for Fe(II) present
in solubilized brush border membrane (BBM) of rat intestine
and isolated three proteins (143, 100, and 77 kD) with iron
binding activity. The microcytic anemia phenotype of rat and
mouse mutants at Nramp222,23 and the known biochemical
properties of Nramp2,25 together with the localization of
Nramp2 to the brush border of the duodenum presented in this
report, clearly establish that Nramp2 is the membrane transporter responsible for apical iron entry into enterocytes. Nramp2
is specific for Fe(II) and does not use Fe(III) as a substrate,25
suggesting that Nramp2 only transports Fe(II) at that site and is
thus functionally linked to ferrireductase activity in the lumen.
The proton gradient (with exterior positive) required for activation of Nramp2 and for the proton cotransport of iron25 by
Nramp2 may be provided by the relatively acidic environment
of the proximal portion of the duodenal lumen.
The intracellular trafficking of iron in enterocytes to reach the
basolateral membrane is the least understood aspect of iron
uptake. It has been suggested that, once released in the
cytoplasm, Fe(II) may bind to specific chelators or iron
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NRAMP2 PROTEIN EXPRESSION IN INTESTINE
chaperones to shuttle to the baso-lateral membrane.64 Conrad et
al68 have proposed a model in which iron would bind to mucin
in the intestinal lumen, then transfer to integrin at the cell
surface, and then transfer to the intracellular protein mobilferrin. However, a precise role for these proteins in iron transport
has yet to be demonstrated. The transport of iron across the
basolateral membrane is most likely a carrier-mediated process.
Indeed, studies in everted membrane vesicles from duodenum
of the mouse mutant sla (sex-linked anemia) indicate that
mucosal acquisition of iron is normal in this mutant, whereas
iron export from these cells would be impaired.69,70 The recent
chromosomal assignment of the sla mutation,71 together with
the noted absence of Nramp2 expression at the basolateral
membrane, suggests that Nramp2 is not involved in this
process. Furthermore, biochemical and genetic studies in copperdeficient animals72,73 have pointed at the serum coppercontaining protein ceruloplasmin (ferroxidase activity) as an
important component of iron release from the enterocyte.74
Recently, Vulpe et al75 have identified a gene in the sla region
that codes for a protein presenting 50% sequence similarity with
the mouse ceruloplasmin, including conservation of multiple
copper binding sites. This protein is highly expressed in mouse
intestine. Interestingly, in sla mice, this gene shows an in-frame
deletion of 194 amino acids, probably resulting in a nonfunctional product. The investigators conclude that this new protein
is a transmembrane-bound ferroxidase necessary for iron export
from the intestine.
Finally, the iron overload syndrome hereditary hemochromatosis (HH) has been shown to be caused by a mutation in a novel
HFE gene (HLA-H), a member of the major histocompatibility
complex (MHC) class I family.76 This syndrome is characterized by a defect in regulation of iron absorption that, ultimately,
leads to iron deposition in several organs.77 A similar phenotype
has been observed in a mouse mutant bearing a null allele at the
␤2-microglobulin locus, and ␤2M⫺/⫺ mice have been proposed as an animal model for the study of HH. Recent studies78
have suggested that the defect observed in ␤2M⫺/⫺ mice
(inability to downregulate intestinal iron absorption) could
reflect a regulatory role of ␤2M/HFE on Nramp2 or on the sla
gene product or alternatively on the activity or expressivity of
ferrireductase.
We have noted dramatic upregulation of the Nramp2 protein
expression in the duodenal mucosa by dietary iron. These
findings are in agreement with the upregulation of Nramp2
RNA previously detected by Northern blotting in the same
tissue.25 This dramatic upregulation of Nramp2 mRNA and
protein may occur through the presence of the IRE element in
the 38 UTR of the Nramp2 mRNA or alternatively may be
caused by transcriptional activation of the gene. We do not have
direct evidence to support either, and discussion of this regulation can only be speculative at this time. However, many
mRNAs encoding proteins involved in metabolism of iron
contain IREs in their UTRs that mediate changes in protein
levels in response to iron availability through posttranscriptional mechanisms.43 Under conditions of low iron availability,
IRE-binding proteins are available for binding to IREs. Binding
of proteins to IREs in the 58UTR of mRNAs such as that
encoding ferritin causes a decreased translation of the message.
Conversely, protein binding to IREs in the 38UTR of mRNAs
4415
like the one encoding the transferrin receptor causes an
increased half-life of the mRNA. This allows for the coordinate
regulation of iron uptake (TfR) and storage (ferritin) in response
to iron availability. Interestingly, we note that the iron depletion
protocol used here had no effect on TfR expression in the
intestine, whereas it caused upregulation only in liver. The lack
of TfR regulation by dietary iron in the intestine is surprising
but has been previously reported.79 Nevertheless, we must
consider that the IRE present in Nramp2 mRNA may function to
regulate expression of Nramp2 protein in response to iron. This
is strongly suggested by the observation that it is the Nramp2
isoform I that is upregulated in the duodenal mucosa (Fig 3) and
by the recent report that both IRP1 and IRP2 bind to the hairpin
structure in the 38end of the Nramp2 IRE mRNA.80 Gunshin et
al25 have observed a gradient of Nramp2 mRNA expression in
the intestinal mucosa, from high in the crypts and at the base of
the villi, to progressive attenuation of expression towards the
distal part of the villi, to complete absence of expression in the
distal third of the villi. On the other hand, we have observed
somewhat of an opposite pattern of Nramp2 protein expression,
with no expression in the crypts and at the base of the villi, and
increasing expression from the proximal half to the distal half of
the villi (Fig 3). This suggests that Nramp2 mRNA expression
may be turned on in precursor cells of the crypts in response to
iron starvation. The mRNA continues to accumulate during
maturation of the cells along the structure of the villus, at which
point Nramp2 isoform I protein begins to accumulate to become
maximum in the most mature cells of the villi. Interestingly,
Oates et al81 reported a similar observation for ferritin gene
expression in intestine of rats with varying iron stores. In
iron-deficient and iron-loaded rats, ferritin mRNA was seen at
highest levels in the epithelial cells of the crypt and at lower
levels in villus epithelial cells. However, the ferritin protein was
not seen in the crypt region but was seen with increasing
staining in the apical two thirds of the villus cells of control and
iron-loaded rats, but not in iron-deficient rats. Therefore, it is
tempting to speculate that expression of ferritin and Nramp2
genes and proteins may be regulated in somewhat opposite
manner in response to body iron level but through a common
mechanism. In undifferentiated crypt cells, the Nramp2 and
ferritin genes are transcribed, but the messages may not be
translated. During and after differentiation of the enterocytes,
Nramp2 and ferritin expression could then be regulated posttranslationally in response to the size of iron stores.
Finally, we noted only a modest increase in Nramp2 isoform I
expression in the kidney, in response to depletion of dietary
iron. This suggests that some component of the IRE or other
regulatory pathway required for Nramp2 isoform I expression is
missing in kidney tissue or that only a subset of Nramp2
positive cells in the kidney are responsive to iron regulation
and, consequently, Nramp2 isoform I overexpression. In addition, we cannot exclude the possibility that regulation of
Nramp2 mRNA expression may be at the transcriptional level in
the kidney and does not involve participation of the IRE
element itself. Finally, Nramp2 has been shown to transport a
number of divalent cations in addition to iron. Thus, it is also
possible that regulation of Nramp2 protein expression in the
kidney involve additional mechanisms associated with metabolisms of these other cations. The answers to these questions
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
4416
CANONNE-HERGAUX ET AL
await the identification of the cell type and subcellular localization of the Nramp2 protein in kidney and further characterization of its transcriptional or posttranscriptional regulation.
ACKNOWLEDGMENT
The authors are indebted to Dr N. Beauchemin (McGill University)
for the generous gifts of the anti-Bgp1 antiserum and to Dr E. Roux
(McGill University) for helpful advice and guidance in the immunochemistry studies.
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1999 93: 4406-4417
Cellular and Subcellular Localization of the Nramp2 Iron Transporter in the
Intestinal Brush Border and Regulation by Dietary Iron
F. Canonne-Hergaux, S. Gruenheid, P. Ponka and P. Gros
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