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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4406-4417
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
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.
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ABSTRACT |
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 3' 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 duodenum 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 mucus-secreting 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.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IRON 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) 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
resistance-associated 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 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 micro-organisms 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 3' exons encoding
distinct C-termini of the protein as well as distinct 3'
untranslated regions (UTRs).40 Interestingly, one
Nramp2 mRNA contains an iron-responsive element (IRE) in its 3'UTR. The IRE is an RNA secondary structure present in the
5'- or the 3'-UTR of animal mRNAs encoding proteins
involved in iron metabolism.41,42 In iron-replete 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
3' 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.
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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 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 (SDS-PAGE), 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 intervals, and
blood (10 mL) was obtained by arterial puncture 11 days after each
boost. Serum was isolated and kept frozen at 20°C.
Affinity 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 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
paraffin-embedded 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 3'-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.
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RESULTS |
Production and characterization of isoform-specific anti-Nramp2
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 3' untranslated region (presence or
absence of iron-response 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.
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| Fig 1.
Detection of the Nramp1 and Nramp2 cMyc-tagged 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 3' end exon
(see Lee et al40). The two mRNAs harbor distinct 3'
untranslated region (3'UTR) 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.
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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). 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
affinity-purified 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 anti-Nramp2 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
anti-Nramp2 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 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 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 either Nramp1 or Nramp2
isoform II were included as controls (Fig 3, lanes 15, 16, and 17).
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| 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.
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| Fig 3.
Effect of dietary iron depletion on Nramp protein
expression in normal tissues. Organs were harvested from mice
maintained on low iron diet (Fe) or on the same diet but
supplemented with iron (+Fe). 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 anti-Nramp antibodies, 25 µg of membrane proteins from control
CHO cells (CHO) or CHO transfectants expressing either a cMyc-tagged
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.
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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, 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 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 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.
View larger version (89K):
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| 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
anti-mouse Bgp1 antibody (C and F). Sections were counterstained with
methylene blue, before microscopic examination and photography. Low
magnification (400×) 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).
|
|
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 rhodamine-coupled 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).
View larger version (89K):
[in this window]
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| 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 × 1,000.)
|
|
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
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 starvation in the latter tissue.25 The
Nramp2 gene encodes two mRNAs generated by alternative splicing
of two 3' terminal exons.40 The two mRNAs code for
two proteins with distinct carboxy termini and also have different
3' 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 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 pH-dependence 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
carrier-mediated 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 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 copper-deficient animals72,73 have pointed at
the serum copper-containing 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 3' 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 5'UTR of mRNAs such as that encoding
ferritin causes a decreased translation of the message. Conversely,
protein binding to IREs in the 3'UTR of mRNAs 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
3'end 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 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.
| |
FOOTNOTES |
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.
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.
Address reprint requests to P. Gros, PhD, Department of Biochemistry,
McGill University, 3655 Drummond, Room 907, Montreal, Quebec, Canada
H3G-1Y6; e-mail: gros{at}med.mcgill.ca.
| |
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TOP
ABSTRACT
INTRODUCTION