ORIGINAL ARTICLE
Expression of iron absorption genes in mouse large intestine
KEN TAKEUCHI1, INGVAR BJARNASON1, ABAS H. LAFTAH2,
GLADYS O. LATUNDE-DADA2, ROBERT J. SIMPSON2 & ANDREW T. MCKIE2
1Division of Internal Medicine, Diabetes and Endocrinology and 2Department of Life Sciences King’s College London, UK
AbstractObjective. The large intestine has been reported to have a capacity for iron absorption and expresses genes for ironabsorption normally found in the duodenum. The importance and function of these genes in the large intestine are notunderstood. We therefore investigated the cellular localization and regulation of expression of these genes in mouse caecumand colon. Material and methods. Gene expression was measured by real-time PCR using RNA extracted from iron-deficient and hypoxic mouse large intestine, compared to controls. Protein localization and regulation were measured byimmunohistochemistry using frozen sections of the large intestine from the same mice. Results. Dcytb (duodenal ferricreductase) was expressed at very low levels in the large intestine, compared to the duodenum, while Ireg1 and DMT1 wereexpressed at significant levels in the large intestine and were increased in iron-deficient caecum, proximal and distal colon,with the most significant increases seen in the distal colon. Hypoxia increased Ireg1 expression in the proximal colon.Immunohistochemistry detected significant levels of only IREG1, which was localized to the basolateral membrane ofcolonic epithelial cells. Conclusions. Iron absorption genes were expressed at lower levels in mouse caecum and colon thanin the duodenum. They are regulated by body iron requirements. Colonic epithelial cells express basolateral IREG1in thesame fashion as in the duodenum and this protein could regulate colonic epithelial cell iron levels.
Key Words: Dcytb, DMT-1, Ireg1, large intestine, mouse
Introduction
Iron is an essential element required by mammals for
a wide spectrum of biologic functions, including
oxygen transport, mitochondrial electron transfer,
and a wide range of enzymatic reactions. However,
excess iron can catalyse the generation of oxygen free
radicals that can be toxic for cellular components.
Therefore, the amount of iron must be strictly
regulated to prevent accumulation of excess.
Iron homeostasis is thought to be maintained
primarily by regulation of iron absorption by the
proximal small intestine [1]. Iron absorption has
traditionally been attributed to the activity of a
highly regulated iron uptake pathway localized to
the duodenum. Recent work has led to the identifi-
cation of several genes for the uptake pathway of
non-haem (inorganic) iron in mice and humans [1].
Duodenal cytochrome b (Dcytb) is highly expressed
in the brush border membrane of enterocytes and
reduces dietary ferric iron to the ferrous form for
transport via the brush border membrane iron
transporter, Nramp2/DCT1 (divalent-cation trans-
porter 1) [2,3]. IREG1/ferroportin is localized in the
basolateral membrane and mediates iron efflux [4].
Hephaestin is the ferroxidase that is postulated to
play a role in the release of iron from the basolateral
membrane of the enterocyte, but there seems to be
little regulation of expression of this gene by iron
status [5,6]. In accordance with earlier physiological
studies, the expression of most of these genes is
highly localized within the small intestine to duode-
num and proximal jejunum. Expression of the genes
declines along the small intestine [6]. This compares
with the report of a sharp fall in transfer of iron to
the body when going distally from duodenum to
jejunum and ileum [7]. However, a surprising
finding was that some of these genes (especially
Ireg1, Hfe and hephaestin) are expressed at signifi-
cant levels in the colon [6], an organ not usually
thought to be important for iron absorption. Phy-
siological studies have occasionally proposed a role
Correspondence: Ken Takeuchi, MD, PhD, Division of Internal Medicine, Diabetes and Endocrinology, King’s College London, Guy’s, King’s and
St Thomas’ School of Medicine, Bessemer Road, London SE5 9PJ, UK. Tel: �/44 020 78 8 4578. Fax: �/44 020 7848 4500. E-mail: [email protected]
Scandinavian Journal of Gastroenterology, 2005; 40: 169�/177
(Received 14 May 2004; accepted 3 September 2004)
ISSN 0036-5521 print/ISSN 1502-7708 online # 2005 Taylor & Francis
DOI: 10.1080/00365520510011489
for the colon in iron homeostasis [8�/10], but this is
not a universal finding [7]. Removal of the colon
may not affect iron absorption, although it is
frequently associated with iron deficiency [11,12].
Iron metabolism in this organ has, however, been
implicated in colon carcinogenesis [13,14]. It was
reported that HFE gene mutations, which are
responsible for hereditary haemochromatosis, are
associated with an increased risk of colon cancer
[13]. Furthermore, some studies showed a positive
association between dietary and body iron stores and
colorectal cancer risk [15�/17]. Studies in rats have
shown that increasing luminal iron levels in the colon
can lead to oxidative damage and proliferative
changes in the colonic mucosa [18,19].
We therefore set out to investigate in more detail
the regulation of expression of iron absorption genes
in mice with altered iron metabolism in order to
clarify a possible function for colonic expression of
these genes.
Material and methods
Animals and treatments
Dietary iron deficiency. Four 3-week-old CD1 male
mice were fed a low iron diet for 3 weeks from
weaning, which consisted of vitamin-free casein
(18%), hydrogenated cotton seed oil (5%), sucrose
(73%), adequate vitamin, and iron-free mineral
mixtures [20]. The diet content has been shown to
satisfy the recommended dietary intake criteria, as
described by the US National Research Council
(1962). Four controls received the same diet with
the exception that it was supplemented with iron as
FeCl3 (125 mg/kg diet).
Hypoxic treatment. CD1 mice (aged about 6 weeks)
were used. Four mice were placed in a hypobaric
chamber at 0.5 atm for each of 24 or 72 h [21]. Four
controls were left at room air pressure. All the mice
were provided with food and water ad libitum . The
mice were fed a standard rodent diet (SDS, Witham,
Essex, UK, diet RM1; this diet contains 106 mg/kg
Fe).
The mice were killed with an overdose of inhaled
anaesthetic and then death was confirmed by dis-
location of the neck. Samples of the duodenum and
the colon were embedded in freezing medium
(Triangle Biomedical Sciences, Durham, N.C.,
USA) and immediately frozen in liquid nitrogen for
immunohistochemistry analysis. At the same time,
the remaining mucosa of the duodenum and the
colon was used for total mRNA extraction. All the
protocols were reviewed and approved by the UK
Home Office following the Animals (Scientific
Procedures) Act 1986.
Liver non-haem iron determination
Non-haem iron levels in liver were measured as
described elsewhere [22].
RNA extraction and cDNA preparation
Total RNA was extracted from the mucosa of the
duodenum, the caecum, the proximal colon and the
distal colon with TRIZOL Reagent (Invitrogen Ltd.,
Paisley, UK), separated from DNA and protein with
chloroform. The RNA samples were precipitated
with isopropyl alcohol and washed with 75% etha-
nol. Following brief drying, the RNA pellets were
dissolved in RNase-free water and stored at �/808Cuntil required. cDNA was synthesized using the
general protocol for first-strand cDNA synthesis.
Total RNA (1 mg) and 2 ml Random hexanucleotides
(Hexanucleotide mix, 10�/; Roche Diagnostics
GmbH, Penzberg, Germany) were denatured for
10 min at 658C and immediately cooled on ice. PCR
nucleotide mix (2 ml of mixture with 10 mM of each
nucleotide), 4 ml of 5�/ Expand Reverse Transcrip-
tase buffer, 2 ml of 100 mM DTT and 1 ml Expand
Reverse Transcriptase (Roche Diagnostics GmbH,
Mannheim, Germany) 50 U/ml was added to the
tube. The mixture was incubated for 10 min at
308C, 45 min at 428C, and finally cooled to 48C.
The samples were then stored at �/208C.
Real-time PCR
For quantification of the target gene expression,
RT-PCR was carried out by means of the Light-
Cycler (Roche Diagnostics GmbH, Penzberg,
Germany). PCR reactions were performed in a final
volume of 20 ml using FastStart DNA Master SYBR
Green I (Roche Diagnostics GmbH, Penzberg,
Germany). Forward and reverse primers for the
genes of interest were designed (Table I) and
purchased from MWG (MWG-Biotech Ltd., Milton
Keynes, UK). The final concentrations of the
primers (0.25 or 0.5 mM) and MgCl2 (3 or 4 mM)
were optimized for each gene (Table I). The PCR
conditions were as follows: a pre-incubation step at
958C for 10 min, followed by 45 cycles or 30 cycles
in the case of 18S of 958C for 10 s, annealing at the
corresponding temperature for each primer set for
5 s and extension at 728C for 1 s per 25 bp of the
amplicon. The efficacy of the amplification was
confirmed by a melting curve analysis and by gel
electrophoresis to confirm the presence of a single
product.
170 K. Takeuchi et al.
For quantitative analysis, a standard curve for
each target gene was established by performing the
above procedure on serially diluted cDNA samples
from total RNA from normal mouse duodenal
mucosa. The relative concentration of the target
gene was calculated based on crossing-point analy-
sis, followed by normalization using the relative
amount of the 18S gene expression (m18s) in the
same sample.
Immunohistochemistry
Pieces of mouse duodenum were excised and placed
in tissue freezing medium (Triangle Biomedical
Sciences) and frozen on dry ice. Tissue sections
(5 mm) were cut at �/208C using a cryostat (Bright
Instrument Co. Ltd., England) and placed on
polylysine-coated microscope slides (Sigma Diagnos-
tics P.O., St. Louis, Mo., USA). Before proceeding
with immunocytochemistry, the sections were fixed
for 20 min in 100% acetone at 48C. Following
blocking with 1% bovine serum albumin (BSA) in
PBS, the sections were incubated with 1:100 diluted
primary antibody for each gene of interest for an hour
at room temperature. Polyclonal antibodies raised
in rabbits against mouse antigenic peptides from
each specific protein, as described in. [2,4] (Dcytb;
[Cys]-DAESSSEGAARKRTLGLADSGQRSTM,
corresponding to the COOH-terminus of mouse
Dcytb, coded 834, IREG1; [Cys]-GKQLTSPKD-
TEPKPLEGTH, corresponding to amino acids
247�/264 of mouse IREG1, coded 3566, Sigma-
Genosis, UK). After the primary antibody was
removed by washing in PBS, the sections were
incubated with 1:100 diluted swine FITC-con-
jugated anti-rabbit secondary (DAKO A/S, Copen-
hagen, Denmark) for an hour at room temperature.
The slides were washed in PBS and then mounted
with VECTASHIELD (Vector Laboratories Inc.,
Burlingame, Calif., USA) for fluorescence micro-
scopy.
Statistical analysis
All values are expressed as means 9/SEM. Statistical
differences between the data were analysed with
StatView for windows (SAS Institute Inc., Cary,
N.C., USA) and Microsoft Excel (Microsoft Cor-
poration, N.Y., USA) using repeated two-way AN-
OVA and Student’s t-test or Welch’s t-test (p B/0.05).
Results
Effect of an iron-deficient diet on gene expression in the
caecum and the colon
Mice fed on a purified low-iron diet had redu-
ced liver non-haem iron levels compared to
controls receiving the same diet supplemented
with iron (respectively, 0.199/0.03 versus 1.539/
0.22 nmol/mg, SEM, n�/4, p B/0.001). RT-PCR
was carried out to determine the expression of iron
metabolism genes in duodenal and colonic mucosa
from these mice. All the genes related to iron
metabolism that we investigated by RT-PCR, i.e.
Dcytb, Ireg1 and DMT1, could be detected in the
large intestine. The amount of Dcytb gene expres-
sion was much lower than that in the duodenum and
no significant change was found between the cae-
cum, proximal and distal colon (Figure 1A). Iron-
deficient diet feeding induced an increase in the gene
expression in both the duodenum and large intes-
tine. However, the increase was of lower magnitude
in the large intestine compared with that seen in the
duodenum. Significant increases were seen in the
caecum and distal colon.
In the caecum and the colon, DMT1 gene
expression was detected (Figure 1B). The amount
of DMT1 gene was also lower in the large intestine
than in the duodenum and no difference in intensity
of the gene expression was found from the caecum to
the distal colon. Cloning of DMT1 cDNAs has
shown that alternative splicing and 3? end processing
yield two DMT1 mRNA variants that differ with
regard to their 3? terminal sequences of the ORF and
Table I. Primer design.
Primer Sequence Annealing
temperature (8C)
MgCl2(mM)
Amplicon
(bp)
mDMT-1 IRE forward 5?-CTG CTG AGC GAA GAT ACC-3? 52.6 4 103
mDMT-1 IRE reverse 5?-GTA AAC CAT AGA AAC ACA CTG G-3?mIREG-1 forward 5?-CCA GCA TCA GAA CAA ACA CG-3? 57.3 3 175
mIREG-1 reverse 5?-ACT GCA AAG TGC CAC ATC C-3?mDcytB forward 5?-ATG TAC AGC CTG CAC AGC-3? 56.4 4 192
mDcytB reverse 5?-TGT CAC TCC CAT GAG AAC C-3?m18s forward 5?-GAA TTC CCA GTA AGT GCG GG-3? 59.4 3 51
m18s reverse 5?-GGG CAG GGA CTT AAT CAA CG-3?
Iron absorption genes in large intestine 171
their 3? UTRs. One of these variants carries a
conserved iron�/responsive element (IRE), where-
as the other does not. The DMT1 mRNA
harbouring an IRE in its 3? UTR was found to be
up-regulated in the duodenum of iron-deficient
animals (Figure 1B). The increase in DMT1-IRE
mRNA was larger in the distal colon compared to
other parts of the large intestine. Significant
increases were seen in the caecum and distal colon.
The Ireg1 gene was expressed at lower levels in the
caecum and the colon than in the duodenum;
however, interestingly, the difference in the amount
of gene expression between the large intestine and
the duodenum was not as great as that seen for the
other two genes (Figure 1C). There was no
significant variation in Ireg1 mRNA between the
caecum and proximal and distal colon. Iron-defi-
cient diet feeding was found to increase Ireg1 mRNA
Figure 1. Effect of an iron-deficient diet on the expression of the iron metabolism-related gene in mouse large intestine. RT-PCR gene
expression of Dcytb (A), DMT1 (B) and Ireg1 (C) in the mucosa of the duodenum, the caecum, the proximal and the distal colon.
Mice were fed with a control diet (open bars) or iron-deficient diet (closed bars) for 3 weeks from weaning. Data are expressed as
means 9/SE (n�/4); p B/0.01; p B/0.05 versus control diet.
172 K. Takeuchi et al.
levels in the distal colon by approximately
4-fold (control 116.39/46.5 versus iron-deficient
443.59/43.7%; p B/0.05). Other regions of the large
intestine showed little or no increase (Figure 1C).
Effect of hypoxia on gene expression in the caecum and
the colon
The effect of hypoxia was studied in mice fed on a
standard rodent laboratory diet. Mice fed on this
diet had much higher expression of Dcytb, DMT1
and Ireg1 in the large intestine than was found in
mice fed the purified diet.
Hypoxia has been known to up-regulate the
expression of genes related to iron metabolism in
the duodenum. In this study, hypoxia augmented the
expression of Dcytb gene expression in the duode-
num (Figure 2A). However, because of inter-animal
variations, the results did not show statistically
significant differences. In the caecum and the colon,
no significant effect of hypoxia was detected after
72 h.
DMT1 IRE in the duodenum tended to increase
in line with the hypoxia time-course, but the
increases did not reach statistical significance.
DMT1 mRNA levels in the caecum were compar-
able with those in the duodenum; however, we found
significantly lower levels in the duodenum compared
with the proximal colon (p B/0.05). DMT1 mRNA
levels were lower in the distal colon than in the
duodenum, caecum and proximal colon. In addition,
the gene expression in the caecum tended to increase
in parallel with the duodenum (Figure 2B).
The results of hypoxic exposure on Ireg1 gene
expression are shown in Figure 2C. After hypoxic
treatment started, Ireg1 gene expression in the
duodenum was increased at 24 h and then decreased
back to levels seen in controls after 72 h. A similar
change could be observed in the caecum and the
proximal colon, the increase in the proximal colon at
24 h hypoxia being significant (p B/0.05). Further-
more, in this model, Ireg1 expression in the caecum
was less than in other parts of the colon (caecum
7.29/1.5, proximal colon 14.89/1.0, distal colon
37.99/6.7; caecum versus proximal colon p B/0.01,
caecum versus distal colon p B/0.05).
Localization of IREG1 in enterocytes of the colon
To determine the subcellular localization of Dcytb
and IREG1 in the enterocyte of the colon, immuno-
fluorescence was carried out using the polyclonal
antibodies that were raised in rabbits against mouse
antigenic peptides from each specific protein.
No immunofluorescence staining of the colonic
enterocytes with Dcytb antibody was seen even in
hypoxic conditions (Figure 3B), despite staining
being visible in duodenal sections from the same
mice (Figure 3A). Presumably, as with the mRNA,
this protein is present at much lower levels in the
colon than are seen in the duodenum.
Using IREG1 polyclonal antibody, immunofluor-
escence was detected in the basolateral membrane of
the enterocyte of the large intestine and no staining
was detected on the apical side. Furthermore, the
staining was predominantly in villous enterocytes
(Figure 3D, F). This staining pattern resembles
the localization of the duodenum enterocytes
(Figure 3C, E). Although immunofluorescence was
also performed to determine an effect of iron-
deficient diet on IREG1 expression, no clear differ-
ence from the control was detected (data not shown).
Discussion
There have been suggestions that iron absorption
might occur in the colon, at least in rodents
[7,9,10,23], dogs [8]or guinea pigs [24]. Colonic
epithelial cells have increased iron levels when diet-
ary iron levels are increased [25,26], suggesting that
the colon can take up iron.
In this study, our data show that some of the genes
responsible for iron absorption in the duodenum are
also present in the large intestine at significant levels
and are regulated by body iron requirements.
Another stimulus for iron absorption, hypoxia, also
showed a tendency to increase expression of the
genes for iron absorption in the colon, although the
effects were not as clear-cut as seen with iron
deficiency. Hypoxia can increase the number
of circulating red blood cells by stimulating erythro-
poiesis in order to compensate for the lower oxygen
tension. Consequently, hypoxia increases the de-
mand for iron and increases iron absorption through
modulation of hepcidin levels [27], which ultimately
alters gene expression of duodenal iron transporters.
However, the effect of hypoxia on iron metabolism
and gene expression appears to be more variable
than deficiency of iron, perhaps because the stimulus
of hypoxia to iron metabolism is more complex.
We could not find evidence for the duodenal ferric
reductase Dcytb being present at significant levels;
the small amounts of mRNA that were detected
could be explained by tissue macrophages, which
express Dcytb (Y. Sanchez and A.T., Mckie, unpub-
lished observation).
In the duodenum, IREG1 is expressed in mature
duodenal enterocytes of the villus and little in
the crypt regions [4,28]. In this study, we found
that immunofluorescence with IREG1 polyclonal
antibody was predominantly detected in the baso-
lateral membrane of the villous enterocytes of the
Iron absorption genes in large intestine 173
large intestine, similar to the staining pattern in the
duodenal enterocytes. This suggests that IREG1 of
enterocytes in the large intestine could have the same
function as in the duodenum; that is, efflux of
cellular iron. The finding that Ireg1 expression is
regulated by iron levels suggests that colonic epithe-
lial cell iron may be regulated by Ireg1.
Conditions in the large intestinal lumen are less
favourable for iron absorption than conditions in the
duodenum, although the presence of a large caecum
in rodents provides the possibility for further release
of nutrients, including iron from partially digested
food, and the transit time of luminal contents in the
caecum is slow, which might facilitate absorption.
Figure 2. Effect of hypoxia on the expression of the iron metabolism-related gene in mouse large intestine. Gene expression of Dcytb (A),
DMT1 (B) and Ireg1 (C) in the mucosa of the duodenum, the caecum, the proximal and the distal colon. Mice were exposed to hypoxia for
0 h (open bars), 24 h (dot bars) or 72 h (closed bars). Data are expressed as means 9/SE (n�/3�/4); p B/0.05 versus control.
174 K. Takeuchi et al.
Iron metabolism in the large intestine may have two
factors, one being absorption of iron as one of
nutrient elements and the other elimination of excess
toxic iron from the intestinal mucosa. The excess
iron may participate in Fenton reactions, which
increase the production of hydrogen peroxide
and hydroxyl radicals at the mucosal surface, in
conjunction with the intraluminal bacteria [29].
Furthermore, hydrogen peroxide or iron may induce
inflammation or enter the colonocytes and increase
the risk of DNA damage and thus act to increase the
risk of mutations occurring either as initiating events
or later in the adenoma carcinoma sequence [30]. It
does not seem that it is necessary for colonocytes
themselves to have the ferric reductase Dcytb for
iron absorption, either because there are significant
numbers of bacteria, which could have ferric
reductases, in the large intestine, or the colon may
possess alternate ferric reductases to Dcytb.
Recently, it has been reported that caecal contents
in mice contain significant levels of ferrous iron
and this can be affected by the bacterial strains
present [31].
It is important that, in order to maintain a
constant concentration of iron in a colonocyte,
both DMT-1, an apical membrane iron transporter,
Figure 3. Localization of Dcytb and IREG1 in enterocytes in the duodenum and the colon. Immunofluorescence imaging was performed
using antibodies specific for Dcytb and IREG1. Nuclei are stained with propidium iodide, and Dcytb and IREG1 are visualized with a
FITC-labelled secondary antibody. Dcytb is detected at the apical side of the duodenal enterocyte (A), but there is no staining in the colon
(B). IREG1 is detected in the basolateral membrane of the enterocytes both in the duodenum (C, E) and the colon (D, F). C. and D. Low
magnification image. E. and F. High magnification image.
Iron absorption genes in large intestine 175
and IREG1, a basolateral iron transporter, must be
expressed in the large intestine at significant levels.
These genes may be necessary only for the purpose
of maintaining the homeostasis of iron in colono-
cytes; consequently, alteration in expression of those
genes in the large intestine is much smaller than in
the duodenum. Further studies are required to test
whether such a mechanism for iron absorption is
important in man and to examine whether iron
species suitable for absorption are present in the
colon. It is noteworthy that a colon-derived human
carcinoma cell line (Caco 2) expresses Ireg1, Hfe,
DMT1 and Dcytb [3,4,32,33].
Iron metabolism in the colon has hardly been
examined and the implication that iron may play a
role in colonic carcinogenesis suggests that further
inquiry into this area is needed. The finding
that genes for colonic iron metabolism can be
regulated by iron level may provide insights into
the mechanism and possible prevention of iron
mediated colonic carcinogensis. The present data
show that iron absorption genes present in mouse
caecum and colon are regulated by body iron
requirements.
Acknowledgements
Ken Takeuchi was supported by the Medical
Corporation Ryowa-kai (Japan), Andrew T. Mckie
received an MRC Career Development award. The
work was supported by the UK MRC.
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