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: ken.takeuchi@kcl.ac.uk

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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