Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan
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ABSTRACT |
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Development and differentiation of the intestinal epithelium appear to be regulated by various growth factors. Using cDNA microarrays, we identified heparin-binding EGF-like growth factor (HB-EGF) as one of the genes induced by intestinal-specific transcription factor Cdx2 in an intestinal undifferentiated rat cell line, intestinal epithetial cell (IEC)-6. Both Cdx2 and HB-EGF stimulated cell proliferation and migration, and their effects were inhibited partially by an EGF receptor-specific tyrosine kinase inhibitor, PD-153035. HB-EGF may function as one of the mediators of Cdx2 and may be associated with the proliferation and migration in the intestinal epithelium. The Cdx2 protein can bind to the Cdx2-binding element of the HB-EGF gene. Reporter gene analyses showed that the HB-EGF gene promoter is Cdx2 responsive and that the activity of the promoter in the IEC-6 cells depends on the number of consensus Cdx2-binding site-like sequences. These data indicate that HB-EGF gene expression can be regulated by Cdx2 and serves to mediate the control of Cdx2 of the proliferation and migration of IEC-6 cells.
enterocyte; intestinal epithelial cell-6
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INTRODUCTION |
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HOMEOBOX GENES ENCODE for transcription factors involved in the regulation of development and differentiation in organisms. Cdx2 is a member of the caudal-related homeobox gene family based on its sequence homology to the caudal gene of Drosophilae melanogaster (11). In the developing mouse embryo, the Cdx2 gene is expressed at 8.5 days after coitum when the visceral endoderm transforms into a simple columnar epithelium with nascent villi, a critical transition during intestinal development (2). In the adult mouse, the Cdx2 gene is expressed in the epithelium of the small intestine and colon with undetectable levels of mRNA in other tissues (11). In experiments with intestinal epithelial cells (IEC-6), an undifferentiated rat intestinal epithelial cell line that does not express Cdx2 protein, the expression of Cdx2 protein was observed to affect cellular proliferation and induce cellular differentiation characterized by multicellular structures with microvilli and tight junctions (21). In addition, mice homozygous for a Cdx2 null mutation die in embryonic life (3.5-5.5 days postcoitum), whereas heterozygotes develop multiple intestinal polyps (4). These findings suggest that Cdx2 is necessary to control the proliferation and differentiation of intestinal epithelial cells. However, it is unclear which Cdx2-induced genes regulate cellular proliferation and differentiation. Cdx2 protein interacts with DNA elements that have an AT-rich consensus sequence (TTTAC/T). Several studies (6, 7, 13, 14, 20, 22, 24) have reported that Cdx2 activates the transcription of various genes, such as sucrase-isomaltase (SI), lactase-phlorizin hydrolase, lactase, apolipoprotein B, carbonic anhydrase 1, calbindin-D9K, and clusterin, but these genes are not known as regulators of cellular proliferation. Therefore, it is unclear how Cdx2 is involved in the control of cellular proliferation.
To identify the gene targets for Cdx2 that may be involved in the proliferation or differentiation processes, we compared the expression of genes that are differentially expressed during Cdx2 induction by means of a cDNA array hybridization technique. One of the mRNAs induced after the expression of Cdx2 was heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), a member of the EGF family and a potent stimulator of cellular proliferation and migration. In this study, we identified HB-EGF as a novel Cdx2-responsive gene. We have shown that Cdx2 can activate transcription from the HB-EGF promoter and that sequences similar to consensus Cdx2-binding sites within the promoter are necessary for transactivation.
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MATERIALS AND METHODS |
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Cell culture. The rat intestinal cell line IEC-6 was obtained from Riken Cell Bank (Tsukuba, Japan). Cells were maintained under an atmosphere of 5% CO2 at 37°C in DMEM containing 5% FCS (P. A. A. Wiener Strasse, Linz, Austria), 0.1 U/ml insulin, 50 U/ml penicillin, and 50 µg/ml streptomycin (1× PSS; Sigma, St. Louis, MO). On reaching 90% confluence, cells were dissociated with 0.25% trypsin and 0.02% EDTA and placed again.
Cell lines with conditional Cdx2 expression. The Ecdysone-Inducible Mammalian Expression System (Invitrogen, Carlsbad, CA) was used in the method for directing the conditional expression of Cdx2 in stably transfected cell lines. The complete open reading frame of the rat Cdx2 gene was obtained by RT-PCR from rat intestine. Primers were selected from the consensus cDNA sequences between human and mouse Cdx2 (5, 11). The 5' region was generated by using the forward primer1, 5'-CCACCATGTACGTGAGCTAC-3', and the reverse primer1, 5'-GTCCTGGTTTTCACTTGGCT-3'; and the 3' region was generated using the forward primer2, 5'-GGACGTGAGCATGTACCCT-3', and the reverse primer2, 5'-GCGTCCATACTCCTCATGG-3'. The PCR products were directly subcloned into pGEM-T Easy Vector (Promega, Madison, WI) and sequenced by using an automated sequencer (PerkinElmer, Norwalk, CT). Both PCR fragments contained a PstI restriction site corresponding to the single site included in the Cdx2 open reading frame. This site was used to reconstitute a fragment overlapping the complete Cdx2 open reading frame, and this fragment was inserted in the EcoRI site of expression plasmids, pIND(Sp1) and pIND(Sp1)/V5-His in frame with COOH-terminal tag. Plasmid DNA was purified with a Qiagen plasmid kit (Qiagen, Hilden, Germany), and DNA concentration was measured by spectrophotometry. The sequence was verified by the chain termination methods by using a PerkinElmer automated sequencer. IEC-6 cells were stably cotransfected with the pVgRXR and pIND-Cdx2 plasmids. Transfections were carried out with Effectene following the manufacturer's instructions. Colonies were picked after 3 wk in selective medium (0.3 mg/ml Zeocin for selection of pVgRXR clones and 0.6 mg/ml G-418 for selection of pIND-Cdx2 clones).
RNA extraction, labeling, and hybridization of cDNA array.
Atlas mouse cDNA expression arrays from Clontech (Palo Alto, CA) were
chosen for this study. Total RNA was extracted from the cell lines by
using an Atlas Pure RNA Isolation kit (Clontech) according to the
manufacturer's instructions. Labeling, hybridization, and washing of
the cDNA array membranes were carried out according to the
instructions. Briefly, 3-4 µg total RNA was used as a template for cDNA synthesis, which was done in the presence of
deoxyadenosine [-32P]triphosphate (dATP; Amersham
Pharmacia Biotech, Piscataway, NJ). The labeled probes were purified by
spin-column centrifugation (Chroma Spin-200; Clontech), and
hybridization was carried out overnight with continuous agitation at
68°C. The membranes were then washed at 68°C (3 times with 2× SSC,
1% SDS, followed by 2 times with 1× SSC, 0.1% SDS) and exposed to
X-ray film at
70°C for 1-3 days.
Gene delivery to the mouse intestine and epithelial cell isolation. Delivery of Cdx2 gene to intestine of 6-wk-old B6C3F1 male mice (Charles River, Yokohama, Japan) was done by tail vein injection of pTRACER plasmid (Invitrogen) including Cdx2 cDNA with pretreatment of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylamonium salts (DOTAP). DOTAP-cholesterol-plasmid DNA complex was made by using an in vivo GeneSHUTTLE kit (Quantum, Montreal, PQ, Canada). Intestinal epithelial cell isolation was adapted from Grossmann et al. (9). The tissue was dissected and thoroughly rinsed in Ca2+- and Mg2+-free HBSS (Sigma) and cut into strips. These were washed in 10 mM DTT for 30 min at room temperature and then incubated for 60 min in 1 mM EDTA in HBSS at 4°C. Epithelial cells were detached as intact crypts by 10 vigorous shakes of the vessel and immediately passed through an 80-µm nylon mesh. The retained crypt on the mesh was rinsed with DMEM and resuspended in keratinocyte serum-free medium (Invitrogen) containing 1× PSS. Cell viability was assessed by trypan blue exclusion and light microscopy.
RT-PCR. RT-PCR was performed on oligo(dT)-primed cDNA. Gene-specific primers, Cdx2, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), HB-EGF, and green fluorescent protein (GFP) primers were designed as follows: Cdx2 forward, 5'-GGACGTGAGCATGTACCCT-3'; Cdx2 reverse, 5'-CTGAGCGCTATCCAAGTTCG-3'; G3PDH forward, 5'-ACCACAGTCCATGCCATCAC-3'; G3PDH reverse, 5'-TCCACCACCCTGTTGCTGTA-3'; HB-EGF forward, 5'-GTGCTGAAGCTCTTTCTGG-3'; HB-EGF reverse, 5'-CGCCCAACTTCACTTTCTC-3'; GFP forward, 5'-GAAGGTGATGCTACATACG-3'; and GFP reverse, 5'-CAGTTACAAACTCAAGAAGG-3'. The PCR product sizes of Cdx2, G3PDH, HB-EGF, and GFP genes were 151, 452, 588, and 577 bp, respectively.
Western blot analysis. Cells were harvested and resuspended in buffer A [10 mM HEPES/KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5% (octylphenoxy)polyethoxyethanol] and protease inhibitor cocktail (Sigma) and were disrupted by passage through a 21-gauge needle five times. The supernatant was removed by centrifugation at 6,500 g for 20 s, and the nuclear pellet was resuspended in buffer B (20 mM HEPES/KOH, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, and protease inhibitor cocktail). Protein concentration was determined by a bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL). Equal amounts of protein were fractionated by 10% SDS-PAGE and transferred to a Hybond enhanced chemiluminescense (ECL) membrane (Amersham Pharmacia). Western blotting was carried out by using an ECL kit as described by the manufacturer (Amersham Pharmacia). Cdx2 with a COOH-terminal polyhistidine tag was detected by the horseradish peroxidase (HRP)-conjugated anti-His6 antibody (Invitrogen). HB-EGF was detected by the anti-HB-EGF antibody and a HRP-conjugated secondary antibody to goat Ig (Santa Cruz Biotechnology, Santa Cruz, CA). The film was scanned into Adobe Photoshop 5.5, and the signal density was measured with NIH Image software.
Assessment of cellular proliferation. To assess the change in cell number after various durations of cell culture, cells were plated in 60-mm-well culture plates at 104 cells/well. At 24 h after plating, medium containing or not containing 5 µM ponasterone A (PA) was added, and the cells were collected by trypsin treatment at various intervals and counted with a hemacytometer. A bromodeoxyuridine (BrdU) incorporation assay for the quantification of cell proliferation was performed by using a cell proliferation ELISA kit as described by the manufacturer (Roche Diagnostics, Mannheim, Germany). PD-153035 was purchased from Calbiochem (San Diego, CA).
Measurement of cell migration. Cells were plated on 35-mm dishes and grown to 95% confluence. Then the cell layer was scratched with a sterile razor blade to initiate migration. After the scratch, care was taken to include some identifying mark on the dish to serve as a future reference point. After wounding, the cells were incubated for 48 h. They were then fixed with 2% formaldehyde and 0.2% glutaraldehyde in phosphate-buffered saline, and cell migration was measured.
Reporter assay.
The 5'-flanking region of the rat HB-EGF gene between +103 and 1587
(15) was the kind gift of Dr. John C. Pascall (Babraham Institute, Cambridge, UK). The DNA fragments of the HB-EGF genomic clone were obtained by PCR, confirmed by sequence analysis, and subcloned into the multiple-cloning site of the promoterless vector pGL3- Basic (Promega) to generate the reporter constructs pGL3/HB-EGF 1587 (
1587/+72), pGL3/HB-EGF 1139 (
1139/+72), pGL3/HB-EGF 451 (
451/+72) and pGL3/HB-EGF 392 (
392/+72). The numbers in parentheses indicate the nucleotide position with respect to the transcription initiation site. Three mutant reporter vectors (Mut
1-3) were constructed by a site-directed mutagenesis kit,
QuickChange (Stratagene, La Jolla, CA) from pGL3/HB-EGF 451. For the
luciferase assay, IEC-6/Cdx2 cells plated 24 h prior to
transfection at 2 × 105 cells/well in 60-mm-well
plates were cotransfected with 1 µg of the firefly luciferase
reporter plasmid and 50 ng of the Renilla luciferase
reporter plasmid pRL-TK (Promega) by using Effectene according to the
manufacturer's instructions (Qiagen). After 24 h of
incubation, the cells were lysed in lysis buffer supplied by the
manufacturer, followed by measurement of the firefly and Renilla luciferase activities on a luminometer (model 1253;
Dainippon, Osaka, Japan). The relative firefly luciferase activities
were calculated by normalizing transfection efficiency according to the
Renilla luciferase activities. The experiments were
performed in triplicate, and similar results were obtained from at
least three independent experiments.
Electrophoretic mobility supershift assay.
Nuclear extract was obtained as described in Western blot
analysis. The Cdx2 binding consensus region probe consisted of a double-stranded 9-bp oligonucleotide (5'-TTTTTACAC-3') from
429 to
421 of rat HB-EGF gene. The sequence of the mutant oligonucleotide was 5'-TTTTGCCAC-3'. Probes were labeled
with a digoxigenin (DIG) oligonucleotide labeling kit (Roche
Diagnostics, Mannheim, Germany) according to the manufacturer's
instructions. Nuclear extract (2 µg) was incubated in the presence of
1-ng DIG-labeled probe with 0.5 µg poly(dI-dC), 0.02 µg salmon
testis DNA, 50 mM Tris · HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 2 mM MgCl2, and 5% glycerol for 30 min at room temperature.
DNA-protein complexes were loaded on 5% polyacrylamide gel. For DIG
detection, gels were transferred by electroblotting for 30 min at 40 mA
to Hybond N+ membranes (Amersham). DIG detection was
performed as described by the manufacturer (Roche Diagnotics). The
polyclonal rabbit affinity-purified antibody raised against Cdx2 was
produced as previously described by Silberg et al. (19).
Statistics. All results are presented as means ± SE. Data were analyzed by using the Mann-Whitney U-test. Statistical values of P < 0.05 were considered significant. Statistical analysis was performed with SPSS software (SPSS, Chicago, IL).
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RESULTS |
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Effects of conditional Cdx2 expression on proliferation of IEC-6
cell lines.
To investigate the target genes that Cdx2 induces in the intestinal
epithelial cells, we first established an inducible system in which
Cdx2 expression could be controlled. The IEC-6/Cdx2 clone cell
conditionally expresses Cdx2 under the control of an ecdisone-regulated nuclear receptor. When an induction medium containing PA, an ecdysone analog, was added to the cultures, there was a marked induction of Cdx2
mRNA and protein (Fig. 1, A and
B). To demonstrate that Cdx2
was functional in IEC-6/Cdx2 cells, we performed transactivation experiments with constructs involving the SI promoter, through which
Cdx2 activates transcription (10, 20). Although the activity of the SI promoter was weak in the basal condition, the induction of Cdx2 expression led to twice the activity (Fig.
1C), strongly suggesting that Cdx2 is indeed expressed in
these cells. In this clone, the growth rate of IEC-6/Cdx2-expressing
cells increased significantly compared with that of nonexpressing cells (Fig. 1D). The IEC-6/Cdx2 cells that were not treated with
PA showed the same proliferation rate as the parental IEC-6 cells treated with PA (data not shown).
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Ectopic Cdx2 expression induces HB-EGF transcripts.
To identify target genes for Cdx2 in the IEC-6 cell line, we used a
cDNA array hybridization technique to compare the expression patterns
of several mRNA between IEC-6 cells immediately after the induction of
Cdx2 and IEC-6 cells in which Cdx2 was not induced. An analysis of cDNA
array hybridization data identified the HB-EGF mRNA whose expression
was stimulated in IEC-6 cells expressing Cdx2. The differential
expression of HB-EGF was characterized further by RT-PCR analysis and
Western blot analysis of IEC-6 cells during Cdx2 induction (Fig. 2,
A and B).
IEC-6/Cdx2 cells cultured in medium that did not contain PA expressed
very low levels of HB-EGF mRNA. IEC-6/Cdx2 cells were stimulated with
PA to express Cdx2 as early as 2 h after induction. By 2 h
after induction with PA, there was already a small increase in HB-EGF mRNA, and by 4 h, the increase had reached its peak. HB-EGF
protein was also detected 24 h after induction with PA. Parental
IEC-6 cells incubated with medium containing PA had no effect on the expression of HB-EGF mRNA (data not shown). To determine whether Cdx2
induced HB-EGF in the intestinal tissue of the mouse, we transfected
Cdx2 by tail vein injection of pTRACER-Cdx2 with pretreatment of
DOTAP plus cholesterol. Although no expression of HB-EGF was detected in the isolated epithelial cells taken from the intestine of
the normal mice, the epithelial cells from the Cdx2-transfected mice
were found to express detectable HB-EGF mRNA (Fig. 2C).
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Effects of Cdx2 and HB-EGF on cellular proliferation and migration.
HB-EGF is a potent stimulator of cellular proliferation and DNA
synthesis in the intestinal cell line (3). We compared BrdU incorporation in IEC-6 cells expressing Cdx2 and in IEC-6 cells
treated with HB-EGF (Fig. 3A).
The expression of Cdx2 stimulated BrdU incorporation significantly
(P < 0.05), as did the administration of HB-EGF. The
EGF receptor-specific tyrosine kinase inhibitor PD-153035
(8) blocked HB-EGF-stimulated BrdU incorporation completely (Fig. 3B). Under these conditions, the effect of
Cdx2 expression on the BrdU incorporation was inhibited (Fig.
3B). HB-EGF also stimulates the cell migration of epithelial
cells (25). We therefore monitored the modulation of IEC-6
cell migration by Cdx2 expression. As shown in Fig.
4, the administration of HB-EGF and the
expression of Cdx2 led to a significantly higher migration rate
(P < 0.05). In the presence of PD-153035 (1 µM), neither HB-EGF nor Cdx2 stimulated the migration of the IEC-6 cells
(Fig. 4). These results suggest that EGF receptor tyrosine kinase
activation is at least partially required for Cdx2-induced cellular
proliferation and migration.
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Effect of Cdx2 expression on rat HB-EGF gene promoter activity.
To examine whether the HB-EGF gene is a potential target of Cdx2
transactivation, the ability of the rat HB-EGF promoter to drive
transcription in the intestinal cell line was tested by using various
lengths of the 5'-flanking region of the rat HB-EGF gene linked to the
luciferase reporter gene (Fig. 5). As
shown in Fig. 6, Cdx2 expression in IEC-6
cells caused a threefold increase in the promoter activity of the
1587-bp promoter (pGL3/HB-EGF 1587), but 1139-bp and 451-bp HB-EGF
reporters (pGL3/HB-EGF 1139 and 451) show slightly weaker induction
(2.5-fold and 2-fold, respectively) in response to Cdx2. In contrast,
we observed no enhanced transcription in cells transfected with the
392-bp 5'-flanking DNA (pGL3/HB-EGF 392) that contained no caudal motif
(TTTAC/T).
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Cdx2 proteins interact with the HB-EGF promoter element.
To determine whether Cdx2 proteins in IEC-6/Cdx2 cells bind to the
caudal motif of the HB-EGF gene, we incubated the DIG-labeled Cdx2
region probe and nuclear extract from the IEC-6/Cdx2 cells in the
absence or presence of polyclonal Cdx2 antibody. The probe alone or the
unbound probe migrated rapidly through the entire gel in this
condition. A DNA-protein complex of slower mobility was detected as
shown in Fig. 8. The increase in the
amount of the probe-protein complex depended on the concentration of PA that induced the Cdx2 expression. Mutant oligonucleotide
(5'-TTTTGCCAC-3') provided in 100-fold excess was not able
to compete for nuclear protein binding to the probe (data not shown).
Cdx2 antibody recognized the nuclear protein bound to the Cdx2 region
probe, resulting in a reduction of gel mobility (Fig. 8).
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DISCUSSION |
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The present study identifies the HB-EGF gene as a target for Cdx2 transcription factor in intestinal epithelial cells. HB-EGF is widely expressed (17), but its mRNA levels seem to be relatively low in normal tissues. Its expression increases in response to tissue damage, such as ischemia-reperfusion injury (16). Because HB-EGF stimulates the survival (23), proliferation (1), and migration (25) of epithelial cells, one physiological role of induced HB-EGF expression would seem to be the promotion of epithelial repair after wounding. Our results in this study showed that conditional Cdx2 expression in IEC-6 cells stimulated BrdU incorporation (Fig. 3) and cell migration after wounding (Fig. 4), as did HB-EGF. These effects were inhibited by an EGF receptor tyrosine kinase-specific inhibitor, PD-153035, suggesting that HB-EGF may act as one mediator of the Cdx2 pathway and may be associated with the proliferation and migration in the intestinal epithelia. Thus the Cdx2 and HB-EGF pathway may be involved in the wound healing in the intestinal epithelia.
Rings et al. (18) reported that the Cdx2-activation domain could be phosphorylated at Ser60 via the mitogen-activated protein kinase pathway and suggested that unphosphorylated Cdx2 might contribute to the epithelial cell differentiation in the intestine. This suggestion seems to account for the development of epithelial cell differentiation by stable Cdx2 transfection in IEC-6 as reported by Suh and Traber (21). In contrast, the role of the phosphorylated Cdx2 in the proliferative compartments of the intestine is less well understood, although the phosphorylated Cdx2 was found mainly in the crypt of the intestine (18). So, it is interesting and necessary to investigate the effects of phosphorylated Cdx2 on the expression of HB-EGF and on the proliferation, differentiation, and migration of the intestinal epithelial cells.
The rapid induction of HB-EGF mRNA on the expression of Cdx2 in IEC-6
cells provides evidence that the regulation of the HB-EGF gene is part
of a cascade of events initiated by Cdx2 expression (Fig.
2A). Transfection experiments using the rat HB-EGF gene promoter show that Cdx2 acts as a regulator of the transcriptional initiation of the HB-EGF gene. The 5'-flanking region of the HB-EGF gene revealed that the activation of the HB-EGF promoter depends on the
number of Cdx2-binding consensus sequences. Reduction of the number of
Cdx2-binding elements led to a decrease in the promoter activity of
HB-EGF in response to the Cdx2 expression. In addition, promoter
activity was further reduced by mutants in which the Cdx2-binding
elements between 451 and
392 were disrupted. Cdx2 protein could
bind to the Cdx2-binding element of the HB-EGF gene. These sites appear
to play an essential role in the regulation of HB-EGF gene by Cdx2.
We assume it is unlikely that Cdx2 is the only transcription factor
involved in HB-EGF gene transcriptional regulation in the intestinal
epithelial cells, because promoter activity of the HB-EGF gene
(pGL3/HB-EGF 1587) was increased in unstimulated (PA)
IEC-6 cells (Fig. 6). The AT-rich regions containing the Cdx2-binding motif in the HB-EGF promoter region are known to be binding sites for a
wide range of homeoproteins. A homeodomain transcription factor,
pancreatic duodenal homeobox-1 (PDX-1), is known to regulate HB-EGF
gene expression during pancreas development (12). The presence of PDX-1 mRNA was detected in IEC-6 cells (data not shown). Therefore, PDX-1 may regulate the HB-EGF gene expression in the basal
condition (Fig. 2A). Although a role for PDX-1 in intestinal gene regulation has not been established, a recent study
(10) indicates that PDX-1 and Cdx2 can physically interact
and bind to each other. Whether such interactions are involved in the
HB-EGF gene transactivation remains to be investigated.
In conclusion, HB-EGF was here shown to be induced by the Cdx2 transcription factor. HB-EGF is likely to be one of many target genes regulated directly by Cdx2 and may be involved in the differentiation or growth of intestinal epithelial cells.
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ACKNOWLEDGEMENTS |
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Technical assistance in the performance of these studies was provided by Noriko Kageyama.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Uesaka, Dept. of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan (E-mail: tochi{at}hiroshima-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 3, 2002;10.1152/ajpgi.00075.2002
Received 20 February 2002; accepted in final form 24 June 2002.
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