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Regulation of TFF3 expression by homeodomain protein CDX2
Regulatory Peptides 140 (2007) 81 – 87 www.elsevier.com/locate/regpep
Regulation of TFF3 expression by homeodomain protein CDX2 Tadahito Shimada ⁎, Takero Koike, Michiko Yamagata, Masashi Yoneda, Hideyuki Hiraishi Department of Gastroenterology, Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan Institute for Medical Science, Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan Received 24 June 2006; received in revised form 3 November 2006; accepted 10 November 2006 Available online 19 December 2006
Abstract Although trefoil factor family 3 (TFF3) plays an important role in protecting the intestinal mucosa, the regulatory mechanisms of its expression are not fully understood. Since homeodomain protein CDX2 has been reported to be critically involved in the development and differentiation of intestinal epithelium, we examined whether CDX2 affects the expression of TFF3. The transcription of human TFF3 reporter genes was significantly up-regulated by the transient overexpression of CDX2 in COS-7 cells and AGS gastric cells. Electrophoretic mobility shift assay revealed the presence of at least two CDX-binding sites within the human TFF3 promoter. Deletion analysis showed the relative importance of the proximal CDX-binding site at − 63. We also detected the up-regulation of endogenous TFF3 mRNA expression in AGS cells stably transfected with CDX2 expression vectors. These results suggest that CDX2 plays a key role in the expression of TFF3 in the intestine and perhaps in intestinal metaplasia of the stomach. © 2006 Elsevier B.V. All rights reserved. Keywords: TFF3; Intestinal trefoil factor; CDX2; Goblet cells; Intestinal metaplasia
1. Introduction The trefoil factor family (TFF) is a group of small secretory peptides that plays critical roles in the protection and repair of the gastrointestinal mucosa [1–3]. Among three TFF members, TFF1 (formerly pS2) and TFF2 (spasmolytic polypeptide, SP) are specifically expressed in gastric epithelial cells, while TFF3 (intestinal trefoil factor, ITF) is expressed at a high level in the goblet cells of the small and large intestines [2]. It is also known that, in intestinal metaplasia of the stomach, the expression of TFF3 is induced and the expression of TFF1 and TFF2 is downregulated [3]. Mashimo et al. [4] generated Tff3-deficient mice and showed that they developed serious colitis in response to dextran sulfate sodium (DSS) treatment, confirming the indispensable role of TFF3 in the protection and repair of the lower intestinal mucosa. Several authors have investigated the regulatory mechanisms of the intestine-specific expression of ⁎ Corresponding author. Department of Gastroenterology, Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan. Tel.: +81 282 86 1111; fax: +81 282 86 7761. E-mail address: [email protected] (T. Shimada). 0167-0115/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.11.014
TFF3. In mice, a combination of the goblet cell-responsive element (GCRE), which is present in the proximal Tff3 promoter, and the goblet cell-silencer inhibitor (GCSI), which is located about 2200 bp upstream of the Tff3 transcriptional start site, has been reported to be important for the goblet cell-specific induction of TFF3 expression [5,6]. Iwakiri and Podolsky [7] also reported the involvement of keratinocyte growth factor (KGF) in the differentiation of goblet cells and KGF-induced upregulation of TFF3 expression. However, the detailed regulatory mechanisms of the expression of TFF3 in the lower intestine and in intestinal metaplasia of the stomach remain to be elucidated. CDX (caudal-related homeobox) proteins (CDX1 and CDX2) are physiologically expressed in intestinal epithelial cells distal to the duodenum and are critically involved in the development, proliferation, and differentiation of intestinal epithelial cells [8]. Chawengsaksophak et al. [9] reported the characteristics of Cdx2-deficient mice, in which the small intestinal crypt–villus architecture was altered and they developed colonic tumors. Intestinal abnormalities have also been reported in Cdx1-deficient mice [10]. Suh and Traber [11] showed that overexpression of CDX2 in IEC-6, an undifferentiated intestinal cell line, leads to the differentiation of the cells to functional
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intestinal epithelial cells. Since CDX proteins are transcription factors, a number of studies identified the involvement of CDX in regulating the expression of genes encoding intestine-specific proteins, such as sucrase-isomaltase [12], lactase [13,14], calbindin-D9K [15,16], apolipoprotein B [17], claudin-2 [18], and MUC2 [19,20]. Although CDX expression is absent in the normal gastric mucosa, the ectopic expression of CDX is detected in the gastric mucosa with intestinal metaplasia [21–23]. The appearance of goblet cells and expression of intestine-specific proteins, such as MUC2, are also characteristics of intestinal metaplasia of the stomach [21,23]. Silberg et al. [24] made Cdx2 transgenic mice using Foxa 3-driven Cdx2, and Mutoh et al. [25] made Cdx2 transgenic mice using H-K ATPase promoter-driven Cdx2. In these Cdx2 transgenic mice, normal gastric mucosa was completely replaced by intestinal metaplasia, confirming the central role of CDX2 in the induction of intestinal metaplasia. The above evidence suggests a possible role of CDX2 in the induction and regulation of TFF3 expression in the lower intestine and in intestinal metaplasia of the stomach. Thus, in this study, we investigated the effect of CDX2 on the expression of TFF3 in COS-7 cells and AGS gastric cells to determine whether TFF3 is a direct target of CDX2.
visualized with LumiGLO chemiluminescent reagent (Cell Signaling Technology; Beverly, MA). 2.3. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR)
COS-7 cells (a SV40-transformed cell line derived from African green monkey kidney) and AGS cells (a cell line derived from human gastric carcinoma) were obtained from the Human Health Resources Bank (Osaka, Japan). COS-7 cells were grown in Dulbecco's Modified Eagle's Medium (Invitrogen; Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen). AGS cells were maintained in Ham's F-12 culture medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen).
Total RNA was extracted from the cells with TRIZOL reagent (Invitrogen). First-strand cDNA was made with You-Prime FirstStrand Beads (GE Healthcare Bio-Sciences) using oligo (dT) primers (Invitrogen). The PCR primers used in this study were made based on the reported sequences as follows: human TFF3 (GenBank accession No. NM_003226); 5′-TGTCTGCAAACCAGTGTGCC-3′ (sense) and 5′-GCATTCTGTCTTCCTAGTCAGGG-3′ (antisense) (product length 158 bp), human MUC2 (NM_002457); 5′-ATTTGTCATGTACTCGGCCAA-3′ (sense) and 5′-CGATGTGGGTGTAGGTGTGTG-3′ (antisense) (product length 137 bp), human CDX2 (NM_001265); 5′ACCGCAGAGCAAAGGAGAGG-3′ (sense) and 5′-CCAGGGACAGAGCCAGACAC-3′ (antisense) (product length 187 bp), human β-actin (NM_001101); 5′-TTCCTGGGCATGGAGTCCT-3′ (sense) and 5′-AGGAGGAGCAATGATCTTGATC-3′ (antisense) (product length 204 bp). Conventional RT-PCR was performed in a GeneAmp PCR System 9700 (Applied Biosystems; Foster City, CA) using HotStar Taq DNA polymerase (Qiagen; Hilden, Germany). Real-time quantitative RT-PCR was performed in an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) using SYBR Green reagents (Applied Biosystems) as described previously [26–28]. After an initial 10 min at 95 °C, the reaction was run for 35 PCR cycles of 15 s at 95 °C and 1 min at 56 °C. To prepare standard templates, conventional RT-PCR was performed using the primers mentioned above and PCR products were purified with Qiaquick PCR Purification Kits (Qiagen). Purified PCR products were diluted and used as standard samples (6 × 102 to 6 × 107 copies) to generate a standard curve for each experiment. β-actin mRNA measurement was always performed for standardization.
2.2. Western blotting of CDX2 proteins
2.4. Plasmid construction and transfection
Cells grown in 10-cm culture dishes were washed with phosphate-buffered saline (PBS) and cell lysates were obtained using Passive Lysis Buffer (Promega; Madison, WI). Supernatants of the whole cell lysates (30 μg protein/lane) were subjected to 5–20% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (HyBond P membrane, GE Healthcare Bio-Sciences; Piscataway, NJ). The membrane was blocked with TTBS [10 mM Tris–HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20] containing 5% non-fat dry milk for 1 h at room temperature and then incubated with monoclonal anti-CDX2 antibody (BioGenex; San Ramon, CA) in TTBS containing 5% non-fat dry milk overnight at 4 °C. After washing with TTBS, the membrane was incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnologies; Santa Cruz, CA) in TTBS containing 5% non-fat dry milk for 1 h at room temperature. The membrane was washed again and the band was
The 5′-flanking regions of human TFF1 (−953 to +34) (Accession No. AB038162), TFF2 (−912 to +24) (AB038162), TFF3 (−957 to +12) (AB038162) and human MUC2 (−1139 to +22) (U67167) were PCR amplified and cloned into the SmaI site of the pGL3-basic luciferase vector (Promega). The nucleotide identity and direction of the insert were verified by sequencing of both strands. GeneEditor in vitro site-directed mutagenesis system (Promega) was employed to modify the TFF3 reporter gene. In order to make a CDX2 expression vector, the coding sequence of the human CDX2 (NM_001265) was PCR amplified and cloned into pcDNA3.1/V5/His vector (Invitrogen). The sense and antisense primers were 5′-GCAGCATGGTGAGGTCTGCTCC-3′ and 5′-CTGGGTGACGGTGGGGTTTAGC-3′, respectively. Since the PCR product does not possess a terminal codon, this expression vector produces CDX2 proteins that contain C-terminal V5/His tags. Similarly, a CDX1 expression vector was also made using the following
2. Materials and methods 2.1. Cell lines and culture
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Fig. 2. Effects of the transient transfection of CDX2 expression vectors on the transcription of MUC2, TFF1, TFF2, and TFF3 reporter genes in COS-7 cells. Control cells were transfected with empty pcDNA3.1/V5/His vectors. pSV-βgalactosidase vectors were co-transfected for standardization. (mean ± S.D., n = 4).
Fig. 1. (A) A representative RT-PCR experiment showing the expression of CDX2 mRNA in COS-7 cells. Lane 1, 100 bp ladder; lane 2, COS-7 cells transfected with empty pcDNA3.1/V5/His vectors; lane 3, COS-7 cells transfected with CDX2 expression vectors. (B) Representative Western blot analysis showing the expression of CDX2 proteins in COS-7 cells. Lane 1, COS-7 cells transfected with empty pcDNA3.1/V5/His vectors; lane 2, COS-7 cells transfected with CDX2 expression vectors.
primers based on the reported sequence (NM_001814): 5′-GGGACCCCGCGGCCACCATGTA-3′ (sense) and 5′-GCACAGGCTGGGCATGGGGCTA-3′ (antisense). The nucleotide identity of the inserts was verified by sequencing of both strands. For transient transfection assays, cells were seeded onto 24well culture plates the day before transfection. On the day of transfection, reporter vectors (0.6 μg/well) together with CDX expression vectors (0.4 μg/well) were transfected into the cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Empty pcDNA3.1/V5/His vectors were transfected to control cells instead of the CDX expression vectors. For standardization, pSV-β-galactosidase vectors (Promega) (0.05 μg/well) were also transfected into the cells. Thirty hours after transfection, cells were harvested using the Passive Lysis Buffer (Promega) and reporter gene assays were performed in a microplate luminometer LB96V (Berthold Technologies; Bad Wildbad, Germany). Luciferase activity was measured using the Luciferase Assay System (Promega) and β-galactosidase activity was measured using the β-Gal Reporter Gene Assay System (Roche Diagnostics; Mannheim, Germany). For stable transfection of the CDX2 expression vectors, AGS cells were seeded on a 6-well culture plate and the CDX2 expression vectors or the empty pcDNA3.1/V5/His vectors described above (2.4 μg/well) were transfected into the cells using Lipofectamine 2000 reagent (Invitrogen). Genetecin (Invitrogen) was used for cell cloning. 2.5. Electrophoretic mobility shift assay (EMSA) Double-stranded DNA oligonucleotides labeled with Cy3 at the 5′-termini of both strands (Tsukuba Oligo, Tsukuba, Japan)
were made for EMSA. Sense strand sequences were as follows: TFF3-A, 5′-ATGTGTACCATGTTTTTACTAACATATTTT-3′, which corresponds to − 624 to − 595 of the human TFF3 promoter, TFF3-B, 5′-CAAACAACGGTGCATAAATGAGGCCTCCTG-3′, which corresponds to − 76 to − 47 of the human TFF3 promoter (putative CDX-binding sequences are underlined). We also made another double-stranded oligonucleotide designated as SIF-1S, the sequence corresponding to CDX-responsive elements within the promoter of the gene encoding human sucrase-isomaltase [12]. The sense strand sequence was 5′-GGCTGGTGAGGGTGCAATAAACTTTATGAGTA-3′. SIF-1S was used as a competitor oligo in EMSA experiments. Nuclear extracts were prepared from CDX1- or CDX-2overexpressed COS-7 cells using Nuclear Extract Kits (Active Motif, Carlsbad, CA) according to the manufacturer's protocol. For DNA binding reactions, nuclear extracts (10 μg protein) were added to 1× DNA binding buffer (Promega) containing 125 pmol of the Cy3-labeled oligonucleotide and incubated for
Fig. 3. Effects of the transient transfection of CDX2 expression vectors on the transcription of MUC2, TFF1, TFF2, and TFF3 reporter genes in AGS gastric cells. Control cells were transfected with empty pcDNA3.1/V5/His vectors. pSVβ-galactosidase vectors were co-transfected for standardization. (mean ± S.D., n = 4).
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3. Results 3.1. CDX2 induces TFF3 expression
Fig. 4. Effects of the transient transfection of CDX1 expression vectors on the transcription of MUC2, TFF1, TFF2, and TFF3 reporter genes in COS-7 cells. Control cells were transfected with empty pcDNA3.1/V5/His vectors. pSV-βgalactosidase vectors were co-transfected for standardization. (mean ± S.D., n = 4).
20 min at room temperature. When the competitor oligo was used, 100 × excess amounts of SIF-1S were added to the reaction 10 min prior to the addition of the Cy3-labeled oligonucleotide. The reactions were electrophoresed on 5–20% native PAGE gels (ATTO, Tokyo, Japan) and the gels were directly imaged using a Pharos FX System (Bio Rad Laboratories, Hercules, CA).
Fig. 5. (A) The sequences of putative CDX-binding sites within the human TFF3 promoter are shown. The caudal-related protein consensus binding sites (TTTAC/T) are underlined. (B) Electrophoretic mobility shift assay. Nuclear extracts prepared from CDX1-overexpressed (lanes 3–6) or CDX2-overexpressed (lanes 7–10) COS-7 cells were incubated with Cy3-labeled doublestranded oligonucleotide probes, TFF3-A (lanes 1, 3, 4, 7 and 8) and TFF3-B (lanes 2, 5, 6, 9 and 10), the sequences corresponding to − 624 to − 595 and − 76 to −47 of the human TFF3 promoter. 100 × excess amount of competitor oligonucleotides (SIF-1S) was present in the incubations in lanes 4, 6, 8, and 10. Nuclear extracts were not added to the incubations in lanes 1 and 2.
The entire coding sequence of human CDX2 was PCR amplified and inserted into the pcDNA3.1/V5/His vector to make a CDX2 expression vector. Although COS-7 cells expressed a negligible level of CDX2, transfection of the CDX2 expression vectors caused a significant increase in the expression of CDX2 mRNA as well as CDX2 proteins, as shown in Fig. 1. Fig. 2 shows the effect of CDX2 overexpression on the transcription of TFF3 reporter genes and MUC2 reporter genes in COS-7 cells. The effects of CDX2 overexpression on the transcription of reporter genes for other TFF members, TFF1 and TFF2 are also shown in Fig. 2. Consistent with previous reports [19,20], MUC2 reporter gene transcription was up-regulated by CDX2 overexpression. TFF3 reporter gene transcription was also found to be significantly up-regulated by CDX2 overexpression and that of TFF1 and TFF2 reporter genes was slightly up-regulated. Fig. 3 shows the effect of CDX2 overexpression on these reporter genes in AGS gastric cells. CDX2 similarly up-regulated the transcription of TFF3 and MUC2 reporter genes. TFF1 reporter gene transcription was not significantly affected and that of TFF2 reporter genes was slightly up-regulated. We also tested the effect of CDX1 overexpression on the transcription of TFF1-3 and MUC2 reporter genes in COS-7 cells, as shown in Fig. 4. Although less potent compared to CDX2, CDX1 also up-regulated the transcription of both MUC2 and TFF3 reporter genes.
Fig. 6. (A) From the wild-type TFF3 reporter genes (TFF3-Luc), putative CDXbinding sites within the human TFF3 promoter at − 610 and − 63 were deleted in the mutA and mutB reporter genes, respectively. In the mutC reporter gene, both sites were deleted. (B) Effects of the transient transfection of CDX2 expression vectors on the transcription of the wild-type TFF3 reporter gene and mutated reporter genes in COS-7 cells. (mean ± S.D., n = 4).
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3.2. Interaction of CDX2 proteins with the TFF3 cis-element The CDX-responsive sequence has been reported as TTTAT/ C [12,29]. We identified 2 potential CDX-binding sites within the 5′-untranslated region of human TFF3, as shown in Fig. 5A. To test whether CDX proteins can bind to these elements, we performed EMSA experiments. Nuclear extracts prepared from CDX1- or CDX2-overexpressed COS-7 cells were incubated with Cy3-labeled double-stranded oligonucleotides, the sequences being identical to the potential CDX-binding sites of the human TFF3 promoter. As shown in Fig. 5B, these oligonucleotides made shift bands that disappeared in the
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presence of an excess amount of competitor oligonucleotides, suggesting that CDX proteins can bind to both of these sequences. To examine the relative importance of these CDXbinding sites, we made mutant-type TFF3 reporter genes, as shown in Fig. 6A. The potential CDX-binding site at −610 was deleted in the mutA reporter gene, the potential CDX-binding site at − 63 was deleted in the mutB reporter gene, and both sites were deleted in the mutC reporter gene. Fig. 6B shows the effect of CDX2 overexpression on the transcription of these reporters in COS-7 cells. As shown in this figure, deletion of the potential CDX-binding site at − 63 had more significant effect on the transcription of reporter genes. 3.3. Effect of CDX2 on the expression of endogenous TFF3 mRNA We also stably transfected CDX2 expression vectors or empty pcDNA3.1/V5/His vectors into AGS cells (AGS-CDX2 and AGS-pcDNA). Since AGS cells express endogenous CDX2 at a certain level, two bands (endogenous CDX2 proteins and expression vector-derived CDX2 proteins with 3′-tags) can be seen in the Western blot analysis shown in Fig. 7A. Fig. 7B and C are real-time quantitative RT-PCR experiments showing the expression of endogenous MUC2 and TFF3 mRNA in AGSCDX2 and AGS-pcDNA cells. Not only MUC2 but also TFF3 mRNA expression was up-regulated in AGS-CDX2 cells compared to AGS-pcDNA cells, suggesting the regulation of endogenous TFF3 mRNA expression by CDX2 proteins. 4. Discussion
Fig. 7. (A) Western blot analysis showing the expression of CDX2 proteins in AGS cells (lane 1, AGS cells stably transfected with empty pcDNA3.1/V5/His vectors; lane 2, AGS cells stably transfected with CDX2 expression vectors). In AGS cells stably transfected with CDX2 expression vectors, the expression of V5/His-tagged CDX2 proteins (arrow) was recognized in addition to endogenous CDX2 proteins. (B) Real-tine quantitative RT-PCR analysis showing the expression of endogenous MUC2 mRNA in AGS cells stably transfected with empty pcDNA3.1/V5/His vectors or CDX2 expression vectors. (mean ± S.D., n = 6, p b 0.001 unpaired t-test). (C) Real-tine quantitative RTPCR analysis showing the expression of endogenous TFF3 mRNA in AGS cells stably transfected with empty pcDNA3.1/V5/His vectors or CDX2 expression vectors. (mean ± S.D., n = 6, p b 0.001 unpaired t-test).
In this study, we transfected CDX2 expression vectors into COS-7 cells and AGS gastric cells and examined its effect on the transcription of the human TFF3 reporter genes. Similar to human MUC2, which was previously identified as a target gene of CDX [19,20], we found that TFF3 reporter gene transcription was up-regulated by CDX2 overexpression. CDX1 also had a similar stimulatory effect on the transcription of the TFF3 reporter gene although the effect was less potent than CDX2. Thus, these results suggest a role of CDX proteins in the induction and regulation of human TFF3 expression. The CDX-binding sequence has been reported as TTTAC/T [12,29]. In order to determine whether the regulation of TFF3 expression by CDX is direct, we examined the interaction between CDX proteins and putative CDX-binding sites (− 610 and − 63) within the human TFF3 promoter. Although EMSA experiments (Fig. 5) revealed the possible binding of CDX proteins to both sites, deletion analysis of the TFF3 reporter gene (Fig. 6) suggests the relative importance of the binding site at − 63. We further explored the effect of CDX2 on the expression of endogenous TFF3 mRNA using AGS cells stably transfected with CDX2 expression vectors. Compared to AGS cells that were stably transfected with empty pcDNA3.1/V5/His vectors, CDX2-transfected AGS cells showed a higher level of TFF3 mRNA expression along with a higher level of MUC2 mRNA expression. Together with the data obtained in the reporter gene assays, this study provides evidence that human
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TFF3 is a target of CDX2 and CDX2 plays an important role in the regulation of TFF3 expression. Although we also cloned COS-7 cells stably transfected with CDX2 expression vectors, we could not observe the up-regulated expression of endogenous TFF3 mRNA (data not shown). Thus, it is likely that other cell-type-specific factor or factors are also required for the induction of TFF3 in addition to CDX proteins. Since previous studies indicated the roles of the goblet cell responsive element, the goblet cell-silencer inhibitor, and KGF in the goblet cellspecific expression of TFF3 [5–7], a combination of these factors and CDX may be important. Among three TFF peptides, TFF1 and TFF2 are expressed in the gastric mucosa but it is known that their expression is downregulated in intestinal metaplasia inversely related to the upregulation of TFF3 in intestinal metaplasia of the stomach [3]. Although the present results suggest a role of CDX2, which is expressed at a high level in intestinal metaplasia of the stomach [21–23], in the induction of TFF3, it is also interesting to determine whether the down-regulation of TFF1 and TFF2 expression is related to the appearance of CDX2 in the gastric mucosa. However, based on the results of the reporter gene assays shown in Figs. 2 and 3, it is not likely that CDX2 negatively regulates the expression of TFF1 and TFF2. Thus, the down-regulation of TFF1 and TFF2 expression in intestinal metaplasia of the stomach appears to be independent of CDX2 but mediated by other factor or factors. The results of this study indicate that, in addition to other intestine-specific genes, CDX homeodomain proteins play a critical role in the regulation of TFF3 expression. Further studies are needed to elucidate the interaction between CDX and other transcriptional regulator proteins, and overall transcriptional control of the human TFF3 in intestinal epithelial cells and intestinal metaplasia of the stomach in vivo. Since TFF3 is an important factor in protecting the intestinal mucosa, a better understanding of the regulatory mechanisms of its expression may benefit the clinical application of this peptide. Acknowledgements We thank Drs. Yoichiro Fujii, Akihiro Tajima, and Akira Terano for their helpful discussion. We also thank Ms. Kyoko Tabei and Mr. Takashi Namatame for technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science. References [1] Wong WM, Poulsom R, Wright NA. Trefoil peptides. Gut 1999;44:890–5. [2] Hoffmann W, Jagla W. Cell type specific expression of secretory TFF peptides: colocalization with mucins and synthesis in the brain. Int Rev Cytol 2002;213:147–81. [3] Taupin D, Podolsky DK. Trefoil factors: initiators of mucosal healing. Nat Rev Mol Cell Biol 2003;4:721–32. [4] Mashimo H, Wu DC, Podolsky DK, Fishman MC. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science 1996;274: 262–5. [5] Ogata H, Inoue N, Podolsky DK. Identification of a goblet cell-specific enhancer element in the rat intestinal trefoil factor gene promoter bound by a goblet cell nuclear protein. J Biol Chem 1998;273:3060–7.
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Regulation of TFF3 expression by homeodomain protein CDX2 Tadahito Shimada ⁎, Takero Koike, Michiko Yamagata, Masashi Yoneda, Hideyuki Hiraishi Department of Gastroenterology, Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan Institute for Medical Science, Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan Received 24 June 2006; received in revised form 3 November 2006; accepted 10 November 2006 Available online 19 December 2006
Abstract Although trefoil factor family 3 (TFF3) plays an important role in protecting the intestinal mucosa, the regulatory mechanisms of its expression are not fully understood. Since homeodomain protein CDX2 has been reported to be critically involved in the development and differentiation of intestinal epithelium, we examined whether CDX2 affects the expression of TFF3. The transcription of human TFF3 reporter genes was significantly up-regulated by the transient overexpression of CDX2 in COS-7 cells and AGS gastric cells. Electrophoretic mobility shift assay revealed the presence of at least two CDX-binding sites within the human TFF3 promoter. Deletion analysis showed the relative importance of the proximal CDX-binding site at − 63. We also detected the up-regulation of endogenous TFF3 mRNA expression in AGS cells stably transfected with CDX2 expression vectors. These results suggest that CDX2 plays a key role in the expression of TFF3 in the intestine and perhaps in intestinal metaplasia of the stomach. © 2006 Elsevier B.V. All rights reserved. Keywords: TFF3; Intestinal trefoil factor; CDX2; Goblet cells; Intestinal metaplasia
1. Introduction The trefoil factor family (TFF) is a group of small secretory peptides that plays critical roles in the protection and repair of the gastrointestinal mucosa [1–3]. Among three TFF members, TFF1 (formerly pS2) and TFF2 (spasmolytic polypeptide, SP) are specifically expressed in gastric epithelial cells, while TFF3 (intestinal trefoil factor, ITF) is expressed at a high level in the goblet cells of the small and large intestines [2]. It is also known that, in intestinal metaplasia of the stomach, the expression of TFF3 is induced and the expression of TFF1 and TFF2 is downregulated [3]. Mashimo et al. [4] generated Tff3-deficient mice and showed that they developed serious colitis in response to dextran sulfate sodium (DSS) treatment, confirming the indispensable role of TFF3 in the protection and repair of the lower intestinal mucosa. Several authors have investigated the regulatory mechanisms of the intestine-specific expression of ⁎ Corresponding author. Department of Gastroenterology, Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan. Tel.: +81 282 86 1111; fax: +81 282 86 7761. E-mail address: [email protected] (T. Shimada). 0167-0115/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.11.014
TFF3. In mice, a combination of the goblet cell-responsive element (GCRE), which is present in the proximal Tff3 promoter, and the goblet cell-silencer inhibitor (GCSI), which is located about 2200 bp upstream of the Tff3 transcriptional start site, has been reported to be important for the goblet cell-specific induction of TFF3 expression [5,6]. Iwakiri and Podolsky [7] also reported the involvement of keratinocyte growth factor (KGF) in the differentiation of goblet cells and KGF-induced upregulation of TFF3 expression. However, the detailed regulatory mechanisms of the expression of TFF3 in the lower intestine and in intestinal metaplasia of the stomach remain to be elucidated. CDX (caudal-related homeobox) proteins (CDX1 and CDX2) are physiologically expressed in intestinal epithelial cells distal to the duodenum and are critically involved in the development, proliferation, and differentiation of intestinal epithelial cells [8]. Chawengsaksophak et al. [9] reported the characteristics of Cdx2-deficient mice, in which the small intestinal crypt–villus architecture was altered and they developed colonic tumors. Intestinal abnormalities have also been reported in Cdx1-deficient mice [10]. Suh and Traber [11] showed that overexpression of CDX2 in IEC-6, an undifferentiated intestinal cell line, leads to the differentiation of the cells to functional
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intestinal epithelial cells. Since CDX proteins are transcription factors, a number of studies identified the involvement of CDX in regulating the expression of genes encoding intestine-specific proteins, such as sucrase-isomaltase [12], lactase [13,14], calbindin-D9K [15,16], apolipoprotein B [17], claudin-2 [18], and MUC2 [19,20]. Although CDX expression is absent in the normal gastric mucosa, the ectopic expression of CDX is detected in the gastric mucosa with intestinal metaplasia [21–23]. The appearance of goblet cells and expression of intestine-specific proteins, such as MUC2, are also characteristics of intestinal metaplasia of the stomach [21,23]. Silberg et al. [24] made Cdx2 transgenic mice using Foxa 3-driven Cdx2, and Mutoh et al. [25] made Cdx2 transgenic mice using H-K ATPase promoter-driven Cdx2. In these Cdx2 transgenic mice, normal gastric mucosa was completely replaced by intestinal metaplasia, confirming the central role of CDX2 in the induction of intestinal metaplasia. The above evidence suggests a possible role of CDX2 in the induction and regulation of TFF3 expression in the lower intestine and in intestinal metaplasia of the stomach. Thus, in this study, we investigated the effect of CDX2 on the expression of TFF3 in COS-7 cells and AGS gastric cells to determine whether TFF3 is a direct target of CDX2.
visualized with LumiGLO chemiluminescent reagent (Cell Signaling Technology; Beverly, MA). 2.3. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR)
COS-7 cells (a SV40-transformed cell line derived from African green monkey kidney) and AGS cells (a cell line derived from human gastric carcinoma) were obtained from the Human Health Resources Bank (Osaka, Japan). COS-7 cells were grown in Dulbecco's Modified Eagle's Medium (Invitrogen; Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen). AGS cells were maintained in Ham's F-12 culture medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen).
Total RNA was extracted from the cells with TRIZOL reagent (Invitrogen). First-strand cDNA was made with You-Prime FirstStrand Beads (GE Healthcare Bio-Sciences) using oligo (dT) primers (Invitrogen). The PCR primers used in this study were made based on the reported sequences as follows: human TFF3 (GenBank accession No. NM_003226); 5′-TGTCTGCAAACCAGTGTGCC-3′ (sense) and 5′-GCATTCTGTCTTCCTAGTCAGGG-3′ (antisense) (product length 158 bp), human MUC2 (NM_002457); 5′-ATTTGTCATGTACTCGGCCAA-3′ (sense) and 5′-CGATGTGGGTGTAGGTGTGTG-3′ (antisense) (product length 137 bp), human CDX2 (NM_001265); 5′ACCGCAGAGCAAAGGAGAGG-3′ (sense) and 5′-CCAGGGACAGAGCCAGACAC-3′ (antisense) (product length 187 bp), human β-actin (NM_001101); 5′-TTCCTGGGCATGGAGTCCT-3′ (sense) and 5′-AGGAGGAGCAATGATCTTGATC-3′ (antisense) (product length 204 bp). Conventional RT-PCR was performed in a GeneAmp PCR System 9700 (Applied Biosystems; Foster City, CA) using HotStar Taq DNA polymerase (Qiagen; Hilden, Germany). Real-time quantitative RT-PCR was performed in an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) using SYBR Green reagents (Applied Biosystems) as described previously [26–28]. After an initial 10 min at 95 °C, the reaction was run for 35 PCR cycles of 15 s at 95 °C and 1 min at 56 °C. To prepare standard templates, conventional RT-PCR was performed using the primers mentioned above and PCR products were purified with Qiaquick PCR Purification Kits (Qiagen). Purified PCR products were diluted and used as standard samples (6 × 102 to 6 × 107 copies) to generate a standard curve for each experiment. β-actin mRNA measurement was always performed for standardization.
2.2. Western blotting of CDX2 proteins
2.4. Plasmid construction and transfection
Cells grown in 10-cm culture dishes were washed with phosphate-buffered saline (PBS) and cell lysates were obtained using Passive Lysis Buffer (Promega; Madison, WI). Supernatants of the whole cell lysates (30 μg protein/lane) were subjected to 5–20% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (HyBond P membrane, GE Healthcare Bio-Sciences; Piscataway, NJ). The membrane was blocked with TTBS [10 mM Tris–HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20] containing 5% non-fat dry milk for 1 h at room temperature and then incubated with monoclonal anti-CDX2 antibody (BioGenex; San Ramon, CA) in TTBS containing 5% non-fat dry milk overnight at 4 °C. After washing with TTBS, the membrane was incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnologies; Santa Cruz, CA) in TTBS containing 5% non-fat dry milk for 1 h at room temperature. The membrane was washed again and the band was
The 5′-flanking regions of human TFF1 (−953 to +34) (Accession No. AB038162), TFF2 (−912 to +24) (AB038162), TFF3 (−957 to +12) (AB038162) and human MUC2 (−1139 to +22) (U67167) were PCR amplified and cloned into the SmaI site of the pGL3-basic luciferase vector (Promega). The nucleotide identity and direction of the insert were verified by sequencing of both strands. GeneEditor in vitro site-directed mutagenesis system (Promega) was employed to modify the TFF3 reporter gene. In order to make a CDX2 expression vector, the coding sequence of the human CDX2 (NM_001265) was PCR amplified and cloned into pcDNA3.1/V5/His vector (Invitrogen). The sense and antisense primers were 5′-GCAGCATGGTGAGGTCTGCTCC-3′ and 5′-CTGGGTGACGGTGGGGTTTAGC-3′, respectively. Since the PCR product does not possess a terminal codon, this expression vector produces CDX2 proteins that contain C-terminal V5/His tags. Similarly, a CDX1 expression vector was also made using the following
2. Materials and methods 2.1. Cell lines and culture
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Fig. 2. Effects of the transient transfection of CDX2 expression vectors on the transcription of MUC2, TFF1, TFF2, and TFF3 reporter genes in COS-7 cells. Control cells were transfected with empty pcDNA3.1/V5/His vectors. pSV-βgalactosidase vectors were co-transfected for standardization. (mean ± S.D., n = 4).
Fig. 1. (A) A representative RT-PCR experiment showing the expression of CDX2 mRNA in COS-7 cells. Lane 1, 100 bp ladder; lane 2, COS-7 cells transfected with empty pcDNA3.1/V5/His vectors; lane 3, COS-7 cells transfected with CDX2 expression vectors. (B) Representative Western blot analysis showing the expression of CDX2 proteins in COS-7 cells. Lane 1, COS-7 cells transfected with empty pcDNA3.1/V5/His vectors; lane 2, COS-7 cells transfected with CDX2 expression vectors.
primers based on the reported sequence (NM_001814): 5′-GGGACCCCGCGGCCACCATGTA-3′ (sense) and 5′-GCACAGGCTGGGCATGGGGCTA-3′ (antisense). The nucleotide identity of the inserts was verified by sequencing of both strands. For transient transfection assays, cells were seeded onto 24well culture plates the day before transfection. On the day of transfection, reporter vectors (0.6 μg/well) together with CDX expression vectors (0.4 μg/well) were transfected into the cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Empty pcDNA3.1/V5/His vectors were transfected to control cells instead of the CDX expression vectors. For standardization, pSV-β-galactosidase vectors (Promega) (0.05 μg/well) were also transfected into the cells. Thirty hours after transfection, cells were harvested using the Passive Lysis Buffer (Promega) and reporter gene assays were performed in a microplate luminometer LB96V (Berthold Technologies; Bad Wildbad, Germany). Luciferase activity was measured using the Luciferase Assay System (Promega) and β-galactosidase activity was measured using the β-Gal Reporter Gene Assay System (Roche Diagnostics; Mannheim, Germany). For stable transfection of the CDX2 expression vectors, AGS cells were seeded on a 6-well culture plate and the CDX2 expression vectors or the empty pcDNA3.1/V5/His vectors described above (2.4 μg/well) were transfected into the cells using Lipofectamine 2000 reagent (Invitrogen). Genetecin (Invitrogen) was used for cell cloning. 2.5. Electrophoretic mobility shift assay (EMSA) Double-stranded DNA oligonucleotides labeled with Cy3 at the 5′-termini of both strands (Tsukuba Oligo, Tsukuba, Japan)
were made for EMSA. Sense strand sequences were as follows: TFF3-A, 5′-ATGTGTACCATGTTTTTACTAACATATTTT-3′, which corresponds to − 624 to − 595 of the human TFF3 promoter, TFF3-B, 5′-CAAACAACGGTGCATAAATGAGGCCTCCTG-3′, which corresponds to − 76 to − 47 of the human TFF3 promoter (putative CDX-binding sequences are underlined). We also made another double-stranded oligonucleotide designated as SIF-1S, the sequence corresponding to CDX-responsive elements within the promoter of the gene encoding human sucrase-isomaltase [12]. The sense strand sequence was 5′-GGCTGGTGAGGGTGCAATAAACTTTATGAGTA-3′. SIF-1S was used as a competitor oligo in EMSA experiments. Nuclear extracts were prepared from CDX1- or CDX-2overexpressed COS-7 cells using Nuclear Extract Kits (Active Motif, Carlsbad, CA) according to the manufacturer's protocol. For DNA binding reactions, nuclear extracts (10 μg protein) were added to 1× DNA binding buffer (Promega) containing 125 pmol of the Cy3-labeled oligonucleotide and incubated for
Fig. 3. Effects of the transient transfection of CDX2 expression vectors on the transcription of MUC2, TFF1, TFF2, and TFF3 reporter genes in AGS gastric cells. Control cells were transfected with empty pcDNA3.1/V5/His vectors. pSVβ-galactosidase vectors were co-transfected for standardization. (mean ± S.D., n = 4).
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3. Results 3.1. CDX2 induces TFF3 expression
Fig. 4. Effects of the transient transfection of CDX1 expression vectors on the transcription of MUC2, TFF1, TFF2, and TFF3 reporter genes in COS-7 cells. Control cells were transfected with empty pcDNA3.1/V5/His vectors. pSV-βgalactosidase vectors were co-transfected for standardization. (mean ± S.D., n = 4).
20 min at room temperature. When the competitor oligo was used, 100 × excess amounts of SIF-1S were added to the reaction 10 min prior to the addition of the Cy3-labeled oligonucleotide. The reactions were electrophoresed on 5–20% native PAGE gels (ATTO, Tokyo, Japan) and the gels were directly imaged using a Pharos FX System (Bio Rad Laboratories, Hercules, CA).
Fig. 5. (A) The sequences of putative CDX-binding sites within the human TFF3 promoter are shown. The caudal-related protein consensus binding sites (TTTAC/T) are underlined. (B) Electrophoretic mobility shift assay. Nuclear extracts prepared from CDX1-overexpressed (lanes 3–6) or CDX2-overexpressed (lanes 7–10) COS-7 cells were incubated with Cy3-labeled doublestranded oligonucleotide probes, TFF3-A (lanes 1, 3, 4, 7 and 8) and TFF3-B (lanes 2, 5, 6, 9 and 10), the sequences corresponding to − 624 to − 595 and − 76 to −47 of the human TFF3 promoter. 100 × excess amount of competitor oligonucleotides (SIF-1S) was present in the incubations in lanes 4, 6, 8, and 10. Nuclear extracts were not added to the incubations in lanes 1 and 2.
The entire coding sequence of human CDX2 was PCR amplified and inserted into the pcDNA3.1/V5/His vector to make a CDX2 expression vector. Although COS-7 cells expressed a negligible level of CDX2, transfection of the CDX2 expression vectors caused a significant increase in the expression of CDX2 mRNA as well as CDX2 proteins, as shown in Fig. 1. Fig. 2 shows the effect of CDX2 overexpression on the transcription of TFF3 reporter genes and MUC2 reporter genes in COS-7 cells. The effects of CDX2 overexpression on the transcription of reporter genes for other TFF members, TFF1 and TFF2 are also shown in Fig. 2. Consistent with previous reports [19,20], MUC2 reporter gene transcription was up-regulated by CDX2 overexpression. TFF3 reporter gene transcription was also found to be significantly up-regulated by CDX2 overexpression and that of TFF1 and TFF2 reporter genes was slightly up-regulated. Fig. 3 shows the effect of CDX2 overexpression on these reporter genes in AGS gastric cells. CDX2 similarly up-regulated the transcription of TFF3 and MUC2 reporter genes. TFF1 reporter gene transcription was not significantly affected and that of TFF2 reporter genes was slightly up-regulated. We also tested the effect of CDX1 overexpression on the transcription of TFF1-3 and MUC2 reporter genes in COS-7 cells, as shown in Fig. 4. Although less potent compared to CDX2, CDX1 also up-regulated the transcription of both MUC2 and TFF3 reporter genes.
Fig. 6. (A) From the wild-type TFF3 reporter genes (TFF3-Luc), putative CDXbinding sites within the human TFF3 promoter at − 610 and − 63 were deleted in the mutA and mutB reporter genes, respectively. In the mutC reporter gene, both sites were deleted. (B) Effects of the transient transfection of CDX2 expression vectors on the transcription of the wild-type TFF3 reporter gene and mutated reporter genes in COS-7 cells. (mean ± S.D., n = 4).
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3.2. Interaction of CDX2 proteins with the TFF3 cis-element The CDX-responsive sequence has been reported as TTTAT/ C [12,29]. We identified 2 potential CDX-binding sites within the 5′-untranslated region of human TFF3, as shown in Fig. 5A. To test whether CDX proteins can bind to these elements, we performed EMSA experiments. Nuclear extracts prepared from CDX1- or CDX2-overexpressed COS-7 cells were incubated with Cy3-labeled double-stranded oligonucleotides, the sequences being identical to the potential CDX-binding sites of the human TFF3 promoter. As shown in Fig. 5B, these oligonucleotides made shift bands that disappeared in the
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presence of an excess amount of competitor oligonucleotides, suggesting that CDX proteins can bind to both of these sequences. To examine the relative importance of these CDXbinding sites, we made mutant-type TFF3 reporter genes, as shown in Fig. 6A. The potential CDX-binding site at −610 was deleted in the mutA reporter gene, the potential CDX-binding site at − 63 was deleted in the mutB reporter gene, and both sites were deleted in the mutC reporter gene. Fig. 6B shows the effect of CDX2 overexpression on the transcription of these reporters in COS-7 cells. As shown in this figure, deletion of the potential CDX-binding site at − 63 had more significant effect on the transcription of reporter genes. 3.3. Effect of CDX2 on the expression of endogenous TFF3 mRNA We also stably transfected CDX2 expression vectors or empty pcDNA3.1/V5/His vectors into AGS cells (AGS-CDX2 and AGS-pcDNA). Since AGS cells express endogenous CDX2 at a certain level, two bands (endogenous CDX2 proteins and expression vector-derived CDX2 proteins with 3′-tags) can be seen in the Western blot analysis shown in Fig. 7A. Fig. 7B and C are real-time quantitative RT-PCR experiments showing the expression of endogenous MUC2 and TFF3 mRNA in AGSCDX2 and AGS-pcDNA cells. Not only MUC2 but also TFF3 mRNA expression was up-regulated in AGS-CDX2 cells compared to AGS-pcDNA cells, suggesting the regulation of endogenous TFF3 mRNA expression by CDX2 proteins. 4. Discussion
Fig. 7. (A) Western blot analysis showing the expression of CDX2 proteins in AGS cells (lane 1, AGS cells stably transfected with empty pcDNA3.1/V5/His vectors; lane 2, AGS cells stably transfected with CDX2 expression vectors). In AGS cells stably transfected with CDX2 expression vectors, the expression of V5/His-tagged CDX2 proteins (arrow) was recognized in addition to endogenous CDX2 proteins. (B) Real-tine quantitative RT-PCR analysis showing the expression of endogenous MUC2 mRNA in AGS cells stably transfected with empty pcDNA3.1/V5/His vectors or CDX2 expression vectors. (mean ± S.D., n = 6, p b 0.001 unpaired t-test). (C) Real-tine quantitative RTPCR analysis showing the expression of endogenous TFF3 mRNA in AGS cells stably transfected with empty pcDNA3.1/V5/His vectors or CDX2 expression vectors. (mean ± S.D., n = 6, p b 0.001 unpaired t-test).
In this study, we transfected CDX2 expression vectors into COS-7 cells and AGS gastric cells and examined its effect on the transcription of the human TFF3 reporter genes. Similar to human MUC2, which was previously identified as a target gene of CDX [19,20], we found that TFF3 reporter gene transcription was up-regulated by CDX2 overexpression. CDX1 also had a similar stimulatory effect on the transcription of the TFF3 reporter gene although the effect was less potent than CDX2. Thus, these results suggest a role of CDX proteins in the induction and regulation of human TFF3 expression. The CDX-binding sequence has been reported as TTTAC/T [12,29]. In order to determine whether the regulation of TFF3 expression by CDX is direct, we examined the interaction between CDX proteins and putative CDX-binding sites (− 610 and − 63) within the human TFF3 promoter. Although EMSA experiments (Fig. 5) revealed the possible binding of CDX proteins to both sites, deletion analysis of the TFF3 reporter gene (Fig. 6) suggests the relative importance of the binding site at − 63. We further explored the effect of CDX2 on the expression of endogenous TFF3 mRNA using AGS cells stably transfected with CDX2 expression vectors. Compared to AGS cells that were stably transfected with empty pcDNA3.1/V5/His vectors, CDX2-transfected AGS cells showed a higher level of TFF3 mRNA expression along with a higher level of MUC2 mRNA expression. Together with the data obtained in the reporter gene assays, this study provides evidence that human
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TFF3 is a target of CDX2 and CDX2 plays an important role in the regulation of TFF3 expression. Although we also cloned COS-7 cells stably transfected with CDX2 expression vectors, we could not observe the up-regulated expression of endogenous TFF3 mRNA (data not shown). Thus, it is likely that other cell-type-specific factor or factors are also required for the induction of TFF3 in addition to CDX proteins. Since previous studies indicated the roles of the goblet cell responsive element, the goblet cell-silencer inhibitor, and KGF in the goblet cellspecific expression of TFF3 [5–7], a combination of these factors and CDX may be important. Among three TFF peptides, TFF1 and TFF2 are expressed in the gastric mucosa but it is known that their expression is downregulated in intestinal metaplasia inversely related to the upregulation of TFF3 in intestinal metaplasia of the stomach [3]. Although the present results suggest a role of CDX2, which is expressed at a high level in intestinal metaplasia of the stomach [21–23], in the induction of TFF3, it is also interesting to determine whether the down-regulation of TFF1 and TFF2 expression is related to the appearance of CDX2 in the gastric mucosa. However, based on the results of the reporter gene assays shown in Figs. 2 and 3, it is not likely that CDX2 negatively regulates the expression of TFF1 and TFF2. Thus, the down-regulation of TFF1 and TFF2 expression in intestinal metaplasia of the stomach appears to be independent of CDX2 but mediated by other factor or factors. The results of this study indicate that, in addition to other intestine-specific genes, CDX homeodomain proteins play a critical role in the regulation of TFF3 expression. Further studies are needed to elucidate the interaction between CDX and other transcriptional regulator proteins, and overall transcriptional control of the human TFF3 in intestinal epithelial cells and intestinal metaplasia of the stomach in vivo. Since TFF3 is an important factor in protecting the intestinal mucosa, a better understanding of the regulatory mechanisms of its expression may benefit the clinical application of this peptide. Acknowledgements We thank Drs. Yoichiro Fujii, Akihiro Tajima, and Akira Terano for their helpful discussion. We also thank Ms. Kyoko Tabei and Mr. Takashi Namatame for technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science. References [1] Wong WM, Poulsom R, Wright NA. Trefoil peptides. Gut 1999;44:890–5. [2] Hoffmann W, Jagla W. Cell type specific expression of secretory TFF peptides: colocalization with mucins and synthesis in the brain. Int Rev Cytol 2002;213:147–81. [3] Taupin D, Podolsky DK. Trefoil factors: initiators of mucosal healing. Nat Rev Mol Cell Biol 2003;4:721–32. [4] Mashimo H, Wu DC, Podolsky DK, Fishman MC. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science 1996;274: 262–5. [5] Ogata H, Inoue N, Podolsky DK. Identification of a goblet cell-specific enhancer element in the rat intestinal trefoil factor gene promoter bound by a goblet cell nuclear protein. J Biol Chem 1998;273:3060–7.
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