Regulation of TFF3 expression by homeodomain protein CDX2

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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.

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], [2], [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 down-regulated [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 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 up-regulation 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 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], [22], [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.

Section snippets

Cell lines and culture

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).

Western blotting of CDX2 proteins

Cells grown in

CDX2 induces TFF3 expression

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

Discussion

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

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 (29)

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