Characterization of the Mouse TFF1 (pS2) Gene Promoter Region.

Abstract

Trefoil peptides (TFFs) are small and stable molecules secreted by the mammalian gastrointestinal tract.1) The term "trefoil", meaning three leaves, is derived from their three intrachain loops (trefoil domain) that are maintained by disulfide bonds. Three family members are found in mammals: TFF1/pS2 and TFF3/ITF contain one trefoil domain, whereas TFF2/SP has two domains.2) It is suggested that the TFFs could be involved in protection and repair of the gastrointestinal tract through stimulation of cell migration and mucus polymerization. Actually, mice lacking TFF3 exhibited impaired mucosal healing and died from extensive colitis after oral administration of dextran sulfate.3) Furthermore, loss of TFF1 induced severe hyperplasia and dysplasia of the gastric mucosa, and the development of adenomas and carcinomas. 4)The results suggest that TFF1 is essential for normal differentiation of the gastric mucosa and that it functions as a tumor suppressor.The three TFFs exhibit strict tissue-specific expression patterns: TFF1 in stomach, TFF2 in stomach and duodenum, and TFF3 in small intestine and colon.2) We are interested in the gene regulation of TFF1 from the viewpoint of stomachspecific transcription, as our previous studies focused on the genes specifically expressed in stomach, such as those of the gastric proton pump, 5,6) intrinsic factor, 7) and H 2 receptor. 8) In this study, we cloned the mouse TFF1 gene 5Ј-upstream sequence, and identified the enhancer and silencer regions by means of a reporter gene assay. Furthermore, significance of the sequence motifs in the upstream regions are discussed in comparison with those in the human gene. MATERIALS AND METHODS Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)Total RNA was prepared from tissues of a 6-weekold male ddY mouse by the guanidine thiocyanate-CsCl method.9) cDNA was synthesized from total RNA (8 mg) with a First-Strand cDNA Synthesis Kit (Amersham-PharmaciaBiotech) and an oligo d(T)12-18 primer, and then subjected to PCR 10) to amplify specific nucleotide sequences for the three TFFs. The pairs of PCR primers used for TFF1 (S29 and S30), TFF2 (S26 and S27) and TFF3 (S3 and S4) are shown in Table 1. The PCR conditions were: preheating (94°C, 3 min), followed by 35 cycles of denaturation (94°C, 0.5 min), annealing (54°C for TFF1, 58°C for TFF2, or 62°C for TFF3, 0.5 min), and extension (72°C, 0.5 min). The 2nd PCR was carried out for TFF3 (54°C annealing). The products were analyzed by agarose gel electrophoresis [1.0% (w/v) agarose (TaKaRa L03) in TAE-buffer 9) ], and visualized by ethidium bromide staining.Total cellular RNA was prepared from GSM10 cells 11,12) as above and then PCR analysis was carried out. The PCR conditions were essentially the same as for tissues except that the annealing temperature and cycle number for the three TFFs were 61°C and 40 cycles, respectively. The GSM10 cells, the generous gift of Daiichi Pharmaceutical Co., Ltd., were cultured in Dulbecco's modified Eagle medium/F12 medium (GIBCO BRL) containing 10% (v/v)...

Stable expression and visualization of Mat-8 (FXYD-3) tagged with a fluorescent protein in Chinese Hamster Ovary (CHO)-K1 cells

“…The Mat-8 cDNA was amplified from total RNA of mouse GSM06 cells (Terada et al 2001) by means of RT-PCR essentially as described previously (Takeda et al 2004). The PCR conditions comprised 35 cycles of denaturation (94°C, 0.5 min), annealing (58°C, 0.5 min), and extension (72°C, 0.5 min) with a primer pair (5¢-CAG-GTCAAGAATTCGACATGC-3¢/5¢-TCAGCAG-TTGTGAGCTGAGCCTGG-3¢) according to the published sequence (Morrison & Leder 1994, Morrison et al 1995.…”

Frequent trefoil factor 3 (TFF3) overexpression and promoter hypomethylation in mouse and human hepatocellular carcinomas

“…These three genes are evolutionarily highly conserved and are clustered on the same chromosomal region in mouse chromosome 17 and human chromosome 21. They act to maintain mucosal integrity in the GI tract by epithelium restitution with rapid cell migration (6) and resistance to apoptosis (7), and they also commit to differentiation during embryonic development of the GI tract (8). It has been suggested that TFF3, originally known as intestinal trefoil factor, enhances cell migration through modulation of E-cadherin/catenin complex function (9).…”

Persistent trefoil factor 1 expression imprinted on mouse vaginal epithelium by neonatal estrogenization

“…TFF1/pS2 was originally identified as an estrogen-induced gene in human breast tumors, and the estradiol responsive element (ERE) in the cis-promoter of the TFF1 gene predominantly controls its transcription (Rio et al 1987). The mouse TFF1 gene, however, lacks a functional ERE (Terada et al 2001), and TFF1 expression is not observed in the mouse mammary gland, even during estrogen-dependent postnatal development (Lefebvre et al 1993). Thus, estrogeninduced TFF1 expression in the developing mouse vagina is caused not by the direct action of ligand-activated ER on the proximal promoter of the TFF1 gene in the vaginal epithelial cells, but rather by complex gene regulation mechanisms that may involve an epithelium-stroma interaction.…”

“…Epidermal growth factor (EGF) is one possible mediator of estrogen-induced TFF1 expression in the proliferated vaginal epithelium. The 5′-flanking region of TFF1 contains an EGF-responsive enhancer region that mediates EGF-induced expression of TFF1 in the gastric tissue (Terada et al 2001), whereas EGF-EGFR signaling is activated in the transiently or permanently hyperplasic epithelium of the estrogenized vagina (Miyagawa et al 2004). These factors alone, however, do not explain why the vaginal epithelium of normal adult mice does not express TFF1 in the presence of estrogen.…”