cDNA cloning of rat pS2 peptide and expression of trefoil peptides in acetic acid-induced colitis

Itoh, Hiroshi; Tomita, Masaki; Uchino, H; Kobayashi, Takahiko; Kataoka, Hiroaki; Sekiya, Ryo; Nawa, Yukifumi

doi:10.1042/bj3180939

Cited by 42 publications

(41 citation statements)

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“…The probes used were DT-A fragment, rITF cDNA (12), PCR products of rpS2 and rSP cDNA (24), and GAPDH (CLONTECH Laboratories Inc.). Rat MUC2 and mouse MUC3 probes corresponding to nucleotide positions 440-657 and 1,062-1,399 respectively were also prepared by PCR (25,26).…”

Section: Figurementioning

confidence: 99%

“…Immediately after death or sacrifice at the end of the experiment, tissues were prepared for histologic examination. For the latter treatment, after saline lavage, 3% acetic acid (10 µL/g of body weight) was instilled through a tube inserted 3 cm into the rectum of 28 DT-A transgenic and 28 normal mice under ether anesthesia (23,24,30). Survival rate was calculated and mice were examined histologically on days 1, 3, and 5 after administration.…”

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“…Samples from stomach, duodenum, jejunum, ileum, and proximal and distal portions of colon were fixed in 10% buffered formalin overnight followed by dehydration in 70% ethanol and embedding in paraffin wax. Serial sections 4 µm thick were deparaffinized, rehydrated, and stained with hematoxylin and eosin for routine histology, or with Alcian blue and periodic acid-Schiff (AB-PAS) to locate goblet cells as described previously (23,24). The ratio of goblet cells to total epithelial cells was obtained by counting AB-PAS-positive cells and total epithelial cells in 10 highpower fields of well-oriented longitudinal sections under light microscopy.…”

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A paradoxical reduction in susceptibility to colonic injury upon targeted transgenic ablation of goblet cells

Itoh¹,

Beck²,

Inoue³

et al. 1999

J. Clin. Invest.

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MethodsPreparation of mITF promoter. To obtain a sufficiently large segment of 5′ flanking promoter region to confer goblet cell-specific expression of the transgene, a Hiroshi Itoh and Paul L. Beck contributed equally to this work.Received for publication January 5, 1999, and accepted in revised form October 21, 1999.Goblet cells are the major mucus-producing cells of the intestine and are presumed to play an important role in mucosal protection. However, their functional role has not been directly assessed in vivo.In initial studies, a 5′ flanking sequence of the murine intestinal trefoil factor (ITF) gene was found to confer goblet cell-specific expression of a transgene. To assess the role of goblet cells in the intestine, we generated transgenic mice in which ∼60% of goblet cells were ablated by the expression of an attenuated diphtheria toxin (DT) gene driven by the ITF promoter; other cell lineages were unaffected. We administered 2 exogenous agents, dextran sodium sulfate (DSS) and acetic acid, to assess the susceptibility of mITF/DT-A transgenic mice to colonic injury. After oral administration of DSS, 55% of control mice died, whereas DT transgenic mice retained their body weight and less than 5% died. Similarly, 30% of the wild-type mice died after mucosal administration of acetic acid, compared with 3.2% of the transgenic mice. Despite the reduction in goblet-cell number, the total amount of ITF was increased in the mITF/DT-A transgenic mice, indicating inducible compensatory mechanisms. These results suggest that goblet cells contribute to mucosal protection and repair predominantly through production of trefoil peptides. Construction of transgene and generation of β-galactosidase ITF transgenic mice. The β-galactosidase gene was obtained from pSV-β-galactosidase vector (Promega Corp., Madison, Wisconsin, USA) by digestion with BamHI and HindIII. The resulting 3.7 kb of β-galactosidase gene was inserted into the BamHI and HindIII sites of pBluescript II KS(+). The 6.3 kb of XhoI-digested 5′ end of the mITF promoter was then inserted into XhoI sites of pBluescript II KS(+) containing the β-galactosidase gene. The resulting plasmid was digested with BamHI, yielding a 10-kb DNA fragment consisting of the 6.35-kb mITF promoter followed by the β-galactosidase gene (3.7 kb). This fragment was gel purified, dialyzed, and injected into the mouse oocytes. The resulting offspring were screened both by Southern blotting (using the above β-galactosidase fragment as a probe) and by PCR using the following primers directed at the inserted β-galactosidase gene: sense: 5′-CCTGAGGCCGATACTGTCGTC-3′; antisense: 5′-CCAGATAACTGCCGTCACTCC-3′. PCR was performed according to the manufacturer's guidelines (Promega Corp.) using 0.5 µL of Taq DNA polymerase (Promega Corp.) in a 50-µL reaction containing 1.5 mM MgCl 2 , 0.2 mM dNTP, 0.8 µM of each primer, and approximately 1 µg of extracted tail DNA. PCR conditions included an initial 10 minutes at 95°C followed by 36 cycles each of 95°C for 1 minute, 58°C for 1 minute, 72°C f...

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A paradoxical reduction in susceptibility to colonic injury upon targeted transgenic ablation of goblet cells

Itoh¹,

Beck²,

Inoue³

et al. 1999

J. Clin. Invest.

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“…SP is a product of the gastric mucous neck cell (15,16), with pS2 expressed by surface (foveolar) mucous cells of the stomach (17,18). ITF is a major peptide product of the intestinal goblet cell, with less abundant expression in gastric glands (16,19,20).…”

Section: Introductionmentioning

confidence: 99%

The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor– and MAP kinase–dependent interregulation

Taupin¹,

Wu²,

Jeon³

et al. 1999

J. Clin. Invest.

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108

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“…A site-directed mutagenesis study demonstrated that the Motif III and Motif IV contributed positively to transcriptional regulation of human TFF1 gene. 20) In this regard, the roles of HNF3/FKH proteins which recognized human Motif IV and triggered transcription of the TFF1 reporter gene 21) are of interest from the viewpoints of TFF1 induction upon injury, inflammation and tumor development 21,22) as well as tissue-specific TFF1 expression. However, it should also be mensioned that DNA methylation of the promoter region of TFF1 gene is another important factor for gene regulation associated with carcinogenesis.…”

Section: Fig 6 Analysis Of the Promoter Region By Means Of The Repomentioning

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

Terada

Sakagami

Tabuchi

et al. 2001

Biological & Pharmaceutical Bulletin

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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)...

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cDNA cloning of rat pS2 peptide and expression of trefoil peptides in acetic acid-induced colitis

Cited by 42 publications

References 42 publications

A paradoxical reduction in susceptibility to colonic injury upon targeted transgenic ablation of goblet cells

A paradoxical reduction in susceptibility to colonic injury upon targeted transgenic ablation of goblet cells

The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor– and MAP kinase–dependent interregulation

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

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