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Publications (9)30.26 Total impact

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    ABSTRACT: TFF1 (pS2) is expressed at a high level in gastric epithelial cells and plays an important role in protecting the gastric mucosa. However, the regulatory mechanisms of TFF1 expression are not fully understood. The aim of this study was to investigate the effect of TNF-alpha, a representative proinflammatory cytokine, on TFF1 expression. MKN45 and AGS cells, derived from human gastric carcinoma, were used. Endogenous TFF1 mRNA expression was analyzed by real-time quantitative RT-PCR. The sequences of the human TFF1 promoter were cloned into the pGL3-basic vector and reporter gene assays were performed. Nuclear factor (NF)-kappaB activity was monitored using a reporter vector that contained multiple copies of NF-kappaB responsive element upstream of the luciferase gene. Interaction between NF-kappaB and TFF1 cis-element was examined by electophoretic mobility shift assay (EMSA). TNF-alpha activated NF-kappaB and up-regulated endogenous TFF1 mRNA expression as well as the transcription of the TFF1 reporter genes in a dose-dependent manner. IL-1beta, another proinflammatory cytokine, also up-regulated TFF1 expression. TNF-alpha responsive element was mapped between -342 and -147 of the human TFF1 promoter and a putative NF-kappaB binding site was identified at -231. When this element was deleted, the reporter genes became almost insensitive to TNF-alpha treatment. EMSA showed binding of NF-kappaB to this element. Inflammatory stimuli that activate NF-kappaB appear to up-regulate TFF1 expression in gastric epithelial cells. This mechanism may aid in the protection of the gastric mucosa under inflammatory conditions.
    Journal of Gastroenterology and Hepatology 07/2007; 22(6):936-42. · 3.33 Impact Factor
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    ABSTRACT: Although trefoil factor family 2 (TFF2) plays a critical role in the defense and repair of gastric mucosa, the regulatory mechanism of TFF2 expression is not fully understood. In this study, we investigated the regulation of TFF2 expression by peroxisome proliferator-activated receptor gamma (PPARgamma) in gastric epithelial cells. MKN45 gastric cells were used. TFF2 mRNA expression was analyzed by real-time quantitative RT-PCR. The promoter sequence of the human TFF2 gene was cloned into pGL3-basic vector for reporter gene assays. Ciglitazone was mainly used as a specific PPARgamma ligand. MKN45 cells expressed functional PPARgamma proteins. Endogenous TFF2 mRNA expression and TFF2 reporter gene transcription was significantly up-regulated by ciglitazone in a dose-dependent manner. Reporter gene assays showed that two distinct cis-elements are involved in the response to PAPRgamma activation. Within one of these element (nucleotides -558 to -507), we identified a functional peroxisome proliferator responsive element (PPRE) at -522 (5'-GGGACAAAGGGCA-3'). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay confirmed the binding of PPARgamma to this sequence. Another element (nucleotides -407 to -358) appeared to be a composite enhancer element indirectly regulated by PPARgamma and a combination of these two cis-elements was required for the full response of the human TFF2 gene expression to PPARgamma. These data demonstrate that human TFF2 gene is a direct target of PPARgamma in gastric epithelial cells. Since TFF2 is a critical gastroprotective agent, PPARgamma may be involved in the gastric mucosal defense through regulating TFF2 expression in humans.
    The International Journal of Biochemistry & Cell Biology 02/2007; 39(3):626-37. · 4.15 Impact Factor
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    ABSTRACT: Trefoil factor family (TFF) is a group of small peptides secreted by gastrointestinal epithelial cells.Among three known TFF peptides, TFF1 (formerly pS2) is expressed at a high level in gastric epithelial cells and plays an essential and critical role in maintaining the integrity of the gastric mucosa. Recent evidence also suggests that TFF1 acts as a tumour suppressor gene in the stomach.TFF1 was originally discovered as an oestrogen-inducible gene in MCF-7 breast cancer cells, and its expression is dependent on oestrogen in MCF-7 and other hormone-dependent breast cancer cells. Although gastric epithelial cells express oestrogen receptors (ERs), gastric TFF1 expression appears to be independent of oestrogen signalling. Instead, several cis-regulatory elements are involved in the regulation of gastric TFF1 expression and balanced signalling from gp130, a common IL-6 family coreceptor, has been shown to be necessary for the proper expression of TFF1 in the stomach.Epigenetic regulation, such as DNA methylation in the promoter region of the TFF1 gene, may be also important for tissue-specific expression of TFF1 in the stomach. However, further studies are still needed to fully understand the detailed regulatory mechanisms of TFF1 expression in gastric epithelial cells.
    Alimentary Pharmacology & Therapeutics Symposium Series 06/2006; 2(1):285 - 291.
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    ABSTRACT: Aim:  Trefoil factor family (TFF) peptides are known to facilitate wound healing in gastric mucosa. However, the regulatory mechanisms of gastric TFF expression are not fully understood yet. In this study, we examined the effect of TNF-α on TFF1 and TFF2 expression in gastric epithelial cells.Methods:  MKN45 cells were used. TFF mRNA expression was analyzed by real-time quantitative RT-PCR. Promoter sequences of TFF1 gene (−956 to +36) and TFF2 gene (−912 to +24) were inserted into pGL3 vector and reporter gene assays were performed. NF-κB activity was monitored by using a NF-κB responsive element-driven reporter vector.Results:  (1) TNF-α(0.1–30 ng/ml) down-regulated TFF1 and TFF2 mRNA expression in a dose-dependent manner. (2) Reporter gene assays also confirmed the down-regulation of TFF1 and TFF2 gene transcription by TNF-α. (3) TNF-α activated NF-κB. (4) Overexpression of dominant negative IκBα prevented both TNF-α-induced NF-κB activation and TNF-α-induced down-regulation of TFF expression.Conclusions:  TNF-α down-regulates gastric TFF expression through NF-κB pathway, suggesting that TFF expression is sensitive to inflammatory stimuli.
    Wound Repair and Regeneration 01/2005; 13(1):A5 - A5. · 2.76 Impact Factor
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    ABSTRACT: Trefoil factor family peptides are expressed in gastrointestinal epithelial cells and play a critical role in maintaining mucosal integrity. Although non-steroidal anti-inflammatory drugs (NSAIDs) are important causative agents of gastric mucosal lesions, few data are available about the effect of NSAIDs on trefoil family peptides in gastric mucosa. To examine whether indometacin, a widely used NSAID, affects trefoil factor family expression in gastric epithelial cells. MKN45, a cell line derived from human gastric cancer, was used. TFF1, TFF2, and TFF3 mRNA expression was assessed by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). TFF2 gene transcription was also examined by luciferase reporter gene assay. Relative expression level of TFF1, TFF2, TFF3 mRNA was 616: 12: 1 in unstimulated MKN45 cells. Although indometacin (1-250 micro mol/L) had no significant effect on the expression of TFF1 and TFF3 mRNA, it up-regulated TFF2 mRNA expression in a dose- and time-dependent manner. Luciferase reporter gene assay confirmed the up-regulation of TFF2 gene transcription by indometacin. Indometacin-induced up-regulation of TFF2 expression was not antagonized by externally applied prostaglandin E2. These results suggest that indometacin up-regulates gastric epithelial cell TFF2 expression through a COX-independent mechanism. Since TFF peptides play an important role in gastric mucosal protection, indometacin-induced TFF2 may reduce the degree of gastric mucosal damage induced by indometacin.
    Alimentary Pharmacology & Therapeutics 08/2004; 20 Suppl 1:171-6. · 4.55 Impact Factor
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    ABSTRACT: Thyroid dysgenesis is the most frequent cause of congenital hypothyroidism, but its molecular pathophysiology is largely unknown. Our hypothesis that some genes downstream to thyroid transcription factor-2 (TTF-2) might be responsible for development of the thyroid prompted us to identify genes whose expression is stimulated by TTF-2. PCR products of cDNA clones obtained by a subtraction PCR method in TTF-2 expressing cell lines were screened with labeled cDNA by microarray analysis. We isolated 17 genes up-regulated by TTF-2, which were subsequently confirmed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). One of them is a novel gene designated T1560 that showed a highly thyroid-specific expression pattern. Luciferase reporter assays showed that expression of all of the 14 genes tested was stimulated by both TTF-2 and TTF-1, another thyroid-specific transcription factor. Our results have important implications for understanding normal thyroid development as well as the molecular defects underlying thyroid dysgenesis.
    Molecular and Cellular Endocrinology 07/2004; 221(1-2):33-46. · 4.04 Impact Factor
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    ABSTRACT: Trefoil factor family (TFF) is a group of peptides that play critical roles in maintaining gastric mucosal integrity. In real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and reporter gene assays, we show that indomethacin and aspirin upregulate TFF2 expression in MKN45 gastric cells. These drugs also activated peroxisome proliferator-activated receptor gamma (PPARgamma) at concentration ranges that increase TFF2 expression, and upregulated TFF2 expression was suppressed by GW9662, a specific inhibitor of PPARgamma. These results suggest that indomethacin and aspirin upregulate gastric expression of TFF2 through activation of PPARgamma. This mechanism may be important in reducing the extent of gastric mucosal injury caused by the administration of non-steroidal anti-inflammatory drugs (NSAIDs).
    FEBS Letters 02/2004; 558(1-3):33-8. · 3.58 Impact Factor
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    ABSTRACT: One of the thyroid-specific transcription factors, thyroid transcription factor-2 (TTF-2), performs a crucial role in the development of the thyroid gland. We performed genetic analysis of the TITF2 gene (encoding TTF-2) in patients with thyroid dysgenesis. By direct sequencing of the PCR products of TITF2, we screened the genomic DNA from 46 patients with thyroid dysgenesis (five had agenesis, six had hypoplasia, 15 had ectopy, and 20 were undetermined). We also studied the transcriptional activities of TITF2 by co-expressing the luciferase gene directed by the human thyroglobulin gene promoter. Human TITF2 consists of a forkhead domain, a polyalanine tract, and unique C-terminal residues. In one of the patients with an ectopic sublingual thyroid, we found a polyalanine tract of 11 alanine residues on one chromosome instead of the 14 alanine residues found in normal controls. In one patient with hypoplasia, the polyalanine tract consisted of 12 heterozygous alanine residues. The reduced polyalanine tracts were not detected in 101 normal individuals. However, the expression study showed that the transcriptional activities of TITF2 with reduced polyalanine-tract lengths were equal to that of TITF2 with an unreduced polyalanine tract. These results suggest that the polymorphism of the polyalanine tract of TITF2 is not a frequent cause of developmental defects of the human thyroid gland.
    European Journal of Endocrinology 11/2001; 145(4):385-9. · 3.14 Impact Factor
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    ABSTRACT: The rdw rat is a hereditary hypothyroid variant initially derived from the Wistar-Imamichi strain. Proteome analysis by two-dimensional gelelectrophoresis showed that molecular chaperones accumulated in the thyroid glands, suggesting retention of abnormal proteins in the endoplasmic reticulum (ER). Anatomical studies indicated that thyroglobulin (Tg) was not secreted into the follicular lumina, but retained in the dilated ER. Sequencing of the entire Tg complementary DNA from the rdw rat revealed a missense mutation (G2320R) in the acetylcholinesterase-like domain at the 2320th amino acid residue. Carbohydrate residues of the G2320R Tg mutant were of the high-mannose ER type, as shown by sensitivity to the treatment with endoglycosidase H. Molecular chaperones, GRP94, GRP78, and calreticulin, were all accumulated in the rdw rat thyroid glands. Computer analysis of protein secondary structure predicted that the mutation would cause extension of the helix where beta-sheet and turns were formed in the normal Tg. Altered folding of Tg might account for the impaired intracellular transport of Tg and activated premature degradation by the same mechanism as in ER storage diseases.
    Endocrinology 12/2000; 141(11):4050-5. · 4.72 Impact Factor