TFF Expression Research Articles

Modulation of Pectin on Mucosal Innate Immune Function in Pigs Mediated by Gut Microbiota.

The use of prebiotics to regulate gut microbiota is a promising strategy to improve gut health. Pectin (PEC) is a prebiotic carbohydrate that enhances the health of the gut by promoting the growth of beneficial microbes. These microbes produce metabolites that are known to improve mucosal immune responses. This study was conducted to better understand effects of PEC on the microbiome and mucosal immunity in pigs. Pigs were fed two diets, with or without 5% apple PEC, for 72 days. Effects of PEC on the microbiota, cytokine expression, short-chain fatty acids (SCFAs) concentration and barrier function were examined in the ileum and cecum of the pigs. An integrative analysis was used to determine interactions of PEC consumption with bacterial metabolites and microbiome composition and host mucosal responses. Consumption of PEC reduced expression of pro-inflammatory cytokines such as IFN-γ, IL-6, IL-8, IL-12 and IL-18, and the activation of the pro-inflammatory NF-κB signaling cascade. Expression of MUC2 and TFF and the sIgA content was upregulated in the mucosa of PEC-fed pigs. Network analysis revealed that PEC induced significant interactions between microbiome composition in the ileum and cecum on mucosal immune pathways. PEC-induced changes in bacterial genera and fermentation metabolites, such as Akkermansia, Faecalibacterium, Oscillibacter, Lawsonia and butyrate, correlated with the differentially expressed genes and cytokines in the mucosa. In summary, the results demonstrate the anti-inflammatory properties of PEC on mucosal immune status in the ileum and cecum effected through modulation of the host microbiome.

Trefoil factor family peptides--friends or foes?

Trefoil factor family (TFF) peptides are a group of molecules bearing a characteristic three-loop trefoil domain. They are mainly secreted in mucous epithelia together with mucins but are also synthesized in the nervous system. For many years, TFF peptides were only known for their wound healing and protective function, e.g. in epithelial protection and restitution. However, experimental evidence has emerged supporting a pivotal role of TFF peptides in oncogenic transformation, tumorigenesis and metastasis. Deregulated expression of TFF peptides at the gene and protein level is obviously implicated in numerous cancers, and opposing functions as oncogenes and tumor suppressors have been described. With regard to the regulation of TFF expression, epigenetic mechanisms as well as the involvement of various miRNAs are new, promising aspects in the field of cancer research. This review will summarize current knowledge about the expression and regulation of TFF peptides and the involvement of TFF peptides in tumor biology and cancerogenesis.

PC.17 Impaired mucosal restitution in neonatal necrotising enterocolitis

BackgroundThe aetiology of necrotizing enterocolitis (NEC) is multifactorial and its pathogenesis poorly understood. The trefoil factor peptides (TFF1–3) contribute to protective mechanisms operating in the gastro-intestinal (GI) mucosa and play...

Expressions of NF-B and TFF3 in gastric cancer and its clinical significance

Objective To study the expressions and correlation of nuclear factor kappa B （ NF-B） p65 and trefoil factor family-3（TFF3） in gastric cancer,in order to evaluate the roles of NF-Bp65 and TFF3 in the occurrence and development of gastric cancer,and their correlation with Helicobacter pylori （Hp） infection.Methods The expressions of NF-Bp65 and TFF3 were detected by immunohistochemistey in 30 normal gastric mucosa specimens ,32 dysplasia specimens and 70 gastric cancer specimens .At the same time,the infection of Hp was detected .Results In normal gastric mucosa ,dysplasia and gastric cancer ,the expression of NF-Bp65 had a increasing tendency （ compared with the normal gastric mucosa group ,χ^2 =18.632,44.291,all P〈0.01）.The expression of NF-Bp65 in gastric cancer group was higher than that in dysplasia group （χ^2 =5.205,P〈0.05）.The expression of TFF3 in three groups also had a increasing tendency （compared with the normal gastric mucosa group ,χ2 =16.944,22.917,all P 〈0.01）.The expression of TFF3 in gastric cancer group was higher than dysplasia group ,but there was no significant difference between the two groups （χ^2 =0.162,P〉0.05）.In dysplasia group,the expressions of TFF3 and NF-κBp65 in Hp positive patients were significantly higher than those in Hp negative patients （P〈0.01 or P〈0.05）.There was significantly positive correlation between expression of NF-Bp65 and TFF3 in gastric cancer group （ r=0.350,P=0.003）.Conclusion High expressions of NF-Bp65 and TFF3 may be involved in an early event of gastric cancer . NF-Bp65 expression is positively correlated with the abnormal expression of TFF 3,both of which may be involved in the development and progression of gastric cancer . Key words: Stomach neoplasms; Transcription factors

Promoter hypomethylation and upregulation of trefoil factors in prostate cancer

Trefoil factors, mucin-associated peptides, are overexpressed in prostate cancer (PC). We hypothesized that promoter methylation contributes to the regulation of trefoil factors (TFF1, TFF2 and TFF3) in human prostate cells. Here we show hypomethylation of promoter regions of TFF1 and TFF3 in PC cell lines with significant TFF expression as compared to benign immortalized prostate cell lines and PC cell lines not expressing trefoil factor. The most striking difference was observed for CpG sites located close to the AUG start codon overlapping several putative binding sites for cellular transcription factors. TFF2 was hypermethylated and had no or very low expression in all prostate cell lines investigated. Treatment of methylated cell lines with 5-aza-2'-deoxycytidine restored TFF expression in cell lines not expressing TFF and increased expression significantly in low-expressing cell lines. In clinical samples, methylation of the promoter/enhancer regions of TFF1 and TFF3 was significantly lower in PC compared to benign prostatic hyperplasia. The present study shows an inverse relation between promoter methylation and expression of trefoil factors. Preliminary analysis on clinical samples suggests that this regulatory mechanism is responsible for the increased levels of TFF1 and TFF3 observed in PC. The overexpression and promoter hypomethylation of trefoil factors may serve as biomarkers in PC.

W1595 Regulation of TFF and MUC Expression By SOX2 in Gastric Epithelial Cells

factors, but their roles in angiogenesis in general, and, specifically in angiogenesis in gastric mucosa remain unexplored. Material andMethods:We isolated gastricmucosal microvascular endothelial cells (GMMECs) from Fisher F-344 rats, 3 months of age using anti-PECAM-1 selection (Chemicon) and magnetic bead separation. PECAM-1 positive cells were separated by using a miniMACS column and grown on collagen coated dishes in endothelial cell growth media. The isolated GMMECs were characterized by immunohistochemical staining for the endothelial markers von Willebrand's factor (Factor VIII -related antigen), PECAM1 (CD31) and VEGFR2. Cells were treated with either: 100nM control siRNA, importinα1 or α3 specific siRNAs (Qiagen) for 48 or 72 hrs. Studies: 1) Importin α and VEGF mRNA expression using reverse transcription real-time PCR method with prevalidated QuantiTect assays (Qiagen, Valencia, CA), and, expression and subcellular localization of importin α and VEGF proteins using immunoflorescence studies; 2) In-vitro angiogenesis on matrigel and quantification of capillary-like endothelial tube formation using Metamorph image analysis system. Results: In control GMMECs, importin α and VEGF were strongly expressed and localized to the cell nuclei, with the former also expressed in the nuclear membrane. GMMECs plated on matrigel exhibited endothelial cell migration and capillary tube network and lumen formation at 6 hrs, reflecting in-vitro angiogenesis. Treatment of young GMMECs with importin α1 and α3 specific siRNAs significantly downregulated expression of importin α 1 and α3 (by 2.7-fold; p <0.05 and 2.0-fold; p <0.05 respectively), significantly reduced VEGF gene expression (by 3.3-fold; p <0.05 and 2.4-fold; p <0.05 respectively), and reduced in-vitro angiogenesis in GMMECs (by 1.7-fold; p<0.05 and 1.5-fold; p<0.05 respectively) vs. controls. Conclusions: 1) Knockdown of importin α1 and importin α3 significantly downregulates expression of VEGF and inhibits angiogenesis in gastric mucosal microvascular endothelial cells. 2) These data demonstrate a mechanistic role of importin α as one of the major players in gastric angiogenesis and also as a potential therapeutic target.

Plasma Levels of Trefoil Factors are Increased in Patients with Advanced Prostate Cancer

Through cDNA array analyses and immunohistochemistry on tissue microarrays, trefoil factor 3 (TFF3) was recently shown to be overexpressed in prostate cancer. The purpose of this study was to test the feasibility of using the levels of trefoil factors as a plasma marker for prostate cancer. In 79 patients with prostate cancer, 23 patients with benign prostatic hyperplasia, and 44 healthy individuals plasma TFF1, TFF2, and TFF3 were determined with ELISAs and compared with clinical stage and prostate-specific antigen (PSA) values. Plasma levels of TFF were compared with the immunohistochemical expression of TFF and chromogranin A in 30 prostate cancer tissue samples. Patients with advanced prostate cancer had significantly higher plasma concentrations of TFF1, TFF2, and TFF3 (P < 0.01) compared with patients with localized disease. Using a cutoff of 200 pmol/L, the sensitivity and specificity of plasma TFF3 in differentiating between patients with localized and advanced disease was 74% (59-85%) and 81% (66-91%). Plasma levels of TFF3 were highest in patients with bone metastases (P = 0.008). Patients with serum PSA >10 microg/L had significantly higher plasma TFF3 values than patients with serum PSA <10 microg/L (P = 0.03) and TFF3 levels were higher in patients with Gleason sums of > or = 7 (P = 0.02). Expression of TFF1 and TFF3 determined by immunohistochemistry was increased in patients with prostate cancer but did not correlate with plasma trefoil factor values. Plasma levels of trefoil factors are increased in patients with advanced prostate cancer. Prospective studies are needed to confirm the predictive utility of trefoil factors in prostate cancer.

Impaired mucosal regeneration in neonatal necrotising enterocolitis

Necrotising enterocolitis (NEC) remains an overwhelming gastrointestinal (GI) emergency in premature infants, with an annual incidence of 350 cases and a mortality of 23% in the United Kingdom. The aetiology of NEC is multifactorial and its pathogenesis poorly understood. It is characterised by severe necrotic damage to the intestine. Mucus is an adherent, viscoelastic gel layer protecting the delicate underlying epithelium from lumenal aggressors such as digestive enzymes and bacterial toxins. The group of trefoil factor peptides (TFF1-3) are part of the protective mechanism operating in the intestinal mucosa and play a fundamental role in epithelial protection, repair, and restitution. These secreted peptides have been identified in a site-specific pattern in the GI mucosa, and their expression has been shown to be upregulated in early stages of mucosal repair. The role of trefoil peptides in neonatal mucosal protection has not been well investigated. Impaired mucosal regeneration due in part to failure of upregulation of TFF expression may contribute to the pathogenesis of NEC. The aim of this study was to investigate TFF1-3 mRNA expression and to identify the gene product in the GI tracts of normal neonatal controls and infants with NEC. Parents of all babies having a laparotomy in the neonatal period (defined as up to 44 weeks' gestation) and bowel resection were approached for written consent. Bowel samples were fixed in formalin and then embedded in paraffin in an RNAse-free manner. In situ hybridisation and immunohistochemistry were performed to examine the pattern of trefoil mRNA expression and to localise the peptides in the neonatal GI tract. Forty neonatal bowel specimens were examined. Twelve patients had NEC, eight were recovering from NEC, and 20 control specimens were obtained. TFF1 and TFF2 mRNA expression were not detected in the majority of NEC specimens, and there was a relative downregulation of TFF3 expression in 83% of NEC patients. TFF1 and TFF2 expression were noted in the recovery phase from NEC. Immunohistochemistry revealed a decrease in TFF3 gene product in sites adjacent to mucosal damage secondary to NEC. In acute NEC there was no apparent expression of TFF1 and 2 protein. In the group of patients recovering from NEC, TFF1 and 2 expression were seen in association with regenerative changes in the mucosa. Previous data has shown TFF1-3 to be upregulated in the acute phase response to mucosal injury in the gut. Trefoil peptides have been shown to promote epithelial cell migration and protect against apoptosis. Our results suggest that there is a lack of TFF expression in response to NEC in the premature gut. This may lead to impaired restitution of the mucosa and contribute to the cascade of bowel necrosis and generalised sepsis characteristic of NEC.

Up-regulation of TFF expression by PPARgamma ligands in gastric epithelial cells.

Although trefoil factor family peptides (TFF peptides) are assumed to play important roles in gastric mucosal protection, the regulatory mechanism of gastric TFF expression has not been fully understood yet. Recent reports showed gastric expression of peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor known to be involved in the regulation of cell growth and differentiation in other cell types, such as adipocytes. To determine whether PPARgamma affects the expression of TFF in gastric epithelial cells. MKN45 gastric cells were used as a model of gastric epithelial cells. DNA synthesis of the cells was determined by the measurement of BrdU incorporation. The effects of PPARgamma ligands, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ) on TFF expression were assessed by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). MKN45 cells expressed a significant amount of PPARgamma. Both 15d-PGJ2 and TGZ suppressed DNA synthesis of the cells in a dose-dependent manner. In the control condition, MKN45 cells most abundantly expressed TFF1 and the relative expression level of TFF1, TFF2, and TFF3 mRNA was 1700:32:1. TFF1 and TFF2 mRNA levels were significantly up-regulated by the incubation of the cells with 15d-PGJ2 (10 micro m) or TGZ (30 micro m), whereas TFF3 mRNA level was not affected. The results of the present study suggest a possible role of PPARgamma in the regulation of TFF expression in gastric epithelial cells.

Histone deacetylase inhibitors suppress TFF expression in gastric epithelial cells

Histone deacetylase inhibitors suppress TFF expression in gastric epithelial cells

Role of TFF in healing of stress-induced gastric lesions.

To determine the changes of pS2 and ITF of TFF expression in gastric mucosa and the effect on ulcer healing of pS2, ITF to Water-immersion and restraint stress (WRS) in rats. Wistar rats were exposed to single or repeated WRS for 4 h every other day for up to 6 days.Gastric mucosal blood flow (GMBF) was measured by LDF-3 flowmeter and the extent of gastric mucosal lesions were evaluated grossly and histologically. Expression of pS2 and ITF mRNA was determined by RT-PCR. Immunohistochemistry was used to further detect the expression of pS2 and ITF. WRS applied once produced numerous gastric mucosal erosions, but the number of these lesions gradually declined and GMBF restored at 2, 4, 8 h after stress. The area of gastric mucosal lesion was reduced by 64.9 % and GMBF was increased by 89.8 % at 8 h. The healing of stress-induced ulcerations was accompanied by increased expression of pS2 (0.51+/-0.14 vs 0.77+/-0.11, P<0.01) and ITF (0.022+/-0.001 vs 0.177+/-0.010, P<0.01). The results were demonstrated further by immunohistochemistry of pS2 (0.95+/-0.11 vs 1.41+/-0.04, P<0.01) and ITF (0.134+/-0.001 vs 0.253+/-0.01,P<0.01). With repeated WRS, adaptation to this WRS developed, the area of gastric mucosal lesions was reduced by 22.0 % after four consecutive WRS. This adaptation to WRS was accompanied by increased GMBF (being increased by 94.2 %), active cell proliferation in the neck region of gastric glands, and increased expression of pS2 (0.37+/-0.02 vs 0.77+/-0.01, P<0.01) and ITF (0.040+/- 0.001 vs 0.372+/-0.010, P<0.01). The result was demonstrated further by immunohistochemistry of pS2 (0.55+/-0.04 vs 2.46+/-0.08, P<0.01) and ITF (0.134+/-0.001 vs 0.354+/-0.070, P<0.01). TFF may not only participate in the early phase of epithelial repair known as restitution (maked by increased cell migration), but also play an important role in the subsequent, protracted phase of glandular renewal(made by cell proliferation).

Biliary epithelial trefoil peptide expression is increased in biliary diseases.

Maintenance of the cellular integrity of the biliary epithelium may involve the production of mucins and mucin-associated peptides. In the luminal gastrointestinal tract, mucins and the mucin-associated trefoil peptides (TFF) are integral to cytoprotection and cellular repair of the mucosa. Samples of normal and diseased human liver tissue were examined using histological and immunohistochemical techniques, for the expression of TFF and mucins. Bile ducts were classified as small, medium or large depending upon the number of biliary epithelial cells. TFF expression was demonstrated in biliary epithelial cells of both normal and diseased liver tissue. TFF expression was greatest in the large bile ducts. In normal liver tissue, expression of at least one TFF was demonstrated in 2-7% of small bile ducts, 5-31% of medium bile ducts and 31-85% of large bile ducts. Seventy-seven percent of large bile ducts secreted mucins and all three TFF concurrently, compared with 3% of medium bile ducts and no small bile ducts. Biliary disease resulted in an increased expression of TFF1 and TFF3 in the medium bile ducts. The biliary epithelial cells in normal and diseased human liver tissue express TFF, particularly in the larger bile ducts. TFF expression may be up-regulated or induced in biliary diseases as a response to injury, as is seen in epithelial damage elsewhere in the gastrointestinal tract.

Down-regulation of TFF Expression in Gastrointestinal Cell Lines by Cytokines and Nuclear Factors

Background and Aims: Trefoil peptides (TFF1, TFF2 and TFF3) are acute phase proteins up-regulated in response to gastrointestinal mucosal damage. They promote cell migration, protect and heal the mucosa and may function as tumorsuppressors. We assumed them to be regulated by the proinflammatory cytokines interleukin-1β (IL1β) and interleukin-6 (IL6), which trigger the transcriptional factors NF-ĸB and C/EBPβ. Methods: Following IL1β and IL6 stimulation, expression of TFF genes was analyzed in gastrointestinal cell lines HT-29 and KATO III by reporter gene assays using TFF promoter constructs and by quantitative real-time PCR. NF-ĸB and C/EBPβ were transiently co-expressed. Results: We have functionally identified transcription factors NF-ĸB and C/EBPβ to inhibit transcription of human TFF genes. Down-regulation of TFF transcription is also observed by IL1β and IL6, suggesting crosstalk with or in response to the immune system. IL1β and IL6 caused a 3- to 11-fold reduction in TFF mRNA expression, displayed in real-time PCR. Conclusions: Down-regulation of intestinal trefoil factor TFF3 due to transcriptional repression by IL1β through NF-ĸB as well as by IL6 through C/EBPβ activation in vitro may reflect the situation in vivo and may contribute to ulceration and decreased wound healing during inflammatory bowel disease. Additionally, IL1 and IL6 over-expression in chronic gastritis may lead to mucosal damage and gastric carcinogenesis through transcriptional repression of TFF1 and TFF2.

Trefoil factor family domain peptides in the human respiratory tract.

Trefoil factor family domain peptides (TFF) are thought to be involved in mucosal epithelial restitution and wound healing of the gastrointestinal tract and are up-regulated in ulceration and in a variety of solid tumours. It was hypothesized that TFFs are also expressed on mucosal surfaces of the human respiratory tract. Lung tissue, nasal polyps, and sputum samples from seven patients with cystic fibrosis (CF), two with chronic and acute bronchitis, and non-dysplastic material from two cases of bronchial adenocarcinoma were analysed for TFF expression by immunohistochemistry, immunofluorescence, western blot and RT-PCR. Expression of TFF1 and TFF3 was observed in material from all patients. TFFs were localized in goblet and ciliated cells, as well as in some submucosal cells of tracheobronchial tissues and nasal polyps from normal and CF individuals. In sputa of patients with CF and with chronic or acute bronchitis, TFF1 and TFF3 were detected by western blotting. Freshly cultivated nasal epithelial cells transcribed and secreted TFFs and mucins, whereas nasal cells cultivated for 6 weeks still expressed mucins, but not TFFs. Secreted TFFs and mucins also bound to the surface of Staphylococcus aureus in infected CF airways. In conclusion, TFF1 and TFF3 are expressed and secreted in normal and inflamed airways. The association of TFFs with bacteria may contribute to the anti-microbial mucociliary defence system.

Trefoil factor family domain peptides in the human respiratory tract

Trefoil factor family domain peptides (TFF) are thought to be involved in mucosal epithelial restitution and wound healing of the gastrointestinal tract and are up-regulated in ulceration and in a variety of solid tumours. It was hypothesized that TFFs are also expressed on mucosal surfaces of the human respiratory tract. Lung tissue, nasal polyps, and sputum samples from seven patients with cystic fibrosis (CF), two with chronic and acute bronchitis, and non-dysplastic material from two cases of bronchial adenocarcinoma were analysed for TFF expression by immunohistochemistry, immunofluorescence, western blot and RT-PCR. Expression of TFF1 and TFF3 was observed in material from all patients. TFFs were localized in goblet and ciliated cells, as well as in some submucosal cells of tracheobronchial tissues and nasal polyps from normal and CF individuals. In sputa of patients with CF and with chronic or acute bronchitis, TFF1 and TFF3 were detected by western blotting. Freshly cultivated nasal epithelial cells transcribed and secreted TFFs and mucins, whereas nasal cells cultivated for 6 weeks still expressed mucins, but not TFFs. Secreted TFFs and mucins also bound to the surface of Staphylococcus aureus in infected CF airways. In conclusion, TFF1 and TFF3 are expressed and secreted in normal and inflamed airways. The association of TFFs with bacteria may contribute to the anti-microbial mucociliary defence system. Copyright © 2000 John Wiley & Sons, Ltd.

5'-flanking motifs control cell-specific expression of trefoil factor genes (TFF).

A group of secreted peptides (trefoil factor family; TFF) is abundantly expressed at mucosal surfaces of the gastrointestinal tract and promote epithelial restitution. They are upregulated around areas of epithelial damage, ulceration and neoplasia. The transcriptional regulation of the three human TFF genes was assayed by multiplex RT-PCR and reporter gene analysis in 8 gastrointestinal carcinoma cell lines. The level of endogenous mRNA matched well the reporter gene activity of all TFFs, indicating that the cis-acting elements located less than 1,000 bp upstream of the TATAA box account for cell-specific gene expression. In HT-29, the endogenous TFF expression profile changed in relation to cell growth conditions. Deletion and mutation analysis of TFF promoter constructs revealed enhancing elements shared within the three TFF promoters that were shown to bind nuclear proteins. Thus such specific DNA-protein interaction may explain the TFF peptides' cell specific expression pattern and altered levels in pathological conditions.