The trefoil factor interacting protein TFIZ1 binds the trefoil protein TFF1 preferentially in normal gastric mucosal cells but the co-expression of these proteins is deregulated in gastric cancer

The gastric tumour suppressor trefoil protein TFF1 is present as a covalently bound heterodimer with a previously uncharacterised protein, TFIZ1, in normal human gastric mucosa. The purpose of this research was firstly to examine the molecular forms of TFIZ1 present, secondly to determine if TFIZ1 binds other proteins apart form TFF1 in vivo, thirdly to investigate if TFIZ1 and TFF1 are co-regulated in normal gastric mucosa and fourthly to determine if their co-regulation is maintained or disrupted in gastric cancer. We demonstrate that almost all human TFIZ1 is present as a heterodimer with TFF1 and that TFIZ1 is not bound to either of the other two trefoil proteins, TFF2 and TFF3. TFIZ1 and TFF1 are co-expressed by the surface mucus secretory cells throughout the stomach and the molecular forms of each protein are affected by the relative abundance of the other. TFIZ1 expression is lost consistently, early and permanently in gastric tumour cells. In contrast, TFF1 is sometimes expressed in the absence of TFIZ1 in gastric cancer cells and this expression is associated with metastasis (lymph node involvement: p=0.007). In conclusion, formation of the heterodimer between TFIZ1 and TFF1 is a specific interaction that occurs uniquely in the mucus secretory cells of the stomach, co-expression of the two proteins is disrupted in gastric cancer and expression of TFF1 in the absence of TFIZ1 is associated with a more invasive and metastatic phenotype. This indicates that TFF1 expression in the absence of TFIZ1 expression has potentially deleterious consequences in gastric cancer.     

Interaction between TFF1, a gastric tumor suppressor trefoil protein, and TFIZ1, a brichos domain-containing protein with homology to SP-C

TFF1 is a gastric tumor suppressor that protects gastric epithelial cells from damage but can promote invasive properties of tumor cells. Antibodies were raised against correctly folded TFF1 protein. These showed that the 6.67 kDa secreted trefoil protein is present as an approximately 25 kDa complex in normal human gastric mucosa. The TFF1 complex was immunopurified from human gastric mucosa and shown to comprise two proteins joined by a disulfide bond. Both were identified by amino-terminal sequencing and MALDI TOF mass spectrometry. The TFF1 protein partner is a previously unknown protein that we have called TFIZ1 for trefoil factor interactions(z) 1. TFIZ1 is expressed and secreted in normal gastric mucosa. TFIZ1 mRNA was cloned from gastric mucosa and sequenced. TFIZ1 is an 18.31 kDa protein and contains an approximately 100 amino acid brichos domain and homology with smart00019.10, SF_P. This is the first demonstration that a member of the trefoil factor family of proteins is bound covalently to a brichos domain-containing protein. The apparent molecular mass of the TFF1:TFIZ1 heterodimer is remarkably close to the theoretical molecular mass of 24.98 kDa. In conclusion, the heterodimer comprises one molecule each of TFF1 and TFIZ1, and the disulfide bond between TFF1 and TFIZ1 is the most important factor stabilizing the heterodimer.     

Co-localization of TFF2 with gland mucous cell mucin in gastric mucous cells and in extracellular mucous gel adherent to normal and damaged gastric mucosa

Trefoil factor 2 (TFF2) is mucin associated peptide that has a mucosal barrier function in addition to participating in repair and healing. We examined the localization of TFF2 and gastric mucins in gastric mucous cells, the surface mucous gel layer (SMGL) adherent to normal gastric mucosa, and in the mucoid cap covering gastric erosions. Carnoy’s solution, or formalin/picric acid-fixed paraffin embedded materials from resected stomachs and formalin-fixed paraffin embedded gastric biopsy materials were used. Sections were immunostained for the TFF2 and histochemically stained for gastric mucins. In addition, thick sectioned gastric mucosa fixed in Carnoy’s solution were stained with FITC-labeled GSA-II lectin specific for gland mucous cell mucin and examined for three-dimensional images of the SMGL using a confocal laser scanning microscope. The TFF2 and gland mucous cell mucin were found intermixed together in the gastric gland mucous cells, in the SMGL in laminated layers, and in the mucoid cap. A laminated arrangement of continuous sheets of gland mucous cell mucin in the SMGL was demonstrated in the three-dimensional images. Co-localization of the TFF2 with gland mucous cell mucin suggests a physical interaction between the TFF2 and gland mucous cell mucin. The TFF2 trapped in the adherent mucins may be responsible for mucosal defense, healing, and repair.     

大鼠实验性胃溃疡期间下颌下腺TFF3基因的变化

目的研究下颌下腺肠三叶因子(intestinal trefoil peptide,ITF即TFF3)基因在大鼠实验性胃溃疡自愈过程的变化,探讨其与胃溃疡自愈的关系。方法通过胃窦前壁黏膜下注射冰乙酸制备大鼠胃溃疡模型:⑴用免疫组织化学SABC法和RT-PCR检测42只溃疡组,21只盐水组,及6只正常组大鼠下颌下腺组织中TFF3肽和TFF3mRNA的表达情况。结果(1)免疫组化显示:溃疡组大鼠下颌下腺的TFF3肽主要表达于导管系统上皮,如闰管、颗粒曲管(granular convoluted tubule,GCT)以及纹状管、小叶间导管上皮细胞、黏液腺泡细胞也有少量分布,浆液腺泡细胞呈阴性。溃疡组手术后第1d时,下颌下腺TFF3表达明显强于盐水组和正常组(P0.01)。术后第2d,积分光密度明显低于1d溃疡组(P0.05),4、6d积分光密度逐渐增强并高于对照组(P0.05),到术后第10d达高峰(P0.01),23d积分光密度仍高于对照组(P0.05)。(2)RT-PCR显示:溃疡1、2、4、6、10、14、23dTFF3/GAPDH光密度比值分别为1.42±0.10,1.18±0.13,1.29±0.15,1.24±0.17,1.57±0.19,1.25±0.14,1.13±0.16明显高于相应盐水对照组的TFF3/GAP-DH光密度比值(P0.01)。结论大鼠胃溃疡时期,下颌下腺TFF3基因上调,推测下颌下腺TFF3通过外分泌或内分泌参与胃溃疡愈合过程     

Polymorphism of estrogen response element in TFF1 gene promoter is associated with an increased susceptibility to gastric cancer

TFF1 is a cysteine-rich protein that forms a characteristic trefoil domain through disulfide bonds, which render it resistant to vigorous conditions and it involves in maintaining the integrity of the gastric mucosa. Decreased expression of TFF1 gene plays a role in the development of gastric cancer. We examined the association between the promoter polymorphisms of the TFF1 gene and the risk of development of gastric cancer, in a case-control study including 199 controls and 141 patients with gastric cancer. Assessment of single nucleotide polymorphisms in the promoter region of the TFF1 gene was performed by sequencing and polymerase chain reaction-based restriction fragment length polymorphism. We found a statistically significant increased risk of gastric cancer associated with − 394 TT genotypes (OR = 8.78, CI = 2.85-27.05, p < 0.001) and CT (OR = 1.64, CI = 1.04-2.60, p = 0.033). This single nucleotide polymorphism occurs naturally in an estrogen response element. According to induction of the TFF1 gene by estrogen, it is possible that the substitution of C to T results in a decreased estrogen receptor binding affinity to the estrogen response element and in turn it decreases the expression of the TFF1 gene that may be involved in development of gastric cancer over a lifetime.     

Copper Promotes TFF1-Mediated Helicobacter pylori Colonization

The trefoil peptides (TFF1, TFF2 and TFF3) are a family of small highly conserved proteins that play an essential role in epithelial regeneration within the gastrointestinal tract, where they are mainly expressed. TFF1 expression is strongly induced after mucosal injury and it has been proposed that tff1 functions as a gastric tumor suppressor gene. Several studies confirm that tff1 expression is frequently lost in gastric cancer because of deletions, mutations or methylation of the tff1 promoter. Infection by Helicobacter pylori (H. pylori) results in chronic gastritis and it can lead to the development of gastric or duodenal ulcers. Moreover, it is known that there is a strong link to the development of gastric cancer. It has been shown that H. pylori interacts with the dimeric form of TFF1 and that the rough form of lipopolysaccharide mediates this interaction. We have previously reported that the carboxy-terminus of TFF1 is able to specifically bind copper ions (Cu) and that Cu binding favours the homodimerization of the peptide, thus enhancing its motogenic activity. Here, we report that the Cu-TFF1 cuprocomplex promotes adherence of H. pylori to epithelial cells. Adherence of H. pylori to gastric adenocarcinoma cells, AGS AC1 cells, induced to hyper-express TFF1 was enhanced compared to noninduced cells. Copper further promoted this interaction. A H. pylori mutant unable to bind TFF1 did not show enhanced infection of induced cells. Cu treatment induced a thickening of the mucus layer produced by the colorectal adenocarcinoma mucus secreting, goblet cells, HT29-E12 and promoted H. pylori colonisation. Finally, SPR analysis shows that the C-terminus of TFF1, involved in the binding of copper, is also able to selectively bind H. pylori RF-LPS.     

Cytosolic levels of an estrogen-induced breast cancer-associated peptide (TFF1/pS2) in colorectal cancer: clinical significance and relationship with steroid receptors

BACKGROUND: The Trefoil Factor 1 (TFF1/pS2), a peptide consisting of 60 amino acids, is the most abundant estrogen-induced messenger RNA in MCF-7 breast cancer cells and is also expressed by colorectal carcinomas. The objective of this work was to evaluate the cytosolic TFF1 content in colorectal carcinomas, its possible relationship with estrogen and progesterone receptors as well as with clinicopathological tumor parameters, and its potential prognostic significance. METHODS: Cytosolic TFF1 levels were examined by immunoradiometric assay in 178 patients with resectable colorectal cancer. The mean follow-up period was 32 months. RESULTS: There was a wide variability of cytosolic TFF1 levels in tumor-surrounding mucosa samples (0.09-42.5 ng/mg protein) as well as in tumors (0.01-270 ng/mg protein). Comparison of paired mucosa and carcinoma samples showed significantly higher TFF1 levels in tumors (mean: 17.1 ng/mg protein) than in mucosa samples (10 ng/mg protein) (p = 0.027). TFF1 levels were significantly higher in mucosa samples surrounding distal colon and rectal tumors (p = 0.0001) and in tumor samples obtained from older patients (p = 0.007). However, there were no significant differences in tumor TFF1 levels with respect to clinicopathological parameters such as the patient's sex, tumor location, stage, histological grade, ploidy, S-phase, or tumor estrogen and progesterone receptors. In addition, there was no significant relationship between tumor TFF1 levels and disease outcome. CONCLUSIONS:TFF1 may play an as yet undetermined role in the tumorigenesis of colorectal carcinomas. However, cytosolic levels of TFF1 do not seem to have any prognostic significance in colorectal carcinomas.     

Amplification and invasiveness of epithelial progenitors during gastric carcinogenesis in trefoil factor 1 knockout mice

Abstract. Objective: It is not known whether or not epithelial progenitors of the pyloric antrum are involved in gastric carcinogenesis. Normally, these progenitors give rise to two main cell lineages: pit and gland mucous cells. This study was designed to examine the changes that occur in pyloric antral mucous cell lineages and their progenitors during development of gastric adenoma and carcinoma in trefoil factor 1 (TFF1) knockout mice. Materials and methods: Pyloric antral mucosal tissues of TFF1 knockout mice at ages from 3 days to 17 months were processed for histochemical analysis using Ulex europaeus and Grifforia simplifolica lectins as markers for pit and gland mucous cells, respectively. The dividing epithelial progenitors were identified by using immunohistochemical and electron microscopy techniques. Results: TFF1 loss was associated with amplification of both mucus‐secreting pit and gland cells. Both lectins examined bound not only to mature mucous cells, but also to most of epithelial progenitors which gradually amplified with age and frequently were seen in mitosis. Analysis of 12‐ to 17‐month‐old TFF1‐deficient stomachs revealed occasional groups of poorly differentiated mucosal cells with features similar to those of epithelial progenitors (or stem cells), in the basal portion of the antral mucosa. These cells eventually invaded the muscularis mucosa while maintaining some capacity to differentiate. Conclusion: This study shows that the progenitors of pit and gland mucous cells contribute to gastric carcinogenesis in the pyloric antrum of TFF1 knockout mice, strongly supporting the concept of stem cell origin of cancer.     

Increased Expression of Protease-Activated Receptor 4 and Trefoil Factor 2 in Human Colorectal Cancer

Protease-activated receptor 4 (PAR4), a member of G-protein coupled receptors family, was recently reported to exhibit decreased expression in gastric cancer and esophageal squamous cancer, yet increased expression during the progression of prostate cancer. Trefoil factor 2 (TFF2), a small peptide constitutively expressed in the gastric mucosa, plays a protective role in restitution of gastric mucosa. Altered TFF2 expression was also related to the development of gastrointestinal cancer. TFF2 has been verified to promote cell migration via PAR4, but the roles of PAR4 and TFF2 in the progress of colorectal cancer are still unknown. In this study, the expression level of PAR4 and TFF2 in colorectal cancer tissues was measured using real-time PCR (n = 38), western blotting (n=38) and tissue microarrays (n = 66). The mRNA and protein expression levels of PAR4 and TFF2 were remarkably increased in colorectal cancer compared with matched noncancerous tissues, especially in positive lymph node and poorly differentiated cancers. The colorectal carcinoma cell LoVo showed an increased response to TFF2 as assessed by cell invasion upon PAR4 expression. However, after intervention of PAR4 expression, PAR4 positive colorectal carcinoma cell HT-29 was less responsive to TFF2 in cell invasion. Genomic bisulfite sequencing showed the hypomethylation of PAR4 promoter in colorectal cancer tissues and the hypermethylation in the normal mucosa that suggested the low methylation of promoter was correlated to the increased PAR4 expression. Taken together, the results demonstrated that the up-regulated expression of PAR4 and TFF2 frequently occurs in colorectal cancer tissues, and that overexpression of PAR4 may be resulted from promoter hypomethylation. While TFF2 promotes invasion activity of LoVo cells overexpressing PAR4, and this effect was significantly decreased when PAR4 was knockdowned in HT-29 cells. Our findings will be helpful in further investigations into the functions and molecular mechanisms of Proteinase-activated receptors (PARs) and Trefoil factor factors (TFFs) during the progression of colorectal cancer.     

The Interaction of Helicobacter pylori with the Adherent Mucus Gel Layer Secreted by Polarized HT29-MTX-E12 Cells

Helicobacter pylori colonises the gastric mucosa of humans. The majority of organisms live in mucus. These organisms are an important reservoir for infection of the underlying epithelium. Cell culture models for H. pylori infection do not normally possess a mucus layer. The interaction of H. pylori with TFF1, a member of the trefoil factor family found in gastric mucin, is mediated by lipopolysaccharide. To test the hypothesis that the interaction of H. pylori with TFF1 promotes mucus colonization we characterised the interaction of H. pylori with a mucus secreting cell line, HT29-MTX-E12. An isogenic mutant of H. pylori with truncated core oligosaccharides was produced and binding to TFF1 and ability to colonise HT29-MTX-E12 cells determined. The adherent mucus layer of HT29-MTX-E12 cells contained the gastric mucin MUC5AC and trefoil factors, TFF1 and TFF3. H. pylori was found within the mucus layer in discrete clusters and in close association with TFF1. It also interacted with the membrane bound mucin MUC1 and replicated when co-cultured with the cells. An isogenic mutant of H. pylori with a truncated LPS core did not interact with TFF1, and colonization of HT29-MTX-E12 cells was reduced compared to the wild-type strain (p<0.05). Preincubation of cells with wild type LPS but not with truncated LPS resulted in reduced colonization by H. pylori. These results demonstrate that the interaction of TFF1 with H. pylori is important for colonization of gastric mucus and the core oligosaccharide of H. pylori LPS is critical for this interaction to occur. HT29-MTX-E12 cells are a useful system with which to study the interaction of bacteria with mucosal surfaces and the effect of such interactions on mediating colonization.     

TFF3-peptide increases transepithelial resistance in epithelial cells by modulating claudin-1 and -2 expression

TFF3 plays an important role in the protection and repair of the gastrointestinal mucosa. The molecular mechanisms of TFF function, however, are still largely unknown. Increasing evidence indicates that apart from stabilizing mucosal mucins TFF3 induces cellular signals that modulate cell–cell junctions of epithelia. In transfected HT29/B6 and MDCK cells stably expressing FLAG-tagged human TFF3 we have recently shown that TFF3 down-regulates E-cadherin, impairs the function of adherens junctions and thus facilitates cell migration in wounded epithelial cell layers. Here we investigate TFF3-induced effects on the composition and function of tight junctions in these cells. TFF3 increased the cellular level of tightening claudin-1 and decreased the amount of claudin-2 known to form cation-selective channels. Expression of ZO-1, ZO-2 and occludin was not altered. The change in claudin-1 and -2 expression in TFF3-expressing HT29/B6 cells was accompanied by an increase in the transepithelial resistance in confluent monolayers of these cells. These data suggest that TFF3 plays a role in the regulation of intestinal barrier function by altering the claudin composition within tight junctions thus decreasing paracellular permeability of the intestinal mucosa.     

灰树花多糖对体外胃粘膜创伤的修复作用

本文采用MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium]法检测灰树花多糖(GFP)对人胃正常粘膜上皮细胞(human gastric epithelial cell,GES-1)细胞增殖影响;使用毛细管对单层融合的GES-1细胞进行划痕,模拟胃粘膜创伤,观察并计算GFP作用后GES-1细胞创伤区域迁移及修复损伤的面积;ELISA(enzyme-linked immunosorbent assay)方法测定培养基上清液中表皮生长因子(epidermal growth factor,EGF),转移生长因子(transforming growth factor-β₁,TGF-β₁)和三叶因子2(trefoil factors 2,TFF2)的含量变化;荧光定量PCR(RT-PCR)检测其mRNA的变化.实验结果表明:GFP通过上调EGF,TFF2,下调TGF-β₁ 表达而促进GES-1细胞增殖,促进其向创伤区域迁移,可以完全覆盖创伤区域达到修复胃粘膜作用.     

木瓜三萜对吲哚美辛致胃黏膜损伤小鼠胃酸分泌及胃黏膜屏障的影响

观察木瓜三萜对吲哚美辛致胃黏膜损伤小鼠胃酸分泌及胃黏膜屏障的影响,在此基础上探讨其可能的机制。实验时,将小鼠随机分为正常组、模型组、木瓜三萜(50、100mg/kg)和奥美拉唑(20mg/kg)组。将给药组灌胃给予相应的药物,正常组和模型组灌胃给予0.5%羧甲基纤维素钠溶液,给药6小时后,除正常组外,灌胃给予20mg/kg的吲哚美辛,每天一次,连续7天。末次给药次日,小鼠用水合氯醛麻醉后,固定,剪开腹腔,进行胃黏膜血流量的测定,然后取胃检测胃液量、胃液酸度和胃结合黏液量;检测胃黏膜中表皮生长因子基因(EGF)和三叶因子1基因(TFF1)的mRNA和蛋白表达。研究发现:吲哚美辛致胃黏膜损伤模型组小鼠胃液分泌量,胃液酸度、胃黏膜血流量、胃结合黏液量及胃黏膜组织中EGF和TFF1的mRNA和蛋白表达明显降低,与正常组比较均具有统计学差异(P<0.01);用木瓜三萜预处理后,上述异常的变化均得到了有效逆转,与模型组比较具有显著性差异(P<0.05,P<0.01)。实验结果表明木瓜三萜(50、100mg/kg)对吲哚美辛致小鼠胃黏膜损伤具有较好的保护作用,通过上调EGF和TFF1的表达水平,增加胃液分泌量、胃液酸度、胃黏膜血流量、胃结合黏液量,恢复胃黏膜防御屏障的功能可能是其治疗吲哚美辛致胃黏膜损伤的机制之一。