Specific secretion of gel-forming mucins and TFF peptides in HT-29 cells of mucin-secreting phenotype

Trefoil factor family (TFF) peptides are typical secretory products of mucin-producing cells, e.g. of the gastrointestinal tract. Here, the expression and secretion of mucins and TFF peptides was studied in the HT-29 cell line throughout cellular growth and differentiation in relation to a mucin-secreting (HT-29 MTX) or an enterocyte-like (HT-29 G(-)) phenotype. mRNAs of several MUC and TFF genes were expressed in both cell subpopulations. However, for most MUC and TFF genes, the expression appeared strongly induced with the differentiation into the mucin-secreting phenotype. On the other hand, TFF2 was specifically expressed in the mucin-secreting HT-29 MTX cells. The differentiation of HT-29 MTX cells into the mucin-secreting phenotype was characterised by secretion of the gel-forming mucins MUC2, MUC5AC, and MUC5B, however, according to a different pattern in the course of differentiation. A significant amount of TFF1 and TFF3 was secreted after differentiation, also according to a different pattern, whereas TFF2 was only faintly detected. Secretagogues, known to induce the secretion of mucus, increased the secretion of all three TFF peptides. In contrast, neither a secretory mucin nor a TFF peptide was found in the culture medium of HT-29 G(-) cells. Overlay assays indicated that HT-29 MTX mucins bound to secretory peptides of HT-29 MTX cells with relative molecular mass similar to TFF peptides. TFF1 and TFF3 were specifically localised in the mucus layer of HT-29 MTX cells by confocal microscopy. Finally, the secretion of TFF peptides and mucins appears as a co-ordinated process which only occurs after differentiation into goblet cell-like phenotype.     

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.     

Self-renewal of the human gastric epithelium: new insights from expression profiling using laser microdissection

The gastric mucosa is subject to continual bidirectional renewal by differentiation from stem and transit amplifying cells. It was the aim of this study to characterize the self-renewal of the human gastric mucosa and its two major types of glands in the fundus and antrum, respectively. Three characteristic regions (pit, proliferative, and lower neck regions) were isolated from fundic and antral units by the use of laser microdissection, and expression profiles concerning 15 marker genes were generated by RT-PCR analysis. The surface mucous cells (SMCs) of fundic and antral units differed in their expression of at least four secretory genes, i.e., gastric lipase, TFF3, FCGBP, and lysozyme. The maturation of mucous neck cells was shown to occur stepwise, first towards a mucous phenotype followed by a serous differentiation step. Also, a stepwise maturation of both the antral SMCs and antral gland cells was observed. Additionally, the presence of gastric lipase was also demonstrated for the first time in antral gland cells. In conclusion, the different expression profiles of SMCs of the fundic and antral units could be the basis for the different self-renewal rates of fundic and antral SMCs and could influence the spatial organization of the bacterial microbiota within the various parts of the gastric mucosa.     

Expression profiles of MUC mucins and trefoil factor family (TFF) peptides in the intrahepatic biliary system: physiological distribution and pathological significance

Mucin secreted by mucosal epithelial cells plays a role in the protection of the mucosal surface and also is involved in pathological processes. So far, MUC1-4, 5AC, 5B, 6-8, 11-13 and 15-17 genes coding the backbone mucin core protein have been identified in humans. Their diverse physiological distribution and pathological alterations have been reported. Trefoil factor family (TFF) peptides are mucin-associated molecules co-expressed with MUC mucins and involved in the maintenance of mucosal barrier and the biological behavior of epithelial and carcinoma cells. Intrahepatic biliary system is a route linking the bile canaliculi and the extrahepatic bile duct for the excretion of bile synthesized by hepatocytes. Biliary epithelial cells line in the intrahepatic biliary system, secreting mucin and other molecules involved in the maintenance and regulation of the system. In this review, the latest information regarding properties, expression profiles and regulation of MUC mucins and TFF peptides in the intrahepatic biliary system is summarized. In particular, we focus on the expression profiles and their significance of MUC mucins in developmental and normal livers, various hepatobiliary diseases and intrahepatic cholangiocarcinoma.     

Immunohistochemical study of the expression of MUC6 mucin and co-expression of other secreted mucins (MUC5AC and MUC2) in human gastric carcinomas.

To investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas.     

Alterations in the composition of the supramucosal defense barrier in relation to disease severity of ulcerative colitis.

Mucin glycoproteins and trefoil peptides play an important role in protection and repair of the gastrointestinal epithelium. This study investigates alterations in mucin and trefoil peptide gene expression and product localization in ulcerative colitis (UC). Product localization and message expression of mucin MUC1 to 6 and trefoil peptide TFF1 to 3 genes was analyzed in rectosigmoid tissue from a cohort of patients with active UC and compared with that of normal colorectal mucosa. MUC1 expression was upregulated in severe UC at the site of rupture of crypt abscesses. Reduction in MUC2 expression occurred in UC adjacent to ulceration. No alteration in MUC3 or MUC4 gene expression was detectable in UC compared with normal colorectal mucosa. No ectopic expression of MUC5AC, MUC5B, or MUC6 was identified in UC. Ectopic TFF1 expression was identified in tissues eliciting histological features of severe disease. Decreased TFF3 localization was demonstrated in UC tissues, but no TFF2 expression was detected in any colorectal specimens. Subtle alterations in composition of the supramucosal defense barrier exist in UC and vary in relation to clinical severity of disease. There is upregulation in mucin MUC1 at crypt abscesses and neo-expression of TFF1 trefoil peptide in severe disease.     

Injected TFF1 and TFF3 bind to TFF2-immunoreactive cells in the gastrointestinal tract in rats

Peptides of the trefoil factor family (TFF1, TFF2 and TFF3) are co-secreted with mucus in most organ systems and are believed to interact with mucins to produce high-viscosity, stable gel complexes. We have previously demonstrated that cells in the GI tract possess binding sites to TFF2 and that injected TFF2 ends up in the mucus layer. In the present study, tissue binding and metabolism of parenterally administered human TFF1 and TFF3 in rats were described and compared to the immunohistochemical localization of the TFF peptides. 125I-TFF1 monomer and 125I-TFF3 mono- and dimer were given intravenously to female Wistar rats. The tissue distribution was assessed by gamma counting of organ samples and by autoradiography of histological sections. The degradation of 125I-TFF3 was studied by means of trichloracetic acid (TCA) precipitation and the saturability of the binding by administration of excess unlabelled peptide. The TFF peptides were localized in histologic sections from the GI tract by immunohistochemistry. Injected TFF3 dimer (12%) was taken up by the GI tract. At autoradiography, grains were localized to the same cells that were immunoreactive to TFF2. The binding could be displaced by excess TFF3. Similar binding was observed for the TFF1 and TFF3 monomers apart from binding in the stomach, where the uptake was only 15% in comparison to the dimer. There was no specific binding outside the GI tract and no binding to TFF1 or TFF3 immunoreactive cells. In conclusion, the TFF2-binding cells in the gastrointestinal tract seem to have basolateral, receptor-like activity to all three TFF peptides. The mucous neck cells of the stomach predominantly take up TFFs with two trefoil domains, indicating a different receptor-like activity in the stomach compared to the rest of the GI tract.     

Hyperplastic polyps and sessile serrated ‘adenomas’ of the colon and rectum display gastric pyloric differentiation

The serrated polyp-neoplasia pathway is a novel concept that has been demonstrated to differ from the conventional adenoma-carcinoma pathway. To characterize the phenotypic patterns of differentiation in colorectal serrated polyps, we examined the immunohistochemical expression profile of gastric (MUC5AC, TFF1, MUC6, GlcNAcα1 → 4Gal → R, and PDX1) and intestinal (MUC2, TFF3, and CDX2) epithelial markers in 15 hyperplastic polyps (HPs), 29 sessile serrated adenomas (SSAs),12 traditional serrated adenomas (TSAs), and 16 conventional adenomas (CAs). MUC5AC and TFF1 were upregulated in the HPs, SSAs, and TSAs. MUC6 was expressed in the HPs and SSAs. GlcNAcα1 → 4Gal → R was expressed only in the SSAs. Although MUC2 expression was preserved, TFF3 was downregulated in the HPs, SSAs, and TSAs. PDX1 was upregulated in the HPs, SSAs, and TSAs. On the other hand, CDX2 was downregulated in the HPs and SSAs. The colorectal serrated polyps showed higher expression of gastric makers than CAs. The HPs and SSAs showed gastric and intestinal mixed phenotype expression with gastric pyloric organoid differentiation and almost identical, but different from the TSAs, marker profile. PDX1 up-regulation and CDX2 down-regulation could be important for the induction of a gastric pyloric pattern of cell differentiation in colorectal serrated polyps.     

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.     

Expression of trefoil factor family 1, 2, and 3 peptide is augmented in hepatolithiasis

Trefoil factor family (TFF) 1, 2, and 3 is a mucin-associated protein involved in the maintenance of mucosal barrier and restitution of lining epithelial cells. In this study, the expression of TFF1, 2, and 3 and MUC mucins were examined immunohistochemically in hepatolithiasis and control livers. The expression of TFF1, 2, and 3 were augmented markedly in biliary mucosa in hepatolithiasis in coordinate with gel-forming mucin. TFF3 was detected in hepatic bile samples of hepatolithiasis. Augmented expression and secretion of TFF in biliary mucosa in hepatolithiasis may play a role in lithogenesis together with gel-forming mucin in addition to mucosal defense and repair.     

Human Trefoil Factor 2 Is a Lectin That Binds α-GlcNAc-capped Mucin Glycans with Antibiotic Activity against Helicobacter pylori

Helicobacter pylori infection is the major cause of gastric cancer and remains an important health care challenge. The trefoil factor peptides are a family of small highly conserved proteins that are claimed to play essential roles in cytoprotection and epithelial repair within the gastrointestinal tract. H. pylori colocalizes with MUC5AC at the gastric surface epithelium, but not with MUC6 secreted in concert with TFF2 by deep gastric glands. Both components of the gastric gland secretome associate non-covalently and show increased expression upon H. pylori infection. Although blood group active O-glycans of the Lewis-type form the basis of H. pylori adhesion to the surface mucin layer and to epithelial cells, α1,4-GlcNAc-capped O-glycans on gastric mucins were proposed to inhibit H. pylori growth as a natural antibiotic. We show here that the gastric glycoform of TFF2 is a calcium-independent lectin, which binds with high specificity to O-linked α1,4-GlcNAc-capped hexasaccharides on human and porcine stomach mucin. The structural assignments of two hexasaccharide isomers and the binding active glycotope were based on mass spectrometry, linkage analysis, ¹H nuclear magnetic resonance spectroscopy, glycan inhibition, and lectin competition of TFF2-mucin binding. Neoglycolipids derived from the C3/C6-linked branches of the two isomers revealed highly specific TFF2 binding to the 6-linked trisaccharide in GlcNAcα1-4Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-3)GalNAc-ol(Structure 1). Supposedly, lectin TFF2 is involved in protection of gastric epithelia via a functional relationship to defense against H. pylori launched by antibiotic α1,4-GlcNAc-capped mucin glycans. Lectin-carbohydrate interaction may have also an impact on more general functional aspects of TFF members by mediating their binding to cell signaling receptors.     

Variation of human mucin gene expression in gastric cancer cell lines and gastric mucous cell primary cultures

Human gastric mucous cells - gastric cancer cell lines mucin gene expression - TNFalpha - RT-PCR immunocytochemistry Little is known on the expression pattern of mucin genes in human gastric cancer cell lines in relation to mucin expression in normal gastric epithelial cells. Thus, the aim of this study was to compare gastric cancer cell lines and non-transformed epithelial cells in their expression of the different mucin genes, in order to use these cells as models for physiological MUC expression in human stomach. Human gastric mucous cell primary cultures which were obtained from surgical specimen by collagenase/pronase treatment and a panel of six human gastric cancer cells were screened for mRNA expression of the mucin genes MUC1, MUC2, MUC5AC, MUC5B, and MUC6. Mucin gene expression was analyzed by semi-quantitative RT-PCR, and by Western blotting and immunocytochemistry. Primary cultured human gastric mucous cells retained the stomach-specific pattern of mRNA expression found in gastric mucosal biopsies (MUC1, MUC5AC, MUC6), whereas any gastric cancer cell line exhibited an aberrant mucin gene expression. Mucin gene expression showed large variations in levels and patterns from cell line to cell line, but MUC2 was aberrantly expressed in all cancer cells. Immunocytochemistry confirmed aberrant MUC2 protein expression in cancer cells. The expression of the secretory mucin genes MUC2 and MUC5AC varied in relation to the length of cultivation of the cancer cell lines. Treatment of the gastric cancer cells with TNFalpha resulted in an enhanced mRNA expression of MUC1, MUC2, and MUC5AC (2-fold increase within 3 hours; p <0.05). In contrast, immunocytochemistry disclosed a decrease in MUC2 and MUC5AC staining intensity. Our results indicate that primary cultured human gastric mucous cells provide a physiological in vitro system for investigations of gastric mucin gene regulation. In gastric cancer cells marked changes in the mucin gene expression pattern are found with coexpression of non-gastric type mucins. Gastric mucin gene expression may be regulated by proinflammatory cytokines which could have implications in gastritis.     

TACC1及TFF3在胃癌组织中的表达及其临床意义(英文)

目的:探讨转化酸性卷曲螺旋蛋白1(TACC1)和肠三叶因子(TFF3)在胃癌组织中的表达及其临床意义。方法:采用免疫组化方法检测胃癌组织中TACC1及TFF3的表达情况。采用x2检验、乘积极限法及单因素、多因素生存分析等统计学方法,分析胃癌组织中TACC1及TFF3的表达与患者临床病理参数及预后的关系。结果:TACC1和TFF3在进展期胃癌、有淋巴结转移及复发的胃癌组织中的表达率较高。此外,TFF3在较大肿瘤(肿瘤大小4 cm)的胃癌组织中的表达较高。单因素生存分析显示年龄、淋巴结转移、TACC1和TFF3表达与低生存期显著相关(P0.05)。多因素分析结果表明,TACC1和TFF3的表达是独立的预后预测因子。结论:TACC1和TFF3的表达可以作为胃癌的独立不良预后因子,而且在胃癌组织中TACC1和TFF3共同表达的患者生存时间更短。     

TACC1及TFF3在胃癌组织中的表达及其临床意义

目的：探讨转化酸性卷曲螺旋蛋白1（TACC1）和肠三叶因子（TFF3）在胃癌组织中的表达及其临床意义。方法：采用免疫组化方法检测胃癌组织中TACC1及TFF3的表达情况。采用x2检验、乘积极限法及单因素、多因素生存分析等统计学方法,分析胃癌组织中TACC1及TFF3的表达与患者临床病理参数及预后的关系。结果：TACC1和TFF3在进展期胃癌、有淋巴结转移及复发的胃癌组织中的表达率较高。此外,TFF3在较大肿瘤（肿瘤大小〉4 cm）的胃癌组织中的表达较高。单因素生存分析显示年龄、淋巴结转移、TACC1和TFF3表达与低生存期显著相关（P〈0.05）。多因素分析结果表明,TACC1和TFF3的表达是独立的预后预测因子。结论：TACC1和TFF3的表达可以作为胃癌的独立不良预后因子,而且在胃癌组织中TACC1和TFF3共同表达的患者生存时间更短。