A carboxy-terminal alpha-helical segment in the rat skeletal muscle voltage-dependent Na+ channel is responsible for its interaction with the amino-terminus

Cytoplasmic segments of the adult rat skeletal muscle sodium channel alpha-subunit (rSkM1) comprise a major portion (approximately 40%) of the total protein and are involved in channel functions both general, such as inactivation, and isoform-specific, for example, protein kinase A modulation. Far ultraviolet circular dichroism measurements of synthetic peptides and overexpressed fusion proteins containing individual channel cytoplasmic segments suggest that cytoplasmic domains of rSkM1 contain ordered secondary structures even in the absence of adjoining transmembrane segments. Intrinsic fluorescence experiments with a nested set of carboxy-terminal deletion proteins confirm a specific interaction between the channel's amino- and carboxy-termini and identify residues 1716-1737 in the carboxy-terminus as the region that binds to the amino-terminus. Circular dichroism measurements suggest that this same region is organized as an alpha-helix and that electrostatic forces may contribute to this association. The interaction of the amino- and carboxy-termini is not accompanied by secondary structure changes detectable by circular dichroism spectroscopy, but a decrease in intrinsic fluorescence indicates that this association is accompanied by a change in the environment of Trp1617.     

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.     

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.     

Transcriptional repression of TFF1 in gastric epithelial cells by CCAAT/enhancer binding protein-beta

TFF1 is a member of a unique family of gastrointestinal peptides. Loss of TFF1 expression has been observed in the majority of human gastric cancers and the biological significance of this loss has been demonstrated in a Tff1 knockout mouse model. However, few TFF1 gene mutations or allelic loss have also been documented. To understand the molecular mechanism repressing the TFF1 gene expression, the 5'-flanking region of the human TFF1 gene was characterized. We found a repressor region (-241 to -84), which is active in MKN45 and IMGE5 cells expressing endogenous TFF1 gene. A consensus binding site for C/EBPbeta was identified and EMSA analysis demonstrated specific binding of CEBPbeta. Mutation of this C/EBPbeta element potentiated the transactivation of TFF1 by 50% and 145% for MKN45 and IMGE5 cells respectively. Furthermore, co-transfection of C/EBPbeta isoforms specifically decreased TFF1 promoter activity. These findings suggest that C/EBPbeta is involved in the down-regulating of TFF1 gene expression and this mode of repression may account at least in part for the loss of TFF1 gene expression in transformed human and mice gastric epithelial cells.     

Isolation and characterization of putative trefoil peptide receptors

Mammalian trefoil factors (TFFs) constitute a group of three peptides (TFF1, TFF2 and TFF3) widely distributed in the gastrointestinal tract. Although a mucosal protection/healing effect of these peptides is well documented the mechanism of action is still unknown. A mucosal membrane extract was prepared from porcine stomach scrapings and incubated with a gel containing immobilized porcine TFF2. The affinity gel material was specifically eluted with a neutral buffer containing a high concentration of the ligand (porcine TFF2). A subsequent SDS–gel electrophoresis showed one protein with a MW of approximately 220 kDa and three proteins with MW around 140 kDa. The proteins were analyzed by trypsin digestion followed by mass spectrometric sequencing of tryptic fragments. In this way a 140-kDa β subunit of fibronectin receptor and a 224-kDa CRP-Ductin gene product were identified. The CRP-Ductin gene product (also named MUCLIN), which is expressed in the intestinal crypts, is characterized by being a membrane protein with a short cytoplasmic region, a transmembrane domain and a large extracellular region. This protein thus fulfils some of the criteria for being a TFF receptor or a TFF binding protein.     

三叶因子:从实验室研究到临床医学

三叶因子（trefoil factor, TFF）家族是具有一个或多个三叶因子结构域的蛋白质多肽,在进化上具有高度的保守性,具有耐热、耐酶消化的理化特性。哺乳动物的TFF有三个成员(TFF1、TFF2和TFF3)。黏膜组织,如胃肠道和呼吸道黏膜等是TFF的主要合成场所。生理条件下,TFF的分布具有组织专一性,具有黏膜保护和创伤修复作用。TFF在肿瘤组织中广泛表达,在肿瘤的发生、发展、侵袭、转移过程中发挥着重要作用,可能是癌基因在对不同刺激做出反应时的共有递质。TFF生物学功能存在一个复杂的调控过程,单链TFF可通过膜受体而激活相关信号通路,TFF也可以通过与其它蛋白质的结合而协同发挥作用。TFF在临床医学领域主要运用在黏膜保护、黏膜损伤相关疾病的预防和治疗及肿瘤病理的诊断与干预等方面。TFF的作用机制和功能行使相关信号通路仍然是目前研究的重点,TFF与βγ-晶状体蛋白天然复合物的发现有助于对TFF的进一步认识和深入研究。     

TFF1 is membrane-associated in breast carcinoma cell line MCF-7

Trefoil factor family (TFF) domain peptides, products of mucin-secreting epithelial cells, are thought to influence mucosal integrity. Molecular studies revealed that mammalian TFFs lack transmembrane domains. Using immunocytochemistry and FACS analysis we demonstrated the association of TFF1 with the cell membrane in MCF-7 (a breast adenocarcinoma cell line), and tested the hypothesis that glycosylphosphatidylinositol (GPI) linkage is the mechanism for this association. Cleavage of GPI anchorage using phospholipase C did not affect TFF1 binding to the cell membrane. Our results demonstrate for the first time that TFF1 is associated with the cell membrane of MCF-7 cells and is not linked via a GPI anchor.     

Expression of Trefoil Factor 1 in the Developing and Adult Rat Ventral Mesencephalon

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides with a characteristic tree-looped trefoil structure. TFFs are mainly expressed in the gastrointestinal tract where they play a critical role in the function of the mucosal barrier. TFF1 has been suggested as a neuropeptide, but not much is known about its expression and function in the central nervous system. We investigated the expression of TFF1 in the developing and adult rat midbrain. In the adult ventral mesencephalon, TFF1-immunoreactive (-ir) cells were predominantly found in the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA) and in periaqueductal areas. While around 90% of the TFF1-ir cells in the SNc co-expressed tyrosine hydroxylase (TH), only a subpopulation of the TH-ir neurons expressed TFF1. Some TFF1-ir cells in the SNc co-expressed the calcium-binding proteins calbindin or calretinin and nearly all were NeuN-ir confirming a neuronal phenotype, which was supported by lack of co-localization with the astroglial marker glial fibrillary acidic protein (GFAP). Interestingly, at postnatal (P) day 7 and P14, a significantly higher proportion of TH-ir neurons in the SNc co-expressed TFF1 as compared to P21. In contrast, the proportion of TFF1-ir cells expressing TH remained unchanged during postnatal development. Furthermore, significantly more TH-ir neurons expressed TFF1 in the SNc, compared to the VTA at all four time-points investigated. Injection of the tracer fluorogold into the striatum of adult rats resulted in retrograde labeling of several TFF1 expressing cells in the SNc showing that a significant fraction of the TFF1-ir cells were projection neurons. This was also reflected by unilateral loss of TFF1-ir cells in SNc of 6-hydroxylase-lesioned hemiparkinsonian rats. In conclusion, we show for the first time that distinct subpopulations of midbrain dopaminergic neurons express TFF1, and that this expression pattern is altered in a rat model of Parkinson’s disease.     

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.     

TFF peptides in the human false vocal folds of the larynx

TFF peptides (formerly P domain peptides, trefoil factors) are typical secretory products of mucin-producing cells and are thought to influence the rheological properties of mucous gels. We investigated the localization of these peptides in the human false vocal folds of the larynx, also known as the ventricular folds or vestibular folds. An analysis of TFF peptide mRNA by RT-PCR and TFF protein by Western blot detected TFF1 and TFF3, but not TFF2. Immunohistochemistry revealed TFF1 to be associated with the secretory product of goblet cells and mucous parts of subepithelial seromucous glands. TFF3 occurred in columnar epithelial cells of the mucosa and in serous cells and excretory duct cells of seromucous glands. These peptides may play a role in the rheological function of mucus secreted onto the true vocal folds and are thus important constituents of vocal production.     

Development of a two-site ELISA assay for the dimeric form of human TFF1

TFF1 is one of three human trefoil proteins expressed principally in the gastrointestinal tract in normal tissues. TFF1 protects the gastric mucosa against damage as a result of its ability to facilitate reconstitution of damaged gastric mucosa and its involvement in the secretion and structure of gastric mucus. The most biologically active molecular form in cell culture and animal models tested is a dimer formed by a disulfide bond between two cysteine residues close to the C terminus of the protein. We have therefore developed an assay for this form of TFF1 which should facilitate its measurement in biological samples.     

Expression and localization of trefoil factor family genes in rat submandibular glands

The trefoil factor (TFF) family, which comprises TFF1, TFF2 and TFF3, plays an essential role in epithelial regeneration within the gastrointestinal tract. All three TFFs are present in human saliva; TFF3 is the predominant trefoil peptide. Little is known about the expression and tissue distribution of TFFs in rats, which are commonly used as a model system for human studies. We investigated the localization of the TFF genes that encode secretory peptides in rat submandibular glands (SMG). All three TFFs were expressed in rat SMG, although their location varied. Substantial amounts of TFF1 were detected only in the cytoplasm of epithelial cells in the SMG granular convoluted tubules (GCT), while TFF2 and TFF3 were widely distributed in the cytoplasm of epithelial cells of intercalated ducts (ID), striated ducts (SD) and interlobular ducts (ILD). The three TFFs also were detected especially in the lumens of the SD and ILD. Semi-quantitative RT-PCR and in situ hybridization experiments confirmed TFF1, TFF2 and TFF3 mRNA expressions in the SMG. Greater expression of TFF peptides and mRNA was observed in male rats than in females. The broad expression of TFFs in rat SMG cells and lumens suggests that TFFs function in this organ by their secretion into the duct lumens. We also found differences in TFF expression profiles between rat and human SMG; therefore, caution should be exercised when using rats as a model for human TFF studies.     

Structural Requirements for Cub Domain Containing Protein 1 (CDCP1) and Src Dependent Cell Transformation

Cub domain containing protein 1 (CDCP1) is strongly expressed in tumors derived from lung, colon, ovary, or kidney. It is a membrane protein that is phosphorylated and then bound by Src family kinases. Although expression and phosphorylation of CDCP1 have been investigated in many tumor cell lines, the CDCP1 features responsible for transformation have not been fully evaluated. This is in part due to the lack of an experimental system in which cellular transformation depends on expression of exogenous CDCP1 and Src. Here we use retrovirus mediated co-overexpression of c-Src and CDCP1 to induce focus formation of NIH3T3 cells. Employing different mutants of CDCP1 we show that for a full transformation capacity, the intact amino- and carboxy-termini of CDCP1 are essential. Mutation of any of the core intracellular tyrosine residues (Y734, Y743, or Y762) abolished transformation, and mutation of a palmitoylation motif (C689,690G) strongly reduced it. Src kinase binding to CDCP1 was not required since Src with a defective SH2 domain generated even more CDCP1 dependent foci whereas Src myristoylation was necessary. Taken together, the focus formation assay allowed us to define structural requirements of CDCP1/Src dependent transformation and to characterize the interaction of CDCP1 and Src.     

High trefoil factor 1 (TFF1) expression in human retinoblastoma cells correlates with low growth kinetics, increased cyclin-dependent kinase (CDK) inhibitor levels and a selective down-regulation of CDK6

Trefoil factor family (TFFs) peptides facilitate epithelial restitution, but also effect cell proliferation and apoptosis of normal and various cancer cell lines. In a recent study by our group, TFF2 expression was demonstrated in the murine retina, where it exhibits pro-proliferative and pro-apoptotic effects. In the present study, we investigated the expression and function of TFF peptides in eight human retinoblastoma cell lines. TFF1 was the only TFF peptide expressed at detectable levels in immunoblots of retinoblastoma cells. TFF1 expression levels were highly variable in different retinoblastoma cell lines and negatively correlated with cell growth curves. Recombinant human TFF1 had a negative effect on cell viability and caused a reduction in cell proliferation. Retinoblastoma cell lines with high TFF1 expression levels exhibited a selective down-regulation of cyclin-dependent kinase (CDK) 6, whereas CDK4 and CDK2 seem to be unaffected by TFF1 expression. In immunocytochemical studies, we observed a nuclear co-localization of TFF1 and CDK2 in Cajal bodies (CBs). In high TFF1 expressing human retinoblastoma cell lines CBs were smaller and higher in number compared to retinoblastoma lines with low TFF1 expression, indicating differences in cell cycle status between the different retinoblastoma cell lines. Our data further support the notion for a potential tumor suppressor function of TFF1. The nuclear localization of TFF1 in CBs—considered to play a role in cell cycle progression, potentially acting as a platform for CDK-cyclin function—offers a new impetus in the ongoing search for potential TFF1 interacting proteins.     

Synergistic effects of systemic trefoil factor family 1 (TFF1) peptide and epidermal growth factor in a rat model of colitis

Novel therapies for the treatment of colitis are required. We therefore examined the potential value of the trefoil factor family 1 (TFF1) peptide and epidermal growth factor (EGF) alone and in combination. Effects of TFF1- Cys58 +/- EGF on an in vitro HT29 cell wounding model of restitution showed synergistic activity when used in combination. In addition, animals had colitis induced by adding 4% dextran sulphate sodium (DSS) to the drinking water for 7 days and they also received twice daily subcutaneous injections of test peptides. Treatment with TFF1-Cys58 alone (100 microg/kg) reduced histological colitis score by 22%, but the TFF1-Ser58 variant was ineffective. In a second study, TFF1-Cys58 reduced histological colitis score by 15%, EGF (600 microg/kg) by 26%, and an additive response (42% reduction) was demonstrated when used together (P < 0.01 versus either peptide given alone). Similar results were found using tissue myeloperoxidase (MPO) activity as a marker of inflammation. Where clinical risk/benefit seems justified, these initial studies suggest that combination therapy of systemic EGF and TFF peptides may prove useful for treatment of colitis in patients with disease extending beyond the reach of topical (enema) therapy.