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.     

Trefoil factor 2 (TFF2) deficiency in murine digestive tract influences the immune system.

BACKGROUND AND AIMS: The gastrointestinal trefoil factor family (TFF1, TFF2, TFF3) peptides are considered to play an important role in maintaining the integrity of the mucosa. The physiological role of TFF2 in the protection of the GI tract was investigated in TFF2 deficiency. METHODS: TFF2-/- mice were generated and differential expression of various genes was assessed by using a mouse expression microarray, quantitative real time PCR, Northern blots or immunohistochemistry. RESULTS: On an mRNA level we found 128 differentially expressed genes. We observed modulation of a number of crucial genes involved in innate and adaptive immunity in the TFF2-/- mice. Expression of proteasomal subunits genes (LMP2, LMP7 and PSMB5) involved in the MHC class I presentation pathway were modulated indicating the formation of immunoproteasomes improving antigen presentation. Expression of one subunit of a transporter (TAP1) responsible for importing degraded antigens into ER was increased, similarly to the BAG2 gene that modulates chaperone activity in ER helping proper loading on MHC class I molecules. Several mouse defensin (cryptdin) genes coding important intestinal microbicidal proteins were up-regulated as a consequence of TFF2 deficiency. Normally moderate expression of TFF3 was highly increased in stomach.     

Biosynthesis of gastrokine-2 in the human gastric mucosa: restricted spatial expression along the antral gland axis and differential interaction with TFF1, TFF2 and mucins.

Gastrokine-2 (GKN2) is a secretory peptide of human gastric surface mucous cells (SMCs). It forms disulfide-linked heterodimers with the trefoil factor family (TFF) peptide TFF1. Binding with TFF2 was also reported. Antral SMCs differ from those of the corpus by their TFF3 expression. The aim of this study was to localize GKN2 expression along the antral gland axis, to characterize the continuous regeneration of antral glands, and to investigate the interactions of GKN2 with TFF1, TFF2 and mucins. Methods: The spatial expression of GKN1, GKN2, TFF1-3, MUC5AC and MUC6 was determined using laser microdissection and RT-PCR analysis. Furthermore, antral extracts were separated by gel chromatography and the association of GKN2 with TFF1, TFF2, and mucins was investigated. Results: Differential GKN2 expression was localized along the rostro-caudal axis of the stomach. Laser microdissection revealed characteristic differential expression profiles of GKN1, GKN2, TFF1-3, MUC5AC and MUC6 along the antral gland axis. Both GKN2 and TFF1 were expressed in superficial SMCs. Surprisingly, the TFF1-GKN2 heterodimer did not associate with the mucin fraction; whereas TFF2 showed exclusive association with mucins. Conclusions: Maturation of antral SMCs occurs stepwise via trans-differentiation of TFF3 expressing progenitor cells. The TFF1-GKN2 heterodimer and TFF2 differ characteristically by their binding to gastric mucins. This points to different physiological functions of TFF1 and TFF2, the latter maybe acting as a 'link peptide' for stabilization of the gastric mucus.     

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.     

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.     

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.     

Regulatory Function of Trefoil peptides (TFF) on Intestinal Cell Junctional Complexes

Abstract

Trefoil peptides (TFF) are constitutively expressed in the gastrointestinal tract and are involved in gastrointestinal defence and repair by promoting epithelial restitution. Although there is a general consensus regarding the pro-motogenic activity of trefoil peptides, the cellular mechanisms through which they mediate these processes are not completely understood. Pertubation of the E-cadherin/catenin complex at intercellular junctions appears to be a functional pathway through which TFF2 and TFF3 promote cell migration. Tight junction complexes seal the paracellular spaces between cells and contribute to epithelial barrier function. TFF3 peptide stimulation stabilises these junctions through upregulation of the tightening protein claudin-1 and redistribution of ZO-1 from the cytoplasm to the intercellular membrane with an increase in binding to occludin. Modulation of the functional activity and subcellular localisation of epithelial junctional adhesion molecules represent important mechanisms by which trefoil peptides may promote migration of intestinal epithelial cells in vitro and healing of mucosal damage in vivo.     

Solution structure of the disulfide-linked dimer of human intestinal trefoil factor (TFF3): the intermolecular orientation and interactions are markedly different from those of other dimeric trefoil proteins

The trefoil protein TFF3 forms a homodimer (via a disulfide linkage) that is thought to have increased biological activity over the monomer. The solution structure of the TFF3 dimer has been determined by NMR and compared with the structure of the TFF3 monomer and with other trefoil dimer structures (TFF1 and TFF2). The most significant structural differences between the trefoil domain in the monomer and dimer TFF3 are in the orientations of the N-terminal 3(10)-helix (residues 10-12) and in the presence in the dimer of an additional 3(10)-helix (residues 53-55) outside of the core region. The TFF3 dimer forms a more compact structure as compared with the TFF1 dimer where the two trefoil domains are connected by a flexible region with the monomer units being at variable distances from each other and in many different orientations. Although TFF2 is also a compact structure, the dispositions of its monomer units are very different from those of TFF3. The structural differences between the dimers result in the two putative receptor/ligand binding sites that remain solvent exposed in the dimeric structures having very different dispositions in the different dimers. Such differences have significant implications for the mechanism of action and functional specificity for the TFF class of proteins.     

Localization of TFF3 peptide to porcine conjunctival goblet cells

TFF-peptides (formerly P-domain peptides, trefoil factors) form a new family of mucin-associated peptides mainly in the gastrointestinal tract. TFF3 is a typical secretory product of intestinal goblet cells and occurs also in the respiratory tract. Here, polyclonal antisera specific for TFF3 were used in Western blot analysis and immunofluorescence to determine the presence and distribution of TFF3 in the porcine conjunctiva, which is the primary source for ocular mucins. Significant accumulation of TFF3 was detected in conjunctival goblet cells but not in the lacrimal glands. This peptide, together with ocular mucins, may play a role in the rheological function of the tear film.     

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.     

The closely related estrogen-regulated trefoil proteins TFF1 and TFF3 have markedly different hydrodynamic properties,overall charge,and distribution of surface charge

The human trefoil proteins TFF1 and TFF3 are expressed predominantly in the gastrointestinal tract. They are also expressed and regulated by estrogens in malignant breast epithelial cells. TFF1 and TFF3 are small cysteine-rich acidic secreted proteins of 60 and 59 amino acids with similar isoelectric points of 4.75 and 3.94, respectively. Each contains one trefoil domain that is characterized by several conserved features including six cysteine residues with conserved spacing. TFF1 and TFF3 form intermolecular disulfide bonds via an extra-trefoil domain cysteine residue and are present in vivo as monomers and homodimers and as complexes with other proteins. The TFF1 dimer is more active than the TFF1 monomer. In the present study the hydrodynamic and charge properties of TFF1 and TFF3 monomers and homodimers have been compared and shown to differ markedly. Notably, TFF1 is significantly more asymmetric than TFF3 (frictional coefficients 1.25 and 1.12, respectively, p < 0.001), and homodimerization of TFF1 results in a greater increase in asymmetry than for TFF3. The overall charges of TFF1 and TFF3 are very different at neutral pH. Titration curves predicted significant differences in charge across a wide pH range that agreed well with experimental data. The locations of charged amino acids in the primary sequences and in the tertiary structures of TFF1 and TFF3 were examined. This revealed interesting divergence in both the distribution and local topology of charged amino acid side chains. The significant differences between the shape, size, and surface charge of these two closely related molecules may account for their divergent biological activities.     

Expression of trefoil peptides in the subtypes of intestinal metaplasia

We studied the expression of trefoil peptides in the different types of intestinal metaplasia of the stomach. Endoscopic biopsy was performed in 132 patients with dyspepsia. Intestinal metaplasia subtype was classified according to the pattern of alcian blue/PAS staining and high iron diamine staining. Expression of trefoil peptides was measured by immunohistochemistry. TFF1 and TFF3 were mainly expressed in goblet cells and TFF2 in columnar cells in all the types of intestinal metaplasia. There was a gradual decrease of TFF1 and TFF3, and increase of TFF2, during the progression of intestinal metaplasia from type I to type III via the type II intermediate.     

Co-localization of TFF3 peptide and oxytocin in the human hypothalamus.

TFF-peptides (formerly P domain peptides, trefoil factors) are typical secretory products of many mucous epithelial cells. TFF3 is also synthesized in the hypothalamus and has anxiolytic or anxiogenic activities when injected into the rat amygdala. Here we show by immunohistochemistry that TFF3 is localized to a distinct population of neurons of the human hypothalamic paraventricular and supraoptic nuclei. Generally, TFF3-positive cells are co-localized in oxytocin-producing cells and not in vasopressin-producing cells. Relatively large amounts of TFF3-but not TFF1 and TFF2-are present in the posterior lobe of the human pituitary, where it is probably released into the bloodstream. Furthermore, TFF3 was also detectable in human postmortem cerebrospinal fluid.     

Ultrastructural co-localization of TFF3-peptide and oxytocin in the neural lobe of the porcine pituitary

TFF-peptides (formerly P-domain peptides, trefoil factors) are typical secretory products of many mucous epithelial cells. TFF3 is also synthesized in oxytocinergic neurons in the paraventricular and supraoptic nuclei of the human hypothalamus. Here, TFF3 and oxytocin are shown to be co-localized within the same secretory vesicles in the neural (posterior) lobe of the procine pituitary by means of immunoelectron microscopy. Relatively large amounts of TFF3, but not TFF1 and TFF2, are present in the neural lobe of the porcine pituitary, where it is probably released into the bloodstream.     

Differential expression of the TFF-peptides xP1 and xP4 in the gastrointestinal tract of Xenopus laevis

TFF-peptides (formerly P-domain peptides, trefoil factors) represent major secretory products of the mammalian gastrointestinal tract. A molecular cloning approach revealed the existence of two TFF-peptides, xP1 and xP4, also in the stomach of Xenopus laevis. Here, the localization of these two peptides by Western blot analysis as well as immunohistochemistry is presented. xP1 is found predominantly in the surface mucous cells of the stomach, whereas xP4 is mainly localized to a specific population of goblet cells in the esophagus, to mucous neck cells of the stomach, and to closely resembling cells in antral glands. xP4 in the esophagus and in the stomach differ by their N-glycosylation patterns. Compared to mammalian TFF-peptides, xP1 obviously represents the frog homologue of human TFF1 (formerly pS2) and xP4 seems to be the amphibian equivalent of human TFF2 (formerly hSP).     

Synthesis and localization of the mucin-associated TFF-peptides in the human uterus

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. Here, the localization of these peptides in the human uterus was investigated. An analysis of TFF-peptides mRNA by the polymerase chain reaction revealed TFF3 mainly in the endocervix and smaller amounts in the endometrium. TFF1 and TFF2 mRNA was detectable occasionally in the endocervix and very rarely in the endometrium. Western blot analysis detected only TFF3 in tissue extracts of the endocervix and as a constituent of human cervical mucus. Immunofluorescence localized TFF3 in the surface epithelium of the endocervix and in gland-like structures of the cervical epithelium.