Trefoil Factor Family Peptides Research Articles

Pharmacokinetics of trefoil peptides and their stability in gastrointestinal contents

Trefoil factor family (TFF) peptides are considered promising for therapeutic use in gastrointestinal diseases, and there is a need to explore the fate of injected TFF and the stability of the peptides in the gastrointestinal tract. We studied the pharmacokinetics of intravenously (i.v.) administered hTFF2 in mice and rats and of hTFF3 administered i.v., intramuscularly, intraperitoneally, and subcutaneously in mice, and estimated by ELISA the decay of the peptides added to rat and human gastrointestinal contents. We found that i.v. injected hTFF2 and hTFF3 were cleared from the circulation within 2–3 h, exhibiting comparable pharmacokinetic profiles. In contents from the rat stomach, hTFF levels remained unchanged for up to 6 days. In the small and large intestine of rats, the hTFF levels decreased markedly after 4 and 1 h, respectively. In small intestinal contents from humans, the levels remained stable for more than 24 h. We conclude that systemically administered hTFF2 and hTFF3 are rapidly eliminated from the circulation and that the stability of hTFF2 and hTFF3 in GI contents appeared higher in the gastric and small intestinal milieu than in the large intestine and feces, suggesting a higher stability toward gastric acid and digestive enzymes than toward microbial degradation.

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.

Cytokine Regulation of the Trefoil Factor Family Binding Protein GKN2 (GDDR/TFIZ1/blottin) in Human Gastrointestinal Epithelial Cells

Trefoil factor family (TFF) peptides are major secretory products of mucous epithelia and play a multifunctional role in cytoprotection, apoptosis, and immune response. Recently, a TFF2-binding protein was discovered in mice and named blottin. It is down-regulated in gastric cancer (GDDR), abundant in human gastric surface (TFIZ1) and its similarity to gastrokine-1 led to the gene’s name GKN2. To investigate the mode of GKN2 regulation activity of a luciferase reporter gene, controlled by the GKN2 promoter, was monitored upon treatment with various pro-inflammatory (TNF-α, IL-1β, IL-6, IFN-γ) and anti-inflammatory (TGF-β1) cytokines using gastric (AGS, KATO III) and colonic (HT-29) cell lines. To assess the direct role of transcription factors (NFĸB, HNF-3β, hGATA6) in regulating GKN2 we performed transient co-transfection of their expression plasmids and the reporter gene construct. GKN2 gene was down-regulated by pro-inflammatory cytokines in all tested cell lines while up-regulated by TGF-β1 only in the colonic cell line. GKN2 expression was significantly reduced in both gastric adenocarcinoma cell lines by the active form of NFĸB transcription factor, whereas in the colonic cell line an up-regulation was noticed. Down-regulation by IL-6 was mediated by C/EBPβ transcription factor in case of HT-29 but not of KATO III cells. We conclude that the regulation of GKN2 parallels that of TFF genes, indicating that together they may play an important role in maintaining the homeostasis of the gastrointestinal tract.

TFF3 and EGF Induce Different Migration Patterns of Intestinal Epithelial Cells In Vitro and Trigger Increased Internalization of E-cadherin

Background/Aims: TFF3, a member of the TFF (trefoil factor family) peptides, and epidermal growth factor (EGF) actively support the repair of mucosal barriers, particularly during restitution. The aim of this study was to compare the motogenic effects of TFF3 and EGF. Methods: The influence of recombinant human TFF3 (dimeric form) and EGF on the migration of IEC-18 cells was characterized in an in vitro restitution model (scratch wound assay) with the help of time-lapse video microscopy, morphometry, and immunocytochemistry including confocal laser scanning microscopy. Results: TFF3- and EGF-treated cells re-populated the wounded area via different migration patterns; TFF3 treatment resulted in the formation of continuous sheets of migrating cells with only a few gaps. In contrast, EGF-treated cells formed a network of migrating cells (often with a fibroblast-like morphology) with numerous gaps and only punctual contacts. TFF3 and EGF treatment also changed the localization of E-cadherin indicating endocytotic recycling and/or degradation of E-cadherin. Conclusion: TFF3, in contrast to EGF, enhanced a collective cell migration ensuring a precise coverage of the re-populated area avoiding gaps.

Trefoil Factor Family 3 Peptide Promotes Human Airway Epithelial Ciliated Cell Differentiation

Human airway surface epithelium is frequently damaged by inhaled factors (viruses, bacteria, xenobiotic substances) as well as by inflammatory mediators that contribute to the shedding of surface epithelial cells. To regain its protective function, the epithelium must rapidly repair and redifferentiate. The Trefoil Factor Family (TFF) peptides are secretory products of many mucous cells. TFF3, the major TFF in the airways, is able to enhance airway epithelial cell migration, but the role of this protein in differentiation has not been defined. To identify the specific role of TFF3 in the differentiation of the human airway surface epithelium, we analyzed the temporal expression pattern of TFF3, MUC5AC, and MUC5B mucins (goblet cells) and ciliated cell markers beta-tubulin (cilia) and FOXJ1 (ciliogenesis) during human airway epithelial regeneration using in vivo humanized airway xenograft and in vitro air-liquid interface (ALI) culture models. We observed that TFF3, MUC5AC, MUC5B, and ciliated cell markers were expressed in well-differentiated airway epithelium. The addition of exogenous recombinant human TFF3 to epithelial cell cultures before the initiation of differentiation resulted in no change in MUC5AC or cytokeratin 13 (CK13, basal cell marker)-positive cells, but induced an increase in the number of FOXJ1-positive cells and in the number of beta-tubulin-positive ciliated cells (P < 0.05). Furthermore, this effect on ciliated cell differentiation could be reversed by specific epidermal growth factor (EGF) receptor (EGF-R) inhibition. These results indicate that TFF3 is able to induce ciliogenesis and to promote airway epithelial ciliated cell differentiation, in part through an EGF-R-dependent pathway.

Induced Trefoil Factor Family 1 Expression by Trans-Differentiating Clara Cells in a Murine Asthma Model

Asthma is a chronic inflammatory disease of the airways that is accompanied by goblet cell metaplasia and mucus hypersecretion. Trefoil factor family (TFF) peptides represent major secretory products of the respiratory tract and are synthesized together with mucins. In the murine lung, TFF2 is mainly expressed, whereas TFF1 transcripts represent only a minor species. TFF peptides are well known for their motogenic and anti-apoptotic effects, and they modulate the inflammatory response of bronchial epithelial cells. Here, an established mouse model of asthma was investigated (i.e., exposure to Aspergillus fumigatus [AF] antigens). RT-PCR analysis of lung tissue showed elevated levels particularly of TFF1 transcripts in AF-sensitized/challenged animals. In contrast, transcripts encoding Clara cell secretory protein (CCSP/CC10) were strongly diminished in these animals. For comparison, the expression of the goblet cell secretory granule marker mCLCA3/Gob-5, the mucins Muc1-Muc6 and Muc19, and the secretoglobins ScgB3A1 and ScgB3A2, as well as the mammalian ependymin-related gene MERP2, were monitored. Immunohistochemistry localized TFF1 mainly in cells with a mixed phenotype (e.g., TFF1-positive cells stain with the lectin wheat germ agglutinin (WGA), which recognizes mucins characteristic of goblet cells). In addition, these cells express CCSP/CC10, a Clara cell marker. When compared with mucins or CCSP/CC10, TFF1 was stored in a different population of secretory granules localized at the more basolateral portion of these cells. Thus, the results presented indicate for the first time that allergen exposure leads to the trans-differentiation of Clara cells toward a TFF1-expressing mucous phenotype.

Identification of blottin: A novel gastric trefoil factor family-2 binding protein

The trefoil factor family (TFF) peptides are important in gastro-intestinal mucosal protection and repair. Their mechanism of action remains unclear and receptors are sought. We aimed to identify and characterise proteins binding to TFF2. A fusion protein of mouse TFF2 with alkaline phosphatase was generated and used to probe 2-D protein blots of mouse stomach. The resulting spots were analysed by MS. The protein identified was characterised by bioinformatics, rapid amplification of cDNA ends, in situ hybridisation (ISH) and immunohistochemistry (IHC). Functional assays were performed in gastrointestinal cell lines. A single major murine protein was identified and named blottin. It was previously unknown as a translated product. Blottin is also present in rat and human; the latter gene is also known as GDDR. The predicted full-length proteins are 184 amino acids long (20 kDa), reducing to 164 amino acids (18 kDa) after signal peptide cleavage. ISH of gastrointestinal tissues shows abundant blottin mRNA in gastric surface and foveolar epithelium. IHC shows cytoplasmic staining for blottin protein, and by immunoelectron microscopy in mucus granules and Golgi stacks. Previous work showed that blottin is down-regulated in gastric cancers. Blottin contains a BRICHOS domain, and has 56% similarity with gastrokine-1. Cultured HT-29 cells express blottin and show increased DNA synthesis with antiblottin antibody; however, this effect is reversed by the immunising peptide. We have identified and characterised a TFF2-binding protein produced by gastric epithelium. Blottin may play a role in gastrointestinal mucosal protection and modulate gut epithelial cell proliferation.

Mucins and TFF peptides of the tear film and lacrimal apparatus

The three-dimensional organization of the tear film, which is produced and drained by the different structures of the ocular adnexa, is essential for maintainance and protection of the ocular surface. This is facilitated by a class of large, highly glycosylated, hydrophilic glycoproteins, the mucins, which are usually expressed in association with a class of peptides having a well-defined, structurally conserved trefoil domain, the mammalian trefoil factor family (TFF) peptides. In this review, the latest information regarding mucin and TFF peptide function and regulation in the human lacrimal system, the tear film and the ocular surface is summarized with regard to mucous epithelia integrity, rheological and antimicrobial properties of the tear film and tear outflow, age-related changes and certain disease states such as dry eye, dacryostenosis and dacryolith formation.

Trefoil factors TFF (trefoil factor family) peptide-triggered signals promoting mucosal restitution.

Rapid repair of mucous epithelia is essential for preventing inflammation which is a critical component of cancer progression. 'Restitution' is an early repair process which can begin within minutes and is achieved via the migration of neighbouring cells into the wounded area. Mucosal restitution is a multistep process which requires continuous blood flow and includes at least (i) the reduction of cell-cell contacts and a shift in the cell shape towards a migratory phenotype (characteristics of the epithelial-mesenchymal transition), (ii) migration of cells, (iii) repolarization and formation of tight junctions (morphological restitution) and (iv) restoration of barrier function (transmucosal epithelial resistance, functional restitution). Secretory TFF (trefoil factor family) peptides TFF1, TFF2 and TFF3 are well known for their potent protective and healing effects after mucosal damage (function as 'luminal surveillance peptides'). Here, the contributions of the TFFs during the different steps of mucosal restitution are discussed, i. e. the modulation of cell-cell contacts, their motogenic activity and synergy with epidermal growth factor, their anti-apoptotic and pro-angiogenic effects. Special emphasis has been given to discussion of the various signal transduction networks triggered by TFFs. It is becoming increasingly clear that these pathways differ depending on the respective TFF.

Trefoil factor family-interacting proteins.

Trefoil factor family (TFF) peptides have many in vivo and in vitro effects on restitution, wound healing, apoptosis, cell motility, adhesion and vectorial ion pumping, amongst others. (125)I-TFF peptides bind to cell membranes with classical saturable ability. It would be surprising if there were not TFF-protein interactions that would explain these actions, but to date no convincing TFF-binding partner has been shown which unambiguously takes part in any of these functions. Nevertheless, several TFF-binding proteins exist, including the small intestinal CRP-ductin (muclin), which binds TFF2, and the recently described gastric foveolar proteins TFIZ1 (TFF1-binding) and blottin (TFF2-binding), any of which may yet interact in novel ways to elicit TFF-mediated events. This review describes the expression and, where known, the functions of such proteins.

Expression of Trefoil Factor Family Members Correlates with Patient Prognosis and Neoangiogenesis

Trefoil factor family (TFF) peptides are thought to contribute to epithelial protection and restitution by virtue of their protease-resistant nature and their strong affinity for mucins. However, they are often overexpressed in tumors, where they seem to be negative prognostic factors, possibly contributing to tumor spread, although the precise mechanisms have not been defined. Tissue sections from 111 patients with curatively resected advanced gastric carcinoma were immunohistochemically stained for TFF2, ITF (TFF3), and CD34. Microvessel density was expressed as number and area of microvessels. Results were correlated with clinicopathological characteristics and patient survival. Forty-nine (44.1%) and 41 (36.9%) tumors were immunohistochemically positive for TFF3 and TFF2, respectively. Among the various clinicopathologic variables, overexpression of TFF3 had a significant correlation with patient age only. In addition, a significantly higher prevalence of positive TFF2 staining was detected in large, diffuse tumors and in tumors with lymph node metastasis. The number of microvessels had a significant correlation with both TFF3 and TFF2 staining, whereas the area of microvessels had a significant correlation only with TFF3 staining. Both TFF3 and TFF2 were independent predictors of a worse disease-free survival. TFF3 had a gender-specific negative survival advantage, with a 91.3% disease-free survival in female patients with TFF3-negative advanced gastric carcinoma. Induction of increased tumor vascularity might be one of the mechanisms by which TFFs confer metastatic phenotype and frequent disease recurrence in gastric carcinomas. Female patients with TFF3-negative advanced gastric carcinoma have comparable survival as that reported for patients with early gastric carcinoma.

Trefoil factor family (TFF) peptides of normal human Vater’s ampulla

Vater's ampulla is of great clinical relevance with regard to the influx of chyme, ascending inflammation, intubation during diagnostic and therapeutic endoscopic maneuvers, therapeutic papillotomy and, especially, the formation of malignancies. Little is known about the distribution of trefoil factor family (TFF) peptides in the ampulla. We have therefore examined TFF peptide distribution in the normal ampulla of Vater and compared it with that in duodenal mucosa and Brunner's glands. Expression and synthesis of TFF peptides in Vater's ampulla and duodenum was investigated by reverse transcription-polymerase chain reaction, Western blot and immunohistochemistry. The samples studied originated from 30 autopsy cases with short postmortem intervals. TFF3 was expressed in the ampulla of Vater. mRNA expression of TFF1 was detected in only approximately 25% of the investigated samples. Western blot revealed the production of TFF3 and immunohistochemistry showed that TFF3 was the product of goblet cells. TFF peptide composition of Vater's ampulla varied in comparison with that in the duodenum regarding TFF2 expression. The ampulla of Vater thus has a unique profile of TFF peptide production, supporting the hypothesis that the ampulla is an autonomous organ. The observed differences in the TFF peptide distribution between the duodenum and Vater's ampulla favour the investigation of TFF peptides as prognostic markers in the classification of ampullary carcinomas.

Expression of human TFF3 in relation to growth of HT-29 cell subpopulations: involvement of PI3-K but not STAT6

The trefoil factor family (TFF) peptides 1 and 2 (TFF1 and 2) are expressed in mucus cells of the stomach, whereas TFF3 is localized in goblet cells of the intestine. In the present study, we aimed to determine whether phosphatidylinositol 3-kinase (PI3-K) or signal transducer and activator of transcription protein 6 (STAT6) is involved in the expression of goblet cell specific markers. TFF3 expression was analyzed by RT-PCR, Northern blot, and radioimmunoassay (RIA) in relation to cell growth in subclones of HT-29 cells including the CL.16E and methotrexate (MTX) cell lines, which both exhibit a phenotype of mucus-secreting intestinal cells. A 30-fold increase in TFF3 mRNA levels and a 10-fold increase in TFF3-cell content were observed between the early proliferative and the late confluency states. The levels of MUC2 and MUC3 mRNA were also increased in the course of the differentiation process. A three to fourfold increase in PI3-K and Akt activities was observed in early post-confluent cells as compared with pre-confluent cells. Exposure of pre- and post-confluent cells to LY294002, a specific PI3-K inhibitor, for 1–4 days profoundly reduced TFF3 and MUC2 expression. A marked reduction in mucin granules content was also observed in LY-treated cells. Inhibition of the mitogen-activated protein (MAP) kinase kinase (MEK) with PD98059 did not modify the course of differentiation of the goblet cell lines. Moreover, stable transfection of HT-29 CL.16E cells with a dominant negative form of STAT6 had no effect on TFF3 induction. Together, these data indicate that PI3-K promotes the expression of TFF3 and MUC2 and that the PI3-K/Akt pathway may play a pivotal role in intestinal goblet cell differentiation.

Frequent trefoil factor 3 (TFF3) overexpression and promoter hypomethylation in mouse and human hepatocellular carcinomas

Expression profiling analysis revealed ectopic high expression of mouse TFF3 in non-tumor liver tissues from the hepatocellular carcinoma (HCC) susceptible PWK/Rbrc strain. TFF3 is a member of the trefoil factor family peptides, which are small secreted proteins regulating mucosal regeneration and repair, and which are overexpressed during inflammatory processes and cancer progression. We, therefore, analyzed the TFF3 expression extensively in mouse and human HCCs. Expression of the mouse TFF3 gene was significantly increased in 6 out of 7 HCCs from a PWK spontaneous tumor model and in all 7 HCCs from an SV40T antigen-induced transgenic MT-D2C57BL/6 model. In humans, 8 of 20 HCCs (40%) had overexpression of TFF3 in both mRNA level and protein level. We then analyzed DNA methylation patterns of the TFF3 promoter region to evaluate expression regulation of promoter methylation. In mouse HCCs, we demonstrated that two CpGs, at positions -992 and +109, were hypomethylated in 13 of 14 mouse HCCs. In human HCCs, hypomethylation at CpG -260 was associated with TFF3 overexpression (p=0.04). These results indicate that TFF3 overexpression may be a critical process in mouse and human hepatocellular carcinogenesis, and the specific promoter CpG hypomethylation may be one of the regulation mechanisms of TFF3 overexpression in HCCs.

Trefoil Factor 2 (Tff2) Deficiency in Murine Digestive Tract Influences the Immune System

Background & 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.

General session II Moderated by Yashihiro Takami: Down‐Regulation of TFF Expression by TNF‐α in Gastric Epithelial Cells

Aim: Trefoil factor family (TFF) peptides are known to facilitate wound healing in gastric mucosa. However, the regulatory mechanisms of gastric TFF expression are not fully understood yet. In this study, we examined the effect of TNF‐α on TFF1 and TFF2 expression in gastric epithelial cells.Methods: MKN45 cells were used. TFF mRNA expression was analyzed by real‐time quantitative RT‐PCR. Promoter sequences of TFF1 gene (−956 to +36) and TFF2 gene (−912 to +24) were inserted into pGL3 vector and reporter gene assays were performed. NF‐κB activity was monitored by using a NF‐κB responsive element‐driven reporter vector.Results: (1) TNF‐α(0.1–30 ng/ml) down‐regulated TFF1 and TFF2 mRNA expression in a dose‐dependent manner. (2) Reporter gene assays also confirmed the down‐regulation of TFF1 and TFF2 gene transcription by TNF‐α. (3) TNF‐α activated NF‐κB. (4) Overexpression of dominant negative IκBα prevented both TNF‐α‐induced NF‐κB activation and TNF‐α‐induced down‐regulation of TFF expression.Conclusions: TNF‐α down‐regulates gastric TFF expression through NF‐κB pathway, suggesting that TFF expression is sensitive to inflammatory stimuli.

Trefoil factor family peptide 3 prevents the development and promotes healing of ischemia-reperfusion injury in weanling rats

Background/Purpose Although the pathogenesis of necrotizing enterocolitis (NEC) is not completely defined, ischemia appears to be one of the most important causative factors. Trefoil factor family peptide 3 (TFF3) is a peptide normally expressed in the small bowel and colon and is involved in the maintenance and repair of mucosal integrity. The authors hypothesized that monomeric (TFF3 Ser 57) and dimeric (TFF3 Cys 57) recombinant TFF3 may prevent the development and accelerate healing of intestinal ischemia-reperfusion injury in weanling rats. Methods Intestinal injury was induced in 18-day-old rats by occlusion of the superior mesenteric vessels for 60 minutes. To examine the protective effect, rats were given 3 μg/g of TFF3 Ser 57 or TFF3 Cys 57 by subcutaneous or enteral administration 30 minutes before the vascular occlusion. To examine the healing effect, rats were given 3 μg/g of TFF3 Ser 57 or TFF3 Cys 57 by subcutaneous or enteral administration 60 minutes after the beginning of reperfusion. Samples from small bowel and colon were collected for morphometric analysis after 3 hours of reperfusion. Mucosal damage was assessed by the Chiu score. Results Both forms of TFF3 reduced the amount of damage when administered before the ischemia. Administration of TFF3 Ser 57 and TFF3 Cys 57 after the beginning of reperfusion significantly increased the villous height and decreased the Chiu score in the small intestine and colon. Conclusions TFF3 Ser 57 monomer and TFF3 Cys 57 dimer prevent the development and promote healing of ischemia-reperfusion injury in weanling rats. There are no differences between the routes of administration of TFF3.

Epidermal growth factor and trefoil factor family 2 synergistically trigger chemotaxis on BEAS-2B cells via different signaling cascades.

Injured areas of the respiratory epithelium are subject to rapid repair by the migration of adjacent epithelial cells, a process termed "restitution". Rapid re-epithelialization is promoted by interactions between migrating cells and the extracellular matrix proteins. Furthermore, epidermal growth factor (EGF) as well as trefoil factor family (TFF) peptides are well known regulators of epithelial restitution due to their motogenic effects. Migration of the human bronchial epithelial cell line BEAS-2B in modified Boyden chambers was used as a model system for airway restitution. EGF or recombinant human TFF2 or TFF3 showed mainly chemotactic activity. The motogenic response was strictly dependent upon a haptotactic substrate, but to different degrees. EGF induced phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, c-Jun-N-terminal kinase, p38, Akt, and p70S6K in BEAS-2B cells. Using specific inhibitors, the signaling cascades responsible for the motogenic response were shown to differ drastically when EGF was compared with TFF2. The motogenic effect of TFF2 was previously demonstrated to depend on ERK1/2 and protein kinase C activation; whereas the EGF-triggered motogenic response was completely independent of ERK1/2 activation but sensitive to the inhibition of phosphoinositide 3-kinase, p38, protein kinase C, or nuclear factor kappaB. However, the motogenic effects of EGF and TFF2 are additive. These data suggest that luminal EGF and TFF peptides can act synergistically in the human respiratory epithelium to enhance rapid repair processes in the course of diseases such as asthma.

Indometacin up-regulates TFF2 expression in gastric epithelial cells.

Trefoil factor family peptides are expressed in gastrointestinal epithelial cells and play a critical role in maintaining mucosal integrity. Although non-steroidal anti-inflammatory drugs (NSAIDs) are important causative agents of gastric mucosal lesions, few data are available about the effect of NSAIDs on trefoil family peptides in gastric mucosa. To examine whether indometacin, a widely used NSAID, affects trefoil factor family expression in gastric epithelial cells. MKN45, a cell line derived from human gastric cancer, was used. TFF1, TFF2, and TFF3 mRNA expression was assessed by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). TFF2 gene transcription was also examined by luciferase reporter gene assay. Relative expression level of TFF1, TFF2, TFF3 mRNA was 616: 12: 1 in unstimulated MKN45 cells. Although indometacin (1-250 micro mol/L) had no significant effect on the expression of TFF1 and TFF3 mRNA, it up-regulated TFF2 mRNA expression in a dose- and time-dependent manner. Luciferase reporter gene assay confirmed the up-regulation of TFF2 gene transcription by indometacin. Indometacin-induced up-regulation of TFF2 expression was not antagonized by externally applied prostaglandin E2. These results suggest that indometacin up-regulates gastric epithelial cell TFF2 expression through a COX-independent mechanism. Since TFF peptides play an important role in gastric mucosal protection, indometacin-induced TFF2 may reduce the degree of gastric mucosal damage induced by indometacin.

Trefoil factor family (TFF) expression in the mouse brain and pituitary: changes in the developing cerebellum

Trefoil factor family (TFF) peptides, besides their prominent expression in mucous epithelia, are also synthesized in the central nervous system. Previously TFF1 expression was observed in mouse brain astrocytes, while oxytocinergic neurons of the hypothalamo-pituitary axis are recognized sites of TFF3 synthesis. Here, the expression of TFF1, TFF2, and TFF3 was systematically studied using reverse transcription-polymerase chain reaction (RT-PCR) analysis of dissected adult mouse brain regions including the pituitary. Additionally, the developmental profile of TFF expression in murine cerebral cortex and cerebellum was monitored. Overall, the expression patterns of the three TFF genes differed. The TFF1 and TFF2 profiles shared some similarities, whereas the TFF3 expression pattern was completely different. TFF1 was nearly uniformly, but weakly expressed in all brain regions tested. The TFF1 and TFF2 expression patterns differed characteristically in the pituitary where abundant TFF2 transcription was detected in the anterior and not the posterior lobe and the expression level in males was higher than in females. In contrast, TFF3 expression was limited to the hippocampus, the temporal cortex, and the cerebellum, the latter being surprisingly the major site of expression. Here, TFF3 mRNA appeared to be restricted mainly to neurons and not glial cells. Cerebellar TFF3 expression is clearly developmentally regulated (maximum at P15), indicating a role for TFF3 during postnatal cerebellar development.