Intestinal trefoil factor promotes invasion in non-tumorigenic Rat-2 fibroblast cell

Intestinal trefoil factor (TFF3) is essential in regulating cell migration and maintaining mucosal integrity in gastrointestinal tract. We previously showed that TFF3 was overexpressed in gastric carcinoma. Whether TFF3 possesses malignant potential is not fully elucidated. We sought to investigate the effects of inducting TFF3 expression in a non-malignant rat fibroblast cell line (Rat-2) on the cell proliferation, invasion and the genes regulating cell invasion. Invasiveness and proliferation of transfected Rat-2 cell line were assessed using in vitro invasion chamber assay and colorimetric MTS assay. Differential mRNA expressions of invasion-related genes, namely, metalloproteinases (MMP-9), tissue inhibitors of metalloproteinases (TIMP-1), beta-catenin and E-cadherin, were determined by quantitative real-time polymerase chain reaction (PCR). We showed that TFF3 did not inhibit the proliferation of Rat-2 cells. We also demonstrated that transfection of TFF3 significantly promoted invasion of Rat-2 cells by 1.4- to 2.2-folds. There was an upregulation of beta-catenin (13.1-23.0%) and MMP-9 (43.4-92.2%) mRNA expression levels, and downregulation of E-cadherin (25.6-33.8%) and TIMP-1 (31.5-37.8%) in TFF3-transfected cells compared to controls during 48-h incubation. Our results suggested that TFF3 possesses malignant potential through promotion of cell invasiveness and alteration of invasion-related genes.     

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.     

Trefoil peptide expression and goblet cell number in rat intestine: effects of KGF and fasting-refeeding

The trefoil factor family peptides TFF1, TFF2, and TFF3 are important for gut mucosal protection and restitution. Keratinocyte growth factor (KGF) stimulates proliferation and differentiation of epithelial cells with potent effects on goblet cells. To investigate interactions between food intake and KGF, rats were fed ad libitum (control), fasted for 72 h, or fasted for 72 h and then refed for 72 h with or without KGF (3 mg. kg(-1). day(-1)). With fasting, goblet cell number in duodenum increased, TFF3 mRNA in duodenum and jejunum decreased, and TFF3 protein did not change or increased. KGF during fasting stimulated colonic growth, normalized TFF3 mRNA in duodenum and jejunum, and broadly upregulated gut goblet cell number and TFF3 protein expression. With fasting-refeeding, KGF increased small bowel and colonic mucosal growth, goblet cell number, and TFF3 protein but had variable effects on TFF3 mRNA. KGF induced TFF2 mRNA and protein in duodenum and jejunum with both nutritional regimens. We conclude that nutrient availability modifies rat intestinal goblet cell number, TFF3 mRNA, and the gut-trophic effects of KGF in a region-specific manner. KGF enhances TFF2 expression in proximal small bowel and increases goblet cell number and TFF3 protein content throughout the intestine independent of food intake.     

Secretion of the trefoil factor TFF3 from the isolated vascularly perfused rat colon

The trefoil factor TFF3 is a peptide predominantly produced by mucus-secreting cells in the small and large intestines. It has been implicated in intestinal protection and repair. The mechanisms that govern TFF3 secretion are poorly understood. The aim of this study was, therefore, to evaluate the influence of neurotransmitters, hormonal peptides and mediators of inflammation on the release of TFF3. For this purpose, an isolated vascularly perfused rat colon preparation was used. After a bolus administration of 1 ml isotonic saline into the lumen, TFF3 secretion was induced by a 30-min intra-arterial infusion of the compounds to be tested. TFF3 was evaluated in the luminal effluent using a newly developed radioimmunoassay. TFF3 was barely detected in crude luminal samples. In contrast, dithiothreitol (DTT) treatment of the effluent revealed TFF3 immunoreactivity, which amounted to about 0.3 pmol min(-1) cm(-1) in the basal state. Gel chromatography of DTT-treated luminal samples revealed a single peak that co-eluted with the monomeric form of TFF3. TFF3 was not detected in the portal effluent. Bethanechol (10(-6)-10(-4) M), vasoactive intestinal peptide (VIP, 10(-8)-10(-7) M) or bombesin (10(-8)-10(-7) M) induced a dose-dependent release of TFF3. In contrast, substance P evoked a modest release of TFF3, whereas calcitonin gene-related peptide (CGRP), somatostatin, neurotensin or peptide YY (PYY) did not modify TFF3 secretion. The degranulator compound bromolasalocid, 16,16-dimethyl PGE2 (dmPGE2) or interleukin-1-beta (IL-1-beta) also evoked a marked release of TFF3. In conclusion, TFF3 in the colonic effluent is present in a complex. This association presumably involves a disulfide bond. Additionally, the present results suggest a role for enteric nervous system and resident immune cells in mediation of colonic TFF3 secretion.     

The protective effect of trefoil factor 3 on the intestinal tight junction barrier is mediated by toll-like receptor 2 via a PI3K/Akt dependent mechanism

Trefoil factor peptides are highly conserved secreted molecules characterized by heat and enzymatic digestion resistance. Intestinal trefoil factor 3 (TFF3) protects and repairs the gastrointestinal mucosa and restores normal intestinal permeability, which is dependent on the integrity of the tight junction (TJ) barrier and the TJ associated proteins claudin-1, zona occludens-1 (ZO-1) and occludin. Despite the important role of intestinal barrier dysfunction in the pathogenesis of inflammatory bowel diseases, the underlying mechanisms and associated molecules remain unclear. In the present study, we show that TFF3 and toll-like receptor 2 (TLR2) are functionally linked and modulate intestinal epithelial permeability via a mechanism that involves the PI3K/Akt pathway. We used the Caco-2 cell model to show that TLR2 and TFF3 inhibit the IL-1β induced increase in permeability and release of proinflammatory cytokines, and that this effect is mediated by activation of PI3K/Akt signaling. TLR2 silencing downregulated the expression of TFF3 and overexpression of TLR2 and TFF3 increased the levels of phospho-Akt. TFF3 overexpression significantly upregulated the expression of ZO-1, occludin and claudin-1 and this effect was abrogated by inhibition of the PI3K/Akt pathway. Taken together, our results indicate that TLR2 signaling selectively enhances intestinal TJ barrier integrity through a mechanism involving TFF3 and the activation of the PI3K/Akt pathway.     

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.     

Trefoil factor 3 stimulates human and rodent pancreatic islet beta-cell replication with retention of function

Both major forms of diabetes involve a decline in beta-cell mass, mediated by autoimmune destruction of insulin-producing cells in type 1 diabetes and by increased rates of apoptosis secondary to metabolic stress in type 2 diabetes. Methods for controlled expansion of beta-cell mass are currently not available but would have great potential utility for treatment of these diseases. In the current study, we demonstrate that overexpression of trefoil factor 3 (TFF3) in rat pancreatic islets results in a 4- to 5-fold increase in [(3)H]thymidine incorporation, with full retention of glucose-stimulated insulin secretion. This increase was almost exclusively due to stimulation of beta-cell replication, as demonstrated by studies of bromodeoxyuridine incorporation and co-immunofluorescence analysis with anti-bromodeoxyuridine and antiinsulin or antiglucagon antibodies. The proliferative effect of TFF3 required the presence of serum or 0.5 ng/ml epidermal growth factor. The ability of TFF3 overexpression to stimulate proliferation of rat islets in serum was abolished by the addition of epidermal growth factor receptor antagonist AG1478. Furthermore, TFF3-induced increases in [3H]thymidine incorporation in rat islets cultured in serum was blocked by overexpression of a dominant-negative Akt protein or treatment with triciribine, an Akt inhibitor. Finally, overexpression of TFF3 also caused a doubling of [3H]thymidine incorporation in human islets. In summary, our findings reveal a novel TFF3-mediated pathway for stimulation of beta-cell replication that could ultimately be exploited for expansion or preservation of islet beta-cell mass.     

Persistent trefoil factor 1 expression imprinted on mouse vaginal epithelium by neonatal estrogenization

Exposure of female mice to estrogenic substances during the neonatal period induces developmental defects in the reproductive tract such as estrogen-independent persistent proliferation of the vaginal epithelium, which often leads to carcinogenesis in adulthood. In this study, several estrogen-regulated genes have been identified in the neonatal mouse vagina by DNA microarray hybridization analysis. Among the genes up-regulated in the developing vagina by a high dose of estrogen, trefoil factor 1 (TFF1), a mucin-associated gastrointestinal growth factor, showed a unique expression pattern in accordance with the irreversible changes induced by neonatal estrogenization in the vagina. Vaginal expression of TFF1 mRNA was markedly increased by estrogen in neonatal mice but not in adults, and pronouncedly intensified expression of the gastrointestinal gene was observed in the vagina of neonatally estrogenized mice even at adulthood. The specific localization of TFF1 protein in the epithelium of neonatally estrogenized vagina was confirmed by immunohistochemistry. Moreover, without any obvious alteration in the expression of gel-forming mucin genes, the lumen of the neonatally estrogenized vagina became filled with periodic-acid-Schiff-stained mucinous gel, which was possibly caused by the overexpression of TFF1. Thus, estrogen acts directly on the developing vagina in the permanent induction of TFF1 gene expression, and the gene induction does not appear to be related to hypermethylation of the cis-promoter of the TFF1 gene. TFF1 may be a useful marker for developmental estrogenization syndrome of the mouse vagina. This work was supported by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports, and Culture, Japan, and grants from the University of Tsukuba to M. M.     

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.     

The effect of atrial natriuretic peptide on intestinal electrolyte transport.

The effect of atrial natriuretic peptide (ANP) on rat small intestinal electrolyte transport was examined. In vivo, intravenous administration of rat ANP(99-126) induced diuresis and natriuresis in conjunction with a significant decrease in intestinal water (basal, 37.1 +/- 5.7 versus ANP 28.5 +/- 6.0 microliters/cm per 20 min, P less than 0.05) and Na+ (4.0 +/- 0.7 versus 2.8 +/- 0.9 mumol/cm per 20 min, P less than 0.05) absorption (n = 9). In vitro, in Ussing chambers, in both jejunum and ileum, addition of 1.0 microM ANP to short circuited, stripped tissue produced a maximal increase in short circuit current and stimulated net Cl- secretion due to a significant increase in the unidirectional serosal to mucosal flux (JCl-sm: jejunum 17.4 +/- 1.3 versus 19.8 +/- 1.3 microEq/cm2 per h, P less than 0.01, n = 6; ileum 13.4 +/- 0.5 versus 17.2 +/- 0.6, P less than 0.01, n = 6) which was inhibited by the calcium channel antagonist verapamil (82 +/- 26%, P less than 0.05) and by the 5-HT2 receptor antagonist cinanserin (72 +/- 44%, P less than 0.05). Guanylate cyclase activity was stimulated by ANP in intact epithelium, but not in isolated crypt and villus enterocytes.     

Promoter polymorphisms in trefoil factor 2 and trefoil factor 3 genes and susceptibility to gastric cancer and atrophic gastritis among Chinese population

The polymorphisms in trefoil factor (TFF) gene family that protect gastrointestinal epithelium might influence individual vulnerability to gastric cancer (GC) and atrophic gastritis. We used the Sequenom MassARRAY platform to identify the genotypes of TFF2 rs3814896 and TFF3 rs9981660 polymorphisms in 478 GC patients, 652 atrophic gastritis patients, and 724 controls. For the TFF2 rs3814896 polymorphism, in the subgroup aged ≤ 50 years, we found that AG + GG genotypes were associated with a 0.746-fold decreased risk of atrophic gastritis [p = 0.023, 95% confidence interval (CI) = 0.580–0.960], a 0.626-fold decreased risk of GC (p = 0.005, 95% CI = 0.451–0.868), and a 0.663-fold decreased risk of diffuse-type GC (p = 0.034, 95% CI = 0.452–0.970) compared with the common AA genotype. For the TFF3 rs9981660 polymorphism, in the male subgroup, individuals with variant AG + AA genotype were associated with a 0.761-fold decreased risk of diffuse-type GC compared with the common GG genotype (p = 0.043, 95% CI = 0.584–0.992). Additionally, we found that in subjects aged ≤ 50 years compared with common AA genotype, TFF2 rs3814896 AG + GG genotypes were associated with increased TFF2 mRNA levels in the total gastric cancer specimens and in the diffuse-type gastric cancer specimens; and in males aged ≤ 50 years compared with common GG genotype, TFF3 rs9981660 AA + AG genotypes were associated with TFF3 mRNA levels in diffuse-type gastric cancer tissues and their corresponding non-cancerous tissues. To our knowledge, this is the first report of an association between the TFF2 rs3814896 AG + GG genotypes and decreased risks of GC, diffuse-type GC, and atrophic gastritis in younger people aged ≤ 50 years, and an association between TFF3 rs9981660 AG + AA genotype and decreased risk of diffuse-type GC in men. Moreover, we found that TFF2 rs3814896 AG + GG genotypes in people aged ≤ 50 years and TFF3 rs9981660 AG + AA genotypes in younger males with diffuse-type GC were associated with higher levels of TFF2 and TFF3 mRNA respectively.     

Dura mater biology: autocrine and paracrine effects of fibroblast growth factor 2

The dura mater, the outermost layer of the meninges, is thought to be essential for calvarial morphogenesis, postnatal suture fusion, and osseous repair of calvarial defects. Despite numerous studies illustrating the fundamental role of the dura mater, there is little information about the autocrine and paracrine mechanisms regulating dural cell biology during calvarial ossification. Previous work conducted in the authors' laboratory demonstrated that non-suture-associated dural cells from 6-day-old rat pups expressed high levels of fibroblast growth factor 2 (FGF-2), whereas dural cells from 60-day-old adult rats expressed very little FGF-2. Because young mammals can successfully heal large calvarial defects, the authors sought to investigate the autocrine and/or paracrine effects of FGF-2 on the proliferation, gene expression, and alkaline phosphatase production of dural cells.Cultures of non-suture-associated dural cells were established from 6-day-old Sprague-Dawley rat pups and then stimulated with recombinant human FGF-2 (rhFGF-2; 10 ng/ml). Dural cells stimulated with rhFGF-2 proliferated significantly faster than untreated dural cells at 24 hours (2.1 x 10(5) +/- 3.2 x 10(4) versus 1.1 x 10(5) +/- 1.8 x 10(4), p < or = 0.001) and 48 hours (2.3 x 10(5) +/- 4.2 x 10(4) versus 1.2 x 10(5) +/- 1.3 x 10(4), p < or = 0.001). Moreover, dural cells stimulated with rhFGF-2 expressed 7-fold more proliferating cell nuclear antigen than did control cultures. Treatment with rhFGF-2 increased dural cell expression of genes important for skeletal repair: FGF-2 (7-fold), transforming growth factor beta 1 (3-fold), transforming growth factor beta 3 (4-fold), and type I collagen (4-fold). Furthermore, rhFGF-2 increased dural cell expression of osteopontin (2-fold), a "late" marker of osteoblastic differentiation. Interestingly, dural cell alkaline phosphatase activity, an "earlier" marker of osteoblast differentiation, was significantly decreased by treatment with rhFGF-2 compared with control cultures at 24 hours (0.005 +/- 0.001 versus 0.01 +/- 0.003, p < or = 0.01) and 48 hours (0.004 +/- 0.0009 versus 0.01 +/- 0.0009). Together these data provide insight into the autocrine and paracrine effects of FGF-2 on the biology of the dura mater.     

Bm-TFF2, a toad trefoil factor, promotes cell migration, survival and wound healing

Toad skin is naked and continually confronted by various injurious factors. Constant skin renewal and repairs occur frequently. However, the mechanisms of the renewal and repair have not clearly elucidated. In our previous work, a trefoil factor (TFF), Bm-TFF2, has been purified from the Bombina maxima skin and characterized as a platelet agonist. The mRNA of TFFs in toad skin was up-regulated greatly during the metamorphosis, indicating a pivotal role of TFFs in amphibian skin. Here, we presented the effects of Bm-TFF2 on the cell migration, apoptosis and proliferation. Bm-TFF2 bound to epithelial cells and showed strong cell motility activity. At the concentrations of 1-100 nM, Bm-TFF2-induced migration of human epithelial AGS and HT-29 cells, and rat intestinal epithelial IEC-6 cell lines. The in vitro wound healing assay also verified the activity of Bm-TFF2. Bm-TFF2 could also inhibit cell apoptosis induced by ceramide and sodium butyrate. The cell migration-promoting activity was abolished by MEK1 inhibitors, U0126 and PD98059, suggesting that ERK1/2 activation is crucial for Bm-TFF2 to stimulate cell migration. Taken together, Bm-TFF2 promoted wound healing by stimulating cell migration via MAPK pathway and preventing cell apoptosis. The potent biological activity of Bm-TFF2 makes it a useful molecular tool for further studies of structure-function relationship of the related human TFFs.     

The therapeutic effect of recombinant human trefoil factor 3 on hypoxia-induced necrotizing enterocolitis in immature rat

Trefoil peptides are a new class of regulatory peptides involved in mucosal protection and repair in the gastrointestinal tract. Among them, trefoil factor 3 (TFF3) (intestinal trefoil factor) is known to be cytoprotective in the gut. The aim of this study was to determine the effect of recombinant human trefoil factor 3 (rhTFF3) on hypoxia-induced necrotizing enterocolitis (NEC) in immature rats. In the present study, thirty-two 1-day-old Wistar rat pups were randomly divided into four groups. Group 1 served as nonhypoxic controls. Group 2 rats were subjected to hypoxia reoxygenation (H/O) and then were returned to their mothers. Groups 3 and 4 rats were subjected to H/O, were returned to their mothers, and were treated with rhTFF3 intraperitoneally (0.5 mg) and subcutaneously (0.2 mg), respectively, for the next 3 days. All animals were killed on day 4, and intestine specimens were obtained to determine the histological changes, tissue level of interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), malondialdehyde (MDA), prostaglandin E2 (PGE2), tromboxane B2 (TXB2), and nitric oxide (NO). In addition, the effects of rhTFF3 on abundance of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) were also investigated. In neonatal NEC (group 2), necrosis of villi and crypts and, in some cases, transmural necrosis was observed under light microscopy. Tissue level of interleukin-8, tumor necrosis factor-alpha, malondialdehyde, prostaglandin E2, tromboxane B2, and nitric oxide were significantly higher than group 1. In addition, abundance of inducible nitric oxide synthase and cyclooxygenase 2 was markedly increased. In groups 3 and 4, only very slight intestinal injury was observed. The tissue level of interleukin-8, tumor necrosis factor-alpha, malondialdehyde, prostaglandin E2, tromboxane B2, and nitric oxide were significantly decreased in comparison to the group 2. Meanwhile, the abundance of inducible nitric oxide synthase and cyclooxygenase 2 was also marked decreased in comparison to group 2. The current study suggests a therapeutic role of TFF3 in an experimental model of NEC. Our findings may open a new insight into the treatment of NEC in newborns.     

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.