Autoinduction of the trefoil factor 2 (TFF2) promoter requires an upstream cis-acting element

Most benign brain tumors are associated with loss of the Nf2 gene tumor suppressor product schwannomin/merlin. Interactions between schwannomin fragments have given rise to hypotheses of in vivo schwannomin folding and dimerization. Previously, we showed that schwannomin with missense mutations L360P, L535P, and Q538P alters interaction with betaII-spectrin and Hrs. Using yeast two-hybrid tests of interaction, we now show the effects of 11 Nf2 missense mutations on schwannomin self-interaction as well as schwannomin interaction with Hrs isoforms 1 and 2, betaII-spectrin, and p110. Missense mutations L46R and K364I significantly decreased affinity of schwannomin for binding all interacting proteins. The schwannomin L46R mutation may result in a complex conformational change that alters folding and denies betaII-spectrin access to an intact binding site in the C-terminal half of schwannomin. We show that unique inter- and intramolecular interactions occur for schwannomin isoform 2, suggesting that this schwannomin isoform has unique functional properties compared to schwannomin isoform 1.     

Intestinal trefoil factor/TFF3 promotes re-epithelialization of corneal wounds

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study using cornea as a model, we demonstrate the importance of trefoil factor 3 (TFF3, also known as intestinal trefoil factor) in re-epithelialization of wounds. In two different models of corneal wound healing, alkali- and laser-induced corneal wounding, we analyzed the wound healing process in in vivo as well as in combined in vivo/in vitro model in wild type (Tff3(+)(/)(+)) and Tff3-deficient (Tff3(-)(/)(-)) mice. Furthermore, we topically applied different concentrations of recombinant human TFF3 (rTFF3) peptide on the wounded cornea to determine the efficacy of rTFF3 on corneal wound healing. We found that Tff3 peptide is not expressed in intact corneal epithelium, but its expression is extensively up-regulated after epithelial injury. Re-epithelialization of corneal wounds in Tff3(-/-) mice is significantly prolonged in comparison to Tff3(+/+) mice. In addition, exogenous application of rTFF3 to the alkali-induced corneal wounds accelerates significantly in in vivo and in combined in vivo/in vitro model wound healing in Tff3(+/+) and Tff3(-/-) mice. These findings reveal a pivotal role for Tff3 in corneal wound healing mechanism and have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.     

Effects of Trichinella spiralis infection on intestinal pathology in mice lacking interleukin-4 (IL-4) or intestinal trefoil factor (ITF/TFF3)

The nematode Trichinella spiralis induces pathological changes in the small intestine of the host, which are known to be controlled by immune and inflammatory mediators. The detail of this control has still to be completely understood. Mice deficient in interleukin 4 (IL-4) or in intestinal trefoil factor/trefoil family factor 3 (ITF/TFF3) were infected with T. spiralis and the resultant changes in the intestinal mucosa followed by quantifying numbers of mucosal mast cells, goblet cells, Paneth cells and by monitoring structural changes in villus length and crypt depth. Mice lacking IL-4 were unable to mount a normal protective response to infection, such that worm survival was increased. These mice failed to mount a mucosal mast cell response, but did make goblet cell and Paneth cell responses comparable to normal controls. Mice lacking ITF/TFF3 similarly made normal levels of goblet cell and Paneth cell responses. They also underwent profound changes in mucosal architecture, with marked villus atrophy and crypt hyperplasia. These results are discussed in relation to known patterns of T cell and cytokine control of protective immunity to T. spiralis. They suggest that increased numbers of goblet cell and Paneth cell are not, by themselves, required for protective immunity. ITF/TFF3 appears not to influence cellular responses and does not alter parasite-induced pathological changes in the small intestine.     

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.     

Effect of ectopic expression of rat trefoil factor family 3 (intestinal trefoil factor) in the jejunum of transgenic mice

To further examine the function of the trefoil factor family (TFF), the expression of which is up-regulated at sites of injury, we have produced transgenic mice that chronically express rat TFF3 within the jejunum (using a rat fatty acid-binding protein promoter). The expression of rat TFF3 was limited to the villi of the jejunum and had no effect on base-line morphology. Rat TFF3 expression did result, however, in a reduced sensitivity to indomethacin (85 mg/kg subcutaneously), which only caused a 29% reduction in villus height in transgenics versus 51% reduction in controls (p < 0.01). Indomethacin increased initial intestinal epithelial cell proliferation and migration, but the presence of rat TFF3 caused no additional change in proliferation (bromodeoxyuridine), cell migration ([(3)H]thymidine and bromodeoxyuridine), apoptosis (terminal deoxyuridine nucleotidyl nick end labeling), or E-cadherin immunostaining. In vitro studies following changes in resistance of intestinal strips in Ussing chambers (voltage-clamp technique) showed increased base-line resistance in the rat TFF3-expressing region (326 +/- 60 versus 195 +/- 48 ohm.cm(2) in controls, p < 0.05) and reduced the fall in resistance following HCl exposure by about 40% (p < 0.01). Overexpression of TFF3 stabilizes the mucosa against noxious agents, supporting its role in mucosal protection/repair. It may therefore provide a novel approach to the prevention and/or treatment of intestinal ulceration.     

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.     

Vangl1 protein acts as a downstream effector of intestinal trefoil factor (ITF)/TFF3 signaling and regulates wound healing of intestinal epithelium

The intestinal trefoil factor (ITF/TFF3) protects intestinal epithelia from a range of insults and contributes to mucosal repair. However, the signaling events that mediate healing responses are only partially understood. To identify ITF signaling pathways, proteins that were Ser/Thr phosphorylated in response to ITF stimulation were immunoprecipitated from human colon carcinoma cell lines and identified by mass spectrometry. We demonstrated that Van Gogh-like protein 1 (also designated Vang-like 1 or Vangl1), a protein with four transmembrane domains, was Ser/Thr phosphorylated in response to ITF stimulation. Vangl1 was present in normal human colon and all intestinal epithelial cell lines (IEC) tested. In transfected IEC, FLAG-Vangl1 was mostly present in the Nonidet P-40 soluble fraction as detected by Western blotting, corresponding to the localization of endogenous protein in cytoplasmic vesicular structures by confocal microscopy with rabbit polyclonal anti-human Vangl1 antibody (alpha-Vangl1). Vangl1 cell membrane association increased with differentiation, as demonstrated by co-localization with E-cadherin in differentiated IEC. Increased Vangl1 phosphorylation after stimulation with ITF corresponded to decreased cell membrane association with E-cadherin. Functionally, Vangl1 overexpression enhanced ITF unstimulated and stimulated wound closure of IEC, whereas siRNA directed against Vangl1 inhibited the migratory response to ITF. Vangl1 protein may serve as an effector mediating the ITF healing response of the intestinal mucosa.     

Down-regulation by nuclear factor kappaB of human 25-hydroxyvitamin D3 1alpha-hydroxylase promoter

1,25-(OH)(2) vitamin D(3) is important for calcium homeostasis and cell differentiation. The key enzyme for the activation of liver-derived 25(OH) vitamin D(3) is 25-hydroxyvitamin D(3) 1alpha-hydroxylase. It is expressed mainly in the kidney but also in peripheral tissues. A 1413-bp fragment of the 1alpha-hydroxylase promoter was cloned into luciferase vectors pGL2basic and pGL3basic. Sequence analyses revealed four base exchanges and three base deletions compared with the published sequence which were identically found in five control persons. In silico promoter analyses revealed 17 putative nuclear factor (NF)kappaB sites, 10 of which were found to bind NFkappaB in EMSA experiments. Cotransfection of NFkappaB p50 and p65 subunits resulted in dramatic reduction of the promoter activity of the full-length construct as well as a series of 5'-deletion constructs. Deletion of the farmost 3'-situated NFkappaB-responsive element almost abolished NFkappaB responsiveness. Treatment of human embryonic kidney 293 cells with sulfasalazine, a NFkappaB inhibitor, resulted in enhanced 1alpha-hydroxylase mRNA production. Down-regulation of 1alpha-hydroxylase promoter through NFkappaB signaling may contribute to the pathogenesis of inflammation-associated osteopenia/osteoporosis.     

Anti-sense trefoil factor family-3 (intestinal trefoil factor) inhibits cell growth and induces chemosensitivity to adriamycin in human gastric cancer cells

Intestinal trefoil factor (ITF), which is normally absent in gastric mucosa, is over-expressed in gastric cancer. However, the functional significance of ITF in gastric cancer is unknown. We examined the effects of blocking ITF expression on the growth of gastric cancer cells and their responses to chemotherapeutic agents. Anti-sense ITF cDNA was cloned into mammalian expression vector pcDNA3 and was transfected into an ITF-expressing gastric cancer cell line SNU-1. We assessed the doubling time and anchorage dependent growth of the transfected cells using growth curve and soft agar assay respectively. Cell cycle analysis and apoptosis were determined by flow cytometry and cell death ELISA. The response to chemotherapeutic agents after transfecting anti-sense ITF was also examined. Anti-sense ITF transfectant (3A-5) had a significantly longer doubling time as compared to control cells which were transfected with empty vector (32.4 hr vs 26.9 hr, p < 0.05). In the soft agar assay, 3A-5 formed fewer colonies than control (3.5 colonies vs 23.5 colonies, p < 0.05). Although there was no significant difference in the cell cycle distribution between 3A-5 and control, anti-sense ITF resulted in marked increase in adriamycin-induced apoptosis. Our results demonstrated that blocking the expression of ITF inhibits growth of gastric cancer cells and enhances the response to chemotherapy.     

Post-transcriptional control of CCAAT/enhancer-binding protein beta (C/EBPbeta) expression: formation of a nuclear HuR-C/EBPbeta mRNA complex determines the amount of message reaching the cytosol

In 3T3-L1 cells, HuR is constitutively expressed and prior to induction of differentiation localized predominantly to the nucleus. Within minutes of induction of differentiation, nuclear HuR binds to its target ligand mRNAs, and the complexes appear to move to the cytosol. One ligand mRNA is the CCAAT/enhancer-binding protein beta (C/EBPbeta) message. To examine the function and importance of the HuR-C/EBPbeta interaction, retroviral expression constructs were created in which the HuR binding site was altered by deletion (betadel) or deletion and substitution (betad/s). Expression of these constructs in murine embryonic fibroblasts resulted in significant adipose conversion relative to those cells expressing wild type C/EBPbeta. C/EBPbeta protein content was increased markedly in both betadel and betad/s, which correlated with the acquisition of the adipocyte phenotype. Analysis of the betad/s cell line demonstrated a robust expression of C/EBPalpha coincident with peroxisome proliferator-activated receptor gamma expression. Total C/EBPbeta mRNA accumulation indicated no difference between cells harboring either the wild type C/EBPbeta cDNA or betad/s construct. However, cytosolic C/EBPbeta mRNA in the cells expressing the betad/s construct was maintained at levels between 2- and 7-fold greater than in the cells expressing the wild type construct. Alteration in mRNA half-life was not responsible for the increased accumulation. Mechanistically, these data suggest that HuR binding results in nuclear retention of the C/EBPbeta mRNA and is consistent with HuR control, at least in part, of mRNA processing.