Distinct pathways of cell migration and antiapoptotic response to epithelial injury: structure-function analysis of human intestinal trefoil factor

The trefoil peptide intestinal trefoil factor (ITF) plays a critical role in the protection of colonic mucosa and is essential to restitution after epithelial damage. These functional properties are accomplished through coordinated promotion of cell migration and inhibition of apoptosis. ITF contains a unique three-looped trefoil motif formed by intrachain disulfide bonds among six conserved cysteine residues, which is thought to contribute to its marked protease resistance. ITF also has a seventh cysteine residue, which permits homodimer formation. A series of cysteine-to-serine substitutions and a C-terminally truncated ITF were made by PCR site-directed mutagenesis. Any alteration of the trefoil motif or truncation resulted in loss of protease resistance. However, neither an intact trefoil domain nor dimerization was required to promote cell migration. This pro-restitution activity correlated with the ability of the ITF mutants to activate mitogen-activated protein (MAP) kinase independent of phosphorylation of the epidermal growth factor (EGF) receptor. In contrast, only intact ITF retained both phosphatidylinositol 3-kinase and the EGF receptor-dependent antiapoptotic effect in HCT116 and IEC-6 cells. The inability to block apoptosis correlated with a loss of trefoil peptide-induced transactivation of the EGF receptor or Akt kinase in HT-29 cells. In addition to defining structural requirements for the functional properties of ITF, these findings demonstrate that distinct intracellular signaling pathways mediate the effects of ITF on cell migration and apoptosis.     

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.     

Trefoil peptides promote restitution of wounded corneal epithelial cells

The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.     

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.     

Alterations in the composition of the supramucosal defense barrier in relation to disease severity of ulcerative colitis.

Mucin glycoproteins and trefoil peptides play an important role in protection and repair of the gastrointestinal epithelium. This study investigates alterations in mucin and trefoil peptide gene expression and product localization in ulcerative colitis (UC). Product localization and message expression of mucin MUC1 to 6 and trefoil peptide TFF1 to 3 genes was analyzed in rectosigmoid tissue from a cohort of patients with active UC and compared with that of normal colorectal mucosa. MUC1 expression was upregulated in severe UC at the site of rupture of crypt abscesses. Reduction in MUC2 expression occurred in UC adjacent to ulceration. No alteration in MUC3 or MUC4 gene expression was detectable in UC compared with normal colorectal mucosa. No ectopic expression of MUC5AC, MUC5B, or MUC6 was identified in UC. Ectopic TFF1 expression was identified in tissues eliciting histological features of severe disease. Decreased TFF3 localization was demonstrated in UC tissues, but no TFF2 expression was detected in any colorectal specimens. Subtle alterations in composition of the supramucosal defense barrier exist in UC and vary in relation to clinical severity of disease. There is upregulation in mucin MUC1 at crypt abscesses and neo-expression of TFF1 trefoil peptide in severe disease.     

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.     

人小肠三叶因子在毕赤酵母中克隆表达及对肠粘膜修复作用的研究

为了研究人肠三叶因子(hITF)对肠粘膜的保护作用,利用RT-PCR从肠粘膜中扩增出hITF基因片段,与诱导分泌型毕赤酵母载体pPIC9连接构建了重组质粒pPIC9hITF,重组质粒转化至宿主菌GS115,经过PCR鉴定和转化子发酵筛选,得到一个重组毕赤酵母高产菌株GS115/pPIC9hITF。在5L发酵罐中用基本盐培养基培养重组菌株,添加甲醇诱导表达hITF,离心收集的上清液通过离心交换层析纯化得到hITF。质谱鉴定结果表明纯化的hITF与天然提取产品在N端序列上完全相同。细胞实验和动物实验结果表明重组hITF能够促进细胞迁移,并可以保护肠粘膜免受有害因子的侵袭,保持了较好的生物学活性。     

Intestinal trefoil factor induces decay-accelerating factor expression and enhances the protective activities against complement activation in intestinal epithelial cells

Mucosal damage induces a massive influx of serum complement components into the lumen. The epithelium produces a number of factors that can potentially ameliorate injury including intestinal trefoil factor (ITF), a small protease-resistant peptide produced and secreted onto the mucosal surface by goblet cells, and decay-accelerating factor (DAF), a protein produced by columnar epithelium which protects the host tissue from autologous complement injury. However, coordination of these intrinsic defensive products has not been delineated. DAF protein and mRNA expression were evaluated by immunoblotting and Northern blotting, respectively. NF-kappaB-DNA binding activity and DAF promoter activity were assessed by an electrophoretic gel mobility shift assay and a reporter gene luciferase assay, respectively. ITF induced a dose- and time-dependent increase in DAF protein and mRNA expression in human (HT-29 and T84) and rat (IEC-6) intestinal epithelial cells. In differentiated T84 cells grown on cell culture inserts, basolateral stimulation with ITF strongly enhanced DAF expression, but apical stimulation had no effects. The C3 deposition induced by complement activation was significantly blocked by the treatment with ITF. In HT-29 cells, ITF increased the stability of DAF mRNA. ITF also enhanced the promoter activity of the DAF gene via NF-kappaB motif and induced activation of NF-kappaB-DNA binding activity. ITF promotes protection of epithelial cells from complement activation via up-regulation of DAF expression, contributing to a robust mucosal defense.     

ITF1697, a stable Lys-Pro-containing peptide, inhibits weibel-palade body exocytosis induced by ischemia/reperfusion and pressure elevation

A number of Lys-Pro-containing short peptides have been described as possessing a variety of biological activities in vitro. Because of limited metabolic stability, however, their efficacy in vivo is uncertain. To exploit the pharmacological potential of Lys-Pro-containing short peptides, we synthesized a series of chemically modified forms of these peptides. One of them, ITF1697 (Gly-(Nalpha-Et)Lys-Pro-Arg) was stable in vivo and particularly efficacious in experimental models of disseminated endotoxemia and of cardiovascular disorders. Using intravital fluorescence microscopy, we studied the peptide cellular and molecular basis of protection in the Syrian hamster cheek pouch microcirculation subjected to ischemia/reperfusion (I/R) and in pressure elevation-induced proinflammatory responses in isolated Sprague-Dawley rat lungs. Continuous intravenous infusion of ITF1697 at 0.1 to 100 mug/kg/min nearly completely protected the cheek pouch microcirculation from I/R injury as measured by decreased vascular permeability and increased capillary perfusion. Adhesion of leukocytes and platelets to blood vessels was strongly inhibited by the peptide. ITF1697 exerted its activity at the early stages of endothelial activation and inhibited P-selectin and von Willebrand factor secretion. Further mechanistic studies in the rat lung preparation revealed that the peptide inhibited the intracellular Ca(2+)-dependent fusion of Weibel-Palade bodies with the plasma membrane. The ability of ITF1697 to inhibit the early functions of activated endothelial cells, such as the exocytosis of Weibel-Palade bodies, represents a novel and promising pharmacological tool in model of pathologies of a variety of microvascular disorders.     

小肠三叶因子在乳酸菌中的表达

实验目的是在乳酸菌中表达小肠三叶因子(ITF) ,并建立兔子胃溃疡模型,口服观察ITF对胃黏膜损伤的再生作用。在实验中利用了分子克隆技术构建携带ITF基因的重组原核表达质粒 pNICE:sec ITF,将重组质粒转化乳酸菌 NZ9000株 ,筛选鉴定阳性菌落,用nisin诱导表达,表达ITF蛋白通过Tricine SDS-PAGE和 Western blot进行鉴定。将重约2㎏的新西兰成年兔分为对照组,预防组,治疗组,用盐酸诱导胃溃疡模型,预防组在模型建立前用携带 pNICE:sec-ITF的乳酸菌灌胃,对照组,治疗组,在溃疡模型建立后,分别用 PBS、携带pNICE:sec-ITF的乳酸菌灌胃。通过溃疡级别及损伤指数的确定携带 pNICE:sec-ITF的乳酸菌灌胃后对胃黏膜损伤再生的作用。实验成功扩增ITF基因并构建了重组原核表达质粒 pNICE:sec-ITF,转化乳酸菌 NZ9000后经 nisin诱导可表达 Mr约 6.0kDa的重组蛋白 ,表达量约占菌体总蛋白量的 5%。动物实验的预防组和治疗组显示在盐酸诱导胃溃疡模型前和后用携带pNICE:sec-ITF的乳酸菌灌胃,能够促进溃疡黏膜的再生。这对新型的基因工程药物的研究开发具有一定的理论意义,为乳酸菌作为药物递送载体的研究和开发打下一定的实验基础。     

Cloning of contiguous genomic fragments from human chromosome 21 harbouring three trefoil peptide genes

A group of small peptides with a typical cysteine-rich domain (termed trefoil motif or P-domain) is abundantly expressed at mucosal surfaces of specific normal and neoplastic tissues. Their association with the maintenance of surface integrity was suggested. The first known human trefoil peptide (pS2) was isolated from breast cancer cells (MCF7). Its oestrogen-inducible gene, and the human homologue to the porcine spasmolytic peptide gene (hSP/SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be localised at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene were described recently. By using suitable oligonucleotide primers and PCR and isolating large (110–250 kb) genomic recombinants cloned in the bacterial artificial chromosome (BAC) system, we present a genomic region from chromosome band 21q22.3 cloned in contiguous sequences and encoding all three members of human P-domain/trefoil peptides proving a physical linkage of all three trefoil peptide genes. Such genomic sequences will provide useful experimental material for analysis of gene regulation, for gene modification experiments and for establishing transgenic cells or animals. Received: 2 January 1996 / Revised: 4 March 1996     

Human colorectal tumours in short-term organ culture A stathmokinetic study

Abstract. Short-term organ culture, using a technique to preserve epithelial/stromal interaction and metabolism, is a useful technique for carrying out kinetic studies on human colorectal carcinoma and adjacent normal mucosa, providing initial perturbations of proliferative indices are allowed to settle. Tumours require 3.0 μg/ml vincristine for complete metaphase arrest compared with mucosa, which needs 0.5 μg/ml, a 6-fold difference. Using a stathmokinetic technique, the birth rate of tumour cells is 10.21 cells/1000 cells per hr, compared with 7.73 cells/1000 cells per hr for mucosa, a statistically significant difference ( P < 0.01).     

Survivin、CyclinD1在大肠癌中的表达及其临床意义

目的观察Survivin和CyclinD1在大肠癌组织中的表达与临床病理指标及预后的关系。方法应用免疫组织化学(S-P)法,检测44例大肠癌组织、30例癌旁组织、10例正常黏膜组织中Survivin、CyclinD1的表达情况。结果大肠癌组织中Survivin、CyclinD1的表达明显高于癌旁组织的表达,差异具有显著性(P<0.05),正常黏膜组织无Survivin、CyclinD1表达;Survivin表达与性别、年龄、肿瘤组织类型及组织分级、大体类型差异均无显著性(P>0.05),而与淋巴结转移、Dukes分期显著相关(P<0.05),CyclinD1表达与大肠癌各临床病理指标差异均无显著性(P>0.05);Survivin阳性生存率显著低于Survivin阴性患者,CyclinD1阳性生存率显著低于CyclinD1阴性患者(P<0.05);Survivin、CyclinD1在大肠癌中表达正相关(P<0.05)。结论Survivin表达与淋巴结转移、Dukes分期密切相关,提示预后不良,可作为大肠癌侵袭性和预后评估的生物学指标;CyclinD1表达提示预后不良,是判断预后的指标之一;Survivin、CyclinD1在大肠癌的发生过程中既有独立的功能,又有协同作用。     

The histone deacetylase inhibitor ITF2357 reduces production of pro-inflammatory cytokines in vitro and systemic inflammation in vivo

We studied inhibition of histone deacetylases (HDACs), which results in the unraveling of chromatin, facilitating increased gene expression. ITF2357, an orally active, synthetic inhibitor of HDACs, was evaluated as an anti-inflammatory agent. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2357 reduced by 50% the release of tumor necrosis factor-alpha (TNFalpha) at 10 to 22 nM, the release of intracellular interleukin (IL)-1alpha at 12 nM, the secretion of IL-1beta at 12.5 to 25 nM, and the production of interferon-gamma (IFNgamma) at 25 nM. There was no reduction in IL-8 in these same cultures. Using the combination of IL-12 plus IL-18, IFNgamma and IL-6 production was reduced by 50% at 12.5 to 25 nM, independent of decreased IL-1 or TNFalpha. There was no evidence of cell death in LPS-stimulated PBMCs at 100 nM ITF2357, using assays for DNA degradation, annexin V, and caspase-3/7. By Northern blotting of PBMCs, there was a 50% to 90% reduction in LPS-induced steady-state levels of TNFalpha and IFNgamma mRNA but no effect on IL-1beta or IL-8 levels. Real-time PCR confirmed the reduction in TNFalpha RNA by ITF2357. Oral administration of 1.0 to 10 mg/kg ITF2357 to mice reduced LPS-induced serum TNFalpha and IFNgamma by more than 50%. Anti-CD3-induced cytokines were not suppressed by ITF2357 in PBMCs either in vitro or in the circulation in mice. In concanavalin-A-induced hepatitis, 1 or 5 mg/kg of oral ITF2357 significantly reduced liver damage. Thus, low, nonapoptotic concentrations of the HDAC inhibitor ITF2357 reduce pro-inflammatory cytokine production in primary cells in vitro and exhibit anti-inflammatory effects in vivo.     

Cytosolic levels of an estrogen-induced breast cancer-associated peptide (TFF1/pS2) in colorectal cancer: clinical significance and relationship with steroid receptors

BACKGROUND: The Trefoil Factor 1 (TFF1/pS2), a peptide consisting of 60 amino acids, is the most abundant estrogen-induced messenger RNA in MCF-7 breast cancer cells and is also expressed by colorectal carcinomas. The objective of this work was to evaluate the cytosolic TFF1 content in colorectal carcinomas, its possible relationship with estrogen and progesterone receptors as well as with clinicopathological tumor parameters, and its potential prognostic significance. METHODS: Cytosolic TFF1 levels were examined by immunoradiometric assay in 178 patients with resectable colorectal cancer. The mean follow-up period was 32 months. RESULTS: There was a wide variability of cytosolic TFF1 levels in tumor-surrounding mucosa samples (0.09-42.5 ng/mg protein) as well as in tumors (0.01-270 ng/mg protein). Comparison of paired mucosa and carcinoma samples showed significantly higher TFF1 levels in tumors (mean: 17.1 ng/mg protein) than in mucosa samples (10 ng/mg protein) (p = 0.027). TFF1 levels were significantly higher in mucosa samples surrounding distal colon and rectal tumors (p = 0.0001) and in tumor samples obtained from older patients (p = 0.007). However, there were no significant differences in tumor TFF1 levels with respect to clinicopathological parameters such as the patient's sex, tumor location, stage, histological grade, ploidy, S-phase, or tumor estrogen and progesterone receptors. In addition, there was no significant relationship between tumor TFF1 levels and disease outcome. CONCLUSIONS:TFF1 may play an as yet undetermined role in the tumorigenesis of colorectal carcinomas. However, cytosolic levels of TFF1 do not seem to have any prognostic significance in colorectal carcinomas.