Molecular biological study on the expression and functions of trefoil factor family(TFF) peptides in gastrointestinal mucosa

三叶因子家族(TFF)是一组主要在胃肠道上皮细胞表达的抗蛋白酶多肽。到目前为止，在人类中已鉴定出三种TFF肽，即TFF1(PS2)、TFF2(SP)和TFF3(LTF)。已有的研究表明，转铁蛋白多肽在胃肠道粘膜的防御和修复中起着重要作用。在本研究中，我们研究了生长因子、细胞因子、多种核受体的配体和非甾体类抗炎药对原代培养的胃肠粘膜细胞以及胃肠上皮细胞系中TFF表达的影响。用实时荧光定量RT-PCR检测内源性TFFmRNA的表达，并进行报告基因检测。主要发现如下：(1)TFF1最初是在MCF-7双向癌细胞中发现的雌激素诱导基因。虽然我们在胃上皮细胞中发现了雌激素受体(ER)(主要是ERβ)的表达，但17β-雌二醇对内源性TFF1mRNA的表达以及TFF1报告基因的转录没有明显的影响，提示TFF1在胃上皮细胞中的表达不依赖于ER。(2)肿瘤坏死因子α和佛波酯可上调TFF1的表达。报告基因分析表明，NF-κB和AP-1分别介导肿瘤坏死因子-α和佛波酯的作用。(3)曲格列酮、15d-PGJ2等PPARγ配体可上调胃上皮细胞TFF2的表达。我们发现在人TFF2基因的启动子区域存在一个功能性的过氧化物体增殖物反应元件(PPRE)。(4)我们还发现非甾体抗炎药，如消炎痛和阿司匹林，通过激活PPARγ上调TFF2的表达。由于TFF2是一种关键的细胞保护剂，这一机制可能会减轻这些非类固醇抗炎药对胃粘膜的损伤程度。

Trefoil factor family(TFF) is a group of protease resistant peptides mainly expressed in gastrointestinal epithelial cells. To date, three TFF peptides are identified in humans, TFF1 (pS2), TFF2 (SP), and TFF3 (LTF). It is well established that TFF peptides play critical roles in the defense and repair of gastrointestinal mucosa. In this study, in order to understand the regulatory mechanisms of TFF expression, we examined the effects of growth factors, cytokines, ligands for various nuclear receptors, and NSAIDs on the expression of TFF in primazy cultured gastrointestinal mucosal cells as well as in cell lines derived from gastrointestinal epithelial cells. Endogenous TFF mRNA expression was analyzed by real-time quantitative RT-PCR and reporter gene assays were also performed. Major findings are as follows : (1)TFF1 was originally discovered as an estrogen-inducible gene in MCF-7 bieast cancer cells. Although we identified the expression of estrogen receptors(ER)(mainly ERβ) in gastric epithelial cells, 17β-estradiol had no significant effect on the expression of endogenous TFF1 mRNA as well as the transcription of TFF1 reporter genes, suggesting that gastric expression of TFF1 is independent of ER. (2)TNFα and phorbol ester up-regulated TFF1 expression. Reporter gene assay showed that NF-κB and AP-1 mediates the action of TNF-α and phorbol ester, respectively. (3)Ligands for PPARγ, such as troglitazone and 15d-PGJ2, were found to up-regulate TFF2 expression in gastric epithelial cells. We identified the presence of a functional peroxisome proliferator responsive element(PPRE) within the promoter region of human TFF2 gene. (4)We also found that NSAIDs, such as indomethacin and aspirin, up-regulates TFF2 expression through activating PPARγ. Since TFF2 is a critical cytoprotective agent, this mechanism may reduce the degree of gastric mucosal damage induced by these NSAIDs.