MECHANISMS OF LEUKOCYTE RECRUITMENT IN IBD

描述(改编自申请人的摘要)：该项目的长期目标 项目是确定上皮中性粒细胞激活的机制 由激活的肠细胞产生的多肽-78(ENA-78)调节中性粒细胞 在炎症性肠病中招募。这方面的实验目标是 建议定义调控ENA-78基因的分子机制 在Caco-2肠上皮细胞中表达。ENA-78是一种C-X-C趋化因子 它与CXCR2受体结合，刺激中性粒细胞趋化。ENA-78 也促进细胞再生，是一种有效的血管生成因子。这个 研究人员此前已经表明，肠道上皮细胞的激活 IL-1β或TNFpha诱导的细胞长时间产生ENA-78。这个 研究人员还表明，肠上皮细胞是ENA-78的主要部位 在正常人结肠中产生ENA-78，结肠粘膜中ENA-78产生 溃疡性结肠炎的发病率显著增加。基于这些发现， 研究人员假设ENA-78基因是针对 激活的肠上皮细胞长时间产生ENA-78蛋白。持续的 炎症的肠上皮细胞产生ENA-78可能是 调节中性粒细胞在IBD结肠上皮层的重新募集。这个 初步研究表明，在ENA-785‘中有一个核因子-kappaB结合位点。 启动子区域在IL-1β诱导基因调控中的重要作用 在Caco-2细胞中转录。调查人员还确认了另一起5起 调控元件(-118至-146个碱基对)指定为“A位”。站点A规范 Caco-2细胞中ENA-78基因的基础转录并结合锌指 转录因子Sp-L和另一个尚未确定的核 因素(S)。第一个具体目标是定义功能性的Sp-L-绑定 ENA-78启动子的扫描和定点突变 A位用EMSA和荧光素酶报告基因分析。我们的第二个具体目标 是描述与A位点结合的另一个反式激活剂(S)的特征。 还将检验我们的假设，即结合到A位点的核因子可以 调节特定细胞类型(肠细胞)ENA-70基因的表达。这个 初步数据表明，ENA-78mRNA是一种转录后机制 稳定性，这是ENA-78蛋白生产动力学延长的原因。 第三个具体目标将检验这样一个假设，即 ENA-78mRNA 3‘非翻译区的富金结合元件 赋予消息稳定性，并通过以下方式确定ENA-78的持续生产 激活的肠道细胞。对规则的深入、机械的理解 ENA-78基因在人肠上皮细胞中的表达可能导致新的治疗方法 治疗IBD的方法。

DESCRIPTION (Adapted from the Applicant's Abstract): The long-term goal of this project is to determine the mechanisms whereby Epithelial Neutrophil-Activating peptide-78 (ENA-78), produced by activated enterocytes, regulates neutrophil recruitment in inflammatory bowel disease. The experimental goals for this proposal are to define the molecular mechanisms that regulate ENA-78 gene expression in Caco-2 intestinal epithelial cells. ENA-78 is a C-X-C chemokine that binds to the CXCR2 receptor and stimulates neutrophil chemotaxis. ENA-78 also facilitates cellular regeneration and is a potent angiogenic factor. The investigator has shown previously that activation of intestinal epithelial cells by IL-1beta or TNFalpha induces prolonged ENA-78 production. The investigator has also shown that enterocytes are the main site of ENA-78 production in normal human colon and that colonic mucosal ENA-78 production is substantially increased in ulcerative colitis. Based on these findings, the investigator hypothesizes that the ENA-78 gene is specifically adapted for the prolonged production of ENA-78 protein by activated enterocytes. The sustained production of ENA-78 by inflamed intestinal epithelial cells is likely to regulate neutrophil recruitment into the colonic epithelial layer in IBD. The preliminary studies demonstrate that an NF-kappaB binding site in the ENA-78 5' promoter region plays a major role in regulating IL-lbeta-induced gene transcription in Caco-2 cells. The investigator has also identified a second 5 regulatory element (-118 to -146 bp) designated "Site A." Site A regulates basal ENA-78 gene transcription in Caco-2 cells and binds the zinc finger transcription factor Sp-l in addition to another, as yet unidentified, nuclear factor(s). The first specific aim is to define the functional Sp-l-binding element in the ENA-78 promoter by scanning and site-directed mutagenesis of Site A using EMSA and luciferase reporter gene assays. Our second specific aim is to characterize the other transactivator(s) that bind to Site A. This aim will also examine our hypothesis that nuclear factor binding to Site A can regulate cell-type-specific (enterocyte) ENA-70 gene expression. The preliminary data indicate that a post-transcriptional mechanism, ENA-78 mRNA stability, accounts for the prolonged kinetics of ENA-78 protein production. The third specific aim will test the hypothesis that the organization of AU-rich binding elements within the 3' untranslated region of ENA-78 mRNA confers message stability and determines the sustained production of ENA-78 by activated enterocytes. An in-depth, mechanistic understanding of the regulation of ENA-78 gene expression in human enterocytes may lead to novel therapeutic approaches to IBD.