MECHANISMS OF LEUKOCYTE RECRUITMENT IN IBD

IBD 中白细胞招募的机制

• 批准号: 6256420
• 负责人: Ciaran P Kelly
• 金额: $ 30.66万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6256420
• 关键词: binding sites; chemokine; chemotaxis; gastrointestinal epithelium; gel mobility shift assay; gene expression; inflammatory bowel diseases; leukocyte activation /transformation; mitogen activated protein kinase; molecular cloning; neutrophil; nuclear factor kappa beta; nuclear runoff assay; polymerase chain reaction; protein biosynthesis; protein structure function; site directed mutagenesis; tissue /cell culture; transcription factor

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.

描述（改编自申请人的摘要）：本发明的长期目标是： 该项目旨在确定上皮细胞中性粒细胞活化 肽-78（ENA-78）由活化的肠细胞产生，调节中性粒细胞 炎症性肠病中的招募。这个实验的目标是 目的是阐明ENA-78基因调控的分子机制 在Caco-2肠上皮细胞中表达。ENA-78是C-X-C趋化因子 与CXCR 2受体结合并刺激中性粒细胞趋化性。ENA-78 也促进细胞再生，是一种有效的血管生成因子。的 研究者先前已经表明肠上皮细胞活化 细胞通过IL-1 β或TNF α诱导延长ENA-78产生。的 研究者还表明肠上皮细胞是ENA-78主要位点 ENA-78在正常人结肠中产生，结肠粘膜ENA-78的产生是 溃疡性结肠炎的发病率显著增加。根据这些发现， 研究人员假设ENA-78基因特异性地适应于 通过活化的肠细胞延长ENA-78蛋白的产生。持续 由发炎的肠上皮细胞产生的ENA-78可能 在IBD中调节中性粒细胞募集到结肠上皮层中。的 初步研究表明，在ENA-78 5'端的NF-κ B结合位点， 启动子区在调节IL-1 β诱导基因中起主要作用 在Caco-2细胞中的转录。调查人员还确定了第二个5 调控元件（-118至-146 bp）命名为“位点A”。“A站点监管 Caco-2细胞中基础ENA-78基因转录并结合锌指 转录因子Sp-l除了另一个尚未鉴定的核转录因子外， 因数。第一个具体的目的是定义功能性Sp-I-结合 ENA-78启动子中的元件， 使用EMSA和荧光素酶报告基因测定的位点A。我们的第二个具体目标 是表征与位点A结合的其他反式激活因子。这一目标 还将检验我们的假设，即核因子结合位点A可以 调节细胞类型特异性（肠上皮细胞）ENA-70基因表达。的 初步数据表明，转录后机制，ENA-78 mRNA， 稳定性，解释了ENA-78蛋白生产的延长动力学。 第三个具体目标将检验这样一个假设， ENA-78 mRNA 3'非翻译区富含AU的结合元件 赋予信息稳定性，并通过以下方式确定ENA-78的持续生产： 激活的肠细胞对法规的深入、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 接近IBD。