MECHANISMS OF LEUKOCYTE RECRUITMENT IN IBD

IBD 中白细胞招募的机制

基本信息

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
项目类别：
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

项目摘要

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）：本项目的长期目标该项目的目的是确定上皮中性粒细胞激活的机制肽 78 (ENA-78)，由激活的肠细胞产生，调节中性粒细胞炎症性肠病的募集。本次实验的目标建议定义调节 ENA-78 基因的分子机制 Caco-2肠上皮细胞中的表达。 ENA-78 是一种 C-X-C 趋化因子与 CXCR2 受体结合并刺激中性粒细胞趋化性。 ENA-78 还促进细胞再生，是一种有效的血管生成因子。这研究人员之前已经表明，肠上皮细胞的激活 IL-1beta 或 TNFalpha 诱导细胞延长 ENA-78 的产生。这研究人员还表明，肠上皮细胞是 ENA-78 的主要位点正常人结肠中的产生，并且结肠粘膜 ENA-78 的产生是溃疡性结肠炎显着增加。根据这些发现，研究人员假设 ENA-78 基因专门适应激活的肠上皮细胞延长 ENA-78 蛋白的产生。持续的发炎的肠上皮细胞可能会产生 ENA-78 调节 IBD 中中性粒细胞募集到结肠上皮层。这初步研究表明 ENA-78 5' 中存在 NF-kappaB 结合位点启动子区在调节IL-1β诱导基因中起主要作用 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）指定为“位点 A”。站点 A 监管 Caco-2 细胞中的基础 ENA-78 基因转录并结合锌指转录因子 Sp-l 以及另一种尚未鉴定的核因子因素。第一个具体目标是定义功能性 Sp-l 结合通过扫描和定点诱变来识别 ENA-78 启动子中的元件站点 A 使用 EMSA 和荧光素酶报告基因检测。我们的第二个具体目标是为了表征与位点 A 结合的其他反式激活因子。这个目的还将检验我们的假设，即与位点 A 结合的核因子可以调节细胞类型特异性（肠细胞）ENA-70 基因表达。这初步数据表明转录后机制 ENA-78 mRNA 稳定性，解释了 ENA-78 蛋白生产的长期动力学。第三个具体目标将检验以下假设：组织 ENA-78 mRNA 3' 非翻译区内富含 AU 结合元件赋予信息稳定性并通过以下方式决定 ENA-78 的持续生产激活的肠上皮细胞。对法规的深入、机械的理解人类肠上皮细胞中 ENA-78 基因表达的改变可能会带来新的治疗方法 IBD 的治疗方法。