MOLECULAR BASIS OF ENTEROCYTE REGULATION DURING ONTOGENY

个体发育过程中肠细胞调节的分子基础

• 批准号: 2883155
• 负责人: MARTIN G MARTIN
• 金额: $ 15.27万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2883155
• 关键词: DNA footprinting; antibody receptor; cellular polarity; developmental genetics; developmental immunology; gastrointestinal epithelium; gel mobility shift assay; gene expression; genetic regulatory element; genetically modified animals; histogenesis; humoral immunity; intracellular transport; laboratory mouse; nucleic acid sequence; receptor binding; transcription factor; transfection

In this grant I propose to study the regulation of both the neonatal intestinal immunoglobulin receptors (fcRn) and polymeric receptor (pIgR) genes, as a model to understand small intestinal epithelial ontogeny and adaptation. The Fc receptor transports IgG from breast milk and is essential for establishing humoral immunity in the neonate. Located on the enterocytes apical membrane, FcRn transport IgG transcellulary to the basolateral membrane where its released into the systemic circulation. In contrast, pIgR transports IgA from the basolateral membrane to the lumen. In rodents, I have shown that both FcRn and pIgR regulation have a unique tissue, developmental and hormonal-pattern of expression. FcRn's expression is developmentally restricted to the suckling phase, during which time the transcript is produced in a proximodistal gradient, and is inhibited by corticosteroids administration. In contrast, pIgR mRNA levels are induced after warning, and may be further enhanced by corticosteroids. To begin addressing at a molecular level the mechanism underlying enterocyte regulation during development, the rat FcRn and murine pIgR genes have been cloned. We have done transient-transfection studies with an intestinal cell line, using chimeric constructs of either the 5' - flanking regions of the FcRn or pIgR genes fused to the reporter luciferase. Transfection of clones derived by nested deletions has been done, and has led to the identification of several activator and repressor regions that appear to modulate basal activity. Moreover, I will assess the response of each gene to corticosteroids using in vitro transfection experiments with cells containing chimeric constructs. Experiments with homologous and heterologous promoters will be used to define enhancer repressors elements, and DNase I footprint and electrophoretic gel mobility shift assays will be done to define the DNA-protein interaction. To enhance our ability to draw more physiologically relevant conclusions from our in vitro experiments, a transgenic mouse model is presented that appears to reflect FcRns normal pattern of expression. Similar lines will be developed with the pIgR 5'-flanking region to define the elements needed to control the genes in vivo expression. The projects long term objectives is to evaluate the transcriptional factor that regulates the intestinal and developmental-specific pattern of expression. Analysis of the FcRn and pIgR genes offers a unique model that may lead to a better understanding of the molecular mechanism of intestinal ontogeny, and specifically the intrinsic clock which controls its regulation.

在这项资助中，我建议研究对新生儿的调节 肠道免疫球蛋白受体(FcRN)和多聚体受体(PIgR) 基因，作为理解小肠上皮个体发育和 适应。Fc受体转运母乳中的免疫球蛋白G， 对建立新生儿的体液免疫至关重要。位于 肠上皮细胞顶膜、FcRN转运免疫球蛋白 释放到全身的基底外侧膜 发行量。相反，pIgR从基底外侧转运IgA 膜到管腔。在啮齿动物中，我已经证明了FcRN和pIgR 调节具有独特的组织、发育和激素模式 表情。FcRN的表达在发育上仅限于 哺乳阶段，在此期间，成绩单在 近端倾斜度，并被皮质类固醇抑制 行政管理。相反，pIgR mRNA水平在以下情况下被诱导 警告，并可能进一步加强皮质类固醇。要开始 在分子水平上解决肠道细胞的潜在机制 在发育过程中，大鼠FcRN和小鼠pIgR基因有 都被克隆了。我们已经做了瞬时转基因研究，用一个 肠道细胞系，使用5‘-嵌合结构 FcRN或pIgR基因侧翼区域与报告基因融合 荧光素酶。通过嵌套缺失获得的克隆的转染已经被 已经完成，并导致确定了几个激活剂和 抑制区似乎调节基础活动。此外，我 将使用体外实验评估每个基因对皮质类固醇的反应 用含有嵌合结构的细胞进行的转基因实验。 使用同源和异源启动子的实验将用于 定义增强子抑制子元件和DNase I足迹和 将进行凝胶迁移率变化分析以确定 DNA-蛋白质相互作用。为了提高我们的绘画能力 我们的体外实验得出了与生理相关的结论， 提供了一种似乎反映FcRns的转基因小鼠模型 正常的表达方式。类似的线路将与 PIgR 5‘-侧翼区域，用于定义控制 基因在体内表达。项目的长期目标是 评估调节肠道和肠道的转录因子 发育特有的表达模式。FcRN和FcRN分析 PIgR基因提供了一个独特的模型，可能会导致更好地理解 肠道个体发育的分子机制，特别是 控制其调节的内在时钟。