Small GTP Binding Proteins in Gastrointestinal Mucosa

胃肠粘膜中的小 GTP 结合蛋白

• 批准号: 8598603
• 负责人: JAMES Richard GOLDENRING
• 金额: $ 33.93万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598603
• 关键词: 3-Dimensional; Alleles; Antibodies; Apical; Attention; Caco-2 Cells; Cell Culture Techniques; Cell Differentiation process; Cell Polarity; Cell membrane; Cells; Colon Carcinoma; Complex; Defect; Development; Digestion; Disease; ETV4 gene; Enterocytes; Epithelial; Epithelial Cells; Epithelium; Event; Family; GTP-Binding Proteins; Genetic Transcription; Human; In Situ; Integrins; Intestines; Intracellular Membranes; Investigation; Ions; Kidney; Lateral; Lead; Lesion; MDCK cell; Maintenance; Malignant Neoplasms; Mediating; Mediator of activation protein; Membrane Protein Traffic; Modeling; Molecular; Mucous Membrane; Mus; Neoplasms; Nutrient; Pathway interactions; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiology; Predisposing Factor; Process; Proteins; Recycling; Regulation; Role; Staging; System; Tissues; Transcript; Transcriptional Regulation; Ulcerative Colitis; Vesicle; absorption; apical membrane; carcinogenesis; gastrointestinal; insight; intestinal epithelium; mouse model; novel; outcome forecast; protein transport; public health relevance; trafficking; two-dimensional

DESCRIPTION (provided by applicant): Epithelial cells assemble into mucosal sheets and act as the barrier between the external and internal milieu. To maintain this functional barrier, intestinal epithelial cells must establish segregated apical and basolateral domains. Alterations in these processes through losses in polarity can lead to neoplasia, while loss of proper microvillar assembly or maintenance can lead to malabsorbtion of nutrients and diarrheal disease. Our recent studies have demonstrated that vesicle trafficking proteins critically regulate the processes required for establishment and maintenance of apical polarity. Rab11-Family Interacting Proteins (Rab11-FIPs) mediate both directional trafficking and the establishment of apical polarity. The establishment of apical polarity involves proper phosphorylation of Rab11-FIP2 by the polarity-associated kinase MARK2/Par1b. We have recently demonstrated that Rab11-FIP1B/C is also a substrate for MARK2. While previous investigations have noted the importance of MARK2 phosphorylation in epithelial polarity, no studies have examined how MARK2- dependent phosphorylation events could influence intestinal epithelial cell polarity. In intestinal cells, we also have demonstrated that loss of Rab25, an epithelial-specific small GTP binding protein, in Rab25 KO mice promotes intestinal and colonic neoplasia and knockdown of Rab25 expression in CaCo-2 cells promotes a loss in polarized function and assumption of a more invasive phenotype. Rab25 expression is decreased in human colon cancers regardless of stage, suggesting a role for Rab25 loss in the early stages of carcinogenesis. In both the Rab25 KO mice and CaCo-2 cells, loss of Rab25 is associated with mis-trafficking of ¿1-integrin. In CaCo-2 cells, knockdown of Rab25 expression induces deficits in polarized function with both mistrafficking of integrins as well as decreases in ¿5-integrin transcription. We have hypothesized that components of plasma membrane recycling systems, Rab25 and Rab11-FIP1B/C and Rab11-FIP2, are critical mediators of both the establishment and maintenance of polarity and that defects in these pathways predispose to early carcinogenesis. To investigate our hypothesis we will pursue three specific aims: First, we will determine how Rab25 regulates intestinal polarity through regulation of ETV4-dependent gene transcription. Second, we will identify the roles of Rab11-FIP proteins in regulating polarity in intestinal cells. These studies will utilize novel cell culture models and phosphorylation site-specific antibodies. Third, we will determine the effects of the loss of Rab11-FIP2 and Rab11-FIP1B/C on intestinal cell polarity and differentiation in mice and intestinal enteroids in culture. These studies will utilize novel mouse models for floxed alleles for both Rab11-FIP1B/C and Rab11-FIP2. These investigations will establish how disruption of vesicle trafficking processes that regulate the establishment and maintenance of intestinal epithelial cell polarity may lead to carcinogenesis. !

描述（由申请人提供）：上皮细胞组装成粘膜片并充当外部和内部环境之间的屏障。为了维持这种功能屏障，肠上皮细胞必须建立分离的顶端和基底外侧区域。通过极性损失而改变这些过程可能导致肿瘤，而适当的微绒毛组装或维护的损失可能导致营养吸收不良和腹泻疾病。我们最近的研究表明，囊泡运输蛋白关键性地调节 建立和维持顶端极性所需的过程。 Rab11 家族相互作用蛋白 (Rab11-FIP) 介导定向运输和顶端极性的建立。顶端极性的建立涉及极性相关激酶 MARK2/Par1b 对 Rab11-FIP2 的适当磷酸化。我们最近证明 Rab11-FIP1B/C 也是 MARK2 的底物。虽然之前的研究已经注意到 MARK2 磷酸化在上皮极性中的重要性，但没有研究探讨 MARK2 依赖性磷酸化事件如何影响肠上皮细胞极性。在肠细胞中，我们还证明，Rab25 KO 小鼠中 Rab25（一种上皮特异性小 GTP 结合蛋白）的缺失会促进肠和结肠肿瘤的形成，CaCo-2 细胞中 Rab25 表达的敲低会促进极化功能的丧失和更具侵袭性的表型的假设。无论处于何种阶段，人类结肠癌中 Rab25 的表达都会降低，这表明 Rab25 缺失在癌发生的早期阶段发挥着作用。在 Rab25 KO 小鼠和 CaCo-2 细胞中，Rab25 的丢失与 ¿1-整合素的错误运输有关。在 CaCo-2 细胞中，Rab25 表达的敲低会导致极化功能缺陷，导致整合素的错误运输以及 5-整合素转录的减少。我们假设质膜回收系统的组成部分 Rab25 和 Rab11-FIP1B/C 以及 Rab11-FIP2 是极性建立和维持的关键介质，并且这些途径的缺陷容易导致早期癌变。为了研究我们的假设，我们将追求三个具体目标：首先，我们将确定 Rab25 如何通过调节 ETV4 依赖性基因转录来调节肠道极性。其次，我们将确定 Rab11-FIP 蛋白在调节肠细胞极性中的作用。这些研究将利用新型细胞培养模型和磷酸化位点特异性抗体。第三，我们将 确定 Rab11-FIP2 和 Rab11-FIP1B/C 的缺失对小鼠和培养物肠类肠细胞极性和分化的影响。这些研究将利用新型小鼠模型来研究 Rab11-FIP1B/C 和 Rab11-FIP2 的 floxed 等位基因。这些研究将确定调节肠上皮细胞极性建立和维持的囊泡运输过程的破坏如何可能导致致癌。 ！