Small GTP Binding Proteins in Gastrointestinal Mucosa

描述（由申请方提供）：上皮细胞组装成粘膜层，并作为外部和内部环境之间的屏障。为了维持这一功能屏障，肠上皮细胞必须建立分离的顶端和基底外侧域。这些过程中通过极性损失的改变可导致瘤形成，而适当的微绒毛组装或维持的损失可导致营养吸收不良和牙周病。我们最近的研究表明，囊泡运输蛋白质的关键调节， 建立和维持顶端极性所需的过程。Rab 11家族相互作用蛋白（Rab 11-FIPs）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MARK 2/Par 1b对Rab 11-FIP 2的适当磷酸化。我们最近证明Rab 11-FIP 1B/C也是MARK 2的底物。虽然以前的研究已经注意到MARK 2磷酸化在上皮极性中的重要性，但没有研究研究MARK 2依赖性磷酸化事件如何影响肠上皮细胞极性。在肠细胞中，我们还证明了Rab 25 KO小鼠中Rab 25（一种上皮特异性小GTP结合蛋白）的缺失促进了肠和结肠肿瘤形成，CaCo-2细胞中Rab 25表达的敲低促进了极化功能的丧失和更具侵袭性表型的假设。Rab 25表达在人类结肠癌中降低，无论阶段如何，这表明Rab 25损失在癌发生的早期阶段中的作用。在Rab 25 KO小鼠和CaCo-2细胞中，Rab 25的丢失与β 1-整联蛋白的错误运输有关。在CaCo-2细胞中，Rab 25表达的敲低诱导极化功能的缺陷，包括整合素的误激活以及5-整合素转录的减少。我们假设质膜再循环系统的组成部分，Rab 25和Rab 11-FIP 1B/C和Rab 11-FIP 2，是建立和维持极性的关键介质，这些途径中的缺陷易导致早期癌变。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Rab 25如何通过调节ETV 4依赖的基因转录来调节肠道极性。其次，我们将确定Rab 11-FIP蛋白在调节肠细胞极性中的作用。这些研究将利用新的细胞培养模型和磷酸化位点特异性抗体。三是 确定Rab 11-FIP 2和Rab 11-FIP 1B/C缺失对小鼠肠细胞极性和分化以及培养物中肠上皮的影响。这些研究将利用Rab 11-FIP 1B/C和Rab 11-FIP 2的floxed等位基因的新型小鼠模型。这些研究将确定如何破坏小泡运输过程，调节建立和维持肠上皮细胞极性可能导致癌变。!

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. !