Small GTP Binding Proteins in Gastrointestinal Mucosa

描述（由申请人提供）：上皮细胞聚集成粘膜片，并作为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底外侧结构域。极性缺失导致这些过程的改变可导致肿瘤形成，而微绒毛适当组装或维护的缺失可导致营养吸收不良和腹泻疾病。我们最近的研究表明，囊泡运输蛋白对建立和维持顶端极性所需的过程起着关键的调节作用。rab11家族相互作用蛋白（Rab11-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。虽然以前的研究已经注意到MARK2磷酸化在上皮细胞极性中的重要性，但没有研究检查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，我们也证明了Rab25（一种上皮特异性小GTP结合蛋白）在Rab25 KO小鼠中的缺失会促进肠道和结肠肿瘤的发生，而在CaCo-2细胞中敲低Rab25的表达会促进极化功能的丧失，并导致更具侵袭性的表型。Rab25在人类结肠癌中表达降低，无论分期如何，这表明Rab25的缺失在癌变的早期阶段起作用。在Rab25 KO小鼠和CaCo-2细胞中，Rab25的缺失与ß - 1整合素的错误转运有关。在CaCo-2细胞中，敲低Rab25的表达会导致极化功能的缺陷，包括整合素的误转运以及α - 5整合素转录的减少。我们假设质膜循环系统的组成部分Rab25、Rab11-FIP1B/C和Rab11-FIP2是极性建立和维持的关键介质，这些途径中的缺陷易导致早期癌变。为了验证我们的假设，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蛋白在肠细胞极性调节中的作用。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。第三，我们将确定Rab11-FIP2和Rab11-FIP1B/C缺失对小鼠肠细胞极性和分化的影响以及培养的肠样肠。这些研究将利用新型小鼠模型检测Rab11-FIP1B/C和Rab11-FIP2的固定等位基因。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 ß1-integrin. In CaCo-2 cells, knockdown of Rab25 expression induces deficits in polarized function with both mistrafficking of integrins as well as decreases in α5-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.