Small GTP Binding Proteins in Gastrointestinal Mucosa

描述(申请人提供)：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侧域。这些过程中由于极性丧失而发生的改变可导致肿瘤，而失去适当的微绒毛组装或维护可导致营养吸收不良和腹泻病。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-FIP)既参与定向转运，又参与心尖极性的建立。顶端极性的建立涉及到极性相关激酶Mark2/Par1b对Rab11-FIP2的适当磷酸化。我们最近证明了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，但还没有研究探讨Mark2依赖的磷酸化事件如何影响肠上皮细胞的极性。在肠道细胞方面，我们还证明了Rab25 KO小鼠中上皮特异性小GTP结合蛋白Rab25的缺失促进了肠道和结肠肿瘤的发生，而Caco-2细胞中Rab25表达的下调促进了极化功能的丧失和更具侵袭性的表型。在人类结肠癌中，Rab25的表达降低，提示Rab25缺失在肿瘤发生的早期阶段起到了作用。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的表达下调导致极化功能的缺陷，导致整合素的错误作用以及α5-整合素转录的降低。我们假设，质膜循环系统的组件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于早期癌症发生。为了验证我们的假设，我们将追求三个具体目标：第一，我们将确定Rab25如何通过调节ETV4依赖的基因转录来调节肠道极性。其次，我们将确定Rab11-FIP蛋白在调节肠道细胞极性中的作用。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。第三，我们将确定Rab11-FIP2和Rab11-FIP1B/C的缺失对小鼠肠道细胞极性和分化的影响以及对培养的肠小管的影响。这些研究将利用新的小鼠模型研究Rab11-FIP1B/C和Rab11-FIP2的等位基因。这些研究将确定调控肠道上皮细胞极性建立和维持的囊泡运输过程的中断可能导致癌症的发生。

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.