Regulation of epithelial cell motility by villin

描述(由申请人提供)：PI建议确定和表征绒毛蛋白及其配体(磷脂酰肌醇4，5-二磷酸(PIP2)和肌动蛋白)在上皮细胞运动中的作用。活跃的细胞运动调节许多重要的肠道上皮细胞功能，包括：离子转运蛋白通过内吞和胞吐作用对营养物质的吸收至关重要；肠道重建对于在大渗透和机械应激的情况下保持动态平衡至关重要；细胞沿隐窝绒毛轴的移动；肠道病原体的侵袭和繁殖；免疫监视和炎症；以及肿瘤细胞的扩散和转移。绒毛蛋白是一种肌动蛋白核化、封端、切断和捆绑的蛋白质。绒毛蛋白结合并调节两种已知的调节细胞运动的配体，磷脂酶C-伽马1(PLC-Gamma1)以及脂肪酶的底物磷脂酰肌醇4，5-二磷酸(PIP2)。最近对绒毛蛋白基因敲除小鼠的研究表明，绒毛蛋白对调节上皮细胞的运动是必要的。此外，Villin与其家族的其他蛋白质包括明胶蛋白具有序列同源性，这些蛋白质已被证明在体内调节细胞的运动。我们的工作假设是，Villin调节磷脂酰肌醇介导的信号转导通路和肌动蛋白细胞骨架的能力对上皮细胞生理学和涉及细胞运动性变化的病理生理学是重要的。为了完成我们的总体研究目标，我们将使用以下方法来表征Villin-PIP2的相互作用：使用重组Villin蛋白进行体外重组；在肠上皮细胞系Caco-2中表达内源性Villin；使用四环素调控系统在Villin缺失的肠细胞系IEC-6中过表达Villin和调节细胞内PIP2水平的酶。为了明确地确定维林蛋白在上皮细胞运动中的作用，我们将使用维林蛋白基因敲除小鼠。这些研究展望了改变运动能力以增强正常生理和改善疾病的前景。上皮细胞运动的抑制在几种疾病中可能是显著的，包括炎症性肠病、乳糜泻和结肠癌。

DESCRIPTION (provided by applicant): The PI proposes to determine and characterize the role of villin and its ligands (phosphatidylinositol 4,5- bisphosphate (PIP2) and actin) in epithelial cell motility. Active cell motility regulates many important intestinal epithelial cell functions, including: ion transport proteins via endocytosis and exocytosis, crucial for absorption of nutrients; intestinal restitution, important to maintain homeostasis in the presence of large osmotic and mechanical stress; the movement of cells along the crypt-villus axis; the invasion and propagation of enteropathogens; immune surveillance and inflammation; as well as neoplastic tumor cell dissemination and metastasis. Villin is an actin nucleating, capping, severing, and bundling protein. Villin binds and regulates two ligands that are known to regulate cell motility, phospholipase C-gamma1 (PLC-gamma1) as well as the substrate of the lipase namely, phosphatidylinositol 4,5-bisphosphate (PIP2). Recent studies with villin knock out mice have demonstrated that villin is necessary to regulate epithelial cell motility. In addition, villin shares sequence homology with other proteins of its family including gelsolin, which have been shown to regulate cell motility in vivo. Our working hypothesis is that villin's ability to regulate phosphoinositide-mediated signal transduction pathways and the actin cytoskeleton is important to epithelial cell physiology and pathophysiology involving changes in cell motility. To accomplish our overall study objectives we will characterize the villin-PIP2 interaction using the following approaches: reconstitution in vitro using recombinant villin proteins; endogenous villin expression in the intestinal epithelial cell line, Caco-2; and over expression of villin and enzymes that regulate intracellular PIP2 levels, in the villin null intestinal cell line, IEC-6 using a tetracycline-regulated system. To determine unequivocally the role of villin in the epithelial cell motility, we will use villin knock out mice. These studies promise the prospect of modifying motility for enhancement of normal physiology and for amelioration of disease. Inhibition of epithelial cell motility can be significant in several diseases, including inflammatory bowel disease, celiac disease, and colon cancer.