Regulation of epithelial cell motility by villin.

描述（由申请方提供）：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（PIP 2）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上皮细胞功能，包括：通过内吞作用和胞吐作用的离子转运蛋白，对营养物质的吸收至关重要;肠道恢复，对在大的渗透和机械应力存在下维持稳态至关重要;细胞沿着隐窝-绒毛轴的运动;肠道病原体的入侵和繁殖;免疫监视和炎症;以及赘生性肿瘤细胞扩散和转移。绒毛蛋白是一种肌动蛋白成核、加帽、切断和捆绑蛋白。 绒毛蛋白结合并调节已知调节细胞运动性的两种配体，磷脂酶C-γ 1（PLC-γ 1）以及脂肪酶的底物，即磷脂酰肌醇4，5-二磷酸（PIP 2）。 最近对绒毛蛋白基因敲除小鼠的研究表明，绒毛蛋白是调节上皮细胞运动所必需的。 此外，绒毛蛋白与其家族的其他蛋白质（包括凝溶胶蛋白）具有序列同源性，已显示凝溶胶蛋白在体内调节细胞运动。 我们的工作假设是，绒毛蛋白的调节磷酸肌醇介导的信号转导途径和肌动蛋白细胞骨架的能力是重要的上皮细胞的生理和病理生理，涉及细胞运动的变化。 为了实现我们的总体研究目标，我们将使用以下方法表征绒毛-PIP 2相互作用：使用重组绒毛蛋白体外重建;肠上皮细胞系Caco-2中的内源性绒毛蛋白表达;以及使用四环素调节系统在绒毛蛋白缺失肠细胞系IEC-6中过表达绒毛蛋白和调节细胞内PIP 2水平的酶。 为了明确绒毛蛋白在上皮细胞运动中的作用，我们将使用绒毛蛋白敲除小鼠。 这些研究为改善运动功能、提高正常生理机能和改善疾病提供了前景。 上皮细胞运动性的抑制在几种疾病中可能是显著的，包括炎性肠病、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.