Regulation of epithelial cell motility by villin.

描述（由申请人提供）：该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，在存在大渗透和机械应力的情况下维持体内平衡的重要因素；细胞沿隐窝-绒毛轴的运动；肠道病原体的入侵与传播；免疫监视和炎症；以及肿瘤细胞的播散和转移。绒毛蛋白是一种肌动蛋白成核、盖帽、切断和捆绑蛋白。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-gamma1 （PLC-gamma1）以及脂肪酶的底物，即磷脂酰肌醇4,5-二磷酸（PIP2）。最近对绒毛蛋白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，绒毛蛋白与其他家族蛋白（包括gelsolin）具有序列同源性，已被证明在体内调节细胞运动。我们的工作假设是，绒毛蛋白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对上皮细胞的生理和病理生理涉及细胞运动的变化是重要的。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蛋白- pip2相互作用：利用重组绒毛蛋白在体外重组；内源性绒毛蛋白在肠上皮细胞系Caco-2中的表达；以及绒毛蛋白和调节细胞内PIP2水平的酶的过度表达，在没有绒毛蛋白的肠细胞系IEC-6中使用四环素调节系统。为了明确确定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。这些研究为改善正常生理机能和改善疾病提供了改善运动的前景。上皮细胞运动的抑制在许多疾病中都很重要，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.