Regulation of epithelial cell homeostasis by actin microfilaments

肌动蛋白微丝对上皮细胞稳态的调节

• 批准号: 7915710
• 负责人: Seema Khurana
• 金额: $ 11.1万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-20 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7915710
• 关键词: Actin-Binding Protein; Actins; Affect; Apoptosis; Apoptotic; Architecture; Atrophic; Binding; Biochemical; Biological Assay; Celiac Disease; Cell Line; Cell Survival; Cell physiology; Cell surface; Cells; Cleaved cell; Colitis; Colon Carcinoma; Colorectal Cancer; Communicable Diseases; Complex; Cytoskeleton; Data; Defect; Developed Countries; Disease; Drug usage; Epithelial; Epithelial Cells; Equilibrium; Eukaryotic Cell; Exocrine Glands; Family; Gastrointestinal tract structure; Gelsolin; Gene Targeting; Genes; Genetic; Goals; Healed; Homeostasis; Homologous Protein; Hyperplasia; Immune; Infection; Inflammatory; Inflammatory Bowel Diseases; Injury; Intervention; Intestinal Mucosa; Intestines; Ischemia; Knockout Mice; Large Intestine; Length; Light; Link; Location; Maintenance; Malignant Neoplasms; Mediating; Microfilaments; Mitochondria; Mitochondrial Proteins; Molecular; Morphology; Mus; N-terminal; Organ; Parasitic infection; Pathogenesis; Persons; Pharmaceutical Preparations; Phenotype; Physiological; Physiology; Play; Predisposition; Protein Family; Proteins; Public Health; Radiation; Radiation therapy; Regulation; Reperfusion Injury; Reporting; Resistance; Risk; Role; Sodium Dextran Sulfate; Stimulus; Structure; Surface; Time; Villus; Viral; Yeasts; base; biological adaptation to stress; cancer cell; chemotherapy; design; economic impact; effective therapy; gastrointestinal; gastrointestinal epithelium; genetic regulatory protein; healing; in vivo; intestinal epithelium; novel; overexpression; prevent; public health relevance; research study; social; tumorigenesis; villin; yeast two hybrid system

DESCRIPTION (provided by applicant): The homeostatic balance between proliferation and apoptosis is essential for the intestinal epithelium to function as a physiological and structural barrier. Intestinal epithelial cells have a high rate of cell turnover accompanied by an equally high rate of apoptosis. This normal apoptosis is essential for the hierarchical organization of the intestinal epithelium and apoptotic epithelial cells have been detected both at the base of the crypt as well as the villus tips of the small and large intestine. Defects in apoptosis are associated with several gastrointestinal conditions including villus atrophy, epithelial hyperplasia, loss of normal absorptive function and increased risk of tumorigenesis. Surprisingly, there are few data available on apoptosis as an important mechanism of action in vivo in the gastrointestinal epithelium. There are very comprehensible data supporting links between actin and apoptosis in eukaryotic cells from studies using drugs that affect actin turnover. However, few studies have established a causal link between microfilament reorganization and cell survival. Villin is an actin regulatory protein expressed in the gastrointestinal epithelial cells of the small and large intestine as well as in exocrine glands associated with the GI tract. We recently determined, for the first time, that villin is an epithelial cell-specific anti-apoptotic protein. Further, our preliminary studies suggest that in the absence of villin, this function may be fulfilled by a related actin- binding protein of the villin family, gelsolin. Villin is also cleaved during the apoptotic cycle and we have previously reported that the cleaved NH2-terminal fragment of villin is pro-apoptotic. Using yeast two hybrid, biochemical and cellular approaches, we have recently identified and validated the novel interaction of villin with the mitochondrial protein, ATP synthase 2-subunit, which now allows us to investigate the cellular and molecular mechanism(s) of microfilament- induced regulation of mitochondrial morphology and function and epithelial cell survival by villin. The novelty of our observation will allow us to demonstrate for the first time a causal relationship between microfilament regulation and cell survival in eukaryotic cells. To achieve this, experiments will be conducted under three major specific aims: (i) to characterize the cellular and molecular mechanism(s) of villin's anti- and pro-apoptotic functions and to identify the physiologic relevance of full-length and cleaved villin fragments respectively, in the maintenance of gastrointestinal homeostasis ; (ii) to characterize the interaction of villin with ATP synthase 2- subunit to examine the relevance of this complex in the maintenance of gastrointestinal homeostasis; (iii) to characterize the villin-gelsolin double knock out mice to establish the role of villin and its homologous protein gelsolin in the regulation of gastrointestinal homeostasis. Our studies are vital for the rational design of new and more effective therapies for major intestinal epithelial diseases such as inflammatory bowel disease and cancer. PUBLIC HEALTH RELEVANCE: The intestinal tract fulfils essential roles as a digestive and absorptive surface and constitutes the largest immune organ. In addition the intestine is the anatomical location of diseases of enormous social and economic impact, like the inflammatory bowel disease (affecting about 1 person in every 500 in USA), the colon carcinoma (the second most frequent malignancy in developed countries) and numerous infectious diseases. Apoptosis plays an important role in determining the architecture of intestinal epithelia and also a part of the stress response of intestinal epithelial cells to toxic stimuli. In disease pathogenesis, apoptosis can either be inappropriately excessive or deficient and both of these have been implicated in bacterial, viral and parasitic infections, ischemia-reperfusion injury, inflammatory bowel disease, celiac disease and colorectal cancer. Abnormalities in apoptotic function contribute to both the pathogenesis of colorectal cancer as well as its resistance to chemotherapeutic drugs and radiotherapy. An understanding of apoptosis would help enhance normal physiology and ameliorate diseases. Example of such intervention include hastening the healing or preventing chemotherapy- and radiotherapy-induced intestinal damage; to overcome apoptosis resistance in cancer cells; to retard the damage induced in inflammatory conditions such as inflammatory bowel disease, celiac disease, enteropathogenic infections and ischemia.

描述(由申请人提供)：增殖和凋亡之间的动态平衡对于肠道上皮作为生理和结构屏障的作用至关重要。肠上皮细胞有很高的细胞周转率，并伴随着同样高的凋亡率。这种正常的细胞凋亡对于肠上皮的分级组织是必不可少的，在隐窝的底部以及小肠和大肠的绒毛尖端都检测到了凋亡的上皮细胞。细胞凋亡的缺陷与几种胃肠道疾病有关，包括绒毛萎缩、上皮增生、正常吸收功能丧失和肿瘤发生风险增加。令人惊讶的是，关于细胞凋亡作为胃肠道上皮细胞在体内的重要作用机制的数据很少。在使用影响肌动蛋白周转的药物的研究中，有非常容易理解的数据支持真核细胞中肌动蛋白和细胞凋亡之间的联系。然而，很少有研究确定微丝重组和细胞存活之间的因果联系。绒毛蛋白是一种肌动蛋白调节蛋白，表达于小肠和大肠的胃肠上皮细胞以及与胃肠道相关的外分泌腺中。我们最近首次确定，绒毛蛋白是一种上皮细胞特异性的抗细胞凋亡蛋白。此外，我们的初步研究表明，在缺乏绒毛蛋白的情况下，这一功能可能由绒毛蛋白家族的一种相关的肌动蛋白结合蛋白明胶蛋白来完成。在细胞凋亡周期中，绒毛蛋白也会被切割，我们之前已经报道过，被切割的绒毛蛋白NH2末端片段是促凋亡的。利用酵母菌的两种杂交、生化和细胞方法，我们最近发现并验证了绒毛蛋白与线粒体蛋白-三磷酸腺苷合成酶2-亚基的新的相互作用，这使得我们能够研究微丝诱导的微丝调节线粒体形态和功能以及绒毛蛋白对上皮细胞生存的细胞和分子机制(S)。我们观察的新颖性将使我们第一次证明微丝调节与真核细胞中细胞存活之间的因果关系。为实现这一目标，我们将在三个主要的特定目标下进行实验：(I)鉴定绒毛蛋白抗和促凋亡功能的细胞和分子机制(S)，并分别确定全长和裂解的绒毛蛋白片段在维持胃肠稳态中的生理学相关性；(Ii)表征绒毛蛋白与三磷酸腺苷合成酶2亚单位的相互作用，以检测该复合体在维持胃肠稳态中的相关性；(Iii)表征绒毛蛋白-明胶蛋白双敲除小鼠，以确定绒毛蛋白及其同源蛋白凝胶在调节胃肠稳态中的作用。我们的研究对于合理设计新的更有效的治疗主要肠道上皮病如炎症性肠病和癌症的方法至关重要。 与公共卫生相关：肠道发挥着消化和吸收表面的基本作用，并构成最大的免疫器官。此外，肠道是具有巨大社会和经济影响的疾病的解剖位置，如炎症性肠病(在美国，每500人中约有1人受到影响)、结肠癌(发达国家第二常见的恶性肿瘤)和许多传染病。细胞凋亡在决定肠上皮细胞构筑中起着重要作用，也是肠上皮细胞对毒性刺激应激反应的一部分。在疾病的发病机制中，细胞凋亡可以不适当地过度或不足，这两者都与细菌、病毒和寄生虫感染、缺血再灌注损伤、炎症性肠病、乳糜泻和结直肠癌有关。细胞凋亡功能异常既是结直肠癌发病机制的重要因素，也是结直肠癌对化疗药物和放疗耐药的重要原因。对细胞凋亡的了解将有助于加强正常的生理和改善疾病。这类干预措施的例子包括加速愈合或防止化疗和放射导致的肠道损伤；克服癌细胞的凋亡抵抗；延缓炎症性肠病、乳糜泻、肠源性感染和缺血等炎症条件下造成的损害。