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: 8235412
• 负责人: Seema Khurana
• 金额: $ 30.18万
• 依托单位: UNIVERSITY OF HOUSTON
• 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 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8235412
• 关键词: Regulation; epithelial; cell; homeostasis; actin

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）：增殖和凋亡之间的稳态平衡对于肠上皮作为生理和结构屏障发挥作用至关重要。肠上皮细胞具有高的细胞更新率，伴随着同样高的细胞凋亡率。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，并且已经在隐窝的基部以及小肠和大肠的绒毛尖端检测到凋亡的上皮细胞。细胞凋亡缺陷与几种胃肠道疾病相关，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，几乎没有关于细胞凋亡作为胃肠道上皮细胞体内重要作用机制的数据。有非常容易理解的数据支持肌动蛋白和细胞凋亡之间的联系，在真核细胞的研究中使用的药物，影响肌动蛋白营业额。然而，很少有研究已经建立了微丝重组和细胞存活之间的因果关系。绒毛蛋白是在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表达的肌动蛋白调节蛋白。我们最近首次确定绒毛蛋白是一种上皮细胞特异性抗凋亡蛋白。此外，我们的初步研究表明，在缺乏绒毛蛋白，这一功能可能是由一个相关的肌动蛋白结合蛋白的绒毛蛋白家族，凝溶胶蛋白。在凋亡周期中绒毛蛋白也被切割，我们以前曾报道过绒毛蛋白的NH 2-末端片段被切割是促凋亡的。利用酵母双杂交、生物化学和细胞方法，我们最近鉴定并验证了绒毛蛋白与线粒体蛋白ATP合成酶2-亚基的新型相互作用，这使我们能够研究绒毛蛋白对线粒体形态和功能以及上皮细胞存活的微丝诱导调节的细胞和分子机制。我们观察的新奇将使我们能够首次证明真核细胞中微丝调节和细胞存活之间的因果关系。为了实现这一点，将在三个主要的具体目标下进行实验：（i）表征绒毛蛋白的抗凋亡和促凋亡功能的细胞和分子机制，并分别鉴定全长和切割的绒毛蛋白片段在维持胃肠道稳态中的生理相关性;（ii）研究绒毛蛋白与ATP合成酶2-亚基的相互作用，以检测该复合物在维持胃肠道稳态中的相关性;（iii）表征绒毛蛋白-凝溶胶蛋白双基因敲除小鼠，以确定绒毛蛋白及其同源蛋白凝溶胶蛋白在调节胃肠道稳态中的作用。我们的研究对于合理设计新的和更有效的治疗主要肠上皮疾病，如炎症性肠病和癌症至关重要。 公共卫生相关性：肠道作为消化和吸收表面发挥重要作用，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人受影响）、结肠癌（发达国家中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的结构中起重要作用，也是肠上皮细胞对毒性刺激的应激反应的一部分。在疾病发病机制中，细胞凋亡可以是不适当的过度或不足，并且这两者都涉及细菌、病毒和寄生虫感染、缺血-再灌注损伤、炎性肠病、乳糜泻和结肠直肠癌。细胞凋亡功能异常不仅是结直肠癌的发病机制之一，也是结直肠癌对化疗和放疗产生耐药性的重要原因。对细胞凋亡的了解将有助于增强正常生理功能和改善疾病。这种干预的实例包括加速愈合或预防化疗和放疗诱导的肠损伤;克服癌细胞中的凋亡抗性;延缓在炎性病症如炎性肠病、乳糜泻、肠致病性感染和局部缺血中诱导的损伤。