E-cadherin Mechanotransduction, Pluripotency and the Warburg Effect

E-钙粘蛋白机械转导、多能性和 Warburg 效应

• 批准号: RGPIN-2016-06506
• 负责人: Rancourt, Derrick
• 金额: $ 2.4万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710312
• 关键词: cadherin; Mechanotransduction; Pluripotency; Warburg; Effect

BACKGROUND. Embryonic stem cells (ESCs) are pluripotent meaning they have the ability to differentiate into any cell type in the body. We have observed that the pluripotency of murine (m)ESCs is promoted when mESC aggregates are stirred in bioreactors. Pluripotency is maintained even when the maintenance factor LIF is removed. Moreover, cellular reprogramming, the process of dedifferentiating cells to pluripotency, occurs 1000-fold more efficiently in the bioreactor. Our preliminary data indicates that the pluripotency transcription factor -catenin is translocated into the nucleus in response to fluid shear caused by stirring. Shear stress can modulate gene expression through mechano-transduction. In one pathway, fluid shear leads to the nuclear translocation of -catenin. Translocation is thought to occur when shear is sensed by E-cadherin, a protein responsible for cell-cell interaction and aggregate formation. In response to shear, -catenin may be displaced by vinculin, whose role is to strengthen cell architecture by tethering E-cadherin to the cytoskeleton. During cellular reprogramming, cellular metabolism shifts from oxidative phosphorylation (i.e. mitochondrial) to aerobic glycolysis (non-mitochondrial). Recently -catenin has also been shown to promote this so called Warburg effect in cancer cells by upregulating glycolytic genes. OBJECTIVES. Using the following objectives, we will test if fluid shear promotes mESC pluripotency and cellular reprograming in the bioreactor via mechanotransduction: 1. -catenin Mechanotransduction. Using flow chambers and molecular assays, in combination with pharmacological and genetic approaches, we will determine if -catenin is released from E-cadherin and translocates into the nucleus in reponse to shear. 2. E-cadherin Mechanoreception. Using magnetic twisting cytometry and molecular assays, in combination with pharmacological and genetic approaches, we will determine if shear applied to E-cadherin results in -catenin nuclear translocation. 3. Pluripotency and Warburg Effect. Using pharmacological and genetic approaches, we will determine if -catenin mechanotransduction promotes pluripotency and aerobic glycolysis during reprogramming and expansion in the bioreactor. IMPACT. Government investment in pluripotent stem cell (PSC) biobanking suggests bioreactors will become a future application standard. As leaders in the bioreactor expansion of PSCs, we are positioned to make a significant impact. Understanding the mechanism behind “mechano-pluripotency” will enable the development of more efficient and affordable bioprocesses for producing PSCs and their cell/tissue derivatives. We will continue to train students and postdocs in the research process, while simultaneously helping them to develop leadership and professional skills through experiential learning and knowledge translation.

背景胚胎干细胞（ESC）是多能性的，这意味着它们有能力分化成体内任何类型的细胞。我们已经观察到，当在生物反应器中搅拌mESC聚集体时，促进了鼠（m）ESC的多能性。即使在去除维持因子LIF时，也维持多能性。此外，细胞重编程，即细胞去分化为多能性的过程，在生物反应器中效率提高1000倍。我们的初步数据表明，多能性转录因子-连环蛋白是易位到细胞核中的流体剪切引起的搅拌反应。 剪切应力可以通过机械力转导调节基因表达。在一个途径中，流体剪切导致β-连环蛋白的核转位。易位被认为发生在剪切被E-钙粘蛋白（一种负责细胞间相互作用和聚集体形成的蛋白质）感知时。在响应剪切，-catenin可能会被纽蛋白，其作用是加强细胞结构的拴系E-钙粘蛋白的细胞骨架。 在细胞重编程期间，细胞代谢从氧化磷酸化（即线粒体）转变为有氧糖酵解（非线粒体）。最近，连环蛋白也被证明通过上调糖酵解基因促进癌细胞中的这种所谓的瓦尔堡效应。 目标.使用以下目标，我们将测试流体剪切是否通过机械转导促进mESC多能性和生物反应器中的细胞重编程： 1. - 连环蛋白机械转导。使用流动室和分子检测，结合药理学和遗传学的方法，我们将确定是否-连环蛋白释放的E-钙粘蛋白和易位到细胞核中的响应剪切。 2. E-钙粘蛋白机械感受性。使用磁扭转细胞仪和分子检测，结合药理学和遗传学的方法，我们将确定是否剪切施加到E-钙粘蛋白的结果在-连环蛋白核转位。 3.多能性和瓦尔堡效应。使用药理学和遗传学的方法，我们将确定是否-连环蛋白mechanotransduction促进多能性和有氧糖酵解过程中的生物反应器中的重编程和扩展。 冲击政府对多能干细胞（PSC）生物库的投资表明，生物反应器将成为未来的应用标准。作为PSC生物反应器扩张的领导者，我们有能力产生重大影响。了解“机械多能性”背后的机制将能够开发更有效和负担得起的生物过程来生产PSC及其细胞/组织衍生物。我们将继续在研究过程中培训学生和博士后，同时帮助他们通过体验式学习和知识翻译来发展领导力和专业技能。