Cell Signalling and Stress Fiber Formation

细胞信号传导和应力纤维形成

• 批准号: RGPIN-2019-06393
• 负责人: WestMays, Judith
• 金额: $ 2.62万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=686537
• 关键词: Cell; Signalling; Stress; Fiber; Formation

***Transformation of polarized epithelial cells to non-polarized mesenchymal cells, also known as epithelial to mesenchymal transition (EMT) is a biological process that imparts cells with enhanced capabilities of migration, evasion of apoptosis, and production of extracellular matrix components. Due to these features, EMT is known to play a critical role in numerous biological mechanisms including embryogenesis and pathologies such as cancer and fibrosis. During EMT transformed cells show extensive cytoskeletal remodeling including the acquisition of stress fibers composed of F-actin and the contractile protein -smooth muscle actin (SMA). These EMT events are often induced by the ligands such as epithelial growth factor and transforming growth factor beta (TGF). The model that we have used to investigate stress fiber formation and EMT is the rodent ocular lens. The ocular lens is a unique and powerful model in that it is an avascular organ consisting of only two cell types, lens epithelial cells and lens fiber cells, permitting easy isolation and culturing of a pure epithelial cell population. Additionally, when lens epithelial cells undergo EMT and stress fiber formation they loose their normal transparent nature, which is a physiological response that can be measured quantitatively. Using lens epithelial cell (LEC), whole lens and in vivo rodent models our lab made the observation that in the absence of matrix metalloproteinase 9 (MMP9), an enzyme that degrades the extracellular matrix, LEC loose their ability to form TGF-induced stress fibers and undergo EMT. From these findings, and additional preliminary results, we hypothesize that MMP9, plays a critical role in cytoskeletal remodeling and stress fiber formation during EMT. We will further explore this question using our established models involving the excised rat lens and rat/mouse lens explants. We will also employ our in vivo approaches including adenoviral delivery of TGF (AdTGF) and transgenic/knockout mouse models, all of which are unique to our laboratory. Our specific aims are: 1. To determine the cell signaling molecules dependent on MMP9 in TGF-induced stress fiber formation and EMT. 2. To investigate how MMP9 and required targets revealed in Aim 1 impact stress fiber formation and EMT. 3. To determine if restoration of MMP9 expression, or expression of candidate targets (from Aim 1) will cause LEC to form stress fibers and undergo EMT. Together these experiments will provide a collective effort to reveal the MMP9-mediated signaling pathways necessary for LEC EMT and associated changes in the cell cytoskeleton. Findings generated will support our long-term goal to understand the signaling cascade(s) required for regulating cytoskeletal remodeling during EMT as it pertains to numerous biological events, both normal and pathological. *****************

***极化上皮细胞向非极化间质细胞的转化，也称为上皮间质转化（EMT）是一个生物过程，它赋予细胞增强的迁移、逃避细胞凋亡和产生细胞外基质成分的能力。由于这些特征，EMT 在许多生物机制中发挥着关键作用，包括胚胎发生和癌症和纤维化等病理学。在 EMT 过程中，转化细胞表现出广泛的细胞骨架重塑，包括获得由 F-肌动蛋白和收缩蛋白 - 平滑肌肌动蛋白 (SMA) 组成的应力纤维。这些 EMT 事件通常由上皮生长因子和转化生长因子 β (TGF) 等配体诱导。我们用来研究应力纤维形成和 EMT 的模型是啮齿动物的晶状体。眼晶状体是一种独特而强大的模型，因为它是一种无血管器官，仅由两种细胞类型（晶状体上皮细胞和晶状体纤维细胞）组成，可以轻松分离和培养纯上皮细胞群。此外，当晶状体上皮细胞经历 EMT 和应力纤维形成时，它们会失去正常的透明性质，这是一种可以定量测量的生理反应。我们的实验室使用晶状体上皮细胞 (LEC)、整个晶状体和体内啮齿动物模型观察到，在缺乏基质金属蛋白酶 9 (MMP9)（一种降解细胞外基质的酶）的情况下，LEC 失去了形成 TGF 诱导的应激的能力纤维并进行 EMT。根据这些发现和其他初步结果，我们假设 MMP9 在 EMT 期间的细胞骨架重塑和应力纤维形成中发挥关键作用。我们将使用我们建立的涉及切除的大鼠晶状体和大鼠/小鼠晶状体外植体的模型进一步探讨这个问题。我们还将采用我们的体内方法，包括 TGF (AdTGF) 的腺病毒传递和转基因/基因敲除小鼠模型，所有这些都是我们实验室独有的。我们的具体目标是： 1. 确定在TGF诱导的应激纤维形成和EMT中依赖于MMP9的细胞信号分子。 2. 研究目标 1 中揭示的 MMP9 和所需目标如何影响应力纤维形成和 EMT。 3. 确定 MMP9 表达或候选靶标（来自目标 1）的表达的恢复是否会导致 LEC 形成应力纤维并经历 EMT。这些实验将共同努力揭示 LEC EMT 所需的 MMP9 介导的信号通路以及细胞骨架的相关变化。所产生的发现将支持我们的长期目标，即了解 EMT 期间调节细胞骨架重塑所需的信号级联，因为它与许多正常和病理性的生物事件有关。 ******************