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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761976
• 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 a-smooth muscle actin (aSMA). These EMT events are often induced by the ligands such as epithelial growth factor and transforming growth factor beta (TGFß). 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 TGFß-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 TGFß (AdTGFß) 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 TGFß-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充质细胞的转化，也称为上皮向间充质转化（EMT），是赋予细胞增强的迁移、逃避凋亡和产生细胞外基质组分的能力的生物学过程。由于这些特征，已知EMT在许多生物学机制中发挥关键作用，包括胚胎发生和病理学，如癌症和纤维化。在EMT期间，转化的细胞显示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α-平滑肌肌动蛋白（aSMA）组成的应力纤维。这些EMT事件通常由配体如上皮生长因子和转化生长因子β（TGF β）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眼透镜。眼透镜是一种独特且有效的模型，因为它是仅由两种细胞类型（透镜上皮细胞和透镜纤维细胞）组成的无血管器官，允许容易地分离和培养纯上皮细胞群。此外，当透镜上皮细胞经历EMT和应力纤维形成时，它们失去了它们正常的透明性质，这是一种可以定量测量的生理反应。我们实验室使用透镜上皮细胞（LEC）、整个透镜和体内啮齿动物模型观察到，在基质金属蛋白酶9（MMP 9）（一种降解细胞外基质的酶）不存在的情况下，LEC失去其形成TGF β诱导的应力纤维并经历EMT的能力。从这些发现，以及额外的初步结果，我们假设MMP 9在EMT期间的细胞骨架重塑和应力纤维形成中起关键作用。我们将进一步探讨这个问题，使用我们建立的模型，涉及切除大鼠透镜和大鼠/小鼠透镜外植体。我们还将采用我们的体内方法，包括腺病毒递送TGF β 1（AdTGF β 1）和转基因/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.探讨MMP 9在TGF β诱导的应力纤维形成和EMT中的作用。2.研究MMP 9和目标1中揭示的所需靶标如何影响应力纤维形成和EMT。3.确定MMP 9表达的恢复或候选靶点的表达（来自目标1）是否会导致LEC形成应力纤维并经历EMT。总之，这些实验将提供一个集体的努力，以揭示MMP 9介导的信号通路所必需的LEC EMT和相关的变化，在细胞骨架。研究结果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，因为它涉及许多正常和病理的生物学事件。