Cell Signalling and Stress Fiber Formation

极化上皮细胞向非极化间充质细胞的转化，也称为上皮细胞向间充质细胞的转变(EMT)，是一种赋予细胞更强的迁移、逃避凋亡和产生细胞外基质成分的生物过程。由于这些特点，EMT在许多生物学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过程。在EMT过程中，转化的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事件通常是由上皮生长因子和转化生长因子β(TGF)等配体诱导的。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。晶状体是一种独特而强大的模型，因为它是一个只由两种细胞类型组成的无血管器官，晶状体上皮细胞和晶状体纤维细胞，允许容易地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，它们失去了正常的透明性质，这是一种可以定量测量的生理反应。本实验室利用晶状体上皮细胞(LEC)、全晶状体和在体啮齿动物模型观察到，在缺乏基质金属蛋白酶9(MMP9)的情况下，LEC失去了形成转化生长因子诱导的应力纤维的能力，并经历了EMT。根据这些发现和其他初步结果，我们假设MMP9在EMT过程中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/小鼠晶状体移植的模型来进一步探讨这个问题。我们还将使用我们的体内方法，包括腺病毒转导转化生长因子(AdTGF)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.确定在转化生长因子诱导的应力纤维形成和EMT过程中依赖于MMP9的细胞信号分子。2.探讨MMP9和AIM 1中揭示的必需靶点对应力纤维形成和EMT的影响。3.确定MMP9表达的恢复或候选靶点的表达(来自Aim 1)是否会导致LEC形成应力纤维并接受EMT。总之，这些实验将为揭示MMP9介导的LEC EMT所必需的信号通路以及细胞细胞骨架的相关变化提供集体努力。研究结果将支持我们的长期目标，即了解EMT过程中调节细胞骨架重塑所需的信号级联(S)，因为它与许多生物事件有关，包括正常和病理的。

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.