The cadherin Desmoglein-2 controls cell spreading and extracellular matrix gene expression.

项目摘要 桥粒细胞-细胞粘附复合体在维持组织结构中起重要作用 以及对机械应力的响应的完整性。除了这种中心粘附作用，桥粒 蛋白质还协调多个过程，如增殖、凋亡、分化和细胞凋亡。 迁移先前的研究表明，桥粒钙粘蛋白，负责介导细胞凋亡的蛋白质， 细胞之间的细胞外附着，可以具有促迁移和抗迁移功能，表明它们的 对迁移的影响比细胞间附着缺陷的简单后果更为复杂。我们 最近的研究表明，钙粘蛋白桥粒芯糖蛋白-2（DSG 2）的缺失触发了细胞外基质的激活， Rap 1 GT3蛋白，导致细胞在细胞外基质（ECM）蛋白（如 纤连蛋白和胶原蛋白）。由于这些实验是在单铺展细胞上进行的，我们有 确定了DSG 2在调节细胞凋亡中的一种新的细胞自主的、细胞-细胞粘附独立的作用。 蔓延然而，DSG 2调节Rap 1活性的信号传导机制仍然存在， 未知我们的初步数据还表明Rap 1激活剂PDZ-GEF 2的定位是显着的， 在DSG 2敲除细胞中改变，并且桥粒蛋白桥粒斑蛋白（DSP）的敲除可以 挽救了在这些细胞中观察到的PDZ-GEF 2的增强的扩散和错误定位。我们还 已经获得证据表明DSG 2的缺失通过Src和NF-kB的失调增加ECM基因表达 发信号。在这个建议中，我们将测试DSG 2协调细胞基质的中心假设， 通过控制由内而外的信号传导（通过Rap 1 GEF）和由外向内的信号传导， 信号传导（通过ECM基因表达）。在目的1中，我们将研究PDZ-GEF 2与PDZ-GEF 2的结合是否与PDZ-GEF 2的结合有关。 DSG 2或DSP降低其活性，从而通过抑制Rap 1诱导细胞扩散的由内而外控制 发信号。在目的2中，我们将探讨NF-kB和Src信号在DSG 2依赖性ECM基因中的作用， 表达，以及这是否促进细胞基质扩散和迁移的由外向内控制。最后在 目的3，我们将探讨DSG 2调节细胞迁移的能力不依赖于其自身的能力的假说。 细胞外结构域和/或定位于细胞膜。这些数据的科学影响将是 为了全面了解桥粒钙粘蛋白 DSG 2协调由内向外和由外向内的信号传导，以控制细胞-基质粘附、铺展和粘附。 迁移，对生物过程如组织形态发生、伤口愈合和再形成至关重要的功能。 上皮化这些数据的培训影响将为弗曼大学的本科生提供 大学（主要是本科院校）在科学方法的基础技能，培养他们的 渴望解决重要的生物学问题，并在生物医学科学领域寻求职业生涯。

PROJECT SUMMARY The desmosome cell-cell adhesion complex plays an important role in the maintenance of tissue structure and integrity in response to mechanical stress. In addition to this central adhesive role, desmosomal proteins also coordinate multiple processes such as proliferation, apoptosis, differentiation and cell migration. Prior studies show that desmosomal cadherins, the proteins responsible for mediating the extracellular attachment between cells, can have both pro- and anti-migratory functions, indicating that their influence on migration is more complex than a simple consequence of defective cell-cell attachment. Our recent work has shown that loss of the cadherin Desmoglein-2 (DSG2) triggers increased activation of the Rap1 GTPase, leading to enhanced cell spreading on extracellular matrix (ECM) proteins (such as fibronectin and collagen). As these experiments were performed on singly spreading cells, we have identified a novel cell-autonomous, cell-cell adhesion independent role for DSG2 in the regulation of cell spreading. Nevertheless, the signaling mechanisms via which DSG2 regulates Rap1 activity remain unknown. Our preliminary data also shows that localization of the Rap1 activator PDZ-GEF2 is dramatically altered in DSG2 knockout cells, and that knockdown of the desmosomal protein Desmoplakin (DSP) can rescue both the enhanced spreading and mis-localization of PDZ-GEF2 seen in these cells. Further, we have obtained evidence that loss of DSG2 increases ECM gene expression via deregulated Src and NF-kB signaling. In this proposal, we will test the central hypothesis that DSG2 orchestrates cell-matrix adhesion and spreading through control of both inside-out signaling (via Rap1 GEFs) and outside-in signaling (via ECM gene expression). In Aim 1, we will investigate whether binding of PDZ-GEF2 to DSG2 or DSP reduces its activity, thereby inducing inside-out control of cell spreading via inhibition of Rap1 signaling. In Aim 2, we will explore a role for NF-kB and Src signaling in DSG2-dependent ECM gene expression, and whether this promotes outside-in control of cell-matrix spreading and migration. Finally, in Aim 3, we will explore the hypothesis that the ability of DSG2 to regulate cell migration is independent of its extracellular domains and/or localization to the cell membrane. The scientific impact of these data will be to produce a comprehensive picture of the molecular mechanisms by which the desmosomal cadherin DSG2 coordinates both inside-out and outside-in signaling to control cell-matrix adhesion, spreading and migration, functions critical for biological processes such as tissue morphogenesis, wound healing and re- epithelialization. The training impact of these data will be to provide undergraduate students at Furman University (a primarily undergraduate institution) with foundational skills in the scientific method, foster their desire to tackle important biological problems and pursue a career in the biomedical sciences.