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

项目总结 桥粒细胞-细胞黏附复合体在维持组织结构中起着重要作用 和完整性，以应对机械应力。除了这种中心粘连作用外，桥粒 蛋白质还协调多个过程，如增殖、凋亡、分化和细胞 迁移。先前的研究表明，桥粒钙粘附素是一种负责调节 细胞间的细胞外附着，可以同时具有促进和反迁移的功能，表明它们的 对迁移的影响比细胞-细胞连接缺陷的简单后果要复杂得多。我们的 最近的研究表明，钙粘附素桥粒蛋白-2(DSG2)的缺失触发了细胞外信号转导通路的激活 Rap1 GTP酶，导致细胞在细胞外基质(ECM)蛋白(如 纤维连接蛋白和胶原蛋白)。由于这些实验是在单个扩散的细胞上进行的，我们有 发现了DSG2在细胞调节中的一种新的细胞自主、细胞间黏附不依赖的作用 正在扩散。然而，DSG2调节Rap1活性的信号机制仍然存在 未知。我们的初步数据还表明，Rap1激活剂PDZ-GEF2的本地化程度很高 在DSG2基因敲除细胞中发生改变，桥粒蛋白桥粒蛋白(DSP)的敲除可以 挽救在这些细胞中看到的PDZ-GEF2的增强扩散和错误定位。此外，我们 已有证据表明，DSG2的缺失通过解除调控的Src和NF-kB增加ECM基因的表达 发信号。在这个提案中，我们将测试DSG2协调细胞基质的中心假设 通过控制内向外信号(通过Rap1 GEF)和自外向内信号来实现粘连和传播 信号传递(通过ECM基因表达)。在目标1中，我们将研究PDZ-GEF2与 DSG2或DSP降低其活性，从而通过抑制RAP1诱导由内而外的细胞扩散控制 发信号。在目标2中，我们将探索核因子-kB和Src信号在依赖DSG2的ECM基因中的作用 表达，以及这是否促进了对细胞基质扩散和迁移的由外向内的控制。最后，在 目的3，我们将探讨DSG2调节细胞迁移的能力独立于其自身的假设。 胞外结构域和/或定位到细胞膜。这些数据的科学影响将是 以全面了解桥粒钙粘附素的分子机制 DSG2协调由内向外和由外向内的信号，以控制细胞-基质的黏附、扩散和 迁移，对组织形态发生、伤口愈合和修复等生物过程至关重要的功能。 上皮化。这些数据的培训影响将为福曼大学的本科生提供 大学(主要是本科院校)具有科学方法方面的基本技能，培养他们的 渴望解决重要的生物学问题，并追求生物医学科学的职业生涯。

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.