Plakoglobin: A Novel Regulator of Motility in Keratinocytes

描述(申请人提供)：在组织形态形成和伤口愈合等过程中，细胞-细胞和细胞-基质黏附的协调在调节上皮细胞重塑过程中的运动性和细胞内信号起着关键作用。桥粒蛋白(PG)在这些过程中是一个重要的调节分子。PG增强了细胞与细胞之间的粘附力，不仅抑制了接触的角质形成细胞的运动，而且出人意料地抑制了不与邻近细胞接触的细胞的运动。格林实验室的初步数据表明，PG缺失的角质形成细胞表现出细胞-底物黏附结构、肌动蛋白组织和扩散的变化，这些变化与单细胞运动性增加相关。基于这些观察，我们假设PG通过调节细胞-底物黏附和细胞骨架重组来抑制角质形成细胞的运动。此外，由于初步数据表明，Src或Src家族激酶参与了PG依赖的运动性抑制，我们建议测试PG通过隔离和/或抑制Src及其相关信号伙伴来抑制运动性的想法。为此，我们建议：(1)确定PG是否调节整合素介导的角质形成细胞-底物相互作用；(2)确定PG是否通过控制细胞-底物依赖的肌动蛋白骨架的参与来调节细胞的运动；(3)阐明PG依赖抑制单细胞运动的细胞内信号机制。这些目标将通过使用小鼠PG缺失和人PG缺乏以及对照角质形成细胞来实现，以生化和可视的方式评估PG在整合素依赖的细胞底物黏附和肌动蛋白细胞骨架重塑中的作用。此外，我们将确定参与这些过程的关键分子的活性和细胞内分布。然后，我们将使用现有的PG突变体来确定黏附调节与细胞骨架和运动抑制之间是否存在机械联系。最后，我们将确定PG介导的对Src的抑制作为细胞内信号机制的一部分，PG通过该机制控制调控细胞-底物黏附、肌动蛋白细胞骨架重塑和细胞运动的关键分子。细胞-细胞和细胞基质黏附负责维持组织的完整性，这一机制的破坏会导致正常过程中的缺陷，如伤口愈合、自身免疫、遗传性和感染性皮肤病和皮肤癌。这些拟议的研究集中在PG，一种新兴的细胞-细胞和细胞-基质黏附调节因子，将为未来的上皮疾病的诊断和治疗策略提供基础。

DESCRIPTION (provided by applicant): The coordination of cell-cell and cell-substrate adhesion plays a key role in regulating motility and intracellular signaling during epithelial cell remodeling that occurs in processes such as tissue morphogenesis and wound healing. The desmosomal protein plakoglobin (PG) is emerging as an important regulatory molecule in these processes. PG strengthens cell-cell adhesion and suppresses motility not only of keratinocytes in contact, but also, unexpectedly, cells not in contact with their neighbors. Preliminary data in the Green lab indicates that PG-null keratinocytes exhibit alterations in cell-substrate adhesion structures, actin organization and spreading that correlate with increased single cell motility. Based on these observations, we hypothesize that PG inhibits keratinocyte motility by regulating cell-substrate adhesion and cytoskeleton reorganization. Further, as preliminary data suggest that Src or Src family kinases are involved in PG-dependent suppression of motility, we propose to test the idea that PG suppresses motility through sequestration and/or inhibition of Src and associated signaling partners. Towards this end we propose: (1) To determine whether PG regulates integrin-mediated keratinocyte cell-substrate interaction; (2) To establish whether PG regulates cell motility by controlling cell-substrate dependent engagement of the actin cytoskeleton; (3) To elucidate the intracellular signaling mechanism of PG-dependent suppression of single cell motility. These aims will be accomplished by using murine PG-null and human PG-difficient as well as control keratinocytes, to biochemically and visually assess the role of PG in integrin-dependent cell substrate adhesion and actin cytoskeleton remodeling. Further, we will determine the activity and intracellular distribution of key molecules involved in these processes. We will then use the existing PG mutants to establish whether there is a mechanistic link between the regulation of adhesion and cytoskeleton and motility suppression. Finally, we will determine the potential contribution of PG-mediated inhibition of Src as a part of the intracellular signaling mechanism by which PG controls key molecules regulating cell-substrate adhesion, actin cytoskeleton remodeling and cell motility. Cell-cell and cell substrate adhesion are responsible for maintaining tissue integrity, and breakdown of this mechanism leads to defects in normal processes such as wound healing as well as autoimmune, inherited and infectious skin disease and cutaneous cancer. The proposed studies focused on PG, an emerging regulator of cell-cell and cell-substrate adhesion, will provide a basis for the future diagnostic and therapeutic strategies in epithelial diseases.