Plakoglobin: A Novel Regulator of Motility in Keratinocytes

描述（由申请人提供）：在组织形态发生和伤口愈合过程中，上皮细胞重塑过程中，细胞-细胞和细胞-底物粘附的协调在调节运动和细胞内信号传导中起关键作用。桥粒蛋白血小板红蛋白（PG）在这些过程中成为一个重要的调节分子。PG不仅能增强细胞间的粘附并抑制角化细胞的运动，出乎意料的是，它还能抑制不与周围细胞接触的细胞的运动。Green实验室的初步数据表明，PG-null角质形成细胞在细胞-底物粘附结构、肌动蛋白组织和扩散方面表现出改变，这与单细胞运动性增加有关。基于这些观察，我们假设PG通过调节细胞-底物粘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PG依赖性的运动性抑制，我们提出验证PG通过隔离和/或抑制Src和相关信号伙伴来抑制运动性的观点。为此，我们提出：(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是否通过控制肌动蛋白细胞骨架的细胞底物依赖性参与来调节细胞运动；(3)阐明pg依赖性单细胞运动抑制的胞内信号传导机制。这些目标将通过使用小鼠PG-null和人类PG-困难以及对照角质形成细胞来实现，生化和视觉评估PG在整合素依赖性细胞底物粘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突变体来确定粘附和细胞骨架的调节与运动抑制之间是否存在机制联系。最后，我们将确定PG介导的Src抑制作为细胞内信号传导机制的一部分的潜在贡献，PG通过该机制控制调节细胞底物粘附、肌动蛋白细胞骨架重塑和细胞运动的关键分子。细胞-细胞和细胞底物的粘附负责维持组织的完整性，这种机制的破坏导致正常过程的缺陷，如伤口愈合以及自身免疫性、遗传性和传染性皮肤病和皮肤癌。PG是一种新兴的细胞-细胞和细胞-底物粘附调节因子，这些研究将为未来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.