Modulation of Signal Transduction by Nano-Topography

描述（由申请人提供）：角膜混浊是世界上第三大致盲原因，可能是内皮营养不良、上皮营养不良、纤维化或因伤口愈合失调引起的基质中的haz的结果。角膜伤口愈合在很大程度上受细胞外环境的影响，通过可溶性信号和生物物理线索，如基质地形和细胞外基质硬度。我们实验室和其他人的研究已经证明，细胞外基质的内在生物物理属性深刻地调节细胞的许多基本表型特征，例如粘附、迁移、形状、大小和增殖，伴随着激活/抑制特异性信号传导途径。尽管生物物理线索的根本重要性，一个显着的知识差距仍然存在于我们的理解，这些生物物理线索如何影响基本的细胞反应和伤口愈合的角膜。在这个提议中，我们将确定介导角膜伤口愈合的信号通路的表达和定位影响。雅普（Yes相关蛋白）和TAZ（具有PDZ结合基序的转录共激活因子）作为一对连接细胞外环境变化（如地形或基质硬度变化）的转换器。这两种蛋白质处于影响伤口愈合的多种信号通路的交叉点。初步数据表明，角膜基质的硬度增加了10倍，在7天内受伤后的光治疗性角膜切除术（PTK）在兔。这与雅普/TAZ显著定位于上皮细胞的细胞核中，导致其转录激活生长因子如CTGF和TGF β有关。在角膜损伤后的前基质细胞中观察到雅普/TAZ的稳健表达。这导致了中心假设，即雅普/TAZ的表达和定位是受外部生物物理刺激影响的角膜细胞中信号传导事件的必要介质，并且是角膜伤口修复的基本部分。该建议将描述雅普/TAZ在体外角膜细胞中的影响（A），在体内兔角膜伤口愈合中的影响（B），以及 (C)雅普、TAZ和体内双基因敲除条件性敲除小鼠角膜伤口愈合情况。这些研究将确定上皮细胞中由外部生物物理线索介导的受雅普/TAZ影响的信号传导途径，还将鉴定伤口愈合期间基质中发生的变化。

DESCRIPTION (provided by applicant): Corneal opacities, the third major cause of blindness in the world, may be a result of endothelial dystrophies, epithelial dystrophies, fibrosis or a haz in the stroma due to dysregulated wound healing. Corneal wound healing is largely influenced by the extracellular environment both through soluble signals and from biophysical cues such as substratum topography and extracellular matrix stiffness. Research from our lab and others have demonstrated intrinsic biophysical attributes of the extracellular matrix profoundly modulate a host of fundamental phenotypic characteristics of cells such as adhesion, migration, shape, size and proliferation accompanied by activation/inhibition specific signaling pathways. Despite the fundamental importance of biophysical cues, a significant knowledge gap remains in our understanding of how these biophysical cues influence basic cellular responses and wound healing in the cornea. In this proposal we will determine the expression and localization impact of signaling pathways that mediate corneal wound healing. YAP (Yes- associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif) serve as a pair of transducers linking changes in the extracellular environment, such as a change in topography or of matrix stiffness. These two proteins are at the intersection of multiple signaling pathways that influence wound healing. Preliminary data demonstrate that the stiffness of the corneal stroma increases by 10-fold within 7 days after wounding by phototherapeutic keratectomy (PTK) in rabbits. This was associated with YAP/TAZ being markedly localized in the nucleus of epithelial cells resulting in their transcriptional activation of growth factors such as CTGF and TGF¿. A robust expression of YAP/TAZ was observed in the anterior stromal cells in vivo following corneal wounding. This leads to the central hypothesis that expression and localization of YAP/TAZ are essential mediators of signaling events in corneal cells influenced by external biophysical stimuli and are a fundamental part of corneal wound repair. This proposal would delineate the influence of YAP/TAZ in corneal cells (A) in vitro, (B) in the healing of corneal wounds in rabbits in vivo, and (C) the healing of corneal wounds in conditional knockout mice for YAP, TAZ and the double knockout in vivo. These studies will define the signaling pathways influenced by YAP/TAZ mediated by external biophysical cues in the epithelial cells and will also identify changes that occur in the stroma during wound healing.