Modulation of Signal Transduction by Nano-Topography

描述（由申请人提供）：角膜混浊是世界上致盲的第三大原因，可能是由于内皮细胞营养不良、上皮细胞营养不良、纤维化或由于伤口愈合失调导致的间质模糊。角膜创面愈合在很大程度上受细胞外环境的影响，包括可溶性信号和生物物理信号，如基质地形和细胞外基质刚度。我们的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了细胞的许多基本表型特征，如粘附、迁移、形状、大小和增殖，并伴随着激活/抑制特定的信号通路。尽管生物物理信号具有基本的重要性，但我们对这些生物物理信号如何影响角膜的基本细胞反应和伤口愈合的理解仍然存在重大的知识差距。在这个建议中，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。YAP （Yes相关蛋白）和TAZ（带pdz结合基序的转录共激活因子）作为一对传感器连接细胞外环境的变化，如地形或基质刚度的变化。这两种蛋白是影响伤口愈合的多种信号通路的交叉点。初步数据表明，兔角膜光疗性角膜切除术（PTK）损伤后7天内角膜基质硬度增加10倍。这与YAP/TAZ明显定位于上皮细胞的细胞核，导致其转录激活生长因子，如CTGF和TGF¿有关。角膜损伤后，YAP/TAZ在角膜前基质细胞中有较强的表达。这导致了一个中心假设，即YAP/TAZ的表达和定位是受外部生物物理刺激影响的角膜细胞信号事件的重要介质，也是角膜伤口修复的基本组成部分。本研究将阐述YAP/TAZ对角膜细胞(A)体外、(B)兔角膜创面愈合的影响，以及YAP/TAZ对角膜创面愈合的影响

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.