P63/IGFBP3/BCL2 Control of Corneal Epithelial Homeostasis

描述（由研究者提供）：角膜上皮是一种自我更新、分层的上皮层，它提供了抵御微生物侵入眼睛的第一道防线，也是视力所必需的光滑屈光表面。临床上，无数的环境损伤和疾病病理可能改变上皮的自然更新过程，导致光学清晰度不足和失明。因此，为了为环境应激和疾病的发展和治疗提供一个合理和健全的方法，识别和了解角膜上皮稳态的细胞和分子机制至关重要。有足够的证据表明，核转录因子Np63的同工型在影响这些过程中具有调节作用；然而，它们的确切功能尚不清楚。利用一种独特的具有正常遗传和分化的人角膜上皮细胞（hTCEpi细胞）的新器官型细胞模型，本应用程序通过三个特定的实验目的来验证Np63通过转录抑制IGFBP3调节BCL2介导的角膜上皮细胞凋亡的新统一假设：(1)表征Np63亚型特异性抑制IGFBP3在亚融合、融合培养和钙诱导分化中的作用（CHiP、WB、real-time PCR和延时共聚焦显微镜报告基因检测）；确定分化和凋亡过程中Np63亚型对IGFBP3转录的影响（siRNA、过表达构建体、延时共聚焦显微镜报告基因检测、器官型培养、TUNEL和Annexin V）；并表征TSA处理后Np63亚型的比例（siRNA、过表达构建体和WB）；(2)建立IGFBP3的膜定位和亚细胞运输（细胞分离和WB，以及生物素化的igfpb3 -链霉亲和素共轭量子点，使用延时共聚焦显微镜）；并表征二聚化和糖基化在亚细胞运输和凋亡中的作用（BiFC，定点突变，细胞分离，WB和Annexin V）(3)确定IGFBP3异位表达和siRNA敲低对BCL2和BAX亚细胞动力学，运动和凋亡效应的影响（FRAP， FLIP， SDM，细胞分离，IP，凋亡测定，WB）。IGFBP3在角膜上皮中的首次直接证明，以及先进成像方法的发展和赞赏：FRAP， FLIP， BiFC和量子点标记，允许在纳米分辨率水平上对单个细胞的核/细胞质事件进行直接的4-D可视化，从而代表了在角膜细胞分子水平上观察基因调控的新范式，支持了本研究的意义。公共卫生关系：保护角膜健康是预防失明必不可少的第一步。这项研究的总体目标是确定负责维持覆盖角膜外表面的细胞的关键基因。从这项工作中获得的信息将有助于为因角膜疾病而视力受损的患者开发新的治疗方法，通过佩戴隐形眼镜改善健康的新策略，以及为遭受细胞损失的患者恢复视力。

DESCRIPTION (provided by Investigator): The corneal epithelium is a self-renewing, stratified epithelial sheet that provides the first-line of defense against microorganisms invading the eye and a smooth refractive surface essential for vision. Clinically, a myriad of environmental insults and disease pathologies may alter the natural renewal process of the epithelium, resulting in a lack of optical clarity and blindness. Therefore, in order to provide a rational and sound approach to the development and treatment of environmental stresses and disease, it is critically important to identify and understand the cellular and molecular mechanism(s) of corneal epithelial homeostasis. Sufficient evidence exists to suggest that isoforms of the nuclear transcription factor Np63 have a regulatory role in effecting these processes; however, their exact function is unknown. Using a unique new organotypic cell model of human corneal epithelium with normal genetics and differentiation (hTCEpi cells), this application tests the novel unifying hypothesis that Np63 regulates BCL2 directed apoptotic cell death in the corneal epithelium by transcriptional repression of IGFBP3 through three specific experimental aims: (1) Characterize Np63 isoform specific repression of IGFBP3 in subconfluent and confluent culture and calcium-induced differentiation (CHiP, WB, real-time PCR and reporter assays using time-lapse confocal microscopy); determine the effect of Np63 isoforms during differentiation and apoptosis on IGFBP3 transcription (siRNA, over-expression constructs, reporter assays using time-lapse confocal microscopy, organotypic culture, TUNEL and Annexin V); and characterize the ratio of Np63 isoforms following TSA treatment (siRNA, over-expression constructs, and WB); (2) Establish membrane localization and subcellular trafficking of IGFBP3 (cell fractionation and WB, and biotinylated IGFPB3-streptavidin-conjugated quantum dots using time-lapse confocal microscopy); and characterize the role of dimerization and glycosylation in subcellular trafficking and apoptosis (BiFC, site-directed mutagenesis, cell fractionation, WB, and Annexin V (3) Determine the effects of ectopic expression and siRNA knockdown of IGFBP3 on the subcellular kinetics, movement, and apoptotic effect(s) of BCL2 and BAX (FRAP, FLIP, SDM, cell fractionation, IP, apoptotic assays, WB). The significance of this research is supported by the first direct demonstration of IGFBP3 in the corneal epithelium, and the development and appreciation of advanced imaging methods: FRAP, FLIP, BiFC, and quantum dot labeling, allowing direct 4-D visualization of nuclear/cytoplasmic events in single cells at nano-resolution levels, thus representing a new paradigm to view gene regulation at the molecular level in corneal cells. PUBLIC HEALTH RELANCE: The preservation of corneal health is an essential first step in preventing blindness. The overall goal of this research is to identify critical genes responsible for maintaining the cells covering the outer corneal surface. Information gained from this work will allow for development of new treatments for patients with compromised vision due to corneal disease, new strategies for better health from contact lens wear, and the restoration of sight to those suffering from cell loss.