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的分层上皮层，是抵御微生物侵入眼睛的第一道防线，并具有对视力至关重要的光滑屈光表面。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，导致缺乏光学清晰度和失明。因此，为了提供一个合理的和健全的方法来发展和治疗的环境压力和疾病，它是至关重要的，以确定和理解的细胞和分子机制的角膜上皮稳态。有足够的证据表明核转录因子Np 63的亚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。使用具有正常遗传学和分化的人角膜上皮的独特的新器官型细胞模型（hTCEpi细胞），本申请通过三个特定的实验目的测试了新的统一假设，即Np 63通过IGFBP 3的转录抑制来调节角膜上皮中BCL 2介导的凋亡性细胞死亡：（1）表征IGFBP 3在亚汇合和汇合培养物中的Np 63同种型特异性抑制以及钙诱导的分化（CHiP、WB、使用延时共聚焦显微镜的实时PCR和报告基因测定）;确定分化和凋亡过程中Np 63亚型对IGFBP 3转录的影响（siRNA、过表达构建体、使用延时共聚焦显微镜的报告基因测定、器官型培养、TUNEL和膜联蛋白V）;并表征TSA处理后Np 63同种型的比率（2）建立IGFBP 3的膜定位和亚细胞运输（细胞分级分离和WB，以及使用延时共聚焦显微镜的生物素化IGFPB 3-链霉亲和素缀合的量子点）;并表征二聚化和糖基化在亚细胞运输和凋亡中的作用（BiFC、定点诱变、细胞分级分离、WB和膜联蛋白V）（3）确定IGFBP 3的异位表达和siRNA敲低对亚细胞动力学的影响，BCL 2和BAX的运动和凋亡效应（FRAP、FLIP、SDM、细胞分级分离、IP、凋亡测定、WB）。这项研究的重要性得到了角膜上皮中IGFBP 3的首次直接证明以及先进成像方法的发展和评价的支持：FRAP，FLIP，BiFC和量子点标记，允许在纳米分辨率水平上直接4-D可视化单细胞中的核/细胞质事件，从而代表了在角膜细胞中分子水平上观察基因调控的新范式。公共卫生保护：保护角膜健康是预防失明的重要第一步。这项研究的总体目标是确定负责维持角膜外表面细胞的关键基因。从这项工作中获得的信息将允许开发新的治疗方法，用于因角膜疾病而视力受损的患者，新的策略，以改善接触透镜佩戴的健康状况，以及恢复那些患有细胞损失的患者的视力。

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.