P63/IGFBP3/BCL2 Control of Corneal Epithelial Homeostasis

描述(由Investigator提供)：角膜上皮是一种自我更新的复层上皮膜，它提供了抵御微生物入侵眼睛的第一道防线，并提供了一个对视力至关重要的平滑屈光表面。在临床上，无数的环境侮辱和疾病病理可能会改变上皮细胞的自然更新过程，导致视觉上皮细胞缺乏透明度和失明。因此，为了为环境应激和疾病的发展和治疗提供一种合理和健全的方法，识别和理解角膜上皮细胞和分子机制(S)至关重要。有足够的证据表明，核转录因子Np63的异构体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。使用一种独特的具有正常遗传和分化的人角膜上皮细胞模型(hTCEpi细胞)，本应用验证了新的统一假设，即Np63通过三个特定的实验目的通过转录抑制IGFBP3来调控BCL2诱导角膜上皮细胞凋亡：(1)在亚融合和融合培养和钙诱导分化中表征Np63异构体对IGFBP3的特异性抑制(ChIP、WB、实时荧光聚合酶链式反应和报告基因分析)；确定Np63异构体在分化和凋亡过程中对IGFBP3转录的影响(siRNA、过表达结构、时间推移共聚焦显微镜报告分析、器官培养、TUNEL和Annexin V)；以及TSA处理后Np63异构体的比率(siRNA、过表达构建体和WB)：(2)建立IGFBP3的膜定位和亚细胞转运(细胞分离和WB，以及生物素化的IGFPB3-链霉亲和素结合的量子点在时间推移共聚焦显微镜下)；二聚化和糖基化在亚细胞转运和凋亡中的作用(BIFC、定点突变、细胞分裂、WB和Annexin V(3)确定异位表达和siRNA敲除IGFBP3对bcl2和bax亚细胞动力学、运动和凋亡效应的影响(S)(FRAP、FLIP、SDM、细胞分离、IP、凋亡检测、WB)。这项研究的意义得到了第一次在角膜上皮细胞中直接展示IGFBP3的支持，以及先进成像方法的开发和评价：FRAP、FliP、BIFC和量子点标记，使得能够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，从而代表了在分子水平上观察角膜细胞基因调控的新范式。公共卫生关系：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，通过配戴隐形眼镜改善健康的新策略，以及为那些遭受细胞丧失的患者恢复视力。

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.