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.
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