The Role of BCL-2 in Corneal Epithelial Cell Shedding

描述（由申请人提供）：本提案的主要目标是获得必要的培训，以发展成为一名独立的研究者，将我在视觉科学方面的背景与分子生物学的基础相结合。这一目标将在导师James V. Jester博士、H.Dwight Cavanagh博士和Jerry W. Shay博士的指导下，通过教学和实践实验室培训相结合来实现。本提案的第二个目标侧重于获取所需的知识和分子技术，以回答以下假设：促生存癌基因BCL-2的核定位负责调节角膜上皮分化和凋亡的最后阶段。表面细胞凋亡是维持角膜上皮稳态的重要组成部分，因此是清晰视力的先决条件。在正常人中，抗凋亡蛋白BCL-2定位于细胞核，并在表面细胞凋亡之前消失，这表明BCL-2在介导表面细胞脱落中起作用。在培养中，端粒化的人角膜上皮细胞（hTCEpi）在凋亡前也表现出核BCL-2的消失，从而为研究体外调节上皮脱屑的机制提供了一种新的模型。在本提案中，该模型将用于检验以下四个特定目的：1)hTCEpi构建在TUNEL标记之前概括了正常人类角膜上皮细胞在BCL-2表达、定位和消失方面的分化和脱落。2) BCL-2表达增强导致细胞存活增加、核定位持续、表面细胞脱落减少，而BCL-2表达降低导致细胞存活降低、核定位丧失、表面细胞脱落增加。3) BCL-2的调控环和丝氨酸70磷酸化位点在调节表面细胞脱落中起关键作用。4)紫外光等导致细胞凋亡增加的事件通过BCL-2的磷酸化介导表面细胞凋亡。

DESCRIPTION (provided by applicant): The primary objective of this proposal is to gain the necessary training to develop into an independent investigator, integrating my background in the vision sciences with the fundamentals of molecular biology. This objective will be met by a combination of didactic and practical laboratory training under the guidance of mentors Dr. James V. Jester, Dr. H.Dwight Cavanagh, and Dr. Jerry W. Shay. The second objective of this proposal focuses on acquiring the knowledge and molecular techniques required to answer the hypothesis that: nuclear localization of the pro-survival oncogene, BCL-2, is responsible for regulating the final stages of corneal epithelial differentiation and apoptosis. Surface cell apoptosis is a critical component to the maintenance of corneal epithelial homeostasis and is thus a prerequisite for clear vision. In the normal human, the anti-apoptotic protein, BCL-2, localizes to the nucleus and disappears prior to surface cell apoptosis, suggesting a role for BCL-2 in mediating surface cell shedding. Telomerized human corneal epithelial (hTCEpi) cells have also been shown in culture to exhibit disappearance of nuclear BCL-2 prior to apoptosis, thus providing a novel model for studying the mechanism(s) responsible for regulating epithelial desquamation in vitro. In this proposal, this model will be used to examine four specific aims: 1) That an hTCEpi construct recapitulates normal human corneal epithelial cell differentiation and shedding regarding expression, localization, and disappearance of BCL-2 prior to TUNEL labeling. 2) That enhanced expression of BCL-2 results in increased cell survival, persistence of nuclear BCL-2 localization, and decreased surface cell shedding, whereas reduced expression of BCL-2 results in decreased cell survival, loss of nuclear BCL-2 localization, and increased surface cell shedding. 3) That the regulatory loop of BCL-2 and the serine 70 phosphorylation site are critical to regulating surface cell shedding. 4) That events leading to increased apoptosis such as UV light signal surface cell apoptosis through the phosphorylation of BCL-2.