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，发展成为一个独立的调查员，结合我在视觉科学的背景与分子生物学的基础。 在导师James V. Jester博士、H.德怀特Cavanagh博士和Jerry W.夏伊 该建议的第二个目标集中于获得回答以下假设所需的知识和分子技术：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关键组成部分，因此是清晰视力的先决条件。 在正常人中，抗凋亡蛋白BCL-2定位于细胞核并且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的作用。端粒化的人角膜上皮（hTCEpi）细胞在培养物中也显示出在凋亡之前表现出核BCL-2的消失，从而提供了用于研究负责调节体外上皮脱落的机制的新模型。在该提议中，该模型将用于检查四个特定目标：1）hTCEpi构建体再现了正常人角膜上皮细胞分化和脱落，关于TUNEL标记之前BCL-2的表达、定位和消失。2)BCL-2的表达增强导致细胞存活增加、核BCL-2定位的持续性和表面细胞脱落减少，而BCL-2的表达降低导致细胞存活降低、核BCL-2定位丧失和表面细胞脱落增加。3)BCL-2的调节环和丝氨酸70磷酸化位点对调节表面细胞脱落至关重要。4)导致细胞凋亡增加的事件如UV光通过BCL-2的磷酸化信号表面细胞凋亡。

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.