Determinants of Phenotype in Retinal Pigment Epithelium

描述(申请人提供)：拟议研究的长期目标是确定视网膜色素上皮(RPE)细胞如何获得和保持其表型，以便可以开发策略，在组织因损伤(如视网膜脱离)或病理(如老年性黄斑变性)受损时，最大限度地恢复表型。RPE细胞支持紧邻的视网膜光感受器的功能和生存，这种相互作用要求RPE细胞保持特定的细胞结构。这种架构是如何产生的还不是很清楚，当它由于受伤或病理而丢失时，它可能无法恢复。钙粘附素细胞-细胞黏附蛋白是重要的上皮形态调节蛋白，RPE细胞的不同寻常之处在于它们至少表达两种钙粘附素，N-钙粘附素和E-钙粘附素，这两种钙粘附素通常不存在于同一上皮细胞中。在当前的项目中，将检查RPE细胞中E-钙粘附素的细胞类型特异性行为。这项研究的基础假设是，E-钙粘蛋白在RPE细胞中连接积累的时间是是否以及如何影响RPE表型的关键，E-钙粘蛋白在连接之前形成N-钙粘附素黏附。它进一步假设，RPE细胞通过高效地分解新合成的蛋白质来减缓E-钙粘附素在连接处的积累。其具体目的是：(1)确定在黏附连接形成期间，E-钙粘附素在RPE细胞中的积累是否受到蛋白降解的抑制。为了确定哪些类别的蛋白水解酶参与，以及在哪些亚细胞隔间中发现了E-钙粘素多肽。(2)在培养细胞中连续或同时过表达E-或N-钙粘蛋白基因，以确定在什么条件下这两种钙粘附素共同分布在连接处，它们是否形成共同的分子复合体，以及形成的连接类型是否取决于细胞类型、RPE细胞表型或首先表达哪种钙粘附素。确定E-钙粘蛋白表达与N-钙粘蛋白连接发展相关的时间是否影响细胞表型，重点是Na-K-ATPase的极性。(3)在转基因小鼠视网膜色素上皮细胞中表达E-钙粘附素，原位分析其对表型的影响，重点分析Na-K-ATPase的极性。以确定RPE细胞中预期的极性丧失是否导致临床或形态上可检测到的视网膜变性。

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to determine how cells of the retinal pigment epithelium (RPE) acquire and maintain their phenotype so that strategies can be developed to maximize phenotype recovery when the tissue is damaged by injury, as can accompany retinal detachment, or in pathologies, such as age-related macular degeneration. RPE cells support the function and survival of immediately adjacent retinal photoreceptors, an interaction which requires that RPE cells maintain a specific cellular architecture. How this architecture comes about is not well understood, and when it is lost as a consequence of injury or pathology, it may not recover. Cadherin cell-cell adhesion proteins are known to be important epithelial morphoregulators, and RPE cells are unusual in that they express at least two cadherins, N-cadherin and E-cadherin, which are not normally found in the same epithelial cells. In the current project, the cell type-specific behavior of E-cadherin in RPE cells will be examined. The hypothesis underlying the proposed research is that the timing of junctional accumulation of E-cadherin in RPE cells, which pre-form an N-cadherin adhesion before E-cadherin localizes to junctions, is key to whether and how E-cadherin affects RPE phenotype. It is further hypothesized that RPE cells slow the accumulation of E-cadherin at junctions by highly effective proteolysis of newly-synthesized protein. The specific aims are: (1) To determine whether E-cadherin accumulation in RPE cells is suppressed by proteolytic degradation during the interval when adherens junctions are forming. To determine which classes of proteases are involved and in which subcellular compartments E-cadherin peptides are found. (2) To overexpress E- or N-cadherin genes in cultured cells, either sequentially or simultaneously, to determine under what conditions the two cadherins co-distribute at junctions, whether they form a common molecular complex, and whether the type of junction that forms depends upon cell type, RPE cell phenotype, or which cadherin is expressed first. To determine whether timing of E-cadherin expression relative to the development of the N-cadherin junction affects cell phenotype, with emphasis on the polarity of Na-K ATPase. (3) To express E-cadherin in the RPE of transgenic mice to analyze the effect on phenotype in situ, with emphasis on the polarity of Na-K ATPase. To determine whether the anticipated loss of polarity in RPE cells leads to a clinically or morphologically detectable retinal degeneration.