Determinants of Phenotype in Retinal Pigment Epithelium

描述（由申请人提供）：拟议研究的长期目标是确定视网膜色素上皮（RPE）细胞如何获得和维持其表型，以便在组织因损伤而受损时，如视网膜脱离或病理，如年龄相关性黄斑变性时，可以制定策略以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存活，这种相互作用需要RPE细胞维持特定的细胞结构。这种结构是如何形成的尚不清楚，当它因受伤或病理而丢失时，它可能无法恢复。众所周知，细胞粘附蛋白是重要的上皮形态调节剂，而RPE细胞的不寻常之处在于它们表达至少两种钙粘蛋白，n -钙粘蛋白和e -钙粘蛋白，这两种钙粘蛋白通常不会在同一上皮细胞中发现。本项目将研究E-cadherin在RPE细胞中的细胞类型特异性行为。本研究提出的假设是，在E-cadherin定位到连接处之前，E-cadherin在RPE细胞中连接积聚的时间是E-cadherin是否以及如何影响RPE表型的关键。进一步假设RPE细胞通过对新合成的蛋白进行高效的蛋白水解来减缓e -钙粘蛋白在连接处的积累。具体目的是：(1)确定在粘附连接形成的间隙，蛋白水解降解是否抑制了E-cadherin在RPE细胞中的积累。确定哪类蛋白酶参与其中，在哪些亚细胞区室中发现e -钙粘蛋白肽。(2)在培养细胞中依次或同时过表达E-或n -钙粘蛋白基因，以确定在什么条件下两种钙粘蛋白在连接处共分布，它们是否形成共同的分子复合物，以及形成的连接类型是否取决于细胞类型，RPE细胞表型，或者哪个钙粘蛋白首先表达。为了确定E-cadherin相对于N-cadherin连接发育的表达时间是否影响细胞表型，重点研究Na-K atp酶的极性。(3)在转基因小鼠RPE中表达E-cadherin，原位分析其对表型的影响，重点研究Na-K atp酶的极性。确定预期的RPE细胞极性丧失是否会导致临床或形态学上可检测的视网膜变性。

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.