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.
