The long term objective of the proposed research is to determine the relationship between cell shape and cell function in the retinal pigment epithelium (RPE) of adult eyes. This information can be used to determine how RPE cells with altered phenotype contribute to ocular pathology, and to develop methods to maximize the restoration of normal epithelial phenotype in an RPE monolayer damaged by injury or disease. In the proposed project, subpopulations of human RPE cells will be used which express stable fusiform or epithelioid phenotypes in vitro. Experiments to address the first specific aim of the proposed research focus on the formation of the adherens junction during the process of in vitro morphogenesis which follows confluency. During morphogenesis epithelioid cells form a detergent-resistant and zonular cadherin- containing junction that is not formed in fusiform cells. The hypothesis to be tested states that epithelialization in a cell type like the RPE that expresses N-cadherin, which is also found in many non- epithelial cells, requires a specific time-dependent sequence of changes in the composition, tyrosine phosphorylation state or turnover ate of molecules comprising the cadherin/catenin complex. Cadherin/catenin complexes in extracts of epithelioid and fusiform RPE will be compared using the methods of immunoprecipitation, protein blotting, phosphatase and kinase inhibitor treatment, and confocal imaging. The second specific aim of the proposed project focusses on epithelioid and fusiform RPE cells late in the postconfluent time course when the phenotypes are well-developed. The goal of this aim is to identify differentially-expressed genes which may contribute of the maintenance of the distinct phenotypes, or which may identify distinct functions of RPE cells expressing different shapes. RNA extracts of the phenotypes will be compared using the method of mRNA differential display, which includes reverse transcription, the polymerase chain reaction and DNA sequence analysis.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY010832-04
Application #
6125119
Study Section
Visual Sciences C Study Section (VISC)
Program Officer
Dudley, Peter A
Project Start
1996-12-01
Project End
2001-11-30
Budget Start
1999-12-01
Budget End
2001-11-30
Support Year
4
Fiscal Year
2000
Total Cost
$198,260
Indirect Cost
Name
Medical College of Wisconsin
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
073134603
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Burke, Janice M (2008) Epithelial phenotype and the RPE: is the answer blowing in the Wnt? Prog Retin Eye Res 27:579-95
Rozanowska, Malgorzata; Pawlak, Anna; Rozanowski, Bartosz et al. (2004) Age-related changes in the photoreactivity of retinal lipofuscin granules: role of chloroform-insoluble components. Invest Ophthalmol Vis Sci 45:1052-60
Sarna, Tadeusz; Burke, Janice M; Korytowski, Witold et al. (2003) Loss of melanin from human RPE with aging: possible role of melanin photooxidation. Exp Eye Res 76:89-98
Rozanowska, Malgorzata; Korytowski, Witold; Rozanowski, Bartosz et al. (2002) Photoreactivity of aged human RPE melanosomes: a comparison with lipofuscin. Invest Ophthalmol Vis Sci 43:2088-96
Burke, J M; Cao, F; Irving, P E (2000) High levels of E-/P-cadherin: correlation with decreased apical polarity of Na/K ATPase in bovine RPE cells in situ. Invest Ophthalmol Vis Sci 41:1945-52
Kaida, M; Cao, F; Skumatz, C M et al. (2000) Time at confluence for human RPE cells: effects on the adherens junction and in vitro wound closure. Invest Ophthalmol Vis Sci 41:3215-24
Burke, J M; Cao, F; Irving, P E et al. (1999) Expression of E-cadherin by human retinal pigment epithelium: delayed expression in vitro. Invest Ophthalmol Vis Sci 40:2963-70
Cao, F; Yanagihara, N; Burke, J M (1999) Progressive association of a ""soluble"" glycolytic enzyme with the detergent-insoluble cytoskeleton during in vitro morphogenesis of MDCK epithelial cells. Cell Motil Cytoskeleton 44:133-42
Pollack, J S; Kim, J E; Pulido, J S et al. (1998) Tissue effects of subclinical diode laser treatment of the retina. Arch Ophthalmol 116:1633-9
Wassell, J; Ellis, S; Burke, J et al. (1998) Fluorescence properties of autofluorescent granules generated by cultured human RPE cells. Invest Ophthalmol Vis Sci 39:1487-92

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