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S. Morales, S. Mareninov, D.G. Telander, J. Braun, L.K. Gordon; Role of Epithelial Membrane Protein 2 (EMP2) in Retinal Pigment Epitheliel Cell–Mediated Gel Contraction . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3829.
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© ARVO (1962-2015); The Authors (2016-present)
Proliferative vitreoretinopathy (PVR) is believed to result in part from de–differentiation of retinal pigment epithelium (RPE) with cellular migration, membrane formation, and contraction in an aberrant wound–healing strategy. Epithelial membrane protein 2 (EMP2) controls recruitment and cell surface delivery of specific integrin isoforms in the RPE cell line ARPE–19. The purpose of this study was to investigate the role of EMP–2 in in vitro gel contraction assays.
Levels of EMP2 were increased in ARPE–19 cells (ATCC) through stable infection of an EMP–2 overexpressing retrovirus construct(ARPE–RV–EMP2). ARPE–19 cells with decreased EMP2 were created through stable transfection with an EMP2 ribozyme(ARPE/ribo). EMP2 levels were quantified using Western blot. An in vitro gel contraction assay was used in which RPE cells were seeded on a collagen gel and the percent contraction measured at specific time intervals. The areas of the gel were obtained using image capture (Gel Doc) and quantified using NIH Image software. Cells were pretreated prior to seeding with PP2 (FAK inhibitor) or anti–EMP2 antibody. Total and phosphorylated FAK levels were quantified.
Modulating EMP2 expression significantly altered gel contraction. In comparison to wild type ARPE–19, ARPE–RV–EMP2 cells increased contraction by about 50% (p<0.029). Concordantly, ARPE–19/ribo cells showed a 66% decrease as compared to controls (p<0.002). Cell surface EMP2 blockade using anti–EMP2 antibody also decreased contraction. Inhibition of FAK led to a decrease in gel contraction (75% decrease; p<0.0001). The ratio of phosphorylated FAK to total FAK doubled in the ARPE–RV–EMP2 cells or by exposure of wild type ARPE–19 cells to soluble collagen.
RPE–mediated collagen gel contraction is enhanced by increased levels of EMP2. This contraction may be mediated by FAK activation. These findings may lead to the development of new strategies for control of RPE behavior and may be relevant to PVR control.
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