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SG Elner, VM Elner, AL Kindzelskii, K Horino, HR Petty; Expression of Functional uPAR (CD87) on Human RPE cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4585.
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Purpose:Previously we reported the presence of uPAR on human RPE (HRPE) cells in vitro and in situ. uPAR is known to congregate at interfaces of cellular adhesion and migration where uPAR participates in adhesion, chemotaxis, and proteolysis. The purpose of this study was to demonstrate functional uPAR on HRPE cells and its physical association with other receptors that might regulate its function. Methods:HRPE cells were seeded onto gel matrices containing Bodipy-BSA or flourescein-conjugated collagen (DQ-collagen), both of which become highly fluorescent upon proteolysis. Pericellular fluorescence was measured in the absence and presence of plaminogen activator inhibitor 1 (PAI-1) or the metalloproteinase inhibitor, phenanthroline. Transmigration of RPE cells through the gel matrices was also measured in the presence or absence of these inhibitors. Double-immunofluorescent staining of live human RPE cells with anti-CR3 and anti-CR4 antibodies was performed to demonstrate the physical proximity of these beta-2 integrins with uPAR, and determine whether associations were dependent upon RPE confluence and polarity. Results:HRPE cells demonstrated active proteolysis as evidenced by the development of pericellular fluorescence due to the cleavage of both Bodipy-BSA and DQ-collagen. PAI-1, but not phenanthroline, significantly (p<0.01) reduced pericellular fluorescence and RPE cell migration through gel matrices. Double immunofluorescent staining of HRPE cells with uPAR, CR3, or CR4 revealed close physical association of CR3 and uPAR in non-polarized, confluent cells as evidenced by resonance energy transfer (RET) between the fluorescent probes. In elongated non-confluent cells, uPAR was located at the leading edge of migrating RPE cells while CR3 was located at the trailing edge. CR4/uPAR proximity was similar, but demonstrated less striking polarization in elongated cells. Conclusion:HRPE cells express functional uPAR, which mediates proteolysis and migration through extracellular matrix and may be specifically inhibited by PAI-1. HRPE uPAR polarization may concentrate HRPE proteolyic activity at the leading edge of migrating HRPE cells.
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