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AM Bernstein, L Taliana, R Greenberg, S Masur; Urokinase Activates a Tight Association Between uPAR and the Actin Cytoskeleton in Corneal Fibroblasts . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1702.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Upon wounding, activated corneal fibroblasts become migratory, secrete increased amounts of extracellular matrix, as well as proteases that degrade this matrix. We are investigating the cellular and molecular mechanisms that control fibroblast migration and matrix remodeling. The purpose of the current work is to determine how the binding of a corneal fibroblast protease, urokinase (uPA), to its GPI-linked receptor (uPAR) signals intracellularly to the actin cytoskeleton. Method: To visualize in primary human corneal fibroblasts the functional interaction of uPAR with the cytoskeleton, we stripped cell surface uPA (pH 3 glycine), followed by neutralization (pH7.0) and addition of uPA-FITC (0.02 units/µl, pH7.0, 10 min, RT). Standard immunocytochemical localization was performed on p-formadehyde fixed cells. Results: In un-stripped cells, pre-incubation with uPA prior to fixation produces punctate uPAR immunostaining that is dispersed throughout the cell surface. Addition of exogenous uPA to stripped cells produced cell projections in which uPA/uPAR co-localized with F-actin, ß3 integrin, and FAK. To test this putative functional relationship between uPA/uPAR and the actin cytoskeleton, we treated the cells with cytochalasin D to disassemble the actin cytoskeleton. In the absence of an organized actin cytoskeleton we immunodetected uPA/uPAR as large aggregates, presumably because uPAR was not "hooked into" the actin cytoskeleton and could be cross-linked (aggregated) by the uPA antibody used for immunocytochemistry . It is notable that in contrast with its association with the actin cytoskeleton, uPA/uPAR did not co-localize with tubulin, nor α5ß1 integrin, nor vinculin. Furthermore the dispersed uPA/uPAR pattern was not disrupted by nocodazole (a microtubule disassembling drug). Conclusions: Our data support a model in which uPAR's localization is dynamically regulated. Binding uPA to its receptor initiates interaction with ß3 integrin, FAK, and the actin cytoskeleton.
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