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A. M. Bernstein, L. Wang, A. Garcia, B. S. Pedroja; Cell-Associated Urokinase Regulates Integrin vβ5 Expression and Myofibroblast Differentiation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6421.
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The persistence of myofibroblasts in corneal scars leads to the accumulation of extracellular matrix (ECM). The uPA/uPAR (urokinase plasminogen activator/receptor) protease system controls ECM through the generation of plasmin. Previously we showed that maintaining cell surface uPA/uPAR expression inhibited myofibroblast differentiation. In this study we demonstrate that integrin αvβ5 is a uPA/uPAR-binding partner through which myofibroblast differentiation is modulated.
Human corneal fibroblasts were grown on collagen in supplemented serum-free media. Western blotting and qPCR determined expression of integrin αvβ5 and α-smooth muscle actin (α-SMA). Transfection with control, WT and non-cleavable (NC) uPAR cDNA, uPA and uPAR siRNA, were performed by Nucleofection. uPA activity was determined using a chromogenic activity assay. TGFβ activity was determined using a luciferase reporter assay. Myofibroblast differentiation was determined by immunostaining for α-SMA.
To determine if uPA activity directly affects β5 expression, uPA activity was inhibited with an anti-uPA antibody, resulting in a 1.7 fold increase in β5 expression. Furthermore, uPA and uPAR siRNA knockdown resulted in a 2-3-fold increase in αvβ5 RNA and protein expression, a 2-fold increase in TGFβ activity and induction of myofibroblast differentiation without TGFβ addition. uPAR cleavage of its Domain 1 abolishes uPA binding. Thus, to determine if maintaining uPA on the cell surface reduced β5, a NC uPAR mutant cDNA was over-expressed, resulting in a 60% reduction of β5. Furthermore, Plasminogen Activator Inhibitor-1 (PAI-1) null fibroblasts in which PA activity is unregulated had a 3-fold decrease in β5 expression in parallel with a 3-fold increase in PA activity. Finally, over 7 days in culture with TGFβ as myofibroblast differentiation is induced, β5 was dramatically upregulated as uPAR was reciprocally downregulated, whereas blocking αvβ5 reduced myofibroblast differentiation.
Together these data demonstrate that maintaining cell-surface uPA controls integrin αvβ5 expression, which in turn modulates TGFβ activity and myofibroblast differentiation.
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