Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
Together these data demonstrate that maintaining cell-surface uPA controls integrin αvβ5 expression, which in turn modulates TGFβ activity and myofibroblast differentiation.
Keywords: cornea: stroma and keratocytes • wound healing • differentiation