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Lingyan Wang, Audrey Bernstein; Corneal Myofibroblast Differentiation on Vitronectin is Regulated by uPA. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3428.
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© ARVO (1962-2015); The Authors (2016-present)
To prevent corneal scars that can obstruct vision and result in blindness. Scar tissue is characterized by strongly adherent α-smooth muscle actin (α-SMA) rich myofibroblasts (Mfs) and the accumulation of extracellular matrix (ECM) including vitronectin (VN). We propose that interactions between the protease, uPA (urokinase plasminogen activator), its receptor (uPAR), and the uPA inhibitor (PAI-1) regulate Mf adhesion on VN.
Primary human corneal fibroblasts (HCFs) were cultured in supplemented serum-free media on VN or collagen with 1ng/ml TGFβ1. Adherent cells were quantified using crystal violet. uPAR was downregulated by siRNA. Protein expression was measured by Western blot, and protein interactions were demonstrated by co-immunoprecipitation. Mf differentiation was identified by immunostaining of α-SMA stress fibers.
In vivo, TGFβ induces Mf differentiation in a fibrotic matrix that includes VN. Paradoxically, in vitro TGFβ1-induced PAI-1 prevents cell attachment on VN by inhibiting binding of integrins αvβ3/β5 to VN. We now report that HCFs seeded in the presence of TGFβ1 had an average of 50% attachment on VN compared to collagen and significantly reduced α-SMA stress fiber organization at 72 hours in culture. We investigated whether addition of uPA enhanced the release of PAI-1 from matrix VN and increased cell-adhesion and Mf formation on VN. Co-immunopreciptation experiments showed that uPA competed with VN for PAI-1 binding. In addition, when uPAR was silenced with targeted siRNA, secreted uPA was increased by 3.3-fold (because it is no longer binding to the cell-surface uPAR) and correspondingly PAI-1 binding to matrix VN was reduced by 4.9-fold. Finally, when TGFβ1-treated HCFs were grown on VN for 3 days, uPA treatment dramatically increased α-SMA stress fiber organization and cell area by 2.4-fold.
Together, our data suggest that downregulation of uPAR promotes Mf adhesion on VN through the continued secretion of uPA and corresponding release of PAI-1 from the matrix. These data extend our previous findings that TGFβ1 treatment over 7 days in culture decreases uPAR expression on Mfs. Modulating the interaction between VN, uPA, uPAR and PAI may prevent strongly adherent Mfs that contribute to scarring.
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