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A. M. Bernstein, S. S. Twining, D. J. Warejcka, E. Tall, S. K. Masur; Urokinase Receptor Cleavage: A Crucial Step in Fibroblast to Myofibroblast Differentiation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1498. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In corneal fibroblasts, uPA binding to uPAR (urokinase plasmingen activator and its receptor) localizes an extracellular protease system that promotes matrix remodeling, growth factor activation, and migration in response to wounding. PAI-1 (plasminogen activator inhibitor) is a multifunctional molecule that promotes the internalization of uPA/uPAR and integrins from the cell surface through the binding of the LRP receptor. We have investigated the role of the uPA/uPAR/PAI-1 pathway in myofibroblast differentiation since control of myofibroblast differentiation from fibroblasts is critical for regenerative wound healing.
Human corneal fibroblasts (HCFs) were cultured on collagen or vitronectin in supplemented serum free media for 3 days with 10µg/ml FGF-2/heparin or 1ng/ml TGFß1 to regulate fibroblast and myofibroblast phenotypes, respectively. PAI-1 and uPAR expression were evaluated by western blot and immunofluoresence. To study the role of uPAR on myofibroblast differentiation, HCFs were transfected with a non-cleavable form of uPAR prior to TGFß1 treatment and evaluated by immunocytochemical detection of α-smooth muscle actin (α-SMA) as a marker of myofibroblast differentiation.
uPAR cleavage and PAI-1 were critical to myofibroblast differentiation. Cleavage of uPAR’s N-terminal domain (D1), resulting in a truncated form of uPAR (D2D3) on the myofibroblast’s surface, was required for myofibroblast differentiation since 1) protease inhibitors that prevented uPAR cleavage, also prevented myofibroblast differentiation and 2) over-expression of a non-cleavable uPAR cDNA inhibited myofibroblast differentiation. In addition, PAI-1 and integrin in combination with matrix played a role in mediating myofibroblast differentiation. Myofibroblast differentiation on vitronectin, as indicated by incorporation of α-SMA into stress fibers, required the removal of PAI-1 from the HCFs’ surface and addition of Mn2+ to activate integrins.
These data support the novel hypothesis that uPAR cleavage and PAI-1 expression contribute to the mechanism underlying the fibroblast to myofibroblast transition.
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