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S.K. Masur, A.M. Bernstein, L. Taliana; Urokinase Activity and ZO-1 Distribution in Corneal Fibroblasts and Myofibroblasts . Invest. Ophthalmol. Vis. Sci. 2003;44(13):873.
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Purpose: In studies of the cellular and molecular mechanisms that regulate the contributions of fibroblasts and myofibroblasts to corneal wound healing we have recently evaluated the urokinase system and ZO-1, a scaffolding protein. We found that activated corneal fibroblasts secrete increased amounts of urokinase (uPA), an extracellular protease. Furthermore, binding uPA causes its receptor (uPAR) to interact with the actin cytoskeleton and co-ordinates the protease activity of uPA with cell migration. In corneal fibroblasts we found that ZO-1, typically associated with cell-cell junctions was also detected at the leading edge of migrating fibroblasts. We asked if uPA and ZO-1 are expressed differently in myofibroblasts than in fibroblasts since myofibroblasts function in wound closure rather than migration. Methods: Human or rabbit corneal fibroblasts were cultured in DME/F12 with FBS alone or plus TGF-b for 3 days to induce myofibroblasts. uPA, uPAR and ZO-1 were immunodetected in p-formaldehyde fixed cells. uPA activity was evaluated in cell lysates. We evaluated ZO-1 in western blots of NP-40 soluble and insoluble fractions (cytoskeleton) of rabbit corneal fibroblasts and myofibroblasts. Results: Compared to control fibroblasts, TGF-b treated human corneal fibroblasts had less immunodetectable and less active uPA. Specifically TGF-b treated cells had immunodetectable uPA primarily in an intracellular compartment and little at the cell membrane. Urokinase protease activity of TGF-b treated cells was reduced by 90% compared to fibroblasts. Whereas comparable quantities of ZO-1 were found in rabbit corneal fibroblasts and myofibroblasts, its distribution differed. In lysates of myofibroblasts, ZO-1 partitioned with the cytoskeleton primarily but in fibroblasts it partitioned equally between cytosolic and cytoskeletal fractions. Immunodetection of ZO-1 in fibroblast lamellipodia was greater than in myofibroblasts. Conclusions: Our data support the phenotypic characterizations of fibroblasts and myofibroblasts in wound healing. Fibroblast migration into the wound and remodeling of the matrix are promoted by uPA activity and lamellipodial ZO-1. In contrast these activities are downregulated in myofibroblasts, the phenotype responsible for promoting wound closure.
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