Degradation of Internalized αvβ5 Integrin Is Controlled by uPAR Bound uPA: Effect on β1 Integrin Activity and α-SMA Stress Fiber Assembly

Myofibroblasts (Mfs) that persist in a healing wound promote extracellular matrix (ECM) accumulation and excessive tissue contraction. Increased levels of integrin αvβ5 promote the Mf phenotype and other fibrotic markers. Previously we reported that maintaining uPA (urokinase plasminogen activator) bound to its cell-surface receptor, uPAR prevented TGFβ-induced Mf differentiation. We now demonstrate that uPA/uPAR controls integrin β5 protein levels and in turn, the Mf phenotype. When cell-surface uPA was increased, integrin β5 levels were reduced (61%). In contrast, when uPA/uPAR was silenced, integrin β5 total and cell-surface levels were increased (2–4 fold). Integrin β5 accumulation resulted from a significant decrease in β5 ubiquitination leading to a decrease in the degradation rate of internalized β5. uPA-silencing also induced α-SMA stress fiber organization in cells that were seeded on collagen, increased cell area (1.7 fold), and increased integrin β1 binding to the collagen matrix, with reduced activation of β1. Elevated cell-surface integrin β5 was necessary for these changes after uPA-silencing since blocking αvβ5 function reversed these effects. Our data support a novel mechanism by which downregulation of uPA/uPAR results in increased integrin αvβ5 cell-surface protein levels that regulate the activity of β1 integrins, promoting characteristics of the persistent Mf

Local control of α1-proteinase inhibitor levels: regulation of α1-proteinase inhibitor in the human cornea by growth factors and cytokines

AbstractAlpha 1-proteinase inhibitor is a major serine proteinase inhibitor in the human cornea involved in the protection of the avascular corneal tissue against proteolytic damage. This inhibitor is upregulated systemically during infection, inflammation and injury. Cytokines that mediate the acute phase response such as IL-1β and IL-2 increased α1-proteinase inhibitor present in corneal organ culture media. This released inhibitor represented mainly newly synthesized protein. However, IL-6, a general inducer of the acute phase response that upregulates α1-proteinase inhibitor in all other tissues and cells tested, failed to alter corneal α1-proteinase inhibitor levels over the tested period of 24 h. In addition to IL-1β and IL-2, α1-proteinase inhibitor levels in the corneal organ culture medium increased following the addition of FGF-2 and IGF-I. The effect of the above growth factors and cytokines was relatively fast with maximal induction observed within the first 5 h. Among the tested growth factors and cytokines, IL-1β was the most potent and increased total corneal α1-proteinase inhibitor levels approximately 2.4-fold in the cornea organ culture medium. Newly, synthesized α1-proteinase secreted into the medium increased 3.9-fold. In addition to the effect on corneal α1-proteinase inhibitor, IL-1β also increased the amount of α1-proteinase inhibitor released by monocytes and macrophages but not by HepG2, CaCo2, and MCF-7 cells within 24 h. These results suggest that the cornea can locally control levels of α1-proteinase inhibitor in response to an inflammatory insult

Expression of Insulin-Like Growth Factor 2 Receptor in Corneal Keratocytes During Differentiation and in Response to Wound Healing

PURPOSE. Insulin-like growth factor 2 receptor (IGF2R) associates with ligands that influence wound healing outcomes. However, the expression pattern of IGF2R and its role in the cornea is unknown. METHODS. Human keratocytes were isolated from donor corneas. Fibroblasts (fibroblast growth factor 2 [FGF2]-treated) or myofibroblasts (TGF-b1-treated) were analyzed for IGF2R and a-smooth muscle actin (a-SMA) expression by Western blotting and immunolocalization. Mouse corneas were wounded in vivo and porcine corneas ex vivo. The IGF2R and a-SMA protein expression were visualized and quantified by immunohistochemistry. The IGF2R gene expression in human corneal fibroblasts was knocked-down with targeted lentiviral shRNA. RESULTS. The IGF2R is expressed in epithelial and stromal cells of normal human, mouse, and porcine corneas. The IGF2R increases (11.2 6 0.4-fold) in the epithelial and (11.7 6 0.9-fold) stromal layers of in vivo wounded mouse corneas. Double-staining with a-SMA-and IGF2R-specific antibodies reveals that IGF2R protein expression is increased in stromal myofibroblasts in the wounded cornea relative to keratocytes in the normal cornea (11.2 6 0.8-fold). Human primary stromal keratocytes incubated with FGF2 or TGF-b1 in vitro demonstrate increased expression (2.0 6 0.4-fold) of IGF2R in myofibroblasts relative to fibroblasts. Conversion of IGF2R shRNA-lentiviral particle transduced corneal fibroblasts to myofibroblasts reveals a dependence on IGF2R expression, as only 40% 6 10% of cells transduced converted to myofibroblasts compared to 86% 6 3% in control cells. CONCLUSIONS. The IGF2R protein expression is increased during corneal wound healing and IGF2R regulates human corneal fibroblast to myofibroblast differentiation