Urokinase-type plasminogen activator negatively regulates alpha-smooth muscle actin expression via Endo180 and the uPA receptor in corneal fibroblasts

Corneal fibroblasts are embedded within an extracellular matrix composed largely of collagen type 1, proteoglycans, and other proteins in the corneal stroma, and their morphology and function are subject to continuous regulation by collagen. During wound healing and in various pathological conditions, corneal fibroblasts differentiate into myofibroblasts characterized by the expression of alpha-smooth muscle actin (alpha-SMA). Endo180, also known as urokinase-type plasminogen activator (uPA) receptor-associated protein (uPARAP), is a collagen receptor. Here we investigated whether targeting of Endo180 and the uPA receptor (uPAR) by uPA might play a role in the regulation of alpha-SMA expression by culturing corneal fibroblasts derived from uPA-deficient (uPA(-/-)) or wild-type (uPA(+/+)) mice in a collagen gel or on plastic. The expression of a-SMA was upregulated, the amounts of full-length Endo180 and uPAR were increased, and the levels of both transforming growth factor -beta (TGF-beta) expression and Smad3 phosphorylation were higher in uPA(-/-) corneal fibroblasts compared with uPA(+/+) cells under the collagen gel culture condition. Antibodies to Endo180 inhibited these effects of uPA deficiency on alpha-SMA and TGF-beta expression, whereas a TGF-beta signaling inhibitor blocked the effects on Smad3 phosphorylation and alpha-SMA expression. Our results suggest that uPA deficiency might promote the interaction between collagen and Endo180 and thereby increase alpha-SMA expression in a manner dependent on TGF-beta signaling. Expression of alpha-SMA is thus negatively regulated by uPA through targeting of Endo180 and uPAR.