The Involvement of the Rho-Kinase Pathway and Its Regulation in Cytokine-Induced Collagen Gel Contraction by Hyalocytes

To investigate the involvement of the Rho-kinase pathway in collagen gel contraction by hyalocytes. An in vitro type I collagen gel contraction assay using cultured bovine hyalocytes was performed to evaluate the effect of PDGF-BB and TGF-beta2. The effect of both cytokines on the phosphorylation of myosin light chain (MLC) was analyzed by Western blot analysis. To confirm the involvement of the Rho-kinase pathway in the collagen gel contraction, the effects of Y27632, a specific Rho-kinase inhibitor were examined. The effect of hydroxyfasudil, another potent Rho-kinase inhibitor, was also evaluated. The expression of alpha-smooth muscle actin (alphaSMA) was analyzed by Western blot analysis to examine the myofibroblast-like transdifferentiation of the hyalocytes. Maximum collagen gel contraction was observed within 24 hours after treatment with PDGF-BB and much stronger contraction with TGF-beta2, whose effect was time dependent, at least up to 5 days. Although transient and maximum MLC phosphorylation by PDGF-BB was observed at approximately 4 hours after stimulation (180.8%, P