The involvement of the Rho-kinase pathway and its regulation in cytokine-induced collagen gel contraction by hyalocytes

PURPOSE. To investigate the involvement of the Rho-kinase pathway in collagen gel contraction by hyalocytes. METHODS. An in vitro type I collagen gel contraction assay using cultured bovine hyalocytes was performed to evaluate the effect of PDGF-BB and TGF-β2. 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 α-smooth muscle actin (αSMA) was analyzed by Western blot analysis to examine the myofibroblast-like transdifferentiation of the hyalocytes. RESULTS. Maximum collagen gel contraction was observed within 24 hours after treatment with PDGF-BB and much stronger contraction with TGF-β2, whose effect was time dependent, at least up to 5 days. Although transient and maximum MLC phosphorylation by PDGF-BB was observed at ∼4 hours after stimulation (180.8%, P < 0.01), TGF-β2-elicited MLC phosphorylation occurred in a time-dependent manner at least up to 24 hours (220.0%, P < 0.01) and was maintained up to 5 days. Y27632 demonstrated significant inhibition of collagen gel contraction induced by both cytokines. Hydroxyfasudil dose-dependently (0.03-20.00 μM) prohibited the phosphorylation of MLC, and inhibited collagen gel contraction at a concentration corresponding to that which inhibited MLC phosphorylation. TGF-β2, but not PDGF-BB, also caused myofibroblast-like transdifferentiation with αSMA overexpression, which was downregulated by hydroxyfasudil in part (P < 0.01). CONCLUSIONS. The hyalocytes have a contractile property in the presence of PDGF-BB and TGF-β2. Whereas PDGF-BB initiates collagen gel contraction by transient activation of the Rho-kinase pathway, sustained activation of the Rhokinase pathway and myofibroblast-like transdifferentiation appears to be involved in the TGF-β2-dependent contractile properties of hyalocytes.