TGF–ß2–Dependent Collagen Gel Contraction by RPEs Predominantly Mediated Through the Rho–Kinase Pathway

Abstract

Abstract: : Purpose:To investigate the signaling mechanisms of TGF–ß2–dependent collagen gel contraction by RPEs. Methods:An in vitro type I collagen gel contraction assay was performed to evaluate the effect of TGF–ß2 on the gel contraction by cultured bovine RPEs. The phosphorylation state of myosin light chain (MLC) was analyzed by Western blotting. Kinase inhibitors (Y27632 and ML7) were used to confirm the contribution of Rho– and MLC–kinase respectively. The effect of hydroxyfasudil, a potent Rho–kinase inhibitor already in clinical use, was also evaluated. We further investigated the contribution of other kinases such as PKC, p44/42 MAPK, p38 MAPK and PI3K to MLC phosphorylation, gel contraction and the expression of α–SMA, a biochemical marker of myofibroblasts. Results: TGF–ß2 caused time– and dose–dependent gel contraction. Significant MLC phosphorylation was initially detected after 4hr treatment (p<0.01) and maintained at least up to 5 days investigated. TGF–ß2–dependent MLC phosphorylation and gel contraction was prominently inhibited in the presence of Y27632 while ML7 showed marginal inhibition. Hydroxyfasudil also abrogated TGF–ß2–dependent MLC phosphorylation (p<0.01) and gel contraction (p<0.01) without affecting cell viability. Furthermore, PKC inhibitor (GF109203X, 5 µM) and p38 MAPK inhibitor (SB203580, 10 µM) significantly attenuated TGF–ß2–elicited α–SMA expression, which was not affected by the Rho–kinase inhibitor, in addition to MLC phosphorylation and gel contraction (p<0.01, respectively). Conclusions: TGF–ß2–dependent MLC phosphorylation and collagen gel contraction by RPEs is dominantly mediated through the Rho–kinase pathway which possibly lies downstream of PKC and p38 MAPK signaling at least in part. Both PKC and p38 MAPK pathways also seem to mediate TGF–ß2–elicited myofibroblastic transdifferentiation of RPEs.

Keywords: retinal pigment epithelium • signal transduction