title: Critical role of the Rho-kinase pathway in TGF-beta2-dependent collagen gel contraction by retinal pigment epithelial cells.
abstract: Retinal pigment epithelial cells (RPEs) are thought to be one of the main components of fibrous membrane observed in eyes with proliferative vitreo-retinopathy. We investigated the signalling mechanisms of TGF-beta2-dependent collagen gel contraction by RPEs. An in vitro type I collagen gel contraction assay was performed to evaluate the effect of TGF-beta2 on gel contraction. The expression of alpha-smooth muscle actin (alpha-SMA) and the phosphorylation state of myosin light chain (MLC) were analyzed by Western blotting. The involvement of protein kinases such as p44/42 mitogen-activated protein kinase (MAPK), protein kinase C (PKC), p38 MAPK and phosphatidylinositol-3 kinase was investigated. The contribution of Rho-kinase and/or MLC-kinase was also evaluated using respective kinase inhibitors (Y27632, hydroxyfasudil and ML7). Additionally, RPEs were immunostained to examine whether the expression of alpha-SMA detected in our western blotting correlated to the stress fiber formation within the cells. TGF-beta2 caused time (0-5 days)-and dose (0 10 ng ml(-1))-dependent gel contraction associated with overexpression of alpha-SMA and phosphorylation of MLC (p < 0.01, respectively). PKC inhibitor (GF109203X, 5 microM) and p38 MAPK inhibitor (SB203580, 10 microM) significantly attenuated TGF-beta2-elicited gel contraction via partial downregulation of both alpha-SMA expression and MLC phosphorylation (p < 0.01, respectively). The gel contraction was prominently inhibited in the presence of Y27632 (10 microM) or hydroxyfasudil (10 microM) with strong suppression of MLC phosphorylation but had no significant effect on alpha-SMA expression. Treatment with ML7, in contrast, resulted in a marginal inhibition of MLC phosphorylation and gel contraction. Finally, pretreatment of the cells with Y27632 or hydroxyfasudil prevented the formation of stress fiber within the cells. These results indicate that TGF-beta2-dependent myofibroblastic transdifferentiation and MLC phosphorylation by RPEs involve both PKC and p38 MAPK pathways at least in part. Myofibroblastic transdifferentiation of RPEs appears to be independent of the Rho-kinase pathway, and the presence of alpha-SMA does not necessarily reflect the contractile potential of a cell. While Rho-kinase inhibitors are incapable of preventing myofibroblastic transdifferentiation itself, this pathway could be one of the critical targets of cell-mediated contraction of the tissue containing fibrillar collagens by transdifferentiated RPEs.
authors.affiliation: Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Muneki
authors.initials: M
authors.lastname: Miura
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Hata
authors.forename: Kumiko
authors.initials: K
authors.lastname: Hirayama
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kita
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Noda
authors.forename: Kimihiko
authors.initials: K
authors.lastname: Fujisawa
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Shimokawa
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Ishibashi
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003094:Collagen / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053781:Transforming Growth Factor beta2; D018630:Vitreoretinopathy, Proliferative / Q000378:metabolism / Q000473:pathology; D054460:rho-Associated Kinases
chemical_list: D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D003094:Collagen; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases