title: Inhibition of RhoA/Rho-kinase pathway suppresses the expression of type I collagen induced by TGF-beta2 in human retinal pigment epithelial cells.
abstract: Proliferative vitreoretinopathy (PVR) is a major cause of the failure of rhegmatogenous retinal detachment surgery. The pathogenesis of PVR includes a fibrotic reaction of retinal pigment epithelial (RPE) cells caused by transforming growth factor (TGF)-beta. The cellular mechanisms by which TGF-beta induces extracellular matrix protein synthesis are not fully understood. In this study, we examined whether the RhoA/Rho-kinase pathway was involved in TGF-beta2-induced collagen expression in a human RPE cell line, ARPE-19. The roles of RhoA and Rho-kinase were evaluated using biochemical inhibitors, RhoA inhibitor, simvastatin and Rho-kinase inhibitor, Y27632. The effects of simvastatin or Y27632 on the type I collagen mRNA (COL1A1 and COL1A2) expression induced by TGF-beta2 were evaluated by real-time RT-PCR. The effects of simvastatin or Y27632 on type I collagen synthesis induced by TGF-beta2 were assessed by immunocytochemical analysis with anti-type I collagen antibody. To examine the effects of simvastatin or Y27632 on COL1A2 promoter activity induced by TGF-beta2, luciferase reporter assays were also performed. Moreover, the role of RhoA itself on COL1A2 promoter activity was assessed using the constructs of constitutively active RhoA and dominant-negative RhoA. RhoA was activated within 5 min after stimulation with TGF-beta2, and its activation persisted for as long as 1 h in a dose-dependent fashion. Preincubation of ARPE-19 with simvastatin (5 microM) or Y27632 (10 microM) significantly prevented TGF-beta2-induced COL1A1 and COL1A2 gene expression. Inhibition of RhoA/Rho-kinase markedly suppressed TGF-beta2-induced type I collagen synthesis in ARPE-19. Moreover, the blockage of RhoA/Rho-kinase inhibited the increase in COL1A2 promoter activity when induced by TGF-beta2. Constitutively active RhoA increased COL1A2 promoter activity in the presence or absence of TGF-beta2. Simvastatin and Y27632 reduced active RhoA-induced COL1A2 promoter activity. The dominant-negative RhoA inhibited COL1A2 promoter activity augmentation induced by TGF-beta2. In the luciferase assay using a mutation construct of the Smad binding site in COL1A2 promoter (Smad-mut/Luc), the treatment with simvastatin and Y27632 significantly reduced TGF-beta2 induction of Smad-mut/Luc promoter activity. On the other hand, both simvastatin and Y27632 significantly reduced CAGA12-Luc activity induced by TGF-beta2. These results indicate that the RhoA/Rho-kinase pathway plays a role in relaying TGF-beta2 signal transduction to type I collagen synthesis in RPE cells in a Smad-dependent and Smad-independent fashion. The RhoA/Rho-kinase pathway may be a therapeutic target for treating PVR.
authors.affiliation: Department of Ophthalmology, Oita University, Hasama-machi, Yufu-shi Oita 879-5593, Japan. itoyuji@med.oita-u.ac.jp
authors.forename: Yuji
authors.initials: Y
authors.lastname: Itoh
authors.forename: Kenichi
authors.initials: K
authors.lastname: Kimoto
authors.forename: Masamoto
authors.initials: M
authors.lastname: Imaizumi
authors.forename: Kazuo
authors.initials: K
authors.lastname: Nakatsuka
mesh_terms: D000577:Amides / Q000494:pharmacology; D001549:Benzamides / Q000494:pharmacology; D002460:Cell Line; D024042:Collagen Type I / Q000032:analysis / Q000378:metabolism*; D003864:Depression, Chemical; D004149:Dioxoles / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D019821:Simvastatin / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D053781:Transforming Growth Factor beta2 / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000032:analysis / Q000378:metabolism*
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D000577:Amides; D001549:Benzamides; D024042:Collagen Type I; D004149:Dioxoles; D004791:Enzyme Inhibitors; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; D053781:Transforming Growth Factor beta2; C108830:Y 27632; D019821:Simvastatin; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein