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Inhibition of RhoA/Rho-kinase pathway suppresses the expression of type I collagen induced by TGF-beta2 in human retinal pigment epithelial cells.
Exp Eye Res. 2007 Mar; 84(3):464-72.EE

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+Show Affiliations

Department of Ophthalmology, Oita University, Hasama-machi, Yufu-shi Oita 879-5593, Japan. itoyuji@med.oita-u.ac.jpNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

17217948

Citation

Itoh, Yuji, et al. "Inhibition of RhoA/Rho-kinase Pathway Suppresses the Expression of Type I Collagen Induced By TGF-beta2 in Human Retinal Pigment Epithelial Cells." Experimental Eye Research, vol. 84, no. 3, 2007, pp. 464-72.
Itoh Y, Kimoto K, Imaizumi M, et al. Inhibition of RhoA/Rho-kinase pathway suppresses the expression of type I collagen induced by TGF-beta2 in human retinal pigment epithelial cells. Exp Eye Res. 2007;84(3):464-72.
Itoh, Y., Kimoto, K., Imaizumi, M., & Nakatsuka, K. (2007). Inhibition of RhoA/Rho-kinase pathway suppresses the expression of type I collagen induced by TGF-beta2 in human retinal pigment epithelial cells. Experimental Eye Research, 84(3), 464-72.
Itoh Y, et al. Inhibition of RhoA/Rho-kinase Pathway Suppresses the Expression of Type I Collagen Induced By TGF-beta2 in Human Retinal Pigment Epithelial Cells. Exp Eye Res. 2007;84(3):464-72. PubMed PMID: 17217948.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Inhibition of RhoA/Rho-kinase pathway suppresses the expression of type I collagen induced by TGF-beta2 in human retinal pigment epithelial cells. AU - Itoh,Yuji, AU - Kimoto,Kenichi, AU - Imaizumi,Masamoto, AU - Nakatsuka,Kazuo, Y1 - 2007/01/10/ PY - 2006/05/09/received PY - 2006/08/12/revised PY - 2006/10/23/accepted PY - 2007/1/16/pubmed PY - 2007/3/29/medline PY - 2007/1/16/entrez SP - 464 EP - 72 JF - Experimental eye research JO - Exp Eye Res VL - 84 IS - 3 N2 - 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. SN - 0014-4835 UR - https://www.unboundmedicine.com/medline/citation/17217948/Inhibition_of_RhoA/Rho_kinase_pathway_suppresses_the_expression_of_type_I_collagen_induced_by_TGF_beta2_in_human_retinal_pigment_epithelial_cells_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0014-4835(06)00426-X DB - PRIME DP - Unbound Medicine ER -