Citation:Zhu J,Nguyen D,Ouyang H,Zhang XH,Chen XM,Zhang K.Inhibition of RhoA/Rho-kinase pathway suppresses the expression of extracellular matrix induced by CTGF or TGF- β in ARPE-19.Int J Ophthalmol 2013;6(1):8-14,doi:10.3980/j.issn.2222-3959.2013.01.02
Inhibition of RhoA/Rho-kinase pathway suppresses the expression of extracellular matrix induced by CTGF or TGF- β in ARPE-19
Received:October 16, 2012  Revised:January 28, 2013
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DOI:10.3980/j.issn.2222-3959.2013.01.02
Key Words:Rho-associated protein kinase inhibitor  Connective tissue growth factor  transforming growth factor-β  proliferative vitreoretinopathy
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Jing Zhu Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu , Sichuan Province, China
;Department of Ophthalmology and Shiley Eye Center, and Institute for Genomic Medicine, University of California San Diego, La Jolla, CA 92093, USA
Duy Nguyen Department of Ophthalmology and Shiley Eye Center, and Institute for Genomic Medicine, University of California San Diego, La Jolla, CA 92093, USA
Hong Ouyang Department of Ophthalmology and Shiley Eye Center, and Institute for Genomic Medicine, University of California San Diego, La Jolla, CA 92093, USA
Xiao-Hui Zhang Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Capital Medical University, Beijing , China
Xiao-Ming Chen Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu , Sichuan Province, China
Kang Zhang Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu , Sichuan Province, China
;Department of Ophthalmology and Shiley Eye Center, and Institute for Genomic Medicine, University of California San Diego, La Jolla, CA 92093, USA
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Abstract:
      AIM: To investigate the role of Rho-associated protein kinase (ROCK) inhibitor, Y27632, in mediating the production of extracellular matrix (ECM) components including fibronectin, matrix metallo-proteinase-2 (MMP-2) and type I collagen as induced by connective tissue growth factor (CTGF) or transforming growth factor-β (TGF-β) in a human retinal pigment epithelial cell line, ARPE-19.

    METHODS: The effect of Y27632 on the CTGF or TGF-β induced phenotype in ARPE-19 cells was measured with immunocytochemistry as the change in F-actin. ARPE-19 cells were treated with CTGF (1, 10, 100ng/mL) and TGF-β (10ng/mL) in serum free media, and analyzed for fibronectin, laminin, and MMP-2 and type I collagen by RT-qPCR and immunocytochemistry. Cells were also pretreated with an ROCK inhibitor, Y27632, to analyze the signaling contributing to ECM production.

    RESULTS: Treatment of ARPE-19 cells in culture with TGF-β or CTGF induced an ECM change from a cobblestone morphology to a more elongated swirl pattern indicating a mesenchymal phenotype. RT-qPCR analysis and different gene expression analysis demonstrated an upregulation in expression of genes associated with cytoskeletal structure and motility. CTGF or TGF-β significantly increased expression of fibronectin mRNA (P=0.006, P=0.003 respectively), laminin mRNA (P=0.006, P=0.005), MMP-2 mRNA (P= 0.006, P= 0.001), COL1A1 mRNA (P=0.001, P=0.001), COL1A2 mRNA (P=0.001, P=0.001). Preincubation of ARPE-19 with Y27632 (10mmol/L) significantly prevented CTGF or TGF- β induced fibronectin (P=0.005, P=0.003 respectively), MMP-2 (P= 0.003, P=0.002), COL1A1 (P=0.006, P=0.003), and COL1A2 (P=0.006, P=0.004) gene expression, but not laminin (P=0.375, P=0.516)#$NL

    CONCLUSION: Our study demonstrated that both TGF-β and CTGF upregulate the expression of ECM components including fibronectin, laminin, MMP-2 and type I collagen by activating the RhoA/ROCK signaling pathway. During this process, ARPE-19 cells were shown to change from an epithelial to a mesenchymal phenotype in vitro. Y27632, a ROCK inhibitor, inhibited the transcription of fibronectin, MMP-2 and type I collagen, but not laminin. The data from our work suggest a role for CTGF as a profibrotic mediator. Inhibiting the RhoA/ROCK pathway represents a potential target to prevent the fibrosis of RPE cells. This might lead to a novel therapeutic approach to preventing the onset of early PVR.

PMC FullText Html:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3580241/
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