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Inhibition of RhoA/Rho-kinase pathway suppresses the expression of extracellular matrix induced by CTGF or TGF- β in ARPE-19
oleh: Jing Zhu
Format: | Article |
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Diterbitkan: | Press of International Journal of Ophthalmology (IJO PRESS) 2013-02-01 |
Deskripsi
<b>AIM:</b> 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.<b>METHODS:</b> 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.<b>RESULTS:</b> 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 (<i>P</i>=0.006, <i>P</i>=0.003 respectively), laminin mRNA (<i>P</i>=0.006, <i>P</i>=0.005), MMP-2 mRNA (<i>P</i>= 0.006, <i>P</i>= 0.001), COL1A1 mRNA (<i>P</i>=0.001, <i>P</i>=0.001), COL1A2 mRNA (<i>P</i>=0.001, <i>P</i>=0.001). Preincubation of ARPE-19 with Y27632 (10mmol/L) significantly prevented CTGF or TGF- β induced fibronectin (<i>P</i>=0.005, <i>P</i>=0.003 respectively), MMP-2 (<i>P</i>= 0.003, <i>P</i>=0.002), COL1A1 (<i>P</i>=0.006, <i>P</i>=0.003), and COL1A2 (<i>P</i>=0.006, <i>P</i>=0.004) gene expression, but not laminin (<i>P</i>=0.375, <i>P</i>=0.516)#$NL<b>CONCLUSION:</b> 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 <i>in vitro</i>. 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.