Involvement of Rho-associated coiled-coil kinase signaling inhibition in TGF-β1/Smad2, 3 signal transduction in vitro

• Published in: International journal of ophthalmology Vol. 10; no. 12; pp. 1805 - 1811
• Main Authors: Feng, Zhao-Hui, Zhang, Xiao-Hui, Zhao, Jia-Qi, Ma, Jun-Ze
• Format: Journal Article
• Language: English
• Published: China International Journal of Ophthalmology Press 18.12.2017; Press of International Journal of Ophthalmology (IJO PRESS)
• Subjects: 1811; Basic Research; human ocular Tenon’s capsule fibroblasts; ROCK; Smad; transforming growth factor-beta1; Y-27632; Smad; ROCK; Y-27632; transforming growth factor-beta1; human ocular Tenon's capsule fibroblasts
• ISSN: 2222-3959; 2227-4898
• DOI: 10.18240/ijo.2017.12.03

To research the effect of Y-27632, a selective Rho-associated coiled-coil kinase (ROCK) inhibitor, on TGF-β1/Smad2, 3 signal transduction in ocular Tenon's capsule fibroblasts (OTFs). Primary ocular Tenon's capsule fibroblasts had been cultured . The effect of Y27632 on proliferation of OTF stimulated by lysophosphatidic acid (LPA) was evaluated by MTT colorimetric assay so as to sift out the proper concentrations range of Y-27632 for the next experiment. Real time-polymerase chain reactor (RT-PCR) was to analyze the changes of Smad2 and Smad3 genes of cells affected by Y-27632, though unaffected by transforming growth factor-beta1 (TGF-β1). Proteins of Smad2, Smad3, phosphorylated Smad2 (Ser245/250/255), and phosphorylated Smad3 (Ser423/425/203) were respectively quantified by Western blot after OTFs were successively incubated by TGF-β1 and Y-27632. Meanwhile, α-smooth muscular actin (α-SMA) protein was also quantified after the small intervening gene fragments of human Smad2 and Smad3 were designed, synthesized, and then transfected to OTFs. Y-27632 significantly inhibited OTFs proliferation stimulated by LPA. Also Y-27632 significantly suppressed the expressions of Smad2 mRNA, Smad2, 3 proteins expressions, Smad3 phosphorylation at the carboxylic terminals of Ser423/425/203 which had been radically promoted by TGF-β1. SiRNA-Smad2, 3 suppressed α-SMA expressions, but less effectively than Y-27632. The inhibition of ROCK signaling may be a potential therapeutic candidate for the treatment of the filtration channel fibrosis.