Involvement of Rho-associated coiled-coil kinase signaling inhibition in TGF-β1/Smad2, 3 signal transduction in vitro
Abstract
Aim: This study aimed to investigate the effect of Y-27632, a selective Rho-associated coiled-coil kinase (ROCK) inhibitor, on the TGF-β1/Smad2, 3 signaling pathway in ocular Tenon’s capsule fibroblasts (OTFs).
Methods: Primary OTFs were cultured in vitro. The impact of Y-27632 on OTF proliferation, induced by lysophosphatidic acid (LPA), was assessed using an MTT colorimetric assay to determine the appropriate concentration range for further experiments. Real-time PCR (RT-PCR) was performed to analyze changes in the gene expression of Smad2 and Smad3 in cells treated with Y-27632, independent of TGF-β1 influence. The levels of Smad2, Smad3, phosphorylated Smad2 (Ser245/250/255), and phosphorylated Smad3 (Ser423/425/203) proteins were quantified using Western blotting after treating OTFs with TGF-β1 and Y-27632. Additionally, α-smooth muscle actin (α-SMA) protein levels were measured following transfection of OTFs with small interfering RNA (siRNA) targeting human Smad2 and Smad3.
Results: Y-27632 significantly inhibited LPA-stimulated OTF proliferation. It also notably reduced the expression of Smad2 mRNA and protein, Smad3 protein expression, and the phosphorylation of Smad3 at Ser423/425/203, which had been enhanced by TGF-β1. Additionally, siRNA targeting Smad2 and Smad3 reduced α-SMA expression, but was less effective than Y-27632 in doing so.
Conclusion: Targeting ROCK signaling through inhibition with Y-27632 may offer a promising therapeutic approach for treating filtration channel fibrosis.