RhoA/ROCK Signaling Regulates TGF-beta 1-Induced Fibrotic Effects in Human Pterygium Fibroblasts through MRTF-A

Purpose: The overexpression of transforming growth factor-beta1 (TGF-beta 1) after surgical excision often leads to excessive fibrosis, indicating the recurrence of pterygium. The aims of the present in vitro study were to investigate the role of RhoA/ROCK signaling in regulating fibrotic effects of primary human pterygium fibroblasts (HPFs), as well as to explore the possible mechanisms of these effects. Methods: Pterygium samples were obtained from surgery, and profibrotic activation was induced by TGF-beta 1. Cell proliferation was detected by CCK-8 assay; cell migration was detected by wound healing assay; quantitative real-time PCR and Western blot were used to detect the effects of TGF-beta 1 and the role of RhoA/ROCK signaling in the synthesis of alpha-smooth muscle actin (a-SMA), type I and Ill collagen (COL1 and COL3), and matrix metalloproteinase-9 (MMP9) in HPFs. The changes of signaling pathways were detected by Western blot; and pharmaceutical inhibition of RhoA/ROCK signaling and its downstream MRFT-A/SRF transcription pathway were used to assess their possible mechanism in HPFs fibrosis. Results: ROCK inhibitor Y-27632 decreased TGF-beta 1-induced cell proliferation and migration, reduced the TGF-beta 1-induced expression of profibrotic markers in HPFs, and suppressed TGF-beta 1-induced nuclear accumulation of Myocardin-related transcription factor A (MRTF-A) as well as accompanied elevation of F/G-actin ratio in HPFs. MRTF-A/Serum response factor (SRF) inhibitor CCG-100602 attenuated the TGF-beta 1-induced alpha-SMA expression and reduced myofibroblast activation in HPFs. Conclusions: RhoA/ROCK signaling played a pivotal role in TGF-beta 1-induced fibrosis and myofibroblast activation in HPFs at least in part by inactivating the downstream MRTF-A/SRF transcriptional pathway.