Rho/Rock cross-talks with transforming growth factor-beta/Smad pathway participates in lung fibroblast-myofibroblast differentiation

The differentiation of fibroblasts, which are promoted by transforming growth factor-beta (TGF-beta)/Smad, is involved in the process of pulmonary fibrosis. The Rho/Rho-associated coiled-coil-forming protein kinase (Rock) pathway may regulate the fibroblast differentiation and myofibroblast expression of a-smooth muscle actin (alpha-SMA), however, the mechanism is not clear. The aim of the present study was to evaluate the role of Rho/Rock and TGF-beta/Smad in TGF-beta 1-induced lung fibroblasts differentiation. Human embryonic lung fibroblasts were stimulated by TGF-beta 1, Y-27632 (inhibitor of Rho/Rock signaling) and staurosporine (inhibitor of TGF-beta/Smad signaling). The a-SMA expression, cell cycle progression, content of the extracellular matrix (ECM) in cell culture supernatants and the expression of RhoA, RhoC, Rock1 and Smad2 were detected. The results demonstrated that a-SMA-positive cells significantly increased following TGF-beta 1 stimulation. Rho/Rock and TGF-beta/Smad inhibitors suppressed TGF-beta 1-induced lung fibroblast differentiation. The inhibitors increased G0/G1 and decreased S and G2/M percentages. The concentrations of the ECM proteins in the supernatant were significantly increased by TGF-beta 1 stimulation, whereas they were decreased by inhibitor stimulation. RhoA, RhoC, Rock1, Smad2 and tissue inhibitor of metalloproteinase-1 were upregulated by TGF-beta 1 stimulation. The Rho/Rock inhibitor downregulated Smad2 expression and the TGF-beta/Smad inhibitor downregulated RhoA, RhoC and Rock1 expression. Therefore, the Rho/Rock pathway and Smad signaling were involved in the process of lung fibroblasts transformation, induced by TGF-beta 1, to myofibroblasts. The two pathways may undergo cross-talk in the lung fibroblasts differentiation in vitro.