162 Exp. Mol. Med. Vol. 44(2), 159-166, 2012 Figure 4. Role of RhoA-Rho kinase pathway in the SPC-induced phos- phorylation of Smad2. (A) hASCs were transfected with the domi- nant-negative mutant of RhoA (DN-RhoA) or control vector and then treated with serum-free medium containing 2 μM SPC or vehicles for 4 days. (C) hASCs were treated with serum-free medium containing 2 μM SPC or vehicles in the absence or presence of 10 μM Y27632 for 4 days. Phosphorylation levels of Smad2 and expression levels of Smad2, α-SMA, and GAPDH were determined by Western blotting. (B, D) The densities of α-SMA were quantified and normalized to those of GAPDH. Data are expressed as mean ± SD (n = 4; *P ＜ 0.05). RhoA was completely abrogated by pretreatment of the cells with simvastatin (Figures 3C and D). To confirm these results, we determined the amounts of GTP-loaded RhoA after treatment with SPC and/or simvastatin. As shown in Figure 3E, SPC treatment increased GTP-RhoA levels and pre- treatment with simvastatin abrogated SPC-induced increase of GTP-RhoA. RhoA-Rho kinase is responsible for the SPC-induced α-SMA expression and Smad2 phosphorylation In order to delineate whether RhoA is involved in the SPC-induced α-SMA expression and Smad2 phosphorylation, we examined the effect of a domi- nant-negative mutant of RhoA (RhoAN19) on SPC-in- duced α-SMA expression and Smad2 phosphor- ylation. As shown in Figures 4A and 4B, over- expression of RhoAN19 attenuated the SPC-in- duced α-SMA expression and Smad2 phosphor- ylation, suggesting that RhoA plays a key role in SPC-induced α-SMA expression through a Smad2- dependent mechanism. To explore whether Rho kinase, a major down- stream target of RhoA, is involved in SPC-induced α-SMA expression and Smad2 phosphorylation in Figure 5. Effect of simvastatin on SPC-induced secretion of TGF-β1. hASCs were treated with serum-free medium containing 2 μM SPC or vehicles (w/o) in the absence or presence of 10 μM Y27632 or 1 μM simvastatin for 2 days. The conditioned medium was subjected to ELISA for determination of TGF-β1 protein levels. Data represent mean ± SD (n = 4). *indicates P ＜ 0.05. hASCs, we examined the effect of Y27632, a Rho kinase-specific inhibitor, on α-SMA expression and Smad2 phosphorylation induced by SPC. As shown in Figures 4C and 4D, SPC-induced α-SMA ex- pression and Smad2 phosphorylation were abro- gated by pretreatment of the cells with Y27632, suggesting that Rho kinase is involved in SPC-in- duced α-SMA expression and Smad2 phosphor- ylation. Moreover, basal expression levels of α-SMA in the absence of SPC treatment were slightly in- hibited by either DN-RhoA overexpression or pre- treatment with Y27632, suggesting a potential role of RhoA in basal expression of α-SMA as well as in the SPC-induced α-SMA expression. Simvastatin inhibits SPC-induced secretion of TGF-β1 through Rho kinase-dependent mechanism We previously reported that autocrine secretion of TGF-β1 is responsible for SPC-induced Smad2 phosphorylation and α-SMA expression (Jeon et al., 2006). To explore whether the inhibitory effects of simvastatin on SPC-induced Smad2 phosphor- ylation and α-SMA expression are due to the de- creased secretion of TGF-β1, we measured the ef- fect of simvastatin on SPC-induced TGF-β1 secretion. As shown in Figure 5, SPC stimulated secretion of TGF-β1 from hASCs, and pretreatment of the cells with simvastatin abrogated SPC-induced secretion of TGF-β1. Furthermore, pretreatment of the cells with Y27632 inhibited SPC-induced TGF-β1 secretion. These results suggest that simvastatin inhibits the SPC-induced autocrine TGF-β1/Smad2 signaling pathway and α-SMA expression by inhibiting Rho kinase-dependent TGF-β1 secretion.