Figure 2. Effects of simvastatin on SPC-induced phosphorylation of Smad2 and ERK. (A) hASCs were treated with serum-free medium con- taining 2 μM SPC or vehicles (0.1% DMSO) in the absence or presence of 1 μM simvastatin for 4 days. Phosphorylation levels of Smad2 and ex- pression levels of Smad2 and GADPH were determined by Western blotting. (B) The densities of p-Smad2 were quantified and normalized to those of Smad2. Data are expressed as mean ± SD (n = 4; *P ＜ 0.05). (C) hASCs were pretreated with serum-free medium containing 1 μM simvastatin or vehicles (0.1% DMSO) for 1 day and then treated with 2 μM SPC or vehicles for 10 min. Phosphorylation levels of ERK and ex- pression levels of ERK and GADPH were determined by Western blotting. Representative data from three independent experiments are shown. Simvastatin inhibits SPC-induced sustained phosphorylation of Smad2 We previously reported that SPC treatment elicited phosphorylation of Smad2 on day 1 that was sus- tained until day 4, and that the sustained phos- phorylation of Smad2 was responsible for the in- creased expression of α-SMA (Jeon et al., 2006). Therefore, we sought to determine the effect of simvastatin on SPC-induced Smad2 phosphorylation on day 4. As shown in Figures 2A and 2B, treat- ment of hASCs with SPC for 4 days induced phos- phorylation of Smad2 and pretreatment of the cells with simvastatin markedly attenuated Smad2 phosphorylation. We have shown that SPC elicited acute ERK phosphorylation in 10 min and ERK phosphor- ylation played a key role in SPC-induced sustained phosphorylation of Smad2 (Jeon et al., 2006). Therefore, we examined the effect of simvastatin in SPC-induced ERK phosphorylation. As shown in Figure 2C, treatment of hASCs with SPC for 10 min induced phosphorylation of ERK, whereas SPC-induced ERK phosphorylation was not attenu- ated by pretreatment with simvastatin. This sug- gests that ERK is not a target of simvastatin-in- duced inhibition of Smad2 phosphorylation and α-SMA expression. Effect of simvastatin on differentiation of MSCs 161 Figure 3. Effect of simvastatin on SPC-induced RhoA activation. (A) hASCs were treated with 2 μM SPC for the indicated time periods. (C) hASCs were pretreated with serum-free medium containing 1 μM sim- vastatin or vehicles (0.1% DMSO) for 1 day and then treated with 2 μM SPC or vehicles for the indicated time periods. The amounts of RhoA in total lysates and membrane fractions were determined by Western blotting. (B, D) The densities of RhoA in membrane fractions were quanti- fied and normalized to those of RhoA in total lysates. Data are expressed as mean ± SD (n = 4; *P ＜ 0.05). (E) hASCs were pretreated with 1 μM simvastatin (0.1% DMSO) or vehicles for 1 day, followed by treat- ment with 2 μM SPC for 5 min. The amounts of RhoA in the whole cell ly- sates (Total RhoA) or the GTP-bound RhoA precipitated from the lysates were revealed by Western blotting. Representative data from three in- dependent experiments are shown. Simvastatin inhibits SPC-induced RhoA activation Increasing body of evidence suggests that RhoA-mediated rearrangement of the actin cy- toskeleton plays a pivotal role in SMC differ- entiation (Cen et al., 2004). It has been reported that activated RhoA translocated from the cytosol to the plasma membrane through geranylger- anylation, and that simvastatin inhibited this trans- location (Laufs et al., 1999). To explore whether SPC can induce RhoA acti- vation, hASCs were treated with SPC for the in- dicated periods, and the amounts of mem - brane-associated RhoA were determined. SPC treatment increased the amounts of RhoA asso- ciated with cell membrane in a time-dependent manner, with a maximal increase at 5 min (Figures 3A and 3B). The SPC-induced translocation of