br Materials and methods br Results br Discussion This

Materials and methods
Results
Discussion This present study and previous works in the literature have shown that mechanical stimulation promotes the proliferation of BMSCs (Luu et al., 2009). In this study, the presented results revealed that pressure promoted BMSC proliferation by facilitating purchase Digoxigenin-11-dUTP initiation. The G1 phase progression was positively regulated primarily by the cyclin-dependent kinase (CDK)/cyclin D and CDK/cyclin E complexes (Dulic et al., 1992; Koff et al., 1992; Matsushime et al., 1994). The G1 phase progression was negatively regulated by the interactions between CDK and the inhibitor of kinase 4A (INK4A) and the interactions between CDK and the members of the CDK-interacting protein/kinase inhibitory protein (Cip/Kip) family (McConnell et al., 1999; Parry et al., 1995, 1999)]. Welsh et al. found that the normal expression of the cyclin D protein required the activities of the RhoA/ROCK pathway (Welsh et al., 2001). RhoA/ROCK stabilized ERK activity in cells through the activation of the downstream protein LIM domain kinase (LIMK) and eventually regulated the concentration of the cyclin D protein. In addition, RhoA/ROCK regulated the activities of the CDK inhibitors p21cip1 and p27kip1 (Hu et al., 1999; Olson et al., 1998; Vidal et al., 2002; Weber et al., 1997). The present study showed that pressure promoted cell cycle initiation. However, the simultaneous suppression of RhoA/ROCK activities and application of pressure resulted in cell cycle arrest, indicating that the pressure-induced cell cycle initiation depended on RhoA/ROCK activities. A western blot analysis showed that pressure inhibited ERK phosphorylation, suggesting that the induction of cell cycle initiation by pressure may not require a modulation of the cyclin D concentration, which involved the regulation of ERK activity through the LIMK protein. Therefore, it was speculated that under the present experimental conditions, mechanical pressure promoted cell cycle initiation through the suppression of the activities of the CDK inhibitors p21cip1 and p27kip1. In addition to RhoA/ROCK, which were capable of regulating the cell cycle, the Rac1 pathways have been found to promote cyclin D mRNA translation through integrin activities (Mettouchi et al., 2001). When the Rho pathways were inhibited, Rac1 regulated the expression of cyclin D precursor proteins (Welsh et al., 2001). In agreement with the literature, the present study found that the down-regulation of Rac1 activity effectively antagonized the pressure-induced cell cycle initiation. It has been reported that mechanical stimulation activated the MAPK signaling cascades in BMSCs and affected their proliferation and differentiation (Riddle et al., 2006; Stanton et al., 2003). Jun Liu et al. applied a combination of hydrostatic pressure and osteogenic agents to BMSCs and found that the exposure to either dynamic or static pressure induced an initial osteodifferentiation of BMSCs. ERK signaling participated in early osteodifferentiation and played a positive but non-critical role in mechanotransduction, whereas p38 MAPK was not involved in this process (Liu et al., 2009). However, the present study showed that pressure promoted the phosphorylation of JNKs but not ERKs in the BMSCs. The discrepancy may be explained by the differences in the types of mechanical forces and biochemical factors applied to the cells. Proteins in the Rho family are closely related to proteins in the MAPK pathways. It was reported that Rac1 functioned as an upstream regulatory factor of JNK (Jin et al., 2006; Kukekov et al., 2006). The present study showed that hydrostatic pressure promoted the phosphorylation of the JNK proteins and up-regulation of both RhoA and Rac1 activities and effectively antagonized the pressure-induced activation of JNK1/2. These results, for the first time, confirmed that the activation of the JNK pathway by hydrostatic pressure may be negatively regulated by RhoA and Rac1. Previous studies have shown that there were associations between active phosphorylated JNK and stress fibers in cells (Hamel et al., 2006; Yang et al., 2007). Furthermore, a proximity ligation assay demonstrated the co-localization of phospho-JNK and F-actin under mechanical stimulation (Mengistu et al., 2011). In the present study, we also observed the increased F-actin cytoskeleton assembly and the up-regulated JNK1/2 phosphorylation in BMSCs upon exposure to hydrostatic pressure. Mengistu et al. reported that the initial co-localization of phospho-JNK with the actin pool outside the nucleus and later with cortical actin at cell peripheries could imply a role of JNK in the transport of actin to form cortical actin (Mengistu et al., 2011). Therefore, we inferred that inhibition of phospho-JNK under mechanical forces by the agonists of RhoA and Rac1, as observed in our work, was highly dependent on the regulation of the cytoskeleton-modulating proteins to the cytoskeletal assembly.