Bone resorption is a process of bone remodeling which is known to be mediated by osteoclasts, which result in bone loss by eliminating the bone's mineralized matrix. It has been well reported that excessive bone resorption can be related to bone loss diseases such as osteoporosis and rheumatoid arthritis (Gough et al., 1994
). To date, various drugs have been developed and applied to treat such bone loss related diseases. Simvastatin is known to be one of these drugs used to suppress bone resorption by inhibiting osteoclastogenesis (Yamashita et al., 2010
). However, the underlying biological mechanism of simvastatin's inhibitory effect on osteoclast differentiation has yet to be elucidated. Therefore, we studied the inhibitory effect of simvastatin on osteoclast differentiation and its underlying mechanism.
In this study, simvastatin-mediated inhibition of RANKL-induced osteoclast differentiation was proven. This was shown by a dose-dependent decrease of the formation of TRAP positive cells, as well as a dose-dependent down-regulation of TRAP gene expression. Simvastatin prevents the production of ROS and this action attenuates the H2O2-induced early signaling activities including AKT, JNK, p38, ERK and NF-κB signaling pathways, which subsequently suppresses osteoclast formation.
It is well known that low concentrations of ROS mediated signals are required for the activation of downstream signaling pathways (Rhee, 1999
; Dröge, 2002
) and the generation of ROS is controlled by the binding of various cytokines to receptors (Bonizzi et al., 1999
). In the osteoclast differentiation pathway, RANKL binds to the RANK receptor and then stimulates intracellular ROS generation. This ROS is known to stimulate osteoclast differentiation and participate in early signaling events associated with osteoclast activation for bone resorption (Hall et al., 1995
). Here, we show that ROS was generated within RAW 264.7 cells during RANKL/M-CSF-simulated osteoclast differentiation. This ROS production during RANKL-simulated osteoclast differentiation decreased distinctly when the RAW 264.7 cells were co-treated with simvastatin. Previous studies have reported on the inhibitory function of simvastatin on osteoclast differentiation by blocking the RANKL-induced signaling pathway (Ahn et al., 2008
; Yamashita et al., 2010
). It has also been reported that simvastatin has antioxidant activity (Giroux et al., 1993
; Girona et al., 1999
; Mason, 1999
; Tomás et al., 2000
; Davignon et al., 2004
). From these findings, we hypothesized that simvastatin might suppress osteoclast differentiation by scavenging the generated intracellular ROS which acts as a secondary messenger in the RANKL osteoclast differentiation signaling pathway.
Previous studies have shown that simvastatin inhibited RANKL-induced NF-κB pathway activation through the suppression of IκBα phosphorylation, IκBα degradation, and IκBα kinase activity (Ahn et al., 2008
). This study confirmed these results that simvastatin down-regulated IκBα phosphorylation and IκBα degradation both of which are known to participate in the RANKL-induced osteoclast differentiation signaling pathway. In this study, the main intracellular signal pathways engaged by RANK were also investigated in order to elucidate the underlying mechanisms of simvastatin's inhibitory effects on this pathway. It has been previously found that RANKL-induced osteoclast differentiation is mediated by RANK. RANK mainly activates NF-κB, AKT, JNK, P38 and ERK signaling pathways (Kim et al., 2009
; Hasegawa et al., 2010
; Lee et al., 2010
). Other drugs have been found to suppress these RANKL-induced signaling pathways. Curcumin, a natural polyphenolic derogate extracted from turmeric, has also been found to suppress osteoclast differentiation and osteoclast function. These effects were accompanied by the inhibition of IκBα phosphorylation and NF-κB activation (Von Metzler et al., 2009
). Luteolin has also been found to inhibit osteoclasts, however, this does not affect RANKL-induced MAPKs expression and activation. Instead, this antioxidant was found to affect the phosphorylation of ATF2, which is downstream of the p38 MAPK signaling pathway (Lee et al., 2009
). Sauchinone also attenuates RANKL-induced NF-κB, ERK and p38 MAPKs activation in osteoclast differentiation. However, the activation of JNK by RANKL was not affected by sauchinone (Han et al., 2007
). In our study, we also detected inhibition effects of RANKL-induced signaling pathways by simvastatin. Simvastatin inhibited not only IκBα signal but also the AKT, and MAPKs such as JNK, p38 and ERK signaling pathways which are normally activated through ROS in osteoclast differentiation. Simvastatin strongly attenuates RANKL-induced AKT and JNK signaling.
In conclusion, we analyzed the effect of osteoclast differentiation by simvastatin and identified one mechanism by which simvastatin acts as an inhibitor. These results show that simvastatin suppresses RANKL-induced osteoclastogenesis through inhibition of ROS-induced signaling pathways. This study elucidated the mechanism by which simvastatin acts as an osteoclast inhibitor by blocking ROS and help contribute to further clarification of the mechanisms underlying osteoporosis and other pathological bone-loss diseases. Also, antioxidants may have therapeutic implications for inhibiting bone resorption.