Simvastatin inhibits osteoclast differentiation by scavenging reactive oxygen species

Abstract

Osteoclasts, together with osteoblasts, control the amount of bone tissue and regulate bone remodeling. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. Reactive oxygen species (ROS) acts as a signal mediator in osteoclast differentiation. Simvastatin, which inhibits 3-hydroxy-3-methylglutaryl coenzyme A, is a hypolipidemic drug which is known to affect bone metabolism and suppresses osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand (RANKL). In this study, we analyzed whether simvastatin can inhibit RANKL-induced osteoclastogenesis through suppression of the subsequently formed ROS and investigated whether simvastatin can inhibit H2O2-induced signaling pathways in osteoclast differentiation. We found that simvastatin decreased expression of tartrate-resistant acid phosphatase (TRAP), a genetic marker of osteoclast differentiation, and inhibited intracellular ROS generation in RAW 264.7 cell lines. ROS generation activated NF-κB, protein kinases B (AKT), mitogen-activated protein kinases signaling pathways such as c-JUN N-terminal kinases, p38 MAP kinases as well as extracellular signal- regulated kinase. Simvastatin was found to suppress these H2O2-induced signaling pathways in osteoclastogenesis. Together, these results indicate that simvastatin acts as an osteoclastogenesis inhibitor through suppression of ROS-mediated signaling pathways. This indicates that simvastatin has potential usefulness for osteoporosis and pathological bone resorption.

Figures

Figure 1

Inhibitory effect of simvastatin on osteoclast differentiation from RAW 264.7 cell-induced by RANKL. RAW 264.7 cells were cultured with the indicated dose of simvastatin osteoclastogenesis for 7 days. Photographs of TRAP-stained osteoclasts generated from RAW 264.7 cells (A). Simvastatin reduced TRAP+ multinuclear osteoclast formation from RAW 264.7 cells (B). TRAP down-regulation of RANKL-induced gene expression with simvastatin in real-time PCR (C). *P < 0.01, significantly different from without simvastatin.

Figure 2

Production of ROS following RANKL stimulation and effects of H2O2 on p-IκBα, p-AKT, p-MAPKs signals activation in RAW 264.7 cells. Cells were stimulated with RANKL/M-CSF and Intracellular ROS production was detected by CM-DCF fluorescence (A). The relative level of H2O2 to produce this process was quantified the Amplex Red reagent (B). The phosphorylation of IκBα and AKT activation was induced in a dose-dependent manner by exogenous treatment with H2O2 (C) and MAPK signals activation also was detected in H2O2 treated cells (D). *P < 0.01, significantly different from control.

Figure 3

Inhibition of ROS by simvastatin following exogenous H2O2 stimulation and RANKL stimulation in osteoclast differentiation. The amount of H2O2 was measured by scavenging activity of simvastatin against exogenous H2O2 at different concentrations with Amplex Red reagent (A). The cells were pretreated with simvastatin for 30 min, and then stimulated with RANKL/M-CSF for 60 min. Intracellular ROS production was detected by CM-DCF fluorescence (B). The relative level of H2O2 to produce was quantified the Amplex Red reagent (C). *P < 0.01, significantly different from control.

Figure 4

Effects of simvastatin on RANKL-induced p-IκBα, p-AKT, MAPKs activation in osteoclasts. RAW 264.7 cells were pretreated with simvastatin for 30 min, and then stimulated with RANKL for the indicated times. Whole cell lysates were immunoblotted to western blot analysis for phosphorylation of IκBα and AKT signals proteins (A) and MAPKs signals proteins such ERK, JNK and p38 (B) as indicated.