Stimulation of Osteoclast Formation by RANKL Requires Interferon Regulatory Factor-4 and Is Inhibited by Simvastatin in a Mouse Model of Bone Loss

Yoshiki Nakashima,Tatsuji Haneji,Junming Yue

doi:10.1371/journal.pone.0072033

Abstract

Diseases of bone loss are a major public health problem. Here, we report the novel therapeutic action of simvastatin in osteoclastogenesis and osteoprotection, demonstrated by the ability of simvastatin to suppress osteoclast formation in vitro and in vivo. We found that in vitro, IRF4 expression is upregulated during osteoclast differentiation induced by RANKL (receptor activator of nuclear factor-κB ligand), while simvastatin blocks RANKL-induced osteoclastogenesis and decreases expression of NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1), IRF4 and osteoclast markers. We also show that IRF4 acts in cooperation with NFATc2 and NF-κB on the promoter region of NFATc1 to accelerate its initial transcription during the early stage of osteoclastogenesis. Moreover, our study using IRF4 siRNA knockdown directly demonstrates the requirement for IRF4 in NFATc1 mRNA transcription and its necessity in RANKL-induced osteoclast differentiation. Our results suggest that the reduction in osteoclastogenesis is partly due to the inhibition of IRF4 production in RANKL-induced osteoclast differentiation. To investigate the in vivo effects of simvastatin in RANKL-treated mice, we examined the bone mineral density (BMD) of a mouse model of bone loss, and found that simvastatin significantly reduced bone loss by suppressing osteoclast numbers in vivo, even in the presence of high concentrations of RANKL. These results suggest that the depletion of osteoclasts is not due to the reduction in RANKL produced by osteoblasts in vivo. The results are consistent with the hypothesis that simvastatin blocks RANKL-induced IRF4 expression in osteoclastogenesis. We propose that the expression of IRF4 by osteoclasts could be a promising new therapeutic target in bone-loss diseases.

Highlights

RANKL/RANK signaling induces osteoclast formation and activation via several transcription factors, such as interferonregulatory factors (IRFs) [1,2], c-Fos, nuclear factor-kB (NF-kB) and NFATc1 [3,4]
Our study shows that IRF4 is a constituent of the signalling pathways that mediate the effect of prenylated GTPases on RANK/RANKL-dependent osteoclastogenesis in vitro and in vivo
IRF4 increases during osteoclastogenesis To assess the expression of IRF4 during osteoclastogenesis, we used RT-PCR and immunoblot analyses to detect IRF4 expression in RAW264.7 cells after RANKL stimulation (Fig. 1A, D; full-length blots in Fig. S1A), and showed that robust induction of NFATc1 by RANKL is a necessary and pivotal step for osteoclast differentiation characterized by enhanced expression of Jmjd3, which is an H3K27me3 demethylase

Summary

Introduction

RANKL/RANK signaling induces osteoclast formation and activation via several transcription factors, such as interferonregulatory factors (IRFs) [1,2], c-Fos, NF-kB and NFATc1 [3,4]. Abundant IRF8 interacts with basally-expressed NFATc1 to suppress its transcriptional activity and prevent its activation of target genes, including autoamplification of its own promoter [15]. In this study, to dissect further these IRF4 functions in osteoclast differentiation, we focused on the transcriptional control of NFATc1 gene expression in RAW264.7 cells. Simvastatin inhibits the isoprenoids farnesyl pyrophosphate and geranylgeranyl pyrophosphate (GGPP) These isoprenoid pyrophosphates serve as essential adjuncts in the posttranslational modification of numerous key proteins that function as molecular switches, including the small GTPases RAS, RAC and RAS homologue (RHO) [17,18]. Our study shows that IRF4 is a constituent of the signalling pathways that mediate the effect of prenylated GTPases on RANK/RANKL-dependent osteoclastogenesis in vitro and in vivo

Methods

Results

Conclusion