These isoprenoid pyrophosphates serve as essential adjuncts in the post-translational modification of numerous key proteins that function as molecular switches, including the small GTPases RAS, RAC and RAS homologue (RHO). Simvastatin inhibits the isoprenoids farnesyl pyrophosphate and geranylgeranyl pyrophosphate (GGPP). As effective cholesterol-lowering agents, statins have been extensively used for prevention of cardiovascular disease. Simvastatin is an orally administered competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, an enzyme that catalyzes the conversion of HMG-CoA to mevalonic acid. Furthermore, we performed a pharmacological experiment to identify inhibitors of IRF4. Therefore, 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. However, our understanding of the function of IRF4 in osteoclastogenesis remains elusive. In osteoclast precursors, abundant IRF8 interacts with basally-expressed NFATc1 to suppress its transcriptional activity and thus prevent its activation of target genes, including autoamplification of its own promoter. Recently, an in vivo and in vitro study indicated that IRF8 suppresses osteoclastogenesis. Both factors can be recruited to the IRF DNA-binding site in target genes through interaction with PU.1 –. IRF4 and interferon consensus sequence-binding protein (ICSBP)/IRF8 are members of the IRF family, which are expressed in bone marrow-derived cells. PU.1 confers specificity to the NFATc1 response in RAW264.7 cells. It has also been shown that NFATc1 cooperates with PU.1 on the Cathepsin K and OSCAR promoters, , and forms an osteoclast-specific transcriptional complex containing AP-1 (Fos/Jun) and PU.1 for the efficient induction of osteoclast-specific genes, such as Atp6v0d2, Cathepsin K, DC-STAMP and TRAP. RANKL/RANK signaling induces osteoclast formation and activation via several transcription factors, such as interferon-regulatory factors (IRFs), , c-Fos, NF-κB and NFATc1. We propose that the expression of IRF4 by osteoclasts could be a promising new therapeutic target in bone-loss diseases. The results are consistent with the hypothesis that simvastatin blocks RANKL-induced IRF4 expression in osteoclastogenesis. These results suggest that the depletion of osteoclasts is not due to the reduction in RANKL produced by osteoblasts in vivo. 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. Our results suggest that the reduction in osteoclastogenesis is partly due to the inhibition of IRF4 production in RANKL-induced osteoclast differentiation.
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.
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.
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. 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. Diseases of bone loss are a major public health problem.