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

Haneji T (2013) Stimulation of Osteoclast Formation by RANKL Requires Interferon Regulatory Factor-4 and Is Inhibited by Simvastatin in a Mouse Model of Bone Loss. PLoS ONE 8(9): e72033. doi:10.1371/journal.pone.0072033 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 0 Tatsuji Haneji 0 Junming Yue, The University of Tennessee Health Science Center, United States of America 0 Department of Histology and Oral Histology, Institute of Health Biosciences, The University of Tokushima Graduate School , Tokushima , Japan 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-kB 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-kB 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 RANKLinduced 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. - Funding: This work was supported by MEXT/JSPS KAKENHI (grant number 21592330). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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]. It has also been shown that NFATc1 cooperates with PU.1 on the Cathepsin K and OSCAR promoters [5,6], and forms an osteoclastspecific 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 [4,7,8]. PU.1 confers specificity to the NFATc1 response in RAW264.7 cells [9]. IRF4 and interferon consensus sequence-binding protein (ICSBP)/IRF8 are members of the IRF family, which are expressed in bone marrow-derived cells [10]. Both factors can be recruited to the IRF DNA-binding site in target genes through interaction with PU.1 [1114]. Recently, an in vivo and in vitro study indicated that IRF8 suppresses osteoclastogenesis. 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 [15]. However, our understanding of the function of IRF4 in osteoclastogenesis remains elusive. 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. Furthermore, we performed a pharmacological experiment to identify inhibitors of IRF4. Simvastatin is an orally administered competitive inhibitor of 3hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, an enzyme that catalyzes the conversion of HMG-CoA to mevalonic acid [16]. As effective cholesterol-lowering agents, statins have been extensively used for prevention of cardiovascular disease. 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]. Osteoclast survival, differentiation and function require the GTPases including RAS [19 21], RAC [22,23] and RHO [24,25]. The membrane attachment and biological activity of these small GTPases require prenylation. The Rho family of GTPases is a large family of proteins, which includes RhoA, Rac1 and Rac2. Rho kinase (ROCK) has been shown to activate the DNA binding of IRF4 [26], while another report showed that simvastatin inhibits IRF4 gene expression via selective inhibition of ROCK in Th17 cells [27]. Therefore, in this study, we used simvastatin as an inhibitor of IRF4, and report the role of IRF4 in osteoclast differentiation in the presence of RANKL. 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. Materials and Methods Reagents Reagents were obtained from the following suppliers: Alphamodified Minimum Essential Medium (a-MEM): Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS): MBL (Nagoya, Japan). Recombinant mouse RANKL: Oriental Yeast Co., Ltd. (Shiga, Japan). Simvastatin: Tokyo Chemical Industry co., (Tokyo, Japan). Y-27632: WAKO (Osaka, Japan). BAY117082: Gentaur (Kampenhout, Belgium). Anti-b-actin antibody: Sigma-Aldrich (St. Louis, MO). Anti-B23 (C-19), anti-Eps15 (C20), anti-IRF4 (M-17), anti-IRF8 (C-19), anti-NFATc1 (7A6), anti-NFATc2 (4G6-G5), anti-NF-kB p65 (C-20) and anti-TRAP (K-17) antibodies: Santa Cruz Biotechnology (Santa Cruz, CA). Anti-EZH2 (AC22) antibodies: Cell Signaling Technology (Boston, MA). Anti-osteopontin (O-17) antibody: ImmunoBiological Laboratories Co., Ltd. (Gunma, Japan). Plastic dishes: IWAKI (Chiba, Japan). Animal care All experimental protocols were in accordance with the guidelines for the care and use of laboratory animals set by the Graduate School of the Institute of Health Biosciences, the University of Tokushima (Tokushima, Japan). The protocol was approved by the Committee on Animal Experiments of the University of Tokushima (permit number: 12052 and 12067). C57BL/6J female mice (...truncated)