Table 1.
Sequences of quantitative PCR primers.
Table 2.
Sequences of siRNA duplexes.
Figure 1.
Expression of IRF4 in osteoclastogenesis.
(A) Western blot analysis of Jmjd3, EZH2, IRF4, NFATc1 and β-actin protein expression in RAW264.7 cells cultured in the presence of 50 ng/mL RANKL at 0, 1, 2 and 4 d. β-actin served as the loading control. (B) ChIP assay of the IRF4 and NFATc1 promoter region in RAW264.7 cells cultured in the presence of 50 ng/mL RANKL at 0, 1, 2 and 4 d. (C) Western blot analysis of NFATc1 and IRF4 protein expression in nuclear and cytoplasmic fractions of RAW264.7 cells cultured in the presence of 50 ng/mL RANKL at 0, 1, 2 and 4 d. Expression levels of B23 and EPS protein were measured as the loading controls for nuclear and cytoplasmic fractions, respectively. (D) Quantitative real-time PCR analysis of Jmjd3, IRF4 and NFATc1 mRNA in RAW264.7 cells cultured in the presence of 50 ng/mL RANKL at 0, 1, 2 and 4 d. Data represent mean ± S.D. * P<0.05, **P<0.01.
Figure 2.
NFATc1 expression in osteoclastogenesis after treatment with IRF4 siRNA.
(A) RAW264.7 cells were transfected with IRF4 siRNA or nontargeting control siRNAs (N.C) in the presence of 50 ng/mL RANKL for 5 d. Expression of IRF4 mRNA (top right) and protein (lower right). Expression levels of GAPDH mRNA and β-actin protein were measured as the loading controls. Numbers of TRAP-positive multinucleated cells (MNCs) were counted (middle). TRAP-positive cells appear red in the photomicrograph (left); n = 8. Data represent mean ± S.D. **P<0.01. Scale bar = 100 µm. (B) Quantitative real-time PCR analysis of NFATc1 mRNA in RAW264.7 cells cultured in the presence of 50 ng/mL RANKL and IRF4 siRNA at 2 d; n = 4. * P<0.05. (C) Western blot analysis of NFATc1 protein in RAW264.7 cells cultured in the presence of 50 ng/mL RANKL and IRF4 siRNA at 0, 1, 2 and 4 d. β-actin served as the loading control. (D) ChIP analysis of the NFATc1 promoter region in RAW264.7 cells cultured in the presence of 50 ng/mL RANKL at 0, 1, 2 and 4 d.
Figure 3.
Simvastatin inhibits osteoclastogenesis.
(A) RAW264.7 cells cultured in the presence of 50 ng/mL RANKL and 2.5 µM simvastatin for 5 days, stained for TRAP. Top, TRAP-positive cells appear red in the photomicrograph. Black arrows indicate multinucleated osteoclasts. Bottom, TRAP-positive multinucleated cells were counted as osteoclasts; n = 8. Data represent mean ± S.D. * P<0.05, **P<0.01. Scale bar = 100 µm. (B) Western blot analysis of NF-κB p65, IRF4, NFATc1, NFATc2 and β-actin proteins in RAW264.7 cells cultured in the presence of 50 ng/mL RANKL, 2.5 µM simvastatin and 5 µM BAY11-7082 at 4 d. β-actin served as the loading control. (C) Quantitative real-time PCR analysis of NFATc1 mRNA in RAW264.7 cells cultured in the presence of 50 ng/mL RANKL and 2.5 µM simvastatin at 2 d; n = 4. Data represent mean ± S.D. * P<0.05. (D) Western blot analysis of NFATc1 protein in RAW264.7 cells cultured in the presence of 50 ng/mL RANKL and 2.5 µM simvastatin at 0, 1, 2 and 4 d. β-actin served as the loading control. (E) Western blot analysis of IRF4 protein in RAW264.7 cells cultured in the presence of 50 ng/mL RANKL and 100 µM Y-27632 at 4 d. β-actin served as the loading control. (F) Quantitative real-time PCR analysis of Atp6v0d2, Cathepsin K, TRAP and DC-STAMP expression in RAW264.7 cells cultured in the presence of 50 ng/mL RANKL and 2.5 µM simvastatin at 0 and 4 d. n = 5. Data represent mean ± S.D. **P<0.01.
Figure 4.
In vivo effects of simvastatin in a mouse model of bone loss.
(A) 3D images of the distal femur showing the protection of bone mass by simvastatin in mice injected with 1 mg/kg RANKL. Upper panels: sagittal plane; lower panels: transverse plane. (B) Trabecular, cortical, total and plane BMD were measured; n = 5. Data represent mean ± S.D. **P<0.01. Bottom, cortical thickness, cortical bone area ratio and trabecular bone area ratio were measured; n = 5. Data represent mean ± S.D. **P<0.01. (C) Left, TRAP and osteopontin immunostaining, and toluidine blue staining of the distal femur showing inhibition of osteoclast differentiation by 10 mg/kg simvastatin in 1 mg/kg RANKL-injected mice. Right, osteoclast numbers were counted; n = 5. Data represent mean ± S.D. **P<0.01. Scale bar = 0.1 mm.
Figure 5.
Model of osteoclastogenesis acceleration by IRF4.
In osteoclast precursors, differentiation is regulated by epigenetic modification of the IRF4 and NFATc1 genes, and demethylation of H3K27me3 by Jmjd3 plays a critical role in this process. RANKL induces upregulation of IRF4, thereby augmenting IRF4 expression in the nucleus. We examined the mechanism of the increase in NFATc1 expression with RANKL. Stimulation of osteoclast precursors by RANKL results in activation of NF-κB which binds the NFATc1 promoter, cooperating with activated IRF4 and NFATc2 to induce initial induction of NFATc1. The increase in NFATc1 and IRF4 expression and decreased H3K27me3 detection could be coincidental and not causal.