Cutting Edge: EZH2 Promotes Osteoclastogenesis by Epigenetic Silencing of the Negative Regulator IRF8

EZH2 is required for osteoclast differentiation and NFATc1 expression

We tested the effects of the selective EZH2 inhibitor GSK126 on osteoclast differentiation using human blood–derived OCPs, which are postmitotic cells thought to correspond to circulating quiescent OCPs that migrate to sites of bone erosion and differentiate into osteoclasts (17). The human system offers the advantage of testing the effects of EZH2 inhibition on osteoclastogenesis separately from the effects on proliferation and survival of progenitors that can occur in mouse bone marrow precursors. GSK126 was described to bind EZH2 and inhibit its methyltransferase activity without affecting total histone H3 or PRC2 amounts (13). A dose-response experiment showed that the inhibitory effects of GSK126 on RANKL-induced differentiation of TRAP⁺ multinucleated osteoclasts became apparent at 0.5 μM and were essentially complete at 2 μM GSK126 (Fig. 1A). Inhibition of the osteoclast-differentiation program by GSK126 was corroborated by inhibition of RANKL-induced expression of genes encoding β3 integrin (ITGB3), calcitonin receptor (CALCR), and cathepsin K (CTSK), which are markers of the intermediate and late stages of osteoclast differentiation and are important for osteoclast function. RANKL induction of NFATc1, which is required for osteoclast differentiation and promotes the expression of many osteoclast genes, such as ITGB3, was also strongly suppressed by GSK126 (Fig. 1B, 1C). Accordingly, osteoclast-mediated resorption on osteoassay substrate was markedly suppressed in GSK126-treated osteoclast cultures compared with vehicle-treated controls. (Fig. 1D). As a specificity control, we found that GSK126 did not affect basal expression or induction of various immune response genes (C.F. and Y.Q., unpublished observations).

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FIGURE 1.

EZH2 is required for osteoclast differentiation and NFATc1 expression. (A) Monocytes pretreated with DMSO or the indicated concentrations of GSK126 were stimulated for 5 d with RANKL and stained for TRAP. Data are mean ± SEM of triplicates from a representative experiment (n = 3). Original magnification ×10. (B) Real-time qPCR analysis of mRNA from cells pretreated with GSK126 or DMSO for 1 d and stimulated with RANKL for 0, 1, or 3 d. Data are mean ± SEM of triplicates from one representative experiment. Percentage inhibition by GSK126 at day 3 was significant (p < 0.05, one tailed t test) for pooled data (n = 4). n.s., not significant. (C) Immunoblot of NFATc1 expression in cells treated with GSK126. Blots shown are representative (n = 5). (D) Bone-resorption assay. Representative images stained by toluidine blue from four experiments (left panels). Percentage of resorption area relative to total area of osteoassay plate (right panel). Original magnification ×10. **p = 0.0065, two-tailed, unpaired t test. (E) Effective knockdown (KD) of EZH2 gene expression using EZH2-specific LNAs relative to control LNAs. (F) Representative photomicrographs (left and middle panels) and cell counts (right panel) of TRAP staining of OCPs transfected with control or EZH2-specific LNAs. Percentage suppression was significant for pooled data (n = 5). Original magnification ×10. ***p = 0.0003, two-tailed, unpaired t test. (G) Real-time qPCR analysis of osteoclast gene expression in cells transfected with control or EZH2-specific LNAs. Data are mean ± SEM of triplicates from one of four experiments. p < 0.05 for percentage suppression in pooled data (n = 4). (H) Immunoblot of EZH2 and NFATc1 expression in cells transfected with control or EZH2-specific LNAs. Blots are representative (n = 5). Statistical analysis was performed on pooled data. *p < 0.05, **p < 0.01, ***p < 0.005, one-way ANOVA with post hoc Tukey test for all data with the exception of (D) and (F).

To confirm that GSK126-induced suppression of osteoclastogenesis was mediated by EZH2 inhibition, we knocked down EZH2 expression using LNAs and analyzed osteoclast differentiation and gene expression. EZH2 knockdown (Fig. 1E) effectively suppressed osteoclast differentiation (Fig. 1F) and, accordingly, suppressed RANKL-induced expression of ITGB3 and CALCR (Fig. 1G). These results support a role for EZH2 in osteoclast differentiation and suggest that GSK126 suppresses osteoclastogenesis by inhibiting EZH2 catalytic activity. Strikingly, LNA-mediated knockdown of EZH2 expression also strongly suppressed RANKL-induced expression of NFATc1 (Fig. 1G, 1H). These results show that EZH2 promotes osteoclast differentiation by enabling RANKL induction of NFATc1 to drive downstream osteoclast differentiation.

Because EZH2 is a negative regulator of gene expression, its positive role in NFATc1 induction suggests an indirect mechanism of action. One possible indirect mechanism is regulation of proteins or signaling molecules in pathways linking RANKL stimulation with induction of NFATc1 expression. We found that inhibition of EZH2 or EZH2 knockdown had minimal effects on RANK expression (Supplemental Fig. 1A, 1B) and RANKL-mediated NF-κB activation (Supplemental Fig. 1C). However, GSK126 attenuated RANKL-mediated induction of Fos protein (Supplemental Fig. 1D), which is required for NFATc1 expression, and inhibited induction of Fos mRNA at the 1-d time point (Supplemental Fig. 1E). Thus, EZH2 promotes osteoclast differentiation, in part, by affecting Fos that is required to activate NFATc1.

EZH2 directly silences IRF8 expression

In addition to signals that promote NFATc1 expression, effective osteoclastogenesis requires downregulation of transcriptional repressors, such as IRF8, BCL6, or MAFB, which oppose the differentiation program (4). Perhaps the most established of these is IRF8, which works, at least in part, by suppressing NFATc1 expression (5). Thus, we tested the hypothesis that EZH2 directly represses IRF8, BCL6, or MAFB by depositing H3K27me3 at these gene loci and that inhibition of EZH2 would restore expression of these negative regulators, thereby suppressing osteoclastogenesis. First, we found, using ChIP assays, that H3K27me3 accumulated at the IRF8 promoter 1 d after RANKL stimulation (Fig. 2A) and was associated with the expected decrease in IRF8 expression after RANKL stimulation (Fig. 2B, white bars). Strikingly, GSK126 abrogated the negative H3K27me3 histone mark at the IRF8 promoter (Fig. 2A), which was associated with a reversal of the RANKL-induced downregulation of IRF8 mRNA and protein (Fig. 2B). We found that EZH2 was recruited to the IRF8 promoter concomitant with deposition of H3K27me3 (Fig. 2C), which supports a direct mechanism of action. EZH2 recruitment to IRF8 was suppressed by GSK126, which is consistent with a previously described positive-feedback loop in which H3K27me3 marks reinforce EZH2 recruitment (11). In contrast to IRF8, neither H3K27me3 accumulation nor EZH2 recruitment was detected at the BCL6 or MAFB promoters after RANKL stimulation (Supplemental Fig. 1F, 1G), although GSK126 had a modest and likely indirect effect on BCL6 expression 1 d after RANKL stimulation (Supplemental Fig. 1H). Collectively, these results show that EZH2 is recruited to IRF8 during the early stages of RANKL-induced osteoclast differentiation and that EZH2-mediated H3K27me3 deposition represses IRF8 to facilitate osteoclastogenesis. The increase in IRF8 expression that occurs when EZH2 is inhibited or knocked down would suppress osteoclast differentiation and helps to explain the diminished osteoclastogenesis that was observed when cells were treated with GSK126 or EZH2-specific LNAs.

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FIGURE 2.

EZH2 directly silences IRF8 expression. (A) ChIP assays for H3K27me3 at the IRF8 or negative-control HBB promoter in cells pretreated with GSK126 or DMSO and stimulated with RANKL. Data are mean ± SEM of triplicates from one representative experiment. Pooled data (n = 4) were analyzed. *p < 0.05, ***p < 0.005, repeated-measures one-way ANOVA with post hoc Tukey test. (B) IRF8 mRNA expression in RANKL-stimulated EZH2 knockdown and control cells (left panel) or GSK126-treated cells (center panel) and IRF8 protein expression in GSK126-treated cells stimulated with RANKL (right panel). PCR data are mean ± SEM of triplicates from one experiment (n = 3). Data are from representative experiments. Blots shown are representative (n = 3). (C) ChIP assays for EZH2 at the IRF8 or negative control TNF promoter in cells pretreated with GSK126 or DMSO and stimulated with RANKL. Data are mean ± SEM of triplicates from one representative experiment (n = 4). (B and C) Statistical analysis was performed on pooled data. *p < 0.05, ***p < 0.005, one-way ANOVA with post hoc Tukey test. n.s., not significant.

GSK126 increases bone mass in OVX mice

We then tested whether GSK126 could suppress pathological bone resorption in vivo using an OVX model of osteoporosis. GSK126 treatment was started 3 wk after OVX and continued for 5 wk. μ-CT analysis revealed that, compared with mice that underwent sham surgery, vehicle-treated OVX mice exhibited a nearly 60% reduction in trabecular bone volume in tibiae (Fig. 3). GSK126 treatment significantly increased tibial bone mass relative to vehicle-treated mice (Fig. 3). GSK126 treatment also significantly increased trabecular number and decreased trabecular spacing in tibiae (Fig. 3B). However, an effect of GSK126 was not observed in femurs. We also tested the effect of GSK126 on bone formation of OVX mice. GSK126 treatment showed minimal effects on MAR and bone-formation rate in OVX mice compared with vehicle-treated OVX mice (Supplemental Fig. 2A, 2B). Of note, EZH2 was implicated in osteoblast differentiation, and it was shown that short-term treatment (3 d) with GSK126 promotes in vitro osteoblast differentiation (18). Consistently, long-term treatment (2 wk) with GSK126 showed a trend toward increased in vitro osteoblast differentiation (Supplemental Fig. 2C–E). Taken together, our results suggest that GSK126 has beneficial effects on pathologic bone resorption.

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FIGURE 3.

GSK126 increases bone mass in OVX mice. (A) μ-CT images (n = 6) of trabecular bone from tibiae of sham, OVX, and GSK126-treated OVX mice. (B) Measurements of bone volume to total volume (BV/TV), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and trabecular number (Tb.N) for the indicated mice (n = 6). **p < 0.01, one-way ANOVA with post hoc Tukey test. n.s., not significant.

The importance of epigenetic mechanisms in regulating osteoclast differentiation is becoming increasingly appreciated and represents an emerging research area. This research has therapeutic implications, because inhibitors of the epigenetic regulators HDACs, BET proteins, and Dnmt3a were shown to effectively suppress pathological bone loss in several disease models, including osteoporosis, arthritis, inflammatory osteolysis, and bony tumor metastasis (5, 6, 8, 9, 19). This study adds to this body of work by identifying a key role for EZH2 and associated H3K27me3 in promoting RANKL-induced osteoclast differentiation. Our work supports a model whereby RANKL induces EZH2 recruitment and H3K27me3-mediated repression of IRF8, thereby releasing the IRF8-mediated “brakes” on osteoclastogenesis. In addition, EZH2 regulates RANKL-induced expression of Fos, an essential transcription factor in osteoclast differentiation, suggesting that EZH2 controls positive and negative regulators of osteoclastogenesis. Inhibition of EZH2 did not affect known mechanisms that regulate Fos expression in OCPs, such as IFN signaling, and did not affect the expression of microRNAs that regulate Fos expression (20, 21). Understanding the mechanisms by which EZH2 regulates Fos expression needs further investigation.

A recent report highlighted the importance of stable epigenetic silencing of Irf8 at a later stage of osteoclastogenesis by Dnmt3a-mediated DNA methylation, but it did not clarify the mechanism that initially repressed IRF8 expression. Our findings indicate that the earlier stages of IRF8 repression are mediated by a chromatin-based H3K27me3-mediated mechanism. Interestingly, EZH2-mediated chromatin regulation and Dnmt3a-mediated DNA methylation were shown to cooperate to silence genes in other systems (11, 22), and EZH2 occupancy and H3K27me3 can serve as a recruitment platform for Dnmt3a (23). Thus, our work suggests that EZH2 and Dnmt3a may cooperate to silence IRF8 expression during osteoclast differentiation.

In conclusion, this study provides evidence for a novel mechanism of epigenetic regulation of osteoclastogenesis by EZH2. The repression of IRF8 by EZH2 further highlights IRF8 as an important negative regulator of NFATc1 expression that must be silenced for RANKL-induced osteoclastogenesis. Effective inhibition of osteoclastogenesis by GSK126 in human OCPs provides a proof of concept supporting potential clinical applications of similar inhibitors and molecules that target chromatin regulators for bone resorptive diseases and perhaps also bone metastases.