STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis

Among the diverse cytokines involved in osteoclast differentiation, interleukin (IL)-3 inhibits RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. Here we demonstrate that the activation of signal transducers and activators of transcription 5 (STAT5) by IL-3 inhibits RANKL-induced osteoclastogenesis through the induction of the expression of Id genes. We found that STAT5 overexpression inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate the expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting a key role of STAT5 in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated the expression of Id1 and Id2, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Importantly, microcomputed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in the STAT5 conditional knockout mice than in the wild-type mice after RANKL injection. Taken together, our findings indicate that STAT5 contributes to the remarkable IL-3-mediated inhibition of RANKL-induced osteoclastogenesis by activating Id genes and their associated pathways.

IL-4, and IL-10, which exert potent inhibitory effects on osteoclast differentiation 8 . IL-3, a cytokine secreted predominantly by activated T lymphocytes, serves as a link between the immune system and the hematopoietic system. Although studies using either organ culture or whole bone marrow cultures have revealed IL-3 to be a stimulator of osteoclastogenesis in vitro 9,10 , recent studies have shown that IL-3 irreversibly inhibits RANKL-induced osteoclast differentiation by downregulation of c-Fos expression, prevention of NF-κ B signaling, and inhibition of RANK expression 11,12 . In addition, inhibitors of differentiation and DNA binding (Ids) are involved in the inhibition of RANKL-induced osteoclast differentiation and are known to be induced by IL-3. Thus, IL-3 negatively regulates osteoclast differentiation by regulating Ids and c-Fos expression 13,14 .
Signal transducers and activators of transcription (STATs) are a family of latent cytoplasmic proteins that are activated to participate in gene control when cells encounter various cytokines 15 . Seven known mammalian STAT proteins such as STAT1, 2, 3, 4, 5A, 5B, and 6 exist. STATs are activated by tyrosine phosphorylation following the binding of specific ligands to cognate receptors, leading to dimerization and subsequent translocation of the STATs to the nucleus 15 . Activated STATs bind to specific DNA motifs in regulatory regions and thereby control the transcription of genes that regulate cell proliferation, differentiation, apoptosis, and immune responses.
Two members of the STAT family, STAT5A and STAT5B (collectively called STAT5), have gained prominence owing to the fact that they are activated by a wide variety of cytokines such as IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF), which are known to play important roles in the growth and differentiation of hematopoietic precursors [16][17][18] . Emerging evidence suggests that the STAT signaling pathway plays an important role in bone development and metabolism 19 . Recently, it was demonstrated that STAT5 negatively regulates bone resorption of osteoclasts by inducing Dusp gene expression 20 . However, although STAT5 has been shown to play a role in bone metabolism, the contribution of STAT5 to osteoclast differentiation has not yet been revealed.
In this study, the role of STAT5 in osteoclast differentiation and the underlying mechanism was investigated. The effect of IL-3 on osteoclast development were investigated in Mx1-Cre transgenic mice in which floxed Stat5 was specifically deleted. We discovered that the inhibitory effect of IL-3 on RANKL-induced osteoclastogenesis was dependent on STAT5 activation. In addition, STAT5 activation induced Id gene expression, resulting in the inhibition of osteoclast differentiation. Thus, we concluded that STAT5 inhibits osteoclast differentiation by controlling negative regulators upon IL-3 stimulation.

STAT5 activation attenuates RANKL-induced osteoclast differentiation.
To understand the role of STAT5 in osteoclast differentiation, we initially examined Stat5 levels during RANKL-induced osteoclastogenesis using RT-PCR. When bone marrow-derived macrophage-like cells (BMMs) were cultured with M-CSF and RANKL, the expression of NFATc1, a master transcription factor for osteoclast differentiation, strongly increased. The mRNA expression of Stat5a and Stat5b was observed throughout osteoclastogenesis (Fig. 1A). Next, the effect of STAT5 on osteoclast differentiation was examined by overexpressing a constitutively active form of STAT5A (STAT5A1* 6) in osteoclast precursors. STAT5A1* 6 overexpression strongly attenuated RANKL-induced osteoclastogenesis (Fig. 1B) and resulted in a significant reduction in the number of TRAP-positive multinucleated cells (Fig. 1C). Consistently, the expression of RANKL-inducible osteoclastic genes including c-fos, Nfatc1, Acp5, and Oscar was significantly reduced by ectopic STAT5A1* 6 expression (Fig. 1D). Correspondingly, c-Fos and NFATc1 protein levels were also decreased (Fig. 1E). Moreover, activated STAT5 attenuated pERK and p38 levels in the early stages of RANKL-induced osteoclast differentiation (Fig. 1F). These results suggest that active STAT5A inhibits osteoclastogenesis, which is accompanied by the reduced expression of genes associated with osteoclast differentiation.

RANKL-induced osteoclast differentiation is not affected by STAT5 deficiency.
Osteoclast differentiation in the absence of STAT5 was examined in cells obtained from MX1-Cre recombinase-mediated Stat5 conditional knockout (cKO) mice, targeting both STAT5 isoforms in osteoclasts. We first confirmed the almost complete absence of Stat5a and Stat5b mRNA and STAT5 protein in the BMMs ( Fig. 2A). Unexpectedly, upon differentiation from BMMs lacking STAT5, osteoclast formation was unimpaired and comparable with that observed with Stat5 fl/fl cells (Fig. 2B,C). Furthermore, the mRNA levels of c-fos, Nfatc1, Acp5, and Oscar were not altered by the absence of STAT5 (Fig. 2D). In addition, c-Fos and NFATc1 protein levels were comparable between the Stat5 fl/fl and Stat5 cKO samples (Fig. 2E). These data suggested the possibility that RANKL signaling does not regulate STAT5 expression. We therefore examined STAT5 phosphorylation upon RANKL stimulation. Upon the stimulation of BMMs with RANKL for a short period of time following starvation, no STAT5 phosphorylation was observed at any time over the duration of RANKL stimulation, whereas Iκ B degradation was clearly evident (Fig. 2F), indicating that BMMs had been sufficiently stimulated by RANKL. Furthermore, STAT5 phosphorylation was never observed during RANKL-mediated osteoclastogenesis, whereas NFATc1 expression was strongly induced during osteoclast differentiation (Fig. 2G). In summary, these data show that STAT5 deficiency did not alter RANKL-induced osteoclast differentiation, suggesting that STAT5 activation does not occur via the RANKL signaling pathway.

IL-3 attenuates RANKL-induced osteoclast differentiation.
Although STAT5 activation is not regulated by RANKL, it did result in significant suppression of osteoclast differentiation. To address this conundrum, we searched for other cytokines capable of activating STAT5 and thereby inhibiting RANKL-induced osteoclast differentiation. IL-3 has been shown to activate STAT5 in a variety of cells 21 , including by BMMs, as shown in this study ( Supplementary Fig. 1A). We observed osteoclast differentiation in the presence of increasing concentrations of IL-3 from 10 pg/mL to 1 ng/mL; IL-3 also suppressed RANKL-induced osteoclast differentiation in a dose-dependent manner ( Supplementary Fig. 1B,C). Thus, a negative effect of IL-3 on osteoclast development would be consistent with reports of STAT5-mediated inhibition of osteoclastogenesis.
Scientific RepoRts | 6:30977 | DOI: 10.1038/srep30977 STAT5 deficiency prevents IL-3-mediated inhibition of osteoclastogenesis. Next, we determined whether the inhibitory effect of IL-3 on osteoclast differentiation is attributable to STAT5 activation. To this end, we tested whether the inhibitory effect of IL-3 on osteoclast differentiation was abrogated by STAT5  deficiency. When osteoclast differentiation from Stat5 fl/fl cells and Stat5 cKO cells in the presence of IL-3 was compared, it was found that STAT5 deficiency partially restored osteoclast formation that was inhibited by IL-3 in the Stat5 fl/fl cells (Fig. 3A,B). This was confirmed by mRNA expression of the osteoclastic genes c-fos, Nfatc1, Acp5, and Oscar. In the Stat5 fl/fl cells, the expression of RANKL-inducible c-fos, Nfatc1, Acp5, and Oscar was strongly suppressed by IL-3 in pre-osteoclasts (pOC). In contrast, in Stat5 cKO cells, the expression of c-fos was recovered to almost the level observed in the absence of IL-3, while the expression of Nfatc1, Acp5, and Oscar was lower than that in the absence of IL-3. However, the expression was still higher than that in the IL-3-treated pOC from the Stat5 fl/fl cells (Fig. 3C). Taken together, these results strongly suggest that STAT5 is a key modulator of the IL-3-mediated inhibition of osteoclast development.
Activation of STAT5A or STAT5B alone is sufficient to inhibit osteoclast differentiation. Since redundancy between STAT5A and STAT5B has been proposed in other cell types, we tested this possibility in osteoclastogenesis. Overexpression of either wild-type STAT5A or STAT5B caused a similar reduction in osteoclastogenesis, although the extent of inhibition was weaker than that observed upon overexpression of the constitutive active STAT5A1* 6 (Supplementary Fig. 2A,B). In addition, downregulation of RANKL-mediated NFATc1 induction in osteoclastogenesis was also comparable when STAT5A and STAT5B were ectopically expressed, but this was significantly less than that observed in the control ( Supplementary Fig. 2C). Furthermore, IL-3 inhibited osteoclast differentiation by approximately 23% and 30% following overexpression of STAT5A and STAT5B, respectively ( Supplementary Fig. 2D,E), indicating overlapping functions of STAT5A and STAT5B in Data are represented as the mean ± SD. * * * P < 0.001 vs. Stat5 fl/fl control; # P < 0.05 vs. Stat5 cKO control; n.s., not significant; n = 4. (C) BMMs were treated with IL-3 (1 ng/mL). Cells were further cultured in the presence (pOC) or absence (BMM) of RANKL for two days, and subjected to semi-quantitative real-time PCR for the indicated genes. Data represent mean ± SD of triplicate samples. * * * P < 0.001; n.s., not significant. Bar: 100 μ m. All results are representative of at least three independent experiments. All data were analyzed using ANOVA. the inhibition of osteoclast differentiation. Since STAT5A and STAT5B exhibited an overlapping inhibitory effect on osteoclast differentiation with similar potentials, we determined whether the absence of either isoform would still inhibit RANKL-induced osteoclast differentiation. To address this, a constitutively active form of STAT5A (STAT5A1* 6) was overexpressed in the BMMs obtained from Stat5 fl/fl and Stat5 cKO mice. Expression of the activated STAT5A in Stat5 cKO BMMs led to the attenuation of TRAP positive multi-nucleated cells to a degree comparable to that observed in the Stat5 fl/fl samples ( Supplementary Fig. 3A,B). This provides compelling evidence indicating that activation of either isoform alone is sufficient to inhibit osteoclast differentiation.

ID1 and ID2 are responsible for the STAT5-mediated inhibition of osteoclast differentiation.
To understand the mechanism underlying the inhibition of osteoclast differentiation by STAT5, we searched for STAT5 target genes that were regulated during both RANKL and IL-3 stimulation. RNA sequencing was performed on the BMMs obtained from the Stat5 fl/fl and Stat5 cKO mice stimulated with M-CSF and RANKL, in the presence or absence of IL-3 (GSE76988). The RNA sequencing data indicated that the mRNA expression of Id1 and Id2 was attenuated upon RANKL stimulation and that the levels were recovered by IL-3 stimulation, but not in the absence of STAT5 (Supplementary Table 1). Therefore, we proposed that Id1 and Id2 are the target genes of STAT5. It has also been previously reported that Id1 and Id2 function as negative regulators of osteoclast differentiation 13 . To examine whether Id1 and Id2 are involved in the STAT5-mediated inhibition of osteoclast differentiation, STAT5A1* 6 was overexpressed during osteoclast differentiation of the BMMs. Although this led to a significant increase in the mRNA expression of Id1 and Id2 (Fig. 4A), STAT5A1* 6 did not affect the decrease in Id gene expression observed following RANKL stimulation. In both BMMs and pOC, the expression of Id1 and Id2, but not that of Id3, was significantly increased in the presence of IL-3 (Fig. 4B). In STAT5-deficient cells, however, IL-3 failed to increase the expression of Id1 and Id2 (Fig. 4B), suggesting that IL-3 induces the expression of Id1 and Id2 through STAT5.
Next, we examined whether the ectopic expression of ID1 and ID2 could restore the IL-3-mediated inhibition of osteoclast differentiation that was abrogated by STAT5 deficiency. Consistent with our previous report 13 , ID1 or ID2 overexpression suppressed osteoclast development (data not shown), and the osteoclasts in STAT5-deficient cells were almost fully developed, even in the presence of IL-3 (Fig. 4C). Strikingly, quantification of the number of osteoclasts revealed that the osteoclast formation rescued from IL-3-mediated inhibition in STAT5-deficient cells was abolished by the ectopic expression of ID1 or ID2, where osteoclast differentiation was returned to almost the levels observed in the controls (Fig. 4C,D).
It has been reported that IL-3 induces dendritic cell differentiation 22 , and it therefore seemed possible that the increase in Id1 and Id2 gene expression by IL-3-mediated inhibition of osteoclast differentiation that occurs via STAT5 activation, as shown in the present study, was due to a change in the fate of the cells. In order to examine the effect of STAT5 activation on dendritic cell differentiation, dendritic cell surface markers, including CD80, CD86, MHC class II, and CD11c were analyzed during the osteoclast differentiation of BMMs overexpressing either an empty vector (pMX-IRES-EGFP) or the constitutively active from of STAT5A (STAT5A1* 6) using fluorescence-activated cell sorting (FACS). Increase in the CD80+ , CD86+ , MHC class II+ , and CD11c+ cell populations was observed in the presence of activated STAT5, indicating a conversion of cell fate from osteoclast precursors to dendritic cells ( Supplementary Fig. 4). These results suggest that STAT5 activation by IL-3 inhibits RANKL-induced osteoclast differentiation via the induction of Id1 and Id2 expression, while converting the cell fate of osteoclast precursors to dendritic cells. STAT5 conditional knockout mice exhibit osteoporotic bone phenotype. To evaluate the physiological functions of STAT5 in mice, the bone quality of the Stat5 fl/fl and Stat5 cKO mice was compared. Microcomputed tomography (μ CT) analysis with three-dimensional reconstruction of the trabecular bones of the distal femurs of 16-week-old male mice revealed a relatively lower bone mass in the Stat5 cKO than in the Stat5 fl/fl mice, but there was no difference between 8-week-old Stat5 cKO and Stat5 fl/fl mice (Fig. 5A). The reduced bone mass in 16-week-old Stat5 cKO mice was accompanied by 32.8%, 10.6%, and 21.5% reductions in the bone volume, trabecular thickness, and trabecular numbers, respectively, and an increase of 15.8% in trabecular separation (Fig. 5B). Meanwhile, there was no significant difference in μ CT parameters between Stat5 cKO and Stat5 fl/fl mice at the age of 8 weeks (Fig. 5B). These results indicate that the Stat5 cKO mice were more osteoporotic than the Stat5 fl/fl mice, and this effect was age-dependent. Furthermore, the number of osteoclasts and osteoblasts in the trabecular bones of the proximal tibia of the same mice at the age of 16 weeks analyzed with μ CT were subjected to quantification using TRAP and H&E staining, respectively. The Stat5 cKO mice exhibited a 5% increase in the number of osteoclasts and exhibited an increasing tendency without significance in the osteoclast surface. However, the number of osteoblasts in the trabecular bones was comparable between the Stat5 fl/fl and Stat5 cKO mice, while the osteoblast surface exhibited decreasing tendency without significance (Fig. 5C,D), demonstrating that STAT5 deficiency in osteoclasts does not affect osteoblast differentiation. These results suggest that the reduced bone mass in the Stat5 cKO mice is likely due to a reduced inhibitory effect of IL-3 on osteoclast differentiation via the STAT5-Id axis, which is different from that observed in vitro.    almost no bone volume recovery in the Stat5 cKO mice, which continued to exhibit an osteoporotic phenotype, even after intraperitoneal IL-3 administration (Fig. 6B,C).

Administration of IL-3 has no inhibitory effect on RANKL-induced bone destruction in
Next, histological analysis was performed to confirm bone loss recovery by intraperitoneal IL-3 administration at the cellular level in the tibiae of the Stat5 fl/fl and Stat5 cKO littermate mice. After RANKL injection, there was an increase in the number of TRAP-positive cells, which indicated that the number of osteoclasts was increased by 116.09% and 54.78% in the Stat5 cKO and Stat5 fl/fl littermates, respectively. Following IL-3 injection, the number of TRAP-positive cells was decreased by 22.41% in the Stat5 fl/fl mice, while there was no significant change in the osteoclast number in the Stat5 cKO mice (Fig. 6D,E). In addition, there was no significant difference in the osteoblast numbers between the RANKL and IL-3-injected groups (Fig. 6F,G). These results indicate that bone loss by RANKL injection and bone mass recovery by IL-3 injection in the control mice were due to an increase and a decrease in the osteoclast numbers, respectively, rather than due to changes in the osteoblast numbers. However, the number of osteoclasts remained relatively high even when IL-3 was intraperitoneally administered in the Stat5 cKO mice. In summary, IL-3 had an inhibitory effect on osteoclast differentiation in vivo in the presence of STAT5, whereas it failed to inhibit osteoclast differentiation in the absence of STAT5, indicating that STAT5 is a critical factor in the IL-3-mediated inhibition of osteoclast differentiation in vivo as well as in vitro.

Discussion
This study showed that IL-3 negatively regulates RANKL-induced osteoclast differentiation through the transcription factors STAT5 and ID1/ID2 and shed light on the underlying mechanism. Although previous studies had reported that IL-3 inhibits human osteoclastogenesis and bone resorption through the downregulation of c-Fms 22 and prevents RANKL-induced nuclear translocation of NF-κ B 11 , the transcriptional circuit was not understood. Our study closes this gap and demonstrates that IL-3-induced STAT5 activation in osteoclast precursors reduces c-Fos expression and osteoclast differentiation. However, the induction of osteoclast differentiation by RANKL was independent of IL-3. Our study provides evidence indicating a direct contribution of STAT5 in the negative effect of IL-3 on osteoclast differentiation.
Previously, we had demonstrated that ID transcription factors are responsible for the negative regulation of RANKL-induced osteoclast differentiation 13 . In addition, IL-3-and STAT5-dependent activation of Id1 transcription has been reported 23 . We chose to focus on IDs, as we believed that they might be directly responsible for the inhibition of osteoclast differentiation. Notably, the expression of Id1 and Id2, but not that of Id3, was induced by IL-3 through STAT5 in the osteoclasts. In support of a critical function of ID1 or ID2, their overexpression significantly inhibited RANKL-induced osteoclast differentiation despite STAT5 deficiency. This indicates that IL-3 negatively regulates osteoclast differentiation through the activation of STAT5 and the subsequent induction of Id1 and Id2 expression. These findings are consistent with those of a previous study showing that IL-3 plays an inhibitory role in osteoclast differentiation by regulating the expression of c-Fos and Ids 14 .
Two independent studies suggested that IL-3 inhibits osteoclast differentiation by diverting the osteoclast precursor cells to either a macrophage or a dendritic cell lineage 11,22 . In the current study, we expanded on these findings and demonstrated that STAT5A1* 6 overexpression increased the expression of dendritic cell markers, such as CD80, CD86, MHC class II, and CD11c. These findings solidify the notion that IL-3-induced STAT5 activation is responsible for the conversion of osteoclast precursor cells into dendritic cells.
Previously, Hirose et al. reported that conditional osteoclast-specific STAT5 mutant mice exhibited an osteoporotic phenotype 20 . Consistent with this, an osteoporotic bone phenotype was observed in the Stat5 cKO mice in this study, indicating that STAT5 is essential for bone homeostasis under physiological conditions. While Hirose et al. demonstrated a significant increase in osteoclast surface but not osteoclast number the present study provided evidence of an increased osteoclast number but no significant change in osteoclast surface. These discrepancies are possibly because of the differences in the method used to delete STAT5 from the cells between the two studies. In this study, MX1-Cre was used to remove Stat5 from hematopoietic stem cells and therefore STAT5 was absent from macrophage precursor cells from a very early stage, further confirming that STAT5 deficiency could mitigate cellular development, proliferation, and differentiation in vivo. In contrast, Hirose et al. utilized Cathepsin K-Cre to eliminate STAT5 in pre-osteoclast cells, which might have been more effective at a later stage, such as during resorption activity, than during cell proliferation and development. We suggest that the cell differential elimination of STAT5 elimination is a primary reason for the different lesions determined in these two studies.
It has been suggested that STAT5 deficiency increases the bone-resorbing activity of osteoclasts, which is related to the activation of ERK through the regulation of the expression of Dusp1 and Dusp2 20 . It is well known that ERK positively regulates osteoclast differentiation as well as bone resorptive activity 24 . Similarly, RANKL-induced ERK was slightly reduced upon overexpression of constitutively active STAT5 in the BMMs. Although further studies are needed to elucidate whether STAT5 activation regulates osteoclast differentiation and function by inhibiting RANKL-induced ERK activation, we propose that the reduced osteoclast differentiation caused by STAT5 activation is at least in part caused by decreased ERK activation.
The bone loss observed in the Stat5 cKO mice seemed to be age-related. When the bone volumes of 8-week-old Stat5 cKO and wild-type littermates were analyzed, there was no significant difference in bone volumes between the Stat5 cKO and wild-type littermates. However, when the bone phenotype was analyzed between the Stat5 cKO and wild-type littermates at the age of 16 weeks, the bone mass in the Stat5 cKO mice was found to be reduced. Therefore, these observations imply that the bone loss observed in Stat5 deficiency may be due to aging. In addition, the reduced bone mass observed in the absence of STAT5 at the age of 16 weeks appears to be associated with increased osteoclast differentiation rather than with the increased bone resorbing activity of osteoclasts. Although osteoclast differentiation remained unchanged when BMMs lacking STAT5 were cultured with RANKL, our in vivo study revealed a significant increase in the number of osteoclasts and a moderate increase in osteoclast surface. Collectively, these results suggest that STAT5 acts as a negative factor in osteoclast differentiation under Scientific RepoRts | 6:30977 | DOI: 10.1038/srep30977 the control of an existing endogenous signaling pathway in vivo, which may stimulate STAT5 activation and block osteoclast differentiation, rather than directly inhibiting the RANKL signaling pathway. IL-3 is a potent cytokine for the activation of STAT5 and the inhibition of osteoclast differentiation. Our results provided evidence suggesting that RANKL-induced bone loss was rescued by IL-3 administration only in the presence of STAT5. We hypothesize that RANKL increases IL-3 expression in osteoclasts and thus stimulates STAT5 activation, which ensures normal osteoclast differentiation and thus maintains bone homeostasis. In addition to IL-3, GM-CSF may contribute to bone homeostasis. Based on the in vivo mouse model, we propose that the IL-3/STAT5 pathway has a therapeutic value for age-related bone diseases.

Methods
Reagents. Recombinant  Plasmid DNA constructs. The expression vector for constitutively active Stat5a (Stat5a1* 6) has been described previously 26 . The full-length coding sequences of mouse Stat5a (NM_011488) and Stat5b (MN_011489) were purchased from 21C Frontier Human Gene Bank (Daejeon, Korea). The genes were subcloned into a retroviral vector (pMX-IRES-EGFP) that included a C-terminal Flag tag. Retrovirus vectors encoding ID1 and ID2 were described previously 13 .

Retroviral gene transduction.
Plat E cells were transfected for retrovirus packaging with FuGENE 6 (Promega, Madison, WI, USA) according to the manufacturer instructions. Retroviral supernatant was collected from the culture media 48 hours after transfection. Murine bone marrow-derived macrophages (BMMs) were subsequently infected with the supernatant for eight hours in the presence of 10 μ g/mL of polybrene (Sigma-Aldrich).
Osteoclast formation. Osteoclasts were generated from murine bone marrow cells as previously described 27 . Bone marrow cells obtained via flushing the long bones of 6-week-old mice were cultured in α -MEM (HyClone Laboratories) containing 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin (Life Technologies, Carlsbad, CA, USA) in the presence of M-CSF (30 ng/mL) for 3 days. After non-adherent cells were removed, adherent BMMs were plated onto 96-well plates (1 × 10 4 cells/well) and further cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL). Cultured cells were fixed and stained for TRAP. TRAP-positive multinuclear cells (> 3 nuclei/cell) [TRAP + MNCs] were counted as osteoclasts. Cells were observed using a Leica DMIRB microscope equipped with an N Plan 10 × 0.25 numerical aperture objective lens (Leica Microsystems, Wetzler, Germany). Images were captured using ProgRes ® Capture Pro software (Jenoptik, Jena, Germany).
Real-time PCR. Quantitative real-time PCR analysis was performed in triplicate with SYBR Green (Qiagen) and with specific primers using Rotor-Gene Q (Qiagen). mRNA expression levels were normalized to Gapdh. The relative quantitation value for each target gene compared to the calibrator for that target was expressed as 2 −(Ct-Cc) (Ct and Cc are the mean threshold cycle differences after normalizing to Gapdh). The relative expression levels for each sample were presented by semi-log plot. Primer sequences were as follows: Gapdh forward, 5′ -TGA CCA