Sirt6 cooperates with Blimp1 to positively regulate osteoclast differentiation

Global deletion of the gene encoding a nuclear histone deacetylase sirtuin 6 (Sirt6) in mice leads to osteopenia with a low bone turnover due to impaired bone formation. But whether Sirt6 regulates osteoclast differentiation is less clear. Here we show that Sirt6 functions as a transcriptional regulator to directly repress anti-osteoclastogenic gene expression. Targeted ablation of Sirt6 in hematopoietic cells including osteoclast precursors resulted in increased bone volume caused by a decreased number of osteoclasts. Overexpression of Sirt6 led to an increase in osteoclast formation, and Sirt6-deficient osteoclast precursor cells did not undergo osteoclast differentiation efficiently. Moreover, we showed that Sirt6, induced by RANKL-dependent NFATc1 expression, forms a complex with B lymphocyte-induced maturation protein-1 (Blimp1) to negatively regulate expression of anti-osteoclastogenic gene such as Mafb. These findings identify Sirt6 as a novel regulator of osteoclastogenesis by acting as a transcriptional repressor.

regulating genomic stability, cellular metabolism, inflammation, stress response and longevity [18][19][20][21][22][23] . Sirt6-deficient (Sirt6 −/− ) mice suffer from a variety degenerative aging phenotypes and die around 4 weeks after birth 18,19 . In addition, Sirt6 −/− mice exhibit osteopenia due to impaired mainly bone formation, with 30% reduction in bone mineral density. Since bones are still developing in mice at this age, early postnatal lethality of Sirt6 −/− mice precludes investigation of Sirt6 function in adult mice 19 and makes it difficult to distinguish developmental versus bone remodeling defects in bone metabolism.
Here we investigated the function of Sirt6 in osteoclastogenesis by disrupting Sirt6 at an adult stage using Mx1-Cre mice. We found that Sirt6 induced by RANKL-NFATc1 axis acted as a transcriptional repressor of negative regulators of NFATc1 during osteoclast differentiation. These findings identify a key role for Sirt6 in promoting RANKL-induced osteoclastogenesis and provide further insight into the mechanisms in fine-tuning the transcriptional regulatory network for osteoclastogenesis.

Results
Increased bone mass in Sirt6 f l/f l Mx1Cre mice. Global loss of Sirt6 expression in mice leads to premature death between 3 and 4 weeks of age after birth 19 . Moreover, myeloid-specific deletion of Sirt6 using LysMCre transgenic mice was shown to have profound liver inflammation 24 . To assess skeletal phenotypes following Sirt6 deletion in adult stage of mice, we examined Sirt6 conditional knockout mice by crossing Sirt6 flox/flox mice (Sirt6 fl/fl ) with inducible Cre system, Mx1Cre mice instead of LysMCre or CtsKCre mice. In the Sirt6 fl/fl Mx1Cre mice, the Sirt6 gene is deleted upon polyinosinic-polycytidylic acid (poly I:C) treatment in osteoclast precursors, which allowed us to examine the effect of Sirt6 depletion on osteoclast formation. We first analyzed the bone phenotype of Sirt6 fl/fl Mx1Cre mice at the age of 16 weeks, which had received polyI:C injection at the age of 10 d. The bone volume, the trabecular numbers, and bone mineral density were significantly increased in the Sirt6 fl/fl Mx1Cre mice, without any change in the trabecular thickness (Fig. 1a). Bone morphometric analysis indicated a decrease in the osteoclast number of cells (Fig. 1b,c), but the osteoblast number was not changed in the Sirt6 fl/fl Mx1Cre mice. These results suggested that Sirt6 in osteoclast precursor cells positively regulated osteoclast numbers in vivo.

Impaired osteoclastogenesis in Sirt6-deficient cells.
In vitro osteoclast differentiation of bone marrow-derived monocyte/macrophage precursor cells (BMMs) derived from Sirt6 fl/fl Mx1Cre mice was investigated by measuring the number of multinucleated cells (MNCs) positive for the osteoclast marker tartrate-resistant acid phosphatase (TRAP + ) after stimulation of with RANKL in the presence of M-CSF. The number of TRAP + MNCs was markedly decreased in the Sirt6 fl/fl Mx1Cre cells compared with the control cells ( Fig. 2a and Supplementary Fig. S1). Further, TRAP staining showed that a decrease in osteoclast size and in the number of nuclei per osteoclast was observed in marrow cultures from Sirt6 fl/fl Mx1Cre mice compared with wild-type cultures, suggesting that Sirt6 regulates the fusion of osteoclast precursors as well as the formation of mature osteoclasts. In Sirt6 fl/fl Mx1Cre cells, the expression of Nfatc1 and its target genes, including Atp6v0d2, Dc-stamp, Acp5, and Cathepsin K was decreased at the mRNA and/or protein levels (Fig. 2b,c). However, there was no difference in bone resorbing activity in Sirt6 fl/fl Mx1Cre osteoclasts when the same number of mature osteoclasts were seeded ( Supplementary Fig. S2), suggesting that the increase in bone volume in the Sirt6 fl/fl Mx1Cre mice was caused by the decreased number of osteoclasts, not by a decrease in their activity.
To ensure that the observed Sirt6 fl/fl Mx1Cre BMMs phenotype is solely a result of Sirt6 deficiency, we determined whether impaired osteoclastogenesis in Sirt6 fl/fl Mx1Cre BMMs could be rescued by reintroduction of Sirt6. BMMs from Sirt6 fl/fl and Sirt6 fl/fl Mx1Cre were infected with a Sirt6-expressing retrovirus or control virus. Expression of Sirt6 protein was confirmed by immunoblotting (Fig. 3a). As expected, re-expression of Sirt6 restored the ability of Sirt6 fl/fl Mx1Cre BMMs to differentiate into mature osteoclasts in the presence of RANKL (Fig. 3b,c), indicating that the Sirt6 −/− phenotype only resulted from the null mutation of Sirt6. Consistently, ectopic expression of Sirt6 increased the sensitivity of osteoclast differentiation to RANKL signaling in osteoclast precursor cells ( Fig. 3d-f). Of note, enhanced osteoclastogenesis was observed in lower concentrations of RANKL (5-16 ng/ml) compared to the concentration of RANKL (100 ng/ml) used for Fig. 2a. This may be due to effect of Sirt6 overexpression, which may cause enhanced osteoclast formation in the lower concentrations of RANKL. It is noteworthy that the expression of NFATc1 was accelerated by Sirt6 overexpression in the presence of RANKL (Fig. 3a,d). These results indicate that Sirt6 deletion in osteoclast precursor cells results in decreased osteoclast differentiation through down-regulation of NFATc1 levels.
To exclude the possibility that impaired osteoclastogenesis in Sirt6 fl/fl Mx1Cre BMMs was due to decreased numbers of osteoclast precursors derived from the hematopoietic lineage, we examined the ratio of the osteoclast precursor cells among the bone marrow cells. The percentage of the most highly osteoclastogenic c-kit + c-fms + cells in the CD11b lo/− CD3ε − B220 − population 25 was similar between the control and Sirt6 fl/fl Mx1Cre mice, indicating that the proportion of osteoclast precursor cells in the bone marrow was unchanged ( Supplementary Fig. S3a). In addition, there was no significant difference in the proliferation rate of CD11b + cells cultured in the presence of M-CSF for 2 d (Supplementary Fig. S3b).
Sirt6 is a target of the NFATc1. Since Sirt6 was only slightly expressed in BMMs, but was markedly induced by RANKL but not by lipopolysaccharide ( Supplementary Fig. S4), we examined whether NFATc1 regulates Sirt6 expression during osteoclastogenesis. It has been shown that cyclosporin A (CsA), an inhibitor of calcineurin activity, inhibits RANKL-mediated osteoclastogenesis by suppressing Nfatc1 gene expression 26 . RANKL-dependent induction of Sirt6 at both the protein and mRNA levels was markedly decreased by CsA-mediated NFATc1 inhibition (Fig. 4a). Conversely, we examined whether overexpression of a constitutively active form of NFATc1 (caNFATc1) in BMMs affected the expression of Sirt6. Sirt6 levels were up-regulated by transduction of ca-NFATc1 alone, even without RANKL stimulation (Fig. 4b). These observations suggested that Sirt6 gene is specifically induced by RANKL in osteoclast precursors through NFATc1. The 0. 35 promoter fragment (-350 to + 1), linked to luciferase reporter construct, was activated in response to NFATc1 expression (Fig. 4c). However, luciferase activities were completely abolished in the Sirt6 0.03-kb construct (-30 to + 1) as compared with the 0.35-kb Sirt6 promoter fragment. Consistently, two putative NFAT-binding DNA element 27 were present in the 5′-flanking region of Sirt6 ( Supplementary Fig. S5). Chromatin immunoprecipitation assays indicated that binding of NFATc1 to the 5′-flanking sequence of Sirt6 promoter increased during osteoclast differentiation (Fig. 4d). Together, these data indicate that Sirt6 is a direct transcriptional target of NFATc1 during osteoclastogenesis.  Fig. S6). Although it has been reported previously that Sirt6 functions as a transcriptional repressor in other cell types 22,23,28 , transcription factors can function as either a positive or a negative transcriptional regulator in a context-dependent manner 29 . To investigate whether Sirt6 functions as a transcriptional regulator during osteoclastogenesis, we examined expression of Blimp1 and Mafb which were shown to function as a positive-and a negative-regulator of osteoclastogenesis, respectively 12,30 . Sirt6 deficiency increased the expression of Mafb significantly with a concomitant decrease in Blimp1 expression at protein and mRNA levels (Fig. 5a,b). Irf8 and Bcl6 expression in Sirt6 fl/fl Mx1Cre BMMs also increased upon RANKL stimulation. We analyzed whether Sirt6 binds to the promoters of Mafb, Irf8, and Bcl6 genes and observed more obvious occupancy of Sirt6 in the promoters in wild-type cells in comparison with Sirt6 fl/fl Mx1Cre cells (Fig. 5c). Furthermore, Sirt6 interacted with Blimp1 in mammalian cells (Fig. 5d) as well as in RANKL-stimulated osteoclast precursors (Fig. 5e).
These results suggest that Sirt6 cooperates with Blimp1, which in turn regulates expression of transcriptional repressors of osteoclastogenesis, such as Mafb (Fig. 5f).

Discussion
Previous studies that were based on global ablation of Sirt6 outlined an important role for Sirt6 in bone homeostasis. Histomorphometric analysis of bone and the bone cell biology of Sirt6 −/− mice revealed that deficiency of Sirt6 caused osteopenia due to mainly impaired function of osteoblasts 19,23 . These studies examined mice at 3 weeks and provided important evidence for the function of Sirt6 in specifying osteoblast differentiation during development. However, the requirement for Sirt6 during osteoclast differentiation in adult skeletal remodeling remained unresolved. Here Mx1-Cre was used to delete Sirt6 in mice at 10 days of age as was previously done to investigate the role of NFATc1 during osteoclastogenesis 31 . We identified a new role of Sirt6 as a key positive regulator of osteoclastogenesis. Sirt6 deficiency in osteoclast precursors inhibited osteoclastogenesis by suppressing The balance between positive-and negative-regulation of osteoclastogenesis is important for bone homeostasis and in order to prevent excessive bone resorption in inflammatory and other diseases. Positive signaling pathways and transcription factors that promote osteoclastogenesis have been extensively studied and are well characterized 2,32 . A typical example is NFATc1, whose activity and expression are maintained at an extremely high level by RANKL stimulation, thereby promoting osteoclastogenesis. Recently, it has been known that osteoclastogenesis is also negatively regulated by a number of transcriptional repressors, including MafB, IRF-8, and Bcl6 11,30,[33][34][35] . These factors suppress transcription of Nfatc1 and its target genes, and their expression is downregulated during osteoclastogenesis to allow the gene expression program associated with osteoclast differentiation to proceed. Interestingly, Sirt6 fl/fl Mx1Cre BMMs formed significantly decreased numbers of osteoclasts in vitro and have decreased nuclei per osteoclast. The decreased fusion of osteoclast precursors most likely reflects the reduced expression of Atp6v0d2 and DC-STAMP in osteoclast precursors from Sirt6 fl/fl Mx1Cre BMMs. Similarly, a previous report also has shown that NFATc1 induces osteoclast fusion via upregulation of Atp6v0d2 and DC-STAMP 9 .
How is the balance between positive-and negative-regulation achieved and maintained within transcriptional network? To this end, a signaling pathway usually may stimulate the negative-feedback regulatory pathways to keep in check any excesses in the cell differentiation program. In this context, the fact that Sirt6 was induced by the RANKL-NFATc1 axis and was involved in the negative regulation of anti-osteoclastogenic gene expression places Sirt6 in a transcriptional regulatory network of osteoclastogenesis. A previous study indicated that Sirt6 interacts with the NF-κ B RelA subunit and deacetylates H3K9 at NF-κ B target gene promoters and the loss of Sirt6 caused activation of NF-κ B dependent gene expression 23 . Sirt6 has also been well characterized as a co-repressor of the transcription factor HIF1α to control the expression of multiple glycolytic genes 22 and c-Jun to inhibit pro-inflammatory gene expression 28 . It remains to be determined how Sirt6 exerts negative effects of anti-osteoclastogenic gene expression. Recently, Blimp1 has been placed upstream of several repressors of osteoclastogenesis, including MafB, IRF8, and Bcl6 during osteoclastogenesis 11,30,35 . An increase in Blimp1 expression after RANKL stimulation serves to down-regulate expression of repressors of osteoclastogenesis 10,12 . The function of Sirt6 in osteoclasts is similar to the role of Blimp1, in that they are induced by the RANKL-NFATc1 axis and repress the genes involved in anti-osteoclastogenesis. In addition, Sirt6 binds to promoters of Mafb, Irf8, and Bcl6 genes. Because Sirt6 interacts with Blimp1 in osteoclast precursors, it is reasonable to hypothesize that Sirt6 in cooperation with Blimp1 serves to switch-off the brakes in osteoclastogenesis by acting as a negative regulator of anti-osteoclastogenic gene expression (Fig. 5f). Further understanding of the gene regulatory programs mediated by Sirt6-Blimp1 axis may provide a novel molecular basis for therapeutic strategies against bone and joint diseases.   USA), anti-NFATc1 and anti-GAPDH (Santa Cruz Biotechnology Inc., Santa Cruz CA, USA) followed by secondary horseradish peroxidase-conjugated antibody. Anti-Atp6v0d2 36 antibody was kindly provided by Y. Choi (University of Pennsylvania, Philadelphia PA, USA). The pCDH-3x-Flag-Sirt6 and pcDNA 3.1-V5-Sirt6 were made as described previously 21 . For retroviral expression, the Flag-Sirt6 DNA was subcloned into pMX-puro to make pMX-puro-Flag-Sirt6. A retroviral vector, pMX-puro was provided by Dr T Kitamura (University of Tokyo, Tokyo, Japan). The pMX-puro-Flag-Blimp1 plasmid 12 was provided by J. Rho (Chungnam National University, Daejeon, Korea). The retroviral vector containing a constitutively active form of NFATc1 (caNFATc1) was previously described 37 .

Reagents and plasmids.
Primary cells and cell line. BMMs were obtained from murine bone marrow precursors of 4-to 6-week-old C57BL/6 mice (The Jackson Laboratory, Bar Harbor ME, USA) as described 38 . BMMs were cultured for 3 days in α -minimum essential medium (α -MEM; HyClone, South Logan UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone) and antibiotics containing M-CSF (30 ng/ml). After 3 days, the non-adherent cells were removed and adherent cells (BMMs) were harvested to obtain osteoclast precursor cells of the monocyte/ macrophage lineage. 293 T cells and RAW 264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone) supplemented with 10% FBS with antibiotics. PLAT-E cells were cultured in DMEM with 10% FBS and antibiotics containing blasticidin (10 mg/ml) (Invitrogen) and puromycin (1 mg/ml) (Sigma-Aldrich). PLAT-E cells were provided by Dr T. Kitamura (University of Tokyo).
In vitro osteoclast differentiation. Osteoclasts were prepared form bone marrow cells using a standard method 39 . In brief, the precursor cells were cultured for 3 days with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for osteoclast differentiation. The cells were stained with TRAP staining kit (Sigma-Aldrich). TRAP positive multinucleated (> 5 nuclei) cells (MNCs) were counted as osteoclast-like cells. TRAP assays were also carried out as previously described 38 . Data are presented as the averages of 3 separate experiments done in triplicate ± S.D.
Immunoprecipitation and immunoblot analysis. 293 T cells were transiently transfected with V5-Sirt6 and Flag-Blimp1 using polyethylenimine reagent. Cells were washed twice with cold-PBS and lysed in RIPA buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.2% sodium deoxycholate) supplemented with protease inhibitors. After incubation for 1 hour on ice, lysates were centrifuged at 14,000 rpm for 20 minutes at 4 °C. Subsequently, protein concentration was measured by Bradford assay (Bio-Rad, Hercules CA, USA). Equivalent amounts of protein were incubated with anti-Flag antibodies overnight at 4 °C, followed by an incubation with protein A agarose beads (Millipore). The beads were washed five times with a washing RIPA buffer containing protease inhibitors, resuspended with 2X sample loading buffer, and immunocomplexes were resolved by SDS-PAGE and analyzed by immunoblot with antibodies.