We hypothesized that ER upregulation by Sirt6 was due to protein stabilization via proteasome inhibition and repeated the experiments in the presence of the proteasome inhibitor MG132. ER stabilization promoted transcription of Fas ligand in preosteoclasts, resulting in apoptosis of osteoclasts. Finally, the level of Sirt6 in human preosteoclasts was correlated positively with bone density and ER but negatively with age. In conclusion, our results suggest that deacetylation and upregulation of ER by Sirt6 in preosteoclasts prevent bone loss by inhibiting osteoclast-mediated bone resorption. Activation of Sirt6 in preosteoclasts may provide a new therapeutic approach to attenuate osteoporosis in older or postmenopausal patients. in osteoclast precursor cells decreases bone mass [15, 16], and pharmacological Acebutolol HCl activation of Sirt1 prevents the bone loss caused by OVX [17] or Acebutolol HCl aging [18]. However, the effects of Sirt6, a nuclear form of sirtuin, on estrogen deficiency- or age-related osteoporosis have not been reported. In this study, we investigated the role of Sirt6 in preosteoclasts on regulation of bone mass in osteoporosis animal models and identified its mechanism of action. Materials and methods Acebutolol HCl Animal experiments Sirt6 transgenic mice (C57BL/6-Tg(RP23-352G18)1Coppa/J), mice (B6;129-mice (B6.129P2-(mutant Sirt6), or using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). To evaluate promoter activity, 1?g each of FasL, ERE, and AP-1 promoter luciferase (Promega, Madison, WI, USA) were used. Antibodies Antibodies against the following proteins were purchased from the manufacturer indicated: ER, Ub, FasL (Santa Cruz Biotechnology, Dallas, TX, USA), Sirt6, cleaved caspase 8, cleaved caspase 3, Bax, Ac-Lys (Cell Signaling Technology, Beverly, MA, USA), NFATc1 (Abcam), HA (Roche Diagnostics, Mannheim, German), HSP90 (Enzo Life Sciences, Plymouth Meeting, PA, USA), -tubulin, and GAPDH (Bioworld Technology, St. Louis Park, MN, USA). Liquid chromatography (LC)-mass spectrometry (MS/MS) analysis Immunoprecipitated proteins were visualized by staining SDS gels with colloidal Coomassie blue. After the proteins were excised from the gel bands, they were subjected to in-gel trypsin digestion. The tryptic peptides were analyzed for acetylation using a high-resolution mass spectrometer (LTQ-Orbitrap Velos, Thermo Fisher Scientific, Waltham, MA, USA) coupled with nano-flow liquid chromatograph (nano-LC) as described previously [27]. Site-directed mutagenesis Acetylation mutants of ER were generated using a site-directed mutagenesis kit (GeneAll, Acebutolol HCl Seoul, Korea) by converting each lysine residue (K171 and K299) to arginine (codon change from AAG to AGG). Mutations were confirmed with DNA sequencing performed at Cosmogenetech (Seoul, Korea). Statistical analysis The data are Acebutolol HCl expressed as the mean??standard error of the mean (SEM). Statistical comparisons were performed using one-way analysis of variance followed by Fishers post hoc analysis. The significance of differences between two groups was determined using Students unpaired and or the FasL receptor (Fig.?4b), implying that posttranscriptional regulation of ER by Sirt6 could be a cause of apoptotic alteration. The staining results for apoptotic and TRAP-positive cells further supported these results (Fig.?4c, d). Open in a separate window Rabbit Polyclonal to CYC1 Fig. 4 Increased resistance to apoptotic cell death in preosteoclasts by Sirt6 deficiency. CD11b+ BMCs from WT and mS6KO mice were treated with M-CSF (10?ng/ml) and RANKL (30?ng/ml) for the indicated time. a Proteins of the extrinsic apoptotic pathway were examined using western blotting. b Two days after osteoclastogenesis induction, the mRNA level of was determined using real-time RT-PCR (expression (Fig.?5b), and osteoclast apoptosis (Fig.?5c). In agreement with these findings, the number of TRAP-positive cells was also significantly decreased by Sirt6 re-expression (Fig.?5d). However, transduction with adenovirus carrying mutant Sirt6 lacking deacetylase activity had no effect on these parameters, suggesting a deacetylase-dependent mechanism for Sirt6. Open in a separate window Fig. 5 Restoration of the Sirt6-ER-FasL axis in preosteoclasts by Sirt6 re-expression. CD11b+ BMCs were transduced with AdLacZ, AdSirt6, or AdmSirt6. a Three days after osteoclastogenesis induction, proteins of the extrinsic apoptotic pathway were examined using western blotting..