Sirtuin 3 (SIRT3) maintains bone homeostasis by regulating AMPK-PGC-1β axis in mice

The mitochondrial sirtuin 3 (SIRT3) is involved in suppressing the onset of multiple pathologies, including cardiovascular disease, fatty liver, age-related hearing loss, and breast cancer. But a physiological role of SIRT3 in bone metabolism is not known. Here we show that SIRT3 is a key regulatory molecule to maintain bone homeostasis. Mice deficient in SIRT3 exhibited severe osteopenia owing to increased numbers of osteoclasts. Osteoclast precursors from Sirt3−/− mice underwent increased osteoclastogenesis in response to receptor activator of nuclear factor-κB ligand (RANKL), an essential cytokine for osteoclast differentiation. SIRT3 expression from RANKL induction depended on the transcription coactivator PGC-1β (peroxisome proliferator-activated receptor-γ co-activator-1β) and the nuclear receptor ERRα (estrogen receptor-related receptor α), and that SIRT3 inhibited the differentiation by interfering with the RANKL-induced expression of PGC-1β. Thus an auto-regulatory feedback mechanism operates to induce its own inhibitor SIRT3 by PGC-1β. Moreover, Sirt3−/− osteoclast precursors reduced AMP-activated protein kinase (AMPK) phosphorylation through down-regulating the expression of AMPK. Our results suggest that a mitochondrial SIRT3 is an intrinsic inhibitor for RANKL-mediated osteoclastogenesis.

(a) BMMs from WT or Sirt3 -/mice were stimulated with RANKL for the indicated times. The cell lysates were subjected to immunoblot analysis using antibodies against phospho-ERK, phospho-p38, phospho-JNK, phosphor-AKT, and phospho-IκBα. Immunoblots were stripped and then reprobed with total ERK, p38, JNK, and AKT. Actin serves as a loading control. (b) BMMs from WT or Sirt3 -/mice were exposed to M-CSF for the indicated times. The cell lysates were subjected to immunoblot analysis as described for panel A. Tubulin served as a loading control. (a) Comparison of RANKL-mediated mitochondrial ROS production. BMMs from WT or Sirt3 -/mice were treated with RANKL for 24 h and then stained with MitoSOX for 10 min and analyzed by flow cytometry. Histograms show the results of one representative experiment out of three independent experiments. (b) RANKL-induced intracellular ROS in WT or Sirt3 -/-BMMs. Cells were stimulated with 200 ng/ml RANKL for 10 min. The intracellular ROS was examined by a fluorescence microscope (Left) and a fluorescence spectrophotometer using DCF-DA (Right). Scale bar, 100 μm. Data are represented as mean ± SD. n.s., not significant. BMMs from WT or Sirt3 -/mice were treated with RANKL for indicated days. The expression of Prkaa1 mRNA encoding AMPKα1 protein was measured using qRT-PCR. BMMs from WT or Sirt3 -/mice were incubated with RANKL for 3 days and mitochondrial proteins were extracted. Mitochondrial lysates were subjected to immunoblot analysis using an anti-Acetyl-lysine antibody. VDAC1 serves as a loading control.

Actin-ring staining.
After osteoclast differentiation, the cells were fixed in 4 % paraformaldehyde in PBS and further permeabilized with 0.1 % Triton X-100 for 20 min. Cells were incubated with Alexa

Evaluation of cell proliferation.
To confirm proliferative effects in WT and Sirt3 -/-BMMs, BrdU cell proliferation kit (Roche Applied Science, Penzberg, Germany) was used. Cells were cultured with 30 ng/ml M-CSF with or without 100 ng/ml RANKL for the indicated time periods. Each sample was assayed in triplicate. After that, BrdU was added and the cells were reincubated for 4 h. After removing the culture medium, cells were fixed and the DNA denatured. Then, peroxidaseconjugated anti-BrdU was added to bind to the BrdU. The immune complexes were detected by the 3,3',5,5' tetramethylbenzidine substrate reaction, and the resultant color was read at 17 370 nm in a microplate spectrophotometer. The absorbance values correlated directly to the amount of DNA synthesis and thereby to the number of proliferating cells in culture.

Detection of intracellular ROS.
Intracellular production of ROS was assayed as described 2 . In brief, 10 min after stimulation with 100 ng/ml RANKL, dishes of confluent cells were washed with α-MEM lacking phenol red and then incubated in the dark for 5 min in Krebs-Ringer solution containing 10 μM DCF-DA. Culture dishes were transferred to a Zeiss Axiovert 135 inverted microscope (Carl Zeiss, Oberkochen, Germany), and DCF (2′,7′-dichlorofluorescein) fluorescence was measured with an excitation wavelength of 488 nm and emission at 515-540 nm. After collection of the fluorescence image, the mean relative fluorescence intensity for each group of cells was then measured by Carl Zeiss vision system (KS400, version 3.0).

Mitochondria purification and Complex I/II activity assay.
Mitochondria from WT or Sirt3 -/-BMMs were isolated using Qproteome Mitochondria Isolation Kit (QIAGEN, Venlo, Netherlands) following manufacture's protocol. Isolated mitochondrial fraction was subjected to enzymatic analysis of mitochondrial complex I and II activity using Mitochondrial Complex I Activity Assay Kit (AAMT001-1KITCN; MERK,