Mitochondrial Sirt3 contributes to the bone loss caused by aging or estrogen deficiency

Abstract

Altered mitochondria activity in osteoblasts and osteoclasts has been implicated in the loss of bone mass associated with aging and estrogen deficiency - the 2 most common causes of osteoporosis. However, the mechanisms that control mitochondrial metabolism in bone cells during health or disease remain unknown. The mitochondrial deacetylase sirtuin-3 (Sirt3) has been earlier implicated in age-related diseases. Here, we show that deletion of Sirt3 had no effect on the skeleton of young mice but attenuated the age-related loss of bone mass in both sexes. This effect was associated with impaired bone resorption. Osteoclast progenitors from aged Sirt3-null mice were able to differentiate into osteoclasts, though the differentiated cells exhibited impaired polykaryon formation and resorptive activity, as well as decreased oxidative phosphorylation and mitophagy. The Sirt3 inhibitor LC-0296 recapitulated the effects of Sirt3 deletion in osteoclast formation and mitochondrial function, and its administration to aging mice increased bone mass. Deletion of Sirt3 also attenuated the increase in bone resorption and loss of bone mass caused by estrogen deficiency. These findings suggest that Sirt3 inhibition and the resulting impairment of osteoclast mitochondrial function could be a novel therapeutic intervention for the 2 most important causes of osteoporosis.

Keywords: Bone Biology; Mitochondria; Osteoclast/osteoblast biology; Osteoporosis.

Figure 1. Deletion of Sirt3 prevents age-associated cortical bone loss.

(A–D) Imaging and quantification of femoral bones from female Sirt3-KO mice and WT littermates by micro-CT after sacrifice (n = 7–10 animals/group). (A) Representative images of femoral cortical bone at midshaft. Scale bar: 100 μm. (B–D) Cortical thickness (B) and cortical perimeters (C and D) at the femoral midshaft. (E) Cortical thickness at the femoral midshaft of the femur from male Sirt3-KO mice and WT littermates by micro-CT (n = 5–7 animals/group). Data are presented as ± SD. P values were determined using 2-way ANOVA. Interaction terms generated by 2-way ANOVA are shown below each graph.

Figure 2. Deletion of Sirt3 attenuates age-associated trabecular bone loss.

(A–F) Imaging and quantification of vertebral bones from female Sirt3-KO mice and WT littermates by micro-CT after sacrifice (n = 7–10 animals/group). Representative images of trabecular bone (A) and bone volume per tissue volume (BV/TV), bone mineral density (BMD), and microarchitecture of trabecular bone in L5 (B–F). Scale bar: 100 μm. (G and H) BV/TV and BMD from male Sirt3-KO mice and WT littermates in L5 bones by micro-CT (n = 5–7 animals/group). Data are presented as ± SD. P values were determined using 2-way ANOVA. Interaction terms generated by 2-way ANOVA are shown below each graph.

Figure 3. Deletion of Sirt3 decreases bone resorption in aged mice.

(A–C) Number of osteoclast (N.Oc/B.Pm) (A) and osteoblast (N.Ob/B.Pm) per endocortical bone surface (C), and representative photomicrographs of nondecalcified femur sections stained for TRAPase activity (red) (B) from 16-month-old female Sirt3-KO mice and WT littermates (n = 9 animals/group). Scale bar: 100 μm. (D) Serum concentration of a collagen degradation product (CTx), osteocalcin, and N-terminal propeptide of type I procollagen (P1NP) in 16-month-old female Sirt3-KO mice and WT littermates by ELISA (n = 6–8 animals/group). Data are presented as ± SD. P values were determined using (A and C) 2-way ANOVA or (D) Student’s t test.

Figure 4. Deletion of Sirt3 decreases osteoclast function in aged mice.

(A–C) BMMs were isolated from 16-month-old female Sirt3-KO mice and WT littermates and cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 5 days (A and B) or 2 days (C). (A) Representative pictures (left) and number (right) of TRAP⁺ multinucleated osteoclasts generated from BMMs (quadruplicates of pooled cultures). Scale bar: 500 μm. (B) Representative pictures (left) and resorbed areas (right) of Von Kossa–stained bone biomaterial surface (quadruplicate cultures). Scale bar: 500 μm. The resorbed areas appear white, and the unresorbed mineralized surface appears black. (C) Osteoclast marker levels in mRNA of cultured osteoclasts measured by qPCR (triplicate cultures). (D) Protein levels by Western blot in BMM cell cultures (triplicate cultures). (E) BMMs were isolated from 6-month-old C57BL/6 WT mice and cultured with M-CSF (30 ng/mL, BMM) or with M-CSF and RANKL (30 ng/mL) for 2 days (pOC) or 5 days (mOC). Sirt3 levels in mRNA during osteoclastogenesis by qPCR assay (triplicate cultures). (F–H) BM stromal cells were isolated from 16-month-old female Sirt3 knockout mice and WT littermate controls (F and G) or 6-month-old C57BL/6 WT mice (H) cultured with ascorbate (50 mg/mL; SCs) or ascorbate and β-glycerophosphate (10 mM) for 3 days (pOB) or 14 days (mOB). (F) Representative pictures (left) and quantification (right) of Alizarin Red staining in mOB (triplicates of pooled cultures). Scale bar: 1 cm. (G and H) Osteoblast marker and Sirt3 levels in mRNA of SCs, pOB, and mOB measured by qPCR (triplicate cultures). pOC, preosteoclasts; pOB, osteoblasts; mOC, mature osteoclasts; mOB; mature osteoblasts. Data are presented as ± SD. P values were determined using Student’s t test (A–C, F, and G) or 1-way ANOVA (E and H). All measures were performed in cultured BMMs or stromal cells pooled from 4–5 mice/group.

Figure 5. Deletion of Sirt3 attenuates respiration in osteoclasts of aged mice.

(A–F) BMMs were isolated from 16-month-old female Sirt3-KO mice and WT littermate controls and cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 3 days. Different fractions of mitochondrial and nonmitochondrial respirations per cell, in osteoclasts, measured by Seahorse (n = 14–15 wells/group). Data are presented as ± SD. P values were determined using Student’s t test. All measures were performed in cultured BMMs pooled from 4–5 mice/group.

Figure 6. Mitophagy is the dominant Sirt3 mechanism contributing to osteoclast function.

(A–F) BMMs were isolated from 16-month-old female Sirt3 knockout mice and WT littermate controls and cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for indicated times (A and C), 36 hours (B), or 3 days (D–F). (A) Representative pictures of MitoBright Green staining in cultures by fluorescence imaging. (B and C) Representative analysis (B) and quantification (C) of MitoBright Green signals by FACS (triplicate cultures). Scale bar: 500 μm. (D–F) Representative mitochondrial protein levels by Western blot and expression levels as the indicated ratio (triplicate cultures). Data are presented as ± SD. P values were determined using Student’s t test. All measures were performed in cultured BMMs pooled from 4–5 mice/group.

Figure 7. Administration of LC-0296 increases bone mass in aging mice by attenuating bone resorption.

(A) Schedule of LC-0296 administration (5 μg/g body weight, 100 μL/each i.p. injection) to 12-month-old female C57BL/6 mice. (B and C) Representative images (B) and cortical thickness (C) of femoral cortical bone at midshaft. Scale bar: 100 μm. (D) BV/TV of trabecular bone in L5 measured by micro-CT (n = 13–15 animals/group). (E) Serum CTx by ELISA (n = 11 animals/group). (F–H) Osteoclasts developed in cultures of BMMs from 16-month-old female C57BL/6 mice with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 5 days (F) or 3 days (G and H), in the presence or absence of LC-0296 (10 nM). (F) Representative pictures (left) and number (right) of TRAP⁺ multinucleated osteoclasts (triplicate cultures). Scale bar: 500 μm. (G) Osteoclast marker levels in mRNA of cultured osteoclasts measured by qPCR (triplicate cultures). (H) Mitochondrial respiration per cell, measured by Seahorse (n = 14–16 wells/group). Data are presented as ± SD. P values determined using Student’s t test. All in vitro assays were performed in cultured BMMs pooled from 3 mice.

Figure 8. Deletion of Sirt3 attenuates ovariectomy-induced bone loss.

(A and B) Sirt3 mRNA by qPCR in BMMs isolated from 6-month-old female C57BL/6 mice (A) or 3-month-old females of the indicated genotype (B) and cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 2 days in the presence or absence of E₂ (1 × 10^–8M) (triplicate cultures). (C–J) Five-month-old female Sirt3-KO mice and WT littermates were sham operated or ovariectomized (OVX) for 6 weeks (n = 9–11 animals/group). (C and D) Percent change in BMD by DXA 1 day before surgery and before sacrifice. (E–H) Cortical thickness and areas at the femoral midshaft (E–G) and L5 bones measured by micro-CT (H) (n = 9–11 animals/group). (I) BV/TV of trabecular bone in L5 by micro-CT (n = 9–11 animals/group). (J) Serum CTx concentration measured by ELISA (n = 9–11 animals/group). Data are presented as ± SD. P values were determined using Student’s t test (A and B) or 2-way ANOVA (C–J). Interaction terms generated by 2-way ANOVA are shown below each graph. All in vitro assays were performed in cultured BMMs pooled from 3–4 mice/genotype.