The Sirtuin1 activator SRT3025 down-regulates sclerostin and rescues ovariectomy-induced bone loss and biomechanical deterioration in female mice

Abstract

Estrogen deficiency leads to rapid bone loss and skeletal fragility. Sclerostin, encoded by the sost gene, and a product of the osteocyte, is a negative regulator of bone formation. Blocking sclerostin increases bone mass and strength in animals and humans. Sirtuin1 (Sirt1), a player in aging and metabolism, regulates bone mass and inhibits sost expression by deacetylating histone 3 at its promoter. We asked whether a Sirt1-activating compound could rescue ovariectomy (OVX)-induced bone loss and biomechanical deterioration in 9-week-old C57BL/6 mice. OVX resulted in a substantial decrease in skeletal Sirt1 expression accompanied by an increase in sclerostin. Oral administration of SRT3025, a Sirt1 activator, at 50 and 100 mg/kg·d for 6 weeks starting 6 weeks after OVX fully reversed the deleterious effects of OVX on vertebral bone mass, microarchitecture, and femoral biomechanical properties. Treatment with SRT3025 decreased bone sclerostin expression and increased cortical periosteal mineralizing surface and serum propeptide of type I procollagen, a bone formation marker. In vitro, in the murine long bone osteocyte-Y4 osteocyte-like cell line SRT3025 down-regulated sclerostin and inactive β-catenin, whereas a reciprocal effect was observed with EX-527, a Sirt1 inhibitor. Sirt1 activation by Sirt1-activating compounds is a potential novel pathway to down-regulate sclerostin and design anabolic therapies for osteoporosis concurrently ameliorating other metabolic and age-associated conditions.

Figure 1.

OVX induces an increase in bone turnover, vertebral and femoral bone loss, and a decrease in bone Sirt1 accompanied by elevated sclerostin. A, Experiment time line. B and C, The changes in serum CTX (B), a bone resorption marker, and in P1NP, a bone formation marker (C), 1, 6, and 12 weeks after OVX or SHAM. D–I, μCT analyses in the L4 and in the distal femoral metaphysis in SHAM vs OVX mice. D, Trabecular bone volume/total volume (BV/TV). E, Trabecular number. F, Trabecular thickness. G, Femoral BV/TV. H, Femoral trabecular number. I, Femoral trabecular thickness; n = 9–10 mice/group. Statistical analysis was performed by two-way-ANOVA with time after operation and type of operation as independent variables followed by Sidak's post hoc correction vs SHAM mice. J and K, Sirt1 and sclerostin protein level in whole tibiae extracts obtained from OVX and SHAM mice 6 weeks after surgical intervention. Immunoblot of a representative image (left) and densitometry (right). HSP90 as a control, n = 3–4 bones/group and n = 6 bones/group for Sirt1 and sclerostin, respectively, analyzed by Student's t test. Results are mean ± SEM; *, P < .05; **, P < .005; ***, P < .001.

Figure 2.

SRT3025 administration restores vertebral bone mass and microarchitecture lost with OVX. A, Images of a representative L4 in the various treatment groups. Scale bar, 0.5 mm. B–H, μCT analyses of L4. B, Trabecular bone volume/total volume (BV/TV). C, Trabecular thickness. D, Trabecular number. E, Trabecular connectivity density. F, Trabecular spacing. G, Trabecular bone pattern factor (TBPf). H, Structure model index (SMI); n = 7–9 mice/group. Analyzed by one-way ANOVA followed by Dunnett's post hoc analysis vs vehicle-treated OVX mice. Results are mean ± SEM. *, P < .05; *, P < .005; ***, P < .001.

Figure 3.

SRT3025 administration restores femoral biomechanical properties and increases femoral cortical periosteal mineralizing surface and serum P1NP, a bone formation marker. A and B, Femoral biomechanical parameters determined by the 3-point bending test: A, Stiffness (Newton/millimeter); B, Young's elasticity modulus (E) measured in Giga Pascal (GPa); n = 4–5 mice/group. Analyzed by one-way ANOVA followed by Dunnett's post hoc analysis vs vehicle-treated OVX mice. C–F, Cortical midshaft femur fluorochrome-derived BFRs on the periosteal surface. C, Representative images. D, Histomorphometric analysis of MS/BS (% MS/BS). E, MAR. F, BFR/BS; n = 5–6 mice/group. Analyzed by Student's t test in vehicle vs SRT3025-treated OVX mice. Scale bar, 0.5 mm. G, Serum P1NP, a bone formation marker (H) CTX, a bone resorption marker (I) RANKL, a bone resorption marker, in vehicle- vs SRT3025-treated OVX mice 3 weeks after treatment initiation; n = 6–9 mice/group. Analyzed by Student's t test. Results are mean ± SEM. *, P < .05; **, P < .005.

Figure 4.

SRT3025 down-regulates sclerostin in vivo and in vitro. A, Sclerostin level in whole tibiae extracts in SRT3025- vs vehicle-treated OVX mice. Immunoblot of a representative image (left) and densitometry (right) are presented with HSP90 as a control; n = 3–4 bones/group. Analyzed by non parametric ANOVA followed by Dunn's post hoc analysis. B, Sclerostin expression in SRT3025- vs vehicle-treated MLO-Y4 murine osteocyte-like cells. Immunoblot of a representative image (left) and densitometry (right) are presented with HSP90 as a control; n = 6 repeats analyzed by nonparametric ANOVA followed by Dunn's post hoc analysis. C, Sclerostin expression in vehicle and EX-527-treated MLO-Y4 murine osteocyte-like cells. n = 4 repeats analyzed by Student's t-test. D–E, The ratio between inactive and active β-catenin in SRT3025- and EX-527- vs vehicle-treated MLO-Y4 cells. Immunoblot of a representative image (left) and densitometry (right) of phosphorylated (Ser33/37/Thr41) β-catenin and dephosphorylated β-catenin (Ser37/Thr41). D, n = 4 repeats analyzed by nonparametric ANOVA followed by Dunn's post hoc analysis. E, n = 5 repeats analyzed by Student's t test. Results are mean ± SEM. *, P < .05 vs vehicle-treated cells.