Sirtuin1 (Sirt1) promotes cortical bone formation by preventing β-catenin sequestration by FoxO transcription factors in osteoblast progenitors

Abstract

A decline of the levels and activity of Sirtuin1 (Sirt1), a NAD(+) class III histone deacetylase, with age contributes to the development of several diseases including type 2 diabetes, neurodegeneration, inflammation, and cancer. The anti-aging effects of Sirt1 evidently result from the deacetylation of many transcription factors and co-factors including members of the Forkhead box O (FoxO) family and β-catenin. Wnt/β-catenin is indispensable for osteoblast generation. FoxOs, on the other hand, sequester β-catenin and inhibit osteoprogenitor proliferation. Here, we have deleted Sirt1 in osteoprogenitors expressing Osterix1 (Osx1)-Cre and their descendants. Sirt1(ΔOsx1) mice had lower cortical thickness in femora and vertebrae because of reduced bone formation at the endocortical surface. In line with this, osteoprogenitor cell cultures from the Sirt1(ΔOsx1) mice exhibited lower alkaline phosphatase activity and mineralization, as well as decreased proliferation and increased apoptosis. These changes were associated with decreased Wnt/β-catenin signaling and expression of cyclin D1 and resulted from increased binding of FoxOs to β-catenin. These findings demonstrate that Sirt1-induced deacetylation of FoxOs unleashes Wnt signaling. A decline in Sirt1 activity in osteoblast progenitors with aging may, therefore, contribute to the age-related loss of bone mass. Together with evidence that Sirt1 activators increase bone mass in aged mice, our results also suggest that Sirt1 could be a therapeutic target for osteoporosis.

Keywords: Animal Model; Apoptosis; Cell Proliferation; Osteoporosis; Wnt Signaling.

FIGURE 1.

Sirt1 deletion in osteoprogenitors decreases cortical bone mass. A, body weight of 12-week-old females (n = 6–8/group). B, GFP⁺ osteoprogenitor cell cultures derived from neonatal calvaria. C, Sirt1 protein levels in bone marrow-derived macrophages cultured in the presence of M-CSF, and osteoblastic cells cultured with ascorbate, from mice of the indicated genotypes. D and E, representative microcomputed tomography images of femoral midshaft (D) and femoral and vertebral cortical thickness (E) of mice described in A. F and G, representative microcomputed tomography images (F) and cancellous BV/TV (G) in vertebrae of mice described in A. H, cortical thickness of femur and BV/TV of vertebrae from 8-week-old females (n = 5–7/group). #, p < 0.05 versus wild-type and Sirt1^f/f mice; †, p < 0.05 versus wild-type, Sirt1^f/f and Osx1-Cre mice by two-way ANOVA. *, p < 0.05 by Student's t test. Bars represent mean and S.D. (error bars).

FIGURE 2.

Sirt1^ΔOsx1 mice have decreased endocortical bone formation. A, representative photomicrographs of cortical bone labeled with tetracycline (yellow). Ps, periosteal surface; Ec, endocortical surface. Scale bar, 50 μm. B–D, mineralizing surface (M. Pm/B. Pm), MAR, and BFR as determined by tetracycline labels at the endocortical (B), periosteal (C), and cancellous (D) bone surfaces in longitudinal undecalcified femur sections from 8-week-old females (n = 5–7/group). E, osteoblast (Ob) and osteoclast (Oc) number in endocortical bone surface. *, p < 0.05 by Student's t test. Bars represent mean and S.D. (error bars).

FIGURE 3.

Sirt1 promotes osteoblastogenesis. A, alizarin red staining of bone marrow-derived osteoprogenitor cells cultured with 1% ascorbate for 21 days. B, gene expression in cells described in A. C, osteoprogenitor cells cultured for 3 days with 1% ascorbate in the presence of vehicle (veh) or SRT2104. D, BrdU incorporation in osteoprogenitor cell cultures. RLU, relative luminescence units. E, caspase-3 activity in osteoprogenitor cells cultured in the presence or absence of NAC and H₂O₂, as indicated. AFU, arbitrary fluorescence units. F and G, reactive oxygen species (ROS) (F) and phosphorylated p66^shc (G) in osteoprogenitor cells. *, p < 0.05 by Student's t test. †, p < 0.05 effect of treatment within each genotype; #, p < 0.05 versus the equivalent treatment in Osx1-Cre by two-way ANOVA. Bars represent mean and S.D. (error bars).

FIGURE 4.

Sirt1 potentiates TCF-mediated transcription by attenuating FoxO/β-catenin association. A, bone marrow-derived osteoprogenitor cells were transduced with a TCF-luc reporter construct and cultured in the presence of vehicle (veh) or SRT2104 for 24 h. RLU, relative luminescence units. B, GFP⁺ cell cultures derived from neonatal calvaria. C, top panel, osteoprogenitor cell culture lysates immunoprecipitated (IP) with an anti-β-catenin or -IgG antibody and probed with anti-FoxO1, anti-FoxO3, and anti-β-catenin antibodies. TL, total cell lysates; WB, Western blot. Bottom panel, relative amount of FoxO1 and FoxO3 in β-catenin immunoprecipitates. D, FoxO3 expression was induced in the OPF-iFoxO3 cell line by withdrawal of doxycycline, and cells were transfected with a TCF-luc reporter construct and with a Sirt1 expression plasmid, as indicated. Twenty-four hours later, vehicle or SRT2104 was added to the cultures for another 24 h. E, ST2 cells transfected with TCF-luc and with empty vector, FoxO1, or FoxO3 expression plasmids and co-transfected with Sirt1, as indicated. F, ST2 cells transfected with TCF-luc and with empty vector, wild-type (WT) FoxO1, acetylation mimic (KQ), or acetylation mutant (KR) and cultured in the presence of vehicle or 25 ng/ml Wnt3a for 24 h. G, acetylated FoxO1 (Ac-FoxO1) in bone marrow-derived osteoprogenitor cell cultures by Western blot. H, mRNA from vertebral bone homogenates of 8-week-old females. †, p < 0.05 versus respective vehicle; #, p < 0.05 versus vehicle in control group by two-way ANOVA. *, p < 0.05 by Student's t test. Bars represent mean and S.D. (error bars).

FIGURE 5.

Sirt1 inhibits FoxO activity. A, FoxO3 expression was induced in the OPF-iFoxO3 cell line by withdrawal of doxycycline, and cells were transfected with a FoxO-luc reporter construct and a Sirt1 expression plasmid, as indicated. Twenty-four hours later, vehicle (veh) or SRT2104 was added to the cultures for another 24 h. RLU, relative luminescence units. B, ST2 cells transfected with an empty vector control (pcDNA), FoxO1, or FoxO3 expression plasmids and co-transfected with a Sirt1 plasmid followed by treatment as in A. C and D, GFP⁺ cell cultures derived from neonatal calvaria of FoxO1,3,4 (C) and Sirt1 (D) conditional deletion mouse models. E, ST2 cells transfected with FoxO-luc and with empty vector, wild-type (WT) FoxO1, acetylation mimic (KQ), or acetylation mutant (KR). †, p < 0.05 versus respective vehicle; #, p < 0.05 versus vehicle in control group by two-way ANOVA. *, p < 0.05 by Student's t test. Bars represent mean and S.D. (error bars).