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. 2017 Jan 6;292(1):196-204.
doi: 10.1074/jbc.M116.764761. Epub 2016 Nov 22.

Inactivation of Regulatory-associated Protein of mTOR (Raptor)/Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Osteoclasts Increases Bone Mass by Inhibiting Osteoclast Differentiation in Mice

Qinggang Dai  1   2 , Furong Xie  1 , Yujiao Han  2 , Xuhui Ma  3 , Siru Zhou  4 , Lingyong Jiang  5 , Weiguo Zou  6 , Jun Wang  7
Affiliations

Inactivation of Regulatory-associated Protein of mTOR (Raptor)/Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Osteoclasts Increases Bone Mass by Inhibiting Osteoclast Differentiation in Mice

Qinggang Dai et al. J Biol Chem. .

Abstract

Mammalian target of rapamycin complex 1 (mTORC1) is involved in anabolic metabolism in both osteoblasts and chondrocytes, but the role of mTORC1 in osteoclast biology in vivo remains to be elucidated. In this study, we showed that deletion of regulatory-associated protein of mTOR (Raptor) in osteoclasts led to an increase in bone mass with decreased bone resorption. Raptor-deficient bone marrow-derived macrophages exhibited lower mTORC1-S6K1 signaling and retarded osteoclast differentiation, as determined by the number of osteoclasts, tartrate-resistant acid phosphatase activity, and expression of osteoclast-specific genes. Enforced expression of constitutively active S6K1 rescued the impaired osteoclast differentiation in Raptor-deficient bone marrow-derived macrophages. Furthermore, pharmacological inhibition of mTORC1 signaling by rapamycin could also inhibit osteoclast differentiation and osteoclast-specific gene expression. Taken together, our findings demonstrate that mTORC1 plays a key role in the network of catabolic bone resorption in osteoclasts and may serve as a potential pharmacological target for the regulation of osteoclast activity in bone metabolic disorders.

Keywords: S6 kinase; bone; cell differentiation; mTOR complex (mTORC); osteoclast; osteoporosis.

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Figures

FIGURE 1.
FIGURE 1.
Deletion of Raptor in osteoclasts led to an increase of bone mass. A, illustration of Raptor deletion in Ctsk-expressing osteoclasts. B, Western blotting assay of Raptor of WT and RapCtsk BMMs cultured with osteoclast differentiation medium for 6 days. C, body weight of male WT and RapCtsk littermates measured at different age points. Data represent mean ± S.D. *, p < 0.05; n = 5. D, three-dimensional reconstruction of micro-CT images of trabecular bone close to the distal growth plate and cortical bone at the middle of femora from 4-week-old WT and RapCtsk littermates. E, representative view of micro-CT of femora from 8-week-old WT and RapCtsk mice. F–J, quantitative parameters of micro-CT. The trabecular bone close to the distal growth plate and cortical bone at the middle of the femur was analyzed. Data represent mean ± S.D. *, p < 0.05; n = 5. K–P, H&E staining of femora from 4-week-old male WT and RapCtsk littermates. M and N, high-power images of the secondary spongiosa marked in K and L, respectively. O and P, high-power images of the primary spongiosa marked in K and L, respectively. Ma, bone marrow.
FIGURE 2.
FIGURE 2.
Raptor deficiency in osteoclasts induced increased bone volume. A–D, H&E staining of femora from 4-week-old female WT and RapCtsk littermates. C and D, high-power images of the primary spongiosa marked in A and B, respectively. E–H, H&E staining of femora from 20-week-old male WT and RapCtsk littermates. G and H, high-power images of the primary spongiosa marked in E and F, respectively. Ma, bone marrow.
FIGURE 3.
FIGURE 3.
Ablation of Raptor in osteoclasts resulted in decreased bone resorption. A–D, representative view of TRAP staining of distal femur trabecular bone of 4-week-old male WT and RapCtsk mice. C and D, high-power images of the marked region in A and B, respectively. Arrowheads indicate TRAP-positive osteoclasts. Ma, bone marrow. E and F, immunohistochemical staining of Ctsk in distal femur trabecular bone of 4-week-old male WT and RapCtsk mice. Arrowheads indicate Ctsk-positive osteoclasts. G and H, numbers of TRAP- and Ctsk-positive osteoclasts on the trabecular bone surface, measured as cells per millimeter of perimeter (/B.Pm). Data are mean ± S.D. *, p < 0.05; n = 3. I, alizarin red and calcein double-labeling of femora showed the bone turnover rate of distal femur trabecular bone in 8-week-old WT and RapCtsk littermates. J and K, quantitative parameters of MAR and BFR of trabecular bone in WT and RapCtsk mice. Data represent mean ± S.D.; n = 3. L–O, representative view of TRAP staining of distal femur cortical bone from of 4-week-old male WT and RapCtsk mice. M and O, high-power images of the marked regions in L and N, respectively. P, numbers of TRAP-positive osteoclasts on the cortical bone surface, measured as cells per millimeter of perimeter (/B.Pm). Data are mean ± S.D. *, p < 0.05; n = 3. Q, alizarin red and calcein double-labeling of distal femur cortical bone from 8-week-old WT and RapCtsk littermates. R and S, quantitative parameters of MAR and BFR in WT and RapCtsk mice. Data represent mean ± S.D.
FIGURE 4.
FIGURE 4.
Raptor deficiency impaired osteoclast differentiation. A, BMMs were seeded in 96-well plates (5 × 104 cells/cm2) and treated with 20 ng/ml M-CSF and 250 ng/ml RANKL for 6 days. Cells were fixed with 4% paraformaldehyde and subjected to TRAP staining. B, relative TRAP activity of culture supernatant of WT and RapCtsk BMMs. Data represent means ± S.D. *, p < 0.05; n = 3. C–G, osteoclast-specific gene expression of WT and RapCtsk BMMs cultured with M-CSF and RANKL. Data represent mean ± S.D. *, p < 0.05; n = 3. H, relative growth of WT and RapCtsk BMMs determined by crystal violet staining. Data represent mean ± S.D., n = 3.
FIGURE 5.
FIGURE 5.
CAS6K1 rescue impaired osteoclast differentiation of RapCtsk BMMs. A, WT and RapCtsk BMMs were cultured with M-CSF and RANKL for 6 days and subjected to a Western blotting assay. B, WT and RapCtsk BMMs were infected with lentiviruses expressing GFP and CAS6K1, which was followed by osteoclast differentiation and Western blotting assay. C, WT and RapCtsk BMMs infected with Lenti-GFP or Lenti-S6K1. Cells were treated with RANKL for 6 days and subjected to TRAP staining. D, relative TRAP activity of culture supernatant. Data represent mean ± S.D. *, p < 0.05; n = 3. E–I, osteoclast-specific gene expression of WT and RapCtsk BMMs infected with Lenti-GFP or Lenti-S6K1. Data represent mean ± S.D. *, p < 0.05; n = 3.
FIGURE 6.
FIGURE 6.
Rapamycin inhibited osteoclast differentiation of BMMs. A, WT BMMs were cultured with 20 ng/ml M-CSF or 250 ng/ml RANKL in the absence or presence of different concentrations of rapamycin for 6 days. Cells were subjected to TRAP staining. B, Western blotting assay of BMMs cultured in the absence or presence of different concentrations of rapamycin. C, relative TRAP activity of culture supernatant. Data represent mean ± S.D. *, p < 0.05; n = 3. D–H, rapamycin decreased the expression of osteoclast-specific genes in BMMs. Data represent mean ± S.D. *, p < 0.05; n = 3.
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