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mTORC1 Prevents Preosteoblast Differentiation through the Notch Signaling Pathway.

Huang B, Wang Y, Wang W, Chen J, Lai P, Liu Z, Yan B, Xu S, Zhang Z, Zeng C, Rong L, Liu B, Cai D, Jin D, Bai X - PLoS Genet. (2015)

Bottom Line: Mechanistically, mTORC1 prevented osteoblast maturation through activation of the STAT3/p63/Jagged/Notch pathway and downregulation of Runx2.Preosteoblasts with hyperactive mTORC1 reacquired the capacity to fully differentiate and maturate when subjected to inhibition of the Notch pathway.Together, these findings identified the role of mTORC1 in osteoblast formation and established that mTORC1 prevents preosteoblast differentiation and maturation through activation of the Notch pathway.

View Article: PubMed Central - PubMed

Affiliation: Academy of Orthopedics, Guangdong Province, Department of Orthopedics, The Third Affiliated Hospital, Southern Medical University, Guangzhou, China.

ABSTRACT
The mechanistic target of rapamycin (mTOR) integrates both intracellular and extracellular signals to regulate cell growth and metabolism. However, the role of mTOR signaling in osteoblast differentiation and bone formation is undefined, and the underlying mechanisms have not been elucidated. Here, we report that activation of mTOR complex 1 (mTORC1) is required for preosteoblast proliferation; however, inactivation of mTORC1 is essential for their differentiation and maturation. Inhibition of mTORC1 prevented preosteoblast proliferation, but enhanced their differentiation in vitro and in mice. Activation of mTORC1 by deletion of tuberous sclerosis 1 (Tsc1) in preosteoblasts produced immature woven bone in mice due to excess proliferation but impaired differentiation and maturation of the cells. The mTORC1-specific inhibitor, rapamycin, restored these in vitro and in vivo phenotypic changes. Mechanistically, mTORC1 prevented osteoblast maturation through activation of the STAT3/p63/Jagged/Notch pathway and downregulation of Runx2. Preosteoblasts with hyperactive mTORC1 reacquired the capacity to fully differentiate and maturate when subjected to inhibition of the Notch pathway. Together, these findings identified the role of mTORC1 in osteoblast formation and established that mTORC1 prevents preosteoblast differentiation and maturation through activation of the Notch pathway.

No MeSH data available.


Related in: MedlinePlus

Inactivation of mTORC1 prevents preosteoblast proliferation but enhances their differentiation in vitro and in vivo.(A) Proliferating MC3T3-E1 cells were treated with vehicle (V) or 0.1 nM rapamycin (R) and underwent immunoblotting to detect proliferative markers (cyclin-D1 and PCNA) on the day of cell plating (day 0) and on the 3rd day. (B) Differentiating MC3T3-E1 cells were treated with vehicle or 0.1 nM rapamycin for the indicated time (3d, 7d) and underwent immunoblotting to detect osteoblastic markers (Osx and Ocn). (C) AR-S staining of differentiated MC3T3-E1 cells on the 14th day of osteogenic induction. (D) Immunohistochemistry staining for S6 phosphorylation (Ser235/236) in sections of distal femur of 10-week-old female C57BL/6 mice treated with vehicle (V) or rapamycin (R). (E) Micro-CT images of metaphyseal trabecular bone of the distal femur. (F) Alkaline phosphatase (ALP) staining of trabecular bone in sections from the distal femora (G) Quantification of osteoblast lineage cells stained with ALP. N.ALP+/B.Pm, number of ALP positive cells per bone perimeter (mm-1). Representative immunohistochemistry staining for (H) BrdU, (J) osterix, and (L) osteocalcin in femur sections. (I) Percentage of BrdU+ osteoblasts out of total osteoblasts on bone surface. (K, M) Number of Osx-positive cells (N.Osx+) and Ocn-positive cells (N.Ocn+) on the bone surface was measured as cells per millimeter of perimeter in sections (/B.Pm) and mean density of the corresponding positive cells was calculated as integrated optical density (IOD) per area of positive cells. All data are mean ± SD (n = 3 mice).Scale bar, 100 μm for (D) and 50 μm for (F), (H), (J), (L). *P < 0.05, **P < 0.01, ***P<0.001 by t test.
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pgen.1005426.g002: Inactivation of mTORC1 prevents preosteoblast proliferation but enhances their differentiation in vitro and in vivo.(A) Proliferating MC3T3-E1 cells were treated with vehicle (V) or 0.1 nM rapamycin (R) and underwent immunoblotting to detect proliferative markers (cyclin-D1 and PCNA) on the day of cell plating (day 0) and on the 3rd day. (B) Differentiating MC3T3-E1 cells were treated with vehicle or 0.1 nM rapamycin for the indicated time (3d, 7d) and underwent immunoblotting to detect osteoblastic markers (Osx and Ocn). (C) AR-S staining of differentiated MC3T3-E1 cells on the 14th day of osteogenic induction. (D) Immunohistochemistry staining for S6 phosphorylation (Ser235/236) in sections of distal femur of 10-week-old female C57BL/6 mice treated with vehicle (V) or rapamycin (R). (E) Micro-CT images of metaphyseal trabecular bone of the distal femur. (F) Alkaline phosphatase (ALP) staining of trabecular bone in sections from the distal femora (G) Quantification of osteoblast lineage cells stained with ALP. N.ALP+/B.Pm, number of ALP positive cells per bone perimeter (mm-1). Representative immunohistochemistry staining for (H) BrdU, (J) osterix, and (L) osteocalcin in femur sections. (I) Percentage of BrdU+ osteoblasts out of total osteoblasts on bone surface. (K, M) Number of Osx-positive cells (N.Osx+) and Ocn-positive cells (N.Ocn+) on the bone surface was measured as cells per millimeter of perimeter in sections (/B.Pm) and mean density of the corresponding positive cells was calculated as integrated optical density (IOD) per area of positive cells. All data are mean ± SD (n = 3 mice).Scale bar, 100 μm for (D) and 50 μm for (F), (H), (J), (L). *P < 0.05, **P < 0.01, ***P<0.001 by t test.

Mentions: We next investigated the role of reduced mTORC1 activity caused by rapamycin in the proliferation and differentiation of preosteoblasts. As shown in Fig 1A, the growth of cells treated with 0.1 nM rapamycin significantly lagged behind control cells, and the decreased level of proliferative markers (cyclin D1 and PCNA) in rapamycin-treated cells revealed the underlying mechanism (Fig 2A). We next determined the role of reduced mTORC1 activity in the differentiation of preosteoblasts. As seen in Fig 2B, a low concentration of rapamycin (0.1nM) increased the expression of osterix and osteocalcin. Separate sets of cells were tested for mineralization capacity, a terminal differentiation parameter for osteoblasts, by staining with alizarin red, and the results confirmed enhanced mineralization of the extracellular matrix (ECM) in MC3T3-E1 cells with impaired mTORC1 activity (Fig 2C). Increased mineralization of ECM was also observed in fetal rat calvarial cells treated with 0.1nM rapamycin (S1 Fig).


mTORC1 Prevents Preosteoblast Differentiation through the Notch Signaling Pathway.

Huang B, Wang Y, Wang W, Chen J, Lai P, Liu Z, Yan B, Xu S, Zhang Z, Zeng C, Rong L, Liu B, Cai D, Jin D, Bai X - PLoS Genet. (2015)

Inactivation of mTORC1 prevents preosteoblast proliferation but enhances their differentiation in vitro and in vivo.(A) Proliferating MC3T3-E1 cells were treated with vehicle (V) or 0.1 nM rapamycin (R) and underwent immunoblotting to detect proliferative markers (cyclin-D1 and PCNA) on the day of cell plating (day 0) and on the 3rd day. (B) Differentiating MC3T3-E1 cells were treated with vehicle or 0.1 nM rapamycin for the indicated time (3d, 7d) and underwent immunoblotting to detect osteoblastic markers (Osx and Ocn). (C) AR-S staining of differentiated MC3T3-E1 cells on the 14th day of osteogenic induction. (D) Immunohistochemistry staining for S6 phosphorylation (Ser235/236) in sections of distal femur of 10-week-old female C57BL/6 mice treated with vehicle (V) or rapamycin (R). (E) Micro-CT images of metaphyseal trabecular bone of the distal femur. (F) Alkaline phosphatase (ALP) staining of trabecular bone in sections from the distal femora (G) Quantification of osteoblast lineage cells stained with ALP. N.ALP+/B.Pm, number of ALP positive cells per bone perimeter (mm-1). Representative immunohistochemistry staining for (H) BrdU, (J) osterix, and (L) osteocalcin in femur sections. (I) Percentage of BrdU+ osteoblasts out of total osteoblasts on bone surface. (K, M) Number of Osx-positive cells (N.Osx+) and Ocn-positive cells (N.Ocn+) on the bone surface was measured as cells per millimeter of perimeter in sections (/B.Pm) and mean density of the corresponding positive cells was calculated as integrated optical density (IOD) per area of positive cells. All data are mean ± SD (n = 3 mice).Scale bar, 100 μm for (D) and 50 μm for (F), (H), (J), (L). *P < 0.05, **P < 0.01, ***P<0.001 by t test.
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pgen.1005426.g002: Inactivation of mTORC1 prevents preosteoblast proliferation but enhances their differentiation in vitro and in vivo.(A) Proliferating MC3T3-E1 cells were treated with vehicle (V) or 0.1 nM rapamycin (R) and underwent immunoblotting to detect proliferative markers (cyclin-D1 and PCNA) on the day of cell plating (day 0) and on the 3rd day. (B) Differentiating MC3T3-E1 cells were treated with vehicle or 0.1 nM rapamycin for the indicated time (3d, 7d) and underwent immunoblotting to detect osteoblastic markers (Osx and Ocn). (C) AR-S staining of differentiated MC3T3-E1 cells on the 14th day of osteogenic induction. (D) Immunohistochemistry staining for S6 phosphorylation (Ser235/236) in sections of distal femur of 10-week-old female C57BL/6 mice treated with vehicle (V) or rapamycin (R). (E) Micro-CT images of metaphyseal trabecular bone of the distal femur. (F) Alkaline phosphatase (ALP) staining of trabecular bone in sections from the distal femora (G) Quantification of osteoblast lineage cells stained with ALP. N.ALP+/B.Pm, number of ALP positive cells per bone perimeter (mm-1). Representative immunohistochemistry staining for (H) BrdU, (J) osterix, and (L) osteocalcin in femur sections. (I) Percentage of BrdU+ osteoblasts out of total osteoblasts on bone surface. (K, M) Number of Osx-positive cells (N.Osx+) and Ocn-positive cells (N.Ocn+) on the bone surface was measured as cells per millimeter of perimeter in sections (/B.Pm) and mean density of the corresponding positive cells was calculated as integrated optical density (IOD) per area of positive cells. All data are mean ± SD (n = 3 mice).Scale bar, 100 μm for (D) and 50 μm for (F), (H), (J), (L). *P < 0.05, **P < 0.01, ***P<0.001 by t test.
Mentions: We next investigated the role of reduced mTORC1 activity caused by rapamycin in the proliferation and differentiation of preosteoblasts. As shown in Fig 1A, the growth of cells treated with 0.1 nM rapamycin significantly lagged behind control cells, and the decreased level of proliferative markers (cyclin D1 and PCNA) in rapamycin-treated cells revealed the underlying mechanism (Fig 2A). We next determined the role of reduced mTORC1 activity in the differentiation of preosteoblasts. As seen in Fig 2B, a low concentration of rapamycin (0.1nM) increased the expression of osterix and osteocalcin. Separate sets of cells were tested for mineralization capacity, a terminal differentiation parameter for osteoblasts, by staining with alizarin red, and the results confirmed enhanced mineralization of the extracellular matrix (ECM) in MC3T3-E1 cells with impaired mTORC1 activity (Fig 2C). Increased mineralization of ECM was also observed in fetal rat calvarial cells treated with 0.1nM rapamycin (S1 Fig).

Bottom Line: Mechanistically, mTORC1 prevented osteoblast maturation through activation of the STAT3/p63/Jagged/Notch pathway and downregulation of Runx2.Preosteoblasts with hyperactive mTORC1 reacquired the capacity to fully differentiate and maturate when subjected to inhibition of the Notch pathway.Together, these findings identified the role of mTORC1 in osteoblast formation and established that mTORC1 prevents preosteoblast differentiation and maturation through activation of the Notch pathway.

View Article: PubMed Central - PubMed

Affiliation: Academy of Orthopedics, Guangdong Province, Department of Orthopedics, The Third Affiliated Hospital, Southern Medical University, Guangzhou, China.

ABSTRACT
The mechanistic target of rapamycin (mTOR) integrates both intracellular and extracellular signals to regulate cell growth and metabolism. However, the role of mTOR signaling in osteoblast differentiation and bone formation is undefined, and the underlying mechanisms have not been elucidated. Here, we report that activation of mTOR complex 1 (mTORC1) is required for preosteoblast proliferation; however, inactivation of mTORC1 is essential for their differentiation and maturation. Inhibition of mTORC1 prevented preosteoblast proliferation, but enhanced their differentiation in vitro and in mice. Activation of mTORC1 by deletion of tuberous sclerosis 1 (Tsc1) in preosteoblasts produced immature woven bone in mice due to excess proliferation but impaired differentiation and maturation of the cells. The mTORC1-specific inhibitor, rapamycin, restored these in vitro and in vivo phenotypic changes. Mechanistically, mTORC1 prevented osteoblast maturation through activation of the STAT3/p63/Jagged/Notch pathway and downregulation of Runx2. Preosteoblasts with hyperactive mTORC1 reacquired the capacity to fully differentiate and maturate when subjected to inhibition of the Notch pathway. Together, these findings identified the role of mTORC1 in osteoblast formation and established that mTORC1 prevents preosteoblast differentiation and maturation through activation of the Notch pathway.

No MeSH data available.


Related in: MedlinePlus