Limits...
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

mTORC1 promotes proliferation of preosteoblasts but prevents their maturation.(A) ALP staining in sections of distal femur from 10-week-old control (C), ΔTsc1 (Δ) and rapamycin treated ΔTsc1 (Δ+R) mice. (B) Number of ALP positive cells per bone perimeter (mm-1) (N.ALP+/B.Pm). Immunohistochemistry staining for (C) BrdU, (E) osterix (Osx) and (G) osteocalcin (Ocn) in distal femur. (D) Percentage of BrdU+ osteoblasts out of total osteoblasts on bone surface. (F, H) Number of osterix positive cells (N.Osx+) and osteocalcin positive (N.Ocn+) on the bone surface was measured as cells per millimeter of perimeter in sections (/B.Pm) and the mean density of the corresponding positive cells was calculated as integrated optical density (IOD) per area of positive cells. (I) Calcin double labeling of cortical bone in distal femora. Boxed area is enlarged in the panel below. Ma.: marrow, Endo.: endosteum, Peri.: periosteum. (J) Mineral apposition rate (MAR). (K) Western blot analysis of differentiating primary calvarial preosteoblasts showed decreased expression of osterix and osteocalcin following doxycycline discontinuation in ΔTsc1 cells (0, 7th, 14th day). (L) Alizarin red staining of differentiated primary calvarial preosteoblasts on the 14th day showed decreased mineralized nodules in ΔTsc1 cells. All data are mean ± SD (n = 5 mice), scale bars represent 50 μm for (A), (C), (E), (G) and 100 μm for (I). *P < 0.05, ** P < 0.01, ***P<0.001 by t test.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4524707&req=5

pgen.1005426.g004: mTORC1 promotes proliferation of preosteoblasts but prevents their maturation.(A) ALP staining in sections of distal femur from 10-week-old control (C), ΔTsc1 (Δ) and rapamycin treated ΔTsc1 (Δ+R) mice. (B) Number of ALP positive cells per bone perimeter (mm-1) (N.ALP+/B.Pm). Immunohistochemistry staining for (C) BrdU, (E) osterix (Osx) and (G) osteocalcin (Ocn) in distal femur. (D) Percentage of BrdU+ osteoblasts out of total osteoblasts on bone surface. (F, H) Number of osterix positive cells (N.Osx+) and osteocalcin positive (N.Ocn+) on the bone surface was measured as cells per millimeter of perimeter in sections (/B.Pm) and the mean density of the corresponding positive cells was calculated as integrated optical density (IOD) per area of positive cells. (I) Calcin double labeling of cortical bone in distal femora. Boxed area is enlarged in the panel below. Ma.: marrow, Endo.: endosteum, Peri.: periosteum. (J) Mineral apposition rate (MAR). (K) Western blot analysis of differentiating primary calvarial preosteoblasts showed decreased expression of osterix and osteocalcin following doxycycline discontinuation in ΔTsc1 cells (0, 7th, 14th day). (L) Alizarin red staining of differentiated primary calvarial preosteoblasts on the 14th day showed decreased mineralized nodules in ΔTsc1 cells. All data are mean ± SD (n = 5 mice), scale bars represent 50 μm for (A), (C), (E), (G) and 100 μm for (I). *P < 0.05, ** P < 0.01, ***P<0.001 by t test.

Mentions: We then analyzed the cellular basis for the increased amounts of immature woven bone in ΔTsc1 mice. ALP staining of 10-week-old ΔTsc1 mouse femurs indicated an increased number of osteoblast lineage cells (Fig 4A and 4B). To determine the underlying molecular mechanism of the increase in osteoblasts in transgenic bone, we cultured postnatal day 3 calvarial osteoblasts and found a significant increase in BrdU-positive cells, indicating increased cellular proliferation in ΔTsc1 calvarial osteoblasts (S4 Fig). In addition, the percentage of proliferative osteoblasts on bone surface was increased in ΔTsc1 mice (Fig 4C and 4D).


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)

mTORC1 promotes proliferation of preosteoblasts but prevents their maturation.(A) ALP staining in sections of distal femur from 10-week-old control (C), ΔTsc1 (Δ) and rapamycin treated ΔTsc1 (Δ+R) mice. (B) Number of ALP positive cells per bone perimeter (mm-1) (N.ALP+/B.Pm). Immunohistochemistry staining for (C) BrdU, (E) osterix (Osx) and (G) osteocalcin (Ocn) in distal femur. (D) Percentage of BrdU+ osteoblasts out of total osteoblasts on bone surface. (F, H) Number of osterix positive cells (N.Osx+) and osteocalcin positive (N.Ocn+) on the bone surface was measured as cells per millimeter of perimeter in sections (/B.Pm) and the mean density of the corresponding positive cells was calculated as integrated optical density (IOD) per area of positive cells. (I) Calcin double labeling of cortical bone in distal femora. Boxed area is enlarged in the panel below. Ma.: marrow, Endo.: endosteum, Peri.: periosteum. (J) Mineral apposition rate (MAR). (K) Western blot analysis of differentiating primary calvarial preosteoblasts showed decreased expression of osterix and osteocalcin following doxycycline discontinuation in ΔTsc1 cells (0, 7th, 14th day). (L) Alizarin red staining of differentiated primary calvarial preosteoblasts on the 14th day showed decreased mineralized nodules in ΔTsc1 cells. All data are mean ± SD (n = 5 mice), scale bars represent 50 μm for (A), (C), (E), (G) and 100 μm for (I). *P < 0.05, ** P < 0.01, ***P<0.001 by t test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4524707&req=5

pgen.1005426.g004: mTORC1 promotes proliferation of preosteoblasts but prevents their maturation.(A) ALP staining in sections of distal femur from 10-week-old control (C), ΔTsc1 (Δ) and rapamycin treated ΔTsc1 (Δ+R) mice. (B) Number of ALP positive cells per bone perimeter (mm-1) (N.ALP+/B.Pm). Immunohistochemistry staining for (C) BrdU, (E) osterix (Osx) and (G) osteocalcin (Ocn) in distal femur. (D) Percentage of BrdU+ osteoblasts out of total osteoblasts on bone surface. (F, H) Number of osterix positive cells (N.Osx+) and osteocalcin positive (N.Ocn+) on the bone surface was measured as cells per millimeter of perimeter in sections (/B.Pm) and the mean density of the corresponding positive cells was calculated as integrated optical density (IOD) per area of positive cells. (I) Calcin double labeling of cortical bone in distal femora. Boxed area is enlarged in the panel below. Ma.: marrow, Endo.: endosteum, Peri.: periosteum. (J) Mineral apposition rate (MAR). (K) Western blot analysis of differentiating primary calvarial preosteoblasts showed decreased expression of osterix and osteocalcin following doxycycline discontinuation in ΔTsc1 cells (0, 7th, 14th day). (L) Alizarin red staining of differentiated primary calvarial preosteoblasts on the 14th day showed decreased mineralized nodules in ΔTsc1 cells. All data are mean ± SD (n = 5 mice), scale bars represent 50 μm for (A), (C), (E), (G) and 100 μm for (I). *P < 0.05, ** P < 0.01, ***P<0.001 by t test.
Mentions: We then analyzed the cellular basis for the increased amounts of immature woven bone in ΔTsc1 mice. ALP staining of 10-week-old ΔTsc1 mouse femurs indicated an increased number of osteoblast lineage cells (Fig 4A and 4B). To determine the underlying molecular mechanism of the increase in osteoblasts in transgenic bone, we cultured postnatal day 3 calvarial osteoblasts and found a significant increase in BrdU-positive cells, indicating increased cellular proliferation in ΔTsc1 calvarial osteoblasts (S4 Fig). In addition, the percentage of proliferative osteoblasts on bone surface was increased in ΔTsc1 mice (Fig 4C and 4D).

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