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 impairs osteoblast differentiation through the Notch pathway upstream of Runx2.(A) Western blot analysis of the expression of Jagged1, NICD and Hes1 during differentiation of MC3T3-E1 cells. Differentiating MC3T3-E1 cells were treated with Notch1 siRNA (S) and then underwent immunoblotting to detect osteocalcin (B) on the 7th and 14th day and alizarin red staining (C) on the 14th day. Differentiating control (C) and ΔTsc1 (Δ) primary calvarial cells were treated with negative control (N) or si-Notch1 (S) and then underwent immunoblotting to detect osteocalcin (D) and alizarin red staining (E) on the 14th day. 6-week old ΔTsc1 mice were administered with DAPT (10mg/kg per day) or equivalent volume of DMSO for 4 weeks, micro-CT images of trabecular bone in secondary ossification center (F) and cortical bone of midshaft (G) of femur were shown. Immunohistochemistry staining for Runx2 (H), osterix (Osx) (J), and osteocalcin (Ocn) (L) in distal femur. Numbers of Runx2 (N.Runx2+) (I), osterix (N.Osx+) (K) and osteocalcin (N.Ocn+) (M) positive cells on the bone surface were measured as cells per millimeter of perimeter in sections (/B.Pm). All data are mean ± SD (n = 5 mice), all scale bars represent 50 μm. *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.g008: mTORC1 impairs osteoblast differentiation through the Notch pathway upstream of Runx2.(A) Western blot analysis of the expression of Jagged1, NICD and Hes1 during differentiation of MC3T3-E1 cells. Differentiating MC3T3-E1 cells were treated with Notch1 siRNA (S) and then underwent immunoblotting to detect osteocalcin (B) on the 7th and 14th day and alizarin red staining (C) on the 14th day. Differentiating control (C) and ΔTsc1 (Δ) primary calvarial cells were treated with negative control (N) or si-Notch1 (S) and then underwent immunoblotting to detect osteocalcin (D) and alizarin red staining (E) on the 14th day. 6-week old ΔTsc1 mice were administered with DAPT (10mg/kg per day) or equivalent volume of DMSO for 4 weeks, micro-CT images of trabecular bone in secondary ossification center (F) and cortical bone of midshaft (G) of femur were shown. Immunohistochemistry staining for Runx2 (H), osterix (Osx) (J), and osteocalcin (Ocn) (L) in distal femur. Numbers of Runx2 (N.Runx2+) (I), osterix (N.Osx+) (K) and osteocalcin (N.Ocn+) (M) positive cells on the bone surface were measured as cells per millimeter of perimeter in sections (/B.Pm). All data are mean ± SD (n = 5 mice), all scale bars represent 50 μm. *P < 0.05, ** P < 0.01, *** P < 0.001 by t test.

Mentions: As mTOR is a positive regulator of the Jagged1/Notch/Hes1 pathway, we next determined whether hyperactive Notch signaling was responsible for the impaired differentiation of preosteoblasts by mTORC1 activation. Firstly, we examined the correlation between the level of Notch activity and differentiation of preosteoblasts. Low activity of Notch signaling is required during differentiation of preosteoblasts, as the expression of Jagged1, NICD and Hes1 was decreased in parallel with an increase in the markers of osteoblast differentiation (Fig 8A), and inhibition of the Notch pathway by DAPT or si-Notch1 promoted osteocalcin expression (S7A Fig and Fig 8B) and mineralized nodules formation in MC3T3-E1 cells (S7B Fig and Fig 8C). Importantly, a reduction in Notch by DAPT (S7C and S7D Fig) or siRNA (Fig 8D and 8E) potentiated the differentiation of ΔTsc1 preosteoblasts.


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 impairs osteoblast differentiation through the Notch pathway upstream of Runx2.(A) Western blot analysis of the expression of Jagged1, NICD and Hes1 during differentiation of MC3T3-E1 cells. Differentiating MC3T3-E1 cells were treated with Notch1 siRNA (S) and then underwent immunoblotting to detect osteocalcin (B) on the 7th and 14th day and alizarin red staining (C) on the 14th day. Differentiating control (C) and ΔTsc1 (Δ) primary calvarial cells were treated with negative control (N) or si-Notch1 (S) and then underwent immunoblotting to detect osteocalcin (D) and alizarin red staining (E) on the 14th day. 6-week old ΔTsc1 mice were administered with DAPT (10mg/kg per day) or equivalent volume of DMSO for 4 weeks, micro-CT images of trabecular bone in secondary ossification center (F) and cortical bone of midshaft (G) of femur were shown. Immunohistochemistry staining for Runx2 (H), osterix (Osx) (J), and osteocalcin (Ocn) (L) in distal femur. Numbers of Runx2 (N.Runx2+) (I), osterix (N.Osx+) (K) and osteocalcin (N.Ocn+) (M) positive cells on the bone surface were measured as cells per millimeter of perimeter in sections (/B.Pm). All data are mean ± SD (n = 5 mice), all scale bars represent 50 μm. *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.g008: mTORC1 impairs osteoblast differentiation through the Notch pathway upstream of Runx2.(A) Western blot analysis of the expression of Jagged1, NICD and Hes1 during differentiation of MC3T3-E1 cells. Differentiating MC3T3-E1 cells were treated with Notch1 siRNA (S) and then underwent immunoblotting to detect osteocalcin (B) on the 7th and 14th day and alizarin red staining (C) on the 14th day. Differentiating control (C) and ΔTsc1 (Δ) primary calvarial cells were treated with negative control (N) or si-Notch1 (S) and then underwent immunoblotting to detect osteocalcin (D) and alizarin red staining (E) on the 14th day. 6-week old ΔTsc1 mice were administered with DAPT (10mg/kg per day) or equivalent volume of DMSO for 4 weeks, micro-CT images of trabecular bone in secondary ossification center (F) and cortical bone of midshaft (G) of femur were shown. Immunohistochemistry staining for Runx2 (H), osterix (Osx) (J), and osteocalcin (Ocn) (L) in distal femur. Numbers of Runx2 (N.Runx2+) (I), osterix (N.Osx+) (K) and osteocalcin (N.Ocn+) (M) positive cells on the bone surface were measured as cells per millimeter of perimeter in sections (/B.Pm). All data are mean ± SD (n = 5 mice), all scale bars represent 50 μm. *P < 0.05, ** P < 0.01, *** P < 0.001 by t test.
Mentions: As mTOR is a positive regulator of the Jagged1/Notch/Hes1 pathway, we next determined whether hyperactive Notch signaling was responsible for the impaired differentiation of preosteoblasts by mTORC1 activation. Firstly, we examined the correlation between the level of Notch activity and differentiation of preosteoblasts. Low activity of Notch signaling is required during differentiation of preosteoblasts, as the expression of Jagged1, NICD and Hes1 was decreased in parallel with an increase in the markers of osteoblast differentiation (Fig 8A), and inhibition of the Notch pathway by DAPT or si-Notch1 promoted osteocalcin expression (S7A Fig and Fig 8B) and mineralized nodules formation in MC3T3-E1 cells (S7B Fig and Fig 8C). Importantly, a reduction in Notch by DAPT (S7C and S7D Fig) or siRNA (Fig 8D and 8E) potentiated the differentiation of ΔTsc1 preosteoblasts.

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