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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

mTORC1 is activated during preosteoblast proliferation but is suppressed during their differentiation.(A) Growth curve of proliferating MC3T3-E1 cells treated with vehicle (Veh) or rapamycin (0.1nM). (B) Western blot analysis of proliferative markers (cyclin D1 and PCNA), Osx and mTORC1 activity (indicated by P-S6K (T389) and P-S6 (S235/236)) during MC3T3-E1 proliferation. (C) Western blot analysis of osteoblastic markers (Osx and Ocn) and mTORC1 activity during differentiation of MC3T3-E1 cells. (D) Western blot analysis of osteoblastic markers (Osx and Ocn) and mTORC1 activity during differentiation of FRC. Osx: osterix; Ocn: osteocalcin; FRC: fetal rat calvarial cells, OGI: osteogenic induction.
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pgen.1005426.g001: mTORC1 is activated during preosteoblast proliferation but is suppressed during their differentiation.(A) Growth curve of proliferating MC3T3-E1 cells treated with vehicle (Veh) or rapamycin (0.1nM). (B) Western blot analysis of proliferative markers (cyclin D1 and PCNA), Osx and mTORC1 activity (indicated by P-S6K (T389) and P-S6 (S235/236)) during MC3T3-E1 proliferation. (C) Western blot analysis of osteoblastic markers (Osx and Ocn) and mTORC1 activity during differentiation of MC3T3-E1 cells. (D) Western blot analysis of osteoblastic markers (Osx and Ocn) and mTORC1 activity during differentiation of FRC. Osx: osterix; Ocn: osteocalcin; FRC: fetal rat calvarial cells, OGI: osteogenic induction.

Mentions: mTORC1 is known to promote cell proliferation in many types of cells. To examine the relationship between preosteoblast proliferation and mTORC1 activity, we analyzed the level of mTORC1 during the proliferative expansion of MC3T3-E1 cells, a murine preosteoblast cell line [31], and subsequent cessation of growth. We counted cells each day to monitor their growth and observed that the cells reached confluence after 6–7 days, when proliferation ceased due to contact inhibition (Fig 1A). Western blot analysis reflected this growth inhibition as a decrease in the levels of the cell cycle markers, cyclin D1 and proliferative cell nuclear antigen (PCNA). As expected, the high level of mTORC1 activity (indicated by P-S6K (T389) and P-S6 (S235/236)) decreased when proliferation of MC3T3-E1 cells slowed down and eventually ceased (Fig 1B). Cells did not differentiate during this growth period, as the level of osterix (Osx, a marker of early osteoblasts) was unchanged throughout. These data indicate that the level of mTORC1 activity was positively correlated with the rate of preosteoblast proliferation.


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 is activated during preosteoblast proliferation but is suppressed during their differentiation.(A) Growth curve of proliferating MC3T3-E1 cells treated with vehicle (Veh) or rapamycin (0.1nM). (B) Western blot analysis of proliferative markers (cyclin D1 and PCNA), Osx and mTORC1 activity (indicated by P-S6K (T389) and P-S6 (S235/236)) during MC3T3-E1 proliferation. (C) Western blot analysis of osteoblastic markers (Osx and Ocn) and mTORC1 activity during differentiation of MC3T3-E1 cells. (D) Western blot analysis of osteoblastic markers (Osx and Ocn) and mTORC1 activity during differentiation of FRC. Osx: osterix; Ocn: osteocalcin; FRC: fetal rat calvarial cells, OGI: osteogenic induction.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005426.g001: mTORC1 is activated during preosteoblast proliferation but is suppressed during their differentiation.(A) Growth curve of proliferating MC3T3-E1 cells treated with vehicle (Veh) or rapamycin (0.1nM). (B) Western blot analysis of proliferative markers (cyclin D1 and PCNA), Osx and mTORC1 activity (indicated by P-S6K (T389) and P-S6 (S235/236)) during MC3T3-E1 proliferation. (C) Western blot analysis of osteoblastic markers (Osx and Ocn) and mTORC1 activity during differentiation of MC3T3-E1 cells. (D) Western blot analysis of osteoblastic markers (Osx and Ocn) and mTORC1 activity during differentiation of FRC. Osx: osterix; Ocn: osteocalcin; FRC: fetal rat calvarial cells, OGI: osteogenic induction.
Mentions: mTORC1 is known to promote cell proliferation in many types of cells. To examine the relationship between preosteoblast proliferation and mTORC1 activity, we analyzed the level of mTORC1 during the proliferative expansion of MC3T3-E1 cells, a murine preosteoblast cell line [31], and subsequent cessation of growth. We counted cells each day to monitor their growth and observed that the cells reached confluence after 6–7 days, when proliferation ceased due to contact inhibition (Fig 1A). Western blot analysis reflected this growth inhibition as a decrease in the levels of the cell cycle markers, cyclin D1 and proliferative cell nuclear antigen (PCNA). As expected, the high level of mTORC1 activity (indicated by P-S6K (T389) and P-S6 (S235/236)) decreased when proliferation of MC3T3-E1 cells slowed down and eventually ceased (Fig 1B). Cells did not differentiate during this growth period, as the level of osterix (Osx, a marker of early osteoblasts) was unchanged throughout. These data indicate that the level of mTORC1 activity was positively correlated with the rate of preosteoblast proliferation.

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