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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 activation in preosteoblasts produces immature woven bone.(A) PCR analysis of Tsc1 allele recombination in tissues from Osx-GFP::CreTG/+;Tsc1flox/flox mice (ΔTsc1 mice). Primers for GAPDH were used as a loading control. (B) Immunohistochemical staining of S6 phosphorylation (Ser235/236) in sections of distal femur from 10-week-old control and ΔTsc1 mice. (C) The 10-week-old ΔTsc1 mouse had a square skull (the result of hyperplasia of the osteoid, a typical manifestation of rickets) compared with the control littermate. (D) Plain X-ray examination of a 4-week-old ΔTsc1 mouse revealed a smaller skeleton and higher bone mass compared with the control littermate. (E) Body weight of 4-week-old control and ΔTsc1 mice. (F) Representative images of micro-CT analyses of the structure of metaphyseal trabecular bone and cortical bone in the distal femur showed increased bone mass, but more porous areas (indicating more hypomineralized areas) in ΔTsc1 mice. (G, H) Goldner’s Masson trichrome staining of distal femur showed more osteoid/hypomineralized areas (stained red) in bone of ΔTsc1 mice. Boxed area is enlarged in the panel below. The dotted line marked boundary of the red osteoid. OS/BS, osteoid per bone surface. Scale bar, 100μm.
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pgen.1005426.g003: mTORC1 activation in preosteoblasts produces immature woven bone.(A) PCR analysis of Tsc1 allele recombination in tissues from Osx-GFP::CreTG/+;Tsc1flox/flox mice (ΔTsc1 mice). Primers for GAPDH were used as a loading control. (B) Immunohistochemical staining of S6 phosphorylation (Ser235/236) in sections of distal femur from 10-week-old control and ΔTsc1 mice. (C) The 10-week-old ΔTsc1 mouse had a square skull (the result of hyperplasia of the osteoid, a typical manifestation of rickets) compared with the control littermate. (D) Plain X-ray examination of a 4-week-old ΔTsc1 mouse revealed a smaller skeleton and higher bone mass compared with the control littermate. (E) Body weight of 4-week-old control and ΔTsc1 mice. (F) Representative images of micro-CT analyses of the structure of metaphyseal trabecular bone and cortical bone in the distal femur showed increased bone mass, but more porous areas (indicating more hypomineralized areas) in ΔTsc1 mice. (G, H) Goldner’s Masson trichrome staining of distal femur showed more osteoid/hypomineralized areas (stained red) in bone of ΔTsc1 mice. Boxed area is enlarged in the panel below. The dotted line marked boundary of the red osteoid. OS/BS, osteoid per bone surface. Scale bar, 100μm.

Mentions: To characterize the role of mTORC1 activation in the regulation of proliferation and differentiation of osteogenic progenitors, we generated mice in which mTORC1 were selectively activated in osteoprogenitor cells committed to the osteoblast lineage. To achieve such cell type-specific knockout, we crossed floxed Tsc1 mice with Osx-GFP::Cre mice (which express a GFP-Cre fusion protein under the direction of the Osx1 promoter) to generate conditional Tsc1 knockout mice. We mated Osx-GFP::CreTG/+;Tsc1flox/+ mice and selected female mice with the genotype Osx-GFP::CreTG/+;Tsc1flox/flox (hereafter, referred to as ΔTsc1) for detailed analysis. Female Osx-GFP::CreTG/+;Tsc1+/+ littermates served as controls. ΔTsc1 mice were born at the expected Mendelian frequency, and recombination of Tsc1 alleles only occurred in skeletal tissues (i.e., legs and skull) as demonstrated by allele specific PCR (Fig 3A). Immunohistochemical staining of distal femur sections showed a dramatic increase in S6 phosphorylation (Ser235/236) in ΔTsc1 mice (Fig 3B), indicating that mTORC1 was activated by Tsc1 disruption.


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 activation in preosteoblasts produces immature woven bone.(A) PCR analysis of Tsc1 allele recombination in tissues from Osx-GFP::CreTG/+;Tsc1flox/flox mice (ΔTsc1 mice). Primers for GAPDH were used as a loading control. (B) Immunohistochemical staining of S6 phosphorylation (Ser235/236) in sections of distal femur from 10-week-old control and ΔTsc1 mice. (C) The 10-week-old ΔTsc1 mouse had a square skull (the result of hyperplasia of the osteoid, a typical manifestation of rickets) compared with the control littermate. (D) Plain X-ray examination of a 4-week-old ΔTsc1 mouse revealed a smaller skeleton and higher bone mass compared with the control littermate. (E) Body weight of 4-week-old control and ΔTsc1 mice. (F) Representative images of micro-CT analyses of the structure of metaphyseal trabecular bone and cortical bone in the distal femur showed increased bone mass, but more porous areas (indicating more hypomineralized areas) in ΔTsc1 mice. (G, H) Goldner’s Masson trichrome staining of distal femur showed more osteoid/hypomineralized areas (stained red) in bone of ΔTsc1 mice. Boxed area is enlarged in the panel below. The dotted line marked boundary of the red osteoid. OS/BS, osteoid per bone surface. Scale bar, 100μm.
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pgen.1005426.g003: mTORC1 activation in preosteoblasts produces immature woven bone.(A) PCR analysis of Tsc1 allele recombination in tissues from Osx-GFP::CreTG/+;Tsc1flox/flox mice (ΔTsc1 mice). Primers for GAPDH were used as a loading control. (B) Immunohistochemical staining of S6 phosphorylation (Ser235/236) in sections of distal femur from 10-week-old control and ΔTsc1 mice. (C) The 10-week-old ΔTsc1 mouse had a square skull (the result of hyperplasia of the osteoid, a typical manifestation of rickets) compared with the control littermate. (D) Plain X-ray examination of a 4-week-old ΔTsc1 mouse revealed a smaller skeleton and higher bone mass compared with the control littermate. (E) Body weight of 4-week-old control and ΔTsc1 mice. (F) Representative images of micro-CT analyses of the structure of metaphyseal trabecular bone and cortical bone in the distal femur showed increased bone mass, but more porous areas (indicating more hypomineralized areas) in ΔTsc1 mice. (G, H) Goldner’s Masson trichrome staining of distal femur showed more osteoid/hypomineralized areas (stained red) in bone of ΔTsc1 mice. Boxed area is enlarged in the panel below. The dotted line marked boundary of the red osteoid. OS/BS, osteoid per bone surface. Scale bar, 100μm.
Mentions: To characterize the role of mTORC1 activation in the regulation of proliferation and differentiation of osteogenic progenitors, we generated mice in which mTORC1 were selectively activated in osteoprogenitor cells committed to the osteoblast lineage. To achieve such cell type-specific knockout, we crossed floxed Tsc1 mice with Osx-GFP::Cre mice (which express a GFP-Cre fusion protein under the direction of the Osx1 promoter) to generate conditional Tsc1 knockout mice. We mated Osx-GFP::CreTG/+;Tsc1flox/+ mice and selected female mice with the genotype Osx-GFP::CreTG/+;Tsc1flox/flox (hereafter, referred to as ΔTsc1) for detailed analysis. Female Osx-GFP::CreTG/+;Tsc1+/+ littermates served as controls. ΔTsc1 mice were born at the expected Mendelian frequency, and recombination of Tsc1 alleles only occurred in skeletal tissues (i.e., legs and skull) as demonstrated by allele specific PCR (Fig 3A). Immunohistochemical staining of distal femur sections showed a dramatic increase in S6 phosphorylation (Ser235/236) in ΔTsc1 mice (Fig 3B), indicating that mTORC1 was activated by Tsc1 disruption.

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