title: DNA-PKcs-SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation.
abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1Gy) induced phosphorylation of DNA-PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA-PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA-PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA-PKcs-SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA-PKcs-SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.
authors.affiliation: The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Xu
authors.affiliation: The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000, China.
authors.forename: Shi-Ji
authors.initials: SJ
authors.lastname: Fang
authors.affiliation: Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and Institute of Neuroscience, Soochow University, Suzhou, Jiangsu 215021, China.
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Zhu
authors.affiliation: The Center of Diagnosis and Treatment for Children's Bone Diseases, The Children's Hospital Affiliated to Soochow University, Suzhou, Jiangsu 215000, China. Electronic address: xiaodongwangsz@163.com.
authors.forename: Lun-Qing
authors.initials: LQ
authors.lastname: Zhu
authors.affiliation: The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000, China. Electronic address: zhouxz@suda.edu.cn.
authors.forename: Xiao-Zhong
authors.initials: XZ
authors.lastname: Zhou
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002454:Cell Differentiation / Q000528:radiation effects*; D002478:Cells, Cultured; D017931:DNA Primers; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D010006:Osteoblasts / Q000201:enzymology / Q000378:metabolism / Q000528:radiation effects*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D048868:Adaptor Proteins, Signal Transducing
chemical_list: D000469:Alkaline Phosphatase; D002352:Carrier Proteins; D017931:DNA Primers; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051057:Proto-Oncogene Proteins c-akt; C561937:Mapkap1 protein, mouse; D048868:Adaptor Proteins, Signal Transducing; C497264:Prkdc protein, mouse
keywords: DNA–PKcs; Low-dose irradiation (LDI); Osteoblast differentiation; mTORC2–SIN1 and Akt