title: Decreased BMP2 signal in GIT1 knockout mice slows bone healing.
abstract: Endochondral ossification, an important stage of fracture healing, is regulated by a variety of signaling pathways. Transforming growth factor β (TGFβ) superfamily plays important roles and comprises TGFβs, bone morphogenetic proteins (BMPs), and growth differentiation factors. TGFβs primarily regulate cartilage formation and endochondral ossification. BMP2 shows diverse efficacy, from the formation of skeleton and extraskeletal organs to the osteogenesis and remodeling of bone. G-protein-coupled receptor kinase 2-interacting protein-1 (GIT1), a shuttle protein in osteoblasts, facilitates fracture healing by promoting bone formation and increasing the secretion of vascular endothelial growth factor. Our study examined whether GIT1 regulates fracture healing through the BMP2 signaling pathway and/or through the TGFβ signaling pathway. GIT1 knockout (KO) mice exhibited delayed fracture healing, chondrocyte accumulation in the fracture area, and reduced staining intensity of phosphorylated Smad1/5/8 (pSmad1/5/8) and Runx2. Endochondral mineralization diminished while the staining intensity of phosphorylated Smad2/3 (pSmad2/3) showed no significant change. Bone marrow mesenchymal stem cells extracted from GIT1 KO mice showed a decline of pSmad1/5/8 levels and of pSmad1/5/8 translocated into the cell nucleus after BMP2 stimulus. We detected no significant change in the pSmad2/3 level after TGFβ1 stimulus. Data obtained from reporter gene analysis of C3H10T1/2 cells cultured in vitro confirmed these findings. GIT1-siRNA inhibited transcription in the cell nucleus via pSmad1/5/8 after BMP2 stimulus but had no significant effect on transcription via pSmad2/3 after TGFβ1 stimulus. Our results indicate that GIT1 regulates Smad1/5/8 phosphorylation and mediates BMP2 regulation of Runx2 expression, thus affecting endochondral ossification at the fracture site.
authors.affiliation: Center for Musculoskeletal Research, University of Rochester, Rochester, NY 14642, USA.
authors.forename: T J
authors.initials: TJ
authors.lastname: Sheu
authors.affiliation: Orthopaedic Department, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, Jiangsu Province, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhou
authors.affiliation: Orthopaedic Department, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, Jiangsu Province, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Fan
authors.affiliation: Orthopaedic Department, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, Jiangsu Province, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Center for Musculoskeletal Research, University of Rochester, Rochester, NY 14642, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Zuscik
authors.affiliation: Center for Musculoskeletal Research, University of Rochester, Rochester, NY 14642, USA.
authors.forename: Chao
authors.initials: C
authors.lastname: Xie
authors.affiliation: Orthopaedic Department, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, Jiangsu Province, China.
authors.forename: Guoyong
authors.initials: G
authors.lastname: Yin
authors.affiliation: Aab Cardiovascular Research Institute and Department of Medicine, University of Rochester, 601 Elmwood Ave, Box CVRI, Rochester, NY 14642, USA.
authors.forename: Bradford C
authors.initials: BC
authors.lastname: Berk
mesh_terms: D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism*; D001861:Bone Regeneration / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D015398:Signal Transduction / Q000502:physiology*; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism
chemical_list: C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D018797:Cell Cycle Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D020690:GTPase-Activating Proteins; C120766:Git1 protein, mouse; C493716:Runx2 protein, mouse; D051785:Smad Proteins; C508961:Tgfb1 protein, mouse; D053773:Transforming Growth Factor beta1