Decreased BMP2 signal in GIT1 knockout mice slows bone healing

• Published in: Molecular and cellular biochemistry 2014-12, Vol.397 (1-2), p.67-74
• Main Authors: Sheu, T. J., Zhou, Wei, Fan, Jin, Zhou, Hao, Zuscik, Michael J., Xie, Chao, Yin, Guoyong, Berk, Bradford C.
• Format: Article
• Language: English
• Subjects: Analysis; Animals; Biochemistry; Biomedical and Life Sciences; Bone density; Bone marrow; Bone Morphogenetic Protein 2 - genetics; Bone Morphogenetic Protein 2 - metabolism; Bone Regeneration - physiology; Cardiology; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Nucleus - genetics; Cell Nucleus - metabolism; Core Binding Factor Alpha 1 Subunit - genetics; Core Binding Factor Alpha 1 Subunit - metabolism; Fractures; Growth factors; GTPase-Activating Proteins - genetics; GTPase-Activating Proteins - metabolism; Life Sciences; Medical Biochemistry; Mice; Mice, Knockout; Mineralization; Oncology; Phosphorylation; Proteins; Signal Transduction - physiology; Smad Proteins - genetics; Smad Proteins - metabolism; Stem cells; Transforming Growth Factor beta1 - genetics; Transforming Growth Factor beta1 - metabolism
• ISSN: 0300-8177; 1573-4919
• DOI: 10.1007/s11010-014-2173-5

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.