Osteoblast differentiation and bone formation are controlled by a wide range of extracellular signals including cell-extracellular matrix interaction, matrix stiffness, biomechanical force, hormones, growth factors and morphogens. To induce differentiation, each stimulus must initiate a signal that can be efficiently dispersed through the genome to induce global changes in gene expression necessary for the osteoblastogenesis while simultaneously suppressing other lineages. The following hypothesis is proposed to explain how this is accomplished: i) After exposure to an osteogenic stimulus, P-ERK1/2 translocates to the nucleus and binds chromatin of osteoblast target genes via specific docking sites on Runx2. ii) By phosphorylating RUNX2 on specific sites, kinases initiate chromatin remodeling and transcription;the overall level of RUNX2 phosphorylation is a reflection of the various extracellular stimuli to which the cell is exposed. iii) Conversely, kinases may phosphorylate other lineage-specific transcription factors on chromatin to suppress non-osteogenic lineages such as adipocytes. This hypothesis will be tested by achieving the following aims: 1. Evaluate the role of MAP kinase phosphorylation of RUNX2 and PPAR? in the reciprocal control of osteogenesis and adipogenesis. 2. Evaluate the role of RUNX2 phosphorylation by ERK1/2 in the control of fluid flow shear stress (FFSS)-induced osteoblast gene expression. 3. Determine the role of Runx2 S301,S319 phosphorylation in skeletal development and remodeling. Experiments include detailed epigenetic analyses of chromatin changes accompanying osteoblast versus adipocyte differentiation as well as genomic analysis using chromatin immunoprecipitation and next generation sequencing. These studies will test a new model for global control of gene expression that may explain how osteogenic stimuli, including extracellular matrix signals and mechanical loading, can reprogram the genome to stimulate osteoblast differentiation while suppressing other lineages such as adipocytes. Once this model is understood, it will be possible to manipulate osteogenic signals to optimize bone formation during remodeling and regeneration. Furthermore, this work will test the validity of using RUNX2 phosphorylation-site specific antibodies to monitor activation of this factor under different physiological conditions. This may have applications for diagnosis of osteoporosis and other bone disorders, such as osteoarthritis and certain cancers.

Public Health Relevance

To successfully treat diseases like osteoporosis and osteoarthritis as well as regenerate bone, it is essential that we understand basic mechanisms controlling bone formation. This project will test a new hypothesis to explain how genes necessary for formation of osteoblasts, the bone-forming cells, become activated by anabolic signals such as mechanical loading and hormone/growth factor treatment. The central concept to be explored is that anabolic signals activate nuclear protein kinases, enzymes that add a phosphate group to transcription factors to either stimulate or inhibit their activity. Transcriptin factors subsequently regulate the activity of specific genes. We propose that protein kinases stimulate transcription factors necessary for osteoblast formation while inhibiting factors involved in fat cell formation. In this way, bone formation is favored. If correct, our hypothesis provides a new route for stimulating bone formation through pharmacological manipulation of protein kinase activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
3R01DE011723-15A1S1
Application #
8723410
Study Section
Skeletal Biology Structure and Regeneration Study Section (SBSR)
Program Officer
Wan, Jason
Project Start
1995-09-01
Project End
2017-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
15
Fiscal Year
2013
Total Cost
$24,841
Indirect Cost
$8,866
Name
University of Michigan Ann Arbor
Department
Dentistry
Type
Schools of Dentistry
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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Phimphilai, Mattabhorn; Zhao, Zhouran; Boules, Heidi et al. (2006) BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype. J Bone Miner Res 21:637-46
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Yang, Shuying; Wei, Daoyan; Wang, Dian et al. (2003) In vitro and in vivo synergistic interactions between the Runx2/Cbfa1 transcription factor and bone morphogenetic protein-2 in stimulating osteoblast differentiation. J Bone Miner Res 18:705-15
Franceschi, Renny T; Xiao, Guozhi (2003) Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways. J Cell Biochem 88:446-54

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