This application is guided by two questions: 1) how is OB-specific transcription achieved? What DNA elements and transcription factors combine to direct expression of a gene on osteoblasts (Obs), but not in other cell types?; and 2) how do osteogenic anabolic signals (PTH/cAMP, IGFs, FGFs, BMPs), acting through kinase cascades, regulate the OB-specific transcriptional machinery during development, bone growth and fracture repair? The two Specific Aims of this proposal are: 1) to understand how Msx2 regulates the osteoblast-specific transcriptional machinery, using the rat OC promoter as a model. Msx2 recognizes and regulates the rat OC promoter via the Hoxbox-1 element, presenting the unique opportunity to examine interactions between a homeodomain transcription factor and an osteoblast-specific promoter. By systematic mutation of Msx2 and co-transfection assays with rat promoter-luciferase reporter in cell lines, structure-function analyses will be performed. Included will be a homeodomain mutation described by others in the human MSX2 gene as responsible for craniosynostosis, Boston type. It is further proposed to purify and characterize positively acting factors, supporting OC promoter activity, using classical biochemical techniques. Nuclear factors that were recently defined as recognizing sequences in the adjacent, interacting FGF2/cAMP response elements will be emphasized. Protein-protein interactions necessary for Msx2 action will be characterized with epitope-tagged recombinant Msx2; 2) to define the role of Msx2 developmental osteobiology, mineralization, and skeletal morphogenesis. Expanded pan-osseous expression of Msx2 will be directed in transgenic mice with the full length 1.8kb rat OC promoter. This system will define the role of Msx2 as a regulator of osteoblast proliferation, differentiation, gene expression, and mineralization.
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