Bone matrix is an organic and mineral composite that confers toughness and hardness to bone. The material properties of this matrix may predict bone fracture risk and could complement clinical fracture risk assessment, which currently relies on bone mineral density. Matrix material properties, like other aspects of bone quality, are biologically determined and anatomically distinct. However, little is known about their regulation in healthy bone or their misregulation in disease. The long-term goal of this research is to identify mechanisms that control matrix material properties, so that ultimately, they can be harnessed therapeutically to prevent skeletal disease and improve craniofacial bone repair. Transforming growth factor- (TGF-) was recently identified as the first growth factor to regulate bone matrix material properties. TGF- inhibits osteoblast gene expression and differentiation, as well as bone matrix material properties, by repressing the function of Runx2, a key osteoblast transcription factor. Although Runx2 integrates signals from many pathways, only TGF- and glucocorticoids have yet been shown to regulate matrix material properties. Therefore, this proposal aims to investigate cellular and molecular mechanisms by which TGF- and Runx2 regulate matrix material properties. Osteoblasts are required for the regulation of matrix material properties by TGF-. Whether osteoblasts modify matrix material properties directly, or indirectly by cooperating with osteoclasts to change bone remodeling activity remains unclear. Using genetic and pharmacologic tools to manipulate osteoclast and osteoblast function in mice, Aim 1 seeks to determine the relative roles of both cell types in the specification of matrix material properties. Bone matrix material properties and mineral concentration will be evaluated by nanoindentation, fracture toughness testing, and X-ray tomographic microscopy. TGF- can repress or activate the expression of Runx2, apparently by differential utilization of downstream signaling pathways. Since the level of Runx2 function affects matrix material properties, Aim 2 seeks to identify mechanisms by which TGF- signaling regulates Runx2 to control osteoblast differentiation and matrix material properties. Novel mouse models and in vitro systems will be used. Furthermore, the targets of TGF- and Runx2 action that result in altered matrix material properties remain to be determined. Several TGF- and Runx2-regulated gene products have the potential to affect matrix material properties, including non-collagenous bone matrix proteins, proteases, and other signaling molecules. Aim 3 seeks to identify essential downstream targets through which TGF- and Runx2 regulate matrix material properties, using a combination of in vivo and in vitro approaches. Overall, the proposed studies seek to test the hypothesis that TGF- utilizes Smad3 and non-Smad3 mechanisms to differentially control Runx2 activation of osteoblast genes, which in turn establish matrix material properties both directly, and indirectly through the regulation of osteoclast-mediated bone remodeling. This research will elucidate the role of bone matrix material properties in skeletal health and disease.