The Mechanistic Control of Bone Matrix Material Properties by TGF-beta and Runx2

描述(申请人提供)：骨基质是一种有机和矿物复合材料，赋予骨骼韧性和硬度。这种基质的材料特性可以预测骨折风险，并可以补充目前依赖于骨密度的临床骨折风险评估。基质材料的特性，就像骨质量的其他方面一样，是由生物决定的，在解剖学上是不同的。然而，人们对它们在健康骨骼中的调控或在疾病中的错误调控知之甚少。这项研究的长期目标是确定控制基质材料性能的机制，以便最终能够在治疗上利用它们来预防骨骼疾病和改善颅面骨修复。近年来，转化生长因子被认为是第一个调节骨基质材料性能的生长因子。转化生长因子β通过抑制成骨细胞转录因子Runx2的功能，抑制成骨细胞基因的表达和分化，以及骨基质材料的特性。尽管Runx2整合了许多途径的信号，但只有转化生长因子-β和糖皮质激素被证明调节基质材料的性质。因此，本研究旨在研究转化生长因子β和Runx2调节基质材料性质的细胞和分子机制。成骨细胞是通过转化生长因子-β调节基质材料性质所必需的。成骨细胞是直接改变基质材料的性质，还是通过与破骨细胞合作改变骨重建活性来间接改变基质材料的性质尚不清楚。使用遗传学和药理学工具来操纵小鼠的破骨细胞和成骨细胞功能，Aim 1试图确定这两种细胞类型在基质材料特性规范中的相对作用。骨基质材料的性能和矿物质浓度将通过纳米压痕、断裂韧性测试和X射线断层显微镜进行评估。转化生长因子β可抑制或激活Runx2的表达，其机制明显是通过对下游信号通路的差异化利用。由于Runx2功能水平影响基质材料特性，Aim 2试图确定转化生长因子β信号调节Runx2以控制成骨细胞分化和基质材料特性的机制。将使用新的小鼠模型和体外系统。此外，导致基质材料性质改变的转化生长因子β和Runx2作用的靶点仍有待确定。一些受转化生长因子β和Runx2调控的基因产物有可能影响基质材料的特性，包括非胶原性骨基质蛋白、蛋白酶和其他信号分子。目的3采用体内和体外相结合的方法，试图确定重要的下游靶点，通过这些靶点，转化生长因子β和Runx2调节基质材料的性质。总之，建议的研究试图验证这样的假设，即转化生长因子β利用SMAD3和非SMAD3机制来不同地控制成骨细胞基因的Runx2激活，这反过来又直接和间接地通过调节破骨细胞介导的骨重建来建立基质材料的特性。这项研究将阐明骨基质材料特性在骨骼健康和疾病中的作用。

DESCRIPTION (provided by applicant): 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.