Regulated Tissue Specific BMP Function in Bone Repair

骨形态发生蛋白(BMPs)是一种有效的形态发生蛋白，已被证明能促进软骨和成骨分化。这已经在体外，在骨髓间充质干细胞的模型系统中，以及当重组BMP被植入或注射到异位、异位或原位位置时在体内得到证实。BMPS在骨修复的所有阶段都有高水平的表达，并被证明在外源性应用时，可以增强愈合反应。假设：BMP在骨折愈合的各个阶段都起着关键的调节作用。为了验证这一假设，本申请中提出的研究将产生转基因小鼠，其中采用了一种新的实验策略 这将转基因的表达限制在软骨或骨骼上，并允许通过系统地给药一个小分子来对转基因进行外源调节。转基因动物将被改造成含有一种人工转录因子，编码在两种蛋白质中，一个是新的DNA结合域，另一个是激活域。这些结构域的表达将由组织特异性启动子(软骨的II型胶原或骨骼的骨钙素)驱动。转录因子将被外源二聚剂(雷帕霉素类似物)激活，从而使这些结构域接近。当被激活时，它将识别一个独特的启动子，该启动子将驱动BMP-2的过度表达或拮抗 BMP通过过度表达Noggin发挥作用。使用这一策略，转基因动物将经历正常的胚胎发育和骨折(或者，在项目1的情况下，牵张成骨)将在存在正常骨骼功能的情况下进行。只有当引入二聚体时，这些转基因的过度表达才会导致功能状态的丧失或获得。然后将在骨形态发生蛋白功能改变的情况下，在特定的组织和特定的时间分析骨折愈合情况。这些研究将提供大量关于BMP在关键阶段扮演的具体角色的新数据 并建立一个强大的模型系统，用于研究广泛的分子及其对骨骼功能的影响。

Bone morphogenetic proteins (BMPs) are potent morphogens that have been shown to promote chondrogenic and osteogenic differentiation. This has been demonstrated in vitro, within model systems of mesenchymal stem cells, and in vivo when recombinant BMPs have been implanted or injected into ectopic, heterotopic or orthotopic sites. BMPs are also expressed at elevated levels throughout all stages of bone repair and have been shown, when exogenously administered, to enhance the healing response. Hypothesis: BMPs are critical in the regulation of all phases of fracture healing. In order to test this hypothesis, the studies proposed in this application will generate transgenic mice in which a novel experimental strategy is employed that restricts the expression of a transgene to either cartilage or bone and allows for the exogenous regulation of that transgene through systemic administration of a small molecule. Transgenic animals will be engineered to contain an artificial transcription factor encoded in two proteins, a novel DNA binding domain and an activation domain. The expression of these domains will be driven by a tissue-specific promoter (type II collagen for cartilage or osteocalcin for bone). The transcription factor will be activated by the exogenous administration of a dimerizing agent (rapamycin analog), which brings these domains into proximity. When activated, it will recognize a unique promoter which will drive the overexpression of BMP-2 or antagonize BMP function by overexpressing Noggin. Using this strategy, transgenic animals will undergo normal embryological development and fractures (or, in the case of Project 1, distraction osteogensis) will be carried out in the presence of normal skeletal function. Only upon introduction of the dimerizing agent will loss or gain of function states be induced through the overexpression of these transgenes. Fracture healing will then be analyzed in specific tissues and at specific times under conditions in which BMP function is altered. These studies will provide extensive new data concerning the specific roles that BMPs play at critical stages of fracture healing and establish a powerful model system for investigating a wide array of molecules and their effects on skeletal function.