Regulation of RUNX-2 Transcriptional Activity

描述（由申请人提供）：Runx2是成骨细胞发育和骨形成的必需转录因子。它还有助于T细胞淋巴瘤发生和乳腺癌相关的骨质溶解和骨骼疾病。Runx2是基因表达的激活子和阻遏子。其活性受与其他蛋白质（包括共激活子和共阻遏子）相互作用的调节。组蛋白脱乙酰酶（Hdacs）是与Runx2相关的共阻遏物之一。我们已经鉴定了四种Hdacs（Hdac3、Hdac4、Hdac6、Hdac7）和与Hdac相关的共阻遏物mSinSA作为Runx2相互作用蛋白。我们最近的研究表明，这些相互作用可能对骨形成很重要，因为Hdac抑制剂和Hdac3 shRNA在体外加速成骨细胞成熟。因此，我们知道负调控Runx2转录活性的几种酶的身份，但我们不知道是什么决定了它们与Runx2的相互作用，它们是否在骨形成过程中合作修饰Runx2活性，或者这些共阻遏物是否是体内骨形成的重要调控因子。我们的下一个步骤，在追求我们的长期目标，了解Runx2调节基因表达的分子机制，以确定是否Hdac 3延迟成骨细胞成熟在体内，因为它在体外，继续定义多蛋白Runx2抑制复合物在体外，并开始确定分子“开关”，从一个阻遏物转换为激活剂，通过防止其与辅阻遏物。本申请的目的是确定Hdac 7如何与Hdac 3合作并响应于成骨因子而自行改变Runx 2活性，并确定Hdac 3消耗是否在体内像在体外那样加速成骨细胞成熟和骨形成。这项研究的中心假设是Hdac3和Hdac7是调节Runx2活性和骨形成的多蛋白复合物的组分，并且对细胞外信号有反应。我们将追求的三个具体目标来测试我们的假设并实现本申请的目标是：1）定义Hdac 3在体内成骨细胞成熟和骨形成中的作用，2）确定Hdac 7如何调节Runx 2并与Runx 2相互作用，以及3）鉴定修饰Runx 2和Hdac 7复合物之间相互作用的刺激和机制。这项工作是创新的，因为它将探索多组分阻遏物复合物如何调节Runx2活性，并确定协同阻遏物Hdac3和Hdac7如何在体内和体外促进骨形成。这些结果将产生集体影响，因为它们有望确定Hdacs如何调节Runx2活性和成骨细胞成熟。我们以前的工作表明，HSP70抑制剂是潜在的合成代谢剂。因此，我们期望这项工作将为骨形成障碍的治疗提供新的视角和知识。

DESCRIPTION (provided by applicant): Runx2 is an essential transcription factor for osteoblast development and bone formation. It also contributes to T cell lymphomogenesis and breast cancer-associated osteolysis and skeletal disease. Runx2 is both an activator and represser of gene expression. Its activity is regulated by interactions with other proteins, including co-activators and co-repressors. Histone deacetylases (Hdacs) are among the co-repressors that associate with Runx2. We have identified four Hdacs (Hdac3, Hdac4, Hdac6, Hdac7) and the Hdac- associated co-repressor, mSinSA, as Runx2 interacting proteins. Our recent studies indicate that these interactions may be important for bone formation because Hdac inhibitors and Hdac3 shRNAs accelerate osteoblast maturation in vitro. Thus, we know the identity of several enzymes that negatively regulate Runx2 transcriptional activity, but we do not know what dictates their interactions with Runx2, whether they cooperate to modify Runx2 activity during bone formation, or if these co-repressors are important regulators of bone formation in vivo. Our next steps in pursuit of our long-term goal of understanding the molecular mechanisms whereby Runx2 regulates gene expression are to determine if Hdac3 delays osteoblast maturation in vivo as it does in vitro, to continue defining the multi-protein Runx2-repression complexes in vitro, and to begin identifying the molecular "switches" that convert Runx2 from a represser to an activator by preventing its interactions with co-repressors. The objectives of this application are to determine how Hdac7 modifies Runx2 activity on its own, in cooperation with Hdac3 and in response to an osteogenic factor, and to determine if Hdac3 depletion accelerates osteoblast maturation and bone formation in vivo as it does in vitro. The central hypothesis of this study is that Hdac3 and Hdac7 are components of a multi-protein complex that regulates Runx2 activity and bone formation and is responsive to extracellular signals. The three specific aims we will pursue to test our hypothesis and accomplish the objectives of this application are to: 1) Define the role of Hdac3 in osteoblast maturation and bone formation in vivo, 2) Determine how Hdac7 regulates and interacts with Runx2, and 3) Identify stimuli and mechanisms that modify interactions between Runx2 and Hdac7 complexes. The proposed work is innovative because it will explore how multi-component represser complexes regulate Runx2 activity and determine how the co-repressors Hdac3 and Hdac7 contribute to bone formation in vivo and in vitro. These results will have collective impact because they are expected to define how Hdacs regulate Runx2 activity and osteoblast maturation. Our previous work indicates that Hdac inhibitors are potential anabolic agents. Thus, it is our expectation that this work will provide new perspectives and knowledge into treatment of bone formation disorders.