Regulation of RUNX-2 Transcriptional Activity

描述(申请人提供)：Runx2是成骨细胞发育和骨形成所必需的转录因子。它还有助于T细胞淋巴瘤的发生，以及乳腺癌相关的骨溶解和骨骼疾病。Runx2既是基因表达的激活因子，也是基因表达的抑制因子。它的活性受与其他蛋白质的相互作用的调节，包括共激活子和共抑制子。组蛋白脱乙酰酶(HDAC)是与Runx2相关的共抑制因子。我们已经鉴定出四个HDACs(HDAC3、HDAC4、Hdac6、HDAC7)和与HDAC相关的共抑制因子mSinSA是Runx2相互作用蛋白。我们最近的研究表明，这些相互作用对骨形成可能是重要的，因为HDAC抑制剂和HDAC3shRNAs在体外促进了成骨细胞的成熟。因此，我们知道了几种负调控Runx2转录活性的酶的身份，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协同修改Runx2的活性，或者这些辅助抑制因子是否是体内骨形成的重要调节因子。为了实现了解Runx2调控基因表达的分子机制的长期目标，我们的下一步是确定HDAC3是否像在体外那样延迟体内成骨细胞的成熟，继续在体外定义多蛋白Runx2-抑制复合体，并开始识别通过阻止Runx2与辅助抑制物相互作用将Runx2从抑制物转变为激活物的分子“开关”。这项应用的目的是确定HDAC7如何与HDAC3合作并对成骨因子做出反应来单独修改Runx2的活性，并确定HDAC3的缺失是否像在体外那样加速体内成骨细胞的成熟和骨形成。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体的组成部分，并对细胞外信号做出反应。为了验证我们的假设并实现这一应用的目标，我们将追求的三个具体目标是：1)确定HDAC3在体内成骨细胞成熟和骨形成中的作用，2)确定HDAC7如何调控Runx2并与其相互作用，以及3)确定改变Runx2和HDAC7复合体之间相互作用的刺激和机制。这项拟议的工作具有创新性，因为它将探索多组分抑制物复合体如何调节Runx2的活性，并确定共抑制物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，因为它们有望定义HDAC如何调节Runx2活性和成骨细胞成熟。我们以前的工作表明，HDAC抑制剂是一种潜在的合成代谢药物。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.