Regulation of Runx2 Function by Twist-1 in Tooth Development

描述(申请人提供)：在这次竞争性更新中，我们建议继续研究Runx2在牙齿发育中的作用。我们在之前获奖期间的工作数据表明，Runx2在引导牙齿上皮在形态发生过程中的命运和控制成牙本质细胞分化的开始方面发挥了关键作用。我们的研究指出，迫切需要了解Runx2活性在牙齿形态发生和细胞分化过程中是如何受到精确调控的，以及它在这些过程中的作用是否通过与其他分子的相互作用来调节。核蛋白Twist-1作为Runx2的调节蛋白伙伴特别令人感兴趣。我们研究细胞分化因子Runx2和细胞存活因子Twist-1是否相互作用的理论基础来自我们和其他实验室的研究，这些研究表明Runx2和Twist-1之间的相互作用发生在蛋白质水平。我们的实验将直接验证这一假设，即Runx2的S在成牙本质细胞分化中的关键功能是由Twist-1在蛋白质-蛋白质相互作用水平上调节的，这些相互作用本质上是功能拮抗的。Twist-1对Runx2功能的选择性和短暂阻断提供了一种抑制成牙本质细胞分化的手段，直到形态发生完成。我们进一步认为，Runx2和Twist-1之间的相互作用并不是相互拮抗的，因为Twist-1可以通过成纤维细胞生长因子介导的上皮间充质信号来调节细胞在形态发生过程中的增殖。因此，人类ccd中多余的牙齿和Runx2(-/-)小鼠的副芽的存在可能反映了Twist-1活性的增加，而不是Runx2水平下降的直接影响。目的1确定Runx2和Twist-1(mRNA和蛋白质)的表达模式是否与它们在牙齿发育过程中的伙伴关系相一致，并将这些模式与牙齿形态发生和成牙本质细胞分化的分子标志物的表达相关联。目的2将用小鼠遗传功能丧失和功能获得的方法评估Twist-1表达的变化是否影响牙齿的形态发生和成牙本质细胞的分化。目的3将研究牙间充质中Runx2-Twist-1蛋白相互作用的分子基础以及这种相互作用对Runx2在成牙本质细胞分化中功能的影响，目的4将测试Twist-1的bHLH域是否可以通过不依赖于与Runx2相互作用的成纤维细胞生长因子信号来调节牙齿的形态发生。这些研究将增加我们对Runx2如何通过与Twist-1的合作实现其在牙齿发育中的选择性功能的理解。重要的是，他们将解释多余的牙齿是如何形成的，以及成牙本质细胞的分化是否取决于抑制的释放。这些数据还将为理解锁骨颅骨发育不良和Saethre-Chotzen综合征的发病机制提供一个框架，这两种人类遗传性疾病威胁着牙列。

DESCRIPTION (provided by applicant): In this competing renewal, we propose to continue our research on the role of Runx2 in tooth development. Data from our work in the previous award period indicated key roles for Runx2 in directing the fate of Dental epithelium during morphogenesis and in controlling the onset of odontoblast differentiation. Our studies point to the critical need to learn how Runx2 activities are precisely regulated during tooth morphogenesis and cell differentiation and whether its role in these processes is modulated through interactions with other molecules. The nuclear protein Twist-1 is of particular interest as a regulatory protein partner for Runx2. Our rationale for studying if Runx2, a cell differentiation factor, interacts with Twist-1, a cell survival factor, is derived from studies in our and other laboratories that suggest that these interactions between Runx2 and Twist-1 occur at the protein level. Our experiments will directly test the hypothesis that Runx2's key functions in odontoblast differentiation are regulated by Twist-1 at the level of protein- protein interactions that are functionally antagonistic in nature. The selective and transient blocking of Runx2 function by Twist-1 provides a means to restrain odontoblast differentiation until morphogenesis is complete. We further propose that interactions between Runx2 and Twist-1 are not mutually antagonistic as Twist-1 can mediate cell proliferation during morphogenesis via FGF-mediated epithelial mesenchymal signaling. Hence, the presence of supernumerary teeth in human CCD and accessory buds in Runx2(-/-) mice likely reflect increased activity of Twist-1 rather than a direct effect of decreased levels of Runx2. Aim 1 will determine if the patterns of Runx2 and Twist-1 (mRNA and protein) expression are compatible with their proposed partnership during tooth development and will correlate these patterns with the expression of molecular markers of tooth morphogenesis and odontoblast differentiation. Aim 2 will assess with mouse genetic loss-of-function and gain-of-function approaches whether alterations in Twist-1 expression affects tooth morphogenesis and odontoblast differentiation. Aim 3 will study the molecular basis of Runx2 - Twist-1 protein interactions in Dental mesenchyme and the functional consequences of this interaction on Runx2 functions in odontoblast differentiation, and Aim 4 will test whether the bHLH domain of Twist-1 can mediate tooth morphogenesis via FGF-signaling that is independent of its interactions with Runx2. These studies will increase our understanding of how Runx2 achieves its selective functions in tooth development through its partnership with Twist-1. Importantly, they will explain how supernumerary teeth form and if odontoblast differentiation is determined by the release of an inhibition. Such data will also provide a framework for understanding the pathogenesis of Cleidocranial Dysplasia and Saethre-Chotzen Syndrome, 2 human genetic disorders that threaten dentition.