Regulation of Runx2 Function by Twist-1 in Tooth Development

描述（由申请人提供）：在本次竞争性更新中，我们建议继续研究Runx 2在牙齿发育中的作用。我们在上一个奖项期间的工作数据表明，Runx 2在形态发生期间指导牙齿上皮的命运和控制成牙本质细胞分化的发生方面起着关键作用。我们的研究指出，迫切需要了解Runx 2活动在牙齿形态发生和细胞分化过程中是如何精确调节的，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Runx 2的调节蛋白伴侣特别令人感兴趣。我们研究Runx 2（一种细胞分化因子）是否与Twist-1（一种细胞存活因子）相互作用的基本原理来自我们和其他实验室的研究，这些研究表明Runx 2和Twist-1之间的相互作用发生在蛋白质水平上。我们的实验将直接测试Runx 2在成牙本质细胞分化中的关键功能在本质上功能拮抗的蛋白质-蛋白质相互作用水平上由Twist-1调节的假设。Twist-1对Runx 2功能的选择性和瞬时阻断提供了一种抑制成牙本质细胞分化直至形态发生完成的方法。我们进一步提出Runx 2和Twist-1之间的相互作用不是相互拮抗的，因为Twist-1可以通过FGF介导的上皮间质信号传导介导形态发生期间的细胞增殖。因此，人CCD中多生牙和Runx 2（-/-）小鼠中副芽的存在可能反映了Twist-1活性的增加，而不是Runx 2水平降低的直接影响。目的1将确定Runx 2和Twist-1（mRNA和蛋白质）表达的模式是否与牙齿发育过程中提出的伙伴关系相容，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记物的表达相关联。目的2将评估与小鼠遗传功能丧失和功能获得的方法是否在Twist-1表达的改变影响牙齿形态发生和成牙本质细胞分化。目标3将研究Runx 2- Twist-1蛋白在牙齿间充质中相互作用的分子基础，以及这种相互作用对Runx 2在成牙本质细胞分化中的功能的影响，目标4将测试Twist-1的bHLH结构域是否可以通过FGF信号传导介导牙齿形态发生，这与Runx 2的相互作用无关。这些研究将增加我们对Runx 2如何通过与Twist-1的合作在牙齿发育中实现其选择性功能的理解。重要的是，他们将解释多生牙是如何形成的，以及成牙本质细胞的分化是否由抑制的释放决定。这些数据也将为理解锁骨颅骨发育不良和Saethre-Chotzen综合征（2种威胁牙列的人类遗传性疾病）的发病机制提供一个框架。

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.