Regulation of Runx2 Function by Twist-1 in Tooth Development

描述（由申请人提供）：在本次竞争更新中，我们建议继续研究 Runx2 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，Runx2 在形态发生过程中指导牙上皮的命运和控制成牙本质细胞分化的开始方面发挥着关键作用。我们的研究表明迫切需要了解 Runx2 活性在牙齿形态发生和细胞分化过程中如何精确调节，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进行调节。核蛋白 Twist-1 作为 Runx2 的调节蛋白伴侣特别令人感兴趣。我们研究细胞分化因子 Runx2 是否与细胞存活因子 Twist-1 相互作用的基本原理源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。我们的实验将直接检验这样的假设：Runx2 在成牙本质细胞分化中的关键功能是由 Twist-1 在本质上功能拮抗的蛋白质-蛋白质相互作用水平上调节的。 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直至形态发生完成的方法。我们进一步提出 Runx2 和 Twist-1 之间的相互作用并不相互拮抗，因为 Twist-1 可以通过 FGF 介导的上皮间质信号传导在形态发生过程中介导细胞增殖。因此，人类 CCD 中多生牙和 Runx2(-/-) 小鼠中副芽的存在可能反映了 Twist-1 活性的增加，而不是 Runx2 水平降低的直接影响。目标 1 将确定 Runx2 和 Twist-1（mRNA 和蛋白质）表达模式是否与其在牙齿发育过程中的拟议伙伴关系兼容，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的表达相关联。目标 2 将通过小鼠遗传功能丧失和功能获得方法评估 Twist-1 表达的改变是否影响牙齿形态发生和成牙本质细胞分化。目标 3 将研究牙齿间充质中 Runx2 - Twist-1 蛋白相互作用的分子基础，以及这种相互作用对成牙本质细胞分化中 Runx2 功能的功能影响，目标 4 将测试 Twist-1 的 bHLH 结构域是否可以通过独立于其与 Runx2 相互作用的 FGF 信号传导介导牙齿形态发生。这些研究将加深我们对 Runx2 如何通过与 Twist-1 的合作来实现其在牙齿发育中的选择性功能的理解。重要的是，他们将解释多生牙是如何形成的，以及成牙本质细胞的分化是否是由抑制的释放决定的。这些数据还将为了解锁骨颅骨发育不良和 Saethre-Chotzen 综合征（两种威胁牙列的人类遗传性疾病）的发病机制提供框架。

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.