The Impact of Fluoride on the Rho Signaling Pathway and the Actin Cytoskeleton

描述（由申请人提供）：氟化物被添加到供应给美国三分之二社区的饮用水中，因为它被证明对减少龋齿有好处。然而，在牙齿发育期间，饮食中氟化物含量升高会导致牙釉质结构缺陷，即牙釉质氟中毒，在一些含氟社区，超过12%的居民会出现牙齿美观问题。大鼠饮水中氟化物含量升高导致正常成釉细胞形态的改变，这些对氟化物敏感的分泌珐琅质细胞受到的影响被认为是造成珐琅质结构缺陷的原因之一。利用牙齿器官培养和特异性抑制剂，我们已经证明RhoA通路负责成釉细胞对氟化钠的反应之一，即丝状肌动蛋白（F-actin）的升高。随着成釉细胞功能的改变，f -肌动蛋白在成釉细胞中的定位通常会在发育阶段发生变化，我们认为RhoA是氟化物诱导的细胞骨架失调的核心。我们建议测试RhoA通路在氟化物反应中的中心位置，有两个特定的目的。在Aim 1中，将产生一种新的转基因小鼠模型，该模型在成釉细胞（分泌珐琅质的上皮细胞层）中表达显性阴性的RhoA。将对野生型和转基因小鼠的组织学、共聚焦和扫描电镜图像进行评价，并在体内分析细胞对饮用水中氟化物的反应。在第2项研究中，将野生型和RhoADN转基因小鼠的牙齿器官与Rho通路抑制剂或激活剂一起培养，并通过共聚焦显微镜和生化方法分析RhoA及其下游靶点对氟化物的反应。这些实验将为发现该环境因子对小G蛋白信号通路的作用机制提供新的动物模型。氟化物通常被添加到公共饮用水中，但有些地理位置自然超过了最大推荐剂量。虽然氟化物水平升高会导致一种不美观的牙齿缺陷，即氟中毒，但稍低的氟化物水平对减少龋齿非常有益。更好地了解与环境氟化物有关的健康益处和问题，将导致更明智的政策决定和对这一环境因素影响的基本认识。

DESCRIPTION (provided by applicant): Fluoride is added to drinking water supplied to two-thirds of the communities in the U.S. because of its proven benefit for reduction of dental caries. However, exposure to elevated dietary fluoride during tooth development leads to the enamel structural defect called enamel fluorosis, with dental aesthetic concerns in greater than 12% of residents of some fluoridated communities. Elevated fluoride in drinking water provided to rats led to alterations in the normal ameloblast morphology, and effects on these fluoride-sensitive, enamel-secreting cells are thought to contribute to the enamel structural defects. Using tooth organ culture and specific inhibitors, we have shown that the RhoA pathway is responsible for one of the responses of ameloblasts to sodium fluoride, that of elevation of filamentous actin (F-actin). F-actin localization in ameloblasts normally changes during developmental stages as ameloblast function changes, and we propose that RhoA is central to fluoride-induced cytoskeletal deregulation. We propose to test the central position of the RhoA pathway in the fluoride response with 2 specific aims. In Aim 1, a novel transgenic mouse model will be generated that expresses a dominant-negative RhoA in ameloblasts, the epithelial cell layer that secretes enamel. Histological, confocal and scanning electron microscopic images of wild-type and transgenic mice will be evaluated, and the cellular response to fluoride in drinking water will be analyzed in vivo. In Aim 2, tooth organs from wild-type and RhoADN transgenic mice will be cultured with Rho pathway inhibitors or an activator and analyzed by confocal microscopy and biochemical approaches to monitor the response of RhoA and downstream targets in the pathway to fluoride. These experiments will provide a new animal model for discovery of the mechanism of action for this environmental agent on a small G protein signaling pathway. Fluoride is commonly added to public drinking water, but there are geographic locations where the maximum recommended dose is naturally exceeded. Although elevated fluoride levels lead to an unsightly dental defect known as fluorosis, slightly lower levels are highly beneficial in reducing dental caries. A better understanding of the health-related benefits and problems associated with environmental fluoride will lead to more informed policy decisions and fundamental knowledge about the impact of this environmental agent.