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

氟化物对 Rho 信号通路和肌动蛋白细胞骨架的影响

• 批准号: 7305731
• 负责人: Carolyn Gibson
• 金额: $ 23.63万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7305731
• 关键词: Actins; Adopted; Ameloblasts; Animal Model; Biochemical; Biological Assay; Biological Models; Cells; Cellular Morphology; Communities; Confocal Microscopy; Cytoskeleton; Data; Defect; Dental; Dental Enamel; Dental Esthetics; Dental caries; Development; Dominant-Negative Mutation; Dose; Electrons; Elevation; Environmental Impact; Epithelial Cells; Exposure to; F-Actin; Fibroblasts; Fluorides; G-Protein Signaling Pathway; Generations; Geographic Locations; Goals; Histologic; Image; Incisor; Knowledge; Lead; Link; Microscopic; Monitor; Monomeric GTP-Binding Proteins; Morphology; Mus; Organ; Organ Culture Techniques; Pathway interactions; Play; Policies; Positioning Attribute; Rattus; Role; Scanning; Signal Pathway; Sodium Fluoride; Staging; Testing; Thinking; Tissues; Tooth structure; Transgenic Mice; Transgenic Organisms; United States; Water Supply; amelogenin; dietary excess; drinking water; environmental agent; fluorosis; in vivo; inhibitor/antagonist; mouse model; novel; public drinking; research study; response; rho; transgene expression

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.

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，因为它被证明有利于减少龋齿。然而，在牙齿发育过程中暴露于高水平的膳食氟化物会导致称为釉质氟中毒的釉质结构缺陷，在一些氟化社区超过12%的居民中存在牙齿美学问题。大鼠饮用水中氟含量升高导致正常成釉细胞形态发生改变，对这些氟敏感的釉质分泌细胞的影响被认为是导致釉质结构缺陷的原因。使用牙齿器官培养和特异性抑制剂，我们已经表明RhoA途径是成釉细胞对氟化钠的反应之一，即丝状肌动蛋白（F-肌动蛋白）升高的原因。F-actin在成釉细胞中的定位通常在发育阶段随着成釉细胞功能的变化而变化，我们认为RhoA是氟诱导的细胞骨架失调的核心。我们建议测试的中心位置的RhoA途径在氟化物的反应有2个具体的目标。在目标1中，将产生一种新的转基因小鼠模型，其在成釉细胞（分泌釉质的上皮细胞层）中表达显性负性RhoA。将评估野生型和转基因小鼠的组织学、共聚焦和扫描电子显微镜图像，并将在体内分析饮用水中氟化物对细胞的反应。在目标2中，来自野生型和RhoADN转基因小鼠的牙齿器官将与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物化学方法进行分析，以监测RhoA和通路中下游靶标对氟化物的反应。这些实验将为发现这种环境因子对小G蛋白信号通路的作用机制提供一种新的动物模型。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难看的牙齿缺陷，称为氟中毒，但略低的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，将有助于做出更明智的政策决定，并对这种环境因子的影响有更基本的了解。