The Role of Stress and pH in Fluorosis

压力和 pH 值在氟中毒中的作用

• 批准号: 8656953
• 负责人: JOHN D BARTLETT
• 金额: $ 56.84万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8656953
• 关键词: Acquired Dental Fluorosis; Affect; Alleles; Ameloblasts; Body Surface; Cell Death; Cell Line; Cell membrane; Cells; Cellular Stress; Cessation of life; Child; Cultured Cells; Data; Dental Enamel; Dental caries; Dermal; Development; Diagnostic; Embryo; Enamel Formation; Endoplasmic Reticulum; Environment; Enzymes; Excision; Exposure to; Fibroblasts; Fluoride Poisoning; Fluorides; Gene Expression; Genes; Goals; Health; Hydrofluoric Acid; Hydrogen Fluoride; Hydroxyapatites; Iceland; In Vitro; Incisor; Individual; Ions; Knockout Mice; Knowledge; Livestock; Maturation-Stage Ameloblast; Mediating; Mediator of activation protein; Messenger RNA; Microarray Analysis; Minerals; Molecular; Mus; Mutant Strains Mice; Mutation; Natural Disasters; Null Lymphocytes; Organism; Oxidative Stress; Pathway interactions; Phosphorylation; Phosphotransferases; Polyribosomes; Population; Precipitation; Predisposition; Prevalence; Production; Protein Synthesis Inhibition; Proteins; Rattus; Ribosomes; Role; Secretory-Stage Ameloblast; Staging; Stimulus; Stress; Testing; Thick; Tissues; Tooth structure; Toxic effect; Translating; Translations; Volcanic Eruption; biological adaptation to stress; cell type; coping; drinking water; endoplasmic reticulum stress; fluorosis; in vivo; mutant; prevent; public health relevance

DESCRIPTION (provided by applicant): The long-term goal of this project is to identify genes that respond to F- exposure and protect against F- toxicity. Fluoride (F-) protects teeth from caries, but F- over-exposure causes dental fluorosis in a large and growing proportion of U.S. children. Importantly, dermal exposure of just 2.5% of body surface to F- as hydrofluoric acid (HF) is lethal. Yet, the molecular pathways and genes involved in the F- stress response are not well characterized. Previously studies demonstrated that F- induces phosphorylation of the stress-response mediator eIF2a (eIF2a-P). This occurs both in vitro and in vivo in mouse and rat incisor ameloblasts. This project tests the hypothesis that fluoride causes a stress response in ameloblasts to alleviate F- toxicity and that this stress response is mediated through eIF2a-P. eIF2a is a component of the ribosome that is necessary to translate proteins from mRNA templates. eIF2a phosphorylation inhibits overall protein translation, but will preferentially translate specific downstream stress response mRNAs that help cells to cope with a given stress. Depending upon the type of initiating stress, eIF2a can be phosphorylated by any one of four different kinases -- Gcn2 (Eif2ak4), Hri (Eif2ak1), Perk (Eif2ak3), and Pkr (Eif2ak2) - each of which responds to different stress stimuli. The objective of this project is to identify F--induced up- and downstream mediators of eIF2a-P so that a F-- induced stress response pathway can be elucidated. AIM 1 is to identify upstream mediators of F- -induced eIF2a phosphorylation. We will identify the responsible kinase and determine if F- activates it directly or indirectly such as by inducing endoplasmic reticulum (ER) stress or oxidative stress. AIM 2 is to identify downstream mediators of phosphorylated eIF2a (eIF2a-P) that alleviate stress. We will assess expression of genes such as Atf4 that are known to be regulated by eIF2a-P and will perform polysome profiling to identify previously unknown F--induced eIF2a-P regulated genes. We will isolate transcribed mRNAs in F--treated wild-type cells and in mutant cells that cannot phosphorylate eIF2a. Mutant cell polysome mRNAs will be eliminated from further analysis because they were not induced by eIF2a-P. Since eIF2a-P inhibits overall translation, microarray analysis should provide a manageable set of eIF2a-P regulated genes to characterize. AIM 3 is to identify up- and downstream mediators of eIF2a-P in mouse ameloblasts in vivo. We will confirm that the F--induced eIF2a-P molecular pathway is the same in both cultured cells in vitro and in vivo in mouse ameloblasts responsible for enamel formation. Preliminary data suggest that F- activates Hri and that Hri phosphorylates eIF2a. Our colony of Hri null mice will be used to determine if Hri mediated phosphorylation of eIF2a protects mouse ameloblasts from F- toxicity. We predict that F- treated Hri-/- mice will have softer than normal enamel due to F- toxicity of ameloblasts. Completion of this study will provide a defined F--induced molecular pathway and will identify stress genes that protect cells from F-.

描述（由申请人提供）：该项目的长期目标是识别对氟暴露做出反应并防止氟毒性的基因。氟化物 (F-) 可以保护牙齿免遭龋齿，但过量接触氟化物会导致大量且不断增长的美国儿童患氟斑牙。重要的是，仅 2.5% 的体表皮肤暴露于氢氟酸 (HF) 形式的 F- 即可致命。然而，参与 F-应激反应的分子途径和基因尚未得到很好的表征。先前的研究表明，F- 会诱导应激反应介质 eIF2a (eIF2a-P) 的磷酸化。这在体外和体内的小鼠和大鼠门牙成釉细胞中都会发生。该项目测试了以下假设：氟化物会在成釉细胞中引起应激反应，以减轻氟毒性，并且这种应激反应是通过 eIF2a-P 介导的。 eIF2a 是核糖体的一个组成部分，是从 mRNA 模板翻译蛋白质所必需的。 eIF2a 磷酸化会抑制整体蛋白质翻译，但会优先翻译特定的下游应激反应 mRNA，帮助细胞应对给定的应激。根据起始应激的类型，eIF2a 可以被四种不同激酶中的任何一种磷酸化 - Gcn2 (Eif2ak4)、Hri (Eif2ak1)、Perk (Eif2ak3) 和 Pkr (Eif2ak2) - 每一种激酶都会对不同的应激刺激做出反应。该项目的目标是确定 F- 诱导的 eIF2a-P 上下游介质，以便阐明 F- 诱导的应激反应途径。目的 1 是鉴定 F- 诱导的 eIF2a 磷酸化的上游介质。我们将识别负责的激酶并确定 F- 是否直接或间接激活它，例如通过诱导内质网 (ER) 应激或氧化应激。目标 2 是确定可缓解压力的磷酸化 eIF2a (eIF2a-P) 下游介质。我们将评估已知受 eIF2a-P 调节的 Atf4 等基因的表达，并将进行多核糖体分析以鉴定以前未知的 F-诱导的 eIF2a-P 调节基因。我们将在 F-处理的野生型细胞和不能磷酸化 eIF2a 的突变细胞中分离转录的 mRNA。突变细胞多核糖体 mRNA 将被排除在进一步分析之外，因为它们不是由 eIF2a-P 诱导的。由于 eIF2a-P 抑制整体翻译，因此微阵列分析应提供一组可管理的 eIF2a-P 调节基因进行表征。目的 3 是鉴定小鼠体内成釉细胞中 eIF2a-P 的上下游介质。我们将确认F-诱导的eIF2a-P分子途径在体外培养的细胞和体内负责牙釉质形成的小鼠成釉细胞中是相同的。初步数据表明，F- 激活 Hri，并且 Hri 磷酸化 eIF2a。我们的 Hri 缺失小鼠群体将用于确定 Hri 介导的 eIF2a 磷酸化是否可以保护小鼠成釉细胞免受 F 毒性。我们预测，由于 F- 成釉细胞的毒性，经 F-处理的 Hri-/- 小鼠将具有比正常牙釉质更软的牙釉质。这项研究的完成将提供明确的 F- 诱导分子途径，并将鉴定保护细胞免受 F- 侵害的应激基因。