THE ROLE OF STRESS AND PH IN FLUOROSIS

压力和 PH 值在氟中毒中的作用

• 批准号: 9233520
• 负责人: JOHN D BARTLETT
• 金额: $ 26.09万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-27 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9233520
• 关键词: ROLE; STRESS; PH; FLUOROSIS

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鉴定对氟暴露有反应并保护免受氟毒性的基因。氟化物（F-）保护牙齿免受龋齿，但F-过度暴露会导致大量且不断增长的美国儿童患氟斑牙。重要的是，皮肤暴露于氟（如氢氟酸）的2.5%的身体表面是致命的。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。以前的研究表明，F-诱导应激反应介质eIF 2a（eIF 2a-P）的磷酸化。这种情况在小鼠和大鼠切牙成釉细胞的体外和体内都发生。该项目测试了以下假设：氟化物引起成釉细胞中的应激反应以减轻F-毒性，并且该应激反应通过eIF 2a-P介导。eIF 2a是从mRNA模板翻译蛋白质所必需的核糖体的组分。eIF 2a磷酸化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，帮助细胞科普给定的应激。根据起始应激的类型，eIF 2a可以被四种不同激酶中的任何一种磷酸化-Gcn 2（Eif 2ak 4），Hri（Eif 2ak 1），Perk（Eif 2ak 3）和Pkr（Eif 2ak 2）-每一种都响应不同的应激刺激。本项目的目的是鉴定F-诱导的eIF 2a-P的上下游介质，以便阐明F-诱导的应激反应途径。目的1是鉴定氟诱导eIF 2a磷酸化的上游介质。我们将确定负责的激酶，并确定F-是否直接或间接激活它，如诱导内质网（ER）应激或氧化应激。目的2：鉴定磷酸化eIF 2a（eIF 2a-P）的下游介质，以减轻应激。我们将评估已知受eIF 2a-P调控的基因（如Atf 4）的表达，并将进行多核糖体分析以鉴定先前未知的F-诱导的eIF 2a-P调控基因。我们将在F-处理的野生型细胞和不能磷酸化eIF 2a的突变细胞中分离转录的mRNA。突变细胞多核糖体mRNA将从进一步分析中消除，因为它们不被eIF 2a-P诱导。由于eIF 2a-P抑制整体翻译，微阵列分析应该提供一组可管理的eIF 2a-P调控基因来表征。目的3：在小鼠成釉细胞中鉴定eIF 2a-P的上下游介质。我们将证实，F-诱导的eIF 2a-P分子途径是相同的，在体外培养的细胞和在体内的小鼠成釉细胞负责釉质形成。初步数据表明，F-激活Hri和Hri磷酸化eIF 2a。我们的Hri缺失小鼠群体将用于确定Hri介导的eIF 2a磷酸化是否保护小鼠成釉细胞免受F-毒性。我们预测，由于成釉细胞的氟毒性，氟处理的Hri-/-小鼠将具有比正常的釉质更柔软的釉质。这项研究的完成将提供一个明确的F-诱导的分子途径，并将确定保护细胞免受F-的应激基因。