The Role of Stress and pH in Fluorosis

描述（由申请人提供）：该项目的长期目标是鉴定对F暴露有反应和防止F毒性的基因。氟化物（F-）可以防止牙齿龋齿，但在美国儿童中，大量且越来越多的儿童因过度接触氟化物而导致氟牙症。重要的是，身体表面仅2.5%的皮肤暴露于F-氢氟酸（HF）是致命的。然而，参与F-应激反应的分子途径和基因尚未被很好地表征。先前的研究表明，F-诱导应激反应介质eIF2a （eIF2a- p）的磷酸化。这发生在小鼠和大鼠门牙成釉细胞的体外和体内。本项目验证了氟在成釉细胞中引起应激反应以减轻氟毒性的假设，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，是翻译mRNA模板中的蛋白质所必需的。eIF2a磷酸化抑制整体的蛋白质翻译，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。根据启动应激的类型，eIF2a可以被四种不同的激酶中的任何一种磷酸化——Gcn2 （Eif2ak4）、Hri （Eif2ak1）、Perk （Eif2ak3）和Pkr （Eif2ak2）——每一种激酶对不同的应激刺激都有反应。本项目的目的是鉴定F诱导的eIF2a-P的上下游介质，从而阐明F诱导的应激反应途径。AIM 1是鉴定F-诱导的eIF2a磷酸化的上游介质。我们将确定负责的激酶，并确定F-是否通过诱导内质网（ER）应激或氧化应激直接或间接激活它。AIM 2是鉴定磷酸化eIF2a （eIF2a- p）的下游介质，以缓解应激。我们将评估已知受eIF2a-P调控的基因（如Atf4）的表达，并将进行多体分析，以鉴定以前未知的F诱导的eIF2a-P调控基因。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mrna。突变细胞多体mrna将从进一步的分析中剔除，因为它们不是由eIF2a-P诱导的。由于eIF2a-P抑制整体翻译，微阵列分析应该提供一组可管理的eIF2a-P调节基因来表征。目的3是在体内鉴定小鼠成釉细胞中eIF2a-P的上下游介质。我们将证实F诱导的eIF2a-P分子通路在体外和体内培养的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， 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磷酸化是否保护小鼠成釉细胞免受F毒性。我们预测，由于F对成釉细胞的毒性作用，经F处理的Hri-/-小鼠的牙釉质会比正常小鼠软。这项研究的完成将提供一个明确的F-诱导分子途径，并将确定保护细胞免受F-影响的应激基因。

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-.