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识别对氟暴露做出反应的基因，并防止氟中毒。氟化物(F-)可以防止牙齿龋齿，但过量接触氟化物会导致大量美国儿童患氟斑牙，而且比例还在不断增长。重要的是，只有2.5%的体表皮肤暴露于氟-AS氢氟酸(HF)是致命的。然而，参与F胁迫反应的分子途径和基因还没有得到很好的描述。先前的研究表明，氟诱导应激反应介质eIF2a(eIF2a-P)的磷酸化。这在体外和体内都发生在小鼠和大鼠切牙成釉细胞中。该项目测试了这样一种假设，即氟化物引起成釉细胞的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，它是从mRNA模板翻译蛋白质所必需的。EIF2a的磷酸化抑制了整个蛋白质的翻译，但会优先翻译特定的下游应激反应mRNAs，帮助细胞应对给定的应激。根据启动压力的类型，eIF2a可以被四种不同的激酶中的任何一种--GCN2(Eif2ak4)、HRI(Eif2ak1)、PERK(EIF2AK3)和PKR(Eif2ak2)--中的任何一种磷酸化，每一种都对不同的应激刺激做出反应。本项目的目的是确定氟诱导的eIF2a-P的上下游介体，以便阐明氟诱导的应激反应途径。目的1是确定F诱导的eIF2a磷酸化的上游介体。我们将确定负责的激酶，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。目的2是确定磷酸化eIF2a(eIF2a-P)的下游介质，以缓解压力。我们将评估已知受eIF2a-P调控的基因，如ATF4的表达，并将进行多聚体分析，以识别先前未知的F-诱导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的mRNAs。突变的细胞多聚体mRNAs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易于管理的eIF2a-P调控基因来表征。目的3是在体内确定eIF2a-P在小鼠成釉细胞中的上下游介体。我们将证实，在体外和体内培养的细胞中，F诱导的eIF2a-P分子通路在负责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氟中毒。我们预测，由于成釉细胞的氟毒性，经氟处理的HRI-/-小鼠的牙釉质将比正常牙釉质更柔软。这项研究的完成将提供一个明确的F诱导的分子途径，并将识别保护细胞免受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-.