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确定对F-暴露并预防F-毒性做出反应的基因。氟化物（F-）可保护牙齿免受龋齿的侵害，但F-暴露会导致大量且不断增长的美国儿童的牙齿氟中毒。重要的是，由于氢氟酸（HF）是致命的，只有2.5％的身体表面暴露于F-。然而，涉及F-应力反应所涉及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-诱导应力反应介质EIF2A（EIF2A-P）的磷酸化。这既发生在小鼠和大鼠牙入的成棉布中的体外和体内。该项目检验了以下假设：氟化物会导致成成木的应力反应以减轻F-毒性，并且通过EIF2A-P介导这种应力反应。 EIF2A是核糖体的一个组成部分，它是从mRNA模板中翻译蛋白质所必需的。 EIF2A磷酸化抑制了总体蛋白质的翻译，但优先翻译了特定的下游应力反应mRNA，这些mRNA可帮助细胞应对给定的应激。根据引发应力的类型，EIF2A可以被四种不同激酶中的任何一种 - GCN2（EIF2AK4），HRI（EIF2AK1），PERK（EIF2AK3）和PKR（EIF2AK2）磷酸化 - 每种都会对不同的压力刺激作出反应。该项目的目的是识别EIF2A-P的F-诱导的上游和下游介质，以便可以阐明F-诱导的应力响应途径。目的1是鉴定F-诱导的EIF2A磷酸化的上游介体。我们将识别负责任的激酶，并确定F-是否直接或间接激活它，例如通过诱导内质网（ER）应力或氧化应激。目标2是鉴定减轻压力的磷酸化EIF2A（EIF2A-P）的下游介体。我们将评估已知受EIF2A-P调节的ATF4之类的基因表达，并将执行多元组分析以识别以前未知的F-诱导的EIF2A-P调节基因。我们将分离在F-处理的野生型细胞和无法磷酸化eIF2A的突变细胞中分离的mRNA。突变细胞多质体mRNA将从进一步的分析中消除，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调节基因来表征。 AIM 3是在体内识别EIF2A-P的上下游介质。我们将确认，在培养的细胞中，F-诱导的EIF2A-P分子途径在体外和体内均相同，在负责牙釉质形成的小鼠成成木细胞中。初步数据表明F-激活HRI，HRI磷酸化EIF2A。我们的HRI无效小鼠的菌落将用于确定HRI介导的EIF2A的磷酸化是否可以保护小鼠的成熟细胞免受F-毒性的影响。我们预测，由于成成布的F-毒性，经过处理的HRI - / - 小鼠将比正常牙釉质柔软。这项研究的完成将提供定义的F-诱导的分子途径，并确定保护细胞免受F-的应力基因。