The Role of ER-stress and pH in Fluorosis

ER 应激和 pH 在氟中毒中的作用

• 批准号: 7817010
• 负责人: JOHN D BARTLETT
• 金额: $ 57.09万
• 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 2011-10-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7817010
• 关键词: Acidosis; Acids; Acquired Dental Fluorosis; Ameloblasts; Antibodies; Apoptosis; Applications Grants; B-Lymphocytes; Beverages; Binding Proteins; Biological Process; Boxing; Cell Culture Techniques; Cell Line; Cells; Cellular Stress; Cerebrum; Chemicals; Cultured Cells; Data; Dental Enamel; Development; Diabetes Mellitus; Diffusion; Disease; Dominant-Negative Mutation; Dose; Embryo; Enamel Organ; Endoplasmic Reticulum; Environment; Equilibrium; Familial Hypercholesterolemia; Fibroblasts; Fluorides; Funding Opportunities; Gene Expression; Gene Mutation; Genes; Genetic; Goals; Golgi Apparatus; Homeostasis; In Vitro; Individual; Ingestion; Injury; Lead; Left; Mediating; Minerals; Molecular; Molecular Chaperones; Molecular Genetics; Mus; NIH Program Announcements; Neurodegenerative Disorders; Pathway interactions; Pharmacogenetics; Phosphotransferases; Plasma Cells; Plasmids; Play; Population; Post-Translational Protein Processing; Precipitation; Predisposition; Prevalence; Process; Protein Biosynthesis; Proteins; Protons; PubMed; Public Health; Reporting; Research; Resistance; Risk; Role; Signal Pathway; Source; Staging; Staining method; Stains; Stress; Symptoms; Testing; Tooth structure; Toothpaste; Toxic effect; Tyrosinemias; base; biological adaptation to stress; blood glucose regulation; coping; diabetic; enamel matrix proteins; endoplasmic reticulum stress; extracellular; fluorosis; improved; in vivo; mouse model; novel; prevent; protein expression; protein folding; protein misfolding; protein transport; response

DESCRIPTION (provided by applicant): The overall goal of this project is to define the role of endoplasmic reticulum (ER) stress in Dental fluorosis by identifying genes and molecular pathways that respond to fluoride (F) exposure. The ER mediates protein synthesis, protein folding, and post-translational modification. Perturbations in ER homeostasis can interfere with these processes resulting in the accumulation of unfolded or misfolded proteins that cause ER distention and trigger ER-stress. ER-stress activates specific signaling pathways, termed the unfolded protein response (UPR). The UPR induces: I) chaperone expression to help fold the accumulated proteins, II) reduces overall protein synthesis to allow the ER to cope with the existing proteins, III) directs the degradation of misfolded proteins, and IV) initiates apoptosis. Previously we have shown that F activates the UPR in vivo and in vitro (1). These data corroborate prior studies demonstrating F-induced ER distention (2) and defective ER to Golgi protein transport (3). Also, UPR-mediated reduction in overall protein synthesis may preclude F exposed ameloblasts from actively removing enamel matrix proteins from maturation stage enamel. This could cause the increased protein content observed in fluorosed enamel. We posit that the UPR pathways initiated in response to F-induced ER-stress play a role in Dental fluorosis. Therefore, Aim 1 is to identify UPR genes induced by F and determine if these genes play a role in fluorosis. In this project, we will determine if gene mutations in specific UPR pathways make cultured cells more susceptible to F or make mice more susceptible or resistant to fluorosis. Studies will be performed in: the ameloblast-derived LS8 cell line; LS8 cells carrying the dominant-negative XBP1 expression plasmid; Xbp1+/+, Xbp1+/-, Xbp1-/-; Perk+/+ and Perk-/- mouse embryo fibroblasts (MEFs); and Xbp1+/+, Xbp1+/-, Perk+/+ and Perk+/- mice. Aim 2 is to characterize the contribution of extracellular pH to F susceptibility and to determine if low-dose F causes ER-stress when ameloblasts are present in an acid environment. We posit that acidification of the enamel matrix during the maturation stage of tooth development drives F into ameloblasts and that this increased concentration of intracellular F induces ER stress that culminates in Dental fluorosis. The preliminary studies demonstrate that an acidic environment reduces the threshold F dose required to: a) inhibit proliferation and induce toxicity in vitro, b) activate UPR pathway genes in cultured cells, and c) activate UPR pathway genes in mouse ameloblasts in vivo. In this project, we will determine if cells are more sensitive to ER-stress at low pH, determine if the UPR pathways are altered as a function of low pH, and confirm the cell culture results in ameloblasts from mice with induced acidosis. Public Health Significance: If ER-stress plays a major role in Dental fluorosis, chemical treatments may be available to help ameloblasts properly fold their ER proteins and prevent fluorosis. Chemical treatment of mice for ER-stress improves diabetic glucose homeostasis (4;5) and protects against cerebral ischemic injury (6). Project Narrative The prevalence of fluorosis among the population is increasing (7) yet we currently know very little about what causes fluorosis. We have previously demonstrated that fluoride elicits a cell stress response which in turn, activates genes to help the cell cope with the stress (1). This application seeks to identify those stress response genes and determine if they play a role in causing Dental fluorosis.

描述（由申请人提供）：该项目的总体目标是通过识别响应氟化物（F）暴露的基因和分子途径来确定内质网（ER）应激在氟斑牙中的作用。 ER 介导蛋白质合成、蛋白质折叠和翻译后修饰。内质网稳态的扰动会干扰这些过程，导致未折叠或错误折叠蛋白质的积累，从而导致内质网扩张并引发内质网应激。内质网应激激活特定的信号通路，称为未折叠蛋白反应（UPR）。 UPR 诱导：I) 伴侣表达以帮助折叠累积的蛋白质，II) 减少整体蛋白质合成，使 ER 能够处理现有蛋白质，III) 指导错误折叠蛋白质的降解，IV) 启动细胞凋亡。之前我们已经证明 F 在体内和体外激活 UPR (1)。这些数据证实了先前的研究，证明 F 诱导的 ER 扩张 (2) 和 ER 到高尔基体蛋白转运的缺陷 (3)。此外，UPR介导的总体蛋白质合成减少可能会阻止暴露于F的成釉细胞主动从成熟阶段牙釉质中去除牙釉质基质蛋白。这可能导致氟牙釉质中蛋白质含量增加。我们假设响应 F 诱导的 ER 应激而启动的 UPR 途径在氟斑牙中发挥作用。因此，目标 1 是鉴定 F 诱导的 UPR 基因，并确定这些基因是否在氟中毒中发挥作用。在这个项目中，我们将确定特定 UPR 途径中的基因突变是否会使培养细胞更容易受到 F 的影响，或者使小鼠更容易受到或抵抗氟中毒。研究将在： 成釉细胞衍生的 LS8 细胞系中进行；携带显性失活XBP1表达质粒的LS8细胞； Xbp1+/+、Xbp1+/-、Xbp1-/-； Perk+/+ 和 Perk-/- 小鼠胚胎成纤维细胞 (MEF)；和 Xbp1+/+、Xbp1+/-、Perk+/+ 和 Perk+/- 小鼠。目标 2 是表征细胞外 pH 对 F 敏感性的影响，并确定当成釉细胞存在于酸性环境中时，低剂量 F 是否会导致 ER 应激。我们假设，在牙齿发育的成熟阶段，牙釉质基质的酸化会驱使 F 进入成釉细胞，并且细胞内 F 浓度的增加会引起 ER 应激，最终导致氟斑牙。初步研究表明，酸性环境降低了以下所需的阈值F剂量：a）在体外抑制增殖并诱导毒性，b）激活培养细胞中的UPR途径基因，以及c）激活体内小鼠成釉细胞中的UPR途径基因。在这个项目中，我们将确定细胞是否在低 pH 条件下对 ER 应激更敏感，确定 UPR 通路是否随着低 pH 的函数而改变，并确认诱导性酸中毒小鼠的成釉细胞的细胞培养结果。公共健康意义：如果 ER 应激在氟斑牙中起主要作用，那么化学治疗可能有助于成釉细胞正确折叠其 ER 蛋白并预防氟斑牙。对小鼠进行内质网应激化学治疗可改善糖尿病葡萄糖稳态 (4;5) 并预防脑缺血损伤 (6)。项目叙述 人群中氟中毒的患病率正在增加 (7)，但我们目前对氟中毒的原因知之甚少。我们之前已经证明，氟化物会引发细胞应激反应，进而激活基因来帮助细胞应对应激 (1)。该应用程序旨在识别这些应激反应基因，并确定它们是否在导致氟斑牙中发挥作用。