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: 7497272
• 负责人: JOHN D BARTLETT
• 金额: $ 57.18万
• 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 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7497272
• 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）：本项目的总体目标是通过鉴定对氟（F）暴露做出反应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。ER稳态的扰动可以干扰这些过程，导致未折叠或错误折叠的蛋白质的积累，导致ER扩张并触发ER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）。普遍定期审议包括：I）伴侣蛋白表达以帮助折叠积累的蛋白质，II）减少总体蛋白质合成以允许ER处理现有蛋白质，III）指导错误折叠的蛋白质的降解，以及IV）启动细胞凋亡。以前我们已经证明F在体内和体外激活UPR（1）。这些数据证实了先前的研究，表明F诱导的ER扩张（2）和ER到高尔基体蛋白转运缺陷（3）。此外，UPR介导的整体蛋白质合成的减少可能会阻止氟暴露成釉细胞主动清除釉基质蛋白从成熟阶段的釉质。这可能导致在氟牙釉质中观察到的蛋白质含量增加。我们认为氟诱导的内质网应激启动的UPR通路在氟斑牙发病中起一定作用。因此，目的1是鉴定氟诱导的UPR基因，并确定这些基因是否在氟中毒中起作用。在这个项目中，我们将确定特定UPR途径中的基因突变是否使培养的细胞对氟更敏感，或者使小鼠对氟中毒更敏感或更有抵抗力。研究将在以下地点进行：成釉细胞衍生的LS8细胞系;携带显性阴性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）;以及Xbp1 +/+、Xbp1 +/-、Perk +/+和Perk +/-小鼠。目的2是表征细胞外pH值对F敏感性的贡献，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。我们认为，在牙齿发育的成熟阶段，釉质基质的酸化驱动F进入成釉细胞，这种细胞内F浓度的增加会诱导ER应激，最终导致氟斑牙。初步研究表明，酸性环境降低所需的阈值F剂量：a）体外抑制增殖和诱导毒性，B）激活培养细胞中的UPR途径基因，和c）体内激活小鼠成釉细胞中的UPR途径基因。在这个项目中，我们将确定细胞是否在低pH值下对ER应激更敏感，确定UPR途径是否作为低pH值的函数而改变，并确认诱导酸中毒小鼠的成釉细胞的细胞培养结果。公共卫生意义：如果ER应激在氟斑牙中起主要作用，化学治疗可能有助于成釉细胞正确折叠其ER蛋白并预防氟中毒。对ER应激的小鼠进行化学治疗可改善糖尿病葡萄糖稳态（4; 5），并防止脑缺血性损伤（6）。氟中毒在人群中的患病率正在增加（7），但我们目前对氟中毒的病因知之甚少。我们之前已经证明，氟化物会激发细胞的应激反应，进而激活基因来帮助细胞应对压力（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。