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鉴定响应氟化物（F）暴露的基因和分子途径来定义内质网（ER）应激在牙齿氟中虫病中的作用。 ER介导蛋白质合成，蛋白质折叠和翻译后修饰。 ER稳态中的扰动会干扰这些过程，从而导致不折叠或错误折叠的蛋白质的积累，从而导致ER扩张并触发ER压力。 ER应力激活特定的信号通路，称为展开的蛋白质反应（UPR）。 UPR诱导：i）伴侣表达有助于折叠累积的蛋白质，ii）降低总体蛋白质合成以使ER应对现有蛋白质，iii）指导错误折叠的蛋白质的降解，而IV）引发了凋亡。以前，我们已经表明F激活了体内和体外的UPR（1）。这些数据证实了先前的研究，证明了F诱导的ER扩张（2）和对高尔基蛋白转运的有缺陷（3）。同样，UPR介导的整体蛋白质合成的降低可能会阻止暴露的成成木细胞从成熟期搪瓷中积极去除牙釉质基质蛋白。这可能导致在荧光搪瓷中观察到的蛋白质含量增加。我们认为，响应F诱导的ER应力启动的UPR途径在牙齿氟中毒中起作用。因此，目标1是确定由F诱导的UPR基因，并确定这些基因是否在氟中毒中起作用。在该项目中，我们将确定特定UPR途径中的基因突变是否使培养的细胞更容易受到F的影响或使小鼠更容易受到氟中毒的影响或抗性。研究将在以下方面进行：成熟的LS8细胞系；带有显性阴性​​XBP1表达质粒的LS8细胞； XBP1+/+，XBP1 +/-，XBP1 - / - ; PERK+/+和PERK - / - 鼠标胚胎成纤维细胞（MEFS）;和XBP1+/+，XBP1 +/-，PERK+/+和PERK +/-小鼠。 AIM 2是表征细胞外pH对F易感性的贡献，并确定当酸环境中存在成熟细胞时，低剂量F是否会导致ER压力。我们认为，在牙齿发育的成熟阶段，牙釉质基质的酸化将F驱动到成熟细胞中，并且细胞内F的浓度增加会诱导ER应力，从而导致牙齿氟中毒。初步研究表明，酸性环境降低了所需的阈值F剂量：在该项目中，我们将确定细胞在低pH值下是否对ER压力更敏感，确定UPR途径是否随着低pH的函数而改变，并确认细胞培养会导致诱导酸中毒的小鼠的成核细胞。公共卫生的意义：如果ER应力在牙齿氟中毒中起主要作用，则可以使用化学治疗，以帮助成熟细胞正确折叠其ER蛋白并预防氟中毒。小鼠的化学处理ER应力可改善糖尿病性葡萄糖稳态（4; 5），并预防脑缺血性损伤（6）。项目叙事人群中氟中毒的流行率正在增加（7），但目前我们对导致氟中毒的原因知之甚少。我们以前已经证明氟化物会引起细胞应激反应，从而激活基因以帮助细胞应对应激（1）。该应用程序旨在识别这些压力反应基因，并确定它们是否在引起牙齿氟中毒作用中起作用。