The Role of ER-stress and pH in Fluorosis

描述（由申请人提供）：该项目的总体目标是通过确定对氟(F)暴露作出反应的基因和分子途径，确定内质网（ER）应激在氟牙症中的作用。内质网介导蛋白质合成、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的蛋白质积累，导致内质网膨胀并引发内质网应激。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：1)伴侣蛋白表达，帮助折叠积累的蛋白质；2)减少整体蛋白质合成，使内质网能够应对现有的蛋白质；3)指导错误折叠蛋白质的降解；4)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表明，f诱导内质网扩张(2)和内质网缺陷高尔基蛋白运输(3)。此外，upr介导的总蛋白合成减少可能会阻止F暴露的成釉细胞主动从成熟阶段的牙釉质中去除牙釉质基质蛋白。这可能导致在氟化牙釉质中观察到的蛋白质含量增加。我们假设，响应f诱导的er应激而启动的UPR通路在氟斑牙中起作用。因此，目的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起作用。在这个项目中，我们将确定特定UPR通路中的基因突变是否使培养细胞更容易受到氟中毒的影响，或者使小鼠更容易或更耐氟中毒。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；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是否会引起内质网应激。我们假设，在牙齿发育成熟阶段，牙釉质基质的酸化驱使F进入成釉细胞，细胞内F浓度的增加诱导内质内质酶应激，最终导致氟斑牙。初步研究表明，酸性环境降低了F的阈值剂量，可以在体外抑制增殖和诱导毒性，在体外激活培养细胞中的UPR通路基因，在体内激活小鼠成釉细胞中的UPR通路基因。在这个项目中，我们将确定细胞在低pH下是否对er应激更敏感，确定UPR通路是否随着低pH的变化而改变，并确认诱导酸中毒小鼠的细胞培养结果。公共卫生意义：如果内质网应激在氟牙症中起主要作用，化学治疗可能有助于成釉细胞正确折叠内质网蛋白并预防氟牙症。化学治疗小鼠内质网应激可改善糖尿病葡萄糖稳态（4;5），并可预防脑缺血损伤(6)。氟中毒在人群中的患病率正在上升(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，氟化物引起细胞应激反应，进而激活基因来帮助细胞应对应激(1)。本应用程序旨在识别这些应激反应基因，并确定它们是否在引起氟斑牙中起作用。

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.