Dentin Sialophosphoprotein (DSPP) and Unfolded Protein Response (UPR) in Dentinogenesis Imperfecta (DGI) and Odontoblast Function

牙本质唾液酸磷蛋白 (DSPP) 和未折叠蛋白反应 (UPR) 在牙本质发育不全 (DGI) 和成牙本质细胞功能中的作用

• 批准号: 10190893
• 负责人: Yongbo Lu
• 金额: $ 35.27万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-30 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10190893
• 关键词: Affect; Age; Alleles; Apoptosis; Autophagocytosis; Binding Proteins; Cell Nucleus; Cell Survival; Cells; Cellular Stress; Chemicals; Chronic; Clinical Management; DSPP gene; Data; Defect; Dental; Dental Enamel; Dentin; Dentin Dysplasia; Dentin Formation; Dentinogenesis; Dentinogenesis Imperfecta; Disease; Endoplasmic Reticulum; Endoribonucleases; Enzymes; Extracellular Matrix; Future; Gene Expression; Genes; Goals; Homeostasis; Homologous Gene; Homologous Protein; Human; Impairment; In Vitro; Individual; Inflammatory Response; Inherited; Inositol; Intervention; Knock-in Mouse; Knowledge; Label; Lead; Messenger RNA; Modality; Molecular; Molecular Chaperones; Mouse Strains; Mus; Mutant Strains Mice; Mutation; Odontoblasts; Pathogenesis; Pathogenicity; Pathologic; Patients; Phenylbutyrates; Play; Protein Biosynthesis; Proteins; Reaction; Regulation; Research Project Grants; Role; Stress; Testing; Tooth Attrition; Tooth Loss; Tooth structure; Transmission Electron Microscopy; Work; biomineralization; deciduous tooth; endoplasmic reticulum stress; in vivo; leucylproline; mRNA Transcript Degradation; mutant; novel; overexpression; permanent tooth; prevent; response; secretory protein; skeletal disorder; therapy development; treatment strategy

The Unfolded Protein Response, (UPR) is defined as the natural reaction of cells to stresses caused by the accumulation of misfolded/unfolded proteins within the endoplasmic reticulum (ER). UPR may cause: 1) degradation of mRNAs encoding secretory proteins, 2) inhibition of global protein synthesis, and 3) degradation of unfolded proteins that accumulate in the ER. While UPR works to restore ER homeostasis and promote cell survival, it changes the gene expression of stressed cells and affects their functions, and chronic (pathologic) UPR may cause various diseases. Non-syndromic dentinogenesis imperfecta (DGI)/dentin dysplasia (DD), a common inherited dentin disorder, is caused by mutations in one allele of the dentin sialophosphoprotein (DSPP) gene. Whether DGI/DD is caused by DSPP haploinsufficiency itself or by the accumulation of mutant DSPP in the ER is not understood. Preliminary in vitro data showed that 1) the secretion of mouse DSPP-P19L, a homolog of the human DSPP mutant, p. P17L, was impaired in the transfected cells, and 2) that the chemical chaperone, 4-phenylbutyrate (4-PBA), accelerated the secretion of mutant DSPP-P19L. Analyses with the recently created DsppP19L/+ knock-in mice showed that odontoblasts in DsppP19L/+ or DsppP19L/P19L mice failed to efficiently secrete mutant DSPP into the dentin matrix and that the tooth defects (chamber enlargement) of younger DsppP19L/+ and DsppP19L/P19L mice resembled human DGI Type III, whereas those at an older age (chamber obliteration) mimicked human DGI Type II. While DsppP19L/+ and DsppP19L/P19L odontoblasts had a markedly reduced level of DSPP mRNA, they showed an increased level of the phosphorylated form of inositol-requiring enzyme 1α (IRE1α), indicating the activation of one UPR branch. These findings lead to the central hypothesis that the mutant DSPP-P19L within the ER causes ER stress and pathologic UPR, resulting in DGI; alleviation of ER stress by facilitating the secretion of ER- retained DSPP-P19L may reduce or correct dentin defects. Three Aims are proposed to test this novel hypothesis: Aim 1 - To determine the pathological effects of intracellularly retained DSPP-P19L on odontoblasts and dentin formation, by a) examining if DSPP-P19L accumulates in the ER and causes ER dilation, b) analyzing the dentin defects, c) assessing the activities of all three UPR branches in the molars of DsppP19L/+ and DsppP19L/P19L mice, and d) determining if UPR associated with DSPP-P19L induces autophagy, apoptosis and pro-inflammatory responses. Aim 2 - To determine if chemical chaperones will relieve ER stress and prevent the dental defects of the DsppP19L/+ and DsppP19L/P19L mice, by treating these mice with 4-PBA. Aim 3 - To determine the roles of IRE1α in DSPP mRNA degradation and odontoblast differentiation and function, by a) assessing the influences of IRE1α activation and inactivation on DSPP mRNA, and b) analyzing the teeth of mice in which IRE1α is conditionally ablated. Completion of the proposed project will elucidate the molecular pathogenesis of DGI and help develop effective and non-invasive treatment modalities for DGI.

展开的蛋白质反应，（UPR）被定义为细胞对由 内质网中（ER）中错误折叠/展开的蛋白质的积累。 UPR可能导致：1） 编码秘密蛋白的mRNA降解，2）抑制全球蛋白质合成和3） 积累在ER中的展开蛋白质的降解。而UPR努力恢复ER稳态和 促进细胞存活，它改变了应力细胞的基因表达并影响其功能，慢性 （病理）UPR可能引起各种疾病。非合成齿状发生Imperfecta（DGI）/牙本质 发育不良（DD）是一种常见的遗传性牙本质疾病，是由牙本质一个等位基因突变引起的 唾液磷蛋白（DSPP）基因。 DGI/DD是由DSPP单倍弥补本身还是由 尚不清楚ER中突变型DSPP的积累。初步的体外数据表明1） 小鼠DSPP-P19L的分泌，人类DSPP突变体的同源物，p。 P17L，在 转染的细胞和2）化学链酮4-苯基丁酸（4-PBA）加速了分泌 突变的DSPP-P19L。与最近创建的DSPPP19L/+敲门鼠的分析显示，Odontoblast在 DSPPP19L/+或DSPPP19L/P19L小鼠未能有效地秘密秘密突变体DSPP进入牙本质矩阵，并且 年轻DSPPP19L/+和DSPPP19L/P19L小鼠的牙齿缺陷（室扩展）类似于人类DGI iii，而那些年龄较大的人（室遗忘）模仿了人类DGI II。而DSPPP19L/+和 DSPPP19L/P19L Odontoblasts的DSPP mRNA水平明显降低，它们显示出升高的水平 磷酸化的肌醇提取酶1α（IRE1α）的磷酸化形式，表明一个UPR分支的激活。 这些发现导致了一个中心假设，即ER内的突变体DSPP-P19L会导致ER应力 和病理UPR，导致DGI；通过支持ER的分泌来缓解ER压力 保留的DSPP-P19L可能会减少或纠正牙本质缺陷。提出了三个目标来测试这本小说 假设：目标1-确定细胞内保留的DSPP -P19L对病理影响 通过a）检查dspp-p19L是否积聚在ER中并导致ER 扩张，b）分析牙本质缺陷，c）评估所有三个UPR分支的活性 DSPPP19L/+和DSPPP19L/P19L小鼠，d）确定UPR是否与DSPP-P19L相关联会诱导自噬， 凋亡和促炎反应。目标2-确定化学伴侣是否会挽救ER应力 并通过用4-PBA治疗这些小鼠，以防止DSPPP19L/+和DSPPP19L/P19L小鼠的牙齿缺陷。目的 3-确定IRE1α在DSPP mRNA降解和ODONTOBLAST分化和功能中的作用， 通过a）评估IRE1α激活和灭活对DSPP mRNA的影响，b）分析牙齿 IRE1α有条件消融的小鼠。拟议项目的完成将阐明分子 DGI的发病机理并有助于开发有效的非侵入性治疗方式DGI。