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

基本信息

批准号：
10404027
负责人：
Yongbo Lu
金额：
$ 34.92万
依托单位：
TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-30 至 2025-05-31
项目状态：
未结题

项目摘要

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）被定义为细胞对由您引起的应力的自然反应内质网中的错误折叠/展开的蛋白质（ER）可能会导致：1）编码分泌蛋白的mRNA降解，2）抑制全球蛋白质合成和3）积累在Theer中的展开蛋白质的降解。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。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。尚不清楚ER中突变型DSPP的积累。小鼠DSPP-P19L的分泌是人类DSPP突变体P17L的同源物。转染的细胞和2）化学伴侣，4-苯基丁酸（4-PBA），加速了秘密突变的DSPP-P19L。 DSPPPP19L/+或DSPPP19L/P19L小鼠无法有效地分泌突变体DSPP进入牙本质矩阵，并且您年轻的DSPP19L/+和DSPPP19L/P19L小鼠的牙齿缺陷（腔室扩大）分解的人类DGI类型 iii，而那些年龄较大的年龄（室内消失）模仿了人类DGI II。 DSPPPP19L/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dspp-p19l在ersworctress erstress中和病理UPR，导致DGI; 保留的DSPP-P19L可以减少或纠正牙本质缺陷。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是否积聚并导致ERER 扩张，b）分析牙本质缺陷，c）评估所有三个UPR分支的活性 DSPPPP19L/+和DSPP19L/P19L小鼠，d）确定UPR是否与DSPP-P19L相关，诱导自噬，细胞凋亡和促进剂的呼吸。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 3-确定IRE1α在DSPP mRNA降解中的作用和Odontoblast的分化和功能，通过a）评估IRE1α激活和灭活对DSPP mRNA的影响，b）分析牙齿 IRE1α有条件地烧毁的小鼠。 DGI的发病机理并有助于开发有效的非侵入性治疗方式DGI。