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

未折叠蛋白质反应（Unfolded Protein Response，UPR）被定义为细胞对由未折叠蛋白质引起的应激的自然反应。 内质网（ER）内错误折叠/未折叠蛋白质的积累。普遍定期审议可能导致：1） 降解编码分泌蛋白的mRNA，2）抑制整体蛋白质合成，和3） 在ER中积累的未折叠蛋白质的降解。虽然UPR可以恢复ER的稳态， 促进细胞存活，它改变应激细胞的基因表达并影响其功能， （病理学）UPR可引起多种疾病。非综合征性牙本质生成障碍（DGI）/牙本质 发育不良（DD）是一种常见的遗传性牙本质疾病，由牙本质的一个等位基因突变引起 唾液酸磷蛋白（DSPP）基因。DGI/DD是否由DSPP单倍不足本身或DSPP单倍不足引起， 突变体DSPP在ER中的积累尚不清楚。初步的体外数据显示，1） 小鼠DSPP-P19 L（人DSPP突变体p.P17L的同源物）的分泌受损， 转染的细胞，和2）化学伴侣，4-苯基丁酸（4-PBA），加速分泌 突变体DSPP-P19 L。对最近建立的DsppP 19 L/+基因敲入小鼠的分析表明， DsppP 19 L/+或DsppP 19 L/P19 L小鼠不能有效地将突变体DSPP分泌到牙本质基质中， 年轻的DsppP 19 L/+和DsppP 19 L/P19 L小鼠的牙齿缺损（腔室扩大）类似于人DGI型 III，而那些在老年人（腔室闭塞）模仿人类DGI II型。而DsppP 19 L/+和 DsppP 19 L/P19 L成牙本质细胞DSPP mRNA水平显著降低，而DsppP 19 L/P19 L成牙本质细胞DSPP mRNA水平升高。 肌醇需要酶1α（IRE 1 α）的磷酸化形式，表明一个UPR分支的激活。 这些发现导致了核心假设，即ER内的突变DSPP-P19 L引起ER应激 和病理性UPR，导致DGI;通过促进ER-1的分泌来缓解ER应激。 保留DSPP-P19 L可以减少或纠正牙本质缺陷。本文从三个方面对这部小说进行了检验 假设：目的1 -确定细胞内保留的DSPP-P19 L对细胞凋亡的病理学作用。 a）检查DSPP-P19 L是否在ER中积累并引起ER 扩张，B）分析牙本质缺陷，c）评估以下磨牙中所有三个UPR分支的活性： DsppP 19 L/+和DsppP 19 L/P19 L小鼠，和d）确定与DsppP 19 L相关的UPR是否诱导自噬， 凋亡和促炎反应。目的2 -确定化学伴侣是否会缓解ER应激 并通过用4-PBA处理这些小鼠来预防DsppP 19 L/+和DsppP 19 L/P19 L小鼠的牙齿缺陷。目的 3.研究IRE 1 α在DSPP mRNA降解及成牙本质细胞分化和功能中的作用， 通过a）评估IRE 1 α激活和失活对DSPP mRNA的影响，和B）分析牙齿 IRE 1 α条件性消融的小鼠。完成拟议的项目将阐明分子 DGI的发病机制，并帮助开发DGI的有效和非侵入性治疗方式。

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.