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

未折叠蛋白反应(UPR)被定义为细胞对由 错误折叠/未折叠的蛋白质在内质网(ER)内堆积。UPR可能导致：1) 编码分泌蛋白的mRNAs的降解，2)全球蛋白质合成的抑制，以及3) 内质网中积累的未折叠蛋白质的降解。而UPR致力于恢复ER动态平衡和 促进细胞存活，它改变应激细胞的基因表达并影响其功能，而慢性 (病理)UPR可引起各种疾病。非综合征性牙本质发育不全(DGI)/牙本质 异型增生(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是由DSPP单倍体不足本身还是由 突变的DSPP在内质网中的积累还不清楚。初步的体外数据显示：1) 小鼠DSPP-P19L是人DSPP突变体p.P17L的同源物，在 2)化学伴侣4-苯基丁酸酯(4-PBA)促进细胞分泌 突变株DSPP-P19L。对最近建立的DsppP19L/Knoke-in小鼠的分析表明，成牙本质细胞在 DsppP19L/或DsppP19L/P19L小鼠未能有效地将突变的DSPP分泌到牙本质基质中，并且 年轻DsppP19L/和DsppP19L/P19L小鼠的牙齿缺陷(腔室扩大)与人类DGI型相似 当DsppP19L/和DsppP19L/和 DsppP19L/P19L成牙本质细胞的DSPP mRNA表达水平明显降低，表现为 肌醇需要酶1α(IRE1α)的磷酸化形式，表明一个UPR分支被激活。 这些发现导致了一个中心假设，即内质网内的突变DSPP-P19L导致内质网应激 和病理性UPR，导致DGI；通过促进ER-1的分泌缓解ER应激 保留DSPP-P19L可减少或纠正牙本质缺陷。为了检验这部小说，我们提出了三个目标 假设：目的1-确定DSPP-P19L细胞内滞留的病理效应 成牙本质细胞和牙本质形成，通过a)检查DSPP-P19L是否在内质网中积聚并导致内质网 B)分析牙本质缺陷，c)评估所有三个UPR分支在磨牙中的活动 DsppP19L/和DsppP19L/P19L小鼠，以及d)确定与DSPP-P19L相关的UPR是否诱导自噬， 细胞凋亡和促炎反应。目标2-确定化学伴侣是否可以缓解内质网应激 用4-PBA处理DsppP19L/和DsppP19L/P19L小鼠，预防这些小鼠的牙齿缺陷。目标 3-确定IRE1α在DSPP mRNA降解和成牙本质细胞分化和功能中的作用 通过a)评估IRE1α的激活和失活对DSPP mRNA的影响，以及b)分析牙齿 在有条件地消融ir1α的小鼠中。拟议项目的完成将阐明分子 DGI的发病机制，有助于开发有效的非侵入性治疗方法。

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.