Identification and Function of nuDMP1 in Odontoblast Differentiation

描述(由申请人提供)：牙本质基质蛋白1(DMP1)在人类和小鼠中功能丧失导致常染色体隐性遗传性低磷血症性软骨病(ARHR)，其特征是牙本质和骨骼中矿化减少，低磷血症和循环中成纤维细胞生长因子23(FGF23)水平升高。Dmp1缺陷者的牙齿和骨骼缺陷是由于Dmp1缺失的成牙本质细胞和成骨细胞/骨细胞的内在分化缺陷(局部效应)和低磷血症(全身效应)共同作用的结果。然而，DMP1功能缺失导致成牙本质细胞和成骨细胞/骨细胞分化缺陷的机制尚不清楚。本研究的目的是鉴定DMP1的核异构体(S)，并研究其在成牙本质细胞分化中的作用。我们推测nuDMP1是从AUG起始密码子(S)下游的替代密码子翻译而来，通过调节Dspp的表达来调控成牙本质细胞的分化。这一假说是基于我们最近在体外和体内的研究结果，其中包括：1)在含有内质网进入信号肽序列的全长DMP1表达的细胞中观察到核DMP1；2)表达全长DMP1的构建体没有内质网进入信号肽序列产生两种蛋白质，全长DMP1和可能的nuDMP1；3)产生假定的nuDMP1的构建体比产生全长DMP1的构建体更有效地刺激Dspp启动子的活性；4)3.6kb的Col1a1启动子驱动的DSPP转基因过表达挽救了Dmp1基因缺失小鼠的牙齿缺陷，但不能挽救DMP1缺失小鼠的其他表现。为了验证我们的中心假设，我们提出了三个具体目标。目标1将确定nuDMP1产生并随后移位到核中的机制。目的建立并鉴定3.6kb的Col1a1-nuDMP1转基因小鼠，探讨nuDMP1在成牙本质细胞分化中的作用。目的3将确定Dspp表达是如何通过nuDMP1激活的，以及这一过程的失败是否是Dmp1基因缺失小鼠牙齿表型的唯一原因。这一新提议的成功完成将为DMP1如何通过nuDMP1调节细胞分化提供独特的见解，nuDMP1独立于其调节基质矿化的功能。这种新的nuDMP1及其功能的鉴定不仅有助于阐明DMP1调控牙齿和骨骼形成的分子机制，而且将为开发治疗低磷性软骨病的药物提供科学依据。

DESCRIPTION (provided by applicant): The loss of dentin matrix protein 1 (DMP1) function in humans and mice results in autosomal recessive hypophosphatemic rickets (ARHR), characterized by hypomineralization in dentin and bone, hypophosphatemia and the elevation of circulating fibroblast growth factor 23 (FGF23) levels. The dental and skeletal defects of Dmp1-deficient subjects have been attributed to the combined effects of the intrinsic differentiation defects of Dmp1-null odontoblasts and osteoblasts/osteocytes (local effect) and hypophosphatemia (systemic effect). However, the way in which the loss of DMP1 function causes the differentiation defects of odontoblasts and osteoblasts/osteocytes remains largely unknown. The goal of this application is to identify a nuclear isoform(s) of DMP1 (referred to as "nuDMP1") and to study its function in odontoblast differentiation. We hypothesize that nuDMP1 is translated from alternative downstream in-frame AUG start codon(s) and controls odontoblast differentiation by regulating Dspp expression. This hypothesis is based on our recent in vitro and in vivo findings, which include: 1) nuclear DMP1 is observed in cells transfected with a construct expressing full-length DMP1 with the endoplasmic reticulum (ER)-entry signal peptide sequence; 2) a construct expressing full-length DMP1 without the ER-entry signal peptide sequence produces two proteins, a full-length DMP1 and a putative nuDMP1; 3) a construct producing the putative nuDMP1 is more potent at stimulating Dspp promoter activity than those producing full-length DMP1 with or without the ER-entry signal peptide; and 4) the transgenic overexpression of DSPP driven by a 3.6 kb Col1a1 promoter rescues the dental defects but not the other manifestations of Dmp1-null mice. To test our central hypothesis, three specific aims are proposed. Aim 1 will determine the mechanisms by which the nuDMP1 is generated and subsequently translocated into the nuclei. Aim 2 will investigate the function of the nuDMP1 in odontoblast differentiation in vivo by generating and characterizing the 3.6 kb Col1a1-nuDMP1 transgenic mice. Aim 3 will determine how Dspp expression is activated through nuDMP1 and if the failure of this process is solely responsible for the tooth phenotype in Dmp1-null mice. Successful completion of this novel proposal will provide unique insights into how DMP1 regulates cell differentiation via the nuDMP1, which is independent of its function in regulating matrix mineralization. The identification of this novel nuDMP1 and its function will not only help elucidate the molecular mechanisms through which DMP1 controls the formation of tooth and bone but will also provide a scientific basis for developing therapies for treatment of hypophosphatemic rickets.