Identification and Function of nuDMP1 in Odontoblast Differentiation

描述（由申请人提供）：人类和小鼠牙本质基质蛋白1 （DMP1）功能的丧失导致常染色体隐性低磷佝偻病（ARHR），其特征是牙本质和骨骼的低矿化，低磷血症和循环成纤维细胞生长因子23 （FGF23）水平升高。缺乏dmp1的受试者的牙齿和骨骼缺陷归因于dmp1缺失的成牙细胞和成骨细胞/骨细胞的内在分化缺陷（局部效应）和低磷血症（全身效应）的综合作用。然而，DMP1功能的丧失是如何导致成牙髓细胞和成骨细胞/骨细胞分化缺陷的，在很大程度上仍然未知。本应用程序的目的是鉴定DMP1的核异构体（称为“nuDMP1”），并研究其在成牙细胞分化中的功能。我们假设nuDMP1是从另一个下游的帧内AUG启动密码子翻译而来，并通过调节Dspp的表达来控制成牙细胞分化。这一假设是基于我们最近在体外和体内的发现，其中包括：1)细胞核DMP1在转染表达全长DMP1和内质网（ER）进入信号肽序列的构建体的细胞中观察到；2)表达全长DMP1而不含er进入信号肽序列的构建体产生两种蛋白，全长DMP1和推定的nuDMP1；3)产生nuDMP1的构建体比产生全长DMP1的构建体更能刺激Dspp启动子活性，无论DMP1是否含有er进入信号肽；(4)由3.6 kb Col1a1启动子驱动的转基因过表达DSPP可修复dmp1缺失小鼠的牙缺损，但不能修复dmp1缺失小鼠的其他表现。为了验证我们的中心假设，提出了三个具体目标。目的1将确定nuDMP1产生并随后转运到细胞核中的机制。目的2将通过生成3.6 kb的Col1a1-nuDMP1转基因小鼠，研究nuDMP1在体内成牙细胞分化中的功能。目的3将确定Dspp表达是如何通过nuDMP1激活的，以及该过程的失败是否是dmp1缺失小鼠牙齿表型的唯一原因。成功完成这一新颖的提议将为DMP1如何通过nuDMP1调节细胞分化提供独特的见解，nuDMP1是独立于其调节基质矿化的功能的。这一新的nuDMP1及其功能的发现，不仅有助于阐明DMP1控制牙齿和骨骼形成的分子机制，也将为开发低磷血症佝偻病的治疗方法提供科学依据。

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.