Identification and Function of nuDMP1 in Odontoblast Differentiation

nuDMP1在成牙本质细胞分化中的鉴定及其功能

• 批准号: 9038177
• 负责人: Yongbo Lu
• 金额: $ 32.85万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9038177
• 关键词: Ablation; Binding; Biochemistry; Biology; Blood; Bone Matrix; Bypass; Cell Cycle; Cell Differentiation process; Cell Nucleus; Cells; Cephalic; Clinical Treatment; Co-Immunoprecipitations; Defect; Dental; Dentin; Deposition; Differentiation Antigens; Disease; Endoplasmic Reticulum; Failure; Familial hypophosphatemic bone disease; Genetic; Goals; Health; Human; Hypophosphatemia; Image; In Vitro; Initiator Codon; Knockout Mice; Length; Mass Spectrum Analysis; Minerals; Molecular; Mus; Mutagenesis; N-terminal; Nuclear; Nucleic Acid Regulatory Sequences; Odontoblasts; Osteoblasts; Osteocytes; Peptide Signal Sequences; Phenotype; Principal Investigator; Process; Production; Protein Isoforms; Proteins; Research; Research Personnel; Secondary to; Serum; Staging; Testing; Time; Tooth structure; Transgenes; Transgenic Mice; Transgenic Organisms; Translating; Wild Type Mouse; Work; base; bone; dentin matrix protein 1; face bone structure; fibroblast growth factor 23; in vivo; inorganic phosphate; insight; mineralization; novel; osteoblast differentiation; overexpression; promoter; skeletal; therapy development; transcription factor

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.

描述（由申请人提供）：人类和小鼠中牙本质基质蛋白 1 (DMP1) 功能的丧失会导致常染色体隐性遗传性低磷血症性佝偻病 (ARHR)，其特征是牙本质和骨骼矿化不足、低磷血症和循环成纤维细胞生长因子 23 (FGF23) 水平升高。 Dmp1 缺陷受试者的牙齿和骨骼缺陷归因于 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）的综合影响。然而，DMP1功能丧失导致成牙本质细胞和成骨细胞/骨细胞分化缺陷的方式仍不清楚。本申请的目的是鉴定 DMP1 的核亚型（称为“nuDMP1”）并研究其在成牙本质细胞分化中的功能。我们假设 nuDMP1 是从替代下游框内 AUG 起始密码子翻译而来，并通过调节 Dspp 表达来控制成牙本质细胞分化。这一假设基于我们最近的体外和体内研究结果，其中包括：1）在用表达全长 DMP1 和内质网（ER）进入信号肽序列的构建体转染的细胞中观察到核 DMP1； 2)表达全长DMP1但没有ER进入信号肽序列的构建体产生两种蛋白质，全长DMP1和推定的nuDMP1； 3) 产生假定的 nuDMP1 的构建体比产生具有或不具有 ER 进入信号肽的全长 DMP1 的构建体更能有效地刺激 Dspp 启动子活性； 4) 由 3.6 kb Col1a1 启动子驱动的 DSPP 转基因过表达可以挽救 Dmp1 缺失小鼠的牙齿缺陷，但不能挽救其​​他表现。为了检验我们的中心假设，提出了三个具体目标。目标 1 将确定 nuDMP1 生成并随后转位到细胞核中的机制。目标 2 将通过生成和表征 3.6 kb Col1a1-nuDMP1 转基因小鼠来研究 nuDMP1 在体内成牙本质细胞分化中的功能。目标 3 将确定 Dspp 表达如何通过 nuDMP1 激活，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。这项新提案的成功完成将为 DMP1 如何通过 nuDMP1 调节细胞分化提供独特的见解，这与其调节基质矿化的功能无关。这种新型nuDMP1及其功能的鉴定不仅有助于阐明DMP1控制牙齿和骨骼形成的分子机制，而且还将为开发低磷血症性佝偻病的治疗方法提供科学依据。