A Critical Role of DMP-1 in Mineralization

DMP-1 在矿化中的关键作用

• 批准号: 6829445
• 负责人: JIAN Q. FENG
• 金额: $ 28.07万
• 依托单位: UNIVERSITY OF MISSOURI KANSAS CITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-08 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6829445
• 关键词: Raman spectrometry; bone density; cell differentiation; cell line; densitometry; developmental genetics; extracellular matrix proteins; fluorescent dye /probe; gene deletion mutation; gene expression; genetic regulation; genetically modified animals; infrared spectrometry; interferometry; laboratory mouse; morphometry; normal ossification; osteoblasts; osteogenesis; protein structure function; proteolysis; transcription factor

DESCRIPTION (provided by applicant): The mechanisms by which bone mineralizes are currently unclear. It has been proposed that mineralization is a passive physicochemical process that is controlled mainly at the level of inhibition by molecules such as Matrix Gla Protein. Conversely, it has been proposed that mineralization is an active process that is regulated and initiated or triggered by extracellular matrix (ECM) proteins. Our data supports the latter hypothesis, as we have shown that mice lacking the bone ECM protein, Dentin matrix protein-1 (DMP-1) exhibit a dramatic osteomalacia/rickets phenotype with impaired mineralization. In these mice, a large percentage of the bone remains unmineralized, leading to bone fragility, deformation, and impaired fracture healing. Recent biochemical studies suggest that DMP-1 must be proteolytically processed to yield fragments of 37kDa and 57kDa that are responsible for its bioactivity. Preliminary data suggest that the 57kDa fragment is a nucleator of hydroxyapatite formation. Therefore, our central hypothesis is that DMP-1, an ECM protein specific for mineralizing tissues, plays a key role in controlling mineralization and consequently bone remodeling through its different, proteolytically processed forms. To test this hypothesis, molecular and transgenic approaches will be used to determine the effects of overexpression of bioactive fragments of DMP-1 as well as to determine the ability of these fragments to rescue the phenotype of Dmp-1 null mice. In aim 1, the mineralization defects in mice lacking DMP-1 will be characterized in detail and in relation to skeletal maturity. In aim 2 the function of intact DMP-1, the 37kDa and 57kDa DMP-1 fragments as well as Dmp-1 that is mutated to prevent cleavage will be determined in osteoblast differentiation and mineralization using in vitro overexpression and rescue approaches. In aim 3 the in vivo function of intact, mutated, and cleaved fragments of DMP-1 will be determined by transgenic overexpression and rescue approaches using the Drop-1 null mice. These studies will identify the important functional domains of DMP-1 essential for its role as a regulator of mineralization and may highlight its potential role as a common mediator for mineralization defects observed in other models of osteomalacia/rickets. Completion of these studies will enhance our understanding of the molecular mechanisms of bone mineralization and will identify novel targets for therapeutic intervention in diseases of abnormal bone mineralization such as osteomalacia, rickets, and metabolic bone disease such as osteoporosis.

描述（由申请人提供）： 骨矿化的机制目前尚不清楚。已经提出矿化是一种被动的物理化学过程，其主要由诸如基质玻璃蛋白的分子在抑制水平上控制。相反，已经提出矿化是由细胞外基质（ECM）蛋白调节和启动或触发的主动过程。我们的数据支持后一种假设，因为我们已经表明，缺乏骨ECM蛋白，牙本质基质蛋白-1（Dentin matrix protein-1，DNT-1）的小鼠表现出显着的骨软化症/佝偻病表型，矿化受损。在这些小鼠中，很大一部分骨保持未矿化，导致骨脆性、变形和骨折愈合受损。最近的生物化学研究表明，β-l必须被蛋白水解加工以产生负责其生物活性的37 kDa和57 kDa的片段。初步数据表明，57 kDa片段是羟基磷灰石形成的成核剂。因此，我们的中心假设是，ECM-1，矿化组织特异性的ECM蛋白，通过其不同的蛋白水解加工形式在控制矿化和骨重塑中起着关键作用。为了检验这一假设，将使用分子和转基因方法来确定过表达Dmp-1生物活性片段的影响，以及确定这些片段拯救Dmp-1缺失小鼠表型的能力。在目标1中，将详细表征缺乏β-l的小鼠中的矿化缺陷并与骨骼成熟度相关。在目标2中，将使用体外过表达和拯救方法确定完整的Dmp-1、37 kDa和57 kDa Dmp-1片段以及突变以防止切割的Dmp-1在成骨细胞分化和矿化中的功能。在目的3中，将使用Drop-1缺失小鼠通过转基因过表达和拯救方法来确定完整的、突变的和切割的Drop-1片段的体内功能。这些研究将确定重要的功能域的β-1作为矿化的调节剂的作用是必不可少的，并可能突出其潜在的作用，作为一个共同的调解人的矿化缺陷中观察到的其他模型的骨软化症/佝偻病。这些研究的完成将提高我们对骨矿化的分子机制的理解，并将确定新的治疗干预异常骨矿化的疾病，如骨软化症，佝偻病和代谢性骨病，如骨质疏松症的目标。