A Critical Role of DMP-1 in Mineralization

描述(由申请人提供)： 骨矿化的机制目前尚不清楚。有人认为，矿化是一个被动的物理化学过程，主要受基质GLA蛋白等分子的抑制作用控制。相反，有人提出矿化是一个由细胞外基质(ECM)蛋白调控和启动或触发的活跃过程。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(DMP-1)的小鼠表现出明显的骨软化/软骨病表型，矿化受损。在这些小鼠中，很大比例的骨骼仍未矿化，导致骨骼脆弱、变形，并损害骨折愈合。最近的生化研究表明，DMP-1必须经过蛋白质降解才能产生37 kDa和57 kDa的片段，这两个片段是其生物活性的关键。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DMP-1，一种矿化组织特有的ECM蛋白，通过其不同的蛋白水解性处理形式，在控制矿化从而控制骨重建中发挥关键作用。为了验证这一假说，将使用分子和转基因方法来确定DMP-1生物活性片段的过度表达的影响，并确定这些片段拯救DMP-1缺失小鼠的表型的能力。在目标1中，将详细描述缺乏DMP-1的小鼠的矿化缺陷及其与骨骼成熟度的关系。目的2采用体外过表达和补救的方法，研究完整的DMP-1、37 kDa和57 kDa的DMP-1片段以及突变后的阻止切割的DMP-1在成骨细胞分化和矿化中的作用。在目标3中，利用Drop-1基因缺失的小鼠，通过转基因过表达和挽救方法来确定完整的、突变的和裂解的DMP-1片段的体内功能。这些研究将确定DMP-1作为矿化调节因子所必需的重要功能结构域，并可能突出其作为其他骨软化/软骨病模型中观察到的矿化缺陷的共同介导物的潜在作用。这些研究的完成将加深我们对骨矿化分子机制的理解，并将为治疗骨矿化异常疾病(如骨软化症、软骨病)和代谢性骨病(如骨质疏松)找到新的治疗靶点。

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.