Vitamin K, Bone, and Arterial Calcification

维生素 K、骨骼和动脉钙化

基本信息

批准号：
7092645
负责人：
REIDAR WALLIN
金额：
$ 31.53万
依托单位：
WAKE FOREST UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-07-01 至 2008-12-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Bone specific proteins, including vitamin K-dependent proteins, are found in calcified tissues outside the skeleton suggesting that the mechanism of ectopic calcification is similar to osteogenesis in the skeleton. Differentiation of cells into bone forming cells (osteoblasts) is central to the calcification process. Therefore, an understanding of the factors involved in osteoblast differentiation and how this process is regulated is essential for therapeutic interventions designed to prevent pathological calcification of the arterial system. The main focus of this grant is to understand the involvement of the vitamin K-dependent protein, Matrix Gla protein (MGP), in bone formation. Recent experiments have demonstrated that MGP is an important calcification inhibitor of the arterial system and cartilage. Importantly, its regulatory role in the calcification process has been shown to be dependent upon the vitamin K modification of the protein, which links its function to vitamin K metabolism, function and vitamin K nutrition. We have been able to show that MGP is a binding protein for bone morphogenetic protein-2 (BMP-2), a growth factor that will transform mesenchymal cells and subpopulations of vascular smooth muscle cells in the arterial wall into bone forming cells. We propose that MGP regulates the growth factor activity of BMP-2. We demonstrate that the Gla region on MGP is a binding site for BMP-2 and hypothesize that the vitamin K modification of MGP is essential for neutralizing the growth factor activity of BMP-2. We propose experiments to test this hypothesis in cell culture and in a rat model where arterial calcification can be induced by the vitamin K antagonist warfarin. Binding interactions between MGP and BMP-2 will be studied with Surface Plasmon Resonance (SPR). MGP is an insoluble 14 kDa matrix protein. We show that an intracellular form of MGP appears as a soluble 50 kDa protein and we propose experiments to reveal how this precursor is processed into its 14 kDa insoluble form. 35S-Met labeling of cell protein combined with a proteomic MS/MS approach will be used. We propose that oxidative stress damages the vitamin K-dependent gamma-carboxylation system in atherosclerotic plaques and the aging vessel wall resulting in inadequate gamma-carboxylation of MGP and onset of pathological calcification. This hypothesis will be tested in a rabbit atherosclerosis model and in aging rats. The proposed work will provide basic knowledge of BMP-2 growth factor mediated calcification as it is linked to the function of a fat-soluble vitamin (vitamin K) and biosynthesis of vitamin K-dependent proteins.

描述（由申请人提供）：在骨骼之外的钙化组织中发现了骨特异性蛋白，包括维生素K依赖性蛋白，这表明异位钙化的机制与骨骼中的骨生成相似。细胞分化为骨形成细胞（成骨细胞）是钙化过程的核心。因此，了解成骨细胞分化涉及的因素以及如何调节该过程对于旨在防止动脉系统病理钙化的治疗干预措施至关重要。该赠款的主要重点是了解维生素K依赖性蛋白基质GLA蛋白（MGP）的参与。最近的实验表明，MGP是动脉系统和软骨的重要钙化抑制剂。重要的是，其在钙化过程中的调节作用已被证明取决于蛋白质的维生素K修饰，该蛋白质将其功能与维生素K代谢，功能和维生素K营养联系起来。我们已经能够证明MGP是一种用于骨形态发生蛋白-2（BMP-2）的结合蛋白，这是一种生长因子，它将转化中充质细胞和动脉壁中血管平滑肌细胞的亚群体变成骨形成细胞。我们建议MGP调节BMP-2的生长因子活性。我们证明，MGP上的GLA区域是BMP-2的结合位点，并假设MGP的维生素K修饰对于中和BMP-2的生长因子活性至关重要。我们提出了实验，以在细胞培养和大鼠模型中检验该假设，在大鼠模型中，维生素K拮抗剂华法林可以诱导动脉钙化。将使用表面等离子体共振（SPR）研究MGP和BMP-2之间的结合相互作用。 MGP是一种不溶性14 kDa基质蛋白。我们表明，MGP的细胞内形式似乎是一种可溶性50 kDa蛋白，我们提出了实验，以揭示该前体是如何将其处理成其14 kDa不溶性形式的。将使用35S-MET的细胞蛋白标记与蛋白质组学MS/MS方法结合使用。我们提出氧化应激会损害动脉粥样硬化斑块中维生素K依赖性的γ-羧化系统，而老化血管壁则导致MGP的γ-羧化和病理钙化的发作。该假设将在兔动脉粥样硬化模型和衰老大鼠中进行检验。提出的工作将提供BMP-2生长因子介导的钙化的基本知识，因为它与脂溶性维生素（维生素K）的功能以及维生素K依赖性蛋白的生物合成有关。