Vitamin K, Bone, and Arterial Calcification

描述（由申请方提供）：在骨骼外的钙化组织中发现骨特异性蛋白，包括维生素K依赖性蛋白，表明异位钙化的机制与骨骼中的骨生成相似。细胞分化成骨形成细胞（成骨细胞）是钙化过程的核心。因此，了解成骨细胞分化的相关因素以及这一过程是如何调节的，对于旨在预防动脉系统病理性钙化的治疗干预至关重要。这项资助的主要重点是了解维生素K依赖蛋白，基质玻璃蛋白（MGP），在骨形成的参与。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，它在钙化过程中的调节作用已被证明依赖于蛋白质的维生素K修饰，这将其功能与维生素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是骨形态发生蛋白-2（BMP-2）的结合蛋白，BMP-2是一种生长因子，可将动脉壁中的间充质细胞和血管平滑肌细胞亚群转化为骨形成细胞。我们建议，MGP调节BMP-2的生长因子活性。我们证明，Gla区MGP是BMP-2的结合位点，并假设维生素K修饰的MGP是必不可少的中和BMP-2的生长因子活性。我们提出实验来测试这一假设在细胞培养和大鼠模型中，动脉钙化可以诱导的维生素K拮抗剂华法林。MGP和BMP-2之间的结合相互作用将用表面等离子体共振（SPR）研究。MGP是一种不溶性的14 kDa基质蛋白。我们表明，MGP的细胞内形式出现作为一个可溶性的50 kDa的蛋白质，我们提出的实验，以揭示这种前体是如何加工成其14 kDa的不溶性形式。将使用细胞蛋白的35 S-Met标记结合蛋白质组学MS/MS方法。我们认为氧化应激损伤了动脉粥样硬化斑块中的维生素K依赖性γ-羧化系统和老化的血管壁，导致MGP的γ-羧化不足和病理性钙化的发生。这一假设将在兔动脉粥样硬化模型和老龄大鼠中进行测试。拟议的工作将提供BMP-2生长因子介导的钙化的基础知识，因为它与脂溶性维生素（维生素K）的功能和维生素K依赖性蛋白的生物合成有关。

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.