Vitamin K, Bone, and Arterial Calcification

描述（由申请人提供）：骨特异性蛋白，包括维生素k依赖蛋白，在骨骼外的钙化组织中被发现，这表明异位钙化的机制与骨骼中的成骨相似。细胞分化成成骨细胞（成骨细胞）是钙化过程的核心。因此，了解参与成骨细胞分化的因素以及这一过程是如何被调节的，对于旨在预防动脉系统病理性钙化的治疗干预至关重要。该基金的主要重点是了解维生素k依赖性蛋白基质Gla蛋白（MGP）在骨形成中的作用。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维生素K对蛋白质的修饰，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，可以将动脉壁的间充质细胞和血管平滑肌细胞亚群转化为骨形成细胞。我们认为MGP调节BMP-2的生长因子活性。我们证明了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的生长因子活性是必不可少的。我们建议在细胞培养和大鼠模型中测试这一假设，其中维生素K拮抗剂华法林可以诱导动脉钙化。利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。我们发现MGP的细胞内形式表现为可溶的50 kDa蛋白，我们提出实验来揭示该前体如何被加工成14 kDa的不溶性形式。将使用35S-Met标记细胞蛋白并结合蛋白质组学MS/MS方法。我们提出，氧化应激破坏动脉粥样硬化斑块和血管壁老化中维生素k依赖性γ -羧基化系统，导致MGP γ -羧基化不足和病理性钙化的发生。这一假设将在兔子动脉粥样硬化模型和衰老大鼠中进行验证。拟议的工作将提供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.