Molecular Mechanism of Matrix GLA Protein (MGP)

描述(由申请人提供)：钙化在血管疾病中普遍存在，对发病率和死亡率有很大影响。基质GLA蛋白(MGP)是一种钙化抑制因子，在动脉粥样硬化病变中表达上调。MGP基因的缺失会导致小鼠和人类的动脉钙化和形态异常。MGP是一种分泌型蛋白质，通过维生素K依赖的g-羧化修饰。当从骨骼中分离出来时，它是经过加工的，缺乏七个C-末端氨基酸。MGP的发病机制目前知之甚少。然而，我们的实验数据表明，转化生长因子-β(转化生长因子-β)和骨形态发生蛋白(BMP)信号转导中起作用。这些数据提示BMP与转化生长因子-β之间存在潜在的联系，即BMP-2和BMP-4可诱导转化生长因子-β受体激活素样激酶受体1(ALK1)的表达。通过ALK1信号通路诱导MGP，为BMP提供负反馈调节。MGP与特异性受体的相互作用也被观察到。三个假设将在这项拨款中得到检验。第一种假设是MGP在受体水平上调节转化生长因子-β和骨形态发生蛋白信号，导致SMAD信号和基因表达的改变。我们将研究MGP对转化生长因子-β、骨形态发生蛋白-2和骨形态发生蛋白-4诱导的SMAD信号及相关报告基因的激活作用，并确定受其调控的转化生长因子-β/骨形态发生蛋白受体。第二个假设是，MGP参与了特定的蛋白质-蛋白质相互作用，这种相互作用可能会被C-末端处理改变。我们将使用特定的免疫沉淀结合一般的蛋白质组学方法来表征相互作用，其中包括转化生长因子-β受体。第三个假设是MGP在发育中的动脉和动脉粥样硬化病变中作为BMP抑制物发挥作用。我们将产生一只转基因小鼠，用于靶向、有条件地表达BMP抑制物Noggin。在Mgp基因缺失的小鼠中将诱导noggin的表达，试图取代Mgp并消除钙化。诺金的作用也将在低密度脂蛋白受体缺失小鼠的动脉粥样硬化病变中确定。我们的结果将增加对血管和心脏瓣膜钙化的洞察，这是心脏病中的一个常见问题。此外，它还可能提供有关如何为不想要的钙化设计新的治疗方法的信息。

DESCRIPTION (provided by applicant): Calcification is ubiquitous in vascular disease, and contributes significantly to morbidity and mortality. Matrix GLA protein (MGP) is an alleged calcification inhibitor that is up-regulated in atherosclerotic lesions. Deletion of the MGP gene results in arterial calcification and in morphological abnormalities in mice and humans. MGP is a secreted protein that is modified by vitamin K-dependent g-carboxylation. When isolated from bone, it is processed and lacks seven C-terminal amino acids. The mechanism of MGP is poorly understood. However, our experimental data points to a role in transforming growth factor-beta (TGF-beta) and bone morphogenetic protein (BMP) signaling. The data suggest a potential link between BMP and TGF-beta in that BMP2 and BMP-4 induce expression of the TGF-beta, receptor Activin-like Kinase receptor 1 (ALK1). Signaling through ALK1 induces MGP, which provides negative feedback regulation for BMP. Interactions between MGP and specific receptors were also observed. Three hypotheses will be tested in this grant. The first hypothesis is that MGP regulates TGF-beta and BMP signaling at the receptor level, resulting in altered SMAD signaling and gene expression. We will characterize activation of SMAD signaling and relevant reporter genes induced by TGF-beta, BMP-2 and BMP-4 in presence of MGP, and identify the TGF-beta / BMP receptors that are regulated by MGP. The second hypothesis is that MGP participates in specific protein-protein interactions that may be altered by C-terminal processing. We will characterize the interactions, which include the TGF-beta receptors, using specific immunoprecipitation in combination with a general proteomic approach. The third hypothesis is that MGP functions as a BMP inhibitor in developing arteries and in atherosclerotic lesions. We will generate a transgenic mouse for targeted, conditional expression of the BMP-inhibitor Noggin. Noggin expression will be induced in MGP null mice in attempt to replace MGP and abolish calcification. The effect of Noggin will also be determined in atherosclerotic lesions in LDL-receptor null mice. Our results will add insight to calcification of vessels and heart valves, a common problem in heart disease. In addition, it may provide information on how to design new treatments for unwanted calcification.