Dynamics of Assembly of Bone Matrix Proteins

描述(申请人提供)：细胞外基质(ECM)一直被视为支持细胞和组织的静态三维支架。然而，我们最近在活的成骨细胞中进行的分子成像研究表明，细胞外基质是高度动态的，并且细胞外基质分子形成的结构在细胞产生的机械力的介导下不断发生运动和变形。这些研究还表明了细胞运动在细胞外基质组装和重组中的新作用。纤维连接蛋白是最早组装到细胞外基质中的蛋白质之一，它促进了其他基质蛋白的组装。在之前的资金周期中，利用纤维连接蛋白缺失的细胞培养模型和成骨细胞的靶向基因缺失，研究表明，纤维连接蛋白对多种骨ECM成分的组装是必不可少的，包括I型胶原、纤维蛋白-1、潜伏的TGF2结合蛋白-1、核心蛋白和双聚糖，也是正常矿化所必需的。纤维连接蛋白的缺失也会抑制成骨细胞的分化。纤维连接蛋白对细胞分化的影响可以通过补充BMP2来挽救，而对ECM组装和矿化的影响则不能，提示纤维连接蛋白可能通过ECM靶向成骨生长因子来调节成骨细胞的分化。基于这些观察，拟议的研究围绕两个主要假设展开。首先，纤维连接蛋白是一种多功能的成骨细胞分化和功能调节剂，其作用是作为骨细胞外基质蛋白组装的中心协调者，并通过其在细胞外基质调节生长因子活性中的作用而发挥作用。第二，细胞的动态运动对于骨细胞外基质蛋白的组装和重组是必不可少的。为了验证这些假说，体外和体内方法将与活细胞成像相结合。目标1将定义骨细胞外基质蛋白组装的级联及其与细胞和基质动力学的整合。这将使用纤维连接蛋白缺失的成骨细胞模型，结合骨细胞外基质蛋白的活细胞分子成像，并通过计算分析来量化细胞和纤维动力学。目的2将进一步明确纤维连接蛋白在成骨细胞分化中的作用，通过调节BMP信号。活细胞成像技术还将用于成骨细胞/骨细胞谱系报告和细胞外基质成分的荧光探针，以确定成骨细胞分化和BMP信号如何与细胞外基质组装和重组动态整合。目的3将使用新型的成像探针来确定胶原在成骨细胞ECM中的组装动态，以及纤维连接蛋白在体外和体内胶原沉积中的作用。这些研究将从动态的角度对骨ECM的组装机制以及ECM如何调节成骨细胞功能提供基本的见解。这些数据将大大提高我们对骨形成的分子和动力学机制的理解，并对骨质疏松症、关节炎、成骨不全和与细胞外基质蛋白相关的骨病具有重要意义。 与公共健康相关：这项研究与公共健康相关，因为它将为骨细胞外基质(即细胞外沉积矿物质的蛋白质支架)组装的动态过程提供非常新颖的见解。我们将首次动态成像关键骨基质蛋白在活的骨细胞中沉积的组装过程，并确定细胞的运动特性如何有助于构建用于矿物质沉积的蛋白质支架。由于这些研究将大大提高我们对骨形成过程的机制的理解，因此该研究对治疗骨质疏松症、成骨不全和代谢性骨病具有重要意义。

DESCRIPTION (provided by applicant): The extracellular matrix (ECM) has been viewed as a static three dimensional scaffold that supports cells and tissues. However, our recent molecular imaging studies in living osteoblasts have shown that the ECM is highly dynamic and that ECM molecules form structures that continually undergo movement and deformation, mediated by cell-generated mechanical forces. These studies also suggest a novel role for cell movement in ECM assembly and reorganization. Fibronectin is one of the earliest proteins to be assembled into the ECM and facilitates assembly of other matrix proteins. In the previous funding cycle, using fibronectin null cell culture models and targeted gene deletion in osteoblasts, it was shown that fibronectin is essential for assembly of multiple bone ECM components, including type I collagen, fibrillin-1, Latent TGF2 binding protein-1, decorin and biglycan and is also required for normal mineralization. Fibronectin depletion also inhibits osteoblast differentiation. Fibronectin's effects on differentiation can be rescued by supplementation with BMP2, whereas its effects on ECM assembly and mineralization cannot, suggesting that fibronectin may regulate osteoblast differentiation via ECM targeting of osteogenic growth factors. Based on these observations, the proposed studies are centered on two main hypotheses. The first is that fibronectin is a multifunctional regulator of osteoblast differentiation and function through its effects as a central orchestrator for assembly of bone ECM proteins and through its role in ECM regulation of growth factor activity. The second is that dynamic cell movement is essential for the assembly and reorganization of bone ECM proteins. To test these hypotheses, in vitro and in vivo approaches will be used in combination with live cell imaging. Aim 1 will define the cascade of assembly of bone ECM proteins and its integration with cell and matrix dynamics. This will be done using fibronectin null osteoblast models in conjunction with live cell molecular imaging of bone ECM proteins and quantification of cell and fibril dynamics by computational analysis. Aim 2 will further define the role of fibronectin in osteoblast differentiation through regulation of BMP signaling. Live cell imaging techniques will also be used with osteoblast/osteocyte lineage reporters and fluorescent probes for ECM components to determine how osteoblast differentiation and BMP signaling is dynamically integrated with ECM assembly and reorganization. Aim 3 will use novel imaging probes to determine the dynamics of collagen assembly into the ECM of osteoblasts and the role of fibronectin in collagen deposition in vitro and in vivo. The studies will provide fundamental insights, from a dynamic perspective, into the mechanisms of assembly of bone ECM and how the ECM regulates osteoblast function. The data generated will significantly advance our understanding of the molecular and dynamic mechanisms underlying bone formation and have key implications for skeletal diseases such as osteoporosis, arthritis, osteogenesis imperfecta and bone diseases related to ECM proteins. PUBLIC HEALTH RELEVANCE: This research is relevant to public health as it will provide highly novel insights into the dynamic processes by which the bone extracellular matrix (i.e. the protein scaffold outside the cells onto which mineral is deposited) is assembled. We will for the first time dynamically image the assembly process by which key bone matrix proteins are deposited in living bone cells and determine how the motile properties of the cells contribute to the building of the protein scaffold for mineral deposition. As these studies will significantly advance our mechanistic understanding the process of bone formation, the research has implications for treatment of osteoporosis, osteogenesis imperfecta and metabolic bone diseases.