Dynamics of Assembly of Bone Matrix Proteins

描述（由申请人提供）：细胞外基质（ECM）被视为支持细胞和组织的静态三维支架。然而，我们最近在活成骨细胞中的分子成像研究表明，ECM是高度动态的，ECM分子形成的结构不断经历运动和变形，由细胞产生的机械力介导。这些研究还表明细胞运动在ECM组装和重组中的新作用。纤连蛋白是最早组装到ECM中的蛋白质之一，并且促进其他基质蛋白的组装。在之前的资助周期中，使用纤连蛋白空细胞培养模型和成骨细胞中的靶向基因缺失，表明纤连蛋白对于多种骨ECM组分的组装是必不可少的，包括I型胶原蛋白、胶原蛋白-1、潜伏性TGF 2结合蛋白-1、核心蛋白聚糖和双糖蛋白聚糖，并且也是正常矿化所需的。纤连蛋白耗竭也抑制成骨细胞分化。纤连蛋白对分化的影响可以通过补充BMP 2来挽救，而其对ECM组装和矿化的影响则不能，这表明纤连蛋白可以通过ECM靶向成骨生长因子来调节成骨细胞分化。基于这些观察，拟议的研究集中在两个主要假设。首先，纤连蛋白是成骨细胞分化和功能的多功能调节剂，通过其作为骨ECM蛋白组装的中央协调器的作用，并通过其在ECM调节生长因子活性中的作用。第二，动态细胞运动对于骨ECM蛋白的组装和重组是必不可少的。为了检验这些假设，将结合活细胞成像使用体外和体内方法。目的1将定义骨ECM蛋白的级联组装及其与细胞和基质动力学的整合。这将使用纤连蛋白空成骨细胞模型结合骨ECM蛋白的活细胞分子成像以及通过计算分析定量细胞和原纤维动力学来完成。目的2进一步阐明纤维连接蛋白通过调控BMP信号通路在成骨细胞分化中的作用。活细胞成像技术也将与成骨细胞/骨细胞谱系报告基因和ECM组分的荧光探针一起使用，以确定成骨细胞分化和BMP信号传导如何与ECM组装和重组动态整合。目的3将使用新型成像探针来确定成骨细胞ECM中胶原组装的动力学以及纤连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为骨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。所产生的数据将显着推进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，骨生成障碍和ECM蛋白相关的骨疾病等骨骼疾病具有关键意义。 公共卫生相关性：这项研究与公共卫生有关，因为它将为骨细胞外基质（即沉积矿物质的细胞外蛋白质支架）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沉积在活骨细胞中的组装过程，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推进我们对骨形成过程的机械理解，因此该研究对骨质疏松症，骨生成障碍和代谢性骨疾病的治疗具有意义。

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.