DYNAMICS OF ASSEMBLY OF BONE MATRIX PROTEINS

描述（由申请人提供）：细胞外基质（ECM）通常被视为为细胞和组织提供支持的静态支架。然而，最近的研究表明，ECM分子形成高度动态的结构，其响应于细胞运动而持续地经历运动和变形。越来越多的证据表明，ECM蛋白也可能是生长因子活性的主要调节因子。纤连蛋白是最早组装到基质中的ECM蛋白之一，并促进其他ECM蛋白的组装。使用纤连蛋白空细胞模型，我们发现纤连蛋白是必不可少的组装多种骨ECM蛋白，并要求成骨细胞矿化，但不分化。纤连蛋白对于将潜在的TGF β结合蛋白-1（LTBP 1）（TGF β的重要调节剂）组装到ECM中也是关键的。此外，我们最近在活成骨细胞中的动态成像研究表明，细胞运动在骨ECM组装和重组中具有新的作用。 拟议的研究围绕两个主要假设。首先是纤连蛋白是成骨细胞功能的多功能调节剂，其作用是作为骨ECM蛋白组装的协调者，并通过调节生长因子活性。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，将使用体外和体内方法。在目的1中，我们将确定纤连蛋白在成骨细胞功能中的作用，通过其作为骨ECM蛋白组装的调节剂。纤连蛋白缺失的成骨细胞培养模型将与条件性敲除方法结合使用，以删除成骨细胞谱系中的纤连蛋白。在目的2中，我们将确定纤连蛋白通过与LTBP 1的相互作用在调节TGF β 1在骨中的活性中的作用。这将使用无纤连蛋白的成骨细胞以及可用于测量体内TGF β活性的新型TGF β报告小鼠系来完成。在目标3中，我们将确定骨ECM蛋白的组装和重组的动力学及其与纤连蛋白的相互作用，并确定细胞运动在ECM组装和重组中的作用。这将使用骨ECM蛋白的动态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。这些研究将为骨ECM蛋白的组装机制提供新的见解，并为ECM调节骨中TGF β的复杂分子途径提供新的见解。所产生的数据将对与TGF β 1失调相关的疾病，如纤维化疾病、骨质疏松症、关节炎和癌症具有重要意义。

DESCRIPTION (provided by applicant): The extracellular matrix (ECM) has classically been viewed as a static scaffold that provides support to cells and tissues. However, recent studies have shown that ECM molecules form highly dynamic structures that continually undergo movement and deformation in response to cell movement. Evidence is accumulating that ECM proteins may also be major regulators of growth factor activity. Fibronectin is one of the earliest ECM proteins to be assembled into the matrix and facilitates assembly of other ECM proteins. Using a fibronectin null cell model we have found that fibronectin is essential for assembly of multiple bone ECM proteins and is required for osteoblast mineralization but not differentiation. Fibronectin is also critical for assembly of latent TGF( binding protein-1 (LTBP1), an important regulator of TGF(, into the ECM. In addition, our recent dynamic imaging studies in living osteoblasts have suggested novel roles for cell movement in bone ECM assembly and reorganization. The proposed studies are centered around two main hypotheses. The first is that fibronectin is a multifunctional regulator of osteoblast function through its effects as an orchestrator of assembly of bone ECM proteins and through 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 complimentary in vitro and in vivo approaches will be used. In Aim 1 we will determine the role of fibronectin in osteoblast function through its role as a regulator of assembly of bone ECM proteins. Fibronectin-null osteoblast culture models will be used in conjunction with a conditional knockout approach to delete fibronectin in the osteoblast lineage. In Aim 2 we will determine the role of fibronectin in regulating TGF( activity in bone via interactions with LTBP1. This will be done using fibronectin null osteoblasts as well as a novel TGF( reporter mouse line that can be used to measure in vivo TGF( activity. In Aim 3 we will determine the dynamics of assembly and reorganization of bone ECM proteins and their interactions with fibronectin and determine the role of cell movement in ECM assembly and reorganization. This will be done using dynamic molecular imaging of bone ECM proteins together with quantification of cell and fibril dynamics by computational analysis. These studies will provide novel insights into the mechanisms of assembly of bone ECM proteins and provide new insights into the complex molecular pathways for ECM regulation of TGF( in bone. The data generated will have important implications for diseases associated with misregulation of TGF(, such as fibrotic diseases, osteoporosis, arthritis and cancer.