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，并随着细胞的运动而不断地运动和变形。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。纤维连接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。使用纤维连接蛋白零细胞模型，我们发现纤维连接蛋白是多种骨ECM蛋白组装所必需的，并且是成骨细胞矿化所必需的，但不是分化所必需的。纤维连接蛋白对于TGF结合蛋白-1 （LTBP1）组装到ECM中也至关重要，LTBP1是TGF的重要调节因子。此外，我们最近对活成骨细胞的动态成像研究表明，细胞运动在骨ECM组装和重组中的新作用。

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.