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.
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