The overall goal of this proposal is to understand the role of signals resulting from cell-extracellular matrix (ECM) interactions in the differentiation of osteoblasts. Considerable data support the idea that the osteoblast lineage is derived from a multipotential mesenchymal stem cell that also has the capacity to form other connective tissue cell types. Several multipotential cell lines have recently been established that can differentiate in vitro into cells with osteoblastic characteristics, given the appropriate stimulus. The recently characterized rat C26 osteoblastic cell line expresses very low levels of early markers of osteoblastic differentiation, such as alkaline phosphatase, and forms myotubes at confluence. However, C26 cells can be stimulated to exhibit a differentiated osteoblastic phenotype either by culture in type I collagen gels or exposure to BMP-2, a TGF-beta-like growth factor. Based on these data, the hypothesis to be tested is the specific cell-ECM interactions, mediated by members of the integrin family of ECM receptors, play a critical role in promoting osteoblastic differentiation. One mechanism by which this might occur predicts that ECM ligands, via their integrin receptors, regulate expression of TGF- beta-like growth factors. Alternatively integrin-ECM interactions and TGF-beta-like growth factors might play a cooperative or synergistic role in osteoblastic differentiation. At present, little is known about the identities of specific cell surface ECM receptors expressed at critical stages in the differentiation of OB, or about their targets in the matrix. In addition, the mechanisms by which such cell-ECM interactions might signal mesenchymal precursors to differentiate along the osteoblastic pathway and to maintain a stable osteoblastic phenotype are poorly understood. This proposal is designed to increase understanding of these important areas. Specifically, we will: 1) Characterize the osteogenic response of C26 cells to selected ECM constituents, with the goal of optimizing ECM-induced osteoblastic differentiation of C26 cells; 2) test the hypothesis that interactions between ECM and specific integrin receptors are required for osteoblastic differentiation; 3) determine whether particular integrin-ECM interactions regulate expression of TGF-beta-like growth factors during osteoblastic differentiation; 4) determine whether selected members of the TGF-beta superfamily and ECM constituents act synergistically to promote osteoblastic differentiation of C26 cells; 5) test the hypothesis that accumulation and stabilization of the characteristic osteoblastic ECM involves regulation of matrix-metalloproteinases and their inhibitors by specific integrin-ECM interactions. Greater understanding of the mechanisms by which osteoblast differentiation is regulated should lead to development of more effective materials for the restoration of skeletal deficiencies resulting from aging, arthritis, surgery, trauma and congenital defects.
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