Bone matrix RGD glycoproteins: immunolocalization and interaction with human primary osteoblastic bone cells in vitro.

The interaction of cells with extracellular matrix is essential for their anchorage, proliferation, migration, and differentiation. In bone matrix there are multiple glycoproteins that contain the integrin-binding RGD sequence: fibronectin (FN), thrombospondin (TSP), osteopontin (OPN), bone sialoprotein (BSP), type I collagen (COLL I), and vitronectin (VN). In this study, the localization of TSP, FN, VN, and several integrins within developing human long bone using immunohistochemical methods was examined, as was the effect of all bone RGD proteins on the adhesion of human osteoblastic cells. Thrombospondin, fibronectin, and vitronectin showed distinct localization patterns within bone tissue. TSP was found mainly in osteoid and the periosteum; VN appeared to be present mainly in mature bone matrix. FN was present in the periosteum as well as within both mature and immature bone matrix. Using a panel of antiintegrin antibodies we found that bone cells in vivo and in vitro express alpha 4, alpha v, alpha 5 beta 1, alpha v beta 3, and beta 3/beta 5 integrins, and these receptors are for the most part expressed on all bone cells at different stages of maturation with quantitative rather than qualitative variations, with the exception of alpha 4, which is expressed mainly by osteoblasts. Cell attachment assays were performed using primary human cells of the osteoblastic lineage under serum-free conditions. COLL I, TSP, VN, FN, OPN, and BSP promoted bone cell attachment in a dose-dependent manner and were equivalent in action when used in equimolar concentrations. In the presence of GRGDS peptide in the medium, the adhesion to BSP, OPN, and VN was almost completely blocked (10, 10, and 15% of control, respectively), and attachment to FN, COLL I, and TSP was only slightly decreased (80, 75, and 55%, respectively). These results suggest that human bone cells may use RGD-independent mechanisms for attachment to the latter glycoproteins.