Thrombospondin (TSP1) mediates in vitro proliferation of human MG-63 osteoblastic cells induced by α-thrombin

Abstract

Thrombospondin (TSP) is a 450-kDa glycoprotein synthesized and secreted by human MG-63 osteoblastic cells. In this study, we have first studied the effect of α-thrombin on TSP expression by human MG-63 cells. In situ hybridization indicated that TSP mRNA level in thrombin-treated MG-63 cells was increased when compared to unstimulated cells. As judged by immunofluorescence, thrombin-treatment of MG-63 cells resulted in increased cell surface expression of TSP when compared to quiescent cells. Because thrombin stimulates proliferation of osteoblastic cells, the involvement of TSP in proliferation of thrombin-stimulated osteoblastic cells was then investigated using a serum-free mitogenesis assay. Both α-thrombin (0.01 to 0.15 U ml ) and TSP (5 to 600 ng ml ) caused a dose-dependent increase in [ 3H]thymidine incorporation by MG-63 cells. Proliferation of osteoblastic cells induced by α-thrombin or TSP was specifically and totally inhibited by anti-TSP monoclonal antibodies (3–10 μg ml ) or by indomethacin (1 μM), an inhibitor of prostaglandin synthesis. Anti-TSP antibodies which inhibited cell proliferation also inhibit TSP expression to the surface of these cells. Our experiments support the existence of a mechanism whereby TSP bound to the cell surface of thrombin-treated MG-63 cells stimulates secretion of prostaglandins which, in turn, allow cell proliferation to proceed.