Search

Thrombospondin (TSP) is a 450-kDa extracellular matrix glycoprotein which supports the growth of human MG-63 osteoblastic cells [Abbadia et al., FEBS Lett., 329 (1993) 341-346]. In this study, we describe the effect of the glucocorticoid, dexamethasone, on cell proliferation and TSP expression by MG-63 cells. Using a serum-free mitogenesis assay, dexamethasone (25 to 500 nM) caused a dose-dependent decrease in [ 3H]ithymidine incorporation by MG-63 cels in culture, reaching 40% inhibition of cell proliferation at a concentration of 250 nM. Similarly, the stimulatory effect of TSP (500 ngml) on proliferation of MG-63 cells was totally abolished in the presence of dexamethasone (250 nM). In situ hybridization indicated that TSP mRNA level in dexamethasone-treated MG-63 cells decreased compared to quiescent cells. As judged by fluorescence-activated cell sorting analysis, dexamethasone treatment of MG-63 cells resulted in a 50 to 70% decrease in TSP cell surface expression compared to quiescent cells. Secretion of TSP in the culture fluid of dexamethasone-treated MG-63 cells also decreased by 40% while, under similar experimental conditions, a 180% increase in alkaline phosphatase activity was observed in dexamethasone-treated cells. Because glucocorticoids induce osteoporosis in vivoand reduce proliferation of osteoblasts in vitro, our results argue for an important role of TSP during bone formation.

The structural and immunological properties of human thrombospondins isolated from platelets and from endothelial cells were compared. Both thrombospondins were digested with either trypsin or thermolysin, in the presence or absence of calcium, then injected onto a Superose 12 gel filtration column. The isolated thermolysin‐generated fragments of thrombospondins were identified by radioimmunoassays using either different monoclonal antibodies or a polyclonal antibody directed against platelet thrombospondin. The results show that platelet and endothelial thrombospondins are both partially protected from trypsin digestion in the presence of calcium but have different trypsin and thermolysin fragmentation patterns. The thermolysin‐generated fragments from platelet and endothelial thrombospondins are recognized differently by a monoclonal antibody whereas all of them are identified by a polyclonal antibody.

Structural differences between platelet and endothelial cell thrombospondin (TBSP) were found in two protease‐resistant domains (70 and 18 kDa). The 70 kDa fragment is involved in the binding of TBSP to fibrinogen and the 18 kDa fragment in the attachment to various cultured cells. Despite these structural differences, platelet and endothelial cell TBSP bound with the same affinity to fibrinogen and mediated the attachment of smooth muscle cells but not of endothelial cells.

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.