The growth-supportive effect of thrombospondin (TSP1) and the expression of TSP1 by human MG-63 osteoblastic cells are both inhibited by dexamethasone.

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]thymidine incorporation by MG-63 cells in culture, reaching 40% inhibition of cell proliferation at a concentration of 250 nM. Similarly, the stimulatory effect of TSP (500 ng/ml) 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 vivo and reduce proliferation of osteoblasts in vitro, our results argue for an important role of TSP during bone formation.

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

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 alpha-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 alpha-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 alpha-thrombin or TSP was specifically and totally inhibited by anti-TSP monoclonal antibodies (3-10 micrograms/ml) or by indomethacin (1 microM), 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.

Fatty acid composition and CD36 expression in breast adipose tissue of premenopausal and postmenopausal women.

UNLABELLED: The mammary gland is an organ under complex hormonal and developmental regulation. Among the mediators involved in this process are polypeptides, steroid hormones and certain lipids and their metabolites. The fatty acid content of the breast adipocytes is investigated because of the importance of their role in the proliferation and differentiation of the breast epithelial cells. MATERIALS AND METHODS: To address the hypothesis that polyunsaturated acid might be a risk factor for breast cancer, we examined the association between arachidonic acid (20:4n-6) and menopausal status in the women using lipid extraction from isolated human adipocytes and CD36 detection on a FACS flow cytometer. RESULTS: Compared with the related trigycerides, phospholipids in postmenopausal women contained a higher proportion of polyunsaturated fatty acids like arachidonic acid, a precursor of eicosanoids, required for many physiological roles in reproduction and implicated in the proliferation of several cell types. In order to determine the mechanisms explaining the difference in the fatty acid and long-chain fatty acid composition, we have studied the relationship between CD 36 protein and menopausal status. Menopause in women seemed to be associated with a diminuation of the amount of CD 36 protein of adipocytes (48.82 +/- 3.54% versus 20.01 +/- 0.04%) and also associated with the increased percentage of arachidonic acid observed in postmenopausal women. Our data and others led us to conclude that the variation in the composition of fatty acids can act directly on the phenomena of growth and differentiation of mammary epithelial cells and the variation in CD 36 may act either on the transport of fatty acids or on the transduction of the signal responsible for the stimulation of enzymes catalysing the conversion of arachidonic acid into different metabolites. These two phenomena could influence the risk of breast cancer in pre- and postmenopausal women.