guanosine-5--o-(2-thiodiphosphate) and Osteosarcoma

Human alpha-thrombin is known to elicit bone resorption in vitro and has been proposed as a mediator of increased bone turnover in inflammatory diseases. We used UMR 106-H5 rat osteoblast-like osteosarcoma cells to explore the signal transduction mechanism utilized by thrombin in bone. Thrombin produced a dose-dependent increase in the accumulation of [3H]inositol phosphates (IPs) in UMR 106-H5 cells prelabeled with [3H]myo-inositol (EC50 15 U/ml). In saponin-permeabilized cells, GTP gamma S increased [3H]IP production, whereas GDP beta S inhibited the response to both GTP gamma S and thrombin, indicating involvement of a G-protein in thrombin action. Thrombin produced a dose-dependent increase in intracellular free calcium (Cai2+) in UMR 106-H5 cells (EC50 1 U/ml; maximal increase 4-fold), as well as a small (20%) increase in [3H]thymidine incorporation. Treatment of UMR 106-H5 membranes with pertussis toxin (PT) and [32P]NAD+ resulted in labeling of a 40-kDa protein. However, pretreatment of cells with a dose of PT sufficient to produce maximal endogenous labeling of this protein failed to influence thrombin action on IP accumulation, Cai2+, or [3H]thymidine incorporation. In contrast, PT treatment of CCL39 hamster lung fibroblasts significantly blunted thrombin-stimulated [3H]IP accumulation and [3H]thymidine incorporation. These results suggest that thrombin raises Cai2+ in UMR 106-H5 cells by activating polyphosphoinositide-specific phospholipase C. Whereas in fibroblasts and platelets, thrombin receptors appear to couple to both PT-sensitive and PT-insensitive G-proteins, only a PT-insensitive G-protein appears to mediate thrombin action in UMR 106-H5 cells. Either these cells lack the relevant PT-sensitive G-protein or they possess thrombin receptors that selectively couple to a pertussis toxin-insensitive G-protein.

Topics: Adenosine Diphosphate Ribose; Animals; Calcium; Cricetinae; Fibroblasts; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Inositol; Inositol Phosphates; Kinetics; Osteosarcoma; Pertussis Toxin; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases; Rats; Signal Transduction; Thionucleotides; Thrombin; Tumor Cells, Cultured; Virulence Factors, Bordetella