Thrombin stimulates inositol phosphate production and intracellular free calcium by a pertussis toxin-insensitive mechanism in osteosarcoma cells

Human α-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, GTPγS increased [³H]IP production, whereas GDPβS inhibited the response to both GTPγS and thrombin, indicating involvement of a G-protein in thrombin action. Thrombin produced a dose-dependent increase in intracellular free calcium (Ca_i ²⁺) 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, Ca_i ²⁺, 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 Ca_i ²⁺ 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.