Thrombin induces proliferation of osteoblast-like cells through phosphatidylcholine hydrolysis.
We examined the effect of thrombin on phosphatidylcholine-hydrolyzing phospholipase D activity in osteoblast-like MC3T3-E1 cells. Thrombin stimulated the formation of choline dose dependently in the range between 0.01 and 1 U/ml, but not the phosphocholine formation. Diisopropylfluorophosphate (DFP)- inactivated thrombin had little effect on the choline formation. The combined effects of thrombin and 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C-activating phorbol ester, on the choline formation were additive. Staurosporine, an inhibitor of protein kinases, had little effect on the thrombin-induced formation of choline. Combined addition of thrombin and NaF, an activator of heterotrimeric GTP-binding protein, did not stimulate the formation of choline further. Pertussis toxin had little effect on the thrombin-induced formation of choline. Thrombin stimulated Ca2+ influx from extracellular space time and dose dependently. The depletion of extracellular Ca2+ by EGTA exclusively reduced the thrombin-induced choline formation. Thrombin had only a slight effect on phosphoinositide-hydrolyzing phospholipase C activity. Thrombin induced diacylglycerol formation and DNA synthesis, and increased the number of MC3T3-E1 cells, but DFP-inactivated thrombin did not. Thrombin suppressed both basal and fetal calf serum-induced alkaline phosphatase activity in these cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, inhibited both the thrombin-induced diacylglycerol formation and DNA synthesis. These results suggest that thrombin stimulates phosphatidylcholine-hydrolyzing phospholipase D due to self-induced Ca2+ influx independently of protein kinase C activation in osteoblast-like cells and that its proliferative effect depends on phospholipase D activation.
Suzuki
A
A
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
Kozawa
O
O
Shinoda
J
J
Watanabe
Y
Y
Saito
H
H
Oiso
Y
Y
D000469:Alkaline Phosphatase / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002455:Cell Division / Q000187:drug effects*; D002794:Choline / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology; D007295:Inositol Phosphates / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000166:cytology*; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism; D010767:Phosphorylcholine / Q000378:metabolism; D011433:Propranolol / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D015398:Signal Transduction; D012969:Sodium Fluoride / Q000494:pharmacology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
D000470:Alkaloids; D004791:Enzyme Inhibitors; D007295:Inositol Phosphates; D010713:Phosphatidylcholines; D010767:Phosphorylcholine; D012969:Sodium Fluoride; D011433:Propranolol; D011493:Protein Kinase C; D000469:Alkaline Phosphatase; D010739:Phospholipase D; D013917:Thrombin; D019204:GTP-Binding Proteins; D019311:Staurosporine; D002794:Choline; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium