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 s timulated the formation of choline dose dependently in the range betwe en 0.01 and 1 U/ml, but not the phosphocholine formation. Diisopropylf luorophosphate (DFP)inactivated thrombin had little effect on the chol ine formation. The combined effects of thrombin and 12-O-tetradecanoyl phorbol-13-acetate, a protein kinase C-activating phorbol ester, on th e choline formation were additive. Staurosporine, an inhibitor of prot ein kinases, had little effect on the thrombin-induced formation of ch oline. Combined addition of thrombin and NaF, an activator of heterotr imeric GTP-binding protein, did not stimulate the formation of choline further. Pertussis toxin had little effect on the thrombin-induced fo rmation of choline. Thrombin stimulated Ca2+ influx from extracellular space time and dose dependently. The depletion of extracellular Ca2by EGTA exclusively reduced the thrombin-induced choline formation. Th rombin had only a slight effect on phosphoinositide-hydrolyzing phosph olipase C activity. Thrombin induced diacylglycerol formation and DNA synthesis, and increased the number of MC3T3-E1 cells, but DFP-inactiv ated thrombin did not. Thrombin suppressed both basal and fetal calf s erum-induced alkaline phosphatase activity in these cells. Propranolol , an inhibitor of phosphatidic acid phosphohydrolase, inhibited both t he thrombin-induced diacylglycerol formation and DNA synthesis. These results suggest that thrombin stimulates phosphatidylcholine-hydrolyzi ng phospholipase D due to self-induced Ca2+ influx independently of pr otein kinase C activation in osteoblast-like cells and that its prolif erative effect depends on phospholipase D activation. (C) 1996 Wiley-L iss, Inc.