Effect of platelet-derived growth factor on phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like cells.
We examined the effect of platelet-derived growth factor (PDGF) on the activation of phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells. PDGF-BB stimulated both the formation of choline (EC50 = 15 ng/ml) and inositol phosphates (EC50 = 5 ng/ml). However, PDGF-BB had little effect on the formation of phosphocholine. The formation of choline stimulated by a combination of PDGF-BB and 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C (PKC)-activating phorbol ester, was additive. H-7, an inhibitor of protein kinases, inhibited 12-O-tetradecanoylphorbol-13-acetate-induced choline formation, whereas HA1004, a control for H-7 as PKC inhibitor, had little effect. Neither H-7 nor HA1004 affected the PDGF-BB-induced formation of choline. Genistein and methyl 2,5-dihydroxycinnamate, inhibitors of protein tyrosine kinases, dose dependently inhibited the PDGF-BB-induced formation of choline. PDGF-BB stimulated Ca2+ influx from extracellular space. PDGF-BB-induced choline formation was significantly reduced by chelating extracellular Ca2+ with EGTA. PDGF-BB stimulated DNA synthesis of MC3YT3-E1 cells, and H-7 inhibited the DNA synthesis. These results strongly suggest that PDGF activates phosphatidyl-choline-hydrolyzing phospholipase D independently from PKC activated by phosphoinositide hydrolysis in osteoblast-like cells, and that both tyrosine kinase activation and Ca2+ influx are essential for this mechanism.
Kozawa
O
O
Department of Biochemistry, Institute for Developmental Research, Aichi, Japan.
Suzuki
A
A
Watanabe
Y
Y
Shinoda
J
J
Oiso
Y
Y
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D002934:Cinnamates / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D019833:Genistein; D007295:Inositol Phosphates / Q000378:metabolism; D007529:Isoflavones / Q000494:pharmacology; D051379:Mice; D010006:Osteoblasts / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002934:Cinnamates; D007295:Inositol Phosphates; D007529:Isoflavones; D010713:Phosphatidylcholines; D010982:Platelet-Derived Growth Factor; C029010:cinnamic acid; D004247:DNA; D019833:Genistein; D010739:Phospholipase D; D002794:Choline; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium