Phorbol ester-induced production of prostaglandin E2 from phosphatidylcholine through the activation of phospholipase D in UMR-106 cells.
To determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on phospholipase D (PLD) activity in osteoblast-like UMR-106 cells, we used cells prelabeled with [3H] myristic acid or [3H] arachidonic acid, which were preferentially incorporated to phosphatidylcholine. The treatment of [3H] myristate-labeled cells with TPA in the presence of 1% ethanol caused a dose-dependent formation of [3H] phosphatidylethanol (PEt), a product specific to PLD, suggesting an activation of this enzyme. Pretreatment of the cells with protein kinase C (PKC) inhibitors (GF109203X, staurosporine or H-7) abolished the TPA-dependent formation of PEt. The PEt formation in response to TPA treatment was not observed after the pretreatment of the cells with TPA to downregulate PKC. These results suggest the involvement of PKC in the TPA-induced activation of PLD. With [3H] arachidonate-labeled cells, TPA treatment in the absence of ethanol resulted in the liberation of [3H] arachidonic acid, which was gradually converted to prostaglandin E2 (PGE2), but the accumulations of [3H] phosphatidic acid (PA) and [3H] diacylglycerol (DAG) were very small and temporary. In contrast, PA was linearly accumulated following TPA treatment, when the cells were pretreated with an inhibitor of phosphatidate phosphohydrolase (PAP), propranolol, with no accumulation of either DAG or arachidonic acid. The TPA treatment of the cells pretreated with a DAG lipase inhibitor, RHC-80267, caused the generation of DAG after a lag period of approximately 5 min, with a very small and temporary accumulation of PA. The TPA treatment of cells pretreated with a cyclooxygenase (COX) inhibitor, indomethacin, blocked the PGE2 production. The TPA-induced PGE2 production was not affected by the pretreatment of cells with a phospholipase A2 inhibitor, p-bromophenacylbromide, or with a phospholipase C inhibitor, D-609. TPA also stimulated PGE2 production in osteoblastic cells that were enzymatically isolated from adult rat calvaria, and the experiments with lipid metabolizing enzyme inhibitors gave the same profile of inhibition of TPA-induced PGE2 production as was observed in UMR-106 cells. These results suggest that PA formed as a consequence of the activation of PLD by TPA is rapidly converted to arachidonic acid via a PAP/DAG lipase pathway, followed by a gradual conversion of arachidonic acid to PGE2 by COX in both UMR-106 cells and isolated adult osteoblastic cells, and that neither phospholipase A2 nor phospholipase C is involved in the TPA-induced PGE2 production. To the best of our knowledge, this is the first report that shows that the activation of PKC in osteoblastic cells leads to the production of PGE2 via a PLD/PAP/DAG lipase/COX pathway.
Kaneki
H
H
Department of Hygienic Chemistry, School of Pharmaceutical Sciences, Toho University, Chiba, Japan.
Yokozawa
J
J
Fujieda
M
M
Mizuochi
S
S
Ishikawa
C
C
Ide
H
H
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002460:Cell Line; D015232:Dinoprostone / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020404:Glycerophospholipids / Q000378:metabolism; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors; D019814:Myristic Acid; D010006:Osteoblasts / Q000187:drug effects*; D010711:Phosphatidate Phosphatase / Q000037:antagonists & inhibitors; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; C051521:phosphatidylethanol; D019814:Myristic Acid; D016718:Arachidonic Acid; D011493:Protein Kinase C; D010741:Phospholipases A; D008071:Lipoprotein Lipase; D054467:Phospholipases A2; D010711:Phosphatidate Phosphatase; D010739:Phospholipase D; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate