Kaneki H, Yokozawa J, Fujieda M, Mizuochi S, Ishikawa C, Ide H
Department of Hygienic Chemistry, School of Pharmaceutical Sciences, Toho University, Chiba, Japan.
Bone. 1998 Sep;23(3):213-22. doi: 10.1016/s8756-3282(98)00100-8.
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.
为了确定12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）对成骨样UMR - 106细胞中磷脂酶D（PLD）活性的影响，我们使用了预先用[3H]肉豆蔻酸或[3H]花生四烯酸标记的细胞，这些脂肪酸优先掺入磷脂酰胆碱。在1%乙醇存在下，用TPA处理[3H]肉豆蔻酸标记的细胞会导致[3H]磷脂酰乙醇（PEt）呈剂量依赖性形成，PEt是PLD的特异性产物，表明该酶被激活。用蛋白激酶C（PKC）抑制剂（GF109203X、星形孢菌素或H - 7）预处理细胞可消除TPA依赖性的PEt形成。在用TPA预处理细胞以下调PKC后，未观察到对TPA处理的PEt形成。这些结果表明PKC参与了TPA诱导的PLD激活。对于[3H]花生四烯酸标记的细胞，在无乙醇的情况下用TPA处理会导致[3H]花生四烯酸释放，其逐渐转化为前列腺素E2（PGE2），但[3H]磷脂酸（PA）和[3H]二酰基甘油（DAG）的积累非常少且是暂时的。相反，当用磷脂酸磷酸水解酶（PAP）抑制剂普萘洛尔预处理细胞时，TPA处理后PA呈线性积累，而DAG或花生四烯酸均无积累。用DAG脂肪酶抑制剂RHC - 80267预处理细胞后，TPA处理在约5分钟的延迟期后导致DAG生成，PA积累非常少且是暂时的。用环氧化酶（COX）抑制剂吲哚美辛预处理细胞后，TPA处理可阻断PGE2的产生。TPA诱导的PGE2产生不受磷脂酶A2抑制剂对溴苯甲酰溴或磷脂酶C抑制剂D - 609预处理细胞的影响。TPA还刺激了从成年大鼠颅骨酶分离的成骨细胞中PGE2的产生，并且用脂质代谢酶抑制剂进行的实验给出了与在UMR - 106细胞中观察到的相同的对TPA诱导的PGE2产生的抑制模式。这些结果表明，TPA激活PLD所形成的PA通过PAP/DAG脂肪酶途径迅速转化为花生四烯酸，随后在UMR - 106细胞和分离的成年成骨细胞中，花生四烯酸通过COX逐渐转化为PGE2，并且磷脂酶A2和磷脂酶C均不参与TPA诱导的PGE2产生。据我们所知，这是第一份表明成骨细胞中PKC的激活通过PLD/PAP/DAG脂肪酶/COX途径导致PGE2产生的报告。
