Bourgoin S G, Harbour D, Poubelle P E
Centre de Recherche en Rhumatologie et Immunologie, C.H.U.L., Ste-Foy, Québec, Canada.
J Bone Miner Res. 1996 Nov;11(11):1655-65. doi: 10.1002/jbmr.5650111109.
The effect of fluoride on phospholipase D (PLD) activation was studied using in vitro culture of Saos-2, MG-63 osteosarcoma cells, and normal osteoblast-like cells derived from human bone explants. Millimolar concentrations of NaF induced a significant accumulation of phosphatidylethanol (PEt) in Saos-2 cells but not in MG-63 and normal osteoblast-like cells. PLD activation was evident at 15 mM and concentration-dependent up to 50 mM. This stimulation was inhibited by deferoxamine, a chelator of Al3+, suggesting that PLD activation involves fluoride-sensitive G proteins. A good correlation was found between the levels of intracellular free Ca2+ and the activation of PLD. The time courses of the two responses were nearly identical. The ability of NaF to induce both responses was largely dependent on the presence of extracellular calcium. The calcium ionophore A23187 reproduced the effect of NaF, and this effect was antagonized by EGTA, suggesting that PLD activation was, at least in part, a calcium-regulated event. Phorbol 12-myristate 13-acetate (PMA) also stimulated PLD activity in human bone cells. Protein kinase C alpha (PKC alpha) and epsilon were expressed in Saos-2 cells. Acute pretreatment of cells with PMA reduced concomitantly the amounts of PKC alpha, but not of PKC epsilon, and the subsequent activation of PLD elicited by PKC activators. The PLD response to NaF was not attenuated but rather enhanced by down-regulation of PKC alpha. Therefore, PKC-alpha-induced PLD activation is unlikely to mediate the effect of NaF. Moreover, PMA and NaF showed a supraadditive effect on PLD activation in Saos-2 cells. This stimulation, in contrast to NaF alone, was not reduced by EGTA. Hence, mobilization of calcium by NaF cannot account for the enhanced PLD activation in response to PMA stimulation. Membrane Arf and RhoA contents were assessed by Western immunoblot analyses. Membranes derived from NaF-stimulated Saos-2 cells contained more Arf and RhoA when compared with membranes derived from control or PMA-stimulated cells. Translocation of the small GTPases was calcium-independent. We conclude that PLD activation by NaF in Saos-2 cells includes a fluoride-sensitive G protein, increases in the levels of intracellular calcium, and Arf/RhoA redistribution to membranes. The results also indicate that the NaF-induced Arf/RhoA translocation exerts in concert with PMA-activated PKC alpha a synergistic effect on the activation of PLD in Saos-2 cells.
利用人骨外植体来源的Saos-2、MG-63骨肉瘤细胞及正常成骨样细胞进行体外培养，研究了氟化物对磷脂酶D（PLD）激活的影响。毫摩尔浓度的氟化钠（NaF）可使Saos-2细胞中磷脂酰乙醇（PEt）显著蓄积，但对MG-63细胞和正常成骨样细胞无此作用。在15 mM时可明显激活PLD，且在50 mM浓度范围内呈浓度依赖性。去铁胺（一种Al3+螯合剂）可抑制这种刺激，提示PLD激活涉及对氟化物敏感的G蛋白。细胞内游离Ca2+水平与PLD激活之间存在良好的相关性。两种反应的时间进程几乎相同。NaF诱导这两种反应的能力很大程度上依赖于细胞外钙的存在。钙离子载体A23187可重现NaF的作用，且这种作用可被乙二醇双四乙酸（EGTA）拮抗，提示PLD激活至少部分是一个钙调节事件。佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）也可刺激人骨细胞中的PLD活性。蛋白激酶Cα（PKCα）和ε在Saos-2细胞中表达。用PMA对细胞进行急性预处理可使PKCα的量同时减少，但PKCε的量未减少，且随后PKC激活剂引起的PLD激活也减少。下调PKCα后，PLD对NaF的反应未减弱反而增强。因此，PKCα诱导的PLD激活不太可能介导NaF的作用。此外，PMA和NaF对Saos-2细胞中PLD激活表现出超加性效应。与单独使用NaF不同，这种刺激不会被EGTA减弱。因此，NaF引起的钙动员不能解释对PMA刺激的PLD激活增强。通过蛋白质免疫印迹分析评估膜上的Arf和RhoA含量。与对照或PMA刺激的细胞来源的膜相比，NaF刺激的Saos-2细胞来源的膜含有更多的Arf和RhoA。小GTP酶的转位不依赖于钙。我们得出结论，NaF在Saos-2细胞中激活PLD包括一个对氟化物敏感的G蛋白、细胞内钙水平升高以及Arf/RhoA重新分布到膜上。结果还表明，NaF诱导的Arf/RhoA转位与PMA激活的PKCα协同作用，对Saos-2细胞中PLD的激活产生协同效应。
