Lahoua Z, Vial H, Michel F, Crastes de Paulet A, Astruc M E
INSERM U.58, Montepellier, France.
Cell Signal. 1991;3(6):559-67. doi: 10.1016/0898-6568(91)90032-p.
We previously demonstrated that the oxysterol potentiation of arachidonic acid release and prostaglandin biosynthesis induced by foetal calf serum activation of normal rat kidney (NRK) cells (fibroblastic clone 49F) was not related to a direct effect of oxysterols on cell free Ca2+ level. Since both Ca2+ variations and protein kinase C are involved in arachidonic acid release in some models, we looked for a possible modulation by protein kinase C in the oxysterol effect on arachidonic acid release. We show that when the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C activator, was added to the culture medium, the oxysterol effect on arachidonic acid release and prostaglandin synthesis clearly increased. Moreover, the effect of TPA was dose-dependent and TPA EC50 (4 x 10(-9) M) was unchanged in the presence of the oxysterol. Preincubation of cells with TPA for 24 h prevented the arachidonic acid release induced by TPA alone, whereas the oxysterol effect was decreased but not abolished. In the absence of serum, TPA and ionomycin added together induced the same noticeable (arachidonic acid) release and PGE2 synthesis as serum alone. Nevertheless, the potentiating effect of cholest-5-ene-3 beta, 25-diol was much higher when serum itself was used to activate NRK cells than it was in the present serum-mimicking experimental conditions. Thus, the presence of growth factors is probably required to obtain a full oxysterol effect. We conclude that the oxysterol effect was synergistic with, but not fully dependent on, protein kinase C and Ca2+ ion fluxes, therefore oxysterols could affect earlier events triggered by serum growth factor binding to their cell membrane receptors.
我们先前证明，胎牛血清激活正常大鼠肾（NRK）细胞（成纤维细胞克隆49F）所诱导的花生四烯酸释放和前列腺素生物合成的氧化甾醇增强作用，与氧化甾醇对游离细胞Ca2+水平的直接作用无关。由于在某些模型中，Ca2+变化和蛋白激酶C都参与了花生四烯酸的释放，我们研究了蛋白激酶C对氧化甾醇在花生四烯酸释放作用上可能的调节作用。我们发现，当蛋白激酶C激活剂佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA）添加到培养基中时，氧化甾醇对花生四烯酸释放和前列腺素合成的作用明显增强。此外，TPA的作用呈剂量依赖性，并且在存在氧化甾醇的情况下，TPA的半数有效浓度（EC50，4×10−9 M）不变。用TPA对细胞进行24小时预孵育可防止单独由TPA诱导的花生四烯酸释放，而氧化甾醇的作用则减弱但未消除。在无血清的情况下，一起添加TPA和离子霉素所诱导的（花生四烯酸）释放和PGE2合成与单独血清诱导的相同。然而，当血清本身用于激活NRK细胞时，胆甾-5-烯-3β,25-二醇的增强作用比在当前模拟血清的实验条件下要高得多。因此，可能需要生长因子的存在才能获得完全的氧化甾醇作用。我们得出结论，氧化甾醇的作用与蛋白激酶C和Ca2+离子通量协同，但并非完全依赖于它们，因此氧化甾醇可能影响血清生长因子与其细胞膜受体结合所引发的早期事件。
