Tournier C, Thomas G, Pierre J, Jacquemin C, Pierre M, Saunier B
Unité de recherches sur la glande thyröide et la régulation hormonale,IFR 21, U96 INSERM, Le Kremlin-Bicêtre, France.
Eur J Biochem. 1997 Mar 1;244(2):587-95. doi: 10.1111/j.1432-1033.1997.00587.x.
Reactive oxygen species modulate major cellular functions by mechanisms which are still poorly understood. Recently, H2O2 has been reported to stimulate the activity of the mitogen-activated protein kinases (MAPKs) ERK and JNK, and the expression of the proto-oncogenes c-fos and c-jun. As their expression is enhanced by H2O2 in astrocytes, we studied whether these MAPKs were stimulated by H2O2 in primary cultured astrocytes. The result was positive, a maximum of stimulation being reached with 200 microM H2O2 (0.3 pmol H2O2/cell) for both ERK and JNK. ERK was previously reported to stimulate cytosolic phospholipase A2 phosphorylation and activity. H2O2 stimulated the release of arachidonic acid in astrocytes, as already reported in other cell types. We found also that cPLA2 phosphorylation was increased by H2O2. Moreover, the stimulation by H2O2 of ERK and JNK was decreased by phospholipase A2 activity inhibitors. When astrocytes were incubated first with eicosatetraynoic acid, a structural analogue competing in arachidonic acid metabolism, the stimulation of JNK by H2O was also inhibited, suggesting the involvement of arachidonic acid metabolites. Cyclooxygenase or cytochrome P450 monooxygenase inhibitors failed in decreasing the MAPK stimulation by H2O2, whereas lipoxygenase inhibitors completely abolished that of JNK. Mitogenicity has been reported to be stimulated by H2O2 in other cell types. Although ERK was strongly and durably stimulated by 200 microM H2O2 in astrocytes, at the same extent as by mitogenic growth factors, basal thymidine incorporation rate was decreased by more than 80% after 12-15 h. Moreover, the stimulation of thymidine incorporation induced by basic fibroblast growth factor was transiently abolished by H2O2. Furthermore, H2O2 likely induced the expression of CL100/PAC1/MKP-1, a dual specificity phosphatase which has been implicated in ERK and JNK inactivation in the nucleus. Finally, the prior treatment of astrocytes with MK886, a 5-lipoxygenase-activating protein inhibitor, prevented JNK from stimulation, but did not prevent thymidine incorporation from inhibition, both induced by H2O2. These results strongly suggest an involvement of arachidonic acid and/or its metabolites in the stimulation of both ERK and JNK following the oxidative stress evoked by H2O2, which induced a cell cycle arrest probably independent of the stimulation of JNK.
活性氧通过仍未完全清楚的机制调节主要细胞功能。最近，有报道称过氧化氢（H₂O₂）可刺激丝裂原活化蛋白激酶（MAPK）ERK和JNK的活性，以及原癌基因c-fos和c-jun的表达。由于星形胶质细胞中它们的表达会被H₂O₂增强，我们研究了在原代培养的星形胶质细胞中这些MAPK是否也会被H₂O₂刺激。结果是肯定的，对于ERK和JNK，200微摩尔/升的H₂O₂（0.3皮摩尔H₂O₂/细胞）可达到最大刺激效果。之前有报道称ERK可刺激胞质磷脂酶A₂的磷酸化和活性。H₂O₂可刺激星形胶质细胞中花生四烯酸的释放，这在其他细胞类型中也有报道。我们还发现H₂O₂可增加cPLA₂的磷酸化。此外，磷脂酶A₂活性抑制剂可降低H₂O₂对ERK和JNK的刺激。当星形胶质细胞先用二十碳四烯酸（一种在花生四烯酸代谢中起竞争作用的结构类似物）孵育时，H₂O对JNK的刺激也会受到抑制，这表明花生四烯酸代谢产物参与其中。环氧合酶或细胞色素P450单加氧酶抑制剂未能降低H₂O₂对MAPK的刺激，而脂氧合酶抑制剂则完全消除了对JNK的刺激。在其他细胞类型中，有报道称H₂O₂可刺激有丝分裂活性。尽管在星形胶质细胞中200微摩尔/升的H₂O₂可强烈且持久地刺激ERK，其程度与有丝分裂生长因子相同，但在12 - 15小时后，基础胸苷掺入率下降了80%以上。此外，H₂O₂可暂时消除碱性成纤维细胞生长因子诱导的胸苷掺入刺激。此外，H₂O₂可能诱导了CL100/PAC1/MKP-1的表达，这是一种双特异性磷酸酶，与细胞核中ERK和JNK的失活有关。最后，用5-脂氧合酶激活蛋白抑制剂MK886预先处理星形胶质细胞，可阻止JNK被刺激，但不能阻止H₂O₂诱导的胸苷掺入抑制。这些结果强烈表明，在H₂O₂引起的氧化应激后，花生四烯酸和/或其代谢产物参与了ERK和JNK的刺激过程，这可能导致细胞周期停滞，且可能与JNK的刺激无关。
