Mediation by arachidonic acid metabolites of the H2O2-induced stimulation of mitogen-activated protein kinases (extracellular-signal-regulated kinase and c-Jun NH2-terminal kinase).
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.
Tournier
C
C
Unité de recherches sur la glande thyröide et la régulation hormonale,IFR 21, U96 INSERM, Le Kremlin-Bicêtre, France.
Thomas
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G
Pierre
J
J
Jacquemin
C
C
Pierre
M
M
Saunier
B
B
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D054638:Dual Specificity Phosphatase 1; D004789:Enzyme Activation / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D017874:Immediate-Early Proteins / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases; D008084:Lipoxygenase / Q000378:metabolism; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism
D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D017382:Reactive Oxygen Species; D016718:Arachidonic Acid; D006861:Hydrogen Peroxide; D008084:Lipoxygenase; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; C517021:Dusp1 protein, rat; D017027:Protein Tyrosine Phosphatases; D013936:Thymidine