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 mechanism s which are still poorly understood. Recently, H2O2 has been reported to stimulate the activity of the mitogen-activated protein kinases (MA PKs) 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 studi ed whether these MAPKs were stimulated by H2O2 in primary cultured ast rocytes. The result was positive, a maximum of stimulation being reach ed with 200 mu M H2O2 (0.3 pmol H2O2/cell) for both ERK and JNK. ERK w as previously reported to stimulate cytosolic phospholipase A(2) phosp horylation and activity. H2O2 stimulated the release of arachidonic ac id in astrocytes, as already reported in other cell types. We found al so that cPLA(2) phosphorylation was increased by H2O2. Moreover, the s timulation by H2O2 of ERK and JNK was decreased by phospholipase A(2) activity inhibitors. When astrocytes were incubated first with eicosat etraynoic acid, a structural analogue competing in arachidonic acid me tabolism, the stimulation of JNK by H2O2 was also inhibited, suggestin g the involvement of arachidonic acid metabolites. Cyclooxygenase or c ytochrome P450 monooxygenase inhibitors failed in decreasing the MAPK stimulation by H2O2, whereas lipoxygenase inhibitors completely abolis hed that of JNK. Mitogenicity has been reported to be stimulated by H2 O2 in other cell types. Although ERK was strongly and durably stimulat ed by 200 mu M H2O2 in astrocytes, at the same extent as by mitogenic growth factors, basal thymidine incorporation rate was decreased by mo re than 80% after 12-15 h. Moreover, the stimulation of thymidine inco rporation induced by basic fibroblast growth factor was transiently ab olished by H2O2. Furthermore, H2O2 likely induced the expression of CL 100/PAC1/MKP-1, a dual specificity phosphatase which has been implicat ed in ERK and JNK inactivation in the nucleus. Finally, the prior trea tment of astrocytes with MK886, a 5-lipoxygenase-activating protein in hibitor, prevented JNK from stimulation, but did not prevent thymidine incorporation from inhibition, both induced by H2O2. These results st rongly suggest an involvement of arachidonic acid and/or its metabolit es in the stimulation of both ERK and JNK following the oxidative stre ss evoked by H2O2, which induced a cell cycle arrest probably independ ent of the stimulation of JNK.