Protein kinase activation by warm and cold hypoxia- reoxygenation in primary-cultured rat hepatocytes-JNK(1)/SAPK(1) involvement in apoptosis |
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Authors: | Crenesse D Gugenheim J Hornoy J Tornieri K Laurens M Cambien B Lenegrate G Cursio R De Souza G Auberger P Heurteaux C Rossi B Schmid-Alliana A |
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Institution: | Laboratoires de Physiologie, Université de Nice-Sophia Antipolis, 06107 Nice cedex 2, France. |
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Abstract: | Ischemia-reperfusion procedures induced severe hepatic damages owing to different processes related to hypoxia and reoxygenation (H/R) phases, including the consecutive oxygen free radical (OFR) release. Stress-activated protein kinases (SAPKs) could be activated by extracellular stimuli. The aim of this study was to show whether H/R stress conditions could stimulate these kinases, and especially c-jun-N-terminal kinase (JNK(1)/SAPK(1)), to reveal a potential role of JNK(1)/SAPK(1) in the control of hepatocyte apoptosis. Primary cultured rat hepatocytes, isolated from other liver cells and blood flow, were subjected to warm and cold hypoxia-reoxygenation phases mimicking surgical and transplant conditions. The activation status of SAPKs was evaluated by immunoprecipitation or Western-blotting experiments, whereas apoptosis was assessed by measuring caspase activation and internucleosomal DNA fragmentation in vitro and by TUNEL reaction, in vivo. Hypoxia, and especially hypoxia-reoxygenation, significantly increased JNK(1)/SAPK(1) activation in cultured hepatocytes. Either in warm or cold conditions, OFR scavengers (N-Acetylcystein, Di-Phenyleneiodonium, Deferoxamine) decreased this stimulation. Warm ischemia-reperfusion also led to JNK activation. Hypoxia and especially hypoxia-reoxygenation induced programmed cell death in vivo and in vitro. This last phenomenon was inhibited when hepatocytes were treated with SB 202190, which was described as a potent inhibitor of p38 and JNK activities. Altogether, these results confirmed that JNK(1)/SAPK(1) was activated during the hypoxia-reoxygenation process, and that this activity participated in the onset of the apoptosis program. |
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