Adaptive concentrations of hydrogen peroxide suppress cell death by blocking the activation of SAPK/JNK pathway
Do Kyun Kim1,
Eun Sook Cho1,
Je Kyung Seong2 and
Hong-Duck Um1,3,*
1 Laboratory of Cell Biology, Yonsei Medical Research Center, Yonsei University College of Medicine, CPO Box 8044, Seoul 120-752, Korea
2 Department of Laboratory Animal Science, Yonsei Medical Research Center, Yonsei University College of Medicine, CPO Box 8044, Seoul 120-752, Korea
3 Brain Korea 21 Center for Medical Sciences, Yonsei University College of Medicine, CPO Box 8044, Seoul 120-752, Korea
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Fig. 1. Both MKK4 and MKK7 mediate H2O2-induced SAPK activation and cell death. (A) U937 cells were exposed to 1 mM H2O2 for the indicated periods. The cells were lysed, and the SAPK, ERK and p38 MAPK phosphorylation levels were analyzed by western blotting using antibodies specific to the phosphorylated forms of each MAPK. The protein levels of these MAPKs were also probed using their specific antibodies. (B) The SAPK, ERK, p38 MAPK, MKK4 and MKK7 activities in the lysates were analyzed by in vitro kinase assay. The substrates were recombinant c-Jun protein for SAPK, PHAS-1 for ERK and p38 MAPK, and SAPK protein for MKK4 and MKK7. (C) The U937 cells were stably transfected with the pcDNA vector containing the dominant negative mutant of either MKK4 or MKK7. These transfectants and the cells that received the empty control vector were exposed to 1 mM H2O2 for 30 minutes. Levels of SAPK activity in the treated and untreated control cells were compared by in vitro kinase assay. (D) The transfectants were treated with the indicated H2O2 concentrations for 48 hours and the cellular viability was analyzed by flow cytometry.
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Fig. 2. MKK7, but not MKK4, is involved in the SAPK activation and cell death induced by serum withdrawal (SW) and C2-ceramide. (A) The U937 cells were exposed to a serum-depleted medium or 0.06 mM C2-ceramide. At the end of the indicated incubation periods, the MKK7 activity was analyzed (top). U937/pcDNA and U937/MKK7 cells received the same treatments for 30 minutes. SAPK activities in the treated and untreated cells were compared (bottom). (B) U937 cells were treated with the indicated stimuli for 15 minutes, and MKK4 activity was measured (top). U937/pcDNA and U937/MKK4 cells received the indicated treatments for 30 minutes, and the SAPK activity was compared (bottom). (C) The U937/pcDNA, U937/MKK4 and U937/MKK7 cells were treated with either the serum-depleted medium or C2-ceramide for 48 hours. The viability of the treated and untreated cells was compared by flow cytometry.
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Fig. 3. H2O2 pretreatment reduces the activation of both the MKK4 and MKK7 pathways induced by diverse stimuli. (A) U937 cells were incubated in the presence or absence of 0.05 mM H2O2 for 24 hours, followed by a challenge with 1 mM H2O2 for 30 minutes. The SAPK phosphorylation and protein levels (top two panels) and its activity (third panel) were compared. Alternatively, the activities of MKK4 and MKK7 were analyzed 15 minutes after the challenge (bottom two panels). (B) The preincubated cells received the indicated challenges for 30 minutes. The SAPK phosphorylation and protein levels (top two panels), and the SAPK and MKK7 activities (bottom two panels) were analyzed.
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Fig. 4. H2O2 requires a time lag to induce the SAPK-suppressing effect. The U937 cells were pre-exposed to 0.05 mM H2O2 for the indicated periods, followed by a challenge with 1 mM H2O2 either for 15 minutes for the analysis of the MKK4 and MKK7 activities, or for 30 minutes for SAPK (top). The intensity of each band was quantified by using Tina 2.0 software. The challenge-induced fold stimulation of each kinase was determined over its basal level obtained from the cells that did not receive H2O2 in the whole incubation (bottom). The values are the mean of three separate experiments. The standard deviations were routinely less than 15% of the means. Circle, SAPK; square, MKK4; triangle, MKK7.
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