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
*Author for correspondence (e-mail: hdum{at}yumc.yonsei.ac.kr)
Accepted August 13, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Adaptation, SAPK/JNK, Hydrogen peroxide, Oxidative stress, Cell death
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In recent years, evidence has been accumulating that this adaptation may not always depend on H2O2-degrading enzymes. It has been shown that H2O2 induced the synthesis of 20-25 new proteins in vascular endothelial and fibroblast cells (Lu et al., 1993; Wiese et al., 1995). Although the nature of these proteins is largely unknown, the induction of numerous proteins by H2O2 implies a system of H2O2 adaptation involving multiple mechanisms. Consistent with this possibility, H2O2 pretreatment was found to confer on human U937 leukemia cells a cross-resistance to other lethal stimuli such as serum withdrawal and C2-ceramide (Lee and Um, 1999). Interestingly, it was observed that C2-ceramide, in the absence of such pretreatment, killed U937 cells in a manner independent of H2O2. A lethal concentration of C2-ceramide did not elevate the cellular levels of H2O2 in U937 cells, and C2-ceramide-induced cell death was not suppressed by the addition of antioxidants (Lee and Um, 1999). Therefore, the H2O2-induced resistance of U937 cells to C2-ceramide cannot be explained by the enhanced cellular capacity to degrade H2O2. Moreover, H2O2 was able to adapt U937 cells without signs of enhanced antioxidant capacity, such as an increase in protein levels and activities of the H2O2-degrading enzymes or an enhancement in the cellular capacity to degrade H2O2 (Lee and Um, 1999). A similar observation was also reported using hamster fibroblast cells (Wiese et al., 1995). Taken together, these observations suggests that H2O2 can impart cells with a survival advantage in a manner independent of H2O2-degrading activity. In the case of U937 cells, this alternative mechanism was selectively induced when the cells were exposed to 0.05 mM H2O2, whereas an increase in H2O2-degrading activity required 0.25 mM H2O2 (Lee and Um, 1999). Therefore, the former mechanism appears to be more sensitive to H2O2 than the latter, at least in U937 cells. On the basis of these observations, it was suggested that relatively low H2O2 concentrations protect the cells by blocking the lethal signaling triggered by subsequent stimuli. It was the purpose of this study to investigate this hypothesis using U937 cells. A prerequisite of this investigation was to define the cell death pathways induced by H2O2, serum withdrawal and C2-ceramide, because H2O2 adaptation controls the degree of lethality of all of these stimuli. The data presented in this report indicates that stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), a member of the mitogen-activated protein kinase (MAPK) family, acts as a common mediator of U937 cell death induced by high H2O2 concentrations, serum withdrawal and C2-ceramide. Moreover, evidence is provided showing that low H2O2 concentrations induce a protective response against all these lethal stimuli by specifically blocking their ability to activate SAPK and its upstream kinases. The mechanism of this interesting phenomenon is discussed.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture, DNA transfection and treatments
U937 cells were cultured in a RPMI 1640 medium supplemented with 10% heat-inactivated FBS and gentamicin (50 µg/ml). Cells at a concentration of 3x105/ml were exposed to H2O2 (1 mM), C2-ceramide (0.06 mM), or washed in PBS and then cultured in a serum-depleted medium. Where indicated, the cells were pretreated with 0.05 mM H2O2 for set periods before receiving the lethal treatments. For DNA transfection, the dominant negative mutants of MKK4 (Sanchez et al., 1994) and MKK7 (Moriguchi et al., 1997) were cloned into the pcDNA vector and delivered into the U937 cells by electroporation. The transfected cells were selected by using 1 mg/ml of G418 sulfate, after which they received the indicated treatments.
Analysis of cellular viability
The treated and untreated control cells received propidium iodide (PI) (5 µg/ml) followed by flow cytometry analysis to simultaneously monitor the PI uptake (FL-2 channel) and cell size (forward light scatter). The cells that displayed both a normal size and a low permeability to PI were understood to be viable cells, as previously defined (Mangan et al., 1991). All other populations were understood to be dead.
Western blot analysis
The cells were lysed in a solution containing 70 mM ß-glycerophosphate (pH 7.2), 0.1 mM sodium orthovanadate, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.5% Triton X-100 and protease inhibitors (2 µg/ml aprotinin, 2 µg/ml leupeptin and 100 µg/ml PMSF). After the removal of the cell debris by centrifugation at 13,000 g for 15 minutes, equal amounts of proteins (50 µg) were separated by 12% SDS-PAGE. The proteins were then electrotransferred to Immobilon membranes (Millipore, Bedford, MA), which were subsequently blotted using the indicated antibodies and visualized by chemiluminescence (ECL; Amersham, Arlington Heights, IL).
In vitro kinase assay
The cells were lysed in a Hepes buffer (50 mM, pH 7.4) containing 100 mM NaCl, 10% glycerol, 1% NP-40, 1 mM EDTA, 20 mM ß-glycerophosphate, 1 mM NaF, 1 mM p-nitrophenyl phosphate, 1 mM sodium orthovanadate and the protease inhibitors listed above. The lysates were clarified by centrifugation at 13,000 g for 15 minutes. Immunoprecipitation was performed by using 400 µg of the lysate proteins and the indicated antibodies. The precipitates were resolved in 20 µl of a kinase buffer containing 20 mM Hepes (pH 7.4), 10 mM MgCl2, 20 mM ß-glycerophosphate, 10 mM NaF, 1 mM DTT, 0.5 mM sodium orthovanadate, 50 µM ATP, and 10 µCi [-32P]ATP. The kinase reactions were initiated by adding 2 µg of the specified substrates to the solution. After a certain incubation time, the reaction was stopped by adding a boiled sample buffer, and the proteins were then separated by 12% SDS-PAGE. The gels were dried, and a PhosphoImager using Tina 2.0 software visualized the radioactive bands.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The expression of MKK4 mutants has been reported to suppress H2O2-induced death in U937 cells (Verheij et al., 1996). To investigate whether this can also be achieved using the MKK7 mutant, the transfectants were exposed to various H2O2 concentrations, and their viability was compared using flow cytometry. Unlike the untransfected U937 cells that were marginally susceptible to 0.1 mM H2O2 (Lee and Um, 1999), the exposure of U937/pcDNA cells to the same H2O2 concentration for 48 hours resulted in a cell death of more than 60% (Fig. 1D). Therefore, the susceptibility of U937 cells to H2O2 appeared to be enhanced during the transfection procedure, as reported previously (Kim et al., 2001). Increasing the concentration of H2O2 to 0.25 mM killed almost all of the U937/pcDNA cells. However, the cell death was dramatically reduced when the U937/MKK7 cells were exposed to the same H2O2 concentrations. A direct comparison of U937/MKK7 and U937/MKK4 cells revealed that the MKK7 mutant was slightly more protective than the MKK4 mutant. These results suggested that both MKK4 and MKK7 act as major mediators of H2O2-induced death in U937 cells.
The MKK7, but not MKK4, pathway is shared by serum withdrawal and C2-ceramide
The role of the SAPK pathways in the cell death induced by serum withdrawal and C2-ceramide was investigated. Both of these stimuli activated SAPK and MKK7 (Fig. 2A), but interestingly MKK4 was not stimulated (Fig. 2B). Activation of SAPK, induced by either serum withdrawal or C2-ceramide, was consistently reduced only by mutant MKK7 expression (Fig. 2A), but not by mutant MKK4 (Fig. 2B). Moreover, although the MKK7 mutant efficiently reduced the cell death induced by serum withdrawal and C2-ceramide, such protection was not detected using the MKK4 mutant (Fig. 2C). This finding contrasts with a previous report showing that the expression of MKK4 mutant rescued U937 cells from C2-ceramide (Verheij et al., 1996). Although the reason for this discrepancy is not clear, our data suggests that serum withdrawal and C2-ceramide selectively utilize MKK7 to activate SAPK and kill U937 cells, at least under the experimental conditions of this study. Considering these results and the above findings together, the route leading to SAPK activation appears to vary in a single cell type depending on the types of stimuli. This has also been reported in other studies (Moriguchi et al., 1997).
|
|
Time course of the inhibitory effect
H2O2 requires a time lag to induce cellular resistance to death stimuli (Wiese et al., 1995; Lee and Um, 1999). In the case of U937 cells, pretreatment with 0.05 mM H2O2 for 4 hours was not sufficient for inducing such resistance. This resistance was initially detected 8 hours after the pretreatment, and was further enhanced as the pretreatment was extended up to 24 hours (Lee and Um, 1999). To investigate whether a similar time lag was also required to inhibit SAPK activation, U937 cells were pre-exposed to 0.05 mM H2O2 for various time periods, challenged with 1 mM H2O2 for 15 minutes and then analyzed for MKK4 and MKK7 activity. Alternatively, SAPK activity was analyzed 30 minutes after the challenge. Pretreatment itself did not significantly influence the activities of all those kinases under any of the tested conditions. Interestingly, the challenge-induced activation of MKK4, MKK7 and SAPK were not significantly suppressed in the cells that had been pretreated for 4 hours (Fig. 4). Pretreatment for 8 hours resulted in a dramatic suppression of MKK7, but not MKK4, activation. Therefore, the MKK4 and MKK7 pathways appear to be differentially regulated by H2O2 pretreatment. Pretreatment for 8 hours also displayed some suppressive effect on SAPK activation, suggesting that the selective MKK7 suppression can influence SAPK activation. When the cells were pretreated for 24 hours, the challenge-induced activation of all of MKK4, MKK7, and SAPK were almost completely blocked. These results revealed that the time course required for the inhibition of SAPK activation co-relates exceedingly well with that observed for the induction of cellular resistance to H2O2. This strongly supports the suggestion that the suppression of SAPK activation is a mechanism whereby H2O2 induces this protection.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The time lag required for inducing the SAPK-suppressing response implies that H2O2 induces macromolecules that can inhibit the activation of the SAPK pathways. Given that the MKK4- and MKK7-suppressing responses are induced by different kinetics, multiple factors may be involved in suppressing the SAPK pathways. Although the identity of such factors is currently unclear, several anti-death proteins such as Bcl-2, Bcl-XL and heat-shock proteins have been eliminated as potential candidates (Lee and Um, 1999). Because SAPK is activated by its phosphorylation, we have investigated the possibility that H2O2 pretreatment (0.05 mM) induces phosphatases such as MKP-1 and MKP-2 that can dephosphorylate SAPK (Chu et al., 1996; Sanchez-Perez et al., 2000). However, no evidence from western blot analysis was found (data not shown). Although it has been reported that the overexpression of p21WAF1/CIP1/Sdi1, an inhibitor of cyclin-dependent kinases, can attenuate UV-induced SAPK activation (Shim et al., 1996), the adaptive concentrations (0.05 mM) of H2O2 in this study did not significantly alter the cellular levels of p21 (data not shown). Exposure of the U937 cells to 0.05 mM H2O2 consistently showed no significant influence on their cycling distribution. Therefore, the adaptation induced by 0.05 mM H2O2 does not appear to depend on the p21 level or the cellular cycling status. The authors have recently reported that nuclear factor B (NF-
B) is a mediator of the U937 cell adaptation induced by 0.05 mM H2O2 (Kim et al., 2001). However, the promotion of H2O2-induced NF-
B activation by the overexpression of I-
B kinase
(IKK
) (Kim et al., 2001) did not enhance the SAPK-suppressing activity of H2O2 (data not shown). This observation suggests that NF-
B is not involved in the SAPK-suppressing pathway induced by H2O2. Therefore, the antioxidant-independent adaptation appears to be induced by at least two different mechanisms: one mediated by NF-
B and the other that suppresses SAPK activation. The authors are currently attempting to establish experimental conditions for proteomics to enable them to identify a cellular factor involved in these phenomena.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chu, Y., Solski, P. A., Khosravi-Far, R., Der, C. J. and Kelly, K. (1996). The mitogen-activated protein phosphatases PAC1, MKP-1, and MKP-2 have unique substrate specificities and reduced activity in vivo toward the ERK2 sevenmaker mutation. J. Biol. Chem. 271, 6497-6501.
Cross, T. G., Scheel-Toellner, D., Henriquez, N. V., Deacon, E., Salmon, M. and Lord, J. M. (2000). Serine/threonine protein kinases and apoptosis. Exp. Cell Res. 256, 34-41.[Medline]
Davies, J. M. S., Lowry, C. V. and Davies, K. J. A. (1995). Transient adaptation to oxidative stress in yeast. Arch. Biochem. Biophys. 317, 1-6.[Medline]
Farr, S. B. and Kogoma T. (1991). Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol. Rev. 55, 561-585.
Guyton, K. Z., Liu, Y., Gorospe, M., Xu, Q. and Holbrook, N. J. (1996). Activation of mitogen-activated protein kinase by H2O2. J. Biol. Chem. 271, 4138-4142.
Kim, D. K., Cho, E. S. and Um, H.-D. (2001). NF-B mediates the adaptation of human U937 cells to hydrogen peroxide. Free Radic. Biol. Med. 30, 563-571.[Medline]
Lee, B. R. and Um, H.-D. (1999). Hydrogen peroxide suppresses U937 cell death by two different mechanisms depending on its concentration. Exp. Cell Res. 248, 430-438.[Medline]
Lu, D., Maulik, N., Moraru, I. I., Kreutzer, D. L. and Das, D. K. (1993). Molecular adaptation of vascular endothelial cells to oxidative stress. Am. J. Physiol. 264, C715-C722.
Mangan, D., Welch, G. R. and Wahl, S. M. (1991). Lipopolysaccharide, tumor necrosis factor , and IL-1ß prevent programmed cell death (apoptosis) in human peripheral blood monocytes. J. Immunol. 146, 1541-1546.
Moriguchi, T., Toyoshima, F., Masuyama, N., Hanafusa, H., Gotoh, Y. and Nishida, E. (1997). A novel SAPK/JNK kinase, MKK7, stimulated by TNF and cellular stresses. EMBO J. 16, 7045-7053.
ODonnell-Tormey, J., DeBoer, C. J. and Nathan, C. (1985). Resistance of human tumor cells in vitro to oxidative cytolysis. J. Clin. Invest. 76, 80-86.[Medline]
Sanchez, I., Hughes, R. T., Mayer, B. J., Yee, K., Woodgett, J. R., Avruch, J., Kyriakis, J. M. and Zon, L. I. (1994). Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun. Nature 372, 794-798.[Medline]
Sanchez-Perez, I., Martinez-Gomariz, M., Williams, D., Keyse, S. M. and Perona, R. (2000). CL100/MKP-1 modulates JNK activation and apoptosis in response to cisplatin. Oncogene 19, 5142-5152.[Medline]
Shim, J., Lee, H., Park, J., Kim, H. and Choi, E. (1996). A non-enzymatic p21 protein inhibitor of stress-activated protein kinases. Nature 381, 804-807.[Medline]
Shull, S., Heintz, N. H., Periasamy, M., Manohar, M., Janssen, Y. M. W., Marsh, J. P. and Mossman, B. T. (1991). Differential regulation of antioxidant enzymes in response to oxidants. J. Biol. Chem. 266, 24398-24403.
Tournier, C., Whitmarsh, A. J., Cavanagh, J., Barrett, T. and Davis, R. J. (1997). Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase. Proc. Natl. Acad. Sci. USA 94, 7337-7342.
Toyokuni, S., Okamoto, K., Yodoi, J. and Hiai, H. (1995). Persistent oxidative stress in cancer. FEBS Lett. 358, 1-3.[Medline]
Verheij, M., Bose, R., Lin, X. H., Tao, B., Jarvis, W. D., Grant, S., Birrer, M. J., Azabo, E., Zon, L. I., Kyriakis, J. M. et al. (1996). Requirement for ceramide-initiated SAPK/JNK signaling in stress-induced apoptosis. Nature 380, 75-79.[Medline]
Wang, Y. Z., Zhang, P., Rice, A. B. and Bonner, J C. (2000). Regulation of interleukin-1 beta-induced platelet-derived growth factor receptor-alpha expression in rat pulmonary myofibroblasts by p38 mitogen-activated protein kinase. J. Biol. Chem. 275, 22550-22557.
Wesselborg, S., Bauer, M. K. A., Schmitz, M. L. and Schulze-Osthoff, K. (1997). Activation of transcription factor NF-kappaB and p38 mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways. J. Biol. Chem. 272, 12422-12429.
Whitmarsh, A. J. and Davis, R. J. (1996). Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J. Mol. Med. 74, 589-607.[Medline]
Wiese, A. G., Pacifici, R. E. and Davies, K. J. A. (1995). Transient adaptation to oxidative stress in mammalian cells. Arch. Biochem. Biophys. 318, 231-240.[Medline]
Yagoda, A. (1989). Chemotherapy of renal cell carcinoma. Semin. Urol. 7, 199-206.
Zhang, P., Wang, Y. Z., Kagan, E. and Bonner, J. C. (2000). Peroxynitrite targets the epidermal growth factor receptor, Raf-1, and MEK independently to activate MAPK. J. Biol. Chem. 275, 22479-22486.