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J Biol Chem, Vol. 275, Issue 15, 11418-11424, April 14, 2000

Stimulation of p38 Mitogen-activated Protein Kinase Is an Early Regulatory Event for the Cadmium-induced Apoptosis in Human Promonocytic Cells^*

Alba Galán^§, María L. García-Bermejo^¶, Alfonso Troyano^§, Nuria E. Vilaboa, Elena de Blas, Marcelo G. Kazanietz^¶, and Patricio Aller^**

From the  Centro de Investigaciones Biológicas, CSIC, 28006 Madrid, Spain and the ^¶ Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Pulse treatment of U-937 promonocytic cells with cadmium chloride (2 h at 200 µM) provoked apoptosis and induced a rapid phosphorylation of p38 mitogen-activated protein kinase (p38^MAPK) as well as a late phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). However, although the p38^MAPK-specific inhibitor SB203580 attenuated apoptosis, the process was not affected by the ERK-specific inhibitor PD98059. The attenuation of the cadmium-provoked apoptosis by SB203580 was a highly specific effect. In fact, the kinase inhibitor did not prevent the generation of apoptosis by heat shock and camptothecin, nor the generation of necrosis by cadmium treatment of glutathione-depleted cells, nor the cadmium-provoked activation of the stress response. The generation of apoptosis was preceded by intracellular H₂O₂ accumulation and was accompanied by the disruption of mitochondrial transmembrane potential, both of which were inhibited by SB203580. On the other hand, the antioxidant agent butylated hydroxyanisole-inhibited apoptosis but did not prevent p38^MAPK phosphorylation. In a similar manner, p38^MAPK phosphorylation was not affected by the caspase inhibitors Z-VAD and DEVD-CHO, which nevertheless prevented apoptosis. These results indicate that p38^MAPK activation is an early and specific regulatory event for the cadmium-provoked apoptosis in promonocytic cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cadmium and other heavy metals are frequent environmental contaminants with well known mutagenic, carcinogenic, and teratogenic effects (1). Another property of heavy metals (and of other physical and chemical agents, such as heat and inhibitors of energy metabolism) is the capacity to induce the stress response, characterized by the synthesis and accumulation of heat-shock proteins (HSPs)¹ (2). The stimulation of HSP gene expression by stress inducers in higher eukaryotes seems to be specifically regulated by heat-shock factor 1 (HSF1). Thus, under stressful conditions, HSF1 undergoes trimerization, translocation to the nucleus, binding to the heat-shock consensus elements, and finally hyperphosphorylation to fully acquire the transactivation capacity (3). In addition, stress inducers may provoke cell death, either apoptotic or necrotic, depending on the intensity of the treatments (4, 5). The morphological characteristics of apoptosis and necrosis are well known (6-8). Thus, during apoptosis the cells undergo nuclear and cytoplasmic shrinkage, the chromatin is partitioned into multiple fragments, and the cells are broken into multiple membrane-surrounded bodies ("apoptotic bodies"), but the cell membrane retains its integrity during the process. By contrast, necrosis is characterized by cell swelling and a rapid loss of membrane integrity. However, the regulation of apoptosis and necrosis and the factors that decide the selection of one or the other mode of death are still poorly known.

One of the most relevant aspects in the regulation of both the stress response and apoptosis is the involvement of mitogen-activated protein kinases (MAPKs), a family of serine/threonine kinases that mediate intracellular signal transduction in response to different stimuli (9). The MAPK family members are themselves activated by reversible dual phosphorylation on a Thr-Xaa-Tyr conserved motif, by specific mitogen-activated protein kinases kinases. To date, three major MAPKs have been identified, namely the extracellular signal-regulated kinases (ERK1/2, p44/p42), the stress-activated protein kinases (c-Jun NH₂-terminal kinases, stress-activated protein kinase 1), and the p38 mitogen-activated protein kinases (p38^MAPK, stress-activated protein kinase 2). ERK1/2 are mainly (although not exclusively) activated by growth factors (10, 11) and are critically involved in the regulation of mitogenesis. On the other hand, c-Jun NH₂-terminal kinases and p38^MAPK are mainly activated by cytotoxic insults and are often associated with apoptosis (12-18).

It was recently reported that cadmium chloride induced the stress response and caused a dose-dependent activation of ERK1/2 and p38^MAPK (but not of c-Jun NH₂-terminal kinases) in association with mitogenesis and apoptosis, respectively, in rat brain tumor cells (19). Whereas it was demonstrated that both kinases were involved in the regulation of the stress response, it is not clear whether they were also required to induce cell death. In the present work we investigated the capacity of cadmium chloride to induce p38^MAPK and ERK1/2 activation and to cause cell death in U-937 human promonocytic cells. It was concluded that p38^MAPK activation is an early and specific requirement for the cadmium-provoked apoptosis, which precedes and probably modulates the expression of other regulatory events.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- All components for cell culture were obtained from Life Technologies, Inc. Butylated hydroxyanisole (BHA), camptothecin, DL-buthionine-SR-sulfoximine (BSO), N-acetyl-L-cysteine (NAC), propidium iodide (PI), and Nonidet-P40 were obtained from Sigma; cadmium chloride was from Merck; 4,6-diamino-2-phenylindole was from Serva (Heidelberg, Germany); RNase A was from Roche Diagnostics S. L. (Barcelona, Spain); and dichlorodihydrofluorescein diacetate and 3,3'-dihexylocarbocyanine iodide were from Molecular Probes, Inc. (Eugene, OR). The MAPK inhibitors SB203580 and PD98059 and the caspase inhibitors Z-Val-Ala-Asp (OMe)-CH2F (Z-VAD) and acetyl-Asp-Glu-Val-Asp aldehyde (DEVD-CHO) were obtained from Calbiochem-Novabiochem. All antibodies against mitogen-activated protein kinases, namely rabbit anti-human p38^MAPK pAb, rabbit anti-human phospho-p38^MAPK (Thr¹⁸⁰/Tyr¹⁸²) pAb, rabbit anti-human p44/42^MAPK pAb, and rabbit anti-human phospho-p44/42^MAPK (Thr²⁰²/Tyr²⁰⁴) pAb, were purchased from New England Biolabs, Inc. (Beverly, MA); mouse anti-human HSP70 monoclonal antibody (clone C92F34-5) and rabbit anti-human HSF1 pAb were from StressGen Biotechnologies Corp., Victoria, Canada; and mouse anti-chicken -tubulin monoclonal antibody was from Amersham Pharmacia Biotech. The goat anti-rabbit and anti-mouse peroxidase-conjugated antibodies were from Dako A/S (Glostrup, Denmark). The Annexin V-fluorescein isothiocyanate kit was purchased from Bender MedSystems (Vienna, Austria).

Cells and Treatments-- U-937 promonocytic leukemia cells (20) were routinely grown in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, as described previously (21). Sixteen hours prior to treatments, cells were seeded at 2 × 10⁵ cells/ml in medium containing 1.5% fetal calf serum. Cadmium chloride and BSO were dissolved in distilled water at 0.1 M before use. SB203580, PD98059, camptothecin, and Z-VAD were dissolved in Me₂SO at the concentrations of 13.2, 20, 10, and 20 mM, respectively, and DEVD-CHO in distilled water at 10 mM. All these solutions were stored at 20 °C. For cadmium treatment, cells were incubated for 2 h with 200 µM cadmium chloride. In some experiments, the cells were washed after treatment with prewarmed (37 °C) RPMI medium and allowed to recover under standard culture conditions. For heat shock, cells were placed in a bath at the desired temperature for the required time period and then allowed to recover under standard culture conditions. As controls, cells were subjected to the same manipulations as treated cells, in the absence of cadmium. For GSH depletion, cells were incubated for 18-24 h with 1 mM BSO, as earlier reported (22). Under these conditions BSO reduced the intracellular GSH level by more than 90% without affecting cell proliferation or viability.

Determination of Apoptosis and Necrosis-- To analyze changes in chromatin structure, cells were collected by centrifugation, washed with phosphate-buffered saline (PBS), resuspended in PBS, and mounted on glass slides. After fixation in 70% (v/v) ethanol, the cells were stained for 20 min at room temperature with 4,6-diamino-2-phenylindole (1 µg/ml) and examined by fluorescence microscopy. Cells with fragmented chromatin were considered as apoptotic. Nucleosome-sized DNA cleavage was determined by agarose gel electrophoresis, as described previously (21).

To measure the loss of DNA, cells were collected by centrifugation and incubated for 30 min in PBS containing 0.5 mg/ml RNase A. After the addition of 50 µg/ml PI and permeabilization with 0.1% (w/v) Nonidet P-40, the cells were analyzed by flow cytometry. Cells with sub-G₁ PI incorporation were considered as apoptotic (23). Within the experimental time periods used here necrotic cells did not exhibit sub-G₁ PI incorporation.

The criteria used to determine necrosis was the loss of membrane integrity, as measured by permeability to trypan blue or by massive influx of PI in nonpermeabilized cells. In the later case, cells were washed with PBS and incubated for 10 min at room temperature in 500 µl of a buffer consisting of 10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1.8 mM CaCl₂, containing 20 µg of PI and 3 µl of fluorescein isothiocyanate-conjugated human annexin V. The cells were then analyzed by flow cytometry using appropriate color filters to determine the PI-derived reddish orange fluorescence (emission peak 590 nm) and the fluorescein isothiocyanate-derived greenish fluorescence (emission peak 530 nm). Apoptotic cells were characterized by annexin V binding but with null or low PI influx, whereas necrotic cells were characterized by annexin V binding and a great increase in red fluorescence because of the massive influx of PI (24).

Measurement of Hydrogen Peroxide and Mitochondrial Transmembrane Potential-- The intracellular H₂O₂ content was determined by flow cytometry after loading the cells with dichlorodihydrofluorescein diacetate, as described earlier (25).

To evaluate mitochondrial transmembrane potential (_m), cells were washed with PBS and then incubated for 20 min at room temperature with PBS containing 0.1 nM 3,3'-dihexylocarbocyanine iodide (3). After washing twice with fetal calf serum, the cells were resuspended in PBS, and the fluorescence was measured by flow cytometry.

Immunoblot Assays-- Whole cell extracts were fractionated by SDS-polyacrylamide gel electrophoresis in 10% polyacrylamide minigels and transferred to Immobilon^TM-P transfer membranes (Millipore Corp., Medford, MA). After blocking with 3% nonfat milk in TTBS buffer (0.1% (v/v) Tween 20, 25 mM Tris, 150 mM NaCl, pH 7.5), the membranes were incubated overnight with the primary antibody, then extensively washed with TTBS, and incubated for 1 h with the secondary antibody. After extensive washing with TTBS, the immune complexes were detected by chemiluminiscence using the Western blotting kit from Pierce.

Gel Retardation Assays-- Nuclear extracts from 10⁷ cells were prepared as earlier described (26) and stored at 70 °C. For HSF1 binding assays, the partially complementary oligonucleotides 5'-GCGAAACCCCTGGAATATTCCCGACCTGGC-3' and 5'-GGGCCAGGTCGGGAATATTCCAGGGGTTTCG-3' were synthesized and used to prepare a heat-shock element-containing double strand oligoprobe, which was labeled with [-³²P]dCTP (27). The binding reactions were carried out for 20 min at 4 °C in 20 µl of binding buffer (60 mM KCl, 1 mM MgCl₂, 12% glycerol, 1 mM 1,4-dithiothreitol, and 20 mM HEPES, pH 7.9) containing 5 ng of the labeled probe, 8 µg of total nuclear proteins, 2 µg of poly(dI·dC), and 2 µg of salmon sperm DNA. Fifty-fold excess heat-shock element, unrelated (AP-1-recognizing) unlabeled probes, or anti-HSF1 antibody were used to check the specificity of the binding reaction. The samples were electrophoresed in 4% polyacrylamide gels that were dried and later autoradiographed.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Apoptosis Induction-- We have previously shown that pulse treatment with high cadmium concentrations (100 µM and above) induced the stress response and caused death in U-937 promonocytic cells (28, 29). To determine the mode of death, the expression of apoptotic and necrotic markers was measured in cells pulse-treated for 2 h with 200 µM cadmium chloride followed by recovery. It was observed that the treatment caused chromatin fragmentation (Fig. 1A), accumulation of cells with sub-G₁ DNA content (Fig. 1B), and nucleosome-sized DNA fragmentation (Fig. 1C), all of which are characteristics of apoptosis. Apoptotic cells were already detected at 1 h of recovery after treatment, reaching approximately 60% at 6 h. In contrast, no significant necrosis was detected during this period of recovery, as revealed by trypan blue staining (Fig. 1A) or free PI uptake (see Fig. 6B). Necrosis was not detected even at a cadmium chloride concentration of 1 mM (result not shown). Nevertheless, after prolonged recovery periods (12 h and thereafter) some trypan blue-stained cells started to be detected (result not shown), which suggests secondary necrosis, derived from apoptosis. Hence, 6 h was the maximum recovery period adopted for further experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Induction of apoptosis by cadmium. U-937 cells were treated for 2 h with 200 µM cadmium chloride (Cd), then washed, and allowed to recover in cadmium-free medium. A, frequency of apoptotic (open symbols) and necrotic (closed symbols) cells at different times of recovery, as determined by chromatin fragmentation and trypan blue permeability, respectively. The frequency of apoptotic and necrotic cells in untreated cultures (Cont) was always below 4% (not represented). B, cell distribution according to their DNA content at 6 h of recovery, as measured by PI incorporation in permeabilized cells. The position of apoptotic cells (Ap) is indicated. C, DNA cleavage at 6 h of recovery, as determined by gel electrophoresis. The lines at the margin indicate the position of simultaneously run markers (from top to bottom: 3.76, 1.93, 1.26, and 0.7 kilobases). The values in A represent the mean ± S.D. of three experiments. The determinations in B were repeated three times with similar results.

Activation of Mitogen-activated Protein Kinases-- Earlier observations indicated that cadmium caused a dose-dependent activation of ERK1/2 and p38^MAPK, as measured by their increased phosphorylation, in rat brain tumor cells (19). Hence, we decided to measure the phosphorylation/activation of those kinases in cadmium-treated U-937 cells. As shown in Fig. 2, cadmium provoked a rapid increase in p38^MAPK phosphorylation, which was already detected after 30 min of treatment, hence preceding the expression of apoptotic markers and followed during the whole treatment and recovery periods. In addition, cadmium provoked a late increase in ERK1/2 phosphorylation, which started at 1-3 h of recovery, coincident with the expression of apoptotic markers.

View larger version (75K): [in this window] [in a new window]   Fig. 2.   Effect of cadmium on p38MAPK and ERK1/2 phosphorylation. The figure shows the relative levels of phosphorylated p38MAPK (p38-P) and ERK1/2 (ERK-P) in untreated cells (Cont), in cells treated for different times with 200 µM cadmium chloride (Treat), and in cells allowed to recover for the indicated time periods after 2 h cadmium treatment (Rec), as demonstrated by immunoblot using antibodies specific against phospho-p38MAPK and phospho-ERK1/2. As controls, blots were also probed with antibodies against total (phosphorylated plus nonphosphorylated) p38MAPK (p38-tot) and total ERK1/2 (ERK-tot). Two bands were detected with the anti-ERK antibodies, corresponding to ERK1 (p44) and ERK2 (p42). All determinations were repeated at least three times with similar results. All other conditions were as in Fig. 1.

Effect of Kinase Inhibitors on Apoptosis-- The rapid increase in p38^MAPK phosphorylation and the delayed increase in ERK1/2 phosphorylation might indicate that these kinase activities are involved in the triggering and the execution of apoptosis, respectively. To investigate this, we analyzed the effect of the p38^MAPK-specific inhibitor SB203580 and the ERK-specific inhibitor PD98059 on the cadmium-provoked apoptosis. It has been shown that PD98059 prevents ERKs phosphorylation/activation by inhibiting mitogen-activated protein kinase/extracellular signal-regulated kinase kinase activity (30). SB203580 was first described as an inhibitor of p38^MAPK activity by competing with ATP for binding (31), but it was later demonstrated that the drug also prevents p38^MAPK phosphorylation/activation (32). We observed that treatment of U-937 cells with 30 µM PD98059 totally abolished ERK phosphorylation, whereas treatment with 5-20 µM SB203580 (because higher concentrations were toxic) only partially inhibited p38^MAPK phosphorylation (Fig. 3A). However, although PD98059 failed to alter (either inhibit or potentiate) the expression of apoptotic markers, SB203580 attenuated apoptosis by approximately 50% (Fig. 3, B and C). These results suggest that p38^MAPK activation is required, at least in part, for the cadmium-provoked apoptosis, whereas ERK activation is irrelevant for the process.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Differential effects of the p38MAPK inhibitor SB203580 and the ERK1/2 inhibitor PD98059 on the cadmium-provoked apoptosis. Cells were treated for 2 h with 200 µM cadmium chloride and allowed to recover. SB203580 (SB) was applied 1 h before cadmium and maintained during the cadmium treatment and recovery periods. PD98059 (PD) was applied at the beginning of the recovery period. A, relative levels of p38MAPK phosphorylation and ERK1/2 phosphorylation in untreated cells (Cont) and in cells treated with cadmium without (Cd) or with (Cd/SB and Cd/PD) the indicated concentrations of the kinase inhibitors. B and C, frequency of apoptotic cells in untreated cultures (Cont), in cultures treated with cadmium alone (Cd), or in combination with 10 µM SB203580 (Cd/SB) or 30 µM PD98059 (CD/Pd), as determined by chromatin fragmentation (B) and accumulation of cells with sub-G1 DNA content (C). All determinations were carried out at 6 h of recovery. The values in B represent the mean ± S.D. of three experiments. The determinations in A and C were repeated twice and three times, respectively, with similar results. All other conditions were as in Figs. 1 and 2.

To rule out the possibility of a general, nonspecific interference of SB203580 with the apoptotic machinery, new experiments were carried out using other apoptotic agents, namely the stress inducer heat shock (2 h at 42.5 °C followed by recovery) (28, 29) and the antitumor drug camptothecin (as a continuous treatment at 0.4 µM). Whereas heat shock did not significantly induce p38^MAPK phosphorylation, this kinase was transiently phosphorylated by camptothecin (Fig. 4A). Nevertheless, SB203580 failed to attenuate the generation of apoptosis by both agents, as demonstrated by chromatin fragmentation (Fig. 4B) and the accumulation of cells with sub-G₁ DNA content (result not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effect of heat shock and camptothecin on p38MAPK phosphorylation and apoptosis and their modulation by SB203580. Cells were heated for 2 h at 42.5 °C (HS) and allowed to recover or continuously treated with 0.4 µM camptothecin (Cpt). A, relative levels of p38MAPK phosphorylation in untreated cells (Cont), and at different times of treatment (treat) and/or recovery (rec). The film in the HS experiment was overexposed to clearly detect the basal level of phosphorylated p38MAPK. B, frequency of apoptotic cells at 6 h of treatment (camptothecin) or recovery (heat shock) in the absence (Cpt and HS) or the presence (Cpt/SB and HS/SB) of 10 µM SB203580. The experiments in A were repeated twice with similar results. The values in B represent the mean ± S.D. of three determinations. All other conditions were as in Figs. 1-3.

Stress Response and Necrosis Induction-- To exclude the possibility of a general, nonspecific cadmium inactivation by SB203580 (e.g. by direct drug-metal interaction) we analyzed the action of this kinase inhibitor on other effects of cadmium, namely the stress response and necrosis induction. The stress response was measured by HSF1 binding at 2 h of treatment and by HSP70 expression at different times of recovery. It was observed that SB203580 did not affect the cadmium-provoked accumulation of HSP70 (Fig. 5A) nor the stimulation of HSF1 binding (Fig. 5B), suggesting that the p38^MAPK is not relevant for the induction of the stress response by cadmium in these cells. Moreover, SB203580 also failed to inhibit the increase in HSP70 caused by heat shock, whereas as expected (33), the increase was abolished or greatly reduced by the antioxidant agents NAC and BHA (Fig. 5C). This excludes the possibility that SB203580 may act as a radical oxygen scavenger in U-937 cells.

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Induction of HSP70 expression and stimulation of HSF1 binding activity. In A and B, cells were treated for 2 h with 200 µM cadmium-chloride and allowed to recover, either in the absence (Cd) or in the presence (Cd/SB) of 10 µM SB203580. A, HSP70 levels in untreated cells (Cont) and at 3 and 6 h of recovery, as determined by immunoblot using an anti-HSP70 antibody. As a control, the membrane was reprobed using an anti--tubulin antibody. B, HSF1 binding activity in untreated cells (Cont) and after 2 h of cadmium treatment. As controls for binding specificity, aliquots from the "Cd" sample were incubated with an anti-HSF1 antibody (HSF1ab) or with 50-fold excess related (heat-shock element) or unrelated (AP-1-recognizing) oligonucleotides. HSF1 indicates the protein-DNA complex, whereas the arrow indicates free oligonucleotide. C, HSP70 levels in untreated cells (Cont) and in cells heated for 1 h at 42 °C and allowed to recover for 3 h, either in the absence or the presence of SB203580, NAC, and/or BHA. SB203580 (10 µM), NAC (15 mM), and BHA (100 µM) were applied 2 h before heat shock and maintained during the treatment and recovery periods. As a control, the membrane was reprobed using an anti--tubulin antibody. The experiments were repeated twice (B and C) or three times (A), with similar results. All other conditions were as in Figs. 1-3.

It was reported that depletion of intracellular GSH may potentiate the lethality of cytotoxic drugs, occasionally switching the mode of death from apoptosis to necrosis (34).² We observed that a 18-24-h incubation with the GSH depletor BSO (1 mM) was innocuous (Fig. 6, A and B) and did not affect p38^MAPK and ERK1/2 phosphorylation (results not shown). However, preincubation with BSO potentiated the cadmium toxicity, causing necrosis instead of apoptosis, as revealed by free uptake of trypan blue (Fig. 6A) and PI (Fig. 6B). Under these conditions p38^MAPK was phosphorylated with the same efficacy, and ERK1/2 with greater efficacy, than in cells treated with cadmium alone (Fig. 6C). However, neither SB203580 nor PD98059 were able to prevent the necrotic response (Fig. 6, A and B). Taken together, the present results indicate that the inhibition by SB203580 of cadmium-provoked apoptosis is a highly specific effect.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Modulation by BSO of the cadmium-provoked cell death and MAPK phosphorylation. Cells were treated for 2 h with 200 µM cadmium chloride and allowed to recover. BSO (1 mM) was applied 18 h before cadmium and maintained during the cadmium treatment and recovery periods. A, the left panel shows the frequency of apoptotic (open symbols) and necrotic (closed symbols) cells at different times of recovery upon treatment with BSO plus cadmium, as determined by chromatin fragmentation and trypan blue permeability, respectively. The frequency of apoptotic and necrotic cells in cultures treated with BSO alone was below 5% (not shown). The right panel shows the frequency of necrotic cells at 6 h of recovery in cultures treated with BSO alone, BSO plus cadmium (BSO/Cd), or BSO plus cadmium in combination with either 10 µM SB203580 (BSO/Cd/SB) or 30 µM PD98059 (BSO/Cd/PD). B, annexin V binding and PI uptake in nonpermeabilized cells at 6 h of recovery, using the same treatments as in A. C, relative levels of p38MAPK and ERK1/2 phosphorylation in untreated cells (Cont) and in cells treated with cadmium alone (Cd) or with BSO plus cadmium (BSO/Cd), measured at 1 h of treatment (1 h tr) or recovery (1 h rec). The values in A represent the mean ± S.D. of three experiments. The determinations in B and C were repeated twice and three times, respectively, with similar results. All other conditions were as in Figs. 1-3.

Caspase Activity, Oxidative Stress, and Mitochondrial Transmembrane Potential-- Finally, we wanted to investigate the possible relationship between p38^MAPK activation and other events that regulate apoptosis. This included caspase activities, intracellular oxidation and changes in mitochondrial transmembrane potential (_m). It is known that apoptosis requires the action of ICE-like proteases (caspases) (for review see Ref. 35). Moreover, caspase activities seem also to mediate p38^MAPK activation by some inducers (14). To approach the problem, we analyzed the action of the caspase-3-specific inhibitor DEVD-CHO and the nonspecific caspase inhibitor Z-VAD on apoptosis and p38^MAPK activation in cadmium-treated cells. As expected, both inhibitors attenuated apoptosis (Fig. 7A). However, they did not affect p38^MAPK phosphorylation (Fig. 7B). This suggests that p38^MAPK plays its regulatory role on apoptosis upstream or independently of the caspase cascade.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effect of caspase inhibitors. Cells were treated for 2 h with 200 µM cadmium chloride and allowed to recover. Z-VAD (20 µM) and DEVD-CHO (40 µM) were applied 10 min before cadmium and maintained during the cadmium treatment and recovery periods. A, frequency of apoptosis in untreated cultures (Cont) and at 6 h of recovery from cadmium treatment, either alone (Cd) or in combination with the caspase inhibitors (Cd/Z-VAD and Cd/DEVD). B, p38MAPK phosphorylation at 0.5 and 2 h of treatment, using the same combinations as in A. The values in A represent the mean ± S.D. of three determinations. The experiment in B was repeated twice with the same result. All other conditions were as in Figs. 1-3.

Earlier reports indicated that cytotoxic insults rapidly cause intracellular oxidation measured by H₂O₂ accumulation, which at least in some apoptotic models is a trigger for cell death (25, 36, 37). In addition, apoptosis involves and is regulated by a disruption of _m (for reviews see Refs. 38 and 39). Hence, we wanted to investigate the possible relationship between these phenomena and p38^MAPK activation. We found that cadmium caused a rapid increase in H₂O₂ accumulation, which was already detected after 30 min of treatment (Fig. 8A), at the same time as the increase in p38^MAPK phosphorylation. The treatment also caused a late disruption of _m, which was first detected at 3 h of recovery (Fig. 9), hence paralleling the expression of apoptotic markers. The administration of the antioxidant agent BHA reduced cell death (Fig. 8B), proving the importance of intracellular oxidation for the cadmium-provoked apoptosis, but it did not inhibit p38^MAPK phosphorylation (Fig. 8C). More potent antioxidants such as NAC and other thiol-containing agents could not be used because of the direct reactivity of -SH groups with Cd²⁺ ions. Interestingly, the administration of SB203580 greatly reduced H₂O₂ accumulation (Fig. 8A) as well as _m disruption (Fig. 9). Taken together, these results indicate that p38^MAPK activation is an early regulatory event in cadmium-provoked apoptosis.

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Hydrogen peroxide accumulation and effect of antioxidants. A, intracellular accumulation of H2O2 in untreated cells (Cont) and after 0.5 and 1 h of treatment with 200 µM cadmium chloride, either alone (Cd) or in combination with 10 µM SB203580 (Cd/SB), as revealed by the increase in fluorescence upon dichlorodihydrofluorescein diacetate loading. B, frequency of apoptosis in untreated cultures (Cont) and after 6 h of recovery from cadmium treatment, either alone (Cd) or in combination with BHA (Cd/BHA). C, p38MAPK phosphorylation after 0.5 and 2 h of cadmium treatment, using the same combinations as in B. BHA (100 µM) was applied 2 h before cadmium and maintained during the treatment and recovery periods. The experiments in A and C were repeated three times with similar results. The values in B represent the mean ± S.D. of four determinations. All other conditions were as in Figs. 1-3.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Modulation of mitochondrial transmembrane potential (m). The figure shows the degree of m disruption at different times of recovery from cadmium treatment (2 h at 200 µM), either in the absence (Cd) or in the presence (Cd/SB) of 10 µM SB203580, as revealed by the decrease in fluorescence upon 3,3'-dihexylocarbocyanine iodide loading. All other conditions were as in Figs. 1-3.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although apoptotic stimuli usually activated p38^MAPK and this activation was required for apoptosis in some models (40-43, among others), it seemed to be irrelevant in others. This was proved by the inability of the p38^MAPK-specific inhibitor SB203580 to prevent apoptosis in Fas- and UV-treated Jurkatt T cells (14), in UV-treated U-937 cells (16), in H₂O₂-treated HeLa cells (17), and in nitric oxide-treated RAW macrophages (18), among other examples. Likewise, a p38^MAPK dominant-negative mutant failed to prevent the induction of apoptosis in UV- and -radiation in 293T human embryonic kidney cells (13). It was reported that heavy metals also induce p38^MAPK activation in different cell types (19, 44, 45), but the actual relevance of such activation for apoptosis was not known. Our present results indicate that treatment of U-937 promonocytic cells with apoptosis-inducing concentrations of cadmium chloride rapidly causes p38^MAPK activation, as judged by its increased phosphorylation, and proves that this activation is required for apoptosis, as revealed by the capacity of SB203580 to reduce both p38^MAPK phosphorylation and apoptosis. Moreover, the regulation by p38^MAPK of the cadmium-provoked apoptosis seemed to be a highly specific effect, which cannot be explained by a general interference of the kinase inhibitor with either the apoptotic machinery or the total cadmium activity. This conclusion is based on the following: (a) SB203580 did not prevent the generation of apoptosis by other agents, such heat-shock and camptothecin, although the later one also caused p38^MAPK activation; (b) SB203580 did not prevent the generation of necrosis induced by the cadmium treatment of BSO-preincubated cells, although this treatment also increased p38^MAPK phosphorylation; and (c) SB203580 did not prevent the cadmium-provoked activation of the stress response. This later result was somewhat surprising, because SB203580 was reported to prevent HSF1 activation and HSP70 induction in rat brain tumor cells treated with high apoptosis-inducing cadmium concentrations (19). Such a discrepancy suggests the existence of marked differences in the regulation of the stress response in different cell types.

The rapid phosphorylation of p38^MAPK suggested that this kinase plays an early role in the regulation of the cadmium-provoked apoptosis. This conclusion was also proved by examining other regulatory events, such as caspase activity, _m disruption, and intracellular oxidation. It is known that apoptosis requires the activation of a cascade of ICE-like proteases, which, at least in myeloid cells, ends with the activation of the effector caspase-3 (46, 47). Moreover, caspases may also regulate p38^MAPK activation by some apoptotic inducers, as in Fas-treated Jurkatt T cells (14). However, our experiments showing that the activation of p38^MAPK by cadmium was insensitive to the pan-caspase inhibitor Z-VAD or the caspase-3-specific inhibitor DEVD-CHO suggest that the regulation by p38^MAPK of the cadmium-provoked apoptosis is either upstream or independent of the caspase cascade. The attenuation by SB203508 of the fall in _m suggests that the regulatory role of p38^MAPK is also upstream of _m disruption, a phenomenon that in some cases is an early event while in others (as in cadmium-treated U-937 cells) occurs simultaneously with the execution cell death (48-50). Finally, the interaction between p38^MAPK activation and intracellular oxidation was more intriguing, because both phenomena took place rapidly upon cadmium administration and could not be separated based on kinetic data. Earlier reports indicate that the toxicity of cadmium is the result at least in part of intracellular oxidation (51, 52). This was confirmed in our experiments showing an increase in H₂O₂ production by cadmium and a reduction in apoptosis by the antioxidant agent BHA. Moreover, it was found that SB203580 attenuated H₂O₂ accumulation, whereas BHA did not affect p38^MAPK phosphorylation. The possibility that SB203580 acts as a radical oxygen scavenger may be excluded, because this agent did not prevent the heat-provoked induction of HSP70, as done by typical antioxidants (33 and Fig. 5C). Hence, we conclude that p38^MAPK activity probably plays a role in the triggering of intracellular oxidation by cadmium. An attractive hypothesis is that p38^MAPK is required for apoptosis only when apoptosis is regulated via intracellular oxidation, a subject that is at present under investigation in our laboratory. Interestingly, a recent report indicates that SB203580 inhibits NADPH oxidase in human neutrophils, suggesting that p38^MAPK kinase regulates the oxidative burst in these cells (53).

In addition to their inducibility by mitogenic stimuli, ERKs may also be activated by heavy metals and other cytotoxic insults (17-19, 44, 45). In most cellular models, ERK activation seems to inversely correlate with apoptosis. For instance, suppression of ERK1/2 activation by PD98059 enhanced apoptosis in H₂O₂-treated HeLa cells (17) and in nitric oxide-treated RAW macrophages (18), and conversely, the activation of the ERK pathway inhibited apoptosis in neuronal PC-12 cells (11). However, in other models PD98059 inhibited apoptosis, e.g. in nerve growth factor-deprived pheochromocytoma cells (40) and in H₂O₂-treated CG4 oligodendrocytic cells (54), suggesting a pro-apoptotic role for ERK1/2 activity. Watabe et al. (55) showed that bufalin caused a late ERK activation in U-937 cells, which could explain the generation of apoptosis by this agent. In our experiments cadmium caused a similar late ERK1/2 activation, as indicated by their increased phosphorylation, which paralleled the execution of apoptosis. Moreover, preincubation with BSO followed by cadmium treatment caused necrosis instead of apoptosis and overincreased ERK1/2 phosphorylation, suggesting that the extent of kinase activation might also be important in determining the mode of death. However, PD98059 failed to prevent apoptosis in cadmium-treated cells and necrosis in BSO plus cadmium-treated cells, indicating that ERK activity is irrelevant for death induction (either apoptotic or necrotic) by cadmium. Studies using other apoptotic agents and different procedures to modulate ERK1/2 activity are in progress to better define the possible role of this kinase in death induction in promonocytic cells.

	FOOTNOTES

^* This work was supported by Grant PB97-0144 from the Dirección General de Enseñanza Superior e Investigación Científica, Grant 08.1/0027/1997 from the Comunidad Autónoma de Madrid, Spain (to P. A.), Grant RPG-97-092-01-CNE from the American Cancer Society, and Grant ROI-CA 74197-01 from the National Institutes of Health (to M. G. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Recipients of predoctoral fellowships from the Ministerio de Educación y Cultura, Spain.

Recipient of a postdoctoral fellowship from the Fundación Ramón Areces, Spain.

^** To whom correspondence may be addressed: Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006 Madrid, Spain. Tel.: 34-915644562 (ext. 4247); Fax: 34-915627518; E-mail: aller@cib.csic.es.

² A. Galán, M. L. García-Bermejo, A. Troyano, N. E. Vilaboa, E. de Blas, M. G. Kazanietz, and P. Aller, unpublished results.

	ABBREVIATIONS

The abbreviations used are: HSP, heat-shock protein; HSF, heat-shock factor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; BHA, butylated hydroxyanisole; BSO, DL-buthionine-SR-sulfoximine; NAC, N-acetyl-L-cysteine; PI, propidium iodide; pAb, polyclonal antibody; PBS, phosphate-buffered saline; GSH, glutathione.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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