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Abstract

Diepoxybutane (DEB) is the most potent active metabolite of butadiene, a regulated air pollutant. We previously reported the occurrence of DEB-induced, p53-dependent, mitochondrial-mediated apoptosis in human lymphoblasts. The present study investigated the role of the extracellular signal–regulated protein kinases 1 and 2 (ERK1/2) pathway in DEB-induced apoptotic signaling in exposed human lymphoblasts. Activated ERK1/2 and mitogen-activated protein (MAP) kinase/ERK1/2 kinase (MEK) levels were significantly upregulated in DEB-exposed human lymphoblasts. The MEK inhibitor PD98059 and ERK1/2 siRNA significantly inhibited apoptosis, ERK1/2 activation, as well as p53 and phospho-p53 (serine-15) levels in human lymphoblasts undergoing DEB-induced apoptosis. Collectively, these results demonstrate that DEB induces apoptotic signaling through the MEK-ERK1/2-p53 pathway in human lymphoblasts. This is the first report implicating the activation of the ERK1/2 pathway and its subsequent role in mediating DEB-induced apoptotic signaling in human lymphoblasts. These findings contribute towards the understanding of DEB toxicity, as well as the signaling pathways mediating DEB-induced apoptosis in human lymphoblasts.

Keywords

Diepoxybutane butadiene apoptosis extracellular signal–regulated protein kinase ERK1/2 p53

Introduction

Diepoxybutane (DEB) is a metabolite of 1,3-butadiene (BD). BD (CH2=CH–CH=CH2, CAS No. 106-99-0) is a regulated hazardous air pollutant, and a known human carcinogen and mutagen.^1

–5 Human exposure to BD occurs mainly in occupational settings, although low-level exposures have been reported in nonoccupational settings.^5,6 BD exposure in rodents and humans results in various toxic responses that include multisite carcinogenesis, bone marrow depletion, ovarian, spleen, and thymic atrophy.^5,7
–9 The toxic effects of BD are mediated through the action of its active metabolites, with DEB being the most potent.^1,2,10,11 However, the molecular mechanisms of BD and DEB toxicity are not completely understood.

DEB is known to be genotoxic, cytotoxic, and carcinogenic. It induces chromosomal aberrations, micronucleus formation, sister chromatid exchanges, and mutations in various animal and human cells.^{1,2,5,10
–12} DEB behaves as a bifunctional, alkylating agent that generates reactive oxygen species and forms DNA–DNA and DNA-protein crosslinks.^12

–15 A number of reports have demonstrated the ability of DEB to induce apoptosis in rat and human cells.^12,16,17

Apoptosis is a highly ordered, cellular suicidal mechanism used to eliminate excess, damaged, or cancerous cells throughout life in a variety of organisms.^18,19 Apoptosis occurs in response to various kinds of stressful stimuli, including exposure to DNA damaging agents and environmental stresses such as DEB.^20
–22 Apoptosis was observed in mouse L929 cells,²³ in Big Blue^® Rat fibroblasts,¹² and in human lymphoblasts following exposure to low concentrations of DEB ranging from 2.5 µM to 10 µM.¹⁷ This apoptosis accounted for the DEB-induced cytotoxicity observed in human lymphoblasts at DEB concentrations up to 10 µM.¹⁷ Furthermore, this apoptosis was reported to be mediated by DEB-induced oxidative stress through the mitochondrial pathway, and was p53-dependent at early times (at 24 h and not at 48 h) post-DEB exposure.^17,24 However, the mechanisms involved in DEB-induced p53-mediated apoptotic signaling in human lymphoblasts are currently unknown.

The tumor suppressor protein p53 is one of the major regulators of the apoptotic process in response to DNA damage and environmental stress.^{19,25

–32} The p53 protein is normally maintained at low levels in unstressed cells.^29,33 In response to apoptotic stress, p53 is stabilized and activated to mediate apoptosis by acting directly at the mitochondria or through transcriptional regulation of Bcl2 family members and other apoptosis genes.^34
–36 This activation of p53 in response to stress occurs through the induction of posttranslational modifications within p53^26,34,37,38; these include acetylation, ubiquitinylation, methylation, sumoylation, and phosphorylation at various residues.³⁸ Phosphorylation at the serine-15 position of p53, however, is the initial step and major focal point in the stabilization and activation of p53 during the DNA damage response, irrespective of the initiating stimulus or cell type.^28,33,36,39 In fact, p53 stabilization in human lymphoblasts undergoing DEB-induced apoptosis is directly proportional to the degree of p53 phosphorylation on the serine-15 residue.⁴⁰ Various signaling pathways, including the ERK pathway, can potentially be activated by specific stresses to bring about serine-15 p53 phosphorylation, thus regulating p53 stability and apoptotic functions.^36,41
–43

Extracellular signal–regulated protein kinases 1 and 2 (ERK1/2) are components of the mitogen-activated protein (MAP) kinase superfamily. They are activated by MAP/ERK1/2 kinases 1 and 2 (MEK1/2) by dual phosphorylation at their threonine 202 and tyrosine 204 residues.^44,45 A variety of biological responses (cell proliferation, migration, differentiation, and apoptosis) are associated with ERK1/2 activation; these differential responses are dependent upon cell type, stimulus, magnitude and duration of activation, subcellular localization, interplay with other pathways, and other unclear factors.^{44

–53} For example, transient ERK activation leads to cell proliferation, while sustained ERK activation lasting longer than 12 h may lead to differentiation or apoptosis, depending on the cell type and the nature of the stimulus.^53
–55 In fact, sustained ERK1/2 activation contributes to apoptotic signaling induced by various toxicants, including those that induce cell death through the induction of DNA damage.^44,51,56,57 Sustained ERK activation has also been found to phosphorylate and stabilize p53, thus mediating apoptosis in various systems.^44,57
–59 For example, Zhang et al. reported that the doxorubicin-induced apoptosis in cardiomyocytes was mediated through the ERK1/2/p53 pathway.⁵⁷ Sustained ERK activation was also shown to mediate resveratrol-induced apoptosis through the stabilization of p53 by phosphorylating it at serine-15.⁶⁰

The upstream signaling pathways responsible for mediating DEB-induced, p53-dependent apoptosis in TK6 human lymphoblasts are currently unknown. Since sustained ERK1/2 activation plays a role in mediating apoptosis in other systems, ^44,58,59 its role in DEB-induced apoptosis in human lymphoblasts was investigated in the studies presented here. The hypothesis that the ERK1/2 pathway is activated to mediate DEB-induced apoptotic effects through p53 as a downstream effector in exposed human TK6 lymphoblasts was tested. The DEB concentration and exposure times utilized were predetermined during our previous studies with this experimental system.^17,24,40

Materials and methods

Chemicals

DEB (11.267M) was purchased from the Sigma-Aldrich Chemical Company, St. Louis, MO; dilutions were made in Roswell Park Memorial Institute (RPMI) 1640 (Thermo Scientific, Waltham, MA). The MEK specific inhibitor PD98059 was purchased from the Promega Corporation in Madison, WI.

Antibodies

The following antibodies were obtained from Cell Signaling Technology, Inc in Danvers, MA: anti-phospho-ERK1/2 (Thr202/Tyr204, catalog # 4370), anti-total ERK1/2 (catalog # 4695), anti-phospho-MEK1/2 (ser217/221, catalog # 9154), and anti-phospho-p53 (serine-15, catalog # 9284). All primary antibodies from Cell Signaling Technology, Inc. were used at 1:1000 dilution. Anti-p53 antibody (catalog # OP43L) and an anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (catalog # MAB374) were purchased from the EMD-Millipore Corporation, Bedford, MA; these antibodies were also utilized at 1:1000 dilution. The fluorescent conjugated antibodies IRDye^® 800CW goat anti-rabbit IgG (P/N 925-32211; used at 1:10,000 dilution) and IRDye^® 680LT goat anti-mouse IgG (P/N 925-68020; used at 1: 15,000 dilution) were obtained from Li-Cor Biosciences, Inc in Lincoln, NE.

Cell culture and enumeration

The human B lymphoblastic TK6 cell line was obtained from the American Type Culture Collection; this cell line expresses wild type endogenous p53. Cells were propagated at 2.0 × 10⁵ cells/mL in RPMI 1640 Medium (Lonza Inc., Walkersville, MD) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA), 2-mM l-glutamine, and penicillin–streptomycin. Cell concentration and viability were determined in triplicates by using the Vi-CELL XR Cell Viability Analyzer (Beckman Coulter, Inc., Indianapolis, IN).

Exposure of cells to DEB

This was performed essentially as previously described.^17,40 Cells were placed in fresh media 24 h prior to each experiment. Cells were then washed and seeded in fresh media at a density of 2.0 × 10⁵ cells/mL, prior to the addition of 10-µM DEB or vehicle for 24 and 48 h. All DEB dilutions were performed in RPMI. The 10-µM DEB concentration utilized in these experiments was predetermined in previous studies on DEB-induced apoptosis utilizing this system.¹⁷ This DEB concentration is optimal for study; it induces an easily measured extent of apoptosis, but not necrosis, that is, linear with respect to various DEB concentrations and exposure time up to 48 h.¹⁷ Exposure to DEB in the presence of 50-µM PD98059 MEK inhibitor was performed by pretreating cells for 1 h with PD98059 diluted in DMSO; the final DMSO concentration in control- and inhibitor-exposed cell cultures was 0.1%. Exposure to DEB in the presence of control and ERK1/2 siRNA was performed after pretreating cells with siRNAs for 12 h. All procedures were performed under class 2 type B3 conditions.

Quantitation of DEB-induced apoptosis

The percentage of apoptotic cells in control- and DEB-exposed cells was quantified by assessing nuclear morphology using dual staining with acridine orange and ethidium bromide as previously described.^17,24,61 At least 150 cells were counted in multiple randomly selected fields using a fluorescence microscope; experiments were repeated three times. The Caspase-Glo^®3/7 assay (catalog # G8091, Promega Corporation, Madison, Wisconsin, USA) was also utilized to assess the extent of apoptosis; this was performed as described by the kit manufacturer. All caspase 3 apoptosis quantifications were performed in triplicates; the experiment was repeated three times.

ERK 1/2 siRNA knockdown of DEB-induced apoptosis

Human TK6 lymphoblasts (8 × 10⁶ cells) were transfected with 2.5-nM Silencer Select ERK siRNA (ID #S11140; catalog # 439082) or Silencer Select control scrambled negative control siRNA (catalog # 4390847) using the Lipofectamine^® RNAiMAX reagent (Thermo Fisher Scientific, Inc., Waltham, MA), according to the vendor’s protocol. Cells were then exposed to control or 10-µM DEB at 12 h post-transfection and kept at 37°C in a 5% CO₂ incubator. Cells were quantified in triplicates for apoptosis at 24 h post-DEB exposure using the Caspase-Glo^®3/7 assay. In addition, cell extracts (15 µg) were quantified for phospho-(Thr202/Tyr204)-ERK, ERK, total p53, and GAPDH levels using the western blot analysis technique and appropriate antibodies. All experiments were repeated three times.

Western blot analysis of p53 and ERK pathway proteins

Western blot analysis was utilized to determine the levels of activated phospho-ERK1/2(Thr202/Tyr204), activated phospho-MEK1/2(ser217/221), phospho-p53 (serine-15), total ERK1/2, p53, and GAPDH in all extracts. The procedure was performed as previously described,^17,40 and as modified for fluorescent antibody imaging on the Li-Cor Odyssey system (Li-Cor, Inc., Lincoln, NE), according to the system manufacturer’s instructions. Briefly, experimental and control cells were harvested and lysed by gentle vortexing at 4°C in TBSTDS (10-mM tris, (pH 7.5), 150-mM sodium chloride, 1-mM EDTA, 1% triton X-100, 0.5% sodium deoxycholate, 0.5% sodium-dodecyl-sulfate (SDS), 0.02% sodium azide, and 0.0004% sodium fluoride) supplemented with protease inhibitors (1-mM phenylmethylsufonyl fluoride, 2-μg/mL aprotinin, and 0.1-mM leupeptin) and a phosphatase inhibitor cocktail (2.5-mM sodium pyrophosphate, 1-mM sodium orthovanadate, 25-mM sodium fluoride, and 2-mM β-glycerol-phosphate). Lysates were cleared by spinning (16,000×g for 30 min). Equal amounts of protein (15–25 µg/lane, depending on the experiment) were then subjected to SDS polyacrylamide gel electrophoresis. The electrophoresed proteins were transferred onto Immobilon-FL polyvinylidine difluoride membranes. The membranes were blocked for nonspecific binding in Odyssey blocking buffer for 1 h, and incubated with appropriate primary antibody in Odyssey blocking buffer. Phosphorylation-specific antibodies for activated ERK1/2 (Thr202/Tyr204), MEK1/2(ser217/221), and p53 proteins were incubated overnight at 4°C. Antibodies for total ERK1/2, p53, and GAPDH proteins were incubated for 1 h at room temperature. The membranes were washed and incubated with an appropriate fluorescent IRDye^® conjugated secondary antibody. Antigen levels were detected after extensive washing by utilizing a Li-Cor Odyssey Imaging System. Quantitation of bands was performed in triplicates using the Odyssey version 3.0 software. Presented western blots are representatives of three independent experiments.

Statistical analysis

All statistical analyses were performed using Graph Pad Prism version 7.1 (San Diego, California, USA). Data are presented as mean ± standard error. The significance of difference was determined by using a t-test to compare means of values between groups. Values of p < 0.05 were considered statistically significant.

Results

DEB induces activation of extracellular signal–regulated kinase (ERK1/2) pathway in human lymphoblasts

The activation of the MEK-ERK pathway in human lymphoblasts undergoing DEB-induced apoptosis was assessed. Control-exposed and 10-µM DEB-exposed TK6 lymphoblasts were analyzed by western blot analysis using activation-specific antibodies targeting ERK1/2 and MEK1/2 phosphorylation at Thr202/Tyr204 and ser217/221, respectively (Figure 1). Under conditions where GAPDH levels remained unchanged, normalized phosphorylated ERK (Thr202/ Tyr204) levels significantly increased by approximately twofold at 24 h, and by fourfold at 48 h postexposure in DEB-exposed cells as compared to the corresponding control-unexposed cells (p < 0.05; Figure 1(a)). Normalized phospho-MEK (P-MEK; ser217/221) levels also significantly increased by 2.7-fold at 24 h, and by 3.9-fold at 48 h postexposure in DEB-exposed cells as compared to the control-unexposed cells (p < 0.05; Figure 1(b)). Normalized phospho-ERK (P-ERK) and P-MEK levels in exposed cells at 48 h were significantly greater than normalized P-ERK and P-MEK levels in exposed cells at 24 h (p < 0.05). The specific elevation of normalized P-ERK (Thr202/Tyr204) and P-MEK (ser217/221) levels at 24 and 48 h implied that the MEK-ERK1/2 pathway is activated in cells undergoing DEB-induced apoptosis.

Figure 1.

DEB induces activation of the ERK pathway in exposed human lymphoblasts. TK6 lymphoblasts were exposed to 0 and 10 µM DEB for 24 and 48 h. Protein extracts (25 µg) obtained were analyzed for activation of ERK and MEK on separate blots utilizing the western blot technique. This was performed by utilizing rabbit antibodies specific for activated P-ERK (Thr202/Tyr204) or activated P-MEK (ser217/221), followed by IRDye-680 conjugated goat anti-rabbit secondary antibody. The P-ERK blots were subsequently reacted with a rabbit antibody specific for total ERK, followed by IRDye-800 conjugated goat anti-rabbit antibody. Finally, the blots were analyzed for GAPDH, a loading control; this utilized a mouse antibody and IRDye-680 conjugated goat anti-mouse secondary antibody. (a) Relative normalized P-ERK levels at each time point were obtained after quantification analysis by comparing P-ERK to total ERK ratios for each sample against the control-unexposed sample value. (b) Relative normalized P-MEK levels were obtained after quantification, followed by normalization to the corresponding GAPDH level for each sample. All quantifications were performed in triplicates, and a representative of three experiments is shown. Significance: *p < 0.05: compared to the corresponding control-unexposed cells at each time point, as well as between exposed cells at 24 h versus 48 h. DEB: diepoxybutane; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; P-ERK: phospho-ERK; P-MEK: phospho-MEK.

Activated extracellular signal–regulated kinase (ERK1/2) pathway mediates DEB-induced apoptosis in human lymphoblasts

The role of the MEK-ERK1/2 pathway in mediating DEB-induced apoptosis in human lymphoblasts was examined (Figure 2). As shown in Figure 2(a), 20% and 59% of the cells exposed to DEB in the absence of PD98059 for 24 and 48 h, respectively, were apoptotic. In the presence of the MEK inhibitor PD98059, 7% and 47% of the DEB-exposed cells were undergoing apoptosis at 24 and 48 h, respectively. Thus, apoptosis in DEB-exposed cells pretreated with PD98059 was reduced by 65% at 24 h, and by 20% at 48 h postexposure as compared to the DEB-exposed cells without PD98059. This represents a significant reduction in apoptosis levels in 24-h DEB-exposed cells with the inhibitor as compared to the corresponding cells at 48 h (p < 0.05). Western blot analysis of activated ERK1/2, total ERK1/2, and GAPDH were performed on the resultant protein extracts (Figure 2(b)). Under conditions where GAPDH levels remained unchanged, activated normalized phospho-ERK1/2 levels significantly increased by 3.6-fold at 24 h, and 7.0-fold at 48 h postexposure in DEB-exposed cells as compared to the unexposed cells (p < 0.05). In the presence of PD98059, the DEB-induced ERK1/2 activation was prevented at 24 h post-DEB exposure, and was inhibited by 40% at 48 h post-DEB exposure. This represents a significant reduction in ERK activation due to inhibitor in 24-h DEB-exposed cells as compared to the corresponding cells at 48 h (p < 0.05). However, integration of apoptosis and ERK activation levels demonstrated that the inhibition of ERK activation by 40% only corresponded to an apoptosis reduction of 20% at 48 h postexposure. In contrast, the almost complete inhibition of ERK activation corresponded to almost complete reduction in the extent of apoptosis at 24 h postexposure (Figure 2(a) and (b)). Total ERK1/2 levels were not significantly altered in the presence of PD98059 and/or DEB under these experimental conditions (p > 0.05). These results demonstrate that ERK activation plays a role in mediating DEB-induced apoptosis in human TK6 lymphoblasts, especially at early times (24 h as compared to 48 h) post-DEB exposure.

Figure 2.

Activated ERK mediates DEB-induced apoptosis in exposed human lymphoblasts. TK6 cells were preincubated with vehicle alone or 50-µM MEK inhibitor PD98059 for 1 h. Control- and PD98059-treated cells were then exposed to 10-µM DEB for 24 and 48 h; a second set of cells was exposed to vehicle without DEB. (a) The percentage of apoptosis was determined by nuclear morphology fluorescence dye staining, as described in “Materials and methods” section. (b) Western blot analysis of activated P-ERK, total ERK (ERK), and GAPDH levels were determined using 25 µg of protein, as described in Figure 1. Relative normalized P-ERK levels for each sample were obtained after quantification analysis (in triplicates) by utilizing the ratio of P-ERK to total ERK. A representation of three experiments is shown. Significance: *p < 0.05: compared to the corresponding control-unexposed cells, as well as between DEB-exposed cells with and without the MEK inhibitor at each time point. *p < 0.05: comparison between 24 h and 48 h inhibition of apoptosis and activated ERK levels by MEK inhibitor PD98059. DEB: diepoxybutane; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; P-ERK: phospho-ERK.

The p53 protein is a downstream target of extracellular signal–regulated kinase (ERK1/2) during DEB-induced apoptosis in human lymphoblasts

The possibility that activated ERK mediates DEB-induced apoptosis in human TK6 lymphoblasts through p53 was investigated. To accomplish this, control and TK6 cells pretreated with the PD98059 MEK inhibitor for 1 h prior to DEB exposure were analyzed for apoptosis and serine-15 phospho-p53 protein levels (Figure 3). DEB-induced apoptosis was significantly inhibited by approximately 65% at 24 h, and by 20% at 48 h postexposure in the presence of the MEK inhibitor as compared to the absence of the inhibitor (p < 0.05; Figure 3(a)). The reduction in the quantity of apoptosis due to the MEK inhibitor was significantly greater at 24 h than at 48 h post-DEB exposure. Under these conditions, western blot analysis (Figure 3(b)) demonstrated that normalized serine-15 phospho-p53 levels significantly decreased by 38% at 24 h, and by 22% at 48 h, in DEB-exposed MEK inhibitor–treated cells as compared to the corresponding control untreated DEB-exposed cells (p < 0.05). Total p53 levels were directly proportional to serine-15 phospho-p53 levels under these conditions (thus, not shown), as we previously reported.⁴⁰ Since the elevation of serine-15 phospho-p53, total p53, and apoptosis were inhibited by the MEK inhibitor PD98059 predominatly at 24 h post-DEB exposure, these results demonstrate that ERK1/2 mediates DEB-induced apoptosis in human lymphoblasts, at least partly, through the activation of p53 by phosphorylating it on serine-15.

Figure 3.

p53 is a downstream target of ERK in human lymphoblasts undergoing DEB-induced apoptosis. TK6 cells were preincubated with vehicle alone or 50-µM PD98059 for 1 h, followed by exposure to vehicle alone or 10-µM DEB for 24 or 48 h. (A) The percentage of apoptotic cells were determined by nuclear morphology fluorescence dye staining, as described in “Materials and methods” section. (b) Levels of phospho-p53 (serine-15) and GAPDH were analyzed by the western blot technique, using 25 µg of extract protein. This was accomplished by utilizing a rabbit antibody specific for phospho-p53 (serine-15) and a mouse antibody specific for GAPDH, a loading control. IRDye-680 conjugated goat anti-rabbit and IRDye-800 conjugated goat anti-mouse secondary antibodies were utilized for the detection and quantitation phospho-p53 (serine-15) and GAPDH levels, respectively. The graph shows relative normalized phospho-p53 (serine-15) levels obtained after quantification analysis (in triplicates) by normalizing each point to the corresponding GAPDH levels. A representation of three experiments is shown. Significance: *p < 0.05: compared to the corresponding control-unexposed cells, as well as between DEB-exposed cells with and without the MEK inhibitor at each time point. *p < 0.05: comparison between 24 h and 48 h inhibition of apoptosis and activated ERK levels by MEK inhibitor PD98059. DEB: diepoxybutane; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Confirmation of the role of extracellular signal–regulated kinase (ERK1/2) in mediating DEB-induced apoptosis in human lymphoblasts

To confirm the role of the ERK1/2–p53 pathway in mediating DEB-induced apoptosis, the effect of ERK1/2 knockdown on ERK1/2 activation, p53 levels, and DEB-induced apoptosis in exposed human lymphoblasts was investigated. This was accomplished by pretreating cells with 2.5-nM ERK1/2 siRNA for 12 h prior to their exposure to 10-μM DEB for 24 h (Figure 4). DEB-induced apoptosis, as assessed by caspase 3/7 activity levels, was significantly reduced by 76% in DEB-exposed cells treated with ERK1/2 siRNA as compared to the DEB-exposed cells that received control siRNA (p < 0.05; Figure 4(a)). Under these experimental conditions, normalized activated phospho-ERK1/2 levels in DEB-exposed cells treated with ERK1/2 siRNA were significantly reduced by 30% as compared to the DEB-exposed cells that received control scrambled siRNA (p < 0.05; Figure 4(b)). Normalized total ERK1/2 levels did not significantly change (p > 0.05). Under these conditions, p53 levels were significantly reduced by 74% in DEB-exposed cells treated with ERK1/2 siRNA as compared to the DEB-exposed cells with control siRNA (p < 0.05; Figure 4(b)). Collectively, these observations confirm our findings that the activated ERK1/2 pathway mediates DEB-induced apoptosis in exposed human lymphoblasts predominantly at early times (24 h) postexposure through the p53 pathway.

Figure 4.

ERK 1/2 siRNA knockdown of DEB-induced apoptosis in human lymphoblasts. Human TK6 lymphoblasts were transfected with 2.5-nM Silencer Select ERK siRNA or control scrambled negative control siRNA, as described in “Materials and methods” section. Control and ERK siRNA–treated cells were then exposed to control media or 10-µM DEB at 12 h post-transfection. Cells were then assayed (in triplicates) for apoptosis, P-ERK, p53, ERK, and GAPDH at 24 h post-DEB exposure. (a) The extent of apoptosis was determined using the Caspase-Glo^®3/7 assay, as described in “Materials and methods” section. (b) Western blot analysis of activated P-ERK, total p53, total ERK, and GAPDH levels were determined using 15 µg of protein, as described in Figure 1. Relative normalized P-ERK levels for each sample were obtained after quantification analysis (in triplicates) by utilizing the ratio of P-ERK to total ERK. Relative normalized p53 and ERK levels were obtained after quantification analysis (in triplicates) by normalizing to GAPDH levels. A representation of three experiments is shown. Significant differences were observed between all values for DEB-exposed samples as compared to the control-unexposed samples, and between DEB-exposed cells with control siRNA versus ERK1/2 siRNA (p < 0.05). DEB: diepoxybutane; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; P-ERK: phospho-ERK.

Discussion

Our previous studies demonstrated the occurrence of DEB-induced p53-dependent mitochondrial-mediated apoptosis in human lymphoblasts at early times post-DEB exposure.^17,24,40 In this report, we investigated the role of the ERK1/2 pathway in DEB-induced apoptotic signaling in this experimental system. We report that ERK1/2 is activated, and mediates DEB-induced apoptotic signaling in these cells, especially at early times (24 h compared to 48 h) post-DEB exposure. In addition, we find that MEK is an upstream regulator, and p53 serves as a downstream mediator of the DEB-activated ERK1/2 apoptotic signaling pathway. Collectively, these findings contribute, for the first time, towards the increased understanding of the mechanisms involved in DEB-induced apoptotic signaling in human lymphoblasts.

ERK1/2 is activated in various cells exposed to DNA damaging agents capable of generating oxidative stress; examples of these compounds include cadmium, arsenic, cypermethrin, BH4, cisplatin, etoposide, and doxorubicin.^44,57,62 Since DEB causes DNA damage and induces oxidative stress,^12,24 our observations that ERK1/2 is activated in DEB-exposed human lymphoblasts is in line with these reports. Normalized phosphorylated ERK1/2 (Thr202/Tyr204) levels were increased twofold in DEB-exposed cells at 24 h, and fourfold at 48 h post-DEB exposure, as compared to the control-exposed cells. The 3.6-fold increase in ERK activation observed at 24 h in control DEB-exposed cells in experiments involving the MEK inhibitor PD98059 is due to the different DEB exposure conditions utilized for the PD98059 experiments. Collectively, these observations imply that sustained ERK1/2 activation, which has been linked to the induction of apoptosis,⁴⁴ occurs in this experimental system.

In this study, sustained ERK1/2 activation was found to mediate DEB-induced apoptotic signaling in exposed human lymphoblasts, especially at earlier times (24 h) post-DEB exposure. ERK1/2 siRNA and the MEK inhibitor PD98059 down-modulated activated ERK1/2 levels; under these conditions, DEB-induced apoptosis in human lymphoblasts was dramatically inhibited (by 76% and 65%, respectively) at 24 h post-DEB exposure (as compared to the corresponding control-exposed cells). These findings are in line with published reports that ERK1/2 is activated to mediate apoptosis in various cell types exposed to various toxicants, such as arsenic, cypermethrin, BH4, cisplatin, and doxorubicin.^{44,57,62
–64} ERK1/2, however, is not the only signaling pathway that can lead to apoptosis. Other signaling pathways, such as ATM, JNK, and p38 have been reported to mediate apoptotic signaling of various toxicants in different cell types.^27,62,65,66 Thus, the action of some of these signaling pathways may explain the inability of the MEK inhibitor to completely prevent DEB-induced apoptosis, especially at 48 h post-DEB exposure.^17,40

The p53 protein was found to serve as a downstream mediator of the apoptotic effects of activated ERK1/2 in human lymphoblasts, especially at early times (24 h) post-DEB exposure. Inhibition of ERK1/2 activation by the MEK inhibitor PD98059 or down-modulation of ERK1/2 by specific siRNA dramatically reduced p53-serine 15 phosphorylation, total p53 levels, as well as DEB-induced apoptosis in exposed human lymphoblasts at early times post-DEB exposure. These findings are in line with the fact that p53 was previously found to be elevated, and mediated DEB-induced apoptosis at earlier times (up to 24 h), but not at later times (48 h), post-DEB exposure in this system.^17,40 Our finding that p53 is a downstream target of ERK1/2 through its phosphorylation on serine-15 is in line with our published observations that this phosphorylation stabilizes p53, and is actually reflective of total p53 levels in this experimental system.⁴⁰ These findings are also in line with the fact that ERK1/2 mediates apoptosis in a p53-dependent manner in various cell types exposed to different toxicants, such as cisplatin, shikonin, resveratol, tetramethylpyrazine, bisabolene, terrein, and benzo[a]pyrene.^{44,57,59,67
–69} Moreover, ERK1/2 has also been reported to activate p53 signaling by phosphorylating p53 at serine-15 in toxicant-induced apoptosis in specific cell types.^58,59 Phosphorylation of p53 on the serine-15 residue is known to activate its transactivation functions, thus facilitating the apoptotic process.^36,39,70 It should be noted, however, that other protein kinases (such as ATM, ATR, Chk1, DNA-PK, ERK1/2, and JNK) can also phosphorylate p53 on serine-15.^36,37,39 Infact, ATM (and other kinases) are also activated in DEB-exposed lymphoblasts⁴⁰; this explains the inability of MEK1/2 specific inhibitors as well as ERK1/2 specific siRNA to prevent the phosphorylation of p53 at serine-15, as well as the subsequent apoptosis, at later times (48 h) post-DEB exposure.

MEK was found to serve as an upstream activator of the ERK1/2-mediated apoptotic signaling pathway in human lymphoblasts undergoing DEB-induced apoptosis. This finding is supported by the observation that activated MEK levels were upregulated by DEB in TK6 cells, and the MEK inhibitor suppressed the induction of apoptosis by DEB under conditions where ERK1/2 activation was inhibited. This finding is in line with other reports on MEK as an upstream activator of ERK1/2 in mediating apoptosis induced by other compounds, such as doxorubicin, quercetin, paraquat, cisplatin, etoposide, and arsenic.^{44,62,67,71
–73} Follow-up studies are in progress to identify signaling components of the ERK1/2 pathway that mediate DEB-induced apoptosis upstream of MEK1/2.

In summary, in this report, we have demonstrated for the first time that ERK1/2 is activated, and mediates apoptotic signaling in DEB-exposed lymphoblasts. We also demonstrated that ERK1/2 is regulated by MEK, and that p53 is a downstream mediator of the DEB-activated ERK1/2-mediated apoptotic pathway. Thus, collectively, our findings demonstrate that DEB induces apoptosis through the MEK-ERK1/2-p53 pathway in exposed lymphoblasts at early times post-DEB exposure. The concentrations of DEB and exposure times utilized in this study have previously been associated with the occurrence of apoptosis, and not necrosis; this apoptosis is p53-dependent, especially at early times post-DEB exposure.^17,23 The DEB concentrations utilized are genotoxic, and are achievable in mice blood after exposure to BD.^74,75 It is thus possible that ERK1/2-mediated apoptotic signaling may explain the bone marrow depletion observed in mice and humans exposed to high concentrations of BD.^76
–78 This report contributes towards the understanding of DEB-induced apoptotic signaling. Since apoptosis accounts for the cytotoxicity of low concentrations of DEB^17,23 this report also contributes towards the understanding of mechanisms involved in DEB-induced apoptosis and BD toxicity. Experiments designed to elucidate components mediating apoptotic signaling at late times (48 h), as well as upstream of MEK1/2 at early (24 h) times, are in progress.

Footnotes

Declaration of Conflicting Interests

The author(s) declare that there is no potential conflict of interest with respect to the research, authorship, or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Institute of General Medical Sciences MBRS SCORE grant GM076530, and in part by the National Institute of Environmental Health Sciences AREA grant ES019306.

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Diepoxybutane-induced apoptosis is mediated through the ERK1/2 pathway

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Antibodies

Cell culture and enumeration

Exposure of cells to DEB

Quantitation of DEB-induced apoptosis

ERK 1/2 siRNA knockdown of DEB-induced apoptosis

Western blot analysis of p53 and ERK pathway proteins

Statistical analysis

Results

DEB induces activation of extracellular signal–regulated kinase (ERK1/2) pathway in human lymphoblasts

Activated extracellular signal–regulated kinase (ERK1/2) pathway mediates DEB-induced apoptosis in human lymphoblasts

The p53 protein is a downstream target of extracellular signal–regulated kinase (ERK1/2) during DEB-induced apoptosis in human lymphoblasts

Confirmation of the role of extracellular signal–regulated kinase (ERK1/2) in mediating DEB-induced apoptosis in human lymphoblasts

Discussion

Footnotes

Declaration of Conflicting Interests

Funding

References