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[Cancer Research 66, 4888-4894, May 1, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

The Role of Mitogen-Activated Protein Kinase Phosphatase-1 in Oxidative Damage–Induced Cell Death

Jun-Ying Zhou1, Yusen Liu2 and Gen Sheng Wu1

1 Program in Molecular Biology and Human Genetics, Karmanos Cancer Institute, Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan and 2 Children's Research Institute, Department of Pediatrics, The Ohio State University, Columbus, Ohio

Requests for reprints: Gen Sheng Wu, Program in Molecular Biology and Human Genetics, Karmanos Cancer Institute, Department of Pathology, Wayne State University School of Medicine, Room E216, 110 East Warren Avenue, Detroit, MI 48201. Phone: 313-833-0715, ext. 2328; Fax: 313-831-7518; E-mail: wug{at}karmanos.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a member of the MAPK phosphatase family that functions as a negative regulator of MAPK signaling. MKP-1 is induced by oxidative stress, but the role of its induction in cell death is not fully understood. Here, we show that hydrogen peroxide (H2O2) induces MKP-1 and activates MAPKs. Induction of MKP-1 by H2O2 correlated with inactivation of p38 and c-Jun-NH2-kinase (JNK). Overexpression of MKP-1 increased cell resistance to H2O2-induced death. Furthermore, we show by small interfering RNA silencing that down-regulation of MKP-1 increases phosphorylated p38 and JNK and subsequent cell death induced by H2O2. More importantly, primary embryonic fibroblasts from mice lacking MKP-1 had a higher level of phosphorylated p38 and JNK and were more sensitive to H2O2-induced cell death compared with corresponding cells with MKP-1, indicating that p38 and JNK pathways may play important roles in H2O2-mediated cell death. Thus, these results suggest that activation of MKP-1 is a survival mechanism against oxidative damage. (Cancer Res 2006; 66(9): 4888-94)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Reactive oxygen species (ROS), including superoxide, hydrogen peroxide (H2O2), and hydroxyl radicals, constantly cause damage to biological molecules, including DNA. ROS can induce a variety of cellular responses, including posttranscriptional or posttranslational modifications of many genes, which lead to diverse outcomes, such as cell growth, differentiation, and cell death (1, 2). Among these responses, activation of the mitogen-activated protein kinases (MAPK) plays an important role in initiating oxidative damage–induced cellular responses.

The MAPK signal pathway mainly consists of three subfamilies; the stress-activated protein [stress-activated protein kinase (SAPK)/c-Jun-NH2-kinase (JNK)], the p38 MAPK, and the extracellular signal-regulated kinase (ERK). The mechanisms of MAPK activation are through the reversible phosphorylation of both threonine and tyrosine residues of the TXY motif in the catalytic domain by upstream dual-specificity kinases, called MAPK kinases. These upstream MAPK kinases include MKK1/2, MKK3/6, and MKK4/7. MKK1/2 and MKK3/6 activate ERK and p38, respectively, whereas MKK4/7 activate JNK/SAPK (3–5). Once activated, MAPKs phosphorylate a number of cellular substrates that can trigger diverse signal cascades, leading to many forms of cellular responses, including proliferation, differentiation, and apoptosis. It is believed that activation of ERK can lead to cell proliferation, whereas activation of JNK and p38 can cause cell differentiation and cell death.

In addition to activation of MAPK signaling, oxidative stress also induces the MAPK phosphatases (6, 7). The MAPK phosphatases are a family of dual-specificity protein phosphatases, which can dephosphorylate both phosphothreonine and phosphotyrosine residues and subsequently inactivate the activities of MAPKs (8). Thus, these phosphatases play an important role in negatively regulating MAPK signaling. This family includes MKP-1, MKP-2, MKP-3, MKP-4, MKP-5, VHR, Pac1, hVH2, hVH3, Pyst1, and Pyst2 (9). MKP-1 was the first member of this family to be identified as a MAPK phosphatase. MKP-1 was originally cloned as a growth factor–inducible gene implicated in the G0-G1 transition (10, 11). Subsequent studies showed that MKP-1 is also a stress-induced gene (6, 7). Although MKP-1 was initially characterized as an ERK-specific phosphatase (12, 13), subsequent studies have determined that MKP-1 preferentially acts on JNK and p38 in response to stresses (7, 14). Because JNK, p38, and ERK are capable of either inducing apoptosis or cell proliferation, activation of MKP-1 has been shown to play a role in regulating the cell cycle (15–18) or apoptosis (19, 20). A recent study indicated that MKP-1 can protect cells from anisomycin-induced apoptosis (21). Although MKP-1 was identified as a H2O2-induced early response gene (6), it is not clear that whether such induction plays a role in H2O2-mediated cell death.

In this article, we found that induction of MKP-1 by H2O2 correlates with inactivation of JNK and p38 activity. Overexpression of MKP-1 increased cell resistance to H2O2-induced death. Using the small interfering RNA (siRNA) silencing, we showed that down-regulation of MKP-1 expression sensitizes cells to H2O2-induced cell death, suggesting that MKP-1 is involved in cell survival. Furthermore, we found that primary mouse embryonic fibroblasts (MEF) from MKP-1 knockout mice are much more sensitive than cells with MKP-1 to H2O2-induced death. Thus, our results show that MKP-1 plays an important role in cell survival in response to oxidative damage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents, cell lines, culture conditions, and treatment. H2O2 was purchased from Sigma (St. Louis, MO). The breast cancer cell line MCF-7 was maintained in Ham's F-12/DMEM (1:1) supplemented with 5% fetal bovine serum (FBS), 0.5 nmol/L estradiol, and antibiotics. H460-Neo (wt p53) and H460-E6 (without p53 protein) were previously described (22). HCT116 (wt p53) and HCT116 p53–/– cell lines were kindly provided by Dr. Bert Vogelstein. These cells were maintained at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air. Cells were treated with the different concentrations of H2O2 at various time points.

Isolation of RNA and cDNA synthesis. Total cellular RNA was purified from cells (H460, H460-Neo, H460-E6, HCT116 p53+/+, and HCT116 p53–/–) using the Trizol method (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer. cDNA was synthesized using the Superscript protocol from the manufacturer (Invitrogen).

Northern blot analysis. Total RNA (20 µg) was separated in a 1.5% formaldehyde agarose gel and blotted to Hybond-N+ membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The blots were hybridized with radiolabeled human MKP-1 cDNA as described previously (16). Radioactive signals were analyzed by autoradiography.

Construction of MKP-1-expressing vector and generation of MCF-7 cells overexpressing MKP-1. A full-length MKP-1 cDNA was amplified from cDNA as described above using a GC-rich PCR system (Roche Applied Science, Indianapolis, IN) and the following primers: 5'-GGAATTCATGGTCATGGAAGTGGGC-3' and 5'-CGGGATCCTCAGCAGCTGGGAGAGG-3'. The PCR conditions were as follows: 95°C for 3 minutes; 10 cycles at 95°C for 30 seconds, 62°C for 30 seconds, and 72°C for 135 seconds; and then 25 cycles at 95°C for 30 seconds, 62°C for 30 seconds, and 72°C for 215 seconds. The amplified fragment was isolated from a 1% agarose gel, digested with EcoRI and BamHI, and subcloned into pIRES2-EGFP (BD Biosciences Clontech, Mountain View, CA) to generate the pIRES2-EGFP-MKP-1–expressing construct. MCF-7 cells were transfected with either the empty vector pIRES2-EGFP, or pIRES2-EGFP-MKP-1 using LipofectAMINE 2000 reagent (Invitrogen), as described previously (23), to produce mock-transfected and MKP-1-overexpressing cells, respectively. Transfected cells were selected with 1 mg/mL G418 (Invitrogen) for 2 weeks, and individual clones were isolated as described previously (24). Clones that overexpressed MKP-1 protein, determined by Western blot analysis as described below, were used in this study.

Isolation of primary MEFs. The generation of MKP-1 knockout mice was described previously (25, 26). E12 to E16 embryos were minced and digested with trypsin/EDTA at 37°C for 30 minutes. After washing with PBS, isolated cells were collected by centrifugation and plated at a density of 5 x 106 in a 150 mm dish in DMEM containing 10% FBS and antibiotics.

siRNA transfection for knockdown of MKP-1. siRNA duplex oligonucleotides were purchased from Dharmacon Research (Lafayette, CO). The targeted sequences for MKP-1 siRNA were 5'-CCAAUUGUCCCAACCAUUUU-3' and 5'-CAACGAGGCCAUUGACUUCUU-3'. The transfection was done as suggested by Dharmacon Research with slight modifications. Briefly, MCF-7 cells were plated at 4 x 105 per well in six-well plates. The next day, cells were transfected with MKP-1 siRNA oligonucleotides or scrambled oligonucleotides using Oligofectamine (Invitrogen). After 2 days, transfected cells were equally split into three wells in 24-well plates and allowed to recover overnight. H2O2 was added to cells and induction of MKP-1 and MAPKs were determined by Western blot. The effect of siRNA knockdown on cell viability was determined by trypan blue exclusion.

Determination of cell viability. Cell viability was determined by trypan blue exclusion as previously described (27). Briefly, cells, including attached and floating cells, were collected and stained with trypan blue, and total cells and dead cells were counted. In some experiments, floating cells were collected for trypan blue staining and attached cells were stained with Coomassie blue as described previously (27).

Western blot analysis. Whole cell lysates were prepared as described previously (23), and protein concentration was determined using the Protein Assay kit (Bio-Rad, Hercules, CA). Cell lysates (50 µg) were electrophoresed through 12% denaturing polyacrylamide gels and transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH). The blots were probed or reprobed with the antibodies, and bound antibody was detected using enhanced chemiluminescence reagent (Amersham Pharmacia Biotech) according to the protocol of the manufacturer. Rabbit polyclonal anti-human MKP-1 (c-19) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal antibodies against total and phosphorylated ERK, p38, and JNK, and poly(ADP)ribose polymerase (PARP) were purchased from Cell Signaling Technology (Beverly, MA). Antiactin antibody (AC-74) was purchased from Sigma.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
H2O2 induces MKP-1 that correlates with inactivation of MAPKs. To determine the role of MKP-1 in H2O2-mediated cellular responses, the breast cancer cell MCF-7 was treated with varying doses of H2O2 for 3 hours, and induction of MKP-1 and activation of ERK, p38, and JNK were determined by Western blot. We chose a 3-hour treatment, because at this time point H2O2 induced MKP-1 and activated MAPKs in preliminary experiments. As shown in Fig. 1A , MKP-1 was induced at 100 µmol/L, increased to a much higher degree at 200 µmol/L, and peaked at 400 µmol/L H2O2 in a dose-dependent manner. To determine the effects of such treatments on MAPK pathways, we analyzed the appearance of phosphorylated (active) forms of ERK, p38, and JNK. Similar to the results obtained with MKP-1, H2O2 activated all three MAPKs by increasing phosphorylated ERK, JNK, and p38 (Fig. 1A). However, the levels of total ERK, JNK, and p38 proteins remained unchanged (data not shown). These data are consistent with the role of oxidative damage in activation of MAPKs and MKP-1.


Figure 1
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  		Figure 1. Induction of MKP-1 and activation of MAPKs with H2O2. A, MCF-7 cells were left untreated or treated with 50, 100, 200, and 400 µmol/L H2O2 for 3 hours, and the total protein was then extracted for assaying MKP-1, phosphorylated ERK (p-ERK), p38 (p-p38), and JNK (p-JNK) proteins by Western blot analysis. B, MCF-7 cells were treated with 200 µmol/L H2O2 for 0, 0.5, 1, 2, 4, and 6 hours. The total protein was extracted and then assayed for the levels of MKP-1, phosphorylated ERK, p38, and JNK by Western blot analysis. Actin was included as a loading control. C, HCT116 p53+/+, HCT116 p53–/–, H460-Neo, and H460-E6 cells were treated with 200 µmol/L H2O2 for 0, 1, 2, 4, and 8 hours, and total RNA was extracted to assay for MKP-1 mRNA expression by Northern blot analysis as described previously (16). rRNA was visualized as a loading control.

 
It is well known that MKP-1 is able to dephosphorylate and inactivate ERK, p38, and JNK. Because both MKP-1 and MAPKs are induced by H2O2, we asked if induction of MKP-1 by H2O2 is able to inactivate MAPK activities. To this end, MCF-7 cells were treated with H2O2 at 200 µmol/L for different time points, and induction of MKP-1 and phosphorylation of ERK, p38, and JNK were analyzed. Figure 1B shows that phosphorylation of p38, JNK, and ERK was detectable at 30 minutes, significant at 1 hour, and then returned to normal around 4 hours. Coincidentally, induction of MKP-1 was detected after a 2-hour exposure to H2O2 and then stayed at a higher level for at least 4 more hours (Fig. 1B). Thus, a correlation of MKP-1 induction with the disappearance of phosphorylated MAPKs suggests that MKP-1 might play a physiologic role in inactivation of oxidative damage–induced MAPK activities.

We previously showed that MKP-1 expression could be induced by p53 overexpression (16). To determine whether H2O2-induced MKP-1 is dependent on p53, we treated both HCT116 p53+/+ (wt p53) and HCT116 p53–/– (no p53; ref. 28) with 200 µmol/L H2O2 for varying time points, and induction of MKP-1 was determined by Northern blot analysis. Figure 1C shows that MKP-1 is induced in both cell lines. However, there was a higher level of MKP-1 in HCT116 p53+/+ than HCT116 p53–/– after 1-hour treatment. In addition, we found that MKP-1 is induced by H2O2 in both H460-Neo and H460-E6 cells. Of note, kinetics of MKP-1 induction was slightly different between H460-Neo and H460-E6 cells (Fig. 1C). Therefore, these results suggest that induction of MKP-1 by H2O2 could be p53 independent.

Overexpression of MKP-1 inhibits H2O2-mediated cell death. We have shown that induction of MKP-1 correlates with inactivation of MAPK activity. We asked if MKP-1 plays an inhibitory role in regulating MAPK signaling in response to H2O2 treatment. To this end, we transfected MCF-7 cells with either the empty vector pIRES2-EGFP, or the MKP-1-expressing vector pIRES2-EGFP-MKP-1 and then selected with G418 for individual clones that stably express MKP-1 by Western blot with MKP-1 antibody. As shown in Fig. 2A , three clones (clones 3, 12, and 15) overexpressed MKP-1 protein compared with cells transfected with the empty vector. To determine whether transfected MKP-1 is functional in cells, we treated these cells with H2O2 to induce MAPK activity and then determined the levels of phosphorylated MAPKs. As shown in Fig. 2B, H2O2 activated ERK, p38, and JNK in vector control cells, which is consistent with the results obtained in Fig. 1A. In contrast, activation of all three MAPKs, particularly phosphorylated JNK, was substantially inhibited in cells overexpressing MKP-1, compared with vector control cells (Fig. 2B). These results suggest that overexpression of MKP-1 is able to inactivate MAPK signaling induced by H2O2.


Figure 2
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  		Figure 2. Effects of MKP-1 overexpression on activation of MAPKs and cell viability. A, protein analysis of transfected MCF-7 cells. MCF-7 cells were transfected with either vector alone (lanes 1-3) or MKP-1-expressing vector (lanes 4-6), followed by selection with G418 (1 mg/mL) for 2 weeks. Individual clones were isolated and their cell lysates were analyzed on a 12% SDS-PAGE, transferred to nitrocellulose, and probed with a polyclonal antibody to MKP-1 (Santa Cruz Biotechnology). Clones 3, 12, and 15 expressed a transfected MKP-1 protein (lanes 4-6 versus lanes 1-3). B, activation of MAPKs by H2O2 in MCF-7 cells stably transfected with either vector (clone 1) or MKP-1 (clones 12 and 15). MCF-7 transfectants were treated with 200 µmol/L H2O2 for the indicated time points. Whole-cell protein lysates were prepared, and phosphorylated ERK, JNK, and p38 proteins were detected with Western blot analysis. Actin expression was used as a loading control. C, morphologic changes in MCF-7 cells transfected with either vector (V1) or MKP-1 (12 and 15) following H2O2 treatment. MCF-7 cells were either left untreated or treated with 300 µmol/L H2O2 for 5 hours and attached cells were stained with Coomassie blue. Original magnification, x100. D, quantification of cell death. Floating cells were collected from MCF-7-V1 or MCF-7-MKP-1#12 and MCF-7-MKP-1#15 treated with H2O2 in (C) and dead cells were determined using the trypan blue exclusion assay. Representative of three independent experiments.

 
It is well known that activation of MAPK signaling can lead to either cell proliferation or cell death in part depending on cell types, which can be inhibited by members of the MAPK phosphatase family, including MKP-1 (8). To determine whether overexpression of MKP-1 affects cell survival in response to oxidative stress, MCF-7 cells were treated with H2O2 at 300 µmol/L for 5 hours, and detached dead cells and attached surviving cells were determined by trypan blue exclusion and Coomassie blue staining, respectively. As shown in Fig. 2C, upon H2O2 treatment, the majority of MKP-1-overexpressing cells (clones 12 and 15) remained attached (alive cells), compared with the vector control cells in which fewer cells remained attached. Furthermore, there were many more dead cells detected in H2O2-treated vector control cells over the MKP-1-overexpressing cells (Fig. 2D). Thus, these data suggest that overexpression of MKP-1 could inhibit MCF-7 cell death induced by oxidative damage.

siRNA knockdown of MKP-1 enhances MCF-7 cell death induced by H2O2. Although overexpression of MKP-1 is able to inhibit H2O2-induced cell death, it is possible that the results obtained from the overexpression system may not reflect the physiologic condition. To directly address the role of MKP-1 in oxidative damage–induced cell death, we used siRNA silencing to knockdown MKP-1 expression and then determined the effects of knockdown of MKP-1 on cell death. Using a computer-designed program, we identified four regions that could be targeted by siRNA and two of which were tested for their ability to knockdown MKP-1 expression (data not shown). Our preliminary data indicated that 5'-CCAAUUGUCCCAACCAUUUU-3' is the better one for knockdown of MKP-1 (data not shown) and thus this duplex was used in this study. As shown in Fig. 3A , induction of MKP-1 by H2O2 in cells transfected with MKP-1 siRNA was abolished compared with cells transfected with control oligos. Consistent with activation of MAPKs by H2O2, p38, JNK, and ERK were activated in cells transfected with control oligos. In contrast, by knockdown of MKP-1, activation of these three MAPKs was obvious, particularly for JNK and p38 (Fig. 3A). These data confirm the role of MKP-1 in negative regulation of MAPK activity and support that MKP-1 could play an important role in inhibition of p38 and JNK activation upon H2O2 treatment.


Figure 3
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  		Figure 3. Silencing of MKP-1 expression with siRNA and H2O2-induced MAPK activation and cell death. A, effect of MKP-1 and MAPK activation by transfecting MKP-1 siRNA. MCF-7 cells were plated at 4 x 105 per well in six-well plates. The next day, cells were transfected with MKP-1 siRNA or control oligonucleotides using Oligofectamine (Invitrogen). After 2 days, transfected cells were equally split into three wells in 24-well plates. Thirty-six hours after transfection, cells were treated with 100 or 200 µmol/L H2O2 for 3 hours, and induction of MKP-1 and activation of MAPKs, including ERK, p38, and JNK, were determined by Western blot. B, effect of cell survival by silencing MKP-1 expression with siRNA. MCF-7 cells were transfected with MKP-1 siRNA or control oligonucleotides as described in (A). After 3 days, cells were treated with 300 µmol/L H2O2 for 5 hours. Total cells including, floating cells, were harvested and stained with trypan blue. Cell survival data are expressed as percentage of total cells. Representative of two independent experiments.

 
We next asked if blockade of MKP-1 by siRNA could sensitize cells to oxidative damage–induced cell death. To this end, MCF-7 cells were transfected with either MKP-1 siRNA or control siRNA and then treated with 300 µmol/L H2O2 for 5 hours, and cell viability was determined by trypan blue exclusion. As shown in Fig. 3B, H2O2 caused ~30% of death in cells transfected with control siRNA. In contrast, there was a 20% increase in death in cells transfected with MKP-1 siRNA than cells transfected with control siRNA (Fig. 3B). These data suggest that MKP-1 plays an inhibitory role in oxidative damage–induced cell death and that inhibition of MKP-1 could enhance H2O2-mediated cell death, possibly through activation of the JNK and p38 death pathways.

MKP-1–/– MEF cells are more sensitive than MKP-1+/+ cells to H2O2-induced cell death. We have shown that down-regulation of MKP-1 by siRNA against MKP-1 sensitizes MCF-7 cells to H2O2-induced cell death. Because siRNA could be a transient effect on MKP-1 knockdown, the results obtained with this approach may not completely reflect the role of MKP-1 in oxidative damage–induced cellular responses. Therefore, we thought to test the role of MKP-1 in oxidative damage–induced cell death using MKP-1 knockout MEF cells. Primary MEFs were obtained from wild-type (MKP-1+/+) embryos and embryos in which MKP-1 gene was disrupted by a neomycin cassette in exon 2 (25). MKP-1+/+ and MKP-1–/– cells were treated with 300 µmol/L H2O2 and expression of MAPKs and MKP-1 was analyzed. As expected, MKP-1 was induced by H2O2 in MKP-1+/+ MEFs but not in MKP-1–/– MEFs (Fig. 4A ), confirming the absence of MKP-1 in MKP-1–/– cells. We then analyzed activation of MAPKs in these cells. As shown in Fig. 4A, the levels of phosphorylated ERK were not significantly different between MKP-1+/+ and MKP-1–/– MEFs upon H2O2 treatment. In contrast, activation of JNK and p38 was significantly different between MKP-1+/+ and MKP-1–/– cells upon H2O2 treatment. In MKP-1–/– cells, JNK activation was detected at 2 hours compared with MKP-1+/+ cells in which the level of phosphorylated JNK returned to the basal level (Fig. 4A). Consistent with the role of MKP-1 in regulating p38, activation of p38 was not only more robust but also prolonged to 4 hours after H2O2 treatment in MKP-1–/– cells, compared with MKP-1+/+ cells in which the levels of phosphorylated p38 started to decrease by 2 hours and returned to basal levels by 3 hours (Fig. 4A). However, total ERK, JNK, and p38 proteins remained unchanged following H2O2 treatment in both cell lines (data not shown). These data further suggest that MKP-1 plays a physiologic role in negatively regulating p38 and JNK pathways in response to oxidative stress.


Figure 4
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  		Figure 4. H2O2-induced MAPK activation and cell death in primary MEFs. A, induction of MKP-1 and MAPKs upon H2O2 treatment. MKP-1+/+ and MKP-1–/– MEFs were treated with 300 µmol/L H2O2 for 0, 1, 2, 4, and 4 hours. The total protein was extracted and then assayed for the levels of MKP-1, phosphorylated ERK, p38, and JNK by Western blot analysis. Actin was included as a loading control. B, morphologic changes in MEFs treated with 300 µmol/L H2O2. Photographs were taken after a 24-hour treatment using a Nikon microscope. Original magnification, x100. C, quantification of cell death induced by H2O2 in MEFs. MKP-1+/+ and MKP-1–/– MEFs were treated with 300 µmol/L H2O2 for 24 hours as shown in (B), and total cells including floating cells were collected and stained with trypan blue. Total cells including attached and floating cells were counted, and cell survival data are expressed as percent of untreated cells. Representative of two independent experiments. D, cleavage of PARP by H2O2. MKP-1+/+ and MKP-1–/– MEFs were treated with 300 µmol/L H2O2 for 24 hours. The total protein was extracted and then assayed for PARP cleavage by Western blot analysis. Actin was included as a loading control.

 
To test the effect of loss of MKP-1 on oxidative damage–induced cell death, we treated MKP-1+/+ and MKP-1–/– MEFs with H2O2 at 300 µmol/L for 24 hours and then determined cell viability. As shown in Fig. 4B, MKP-1–/– MEF underwent rapid cell death as evidenced by their rounding up and detaching from the plate. In contrast, the majority of MKP-1+/+ MEFs remained attached and alive (Fig. 4B). Cell viability of MKP-1–/– MEFs decreased to 33% whereas >89% of MKP-1+/+ MEF remained alive as determined by trypan blue exclusion (Fig. 4C). These results indicate that blockade of p38 and JNK signaling by MKP-1 may be critical for protecting cells from oxidative damage–induced death. To determine if loss of MKP-1 enhances H2O2-induced cell death through apoptosis, we tested cleavage of PARP, a hallmark of apoptosis, in cells with or without H2O2 treatment. As shown in Fig. 4D, in MKP-1–/– cells, H2O2 treatment increased PARP cleavage compared with untreated cells. In contrast, cleavage of PARP was not detected in MKP-1+/+ cells treated with 300 µmol/L H2O2. Thus, these results suggest that loss of MKP-1 sensitizes cells to oxidative damage–induced death in part through apoptosis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MKP-1 is an early response gene, which was cloned from cells treated with growth factors and stresses, including oxidative stress (6, 10–13), but the role of MKP-1 in oxidative stress in cells has not been established. In this study, we provide evidence that induction of MKP-1 protects cells from oxidative damage–induced cell death probably through inactivation of the p38 and JNK pathways. These findings suggest that activation of the JNK and p38 pathways plays a critical role in oxidative stress–induced cell death.

MAPK signaling can be activated by a variety of stimuli and stresses, including oxidative stress. We have shown that all three MAPK pathways, including ERK, p38, and JNK, are activated upon H2O2 treatment in MCF-7 (Fig. 1A). It has been known that activation of ERK can lead to cell proliferation, whereas activation of JNK and p38 causes cell death. The net outcome of MAPK activation is dependent on many factors, including the type of stimulus or cell context. Although H2O2 activates all three MAPK kinases (Fig. 1A and B), H2O2 caused MCF-7 cell death (Fig. 2), suggesting that ERK pathway is not important for H2O2-induced cell death. In fact, we found that inhibition of ERK by the MEK1/2 inhibitor U0126 enhances H2O2-induced cell death (data not shown). Therefore, H2O2-induced cell death is primarily through activation of the p38 and JNK pathways. Consistent with the role of JNK and p38 in H2O2-induced cell death, we have shown that both p38 and JNK were activated upon H2O2 treatment (Figs. 1, 2, and 4). Although the original study suggested the ERK is the substrate of MKP-1 (12), several studies indicated that p38 and JNK are the preferential substrates of MKP-1 in cells in response to a variety of stimuli (14, 19, 26). In this study, we found that JNK and p38 were the preferential targets for MKP-1 because loss of MKP-1 by siRNA-mediated down-regulation of MKP-1 or MKP-1 knockout MEFs resulted in a higher level of phosphorylated p38 and JNK than cells with MKP-1. We have also shown that loss of MKP-1 in MCF-7 or MEFs results in an increase in the levels of phosphorylated ERK but such increase was not significant (Figs. 3 and 4). This suggests that ERK is still a physiologic target of MKP-1 in cells in response to oxidative stress. Therefore, we conclude that under oxidative stress, induction of MKP-1 preferentially inactivates JNK and p38 pathways, leading to cell survival.

Because activation of MAPKs could lead to either cell proliferation or apoptosis, it is expected that modulation of MAPK activity could affect cell proliferation or apoptosis. It is well known that MAPKs are negatively regulated by the dual-specificity MAPK phosphatases, including MKP-1. Therefore, it is reasonable to assume that any changes in the protein level of MAPK phosphatases could lead to inactivation or activation of MAPKs, resulting in an increase or decrease in the cellular responses. We have shown that overexpression of MKP-1 inhibits H2O2-induced activation of JNK and p38 and increases cell resistance to H2O2-induced death. Consistent with this, it has been shown that conditional expression of MKP-1 confers human leukemia U937 resistant to UV-induced apoptosis (19). Because UV-induced apoptosis involves activation of the JNK pathway, overexpression of MKP-1 blocks UV-mediated JNK activation, reduces apoptosis, and thus increases cell survival (19). It has also been shown that overexpression of MKP-1 can inhibit Fas ligand–induced apoptosis in human prostate DU145 cells (29). Because a number of anticancer drugs kill cancer cells via the JNK apoptotic pathway, it is expected that blockade of JNK activation could inhibit anticancer activity. In addition, overexpression of MKP-1 has been shown to inhibit cisplatin-induced apoptosis via inactivation of the JNK pathway in human embryonic kidney 293 cells (20).

There are three forms of cell death, including apoptosis, necrosis, and autophagy (30). It has been shown that overexpression of MKP-1 can inhibit apoptosis by inhibiting p38 and JNK apoptotic pathways (19–21). We have shown that overexpression of MKP-1 protects MCF-7 cells from H2O2-induced cell death probably through inhibition of JNK and p38 activity (Fig. 2). However, it is not clear whether MKP-1 blocks JNK- and p38-mediated apoptosis. Because MCF-7 cells contain abnormal caspase-3 owing to a 47 bp deletion within exon 3 of the caspase-3 gene (31), we suspect that H2O2 kills MCF-7 cells through a caspase-independent mechanism. However, we have shown that PARP is cleaved in MKP-1–/– cells by H2O2 (Fig. 4D), which suggests that loss of MKP-1 could sensitize cells to H2O2-induced apoptosis. Because JNK has been linked to autophagic cell death (32), it is possible that MKP-1 protects cells from necrosis/autophagy induced by H2O2 and this issue is under investigation. Nevertheless, MKP-1 plays an important role in protecting cells from oxidative damage–induced death.

Although overexpression of MKP-1 increases cell resistance to UV-, FAS ligand–, and cisplatin-induced cell death (19, 20, 29), it is possible that the levels of MKP-1 in these systems do not reflect the physiologic conditions. To this end, Wu and Bennett (21) have recently shown that using MKP-1 knockout MEFs, MKP-1 promotes cell survival in response to serum starvation, anisomycin, and osmotic stress, and the underlying mechanism of such resistance is believed to activation of the p38 apoptotic pathway. Consistent with this, we provide evidence that MKP-1 plays an important role in protecting cells from oxidative stress–induced cell death. Our conclusion was supported by the following three experiments. First, overexpression of MKP-1 increases MCF-7 cell survival in response to H2O2 treatment (Fig. 2). Second, knockdown of MKP-1 by MKP-1 siRNA sensitized cells to H2O2-induced cells death (Fig. 3). Lastly and most importantly, MKP-1–/– MEF cells were much more sensitive than MKP-1+/+ cells to H2O2-induced cell death (Fig. 4). Collectively, our results, along with other studies, suggest that MKP-1 is a general survival factor to protect cells from a variety of stresses.

In conclusion, we have found that MKP-1 protects cells from H2O2-mediated cell death. We showed that induction of MKP-1 by H2O2 correlates with inactivation of MAPKs. We also showed that overexpression of MKP-1 renders MCF-7 cells resistant to H2O2-induced cell death by inhibition of p38 and JNK activation. Importantly, we showed that loss of MKP-1 by down-regulation via siRNA or deletion of MKP-1 using MKP-1 knockout MEFs sensitizes cells to H2O2-induced cell death. Further studies are needed to determine the mechanisms by which MKP-1 inhibits JNK and p38 pathways upon oxidative damage.


   Acknowledgments
 
Grant support: NIH grant R01 CA100073 and the Elsa U. Pardee Foundation (G.S. Wu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Huanjie Yang for technical assistance, Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD) for HCT116 cells, Dr. Larry H. Matherly for proofreading the manuscript, and Bristol-Myers Squibb Pharmaceutical Research Institute (Princeton, NJ) for providing MKP-1 knockout mice.

Received 11/28/05. Revised 1/24/06. Accepted 2/14/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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