Free Radical Biology & Medicine, Vol. 36, No. 8, pp. 985 – 993, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter
doi:10.1016/j.freeradbiomed.2004.01.009
Original Contribution CELLULAR DEFENSE AGAINST H2O2-INDUCED APOPTOSIS VIA MAP KINASE–MKP-1 PATHWAY QIHE XU,* TSUNEO KONTA,* KENJI NAKAYAMA,* AKIRA FURUSU,* VICTORIA MORENO-MANZANO, y JAVIER LUCIO-CAZANA, y YOSHIHISA ISHIKAWA,* LEON G. FINE,* JIAN YAO , z and MASANORI KITAMURA *,z * Department of Medicine, Royal Free and University College Medical School, University College London, London, England, United Kingdom; y Department of Physiology, Faculty of Medicine, University of Alcala, Madrid, Spain; and z Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho, Yamanashi 409-3898, Japan (Received 13 August 2003; Revised 1 December 2003; Accepted 15 January 2004)
Abstract—Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is an oxidative stress-inducible gene. In this study, we investigated signaling pathways involved in oxidative stress-induced MKP-1 expression and its role in apoptosis of rat mesangial cells. Northern and Western blot analyses showed that H2O2 induced expression of MKP-1 mRNA and protein in a dose-dependent manner, without affecting the stability of the transcript. H2O2 induced phosphorylation of extracellular signal-regulated kinase, p38 MAP kinase, and c-Jun N-terminal kinase and consequently activated activator protein 1 (AP-1). Selective inhibitors of individual MAP kinases or a dominant-negative mutant of cjun significantly suppressed the expression of MKP-1 by H2O2. Inhibition of MKP-1 by a protein tyrosine phosphatase inhibitor (vanadate) enhanced H2O2-triggered apoptosis. Consistently, transfection with a wild-type MKP-1, but not its catalytically inactive mutant MKP-1CS, attenuated H2O2-induced apoptosis. These data elucidate, for the first time, that induction of MKP-1 by H2O2 is mediated by the MAP kinase – AP-1 pathway and that the induced MKP-1 is involved in cellular defense against oxidative stress-induced apoptosis of mesangial cells. D 2004 Elsevier Inc. All rights reserved. Keywords—Mesangial cell, Hydrogen peroxide, Apoptosis, Signal transduction, Extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, Mitogen-activated protein kinase phosphatase 1, Free radicals
have shown that p38 MAP kinase and JNK1/2 are preferentially inactivated by MKP-1 [3,4]. Other MAP kinases, including ERK1/2 and ERK5 MAP kinases, are also inactivated by MKP-1 in vitro and in vivo [5,6]. MKP-1 is induced by various stresses and mitogenic/ nonmitogenic stimuli. Some protein kinases and other signaling molecules are involved in the expression of the MKP-1 gene. These include tyrosine kinases, protein kinase Cq, MAP kinases, phosphatidylinositol-3 (PI3) kinase, Akt, protein kinase A, calcium, cAMP/cGMP, and oxygen radical species [7– 14]. The signal transduction pathways involved in MKP-1 expression are stimulus-specific. For example, in Rat-1 fibroblasts, inhibition of protein kinase C prevented expression of MKP-1 induced by phorbol 12-myristate 13-acetate (PMA) but did not affect lysophosphatidic acid (LPA)-, ionomycin-, and epidermal growth factor (EGF)-induced MKP-1
INTRODUCTION
Mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1), also termed CL100, 3CH134, HVH1, and ERP, is a prototypic member of the family of inducible dual-specificity phosphatases [1]. It selectively binds MAP kinases, including extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) 1/2, p38 MAP kinase, and ERK5, and inactivates these kinases via dephosphorylation of their tyrosine and threonine residues [2]. MKP-1 has different binding sites for different MAP kinases [2]. Previous reports
Address correspondence to: Masanori Kitamura, Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho, Yamanashi 409-3898, Japan; Fax: +81-55-273-8054; E-mail:
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expression. Inhibition of ERK1/2 completely prevented PMA- and ionomycin-induced MKP-1 expression, whereas it only partially inhibited LPA- or EGF- induced MKP-1 [7]. MKP-1 is known to be an oxidative stress-inducible gene [1]. However, signal transduction pathways involved in oxidant-induced expression of MKP-1 are largely unknown. The 5V flanking region of the MKP-1 gene contains an activator protein 1 (AP-1) site [11,15]. We previously reported that hydrogen peroxide (H2O2), a known inducer of MKP-1 [16,17], triggered phosphorylation of MAP kinases, leading to activation of AP-1 [18 – 21]. We hypothesized that the MAP kinase – AP-1 pathway may play a role in mediating H2O2-induced MKP-1 expression. As we previously reported, the MAP kinase – AP-1 pathway plays a crucial role in mediating apoptosis of mesangial cells triggered by H2O2. It is based on experimental evidence that (1) H2O2 induces activation of the MAP kinase – AP-1 pathway, (2) inhibition of AP-1 by overexpression of a dominant-negative mutant of c-Jun attenuates H2O2-induced apoptosis, and (3) suppression of MAP kinases either by dominant-negative mutants or by pharmacological inhibitors also attenuates H2O2induced apoptosis [18 – 22]. Because MKP-1 is a specific inhibitor of MAP kinases, it may be involved in selfdefense mechanisms against oxidative stress-induced apoptosis in mesangial cells. In this report, we examined (1) involvement of the MAP kinase –AP-1 pathway in H2O2-induced MKP-1 expression and (2) a cytoprotective role for MKP-1 in H2O2-induced apoptosis of mesangial cells. Our data show, for the first time, that expression of MKP-1 by H2O2 is mediated by the MAP kinase –AP-1 pathway and that the induced MKP-1 is involved in the cellular defense against oxidative stressinduced apoptosis of mesangial cells.
MATERIALS AND METHODS
Cells Mesangial cells (SM43) were established from isolated glomeruli of a male Sprague Dawley rat and identified as being of the mesangial cell phenotype as described previously [23]. Cells were maintained in DMEM/Ham’s F-12 (Life Technologies, Gaithersburg, MD, USA) supplemented with 100 U/ml penicillin G, 100 Ag/ml streptomycin, 0.25 Ag/ml amphotericin B, and 10% fetal calf serum (FCS). Medium containing 1% FCS was generally used for experiments. SM/JUNDN1 cells in which AP-1 is selectively inactivated were established by stable transfection of SM43 mesangial cells with a dominant-negative mutant of c-Jun, TAM-67 [24]. SM/JUNDN1 cells exhibit de-
pressed activity of AP-1 under both unstimulated and stimulated conditions [24,25]. Pharmacological manipulation Confluent cells were preincubated in 1% FCS for 24 h, treated with H2O2 (50 – 250 AM; Sigma, St. Louis, MO, USA) for 0.5 –6 h, and subjected to Northern and Western blot analyses. Incubation with 100 – 150 AM H2O2 for 1 h was generally used for induction of MKP-1 expression. In some experiments, cells were pretreated with 50 AM MEK inhibitor PD98059 [26], 25 AM p38 MAP kinase inhibitor SB203580 [19] (Calbiochem – Novabiochem Ltd., Nottingham, UK), 20 AM JNK inhibitor curcumin [19,27,28] (Sigma), or 50 –250 nM PI3 kinase/Akt inhibitor wortmannin [29] (Sigma) for 1 h before H2O2 stimulation. Northern blot analysis and evaluation of mRNA stability Total RNA was extracted by a single-step method [30], and Northern blot analysis was performed as described before [31]. cDNAs for MKP-1 [32], c-fos [33], and c-jun [34] were used for radiolabeled probes. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. The intensity of mRNA was evaluated quantitatively by densitometric analysis. The effect of H2O2 on the stability of MKP-1 mRNA was assessed using the RNA synthesis inhibitor actinomycin D [35]. In brief, mesangial cells were treated with or without H2O2 (150 AM) for 1 h in the presence of actinomycin D (5 Ag/ml; Serva, Heidelberg, Germany) for the last 0 – 60 min. Northern blot analysis was performed to examine the level of MKP-1 mRNA and GAPDH mRNA. Western blot analysis After exposure of the cells to H2O2, total protein was extracted with SDS sample buffer (62.5 mM Tris – HCl, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.1% w/v bromphenol blue) and subjected to electrophoresis using 10% SDS – PAGE gels. After transfer onto nitrocellulose membranes, Western blot analysis was performed using a rabbit anti-MKP-1 antibody (sc-1199, 1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), a rabbit anti-h-actin antibody (Sigma), and a secondary anti-rabbit IgG antibody conjugated to horseradish peroxidase (New England Biolabs, Hertfordshire, UK). Kinase assays To examine the effect of H2O2 on the inducible activation of MAP kinases, confluent mesangial cells were incubated in 1% FCS for 24 h and exposed to 100 AM H2O2 for 15 min to 1 h. Phosphorylated forms of ERKs and p38 MAP kinase were detected by Western
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blot analysis using the PhosphoPlus MAP Kinase Antibody Kit and the PhosphoPlus p38 MAP Kinase Antibody Kit (New England Biolabs) following protocols provided by the manufacturer [19]. Activity of JNK was evaluated by phosphorylation of c-Jun using the SAPK/ JNK Assay Kit (New England Biolabs), as described previously [19,21].
ly, as described previously [21,36]. In brief, cells were fixed with 4% formaldehyde for 10 min, stained with Hoechst 33258 (10 Ag/ml; Sigma) for 1 h, and subjected to fluorescence microscopy. Apoptosis was identified using morphological criteria, i.e., nuclear condensation and/or fragmentation. Both attached cells and detached cells were used for evaluation.
Assessment of apoptosis
Transient transfection
Cells were treated or not with H2O2 (250 AM) for 6 – 8 h. To examine the role of phosphatases in the H2O2induced apoptosis, cells were pretreated or not with the protein tyrosine phosphatase inhibitor sodium orthovanadate (vanadate; 100 AM; Sigma) for 1 h and treated with H2O2 for 6 h. Apoptosis was assessed quantitative-
Mesangial cells cultured in 24 well plates were cotransfected with pCI-hGal (170 ng/well) encoding hgalactosidase (a gift from Promega, Madison, WI, USA) and pSG5MKP-1 or pSG5MKP-1CS (500 ng/well; gifts from Dr. N.K. Tonks) [5], encoding a wild-type MKP-1 or a catalytically inactive mutant of MKP-1 (MKP-1CS),
Fig. 1. Expression of MAP kinase phosphatase 1 (MKP-1) in mesangial cells in response to hydrogen peroxide. (A) Rat mesangial cells were treated with H2O2 (150 AM) for up to 6 h, and the level of MKP-1 mRNA was examined by Northern blot analysis. Expression of GAPDH is shown at the bottom as a loading control. (B) Cells were treated with various concentrations of H2O2 (0 – 150 AM) for 1 h, and Northern blot analysis was performed. (C) Cells were treated with (+) or without () H2O2 (150 AM) for 1 h in the presence of actinomycin D (ActD; 5 Ag/ml) for the last 0 – 60 min. The level of MKP-1 mRNA was examined by Northern blot analysis. *Short exposure, **long exposure. (D) Densitometric analysis of the MKP-1 mRNA level. Intensity of each MKP-1 mRNA was evaluated quantitatively by densitometric analysis. Each value was normalized to the level of GAPDH, and relative intensity of each message against ActD () was expressed as normalized MKP-1 (%). Open circle, H2O2 (); closed circle, H2O2 (+). (E) Cells were treated with H2O2 (100 AM) for up to 6 h, and the level of MKP-1 protein (39 kDa) was examined by Western blot analysis. The level of h-actin (42 kDa) was used as a loading control. (F) Cells were treated with several concentrations of H2O2 (0 – 200 AM) for 3 h, and Western blot analysis of MKP-1 was performed.
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respectively. An empty vector, pSG5, was used as a control. After incubation overnight, medium was replaced with 1% FCS. After 24 h, cells were treated with H2O2 (250 –300 AM, 6 h) and subjected to 5-bromo4-chloro-3-indolyl-h-D-galactopyranoside (X-gal) assay [37]. Percentage of shrunk/rounded blue cells against the total number of blue cells was calculated for each well, and the mean value of 4 wells was used to compare data in different groups. Assays were performed in quadruplicate. The effect of H2O2 on the activity of AP-1 was evaluated by reporter assay, as we described previously [21,38,39]. In brief, using the calcium phosphate coprecipitation method, we transiently transfected mesangial cells cultured in 24 well plates (1 105 cells/well) with an AP-1 reporter plasmid, pTRE-LacZ (330 ng/well) [40], or a control plasmid, pCI-hGal (330 ng/well). pTRE-LacZ introduces a h-galactosidase gene (lacZ) under the control of the immediate-early enhancer/promoter of human cytomegalovirus. Forty-eight hours after the transfection, medium was changed to 1% FCS. Cells were incubated for 16 h in the presence or absence of 100 AM H2O2 and subjected to X-gal assay to evaluate AP-1 activity.
without H2O2 for 1 h in the presence of actinomycin D for the last 0 –60 min. Northern blot analysis showed that the increase in the level of MKP-1 mRNA in H2O2stimulated cells was abrogated by the treatment with actinomycin D (Fig. 1C; H2O2 (+)/ActD 60 min vs. H2O2 ()/ActD 0 min). In contrast, the stability of MKP-1 mRNA in the presence of H2O2 was not different from that in the absence of H2O2 (Fig. 1D). This result suggested that the increase in the level of MKP-1 mRNA by H2O2 is due to transcriptional induction. The induction of MKP-1 by H2O2 was further examined at a protein level. Mesangial cells were stimulated
Statistical analysis Data are expressed as means F SE. Statistical analysis was performed using the nonparametric Mann – Whitney U test to compare data in different groups. A p value < .05 was used to indicate a statistically significant difference.
RESULTS
Expression of MKP-1 in mesangial cells in response to H2O2 Expression of MKP-1 is induced in mesangial cells in response to H2O2 [16,17]. We first examined dose- and time-dependent effects of H2O2 on the level of MKP-1 mRNA. Mesangial cells were stimulated with H2O2 (150 AM) for up to 6 h, and Northern blot analysis was performed. As shown in Fig. 1A, expression of MKP-1 was induced within 30 min, peaked to maximum at 1 –2 h, and returned to the basal level after 6 h. To examine a dose-dependent effect of H2O2, mesangial cells were stimulated with 0 – 150 AM H2O2 for 1 h. We found that relatively low concentrations of H2O2 were effective, and the maximum effect was observed at 75 – 100 AM (Fig. 1B). The increased level of MKP-1 mRNA may be caused by increased transcription or increased stability of mRNA. To test the latter, a chemical inhibitor of RNA synthesis was used. Mesangial cells were treated with or
Fig. 2. Involvement of AP-1 in mediating H2O2-induced MKP-1 expression. (A) Mesangial cells were exposed to H2O2 (100 – 150 AM) for up to 2 h, and expression of c-fos and c-jun was examined by Northern analysis. (B) Cells were transfected with an AP-1 reporter plasmid, pTRE-LacZ, treated with (+) or without () H2O2 (100 AM) and subjected to X-gal assay. Activity of AP-1 was evaluated as described under Materials and Methods. Assays were performed in quadruplicate. Data are shown as means F SE. *p < .05. (C) Mesangial cells stably expressing a dominant-negative mutant of c-Jun (SM/ JUNDN1) and control transfectants (SM/control) were treated with (+) or without () H2O2 for 1 h, and Northern blot analysis was performed to evaluate MKP-1 expression.
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with 100 AM H2O2 for up to 6 h and subjected to Western blot analysis. The result showed that, after the exposure to H2O2, MKP-1 protein was rapidly accumulated in the cells within 1 h, and the increased level of MKP-1 was sustained for at least 6 h (Fig. 1E). Figure 1F shows a dose-dependent effect of H2O2 on the level of MKP-1 protein. As demonstrated here, a modest increase in MKP-1 was observed at 100 AM, and its level was increased dose dependently at up to 200 AM. Involvement of AP-1 in mediating H2O2-induced MKP-1 expression The 5Vflanking region of the MKP-1 gene contains an AP-1 site. However, the role of AP-1 in the regulation of the MKP-1 gene is not well understood. To examine the involvement of AP-1 in mediating H2O2-induced MKP-1 expression, we first examined expression of c-fos and c-jun in H2O2-stimulated mesangial cells. Northern blot analysis showed that expression of c-fos and c-jun was markedly induced by H2O2 with a peak at 1 h (Fig. 2A). Consistently, reporter assay showed that activity of AP-1 was increased in mesangial cells after the treatment with H2O2 (Fig. 2B). The role of AP-1 in the induction of MKP-1 was examined using SM/JUNDN1 cells that stably express a dominant-negative mutant of c-Jun. As we previously showed, SM/JUNDN1 cells exhibit depressed activity of AP-1 under both unstimulated and stimulated conditions [24,25]. SM/JUNDN1 cells and control transfectants were stimulated by H2O2, and expression of MKP-1 was examined. As expected, expression of MKP-1 was
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significantly induced by H2O2 in control transfectants. In contrast, the induction of MKP-1 was markedly attenuated in SM/JUNDN1 cells (Fig. 2C). Of note, the basal level of MKP-1 was also suppressed in SM/JUNDN1 cells. Involvement of MAP kinases in mediating H2O2-induced MKP-1 expression The transacting potential of AP-1 depends on induction and phosphorylation of AP-1 components by the MAP kinase family of molecules [41]. We examined the roles of ERK, p38 MAP kinase, and JNK in mediating H2O2-induced MKP-1 expression. Figure 3A shows the kinetics of MAP kinase activation in H2O2-stimulated mesangial cells. Rapid phosphorylation of all three MAP kinases was observed after the stimulation with H2O2 (100 AM). The phosphorylation occurred within 15 min, peaked at 30 min, and declined after 60 min. Involvement of MAP kinases in mediating H2O2induced MKP-1 expression was further examined using selective inhibitors of MAP kinases. Mesangial cells were pretreated with PD98059, SB203580, or curcumin for 1 h and stimulated with H2O2 for 1 h. Northern blot analysis showed that individual MAP kinase inhibitors suppressed the induction of MKP-1 expression in response to H2O2 (Fig. 3B). MKP-1-mediated self-defense against H2O2-induced apoptosis The MAP kinase –AP-1 pathway plays a crucial role in mediating apoptosis of mesangial cells triggered by
Fig. 3. Involvement of MAP kinases in mediating H2O2-induced MKP-1 expression. (A) Mesangial cells were exposed to H2O2 (100 AM) for up to 60 min and subjected to kinase assays for ERK1/2, p38 MAP kinase (p38), and JNK, as described under Materials and Methods. (B) Cells were pretreated with (+) or without () PD98059 (ERK inhibitor; 50 AM), SB203850 (p38 inhibitor; 25 AM), or curcumin (JNK inhibitor; 20 AM) for 1 h. The cells were then exposed to H2O2 (150 AM) for 1 h and subjected to Northern blot analysis.
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H2O2 [19,21]. Because MKP-1 is a specific inhibitor of MAP kinases, it may be involved in the self-defense against oxidative stress-induced apoptosis. To examine this possibility, we tested the effect of vanadate, a known inhibitor of MKP-1 [42], on H2O2-induced apoptosis. Mesangial cells were pretreated or not with vanadate for 1 h and stimulated by H2O2 for 6 h. Apoptosis was evaluated by Hoechst 33258 staining. As shown in Fig. 4A, a modest induction of apoptosis was observed in H2O2-stimulated cells (11.3 F 0.3% vs. 1.6 F 0.1% in unstimulated cells). This induction was markedly enhanced by the pretreatment with vanadate (52.1 F
1.4% in vanadate-treated, H2O2-stimulated cells vs. 11.3 F 0.3% in vanadate-untreated, H2O2-stimulated cells; p < .05). Vanadate alone did not induce apoptosis of mesangial cells. To further examine the antiapoptotic role of MKP-1 in H2O2-induced apoptosis, transient transfection was used. Mesangial cells were cotransfected with empty vector, MKP-1, or MKP-1CS (catalytically inactive mutant) together with a plasmid encoding h-galactosidase. The transfected cells were treated with H2O2 for 6 h and subjected to X-gal assay. As shown in Fig. 4B, significant induction of apoptosis by H2O2 was observed in vector-transfected cells (28.2 F 0.8% in H2O2-stimulated cells vs. 5.2 F 0.4% in unstimulated cells). This induction was abrogated when the cells were transfected with the wild-type MKP-1 (15.1 F 1.5% in H2O2stimulated cells vs. 12.5 F 1.8% in unstimulated cells; not significant). The suppression of H2O2-induced apoptosis by MKP-1 was not observed when the cells were transfected with the catalytically inactive mutant of MKP-1, MKP-1CS (20.6 F 1.7% in H2O2-stimulated cells vs. 5.6 F 0.5% in unstimulated cells). Transfection with MKP-1 significantly attenuated H2O2-triggered apoptosis compared with H2O2-stimulated, vector-transfected cells. Although transfection with MKP-1CS also mildly decreased H2O2-triggered apoptosis, the difference from H2O2-stimulated, vector-transfected cells was not significant. It is worthwhile to note that, under the unstimulated condition, transfection with MKP-1, but not MKP-1CS, modestly induced apoptosis. This is consistent with our previous finding that treatment with MAP kinase inhibitors significantly induced apoptosis of unstimulated mesangial cells [20]. The basal level of MAP kinase activity observed in Fig. 3A may be required for survival of mesangial cells. DISCUSSION
Fig. 4. MKP-1-mediated self-defense against H2O2-induced apoptosis. (A) Mesangial cells were pretreated or not with vanadate (protein tyrosine phosphatase inhibitor; 10 AM) for 1 h and then stimulated by H2O2 (150 AM) for 6 h. Apoptosis was evaluated by Hoechst staining. Assays were performed in quadruplicate, and data are presented as means F SE. *p < .05. (B) Cells were cotransfected with pSG5 (vector), pSG5-MKP-1 (MKP-1), or pSG5-MKP-1CS (MKP-1CS) together with a plasmid encoding h-galactosidase. Cells were then treated with (+) or without () H2O2 for 6 h and subjected to X-gal assay. Percentage of shrunk/rounded blue cells against the total number of blue cells was calculated for each well, and the mean value of four wells was used to compare data in different groups. Data are presented as means F SE. *p < .05. NS, not significant.
In the present study, we demonstrated, for the first time, that the MAP kinase –AP-1 pathway plays a crucial role in mediating H2O2-induced expression of MKP-1. H2O2 induced phosphorylation of ERK, p38 MAP kinase, and JNK, leading to induction and activation of AP-1. Inhibition of MAP kinases by pharmacological inhibitors or inhibition of AP-1 by a dominant-negative mutant of c-Jun attenuated H2O2-induced MKP-1 expression. We also demonstrated that the induction of MKP-1 is involved in the self-defense of mesangial cells against H2O2-induced apoptosis. A previous report showed that activation of either p38 MAP kinase or JNK by specific stimulators may be sufficient to induce MKP-1 in NIH3T3 cells [9]. However, we found that inhibition of individual MAP kinases
Cellular defense via MKP-1
similarly abrogated H2O2-induced MKP-1 expression. This result raises the possibility that three MAP kinases cooperate to induce MKP-1 in mesangial cells. Activation of each MAP kinase may be necessary but not sufficient to induce MKP-1 expression. This is consistent with some previous reports which showed that ERK activation is not sufficient to induce MKP-1 in Rat-1 fibroblasts [7] and that activation of p38 MAP kinase and JNK is not sufficient to induce MKP-1 in a human leukemia cell line [3]. Signaling pathways other than MAP kinases may also be involved in the induction of MKP-1 by H2O2. A possible candidate is the PI3 kinase – Akt pathway. It has been reported that, in some cell types, the PI3 kinase – Akt pathway is activated in response to H2O2 [43,44]. Akt transduces antiapoptotic signals [45] and is involved in insulin-induced MKP-1 expression in vascular smooth muscle cells [8,10]. We tested the role of the PI3 kinase – Akt pathway in the induction of MKP-1 by H2O2 in mesangial cells. In our experimental setting, H2O2 did not induce Akt activation. A specific inhibitor of PI3 kinase, wortmannin, did not inhibit basal and H2O2induced expression of MKP-1 (our unpublished data). These results excluded possible involvement of the PI3K – Akt pathway in the H2O2-induced expression of MKP-1 in mesangial cells. Previous reports showed that H2O2 increased the mRNA level of some genes via transcriptional and/or posttranscriptional mechanisms [46,47]. In this report, we found that the stimulatory effect of H2O2 on MKP-1 was at the transcriptional level. We further identified that the transcription factor AP-1 was required for the expression of MKP-1, which contributed to attenuation of H2O2induced apoptosis. Based on our previous and current findings, the role of AP-1 in the H2O2-induced apoptosis of mesangial cells seems to be a little paradoxical. Although AP-1 plays an important role in the induction of apoptosis by H2O2 [38,39], it may also be involved in the cytoprotective machinery against the apoptotic event. Like the binary role of AP-1, the role of MAP kinases in H2O2-induced apoptosis is also complicated. As we previously showed, MAP kinases play significant roles in mediating H2O2-induced apoptosis [19,21]. In addition to this proapoptotic role, our present data also suggested the antiapoptotic role of MAP kinases via induction of the potentially antiapoptotic gene, MKP-1. This mechanism may be involved in the self-defense of mesangial cells against oxidative stress. Our findings suggested that the MAP kinase –AP-1 pathway possesses both proapoptotic and antiapoptotic properties. Apoptosis of mesangial cells is observed in glomerular diseases in which reactive oxygen species play pathogenic roles. It has been proposed that mesangial cell apoptosis contributes to the generation of glomerular
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damage, especially glomerulosclerosis [48 –51]. Based on this, induction of MKP-1 in response to oxidative stress may play a beneficial role in preventing both mesangial cell death and glomerulosclerosis. Further investigation will be required to clarify the in vivo roles of MKP-1 in glomerular pathophysiology. Acknowledgments —We thank Dr. N.K. Tonks (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) for the kind gifts of MKP-1 expression plasmids. This work was supported, in part, by grants from the Wellcome Trust and the National Kidney Research Fund to M. Kitamura. Q. Xu (Department of Nephrology, General Hospital of Chinese PLA, Beijing, P.R. China) was a training fellow supported by the International Society of Nephrology. REFERENCES [1] Keyse, S. M.; Emslie, E. A. Oxidative stress and heat shock induce a human gene encoding a protein tyrosine phosphatase. Nature 359:644 – 647; 1992. [2] Slack, D. N.; Seternes, O. M.; Gabrielsen, M.; Keyse, S. M. Distinct binding determinants for ERK2/p38a and JNK MAP kinases mediate catalytic activation and substrate selectivity of MAP kinase phosphatase-1. J. Biol. Chem. 276:16491 – 16500; 2001. [3] Franklin, C. C.; Kraft, A. S. Conditional expression of the mitogen-activated protein kinase (MAPK) phosphatase MKP-1 preferentially inhibits p38 MAPK and stress-activated protein kinase in U937 cells. J. Biol. Chem. 272:16917 – 16923; 1997. [4] Lim, H. W.; New, L.; Han, J.; Molkentin, J. D. Calcineurin enhances MAPK phosphatase-1 expression and p38 MAPK inactivation in cardiac myocytes. J. Biol. Chem. 276:15913 – 15919; 2001. [5] Sun, H.; Charles, C. H.; Lau, L. F.; Tonks, N. K. MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell 75:487 – 493; 1993. [6] Kamakura, S.; Moriguchi, T.; Nishida, E. Activation of the protein kinase ERK5/BMK1 by receptor tyrosine kinases: identification and characterization of a signaling pathway to the nucleus. J. Biol. Chem. 274:26563 – 26571; 1999. [7] Cook, S. J.; Beltman, J.; Cadwallader, K. A.; McMahon, M.; McCormick, F. Regulation of mitogen-activated protein kinase phosphatase-1 expression by extracellular signal-regulated kinase-dependent and Ca2+-dependent signal pathways in Rat-1 cells. J. Biol. Chem. 272:13309 – 13319; 1997. [8] Begum, N.; Ragolia, L.; Rienzie, J.; McCarthy, M.; Duddy, N. Regulation of mitogen-activated protein kinase phosphatase-1 induction by insulin in vascular smooth muscle cells: evaluation of the role of the nitric oxide signaling pathway and potential defects in hypertension. J. Biol. Chem. 273:25164 – 25170; 1998. [9] Bokemeyer, D.; Sorokin, A.; Yan, M.; Ahn, N. G.; Templeton, D. J.; Dunn, M. J. Induction of mitogen-activated protein kinase phosphatase 1 by the stress-activated protein kinase signaling pathway but not by extracellular signal-regulated kinase in fibroblasts. J. Biol. Chem. 271:639 – 642; 1996. [10] Takehara, N.; Kawabe, J.; Aizawa, Y.; Hasebe, N.; Kikuchi, K. High glucose attenuates insulin-induced mitogen-activated protein kinase phosphatase-1 (MKP-1) expression in vascular smooth muscle cells. Biochim. Biophys. Acta 1497:244 – 252; 2000. [11] Sommer, A.; Burkhardt, H.; Keyse, S. M.; Luscher, B. Synergistic activation of the MKP-1 gene by protein kinase A signaling and USF, but not c-Myc. FEBS Lett. 474:146 – 150; 2000. [12] Valledor, A. F.; Xaus, J.; Comalada, M.; Soler, C.; Celada, A. Protein kinase C epsilon is required for the induction of mitogen-activated protein kinase phosphatase-1 in lipopolysaccharide-stimulated macrophages. J. Immunol. 164:29 – 37; 2000. [13] Metzler, B.; Hu, Y.; Sturm, G.; Wick, G.; Xu, Q. Induction of
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Cellular defense via MKP-1 ABBREVIATIONS
MAP kinase — mitogen-activated protein kinase MKP-1 — MAP kinase phosphatase 1 ERK — extracellular signal-regulated kinase JNK — c-Jun N-terminal kinase PI3 kinase — phosphatidylinositol 3-kinase
993
PMA — phorbol 12-myristate 13-acetate LPA — lysophosphatidic acid EGF — epidermal growth factor AP-1 — activator protein 1 FCS — fetal calf serum MKP-1CS — catalytically inactive mutant of MKP-1 GAPDH — glyceraldehyde-3-phosphate dehydrogenase
