Cellular defense against H2O2-induced apoptosis via MAP kinase–MKP-1 pathway

transcript

Free Radical Biology & Medicine, Vol. 36, No. 8, pp. 985 –993, 2004Copyright D 2004 Elsevier Inc.

Printed in the USA. All rights reserved0891-5849/$-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; yDepartment of Physiology, Faculty of Medicine, University of Alcala, Madrid, Spain; and

zDepartment 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)

Molecu

Engine

Japan;

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 c-

jun 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

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

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

dress correspondence to: Masanori Kitamura, Department of

lar Signaling, Interdisciplinary Graduate School of Medicine and

ering, University of Yamanashi, Tamaho, Yamanashi 409-3898,

Fax: +81-55-273-8054; E-mail: masanori@yamanashi.ac.jp.

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 transduc-

tion pathways involved in MKP-1 expression are stimu-

lus-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

Q. XU et al.986

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 in-

volved 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 phosphory-

lation 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 exper-

imental 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 H2O2-

induced apoptosis [18–22]. Because MKP-1 is a specific

inhibitor of MAP kinases, it may be involved in self-

defense 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 cytoprotec-

tive 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 stress-

induced apoptosis of mesangial cells.

MATERIALS AND METHODS

Mesangial cells (SM43) were established from isolat-

ed 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) sup-

plemented with 100 U/ml penicillin G, 100 Ag/ml strep-

tomycin, 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 AMH2O2 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] (Cal-

biochem–Novabiochem Ltd., Nottingham, UK), 20 AMJNK 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 dehydroge-

nase (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 actino-

mycin 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 per-

oxidase (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

Cellular defense via MKP-1 987

blot analysis using the PhosphoPlus MAP Kinase Anti-

body Kit and the PhosphoPlus p38 MAP Kinase Anti-

body 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].

Assessment of apoptosis

Cells were treated or not with H2O2 (250 AM) for 6–

8 h. To examine the role of phosphatases in the H2O2-

induced apoptosis, cells were pretreated or not with the

protein tyrosine phosphatase inhibitor sodium orthova-

nadate (vanadate; 100 AM; Sigma) for 1 h and treated

with H2O2 for 6 h. Apoptosis was assessed quantitative-

Fig. 1. Expression of MAP kinase phosphatase 1 (MKP-1) in mesangiawere treated with H2O2 (150 AM) for up to 6 h, and the level of MKP-1GAPDH is shown at the bottom as a loading control. (B) Cells were trand Northern blot analysis was performed. (C) Cells were treated withactinomycin D (ActD; 5 Ag/ml) for the last 0–60 min. The level of Mexposure, **long exposure. (D) Densitometric analysis of the MKP-1quantitatively by densitometric analysis. Each value was normalized tagainst ActD (�) was expressed as normalized MKP-1 (%). Open circlH2O2 (100 AM) for up to 6 h, and the level of MKP-1 protein (39 kDa)kDa) was used as a loading control. (F) Cells were treated with severaanalysis of MKP-1 was performed.

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.

Transient transfection

Mesangial cells cultured in 24 well plates were

cotransfected with pCI-hGal (170 ng/well) encoding h-galactosidase (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),

l cells in response to hydrogen peroxide. (A) Rat mesangial cellsmRNAwas examined by Northern blot analysis. Expression of

eated with various concentrations of H2O2 (0–150 AM) for 1 h,(+) or without (�) H2O2 (150 AM) for 1 h in the presence ofKP-1 mRNA was examined by Northern blot analysis. *ShortmRNA level. Intensity of each MKP-1 mRNA was evaluatedo the level of GAPDH, and relative intensity of each messagee, H2O2 (�); closed circle, H2O2 (+). (E) Cells were treated withwas examined by Western blot analysis. The level of h-actin (42l concentrations of H2O2 (0–200 AM) for 3 h, and Western blot

Q. XU et al.988

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-bromo-

4-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 copre-

cipitation 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/pro-

moter 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.

Statistical analysis

Data are expressed as means F SE. Statistical anal-

ysis 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.

Fig. 2. Involvement of AP-1 in mediating H2O2-induced MKP-1expression. (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 byNorthern analysis. (B) Cells were transfected with an AP-1 reporterplasmid, pTRE-LacZ, treated with (+) or without (�) H2O2 (100 AM)and subjected to X-gal assay. Activity of AP-1 was evaluated asdescribed under Materials and Methods. Assays were performed inquadruplicate. Data are shown as means F SE. *p < .05. (C) Mesangialcells 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 performedto evaluate MKP-1 expression.

RESULTS

Expression of MKP-1 in mesangial cells in response to

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

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 H2O2-

stimulated 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 exam-

ined at a protein level. Mesangial cells were stimulated

with 100 AMH2O2 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

Fig. 3. Involvement of MAP kinases in mediating H2O2-induced MKPAM) for up to 60 min and subjected to kinase assays for ERK1/2, p38Methods. (B) Cells were pretreated with (+) or without (�) PD98059curcumin (JNK inhibitor; 20 AM) for 1 h. The cells were then exposed t

significantly induced by H2O2 in control transfectants. In

contrast, the induction of MKP-1 was markedly attenu-

ated 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 induc-

tion 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 H2O2-

induced 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 re-

sponse 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

-1 expression. (A) Mesangial cells were exposed to H2O2 (100MAP kinase (p38), and JNK, as described under Materials and(ERK inhibitor; 50 AM), SB203850 (p38 inhibitor; 25 AM), oro H2O2 (150 AM) for 1 h and subjected to Northern blot analysis.

Q. XU et al.990

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 en-

hanced by the pretreatment with vanadate (52.1 F

Fig. 4. MKP-1-mediated self-defense against H2O2-induced apoptosis.(A) Mesangial cells were pretreated or not with vanadate (proteintyrosine phosphatase inhibitor; 10 AM) for 1 h and then stimulated byH2O2 (150 AM) for 6 h. Apoptosis was evaluated by Hoechst staining.Assays were performed in quadruplicate, and data are presented asmeans 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 thentreated with (+) or without (�) H2O2 for 6 h and subjected to X-galassay. Percentage of shrunk/rounded blue cells against the total numberof blue cells was calculated for each well, and the mean value of fourwells was used to compare data in different groups. Data are presentedas means F SE. *p < .05. NS, not significant.

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-galactosi-dase. 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-stim-

ulated 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 H2O2-

stimulated cells vs. 12.5 F 1.8% in unstimulated cells;

not significant). The suppression of H2O2-induced apo-

ptosis 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-trans-

fected cells. Although transfection with MKP-1CS also

mildly decreased H2O2-triggered apoptosis, the differ-

ence 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 inhib-

itors 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

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 ki-

nase, 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 ex-

pression. 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]. How-

ever, we found that inhibition of individual MAP kinases

similarly abrogated H2O2-induced MKP-1 expression.

This result raises the possibility that three MAP kinases

cooperate to induce MKP-1 in mesangial cells. Activa-

tion 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 H2O2-

induced 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 H2O2-

induced 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 glomeru-

lar diseases in which reactive oxygen species play

pathogenic roles. It has been proposed that mesangial

cell apoptosis contributes to the generation of glomerular

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 HarborLaboratory, Cold Spring Harbor, NY, USA) for the kind gifts of MKP-1expression plasmids. This work was supported, in part, by grants fromthe Wellcome Trust and the National Kidney Research Fund to M.Kitamura. Q. Xu (Department of Nephrology, General Hospital ofChinese PLA, Beijing, P.R. China) was a training fellow supported bythe International Society of Nephrology.

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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

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

Cellular defense against H2O2-induced apoptosis via MAP kinase–MKP-1 pathway

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