Amphiregulin is a potent mitogen for the vascularsmooth muscle cell line, A7r5

Masayuki Kato,a Tetsuya Inazu,b,* Yasuyuki Kawai,a,1 Katsuhiko Masamura,a

Masahiro Yoshida,a Nobuyoshi Tanaka,a Kaoru Miyamoto,b and Isamu Miyamoria

a The Third Department of Internal Medicine, Fukui Medical University, 23-3 Shimoaizuki, Matsuoka, Fukui 910-1193, Japanb Department of Biochemistry, Fukui Medical University, 23-3 Shimoaizuki, Matsuoka, Fukui 910-1193, Japan

Received 18 December 2002

Abstract

The regulation of amphiregulin, an epidermal growth factor (EGF) family member, and its effect on vascular smooth muscle cells

(VSMC) were examined. Amphiregulin mRNA was upregulated by amphiregulin itself as well as a-thrombin. Amphiregulin caused

an approximate 3-fold increase in DNA synthesis. Its effect on growth was compared with those of other mitogens, and was found to

be approximately 3.5-, 2.4-, and 1.0-fold greater than those of endothelin-I (ET-I), a-thrombin, and platelet-derived growth factor-

AB (PDGF-AB), respectively. As evidenced by Western blot analysis, amphiregulin stimulated the phosphorylation of p42/p44-

mitogen-activated protein kinase (MAPK), p38-MAPK, c-Jun NH2-terminal protein kinase (JNK), and Akt/protein kinase B

(PKB), respectively. By statistical analysis, the amphiregulin-induced growth effect was significantly decreased by the MAP kinase/

extracellular regulated kinase kinase-1 (MEK-1) inhibitor PD98059, p38-MAPK inhibitor SB203580, and phosphatidylinositol 3-

kinase (PI-3 kinase) inhibitor wortmannin, respectively, but was not decreased by JNK inhibitor SP600125. These results suggest

that amphiregulin is the most potent mitogen of the mitogens tested, and its growth effect is mediated at least in part through the

p42/p44-MAPK, p38-MAPK, and PI-3 kinase-Akt/PKB pathways in VSMC.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Amphiregulin; Vascular smooth muscle cells; Proliferation; Mitogen-activated protein kinases; Phosphatidylinositol 3-kinase

Vascular smooth muscle cell (VSMC) proliferation is

a component of arterial remodeling in vasculature dis-eases. The accumulation of VSMC in neointima, as a

result of proliferation and migration, is thought to be a

central feature of atherosclerosis and restenosis after

angioplasty [1]. Mechanistically, growth factors have

been proposed to play a role in this process [2,3]. For

example, VSMC produce and secrete a variety of auto-

crine growth factors after stimulation by thrombin, in-

cluding platelet-derived growth factor-AA (PDGF-AA),basic fibroblast growth factor (bFGF), heparin binding

epidermal growth factor-like growth factor (HB-EGF),

and transforming growth factor-b (TGF-b) [4].

The epidermal growth factor (EGF) family plays an

important role in cell processes, including cell prolifer-ation, survival, differentiation, and migration. The EGF

family consists of EGF, amphiregulin, HB-EGF,

transforming growth factor-a (TGF-a), betacellulin,

epiregulin, and neuregulins (including heregulin/neu-

regulin 1). Amphiregulin was originally purified from

conditioned medium of 12-O-tetradecanoylphorbol-13-acetate-treated MCF-7 human breast carcinoma cells

[5]. The carboxyl terminal half of the amphiregulin ex-hibits a striking homology to EGF and therefore it can

be classified as a member of the EGF family. Like EGF

and TGF-a, amphiregulin is also synthesized in the form

of a transmembrane precursor, with the secreted protein

being released by proteolytic cleavage.

The mitogen-activated protein kinase (MAPK)

pathway is involved in cellular responses such as pro-

liferation, survival, differentiation, and migration. TheMAPK superfamily is divided into three members, p42/

Biochemical and Biophysical Research Communications 301 (2003) 1109–1115

www.elsevier.com/locate/ybbrc

BBRC

* Corresponding author. Fax: +81-776-61-8102.

E-mail addresses: TINAZU@fmsrsa.fukui-med.ac.jp (T. Inazu),

Kawai@fmsrsa.fukui-med.ac.jp (Y. Kawai).1 Also corresponding author.

0006-291X/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0006-291X(03)00093-7

p44-MAPK/extracellular regulated kinase (ERK) 1 and2, p38-MAPK, and c-Jun N-terminal kinase (JNK). In

general, p42/p44-MAPK contribute to cell growth and

proliferation, while p38-MAPK and JNK contributes to

the protection of cells from the stress of inflammation,

osmotic and UV stimulation [6,7].

Phosphatidyl inositol 3-kinase (PI-3 kinase) is in-

volved in the intracellular signal transduction of many

receptors and has been implicated in the transduction ofsurvival signals [8]. Akt, also referred to as protein ki-

nase B (PKB) or that related to A and C kinases (Rac)

[9], which lie downstream from PI-3 kinase, plays a

critical role in controlling the balance between survival

and apoptosis [10]. Most growth factors are able to

prevent cell death by activating a survival pathway [11].

In a previous study, we demonstrated that the one (or

more) autocrine factor is produced by brief exposure ofVSMC to glucocorticoid, which then stimulates VSMC,

[12] and concluded that this factor(s) must be a protein

(unpublished observation). Since this observation, we

continued our studies of growth factors that are derived

from VSMC. According to recent findings, VSMC

produce epiregulin, a member of the EGF family, as the

result of endothelin-I (ET-I), angiotensin-II (AT-II),

and thrombin stimulation [13]. Moreover, VSMC pro-duce and secrete HB-EGF, another member of the EGF

family [4]. These observations suggest that the EGF

family might be important for VSMC as well and we

therefore began a survey of the number of types of this

family that are expressed and examined their role in

VSMC. In this study, we report on several members of

the EGF family that are expressed in VSMC. Further-

more, this is the first observation to show that amphi-regulin mRNA is regulated by amphiregulin itself and

a-thrombin in VSMC, that amphiregulin is a potent

mitogen among those tested, and that its effect is med-

iated at least in part through the p42/p44-MAPK, p38-

MAPK, and PI-3 kinase-Akt/PKB pathways.

Methods

Cell line and Cell culture. A7r5, immortal embryonic rat thoracic

aorta smooth muscle cell line, was obtained from the American Type

Culture Collection (ATCC No. CRL1444). The A7r5 cell line was

grown in Dulbecco�s modified Eagle�s medium (DMEM) with 10%

fetal calf serum (FCS) under 5% CO2 in air at 37 �C in a humidified

incubator.

Reagents. p42/p44-MAPK, p38-MAPK, JNK, phospho-p42/p44-

MAPK, phospho-p38-MAPK antibodies, and phospho and non-

phospho-Akt/PKB antibodies were from Cell Signaling Technology

and phospho-JNK was from BioSource International. Recombinant

human amphiregulin was purchased from R&D systems and a-thrombin was obtained from Sigma. MAP kinase/extracellular regu-

lated kinase kinase-1(MEK-1) inhibitor PD98059 was purchased from

Promega, p38-MAPK inhibitor SB203580, PI-3K inhibitor wortman-

nin was from Sigma, and JNK inhibitor SP600125 was from Calbio-

chem. ½3H�Thymidine was purchased from American Radiolabeled

Chemicals.

RT-PCR analysis. Total cellular RNA was isolated from cultured

A7r5 by the Trizol reagent (Gibco BRL). Reverse transcription-poly-

merase chain reactions (RT-PCRs) were performed using Superscript

II (Gibco BRL) following the protocol recommended by the manu-

facturer. Briefly the total RNA was reverse-transcribed with the Su-

perscript II kit using an oligo(dt) primer for 60min at 37 �C. Using the

specific primers shown below, amplification was performed using a

Gene Amp PCR System 9700 (Perkin–Elmer) for 30 cycles. A cycle

profile consisted of 15 s at 94 �C for denaturation, 30 s at 55 �C for

annealing, and 1min at 72 �C for primer extension. A 2% agarose gel

was used for separating the PCR products, stained by ethidium bro-

mide, and then photographed. The specific primers for rat amphireg-

ulin, epiregulin, HB-EGF, EGF, TGF-a, heregulin, and mouse

betacellulin, and rat EGFR, ErbB2, ErbB3, and ErbB4, respectively,

were prepared by selecting the specific nucleotide sequence. The

primers used were as follows: amphiregulin-sense primer 50-TGGTGG

ACTTGTGGTTTC-30 (corresponding nt 313–332) and antisense pri-

mer 50-TCATTACCGCCGTCCTGCTT-30 (nt 801–820), epiregulin-

sense primer 50-GGTGTTGATTACAAAGTGTAG-30 (nt 172–191)

and antisense primer 50-TCACAGAAAGAAGTGTTCACA-30 (nt

697–716), HB-EGF-sense primer 50-TCTTGGCTGTGGTGGCTG

TA-30 (nt 516–535) and antisense primer 50-ACGGATGAGTGGT

TTATGGA-30 (nt 1027–1046), EGF-sense primer 50-CTGACTCCGC

CTGCTCCAAG-30 (nt 291–310) and antisense primer 50-CGTGTTCT

TCTGAGTTCCT-30 (nt 842–861), TGF-a-sense primer 50-GCAGGA

AGAGAAGCCAGCAT-30 (nt 333–352) and antisense primer 50-CTT

CTTGCTAACCCACACCA-30 (nt 886–905), heregulin-sense primer

50-GCCTCTGCCAACATCACCAT-30 (nt 411–430) and antisense

primer 50-TCGGCTTCGGCAGAGTCTTC-30 (nt 872-891), betacell-

ulin-sense primer 50-GTGAGCGAGTGGACCTGTTT-30 (423–442)

and antisense primer 50-GTTCCAAAATAGCCAAGACT-30 (nt

1068–1088), EGFR-sense primer 50-GCCATGAACATCACCTGTA

CA-30 (nt 1855–1875) and antisense primer 50-TGGTTTGGAGCTT

CTCCGCT-30 (nt 2247–2267), ErbB2-sense primer 50-ATACAGGTA

CATCCAGGCC-30 (nt 3437–3457) and antisense primer 50-CAGC

CCGAGTATGTGAACCAA-30(nt 3778-3799), ErbB3-sense primer

50-GGTTACGAGTACATGGATGTG-30 (nt 3790–3811) and anti-

sense primer 50-TTAGCCTTGGGGAAAAGCCTG-30 (nt 4121–

4142), and ErbB4-sense primer 50-TTGTGTCCCGGAGGAAGAA

TG-30 (nt 3546-3566) and antisense primer 50-CACCACAGTATTCC

GGTGTCT-30 (nt 3937–3958)

Northern blotting. Total cellular RNA was isolated from cultured

A7r5 cells that had been treated with amphiregulin and a-thrombin for

the indicated times. RNA samples were fractionated on 1% agarose–

formaldehyde gel, and transferred to nylon membranes (Immobilon-

N+, Millipore). Probes for amphiregulin (corresponding nt 313–820)

and b-actin were prepared by means of a random primer using a

BcaBEST Labeling Kit (TaKaRa Biomedicals). Hybridizations,

washing, exposure, and analysis were performed as described previ-

ously [14].

Mitogenesis assay. To evaluate the potency of amphiregulin-in-

duced DNA synthesis, ½3H�thymidine incorporation into DNA was

measured as described previously [12]. Cells were plated in 24-well

plates and grown in DMEM with 10% FCS under 5% CO2 in air until

cells reached a subconfluent state. The cells were then maintained in

serum-free medium for 24 h before stimulation and then treated with

an appropriate dose of amphiregulin. To determine the effect of

pharmacological inhibitors of MEK-1, p38-MAPK, JNK, and PI-3

kinase, cells were pretreated with inhibitors for 1 h and then treated

with amphiregulin (10 nM).

Western blotting. Cells were plated on six-well plates in DMEM

with 10% FCS until the cells reached a subconfluent state. The medium

was changed to serum-free DMEM for 24 h, then preincubated in the

presence or absence of inhibitors for 1 h, and stimulated with amphi-

regulin (10 nM) for the indicated times. To stop this reaction, the cells

were washed three times with phosphate buffered saline (PBS) and cell

extracts were then obtained by applying 1� sample buffer following by

1110 M. Kato et al. / Biochemical and Biophysical Research Communications 301 (2003) 1109–1115

boiling for 5min. They were then loaded onto a 10% SDS–PAGE.

After electrophoresis, the proteins were transferred to Immobilon P

membranes (Millipore). The filters were blocked with 0.05% Tween-20,

5% skim milk and then incubated with p42/p44-MAPK, phospho-p42/

p44-MAPK, p38-MAPK, phospho-p38-MAPK, JNK, phospho-JNK,

Akt/PKB, and phosphpo-Akt/PKB antibodies, respectively. After in-

cubation with anti-rabbit secondary antibody, the blots were visualized

with an ECL Western blotting kit (Amersham-pharmacia biotech).

Statistical analysis. The levels of phosphorylation were determined

by densitometric analysis (ATTO Densitograph). Data are presented

as means�SE. Statistical analyses were performed with the use of

ANOVA. p values were adjusted by the Bonferroni method. A value of

p < 0:05 was considered to be statistically significant.

Results

Expression of the EGF family and the EGF receptor

family in A7r5 cells

It is generally believed that the EGF family plays an

important role in cell proliferation, survival, migration,

and differentiation. These factors exert their cellular ef-

fects through binding to cell surface receptors. The EGF

family of receptors include epidermal growth factor re-

ceptor (EGFR also called ErbB1/HER1) [15], ErbB2

(Neu/HER2) [16], ErbB3 (HER3) [17], and ErbB4

(HER4) [18]. We initially addressed the issue of whetherthe EGF family and its receptor family were expressed

in A7r5 cells. Total RNAs from A7r5 were reverse

transcribed and analyzed by RT-PCR.

Among the EGF family members, amphiregulin,

epiregulin, heregulin, and HB-EGF were detectable but

EGF, TGF-a and betacellulin were not. On the other

hand, all members of the EGF receptors were observed,

consistent with a previous report [19] (Fig. 1A). We alsoconfirmed that these products were truly coded for

factors and receptors themselves by DNA sequence

analysis (data not shown). In addition, we also found

that the EGF family members and their receptors were

expressed in primary human aortic smooth muscle cells.

In this cell, amphiregulin, HB-EGF, TGF-a, and all

members of the EGF receptor family were detectable by

RT-PCR analysis (data not shown). This suggests thatseveral members of the EGF family and all members of

the EGFR family were expressed, not only by immor-

talized smooth muscle cells, but by primary smooth

muscle cells as well.

Amphiregulin mRNA is regulated by amphiregulin itself

and a-thrombin

Since amphiregulin is commonly expressed both inA7r5 and primary human aortic smooth muscle cells as

evidenced by RT-PCR analysis, we presumed that

amphiregulin must be an important growth factor and

chose this as a stimulator for these cells. We initially

explored the issue of whether amphiregulin mRNA is

modulated in the A7r5 smooth muscle cell line. It is

well known that G-protein coupling receptor agonistsare agonists for VSMC, so a-thrombin was chosen as a

representative example. We examined the regulation of

amphiregulin mRNA by amphiregulin itself as well as

a-thrombin by Northern blotting. Total RNA was

isolated at 0, 1, 3, and 6 h after the addition of

amphiregulin (10 nM). The presence of amphiregulin

caused an increase in its own mRNA, the message size

of which was 1.4-kb, and the maximal induction was 12-fold at 3 h after stimulation (Fig. 1B). Similarly,

a-thrombin (3U/ml) was found to up-regulate amphi-

regulin mRNA at a maximal 4-fold, 1 h after stimula-

tion (Fig. 1C).

Amphiregulin induces the proliferation of A7r5 cell

We next examined the effect of amphiregulin on the

growth of the A7r5 smooth muscle cell line. The in-corporation of ½3H�thymidine into DNA was deter-

mined. As shown in Fig. 2A, amphiregulin (10 nM)

caused approximately a 3-fold increase in DNA syn-

thesis in the A7r5 cells. We then examined the dose

dependency of amphiregulin. The effect of amphiregulin

on the proliferation of VSMC was dose dependent at

lower concentrations with a maximal effect at 10 nM.

To evaluate the mitogenic ability of amphiregulin, wecompared the effect of amphiregulin and those of other

Fig. 1. Expression of the EGF family and the EGFR family in A7r5

VSMC (A) and induction of amphiregulin mRNA by amphiregulin

and a-thrombin in A7r5 VSMC (B,C) (A) Confluent A7r5 cells were

treated with serum-free medium for 24 h and total RNA was isolated.

EGF family and EGFR family mRNAs were amplified by RT-PCR

using specific primers. The PCR products were electrophoresed on a

2.0% agarose gel and stained with ethidium bromide. Lanes 1, EGF; 2,

betacellulin; 3, TGF-a; 4, heregulin; 5, HB-EGF; 6, epiregulin; 7,

amphiregulin; 8, EGFR; 9, ErbB2; 10, ErbB3; and 11, ErbB4. Total

RNA was prepared from serum-starved VSMC that have been stim-

ulated with amphiregulin (10 nM) (B) or a-thrombin (3U/ml) (C) for

the indicated times, respectively. RNA blots (50lg per lane) were

probed with random-primed 32P-labeled cDNAs containing amphi-

regulin and b-actin. AR: amphiregulin.

M. Kato et al. / Biochemical and Biophysical Research Communications 301 (2003) 1109–1115 1111

well-known mitogens on DNA synthesis. The effect of

amphiregulin on growth was approximately 3.5-, 2.4-,

and 1.0-fold greater than those of ET-1 (100 nM), a-thrombin (3U/ml), and PDGF-AB (15 lM), respec-

tively (Fig. 2B). However, the effect of amphiregulin on

the growth of human aortic smooth muscle cells was

weaker than that of A7r5. Amphiregulin increased this

cell proliferation by approximately 1.3-fold, even at thestrongest stimulation (concentration 1 nM, data not

shown).

Amphiregulin activates p42/p44-MAPK, p38-MAPK, and

JNK

We next examined the issue of whether the MAPK

superfamily is activated by amphiregulin by measure-

ment of their activated state by phospho-specific

MAPK antibodies, which detect only activated

MAPKs. Amphiregulin-activated p42/p44-MAPK.Maximum level of activated p42/p44-MAPK was ob-

served 10min after stimulation and then declined (Fig.

3A). We next attempted to determine whether p38-

MAPK and JNK are activated by amphiregulin using

phospho-specific p38-MAPK and phospho-specific

JNK antibodies, respectively. p38-MAPK was tran-

siently activated by amphiregulin with a peak at 10min

and JNK was also activated with a peak at 20min (Figs.3B and C). Since p38-MAPK and JNK were activated

by amphiregulin, these result suggests that, not only

stress, but growth factor as well stimulate these protein

kinases in VSMC.

Amphiregulin activates Akt/PKB

The PI-3 kinase-Akt/PKB pathway plays a critical

role in controlling the balance between survival and

apoptosis [8]. Therefore, we examined the issue ofwhether Akt/PKB is activated by amphiregulin. Its ac-

tivated state was determined using the phospho-specific

Akt/PKB antibody, which detects only the activated

Fig. 2. The effect of amphiregulin on DNA synthesis in A7r5 VSMC. (A) Subconfluent VSMC were exposed to various concentrations of amphi-

regulin for 24 h and pulse-labeled for 3 h with 0.5 lCi/ml ½3H�thymidine. The ½3H�thymidine incorporated into cells was determined as described in

Methods. (B) Subconfluent VSMC were either left untreated (serum-free control) or stimulated for 24 h with ET-I(100 nM), a-thrombin(3U/ml), and

PDGF-AB(15 lM). The results are shown as 100 arbitary units for the control, untreated VSMC. Asterisks show statistically significant differences

from the control (*p < 0:05 compared with control). AR: amphiregulin.

Fig. 3. Phosphorylation of MAPKs and Akt/PKB by amphiregulin in

A7r5 VSMC. Cultured VSMC were treated with amphiregulin (10 nM)

for the indicated times and cell lysates were analyzed by immuno-

blotting with antibodies for p42/p44-MAPK, phospho-p42/

p44-MAPK (A); p38-MAPK, phospho-p38-MAPK (B); JNK, phos-

pho-JNK (C); and Akt/PKB, phospho-Akt/PKB (D), respectively.

1112 M. Kato et al. / Biochemical and Biophysical Research Communications 301 (2003) 1109–1115

state of Akt/PKB. As shown in Fig. 3D, the maximumlevel of phosphorylated Akt/PKB was observed at

10min after stimulation and then declined (Fig. 3D).

The effect of amphiregulin-induced growth is decreased by

protein kinase inhibitors

We next tested the effect of MEK-1 inhibitor

PD98059, p38-MAPK inhibitor SB203580, JNK inhibi-

tor SP600125, and PI-3 kinase inhibitor wortmannin onamphiregulin-induced VSMC proliferation by means of

½3H�thymidine incorporation measurements. By pre-

treatment with PD98059 (25 lM) and SB203580 (25 lM),

amphiregulin-induced VSMC proliferation was signifi-

cantly decreased (320:1� 5:9 vs 219:4� 5:7, 320:1� 5:9vs 248:8� 6:1; p < 0:05). And wortmannin (50 nM) in-

hibited modestly but significantly amphiregulin-induced

VSMC proliferation (320:1� 5:9 vs 278:0� 5:6; p <0:05). However, pretreatment with JNK inhibitor

SP600125 (3 lM) showed no effect on amphiregulin-in-

duced VSMC proliferation (320:1� 5:9 vs 331:9� 10:6)(Fig. 4A). The effect of inhibitors was also confirmed by

Western blot analysis. The activation state of phos-

phorylation was decreased by approximately 64%, 75%,and 69% by PD98059, SB203580, and SP600125, re-

spectively, and by approximately 100% by wortmannin

(Fig. 4B). This result suggests that the growth signal of

amphiregulin is mediated at least in part through p42/

p44-MAPK, p38-MAPK, and PI-3 kinase-Akt/PKB

pathways.

Discussion

In the present study, we demonstrated that amphi-

regulin, epiregulin, heregulin, and HB-EGF are ex-

pressed as evidenced by RT-PCR analysis. The EGF

family can be divided into two subgroups on the basis of

their direct binding to specific receptors [20,21]. Mem-

bers of the first group, which includes amphiregulin,epiregulin, HB-EGF, EGF, TGF-a, and betacellulin,

bind directly to the classic EGF receptor. Members of

the other group, which includes heregulin, bind directly

to the ErbB3 and ErbB4. Considering the observation

that all EGFR family members are expressed by A7r5

cells, in addition to amphiregulin, epiregulin, heregulin,

Fig. 4. Effect of protein kinase inhibitors on amphiregulin-stimulated growth and phosphorylation of MAPKs and Akt/PKB in A7r5 VSMC.

Subconfluent VSMC were pretreated with or without PD98059(25 lM), SB203580 (25lM), SP600125(3lM), and wortmannin (50 nM) for 1 h and

then stimulated with amphiregulin (10 nM). (A) Cell growth was measured by ½3H�thymidine incorporation into the cells as described in Methods.

The results are shown as 100 arbitary units for the control. (*p < 0:05 compared with amphiregulin stimulation) (B) Whole cell lysates were analyzed

by immunoblotting with antibody for p42/p44-MAPK, phospho-p42/p44-MAPK, p38-MAPK, phospho-p38-MAPK, JNK, phospho-JNK, Akt-

PKB, and phospho-Akt/PKB, respectively. AR: amphiregulin.

M. Kato et al. / Biochemical and Biophysical Research Communications 301 (2003) 1109–1115 1113

and HB-EGF are able to affect A7r5 VSMC via theirreceptors. In fact, epiregulin is produced and secreted by

the G-protein coupling receptor stimulator in VSMC

and functions as an autocrine growth factor [13]. In

addition, HB-EGF functions as an autocrine growth

factor for VSMC [4]. In this experiment, although we

did not observe the production and secretion of am-

phiregulin by an agonist, it is possible that amphiregulin

as well as epiregulin and HB-EGF are produced andsecreted. Therefore, these factors may function as an

autocrine growth factor in VSMC.

A question arises as to why these cells express several

factors of the EGF family and their receptors. One

possible explanation is that several growth factors do

not act independently, but rather, they interact with

each other, and then cooperatively induce cell growth.

In fact, PDGF-AA, bFGF, or PDGF-BB alone givesrelatively weak mitogenic responses in VSMC, but when

PDGF-AA is added in combination with bFGF or

PDGF-BB, the mitogenic responses are markedly en-

hanced [22].

We showed that amphiregulin mRNA is modulated

by amphiregulin itself as well as by a-thrombin stimu-

lation in A7r5 VSMC. Consistent with our observation,

two similar observations that the levels of HB-EGFmRNA transiently increased by HB-EGF, PDGF,

bFGF, or FCS have been documented [23]. In addition,

epiregulin mRNA is also regulated by the G-protein

coupling receptor stimulator [13]. These observations

suggest that several members of EGF family mRNA are

modulated by the growth factor itself as well as other

stimulants such as the G-protein coupling receptor

stimulator.The MAP kinase pathway plays a central role in the

transduction of signals for growth, differentiation, and

other cellular responses [6,7]. Therefore, we confirmed

that this signal transduction pathway is involved in the

growth effect of amphiregulin. The results herein show

that p42/p44-MAPK, p38-MAPK and JNK were acti-

vated by amphiregulin. Pretreatment with the MEK1

inhibitor PD98059 and p38-MAPK inhibitor SB203580led to a suppression of the growth effect of amphiregulin

and the state of phosphorylation of p42/p44-MAPK and

p38-MAPK. On the other hand pretreatment with JNK

inhibitor SP600125 suppressed the state of phosphory-

lation of JNK, but showed no effect on amphiregulin-

induced VSMC proliferation. These results suggest that

p42/p44-MAPK and p38-MAPK are involved at least in

part in the amphiregulin-induced growth effect.Ohashi et al. [24] showed that p38-MAPK is rapidly

activated in the medial cells of the balloon injured rat

carotid artery and the pharmacological inhibition of

p38-MAPK decreased neointimal hyperplasia and the

percentage of proliferating nuclear antigen-positive

cells. This observation suggests that p38-MAPK is ac-

tivated in the case of VSMC proliferation in vivo.

Yamanaka et al. [19] showed that all of the EGF familyligands examined potently activated the JNK signaling

pathway in cultured visceral smooth muscle cells.

However, they observed that heregulin, which potently

phosphorylated JNK, did not induce phenotypic mod-

ulation. Based on this observation, they concluded that

JNK might not be involved in the phenotypic modu-

lation of smooth muscle cells. These observations are

consistent with our observation that both p42/p44-MAPK and p38-MAPK are involved in the amphi-

regulin-stimulated VSMC proliferation, but JNK is

not. Furthermore, the mouse embryo fibroblast in

which the gene for MEK kinase-1 (MEKK1) is dis-

rupted, which is the upstream signal of JNK, showed a

loss of cell motility [25]. Therefore, we speculate that

JNK may be involved the migration of smooth muscle

cells.Furthermore, we observed that Akt/PKB was phos-

phorylated by amphiregulin, and wortmannin, specific

inhibitor of PI-3 kinase, suppressed amphiregulin-in-

duced VSMC growth. Most growth factors prevent cell

death by activating survival pathways, in addition to

stimulating cell proliferation [11]. Therefore, amphireg-

ulin may stimulate VSMC proliferation not only by di-

rectly increasing cell growth, but also by decreasingapoptosis.

In summary, the findings herein clearly show that (i)

amphiregulin is expressed in VSMC as evidenced by RT-

PCR analysis. A similar observation as the result of a

study of the relationship between amphiregulin and

prostate interstitial smooth muscle cells has also been

reported [26]. However, we provided evidence, for the

first time, that (ii) its mRNA was regulated by amphi-regulin itself as well as by a-thrombin, (iii) amphiregulin

was found to be a powerful mitogen, as evidenced by

½3H�thymidine incorporation into the cells, and its effect

was more potent than those of ET-I and a-thrombin,

and nearly equal to that of PDGF-AB, (iv) amphireg-

ulin was found to stimulate both MAPKs and Akt/PKB

pathways. Finally, (v) the growth effect of amphiregulin

was suppressed by MEK-1 inhibitor PD98059, p38-MAPK inhibitor SB203580, and PI-3 kinase inhibitor

wortmannin.

In several members of the EGF family, it is known

that their mRNA and protein are upregulated after

vascular injury and that this may contribute to vascular

restenosis [27,28]. Therefore, further investigations will

be needed to clarify the implication of amphiregulin to

pathological conditions in vivo such as atherosclerosisand post-PTCA restenosis.

Acknowledgments

We are grateful to Dr. Takio Hayashi for encouragement and Mrs.

Mari Kurata for excellent technical assistance.

1114 M. Kato et al. / Biochemical and Biophysical Research Communications 301 (2003) 1109–1115

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