Báo cáo khoa học: Sustained activation of ERK1/2 by NGF induces microRNA-221 and 222 in PC12 cells pdf

Sustained activation of ERK1/2 by NGF induces
microRNA-221 and 222 in PC12 cells
Kazuya Terasawa
1
, Atsuhiko Ichimura
1
, Fumiaki Sato
2
, Kazuharu Shimizu
2
and Gozoh Tsujimoto
1
1 Department of Pharmcogenomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Japan
2 Department of Nanobio Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Japan
MicroRNAs (miRNAs) are evolutionally conserved
small non-coding RNAs that regulate gene expres-
sion at the post-transcriptional level and play impor-
tant roles in a wide variety of biological functions,
including cell differentiation, tumorigenesis, apoptosis
and metabolism [1–3]. Approximately 30% of human
protein-coding genes are predicted to be targets of
miRNA [4,5]. Biogenesis of miRNA and the mecha-
nism for regulation of target gene expression by
miRNA are relatively well characterized. miRNA
genes are initially transcribed mainly by RNA poly-
merase II as long primary transcripts (pri-miRNAs),
processed by the nuclear RNase Drosha to produce
precursor miRNAs (pre-miRNAs), and then exported
to the cytoplasm. Pre-miRNAs are further processed
into mature miRNAs by the cytoplasmic RNase
Dicer [6]. miRNAs recognize and bind to partially

complementary sites in the 3¢ UTRs of target
mRNAs, resulting in either translational repression
or target degradation [7]. To further understand the
functional significance of miRNA, the regulatory
mechanism of miRNA expression needs to be better
understood.
The mitogen-activated protein kinase (MAPK)
cascades play an essential role in transducing extra-
cellular signals to cytoplasmic and nuclear effectors,
and regulate a wide variety of cellular functions,
including cell proliferation, differentiation and stress
responses [8,9]. Extracellular signal-regulated kinases
1 and 2 (ERK1 ⁄ 2) were the first recognized members
of the MAPK family of proteins. These kinases are
primarily activated by mitogenic factors, differentiation
Keywords
Bim; ERK1 ⁄ 2; microRNA; NGF; PC12
Correspondence
G. Tsujimoto, 45-29 Yoshida-Shimo-Adachi-
cho, Sakyo-ku, Kyoto 606-8501, Japan
Fax: +81 75 753 4523
Tel: +81 75 753 4544
E-mail:
(Received 18 December 2008, revised 13
March 2009, accepted 6 April 2009)
doi:10.1111/j.1742-4658.2009.07041.x
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene
expression by inhibiting translation and ⁄ or inducing degradation of target
mRNAs, and they play important roles in a wide variety of biological func-
tions including cell differentiation, tumorigenesis, apoptosis and metabo-

lism. However, there is a paucity of information concerning the regulatory
mechanism of miRNA expression. Here we report identification of growth
factor-regulated miRNAs using the PC12 cell line, an established model of
neuronal growth and differentiation. We found that expression of miR-221
and miR-222 expression were induced by nerve growth factor (NGF) stim-
ulation in PC12 cells, and that this induction was dependent on sustained
activation of the extracellular signal-regulated kinase 1 and 2 (ERK1 ⁄ 2)
pathway. Using a target prediction program, we also identified a pro-apo-
totic factor, the BH3-only protein Bim, as a potential target of miR-
221 ⁄ 222. Overexpression of miR-221 or miR-222 suppressed the activity of
a luciferase reporter activity fused to the 3¢ UTR of Bim mRNA. Further-
more, overexpression of miR-221 ⁄ 222 decreased endogenous Bim mRNA
expression. These results reveal that the ERK signal regulates miR-221 ⁄ 222
expression, and that these miRNAs might contribute to NGF-dependent
cell survival in PC12 cells.
Abbreviations
EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase;
miRNA, microRNA; NGF, nerve growth factor.
FEBS Journal 276 (2009) 3269–3276 ª 2009 The Authors Journal compilation ª 2009 FEBS 3269
stimuli and cytokines such as nerve growth factor
(NGF) and epidermal growth factor (EGF) [10–12].
Because both ERK signaling and miRNA function are
involved in a variety of important biological responses,
the significance of ERK signaling in terms of regulating
miRNA expression is of great interest.
To study the role of the ERK1 ⁄ 2 pathway in the
regulation of miRNA expression, we first determined
the expression profile of miRNAs by using the
growth factor-induced neural differentiation process
of PC12 as a model. It is well known that NGF,

but not EGF, induces neural differentiation in PC12
cells, although both growth factors potently activate
ERK1 ⁄ 2 [13–16]. We identified miR221 and 222 as
differentially regulated miRNAs. Although the
expression of these miRNAs was found to be ERK-
dependent, the effect of NGF and EGF on their
expression was different; thus, only NGF, but not
EGF, can induce their expression. Further, our study
showed that the sustained activation of ERK1 ⁄ 2by
NGF, but not the transient activation of ERK,
could effectively induce miR221 and 222 in PC12
cells. Finally we identified the BH3-only protein
Bim, which is involved in NGF-dependent neuronal
survival [17–19], as a potential target of miR-221
and 222.
Results
NGF stimulation induces miR-221/222 in
PC12 cells
Treatment of PC12 cells with NGF for 48 h induced
readily detectable neurite outgrowth (Fig. 1A), so we
selected two points at 0 and 48 h after stimulation to
compare the expression of miRNAs. We used a TaqMan
miRNA assay, featuring reverse transcription using
stem-loop RT-PCR primers followed by real-time PCR
using TaqMan probes [20], to examine the expression of
156 rat miRNAs. We identified only two miRNAs,
miR-221 and 222, as drastically up-regulated 48 h after
NGF stimulation (data not shown). These miRNAs are
encoded in tandem on the chromosome X (Fig. 1B).
Next, we examined the time course of expression of

miR-221 and 222. Quantitative RT-PCR analysis
showed that these miRNAs had a very similar profile
(Fig. 1C). An alternative RT-PCR analysis, using a set
of primers that amplify a fragment located between
these two miRNAs, demonstrated transcriptional
induction of this region upon NGF stimulation (data
not shown). These data support the notion that miR-
221 and 222 derive from the same pri-miRNA [21].
Following NGF stimulation, the expression level of
Fig. 1. NGF induces expression of the
microRNAs miR-221 and 222 in PC12 cells.
(A) NGF-induced differentiation of PC12
cells. (B) Schematic representation of the
genomic structure of miR-221 and 222 and
their corresponding sequences. (C, F) PC12
cells were treated with 100 ngÆmL
)1
NGF or
30 n
M EGF for the indicated times. Cells
were harvested and total RNA was pre-
pared. The RNA was subjected to quantita-
tive RT-PCR to assess the levels of mature
miR-221 ⁄ 222. The data represent means
of the C
t
values (± SD, n = 3). In (C),
*P < 0.01 for miR-221 versus 0 h point, and

P < 0.01 for miR-222 versus 0 h point. In

(F), P < 0.01 for comparisons indicated by
asterisks (Tukey’s test). (D, E) Cell extracts
were subjected to immunoblotting with
a-phospho-ERK1 ⁄ 2 and a-ERK1 ⁄ 2 IgGs.
NGF induces miR-221 and 222 expression K. Terasawa et al.
3270 FEBS Journal 276 (2009) 3269–3276 ª 2009 The Authors Journal compilation ª 2009 FEBS
both miRNAs rapidly increased and reached a
maximum at 3–6 h, which was then sustained to the
last time point assayed at 48 h. Over this time course,
expression of an internal control U6 remained
unchanged (Fig. 1C). Based on the threshold cycle (C
t
)
changes, we estimate that the expression of miR-221
and 222 had increased by approximately 2
6
-fold. NGF
induced sustained activation of ERK1 ⁄ 2 over this time
course (Fig. 1D). We further examined whether EGF
stimulation also induces miR-221 ⁄ 222 in PC12 cells.
Our analysis confirmed that EGF transiently activated
ERK1 ⁄ 2, unlike NGF [13–16] (Fig. 1E). Because
NGF-mediated expression of miR-221 ⁄ 222 peaked
approximately 3 h after stimulation, we examined the
expression of miR-221⁄ 222 at this time point in all
further experiments. In contrast to NGF stimulation,
EGF did not induce any detectable up-regulation of
miR-221 and 222 at 3 h (Fig. 1F). Moreover, no such
stimulation was found even after monitoring for up to
48 h (data not shown).

Sustained activation of ERK1/2 is necessary for
induction of miR-221/222
We next studied whether the NGF-induced expression
of miR-221 ⁄ 222 depends on ERK1 ⁄ 2 activation. We
first examined the effect of a specific inhibitor (U0126)
for MAPK ⁄ ERK kinase (MEK) 1 ⁄ 2, which is a direct
activator of ERK1 ⁄ 2 [22]. As shown in Fig. 2A, pre-
treatment of U0126 potently inhibited NGF-induced
ERK1 ⁄ 2 activation. The same pre-treatment with
U0126 completely blocked induction of miR-221 and
222 (Fig. 2B). Moreover, we found that expression of
miR-221 ⁄ 222 dramatically increased when constitu-
tively active MEK1 (MEK1SDSE) [23] was transiently
expressed in PC12 cells (Fig. 2C,D).
Taken together, these results indicate that induction
of miR-221 ⁄ 222 depends on the activation of ERK1 ⁄ 2.
However, transient activation of ERK ⁄ 2 upon EGF
stimulation did not induce miR-221 ⁄ 222 expression.
This observation prompted us to hypothesize that
induction of these miRNAs requires sustained activa-
tion of ERK1 ⁄ 2. To verify this hypothesis, we blocked
NGF-induced sustained ERK1 ⁄ 2 activation by add-
ing U0126 10 min after NGF treatment (Fig. 3A). As
shown in Fig. 3B, addition of U0126 completely inhib-
ited the sustained activation of ERK1 ⁄ 2. In this situa-
tion, the induction of miR221 ⁄ 222 was also completely
suppressed (Fig. 3C). These results clearly demonstrate
that sustained activation of ERK1 ⁄ 2 is required for
induction of miR-221 and 222.
However, the apparent induction of these miRNA

molecules was only observed approximately 3 h
after NGF stimulation (Fig. 1C), which implies that
miR-221 and 222 are not the primary target genes
regulated by NGF. We reasoned that this induction
requires de novo protein synthesis. As shown in
Fig. 4A, treatment with cycloheximide completely
inhibited induction of these miRNAs, but had no
significant effect on ERK1 ⁄ 2 activation (Fig. 4B). We
also confirmed inhibition of protein synthesis by
monitoring expression of c-Fos protein, which is a
Fig. 2. Activation of ERK1 ⁄ 2 pathway is involved in miR-221 ⁄ 222
induction. (A, B) PC12 cells were pre-treated with 10 l
M U0126 for
10 min before treatment with 100 ngÆmL
)1
NGF for 3 h. Inhibition
of ERK1 ⁄ 2 activity by U0126 was confirmed by immunoblotting (A).
The expression levels of miR-221 ⁄ 222 were examined as described
in Fig. 1C (*P < 0.05, Tukey’s test) (B). (C, D) PC12 cells were
transfected either with empty vector or HA-tagged MEK1SDSE.
The expression levels of miR-221 ⁄ 222 were examined 24 h after
transfection as described in Fig. 1C (*P < 0.05 versus empty vec-
tor, Student’s t test) (C). Expression of HA-tagged MEK1SDSE was
confirmed by immunoblotting (D). DMSO, dimethylsulfoxide.
K. Terasawa et al. NGF induces miR-221 and 222 expression
FEBS Journal 276 (2009) 3269–3276 ª 2009 The Authors Journal compilation ª 2009 FEBS 3271
well-known NGF-induced immediate early gene [24].
Cycloheximide treatment completely blocked c-Fos
protein synthesis (Fig. 4C, compare with control).
These data indicate that de novo protein synthesis is

required for the induction of miR-221 ⁄ 222.
Pro-apototic Bim is a plausible target of
miR-221/222
We used TargetScan [4] to identify the likely target
genes of miR-221 ⁄ 222. Specifically, we focused on
the pro-apototic Bim gene because Bim has been
reported to be involved in NGF-dependent survival
of PC12 cells [19]. The predicted target site for these
miRNAs is conserved in human, mouse, rat, dog
and chicken (Fig. 5A). The rat Bim gene had no
annotated 3¢ UTR, and so in the TargetScan pro-
gram this is computationally determined based on
the human Bim 3¢ UTR sequence. Initially, we used
RACE to verify whether the predicted 3¢ UTR
region is transcribed. 3¢ RACE analysis detected
products containing the terminal portion of the pre-
dicted Bim 3¢ UTR. Moreover, RT-PCR analysis
showed that a fragment containing the predicted tar-
get site was amplified (data not shown). To examine
whether these miRNAs can target the Bim gene, we
generated a luciferase construct harboring a fragment
of the Bim 3¢ UTR containing the target sequence
(Fig. 5A). Co-expression of either miR-221 or miR-
222 significantly (P < 0.05) suppressed the reporter
activity compared to the control (Fig. 5C, wt). Muta-
tions introduced into the predicted binding site
almost eliminated this suppression. These results sug-
gest a direct interaction of these miRNAs with the
predicted target site of the Bim 3¢ UTR (Fig. 5B,C,
mt). Furthermore, we investigated the effect of expres-

sion of miR-221 ⁄ 222 on endogenous Bim mRNA
expression. Because the Bim gene has three alternative
splice variants [25], we designed a set of primers to
detect all three products. Overexpression of either
miR-221 or miR-222 resulted in significant (P < 0.05)
A

B
C
Fig. 3. Sustained activation of ERK1 ⁄ 2 is required for NGF-induced
miR-221 ⁄ 222 expression. (A) Schematic diagram of the experimen-
tal design. (B, C) PC12 cells were pre-treated with 10 l
M U0126
for 10 min after treatment with 100 ngÆmL
)1
NGF for 3 h. Inhibition
of sustained ERK1 ⁄ 2 activation by U0126 was confirmed by immu-
noblotting (B). The expression levels of miR-221 ⁄ 222 were exam-
ined as described in Fig. 1C (*P < 0.05, Tukey’s test) (C). DMSO,
dimethylsulfoxide.
Fig. 4. Protein synthesis is required for NGF-induced miR-221 ⁄ 222
expression. PC12 cells were pre-treated with 10 lgÆmL
)1
cyclohexi-
mide for 30 min before treatment with 100 ngÆmL
)1
NGF for 3 h.
The expression levels of miR-221 ⁄ 222 were examined as described
in Fig. 1C (*P < 0.05, Tukey’s test) (A). ERK1 ⁄ 2 activation in the
presence of cycloheximide (B) and inhibition of protein synthesis by

cycloheximide (C) were confirmed by immunoblotting. a-Tubulin
was used as a loading control. DMSO, dimethylsulfoxide.
NGF induces miR-221 and 222 expression K. Terasawa et al.
3272 FEBS Journal 276 (2009) 3269–3276 ª 2009 The Authors Journal compilation ª 2009 FEBS
down-regulation of Bim mRNA (Fig. 5D). To show that
this down-regulation is a specific effect for Bim mRNA,
we examined the mRNA level of an apoptosis-related
gene, Bax, because the 3¢ UTR of Bax mRNA has no
predicted target site for miR-221 ⁄ 222. We found that
overexpression of either miR-221 or miR-222 had no
significant effect on the Bax mRNA level (data not
shown). Taken together, our data suggest that miR-221
and 222 can target the Bim gene.
Discussion
The present study has demonstrated that, in PC12
cells, miR-221 and 222 are transcriptionaly induced
upon stimulation by NGF, and that this induction
requires sustained ERK1 ⁄ 2 activation and de novo pro-
tein synthesis. Presumably, sustained ERK1 ⁄ 2 activa-
tion is required for the induction of transcriptional
regulatory protein(s) that regulates miR-221 ⁄ 222
expression. Recently, miR-155 expression has been
shown to be regulated by two MAPK pathways, the
ERK1 ⁄ 2 and c-Jun N-terminal kinase pathways,
through AP-1 signaling [26]. AP-1 family proteins
are good candidates for mediating NGF-induced
miR-221 ⁄ 222 expression in PC12 cells.
A previous study showed that miR-221 and 222
are up-regulated in quiescent cells that have been
stimulated to proliferate by serum stimulation [27].

ERK1 ⁄ 2 is known to play a critical role in growth
factor-stimulated cell-cycle progression from G
0
⁄ G
1
to S phase, and sustained activation is required for
this progression [28]. Our finding that sustained
ERK1 ⁄ 2 activation induces miR-221 ⁄ 222 expression
is entirely consistent with this observation. miR-221
Fig. 5. The Bim 3¢ UTR is a target of miR-221 ⁄ 222. (A) Schematic
representation of the reporter construct and conservation of the tar-
get site of miR-221 ⁄ 222 in the Bim 3¢ UTR in vertebrates. The
boxed region indicates the sites complementary to the seed
sequence of miR-221 ⁄ 222. (B) Predicted base pairing between
miR-221 and 222 and their target sites in the Bim 3¢ UTR. Underlin-
ing indicate the seed sequences. The calculated free energy of
hybridization of each miRNA with the target site is also indicated.
These data were prepared using RNAhybrid. Lower-case letters
indicate the sites introduced to the Bim 3¢ UTR by mutation. The
indicated bases were substituted with complementary nucleotides
in the mutation construct. (C) The indicated RNA oligonucleotides
(10 pmol per well) and reporter plasmid (200 ng per well) were
co-transfected with the internal control plasmid (20 ng per well) into
PC12 cells. After 24 h, the cells were harvested, and the lysates
were subjected to a luciferase assay. The results are the ratio of
firefly to renilla luciferase activity (mean ± SD, n = 3). The lucifer-
ase activity ratio for control RNA transfection (mock) for each repor-
ter was set at 1 (*P < 0.01 versus mock; Student’s t test). (D)
PC12 cells were transfected with the indicated RNA oligonucleo-
tides. After 24 h, the cells were harvested and total RNA was pre-

pared. The RNA was subjected to quantitative RT-PCR to assess
the levels of Bim mRNA. The Bim mRNA expression was normal-
ized against GAPDH mRNA expression (mean ± SD, n = 3). The
normalized Bim expression of control RNA transfection (mock) was
set at 1 (*P < 0.05 versus mock; Student’s t test).
K. Terasawa et al. NGF induces miR-221 and 222 expression
FEBS Journal 276 (2009) 3269–3276 ª 2009 The Authors Journal compilation ª 2009 FEBS 3273
and 222 have also been reported to be up-regulated
in some cancer cell lines and to function as onco-
genic miRNAs by targeting the cyclin-dependent
kinase inhibitor p27
Kip1
[29–32]. These studies
strongly suggest that miR-221 and 222 are involved
in the regulation of cell growth. In PC12 cells, NGF
stimulation of starved cells promotes cell survival
and differentiation, and inhibits cell-cycle progression
[16]. However, NGF-induced miR-221 and miR-222
expression in PC12 cells is probably not involved in
cell-cycle progression. These apparently contradictory
effects might be attributed to cell type-dependent
differences.
We could not observe any detectable effect of
NGF-induced neurite outgrowth when miR-221
and ⁄ or 222 were overexpressed in PC12 cells (data
not shown). However, we show that the pro-apototic
protein Bim is a plausible target of miR-221⁄ 222.
Bim is known to be regulated at both the transcrip-
tional and post-transcriptional level [33]. Here, we
have confirmed that Bim is regulated at the transla-

tional level. In PC12 cells, the Bim gene is induced
upon withdrawal of serum. When NGF stimulation
occurs, activation of ERK1 ⁄ 2 causes phosphorylation
of Bim proteins and inhibits their function, resulting
in cell survival. This translational regulation of Bim
ensures the down-regulation of Bim function and
hence cell survival. These data indicate that NGF-
induced miR-221 ⁄ 222 expression is involved in NGF-
dependent cell survival. Interestingly, recent reports
have shown that Bim is regulated by miR-32 and
the miR-17–92 cluster of miRNAs, which are
known to be up-regulated in several cancers [34–36].
Up-regulation of these miRNAs, which results in
down-regulation of pro-apoptotic Bim mRNA, exerts
an anti-apoptotic effect in some cancers [35,36]. The
miR-17–92 cluster has also been shown to be
involved in B-cell development and to target Bim
mRNA [37,38]. In PC12 cells, the expression level of
these miRNAs is moderate and remains unchanged
upon NGF stimulation (data not shown). Thus,
induced miR-221 and 222 might work cooperatively
with these miRNAs.
miR-221 and 222 are highly conserved in verte-
brates and have the same seed sequence. Moreover,
they are encoded in tandem on the same chromosome
in human, mouse and rat, indicating that they have
important roles in biological processes. In this study,
we found that miR-221 ⁄ 222 expression is regulated
by the ERK1 ⁄ 2 pathway in PC12 cells. This regula-
tion might also function in different biological pro-

cesses. Our findings provide new insights into the
MAPK signaling pathway.
Experimental procedures
Cell culture and transfection
PC12 cells were maintained in Dulbecco’s modified Eagle’s
medium plus 10% fetal bovine serum, 5% donor horse
serum and antibiotics at 37 °Cin5%CO
2
. The cells were
seeded onto poly-l-lysine-coated 60 mm dishes (AGC
Techno Glass Co. Ltd, Chiba, Japan) and incubated in a
low concentration of serum (1% horse serum) for 24 h
prior to treatment with 100 ngÆmL
)1
NGF (Alomone Labs
Ltd, Jerusalem, Israel) or 30 nm EGF (PeproTech EC Ltd,
London, UK). Transfections were performed according to
the manufacturer’s instructions using LipofectAMINE 2000
(Invitrogen, Carlsbad, CA, USA). The miRNA precursors
miR-221 and 222 and control RNA were purchased from
Ambion (Austin, TX, USA).
RNA isolation and RT-PCR analysis
Total RNA was isolated using ISOGEN reagent (Nippon
Gene Co. Ltd, Tokyo, Japan). miRNA expression was mea-
sured using TaqMan MicroRNA Assays (Applied Biosys-
tems, Lincoln, CA, USA) according to the manufacturer’s
protocol, except that all reactions were carried out at half
scale. The rat miRNAs assayed in this study are listed in the
microrna assay index file version 1 (Applied Biosystems).
U6 snRNA was used as an internal control. For detection of

Bim mRNA, reverse transcription was performed using a
QuantiTect reverse transcription kit (Qiagen, Hilden, Ger-
many). Prepared cDNAs were then subjected to quantitative
PCR analysis using Power SYBR Green PCR Master Mix
(Applied Biosystems). The primers for the PCR analysis were
5¢-CTTCCATAAGGCAGTCTCAG-3¢ and 5¢-CGGAAGA
TGAATCGTAACAG-3¢ for Bim, and 5¢-TTGCTGACAA
TCTTGAGGGAG-3¢ and 5¢-GAGTATGTCGTGGAGTC
TACTG-3¢ for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). The data were obtained and analyzed using an
ABI 7300 real-time PCR system (Applied Biosystems).
Plasmid construction
The plasmid pcDNA3-HA encoding Xenopus MEK1SDSE
was supplied by E. Nishida (Graduate School of Biostudies,
Kyoto University, Japan). A partial fragment of the 3¢
UTR of rat Bim was amplified by PCR using the primers
5¢-CCTGCCTCTTGAGGTACTGC-3¢ and 5¢-AGCTAGT
CGCAAGTTTTA-3¢ following reverse transcription from
total RNA isolated from PC12 cells, and cloned into the
pCR-Blunt vector (Invitrogen). Mutagenesis of the Bim 3¢
UTR was performed using a QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA, USA). An
EcoRI site was introduced into the XbaI site of the lucifer-
ase reporter vector pGL4.23 (Promega, Madison, WI,
USA) by ligation with the oligonucleotides 5¢-CTAGACT
NGF induces miR-221 and 222 expression K. Terasawa et al.
3274 FEBS Journal 276 (2009) 3269–3276 ª 2009 The Authors Journal compilation ª 2009 FEBS
GAATTC-3¢ and 5¢-CTAGGAATTCAGT-3¢, yielding the
pGL4.23EcoRI vector. EcoRI fragments of the wild-type
(wt) and mutated (mt) Bim 3¢ UTR forms of the pCR-

Blunt vector were ligated into the EcoRI site of the
pGL4.23EcoRI vector. The identity of all constructs was
confirmed by DNA sequencing.
Immunoblotting
Cells were harvested by scraping from culture dishes in hot
1· SDS sample buffer, and the lysates were separated by
SDS–PAGE and analyzed by immunoblotting. Anti-HA
(3F10) rat monoclonal IgG was purchased from Roche
(Basel, Switzerland). Anti-p44 ⁄ 42 MAP kinase, anti-phos-
pho-p44 ⁄ 42 MAP kinase IgGs (numbers 9101 and 9102,
respectively) and anti-c-Fos IgG (number 4384) were
obtained from Cell Signaling (Danvers, MA, USA). Anti-a-
Tubulin (B-5-1-2) mouse monoclonal IgG was purchased
from Sigma (St Louis, MO, USA). Peroxidase-linked sec-
ondary antibodies were purchased from GE Healthcare
(Chalfont St Giles, UK). An LAS3000 CCD imaging sys-
tem (Fujifilm, Tokyo, Japan) was used for detection.
Reporter assay
Cells grown in 24-well plates (1.0 · 10
5
cells per well) were
harvested for assays 24 h after transfection. The luciferase
activity was measured using a dual-luciferase reporter assay
system (Promega) with a Lumat LB9507 luminometer
(Berthold Technologies, Bad Wildbad, Germany). As an
internal control, a renilla luciferase vector pGL4.70 (Pro-
mega) was used. The data represent means and standard
deviations of three independent experiments.
Statistical analysis
The data were analyzed using Student’s t test or ANOVA

followed by Tukey’s test as indicated.
Acknowledgements
This work was supported in part by a grant from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan (to K.T., F.S., K.S. and G.T.),
the New Energy and Industrial Technology Develop-
ment Organization (to K.T. and G.T.), and the Uehara
Memorial Foundation (to G.T.).
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