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

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,

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

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

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-

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].

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-

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

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

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