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つくば生物ジャーナル Tsukuba Journal of Biology (2013) 12, 51
ERK activation in murine fibroblasts under various growth conditions
森島仁美（筑波大学 生物学類） 指導教員：林 純一（筑波大学 生命環境系）
Raf/MEK/ERK pathway is known to regulate numerous
Using the fibroblasts NIH3T3 and 3T3BXB-ER, ERK
cellular events such as cellular proliferation, senescence,
activation for the growth induction was analyzed. It was
differentiation, apoptosis, and transformation. It is known that
tested to see if the cellular outcomes could be controlled by
differences in duration and intensity of ERK activation signals
changing intensity of ERK activation, investigating
effect these decisions of different biological responses in
parameters which may stimulate accelerated cell growth by
fibroblasts (1). For intensity, it is known that strong prolonged
BXB-ER activation. Using Western blots and cell counting,
activation of ERK leads the cells to go in senescence (2). On the
differences of ERK signals between activation by serum and
other hand, weaker ERK signal leads cellular proliferation.
by BXB-ER were compared. As expected, the induction of
Even though it remains unclear how the activation of one
ERK seemed to be optimal in fibroblasts at various
signaling molecule ERK leads to specific cellular responses (3),
concentrations of serum, but BXB-ER activation by 100nM
accurate regulation of ERK signal could be the key to balance
tamoxifen induced robust ERK activation signal which led
these responses (1). By regulating the ERK activation signals,
cells to go in senescence. To change the cellular outcome,
the cells could have totally different cellular outcomes such as
activation levels of ERK were adjusted using different
senescence or cell growth. For further research of the pathway,
concentrations of tamoxifen/estrogen which acutely regulate
analysis of ERK activation for the growth induction of murine
BXB-ER activation. First, tamoxifen/estrogen concentrations
fibroblasts was done. The aim of the research was to see if the
were decreased to see the signal intensity and cell growth. By
cellular outcomes activated by ERK could be controlled by
lowering, it produced the similar growth rates as the control,
conditional Raf using different activation kinetics, and to adjust
but BXB-ER activation could not induce additional growth.
parameters to induce accelerated cell growth in 3T3BXB-ER
Second, MEK inhibitor, PD184352, was used to regulate the
intensity of ERK activation with various serum
concentrations. When ERK activation was inhibited by
PD184352 (0.2µM-4µM), the cells grew slower than the
control. Then, BXB-ER activation was induced by tamoxifen
Murine immortalized fibroblasts (NIH3T3) and engineered
100nM in addition to PD184352.Under the condition, the
cells (3T3BXB-ER) were used. 3T3BXBER cells are NIH3T3
additional BXB-ER activation could restore ERK
cells, expressing Raf-1 kinase domain-estrogen receptor fusion
phosphorylation levels, and also growth rates to the levels
proteins, which could be regulated by tamoxifen or estrogen (4).
they were before MEK inhibition, but not more. Therefore,
The cells were kept in a humidified incubator at 37°C with
the cells seemed to control the level of ERK activity very
5.0% carbon dioxide in the DMEM medium supplemented
tightly, to be optimal for growth. It is possible that additional
with 10% fetal bovine serum (SIGMA), 1% 200mM
research should have been completed with smaller intervals
L-glutamine (SIGMA), and 1% penicillin streptomycin.
for PD184352 concentrations. Although the parameter, which
induces accelerated cell growth, was not found with the
The cells were counted on the 4th to 5th day of the experiments. research, I showed the trends of how cells react with different Images were taken with Canon IXY 14.1 mega pixels with an
activation kinetics. For future research, I wish to find out
about the mechanism of how ERK activation gives the
specific cellular outcomes by the precise investigation of ERK
Samples were loaded to 12% acrylamide gels for sodium
dodecyl sulfate polyacrylamide gel electrophoresis. The
following antibodies were used.［Primary antibody: P-p44/42
MAPK (T202/Y204) (Cell Signaling) / Anti-MAP Kinase
(1) Wellbrock C, et al. Nat Rev Mol Cell Biol. 2004;5:875-886.
(ERK-1, 2) (SIGMA), Secondary antibody: antibody rabbit
(2) Martin, C, et al. PLoS ONE 2010;5(10):e13322.
(Stabilized Peroxidase Conjugated Goat Rabbit (Thermo)) ］
(3) Ebisuya, M, et al. J Cell Sci 2005;118:2997-3002.
Luminata Crescendo/Classico Western HRP Substrate
(4) Lovric, J., et al. J Biol Chem 1998;273:22848-22855.
(Milipore) was used for protein detection.
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