p38 Mitogen-activated Protein Kinase Regulation of JB6 Cl41 Cell Transformation Promoted by Epidermal Growth Factor*
Zhiwei He,
Yong-Yeon Cho,
Guangming Liu,
Wei-Ya Ma,
Ann M. Bode and
Zigang Dong
From the
Hormel Institute, University of Minnesota, Austin, Minnesota 55912
Received for publication, April 14, 2003
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ABSTRACT
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The relationship between cell transformation and p38 MAP kinase, a major
mitogen-activated protein (MAP) kinase pathway converting signals of various
extracellular stimuli into expression of specific target genes through
activation of transcription factors, still remains unclear. The aim of the
present study was to investigate the role of the p38 MAP kinase pathway in
epidermal growth factor (EGF)-induced cell transformation in JB6 cells. Our
data show that a dominant negative mutant of p38 MAP (DN-p38) kinase inhibits
EGF-promoted JB6 Cl41 cell transformation and that SB202190, an inhibitor of
p38 MAP kinase, also inhibits JB6 Cl41 cell transformation in a dose-dependent
manner. Moreover, our results show that DN-p38 MAP kinase inhibits the
phosphorylation of EGF-stimulated activating transcription factor-2 (ATF-2)
and signal transducer and activator of transcription 1 (STAT1). Additionally,
DN-p38 MAP kinase inhibits EGF-induced phosphorylation of c-Myc
(Thr58/Ser62). Gel shift assays indicate that DN-p38 MAP
kinase inhibits EGF-induced activator protein-1 (AP-1) DNA binding in a
dose-dependent manner. These results show that p38 MAP kinase plays a key role
in the regulation of EGF-induced cell transformation in JB6 cells through
regulation of phosphorylation of p38 MAP kinase and activation of its target
genes in phosphorylation, c-Myc cell transformation-related genes, and AP-1
binding ability.
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INTRODUCTION
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The mitogen-activated protein kinases
(MAPKs),1 including
p38 kinase, c-Jun NH2-terminal kinases (JNKs), extracellular
signal-regulated kinases (ERKs), and signal transduction cascades, are vital
mediators of many cellular functions such as growth, development,
proliferation, differentiation, malignant transformation, inflammation, and
apoptosis
(13).
p38 MAP kinase is activated by cellular stresses including inflammatory
cytokines, ultraviolet light, and growth factors, and the activated p38 MAP
kinase has been shown to phosphorylate several transcription factors including
ATF-2, STAT1, the Max/Myc complex, and myocyte enhancer factor 2 (MEF2)
(1,
46).
On the other hand, ERKs are predominantly activated by mitogenic stimuli,
including mitogens and growth factors, and activated ERKs are involved in cell
differentiation and development
(2,
3). Although one major function
of the p38 kinase and JNKs pathways is regulation of inflammation and
apoptosis, in many cases the biological consequences of p38 kinase and JNK
activation overlap with those of ERKs in mediation of cell growth and
differentiation (2,
3,
6,
7).
Previously, we studied ERKs regulation of epidermal growth factor
(EGF)-induced cell transformation in promotion sensitive (P+)
derivatives of the mouse epidermal JB6 cell line
(8). Results showed that
inhibition of ERKs appeared to be an important contributor to the tumor
promotion-resistant phenotype in JB6 cells. A recent study showed that the p38
kinase and JNKs pathways cooperate to transactivate the vitamin D receptor
through the c-Jun/activator protein-1 (AP-1)
(9). This result indicates that
a connection exists between p38 MAP kinase and AP-1. But AP-1 activation is
involved in JB6 cell transformation promoted by EGF and the phorbol ester
12-O-tetradecanoyl-phorbol-13-acetate (TPA)
(10,
11). AP-1 is a dimeric complex
consisting of proteins encoded by the jun and fos gene
families, and the AP-1 binding region in these genes is referred to as the TPA
response element (TRE). AP-1 induces the transcription of genes that are
related to cell proliferation, metastasis, and metabolism
(1214).
Increased AP-1 activity is associated with malignant transformation, and
repression of AP-1 activity has been shown to lead to suppression of cell
transformation and tumor promotion
(15).
Activation of MAP kinase cascades is known to play a considerable role in
malignant transformation (3,
8). However, whether p38 MAP
kinase is involved in the regulation of cell transformation is not known. A
recent study showed that only p38 MAP kinase, a MAP kinase usually associated
with stress responses, growth arrest, and apoptosis, was consistently
increased in human non-small cell lung cancer samples compared with the normal
tissues examined (16). More
interesting are the results showing that ERKs and JNKs, the MAP kinase
pathways traditionally associated with cell growth and perhaps malignant
transformation, were not activated in the human non-small cell lung tumor
samples. These results indicate that p38 MAP kinase is probably involved in
malignant cell transformation. The purpose of this research project was to
explore the mechanism of p38-mediated JB6 cell transformation using CMV-neo
plasmid-transfected JB6 cells and dominant negative mutant p38 (DN-p38)
plasmid-transfected JB6 cells following EGF stimulation. The results obtained
show that p38 MAP kinase plays a critical role in regulation of JB6 Cl41 cell
transformation promoted by EGF through the inhibition of the phosphorylation
of ATF-2, STAT1, and c-Myc and the repression of AP-1 binding ability.
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EXPERIMENTAL PROCEDURES
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MaterialsEagle's minimal essential medium (MEM),
L-glutamine, and LipofectAMINETM 2000 reagent were from
Invitrogen; EGF and SB202190 were from Calbiochem-Novabiochem. Fetal bovine
serum (FBS) was from Gemini Bio-Product (Calabasas, CA). Penicillin,
streptomycin, and gentamicin sulfate were from BioWhittaker, Inc.
(Walkersville, MD). Folin & Ciocalteu's phenol reagent was from Pierce,
and polyvinylidene difluoride (PVDF) membrane was from Millipore (Bedford,
MA). Antibodies to detect the phosphorylation of p38 kinase, ERKs, JNKs,
ATF-2, STAT1, and c-Myc and the non-phosphorylated levels of p38 kinase, ERKs,
JNKs, ATF-2, STAT1 and the c-Myc protein were from Upstate Biotechnology (Lake
Placid, NY). [
-32P]ATP was from Amersham Biosciences.
Cell Culture and Establishment of Stably Transfected JB6
Cells Using the CMV-neo plasmid and DN-p38 kinase plasmid, we
established stable transfectants according to the protocol from Invitrogen.
All of these cells were selected in media containing 400 µg/ml of G-418 for
2 weeks, and then the G418 concentration was decreased to 200 µg/ml and
maintained. Empty vector CMV-neo plasmid-transfected JB6 cells (Cl41 CMV-neo)
and DN-p38 plasmid-transfected JB6 cells (Cl41 DN-p38) were cultured as
adherent monolayers in MEM supplemented with 5% (v/v) heat-inactivated FBS and
glutamine (2 mM) at 37 °C in a humidified atmosphere of 5%
CO2.
ImmunoblottingCl41 CMV-neo and Cl41 DN-p38 cells were
cultured as described above, and immunoblotting was carried out as described
previously (17). In brief,
cells were cultured to 80% confluence and then starved in 0.1% FBS/MEM for 24
h at 37 °C in a 5% CO2 incubator. The media were changed to
fresh 0.1% FBS/MEM, the cells were incubated for another 24 h at 37
°C, and the cells were then treated with EGF for different time periods at
the concentrations indicated. Cells were then disrupted with lysis buffer
(62.5 mM Tris-HCl, pH 6.8, 50 mM dithiothreitol, 2%
(w/v) SDS, 10% (v/v) glycerol, and 0.1% bromphenol blue). The lysed samples
were transferred into fresh 1.5-ml tubes and sonicated for 510 s.
Samples containing an equal amount of protein were loaded into each lane of an
SDS-polyacrylamide gel for electrophoresis and subsequently transferred onto a
polyvinylidene difluoride membrane. Phosphorylation of ERKs, JNKs, ATF-2,
STAT1, and c-Myc were selectively detected by Western immunoblotting using a
chemiluminescent detection system and a phospho-specific antibody against
phosphorylation of p38 kinase (Thr180/Tyr182), ERKs
(Thr202/Tyr204), JNKs
(Thr183/Tyr185), ATF-2 (Thr71), STAT1
(Ser727), and c-Myc (Thr58/Ser62). Antibodies
against non-phosphorylated levels of p38 kinase, ERKs, JNKs, ATF-2, STAT1, and
c-Myc were used as internal controls to determine loading efficiency.
p38 MAP Kinase Activity AssayA p38 MAP kinase activity
assay was carried out following the instructions from Cell Signaling
Technology Inc. Cl41 CMV-neo cells and Cl41 DN-p38 cells were starved in 0.1%
FBS/MEM at 37 °C in a 5% CO2 incubator for 24 h and then
treated with EGF (10 ng/ml) for various times as indicated. Cells were
harvested with lysis buffer (20 mM Tris, pH 7.5, 5 mM
-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM
Na3VO4, and 10 mM MgCl2), and
protein concentration was determined by the Modified-Lowry protein assay.
Total cell lysates were centrifuged at 10,000 rpm for 10 min at 4 °C. The
cell extracts were incubated overnight at 4 °C with 20 µlofan
immobilized dual phospho-specific p38 MAPK
(Thr180/Tyr182) monoclonal antibody. After extensive
washing, the kinase reaction was performed at 37 °C for 10 min in the
presence of 100 µM ATP and 2 µg of an ATF-2 fusion protein.
Phosphorylation of ATF-2 at Thr71 was measured by Western blot
using a specific antibody against phosphorylation of ATF-2
(Thr71).
AP-1 DNA Binding StudyNuclear protein extracts were
prepared from cells by a modification of the method of Ye et al.
(18). Briefly, Cl41 CMV-neo
cells and Cl41 DN-p38 cells were cultured in 10-cm dishes and starved in 0.1%
FBS/MEM at 37 °C in a 5% CO2 incubator. After 24 h of
starvation, the cells were exposed to different concentrations of EGF for
another 12 h. The cells were then harvested and disrupted in 500 µl of
lysis buffer A (50 mM KCl, 0.5% Nonidet P-40, 100 µM
dithiothreitol, 25 mM HEPES, pH 7.8, 10 µg/ml leupeptin, 25
µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). After
a 1-min centrifugation (16,000 x g at 4 °C), the pellets
containing the nuclei were washed once with 500 µl of Buffer B (Buffer A
without Nonidet P-40). The pellets were then resuspended in 100 µl of
extraction buffer (Buffer B, but with 500 mM KCl and 10% glycerol)
and strongly shaken at 4 °C for 30 min. After a 10-min centrifugation
(16,000 x g at 4 °C), the supernatant solutions were moved
into fresh tubes and stored at 70 °C until analysis. The DNA
binding reaction was incubated at room temperature for 30 min in a mixture
containing 5 µg of nuclear protein, 1 µg of poly(dI·dC), and
15,000 cpm of a
-32P-labeled double-stranded AP-1
oligonucleotide (5'-CGCTTGATGAGTCAGCCGGAA-3'). The samples were
separated on a 5% polyacrylamide gel, and the gels were analyzed using the
Storm 840 phosphorimaging system (Amersham Biosciences).
Anchorage-independent Transformation AssayThe role of p38
MAP kinase in EGF-promoted cell transformation was investigated in Cl41
CMV-neo cells and Cl41 DN-p38 cells. The effect of the inhibitor SB202190 on
EGF-promoted cell transformation was explored in JB6 Cl41 cells. In brief, 8
x 103/ml cells were exposed to EGF (510 ng/ml) with or
without SB202190 (0.10.5 µM) in 1 ml of 0.3% basal medium
Eagle (BME) agar containing 10% FBS. The cultures were maintained at 37 °C
in a 5% CO2 incubator for 10 days, and the cell colonies were
scored as described by Colburn et al.
(19). The effect of DN-p38 on
JB6 Cl41 cell transformation is presented as colony number per 10,000 seeded
Cl41 CMV-neo cells or Cl41 DN-p38 cells in soft agar, and the effect of
SB202190 on JB6 cell transformation is also presented as the number of
colonies per 10,000 cells.
Amplification, Mutagenesis, and Expression Vector Construction of the
STAT1To further explore the role of transcriptional factor STAT1,
one of the targets of p38 MAP kinase in JB6 Cl41 cell transformation, we
gained stable expression of the point mutation at S727A (STAT1-S727A) and
expression of only plasmid pcDNA 3.1. The JB6 Cl41 cell lines used included
JB6 Cl41-STAT1-mt (S727A) and JB6 Cl41-pcDNA3.1 cells. All of these cells were
employed to detect cell transformation ability promoted by EGF. In brief, the
cDNA fragment of STAT1, including a 2253-base pair open reading frame (ORF),
was reverse-transcribed with an oligo(dT) primer and SuperScript II RNase
H reverse transcriptase (Invitrogen), amplified by PCR with
primers 5'-GCA GGA TCC TGT CTC AGT GGT ACG AAC T-3'
(BamHI site underlined) and 5'-TCG ACG CGT TGC TCT
ATA CTG TGT TCA TC-3' (MluI site underlined), and then cloned
into the pACT vector (Promega, Madison, WI) via the BamHI and
MluI restriction sites. The STAT1 coding fragment digested with
BamHI/EcoRV from the pACT was cloned into the
BamHI/SmaI sites of the pUC19 vector. To introduce the point
mutation in the position of the Ser727 residue, a pair of sense and
antisense primers for STAT1 S727A, 5'-C CTG CTC CCC ATG GCT
CCT GAG GAG-3' and 5'-CTC CTC AGG AGC CAT GGG GAG CAG
G-3' (underlined nucleotides indicate the codon coding a mutated amino
acid) were synthesized (Sigma). Then, the nucleotide substitutions were
accomplished using the QuikChange site-directed mutagenesis kit (Stratagene).
To construct the mammalian expression vector, the PCR products of wild type
STAT1 and STAT1-S727A were amplified using primers 5'-CGG GAT
CCA TGT CTC AGT GGT ACG AAC TTC A-3' (BamHI site
underlined) and 5'-GGG ATA TCC TAT ACT GTG TTC ATC ATA
CTG-3' (EcoRV site underlined) and then introduced into same
restriction sites of pcDNA3.1-neo (Invitrogen). All of products, including
cloning, mutagenesis, or expression vectors, were confirmed by DNA sequence
analysis (Sigma).
Statistical AnalysisSignificant differences between groups
were determined by the Student's and Welch's t tests.
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RESULTS
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EGF Induces Phosphorylation of p38 Kinase and ERKs but Not
JNKsMAPKs play very important roles in the transformation of mouse
JB6 Cl41 cells stimulated by EGF or TPA
(8). Our results showed that
EGF induced phosphorylation of ERKs in both time- and dose-dependent manners
(Fig. 1, A and
G). We further investigated whether EGF also contributed
to phosphorylation of other members of MAPKs such as p38 kinase and JNKs. Our
results showed that EGF also induced phosphorylation of p38 kinase in both
time- and dose-dependent manners (Fig. 1,
C and I), but not JNKs
(Fig. 1, E and
K). These data correspond with other findings that show
that EGF mediates cell growth through both MEK/ERK and p38 kinase signal
transduction pathways (20,
21).

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FIG. 1. EGF induces phosphorylation of p38 kinase and ERKs but not JNKs. JB6
Cl41 cells were cultured in 5% FBS/MEM and starved in 0.1% FBS/MEM at 37
°C in a 5% CO2 atmosphere for 24 h. Cells were then incubated
in fresh 0.1% FBS/MEM for another 2 h before being treated with EGF (10 ng/ml)
for 560 min or EGF (520 ng/ml) for 30 min. Phosphorylation of
p38 kinase (C and I), ERKs (A and G), and
JNKs (E and K) were determined by Western blot analysis as
described under "Experimental Procedures" using specific
antibodies against phosphorylation of p38 kinase
(Thr180/Tyr182), ERKs and JNKs. Total protein levels of
p38 kinase (D and J), ERKs (B and H), and
JNKs (F and L) were determined as described under
"Experimental Procedures" using corresponding antibodies against
non-phosphorylated proteins.
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The p38 Kinase Inhibitor SB202190 Inhibits the EGF-induced
Phosphorylation of p38 Kinase but Not ERKsTo explore the role of
p38 kinase in the EGF-stimulated signal transduction pathway, SB202190, a p38
kinase inhibitor
(2225),
was used to pre-treat JB6 cells at different doses for 1 h. Our results showed
that SB202190 inhibited the EGF-induced phosphorylation of p38 kinase in a
dose-dependent manner (Fig.
2B) but had no effect on EGF-induced ERK phosphorylation
(Fig. 2A). These
results indicate that SB202190 can specifically inhibit EGF-induced p38 kinase
phosphorylation.

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FIG. 2. SB202190 inhibits phosphorylation of p38 kinase but not ERKs. JB6
cells were cultured and starved as described above. SB202190 (0.251.0
µM) was used to pretreat JB6 cells for 1 h, and then JB6 cells
were treated with EGF (10 ng/ml) for 30 min. Phosphorylation of ERKs
(A) and p38 kinase (B) were determined by Western blot
analysis as described under "Experimental Procedures" using
specific antibodies against phosphorylation of p38 kinase and ERKs. Total
protein levels of p38 kinase and ERKs were determined as described above.
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The p38 Kinase Inhibitor SB202190 Suppresses JB6 Cl41 Cell
Anchorage-independent Transformation Promoted by EGFTo confirm the
role of p38 MAP kinase in JB6 cell transformation, we explored EGF-promoted
cell transformation in JB6 Cl41 cells using the p38 kinase inhibitor SB202190.
JB6 cells were treated with EGF (10 ng/ml) alone or with SB202190
(0.10.5 µM) in a soft agar matrix at 37 °C in a 5%
CO2 incubator for 10 days. The colony number was determined as
described previously, and experiments were repeated three times. Compared with
JB6 Cl41 cells stimulated only by EGF, the colony numbers were greatly
decreased after EGF and SB202190 treatment, and this inhibition occurred in a
dose-dependent manner (Fig. 3,
AE). These data provide more evidence
that p38 MAP kinase contributes to JB6 cell transformation promoted by EGF,
and this study is the first to report that SB202190 inhibits EGF-induced JB6
Cl41 cell transformation.

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FIG. 3. SB202190 inhibits JB6 Cl41 anchorage-independent transformation promoted
by EGF. JB6 Cl41 anchorage-independent transformation promoted by EGF was
performed as described previously. Final concentrations of 0.1 or 0.5
µM SB202190 were added into soft agar, and the colonies were
counted automatically after 10 days of incubation at 37 °C in a 5%
CO2 atmosphere. Almost no colony formation was observed in JB6
cells without EGF stimulation (A), and many colonies were observed in
JB6 Cl41 cells treated only with EGF (B). SB202190 suppressed
EGF-stimulated JB6 Cl41 cell colony formation in a dose-dependent manner
(C and D). The average number of colonies was determined
from three separate experiments; ctrl, control (E).
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DN-p38 MAP Kinase Inhibits Phosphorylation of p38 Kinase but Not ERKs
or JNKsBecause SB202190 may affect other molecular targets besides
p38 kinase, we used stable transfectants of Cl41 CMV-neo cells and Cl41 DN-p38
cells to further study the biological activity of p38 kinase
(24,
25). Cells were treated with
EGF at various concentrations for different times, and Western blot analysis
was used to determine the level of phosphorylation of p38 kinase, ERKs, and
JNKs using specific antibodies. Our data showed that EGF induced
phosphorylation of p38 kinase and ERKs in a dose-
(Fig. 4, A and
C) and time-dependent
(Fig. 4, G and
I) manner but had no effect on phosphorylation of JNKs
(Fig. 4, E and
K). Total protein levels of these kinases did not change
(Fig. 4, B, D, F, H, J, and
L). These data also showed that DN-p38 inhibited the
EGF-induced phosphorylation of p38 kinase in a time- and dose-dependent manner
but had no effect on phosphorylation of ERKs or JNKs. Results indicate that
ERKs and p38 kinase, but not JNKs, are involved in EGF-stimulated cell signal
transduction.

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FIG. 4. DN-p38 MAP kinase inhibits phosphorylation of p38 kinase but not ERKs or
JNKs. Empty vector CMV-neo plasmidtransfected JB6 Cl41 cells (Cl41
CMV-neo) and dominant negative p38 plasmid-transfected JB6 Cl41 cells
(Cl41 DN-p38) were starved in 0.1% FBS/MEM at 37 °C in a 5%
CO2 atmosphere for 24 h. Cells were then incubated in fresh 0.1%
FBS/MEM for another 2 h before being treated with EGF (10 ng/ml) for
1560 min or EGF (520 ng/ml) for 30 min. Phosphorylation of p38
kinase (A and G), ERKs (C and I), and JNKs
(E and K) was determined by Western blot analysis as
described under "Experimental Procedures" using specific
antibodies against phosphorylation of p38 kinase
(Thr180/Tyr182), ERKs, and JNKs. Total protein levels of
p38 kinase (B and H), ERKs (D and J), and
JNKs (F and L) were determined as described under
"Experimental Procedures" using corresponding antibodies against
non-phosphorylated proteins.
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DN-p38 Inhibits Anchorage-independent Transformation Promoted by
EGFFrom the results described above, we hypothesized that p38
kinase could have a role in regulating JB6 cell transformation. Cell
transformation was assessed using our previously developed methods
(26,
27). Cl41 CMV-neo cells and
Cl41 DN-p38 cells were treated separately with EGF (510 ng/ml) in a
soft agar matrix at 37 °C in a 5% CO2 incubator for 10 days.
The number of colonies formed was counted automatically by computer, and the
same experiment was repeated three times. Our results showed that DN-p38
strongly inhibited the formation of EGF-induced colonies
(Fig. 5D) compared
with control cells (Fig.
5B). The inhibition was evident not only in colony number
(Fig. 5E) but also in
colony size (Fig. 5, B versus
D). However, untreated Cl41 CMV-neo cells and Cl41 DN-p38
cells showed no colony formation (Fig. 5,
A and C). These data indicate that p38 MAP
kinase is involved in JB6 cell transformation as a positive regulator rather
than an inhibitor.

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FIG. 5. DN-p38 MAP kinase inhibits EGF-induced JB6 Cl41 anchorage-independent
transformation. Cl41 CMV-neo cells and Cl41 DN-p38 cells were used to
study cell transformation as described under "Experimental
Procedures." Cells (8 x 103/ml) were incubated in soft
agar at 37 °C in a 5% CO2 atmosphere for 10 days. Colonies of
Cl41 CMV-neo cells (A and B) and Cl41 DN-p38 cells
(C and D) were counted automatically as described
previously. The average colony number was calculated from three separate
experiments; ctrl, control (E).
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DN-p38 Inhibits EGF-induced p38 MAP Kinase ActivityTo
further confirm that the Cl41 DN-p38 stable transfectant cells work as
expected, p38 MAPK activity toward its substrate, ATF-2, was assayed. Cells
were treated with EGF (10 ng/ml) for the indicated times, and the cell
extracts were incubated with an immobilized p38 kinase antibody. An ATF-2
fusion protein was used as a substrate of p38 MAP kinase, and phosphorylation
of ATF-2 at Thr71 was detected by Western blot. Our results showed
that EGF-induced phosphorylation of ATF-2 at Thr71 was decreased
distinctly in Cl41 DN-p38 cells compared with Cl41 CMV-neo cells
(Fig. 6). These data indicate
that p38 kinase has a role in EGF-induced phosphorylation of ATF-2.

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FIG. 6. DN-p38 MAP kinase inhibits p38 MAP kinase activity in vivo.
Cl41 CMV-neo cells and Cl41 DN-p38 cells were seeded in 100-mm dishes with 5%
FBS/MEM and incubated at 37 °C in a 5% CO2 atmosphere for 24 h.
Cells were starved as described previously. Cells were then incubated in fresh
0.1% FBS/MEM for another 2 h before being treated with EGF (10 ng/ml) for the
indicated time periods. Cells were added to lysis buffer as described under
"Experimental Procedures," and the same amount of extracted
proteins was immunoprecipitated with a monoclonal phospho-specific antibody
against p38 MAP kinase (Thr180/Tyr182). The resulting
immunoprecipitate was incubated with an ATF-2 fusion protein in the presence
of ATP and kinase buffer. Phosphorylation of ATF-2 at Thr71 was
measured by Western blotting using the phospho-ATF-2 (Thr71)
antibody (A), and total ATF-2 protein level was determined by Western
blot using an antibody against nonphosphorylated ATF-2 protein
(B).
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DN-p38 Inhibits EGF-induced Phosphorylation of STAT1 at
Ser727 in a Time- and Dose-dependent MannerSTAT1 is
downstream of p38 MAP kinase and is a transcription factor that mediates
cytokine and growth factor-induced signals that culminate in various
biological responses, including proliferation and differentiation
(28). Cl41 DN-p38 cells and
Cl41 CMV-neo cells were treated separately for various times and at various
doses with EGF. We found that EGF (10 ng/ml) strongly induced phosphorylation
of STAT1 at Ser727 in Cl41 CMV-neo cells, but the phosphorylation
of STAT1 at Ser727 in Cl41 DN-p38 cells was relatively weak.
However, phosphorylation of STAT1 gradually decreased within 120 min in both
cell lines (Fig. 7A).
Non-phosphorylated levels of STAT1 were unchanged throughout the time course
and dose course (Fig. 7, B and
D). EGF-induced phosphorylation of STAT1 at
Ser727 gradually increased with increasing EGF concentrations
(Fig. 7C) with no
change in non-phosphorylated levels of STAT1
(Fig. 7D).

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FIG. 7. DN-p38 MAP kinase inhibits phosphorylation of STAT1
(Ser727). Culture and starvation of Cl CMV-neo cells and Cl41
DN-p38 cells were performed as described above. In the time course study
(A and B), cells were treated with EGF (10 ng/ml) for the
indicated time periods and then harvested with lysis buffer, and the protein
concentration of each sample was determined. Equal amounts of protein were
separated by 8% SDS-PAGE gel and analyzed by Western blotting. Phosphorylation
of STAT1 at Ser727 was detected using a specific phospho-STAT1
(Ser727) antibody (A). Total STAT1 protein levels were
detected by a non-phospho-STAT1 antibody (B). In the dose course
study (C and D), Cl41 CMV-neo cells, and Cl41 DN-p38 cells
were treated with EGF at the indicated concentration for 15 min.
Phosphorylation of STAT1 at Ser727 (C) and total STAT1
protein levels (D) were detected as described above.
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DN-p38 Inhibits Phosphorylation of c-Myc at
Thr58/Ser62To understand the mechanism of
p38 MAP kinase in the regulation of JB6 cell transformation, we investigated
the phosphorylation of c-Myc by Western blot using a specific antibody against
phosphorylation of c-Myc at Thr58/Ser62. The c-Myc
oncoprotein is associated with cell growth, development, and malignant
transformation in human tumors and cell lines
(29,
30). The active c-Myc also
contributes to cell transforming potencies in rat embryo cells
(31). In this study, our
results showed that phosphorylation of c-Myc at
Thr58/Ser62 was increased after CMV-neo JB6 cells were
treated from 14 h with EGF (10 ng/ml), but phosphorylation of c-Myc at
Thr58/Ser62 in DN-p38-JB6 cells was relatively less
within the same time course (Fig.
8A). EGF had no effect on total c-Myc protein levels in
Cl41 CMV-neo cells or Cl41 DN-p38 cells
(Fig. 8, B and
D). Moreover, we studied the effects of various doses of
EGF on phosphorylation of c-Myc at Thr58/Ser62. Results
showed that phosphorylation of c-Myc at Thr58/Ser62
increased in Cl41 CMV-neo cells but was only slightly changed in Cl41 DN-p38
cells after cells were treated with EGF (520 ng/ml)
(Fig. 8C). These data
indicate that phosphorylation of c-Myc at Thr58/Ser62 is
induced by EGF in a time- and dose-dependent manner in Cl41 CMV-neo cells and
that DN-p38 inhibits the phosphorylation of c-Myc at
Thr58/Ser62.

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FIG. 8. DN-p38 MAP kinase inhibits phosphorylation of c-Myc at
Thr58/Ser62. Culture and starvation of Cl41 CMV-neo
cells and Cl41 DN-p38 cells were performed as described above. In the time
course study (A and B), cells were treated with EGF (10
ng/ml) for the indicated time periods and then harvested with lysis buffer,
and the protein concentration of each sample protein was determined. Western
blotting was employed to analyze the phosphorylation of c-Myc at serine 63
using a specific phospho-c-Myc (Thr58/Ser62) antibody
(A). Total c-Myc protein levels were detected by a non-phospho-c-Myc
antibody (B). In the dose course study (C and D),
Neo-JB6 Cl41 and DN-p38-JB6 Cl41 cells were treated with EGF at the indicated
concentration for 4 h. Phosphorylation of c-Myc at
Thr58/Ser62 (C) and total c-Myc protein levels
(D) were detected as described above.
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A Mutant of STAT1 (S727A) Suppresses JB6 Cl41 Cell
Anchorage-independent Transformation Promoted by EGFStable
transfectant JB6 Cl41-STAT1 (S727A) cells expressing of STAT1 (S727A) and JB6
Cl41-pcDNA3.1 cells expressing plasmid pcDNA3.1 cells were employed to detect
the cell transformation ability promoted by EGF. Cells were treated with EGF
(10 ng/ml) in a soft agar matrix at 37 °C in a 5% CO2 incubator
for 10 days. The colony number was determined as described previously
(8). The colony numbers were
greatly decreased in JB6 Cl41-STAT1 (S727A) cells promoted by EGF, but there
were many more colonies in JB6 Cl41 cells and JB6 Cl41-pcDNA 3.1 stimulated by
EGF under the same condition (Fig. 9,
AF). These data indicate that STAT1, one of the
targets of p38 MAP kinase, also contributes to JB6 Cl41 cell transformation
promoted by EGF.

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FIG. 9. Mutant of STAT1 (S727A) suppresses JB6 Cl41 cell anchorage-independent
transformation promoted by EGF. Cellular anchorage-independent
transformation promoted by EGF was performed as described previously. Final
concentrations of EGF (10 ng/ml) were added into soft agar, and the colonies
were counted automatically after 10 days of incubation at 37 °C in a 5%
CO2 atmosphere. Almost no colony formation was observed in JB6
cells and JB6 Cl41-pcDNA3.1 cells without EGF stimulation (A and
B), and many colonies were observed in JB6 Cl41 and JB6 Cl41-pcDNA3.1
cells treated only with EGF (C, D, and E). But very few
colonies were counted in JB6 Cl41-pcDNA 3.1-STAT1 (S727A) cells (E).
The average number of colonies was determined from three separate experiments;
ctrl, control (F).
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DN-p38 Inhibits EGF-induced AP-1 Binding ActivityAP-1
induces gene transcription by binding to the TPA response element site in the
promoter region of its target gene and is required for cell proliferation,
differentiation, and malignant transformation
(32,
33). Double-stranded AP-1
oligonucleotides were labeled with
-32P, and electrophoretic
mobility-shift assays were used to measured AP-1 DNA binding activity. Our
results showed that AP-1 binding was decreased in Cl41 DN-p38 cells
(Fig. 10, A and
B, lanes 3, 4, and 5) compared with
that in Cl41 CMV-neo cells (Fig. 10,
A and B, lanes 7, 8, and 9)
after cells were treated with EGF (520 ng/ml) for 12 h. However no
significant change was observed between DN-p38-JB6 cells and Neo-JB6 cells
without EGF stimulation (Fig. 10,
A and B, lanes 2 and 6). To
confirm that the electrophoretic mobility-shift band was specific for AP-1
binding, a 10-fold amount of unlabeled AP-1 oligonucleotide was added, and
results showed that the AP-1 binding band was completely removed
(Fig. 10, A and
B, lane 1). These data indicate that DN-p38
inhibits EGF-induced AP-1 binding ability.

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FIG. 10. DN-p38 MAP kinase inhibits EGF-induced AP-1 DNA binding. Cl41
CMV-neo cells and Cl41 DN-p38 cells were treated with EGF, and nuclear
proteins were extracted as described under "Experimental
Procedures." A, Cl41 DN-p38 cells effectively blocked
EGF-induced AP-1 DNA binding (lanes 35) compared with
EGF-induced AP-1 DNA binding in Cl41 CMV-neo cells (lanes 79).
B, quantitation analysis of panel A from three separate
electrophoretic mobility shift assays performed using nuclear extracts from
different cell preparations.
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DISCUSSION
|
---|
Previous studies show that ERKs are involved in the JB6 cell transformation
promoted by EGF or TPA (8), but
an understanding of the role of p38 MAP kinase in cell transformation still
remains unclear. In this study, we found that p38 MAP kinase plays a critical
role in JB6 Cl41 cell transformation promoted by EGF. Both DN-p38 and the p38
MAP kinase inhibitor SB202190 suppressed JB6 Cl41 cell transformation promoted
by EGF. DN-p38 inhibited EGF-stimulated phosphorylation of ATF-2, STAT1, and
c-Myc, and gel shift assay results showed that DN-p38 inhibits AP-1 binding
ability. These results indicate that p38 MAP kinase is involved in regulation
of JB6 Cl41 cell transformation promoted by EGF through the inhibition of
phosphorylation of its downstream factors, including ATF-2, STAT1, c-Myc, and
also AP-1 DNA binding.
Many studies indicate that EGF induces the phosphorylation of ERKs and p38
MAP kinase (21,
3436).
The ERK pathway is associated with activation of the EGF receptor and has been
shown to play a major role in promoting several tumor phenotypes. However, the
JNKs pathway has not been shown to be activated through the EGF receptor but
is instead more uniformly stimulated by cellular stresses and cytokines
(21). Our present data also
show that EGF only induces the phosphorylation of ERKs and p38 MAP kinase but
not JNKs (Fig. 1). SB202190 and
DN-p38 only inhibit EGF-induced phosphorylation of p38 MAP kinase but not ERKs
(Figs. 2 and
4). These results indicate that
ERKs and p38 MAP kinase, but not JNKs, independently participate in
EGF-stimulated cell signal transduction.
The p38 MAP kinase is usually associated with stress responses, growth
arrest, and apoptosis (1,
2,
4). But a recent study
(16) showed that it was
activated in human lung cancer samples, suggesting an additional role for this
pathway in malignant cell growth or transformation. The present study is the
first to report that p38 MAP kinase is involved in JB6 Cl41 cell
transformation promoted by EGF. SB202190, a p38 MAP kinase inhibitor,
distinctly inhibited colony formation in EGF-promoted JB6 Cl41 cell
transformation (Fig. 3). To
eliminate the effects of SB202190 on other molecular targets, we successfully
constructed a stable transfectant of JB6 Cl41 cells expressing a dominant
negative mutant of p38 kinase and DN-p38 according to the method described
previously (37,
38). Our results showed that
the number of colonies in EGF-treated Cl41 DN-p38 cells was significantly
decreased compared with the number of colonies in Cl41 CMV-neo cells
(Fig. 5). These data indicate
that p38 MAP kinase plays a critical role in JB6 Cl41 cell transformation
promoted by EGF.
To explore the mechanism of p38 kinase in the regulation of JB6 cell
transformation, we first determined whether p38 MAP kinase activity was
inhibited in Cl41 DN-p38 cells. Our results show that DN-p38 distinctly
inhibits EGF-induced phosphorylation of ATF-2 at Thr71
(Fig. 6). Thr71 is a
major ATF-2 phosphorylation site required for transcriptional activity, and a
high level of phosphorylated ATF-2 protein correlates with malignant
phenotypes in the multistage mouse skin carcinogenesis model
(39). One recent study showed
that EGF activates transcription factor ATF-2 through a two-step mechanism.
The Raf-MEK-ERK pathway induces phosphorylation of ATF-2 (Thr71),
whereas subsequent ATF-2 Thr69 phosphorylation requires the
Ral-RalGDS-Src-p38 MAP kinase pathway
(40). Moreover, cooperation
between ERKs and p38 kinase was found to be essential for ATF-2 activation by
EGF (41).
Some of the STAT family members have a role in the regulation of cellular
transformation (42). A recent
study showed that STAT3 activation is required for interleukin-6-induced
transformation in tumor promotion-sensitive mouse skin epithelial cells
(43). Another report showed
that transformation of normal fibroblasts cells with E1A +
Ha-Ras oncogenes causes a constitutive activation of STAT1 and STAT3
transcription factors (44). In
this study, we found that EGF-induced phosphorylation of STAT1
(Ser727) also decreased in Cl41 DN-p38 cells but increased in Cl41
CMV-neo cells after the cells were separately treated with EGF (10 ng/ml)
(Fig. 7). We successfully
employed directed point mutation technology to obtain a mutant of STAT1 at
Ser727 (Ser
Ala). Stable transfectant of JB6 Cl41-STAT1
(S727A) cells expressing mutant of STAT1 at Ser727
(Ser727 (Ser
Ala) displayed its ability to inhibit cell
transformation in JB6 Cl41 cells promoted by EGF
(Fig. 9). These results
indicate that EGF-induced phosphorylation of STAT1 at Ser727 is at
least partially dependent on p38 MAP kinase and that STAT1 is involved in JB6
cell transformation promoted by EGF.
Studies show that the c-Myc oncoprotein is associated with cancer cell
proliferation and transformation
(45,
46). In this study, we also
found that DN-p38 inhibited EGF-induced phosphorylation of c-Myc, indicating
that p38 MAP kinase mediates EGF-induced c-Myc phosphorylation
(Fig. 8). Thus, p38 MAP kinase
may be involved in cell transformation through phosphorylation of c-Myc. On
the other hand, AP-1, a heterodimer commonly comprised of the basic leucine
zipper proteins Fos and Jun, plays a very important role in the induction of
neoplastic transformation and the multiple genes involved in cell
proliferation, differentiation, and inflammation
(47,
48). Blocking the tumor
promoter-induced activation of AP-1 was shown to inhibit neoplastic
transformation in JB6 mouse epidermal cells
(49). One recent study
(47) showed that p38 MAPK and
ERK inhibition with SB203580 and PD98059, respectively, significantly
inhibited silica-induced AP-1 activation. These findings demonstrate that AP-1
activation may be mediated through the p38 MAPK and ERKs pathways
(47). In the present research,
EGF-induced AP-1 binding ability decreased in Cl41 DN-p38 cells compared with
Cl41 CMV-neo cells (Fig. 10).
These data provide further evidence that EGF-induced AP-1 binding ability is
mediated through p38 MAP kinase and that DN-p38 MAP kinase represses
EGF-induced JB6 Cl41 cell transformation through inhibition of AP-1 DNA
binding ability.
In summary, we report for the first time that p38 MAP kinase mediates
EGF-promoted JB6 Cl41 cell transformation through regulation of the
phosphorylation of its downstream factors, such as STAT1, ATF-2, and c-Myc,
and also AP-1 DNA binding ability (Fig.
11). This study illustrates that p38 MAP kinase is another new
pathway involved in JB6 Cl41 cell transformation promoted by EGF.

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FIG. 11. Proposed signal transduction pathways of EGF-promoted JB6 Cl41 cell
transformation. We reported previously that ERKs are necessary factors in
EGF-promoted JB6 Cl41 cell transformation. In the present study, our data show
that DN-p38 MAP kinase effectively blocks EGF-promoted JB6 Cl41 cell
transformation through inhibition of the phosphorylation of transcription
factors, ATF-2 and STAT1, oncogenes, c-Myc, and repression of AP-1 DNA
binding. Both p38 kinase and ERKs are involved in the EGF-promoted JB6 Cl41
cell transformation.
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FOOTNOTES
|
---|
* This work was supported in part by The Hormel Foundation and National
Institutes of Health Grants CA81064, CA88961, and CA77646. The University of
Minnesota is an equal opportunity educator and employer. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed: Hormel Institute, University of
Minnesota, 801 16th Avenue NE, Austin, MN 55912. Tel.: 507-437-9600; Fax:
507-437-9606; E-mail:
zgdong{at}hi.umn.edu.
1 The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK,
extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; JNK, c-Jun
NH2-terminal kinase; ATF-2, activating transcription factor 2;
STAT1, signal transducer and activator of transcription 1; AP-1, activator
protein-1; EGF, epidermal growth factor; TPA,
12-O-tetradecanoyl-phorbol-13-acetate; DN-p38, dominant negative
mutant p38; MEM, Eagle's minimal essential medium; FBS, fetal bovine
serum. 
 |
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