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INTRODUCTION |
Okadaic acid is a polyether compound of a C38 fatty
acid and is produced by certain dinoflagellates that concentrate in
marine sponges and shellfish. It is the major toxic component
responsible for diarrhetic shellfish poisoning (1, 2). Okadaic acid inhibits serine-threonine phosphatases 1, 2A (3), and 3 (4) by binding
to their catalytic subunit and leads to the accumulation of
hyperphosphorylated proteins (5, 6). Okadaic acid is also a potent
tumor promoter in mouse skin initiated with
7,12-dimethylbenz[a]anthracene (7).
Tumor promoters lead to alterations in gene expression modulating
biological processes, such as proliferation, differentiation, and cell
death, which are involved in expansion of "initiated" cells and
tumor development. Gene expression is regulated by transcription factors that bind to specific cis-elements in the promoter
region of genes, and these factors either induce or repress their transcription.
Several lines of evidence indicate the transcription factor complex
AP-1 1 as a critical
component in epidermal tumor development in mouse skin. In this system,
elevated AP-1 activity has been correlated with increased neoplastic
transformation. In mouse epidermal JB6 cells, AP-1 activity increases
with progression from a tumor promotion-resistant to a tumor
promotion-sensitive phenotype (8). Tumor promoters stimulate AP-1
expression and modulate AP-1-regulated gene expression in cultured
mouse keratinocytes and in mouse skin (9). More direct evidence for a
causative role of AP-1 activation in tumor promotion comes from the
finding that acquisition of a tumor promotion-resistant phenotype is
consistent with a loss of responsiveness to tumor promoter-induced AP-1
activation (10). Furthermore, down-regulation of AP-1 by "anti-tumor
promoters" such as aspirin and aspirin-like salicylates (11) or
retinoic acid (12) correlates with inhibition of tumor promoter-induced
transformation and tumor development. The same effects are observed
when AP-1 activity is blocked by the dominant-negative c-Jun mutant
TAM67 (13), whereas overexpression of c-Jun leads to increased
neoplastic transformation (14).
The transcription factor AP-1 (15) is a dimeric complex formed by Jun
or Jun and Fos proteins and binds to a promoter element that is
referred to as the
12-O-tetradecanoylphorbol-13-acetate-responsive element
(TRE). There are three different Jun proteins, c-Jun, JunB, and JunD,
and four different Fos proteins, c-Fos, FosB, Fra-1, and Fra-2. AP-1
activity is regulated at various levels, including transcriptional and
post-transcriptional mechanisms leading to increased AP-1 expression
and post-translational modifications, such as phosphorylation and
oxidation/reduction, altering DNA binding affinity and transactivation
potential. Since DNA binding affinity and transactivation potential are
different for the various AP-1 proteins, AP-1 activity is also
determined by its composition. AP-1 composition, on the other hand, is
dependent not only on the sequence of the actual TRE, but also on the
sequence of adjacent bases (16).
Previously, we presented experimental evidence that in the
papilloma-producing mouse keratinocyte cell line 308, the principal mechanism of okadaic acid-stimulated AP-1 DNA binding activity is
increased AP-1 expression (17). Furthermore, we were able to show that
this increased AP-1 expression is at least partly due to increased AP-1
transcription. This increased AP-1 transcription could be the result of
post-translational modifications of transcription factors regulating
AP-1 gene expression. Mitogen-activated protein kinases (MAPKs) have
been discussed as important mediators of signal transduction regulating
gene expression, and activation of these enzymes has been implicated in
altered phosphorylation of several transcription factors, including
Elk-1 (18), c-Jun (19), and ATF-2 (20). MAPKs are serine-threonine
kinases that are activated in response to extracellular stimuli by a
phosphorylation cascade involving multiple kinases. To date, there are
three distinct groups of MAPKs: ERK-1/2, JNKs/stress-activated protein
kinases, and p38 MAP kinases. ERK-1/2 is predominantly stimulated by
mitogens and hormones, inducing either proliferation and cell growth or differentiation (21). JNKs/stress-activated protein kinases and p38 MAP
kinases are considered part of stress response pathways mediating
inhibition of proliferation or cell death (22).
The purpose of this study was to test whether okadaic acid-induced AP-1
DNA binding activity correlated with increased AP-1 transactivation and
to determine the role of MAPKs in this process. Such studies might
advance our understanding of the complex process of tumor development
and could help to develop molecular and pharmacological tools to
manipulate neoplastic cell growth.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Treatment--
The papilloma-producing 308 mouse keratinocytes were kindly provided by Dr. Stuart H. Yuspa (23).
The cells were maintained in minimal essential medium (MEM)
supplemented with 7.5% fetal calf serum, 2.5% calf serum, and 100 units/ml penicillin/streptomycin (all purchased from Life Technologies,
Inc.) at 37 °C in a humidified atmosphere containing 7.5%
CO2. For experiments, cells were grown to 75-90%
confluency. Before treatment, cells were washed once with serum-free
MEM. Cells were treated with okadaic acid (sodium salt, LC
Laboratories) dissolved in dimethyl sulfoxide added to serum-free MEM
to a final concentration of 100 ng/ml; controls were treated with
equivalent amounts of Me2SO (0.01%, v/v). In the indicated
experiments, prior to the addition of okadaic acid/Me2SO, cells were pretreated for 1 h with 50 µM PD 98059 (2'-amino-3'-methoxyflavone, Alexis Corp.), a specific inhibitor of the
ERK-1/2 activators MEK-1/2, dissolved as 50 mM stock in
Me2SO; controls were treated with Me2SO (0.1%,
v/v).
Plasmids--
To study TRE-dependent
transactivation, a luciferase reporter construct driven by region
74
to +63 of the human collagenase gene containing a TRE was used. This
construct was kindly provided by P. Brown and L. M. Yang. To
determine the role of JunD and FosB in okadaic acid-mediated
TRE-dependent transactivation and to analyze the
involvement of ERK-1/2-mediated phosphorylation in this process, we
generated the fusion protein constructs pBindJunD and pBindFosB.
pBindJunD was generated by PCR using 30 ng of upstream sense primer
5'-CGAACGCGTCGGGGGAGGTGGGGATGGAAAC-3' (mismatches
shown in boldface; base pairs 9-39; GenBankTM X15358),
creating a MluI site, and 150 ng of downstream antisense primer 5'-GCACTGGTACCGCGGCGAAAGCCACGGTGG-3'
(base pairs 633-662), creating an Acc65I site. PCR
was carried out in 50 µl of UlTma buffer with 50 µM
dNTPs, 2 mM Mg2+, 15 ng of junD-KSBluescript
(XHJ-12.4, American Type Culture Collection 63024), and 6 units of
UlTma polymerase (Perkin-Elmer) (35 cycles for 1 min at 98 °C and 2 min at 73 °C). The PCR product was digested with MluI and
Acc65I, gel-purified, and ligated into the
MluI/Acc65I-linearized pBind plasmid (Promega),
creating a fusion product of the Gal4-binding domain and the JunD
transactivation domain. pBindFosB was generated by PCR using 30 ng of
upstream sense primer
5'-GGCTGCAAGAACGCGTACGAAGAGGGG-3' (base pairs 1862-1888; GenBankTM X14897), creating a
MluI site, and 30 ng of downstream antisense primer
5'-CCCTCGCGGCCGCATCTTCCTCCTCC-3'
(base pairs 2260-2285), creating a NotI site. PCR was
carried out in 60 µl of UlTma buffer with 50 µM dNTPs,
1.6 mM Mg2+, 15 ng of fosB-pGem1 (kindly
provided by R. Bravo), and 6 units of UlTma polymerase (35 cycles for 1 min at 97 °C, 1 min at 65 °C, and 1 min at 72 °C). The PCR
product was digested with MluI and NotI,
gel-purified, and ligated into the
MluI/NotI-linearized pBind plasmid, creating a
fusion product of the Gal4-binding domain and the FosB transactivation domain.
Transactivation Analysis--
308 cells were transiently
transfected using
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
methyl sulfate liposomal transfection reagent (Boehringer Mannheim).
Cells (1-1.5 × 105) were seeded in a six-well plate
and transfected after 24 h under serum-free conditions with 5 µg
of the human collagenase TRE-luciferase construct or 5 µg of the
various pBind constructs together with 5 µg of the pG5luc luciferase
reporter containing five Gal4-binding sites upstream of a minimal TATA
box fused to the luciferase gene (Promega). After 12 h, cells were
transferred to serum-containing MEM. After another 12 h, the cells
were treated. At various times, cells were lysed in 1% Triton X-100,
25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, and 1 mM DTT, and protein
concentration was determined with Bio-Rad DC reagent. 180 µl of assay buffer (25 mM glycylglycine, 15 mM K3PO4, 15 mM
MgSO4, 4 mM EGTA, 1 mM DTT, and 2 mM ATP) were added to 40 µg of protein, and the
luminescence reaction was initiated in a Monolight 2010 luminometer
with the injection of 100 µl of 0.2 mM
D-luciferin (in 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, and 2 mM DTT). After 2 s, emission was integrated over a
10-s interval and expressed as relative light units.
Protein Kinase Assay--
Cells were lysed in 20 mM
Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium
pyrophosphate, 1 mM
-glycerol phosphate, 1 mM sodium vanadate, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. 100 µg of protein were incubated with phospho-specific ERK-1/2 antibody, phospho-specific JNK
antibody, or phospho-specific p38 MAPK antibody (New England Biolabs
Inc.) with gentle rocking overnight at 4 °C. Protein A-Sepharose beads were added, and the mixture was rotated at 4 °C for 3 h and washed twice in the lysis buffer and twice in kinase buffer (25 mM Tris (pH 7.5), 5 mM
-glycerol phosphate,
2 mM DTT, 0.1 mM sodium vanadate, and 10 mM MgCl2). Beads were suspended in 50 µl of
kinase buffer with 100 µM ATP and 0.5 µg of substrate (ERK-1/2: Elk-1, New England Biolabs Inc.; JNK: c-Jun, Santa Cruz Biotechnology; and p38 MAPK: ATF-2, Santa Cruz Biotechnology) and
incubated at 30 °C for 30 min. Samples were boiled in SDS sample
buffer (60 mM Tris (pH 6.8), 2% SDS, 5%
-mercaptoethanol, 10% glycerol, and 0.001% bromphenol blue) and
resolved on 12.5% SDS-polyacrylamide gels. Phosphorylated proteins
were analyzed by Western blotting using phospho-specific antibodies for
the different substrates (New England Biolabs Inc.).
Isolation of Nuclear Protein--
Cells were rinsed once with
phosphate-buffered saline and once with buffer A (10 mM
HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM
KCl, and 0.5 mM DTT). Cells were scraped, pelleted by
centrifugation at 1000 rpm for 2 min at 4 °C, and lysed in buffer A
containing 0.1% Nonidet P-40 for 30 min at 4 °C. Crude nuclear
protein extracts were prepared by the method of Dignam et
al. (24). Nuclei were pelleted by centrifugation at 10,000 rpm for
15 min at 4 °C; resuspended in 20 mM HEPES (pH 7.9),
25% glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.5 mM DTT; and
incubated for 15 min at 4 °C. The extracts were centrifuged at
10,000 rpm for 15 min at 4 °C, and the supernatant was diluted 1:6
with 20 mM HEPES (pH 7.9), 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.5 mM DTT. The protein
concentrations of the extracts were determined with Bio-Rad reagent.
Electrophoretic Mobility Shift Assay--
Oligonucleotide probes
were labeled by incorporation of [32P]dCTP (NEN Life
Science Products) into the 5'-overhangs of the annealed human
collagenase TRE oligonucleotide 5'-gcttcaTGAGTCAgacacc-3' with Klenow
DNA polymerase. The oligonucleotide binding assay was performed by
mixing 1 × 105 cpm probe with 5 µg of nuclear
protein extract in the presence of 2 µg of poly(dI-dC)·poly(dI-dC)
(Amersham Pharmacia Biotech) and gel shift buffer (10 mM
Tris (pH 7.5), 50 mM NaCl, 1 mM
MgCl2, 0.5 mM DTT, and 4% glycerol) at room
temperature for 30 min. In antibody clearing experiments, various
amounts of antibodies specific for the various Jun and Fos proteins
(Santa Cruz Biotechnology) were preincubated with nuclear extracts at
room temperature for 2 h prior to the binding assay. An antibody
specific for Nm23-H1 (Santa Cruz Biotechnology) was used as a control;
volumes were adjusted with antibody buffer (phosphate-buffered saline,
0.1% sodium azide, and 0.2% gelatin) containing 100 µg/ml bovine
serum albumin. DNA-binding complexes were resolved by gel
electrophoresis on 5% polyacrylamide and 1× Tris borate
electrophoresis buffer (89 mM Tris, 89 mM boric
acid, and 2 mM EDTA) gels. The gels were dried and exposed
to Kodak X-AR film at
80 °C for 2-96 h using an intensifying screen.
Western Analysis--
20 µg of crude nuclear extract were
boiled in SDS sample buffer and resolved on 12.5% SDS-polyacrylamide
gels. The proteins were transferred to a polyvinylidene difluoride
nylon membrane (Millipore Immobilon P) by electroblotting at 50 V at
4 °C for 12-16 h using a transfer buffer containing 25 mM Tris, 190 mM glycine, and 20% methanol. The
membrane was blocked in 5% nonfat dry milk in TBST (10 mM
Tris (pH 8.0), 150 mM NaCl, and 0.05% Tween-20) at room
temperature for 1 h. Primary antibody was diluted 1:1000 (c-Fos,
Fra-1, and Fra-2) or 1:3000 (JunB, JunD, and FosB) in dry milk/TBST and
added. The membrane was incubated at room temperature for 1 h and
then washed three times with TBST. Horseradish peroxidase-conjugated
goat anti-rabbit IgG (heavy and light chains; New England Biolabs Inc.)
was added at a dilution of 1:2000 in 5% dry milk/TBST. The membrane
was incubated at room temperature for 1 h and then washed three
times with TBST. Antigen-antibody complexes were detected using the
Phototope®-HRP Western blot Detection kit (New England
Biolabs Inc.) according to the manufacturer's instructions.
Orthophosphate Labeling--
Cells (7 × 105)
were seeded in a 10-cm plate. After 48 h, cells were treated and,
during the last hour of the treatment following incubation in
phosphate-free MEM for 1 h, labeled with 100 µCi/ml [32P]orthophosphate. Cells were lysed in radioimmune
precipitation assay buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1% sodium
deoxycholate). The lysates were precleared with 50% protein
A-Sepharose beads in 50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.5% Nonidet P-40 for
10 min at 4 °C. Equal counts (1 × 108 cpm) or
equal amounts (250 µg) of protein were incubated with 1:100 dilutions
of AP-1 antibodies with gentle rocking overnight at 4 °C. Protein
A-Sepharose beads were added, and the mixture was rotated at 4 °C
for 2 h and washed three times in 50 mM Tris (pH 7.4),
0.5 M NaCl, 5 mM EDTA, 1% Nonidet P-40, and
5% sucrose and twice in radioimmune precipitation assay buffer.
Samples were boiled in SDS sample buffer and analyzed on 12.5%
SDS-polyacrylamide gels.
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RESULTS |
Okadaic Acid Increases AP-1 Transactivation--
Previously, we
reported that in papilloma-producing 308 mouse keratinocytes, okadaic
acid increases AP-1 binding to a consensus 12-O-tetradecanoylphorbol-13-acetate-responsive element
(17). To analyze whether this okadaic acid-induced increase in AP-1 DNA
binding activity results in increased AP-1 transactivation, we
transiently transfected 308 cells with a luciferase reporter construct
driven by region
74 to +63 of the human collagenase gene containing a
TRE. We treated the transfected cells for various times with 100 ng/ml
okadaic acid or with the solvent Me2SO. Crude cellular
extracts were prepared and assayed for luciferase activity. The results
are shown in Fig. 1. 308 cells showed a
basal luciferase activity that was significantly increased by okadaic
acid. The okadaic acid-induced increase in luciferase activity was
already detectable at 4 h and gradually increased further at 6 and
12 h. The level of induction was 1.6 ± 0.3-fold at 4 h,
3.1 ± 1.1-fold at 6 h, and 11.2 ± 3.0-fold at 12 h.

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Fig. 1.
Okadaic acid increases AP-1
transactivation. 308 cells were transiently transfected with a
luciferase reporter construct driven by a fragment of the human
collagenase gene containing a TRE. Cells were treated with 100 ng/ml
okadaic acid (OA) or the solvent dimethyl sulfoxide
(DMSO). At the indicated times, cells were lysed, and 40 µg of total protein were analyzed for luciferase activity. The
results shown are representative of three independent experiments.
RLU, relative light units.
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Okadaic Acid Activates ERK-1/2, JNK, and p38 MAPK--
Through
their ability to phosphorylate transcription factors and modulate their
activity, MAPKs have established themselves as key regulators of gene
expression. To test whether okadaic acid-increased AP-1-mediated gene
expression correlates with increased MAPK activity, we isolated
cellular protein from exponentially growing 308 cells that had been
treated with 100 ng/ml okadaic acid or with the solvent
Me2SO. Using phospho-specific antibodies for the three
groups of MAPKs (ERK-1/2, JNK, and p38 MAPK), the activated forms of
these kinases were immunoprecipitated. We then performed in
vitro kinase assays with recombinant proteins using their major
substrates: Elk-1 for ERK-1/2, c-Jun for JNK, and ATF-2 for p38 MAPK.
The phosphorylated forms of these substrates were detected by Western
blot analyses using phospho-specific antibodies. As shown in Fig.
2, all three MAPKs were efficiently activated by okadaic acid. Okadaic acid stimulated ERK-1/2 activity starting at 1 h and progressively increasing up to 12 h (Fig. 2A). For JNK, a high basal activity was observed. Treatment
with okadaic acid led to a further increase in JNK activity. This
increase was detectable at 1 h and did not change significantly
throughout the time course (Fig. 2B). Okadaic acid-mediated
stimulation of p38 MAP kinase was detectable within 2 h, reached a
maximum at 4 h, and remained constant up to 12 h (Fig.
2C).

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Fig. 2.
Okadaic acid activates ERK-1/2, JNK, and p38
MAPK. Exponentially growing 308 cells were treated with 100 ng/ml
okadaic acid (OA) or the solvent dimethyl sulfoxide
(DMSO). At the indicated times, cells were lysed, and 100 µg of total protein were immunoprecipitated with phospho-specific
antibodies for ERK-1/2 (A), JNK (B), and p38 MAPK
(C). In vitro kinase assays were performed, and
phosphorylated substrates were detected by Western blot analysis using
phospho-specific antibodies. The results are representative of three
independent experiments.
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Inhibition of ERK-1/2 Abrogates Okadaic Acid-stimulated AP-1
Transactivation--
To further investigate the role of MAPKs in
okadaic acid-mediated AP-1 activation, we tested whether the observed
increases in AP-1-dependent transcription are a direct
result of MAPK activation. To achieve this goal, we transiently
transfected 308 cells with the human collagenase TRE-luciferase
construct and inhibited ERK-1/2 activity with PD 98059, which has been
described as a specific inhibitor of the ERK-1/2-activating kinases
MEK-1/2 (25). The transfectants were treated with this inhibitor for
1 h prior to the addition of 100 ng/ml okadaic acid. At various
times, crude cellular extracts were prepared, and luciferase activity
was determined. To assure that, under the experimental conditions used,
PD 98059 inhibited ERK-1/2 in our system, cellular protein was isolated from control cells and analyzed for ERK-1/2 activity. Activated ERK-1/2
was immunoprecipitated from cellular lysates, and in vitro kinase assays with recombinant Elk-1 as exogenous substrate were performed. As shown in Fig.
3A, PD 98059 inhibited basal
ERK-1/2 activity totally. Okadaic acid-induced ERK-1/2 activity was
completely inhibited at 4 h, whereas at 6 and 12 h, a low
amount of ERK-1/2 activity was still detectable. As a result of this
inhibition, the effect of okadaic acid on AP-1-mediated transcription
was almost completely abrogated. Induction levels of okadaic
acid-treated cells over Me2SO-treated controls dropped from
3.1 ± 1.1-fold to 1.5 ± 0.5-fold at 6 h and from
11.2 ± 3.0-fold to 1.6 ± 0.4-fold at 12 h (Fig.
3B).

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Fig. 3.
Inhibition of ERK-1/2 abrogates okadaic
acid-stimulated AP-1 transactivation. A, exponentially
growing 308 cells were treated with 50 µM MEK-1/2
inhibitor PD 98059 or Me2SO for 1 h. 100 ng/ml okadaic
acid (OA) or the solvent dimethyl sulfoxide
(DMSO) was added. At the indicated times, cells were lysed,
and 100 µg of total protein were immunoprecipitated with a
phospho-specific antibody for ERK-1/2. In vitro kinase
assays were performed using recombinant Elk-1 as substrate, and
phosphorylated Elk-1 was detected by Western blot analysis using a
phospho-specific antibody. B, 308 cells were transiently
transfected with a luciferase reporter construct driven by a fragment
of the human collagenase gene containing a TRE. Prior to the addition
of 100 ng/ml okadaic acid or the solvent Me2SO, cells were
pretreated with 50 µM MEK-1/2 inhibitor PD 98059 or
Me2SO. At the indicated times, cells were lysed, and 40 µg of total protein were analyzed for luciferase activity. Shown is
the -fold increase in luciferase activity of okadaic acid-treated cells
over Me2SO-treated cells. The results are a combination of
three independent experiments. The error bars represent 95%
confidence.
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Inhibition of ERK-1/2 Does Not Alter AP-1 Protein
Levels--
Increased AP-1 expression not only has been found to be a
prerequisite for okadaic acid-increased AP-1 DNA binding in 308 cells
(17), but is also the predominant mechanism of ERK-1/2-mediated AP-1
activation (26). For this reason, we examined whether okadaic acid-induced increases in AP-1 expression are dependent on ERK-1/2 activity. To address this question, we inhibited ERK-1/2 activity with
the MEK-1/2 inhibitor PD 98059; treated the cells with 100 ng/ml
okadaic acid or Me2SO for 6 h; isolated crude nuclear
extracts; and performed Western blot analyses for c-Jun, JunB, JunD,
c-Fos, FosB, Fra-1, and Fra-2. As shown in Fig.
4, inhibition of ERK-1/2 did not lead to
any significant changes in the steady-state levels of the various AP-1
proteins.

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Fig. 4.
Inhibition of ERK-1/2 does not alter AP-1
protein levels. Western analyses were performed with 20 µg of
nuclear protein isolated from exponentially growing 308 cells
pretreated with 50 µM MEK-1/2 inhibitor PD 98059 or the
solvent dimethyl sulfoxide (DMSO) for 1 h and treated
with 100 ng/ml okadaic acid (OA) or Me2SO for
6 h. 1:1000-1:3000 dilutions of antibodies specific for the
various Jun and Fos proteins were used. Horseradish
peroxidase-conjugated goat anti-rabbit IgG (1:2000) was used as
secondary antibody. Antigen-antibody complexes were visualized by
chemiluminescence. The results shown are representative of three
independent experiments.
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Inhibition of ERK-1/2 Does Not Reduce AP-1 DNA Binding
Activity--
Since okadaic acid-induced accumulation of AP-1 proteins
occurs through an ERK-1/2-independent pathway, we then considered the
possibility that ERK-1/2 activity is responsible for post-translational modifications of AP-1 proteins, increasing their DNA binding activity. To test this hypothesis, we performed electrophoretic mobility shift
assays of crude nuclear extracts from exponentially growing 308 cells
that had been pretreated for 1 h with the MEK-1/2 inhibitor PD
98059 prior to the addition of 100 ng/ml okadaic acid or
Me2SO. At 4, 6, and 12 h, TRE binding activity was
analyzed using a radiolabeled oligonucleotide of the human collagenase
promoter including the TRE. The results are presented in Fig.
5. Basal and okadaic acid-increased TRE
DNA binding activities were not compromised by inhibition of ERK-1/2.
In contrast, at 4 h, inhibition of these MAPKs induced an increase
in DNA binding activity.

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Fig. 5.
Inhibition of ERK-1/2 does not reduce AP-1
DNA binding activity. Shown are the results from an
electrophoretic mobility shift assay of crude nuclear extracts (5 µg)
from exponentially growing 308 cells using a radiolabeled TRE
oligonucleotide. Cells were pretreated with 50 µM MEK-1/2
inhibitor PD 98059 or the solvent dimethyl sulfoxide (DMSO)
for 1 h and treated with 100 ng/ml okadaic acid (OA) or
Me2SO. The results shown are representative of three
independent experiments.
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Inhibition of ERK-1/2 Does Not Alter AP-1 Composition--
Since
we could not demonstrate a causative relationship between ERK-1/2
activation and increased AP-1 DNA binding, we then hypothesized that
ERK-1/2 may increase AP-1-dependent transactivation by
modifying interactions between AP-1 proteins and/or between AP-1
proteins and AP-1 inhibitors such as IP-1 (27) and Jif-1 (28). Those
altered interactions could then lead to changes in the composition of
AP-1 complexes and allow, without altering their DNA binding affinity,
for the formation of complexes with higher transactivation potential.
To address this idea, we used antibody clearing experiments to analyze
the composition of AP-1 complexes bound to the human collagenase TRE
oligonucleotide. Nuclear extracts of cells treated for 6 h with
okadaic acid with (Fig. 6B) or
without (Fig. 6A) prior inhibition of ERK-1/2 by the MEK-1/2
inhibitor PD 98059 were incubated for 2 h with varying amounts of
antibodies for the different Jun and Fos proteins and then analyzed in
electrophoretic mobility shift assays. An Nm23-H1-specific antibody was
used as a control, and protein amounts added to the different samples
were adjusted with bovine serum albumin since unspecific inhibition
increased with increasing amounts of antibody. The presence of the
various AP-1 proteins was evaluated by the ability of the antibodies to
form with the AP-1 complexes a more slowly migrating form or a
supershift and/or to reduce TRE binding activity. As shown in Fig. 6,
JunD and FosB were identified as major components of the AP-1 complexes
binding to the human collagenase TRE at 6 h. Inhibition of ERK-1/2
did not alter this composition.

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Fig. 6.
Inhibition of ERK-1/2 does not alter AP-1
composition. Crude nuclear extracts (5 µg) of exponentially
growing 308 cells pretreated with 50 µM MEK-1/2 inhibitor
PD 98059 (B) or the solvent Me2SO (A)
for 1 h and treated with 100 ng/ml okadaic acid or
Me2SO for 6 h were preincubated for 2 h at room
temperature with varying amounts of antibodies specific for the various
Jun and Fos proteins. Controls were preincubated with an antibody
specific for Nm23-H1. Electrophoretic mobility shift assays were
performed using a radiolabeled TRE oligonucleotide. The results shown
are representative of three independent experiments.
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Inhibition of ERK-1/2 Reduces Phosphorylation of JunD and
FosB--
Since increased AP-1 expression, increased DNA binding, and
formation of more potent AP-1 complexes were excluded as mechanisms of
ERK-1/2-mediated AP-1 transactivation, we then tested the possibility of ERK-1/2-induced increases in AP-1 transactivation potential through
altered phosphorylation of the major components, JunD and FosB. Cells
(pretreated with the MEK-1/2 inhibitor PD 98059 and treated with
okadaic acid or Me2SO for 6 h) were labeled with orthophosphate, and phosphorylation of JunD and FosB was examined by
immunoprecipitation analyses. Similar results were obtained when equal
counts or equal amounts of protein (data not shown) were
immunoprecipitated. As shown in Fig. 7,
okadaic acid increased phosphorylation of JunD and FosB. Pretreatment
with the MEK-1/2 inhibitor significantly reduced okadaic acid-mediated
phosphorylation.

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Fig. 7.
Inhibition of ERK-1/2 reduces phosphorylation
of JunD and FosB. Exponentially growing 308 cells were pretreated
with 50 µM MEK-1/2 inhibitor PD 98059 or the solvent
dimethyl sulfoxide (DMSO) for 1 h and treated with 100 ng/ml okadaic acid (OA) or Me2SO. During the
last hour of treatment, they were labeled with 100 µCi/ml
[32P]orthophosphate. At 6 h, cells were lysed, and
equal counts were immunoprecipitated with antibodies specific for JunD
or FosB. The immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis. The results shown are representative of three
independent experiments.
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Both JunD and FosB Are Required for Okadaic Acid-induced
Transcription--
To examine the role of JunD and FosB in okadaic
acid-induced AP-1 transactivation more closely, we generated fusion
proteins of the DNA-binding domain of the yeast transcription factor
Gal4 and the transactivation domain of JunD (pBindJunD) or FosB
(pBindFosB) and tested directly the ability of okadaic acid to increase
the transactivation potential of these AP-1 proteins. Cells were
cotransfected with pBindJunD, pBindFosB, or pBindJunD and pBindFosB and
a luciferase reporter construct containing five Gal4-binding sites. As
shown in Fig. 8A, okadaic acid
affected JunD- or FosB-mediated transcription only slightly, but
substantially increased transcription mediated by a combination of JunD
and FosB. Treatment with the MEK-1/2 inhibitor PD 98059 had no effect
on JunD- or FosB-mediated transcription, but resulted in a
statistically significant (p < 0.05) reduction of
JunD/FosB-dependent transcription (Fig. 8B).

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Fig. 8.
Both JunD and FosB are required for okadaic
acid-induced transcription. A, 308 cells were
transiently transfected with a construct generating a fusion protein of
the Gal4 DNA-binding domain and the transactivation domain of JunD
(pBindJunD) or FosB (pBindFosB) and a luciferase reporter construct
containing five Gal4-binding sites. Cells were treated with 100 ng/ml
okadaic acid (OA) or the solvent dimethyl sulfoxide
(DMSO). At 6 h, cells were lysed, and 40 µg of total
protein were analyzed for luciferase activity. Shown are relative light
units (RLU) of okadaic acid-treated cells versus
Me2SO-treated cells. The results are representative of four
independent experiments. B, prior to the addition of 100 ng/ml okadaic acid or the solvent Me2SO, cells were
pretreated with 50 µM MEK-1/2 inhibitor PD 98059 or
Me2SO. Shown is the -fold increase in luciferase activity
of okadaic acid-treated cells over Me2SO-treated cells. The
results are a combination of six experiments. The error bars
represent 95% confidence.
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DISCUSSION |
In this study, we were able to show that in papilloma-producing
308 mouse keratinocytes, the previously reported okadaic acid-increased AP-1 DNA binding to a consensus TRE (17) correlates with increased AP-1 transactivation. Okadaic acid was able to induce transcription of a luciferase reporter construct driven by region
74
to +63 of the human collagenase gene containing a TRE.
This result further supports the idea that increased AP-1 activity
plays a critical role in epidermal tumor development in mouse skin.
Increased AP-1 activity has been suggested as a prerequisite for tumor
promoter sensitivity, and further tumor promoter-induced elevation of
AP-1 activity as a requirement for benign tumor development (8). A
constitutive increase in AP-1 activity might be necessary for
development and maintenance of malignant squamous cell carcinomas (29).
Although we could show that okadaic acid activates all three families
of MAPKs (ERK-1/2, JNK, and p38 MAPK), specific inhibition of ERK-1/2
with PD 98059 was sufficient to abrogate okadaic acid-increased AP-1
transactivation. This finding indicates that ERK-1/2 is a critical
component in okadaic acid-mediated AP-1 regulation. A similar
observation has been made by Frost et al. (30), who demonstrated a requirement for ERK-1/2 in AP-1 activation by Ha-Ras, 12-O-tetradecanoylphorbol-13-acetate, and serum.
ERK-1/2-mediated AP-1 activation appears to occur predominantly at a
transcriptional level. c-fos transcription is induced by
phosphorylation of Elk-1, which forms, with the dimeric serum response
factor, a prebound complex at the serum response element (31, 18). A
similar mechanism could be postulated for induction of other AP-1
genes, e.g. junD and fosB, where
Ets-binding sites and serum response elements have been identified and
implicated in transcriptional regulation (32, 33).
In contrast to these reports, our data suggest that ERK-1/2 acts
through a post-translational mechanism, stimulating the transactivation potential of pre-existing AP-1 complexes by alterations in their phosphorylation pattern. We found that inhibition of ERK-1/2 had only
slight effects on overall levels of AP-1 proteins. These minor
decreases in AP-1 expression were not sufficient to decrease AP-1 DNA
binding activity. Using antibody clearing experiments, we demonstrated
that the composition of the AP-1 complexes constituting this DNA
binding activity was the same in ERK-1/2 inhibitor-treated and
untreated cells. Surprisingly and in contrast to our previous study
(17), where JunB was identified as major component of the AP-1 complex,
JunB did not appear to bind to the human collagenase TRE. A possible
explanation for this discrepancy could be the use of a different TRE
oligonucleotide. In the present study, we used an oligonucleotide for
which the sequence was taken from the human collagenase gene to
correspond to the sequence in the luciferase reporter construct used
for transactivation studies as opposed to the TRE oligonucleotide
described by Angel et al. (34). As reported by Ryseck and
Bravo (16), the various AP-1 proteins bind with different
affinities to different oligonucleotides that contain identical
AP-1-binding sites, implying that adjacent sequences influence the
composition of AP-1 complexes.
Our experiments using fusion proteins of the DNA-binding domain of the
yeast transcription factor Gal4 and the transactivation domain of
either JunD or FosB demonstrated the ability of okadaic acid to
increase the transactivation potential of these AP-1 proteins and
directly implicated them in okadaic acid-mediated
TRE-dependent transactivation. Although JunD and FosB each
by itself acted as transcriptional activators, only a combination of
both was effectively inducible by okadaic acid and mediated okadaic
acid effects on transcription. These findings indicate that okadaic
acid-induced activation of JunD and FosB may require interactions
between their transactivation domains. In the Gal4-luciferase reporter,
the five Gal4-binding sites are in close proximity, thus very likely allowing for interactions between monomeric bound Gal4-AP-1 fusion proteins that normally occur through heterodimerization. These interactions may be necessary to introduce activating phosphorylations by recruiting okadaic acid-responsive kinases to their respective phosphorylation sites. A similar observation has been made by Kallunki
et al. (35), who reported that JunD becomes a substrate for
JNK by heterodimerization with c-Jun or JunB that possesses a
JNK-docking site.
Treatment with the MEK-1/2 inhibitor PD 98059 significantly reduced
okadaic acid-mediated JunD/FosB-dependent transcription. These data, together with our phosphorylation analyses demonstrating that okadaic acid-increased phosphorylation of JunD and FosB was significantly reduced by inhibition of ERK-1/2, suggest a potential role of increased JunD and FosB phosphorylation in okadaic acid-induced AP-1 transactivation and indicate ERK-1/2 as the responsible kinase. In
contrast to c-Jun, whose regulation by phosphorylation is well documented, less is known about phosphorylation changes modulating the
activity of JunD and FosB. Nikolakaki et al. (36) reported that glycogen-synthase kinase 3 phosphorylates JunD in a region proximal to its DNA-binding domain and attenuates its DNA binding capacity. In contrast, the finding that serum-induced phosphorylation of JunD does not affect its ability to bind DNA provides evidence that
phosphorylation may also regulate the transactivation potential of this
factor (37). Serine 100, homologous to serine 73, one of two
serines whose phosphorylation increases the transactivation potential
of c-Jun, has been found to be phosphorylated in response to activation
of JNKs. In a similar fashion, phosphorylation of several serine
residues in the C-terminal transactivation domain of FosB has been
shown to increase its transcriptional activity (38).
In conclusion, these results, together with our previous observations,
indicate that in papilloma-producing 308 mouse keratinocytes, okadaic
acid stimulates AP-1 activity by two different mechanisms that appear
to be mediated by independent signal transduction pathways. Okadaic
acid increases TRE DNA binding activity by stimulating expression of
AP-1 proteins. This increased TRE DNA binding activity does not
necessarily result in increased TRE-dependent
transactivation. Further modifications of the pre-existing AP-1
proteins are necessary. In contrast to okadaic acid-increased AP-1 DNA
binding activity, which occurs independently of okadaic acid-mediated
ERK-1/2 activation, ERK-1/2 activity is required for increased transactivation.