Analysis of the Role of the Mitogen-Activated Protein Kinase in Mediating Cyclic-Adenosine 3',5'-Monophosphate Effects on Prolactin Promoter Activity
Paul Kievit,
Jeffrey D. Lauten and
Richard A. Maurer
Department of Cell and Developmental Biology Oregon Health
Sciences University Portland, Oregon 97201
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ABSTRACT
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The mechanisms mediating cAMP effects to stimulate
transcription of the PRL gene have been examined. Treatments that
elevate intracellular cAMP concentrations were found to stimulate the
mitogen-activated protein kinase (MAPK) in GH3
cells. Elevated cAMP was also found to stimulate activation of the
GTP-binding protein, Rap1. Rap1GAP1 reduced cAMP-induced
phosphorylation of MAPK, offering evidence that Rap1 may play a role in
mediating activation of MAPK. Treatment of GH3
cells with PD98059, an inhibitor of the MAPK pathway, reduced the
ability of forskolin to activate a PRL reporter gene, providing
evidence that MAPK contributes to cAMP-mediated effects on the PRL
promoter. As previous studies have implicated Ets factor binding sites
within the PRL promoter in mediating responses to MAPK, we expected
that the Ets sites would also play a role in cAMP responsiveness.
Surprisingly, mutation of all of the consensus Ets factor binding sites
in the proximal PRL promoter greatly reduced responsiveness to
epidermal growth factor (EGF) and TRH but did not reduce cAMP
responsiveness. Experiments using an expression vector for adenovirus
12S E1a provided evidence that the coactivators, CREB binding protein
and/or p300, probably play a role in cAMP responsiveness of the PRL
promoter. Interestingly, the ability of a GAL4-p300 fusion protein to
enhance reporter gene activity was stimulated by cAMP in a
MAPK-dependent manner. These findings provide evidence for a model for
cAMP-induced PRL transcription involving Rap1-induced MAPK activity
leading to stimulation of the transcriptional coactivators, CBP and
p300.
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INTRODUCTION
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Although it has been known for some time that cAMP can stimulate
PRL gene expression (1, 2), it has been difficult to determine the
mechanisms mediating this response. Studies utilizing expression
vectors for the catalytic subunit of the cAMP-dependent protein kinase
[protein kinase A (PKA)] or the heat-stable inhibitor of PKA (PKI)
have provided evidence that PKA is sufficient and necessary for
cAMP-induced activation of the PRL gene (3, 4). However, the events
downstream of PKA have been elusive. Unlike many PKA-regulated genes,
stimulation of PRL gene expression by cAMP probably does not involve
CREB (5, 6). Several studies have provided evidence that the
pituitary-specific POU transcription factor, Pit-1, may play a role in
mediating the effects of cAMP (6, 7, 8) as well as other signaling
pathways (9, 10) on PRL transcription. The finding that Pit-1 is
phosphorylated in response to elevated cAMP levels in
GH3 cells (11, 12) rather strongly supported the
view that Pit-1 may function as a cAMP-regulated transcription factor.
However, studies using Pit-1 mutants in which the phosphorylation sites
are removed (13, 14) have provided evidence that cAMP-induced
activation of the PRL promoter probably does not require
phosphorylation of Pit-1. Although this does not exclude Pit-1 from
contributing to cAMP-mediated effects on PRL transcription, it is by no
means clear that phosphorylation of Pit-1 mediates cAMP effects, and
other mechanisms need to be considered. We have obtained evidence that
cAMP can activate the mitogen-activated protein kinase
(MAPK)-responsive transcription factor, Elk1, providing indirect
evidence that cAMP may stimulate MAPK in GH3
cells (12), raising the possibility that the MAPK pathway may be
involved in mediating effects of cAMP on transcription of the PRL gene.
We have also recently obtained evidence that TRH effects on PRL
transcription may be mediated, at least in part, through activation of
MAPK (15). Thus it is possible that both the TRH and cAMP signaling
pathways may utilize the MAPK cascade to stimulate PRL gene
expression.
In the present study we have examined the ability of cAMP to stimulate
MAPK activity and tested the role that cAMP-induced MAPK activation
plays in modulating PRL transcription. We have found that cAMP can
activate MAPK in GH3 cells and that the increase
in MAPK is required for full effects of cAMP on PRL transcription.
Analysis of DNA sequences of the PRL gene, which are required for cAMP
responsiveness, reveals both similarities and differences in the role
of specific Ets factor binding sites for TRH and cAMP responsiveness.
We have also assessed the possible role that the transcriptional
coactivators, CREB binding protein (CBP) and p300, play in mediating
cAMP-induced activation of the PRL promoter.
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RESULTS
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Elevation of cAMP Induces MAPK Activity in
GH3 Cells
Previous studies from this laboratory used an indirect assay
involving transcriptional activation of a GAL4-Elk1 fusion protein to
test for cAMP effects on MAPK activation (16). To directly assess MAPK
activation, we used an immunocomplex assay (Fig. 1
, A, B, and C). For
this assay, cell lysates were immunoprecipitated with an antibody to a
specific MAPK family member after which the immunoprecipitated proteins
were incubated with [
-32P]ATP and
glutathione-S-transferase (GST)-Elk1 as a substrate. This
assay demonstrated that treatment with chlorophenylthio-cAMP stimulated
the activity of MAPK family members, extracellular signal-regulated
kinases 1 and 2 (ERK1 and ERK2) (Fig. 1
, A and B), with only a minor
effect on the activity of the stress-activated MAPK, JNK1 (c-Jun
N-terminal protein kinase-1) (Fig. 1C
). ERK1 and ERK2 are activated by
phosphorylation of specific tyrosine and threonine residues (17), and
an antibody that can detect this phosphorylation was also used to
assess ERK activation (Fig. 1D
). Treatment of cells with forskolin, an
agent that increases intracellular cAMP levels, resulted in the rapid
stimulation of the phosphorylation of ERK1 and ERK2 (Fig. 1D
). There
was little or no change in the level of total immunoreactive ERK1 and
ERK2, suggesting that the increased phosphorylation is not due to a
change in the total amount of these kinases (Fig. 1E
). Experiments were
then performed to compare the time course of ERK activation by
forskolin and epidermal growth factor (EGF). Cells were treated with
either forskolin or EGF for varying times, and ERK activation was
determined using a phospho-specific antibody and the results
quantitated using a Lumi-Imager system (Roche Molecular Biochemicals, Indianapolis, IN) (Fig. 1
, F and G). EGF effects
on ERK phosphorylation appeared to reach maximal levels more quickly
and also appeared to decline somewhat more rapidly. Several different
experiments supported the view that EGF effects on ERK activity reach a
maximum more quickly than cAMP effects, but decay more rapidly (data
not shown). Overall, the analysis of cAMP effects on MAPK confirm our
earlier results (16) and studies from Jacob et al. (18),
which demonstrated cAMP-induced activation of MAPK in the
GH4 cell line. The present studies also provide
information about the time course and specificity of MAPK
activation.

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Figure 1. Time Course of cAMP-Induced MAPK Activation
AC, Immunocomplex kinase assays of MAPK activation. GH3
cells were treated with 0.5 mM CPT-cAMP for various periods
of time. ERK1 (A), ERK2 (B), or JNK1 (C) was immunoprecipitated from
500 µg of cell lysates using antibodies conjugated to protein A/G
agarose. MAPK activity was assessed by incubating the immunoprecipitate
with [32P]- -ATP and a GST-Elk1 fusion protein as
substrate. The phosphorylated proteins were resolved on a denaturing
polyacrylamide gel and detected by autoradiography. EGF (10
nM) and anisomycin (10 ng/ml) were used as positive controls
for activity of ERK and JNK, respectively. DG, Immunoblot analysis of
ERK activation. GH3 cells were cultured in serum-free
medium for 24 h and treated with 10 µM forskolin or
10 nM EGF for the indicated times. Cell lysates (50 µg)
were resolved on a denaturing polyacrylamide gel, transferred to a
membrane, and phosphorylated ERK1 and ERK2 were detected with a
phosphorylation-specific antibody (D). To determine the total amount of
ERK present, the membrane was stripped and reprobed with an antibody
directed against ERK-1 protein (E). This antibody also detects ERK-2,
although to a lesser extent. In a separate experiment GH3
cells were treated with 10 µM forskolin or 10
nM EGF for varying times and ERK activation was detected by
a phosphorylation-specific antibody and then quantitated by Lumi-Imager
analysis (F and G).
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The major signaling pathway that mediates responses to cAMP involves
activation of the cAMP-dependent protein kinase (PKA). An expression
vector for PKI, the heat-stable inhibitor of PKA (4), was used to
determine whether PKA is required for cAMP-induced activation of MAPK.
For this study, MAPK induction was assessed indirectly through analysis
of the activation of a GAL4-Elk1 fusion protein (19).
GH3 cells were transfected with a GAL4-Elk1
expression vector and a GAL4-dependent luciferase reporter gene either
in the presence or absence of a PKI expression vector (Fig. 2A
). PKI strongly reduced the ability of
forskolin to stimulate GAL4-Elk1 activity as measured by the reporter
gene assay. Although PKI reduced EGF-stimulated GAL4-Elk1 activation,
PKI also had an effect on basal activity in the absence of hormonal
treatment, and the fold activation remained similar. The
pharmacological inhibitor H-89 was also used to determine whether
inhibition of PKA led to a decrease in MAPK activation (Fig. 2
, B and
C). GH3 cells were treated with H-89, after which
forskolin was added for 5 min and the cells were collected. Activation
of MAPK was determined by immunological detection of phosphorylated
ERK1 and ERK2 as described above. Addition of H-89 substantially
blocked the ability of forskolin to activate ERK1 and ERK2, whereas
EGF-induced phosphorylation was essentially unchanged. In addition, the
tyrosine kinase inhibitor genistein had relatively little effect on the
induction of ERK phosphorylation by forskolin, but greatly reduced
EGF-induced ERK phosphorylation. The ability of PKA inhibitors to
attenuate both cAMP-induced activation of GAL4-Elk1 and phosphorylation
of ERK1 and ERK2 suggests that PKA is required for cAMP-induced
stimulation of MAPK activity.

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Figure 2. The cAMP-Dependent Protein Kinase Is Required for
Full cAMP-Induced ERK1 and ERK2 Activation
A, Effects of inhibition of PKA by PKI on GAL4-Elk1 activity.
GH3 cells were transfected with a GAL4-dependent luciferase
reporter gene and an expression vector encoding a GAL4-Elk1 fusion
protein. The cells were also transfected with either an empty vector
(control), or a vector encoding PKI. The cells were treated with EGF
(10 nM) or forskolin (10 µM) for 6 h and
collected for analysis of luciferase activity. Reporter gene activity
is reported as luciferase activity from three independent
transfections ± SEM normalized to ß-galactosidase
activity. B and C, Effects of inhibition of PKA or tyrosine kinases on
MAPK activation. GH3 cells were treated with 20
µM of the PKA inhibitor H-89 or 40 µM of
the tyrosine kinase inhibitor, genistein, for 30 min. Cells were
stimulated for 5 min with forskolin or EGF and harvested. Cell lysates
(50 µg) were separated on a denaturing polyacrylamide gel and
transferred to a membrane. Activated ERK was detected with the antibody
directed against the phosphorylated form of ERK1 and ERK2 (B). To test
for equal loading, the membrane was stripped and probed for total
amount of immunoreactive ERK1 and ERK2 (C).
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Analysis of a Possible Role of the GTP Binding Protein, Rap1, in
Mediating Responses to cAMP
Recent studies have provided new insights into cAMP signal
transduction and demonstrated that, in specific cell types, Rap1 can
play a crucial role in mediating cAMP-induced MAPK activation (20).
These studies have shown that cAMP can stimulate Rap1 activation
leading to activation of B-Raf, MEK1, and MAPK. To determine whether
this pathway might be present in GH3 cells,
cAMP-induced activation of Rap1 was investigated (Fig. 3
). This assay is based on the ability of
RalGDS to interact with activated, GTP-bound Rap1, but not with
inactive, GDP-bound Rap1 (21). Lysates from forskolin-treated
GH3 cells were incubated with the
Rap1-binding domain of RalGDS and then bound Rap1 was detected
immunologically (Fig. 3A
). Activation of Rap1 was detected after 1 min,
the earliest tested time point, and maximum activation was achieved in
510 min. In the same experiment, the time course of MAPK activation
was also examined using antibodies for phospho-ERK (Fig. 3C
). There
appear to be some differences in the time course of Rap1 and ERK
activation. At the 2-min time point, Rap1 was only modestly activated,
whereas ERK phosphorylation appears to be near maximal. This finding
suggests that mechanisms other than Rap1 activation may contribute to
the ability of cAMP to stimulate MAPK activity. Alternatively, the
activation of Rap1 in the early time points may be sufficient for
maximal ERK activation. In any case, the ability of cAMP to stimulate
Rap1 activation suggests a possible role in at least partially
mediating effects on MAPK.

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Figure 3. cAMP Activates Rap1 in GH3 Cells
GH3 cells were cultured in serum-free medium for 24 h
before treatment with 10 µM forskolin. Cells were lysed,
and activated Rap1 was isolated with a GST-RalGDS fusion protein bound
to beads. The Rap1-RalGDS complex was washed, and immobilized proteins
were resolved on a denaturing polyacrylamide gel. The proteins were
transferred to a membrane, and bound Rap1 was visualized with a
polyclonal antibody (A). To verify that equal amounts of Rap1 were
present in the lysates, 2% of the input was separated on a denaturing
polyacrylamide gel and transferred to a membrane (B). Rap1 was detected
using the polyclonal antibody. For the ERK phosphorylation time course,
50 µg of the GH3 lysate was resolved on a denaturing
polyacrylamide gel, and phosphorylated ERK1 and ERK2 were detected
using a phosphorylation-specific antibody (C). Equal loading was
determined by stripping the blot and reprobing with an ERK1 antibody
(D).
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To test whether the activation of Rap1 is necessary for MAPK
activation, an expression vector for the Rap1 GTPase activating protein
(Rap1GAP1) was used (22). Forced expression of Rap1GAP1 should maintain
Rap1 in the inactive, GDP-bound state, and thus act as an antagonist to
this signaling pathway. Therefore, a Rap1GAP1 expression vector was
transfected with an expression vector for FLAG-tagged ERK2 (Fig. 4
). Tagged ERK2 was immunoprecipitated,
and activated ERK2 was detected immunologically using a
phospho-specific antibody (Fig. 4A
). Rap1GAP1 reduced both basal and
forskolin-induced levels of phosphorylated ERK2 at both time points
tested. The ability of Rap1GAP1 to reduce ERK2 phosphorylation offers
evidence that Rap1 may contribute to cAMP-induced MAPK activation in
GH3 cells. To determine whether Rap1 contributes
to cAMP-mediated gene expression, GH3 cells were
transfected with the Rap1GAP1 expression vector and a PRL reporter gene
(Fig. 4D
). At the higher tested concentrations, Rap1GAP1 was capable of
reducing both cAMP-induced and basal PRL reporter gene activity. As was
observed with the effects of Rap1GAP1 on cAMP-induced ERK2
phosphorylation, the effects of cAMP were not completely blocked.
It is not clear whether the remaining cAMP response is a reflection
of limited efficacy of the transfected Rap1GAP1 expression vector or if
it indicates that a component of the cAMP response involves a
Rap1-independent pathway.

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Figure 4. Rap1 Is Required for Full Effects of cAMP to
Activate MAPK and Gene Expression
AC, Analysis of the effects of Rap1GAP1 on ERK activation in
GH3 cells. GH3 cells were transfected with
FLAG-tagged ERK2 (4 µg) and either FLAG-tagged Rap1GAP1 (6 µg) or
empty vector (6 µg) as indicated. After serum deprivation, 10
µM forskolin was added for the indicated times.
FLAG-tagged proteins were precipitated from the cell lysates (1 mg),
and phospho-ERK2 was detected with a phosphorylation-specific
polyclonal antibody (A). Expression levels of Rap1GAP1 (B) and ERK2 (C)
were verified by immunoblot analysis of cell lysates (100 µg) using
the FLAG monoclonal antibody. D, Analysis of the effects of Rap1GAP1
expression on PRL reporter gene activity. GH3 cells were
transfected with increasing amounts of the FLAG-Rap1GAP1 construct and
a reporter construct containing the -255 to +34 region of the PRL
promoter. The total amount of transfected DNA was normalized with empty
expression vector (pcDNA3). At 24 h after transfection, the cells
were treated with 10 µM forskolin for 6 h and
collected for analysis of luciferase activity. Reporter gene activity
is reported as luciferase activity from three independent
transfections ± SEM normalized to ß-galactosidase
activity.
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We also tested the ability of a different reagent to interfere with the
Rap1 signaling pathway. Vossler et al. (20) found that
Rap1-N17 can function as a dominant negative mutant to block
cAMP-induced ERK and GAL4-Elk1 activation in PC12 cells. Therefore, we
tested the effects of Rap1-N17 on ERK activation in
GH3 cells. Surprisingly, we found that Rap1-N17
had no detectable effect on cAMP-induced ERK activation or GAL4-Elk1
activation (data not shown). Another group has also failed to detect an
effect of Rap1-N17 on cAMP-induced ERK activation in a different cell
type (23). It is possible that the differing ability of Rap1GAP1 and
Rap1-N17 to block cAMP-induced ERK activation reflects a different
efficacy of these reagents in blocking endogenous Rap1 signaling.
Alternatively, this observation may indicate that the Rap1GAP1 and
Rap1-N17 interfere with different signaling pathways. We cannot
exclude the possibility that Rap1GAP1 may block a signaling pathway
other than Rap1, perhaps a related small GTPase.
MAPK Activation Is Required for cAMP-Stimulated Transcriptional
Activity
To determine whether cAMP-stimulated MAPK activity is required for
specific gene expression, we used the MEK1 inhibitor, PD98059. PD98059
has a relatively high affinity for MEK1 and can block the ability of
MEK1 to activate ERK1 and ERK2 (24). GH3 cells
were pretreated with 100 µM PD98059 for 30 min and then
treated with forskolin for 5 min and assayed for ERK phosphorylation
(Fig. 5A
). PD98059 treatment greatly
reduced cAMP-stimulated phosphorylation of ERK1 and ERK2.

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Figure 5. MAPK Activation Is Required for Full Effects of
cAMP on PRL Gene Expression
A, Analysis of the effects of the MAPK inhibitor, PD98059, on
forskolin-induced ERK activity in GH3 cells.
GH3 cells were cultured in serum-free medium for 24 h,
pretreated for 30 min with the vehicle, dimethylsulfoxide (DMSO)
(control), or 100 µM PD98059 followed by 10
µM forskolin treatment for 5 min. Cell lysates were
resolved on a denaturing polyacrylamide gel, transferred to a membrane,
and probed with an antibody specific to the phosphorylated form of ERK.
B and C, Analysis of the effects of the MAPK inhibitor, PD98059, on
forskolin-induced reporter gene activity. GH3 cells were
transfected with either a GAL4-dependent luciferase reporter gene and
an expression vector encoding a GAL4-Elk1 fusion protein (B) or a PRL
promoter containing the proximal 255 bp fused to the luciferase
reporter gene (C). At 24 h after transfection, GH3
cells were pretreated for 30 min with either the vehicle, DMSO
(control), or 100 µM PD98059. The cells were then treated
with either 10 µM forskolin or 10 nM EGF for
6 h, after which time the cells were lysed and analyzed for
luciferase activity. Reporter gene activity is reported as light units
from three independent transfections ± SEM normalized
to ß-galactosidase activity.
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The ability to PD98059 to block cAMP-induced transcriptional activation
was then tested (Fig. 5
, B and C). PD98059 strongly reduced the ability
of forskolin to stimulate GAL4-Elk1 activity as assessed by a
GAL4-dependent reporter gene (Fig. 5B
). This finding is consistent with
our observation that cAMP induces MAPK activity and the known ability
of MAPK to induce Elk1 activity (19). PD98059 reduced, but did not
completely block, forskolin effects to induce a PRL reporter gene (Fig. 5C
). Similar results were obtained when a kinase-defective MEK1 was
used to inhibit activation of the MAPK pathway (data not shown). These
findings provide evidence that cAMP-induced activation of MAPK plays a
key role in stimulating Elk1 transcriptional activation and also
contributes to cAMP-induced enhancement of PRL transcription.
Analysis of the Role of Ets Binding Sites in Mediating cAMP-Induced
Activation of the PRL Promoter
The proximal region of the PRL gene contains a number of consensus
sites for members of the Ets family of transcription factors (Fig. 6A
). As MAPK has been shown to stimulate
the transcriptional activity of several members of the Ets family (19, 25), the Ets sites appear to be excellent candidates for mediating the
transcriptional response to cAMP-induced MAPK activation. Indeed,
previous studies have shown that disruption of some of the Ets sites of
the PRL gene can reduce transcriptional responses to Ras (26) or TRH
(15). To determine whether any of the Ets sites play a role in
cAMP-dependent PRL gene expression, PRL reporter constructs were tested
for cAMP, TRH, and EGF responsiveness (Fig. 6B
). Some of the Ets site
mutants were previously described (15), and several Ets site mutants
were newly prepared for this study. The only mutation that reduced
cAMP, TRH, and EGF responsiveness to less than 50% of wild type was
mutation of the Ets site located at position -211, a site that
previously has been shown to be important for MAPK responsiveness (15, 26). Disruption of other Ets sites either reduced responsiveness only
modestly or, in some cases, stimulated reporter gene activation in
response to cAMP, TRH, or EGF.

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Figure 6. Mutational Analysis of Putative Ets Factor Binding
Sites in the Proximal PRL Promoter
Specific Ets factor binding sites within the proximal region of the PRL
gene were disrupted as indicated by the schematic diagram for the PRL
promoter (A) where Ets sites are indicated by black
rectangles and Pit-1 binding sites are indicated by gray
rectangles. GH3 cells were transfected with PRL
promoter reporter constructs in which individual Ets factor binding
sites were disrupted (B) or a reporter gene in which all of the
consensus Ets sites were disrupted (C). After serum deprivation, the
cells were treated with 10 µM forskolin, 100
nM TRH, or 10 nM EGF for 6 h. Cells were
collected and assayed for luciferase activity. The basal levels for the
various promoters were as follows (in arbitrary light units): wild
type, 446 ± 93.1; mut1, 617 ± 66.1; mut2, 2,096 ±
253; mut3, 444 ± 36.7; mut4, 798 ± 78.9; mut5, 293 ±
20.6. The fold induction of the wild-type promoter by forskolin, TRH,
and EGF was 13, 3.2, and 8.7, respectively. Reporter gene activity is
reported as the light units from three independent transfections
± SEM normalized to ß-galactosidase activity.
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As there are multiple Ets sites in the proximal region of the PRL gene,
it seemed possible that the modest effects of mutating single Ets sites
reflected some redundancy in their ability to confer MAPK
responsiveness. To assess the overall contribution of Ets sites within
the proximal region of the PRL gene, a reporter construct was created
in which all the consensus Ets sites were mutated. This construct was
tested for its ability to be activated by the activators forskolin,
EGF, and TRH (Fig. 6C
). Surprisingly, the construct in which all of the
Ets sites were mutated was more responsive to forskolin treatment than
the wild-type PRL promoter. In contrast, and as expected, reporter gene
responses to TRH and EGF were reduced in the Ets mutant. Thus, although
cAMP, TRH, and EGF all lead to activation of MAPK, the requirements for
downstream transcription factor targets of these signaling pathways
appear to differ.
Elevation of cAMP Enhances p300 Transcriptional Activity in a
MAPK-Dependent Manner
As the preceding studies suggest that Ets factors are probably not
required for transcriptional responses to cAMP, we considered a
possible role for other factors. Recent studies have provided evidence
that the ability of cAMP to stimulate PRL promoter activity in
heterologous cells probably involves the closely related coactivator
proteins, CBP and p300 (27, 28). To test the role of endogenous CBP
and/or p300 in mediating responsiveness of the PRL promoter to cAMP, we
used an expression vector for the adenovirus 12S E1a protein, which
blocks CBP and p300 activity (29, 30). We found that increasing amounts
of the E1a expression vector substantially reduced the ability of
forskolin to stimulate PRL promoter activity in
GH3 cells (Fig. 7A
). To test for possible nonspecific
effects of E1a, we compared the wild-type E1a to an E1a mutant
(
235) that is defective for interaction with CBP/p300 (31). The
mutant E1a had much smaller effects on cAMP- and EGF-stimulated PRL
gene expression (Fig. 7B
). These findings offer support for the view
that CBP/p300 plays a role in transcriptional regulation of the PRL
gene. To further explore this topic, we elected to test the ability of
cAMP to stimulate the transcriptional activity of p300. We prepared an
expression vector for a GAL4-p300 fusion that was cotransfected with a
GAL4-dependent reporter gene. The results demonstrate that GAL4-p300
activity is substantially increased by treatment with forskolin (Fig. 7C
). Interestingly, treatment with the MEK1 inhibitor, PD98059, almost
completely blocked the ability of forskolin to enhance GAL4-p300
activity. These findings offer evidence that in
GH3 cells, cAMP stimulates p300 activity in a
MAPK-dependent manner.

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Figure 7. Analysis of a Possible Role for the Coactivator
CBP/p300 in cAMP-Stimulated Activation of the PRL Promoter
A, Analysis of the effects of increasing amounts of an adenovirus E1a
expression vector on forskolin- and EGF-induced PRL reporter gene
activity. GH3 cells were transfected with a PRL reporter
construct and either an empty vector (pcDNA3) or increasing amounts of
E1a expression vector as indicated. The cells were treated with 10
µM forskolin or 10 nM EGF for 6 h after
which the cells were collected and assayed for luciferase activity. B,
Comparison of wild-type and mutant E1a on activation of the PRL
promoter. GH3 cells were transfected with a PRL reporter
construct and 1 µg of an expression vector for either the wild-type
E1a or mutant E1a in which residues 235 have been deleted ( 235).
The cells were treated with 10 µM forskolin or 10
nM EGF for 6 h, and the cells were then collected and
assayed for luciferase activity. C, Analysis of PD98059 effects on
forskolin-induced activation of a GAL4-p300 fusion protein.
GH3 cells were transfected with an expression vector
encoding a GAL4-p300 fusion protein and a reporter construct containing
five GAL4 binding sites. The cells were incubated in serum-free medium
for 24 h and then treated with 100 µM PD98059 for 30
min. Forskolin (10 µM) was added and cells were collected
and assayed 6 h later. Reporter gene activity is reported as light
units from three independent transfections ± SEM
normalized to ß-galactosidase activity.
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DISCUSSION
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These studies provide evidence that cAMP can activate the MAPK
signaling pathway in GH3 cells and that MAPK
activation contributes to the ability of cAMP to stimulate PRL promoter
activity. Analysis of ERK phosphorylation as well as immunocomplex
assay of ERK activity have provided substantial evidence that elevated
cAMP can stimulate MAPK activity. Inhibition of the MAPK pathway was
found to reduce the ability of cAMP to activate the PRL promoter and a
GAL4-Elk1 fusion protein. These findings as well as previous studies
(15, 16, 18, 32) suggest that the cAMP, EGF, and TRH signaling pathways
all converge in GH3 cells to stimulate MAPK
activity, which then plays a role in stimulating PRL gene
expression.
The signaling pathway that mediates cAMP effects on MAPK in
GH3 cells may involve the small GTP-binding
protein, Rap1, although other pathways may be involved. Rap1 has been
shown to play an important role in mediating cAMP-induced MAPK
activation in PC12 cells (20). Rap1 can respond to cAMP through two
different mechanisms. One mechanism involves phosphorylation of Rap1 by
PKA, leading to changes in activity (33). The other mechanism involves
direct binding of cAMP to the Rap1 GDP exchange protein, Epac (34). We
found that elevation of cAMP in GH3 cells leads
to rapid activation of Rap1. An expression vector for Rap1GAP1, which
should inactivate Rap1, reduced cAMP-induced MAPK activation and PRL
promoter activity. On the other hand, Rap1-N17, which should also block
the Rap1 pathway (20), did not reduce cAMP effects on MAPK or the PRL
promoter. It is possible that Rap1-N17 is simply not as effective in
blocking signaling through the endogenous Rap1 pathway as Rap1GAP1.
However, we cannot rule out the possibility that Rap1GAP1 may have
effects on signaling pathways other than Rap1, perhaps blocking the
activity of a related, small GTPase. In some cells, cAMP can modulate
MAPK activity through activation of Ras (35). However, cAMP has little
if any ability to activate Ras in GH3 cells, and
a dominant negative form of Ras did not block cAMP induction of MAPK in
GH3 cells (P. Kievit, unpublished observations).
Another mechanism that might mediate cAMP effects on MAPK would involve
modulation of a protein phosphatase. It has been shown that PKA can
phosphorylate the protein tyrosine phosphatase, PTP-SL, leading to
decreased interaction between the phosphatase and ERK1 and ERK2, thus
allowing for an increase in ERK phosphorylation and activation (36). We
have not tested a possible role for PTP-SL in mediating PKA effects in
GH3 cells.
Concerning the events downstream of MAPK, it seemed likely that members
of the Ets family of transcription factors would be involved in
mediating transcriptional regulation of the PRL gene. Previous studies
have led to a model in which Ras, TRH, or EGF can stimulate MAPK
activity leading to Ets factor phosphorylation and transcriptional
activation involving several specific DNA elements in the PRL gene (15, 16, 26, 37, 38). As the present studies demonstrate that cAMP can
stimulate MAPK activity, it seemed probable that Ets sites would also
play a role in mediating transcriptional responses to cAMP. To test
this possibility, Ets binding sites within the proximal region of the
PRL promoter were mutated, including several Ets sites that have not
previously been studied. Similar to previous studies examining Ras- or
TRH-responsiveness (15, 38), mutation of the Ets site at position -211
decreased cAMP responsiveness to less than 50% of wild-type activity.
Interestingly, this region of the PRL gene has been shown to interact
with the LIM homeodomain transcription factor, Lhx3 (39), and Lhx3
has been shown to enhance Ras responsiveness of the PRL promoter (40).
Mutation of other single Ets sites either had smaller effects or
actually stimulated cAMP responsiveness. The increases in
responsiveness that were observed at some sites may be due to
disruption of the binding of a repressor. It has been reported that
some Ets factors can function as repressors, and one has been shown to
inhibit PRL promoter activity (41, 42). To further explore the overall
role of Ets sites, a PRL promoter reporter gene construct in which all
of the consensus Ets sites were disrupted was prepared. Surprisingly,
the promoter construct with all Ets sites disrupted was even more
responsive to cAMP than the wild-type reporter gene. In contrast,
disruption of all of the consensus Ets sites substantially reduced EGF
responsiveness and partially reduced TRH responsiveness. Thus, it
appears that there is a differential requirement for Ets sites in
mediating regulation of the PRL promoter. The consensus Ets sites are
not required for cAMP responsiveness, but are necessary for full
responses to EGF or TRH. This observation is somewhat surprising in
view of the fact that all of these signaling pathways appear to
converge to activate MAPK. One explanation may involve differing
kinetics of MAPK activation. We found that cAMP- and EGF-induced
activation of ERKs have somewhat different time courses. The effects of
cAMP were observed to have a slower onset and appeared to persist
somewhat longer than EGF effects on MAPK activation. Studies in other
systems have provided evidence that the kinetics of MAPK induction may
have a profound effect on the induced response. For instance, in PC12
cells, transient MAPK activation results in proliferation while
sustained MAPK activation is associated with differentiation (43).
Alternatively, the differential requirement for Ets sites may result
from differences in the combined action of several signaling pathways.
The ability of cAMP to stimulate both PKA and MAPK may lead to
activation of different transcription factors than occurs after
activation of MAPK alone or MAPK combined with other signaling
pathways.
The ability of the PRL promoter to respond to cAMP appears to involve
the transcriptional coactivators, CBP and p300. Previous studies have
used antibody blocking experiments to provide evidence that CBP and
p300 play a role in mediating the ability of PRL promoter to respond to
cAMP (27, 28). We have used an expression vector for adenovirus E1a as
a CBP/p300 antagonist and confirmed that these coactivators appear to
be required for full responsiveness to cAMP. As CBP and p300 can bind
to either Pit-1 or Ets-1 in a constitutive manner (27, 28, 44, 45),
both transcription factors may constitutively recruit CBP/p300 to the
PRL promoter. Our studies with a GAL4-p300 fusion provide evidence that
the transcriptional activity of p300 can be stimulated by elevated cAMP
in a MAPK-dependent manner. Thus, it is possible that CBP/p300 is
constitutively present at the PRL promoter and that the transcriptional
activity of CBP/p300 is modulated by several signaling pathways,
including cAMP, that converge on the MAPK pathway. Alternatively, Xu
et al. (27) have suggested that activation of growth factor
or cAMP pathways leads to recruitment of CBP/p300 to the PRL promoter.
Additional studies assessing the recruitment of CBP/p300 to the PRL
promoter in GH3 cells are required to distinguish
between these two models. The recent development of chromatin
immunoprecipitation assays should provide an appropriate technology to
address this question.
 |
MATERIALS AND METHODS
|
---|
Materials
Tissue culture reagents and media were purchased from Life Technologies, Inc. (Gaithersburg, MD). Antibodies to Rap1, ERK1,
ERK2, phosphorylated ERK, antimouse and antirabbit
horseradish-peroxidase-conjugated IgG and immunoprecipitation reagents
were acquired from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). A polyclonal antibody to phosphorylated ERK was purchased
from New England Biolabs, Inc. (Beverly, MA). Antibodies
to the FLAG epitope and anti-FLAG M2 affinity resin were purchased from
Sigma (St. Louis, MO). The chemical inhibitor PD98059 was
obtained from Alexis Corp. (San Diego, CA), and H-89 and genistein were
purchased from Calbiochem (La Jolla, CA). Forskolin and
chlorophenylthio-cAMP (CPT-cAMP) were purchased from
Sigma. EGF was from Roche Molecular Biochemicals (Indianapolis, IN). TRH was purchased from
Peninsula Laboratories, Inc. (San Carlos, CA).
Radioisotopes and chemiluminescence reagents were obtained from
Dupont-NEN Life Science Products (Boston, MA).
Reporter Genes and Expression Vectors
A PRL reporter construct containing sequences representing the
-255 to +34 region of the rat PRL gene (46) was obtained by PCR and
inserted upstream of the firefly luciferase coding sequence in the
pLuc-Link vector (47). Individual consensus Ets factor binding sites in
the PRL 5'-flanking region were disrupted by oligonucleotide-directed
mutagenesis using PCR. The specific mutations for each site were as
follows: the Ets sites at -211 to -208 and -183 to -180 were
mutated from TTCC to TGAA, the site at -162 to -159 from TTCC to
GGCC, the site at -95 to -92 from GGAA to GTTC, and the site at -75
to -66 from GGAAGAGGAT to GGCCGATTAT.
A GAL4-p300 construct was generated by in-frame subcloning of the full
coding sequence of p300 into a vector containing the GAL4 (1147) DNA
binding domain downstream of the cytomegalovirus promoter (48). The
RSV-PKI vector (4), RSV-Pit-1 vector (8), GAL4-Elk1 (49) construct, and
the luciferase reporter containing five GAL4 binding sites (48) have
been described previously. Dr Philip Stork kindly provided GST-RalGDS
protein and the FLAG-Rap1GAP1 and FLAG-ERK2 expression vectors. The E1a
deletion mutant,
235 (50), was obtained from Dr. James
Lundblad.
Cell Culture and Transfection
GH3 cells were cultured in DMEM
supplemented with 2.5% FBS and 15% equine serum. For transfection,
GH3 cells were seeded into six-well dishes 1 day
before transfection at an approximate density of 500,000 cells per
well. DNA (13 µg total) was transfected using the Lipofectamine
reagent (Life Technologies, Inc.) according to the
manufacturers protocol. After overnight incubation, the cells were
treated with agonists for 6 h, after which the cells were washed,
lysed, and analyzed for luciferase activity. All experiments with the
PD98059 compound were performed in DMEM supplemented with BSA (6.6
mg/ml) to prevent precipitation of PD98059. Cells were transfected with
CMV-ß-galactosidase expression vector as an internal standard
(51).
Immuno-Complex Kinase Assay
MAPK immunoprecipitation assays were performed as previously
described (49). Briefly, GH3 cells were treated
with CPT-cAMP for the indicated time intervals, washed twice with PBS,
and resuspended in lysis buffer (20 mM HEPES-KOH, pH 7.4, 2
mM EGTA, 50 mM ß-glycerophosphate, 10%
glycerol, 1% Triton X-100, 1 mM EDTA, 2 mM
sodium vanadate). ERK1, ERK2, and JNK1 were precipitated using the
appropriate antibody linked to protein A/G agarose, and MAPK activity
was determined by incubating the precipitates with a bacterially
expressed glutathione-S-transferase-Elk1 fusion protein in
the presence of 1 µCi [
-32P]-ATP. The
reactions were stopped with loading buffer, resolved on a 10%
polyacrylamide, denaturing gel, and phosphorylated proteins were
detected by autoradiography.
Immunoblot Analysis
For immunoblot analysis, GH3 cells were
grown to approximately 80% confluency, cultured in serum-free medium
for 24 h, and treated with agonist for specific times. Cells were
lysed in BOS buffer (50 mM Tris-HCl, pH 8.0, 10% glycerol,
1% Nonidet-P40, 200 mM NaCl, 2.5 mM
MgCl2, 2 mM sodium vanadate)
supplemented with a mixture of protease inhibitors (Complete Proteinase
Inhibitor, Roche Molecular Biochemicals). Equal amounts of
protein (50 µg) were loaded on a 10% polyacrylamide, denaturing gel.
Proteins were transferred to a polyvinylidene difluoride membrane
(Millipore Corp., Bedford, MA) before incubation with
antibodies to detect specific proteins. In many cases, membranes were
treated to remove bound antibody to permit immunoblot analysis of a
second protein. The membrane was incubated for 30 min in stripping
buffer (100 mM ß-mercaptoethanol, 1% SDS, 62.5
mM Tris-HCl, pH 6.8), washed in TBS-T (50 mM
Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20), and reprobed
with the appropriate antibody. All antibodies were used at a
concentration of 1:1,000 in TBS-T, 5% nonfat milk, except the FLAG
antibody was used at a dilution of 1:10,000. The proteins were
visualized on radiography film using a secondary antibody conjugated to
horseradish peroxidase and a chemiluminescence detection kit (NEN Life Science Products, Boston, MA). Quantitation of immunoblot
analysis was performed using the Lumi-Imager F-1 system (Roche Molecular Biochemicals).
Affinity Assay for Rap1 Activation
Rap1 activation assays were performed according to Franke
et al. (21). GH3 cells were treated
with forskolin for the indicated times and harvested in BOS buffer
supplemented with complete protease inhibitor. Cell lysates were
cleared by brief centrifugation and adjusted with lysis buffer to a
final concentration of 1 mg/ml. Equal amounts of protein were incubated
for 1 h in the presence of GST-RalGDS Rap1 binding domain
precoupled to glutathione beads. Precipitates were washed three times
with lysis buffer before separation on a denaturing polyacrylamide gel
and immunoblot analysis as described.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Philip Stork and James Lundblad for helpful advice
and generous gifts of a number of reagents. We thank B. Maurer for aid
with these studies.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Richard A. Maurer, Department of Cell and Developmental Biology, L215 Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97201. E-mail:
maurerr{at}ohsu.edu
This research was supported by Public Health Service Grant DK-40339 to
R.A.M.
Received for publication August 8, 2000.
Revision received November 17, 2000.
Accepted for publication December 6, 2000.
 |
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