A Role for the Mitogen-Activated Protein Kinase in Mediating the Ability of Thyrotropin-Releasing Hormone to Stimulate the Prolactin Promoter
Ying-Hong Wang 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 hypothalamic hormone, TRH, stimulates PRL
secretion and gene transcription. We have examined the possibility that
the mitogen-activated protein kinase (MAPK) may play a role in
mediating TRH effects on the PRL gene. TRH was found to stimulate
sustained activation of MAPK in PRL-producing,
GH3 cells, consistent with a possible role in
transcriptional regulation. A kinase-defective, interfering MAPK kinase
(MAPKK) mutant reduced TRH induction of the PRL promoter. Treatment
with the MAPKK inhibitor, PD98059, blocked TRH-induced activation of
MAPK and also reduced TRH induction of a PRL-luciferase reporter gene,
confirming that MAPK activation is necessary for TRH effects on PRL
gene expression. Previous studies have demonstrated that the PRL
promoter contains binding sites for members of the Ets family of
transcription factors, which are important for mediating MAPK
responsiveness of the PRL promoter. Mutation of specific Ets sites
within the PRL promoter reduced responsiveness to both TRH and MAPK.
The finding that DNA elements required for MAPK responsiveness of the
PRL gene colocalize with DNA elements required for TRH responsiveness
further supports a role for MAPK in mediating TRH effects on the PRL
gene. We also explored the signaling mechanisms that link the TRH
receptor to MAPK induction. Occupancy of the TRH receptor results in
activation of protein kinase C (PKC) as well as increases in the
concentration of Ca2+ due to release from
intracellular stores and entry of Ca2+ through
Ca2+ channels. A PKC inhibitor, GF109203X, and
an L-type Ca2+ channel blocker, nimodipine,
both partially reduced TRH-induced MAPK activation and PRL promoter
activity. The effects of the two inhibitors were additive. These
studies are consistent with a signaling pathway involving PKC- and
Ca2+-dependent activation of MAPK, which
leads to phosphorylation of an Ets transcription factor and activation
of the PRL promoter.
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INTRODUCTION
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The ability of TRH to stimulate PRL synthesis and secretion
involves the interaction of the hormone with a G protein-coupled
receptor at the plasma membrane (1, 2). The TRH receptor couples to
Gq to stimulate the activity of phospholipase Cß (3),
leading to increased intracellular levels of diacylglycerol and
inositol 1,4,5-trisphosphate (4). Diacylglycerol activates protein
kinase C (PKC), which is accompanied by redistribution of PKC from a
soluble to a particulate subcellular fraction (5). Inositol
1,4,5-trisphospate stimulates the release of Ca2+ from
intracellular stores, resulting in a first phase of Ca2+
elevation (6). A second phase of Ca2+ elevation is due to
membrane depolarization, which leads to Ca2+ influx through
L-type voltage-sensitive Ca2+ channels (7). Increases in
intracellular Ca2+ lead to activation of a
Ca2+/calmodulin-dependent protein kinase (8). It has also
been shown that TRH can activate the mitogen activated protein kinase
(MAPK)-signaling pathway, also known as the extracellular regulated
kinase (ERK) in GH3 pituitary tumor cells (9). Thus
multiple kinase pathways including PKC,
Ca2+/calmodulin-dependent kinase, and MAPK are activated by
TRH.
The specific signaling pathways that permit TRH to rapidly stimulate
PRL gene transcription (10, 11) have not been clearly defined. There is
evidence that activation of PKC (12),
Ca2+/calmodulin-dependent protein kinases (13, 14), or MAPK
(15) can stimulate the PRL promoter. However, the role that these
pathways play in mediating TRH effects has not been resolved. There is
evidence that PKC may not be required for TRH-induced activation of the
PRL promoter (16, 17). Although inhibition of
Ca2+/calmodulin-dependent protein kinases was found to
reduce the ability of TRH to activate the PRL promoter, the DNA
elements that are required for responsiveness to specific
Ca2+/calmodulin-dependent protein kinases do not colocalize
with TRH-responsive DNA elements (14). Thus, there are questions
concerning the role that PKC and Ca2+/calmodulin-dependent
protein kinases play in mediating TRH effects on the PRL promoter.
In the present study we have examined a possible role for the MAPK
pathway in mediating TRH effects on the PRL promoter. We have used
several approaches to determine whether MAPK activity is required for
TRH effects on the PRL promoter. We have also assessed the role that
PKC activation and Ca2+ signaling play in activating the
MAPK pathway in GH3 cells.
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RESULTS
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Activation of MAPK Can Stimulate PRL Promoter Activity
Previous studies have shown that constitutively active forms of
Ras or Raf can activate the PRL promoter, suggesting that activation of
the MAPK signaling pathway is sufficient to stimulate PRL transcription
(15, 18). While perhaps unlikely, it is possible that Ras or Raf may
lead to signaling events other than activation of MAPK (19, 20). To
further assess the ability of the MAPK pathway to alter PRL promoter
activity, we tested the effects of a constitutively active form of MAPK
kinase (MAPKK), also known as MAPK/ERK kinase (MEK). As the MAP
kinases, ERK1 and ERK2, are the only known substrates of MEK (21), this
approach allows a further test of the ability of MAPK to regulate the
PRL promoter. The expression vector for constitutively active MEK
produced substantial activation of the PRL reporter gene (Fig. 1A
) and had little or no effect on the
thymidine kinase minimal promoter (Fig. 1B
). Thus, MAPK activation
selectively stimulates PRL promoter activity.

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Figure 1. Activation of the MAPK Pathway Can Stimulate PRL
Promoter Activity
Reporter genes containing 255 bp of the proximal region and promoter of
the rat PRL gene linked to firefly luciferase (A) or the herpes simplex
virus thymidine kinase promoter linked to luciferase (B) were
transfected with a control empty vector (control) or an expression
vector for constitutively active MEK (Activated MEK). The cells also
received a control reporter gene that expresses Renilla
luciferase. The cells were collected 48 h after transfection, and
firefly and Renilla luciferase activity was determined.
The values obtained for Renilla luciferase activity were
used to correct for transfection efficiency. Firefly luciferase values
are the average ± SE of three separate
transfections.
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TRH Induces Prolonged MAPK Activation
In some systems, sustained rather than transient activation of a
signaling pathway is required to induce long-term responses. For
instance, in PC12 cells, sustained MAPK activation is associated with
differentiation (22). Although previous studies have shown that TRH can
rapidly activate MAPK (9), these studies examined very early time
points and did not determine whether TRH has more prolonged effects on
the kinase activities. To determine whether TRH-induced MAPK activation
is a reasonable candidate for mediating long-term transcriptional
effects of TRH, the time course of MAPK activation by TRH in
GH3 cells was determined (Fig. 2
). To assess MAPK activation, an
immuno-complex assay was used. For this assay, cell lysates were
immunoprecipitated with an antibody to ERK2, and then the
immunoprecipitated proteins were incubated with [32P]ATP
and GST-Elk1 as the kinase substrate. The immuno-complex assay
demonstrated that TRH-induced ERK2 activation was maximal at the
earliest time point examined, 2.5 min, and declined at later time
points (Fig. 2A
). However, it is important to note that at all TRH time
points, including the 75-min treatment, ERK2 activity of TRH-treated
samples was greater than that of the untreated control. This activation
at later time points has been observed in several different experiments
(data not shown). The use of anti-phospho-ERK antibodies to detect the
activated form of ERKs also yielded results consistent with sustained
activation of MAPK. MAPK is activated by phosphorylation of both
threonine and tyrosine residues (23). Antibodies directed specifically
against the phosphorylated forms of MAPK detect both ERK1 and ERK2.
Increased phosphorylation of ERK1 and ERK2 was detectable for at least
1 h (Fig. 2B
). Analysis of total, immunoreactive ERK1 and ERK2
indicated that TRH-induced increases in ERK phosphorylation were not
due to increases in the amount of these kinases (Fig. 2C
). These
results provide evidence that TRH induces an initial burst of MAPK
activation followed by a lower, but clearly detectable, prolonged phase
of activation.

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Figure 2. Time Course of TRH-Stimulated MAPK Activation
MAPK activation was determined by an immunocomplex assay (A) or by
immunoblotting with an antibody to phospho-ERK (B). GH3
cells were treated with 100 nM TRH for the indicated times,
and cell lysates were prepared. For the immunocomplex kinase assay, the
cell lysates were immunoprecipitated with agarose bead-conjugated
anti-ERK2 antibody. The ERK2 immunoprecipitate was then incubated with
[32P]ATP, and a GST-Elk1 fusion protein was used as a
substrate. The phosphorylated proteins were then resolved by denaturing
gel electrophoresis. For the immunoblot analysis of MAPK activation,
cell lysates were resolved by denaturing gel electrophoresis,
transferred to a membrane, and phospho-ERK visualized by
immunostaining. To assess total levels of MAPK, the blot was reprobed
with an antibody that detects both the activated and nonactivated forms
of ERK2 and more weakly detects both forms of ERK1.
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Activation of MAPK has been shown to lead to phosphorylation and
activation of Elk1, a member of the Ets family of transcription
factors, and a component of the serum response factors (24). To
determine whether TRH-induced MAPK activation is sufficient in
magnitude and duration to alter a transcriptional event, we examined
the activation of a GAL4-Elk1 fusion (Fig. 3
). TRH stimulated expression of the
GAL4-luciferase reporter gene more than 20-fold. Substitution of serine
383 of Elk1 with alanine, which disrupts a critical MAPK
phosphorylation site (24, 25), strongly diminished TRH-induced
activation of Elk1, suggesting that the transcriptional response likely
involves direct phosphorylation of Elk1 by MAPK. These results suggest
that TRH treatment of GH3 cells activates MAPK in a manner
that is sufficient to modulate a transcriptional response.

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Figure 3. TRH Treatment of GH3 Cells Can Increase
the Activity of a MAPK-Responsive Transcription Factor
GH3 cells were transfected with plasmids encoding either
GAL4-Elk1 or mutant GAL4-Elk1 in which serine-383, which is part of an
important MAPK phosphorylation site, was mutated to alanine
(GAL4-ElkS383A). The cells also received a firefly luciferase reporter
gene containing five GAL4-binding sites upstream of a minimal promoter
as well as a control reporter gene that expresses
Renilla luciferase. At 48 h after transfection, the
cells were treated with 100 nM TRH or 10 nM EGF
and then collected 6 h later for analysis of luciferase activity.
Firefly luciferase values are the average ± SE of
three separate transfections that have been corrected for transfection
efficiency.
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MAPK Activation Is Necessary for TRH-Induced Increases in PRL
Promoter Activity
A kinase-defective, interfering mutant form of MEK was used to
investigate the functional role of MAPK. MEK activates MAPK by
phosphorylating MAPK at both threonine and tyrosine residues (23). The
mutant MEK was made by substituting an alanine for a lysine residue
within the ATP-binding site of the enzyme. Although the mutant MEK can
be phosphorylated by Raf, it cannot phosphorylate and activate MAPK
(26, 27) and therefore interferes with signal transduction. To
determine an effective concentration of mutant MEK expression vector,
increasing concentrations of the expression vector were tested for
their ability to reduce TRH-induced GAL4-Elk activation (Fig. 4A
). The MEK mutant reduced both basal
and TRH-induced activation of Gal4-Elk1 in a dose-dependent manner
(Fig. 4A
), suggesting that the MEK mutant can interfere with endogenous
MEK signaling. Based on the titration study, the ability of 2 µg of
the mutant MEK expression vector to inhibit TRH effects on
PRL-luciferase (Fig. 4B
), GAL4-Elk (Fig. 4C
), or the thymidine
kinase-luciferase reporter gene (Fig. 4D
) was compared. This
concentration of the mutant MEK vector had similar effects to reduce
TRH-induced activation of a PRL-luciferase reporter gene and GAL4-Elk1.
In contrast, the mutant MEK had little or no effect on the thymidine
kinase promoter (Fig. 4D
), suggesting that the inhibitory effects on
the PRL promoter are not due to nonspecific inhibition of
transcription.

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Figure 4. Inhibition of MAPK Reduces TRH-Induced PRL Promoter
Activity
GH3 cells were transfected with an expression vector for an
interfering, kinase-deficient mutant MEK (MEK mutant) to inhibit
activation of MAPK. To determine an effective concentration of mutant
MEK expression vector, increasing concentrations of the expression
vector were tested for their ability to reduce TRH-induced GAL4-Elk
activation (A). Based on the titration study, the ability of 2 µg of
the mutant MEK expression vector to inhibit TRH effects on
PRL-luciferase (B), GAL4-Elk (C), or thymidine kinase-luciferase
reporter gene (D) was examined. Cells were treated with TRH at 48
h after transfection and then collected for analysis of luciferase
activity 6 h later. All values are means ± SE
for three separate transfections.
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PD98059 is a selective inhibitor of MEK1 and MEK2 (28, 29). This
inhibitor blocks MAPK activation in several cell types and blocks
processes such as neurite outgrowth in nerve growth factor-treated PC12
cells (30). We first tested whether this inhibitor was able to block
TRH-induced MAPK activation in GH3 cells (Fig. 5
). Treatment of GH3 cells
with 100 µM PD98059 almost completely blocked TRH-induced
ERK2 activation. The effects of PD98059 on TRH-induced activation of
the PRL promoter and GAL4-Elk were then examined (Fig. 6
). PD98059 substantially reduced both
PRL promoter and Gal4 promoter activation by TRH. The results of
studies using an expression vector for an interfering, kinase-defective
MEK as well as the PD98059 studies suggest that MAPKs are required for
TRH-induced activation of the PRL promoter.

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Figure 5. The MEK Inhibitor, PD98059, Blocks TRH-Induced
Activation of MAPK
GH3 cells were untreated or pretreated with 100
µM PD98059 for 1 h and then treated with 100
nM TRH or no additional treatment for 6 h. The cells
were lysed, and ERK2 activity was assessed by immunocomplex kinase
assay using GST-Elk1 as a substrate.
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Figure 6. The MEK Inhibitor, PD98059, Reduces TRH-Induced
Activation of the PRL Promoter
GH3 cells were transfected with either the
255PRL-luciferase construct (A) or an expression vector for GAL4-Elk1
plus a reporter gene containing five GAL4- binding sites upstream of a
minimal promoter linked to luciferase (B). At 48 h after
transfection, the cells were untreated (control) or treated with 100
µM PD98059 as indicated, and after incubation for an
additional hour, half of the cultures were treated with 100
nM TRH. Cells were harvested 6 h after TRH treatment
and luciferase activity was determined. All values are means ±
SE for three separate transfections that have been
corrected for transfection efficiency.
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DNA Elements That Contribute to TRH Responsiveness of the PRL
Promoter Colocalize with MAPK-Responsive Elements
Several binding sites for members of the Ets family of
transcription factors have previously been identified as important for
mediating responses to activation of the MAPK pathway (15, 18). To
determine whether Ets binding sites are also important for TRH
responsiveness, we prepared reporter genes containing block mutations
that disrupt specific Ets-binding sites within the PRL promoter (Fig. 7A
). The wild-type and mutant PRL
reporter constructs were transfected into GH3 cells and
compared for responses to TRH (Fig. 7B
) or an expression vector for
activated MEK (Fig. 7C
). In general, the specific Ets mutations had a
similar pattern of effects on responsiveness to TRH and activated MEK.
Consistent with previous reports (31), disruption of an Ets site
located at -211 to -208 had the greatest effect on reducing
responsiveness to MEK and also had the greatest effect on reducing
TRH-induced reporter gene activity. The Ets sites that were tested are
those that have been reported in previous work to have a possible
involvement in regulating the PRL gene. There are other possible
Ets-binding sites within the proximal region of the PRL gene. At
present, it would be premature to make conclusions about the role of
non-Ets factors in mediating responses to TRH and MAPK. Nonetheless,
the similar pattern of effects of Ets mutations provides evidence that
DNA elements necessary for TRH responsiveness colocalize with DNA
elements required for MAPK responsiveness.

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Figure 7. Colocalization of TRH- and MAPK-Responsive DNA
Elements in the PRL Promoter
Specific Ets sites (solid boxes) that are adjacent to
binding sites for Pit-1 (gray boxes) in PRL promoter
were mutated as indicated (A). Reporter genes containing Ets site
mutations in the context of the 255PRL-luciferase reporter gene (100
ng) were transfected into GH3 cells and tested for
responsiveness to 100 nM TRH treatment for 6 h (B) or
transfection of 150 ng of an expression vector encoding a
constitutively active form of MEK (C). All values are means ±
SE for three separate transfections that have been
corrected for variations in transfection efficiency.
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TRH-Induced Activation of MAPK and the PRL Promoter Is Mediated by
PKC and Ca2+ Influx
Previous studies have used chronic treatment with phorbol esters
to provide evidence that PKC plays a role in TRH-induced MAPK induction
(9). To further test the role of PKC in mediating TRH effects on MAPK
activity, we selected the PKC inhibitor, GF109203X (32), and determined
ERK2 activity using an immunocomplex assay (Fig. 8
). As expected, GF109203X substantially
blocked phorbol myristate acetate (PMA)-induced ERK2 activation,
indicating that the inhibitor successfully blocked PKC-dependent MAPK
activation. The inhibitor partially reduced TRH-stimulated ERK2
activation and had little or no effect on the ability of epidermal
growth factor (EGF) to activate ERK2. While the ability of GF109230X to
reduce TRH-induced MAPK activation is somewhat modest, it has been
consistently observed in several experiments (for instance, see also
Fig. 9
). These results suggest that TRH
appears to induce MAPK activation through both PKC-dependent and
PKC-independent pathways.

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Figure 8. The PKC Inhibitor, GF109203X, Reduces TRH-Induced
Activation of MAPK
GH3 cells were untreated or treated with 5 µM
GF109203X for 20 min, and subsequently treated with 100 nM
TRH for 2.5 min, 10 nM EGF for 5 min, or 100 nM
PMA for 10 min. ERK2 activity was assessed by immunocomplex kinase
assay.
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Figure 9. Analysis of the Time Course of TRH-Induced MAPK
Activation after Treatment with the Ca2+ Channel Blocker,
Nimodipine, or the PKC Inhibitor, GF109203X
GH3 cells were pretreated with 500 nM
nimodipine or 1 µM GF109203X for 20 min and then treated
with 100 nM TRH for the indicated time, and cell lysates
were prepared. ERK2 activity was determined by an immunocomplex kinase
assay.
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TRH also leads to the entry of extracellular Ca2+ through
L-type voltage-sensitive Ca2+ channels resulting in a
sustained, plateau-like elevation of intracellular Ca2+
(33). A role for Ca2+ influx in TRH-mediated activation of
the PRL promoter has previously been described (34, 35, 36). As
Ca2+ influx has been shown to lead to MAPK activation in
some cells (37, 38, 39, 40, 41), it seemed possible that Ca2+ influx
might contribute to TRH-induced MAPK induction. To explore this
possibility, GH3 cells were treated with nimodipine, a
blocker of L-type Ca2+ channels (7). Nimodipine slightly
reduced ERK2 activation at all time points examined (Fig. 9
). At the
later time points, from 10 min to 60 min, ERK2 activation was
approximately half that of control cells. Similar results have been
observed in several different experiments (data not shown). The PKC
inhibitor, GF109203X, had a different time course of effects on ERK2
phosphorylation. GF109203X substantially reduced TRH-induced ERK2
activation at the earliest time point, 2.5 min, and had relatively
little effect at the later time points. The combination of nimodipine
plus GF109203X appeared to reduce ERK2 activation more than either
treatment alone. Similar results were obtained when ERK1 and ERK2
activation was assessed by immunoblotting with a phospho-ERK antibody
(data not shown). These findings suggest that both PKC activation and
Ca2+ influx may play a role in TRH-induced MAPK activation.
PKC appears to play a major role at early times after TRH stimulation
while Ca2+ influx plays a more important role at later time
points.
Inhibitor studies were then used to test the role of Ca2+
influx and PKC activation in mediating TRH-induced PRL promoter
activation. GH3 cells were pretreated with nimodipine,
GF109203X, or a combination of the inhibitors, and the ability of TRH
to stimulate the expression of a PRL-luciferase reporter gene was
examined (Fig. 10
). Both GF109203X and
nimodipine treatment rather strongly blunted the ability of TRH to
stimulate PRL promoter activity, and the combination of the two
inhibitors almost completely blocked TRH effects. These experiments
provide evidence that TRH-induced PKC activation and Ca2+
influx contribute to MAPK induction and activation of the PRL promoter.
To further test the role of Ca2+ influx-induced MAPK
activation in modulating PRL gene expression, the effects of the MEK
inhibitor, PD98059, on Ca2+ influx-stimulated PRL promoter
activity was examined (Fig. 11
).
Control or PD98059-treated GH3 cells were stimulated with
the Ca2+ channel agonist, Bay K8644, which has been
demonstrated to stimulate PRL promoter activity (35, 42). The ability
of PD98059 to substantially reduce Ca2+ influx-stimulated
PRL promoter activity provides evidence that MAPK plays a role in
mediating Ca2+ effects on transcription in this system.

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Figure 10. TRH-Induced Activation of the PRL Promoter Is
Inhibited by Treatment with GF109203X and Nimodipine
GH3 cells were transfected with the 255PRL-luciferase
reporter gene. At 48 h after transfection, the cells were treated
with 500 nM nimodipine or 1 µM GF109203X for
20 min, and then half of the cultures were treated with 100
nM TRH for 6 h. All values are means ±
SE for three separate transfections that have been
corrected for transfection efficiency.
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Figure 11. Ca2+ Influx-Induced Activation of the
PRL Promoter Is Inhibited by Treatment with the MEK Inhibitor, PD98059
GH3 cells were transfected with the 255PRL-luciferase
reporter gene. At 48 h after transfection, the cells were treated
with 100 µM PD98059 for 1 h, and then half of the
cultures were treated with 1 µM Bay K8644 for 6 h.
All values are means ± SE for three separate
transfections that have been corrected for transfection efficiency.
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DISCUSSION
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These studies provide evidence that TRH-induced MAPK activation
likely plays a role in regulating transcription of the PRL gene.
Previous studies have shown that activated Ras can stimulate PRL
promoter activity (15, 18), implying that activation of the MAPK
pathway is sufficient to stimulate PRL gene expression. Our studies
demonstrate that an expression vector for constitutively active MEK
also increases PRL promoter activity. This result offers additional
evidence that it is indeed the MAPK cascade which is sufficient to
activate the PRL promoter. Our work also extends previous studies
demonstrating that TRH can activate MAPK. Our findings demonstrate that
TRH can induce prolonged activation of MAPK. TRH-induced MAPK
activation was observed for at least 1 h, consistent with the
ability of TRH to stimulate prolonged activation of PRL gene
transcription, which persists for hours (10). The finding that TRH can
lead to activation of the GAL4-Elk1 transcription factor offers
additional evidence that TRH can alter a transcriptional event and also
implies that TRH probably induces nuclear localization of MAPK.
Importantly, transfection of a kinase-defective, interfering MEK mutant
or addition of the MEK inhibitor, PD98059, was found to suppress
TRH-induced PRL promoter activity. Our studies thus provide evidence
that TRH activates the MAPK pathway and that MAPK activation is
sufficient and necessary for activation of the PRL promoter.
The finding that DNA sequences of the PRL promoter that are required
for TRH responsiveness colocalize with DNA sequences required for
MAPK-responsiveness reinforces the view that MAPK plays a role in
mediating transcriptional responses to TRH. Previous studies have
provided evidence that binding sites for members of the Ets family of
transcription factors are important for MAPK- and multihormonal
responsiveness of the PRL promoter (18, 31, 43, 44). Therefore, we
tested TRH- and MAPK responsiveness of reporter genes with clustered
point mutations that disrupted several consensus Ets-binding sites.
Disruption of an Ets site at position -211 to -208 of the PRL
promoter had a substantial effect to reduce both TRH- and
MAPK-responsiveness. This Ets site was previously found to be important
for ras-responsiveness (43). Interestingly, although we previously used
multimers of synthetic binding sites to demonstrate that an Ets site at
position -162 to -159 is capable of responding to MAPK activation,
this element was not required for TRH- or MAPK responsiveness within
the context of the normal PRL promoter. It is not clear why the
upstream Ets binding site at -211 to -208 plays the predominant role
in mediating MAPK respon- siveness.
It is probably important that TRH effects on MAPK persist for at least
1 h. The kinetics of MAPK activation can influence nuclear
translocation of MAPK and therefore alter access to nuclear substrates
(22). Therefore, the kinetics of MAPK activation can have important
effects on physiological responses. For instance, in PC12 cells, NGF
causes sustained MAPK activation and nuclear translocation of MAPK and
induces differentiation (45). In contrast, EGF results in a transient
cytoplasmic activation of MAPK that fails to induce differentiation of
PC12 cells (22).
Our studies provide evidence that a PKC-dependent pathway contributes
to TRH effects on both MAPK activation and stimulation of the PRL
promoter. The PKC inhibitor, GF109203X, partially inhibited TRH-induced
activation of the MAPK and PRL promoter activity, providing evidence
that both PKC-dependent and PKC-independent pathways mediate TRH
effects. Our finding that PKC appears to play a role in mediating TRH
effects on PRL gene expression differs from earlier studies, which
concluded that PKC was not involved (35). The previous studies used
chronic phorbol ester treatment to deplete cells of PKC activity while
we used GF109203X. Phorbol ester treatment is a very potent activator
of the PRL promoter, which stimulates transcription for more than
24 h, making it difficult to interpret the effects of PKC
depletion (17). The use of GF109203X avoids the complication of chronic
phorbol ester treatment, which both activates and depletes PKC
activity. GF109203X inhibits the activity of most PKC isoforms (32, 46). It is possible that differences in the effects of GF109203X, as
compared with chronic PMA treatment, reflect differential inhibition of
specific subsets of PKC isozymes.
The present studies also provide evidence that Ca2+ influx
plays a role in mediating TRH effects on both MAPK activation and PRL
gene expression. TRH treatment stimulates a rapid transient increase in
cytosolic Ca2+ as well as a sustained, plateau-like
elevation in Ca2+ levels, which is dependent on influx
through Ca2+ channels (4). Although previous studies have
shown that the L-type Ca2+ channel blockers can inhibit the
ability of TRH to activate the PRL promoter (35, 36), the signaling
mechanisms that permit Ca2+ influx to regulate
transcription have not been identified. Our finding that nimodipine
reduces TRH-induced MAPK activation suggests that MAPK is likely at
least part of the mechanism mediating Ca2+ responsiveness.
Nimodipine effects on TRH-induced MAPK activation were most prominent
at later time points, consistent with the known contribution of
Ca2+ channels to the later, plateau phase of
Ca2+ elevation (4). Furthermore, the MEK inhibitor,
PD98059, blunted the ability of Ca2+ influx to increase PRL
promoter activity, providing evidence that MAPK plays a role in
mediating Ca2+ effects on PRL transcription.
There are many possible mechanisms that may permit Ca2+
influx to activate the MAPK pathway in GH3 cells. In some
cells Ca2+ influx leads to tyrosine phosphorylation of the
EGF receptor in a ligand-independent manner and subsequent activation
of the MAPK by Shc, Grb2, the guanine nucleotide exchange factor Sos1,
and Ras (40). The mechanism responsible for ligand-independent tyrosine
phosphorylation of the EGF receptor is not well established. A
Ca2+-responsive, tyrosine kinase such as PYK2 (38) would be
a candidate for mediating this response. However, we have been unable
to demonstrate the presence of PYK2 in GH3 cells (Y.-H.
Wang, unpublished studies). Src transformation of fibroblasts does lead
to tyrosine phosphorylation of the EGF receptor (39). In addition,
targeted gene disruption of the Src family member Fyn suggests that the
Fyn protein may play a role in calcium-dependent responses in the
nervous system, such as synaptic potentiation and memory formation
(47). Thus, it remains quite possible that some member of the Src
family of tyrosine kinases may contribute to Ca2+ effects
on MAPK activation. Another mechanism that might mediate
Ca2+ effects on MAPK activation would involve a
Ca2+-sensitive guanine nucleotide exchange factor, such as
Ras-GRF (37). Finally, Ca2+ influx may activate MAPK
through Ca2+/calmodulin-dependent kinases (41).
Our studies suggest a model of PRL gene expression in which TRH
generates two different signals, PKC activation and increased
intracellular Ca2+ levels, which converge to activate MAPK.
MAPK likely influences transcription of the PRL gene through
phosphorylation of an Ets transcription factor, probably Ets-1 (31, 48). While this broadly outlines a signaling pathway, there are many
aspects of this pathway that have not been identified. In addition, it
remains possible that pathways other than MAPK activation also
contribute to TRH effects on the PRL promoter. For instance, TRH
activates Ca2+/calmodulin-dependent kinase type II in
GH3 cells (8), and inhibitor studies using KN-62 suggest
that a Ca2+/calmodulin-dependent protein kinase
participates in TRH-induced PRL gene expression (14). However, KN-62
has little or no effect on TRH-induced MAPK activation (Y.-H. Wang,
unpublished result). Thus, a Ca2+/calmodulin-dependent
protein kinase pathway that is independent of the MAPK may also
contribute to TRH-induced PRL gene expression. Although an expression
vector for a constitutively active form of
Ca2+/calmodulin-dependent protein kinase type II can
activate PRL reporter genes that contain the distal enhancer region,
removal of this region almost completely eliminates responsiveness
(14). In contrast, the distal enhancer is not required for TRH
responsiveness (35, 42). Although Ca2+/calmodulin-dependent
protein kinase type II may not be sufficient to activate the proximal
promoter, it remains possible that it acts in concert with the MAPK
pathway to modulate the PRL promoter. Thus, the ability of TRH to
modulate transcription of the PRL gene may depend on a complex
interaction between PKC, MAPK, and perhaps of
Ca2+/calmodulin-dependent protein kinase-signaling
pathways.
 |
MATERIALS AND METHODS
|
---|
Materials
Cell culture media and supplies were purchased from Gibco BRL (Gaithersburg, MD). Radioisotopes and reagents for enhanced
chemiluminescent detection of immunocomplexes were purchased from
DuPont NEN Nuclear (Boston, MA). Anti-ERK2 antibody,
anti-phospho-ERK antibody, agarose beads-conjugated anti-ERK2 antibody,
and horseradish peroxidase-coupled anti-mouse IgG antibody were
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). The MEK inhibitor, PD98059, was purchased from Alexis Corp. (San
Diego, CA); PKC inhibitor, GF109203X, from
Calbiochem (La Jolla, CA); nimodipine from Research Biochemicals International (Natick, MA); EGF from
Boehringer Mannheim (Indianapolis, IN); TRH from
Peninsula Laboratories, Inc. (Belmont, CA).
Reporter Genes and Expression Vectors
A reporter gene containing the proximal 255 bp of the
5'-flanking region of the rat PRL gene was prepared by PCR
amplification of the appropriate region from the PRL gene (49), which
was inserted upstream of the firefly luciferase-coding sequence in the
promoterless construct, pLuc-Link (50). PRL reporter genes containing
mutations in specific Ets factor-binding sites were prepared by
oligonucleotide-directed mutagenesis. The specific mutations involved
mutation of the Ets site at -211 to -208 with the sequence TTCC to
the sequence TGAA, the Ets site at -162 to -159 from TTCC to AGGC,
and the dual Ets site at -75 to -66 from GGAAgaGGAT to GGCCgaTTAT. A
luciferase reporter gene containing 5 GAL4 binding sites upstream of a
minimal promoter, as well as an expression vector for a Gal4-Elk1
fusion protein, have been described previously (51, 52). A reporter
gene, pRL, expressing Renilla luciferase, was purchased from
Promega Corp. (Madison, WI).
A mammalian expression vector for kinase-defective MEK1 mutant (53) was
generously provided by Dr. Edwin G. Krebs. An expression vector
encoding a constitutively active form of MEK1 was generated by
replacing Ser 218 and Ser 222 by glutamic acid and cloned into pCDNA3
vector (45).
Culture and Transfections
GH3 cells were cultured in DMEM supplemented with
15% horse serum, 2.5% FBS, 100 U/ml penicillin, and 100 µg/ml
streptomycin. For transient transfection assays, 2.5 x
105 cells per well were planted in six-well plates 1 day
before the transfection. DNA was introduced into these cells using
lipofectamine (Gibco BRL) in serum-free medium according
to a protocol provided by the manufacturer. In each experiment the
total amount of transfected DNA per culture dish was constant (usually
at 2 µg). After 5 h of treatment with the lipofectamine/DNA
mixture, an equal volume of serum-containing culture medium was added.
After an additional 18 h, the cells were transferred to serum-free
medium. TRH treatments were applied 24 h after transfer to
serum-free medium, and the cells were then lysed 56 h later for
reporter gene analysis. In most experiments, cells were transfected
with a firefly luciferase reporter gene and, as an internal standard
for transfection efficiency, a control plasmid that expresses
Renilla luciferase. Renilla luciferase requires a
different substrate than firefly luciferase and can be assayed
independently. Cells were lysed, and the activities of firefly and
Renilla luciferase were determined using a protocol and
reagents from Promega Corp.. Total firefly luciferase
light units were normalized to total Renilla luciferase
activity. The results of transfection studies are reported as
means ± SE of the mean for several separate
transfections (individual culture dishes) that were performed as part
of the same experiment.
Immunoblotting
For analysis of protein expression, cells were grown to
approximately 80% confluency, transferred to serum-free medium, and
then treated with inhibitors and agonists as indicated. After
treatment, the cells were washed twice with ice-cold 0.15 M
NaCl, 0.01 M NaPO4 (pH 7.4). The cells were
lysed for 10 min on ice in 20 mM Tris, pH 7.5, 1% Triton
X-100, 10% glycerol, 50 mM ß-glycerolphosphate, 2
mM EGTA, and 1 mM dithiothreitol. The lysates
were centrifuged for 20 min at 12,000 x g. The
supernatants (100 µg protein) were adjusted to contain 1% SDS and
5% 2-mercaptoethanol and heated for 3 min in a boiling water bath
before electrophoresis on an SDS-containing polyacrylamide gel (54).
Proteins were then transferred by electroblotting to a polyvinylidene
difluoride membrane. The membranes were incubated in 0.15 M
NaCl, 0.01 M NaPO4 (pH 7.4) containing 3% BSA
and 0.01% Tween 20 for 1 h and then incubated with a primary
antibody for 1 h. After six washes with 0.15 M NaCl,
0.01 M NaPO4 (pH 7.4), 0.01% Tween 20,
horseradish peroxidase-conjugated secondary antibody was incubated with
the blot for 30 min. After extensive washing, the proteins were
detected by using the enhanced chemiluminescence system (DuPont NEN).
ERK Immunocomplex Kinase Assay
Cells were treated and lysed as described for immunoblotting.
Cell extracts were incubated with agarose-conjugated anti-ERK2 antibody
for 12 h. Immunoprecipitates were washed once in lysis buffer, twice
in 500 mM NaCl, 100 mM Tris-HCl, pH 7.5, 0.1%
Triton X-100, and 2.5% sucrose and once in kinase assay buffer (20
mM Tris-HCl, pH 7.5, 10 mM MgCl2,
0.1% Triton X-100, and 2 mM EGTA). In vitro
kinase assays were carried out for 25 min at 30 C in 20 µl of kinase
assay buffer supplemented with 1 µCi of [
-32P]ATP
and 5 µg glutathione S-transferase fusion protein, which
includes the carboxy-terminal transcriptional activation of Elk1
(residues 307428) including several MAPK phosphorylation sites (24).
The GST-Elk1 fusion protein was expressed in Escherichia
coli and purified using glutathione-Sepharose beads (55). The
kinase reactions were stopped by adding SDS to 1% (wt/vol), the
reactions were resolved on a denaturing, 10% polyacrylamide gel, and
the phosphorylated proteins were detected by autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We thank Denis Glenn for preparing an expression vector for
constitutively active MEK, Paul Kievit for preparing some of the PRL
reporter constructs used in this study, and Bobbi Maurer for aid in
preparing this manuscript.
 |
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.
This research was supported by NIH Grant DK-40339 to R. Maurer.
Received for publication February 26, 1999.
Revision received April 16, 1999.
Accepted for publication April 20, 1999.
 |
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