Serotonergic Repression of Mitogen-Activated Protein Kinase Control of the Calcitonin Gene-Related Peptide Enhancer
Paul L. Durham and
Andrew F. Russo
Department of Physiology and Biophysics University of Iowa
Iowa City, Iowa 52242
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ABSTRACT
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We have investigated the mechanisms underlying
regulation of the calcitonin gene-related peptide (CGRP) cell-specific
enhancer. Recently, we reported that this enhancer is inhibited by
serotonin type-1 (5-HT1) agonists, similar to
currently used antimigraine drugs. We have now tested whether this
repression involves a mitogen-activated protein (MAP) kinase pathway.
We first demonstrate that the CGRP enhancer is strongly (10-fold)
activated by a constitutively active MAP kinase kinase (MEK1), yielding
reporter activities 100-fold above the enhancerless control. The
involvement of a MAP kinase pathway was confirmed by down-regulation of
reporter activity upon cotransfection of a dominant negative Ras.
Activation of the enhancer by MEK1 was blocked in a dose-dependent
manner by the 5-HT1 receptor agonist CGS 12066A
(CGS). Since it is not known whether the CGRP enhancer factors are
immediate targets of MAP kinases, we then used Elk-1- and
c-Jun-dependent reporter genes that are directly activated by the ERK
(extracellular signal-regulated kinases) and JNK (c-Jun N-terminal
kinase) MAP kinases. CGS treatment repressed the activation of both of
these reporters, suggesting that at least two MAP kinases are the
immediate targets of CGS-mediated repression. We further demonstrate
that 5-HT1 agonists inactivate ERK by
dephosphorylation, even in the presence of constitutively activated
MEK1. This inactivation appears to be due to a marked increase in the
level of MAP kinase phosphatase-1. These results have defined a novel
and general mechanism by which 5-HT1 receptor
agonists can repress MAP kinase activation of target genes, such as
CGRP.
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INTRODUCTION
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The calcitionin/calcitonin gene-related peptide (CT/CGRP) gene
encodes two biologically active peptides (1). The hormone CT is
produced in thyroid C cells, while CGRP is expressed in neurons. CT
acts to lower serum calcium levels (2), and CGRP is the most potent
peptide vasodilator known and helps to maintain cardiovascular
homeostasis (3, 4). High serum levels of CGRP are associated with
several pathological conditions, including migraines (5, 6). Recently,
a serotonin type-1 (5-HT1) antimigraine drug was shown to
selectively reduce CGRP levels and alleviate pain (7, 8). However,
little is known about the mechanisms that cause the elevated CGRP
levels or by which the 5-HT1 drugs might down-regulate CGRP
levels.
Regulation of CT/CGRP gene expression in response to extracellular
stimuli is controlled exclusively at the transcriptional level. The
cell-specific HLH-OB2 (HO) enhancer is synergistically activated by a
helix-loop-helix (HLH) protein, USF (upstream stimulatory factor), and
an octamer-binding protein, OB2 (9, 10). Activation of this regulatory
element is sufficient and required for cell-specific expression (10).
Inhibition of gene transcription by glucocorticoids (11), retinoic acid
(12), and possibly vitamin D (13) have all been shown to involve the
cell-specific enhancer. Recently, we demonstrated that
5-HT1 agonists inhibited CGRP mRNA levels and repressed
promoter activity through two discrete elements: the cAMP-responsive
element (CRE) and the HO enhancer in CA77 rat medullary thyroid
carcinoma cells (14). CA77 cells provide a useful neuronal model system
that is characterized by high levels of CGRP and expression of
5-HT1B receptor mRNA (15). We also reported that activation
of the 5-HT1 receptors caused a robust and sustained
increase in intracellular calcium that is likely responsible for
mediating repression of the CGRP promoter (14). Elevated levels of
calcium have been reported to control transcription by phosphorylation
of DNA-binding proteins by kinases, including the mitogen-activated
protein (MAP) kinase family (16).
The MAP kinases are the focal points of multiple signaling cascades
that are important in transmitting extracellular signals to the nucleus
(17). The MAP kinase family includes the extracellular signal-regulated
kinases (ERKs), and the stress activated kinases, c-Jun N-terminal
kinase (JNK) and p38 kinase (18). After activation by kinases that
phosphorylate MAP kinases, the MAP kinases are generally translocated
into the nucleus where they activate transcription factors (19).
Previous studies have provided evidence that CT/CGRP gene expression is
regulated by factors that activate the MAP kinase pathways. Nerve
growth factor (NGF), a known activator of the ERK pathway (19), has
been shown to increase CGRP levels in neurons and cell lines (20, 21).
Similarly, treatment with phorbol esters, which can also stimulate the
ERK pathway (22), caused an increase in CT/CGRP expression (23).
Recently, Nelkin and colleagues (24) demonstrated that the Ras/MAP
kinase pathway can increase CT/CGRP transcription through an element
near the CRE that binds a novel zinc-fingered protein. Those studies
did not test promoter sequences containing the HO enhancer; hence, the
question of whether the enhancer is NGF- or Ras-responsive has been
left open.
In this study, we investigated the mechanism by which the
5-HT1 receptor agonist CGS 12066A (CGS) causes repression
of the cell-specific HO enhancer. We determined that activity of the
cell-specific enhancer is positively regulated by a MAP kinase pathway
and that CGS can greatly repress this activation. The inhibition is
mediated by a decrease in ERK phosphorylation, apparently due to an
increase in MAP kinase phosphatase levels. Results from our studies
provide evidence for a new mechanism by which 5-HT1
receptor agonists can regulate gene expression by repressing MAP kinase
activity.
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RESULTS
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Regulation of the HO Enhancer by a Ras/ERK Pathway
CA77 cells were cotransfected with a reporter plasmid containing
the CGRP enhancer and a second plasmid encoding constituitively active
MAP kinase kinase (MEK1). The reporter plasmid contained three copies
of the 18-bp HO enhancer linked to the thymidine kinase (TK) promoter
(Fig. 1
). As mentioned above, the HO
enhancer contains HLH and octamer-binding motifs. It does not include a
CRE and is not cAMP-responsive (14). MEK1 is a highly specific kinase
that has been reported to activate only the ERK MAP kinase (25).
Overexpression of MEK1 resulted in a 10-fold increase in activity (Fig. 1
). As a control, MEK1 had no effect on the parental TK vector. Further
confirmation of ERK activation of the enhancer was provided by
cotransfection of an expression vector encoding a mutant-signaling
protein, dominant negative Ras (N17Ras). Expression of N17Ras caused a
greater than 2-fold decrease in HO enhancer activity (Fig. 1
). The
parental TK vector was not inhibited by N17Ras. These data demonstrate
that the CGRP enhancer is regulated by a signaling pathway involving
Ras and the downstream ERK MAP kinase.

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Figure 1. Regulation of the CGRP Enhancer by an ERK MAP
Kinase Pathway
A, CA77 cells were transfected with luciferase reporter genes
containing the HO enhancer-TK promoter or TK promoter alone. A
constituitively activated MEK1 expression vector or a dominant negative
Ras (N17Ras) expression vector were cotransfected with the reporter
plasmids. After incubation for 2024 h, luciferase activity was
determined and expressed as relative light units per 20 µg of
protein. The means and SE from at least three independent
experiments are shown. Statistically significant inhibition by N17Ras
(*, P < 0.05) and stimulation by MEK1 (#,
P < 0.01) are denoted. B, Sequence of the HO
enhancer. The overlapping USF- and OB2-binding sites are indicated.
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CGS Repression of MEK1-Stimulated HO Enhancer Activity
Having shown that the cell-specific enhancer is stimulated by a
MAP kinase pathway, we then asked whether the 5-HT1
receptor agonist CGS could repress this activation. CGS treatment of
transiently transfected CA77 cells caused repression of MEK1-stimulated
CGRP enhancer activity by almost 5-fold (Fig. 2A
). This repression was specific to the
enhancer since the reporter containing only the TK promoter was
relatively unaffected (Fig. 2A
). For comparison, CGS treatment
repressed the basal HO enhancer activity about 5-fold, but had little
effect on the TK-luciferase reporter, as previously reported (14) (Fig. 2A
). CGS repression of the HO enhancer was dose-dependent with
half-maximal inhibition at approximately 3 µM (Fig. 2B
).
This is similar to the value we previously reported for CGS repression
of basal enhancer activity (14). In addition, our preliminary data
indicate that CGS can repress phorbol ester stimulation of the enhancer
(data not shown). These results indicate that
5-HT1-mediated repression of the CGRP enhancer is due to
inhibition of an ERK MAP kinase pathway.

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Figure 2. Repression of MEK1-Stimulated HO Enhancer Activity
by CGS
A, CA77 cells were transfected with luciferase reporter genes
containing the HO enhancer-TK promoter or TK promoter with or without a
CMV-MEK1 expression vector. The cells were pooled and divided into
parallel dishes that were treated with the vehicle (-) or 10
µM CGS (+). After a 6-h treatment with CGS, the medium
was removed, replaced with fresh medium (without CGS), and the cells
were allowed to incubate for an additional 18 h. The mean reporter
activity per 20 µg of protein with the SE is shown from three
independent experiments. B, The effect of varying concentrations of CGS
(6 h incubation) on MEK1-stimulated enhancer activity is shown. The
activities were normalized to the untreated, MEK1-stimulated cells
(control). The means and SE from three independent
experiments are shown.
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CGS Repression of MAP Kinase Activity
To address the step at which 5-HT1 agonists repress
the ERK MAP kinase pathway, we used a set of reporter genes controlled
by factors that are known to be directly phosphorylated by MAP kinases.
These reporter genes were chosen since we do not know whether ERK
directly activates the HO enhancer proteins, or whether there are
additional downstream steps. CA77 cells were cotransfected with three
plasmids: 1) a transactivator gene encoding the transactivation domains
of either Elk-1 or c-Jun linked to the yeast Gal4 DNA-binding domain,
2) a luciferase reporter gene containing Gal4 DNA-binding sites, 3) an
upstream MAP kinase activator gene, encoding constituitively activated
forms of either MEK1 or MEK kinase (MEKK). MEK1 activates only the ERK
kinase, while MEKK activates kinases that in turn activate ERK (via
MEK1 activation) and JNK (via MEK4/7 activation) (26). Since there is
no endogenous Gal4 in mammalian cells, this assay is very specific for
measuring transactivation of the fusion proteins.
CGS treatment caused significant repression of both Elk-1 and
c-Jun-dependent reporter genes even in the presence of
constitutively activated MEK1 or MEKK (Fig. 3
). Because of the specificity of MEK1,
we can conclude that CGS is repressing ERK activity. Similarly, because
the c-Jun-dependent reporter is preferentially activated by JNK and not
ERK (18), this indicates that CGS can also repress JNK. There was
little or no detectable reporter activity above background in the
absence of MEK1 or MEKK activation, so it was not possible to determine
whether basal activities were repressed by CGS. Thus, our data indicate
that CGS represses the action of two MAP kinases, ERK and JNK, even in
the presence of constitutively activated upstream activators.

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Figure 3. CGS Repression of c-Jun and Elk-1 Activation of
Reporter Genes
CA77 cells were transfected with a Gal4-luciferase reporter plasmid and
plasmids encoding fusion proteins of the Gal4 DNA-binding domain and
transactivation domains of either c-Jun or Elk-1. Constituitively
activated MEKK or MEK1 expression vectors were cotransfected with the
reporter plasmids, and the cells were incubated in the absence (-) or
presence (+) of 10 µM CGS for 6 h as described in
Fig. 2 . The mean luciferase activity per 20 µg protein ±
SE from three experiments is shown.
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CGS-Mediated Decrease in the Level of Phosphorylated ERK
The repression of MEK1 activation of the Elk-1-responsive reporter
gene suggested that CGS treatment was inhibiting ERK activity.
Activation of ERK is mediated by phosphorylation of specific threonine
and tyrosine residues by MEK1. To test whether CGS was
mediating a decrease in phosphorylated ERK, we used Western
blot analysis of cell lysates with phospho-specific antibodies directed
against ERK1 (44 kDa) and ERK2 (42 kDa). In untreated control cells, a
relatively low level of phosphorylated ERK proteins was detected (Fig. 4A
). The basal level of phosphorylated
ERK was markedly inhibited by CGS treatment. This finding is in
agreement with CGS-mediated repression of basal CGRP enhancer activity
(Fig. 2
). Osmotic shock with sorbitol, an agent known to increase
phosphorylation of MAP kinases (17), caused a robust increase in ERK
phosphorylation (Fig. 4
). Similarly, overexpression of MEK1 resulted in
a large increase in the level of phosphorylated ERK1 and ERK2. In
contrast, CGS treatment repressed MEK1-induced levels of phosphorylated
ERK. Interestingly, the inhibitory effect was maintained for at least
18 h after the removal of CGS. This correlates well with the time
course of CGS repression of CGRP promoter activity (14). Thus, the
inhibitory effect of CGS on MEK1-stimulated HO enhancer activity is
mediated by reducing the level of active ERK.

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Figure 4. Inhibition of ERK Phosphorylation by
5-HT1 Receptor Agonists
CA77 cells were untreated (con), treated with 10 µM CGS
for 6 h as described in Fig. 2 (CGS), or stimulated with 0.6
M sorbitol (sorb) for 30 min. Cells transfected with the
MEK1 expression vector were either not treated (MEK1) or treated with
10 µM CGS (6 h), 10 µM mCPP (24 h), or 10
µM TFMPP (24 h). A, Cell lysates were analyzed by Western
blots probed with the antiactive ERK antibodies that recognize only the
phosphorylated ERK proteins. The positions of ERK1 (44 kDa) and ERK2
(42 kDa) are indicated. B, The same blots as in panel A were stripped
and reprobed with antibodies that recognize both the unphosphorylated
and phosphorylated forms of ERK.
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In addition, the effect on the ERKs was investigated using two other
5-HT1 agonists, N-(3-trifluoromethylphenyl)
piperazine hydrochloride (TFMPP), and 1-(3-chlorophenyl) piperazine
hydrochloride (mCPP). These agents were tested since we have previously
shown that they repress basal activity of the HO enhancer (14). Similar
to CGS, both TFMPP and mCPP caused a decrease in MEK1-stimulated ERK
levels (Fig. 4A
).
As a control, the same blots were stripped and reprobed with antibodies
that recognize both phosphorylated and unphosphorylated forms of ERK1
and ERK2. There was little or no change in the total levels of ERK1 and
ERK2 after the various treatments (Fig. 4B
). Hence, the decrease in
active ERK levels is not due to changes in the amount of ERK proteins.
These results provide evidence that 5-HT1 receptor
activation is coupled to decreased levels of active phosphorylated
ERK.
CGS Increases the Level of MAP Kinase Phosphatase-1
The activity of MAP kinases is controlled by dual-specific protein
phosphatases that dephosphorylate both the regulatory threonine and
tyrosine residues (27). To determine whether CGS was mediating an
increase in phosphatase level, CA77 cell lysates were analyzed by
Western blots using MAP kinase phosphatase-1 (MKP-1)-specific
antibodies. MKP-1 has been shown to dephosphorylate multiple MAP
kinases, including ERK (27). A relatively low level of basal MKP-1 was
detected in untreated cells (Fig. 5
).
Similarly, low levels of MKP-1 were detected in cells overexpressing
MEK1. However, CGS treatment caused a marked increase in MKP-1 levels,
even in the presence of activated MEK1 (Fig. 5
). This increase was
detected after only 2 h of CGS treatment (not shown) and was
maintained for at least 18 h after removal of CGS-containing media
(Fig. 5
), which agrees with the prolonged repression of ERK
phosphorylation (Fig. 4
) and CGRP enhancer activity (14). Based on data
from our ERK and MKP-1 studies, we conclude that CGS decreases the
level of phosphorylated ERK due to an increase in MKP-1 activity.

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Figure 5. Induction of MKP-1 by CGS
CA77 cells were transfected with the MEK1 expression vector or an
equivalent amount of a CMV control vector (con). Cells were treated
with 10 µM CGS for 6 h. The expression of MKP-1 was
determined by Western blot analysis using specific anti-MKP-1
antibodies. The MKP-1 immunoreactive band is indicated by an
arrow. The same cell lysates used in Fig. 4 are shown in
this figure.
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DISCUSSION
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The focus of the present study was to elucidate the molecular
mechanism of serotonergic repression of the CT/CGRP cell-specific
enhancer. We first demonstrated that enhancer activity was up-regulated
by ERK, a MAP kinase generally responsive to mitogenic signals. One
significant aspect of MAP kinase activation of CGRP transcription is
that it allows a means for coordinate regulation of CGRP with other
neuropeptide genes (28). This coordinated response may be particularly
relevant to diseases, such as migraine, which involve neurogenic
inflammation (29). In the neurogenic model of migraine, an undefined
stimulus causes release of the inflammatory neuropeptides CGRP and
substance P from trigeminal nerve terminals. In this regard, two agents
known to activate MAP kinases, bradykinin (30) and phorbol esters (22),
have also been shown to increase the release of CGRP and substance P
from cultured sensory neurons (31), and inflammation of peripheral
joints leads to increased CGRP peptide and mRNA levels in the dorsal
root ganglia (32).
Since neuronal activity can activate MAP kinases (33, 34), it is likely
that activation of sensory trigeminal neurons increases CGRP synthesis
during migraine. Inflammatory signals such as bradykinin that activate
MAP kinase pathways would then be predicted to perpetuate the
stimulated synthesis of CGRP. While CGRP levels throughout the duration
of migraine are not known, long-term elevation of CGRP synthesis is
consistent with episodes lasting for periods as long as 72 h (8).
MAP kinase activation may also be important for homeostatic control of
CGRP levels. NGF, which activates ERK (19), increases CGRP gene
expression in adult dorsal root ganglia in vivo (20). In
cell lines, NGF acts in a cell-specific manner through the complex CRE
and upstream sequences that include the HO enhancer (21). Indeed, NGF
caused a small (
2-fold) increase in HO enhancer activity and a
slight elevation in the level of phosphorylated ERK in CA77 cells (data
not shown). These observations support the physiological relevance of
MAP kinase control of the CT/CGRP gene.
We have previously reported that 5-HT1 receptor agonists
inhibit the CGRP cell-specific HO enhancer (14). In this study, we show
that the 5-HT1 agonist CGS can repress MEK1-stimulated
enhancer activity by mediating a decrease in the level of activated
ERK. Thus, repression of MAP kinase activity provides a plausible
mechanism for the regulation of CGRP levels by 5-HT1 drugs
in inflammatory diseases, such as migraine. In addition, CGS also
inhibited the activity of another MAP kinase, JNK. It is possible that
the CGRP enhancer might also be activated by other MAP kinase pathways,
including JNK. There is precedence for single transcription factors
being regulated by multiple MAP kinases (18). To our knowledge, this is
the first demonstration of 5-HT1 receptors coupling to
repression of MAP kinase pathways.
We have shown that CGS treatment of CA77 cells resulted in a sustained
increase in the level of the MAP kinase phosphatase MKP-1. MKP-1 is a
member of a growing family of dual-specificity protein phosphatases
that inactivate MAP kinases by dephosphorylating the regulatory
threonine and tyrosine residues (27). MKP-1 can dephosphorylate
multiple MAP kinases (27). Hence, MKP-1 is capable of mediating
5-HT1 repression of ERK and JNK activity, although it is
possible that additional MKPs may also be involved. Several lines of
evidence support the prediction that induction of an MKP is responsible
for the repression of CGRP activity. First, the fact that CGS
counteracted the constitutively activated MEK1, as measured by reporter
gene activity and ERK phosphorylation, shows that the CGS-mediated
increase in MKP-1 levels exerts a dominant effect. This type of
repression has recently been reported for angiotensin type 2 repression
of MAP kinase activity (35) and possibly, insulin action (36). Second,
since MEK1 is constitutively activated, then it is highly unlikely that
CGS could be repressing it, which leaves direct dephosphorylation of
ERK by an MKP as the most likely consequence. Third, the long-term
effect of MKP-1 induction is consistent with the time course of CGS
repression (14). The role of phosphatases in regulating CGRP expression
has not been previously addressed, although it is interesting that
treatment of dorsal root ganglia neurons with the phosphatase inhibitor
okadaic acid increased the release of CGRP (37).
How does activation of 5-HT1 receptors increase MKP-1
levels? A likely mechanism is that 5-HT1 receptor
activation increases MKP-1 gene expression via a calcium-dependent
pathway. While the 5-HT1 receptors are classically viewed
as Gi-coupled proteins that decrease adenylate cyclase
activity (38), they have also been reported to elevate intracellular
calcium (14, 39, 40). We have previously shown that both CGS and
sumatriptan cause a prolonged rise in intracellular calcium levels in
the CA77 cell line and primary cultures of trigeminal neurons (Ref. 14
and our unpublished observations). Direct support for the role of
calcium has been provided by Meloche and colleagues (41), who have
recently shown that increased intracellular calcium was necessary and
sufficient for induction of MKP-1 mRNA and protein expression in a
fibroblast cell line.
We propose the following model to describe how 5-HT1
agonists repress activity of the CGRP cell-specific enhancer (Fig. 6
). Activation of a MAP kinase pathway by
extracellular stimuli initiates a cascade involving ERK, and possibly
other MAP kinases, that ultimately leads to an increase in HO enhancer
activity. It is not known whether MAP kinases can directly
phosphorylate the HO enhancer-binding proteins, USF and OB2. Repression
of enhancer activity is initiated by the binding of an agonist to
5-HT1 receptors, which causes a sustained increase in
intracellular calcium. The rise in calcium might cause an initial
activation of the ERK pathway (34), but the dominant consequence is the
subsequent elevation of MKP-1. MKP-1 then dephosphorylates ERK and
other MAP kinases, thus diminishing the stimulatory effect on the CGRP
enhancer. This mechanism might also account for our previously observed
cAMP-independent repression of the CGRP CRE (14), since the CRE binding
protein, CREB, can also be activated by a MAP kinase pathway (42).
Based on our model, this repression would be shared by all genes
regulated by MAP kinases, as shown with the Elk-1 and c-Jun reporters.
Hence, 5-HT1 induction of MKP-1 is predicted to underlie a
general mechanism of gene regulation. For example, this pathway may be
an important component of the inhibitory feedback role of
5-HT1 receptors on serotonergic neurons. In conclusion, the
results of our studies have defined a novel mechanism by which
5-HT1 receptor agonists can regulate gene expression by
repressing MAP kinase activity.

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Figure 6. Model of Extracellular Control of CGRP Enhancer
Activity
Activation of the enhancer by extracellular stimuli that signal via the
ERK MAP kinase pathway is schematically shown. Whether ERK directly
phosphorylates the HO enhancer proteins is not known. The enhancer is
repressed by 5-HT1 receptor agonists that cause a prolonged
elevation of intracellular calcium and increased levels of MKP-1
protein. ERK and other MAP kinases are then deactivated by MKP-1,
leading to repression of HO enhancer activity. The mechanism of
inhibition of cAMP stimulation of the CRE is not known, but may also
involve MKP-1.
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MATERIALS AND METHODS
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Cell Culture
CA77 cells were maintained as previously described (14). Cells
used for Western blots and transient transfection assays were
subcultured in serum-free medium supplemented with insulin,
transferrin, and selenium (ITS, Collaborative Biomedical Products,
Bedford, MA) 24 h before treatment, as described (11, 14). The
pyrroloquinoxaline CGS 12066A monomaleate, TFMPP, and mCPP were
obtained from Research Biochemicals International (RBI, Natick, MA) and
prepared as described (14). In all studies, the cells were treated with
equivalent amounts of vehicle. NGF (2.5 S) was purchased from Life
Technologies (Grand Island, NY).
Plasmids and Transfection Assays
The rat CT/CGRP and TK promoter luciferase reporter plasmids
have been described previously (11, 14). The HO-TK enhancer plasmid
used in our study contains three tandem repeats of an 18-bp sequence
with flanking BamHI ends (in lower case)
(ggatccGGCAGCTGTGCAAATCCTggatcc) (-1043 to -1025 of the rat CT/CGRP
promoter) (9). The plasmid containing dominant negative Ras mutant,
N17Ras (43), was provided by J. Pessin (University of Iowa, Iowa City,
IA). The following plasmids were obtained from Stratagene (La Jolla,
CA): MEK1 (S218/222E,
3251) (44) and MEKK (380672) (45)
expression vectors, the transactivator plasmids containing ELK1
(307428) and c-JUN (1223) activation domains fused to the Gal4
DNA-binding domain (1147), and the Gal4 reporter plasmid.
CA77 cells were transiently transfected by electroporation essentially
as described previously (14). Approximately 24 x
106 cells were transfected with 1015 µg luciferase
reporter plasmid DNA, 20 µg N17Ras plasmid, and/or 25 µg kinase
expression plasmid DNA using a Bio-Rad gene pulsar apparatus.
Transfected cells were equally divided between 60-mm dishes containing
serum-free medium and typically treated with either CGS or vehicle
control (0.0001 N HCl) for 6 h, after which the media
were replaced with serum-free media and cells were allowed to incubate
for an additional 18 h. This experimental protocol ensured that
the control and drug-treated cells had equal transfection efficiencies.
The cells were incubated with CGS for only 6 h since this time was
sufficient for maximal inhibition (data not shown), as previously shown
for the 1.3-kb CT/CGRP-luciferase reporter (14). In experiments
designed to determine the effect of CGS on MEK1-stimulation, CA77 cells
were transiently transfected with a luciferase reporter plasmid and
either MEK1, MEKK, or N17Ras. Cotransfected cells were then incubated
for 6 h in medium containing CGS (10 µM) or vehicle,
after which the media were replaced and the cells were allowed to
continue to incubate overnight. Luciferase activity was measured using
reagents from Promega (Madison, WI). Each experimental condition was
repeated in at least three independent experiments done in duplicate.
Transfection efficiencies were estimated to be 3060% based on
X-gal staining of cells transfected with a cytomegalovirus
(CMV)-ß-galactosidase reporter gene. Statistical analyses were done
using Students t test (unpaired samples).
Western Blot Analysis
CA77 cells were treated as described for the reporter assays
with CGS treatments for 6 h. The other 5-HT receptor agonists (10
µM), TFMPP and mCPP, were added immediately after
transfection and remained in the media until the cells were harvested
after 24 h. Sorbitol (0.6 M) and NGF (50 ng/ml)
treatments were for 30 min before harvesting. After the various
treatments, CA77 cells were rinsed once with ice-cold PBS, removed from
the plate by scraping, transferred to microcentrifuge tubes, and spun
for 1 min at 4 C to pellet the cells. After removal of the supernatant,
the cells were resuspended in lysis buffer (20 mM Tris, pH
7.5), 150 mM NaCl, 1 mM EDTA and 1
mM EGTA, 1% Triton X-100, 2.5 mM sodium
pyrophosphate, 1 mM ß-glycerophosphate, 1 mM
sodium vanadate, 1 µg/ml leupeptin) and allowed to incubate for 5 min
on ice. The cells were lysed by sonication, microcentrifuged for 10 min
at 4 C, and transferred to a new tube. The amount of protein in each
sample was determined using the Bradford method. Cell lysates were
stored at -80 C.
Equal amounts of cell lysate (10 µg) were subjected to SDS-PAGE and
transferred to Immobilon-P membranes as recommended (Millipore Corp.,
Bedford, MA). Membranes were blocked in Tris-buffered saline containing
0.1% Tween-20 (TBST) plus 5% nonfat dry milk for 1 h before
incubation with primary antibodies for 1 h at room temperature.
The anti-active MAPK polyclonal antibodies directed against ERK1 and
ERK2 (Promega, Madison, WI) were diluted 1:5,000 in TBST plus 3% BSA.
The MKP-1-specific antibodies (V-15, Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) were used at 1 µg/ml in the same buffer. After
extensive washing with TBST, the membranes were incubated with
horseradish peroxidase-conjugated donkey antirabbit IgG (Promega)
diluted 1:10,000 in TBST plus 3% BSA. After thorough washing with
TBST, the immunoreactive bands were visualized using the enhanced
chemiluminescence reagents (Amersham, Arlington Heights, IL). Blots
probed for active ERK were stripped following manufacturers
instructions (Amersham) and reprobed using antibodies that recognize
the inactive (unphosphorylated) and active (phosphorylated) forms of
ERK1 and ERK2 (ERK1, K-23, 1 µg/ml, Santa Cruz Biotechnology). In
general, the phospho-ERK exposure times were 45 min, whereas the total
ERK exposure times were only 12 min. Each experimental condition was
repeated in at least two independent experiments.
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ACKNOWLEDGMENTS
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We wish to thank Dr. Jeff Pessin for his helpful advice and for
generously providing reagents, and Sarah Shoesmith and Emily Kuhn for
excellent assistance.
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FOOTNOTES
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Address requests for reprints to: Andrew F. Russo, Department of Physiology and Biophysics 5632 BSB, University of Iowa, Iowa City, Iowa 52242. e-mail: andrew-russo@uiowa.edu.
This work was supported by grants from the NIH (HD-25969), American
Heart Association (96013860), and National Headache Foundation, with
tissue culture support provided by the Diabetes and Endocrinology
Center (DK-25295), and an Iowa Cardiovascular Interdisciplinary
Research Fellowship (HL-07121) to P.L.D.
Received for publication February 3, 1998.
Revision received March 11, 1998.
Accepted for publication March 17, 1998.
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