Differential Regulation of Mitogen-Activated Protein Kinase-Responsive Genes by the Duration of a Calcium Signal
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 cellular mechanisms
by which changes in intracellular calcium
(Ca2+) can differentially regulate gene
expression. Two Ca2+ paradigms, involving
prolonged and transient Ca2+ increases, were
used. As a starting point, we studied the slow, prolonged elevation of
Ca2+ caused by activation of
5-HT1 receptors. We had previously shown that
5-HT1 agonists inhibit calcitonin gene-related
peptide (CGRP) transcription and secretion. The
Ca2+ ionophore, ionomycin, was used to produce
a prolonged elevation of the Ca2+ signal
similar to that generated by 5-HT1 receptor
agonists. Ionomycin treatment of the neuronal-like CA77 cell line
specifically inhibited mitogen-activated protein (MAP) kinase
stimulation of the CGRP enhancer and two synthetic MAP
kinase-responsive reporter genes (4- to 10-fold). We then showed that
ionomycin repression of promoter activity involved selective induction
of MAP kinase phosphatase-1 (MKP-1), but not MKP-2, and that
overexpression of MKP-1 was sufficient to repress CGRP enhancer
activity. These effects were then compared with a
Ca2+ paradigm involving a transient elevation
in Ca2+ as seen after depolarization. At 4
h after the transient increase in Ca2+, the
CGRP enhancer and synthetic MAP kinase-responsive reporter genes were
stimulated. In contrast, exposure to depolarizing stimuli overnight
caused only a less than 2-fold inhibition of promoter activity. We
propose that the duration of the Ca2+ signal
can determine the magnitude of a negative feedback loop that leads to
differential regulation of MAP kinase-responsive genes.
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INTRODUCTION
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Changes in intracellular Ca2+ levels
regulate many important cellular processes, including motility,
neurotransmitter release, proliferation, and gene expression (1, 2).
Ca2+ has been shown to regulate gene expression
via multiple signaling pathways by activating
Ca2+-sensitive kinases such as calmodulin (CaM)
kinases (3, 4) and mitogen-activated protein (MAP) kinases (5). The MAP
kinases, extracellular-regulated kinase-1 (ERK1), ERK2, and c-Jun
N-terminal kinase (JNK) have been reported to be activated by increases
in intracellular Ca2+ that activate upstream
kinases (6, 7). Activity of the MAP kinases is repressed by
dual-specific protein phosphatases (8). MAP kinase phosphatase-1
(MKP-1) is a Ca2+-induced protein phosphatase (9)
that can dephosphorylate multiple MAP kinases (8). Hence, regulation of
MAP kinase pathways by Ca2+ involves a balance
between stimulatory kinases and inhibitory phosphatases.
The calcitonin/calcitonin gene-related peptide (CT/CGRP) gene has been
shown to be MAP kinase responsive (10, 11). Alternative splicing of the
CT/CGRP gene generates CGRP in a subset of peripheral and central
neurons (12). CGRP is the most potent peptide vasodilator known and
plays an important role in regulating peripheral and cerebral blood
flow (13, 14). CGRP is also involved in mediating neurogenic
inflammation (15) and functions to convey nociceptive information from
the periphery to the central nervous system (16). Elevated serum
levels of CGRP are associated with several pathological conditions,
including migraines (17, 18). MAP kinase stimulation of CT/CGRP gene
expression is mediated through two regulatory sites, a distal
cell-specific enhancer (11) and a proximal Ras-responsive region (10).
The cell-specific HLH-OB2 (HO) enhancer is synergistically activated by
a helix-loop-helix (HLH) protein, USF, and an unidentified
octamer-binding protein, OB2 (19, 20). Recently, HO enhancer activity
was shown to be repressed by serotonergic antimigraine drugs (21).
We reported that activation of the serotonin class 1
(5-HT1) receptors by currently used antimigraine
drugs caused a prolonged increase in intracellular
Ca2+ (21). The type 1 class of 5-HT receptors
includes the 5-HT1A,
5-HT1B, 5-HT1D, and
5-HT1F receptors (22). The antimigraine drug
sumatriptan has been reported to preferentially interact with the human
and rat 5-HT1B, 5-HT1D, and
5-HT1F receptors (22). Expression of each of
these G protein-coupled receptors in nonneuronal cell lines has been
shown to mediate a decrease in intracellular cAMP levels and cause a
transient increase in calcium (23, 24). In contrast, we found that
activation of 5-HT1 receptors by serotonergic
antimigraine drugs did not couple to decreases in cAMP but, rather,
resulted in a prolonged elevation in intracellular
Ca2+ in both cultured trigeminal ganglia neurons
and the neuronal-like CA77 cell line (21, 25). These increases in
Ca2+ correlated with inhibition of CGRP release,
decreased CGRP messenger RNA levels, and repression of basal and MAP
kinase-activated HO enhancer activity (11, 21). Based on these data, we
hypothesized that the sustained increase in Ca2+
may be responsible for mediating the inhibitory effects of serotonergic
antimigraine drugs on CGRP gene expression. However, the role of
Ca2+ in regulating CGRP gene expression is not
well understood.
In this study we investigated the mechanism by which
Ca2+ can differentially regulate expression of
MAP kinase-responsive genes. Initially, we show that a sustained
increase in intracellular Ca2+ is sufficient to
repress the activity of the CT/CGRP enhancer and two synthetic MAP
kinase-responsive reporter genes. We then provide evidence that this
repression is at least partially due to induction of MKP-1. In
contrast, the activity of these genes was stimulated by transient
elevations in Ca2+ after depolarization of CA77
cells. Based on our results, we propose that the duration of the
Ca2+ signal can selectively regulate expression
of MAP kinase-responsive genes.
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RESULTS
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Ionomycin Treatment Mimics the Effect of Antimigraine Drugs on
Intracellular Ca2+ Levels
To directly test the role of Ca2+ in
regulating CGRP promoter activity, we used the
Ca2+ ionophore ionomycin and the neuronal-like
cell line CA77 (26). Ionomycin preferentially binds
Ca2+ ions in a 1:1 stoichiometric ratio and then
acts as a carrier to transport Ca2+ ions across
the plasma membrane to increase the intracellular concentration of
Ca2+. Treatment of CA77 cells with 1
µM ionomycin resulted in a sustained elevation in
Ca2+ that was maintained for at least 20 min
(Fig. 1
). Ionomycin initially caused a
rapid increase in Ca2+ levels that reached a peak
of 200 nM about 1 min after addition.
Ca2+ levels decreased slightly over the next
couple of minutes, then reached an equilibrium of about 180
nM for the duration of the experiment. For comparison, we
determined the effect of the selective 5-HT1
receptor agonist, CGS 12066A (CGS), on intracellular
Ca2+ levels. The selectivity of CGS for
5-HT1 receptors has previously been demonstrated
using the 5-HT1 antagonist, methiothepin, which
blocked the CGS effect on calcium amplitude in CA77 cells (11).
Furthermore, we have shown that other selective
5-HT1 receptor agonists, such as sumatriptan,
TFMPP, and L-694,247, mediate physiological effects very similar to
that of CGS (11). As shown in this study, addition of 10
µM CGS caused a relatively slow, steady increase in
intracellular Ca2+ (Fig. 2
). Ca2+ levels
increased from a basal level of 75 nM to a peak of 180
nM that remained relatively unchanged for at least 22 min
after CGS treatment. Hence, ionomycin treatment generated a
Ca2+ signal that was similar in long-term
amplitude and duration to that observed after activation of
5-HT1 receptors.

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Figure 1. Ionomycin Causes a Sustained Increase in
Intracellular Ca2+ Levels in CA77 Cells
Intracellular Ca2+ concentrations
([Ca2+]i) were measured using fura-2 and a
microscopic digital imaging system. A, Basal Ca2+ levels in
cells 1 min before addition of 1 µM ionomycin at time
zero. B, Levels were elevated 1 min after addition of ionomycin. C,
Levels remained elevated 20 min after the addition of ionomycin. D,
Mean change with SE in [Ca2+]i
after treatment with 1 µM ionomycin (n = 25 cells).
The small decrease in [Ca2+]i at time zero
was due to a volume change after the addition of ionomycin. The
pseudo-color scale indicates the 340/380 nm excitation wavelength ratio
and corresponding [Ca2+]i.
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Figure 2. CGS Causes a Sustained Increase in Intracellular
Ca2+ Levels in CA77 Cells
Intracellular Ca2+ concentrations were measured as
described in Fig. 1 . A, Basal Ca2+ levels in cells 1 min
before the addition of 10 µM CGS at time zero. B, Levels
were relatively unchanged 1 min after the addition of CGS. C, Levels
were elevated 20 min after the addition of CGS. D, Mean change with
SE in [Ca2+]i after treatment
with 10 µM CGS (n = 27 cells).
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Inhibition of CGRP Promoter Activity by Ionomycin
We then asked whether the sustained increase in
Ca2+ mediated by ionomycin could inhibit CT/CGRP
promoter activity. A firefly luciferase reporter gene containing a
1250-bp fragment of the 5'-flanking sequences of the rat CT/CGRP
promoter was transiently transfected into CA77 cells. This promoter
fragment contains regulatory sequences responsible for the
cell-specific and cAMP-responsive activities of the CT/CGRP gene.
Promoter activity was decreased in a dose-dependent manner by overnight
treatment of CA77 cells with ionomycin (Fig. 3A
). At the highest concentration of
ionomycin tested (1 µM), CT/CGRP promoter activity was
decreased to approximately 15% of control levels. In contrast,
ionomycin treatment had only a minor inhibitory effect on the activity
of a cotransfected cytomegalovirus (CMV) promoter-ß-galactosidase
reporter gene (Fig. 3A
) and a cotransfected CMV-Renilla
luciferase reporter gene (Fig. 3B
). To further demonstrate the
specificity of the ionomycin effect on CT/CGRP promoter activity, a
CT/CGRP promoter-ß-galactosidase reporter gene was cotransfected
with a CMV promoter-firefly luciferase reporter gene. Although
galactosidase activity of the CT/CGRP promoter was greatly inhibited by
ionomycin, control CMV-firefly luciferase activity was relatively
unaffected (Fig. 3C
). The decreases seen in CT/CGRP promoter activity
by 1 µM ionomycin were not due to toxic
effects, as cell viability was similar for the ionomycin- and
vehicle-treated cells (>98% under all conditions; n = 3). This
level of repression and specificity seen with ionomycin treatment is
very similar in magnitude to that caused by the
5-HT1 receptor agonist CGS (21).

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Figure 3. Ionomycin Repression of CGRP Promoter Activity
The -1250 bp CT/CGRP promoter fragment contains a proximal cAMP
response region (gray box), and a distal enhancer that
contains both cell-specific (black box) and
noncell-specific (striped box) elements. A, CA77 cells
were cotransfected with the CT/CGRP-luciferase and
CMV-ß-galactosidase reporter genes. The cells were pooled and divided
into parallel dishes that were treated overnight with the indicated
doses of ionomycin or the maximum volume of vehicle. The mean reporter
activity and SE are shown from four independent
experiments. The activities were normalized to the vehicle activities,
which were 51,000 ± 4,000 light units/20 µg protein (CT/CGRP)
and 178,000 ± 16,000 light units/20 µg protein (CMV-ß-gal).
B, Cells were cotransfected with the CT/CGRP-luciferase and
CMV-Renilla luciferase (CMV-R-luc)
reporter genes and treated as described. The mean reporter activity and
SE are shown from two independent experiments, each
performed in duplicate. The activities were normalized to the vehicle
activities that were 49,700 ± 5,700 light units/20 µg protein
(CT/CGRP-luc) and 356,000 ± 35,000 light units/20 µg protein
(CMV-R-luc). C, Cells were cotransfected with the
CT/CGRP-ß-galactosidase and CMV-firefly luciferase (CMV-luc) reporter
genes and treated as described. The mean reporter activity and
SE are shown from two independent experiments, each
performed in duplicate. The activities were normalized to the vehicle
activities that were 54,700 ± 7,000 light units/20 µg protein
(CT/CGRP-ß-gal) and 38,800 ± 3,600 light units/20 µg protein
(CMV-luc).
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We had previously reported that CGS repressed the CT/CGRP cell-specific
HO enhancer that contains regulatory sites for HLH and octamer-binding
transcription factors. To test whether ionomycin treatment would also
repress HO enhancer activity, we compared the wild-type 1250-bp
promoter activity with the same fragment in which the HLH site was
mutated by insertion of a BamHI linker (20). In contrast to
the wild-type CT/CGRP promoter, ionomycin treatment did not appreciably
repress activity of the mutant enhancer (Fig. 4
). Likewise, ionomycin greatly repressed
activity of reporter genes containing the cell-specific HO enhancer
linked to a minimal thymidine-kinase (TK) promoter 5- to 10-fold (Fig. 4
). The -920 to -1250 bp region contains the 18-bp cell-specific
enhancer as well as flanking noncell-specific regulatory sequences
(19). The octamer-binding site within the HO enhancer is also involved
in mediating the inhibitory effect of ionomycin, as mutation of this
site by addition of a single adenosine residue effectively abolished
the ionomycin effect (Fig. 4
). As luciferase activities for the CT/CGRP
control constructs were more than 10-fold higher than values determined
from cells transfected with promoterless vectors, reporter activities
would be high enough to detect repression with ionomycin. In contrast
to the 20-h treatment times, treatment with ionomycin for a shorter 4-h
period did not appreciably affect luciferase activity (Fig. 4
). As a
control, we showed that ionomycin treatment had relatively no effect on
thymidine kinase promoter activity (Fig. 4
). These studies demonstrate
that the ionomycin repression of CT/CGRP promoter activity is time
dependent and requires both the HLH and octamer-binding sites within
the enhancer.

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Figure 4. Ionomycin Repression of CGRP Enhancer Activity
CA77 cells were transfected with a series of reporter genes containing
the cell-specific CT/CGRP HO enhancer. Ionomycin responsiveness was
also tested using reporter genes that contain site-directed mutations
in the cell-specific enhancer by insertion of a BamHI
linker or a single adenosine residue (HO+A). Cells were treated with 1
µM ionomycin for either 20 h or 4 h as
indicated, and then assayed for luciferase activity. The mean reporter
activity per 20 µg protein with the SE is shown from at
least four independent experiments.
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Ionomycin Represses MEK1 Activation of the HO Enhancer
Having demonstrated that ionomycin could inhibit basal CT/CGRP HO
enhancer activity, we then wanted to determine whether ionomycin could
repress MAP kinase-stimulated enhancer activity. We had previously
shown that overexpression of the MAP kinase kinase MEK1 in CA77 cells
increased HO enhancer activity approximately 10-fold (11). In this
study cotransfection of the HO enhancer reporter plasmid and an
expression vector encoding MEK1 resulted in about a 4-fold increase in
enhancer activity (Fig. 5
). The
difference in fold stimulation with MEK1 in this study is probably
attributable to the use of FBS in the culture medium, which is known to
contain factors that can stimulate MAP kinases. Ionomycin repressed
MEK1-activated enhancer activity to 10% of the control value. This
level of repression was similar to that which we reported for CGS
repression (21). These results demonstrate that the sustained increase
in intracellular Ca2+ is sufficient to cause
repression of MAP kinase activation of the HO enhancer.

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Figure 5. Repression of MEK-1-Stimulated CGRP Enhancer
Activity by Ionomycin
CA77 cells were transfected with luciferase reporter genes containing
the HO enhancer-TK promoter or TK promoter with or without a
constitutively activated MEK1 expression vector as indicated. The cells
were pooled and divided into parallel dishes that were treated for
20 h with the vehicle (-) or 1 µM ionomycin (+).
The mean reporter activity per 20 µg protein with the SE
is shown from three independent experiments.
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Ionomycin Repression of Other MAP Kinase-Responsive Genes
To determine whether ionomycin treatment could lead to repression
of other MAP kinase-responsive genes, we used reporter genes known to
be directly regulated by MAP kinases. In this study we focused on the
ubiquitous transcription factor, Elk-1, that is a substrate for all
three MAP kinase pathways and plays an important role in regulating
immediate-early gene transcription. CA77 cells were cotransfected with
three plasmids containing 1) a trans-activator gene that
encodes the trans-activation domains of either Elk-1 or
c-Jun fused to the yeast Gal4 DNA-binding domain, 2) a luciferase
reporter gene containing Gal4 DNA-binding sites, and 3) an upstream MAP
kinase activator gene encoding constitutively activated forms of either
MEK1 or MEK kinase (MEKK). Although MEK1 selectively activates ERK,
MEKK has been shown to activate kinases that lead to phosphorylation of
ERK (via MEK1 activation) and JNK (via MEK4/7 activation) (27).
Overnight ionomycin treatment repressed both the Elk-1- and
c-Jun-dependent reporter genes in a dose-dependent manner even in the
presence of constitutively activated MEK1 (Fig. 6A
) or MEKK (Fig. 6B
). This dose curve
was very similar to the ionomycin inhibition of CT/CGRP promoter
activity (Fig. 3A
). In contrast to the long-term treatment, activity of
the Elk-1 reporter gene was relatively unaffected by short-term (4-h)
ionomycin treatment (<1.5-fold; n = 3). Because of the
specificity of MEK1, we can conclude that ionomycin treatment is
repressing ERK activity. Similarly, as the c-Jun-dependent reporter is
preferentially activated by JNK and not ERK (28), it is likely that
ionomycin treatment can also repress JNK. Our data provide evidence
that sustained increases in intracellular Ca2+
are responsible for repressing the action of the MAP kinases, ERK and
JNK.

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Figure 6. Ionomycin Repression of Elk-1 and c-Jun 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
trans-activation domains of either Elk-1 or c-Jun.
Constitutively activated MEK1 and MEKK expression vectors were
cotransfected with the ELK-1/Gal4 and c-Jun/Gal4 reporter plasmids,
respectively, and the cells were incubated in the absence or presence
of increasing doses of ionomycin for 20 h. A, The effect of
varying concentrations of ionomycin on MEK1-activated Elk-1/Gal4
promoter activity. B, The effects of varying concentrations of
ionomycin on MEKK-activated c-Jun/Gal4 promoter activity. The mean
luciferase activity per 20 µg protein ± SE from
three independent experiments is shown.
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Ionomycin-Mediated Decrease in the Level of Phosphorylated ERK
As ionomycin treatment repressed MEK1 activation of the CGRP
enhancer and Elk-1-responsive reporter genes, we predicted that
ionomycin may be inhibiting ERK activity. Phosphorylation of specific
threonine and tyrosine residues by MEK1 leads to activation of ERK. We
used Western blot analysis and phospho-specific ERK antibodies to
determine whether ionomycin was inhibiting ERK phosphorylation. In
untreated control cells, the phosphorylated forms of ERK1 (44 kDa) and
ERK2 (42 kDa) were easily detected (Fig. 7A
). Ionomycin treatment caused a marked
decrease in the levels of phosphorylated ERK1 and ERK2. This result is
in agreement with the ionomycin-mediated repression of basal CGRP
enhancer activity (Fig. 3A
). As a control, the phospho-specific
antibodies were removed, and Western blot analysis was performed using
antibodies that recognize both the phosphorylated and unphosphorylated
forms of ERK1 and ERK2. There was no appreciable change in the total
levels of ERK1 and ERK2 after ionomycin treatments (Fig. 7B
). These
data suggest that ionomycin repression of MEK-1-stimulated CGRP
enhancer and Elk-1-responsive reporter genes is mediated by inhibition
of active ERK levels.

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Figure 7. Inhibition of ERK Phosphorylation by Ionomycin
CA77 cells were untreated (con) or treated with 1 µM
ionomycin (iono) for 20 h. A, Cell lysates were analyzed by
Western blot analysis using antiactive ERK antibodies that recognize
only the phosphorylated ERK proteins. The immunoreactive bands for ERK
1 (44 kDa) and ERK2 (42 kDa) are indicated. B, The same blot as that
shown in A was stripped and reprobed with antibodies that recognize
both the unphosphorylated and phosphorylated forms of ERK.
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MKP-1 Is Sufficient to Inhibit CGRP Enhancer Activity
The phosphatase MKP-1 has been reported to dephosphorylate several
MAP kinases, including ERK (8). To determine whether the
ionomycin-mediated decrease in active ERK levels might involve
up-regulation of MKP-1 expression, cell lysates from CA77 cells were
analyzed by Western blot analysis using antibodies directed against
MKP-1. Initially, we showed that the level of MKP-1 was elevated upon
activation of 5-HT1 receptors by CGS in CA77
cells (Fig. 8A
), in agreement with our
previously published finding (21). However, the connection between the
Ca2+ increase and MKP-1 in these cells was not
established. We demonstrated that ionomycin treatment markedly induced
the expression of MKP-1 in CA77 cells (Fig. 8A
). To test whether MKP-1
expression correlated with repression of stimulated CGRP enhancer
activity, MKP-1 levels were determined in the presence of
constitutively active MEK1. Consistent with the functional data, MKP-1
was also increased in ionomycin-treated cells transfected with the MEK1
expression vector (Fig. 8A
). In contrast to MKP-1, treatment of CA77
cells with CGS or ionomycin decreased the level of another MAP kinase
phosphatase, MKP-2 (Fig. 8B
). To directly demonstrate that the increase
in MKP-1 levels was involved in repressing CT/CGRP promoter activity,
CA77 cells were cotransfected with reporter plasmids and an MKP-1
expression plasmid. Overexpression of MKP-1 greatly reduced the basal
activity of both the -1250 bp fragment of the CT/CGRP promoter and the
cell-specific HO enhancer (Fig. 8C
). As a control, MKP-1 had no effect
on TK-luciferase activity (Fig. 8C
). These data provide direct evidence
for a role for MKP-1 in regulating the MAP kinase-responsive CGRP
enhancer.

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Figure 8. MKP-1-Mediated Repression of CGRP Enhancer Activity
CA77 cells were untreated (con) or transfected with MEK-1 expression
plasmid (MEK1) and then treated with 10 µM CGS or 1
µM ionomycin (iono) for 20 h. The expression of
MKP-1 (A) or MKP-2 (B) was determined by Western blot analysis using 10
µg cell lysates and specific anti-MKP-1 or-MKP-2 antibodies,
respectively. The 38-kDa MKP-1 and 42-kDa MKP-immunoreactive bands are
indicated. A 50-kDa nonspecific band was also detected with the MKP-2
antibody. C, CA77 cells were transfected with CT/CGRP- or TK-luciferase
reporter plasmids and a control expression plasmid CMV-5 (-) or
cotransfected with a CMV-MKP-1 expression vector (+) in DMEM/F-12/ITS
medium. After 20 h, luciferase activity was measured and expressed
as the mean light units per 20 µg protein ± SE from
at least three independent experiments.
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Depolarization Leads to a Transient Increase in
Ca2+ and Stimulation of Reporter Genes
It has been reported that depolarization of neuronal cells leads
to an increase in intracellular Ca2+ and
activation of MAP kinase pathways (29). However, we have shown that
several MAP kinase-responsive reporter genes are repressed by an
elevation in Ca2+ levels after
5-HT1 receptor activation (11) or ionomycin
treatment. In an attempt to understand this apparent paradox,
intracellular Ca2+ levels were measured in CA77
cells after depolarization with potassium chloride (KCl).
Ca2+ levels rapidly increased from a basal level
of about 75 nM to a peak of about 210 nM after
depolarization (Fig. 9
). After the
initial spike in Ca2+ levels, the
Ca2+ concentration within the cell slowly
returned to near-basal levels by 20 min. This transient increase in
Ca2+ is in stark contrast to the prolonged
increase observed with CGS or ionomycin treatment (Figs. 1
and 2
).
Interestingly, the fold change in Ca2+ levels
(
3-fold) was similar for all three stimuli used in this study.

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Figure 9. Transient Elevation in Intracellular
Ca2+ Levels after Depolarization
Intracellular Ca2+ concentrations were measured as
described in Fig. 1 . A, Basal Ca2+ levels in cells 1 min
before the addition of 60 mM KCl at time zero. B, Levels
were elevated 1 min after the addition of KCl. C, Levels returned to
near basal levels 20 min after the addition of KCl. D, Mean change with
SE in [Ca2+]i after treatment
with 60 mM KCl (n = 26 cells).
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To determine the effect of the transient increase in
Ca2+ after depolarization on the CGRP enhancer
and Elk-1 reporter genes, CA77 cells were treated with 60
mM KCl for 4 or 20 h before measuring luciferase
activity. Chemical depolarization with KCl for 4 h caused an
almost 3-fold increase in CGRP enhancer activity (Fig. 10A
) and about a 6-fold increase in
Elk-1 reporter gene activity. These findings are in agreement with
studies in other systems that have reported depolarization-activated
MAP kinases that can phosphorylate Elk-1 (30, 31). In contrast,
overnight treatment with KCl mildly repressed activation of both the
CGRP enhancer and Elk-1 reporter gene less than 2-fold. The activity of
the control reporter plasmid containing only the TK promoter was
relatively unchanged by the 4-h or overnight KCl treatment. Thus,
depolarizing stimuli can only transiently stimulate MAP
kinase-responsive gene expression.

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Figure 10. Stimulation of CGRP Enhancer and Elk-1 Reporter
Activities after Depolarization
The HO enhancer, TK promoter, and Elk-1/Gal4 reporter genes were
transfected into CA77 cells incubated in the absence (-) or presence
(+) of 60 mM KCl for 20 or 4 h before measuring
luciferase activity. The effects of KCl on the HO-TK promoter and
control TK promoter luciferase activities (A) and the Elk-1 reporter
gene (B) are shown. The data in each experiment were normalized to the
luciferase reporter activity in the absence of KCl, which was set at 1.
The mean reporter activities ± SE for the untreated
HO-TK, TK, and Elk-1/Gal4-transfected cells were 56,000 ± 9,600,
3,930 ± 1,400, and 1,770 ± 450 light units/20 µg protein.
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The stimulation of CGRP enhancer and Elk-1responsive reporter
genes suggested that depolarization was increasing the level of active
ERK. To test this prediction, we measured the level of phosphorylated
ERK by Western blot analysis using phospho-specific ERK antibodies. In
untreated control cells, the phosphorylated forms of ERK1 (44 kDa) and
ERK2 (42 kDa) were detected (Fig. 11A
).
Depolarization of CA77 cells caused a time-dependent increase in the
levels of phosphorylated ERK1 and ERK2 (Fig. 11A
). The active level of
ERK2 increased more rapidly than that of ERK1, but the levels of both
proteins were greatest at the 60 min treatment time. Interestingly,
phosphorylated ERK1 and ERK2 levels returned to near-basal levels by
240 min. As a control, the phospho-specific antibodies were removed,
and Western blot analysis was performed using antibodies that recognize
both the phosphorylated and unphosphorylated forms of ERK1 and ERK2.
The total levels of ERK1 and ERK2 were relatively unaffected by KCl
treatment, suggesting that the effect of KCl was not due to changes in
the amount of total ERK proteins (Fig. 11B
). Based on these data, it is
likely that the KCl-mediated increases in CGRP and Elk-1 reporter gene
activities are mediated at least in part by activation of ERK.

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Figure 11. Stimulation of ERK Phosphorylation by KCl
Treatment
CA77 cells were untreated (con) or treated with 60 mM KCl
for the indicated times (minutes) before harvesting. A, Cell lysates
were analyzed by Western blot analysis using antiactive ERK antibodies
that recognize only the phosphorylated ERK proteins. The immunoreactive
bands for ERK1 (44 kDa) and ERK2 (42 kDa) are indicated. B, The same
blot as that shown in A was stripped and reprobed with antibodies that
recognize both the unphosphorylated and phosphorylated forms of ERK.
The active ERK1 or ERK2 levels were normalized to the density of the
corresponding total ERK band and are reported as the fold change
relative to control ERK levels, which were made equal to 1.
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DISCUSSION
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Ca2+ is known to act within the contexts of
space, time, and amplitude to regulate gene expression. For example,
the amplitude and duration of the Ca2+ increase
(32), the subcellular location of Ca2+ (33, 34),
and the frequency of Ca2+ oscillations (35) have
been shown to differentially activate different transcriptional
regulatory proteins. In this study we focused our studies on
understanding the role of duration in regulating MAP kinase-responsive
genes in a neuronal-like cell line.
We found that the duration of a Ca2+ signal can
differentially regulate the expression of both complex and synthetic
MAP kinase-responsive enhancers. The foundation of the differential
regulation is the dynamic balance between kinase and phosphatase
activities that control MAP kinase pathways (Fig. 12
). A transient increase in
intracellular Ca2+, such as that caused by
depolarizing stimuli, stimulates MAP kinase activity, leading to
activation of the CT/CGRP HO enhancer and Elk-1 reporter genes. In the
CA77 cells, this increase was maximal after shorter treatment times,
and in fact a slight repression was seen after longer treatments. This
is in agreement with studies in other systems demonstrating that
neuronal depolarization activates MAP kinases and gene expression (6, 30, 31), and that MAP kinase activation can also induce MKPs to create
a negative feedback loop (36, 37). A key point of our model is that a
prolonged Ca2+ signal results in a much greater
magnitude of MKP induction than that seen after the transient signal
(Fig. 12
). Hence, after activation of 5-HT1
receptors and ionomycin treatment, the prolonged
Ca2+ signal leads to repression of MAP
kinase-responsive genes due to induction of MKP-1. The induction of
MKP-1 under these conditions is consistent with previous studies by
ourselves (11) and by Meloche and colleagues (9). The requirement for
synthesis of MKP-1 would account for the lack of stimulation at the
early time points and the lag before repression.

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|
Figure 12. Model of Differential Regulation of MAP
Kinase-Responsive Genes
A transient elevation of intracellular Ca2+, as seen after
KCl-induced depolarization, stimulates the activity of the CGRP HO
enhancer and the Elk-1 reporter gene via activation of the MAP kinase
pathways. Whether the HO enhancer factors are directly phosphorylated
by ERK is not known; hence, multiple arrows are shown. In contrast,
prolonged elevation of intracellular Ca2+, as seen after
5-HT1 receptor activation or ionomycin treatment, induces
expression of MKP-1 that represses MAP kinase-responsive gene
expression.
|
|
Our study has shown activation and repression of a single transcription
factor (Elk-1) by different Ca2+ signals. This
result extends the important findings of Dolmetsch et al.
(32), who showed that the type of Ca2+ signal
leads to differential activation of different transcriptional
regulators (nuclear factor-
B, JNK, and nuclear factor-AT). Similar
to our findings, data from recent studies have shown that
Ca2+-dependent control of Elk-1 involves a
dynamic balance between kinase and phosphatase activities (38, 39). We
have been able to key on the duration of the Ca2+
signal because the transient and prolonged Ca2+
signals had very similar amplitudes, with little or no oscillation. The
rate of Ca2+ increase also did not appear to play
a key role in mediating the inhibition of MAP kinase-responsive genes,
as ionomycin caused a very rapid increase compared with the slow
gradual increase seen after CGS treatment. This contrasts with other
systems in which gene activation was dependent on the rate of
Ca2+ increase (40). Also, we did not see any
difference in the localization of the Ca2+ signal
after KCl, CGS, or ionomycin treatments, although we cannot completely
rule out this possibility due to the resolution of the assay. Taken
together, our results demonstrate the importance of the duration of the
Ca2+ signal in regulating MAP kinase-responsive
gene expression.
An intriguing finding was that prolonged Ca2+
signals lowered the levels of a different MAP kinase phosphatase,
MKP-2. Evidence for differential induction of MKP-1 and MKP-2 in
response to growth factors, stress-inducing agents, and other agents
has been reported, suggesting that these phosphatases may perform
distinct physiological functions (36, 41). MKP-2 is coexpressed with
MKP-1 in a variety of tissues, but the relative levels of messenger RNA
can vary (41). Thus, it is interesting to speculate that
5-HT1 receptors might either stimulate or inhibit
MAP kinase pathways depending on the relative complement of MKP-1 and
MKP-2 in the cell. In the CA77 cells, an increase in MKP-1 levels was
sufficient to inhibit CT/CGRP HO enhancer activity. However, as
overexpression of MKP-1 repressed HO enhancer activity to a lesser
degree than that seen using ionomycin, it seems likely that other
phosphatases may be recruited by the prolonged
Ca2+ signal. Another phosphatase known to be
activated by Ca2+ (42) and to regulate MAP
kinase-responsive genes is calcineurin (PP2B) (38, 39), although in
preliminary studies the calcineurin inhibitor cypermethrin had no
effect on HO enhancer activity.
The role of Ca2+ in regulating CGRP gene
expression is of particular interest, because we have recently shown
that 5-HT1 receptor agonists, including
sumatriptan and CGS, cause a markedly prolonged
Ca2+ increase in trigeminal ganglia neurons (21, 25). Sumatriptan is an antimigraine drug that lowers elevated serum
levels of CGRP in migraine patients (43, 44) and inhibits the release
of CGRP from cultured trigeminal neurons (25). Based on our studies, we
propose that activation of 5-HT1 receptors by the
currently used antimigraine drugs such as sumatriptan could lead to an
induction of specific phosphatases that repress CGRP gene expression.
The release of inflammatory agents during the neurogenic inflammation
period of migraine as well as neuronal activity would be able to
stimulate MAP kinase pathways (45, 46). Hence,
5-HT1 agonists could potentially be providing
long-term repression of CGRP and other MAP kinase-responsive genes.
Based on these data, 5-HT1 agonists and other
agents that cause prolonged Ca2+ signals may have
general applications for regulating MAP kinase pathways and MAP-kinase
responsive genes.
 |
MATERIALS AND METHODS
|
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Cell Culture
CA77 cells were maintained in Hams F-12/DMEM (low glucose;
1:1) and 10% FBS at 37 C in 7% CO2. Penicillin
and streptomycin were added to all media. CA77 cells were subcultured
in serum-free medium supplemented with insulin, transferrin, and
selenium (ITS; Collaborative Biomedical Products, Bedford, MA) 24
h before treatment, as previously described (21). Serum-free medium was
used for all experiments except those involving ionomycin. We found
that the effects of 5-HT1 receptor activation and
KCl stimulation on promoter activities were maximal when CA77 cells
were incubated in serum-free medium. The reason for this is not known,
but it may be due to factors in the serum that modulate signaling
pathways that confound the effects of 5-HT1
agonists and depolarization. Cells used in the ionomycin experiments
were maintained in serum-containing medium, because cell viability was
greatly reduced after long-term ionomycin treatment in serum-free
medium. Cell viability after CGS or ionomycin treatments was determined
by trypan blue exclusion (Life Technologies, Inc.,
Gaithersburg, MD). The pyrroloquinoxaline CGS 12066A monomaleate was
obtained from Research Biochemicals International (Antic,
MA) and was prepared and used as previously described (21). The
Ca2+ ionophore ionomycin was purchased from
Calbiochem-Novabiochem (La Jolla, CA) and was prepared in
100% dimethylsulfoxide. In all studies the cells were treated with
equivalent amounts of vehicle.
Ca2+ Measurements
Intracellular Ca2+ levels in CA77 cells
were measured using a video microscope digital image analysis system
(Photon Technology International, Inc., South Brunswick, NJ) as
described previously (21). Briefly, CA77 cells grown on laminin-coated
25-mm glass coverslips were maintained at 37 C in phenol- and
serum-free medium supplemented with ITS 24 h before the start of
the Ca2+ imaging procedure. Cells were incubated
in DMEM (high glucose) containing 0.2% BSA and 1 µM
fura-2/AM (Molecular Probes, Inc., Eugene, OR) for 30 min
at 37 C in 7% CO2. After washing the cells twice
with DMEM/BSA, the cells were incubated in DMEM/F-12/ITS medium for at
least 30 min to allow complete hydrolysis of fura-2/AM before
measurement using a Nikon Diaphot microscope. Basal
Ca2+ levels were measured for a minimum of
120 s in cells incubated in DMEM/F-12/FBS medium for the ionomycin
treatments or DMEM/F-12/ITS medium for the CGS-treated cells. An equal
volume of medium containing CGS, ionomycin, or vehicle (at 2 times the
final concentration) was added directly to the cells, and measurements
were recorded every 10 sec for more than 20 min on a heated stage at 37
C. Ca2+ levels within the entire cell were
averaged from masked images to obtain the whole cell
Ca2+ concentration. The concentration of
Ca2+ in intact cells was determined for each
buffer system based on the original equation described by Grykyewicz
et al. (47) and assuming a Kd of
Ca2+-fura-2 interaction to be 225
nM. Each experimental condition was repeated a
minimum of three times.
Plasmids and Transfection Assays
The rat CT/CGRP and TK promoter luciferase reporter plasmids,
CT/CGRP-ß-galactosidase reporter plasmid, and the CMV
ß-galactosidase plasmid have been described previously (19, 20, 21). The
Renilla luciferase control vector (pRL-CMV) was purchased
from Promega Corp. The Myc-tagged MKP-1 expression vector,
controlled by the CMV promoter and containing sequence for the first
314 amino acids, was provided by Dr. Jeffrey Pessin (48). The plasmids
containing CMV-MEK1 (S218/222E,
3251),
CMV-MEKK380672, Elk-1307428, and
c-Jun1223 activation domains fused to the Gal4
DNA-binding domain (1147), and Gal4 promoter-luciferase have been
described previously (11).
CA77 cells were transiently transfected by electroporation essentially
as described previously (21). Approximately 12 x
106 cells were transfected with 510 µg
luciferase reporter plasmid DNA, 50 µg MKP-1 plasmid, and/or 25
µg MEK1 or MEKK expression plasmid DNA using a Bio-Rad Laboratories, Inc., gene pulsar apparatus (Richmond, CA). The
amount of DNA transfected into the cells was kept constant by addition
of the empty expression vector CMV-5 (11). Transfected cells were
equally divided among 60-mm dishes for controls or treatment with
regulatory agents. This experimental protocol insured that the control
and treated cells had equal transfection efficiencies. After
transfection, cells were incubated in DMEM/F-12/FBS medium with either
ionomycin or vehicle control (dimethylsulfoxide) for 20 or 4 h
before measuring luciferase activity. Transfected cells used in the
depolarization studies were incubated in DMEM/F-12/ITS medium only
(control) or with 60 mM KCl as described for the ionomycin
treatment. To control for changes in osmolarity, 60 mM NaCl
was substituted for KCl in the control medium. In all experiments, NaCl
treatment had no effect on promoter activity. For the time-course
study, transfected cells were incubated in 1 µM ionomycin
for 20 or 4 h before harvesting the cells. Firefly and
Renilla luciferase activities were measured using reagents
from Promega Corp., and ß-galactosidase activity was
determined using Galacto-Light reagents (Tropix, Bedford, MA). Each
experimental condition was repeated in at least three independent
experiments in duplicate. In some experiments, transfection
efficiencies were normalized to ß-galactosidase activity. The
ß-galactosidase normalization did not significantly alter the
relative activities.
Western Blot Analysis
Cells were treated as detailed for the reporter assays, and
Western blot analysis was performed as previously described (11). 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, 1 mM EGTA, 1% Triton X-100, 2.5 mM
sodium pyrophosphate, 1 mM ß-glycerophosphate, 1
mM sodium vanadate, and 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, transferred to a new tube, and
stored at -80 C. Protein was determined by the Bradford method.
Equal amounts of cell lysate (10 µg) were subjected to SDS-PAGE
and transferred to Immobilon-P membranes as recommended by the
manufacturer (Millipore Corp., Bedford, MA). Membranes
were blocked in 20 mM Tris/140 mM sodium
chloride 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 antiactive MAPK polyclonal antibodies (Promega Corp.) directed against phosphorylated ERK1 and ERK2 were
diluted 1:5,000 in TBST. The MKP-1- or MKP-2-specific antibodies (V-15
or S-18, Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
were used at 1 µg/ml in TBST containing 3% BSA. After extensive
washing with TBST, the membranes were incubated with horseradish
peroxidase-conjugated donkey antirabbit IgG (Promega Corp.) 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 Pharmacia Biotech, Arlington Heights, IL). Antibodies bound to active ERK
were removed by incubation in 62.5 mM Tris (pH 6.8)
containing 100 mM ß-mercaptoethanol and 2% SDS for 30
min at 50 C. The membranes were then probed using antibodies that
recognize both the phosphorylated and unphosphorylated forms of ERK1
and ERK2 (ERK1, K-23; 1 µg/ml; Santa Cruz Biotechnology, Inc.). The signals were quantitated using NIH Image software.
Each condition was repeated in at least two independent
experiments.
 |
ACKNOWLEDGMENTS
|
---|
We thank lab members for discussions, and Dr. Jeffrey Pessin for
providing reagents.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Andrew F. Russo, Department of Physiology and Biophysics, 5632 BSB, University of Iowa, Iowa City, Iowa 52242. E-mail: andrew-russo{at}uiowa.edu
This work was supported by NIH Grants HD-25969, NS-37386, and HL-14388,
with tissue culture support provided by the Diabetes and Endocrinology
Center (DK-25295) and National Headache Foundation (to P.D.).
Received for publication January 18, 2000.
Revision received June 12, 2000.
Accepted for publication June 15, 2000.
 |
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