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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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. 2Go). 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. 1Go. 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).

 
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. 3AGo). 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. 3AGo) and a cotransfected CMV-Renilla luciferase reporter gene (Fig. 3BGo). 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. 3CGo). 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).

 
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. 4Go). 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. 4Go). 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. 4Go). 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. 4Go). As a control, we showed that ionomycin treatment had relatively no effect on thymidine kinase promoter activity (Fig. 4Go). 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.

 
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. 5Go). 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.

 
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. 6AGo) or MEKK (Fig. 6BGo). This dose curve was very similar to the ionomycin inhibition of CT/CGRP promoter activity (Fig. 3AGo). 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.

 
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. 7AGo). 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. 3AGo). 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. 7BGo). 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.

 
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. 8AGo), 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. 8AGo). 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. 8AGo). In contrast to MKP-1, treatment of CA77 cells with CGS or ionomycin decreased the level of another MAP kinase phosphatase, MKP-2 (Fig. 8BGo). 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. 8CGo). As a control, MKP-1 had no effect on TK-luciferase activity (Fig. 8CGo). 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.

 
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. 9Go). 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. 1Go and 2Go). 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. 1Go. 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).

 
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. 10AGo) 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.

 
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. 11AGo). Depolarization of CA77 cells caused a time-dependent increase in the levels of phosphorylated ERK1 and ERK2 (Fig. 11AGo). 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. 11BGo). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 12Go). 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. 12Go). 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-{kappa}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
CA77 cells were maintained in Ham’s 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, {Delta}32–51), CMV-MEKK380–672, Elk-1307–428, and c-Jun1–223 activation domains fused to the Gal4 DNA-binding domain (1–147), and Gal4 promoter-luciferase have been described previously (11).

CA77 cells were transiently transfected by electroporation essentially as described previously (21). Approximately 1–2 x 106 cells were transfected with 5–10 µg luciferase reporter plasmid DNA, 50 µg MKP-1 plasmid, and/or 2–5 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, 5–632 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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