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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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. 1Go). 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. 1Go). 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 20–24 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.

 
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. 2AGo). This repression was specific to the enhancer since the reporter containing only the TK promoter was relatively unaffected (Fig. 2AGo). 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. 2AGo). CGS repression of the HO enhancer was dose-dependent with half-maximal inhibition at approximately 3 µM (Fig. 2BGo). 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.

 
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. 3Go). 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. 2Go. The mean luciferase activity per 20 µg protein ± SE from three experiments is shown.

 
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. 4AGo). 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. 2Go). Osmotic shock with sorbitol, an agent known to increase phosphorylation of MAP kinases (17), caused a robust increase in ERK phosphorylation (Fig. 4Go). 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. 2Go (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.

 
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. 4AGo).

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. 4BGo). 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. 5Go). 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. 5Go). 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. 5Go), which agrees with the prolonged repression of ERK phosphorylation (Fig. 4Go) 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. 4Go are shown in this figure.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 6Go). 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.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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, {Delta}32–51) (44) and MEKK (380–672) (45) expression vectors, the transactivator plasmids containing ELK1 (307–428) and c-JUN (1–223) activation domains fused to the Gal4 DNA-binding domain (1–147), and the Gal4 reporter plasmid.

CA77 cells were transiently transfected by electroporation essentially as described previously (14). Approximately 2–4 x 106 cells were transfected with 10–15 µg luciferase reporter plasmid DNA, 20 µg N17Ras plasmid, and/or 2–5 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 30–60% based on X-gal staining of cells transfected with a cytomegalovirus (CMV)-ß-galactosidase reporter gene. Statistical analyses were done using Student’s 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 manufacturer’s 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 1–2 min. Each experimental condition was repeated in at least two independent experiments.


    ACKNOWLEDGMENTS
 
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.


    FOOTNOTES
 
Address requests for reprints to: Andrew F. Russo, Department of Physiology and Biophysics 5–632 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.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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