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Originally published In Press as doi:10.1074/jbc.M409090200 on September 7, 2004

J. Biol. Chem., Vol. 279, Issue 46, 47856-47865, November 12, 2004
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Activity-dependent Transcriptional Activation and mRNA Stabilization for Cumulative Expression of Pituitary Adenylate Cyclase-activating Polypeptide mRNA Controlled by Calcium and cAMP Signals in Neurons*

Mamoru Fukuchi{ddagger}, Akiko Tabuchi{ddagger}§, and Masaaki Tsuda{ddagger}§

From the {ddagger}Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 931-0194 and §Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corp., Shibuya 3-13-11, Tokyo 150-0002, Japan

Received for publication, August 9, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although it has been established that an activity-dependent gene transcription is induced by the calcium (Ca2+) signals in neurons, it is unclear how the specific mRNA moieties are transiently accumulated in response to synaptic transmission which evokes multiple intracellular signals including Ca2+ and cAMP ones. The expression of pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide, is controlled by Ca2+ signals evoked via membrane depolarization in neurons, and, in cultured rat cortical neuronal cells, we found that the Ca2+ signal-mediated activation of the PACAP gene promoter was critically controlled by a single cAMP-response element (CRE) located at around –200, to which the CRE-binding protein predominantly bound. The Ca2+ signal-induced expression of PACAP mRNA was enhanced by forskolin, which evokes cAMP signals. In support, the PACAP gene promoter was synergistically enhanced by Ca2+ and cAMP signals through the CRE, accompanying a prolonged activation of extracellular signal-related protein kinase 1/2 and CRE-binding protein. On the other hand, sole administration of forskolin markedly reduced the cellular content of PACAP mRNA, which was restored by the addition of Ca2+ signals. We found that the stability of PACAP mRNA was increased in response to Ca2+ signals but not that of activity-regulated cytoskeleton-associated protein (Arc) mRNA, indicating an activity-dependent stabilization of specific mRNA species in neurons, which can antagonize the regulation mediated by cAMP signals. Thus, the transcriptional activation and mRNA stabilization are coordinately regulated by Ca2+ and cAMP signals for the cumulative expression of PACAP mRNA in neurons.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It is widely accepted that activity-dependent gene transcription in neurons is transiently induced by the Ca2+ signals evoked via glutamatergic synaptic transmission and plays a key role in establishing a long-term neuronal plasticity. Such a Ca2+ signal-mediated activation of gene transcription has fully been demonstrated with the brain-derived neurotrophic factor (1, 2) and c-fos (3, 4) genes, in which the cAMP-response element (CRE)1-binding protein (CREB) is commonly involved. For this Ca2+ signaling in neurons, the ERK/mitogen-activated protein (MAP) kinase pathway is a major route that leads to the activation of CREB through phosphorylation of Ser-133 (5, 6). In addition, Impey et al. (7) have reported that cross-talk between ERK1/2 and cAMP-dependent protein kinase (PKA), which are activated by Ca2+ and cAMP signals, attains a synergistic activation of CREB-dependent transcription in neurons. This coupling of both signaling pathways has also been suggested to contribute to the formation of long-lasting synaptic plasticity (8). However, it is still unclear how the cumulative expression of particular mRNA species responsible for synaptic plasticity is controlled transcriptionally and/or post-transcriptionally in response to synaptic transmission.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the vasoactive intestinal polypeptide/secretin/glucagon family and was first isolated from the ovine hypothalamus (9). PACAP, which is synthesized as a precursor of 175 amino acids, is processed to its bioactive forms, PACAP38 and PACAP27 (10), which bind to at least two types of receptors, PAC1 and VPAC1/2 (11, 12). The binding of PACAP to these receptors triggers several intracellular signaling pathways, resulting in versatile biological functions (13) such as neuronal survival (14, 15) and synaptic plasticity (16). The rat PACAP gene consists of six exons including an alternative exon IA and IB and four exons encoding the coding region, and a variety of alternative transcripts are produced in neurons (17–19).

It has been established that the expression of the PACAP gene is activated by the Ca2+ signals in neurons (15, 20). On the other hand, the stabilization and de-stabilization of mRNA moieties are critical to control the level of PACAP mRNA in cells (21). In this study, therefore, we first assigned the CRE located on the PACAP gene promoter as a Ca2+ signal-response element and then investigated whether or not synergistic activation could be induced by Ca2+ and cAMP signals through the CRE and, furthermore, whether the stabilization of PACAP mRNA was affected by these signals. We found an activity-dependent stabilization of mRNA that contributed to the cumulative expression of PACAP mRNA in combination with the transcriptional activation synergistically induced by Ca2+ and cAMP signals.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Forskolin, isobutylmethylxanthine (IBMX), nicardipine, actinomycin D, n-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide dihydrochloride (H89), 1,4-diamino-2,3-dicyano-1,4-bis(oaminophenylmercapto)butadiene (U0126), (9S, 10S,12R)-2,3,9,10, 11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo [1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i](1, 6)benzodiazocine-10-carboxylic acid hexyl ester (KT5720), and DL-amino-5-phosphonovalerate (APV) were purchased from Sigma.

Antibodies—Antibodies used for the super-shift assay were purchased from Santa Cruz and included rabbit polyclonal antibody against ATF2 (N-96) and mouse monoclonal antibody against ATF1 (C41–5.1), which specifically reacts with ATF1 but not with other ATF/CREB transcription factors. Additionally, mouse monoclonal antibody against the CREB family (25C10G) (not specific for ATF1) reacts with ATF1, CREB, and CREB modulator (CREM), and furthermore, mouse monoclonal antibody against CREB (24H4B) specifically reacts with CREB but not with other ATF/CREB transcription factors. Rabbit polyclonal antibody, which specifically cross-reacts with phosphorylated CREB at the 133rd Ser residue (#17926 Upstate Biotechnology), was also used. Normal IgG was used as a control.

Cell Culture—Primary cultures of rat cortical neurons were prepared from the cerebral cortexes of 17–18-day-old Sprague-Dawley rat embryos as described previously (22). Briefly, small pieces of cerebral cortex were dissected by enzymatic (DNase I (Sigma) followed by trypsin (Sigma)) treatment and mechanical dissociation, and the cells were seeded at 5 x 106 cells in a 60-mm culture dish (Iwaki). The cells were grown for 48 h in Dulbecco's modified Eagle's medium (DMEM) (Nissui) containing 10% fetal calf serum, and then the medium was replaced with serum-free DMEM containing glucose (4.5 mg/ml), transferrin (5 µg/ml), insulin (5 µg/ml), sodium selenite (5 ng/ml), bovine serum albumin (1 mg/ml), and kanamycin sulfate (100 µg/ml) (TIS medium). Cytosine arabinoside (Ara-C) (Sigma) was also added at 2 µM to prevent the proliferation of glial cells. The medium was replaced with fresh TIS medium but devoid of Ara-C 2 h before DNA transfection.

RNA Isolation and Measurements of PACAP Transcripts by Reverse Transcriptase (RT)-PCR—Total RNA was extracted from the cultured cells using ISOGEN (NipponGene). RT-PCR was performed as described (15). Briefly, total RNA (1 µg) was reverse-transcribed into cDNA in 20 µl of 1x first strand buffer containing 0.5 µM oligo(dT)15 (5'-AGCTTTTTTTTTTV-3') as a primer, 200 units of SuperScript II reverse transcriptase (Invitrogen), 400 µM dNTPs, and 10 units of RNase inhibitor (Invitrogen) as recommended by the manufacturer. After reverse transcription, the reaction mixture was treated with 1.1 units of RNase H (Invitrogen) at 37 °C for 20 min and used for PCR as cDNA solution. PCR was performed in 50 µl of 1x PCR buffer containing 1 µl of cDNA solution, 1.25 units of AmpliTaq Gold DNA polymerase (PerkinElmer Life Sciences), 1.5 mM MgCl2, 200 µM dNTPs, and 0.5 µM of the primer pairs. To distinguish the four alternative exons (exon IA, exon IB, exon II) of the rat PACAP gene, PACAPE1A (5'-CTCTGTCAGCAGCAGCAGAA-3'), PACAPE1B (5'-TGTCACGCTCCCTCCTAGTT-3'), and PACAPE2as (5'-AGGTGAACAGGAGACGCTGT-3') were used (see Fig. 1). To investigate cDNA corresponding to the coding region, PACAPE2s (5'-GGACTCAGCTTCCCTGGGAT-3') and rPACA-PE5as (5'-AAGTACGCTATTCGGCGTCC-3') were used. For the amplification of rat activity-regulated cytoskeleton-associated protein (Arc) cDNA, Arc sense (5'-CGCTGGAAGAAGTCCATCAA-3') and rArc antisense (5'-GGGCTAACAGTGTAGTCGTA-3') were used. For the internal control, rat glyceraldehyde-3-phosphate dehydrogenase (GA-PDH) cDNA was amplified using GAPDH sense (5'-TCCATGACAACTTTGGCATCGTGG-3') and antisense (5'-GTTGCTGTTGAAGTCACAGGAGAC-3') primers. For amplification of preproPACAP cDNA, the PCR conditions after preheating at 95 °C for 10 min were denaturation at 96 °C for 45 s, annealing at 57 °C for 45 s, extension at 72 °C for 1 min for 30 cycles, and a final extension at 72 °C for 10 min. The amplification of GAPDH was carried out for 31 cycles under the same conditions. PCR products were separated by electrophoresis on 2% agarose gels, and the densities of the DNA bands stained with ethidium bromide were analyzed using a Bit-Map loader (ATTO, Japan) and software (NIH image 1.52).



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  		FIG. 1.
Changes in expression of exon IA-II, IB-II, or II-V transcripts upon membrane depolarization. A, structure of the PACAP gene and positions of forward and reverse primers designed to detect the transcripts covering exon IA to exon II (exon IA-II), exon IB to exon II (exon IB-II), and exon II to exon V (exon II-V). B, after 5 days in culture, the cortical cells were stimulated with 25 mM KCl or vehicle for 0, 3, 6, 9, 12, and 24 h, and total RNA was extracted for RT-PCR. After PCR products were separated by an electrophoresis on agarose gel, the densities of ethidium-stained bands were visualized with a Bit-Map loader (ATTO, Japan) and analyzed using software (NIH image 1.52). GAPDH was analyzed as a control for RNA input and reverse transcription efficacy. The data represent the mean ± S.E. from experiments performed in triplicate, and the same tendency was observed in at least three independent experiments. p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) versus 5 mM KCl at the same time.

 
Quantitative real-time RT-PCR was carried out in an ABI PRISM 7700 sequence detection system using SYBR Green PCR master mix. PCR was performed in 25 µl of 1x SYBR system using SYBR Green PCR master mix (Applied Biosytems) containing 1 µl of cDNA solution, which was amplified as described above, and 0.5 µM primer pairs. The PCRs were carried out as follows. After preheating at 95 °C for 10 min, the samples were denatured at 95 °C for 45 s, annealed at 57 °C for 45 s, and extended at 72 °C for 1 min for 45 cycles. To generate a standard curve, known amounts of plasmid DNA with a cDNA fragment of interest were used. The threshold cycle for each sample was taken from the linear range and converted to the starting amount by interpolation from a standard curve. For an internal control, GAPDH cDNA was amplified, and the levels of PACAP transcripts were normalized to GAPDH.

Reporter Vectors Used for Promoter Analysis and Overexpression Experiments—The rat PACAP promoter gene (–787~+376) was amplified by PCR. The genomic gene was isolated from Sprague-Dawley rat cortical neurons. The PCR with rat genomic DNA was performed in 50 µl of 1x PCR buffer containing 1.5 units of Pfu polymerase (Promega), 2 mM MgSO4, 1 µM dNTPs, and 1 µM of primer pair (sense, 5'-GAGGCTGTGGTGAAAATTAAACCAGTGG-3', and antisense, 5'-GGGGGACTTGTTTGCCGAAGCTAAAATTCC-3'). The PCR product was ligated into the EcoRV site of pBluescript II (KS+) and sequenced. The nucleotide sequence of the cloned rat PACAP gene promoter was identical to the one that other groups reported (23, 24).

For the promoter analysis, the luciferase reporter vector, pGL3-rat PACAP promoter (pPACAPp-1AB), was constructed by ligation of pGL3-Basic vector (Promega) with a KpnI/SacI-digested fragment including the region (–787~+376) of the rat PACAP gene promoter from the pBluescript II (KS+)-rat PACAP promoter. As an internal control vector for the luciferase experimental vector, we used a Renilla luciferase vector carrying the human elongation factor 1{alpha} promoter (pRL-EF1{alpha}) (2). For the overexpression experiments, we used a {beta}-galactosidase vector carrying the human elongation factor 1{alpha} promoter (EF1{alpha}-{beta}-Gal) as an internal control (25).

The site-directed mutagenesis for the substitution of 4–8 bases within pPACAPp-1AB was performed using a QuikChange site-directed mutagenesis kit (Stratagene). Left-side CRE-Lmut, all CRE-Cmut, and right-side CRE-Rmut were generated using as a forward primer, 5'-CCTTGAGGGACTAGGGCAGTGACGTCTTTTACTGATACCGGATC-3', 5'-CCTTGAGGGACTAGGATGCCACGACTGTTTACTGATACCGGATC-3', and 5'-CCTTGAGGGACTAGGATGCTGACGTCTAGATCTGATACCGGATC-3', respectively, and the complementary primer pair (the underline means substituted nucleotide bases). The expression vector A-CREB, was generously provided by Dr. C. Vinson (National Cancer Institute, National Institutes of Health).

DNA Transfection, Luciferase, and {beta}-Galactosidase Assays—DNA transfection was carried out over 3 days in culture using calcium phosphate/DNA precipitation as already described (22). Briefly, the calcium phosphate/DNA precipitates were prepared by mixing one volume (100 µl) of plasmid DNA (8 µg, pPACAPp:pRL-EF1{alpha} = 50: 1) in a 250 mM CaCl2 solution with an equal volume of 2x HBS (50 mM HEPES-NaOH (pH 7.05), 280 mM NaCl, 1.5 mM Na2HPO4) and added to a 60-mm dish. For overexpression experiments, we used plasmid DNA (8 µg, pPAC-APp:EF1{alpha}-{beta}-Gal:expression vector = 5:1:10). The dish was washed three times with phosphate-buffered saline, and fresh TIS medium was added. After 40 h, the transfected cells were stimulated with 25 or 50 mM KCl (25 or 50 mM KCl stimulation) or vehicle for 6~9 h, and cell lysates were prepared.

For the dual (firefly and Renilla) luciferase assay, cell lysates were extracted with passive lysis buffer (Promega) and used as described previously (2, 25). For the measurement of {beta}-galactosidase activity as an internal control, cell lysates were extracted with 250 µl of cell lysis buffer containing 1 mM potassium phosphate (pH 7.8), 1 mM dithiothreitol, and 0.5% Triton X-100; 20 µl of the lysate was used for the chemiluminescence-based {beta}-galactosidase assay (Clontech) and 20 µl was used for the firefly luciferase assay (26).

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay—Forty hours after the medium exchange at 3 days in vitro, cells were stimulated with 25 mM KCl or vehicle and incubated for 6 h. Then nuclear extracts were prepared as described with minor modifications (27). Electrophoretic mobility shift assays were carried out using probes corresponding to the PACAP sequence, as reported previously (25). Briefly, end-labeling of DNA probes was performed at 37 °C for 30 min in 10 µl of reaction mixture (6.6 mM Tris-HCl (pH 7.4), 50 mM NaCl, 6.6 mM MgCl2, 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dTTP, and 1 mM dithiothreitol) containing 200 ng of DNA, 2 µl of [32P]dCTP, and 1 unit of Klenow fragments. Then DNA probes were recovered with a Sephadex G-50 column. The DNA-protein binding reaction was carried out at 25 °C for 15 min in 20 µl of binding buffer (20 mM Hepes-NaOH, pH 7.9, 80 mM NaCl, 0.3 mM EDTA, 0.2 mM EGTA, 0.2 mM phenylmethanesulfonyl fluoride, 10% glycerol, 2 µg of poly [dI-dC], 0.2–0.4 ng of 32P-labeled DNA probes, and 5 µg of nuclear extract). Then, DNA-protein complexes were separated on 4% polyacrylamide gel at 132 V for 2.5 h. The protein concentration was determined by the method of Lowry. For supershift electrophoretic mobility shift assay, a series of antibodies (2 mg/ml) was used at a dilution of 1:20.

Western Blotting—After preparing the cell lysates and denaturing in 1x sample buffer (10 mM Tris-HCl, pH 6.8, 1% SDS, 1% {beta}-mercaptoethanol, 20% glycerol) for 5 min at 95 °C, 10 µg of proteins were separated on 10% SDS-polyacrylamide gel at 20 mA. Sample proteins were transferred onto a polyvinylidene difluoride membrane filter (Bio-Rad), and the filter was blocked with 5% skim milk in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween. The filter was incubated with rabbit anti-phospho-CREB (1:1000, New England Biolabs (NEB)), rabbit anti-CREB (1:1000, NEB), rabbit anti-active-MAP kinase (1:2500, NEB), or rabbit anti-MAP kinase (1:5000, NEB) overnight at 4 °C and then with horseradish peroxidase-conjugated rabbit anti-IgG (1:15,000, Amersham Biosciences) for 1 h at room temperature. CREB and ERK1/2 were finally detected with the enhanced chemiluminescence (ECL Western blotting detection reagents, Amersham Biosciences).

Statistical Analysis—All values represent the means ± S.E. of a number of separate experiments performed in duplicate, as indicated in the corresponding figures. Comparisons between groups were made using Student's t test followed by the F-test, with p < 0.05 as the minimum level of significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Increases in Expression of PACAP Multiple Transcripts Induced by Membrane Depolarization—We first examined which transcripts encoding PACAP were expressed and increased in amount with membrane depolarization induced by high K+ (25 mM KCl) in rat cortical neurons. The combinations of primers were designed to examine the transcripts containing exon IA-II or IB-II and the exon II-V transcripts containing the coding sequences (Fig. 1A). As shown in Fig. 1B, the expression of all the transcripts began to increase 3 h after the exposure of cells to 25 mM KCl, and this increase was maintained for at least 24 h. Three amplified DNA bands of different lengths were observed with the transcripts of exon IA-II, whereas only a single amplified band was observed with those of exon IB-II and exon II-V (Fig. 1B). The expression patterns of these transcripts were the same as those expressed in mouse cerebellar granule cells (18), and in an immunostaining analysis, the PACAP-positive signals were found to increase with most of the neurons in culture (data not shown but see the Tabuchi et al. (15)).

Dependence of the Promoter Activation upon Ca2+ Influx into Neurons—To detect the promoter activity of the PACAP gene, we constructed a plasmid containing both exon IA and exon IB in front of a luciferase reporter gene (pPACAPp-1AB) (Fig. 2A). The DNA transfection experiments of pPACAPp-IAB revealed that the luciferase activity increased upon KCl elevation and the increase in luciferase activity was higher at 50 mM KCl than at 25 mM KCl (Fig. 2A). However, the plasmids containing the region covering either exon IA or IB revealed only low basal and stimulatory promoter activity (data not shown), suggesting the presence of some cis-elements controlling the basal promoter activity of the PACAP gene. From these results, it is evident that cis-elements largely responsible for the inducibility of the PACAP promoter are located in the 5'-flanking region of exon IA.



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  		FIG. 2.
Activation of PACAP gene promoter-IA induced by membrane depolarization and pinpoint usage of a CRE-LE. A, effect of nicardipine (Nica) and AP-V on the activation of the PACAP promoter. After DNA transfection of pPACAPp-IAB and a 40-h incubation, a 2 M KCl solution or vehicle was administered to adjust the KCl concentration to 5, 25, and 50 mM. Then, 5 µM nicardipine and/or 200 µM AP-V was added 10 min before the stimulation with KCl. Cells were collected 9 h after stimulation, and luciferase activity was measured according to the methods described under "Experimental Procedures." Transcriptional activity was shown as a multiple of the value at 5 mM KCl. The data represent the mean ± S.E. from experiments conducted in triplicate, and the same tendency was observed in two independent experiments. p < 0.05 (*) and p < 0.01 (**) versus the same sample (50 mM KCl) without Ca2+ channel blockers. B, effect of replacement of the whole CRE-LE or its 5'- and 3'-surrounding nucleotide sequences upon the activation of the PACAP promoter. The construction of the mutated promoter was described under "Experimental Procedures." CRE-Cmut, CRE-Lmut, and CRE-Rmut mean the mutation of CRE-LE and the 5'- and 3'-flanking nucleotide sequences adjacent to the CRE-LE, respectively. The data represent the mean ± S.E. from experiments performed in triplicate, and the same tendency was observed in three independent experiments.

 
Using the plasmid pPACAPp-1AB, we examined the effect of blockade of a Ca2+ influx into neurons, which was attained with nicardipine and APV, potent antagonists for L-type voltage-dependent Ca2+ channel and N-methyl-D-aspartate glutamate receptor, respectively, on the activation of PACAP promoter IA. As shown in Fig. 2A, the residual activation of promoter IA was detected when either nicardipine or APV was added to the medium containing 50 mM KCl, but a simultaneous addition of these antagonists completely abolished the activation. Thus, both the Ca2+ influx through L-type voltage-dependent Ca2+ channel and N-methyl-D-aspartate glutamate receptor were effective for inducing the activation of the PACAP promoter.

Pinpoint Usage of a Single CRE for the Activation of the Promoter—To assign the region responsible for the Ca2+ responsiveness of the promoter, we first examined the effect of a series of internal deletions of the promoter and finally found that the 30-bp deletion between –213 and –184 markedly reduced the activation of the promoter activity (data not shown), in which a CRE-like element (CRE-LE) is present. To further examine whether the activation of the promoter is attributable to the CRE-LE or not, we exchanged all the nucleotides covering the CRE-LE. In addition, we exchanged four nucleotides surrounding the CRE-LE to examine the possible contribution of these sequences to the promoter activation. As shown in Fig. 2B, the exchange of all nucleotides of CRE-LE markedly reduced the basal promoter activity and almost completely abolished the activation, whereas the mutations introduced into the 5'- and 3'-surrounding regions of CRE-LE did not reduce the activation.

Predominant Binding of CREB to CRE-LE—To specify the proteins binding to CRE-LE, we performed a supershift gelmobility assay using probe C, which contains CRE-LE and its surrounding nucleotide sequences (nucleotide position –211 ~ –187) (Fig. 3A). When the anti-CREB/ATF family antibody was added to the DNA binding reaction mixture, the bands, which were formed specifically (data not shown), were markedly supershifted (Fig. 3A, compare lane 2 with lane 1). The addition of anti-ATF1 or anti-ATF2 antibody did not shift the bands (lanes 3 and 4), whereas that of anti-CREB or antiphosphorylated CREB antibody markedly shifted the bands (lanes 5 and 6).



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  		FIG. 3.
Specific binding of CREB to the CRE-LE of PACAP promoter. A, analysis of transcription factors binding to CRE-LE using supershift EMSA. Nucleotide sequences of the probes are shown, and the underlined sequences indicate CRE-LE. Nuclear extracts prepared from the cells stimulated with 25 mM KCl for 6 h were used for the binding reaction. Antibodies (80 µg/ml) against the CREB/ATF family, ATF1, ATF2, CREB, and CREB phosphorylated at Ser-133, and normal IgG were added to the reaction mixture before the reaction was started at 25 °C (see "Experimental Procedures"). Supershifted means the positions of supershifted bands. The experiments were done twice, and the same results were obtained. B, the plasmid vectors for overexpression of wild type CREB or the dominant negative CREB, A-CREB and K-CREB, were co-transfected with pPACAPp-IAB and pEF1{alpha}-{beta}-Gal reporter vectors into rat cortical neurons (see "Experimental Procedures"). Empty vector plasmids were also co-transfected as a control. Forty hours after transfection, cells were stimulated with 25 mM KCl (+) or vehicle (–) and incubated for another 9 h. Cell lysates were then extracted for measuring luciferase and {beta}-galactosidase activities. The value for the transcriptional activation of pPACAPp-IAB obtained with the empty vector at 5 mM KCl was standardized to one and shown as a multiple of the control value (-fold increase). The data represent the mean ± S.E. from at least three independent experiments. *, p < 0.05 versus the control with empty vector at 25 mM KCl.

 
To further address the crucial role of CREB in the Ca2+ signal-mediated activation of promoter IA, mutant plasmid vectors of dominant negative CREB were introduced into rat cortical neurons with a reporter plasmid, pPACAPp-1AB. Although overexpression of wild type CREB did not alter the activation level, that of A-CREB, a mutant with an acidic extension at the N terminus of the CREB leucine zipper domain that could interfere with the binding to DNA and transcriptional activation of wild type CREB (28), or K-CREB, a mutant with a substitution in the DNA binding domain that could inhibit the binding of wild type CREB (29), significantly decreased the promoter activation induced by depolarization (Fig. 3B).

Changes in Cellular Content of PACAP mRNA Induced by Ca2+ and cAMP Signals—Using a quantitative real-time RT-PCR, we measured the expression levels of PACAP exon II-V transcripts when the cells were treated with depolarization and/or forskolin for 12 h. As shown in Fig. 4A, the level of the transcripts began to increase 3 h and peaked 6 h after the onset of depolarization. In contrast to this, the level of the transcripts rapidly decreased upon the addition of forskolin at 5 mM KCl even at 1 h after the treatment and remained reduced for at least 12 h. Simultaneous addition of depolarization with forskolin, however, markedly increased the expression levels of transcripts more than the sole addition of depolarization at 6 and 12 h after the stimulation (Fig. 4A). The expression levels of transcripts were not changed at 5 mM KCl.



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  		FIG. 4.
Changes in the levels of exon II-V transcripts induced by depolarization and/or forskolin. A, time courses of expression of exon II-V transcripts. After being cultured in vitro for 5 days, the cortical cells were treated with 50 mM KCl and 10 µM forskolin (plus 300 µM IBMX) alone or in combination. At the indicated times (h), the cells were collected, and the total RNA was prepared for measuring exon II-V using real-time RT-PCR (see the "Experimental Procedures"). Open triangle, 5 mM KCl. Open circle, 50 mM KCl. Closed triangles, 5 mM KCl plus 10 µM forskolin plus 300 µM IBMX. Solid circles, 50mM KCl plus 10 µM forskolin (Forsk.) plus 300 µM IBMX. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) versus 5 mM KCl at the same time. p < 0.05 (#) and p < 0.01 (##) versus 50 mM KCl at the same time. B, expression levels of exon II-V transcripts. After the cells were treated with 50 mM KCl and/or forskolin plus IBMX for 6 h, total RNA was prepared for measuring the levels of exon II-V by real-time RT-PCR. **, p < 0.01 versus the same sample in the absence of APV and nicardipine (Nica). #, p < 0.05 versus 50 mM KCl in the presence of nicardipine and APV.

 
Because the addition of nicardipine and APV markedly decreased the expression level of transcripts attained upon depolarization (Fig. 4B), the elevation of exon II-V transcripts induced by depolarization is caused by the Ca2+ influx into neurons through L-type voltage-dependent Ca2+ channel and N-methyl-D-aspartate glutamate receptor. Co-administration of forskolin with depolarization further enhanced the expression level compared with that attained by depolarization only, the level of which was also reduced in the presence of nicardipine and APV, and furthermore, more extensively reduced than that obtained by depolarization only (Fig. 4B). Thus, these observations indicate that the addition of forskolin effectively decreases the level of PACAP transcript in the absence of Ca2+ signals, but its inhibitory effect can be erased upon depolarization.

Synergistic Activation of the Promoter Attained by the Treatment of Cells with Depolarization and Forskolin—Because the highest expression of exon II-V transcripts was obtained by depolarization and forskolin (Fig. 4, A and B), we investigated the effect of simultaneous administration of depolarization and forskolin on the PACAP promoter activation because the ERK/MAP kinase pathway contributes to the activation of CREB-dependent transcription in concert with the PKA pathway (7). As shown in Fig. 5A, simultaneous addition of depolarization and forskolin (plus IBMX) synergistically increased the promoter activity compared with that induced by either stimulus alone. This synergistic promoter activation was completely inhibited by addition of both U0126 and H89 or KT5720, a potent inhibitor for mitogen-activated extracellular signal-regulated kinase (MEK1/2) and for PKA, respectively, but incompletely by either (Fig. 5A), indicating an involvement of both the ERK/MAP kinase and PKA pathways in the activation. In support of the involvement of the PKA pathway, the overexpression of the catalytic protein kinase A subunit (cPKA), which was attained by co-transfection of pcPKA plasmid DNA, also induced the synergistic activation of the promoter when the depolarization was co-administered (Fig. 5B). The mutation of the CRE-LE sequence completely reduced the level of synergistic activation, indicating a predominant contribution of CRE-LE to the synergistic activation of PACAP promoter IA (Fig. 5C).



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  		FIG. 5.
Synergistic activation of PACAP promoter-IA by depolarization and forskolin through CRE-LE. A, 40 h after the DNA transfection with pPACAP-pIAB, the cells were stimulated with depolarization (50 mM KCl) and/or forskolin (Forsk.; 10 µM) plus IBMX (300 µM). Ten minutes before the stimulation, 20 µM U0126, 2 µM KT5720, or 10 µM H89 was added, and the incubation was continued for 6 h before preparing the cellular extracts for the luciferase assay. ***, p < 0.001 versus 5 mM KCl. ###, p < 0.001 versus the same sample (50 mM KCl and 10 µM forskolin plus 300 µM IBMX) without inhibitors. ++, p < 0.01 versus the same sample with U0126. B, after the pcPKA plasmid (3 µg) was transfected with pPACAP-pIAB (2.73 µg) plus the internal control vector (0.27 µg), the incubation was continued for 40 h before stimulation with depolarization (50 mM KCl) or vehicle. The cells were further cultured for 6 h before preparing cellular extracts. p < 0.01 (**) and p < 0.001 (***) versus 5mM KCl. ###, p < 0.001 versus 50 mM KCl or cPKA plasmid alone. C, the plasmids, pPACAP-pIAB and CRE-LE-mutated pPACAP-pIAB, were transfected with the internal control vector. After the stimulation of cells with depolarization and forskolin (plus IBMX), the incubation was continued for 6 h before the cellular extracts were collected. The experiments were performed twice, and the same result was obtained.

 
Continuous Activation of ERK1/2 and CREB upon Depolarization and Forskolin—Phosphorylation of ERK1/2 was markedly induced by the treatment of cells with depolarization and less by that with forskolin (Fig. 6A). Simultaneous addition of both stimuli did not increase the strength of phosphorylated ERK1/2 bands at 5 min after the stimulation compared with that obtained by depolarization only, but did after 60 min. On the other hand, the phosphorylation at Ser-133 of CREB increased 5 min after the onset of simultaneous stimuli, the level of which was higher than either the depolarization or forskolin, and remained elevated till at least 60 min after the stimulation (Fig. 6B).



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  		FIG. 6.
Activation of ERKs and CREB in cortical cells induced by depolarization and/or forskolin. Cortical neuronal cells in culture were treated with 50 mM KCl and 10 µM forskolin plus 300 µM IBMX alone or in combination for 5 and 60 min. After preparing the cell lysates, immunoblotting analyses were performed using phosphorylated or unphosphorylated (total) ERK1/2-specific (A) or CREB (B) antibodies. The intensities of immunostained bands were scanned and quantified with NIH Image version 1.52. The experiments were performed twice, and the same tendency was observed.

 
Ca2+ Signal-dependent Stabilization of Exon II-V Transcripts—Because the treatment of cells with depolarization and/or forskolin drastically changed the expression level of exon II-V transcripts (Fig. 4A), which at least in part did not correspond to that of promoter activity (Figs. 4A and 5A), it was considered that the Ca2+ and/or cAMP signals could affect the degradation of exon II-V transcripts. To examine this possibility, we added actinomycin D to stop the de novo mRNA synthesis and subsequently prepared total RNA for measuring the level of transcripts. As shown in Fig. 7A, the levels of transcripts gradually decreased under 5 mM KCl upon the addition of actinomycin D, the half-life (t1/2) of which was calculated as about 1.82 h. On the other hand, the half-life of transcripts obtained at 50 mM KCl was 3.41 h, which was longer than that obtained at 5 mM KCl (Fig. 7A). This prolongation of the half-life was abolished by administration of APV and nicardipine, the half-life of which changed from 3.41 (50 mM KCl) to 2.19 h (50 mM KCl plus APV and nicardipine) (Fig. 7A). In contrast, there was no change in the half-life of Arc mRNA, although the levels of transcripts rapidly decreased after the addition of actinomycin D (Fig. 7A). Upon depolarization, the Arc mRNA expression was elevated to a level about 10-fold higher than that of the control 1 h later (data not shown). It was not clear whether the transcript of the GAPDH gene changes its degradation rate by depolarization or not because of its intrinsic long-term stability (Fig. 7A).



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  		FIG. 7.
Changes of half-life of PACAP exon II-V transcripts controlled by depolarization and/or forskolin. A, effect of actinomycin D on the expression of PACAP exon II-V, Arc, and GAPDH transcripts. After culturing the cells at 5 mM KCl for 5 days, the cells were treated with 50 mM KCl or vehicle. After 2 h of treatment with KCl, 10 µg/ml actinomycin D was added, and incubation was carried out for a further 6 h to collect RNA at the indicated times (h) for a measurement of the transcripts by real-time RT-PCR. 200 µM APV and 5 µM nicardipine (Nica) were added 10 min before 50 mM KCl, and/or forskolin was administered. Open triangles and circles, PACAP exon II-V transcripts at 5 and 50 mM KCl, respectively. Squares, PACAP exon II-V at 50 mM KCl plus nicardipine and APV. Gray triangles and circles, Arc transcripts at 5 and 50 mM KCl. Dotted gray triangles and circles, GAPDH transcripts at 5 and 50 mM KCl. The half-life (h) of exon II-V calculated in each case is shown under the figure. p < 0.05 (*) and p < 0.01 (**) versus the control at 5 mM KCl at the same time. ##, p < 0.01 versus 50 mM KCl at the same time. B, changes in the half-lives of PACAP exon II-V transcripts caused by depolarization and/or forskolin (Forsk.). After culturing at 5 mM KCl for 5 days, the cells were treated with 50 mM KCl and forskolin alone or in combination. The experimental schedule was the same as described in A. Open triangles and circles, PACAP exon II-V transcripts at 5 and 50 mM KCl, respectively. Solid triangles and circles, PACAP exon II-V transcripts at 10 µM forskolin and IBMX in the absence and presence of 50 mM KCl, respectively. The half-life (h) of exon II-V calculated in each case is shown under the figure. p < 0.05 (*) and p < 0.01 (**) versus the control at 5 mM KCl at the same time.

 
Although the addition of forskolin with 5 mM KCl tended to decrease the levels of transcripts rapidly after the addition of actinomycin D, no change was observed with the degradation rate of exon II-V transcripts (t1/2 = 1.81) compared with the addition of 5 mM KCl alone (t1/2 = 1.82) (Fig. 7B). In any case, co-administration of depolarization and forskolin recovered the half-life of transcripts to almost the same extent as was obtained by depolarization only, the value of which was calculated to be 3.13 h (Fig. 7B). These results indicate that the cAMP signals effectively reduce the expression level of PACAP mRNA, which can be antagonized by the Ca2+ signals evoked via depolarization through a stabilization of PACAP mRNA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we demonstrated that the PACAP promoter-IA was activated by Ca2+ signals through only one CRE (CRE-LE) located between position –205 and –198 of the promoter (Fig. 2B). This CRE was also responsible for the activation of promoter-IA induced by glutamate (50 µM) (data not shown) and predominantly served in the binding of CREB (Fig. 3, A and B), indicating that the CRE located at around –200 of PACAP promoter-IA is predominantly responsible for the responsiveness of the promoter to the Ca2+ signal. In the Ca2+ signal-mediated activation of brain-derived neurotrophic factor and c-fos gene promoters, not only the CRE but also other cis elements, such as the upstream stimulatory factor binding element (25, 30), Ca2+-response element 1 (CaRE1) (31), and serum-response element (32), are involved. In the activation of the promoter of the nitric-oxide synthase gene, whose mRNA can be controlled in an activity-dependent manner, however, two CREs, which are separated by three nucleotides, are responsible for the responsiveness to the Ca2+ signal (26). Thus, the PACAP gene promoter is peculiar in terms of the pinpoint usage of a single CRE for the Ca2+ signal-mediated activation.

Using a CRE-reporter plasmid vector, Impey et al. (7) reported that a synergistic activation of CREB-dependent transcription was attained by cross-talk between ERK/MAP kinase and PKA in PC12 and rat hippocampal neuronal cells, which can be induced by depolarization and forskolin. This synergism has also been suggested to contribute to the formation of long-lasting neuronal plasticity and memory storage (7, 8, 33). In the present study, we also detected the synergistic activation of promoter-IA induced by Ca2+ and cAMP signals evoked via depolarization and forskolin (Fig. 5A), depending upon the CRE (Fig. 5C). In good agreement with the transcriptional activation of the promoter, the highest expression level of exon II-V transcripts was obtained by the co-administration of depolarization and forskolin (Fig. 4A).

Because this expression of transcripts was completely diminished by the addition of nicardipine and APV (Fig. 4B), it is evident that the Ca2+ signals evoked via Ca2+ influxes into neurons are basically responsible for the highest levels of expression and the cAMP signals play a role in modulating the Ca2+ signal-mediated signaling pathways. The synergistic activation of the promoter was completely repressed by co-administration of U0126 and H89 or KT5720 but incompletely by either (Fig. 5A), suggesting a requirement of coordinated activation of ERK/MAP kinase and PKA pathways for the synergistic promoter activation and, hence, for the highest expression of exon II-V transcripts induced by depolarization and forskolin (Fig. 4A). The involvement of the PKA pathway was supported by the observation that the expression of catalytic subunits of PKA resulted in the synergistic activation of the promoter-IA in the presence of Ca2+ signals (Fig. 5B). Impey et al. (7) already demonstrated that the PKA activation induced by forskolin was required for the Ca2+ signal-induced nuclear translocation of ERK. This coupling of the ERK/MAP kinase and PKA pathways may involve Rap1 (Ras proximate 1), a member of the Ras superfamily of GTP-binding proteins, which is activated by PKA (33) and has been demonstrated to be involved in neuronal plasticity (8).

Nevertheless, the extent of the changes in ERK1/2 and CREB phosphorylation induced by depolarization and forskolin seemed to be too small to explain the marked synergistic activation of the PACAP promoter (Fig. 6), suggesting that factors other than CREB could be involved in the synergistic activation. As one of the most likely candidates responsible for the synergistic activation, the co-activator CREB-binding protein should be considered, which is activated by Ca2+ signals through the phosphorylation of Ser-301-mediated by Ca2+/calmodulin-dependent protein kinase IV and promotes gene transcription via direct interaction with basal transcriptional machinery (34). Actually, Hansen et al. (35) quite recently reported that CREB-binding protein could be involved in the synergistic activation of the human cholecystokinin gene promoter induced by depolarization and forskolin in PC12 cells.

It is of particular interest that the rapid decrease in the expression of exon II-V transcripts is induced by cAMP signals but attenuated by the addition of Ca2+ signals evoked via depolarization (Fig. 4, A and B) because these phenomena indicate that the cellular content of PACAP mRNA can be dynamically regulated not only at the level of transcription but also at that of mRNA degradation in an activity-dependent manner. In support of this, we found that the half-life of PACAP mRNA was prolonged by depolarization, which was inhibited by the blockade of Ca2+ influx into neurons with nicardipine and APV (Fig. 7A), indicating that the Ca2+ signals are responsible for the prolongation of PACAP mRNA stability. The same prolongation of stability was also detected with brain-derived neurotrophic factor mRNA (data not shown) but not with the Arc mRNA (Fig. 7A), which can be selectively recruited to active regions of the dendritic arbor and be translated there (36). These observations clearly indicate an activity-dependent stabilization of particular mRNA species in neurons, which can be controlled by Ca2+ signals. On the other hand, the mechanism for the marked reduction of cellular content of PACAP mRNA induced by forskolin (Fig. 4, A and B) is still unclear because the degradation rate of exon II-V transcripts was not changed by administration of forskolin (Fig. 7B). In PC12 cells, administration of forskolin increased the level of PACAP mRNA (37), suggesting that the mechanisms for the expression of PACAP mRNA controlled by cAMP signals might be different among subpopulations of neurons. Further studies are needed to clarify the mechanisms for the cAMP-mediated reduction of the cellular content of PACAP mRNA.

Despite this reduction controlled by cAMP signals, it was clearly observed that co-administration of depolarization with forskolin restored the cellular content of PACAP mRNA to its highest level (Fig. 4, A and B). This restoration does not seem to be solely attributable to the synergistic activation of promoter-IA (Fig. 5A) because the half-life of the transcripts was prolonged from 1.82 to 3.13 h by depolarization even in the presence of forskolin (Fig. 7B), suggesting that the retardation of mRNA degradation should at least in part contribute to the effective accumulation of transcripts. This fact also indicates a dominant effect of Ca2+ signals over the cAMP signals on the regulation of stabilization and de-stabilization of PACAP mRNA in neurons, which is considered important to avoid a simultaneous acceleration of mRNA synthesis and degradation in the same neurons.

Once neurons are deprived of the Ca2+ signals in the presence of cAMP signals, however, the accumulated PACAP mRNA would immediately be degraded, as supported by the marked reduction in the cellular content of PACAP mRNA when nicardipine and APV were added in the presence of forskolin (Fig. 4B), probably giving rise to a temporal expression of PACAP mRNA in neurons in response to the Ca2+ signals. Thus, the cAMP signal could exert its effect on modulating the transcriptional activation if the Ca2+ signals are transduced in neurons but play a role in reducing the cellular content of PACAP mRNA by unknown mechanisms once the Ca2+ signals are blocked. Based on these observations, it can be considered that the coordination of activity-dependent transcriptional activation and mRNA stabilization, which is mainly controlled by Ca2+ signals but also modulated by cAMP signals, plays an important role in acutely changing the cellular content of specific mRNA species in response to synaptic transmissions, such as glutamatergic and adrenergic inputs, possibly leading to the formation of long-lasting neuronal plasticity.

This study is the first to demonstrate an activity-dependent stabilization of specific mRNA species. Because not only the activity-dependent gene transcription but also the activity-dependent dendritic targeting of brain-derived neurotrophic factor mRNA (38) has been reported, the metabolism of particular mRNA species might be dynamically regulated in neurons in an activity-dependent manner. Although such an activity-dependent mRNA stabilization had not been reported, the mechanisms for this might be related to the AU-rich elements located in the 3'-untranslated region of mRNAs (39), which are commonly found in mRNAs with short half-lives and prominently in the group of early response gene products, including cytokines and lymphokines (40). The PACAP gene can be clarified as an early-response gene and we found one AU-rich element in the 3'-untranslated region of the PACAP mRNA (data not shown). Elucidation of the molecular mechanisms for the activity-dependent mRNA stabilization would provide good insight into the formation of synaptic plasticity.


   FOOTNOTES
 
* This study was supported by a grant-in-aid for Core Research for Evolutional Science and Technology (CREST) from the Science and Technology Corporation of Japan, Japan and a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 81-76-434-7535; Fax: 81-76-434-5048; E-mail: tsuda{at}ms.toyama-mpu.ac.jp.

1 The abbreviations used are: CRE, cAMP-response element; PACAP, pituitary adenylate cyclase activating polypeptide; CREB, cAMP-responsive element-binding protein; RT, reverse transcription; APV, DL-amino-5-phosphonovalerate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Arc, activity-regulated cytoskeleton-associated protein; EF1{alpha}, human elongation factor 1{alpha}; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; PKA, cAMP-dependent protein kinase; IBMX, isobutylmethylxanthine; CRE-LE, CRE-like element; cPKA, catalytic protein kinase A subunit. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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