Molecular mechanisms involved in gastrin-mediated regulation of cAMP-responsive promoter elements

Liv Thommesen1, Eva Hofsli1, Ruth H. Paulssen3, Marit W. Anthonsen2, and Astrid Lægreid1

1 Department of Physiology and Biomedical Engineering, 2 Faculty of Chemistry and Biology, Norwegian University of Science and Technology, N-7489 Trondheim; and 3 Department of Clinical Medicine, Tromsø University, N-9037 Tromsø, Norway


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we explore the role of cAMP-responsive (CRE) promoter elements in gastrin-mediated gene activation. By using the minimal CRE promoter reporter plasmid, pCRELuc, we show that gastrin can activate CRE. This activation is blocked by H-89 and GF 109203x, which inhibit protein kinases A and C, respectively. Moreover, Ca2+-activated pathways seem to be involved, because the calmodulin inhibitor W-7 reduced gastrin-mediated activation of pCRELuc. Deletion of CRE from the c-fos promoter rendered this promoter completely unresponsive to gastrin, indicating that CRE plays a central role in c-fos transactivation. Interestingly, gastrin-induced expression of the inducible cAMP early repressor (ICER), a gene that is known to be regulated by CRE promoter elements, was not reduced by H-89, W-7, or GF 109203x. Furthermore, bandshift analyses indicated that the region of the ICER promoter containing the CRE-like elements CARE 3-4 binds transcription factors that are not members of the CRE-binding protein-CRE modulator protein-activating transcription factor, or CREB/CREM/ATF-1, family. Our results underline the significance of the CRE promoter element in gastrin-mediated gene regulation and indicate that a variety of signaling mechanisms are involved, depending on the CRE promoter context.

3',5'-cyclic adenosine monophosphate; inducible cyclic adenosine monophosphate early repressor; signal transduction; gastrin;


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE PEPTIDE HORMONE GASTRIN plays a central role in regulation of growth and function of the gastrointestinal tract (16, 23). Gastrin is a potent inducer of gastric acid secretion, and this physiological response is regulated through the interaction of a variety of neuroendocrine cell types in the stomach mucosa (65). Gastrin is also a potent growth factor for both normal and malignant gastrointestinal tissues (41, 48, 65). Cellular effects of gastrin are transmitted via a specific transmembrane Gq/G11 protein-coupled receptor, the CCK-B/gastrin receptor, and the signals transmitted via this receptor are known to target a cascade of intracellular mediators initially described in response to activation of receptor tyrosine kinases by growth factors (21, 38). This response includes phospholipase C-beta and protein kinase C (PKC), as well as activation of Shc, Grb and Sos, Ras and Raf proteins, and mitogen-activated protein kinases (MAPK) (9, 10, 24).

A number of genes whose gene products play a central role in the maintenance and function of normal stomach mucosa contain cAMP-responsive (CRE) promoter elements. These genes include somatostatin, glucagon, gastric inhibitory polypeptide, chromogranin A (CGA), and insulin (31, 34, 37, 54, 69). Gastrin is known to modulate expression of CGA in enterochromaffin-like cells and somatostatin in D cells (13, 45) in the stomach mucosa. We have recently shown that gastrin induces expression of the CRE-responsive gene inducible cAMP early repressor (ICER) (59), which is suggested to play a role in negative feedback regulation of genes activated via CRE (4, 32, 44). Thus CRE promoter elements may play a central role in gastrin-mediated modulation of gene expression involved in physiological effects of this gastric hormone.

Factors involved in regulation of CRE-promoter elements include CRE-binding protein (CREB), CRE-modulator (CREM), and activating transcription factor (ATF) families of transcription factors (1, 15, 19). ICER is generated from an internal promoter within the CREM gene. CREB, CREM, and ATF proteins have been postulated to be of importance in a variety of neuroendocrine processes, such as stress responses, diurnal rhythm regulation, and thyroid-stimulating hormone receptor expression (19, 27, 55). CREB activity was first identified, purified, and cloned in studies investigating the expression and regulation of the somatostatin gene (35). The intracellular signaling pathways leading to activation of the CREB/CREM/ATF family of transcription factors are known to include activation of protein kinase A (PKA) in response to increased cAMP levels (18, 28), as well as other kinases like PKC, calmodulin-dependent kinases (CaM kinases), casein-kinase, MAPK-activated protein kinases, receptor-activated signal kinases, and protein kinase B (3, 5, 14, 58, 70).

The ICER promoter contains four different CRE-responsive elements, termed CARE 1 to CARE 4, known to be critical in its activation (32). The CARE 3 promoter element (TGACGTCA) is identical to somatostatin consensus CRE, whereas CARE 1, 2, and 4 differ from the CRE consensus sequence in one or three basepairs. Induction of ICER gene expression is assumed to be triggered by CRE-binding transcription factors via these promoter elements (32).

Even though it is well documented that gastrin can activate genes that are regulated via CRE promoter elements, there are until now no reports in the literature on whether gastrin directly activates CRE promoter elements or transcription factors known to regulate such elements. However, recently, gastrin-mediated transcriptional activity of rat vesicular monoamine transporter 2 was shown to depend on CRE consensus sites (67). The purpose of the present study was to investigate whether gastrin can activate gene expression via CRE promoter elements and, in case of a positive answer, to characterize signaling mechanisms involved in this gastrin response. We show that gastrin can indeed induce transcriptional activation of a minimal promoter containing somatostatin consensus CRE promoter elements and that the CRE element is indispensible for gastrin-mediated transactivation of the c-fos promoter. Gastrin-mediated activation of the minimal CRE promoter involves different signaling mechanisms from gastrin-induced activation of the CRE-regulated ICER promoter. Our study indicates that the intracellular signaling mechanisms and transcription factors involved in gastrin-mediated transactivation via CRE promoter elements depend on the promoter context in which the CRE is located.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cells and reagents. AR42J (rat pancreatic acinar cell derived, ATCC) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/l glucose (GIBCO-BRL, Life Technologies, Paisley, Scotland), 1 mM Na-pyruvate, 0.1 mg/ml L-glutamine (GIBCO), 10 U/ml penicillin-streptomycin (GIBCO), and 1 µg/ml fungizone (Sigma Chemical, St. Louis, MO) supplemented with 15% fetal calf serum (FCS; Biological Industries, Bat Haemek, Israel).

Cholecystokinin octapeptide (CCK-8) and pituitary adenylate cyclase-activating polypeptide (PaCaP-38) were purchased from Bachem (Bobendorf, Switzerland). Gastrin-17 was obtained from Sigma. The peptides were stored, dried, and frozen (-20°C). H-89 and W-7 (Calbiochem, La Jolla, CA) were both dissolved in water at 10 mM, and GF 109203x (bisindolylmaleimide I, Calbiochem) was dissolved in DMSO at 4.8 mM. CREB consensus oligonucleotide (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3') was from Promega (Madison, WI); CREB mutant oligonucleotides (5'-AGAGATTGCCTGTGGTCAGAGAGCTAG-3') and polyclonal anti-CREM antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-CREB antibody was purchased from New England Biolabs (Beverley, UK).

Reporter plasmids. The plasmid pCRELuc containing 4x CRE somatostatin consensus promoter elements (TGACGTCA) was obtained from Stratagene (La Jolla, CA). The plasmids human wild-type (WT) c-fosLuc (nucleotides -362 to +13) and the mutated c-fos promoter constructs c-fosDelta CRE, c-fosDelta FAP-1, and c-fosDelta SRE (47) were a generous gift from Dr. Ugo Moens (University of Tromsø, Tromsø, Norway).

Transfection and luciferase assay. Cells (2 × 104/well) were seeded out in 96-well plates and transfected after 24 h with 0.12 µg luciferase reporter plasmid DNA per well, by use of 0.35 µl Fugene transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany). After cultivation for 2 days in the presence of plasmid and transfection agent, cells were treated with agonists and inhibitors followed by PBS wash (twice) and lysis in 15 µl of Promega lysis buffer. Luciferase activity was measured by luminometer (model TD-20/20, Turner Designs) with the Luciferase Reporter Assay System (Promega, Madison, WI), as recommended by the manufacturer.

Detection of PKC isoforms by RT-PCR. Before PCR amplification, total RNA was isolated with TRIzol LS reagent (GIBCO-BRL) according to the manufacturer's protocol and treated with deoxyribonuclease I, amplification grade (GIBCO-BRL, Gaithersburg, MD). cDNA synthesis and amplification were performed with Ready-to-Go RT-PCR beads (Amersham Pharmacia Biotech, Oslo, Norway) according to the manufacturer's protocol. The PCR was carried out as previously described (39), with primers which direct specific amplification of each of the PKC isoforms alpha , beta 1, beta 2, delta , epsilon , lambda , zeta , (68), gamma , eta , theta , (17), and µ (60). Preparations of total RNA isolated from different rat tissues, i.e., brain and skeletal muscle, served as positive controls. PCR amplification was conducted in a reaction volume of 50 µl by use of a PTC-100 programmable thermal cycler (MJ Research, Watertown, MA). The resulting PCR products were separated on 1.8% agarose gels and visualized by staining with ethidium bromide.

RT-PCR. RT-PCR analysis was performed as previously described (59). Briefly, after treatment, cells were washed with phosphate-buffered saline (PBS) and lysed in 500 µl of lysis/binding buffer. Poly(A+) RNA was isolated by using oligo (dT) Dynabeads (Dynal, Oslo, Norway). RT-PCR was performed with rTth DNA polymerase (Perkin-Elmer, Branchburg, NJ). cDNA synthesis was performed at 61°C for 40 min, followed by 35 cycles of PCR with an annealing temperature of 61°C. The following PCR primers were used: ICER-S: 5'-GTAACTGGAGATGAAACTGA-3'; ICER-AS: 5'-GACACTTGACATACTCTTTC-3'. To check whether comparable amounts of poly(A+) RNA from each sample were used, RT-PCR reactions for the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed using the following primers: GAPDH-S: 5'-CCCATCACCATCTTCCAG-3' and GAPDH-AS: 5'-ACAGTCTTCTGAGTGGCA-3' (annealing temperature 55°C, 25 cycles). PCR products were run out on a 1.0% agarose gel.

Gel shift assay. Preparation of nuclear extracts and gel shift analysis were performed as described previously (26). Briefly, cells were washed with PBS, incubated in buffer A [10 mM HEPES, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM dithiothreitol (DTT); 1 mM benzamidine; and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] for 10 min before lysis with 0.05% Igepal (Sigma). After centrifugation, supernatants were removed, and nuclear proteins were extracted from the pellets by continuously shaking in buffer C (20 mM HEPES, pH 7.9; 400 mM NaCl; 1 mM EDTA; 1 mM EGTA; 25% glycerol; 1 mM DTT; 1 mM benzamidine; and 0.5 mM PMSF) for 1 h. After another centrifugation, supernatants were examined for protein concentration, and equal amounts of nuclear protein from each sample were incubated with 1 µg poly(dI-dC) (Pharmacia Fine Chemicals, Upsala, Sweden) in binding buffer (20 mM HEPES, pH 7.9; 50 mM KCl; 1 mM EDTA; 1 mM DTT; 0.25 mg/ml BSA; and 2% Ficoll, to a final volume of 20 µl) for 10 min at room temperature. Then, 17 fmol of 33P-labeled oligonucleotide probe were added, and the mixture was incubated for 30 min at room temperature. The samples were applied on nondenaturing polyacrylamide gels (7% acrylamide, 0.25× Tris borate-EDTA, and 2.5% glycerol) and run at 80 V for 1 h and then at 160 V for 2-2.5 h, after which the gels were dried and exposed to X-ray film (Biomax, Kodak, Rochester, NY) for 48-72 h. For supershift analysis, nuclear extracts were first incubated at room temperature with 33P-labeled CREB probe for 30 min, then 2 µg of antibody were added, and the mixture was incubated for another 45 min on ice before electrophoresis. The CARE 1-4 probe consists of the following sense-strand sequence corresponding to position -167 to -96 in the ICER promoter (32): 5'-TACAGGGCTTTGCTTTCAGTGAGCTGCACATTGATGGCAGTGATAGGCTGGTGACGTCACTGTGATGTCAGT-3'. The CARE 3-4 consists of the following sense-strand sequence: 5'-GGCTGGTGACGTCACTGTGATGTCAGTGCTC-3' (position -122 to -92).

Proliferation assay. Cells (2 × 103/well) were seeded out in 96-well plates and cultured for 24 h. Then the cells were washed once with 180 µl of serum-free medium before addition of new medium with or without agonists and inhibitors. After 2 h, 5-bromo-2'-deoxyuridine (BrdU)-labeling solution (Roche Molecular Biochemicals, Mannheim, Germany) was added, and the cells were cultured for an additional 18 h before incorporation of BrdU was measured as described by the manufacturer. Briefly, the labeling medium was removed, and the cells were fixed and DNA was denatured by adding 150 µl of FixDenat per well for 30 min at room temperature. FixDenat solution was removed, and 100 µl of anti-BrdU-peroxidase working solution were added per well, followed by incubation at room temperature for 90 min. The cells were then rinsed three times with 200 of µl washing solution before 100 µl of substrate solution were added. After 3 min, the light emission of the samples (relative luminiscence units or RLU) was measured in a microplate luminometer (Fluoroskan Ascent FL, Labsystems).

Data presentation. Reporter gene experiments were repeated four times (n = 4), with four wells per condition per experiment. Proliferation experiments were repeated three times, with four wells per experiment. Results are shown as the mean value for separate transfections ± SE. Significant differences were calculated using Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Gastrin activates CRE promoter elements by a mechanism involving PKA. We have recently reported that gastrin can activate expression of the CRE-regulated gene ICER in AR42J cells (59). To examine whether gastrin-induced gene expression can be mediated via CRE promoter elements, we analyzed the gastrin response in AR42J cells transfected with the plasmid pCRELuc, which contains the luciferase reporter gene driven by a basic promoter element (TATA-box) joined to four repeats of the CRE-somatostatin consensus elements. Gastrin (gastrin-17) was found to induce moderate, but highly reproducible, activation of pCRELuc (Fig. 1). Similarly, CCK (CCK-8) induced activation of pCRELuc (Fig. 1). CCK binds with similar affinity to CCK-A(1) and CCK-B(2) receptors (52) and activates ICER via both receptors (59); thus it is possible that both receptors are involved in CCK-mediated CRE activation. Because cAMP is known to be a potent inducer of CRE elements, we included as positive controls 1) forskolin, which directly activates adenylyl cylase, and 2) PaCaP, which activates signaling in AR42J cells via the Gs- and Gq-coupled PaCaP I receptor (12).


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Fig. 1.   Gastrin (G) activates somatostatin consensus cAMP-responsive promoter elements (CRE). AR42J cells were transfected with pCRELuc and treated with 50 nM G-17, 10 nM cholecystokinin (CCK)-8, 50 µM forskolin (F), 200 nM pituitary adenylate cyclase-activating polypeptide (PaCaP), or 100 ng/ml phorbol 12-myristate 13-acetate (PMA) for 6 h. Results are means ± SE and are expressed as degree of induction compared with control.

PKA is known to be a central signal transduction component in the activation of CRE elements. Thus it was of interest to examine the possible involvement of PKA in gastrin-induced activation of the minimal CRE reporter plasmid. We found that the specific PKA inhibitor H-89 strongly reduced gastrin- and CCK-induced activation of pCRELuc (Fig. 2A; P < 0.05), indicating that PKA plays a role in gastrin and CCK-mediated activation of CRE promoter elements. The forskolin-mediated response was completely blocked by H-89, and the PaCaP-mediated induction was reduced by ~80% (Fig. 2A; P < 0.05).


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Fig. 2.   Effect of H-89, GF 109203x, or W-7 on gastrin-mediated activation of CRE reporter plasmid. AR42J cells were transfected with pCRELuc and pretreated with 10 µM H-89 (A), 3.5 µM GF 109203x (B), or 1 µM W-7 (C) for 1 h before treatment with 50 nM G-17, 10 nM CCK-8, 50 µM forskolin, or 200 nM PaCaP for 6 h. Results are means ± SE and are expressed as degree of induction compared with control (significant differences with inhibitors, *P < 0.05).

Gastrin-induced activation of CRE elements involves phorbol 12-myristate 13-acetate-insensitive PKC- and Ca2+-calmodulin-dependent mechanisms. Gastrin-activated intracellular signaling pathways leading to proliferation and transactivation of c-fos have been reported to involve PKC (10, 11, 57, 62). To investigate a possible role of PKC in gastrin-induced transcriptional activation via CRE promoter elements, pCRELuc reporter gene analyses were performed in the presence of the selective PKC inhibitor bisindolylmaleimide, GF 109203x. This agent is known to inhibit both diacylglycerol (DAG)-sensitive and -insensitive PKC isoforms (29). We found that GF 109203x strongly reduced gastrin- and CCK-induced activation of pCRELuc (Fig. 2B). Forskolin-induced pCRELuc activation was unchanged, indicating that PKA-mediated signaling to CRE was not affected by GF 109203x. PaCaP-induced activation of pCRELuc was strongly reduced by GF 109203x (Fig. 2B), indicating that both PKA and PKC may be involved in PaCaP-activated intracellular signaling in a manner similar to gastrin-activated signaling. RT-PCR profiling of PKC isoenzymes indicated that AR42J cells express both the DAG-responsive PKC isoforms alpha , beta I, beta II, gamma  (classical), delta , and epsilon  (novel), as well as the DAG-insensitive forms PKC lambda , µ, and zeta  (Fig. 3). Phorbol 12-myristate 13-acetate (PMA), which is known to activate both classical and novel DAG-responsive PKCs (36), did not activate pCRELuc in AR42J (Fig. 1), indicating that these PKCs are unable to activate the somatostatin CRE promoter element in these cells. This suggests that the PKC subtype(s) involved in gastrin-mediated activation of somatostatin CRE elements belongs to the group of DAG-insensitive isoforms.


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Fig. 3.   PCR amplification of protein kinase C (PKC) isoforms in AR42J cells and rat tissue. RT-PCR was performed using RNA from AR42J cells (top) and rat tissues (bottom), as indicated and as described in EXPERIMENTAL PROCEDURES. The amplified products were resolved on 1.8% agarose gels and subsequently stained with ethidium bromide. PKC isoforms are indicated. The RT-PCR product sizes for PKC isoforms are alpha  (443 bp), beta 1 (404 bp), beta 2 (309 bp), delta  (260 bp), epsilon  (464 bp), gamma  (543 bp), eta  (383 bp), lambda  (547 bp), µ (1,317 bp), theta  (178 bp), and zeta  (359 bp). The marker (M) is a 100-bp DNA ladder molecular weight standard (GIBCO-BRL).

Activation of the gastrin/CCK-B receptor has been reported to induce rapid and transient increase of intracellular Ca2+ (22, 53). One of the best characterized intracellular targets for Ca2+ is calmodulin, a protein that binds Ca2+ and that can regulate the function of several intracellular pathways. We found that the calmodulin inhibitor W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] inhibited gastrin-, CCK-, and PaCaP-induced activation of pCRELuc by ~50% (Fig. 2C).

PKA is involved in gastrin-induced proliferation. The roles of PKC, as well as of the ras and MAPK signaling pathways in gastrin-induced proliferation and c-fos activation in AR42J cells, have been well characterized (49, 50, 56, 61). However, it is not known whether PKA may also participate in these gastrin responses. Because we found that gastrin-induced CRE activation in AR42J cells involves PKA (Fig. 1), and because PKA has been found to participate in other signaling pathways involved in proliferation (63, 71), it was of interest to explore whether PKA plays a role in gastrin-induced proliferation. Measurement of BrdU incorporation in the presence of H-89 showed that gastrin-induced proliferation was markedly reduced (Fig. 4), thus suggesting a role for PKA in signaling pathways mediating gastrin-induced growth. The calmodulin inhibitor W-7 also inhibited gastrin-induced proliferation, which is in accord with results previously reported (56).


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Fig. 4.   Effect of H-89 and W-7 on gastrin-induced proliferation. Proliferation assay was performed as described in EXPERIMENTAL PROCEDURES. AR42J cells were pretreated for 1 h with either H-89 (10 µM) or W-7 (1 µM) before stimulation with gastrin (0.1, 1, and 10 nM); significant differences with inhibitors, *P < 0.05. Inhibitors alone (gastrin 0 nM) had no effect.

CRE is indispensible in gastrin-mediated activation of the c-fos promoter. To investigate the significance of the CRE promoter element in gastrin-mediated gene regulation of an authentic promoter, we measured the gastrin response in a reporter gene assay with the c-fos promoter deleted in CRE (c-fosDelta CRE). We found that c-fosDelta CRE was completely unresponsive to gastrin (Fig. 5A). This indicates that CRE is indispensible in gastrin-mediated activation of the c-fos promoter. The c-fosDelta SRE reporter gene plasmid, which is deleted in the promoter element SRE (serum-responsive element), served as a negative control, because SRE has been shown by others to be indispensible for gastrin-mediated c-fos transactivation (57). Deletion of the c-fos activating protein (AP)-1-binding site (FAP) did not impair gastrin-induced c-fos transactivation (Fig. 5A), indicating that this promoter element is not necessary for the gastrin response. The c-fosDelta CRE, c-fosDelta SRE, and c-fosDelta FAP promoters are all responsive to forskolin, demonstrating that the mutated promoters are functional (47).


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Fig. 5.   Transcriptional activity of the c-fos promoter. AR42J cells were transfected with plasmids containing the luciferase reporter gene driven by the wild-type (WT) c-fos promoter (-362 to +13) or by promoter mutants deleted in CRE, c-fos activated protein (FAP), or serum-responsive element (SRE), respectively. Serum-starved cells were stimulated for 5 h with gastrin (50 nM; A), CCK (10 nM; B), PaCaP (50 nM; C), or epidermal growth factor (EGF, 10 ng/ml; D). The activity of c-fos reporter gene plasmids (WT or mutated) in unstimulated cells was arbitrarily set to 1.0. Values (expressed as degree of induction) are means ± SE (n = 4).

We also examined the response of the c-fos promoter deletion variants to other growth factors. CCK, known to signal via both CCK-A (CCK-1) and CCK-B receptors in AR42J cells (7), was found to exhibit a dependence on CRE similar to that of gastrin (Fig. 5B). When the studies were extended to include PaCaP (Fig. 5C), we found results similar to the result with CCK. PaCaP activates transcription of the c-fosDelta CRE 3.6-fold compared with 13.6-fold induction of the wt c-fos reporter plasmid, indicating a crucial role of CRE in PaCaP-mediated c-fos activation. Similarly, also epidermal growth factor (EGF)-mediated activation of c-fosDelta CRE was reduced ~80% compared with wt c-fos (Fig. 5D). Even though EGF has been shown to promote gene transcription via CRE in other promoters (30, 51), the involvement of CRE in EGF-mediated c-fos activation has not been reported earlier.

CCK-, PaCaP-, and EGF-induced activation of the c-fosDelta FAP was reduced by ~50% compared with the responses of the wild-type promoters (Fig. 5, B-D), indicating that the FAP promoter element is essential for full activation of c-fos by these agonists. PaCaP has been reported to transactivate AP-1 reporter plasmid in PC-12 and AR42J cells (33, 46), and mutation of the AP-1 promoter element abolished EGF-mediated fibroblast growth factor-binding protein transactivation (20). However, the role of the c-fos AP-1 promoter element (FAP) in EGF-mediated c-fos transactivation has to our knowledge not been examined.

The transcription factor c-fos is involved in growth control in a variety of cell types (8, 42), and our results show that the CRE promoter element is indispensible for transactivation of c-fos (Fig. 5). Our observation that PKA is necessary for gastrin-induced proliferation in AR42J (Fig. 4) could imply that the proliferation-promoting effect of PKA is mediated via CRE-regulated activation of the c-fos promoter. However, when transcriptional activation of the WT c-fos promoter was measured in the presence of the PKA inhibitor H-89, we found that neither gastrin- nor CCK-induced c-fos transactivation was inhibited by H-89 (Fig. 6). In contrast, PaCaP-induced transactivation was clearly reduced, as has been found by others (64). The results indicate that signal transduction components other than PKA are involved in gastrin-mediated activation of the c-fos CRE promoter element.


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Fig. 6.   Activation of c-fos reporter plasmid. AR42J cells were transfected with WT c-fos reporter plasmid and pretreated with 10 µM H-89 for 1 h before treatment with 50 nM G-17, 10 nM CCK, 200 nM PaCaP, or 100 ng/ml PMA for 6 h. Results are means ± SE and are expressed as degree of induction compared with control (significant difference with inhibitor, *P < 0.05).

The ICER promoter is activated by mechanisms that are different from those involved in activation of the minimal CRE promoter. ICER is reported to be regulated via CRE promoter elements (32). Gastrin-induced activation of the minimal CRE promoter is dependent on PKA (Fig. 2A), whereas gastrin-induced activation of ICER is not (59), indicating that distinct or additional signaling mechanisms are involved in activation of the ICER promoter compared with a minimal CRE promoter. To further examine these differences, we measured gastrin-induced activation of ICER gene expression in the presence of the Ca2+/calmodulin inhibitor W-7 or the specific PKC inhibitor bisindolylmaleimide, GF 109203x. Figure 7 shows that neither of these inhibitors interfered with gastrin-induced ICER expression as measured by RT-PCR. Forskolin-induced ICER expression, on the other hand, was completely abolished with the PKA inhibitor H-89, showing that inhibition of ICER gene expression can be measured in this assay. These results indicate that gastrin-mediated activation of ICER transcription does not involve PKC- or Ca2+/calmodulin-dependent mechanisms. This is in strong contrast to gastrin-mediated activation of the minimal CRE promoter, which depends on each of these signaling components. We thus speculate that gastrin-mediated activation of the ICER promoter may involve promoter elements other than CRE, or that the CRE-like elements in the ICER promoter respond to signaling mechanisms different from those involved in activation of the minimal CRE promoter.


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Fig. 7.   Effect of W-7 and GF 109203x on gastrin-induced inducible cAMP early repressor (ICER) gene expression. AR42J cells were treated for 2 h with gastrin (G, 50 nM) in the presence or absence of W-7 (10 µM) or GF 109203x (GF, 10 µM) and analyzed for ICER gene expression by RT-PCR. F, forskolin. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Results are shown for the 2 alternative splice variant products ICER I and II (32). Inhibitors were added 1 h before gastrin. Similar results were obtained in 2 other experiments.

Proteins binding to the ICER promoter include proteins other than those of the CREB/CREM/ATF-1 family. We have previously shown that AR42J proteins binding to the somatostatin consensus CRE are members of the CREB/CREM/ATF-1 family (59). This family of transcription factors is known to be activated by PKA-, PKC-, and Ca2+/calmodulin-activated kinases (15). Because our data indicate that none of these signaling components is necessary for gastrin-induced activation of the ICER, it was of interest to examine the proteins binding to the ICER promoter. To this end, we used DNA probes identical to the ICER promoter regions that contained either the CRE-like CARE 1-4 promoter elements or only the CARE 3-4 elements. The CARE 1-4 probe specifically bound at least two protein complexes (Fig. 8A). These complexes were strongly reduced in the presence of excess unlabeled consensus CRE oligonucleotide (CREB), indicating the presence of CRE-binding proteins. To further identify these proteins, supershift analyses with anti-CREB and anti-CREM antibodies were performed. The appearance of distinct supershifted complexes (Fig. 8A) indicated that the proteins binding to the CARE 1-4 region of the ICER promoter include members of the CREB-CREM-ATF-1 family of transcription factors. Interestingly, the proteins binding to the CARE 3-4 probe were not supershifted with anti-CREB or anti-CREM antibodies (Fig. 8B). This suggests that the region containing the promoter elements CARE 3-4 can bind proteins that are not members of the CREB-CREM-ATF-1 family. These proteins may be identical to the protein CARE 1-4 complexes, which remained unshifted by anti-CREB and by anti-CREM (Fig. 8A), and may play a role in regulation of ICER gene expression.


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Fig. 8.   Gel shift analysis of proteins binding to CRE-like elements (CARE) 1-4 and CARE 3-4. A: nuclear extracts from AR42J cells were analyzed for factors binding to an oligonucleotide containing the CARE 1-4 ICER promoter region (described in EXPERIMENTAL PROCEDURES). Complexes I and II represent specific CRE-binding factors, as they are competed out in the presence of 50-fold excess of unlabeled CRE-binding protein (CREB) consensus oligonucleotide (lane 4). Complexes were supershifted with 2 µg of anti-CREB and 2 µg of anti-CRE modulator protein (CREM) antibodies (termed a and b, lanes 6 and 7). -NE, without nuclear extract. B: gel shift analysis of proteins binding the CARE 3-4 region of the ICER promoter (see EXPERIMENTAL PROCEDURES). These complexes were competed out by 50-fold excess of unlabeled CARE 3-4 (lane 3) but not by unlabeled CREB consensus oligonucleotide (lane 4). Two micrograms of alpha -CREB or alpha -CREM antibodies were added in lanes 5 and 6, respectively. Results are representative of 3 independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Several genes that are known to be regulated by gastrin contain CRE promoter elements (31, 34, 54, 69), but the role of these in gastrin-mediated gene activation has not been characterized. The present study was initiated to address this aspect. We show for the first time that gastrin can mediate transactivation of somatostatin consensus CRE promoter element, and that this activation involves several distinct signaling components, including PKA, PKC, and Ca2+/calmodulin. We show that PKA, the classical mediator of cAMP signaling, is also involved in gastrin-induced proliferation. This implies that AR42J cells may be growth stimulated via cAMP/PKA-dependent mechanisms. Recently, gastrin was reported to stimulate cAMP production in AR42J cells (2). The direct involvement of PKA in mediating gastrin-induced cellular responses has not been shown earlier.

Transcriptional activation of c-fos involves a concerted action of multiple promoter elements, which are regulated in a cell- and agonist-specific manner (10, 56). Several studies have focused the importance of the SRE promoter element, which is indispensible for gastrin- as well as for EGF-mediated activation (57). The present study demonstrates that, in the neuroendocrine cell line AR42J, CRE is also an indispensible promoter element in gastrin-mediated c-fos activation. This implies that activation of the c-fos promoter is dependent on cooperation between proteins binding to SRE and proteins binding to CRE. Parathyroid hormone-induced c-fos transactivation in osteoblastic cells has been reported to depend on an intact CRE promoter element (40). Our observation that CRE is also necessary for gastrin-, CCK-, EGF-, and PaCaP-mediated activation of the c-fos promoter indicates a more central role of CRE in c-fos transactivation than previously shown. However, the fact that inhibition of PKA did not reduce gastrin-mediated WT c-fos transactivation suggests that the CRE promoter element in the c-fos promoter is activated via PKA-independent mechanisms. Furthermore, these results imply that the PKA-dependent mechanisms involved in gastrin-induced proliferation are not mediated via c-fos.

Because the ICER promoter is mainly regulated via CRE elements (32), it was somewhat surprising to find that signaling mechanisms participating in gastrin-mediated ICER activation differ from those involved in gastrin-mediated CRE activation. The ICER promoter contains four different CRE-like promoter elements, and only one of these corresponds to the palindromic TGACGTCA, the somatostatin consensus CRE element that is present in pCRELuc. Thus our results may indicate that the somatostatin consensus CRE elements in the minimal CRE promoter are regulated in a manner different from the CRE promoter elements within the ICER promoter, in that alternative and/or additional signaling pathways are involved in ICER gene expression. The spacing of the CARE 1-4 elements in the ICER promoter, as well as their neighboring DNA sequences, differs from spacing of the four consensus CRE elements in the minimal CRE promoter. It has been reported that the Ca2+/calmodulin responsiveness of CRE elements can vary substantially, depending on the number of basepairs separating the CREs (25). Thus the specific DNA sequence of the ICER CARE 1-4 promoter region may facilitate binding of transcription factors other than those binding to the CRE minimal promoter. Indeed, we found that the CARE 1-4 region of the ICER promoter can bind both proteins of the CREB/CREM/ATF-1 family of transcription factors and proteins that are not recognized by anti-CREB or anti-CREM antibodies. The latter may constitute other members of the extensive CREB-ATF family (19). Alternatively, transcription factors that do not belong to the CREB-ATF family may bind to the CARE 1-4 region. These factors may be members of the C/EBP family, because such proteins have recently been reported to bind with high affinity to the nonconsensus CRE in the phosphoenolpyruvate carboxykinase promoter and to functionally substitute for CREB (43). More specifically, the CREB/CREM/ATF-1 family of transcription factors binding to the CARE 1-4 promoter region of ICER may interact with C/EBP or other proteins that bind to the same region and that are not dependent on activation by PKA, PKC, or Ca2+/calmodulin.

Even though this has not been discussed by others, it cannot be excluded that parts of the ICER promoter other than the CARE 1-4 region may play a role in gastrin-mediated induction of ICER gene expression. Upstream of CARE 1-4, the ICER promoter contains binding sites for transcription factors like STAT (signal transducers and activators of transcription) and Ets (E26 transformation specific)06221. Both STAT and Ets are transcription factors involved in growth control (6, 66), and Ets is one of the factors known to play a role in gastrin-induced c-fos transactivation via the SRE element (57). Thus the involvement of other transcription factors, like STAT or Ets, in gastrin-mediated activation of ICER gene expression cannot be excluded. Further studies will be necessary to elucidate this aspect.


    ACKNOWLEDGEMENTS

We thank Dr. Ugo Moens for providing us with the plasmids WT c-fosLuc, c-fosLucDelta CRE, c-fosLucDelta SRE, and c-fosLucDelta FAP.


    FOOTNOTES

This work was made possible by support from the Norwegian Cancer Society and the Norwegian Research Council.

Address for reprint requests and other correspondence: A. Lægreid, Dept. of Physiology and Biomedical Engineering, Faculty of Medicine, Norwegian Univ. of Science and Technology, Medisinsk Teknisk Senter, N-7489 Trondheim, Norway (E-mail: astridl{at}medisin.ntnu.no).

1 According to analysis with Transfac-The Transcription Factor Database; http://transfac.gbf.de/TRANSFAC/index.html.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 May 2001; accepted in final form 8 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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