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J Biol Chem, Vol. 275, Issue 6, 4244-4250, February 11, 2000

Regulation of Inducible cAMP Early Repressor Expression by Gastrin and Cholecystokinin in the Pancreatic Cell Line AR42J^*

Liv Thommesen, Kristin Nørsett, Arne K. Sandvik, Eva Hofsli, and Astrid Lægreid

From the Department of Physiology and Biomedical Engineering, Norwegian University of Science and Technology, N-7489 Trondheim, Norway

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The CREM gene encodes both activators and repressors of cAMP-induced transcription. Inducible cAMP early repressor (ICER) isoforms are generated upon activation of an alternative, intronic promoter within the CREM gene. ICER is proposed to down-regulate both its own expression and the expression of other genes that contain cAMP-responsive elements such as a number of growth factors. Thus, ICER has been postulated to play a role in proliferation and differentiation. Here we show that ICER gene expression is induced by gastrin, cholecystokinin (CCK), and epidermal growth factor in AR42J cells. The time course of gastrin- and CCK-mediated ICER induction is rapid and transient, similar to forskolin- and phorbol 12-myristate 13-acetate-induced ICER expression. The specific CCK-B receptor antagonist L740,093 blocks the gastrin but not the CCK response, indicating that both the CCK-B and the CCK-A receptor can mediate ICER gene activation. Noteworthy, CREB is constitutively phosphorylated at Ser-133 in AR42J cells, and ICER induction proceeds in the absence of increased CREB Ser(P)-133. Gastrin-mediated ICER induction was not reduced in the presence of the protein kinase A inhibitor H-89, indicating a protein kinase A-independent mechanism. This is the first report on ICER inducibility via G_q/G₁₁ protein-coupled receptors.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inducible cAMP early repressor (ICER)¹ is a member of the CREB and CREM family of transcription factors, which bind to cAMP-responsive promoter elements (CREs) (1-3). CREB was first identified as an activator of gene expression in response to increased intracellular concentration of cAMP (4, 5). The CRE modulator (CREM) gene encodes multiple members of the CRE-binding protein family by alternative splicing as well as by internal transcriptional initiation (6-8). These proteins include both activators (CREM ,) and repressors (CREM , , ) of cAMP-induced transcription. Upon increased levels of cAMP, CREB is phosphorylated at Ser-133 by the catalytic subunit of protein kinase A (PKA) (9, 10), which leads to a dramatic increase in its transactivating potential. Similarly, the activity of CREM  isoforms are increased by PKA-catalyzed phosphorylation (11). However, it has been shown that phosphorylation of both CREM and CREB transcription factors also can involve other kinases like protein kinase C (PKC), calmodulin-dependent kinases, casein kinase, and mitogen-activated protein kinases (MAPKs) (12-15). The discovery of CREM proteins suggests that the activity of the functional CREB dimer is regulated not only by its phosphorylation state, but also by protein/protein interactions like formation of heterodimers, which may either activate or repress transcription, or by competition between CREM and CREB homodimers (11, 16, 17).

ICER isoforms represent a unique CREM subfamily of transcription factors, which are generated by alternative splicing of a transcript generated from an internal CREM gene promotor (P2) containing cAMP response elements (Fig. 1) (1, 2, 17). The kinetics of cAMP-induced ICER expression are characteristic of early response genes, reaching maximum expression after approximately 2 h of stimulation. ICER is proposed to down-regulate both its own expression and the expression of other CRE-containing genes (17).

A large number of genes that are modulated by regulatory peptides contain CRE-like sequences in their promoters, and CRE-binding proteins have been postulated to play an important role in a variety of neuroendocrine processes. cAMP serves as a mitogenic signal for the somatotroph cells, and transgenic mice that express a CREB mutant, which cannot be phosphorylated by PKA, exhibit atrophied pituitary glands and a dwarf phenotype (18). CREM appears to play a decisive role in the regulation of the spermatogenetic process (7), and studies with CREM gene knock-out mice display animals that completely lack spermatozoa and are sterile (19). In particular, inducibility of the ICER repressor has been reported to occur in several tissues in response to hormone stimulation, first described in the hypothalamic-pituitary-gonadal axis and in the pineal gland (1, 17, 20, 21).

Gastrin and CCK are gastrointestinal regulatory peptides, which play a crucial role in differentiation and proliferation of the gastrointestinal tract. While CCK is known to stimulate pancreatic secretion and proliferation, gastrin is a potent inducer of gastric acid secretion. This physiological response is regulated through an interplay of a variety of neuroendocrine cell types in the stomach mucosa (22). Gastrin is also known to act as a growth factor, stimulating proliferation of normal and neoplastic gastrointestinal cells in rats (23), colon carcinoma cell lines (24), fibroblasts (25), and the pancreatic acinar cell-derived cell line, AR42J (26-28). Gastrin has been found to transmit its cellular effects via a specific transmembrane G_q/G₁₁ protein-coupled receptor, the CCK-B/gastrin receptor, while CCK-mediated signaling can occur via both the CCK-B/gastrin and via another receptor within the same family, the CCK-A receptor (29). Both gastrin and CCK-mediated intracellular signaling mechanisms have been reported to include activation of the phospholipase C- (PLC-), as well as activation of the adaptor proteins Shc, Grb, and Sos; Ras and Raf proteins; PKC; and MAPK (30-33). In addition, CCK-A receptors can couple to adenylyl cyclase (34).

In the present study we show that ICER is induced in a variety of rat neuroendocrine cell lines by agents known to activate either PKA or PKC signaling pathways. Furthermore, both gastrin and CCK, whose predominant effector pathway is linked to activation of PLC-  and MAPK, induce ICER gene expression in the AR42J cell line. Gastrin-mediated ICER induction was not reduced in the presence of the PKA inhibitor H-89, indicating a PKA-independent mechanism. This is the first report on ICER inducibility via G_q/G₁₁ protein-coupled receptors. Moreover, ICER is induced in a rat fibroblast and a rat epithelial cell line, but not in human HeLa (epithelial) and HaCaT (keratinocyte) cells, indicating cell specific differences in ICER inducibility.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells and Reagents-- RIN5F cells (rat insulinoma, ATCC) were grown in RPMI 1640 (Life Technologies, Inc., Paisley, Scotland), with 2 g/liter glucose supplemented with 10% (v/v) fetal calf serum (FCS) (Biological Industries, Beit Haemek, Israel), 0.1 mg/ml L-glutamine (Life Technologies, Inc.), and 0.04 mg/ml garamycin (Schering-Plow, Labo, Belgium). PC-12 cells (rat pheochromocytoma, ATCC) were cultivated in Dulbecco's modified Eagle's medium with 4.5 g/liter glucose, supplemented with 10% FCS, 1 mM sodium pyruvate, 0.1 mg/ml L-glutamine, 10 units/ml penicillin/streptomycin (Life Technologies, Inc.), and 1 µg/ml fungizone (Sigma). AR42J (rat pancreatic acinar cell-derived, ATCC), Rat-2 (rat fibroblast, ATCC), and NRK-52E (rat epithelial, ATCC) cells were maintained in Dulbecco's modified Eagle's medium with 4.5 g/liter glucose, 15% FCS (AR42J) or 5% FCS (Rat-2 and NRK52E), 1 mM sodium pyruvate, 0.1 mg/ml L-glutamine, 10 units/ml penicillin/streptomycin, and 1 µg/ml fungizone. HeLa (ATCC) and HaCaT (human keratinocyte cell line, provided by Prof. N. E. Fusenig, Heidelberg, Germany) were cultivated in Dulbecco's modified Eagle's medium with 1 g/liter glucose, 10% FCS, 1 mM sodium pyruvate, 0.1 mg/ml L-glutamine, 10 units/ml penicillin/streptomycin, and 1 µg/ml fungizone. For reverse transcription-polymerase chain reaction (RT-PCR) and gel shift analysis, cells were seeded out in growth medium at 0.9 × 10⁶ (AR42J) or 1 × 10⁶ (RIN5F, PC-12, Rat-2, and NRK52E) cells/well in six-well plates and cultivated for 3 days (subconfluent) before treatment. For Western blot analysis, 4 × 10⁶ cells were seeded out in bottles (75 cm²) and cultivated for 3 days (subconfluent). Cells to be used for Western blot analysis were kept in serum-free medium the last 18-20 h before stimulation.

Cholecystokinin octapeptide (CCK-8) (stored dried and frozen) was purchased from Bachem (Bobendorf, Switzerland). Gastrin-17 (stored dried and frozen) was obtained from Sigma. The peptides were stable for months (20 °C). CREB consensus oligonucleotide (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3') was from Promega Corp. (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 antibodies and anti-phosphorylated CREB were purchased from New England Biolabs (Beverly, United Kingdom). Peroxidase-conjugated swine-anti-rabbit immunoglobulins (1.3 g/liter) were obtained from DAKO (Glostrup, Denmark).

Benzamidine (Sigma) was dissolved in 50% ethanol at 0.5 M; PMA (Sigma) in 96% ethanol at 1 mg/ml and phenylmethylsulfonyl fluoride (PMSF) (Sigma) in isopropyl alcohol at 0.1 M. Dithiothreitol (DTT) (Sigma) was dissolved in 0.01 M sodium acetate at 1 M; H-89 (Calbiochem) was dissolved in water at 10 mM. The reagents were stable for months (PMA, 20 °C; benzamidine, 20 °C; H-89, 20 °C; PMSF, 4 °C).

RT-PCR-- After treatment, cells were washed twice with phosphate-buffered saline, 500 µl of lysis/binding buffer (100 mM Tris, pH 8.0, 500 mM LiCl, 10 mM EDTA, pH 8.0, 1% LiDS, 5 mM DTT was added, and the lysate was pressed three times through a 21-gauge needle by a 1-2-ml syringe to reduce viscosity. Poly(A)⁺ RNA was isolated from lysate (2.5 × 10⁵ cells) with 125 µl of oligo(dT) Dynabeads (Dynal A/S, Norway) according to the protocol of the manufacturer and eluted from the beads in 20 µl of Tris (10 mM, pH 7.5). RT-PCR was performed with 0.5 µl of eluate with rTth DNA polymerase (Perkin-Elmer) according to the procedure recommended by the manufacturer. cDNA synthesis was performed at 61 °C, for 40 min, followed by 35 cycles of PCR with annealing temperature 61 °C, 300 µM dNTP, 50 nM primers, and 3.0 mM Mn (OAc)₂. The number of PCR cycles was selected on the basis of experiments with 20, 25, 30, 35, 40, and 45 cycles, which showed that 35 cycles yielded quantitative results within the linear range. The following PCR primers were used: CREM (ICER)-a, 5'-GTAACTGGAGATGAAACTGA-3'; CREM (ICER)-b, 5'-GACACTTGACATACTCTTTC-3' (Fig. 1). To check whether comparable amounts of poly(A)⁺ RNA from each sample were used, RT-PCR reactions for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed using the following primers: GAPDH-A, 5'-CCCATCACCATCTTCCAG-3'; and GAPDH-B, 5'-ACAGTCTTCTGAGTGGCA-3'. PCR products were run out on a 1.2% or 2% agarose gel and quantitated with Electronic Multi Wave Transilluminator (BRP) and Gelpro-analyser software (Media Cybernetics). The 257- and 700-bp PCR products were cloned into pCR-II (Invitrogen Corp.) and sequenced. Southern blot analysis of the RT-PCR products was performed according to standard procedures (35) with the cloned 257-bp ICER PCR product as a probe.

View larger version (6K): [in this window] [in a new window]   Fig. 1.   Schematic presentation of CREM gene, with positions of PCR primers (a and b) indicated. Positions and orientations of oligonucleotide primers used in PCR are shown by horizontal arrows. Start sites for CREM transcripts (25) and for ICER transcript (P2) are indicated by broken arrows, while translational stop codons are shown by vertical arrows. The functional domains, two glutamine-rich transactivating regions (Q1 and Q2), the phosphorylation domain (P-Box), and two alternative dimerization and DNA binding domains (DBI and DBII) are indicated.

Western Blot Analysis-- Whole cell lysates were prepared from 5-7 × 10⁶ cells, which were washed twice in phosphate-buffered saline, scraped, and harvested directly in 500 µl of SDS-sample buffer (62.5 mM Tris-HCl, pH 6.8, 8.7% glycerol, 2% w/v SDS, 5% v/v 2--mercaptoethanol, 0.09% w/v bromphenol blue). Viscosity was reduced by drawing the suspension through a 21-gauge needle, cell debris were removed by centrifugation (15,000 × g, 10 min), and the supernatant was stored at 80 °C. 15 µl of each extract was boiled and separated on a 10% SDS-polyacrylamide gel (running buffer: 25 mM Tris-HCl, pH 8.3, 190 mM glycine, 0.1% w/v SDS) prior to electrotransfer onto Hybond-P membranes (Amersham Pharmacia Biotech). The transfer was performed in 25 mM Tris-HCl, 190 mM glycine, and 20% methanol, pH 8.3, for 1 h at 175 mA. The membranes were treated with 5% nonfat dry milk (Nestlé) in TBS (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl) for 1 h at room temperature and incubated with primary antibodies diluted 1:1000 in TBS with 1% bovine serum albumin and 0.05% Tween 20 overnight at 4 °C. The blots were then incubated with peroxidase-conjugated secondary antibodies in TBS with 1% bovine serum albumin and 0.05% Tween 20 for 1.5 h at room temperature. After washing (four 15-min washes in TBS with 0.05% Tween 20), binding of secondary antibodies (1:1000) was visualized by the ECL detection system (Amersham Pharmacia Biotech).

Measurement of PKA Activity-- Whole cell extracts (10 mM potassium phosphate, pH 6.8, 5 mM EDTA, 250 mM sucrose, 0.5% Triton X-100, 50 mM NaF, 30 mM Na₄P₂O₇, 100 µM Na₃VO₄, 1 mM DTT, 5 µg/ml pepstatin A, 0.5 mM benzamidine, 0.5 mM PMSF) were prepared from cells treated with isobutylmethylxanthine (50 µM) for 30 min. before stimulation for 30 min. with forskolin (100 µM), gastrin (10 nM), or CCK (10 nM) in the presence of isobutylmethylxanthine. PKA activity was determined as described (36). Briefly, 1.5 µl of extract was incubated in a total volume of 20 µl containing 20 mM potassium phosphate, pH 6.8, 10 mM MgCl₂, 100 µM [-³²P]ATP, 0.25 mg/ml bovine serum albumin, and 50 µM CREB-tide (Sigma). After incubation for 10 min at 30 °C, 5 µl of the reaction mixture was spotted onto nitrocellulose filters, washed with 1% phosphoric acid and water, and counted in a scintillation counter. Total PKA activity was determined in the presence of 10 µM cAMP. PKA activity was defined as that sensitive to 10 µM PKA inhibitor.

Gel Shift Assay-- Preparation of nuclear extracts and gel shift analysis was performed essentially as described previously (37). Briefly, cells were washed with phosphate-buffered saline, incubated in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM benzamidine, 0.5 mM 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, 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 of poly(dI-dC) (Amersham Pharmacia Biotech) in binding buffer (20 mM Hepes, pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 0.25 mg/ml bovine serum albumin, 2% Ficoll) (20 µl final volume) for 10 min at room temperature. Then, 17 fmol of ³³P-labeled oligonucleotide probe was added, and the mixture was incubated for 30 min at room temperature. The samples were applied on non-denaturing polyacrylamide gels (7% acrylamide, 0.25 × Tris borate-EDTA, 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, Eastman Kodak Co.) 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 was added and the mixture was incubated for another 45 min on ice before electrophoresis.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ICER Gene Expression in the AR42J Cell Line-- ICER gene expression is known to be rapidly inducible via cAMP in the neuroendocrine-derived cell line PC-12 (17). We found that forskolin could induce ICER expression in the neuroendocrine cell line RIN5F and in AR42J cells in a manner similar to that for PC-12 (Fig. 2, A and B). Sequencing analysis of the 257- and 700-bp PCR products, and Southern blot analysis showed that the 700-, 661-, 262-, and 223-bp RT-PCR products are derived from the transcripts ICER I, ICER I, ICER II, and ICER II, respectively (1, 17). In all cells, low levels of ICER were detected in untreated cells. Moreover, we found that PMA could also induce ICER in AR42J, RIN5F, and PC-12 cells albeit at lower levels compared with forskolin (Fig. 2B). These results indicate that several signaling pathways can activate ICER gene expression in these cell lines.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   ICER expression in forskolin- and PMA-treated cells analyzed by RT-PCR of poly(A)+ RNA and visualized in ethidium bromide-stained agarose gels. GAPDH RT-PCR was performed as a control of the RNA amount in each sample. A, AR42J cells, untreated or treated for 2 h with forskolin (25 µM). B, AR42J, RIN5F, and PC-12 cells untreated or treated for 2 h with forskolin (25 µM) or PMA (100 ng/ml). C, the time course of ICER expression in AR42J cells treated with forskolin (25 µM) or PMA (100 ng/ml). D, Rat-2 and NRK52E cells untreated or treated for 2 h with forskolin (25 µM). A 100-bp ladder was used as a marker. The results shown are representative of three independent experiments with duplicate samples. A, 2.2% gel, B-D, 1.2% gel.

Analysis of AR42J cells after varying periods of forskolin treatment showed that a rise in ICER transcript levels is clearly detectable after 0.5 h of stimulation, while a peak was reached after approximately 2 h. ICER levels were strongly reduced after 6 h (Fig. 2C). A similar time course was observed in PMA-treated AR42J cells (Fig. 2C). Similar time courses were observed in forskolin-treated RIN5F and PC-12 cells.²

ICER activation has been linked to regulation and control of development in neuroendocrine cells and tissue (1, 17), but several studies over the last years have suggested a role for ICER as a general transcriptional repressor (38, 39). It was therefore of interest to include both a fibroblast (Rat-2) and an epithelial rat (NRK52E) cell line in our studies of ICER expression. We found that ICER gene expression was induced by forskolin both in Rat-2 and in NRK52E cells (Fig. 2D). However, ICER expression could not be detected in forskolin-treated human cell lines HaCaT or HeLa,² even though the PCR primers used are derived from regions of the CREM gene that are highly conserved in rat and human and that will prime the PCR reaction of human ICER equally well as rat ICER. This indicates that ICER is not expressed in all cell lines.

EGF and NGF Mediate Induction of ICER Expression-- Since we had shown that ICER gene expression could be induced by the PKC activator PMA, we wanted to investigate whether other signaling mechanisms distinct from cAMP-coupled pathways could also regulate ICER gene expression. We therefore analyzed ICER gene expression in AR42J cells treated with epidermal growth factor (EGF) whose signaling pathways are linked to intracellular receptor tyrosine kinase activity. We found that EGF could induce ICER gene expression to a similar extent as forskolin (Fig. 3A).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   ICER expression in EGF- and NGF-treated cells. AR42J cells treated for 2 h with EGF (10 ng/ml) (A) and PC-12 cells treated for 2 h with NGF (10 ng/ml) or EGF (10 ng/ml) (B). Forskolin treatment (25 µM, 2 h) was used as a positive control. The results shown are representative of three independent experiments with duplicate analysis.

Elevated ICER mRNA expression could also be detected in PC-12 cells upon nerve growth factor (NGF) and EGF stimulation (Fig. 3B). These results indicate that tyrosine kinase-coupled receptors can mediate induction of ICER gene expression in different cell lines.

Gastrin-17 and CCK-8 Activate Transcription of ICER in AR42J-- AR42J cells express both cholecystokinin and gastrin receptors (26). The G protein-coupled gastrin receptor CCK-B signal transduction mechanism is known to involve PLC-, PKC, and MAPK (30, 31). Since our results so far indicated that PKC could induce ICER gene expression, it was of interest to investigate whether gastrin had a similar effect. We found that gastrin-17 increased the level of ICER transcripts in a dose-dependent manner (Fig. 4A). Maximum activation of ICER was reached after approximately 2 h and was clearly reduced after 6-10 h of stimulation (Fig. 4B). The results indicate that activation of the CCK-B receptor can induce an increase in ICER gene expression with a similar time course as forskolin and PMA.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   ICER expression induced by gastrin-17 and CCK-8 in AR42J cells treated for 2 h with increasing concentrations of gastrin (0 - 500 nM) (A) or CCK (0-500 nM) (C), or treated with gastrin (50 nM) (B) or CCK (10 nM) (D) for varying time periods between 0.5 and 10 h. Similar results were obtained in two other experiments. Quantitation was performed with Electronic Multi Wave Transilluminator and Gelpro-analyser software. Results are shown for ICER II transcripts. The time course for ICER I transcripts was similar.

We also measured ICER expression in AR42J cells treated with cholecystokinin octapeptide (CCK-8), which can activate both the CCK-A and CCK-B receptors. CCK-8-induced ICER gene expression in a dose-dependent manner (Fig. 4C) with similar kinetics as gastrin-17 (Fig. 4D). However, CCK-8 seems to be a more potent agonist than gastrin-17, since 0.1 nM CCK-8 was sufficient to induce ICER expression while 10-fold amounts of gastrin-17 had to be applied in order to reach similar ICER levels.

The CCK-B Antagonist L740,093 Blocks Gastrin-induced but Not CCK-induced Increase in ICER Gene Expression-- CCK binds CCK-A and CCK-B receptors with similar affinity (26). To elucidate whether CCK-8 mediated its ICER -inducing effect through CCK-B or through both CCK-A and CCK-B receptors, we stimulated cells with either CCK-8 or gastrin-17 in the presence of increasing amounts of L740,093, a selective CCK-B antagonist (40). Fig. 5 shows that, while gastrin-17-mediated induction of ICER expression was completely inhibited in the presence of 0.1 nM L740,093, CCK-8-mediated ICER induction was not affected by the antagonist, even at a concentration of 10 nM. The results indicate that gastrin-mediated ICER induction is completely dependent on CCK-B receptors, while CCK-8 can induce ICER expression independently of CCK-B receptors. Taken together, these experiments suggest that activation of either CCK-A or CCK-B receptors can induce ICER gene expression in AR42J cells.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effect of L740,093 on CCK- and gastrin-induced ICER expression. AR42J cells treated for 2 h with gastrin (10 nM) or CCK (10 nM) in the presence of increasing concentrations of L740,093 (0-10 nM). Results are shown for the ICER II transcript. L740,093 was added immediately prior to gastrin or CCK. Similar results were obtained in two other experiments.

CREB Is Constitutively Phosphorylated at Ser-133 in AR42J-- Since transcription of ICER is controlled by CRE elements in the CREM gene P2 promoter (17), CREB is thought to play an important role in ICER gene expression. Phosphorylation of CREB at Ser-133 is necessary for its transcription promoting activity (41) and can be catalyzed by several kinases like PKA, PKC, and calmodulin-dependent kinase (13, 42-44). It was therefore of interest to explore whether gastrin and/or CCK can induce CREB Ser-133 phosphorylation, in order to clarify whether this transcription factor can play a role in gastrin/CCK-mediated induction of the ICER promoter. In PC-12 cells, the level of phosphorylated CREB is low in unstimulated cells, whereas forskolin treatment strongly induces CREB phosphorylation (9) (Fig. 6, lanes 6 and 7). In AR42J cells, however, CREB was found to be constitutively phosphorylated at Ser-133, and no increase in CREB phosphorylation was observed in lysates from cells stimulated with either forskolin, gastrin-17, or CCK-8 for 30 min (Fig. 6, lanes 3-5), or in lysates after stimulation periods up to 2 h.² Likewise, costimulation of AR42J with forskolin and gastrin-17, CCK-8, or PMA did not result in a detectable change in CREB Ser-133 phosphorylation.²

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Western blot analysis of CREB Ser-133 phosphorylation. Whole cell lysates from AR42J and PC-12 cells were treated for 30 min with forskolin (25 µM), CCK-8 (10 nM), or gastrin-17 (50 nM), and PC-12 cells were treated with forskolin (25 µM, 30 min) before analysis by Western blot as described under "Experimental Procedures." The mobilities of mass markers 41.8 and 47.5 kDa are indicated. The results shown are representative of three independent experiments.

Gastrin Can Induce ICER Gene Expression Independently of PKA-- Since PKA is known to be involved in activation of the ICER promotor, it was of interest to investigate whether gastrin and CCK could activate this kinase. We could not detect gastrin- or CCK-induced activation of PKA in AR42J cells, while forskolin treatment of the cells resulted in a 2-4-fold increase in PKA activity.² The well known PKA inhibitor H-89 (45) completely abolished forskolin-mediated ICER induction, but did not affect gastrin-induced ICER up-regulation (Fig. 7). This observation indicates that PKA is not necessary for gastrin-mediated ICER induction.

View larger version (63K): [in this window] [in a new window]   Fig. 7.   Effect of H-89 on gastrin-induced ICER expression. AR42J cells treated for 2 h with forskolin (25 µM) or gastrin (10 nM) with or without H-89 (10 µM). Results are shown for the ICER I and II transcript. H-89 was added 1 h prior to gastrin or forskolin. Similar results were obtained in two other experiments.

Gel Shift Analysis Displays Cell-specific Differences in CRE-binding Proteins-- Our studies so far show that ICER gene expression is inducible in Rat-2, NRK52E, AR42J, RIN5F, and PC-12 cells. However, the relative levels of ICER transcripts vary with different stimulations and cell lines. Since the CRE elements in the CREM P2 promoter are of major importance in the regulation of ICER gene expression (17), we wanted to compare CRE-binding proteins in the various cell lines. Fig. 8A shows gel shift analysis of nuclear extracts from AR42J cells, demonstrating specific binding of five protein complexes to an oligonucleotide containing the somatostatin consensus CRE sequence. To further identify the CRE-binding proteins, supershift analysis was performed. Addition of anti-CREB and anti-CREM antibodies led to the appearance of two distinct supershifted complexes (termed a and b, respectively) (Fig. 8A). Anti-CREB antibody, which is generated against exon E (kinase-inducible domain) (amino acids 123-137) supershifted complex III and IV more efficiently than complex V. With anti-CREM antibody, generated against a full-length fusion protein, bands III, IV, and V disappear completely. Anti-CREM cross-reacts partially with other activating transcription factor (ATF)/CREB proteins, while anti-CREB would be expected to detect all CREB and CREM proteins containing exon E, since amino acids 123-137 are identical in these proteins. This indicates that complexes III, IV, and V contain CREM or CREB or ATF proteins. Since complex V was mainly supershifted by anti-CREM and not by anti-CREB antibodies, it may contain a CREM/CREB/ATF protein that lacks exon E. Complex I and II were not supershifted and thus represent CRE-binding factors immunologically unrelated to the CREB/CREM/ATF family. Further gel shift analysis showed that similar CRE- binding proteins can be detected in RIN5F and PC-12 (Fig. 8B). However, the relative amounts of each complex differ. Complex III, which appeared to be most abundant in AR42J cells, was barely detectable in PC-12 and RIN5F cells, where complex V constituted a relatively higher proportion of CRE-binding proteins (Fig. 8B). Rat-2 and NRK52E displayed a similar pattern of CRE-binding proteins as AR42J.² The fact that CREM proteins have been reported to show cell-specific expression patterns (17) may indicate that complexes III and/or V are derived from CREM proteins.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   CRE gel shift analysis of AR42J, RIN5F and PC-12. A, nuclear extracts from the untreated AR42J cells were analyzed for factors binding to an oligonucleotide containing the somatostatin promoter CRE sequence. Complexes I, II, III, IV, and V represent specific CRE-binding factors, as they are displaced by 20-, 60-, and 80-fold excess of unlabeled CRE oligonucleotide (wt) (lanes 3, 4, and 5), while oligonucleotides mutated in the CRE consensus sequence (M) were not able to compete for binding (lanes 6, 7, and 8). Complexes III, IV, and V were partially supershifted with 2 µg of anti-CREB antibody (lane 9) and completely supershifted with 2 µg of anti-CREM antibody (lane 10). Complex a represents the specific supershift with anti-CREB antibody and complex b the anti- CREM antibody supershift. NE, without nuclear extract. B, CRE gel shift of nuclear extracts from AR42J (lanes 2-4), PC-12 (lanes 5-7), and RIN5F (lanes 8-10). Competition was performed with 60-fold excess of either unlabeled CREB consensus oligonucleotide (wt) (lanes 3, 6, and 9), or with mutated oligonucleotide (M) (lanes 4, 7, and 10). The results shown are representative of three independent experiments.

Comparison of untreated and treated cells revealed no difference in the pattern of CRE-binding proteins, indicating that these proteins are not inducible. This, and our failure to observe CRE-binding proteins of low molecular weight, indicates that we did not detect ICER proteins by gel shift analyses. However, others have reported detection of inducible ICER proteins in other neuroendocrine cells by band shift analysis (17, 46). The discrepancy between these reports and our results may be due to low levels of ICER protein in AR42J cells or to the fact that we used an oligonucleotide containing the somatostatin promoter CRE element, while the CRE elements from the CREM gene P2 promoter may be more optimal for detection of ICER proteins by gel shift (17).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present study shows that ICER gene expression can be induced in AR42J cells by variety of agents including EGF, gastrin, and CCK. This has not been reported earlier. The AR42J cell line, derived from an azaserine-induced tumor of rat pancreas, possesses both exocrine and neuroendocrine characteristics (47). Due to the fact that AR42J expresses both CCK-B/gastrin and CCK-A receptors, the growth-promoting effect of gastrin and CCK and their intracellular signaling pathways has been extensively studied in these cells (30, 31, 48, 49). Since ICER is thought to play a pivotal role in regulation of growth and differentiation, we consider AR42J cells an interesting model for the study of a possible involvement of ICER in gastrin- and CCK-mediated cellular responses.

AR42J cells may also be suited for comparison of the signaling mechanisms of CCK-A and CCK-B, since our results indicate that induction of ICER gene expression can be mediated by either of these receptors. Although CCK-A and CCK-B have been found to activate similar downstream signaling events, more detailed studies may reveal differences in the manner that these intracellular events are set into play by a given receptor in a given cellular response. This would be analogous to the two types of tumor necrosis factor receptors, which we have found to mediate activation of transcription factor NFB through different, but partially overlapping signal transduction pathways (37, 50).³

CCK-A and CCK-B receptors are G protein-coupled receptors where the main G-protein has been found to be of the G_q/G₁₁ type (51-53). Thus, the predominant effector pathway of gastrin and CCK is linked to activation of PLC-, formation of the second messengers inositol triphosphate and diacylglycerol with subsequent mobilization of intracellular Ca²⁺ and activation of PKC (54-56). Furthermore, gastrin and CCK induce activation of Ras and Raf proteins and the MAPK cascade via adaptor proteins Shc, Grb, and Sos (30-33, 57, 58). Recently, gastrin was reported to cause cAMP accumulation in AR42J cells (59), which may indicate that the CCK-B receptor can also couple to G proteins of the G_s type. Alternatively, cAMP production may be caused by the gastrin-induced rise in intracellular Ca²⁺ via calmodulin-activated adenylyl cyclase. However, we could not detect a rise in PKA activity in cells stimulated with gastrin or CCK. Furthermore, experiments with the PKA inhibitor H-89 show that PKA is not necessary for gastrin-mediated ICER induction. Thus our data imply that gastrin-induced ICER gene expression is mediated by cAMP-independent signaling mechanisms.

We found that EGF could induce ICER gene expression in AR42J, while both EGF and NGF could cause the same effect in PC-12 cells. NGF-mediated ICER induction via a Ras-dependent pathway was also recently reported by others (44). In the same study, however, ICER expression could not be detected upon EGF stimulation of PC-12 cells, a discrepancy that could be due to different analytical methods. Our results support the hypothesis that growth factors which transmit their signals via tyrosine kinase receptors can also induce ICER gene expression. A central, downstream signaling pathway activated by tyrosine kinase receptors is the MAPK cascade (60). This pathway can also be activated via PKC (61). Our results showing that ICER expression can be induced by treatment of cells with either tyrosine kinase receptor stimulating growth factors or with PMA, which acts via PKC, may indicate that the MAPK cascade can be involved in activation of ICER gene expression. Taken together with our observation that gastrin can induce ICER in a PKA-independent manner, we therefore speculate that activation of the MAPK cascade plays a major role in the gastrin-activated signaling mechanisms involved in induction of ICER gene expression.

Activation of the ICER promotor is thought to be controlled by CREB, a transcription factor that is known to depend on phosphorylation at Ser-133 (41). In contrast to PC-12 cells, where CREB phosphorylation only occurs upon stimulation with agents like NGF or forskolin (9, 13), we found that CREB was constitutively phosphorylated at Ser-133 in AR42J cells and no increase in CREB Ser-133- P was detected after treatment with forskolin or other agents. In NIH 3T3 cells, the transcriptional potential of CREB is reported to be moderated by signals independent of Ser-133, although Ser-133 phosphorylation seems necessary for activation (62). Another study has shown that CREB Ser-133 phosphorylation is necessary but not sufficient to induce c-fos expression upon CCK injection (63). Similarly, our results suggest that signaling events additional to CREB Ser-133 phosphorylation are necessary in order to induce ICER transcription in AR42J cells. The spectrum of agents found to induce ICER in AR42J suggest that these additional signaling events can be supplied by PKA or PKC, or by PLC- -associated signaling pathways, one of which may be the MAPK cascade. Furthermore, it is likely that these additional signals function through sites in CREB other than Ser-133 or that they involve other transcription factors than CREB.

Recent studies have brought to light ICER as a general repressor in non-neuroendocrine cells as well. In the myeloid leukemia cell line IPC-81, high levels of ICER proteins protected the cells from cAMP-induced apoptosis (38). Furthermore, ICER has been found to repress cytokine gene expression (39), as well as human T-cell lymphotrophic virus, type 1 promotor activity (64), in human medullary thymocytes. Our observation that ICER can be induced also in fibroblast and epithelial cell lines, has to our knowledge, not been previously reported and indicates a role for the transcriptional repressor ICER in these cell types as well.

An interesting aspect of our study is the putative role of the transcriptional repressor ICER in the mitogenic cellular responses mediated by gastrin and CCK. Further studies to elucidate the mechanisms of gastrin and CCK-mediated ICER induction and possible biological implications are under way in our laboratory.

	ACKNOWLEDGEMENTS

We thank Dr. Ole Morten Seternes and Dr. Ketil Taskén for valuable advice and support with the PKA assays.

	FOOTNOTES

^* This work was supported by the Norwegian Cancer Society, The Research Council of Norway, and the Cancer Foundation at the Trondheim University Hospital.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.

To whom correspondence should be addressed: Dept. of Physiology and Biomedical Engineering, Norwegian University of Science and Technology, Medisinsk Teknisk Senter, N-7005 Trondheim, Norway. Tel.: 47-73-59-86-16; Fax: 47-73-59-89-86; E-mail: astridl@medisin.ntnu.no.

² L. Thommesen, K. Nørsett, A. K. Sandvik, E. Hofsli, and A. Lægreid, data not shown.

³ A. Laegreid, W. Rikardsen, L. Thommesen, A. E. Medvedev, A. Sundan, and T. Espevik, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: ICER, inducible cAMP early repressor; CRE, cAMP-responsive element; CREB, cAMP-responsive element binding protein; P-CREB, phosphorylated CREB; CREM, cAMP-responsive element modulator protein; MAPK, mitogen-activated kinase; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol myristate acetate; CCK, cholecystokinin; EGF, epidermal growth factor; NGF, nerve growth factor; ATF, activating transcription factor; PCR, polymerase chain reaction; RT, reverse transcription; FCS, fetal calf serum; PMSF, phenylmethylsulfonyl fluoride; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TBS, Tris-buffered saline; FCS, fetal calf serum; DTT, dithiothreitol.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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