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Originally published In Press as doi:10.1074/jbc.M407485200 on December 14, 2004

J. Biol. Chem., Vol. 280, Issue 9, 7976-7984, March 4, 2005
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Essential Interaction of Egr-1 at an Islet-specific Response Element for Basal and Gastrin-dependent Glucagon Gene Transactivation in Pancreatic {alpha}-Cells*

Stéphane Leung-Theung-Long{ddagger}§, Emmanuelle Roulet¶, Pascal Clerc{ddagger}, Chantal Escrieut{ddagger}, Sophie Marchal-Victorion{ddagger}||, Beate Ritz-Laser¶, Jacques Philippe¶, Lucien Pradayrol{ddagger}, Catherine Seva{ddagger}, Daniel Fourmy{ddagger}, and Marlène Dufresne{ddagger}**

From the {ddagger}Inserm U531, IFR31, Hospital Rangueil, 31059 Toulouse Cedex 9, France, and the Diabetes Unit, University Hospital Geneva, 1211 Geneva 14, Switzerland

Received for publication, July 6, 2004 , and in revised form, December 3, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The peptide hormone gastrin is secreted from G cells of the gastric antrum and is the main inducer of gastric acid secretion via activation of its receptor the cholecystokinin 2 (CCK2) receptor. Both gastrin and CCK2 receptors are also transiently detected in the fetal pancreas and believed to exert growth/differentiation effects during endocrine pancreatic development. We demonstrated previously that whereas gastrin expression is extinguished in adult pancreas, CCK2 receptors are present in human glucagon-producing cells where their activation stimulates glucagon secretion. Based on these findings, we investigate in the present study whether gastrin regulates glucagon gene expression. To this aim, the CCK2 receptor was stably expressed into a glucagon-producing pancreatic islet cell line, and a glucagon-reporter fusion gene was transiently transfected in this new cellular model. We report that gastrin stimulates glucagon gene expression in glucagon-producing pancreatic cells. By using progressively 5'-increased sequences of the glucagon gene, gastrin responsiveness was located within the minimal promoter. Moreover, we clearly identified early growth response protein 1 (Egr-1) as an essential transcription factor interacting with the islet cell-specific G4 element. Egr-1 was shown to be essential for basal and gastrin-dependent glucagon gene transactivation. Furthermore, our results demonstrate that the MEK1/ERK1/2 pathway couples the CCK2 receptor to nuclearization and DNA binding of Egr-1. In conclusion, our data provide new information concerning the transcriptional regulation of the glucagon gene. Moreover they open new working hypothesis with reference to a potential role of gastrin in glucagon-producing pancreatic cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Gastrin, an important gastrointestinal regulatory peptide, was recognized initially as the main inducer of gastric acid secretion. Gastrin is secreted from endocrine G cells of the gastric antrum during postnatal and adult life in response to a meal (1). The cholecystokinin 2 (CCK2)1 receptor, a G protein-coupled receptor, specifically mediates the actions of gastrin on target cells via activation of multiple signaling enzymes including phospholipase C with subsequent phosphoinositide breakdown, intracellular calcium mobilization, and protein kinase C stimulation (2), but also intracellular mediators classically described in the regulation of mitogenesis and cellular adhesion by growth factors. The mitogen-activated protein kinases, ERK1/2, Jun kinase, and p38 MAPK, as well as phosphatidylinositol 3-kinase, are known targets of gastrin. Several groups, including ours, have documented the contribution of upstream Src and Fak family tyrosine kinases in the activation of these pathways (3–6).

Besides a role in the stomach, several lines of evidence, including our data, raise the possibility that the endocrine pancreas is also a potential physiological target of gastrin. Indeed, because of the presence of responsive elements that account for islet expression on its gene promoter, the major site of expression of amidated gastrin is the pancreas of mammals during fetal life (7, 8). After birth, gastrin ceases to be expressed in the pancreas, supporting a specific role played in this organ during the fetal period (9). We confirmed the presence of amidated gastrin in human fetal pancreas together with localization of CCK2 receptors partly in undifferentiated cells and in glucagon-producing cells (10).

Additionally, and further supporting a role in the endocrine pancreas, we demonstrated previously that gastrin may contribute to glucose homeostasis in adult via stimulation of glucagon secretion. Indeed, expression of CCK2 receptors on adult pancreatic glucagon cells was linked to a physiological secretion of glucagon from isolated human islets in response to gastrin (10). Of importance, the essential role of gastrin in the normal islet glucagon counterregulatory response to hypoglycemia has been confirmed recently in mice in which its gene has been knocked out (11). Although glucagon is the second major hormone in the control of glucose homeostasis, acting as a counterregulator to insulin, the regulation of its secretion, biosynthesis, and gene expression by nutrients and other factors has not been investigated as intensively as in the case of insulin. However, the metabolic consequences of abnormal {alpha}-cell function are well defined. Indeed, chronic hyperglucagonemia is a common feature of type II diabetic patients and is partially responsible for hyperglycemia (12). Conversely, loss of the glucagon response impairs recovery from hypoglycemia in nondiabetic patients but is also a major factor in the susceptibility of patients with type I diabetes to prolonged and severe hypoglycemia (13). There is evidence that increased glucagon gene expression accompanies hyperglucagonemia but also that regulation of glucagon gene expression may differ from that of glucagon secretion (14). Effects of gastrin on the expression of glucagon are therefore important to consider.

In the present study, we investigated whether gastrin regulates glucagon gene expression. To this aim and to characterize the molecular mechanisms involved in this effect, we engineered a new cellular model derived from the glucagonoma cell line InR1G9 (15) stably expressing the human CCK2 receptor. We now report that gastrin stimulates glucagon gene expression and identify early growth response protein 1 (Egr-1) as an essential transcription factor for basal and gastrin-stimulated glucagon gene expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—The glucagon-producing hamster InR1G9 cell line was stably transfected with a vector (PRFEneo) expressing the human CCK2 receptor using FuGENE 6 reagent (Roche Applied Science) as described previously (6) and grown at 37 °C in a 5% CO2 humidified atmosphere, in RPMI 1640 with Glutamax-I (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (BioWhittaker, Inc.), 50 units/ml penicillin (Invitrogen), 50 µg/ml streptomycin (Invitrogen), and 200 µg/ml geneticin (Sigma).

Receptor Binding Assay—Approximately 24 h after their transfer to 24-well plates (20,000 cells/well), cells were washed with phosphate-buffered saline (PBS), pH 6.95, and 0.1% BSA and then incubated for 60 min at 37 °C in 0.5 ml of RPMI, 0.1% BSA with 60 pM sulfated 125I-CCK9 in the presence of competing CCK9. Cells were washed twice with cold PBS, pH 6.95, containing 2% BSA, and cell-associated radioligand was collected with 0.1 N NaOH added to each well. The radioactivity was counted in a {gamma}-counter (Auto-Gamma, PerkinElmer Life Sciences).

CCK2 Receptor Fluorescent Probe—The [Thr28,Ahx31]CCK-(25–33) peptide (CCK9) was derivatized with fluorescein isothiocyanate (Sigma). The peptide was dissolved in 0.1 M borate buffer, pH 9, and reacted overnight with an equimolar quantity of fluorescent label. The fluorescent probe was purified by reverse phase high performance liquid chromatography.

CCK2 Receptor Localization—Cells were cultured on glass slides overnight at 37 °C. Cells were washed twice with PBS containing 0.2% BSA and incubated with 50 nM fluorescein isothiocyanate-coupled CCK9 for 1 h at 4 °C. Cells were then fixed with 2% paraformaldehyde in PBS for 15 min at room temperature, and slides were mounted.

Glucagon Secretion and Radioimmunoassay—Cells were cultured for 1 h in serum-free medium, containing 0.1% BSA, in 24-well plates (50,000 cells/well). Cells were incubated in 1 ml of RPMI, 0.1% BSA, containing CCK or gastrin at 37 °C for 1 h. Media were then collected and stored at -20 °C until use. Secretion of glucagon was quantified on 50 µl of medium by radioimmunoassay using 16 pM 125I-glucagon, and an antibody was raised against the carboxyl terminus of glucagon (Gan8, 1:75,000, generously provided by Dr. D. Bataille, Inserm U376, Montpellier, France). Free glucagon was separated from antibody-bound glucagon adding 4 mg/ml activated charcoal and 5 mg/ml dextran T70 for 15 min at 4 °C. After centrifugation at 2,000 x g for 20 min, supernatants, containing glucagon-antibody complexes, were collected, and radioactivity was counted in a {gamma}-counter.

Northern Blot Analysis—Cells (1.5 x 106) were cultured in 60-mm culture dishes overnight in serum-free medium, containing 0.1% BSA, for 24 h at 37 °C before stimulation with 1 µM gastrin. Total cellular RNA was extracted by TRIzol reagent (Invitrogen). RNA samples (10 µg/lane) were separated on formaldehyde-containing 1% agarose gel and blotted onto nylon membrane. The blot was hybridized with glucagon cDNA probe labeled with [{alpha}-32P]dCTP by random primer extension. Specific hybridization was visualized after exposure to a PhosphorImager (Molecular Dynamics). To ensure RNA integrity and to confirm equal loading of lanes, the membrane was rehybridized with a probe for 18 S rRNA.

Glucagon mRNA Stability—Glucagon mRNA half-life was determined from an actinomycin D decay curve. Actinomycin D (5 µg/ml) (Sigma) was added to the medium of both control and gastrin-stimulated cells. Total cellular RNA was extracted 4, 8, and 12 h after addition of gastrin. Levels of glucagon mRNA were then analyzed by Northern blot.

DNA Transfection—Cells (4 x 106) were plated on 100-mm culture dishes 24 h before cotransfection. They were transfected using the DEAE-dextran method, with 3 µg of reporter gene plasmid (pOCAT) and 1 µg of pRSV-Luc, a transfection control plasmid. Reporter gene plasmid consisted of either rat wild-type glucagon gene -2.5-kb fragment (nucleotides -2.5 kb to +58) or 292-bp fragment (nucleotides -292 to +58), 169-bp fragment (nucleotides -169 to +58) or 138-bp fragment (nucleotides -138 to +58) of the 5'-flanking sequence of the rat glucagon gene, linked to the reporter gene CAT (-2.5 kb CAT, -292 CAT, -169 CAT, and -138 CAT, respectively). When indicated, -138 CAT was mutated at nucleotides -135/-130, -120/-115, -107/-102, -89/-88, -72/-71, or -56/-55 (called E47/{beta}2 mut, 120 mut, {beta}-TF1 mut, Pax-6 mut, Cdx-2/3 mut, and Isl-1 mut, respectively).

Chloramphenicol Acetyltransferase (CAT) Assay—24 h after transfection, cells were transferred into 60-mm culture dishes and cultured in serum-free medium with 0.1% BSA overnight at 37 °C. Cells were then stimulated or not with gastrin for 24 h in RPMI 1640, 0.1% BSA. When indicated, inhibition of MEK1 was achieved incubating serum-starved cells with the specific inhibitor 50 µM PD98059 for 1 h at 37 °C before stimulation with gastrin. Cell extracts were prepared and analyzed for CAT activity as described previously (16). Quantification of acetylated and nonacetylated forms of chloramphenicol was done by PhosphorImager analysis. For each experiment, three independent transfections were performed, each of them carried out in duplicate.

Site-directed Mutagenesis—Nucleotide substitution mutations of binding domains for transcription factors were performed by oligonucleotide-directed mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene) using -138 CAT clone as template. The presence of desired mutations was confirmed by automated sequencing of both strands (Applied Biosystems).

Nuclear Extracts—Cells (4 x 106) were plated on 100-mm culture dishes. Before stimulation with 1 µM gastrin for the indicated time periods, cells were cultured overnight in serum-free medium with 0.1% BSA. When indicated, inhibition of MEK1 was achieved by incubating serum-starved cells with 50 µM PD98059, the specific inhibitor of MEK1, for 1 h at 37 °C before stimulation with gastrin. Cells were washed with cold PBS, treated with trypsin, and centrifuged for 5 min at 100 x g at 4 °C. Cells were washed twice with cold PBS, resuspended carefully in 400 µl of a 10 mM HEPES buffer, pH 7.9, containing 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2.5 mM dithiothreitol, and 1.2 mM phenylmethylsulfonyl fluoride, and incubated for 15 min on ice. 30 µl of 10% Nonidet P-40 was added, and the mixture was vortexed and centrifuged for 1 min at 4 °C at 10,000 x g. Pellets were incubated and shaken at 4 °C for 15 min with 60 µl of a 20 mM HEPES buffer, pH 7.9, containing 25% glycerol, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM dithiothreitol, and 1.2 mM phenylmethylsulfonyl fluoride, centrifuged for 10 min at 15,000 x g at 4 °C, and supernatants were stored at -80 °C until use.

Electrophoretic Mobility Shift Assays (EMSAs)—Double-stranded oligonucleotide probe 120 corresponding to the nucleotides -129/-108 (5'-AGCAGAGTGGGCGAGTGAAAT-3') of the glucagon gene was 5'- end labeled with [32P]dATP using T4 kinase (Invitrogen). Binding reactions were performed at room temperature for 20 min in a final volume of 20 µl of binding buffer containing 20 mM HEPES, pH 7.9, 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol, 50 mM KCl, 50 µg/ml BSA, 10 mM dithiothreitol, 10 µM ZnSO4, 1 µg of polydeoxyinosinic-deoxycytidylic acid, 10 fmol of oligonucleotide probe, and 10 µg of nuclear proteins. For supershift experiments, 1 µg of a rabbit anti-Egr-1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was incubated with nuclear proteins for 20 min at room temperature followed by an incubation period of 10 min on ice before the addition of the radiolabeled probe. For competition experiments, a 50-, 100-, or 200-fold excess of nonradioactive probe 120 mutated (5'-AGCAGAGTTCGATAGTGAAAT-3') or not, or nonradioactive Egr-1 consensus site probe (5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3'), was incubated with nuclear proteins for 10 min at room temperature before the addition of the radiolabeled probe 120. DNA-protein complexes were separated on a 6% nondenaturing polyacrylamide gel containing 0.1 M Tris, 90 mM boric acid, and 1 mM EDTA at 150 V for 6 h at 4 °C, dried, and analyzed with PhosphorImager scanning.

Cloning of Glucagon Gene Promoter from InR1G9-CCK2 Cells— InR1G9-CCK2 genomic DNA was purified according the manufacturer's procedures (QIAamp DNA mini kit, Qiagen). A primer pair for PCR amplification (5'-ATCAAGGGATAAGACCCTCAAATGA-3' as the forward and 5'-GGGAACTTTGAGTGTGTTCTGCG-3' as the reverse) was developed from conserved structures in the DNA sequences of rat, mouse, and human glucagon gene promoters in databases. PCR was carried out in a total volume of 100 µl according to the manufacturer's procedures (Invitrogen). The amplification reaction involved denaturation at 95 °C for 5 min followed by 30 cycles as follows: 95 °C for 1 min, primer annealing at 50 °C for 1 min, and extension at 72 °C for 1.5 min. After cycling, a terminal elongation of 10 min at 72 °C was performed, and PCR products were cloned into the pCR2.1-TOPO vector (Invitrogen). Plasmids DNA were transferred into Escherichia coli and isolated by mini-prep (Macherey-Nagel). Both strands of PCR products were sequenced using M13 reverse and M13 forward primers (Applied Biosystems). The partial hamster glucagon gene promoter sequence is available under the GenBank accession no. AY842856 [GenBank] .

Chromatin Immunoprecipitation Assays—Cells (20 x 106) were plated on 150-mm culture dishes. Before stimulation with 1 µM gastrin for 1 h, cells were cultured overnight in serum-free medium with 0.1% BSA. Chromatin cross-linking was performed by adding 1% formaldehyde to the InR1G9-CCK2 cells at room temperature for 8 min. The reaction was stopped by adding glycine to a final concentration of 0.125 M. Cells were then washed twice with ice-cold PBS, collected in 5 ml of PBS, and harvested by brief centrifugation. Cells were resuspended in ice-cold cell lysis buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% Nonidet P-40, protease inhibitors: 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM benzamidine), incubated on ice for 5 min, and briefly centrifuged. Cells were then resuspended in nuclear lysis buffer (10 mM Tris-HCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.5% sarcosyl, 0.5 M NaCl, protease inhibitors). Sonication of the cells was performed 30 times for 30 s in TEN buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl) with a Branson 250 sonifier. Chromatins were immunoprecipitated with 10 µg of rabbit polyclonal anti-Egr-1. A normal rabbit IgG (Santa Cruz Biotechnology, Inc.) was used as the nonspecific antibody control. After incubation with 12 µl of protein A-Sepharose (Amersham Biosciences) for 1.5 h at room temperature, the beads were washed seven times in radioimmune precipitation assay buffer (50 mM HEPES, pH 7.6, 1 mM EDTA, 0.5 M LiCl, 0.7% sodium deoxycholate, 1% Nonidet P-40) and once in TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and extracted twice with 100 µl of elution buffer (100 mM Tris-HCl, pH 8.0, 1% SDS). Eluates were pooled, treated with 100 µg/ml proteinase K for 1 h at 42 °C, heated at 65 °C overnight to reverse formaldehyde crosslinking, and purified using a QIAquick PCR purification kit (Qiagen). Purified immunoprecipitated DNA was analyzed by real time PCR using a Light-Cycler (Roche Diagnostics). Primers used for the analysis of the hamster glucagon promoter were 5'-CAAAGCGAGTGGGTGAGTG-3' and 5-GCCACGCAGATATTACGCTG-3', yielding an amplification product of 133 bp. The amount of PCR product was calculated from standard curves obtained from PCR with the same primers and serially diluted total DNA. Data were expressed as -fold differences relative to control conditions, in which normal rabbit serum was used instead of specific anti-Egr-1 antibody.

Western Blot Analysis—For study of nuclear Egr-1 accumulation, 20 µg of nuclear protein extracts were separated by SDS-PAGE and blotted on a polyvinylidene difluoride membrane (PerkinElmer Life Sciences). Western blot analysis was performed using a rabbit anti-Egr-1 antibody (1:10,000). To demonstrate equivalent protein loading, membranes were reprobed with a mouse anti-glyceraldehyde-3-phosphate dehydrogenase antibody (1:500, Chemicon International Inc., Temecula, CA). For study of ERK1/2 activation, 50 µg of cytoplasmic protein extracted as described previously (5) was separated by SDS-PAGE and blotted on membranes. Western blot analysis was performed using a goat anti-ERK1/2 MAPKs (1:10,000, Santa Cruz Biotechnology, Inc.) and a mouse antibody that specifically recognized the phosphorylated forms of ERK (1:10,000, Cell Signaling Technology, Inc., Beverly, MA). Membranes were incubated with peroxidase-coupled secondary antibodies (1:10,000, Pierce) and proteins were detected using the enhanced chemiluminescence system (ECL, Amersham Biosciences).

Phosphorylation—Cells (4 x 106) were plated on 100-mm culture dishes overnight at 37 °C in serum-free RPMI 1640 medium without phosphate (ICN), containing 0.1% BSA. 200 µCi of 32Pi (PerkinElmer Life Sciences) was added to medium 2 h before stimulation with 1 µM gastrin. After 1 h of stimulation with gastrin, whole nuclear extracts were prepared and were subjected to immunoprecipitation overnight at 4 °C, as described previously (3) using a rabbit anti-Egr-1 antibody preadsorbed on protein A-Sepharose. Whole immunoprecipitates were separated by SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane (PerkinElmer Life Sciences), which was then exposed to a PhosphorImager. To confirm the identity of the immunoprecipitated radiolabeled proteins, the membrane was blotted with a rabbit anti-Egr-1 antibody (1:10,000) and revealed using the ECL system.

Immunocytochemistry—Cells were cultured on glass slides. Before stimulation with 1 µM gastrin for 1 h, cells were cultured in serum-free medium with 0.1% BSA overnight at 37 °C and preincubated with the MEK1 inhibitor, PD98059, when indicated. After stimulation, cells were fixed with 2% paraformaldehyde in PBS for 15 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature. After blocking nonspecific background staining (Protein Block, DAKO S.A., Trappes, France) for 10 min at room temperature, cells were incubated with anti-Egr-1 antibody (1:5,000) in PBS overnight at 4 °C. Egr-1 was localized using a Cy2-coupled anti-rabbit secondary antibody (1:100, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). For visualizing nuclei, cells were incubated with 0.4 µg/ml DAPI (4',6-diamidino-2-phenylindole) (Sigma) in PBS for 2 min at room temperature before mounting the slides.

Data Analysis—Data are presented as the means ± S.E., and statistical significance was determined using the Student's t test. The threshold for statistical significance was a p value of less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characteristics of InR1G9-CCK2 Cells—Studies that determined the factors important for the regulation of glucagon gene expression mostly used the InR1G9 cells. Indeed, this cell line has characteristics similar to native islet {alpha}-cells, producing a stable and high level of glucagon and possessing the cellular machinery controlling glucagon gene expression. Therefore, to assess whether the CCK2 receptor regulates glucagon gene in pancreatic {alpha}-cells, we stably transfected the InR1G9 cells with a plasmid encoding the human CCK2 receptor cDNA. Selection of InR1G9 cell clones was based on their capacity to secrete glucagon in response to the CCK2 receptor agonists, CCK and gastrin, but also on the biphasic dose-response pattern of secretion as observed previously on isolated human islets of Langerhans (10). One clone was selected, InR1G9-CCK2, which responded dose-dependently to gastrin and CCK, with glucagon secretion being detectable at 10 pM agonists and reaching a maximum at 1 nM (18.1 ± 4.4 and 18.9 ± 4.7 pmol/106 cells/h for CCK and gastrin, respectively) (Fig. 1A). Concentrations of CCK and gastrin eliciting half-maximal levels of glucagon secretion were nearly identical (0.09 ± 0.01 nM and 0.14 ± 0.01 nM for CCK and gastrin, respectively). At supramaximal concentrations of agonists, glucagon secretion decreased to nearly 50% of maximal secretion for both agonists. Non-transfected InR1G9 cells did not respond to gastrin or CCK (data not shown) in accordance with the absence of CCK2 receptor expression (Fig. 1B). In agreement with expression of a CCK2 receptor with typical binding characteristics in InR1G9-CCK2 cells, the binding affinity of sulfated CCK was in the nanomolar range (Kd = 3.1 ± 0.9 nM). The maximum number of CCK2 receptor binding sites seen by CCK was 2.1 ± 0.4 pmol/106 cells. Taken together, these results show that the selected InR1G9-CCK2 cells provide a new suitable experimental model for the study of the regulation of the glucagon gene by gastrin in pancreatic {alpha}-cells.



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  		FIG. 1.
Characteristics of InR1G9 cells expressing the CCK2 receptor. A, glucagon secretion studies from InR1G9-CCK2 cells. Cells were stimulated for 1 h with increasing concentrations of either CCK ({blacksquare}) or gastrin ({diamondsuit}), and the amounts of secreted glucagon were measured by radioimmunoassay as described under "Experimental Procedures." Results are expressed as a percentage of maximal secretion with basal secretion subtracted (6.4 ± 2.3 pmol/106 cells/h). Values are the mean ± S.E. from four separate experiments, each performed in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with maximal secretion. B, localization of CCK2 receptor on InR1G9 cells expressing the CCK2 receptor (InR1G9-CCK2) or not (InR1G9) using a fluorescein isothiocyanate-coupled CCK9 ligand.

 
Gastrin Stimulates Glucagon Gene Expression—We first determined whether the level of glucagon mRNA varied in response to CCK2 receptor activation by performing Northern blot analysis. Glucagon mRNA and 18 S rRNA were quantified from InR1G9-CCK2 cells stimulated with gastrin for up to 24 h. Gastrin increased glucagon mRNA abundance in a dose- and time-dependent manner. Indeed, stimulation was detectable with 1 nM gastrin from 6 h after induction (data not shown). Maximum stimulation (2.9-fold compared with nonstimulated cells) was observed at 24 h after stimulation with 1 µM gastrin (Fig. 2A).



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  		FIG. 2.
Gastrin stimulates glucagon gene expression. A, effect of gastrin on glucagon mRNA level. Cells were stimulated for 24 h with or without 1 µM gastrin, and total RNA was extracted for Northern blot analysis as described under "Experimental Procedures." Blots were quantified by PhosphorImager scanning. Values are the mean ± S.E. from three separate experiments, each performed twice. **, p < 0.01. B, effect of gastrin on glucagon mRNA half-life. Actinomycin D (5 µg/ml) was added to control and gastrin-treated cells, and RNA was extracted after 4, 8, and 12 h. Glucagon mRNA was quantified by Northern blot analysis, using the 0 time of actinomycin treatment as a reference for 100% survival. Values are the mean ± S.E. from three separate experiments, each performed twice.

 
To eliminate the possibility that the increased level of glucagon mRNA resulted from a gastrin-dependent glucagon mRNA stabilization, we compared glucagon mRNA half-life in both control and gastrin-stimulated cells. For this purpose, actinomycin D, which completely blocks RNA synthesis, was added to InR1G9-CCK2 cells before incubation in the presence or absence of gastrin for 4, 8, and 12 h. In these conditions, the half-life of glucagon mRNA was nearly identical in both control and gastrin-treated cells (18.1 ± 1.2 h and 17.6 ± 1.1 h, respectively) (Fig. 2B), showing that gastrin does not modify the stability of glucagon mRNA in InR1G9-CCK2 cells and thus likely stimulates glucagon gene expression at the level of transcription.

The Proximal Promoter of the Glucagon Gene Confers the Response to Gastrin—The proximal 350 bp of the rat glucagon 5'-flanking sequence has been well characterized and shown to contain the {alpha}-cell-specific G1 and the islet-specific G4 elements as well as the G5, G2, and G3 enhancer elements and a cAMP response element (Fig. 3A). To examine how gastrin stimulates glucagon gene expression, various plasmids progressively 5'-increased for the different elements of the rat promoter/enhancer region and fused to the CAT gene were transiently transfected into InR1G9-CCK2 cells. Gastrin stimulation of cells transfected with a plasmid containing the G1 and G4 promoter elements (-138 CAT) induced a significant 1.6-fold increase of the transcriptional activity (Fig. 3B). The addition of G5 element (-169 CAT) did not significantly modify the response to gastrin. The identical response was also obtained after the addition of the G2 and G3 elements (-292 CAT). Finally, a significant stimulatory effect of gastrin on the glucagon gene transactivation was also observed after transfection of a plasmid containing the 2.5 kb of the glucagon 5'-flanking sequence (-2.5 kb CAT). These results demonstrate that gastrin responsiveness depends on elements present on the promoter, composed of the {alpha}-cell-specific G1 element and the islet-specific G4 element.



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  		FIG. 3.
Identification of the minimal sequence of the glucagon gene responding to gastrin. A, schematic representation of control elements and transcription factors regulating the glucagon gene. CRE, cAMP response element; CREB, CRE-binding protein; WiHe, winged helix family protein; HNF-3{beta}, hepatocyte nuclear factor-3{beta}; Pbx, PBC homeoprotein; Prep1, homeodomain protein; E47/{beta}2, basic helix-loop-helix family protein; {beta}-TF1, {beta}-cell transcription factor 1; Cdx-2/3, caudal-type homeobox protein 2/3; Ets, Ets family protein; Isl-1, insulin factor 1; Pax, paired homeobox protein. B, minimal glucagon promoter analysis. The indicated constructs, containing increasing 5'-flanking sequences of the glucagon gene linked to CAT and a plasmid encoding luciferase, were cotransfected into InR1G9-CCK2 cells, and the cells were treated (black bars) or not (white bars) with 1 µM gastrin for 24 h. After protein extraction, CAT and luciferase activities were measured. Results represent relative CAT/luciferase activities over control. Values are the mean ± S.E. from three separate experiments, each performed twice. *, p < 0.05; **, p < 0.01 compared with nontreated cells.

 
Identification of a Gastrin Response Element on the G4 Element—To map more precisely the gastrin-responsive region of the glucagon gene promoter, we measured the transcriptional activity after mutation of the nucleotide sequences required for binding of transcription factors known to interact with this region (Fig. 4A). Isl-1, Cdx-2/3, and Pax-6 have been characterized for their ability to bind the G1 element, whereas IEF-1 or the E47/{beta}2 heterodimer and the uncharacterized {beta}-TF1 were identified to interact with G4 element (Fig. 3A). Furthermore, it has been described that the G4 element possesses, in addition to E47/{beta}2 and {beta}-TF1 binding sites, a cis-acting sequence (region -120), interacting with an as yet unidentified transcription factor (Fig. 4A) (17). Results obtained after transfection of the different mutant plasmids into InR1G9-CCK2 cells show that whereas basal transcriptional relative activity was unchanged after mutations of Isl-1 or Cdx-2/3 binding sequences, it decreased from 5.64 ± 0.66 to 1.04 ± 0.20 when the Pax-6 binding sequence was mutated, confirming that Pax-6 is an essential transcription factor for the glucagon gene (Table I). Mutations on the {beta}-TF1 or E47/{beta}2 binding sequences did not affect basal transcriptional relative activity either. Noteworthy, the mutation at position -120 (120 mut) dramatically decreased the basal promoter transcriptional relative activity from 5.64 ± 0.66 to 1.80 ± 0.64 (Table I).



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  		FIG. 4.
Mapping of a gastrin cis-regulating sequence on the glucagon proximal promoter. A, wild-type (WT) and mutated (mut) nucleotide sequences of binding domains for transcription factors known to interact with the glucagon gene promoter. Binding domains are shown in gray boxes, and mutated nucleotides abolishing binding of transcription factor are underlined. A putative binding domain in the 120 region for an unknown factor is also indicated. B, effect of mutations within binding domains on gastrin-stimulated glucagon gene expression. The wild-type minimal promoter, containing the first 138 bp of the promoter (G1 and G4 elements) or the indicated mutated promoter, were cotransfected with a plasmid encoding luciferase into InR1G9-CCK2 cells. Cells were treated (black bars) or not (white bars) with 1 µM gastrin for 24 h. After protein extraction, CAT and luciferase activities were measured. Results represent relative CAT/luciferase activities over basal for each construct. Values are the mean ± S.E. from three separate experiments, each performed twice. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with control.

 


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  		TABLE I
Comparative effects of transcription factor binding site mutations on transcriptional activity of the glucagon gene proximal promoter Basal and gastrin-stimulated transcriptional relative activities of either wild-type (WT) proximal glucagon gene promoter plasmid or plasmids mutated (mut) on the nucleotide sequences binding the different transcription factors interacting with this region are shown. Results represent basal relative CAT/luciferase activities (basal), gastrin-stimulated relative CAT/luciferase activities (gastrin-stimulated), or gastrin-stimulated relative CAT/luciferase activities over basal (gastrin-stimulated -fold basal) after gastrin stimulation of each construct. Values are the means ± S.E. from three separate experiments, each performed twice. {ddagger}, p < 0.05; {ddagger}{ddagger}, p < 0.01 compared with basal relative activity of WT glucagon proximal promoter; *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with unstimulated relative activity for each construct.

 
Stimulation with gastrin after transfection of the different mutants shows that glucagon gene transactivation by gastrin was not modified by mutations preventing binding of either Isl-1, Cdx-2/3, Pax-6, {beta}-TF1, or E47/{beta}2 on the promoter (Table I, Fig. 4B). Remarkably, the mutation at position -120 reduced significantly the effect of gastrin on glucagon promoter transcriptional activity by 60% compared with wild-type promoter (1.65 ± 0.22 and 2.66 ± 0.25-fold basal, respectively) (Table I and Fig. 4B), suggesting that this sequence contains a response element to gastrin. Taken together, these data demonstrate that gastrin stimulation of glucagon gene expression involves an unidentified critical trans-acting transcription factor. This factor binds to the nucleotide region -120 of the G4 element of the glucagon gene and is essential for basal and gastrin-stimulated glucagon gene expression.

Egr-1 Interacts with the G4 Element of the Glucagon Gene Promoter after Gastrin Stimulation of Nuclear Accumulation— The molecular mechanisms of glucagon gene transactivation by gastrin were investigated next. First, to identify the transcription factors that interact with the G4 element and mediate the gastrin-stimulated glucagon gene transactivation, we performed EMSAs using the 120 probe, corresponding to a partial sequence of the G4 element (nucleotides -129/-108). Formation of a major DNA-proteins complex was observed after incubation of the 32P-labeled sequence with nuclear extracts prepared from InR1G9-CCK2 cells treated with gastrin for up to 24 h (Fig. 5A). Time course analysis showed that gastrin-dependent formation of this complex was transient, with a binding peak after 1 h of agonist stimulation, which decreased between 1 and 3 h of stimulation, whereas the complex disappeared after 6 h of treatment.



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  		FIG. 5.
Egr-1 binds to the G4 element of the glucagon gene promoter. A, EMSAs were performed with 10 µg of nuclear extracts from InR1G9-CCK2 cells, stimulated or not with 1 µM gastrin for the times indicated, and incubated with the 32P-labeled oligonucleotide encoding partially the G4 element (sequence of the 120 probe is shown under "Experimental Procedures"). B, EMSA competition studies were carried out with 10 µg of nuclear extracts from 1-h gastrin-stimulated InR1G9-CCK2 cells using a 50-, 100-, and 200-fold molar excess of unlabeled 120 probe or mutated 120 probe (the sequence is shown under "Experimental Procedures") as indicated. C, 50-, 100-, and 200-fold molar excess of unlabeled Egr-1 consensus binding site oligonucleotide (sequence is shown under "Experimental Procedures") was used for EMSA competition experiment. D, for supershift assays, nuclear extracts were preincubated with an anti-Egr-1 antibody (Ab x Egr-1) before the addition of the radiolabeled probe. The data shown are representative of three independent experiments.

 
To characterize the specificity of the complex of the -120 region DNA sequence, competition studies were performed with increasing concentrations of unlabeled 120 probe. The intensity of the radioactive signal diminished progressively until complete extinction with 200-fold molar excess, indicating that the proteins complex specifically interacted with the 120 probe (Fig. 5B). We next assessed whether mutations on the nucleotide region -120 that altered gastrin stimulation of the transcriptional activity also prevented formation of the DNA-protein complex. Results presented in Fig. 5B show that an excess of unlabeled 120 mut probe did not affect the signal intensity, thus indicating that gastrin responsiveness is linked to the formation of a specific DNA-proteins interaction in this region of the promoter.

Analysis of the nucleotide region -120 on the G4 element shows that it contains a GC-rich motif, GAGTGGGCG, which constitutes a putative binding domain for Egr-1. Indeed, Egr-1 protein is a zinc finger-containing transcription factor that specifically binds the DNA sequence GCG(G/T)GGGCG (18). Because of its unknown importance we first investigated whether the DNA-protein complex contained the transcription factor Egr-1 by performing EMSA competition experiments with an oligonucleotide containing the Egr-1 binding consensus site (Fig. 5C). Formation of the complex disappeared completely with an excess of this unlabeled oligonucleotide, suggesting that Egr-1 is present in this complex. To confirm further the presence of Egr-1, we carried out EMSA supershift experiments employing an anti-Egr-1 antibody (Fig. 5D). The complex was supershifted, demonstrating that Egr-1 binds to G4 element of the glucagon gene promoter. Moreover, we also assessed whether this complex contains other transcription factors in addition to Egr-1 by performing EMSA supershift experiments using anti-Sp1, anti-E47, anti-BETA2, or anti-p300 antibodies, but none of these antibodies supershifted the complex (data not shown).

We also determined whether gastrin regulates nuclear translocation of Egr-1 in InR1G9-CCK2 cells by Western blot analysis of its nuclear abundance in response to gastrin stimulation for up to 24 h. Results of these experiments show a time-dependent increase of Egr-1 in the nucleus, being maximum after 1 h and reaching basal level after 6 h of gastrin stimulation (Fig. 6A), which is identical to the time course of association of Egr-1 to G4 element which was observed with EMSA experiments. Given that the phosphorylated form of Egr-1 binds to DNA more efficiently than nonphosphorylated forms, suggesting that its phosphorylation is critical for its transcriptional activity (19), we determined whether Egr-1 is phosphorylated in the nuclei of InR1G9-CCK2 cells after stimulation with gastrin. Results of radiolabeling experiment followed by Egr-1 immunoprecipitation show an accumulation of 32P-labeled Egr-1 in the nuclei of gastrin-stimulated InR1G9-CCK2 cells (Fig. 6B), demonstrating that gastrin phosphorylates Egr-1, in addition to its nuclearization.



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  		FIG. 6.
Gastrin stimulates nuclear accumulation and phosphorylation of Egr-1. A, nuclear extracts were prepared from InR1G9-CCK2 cells stimulated or not with 1 µM gastrin for the indicated time periods, and Western blot studies were performed using Egr-1 antibody. To demonstrate equivalent protein loading, the blot was reprobed with an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody. The data shown represent typical results of three independent experiments. B, InR1G9-CCK2 cells were metabolically labeled with 32Pi, and nuclear extracts were prepared after stimulation or not with 1 µM gastrin for 1 h, analyzed on SDS-PAGE, and radioactivity revealed by PhosphorImager (upper panel). The membrane was immunoblotted (IB) with an anti-Egr-1 antibody to demonstrate that the radiolabeled immunoprecipitated proteins are Egr-1 (lower panel).

 
The Proximal Glucagon Gene Promoter of InR1G9-CCK2 Cells Interacts with Egr-1 in a Gastrin-dependent Manner—To demonstrate that Egr-1 proteins are indeed recruited to the endogenous proximal glucagon gene promoter of InR1G9-CCK2 cells in response to gastrin, we performed a chromatin immunoprecipitation assay. However, given that the nucleotide sequence of the hamster glucagon gene promoter was unknown, prior cloning of this sequence from InR1G9-CCK2 cells was necessary, and a partial sequence is shown in Fig. 7A. After stimulation by gastrin, cross-linked DNA-protein complexes were immunoprecipitated with the specific antibody recognizing Egr-1. The DNA fragments containing the G4 element were amplified by real time PCR using primers flanking the G4 element of the hamster glucagon gene promoter. As shown in Fig. 7B, there was a 2.9-fold enrichment of immunoprecipitated glucagon gene promoter in the gastrin-stimulated InR1G9-CCK2 cells compared with the untreated cells, thus demonstrating gastrin-dependent binding of Egr-1 to this sequence.



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  		FIG. 7.
Egr-1 binds to the endogenous glucagon gene promoter in InR1G9-CCK2 cells. A, partial sequence of the glucagon gene promoter of InR1G9-CCK2 cells. Conserved DNA sequences that interact with Isl-1, Cdx-2/3, Pax-6, {beta}-TF1, and E47/{beta}2 transcription factors are shown in gray boxes. The putative nucleotide sequence interacting with Egr-1 is shown in a blank box. B, results of real time PCR after chromatin immunoprecipitation. The data shown are derived from quantitative real time PCR analysis of the glucagon gene promoter after a chromatin immunoprecipitation assay with the specific anti-Egr-1 antibody. Data are expressed as -fold differences between gastrin-stimulated cells (black bar) versus gastrin-unstimulated cells (white bar). Values are the mean ± S.E. from three independent experiments performed in duplicate. **, p < 0.01 compared with nontreated cells.

 
The MEK1/ERK1/2 Pathway Is Critical for Gastrin-dependent Glucagon Gene Transactivation—Egr-1 binding to DNA is increased significantly by inhibitors of protein serine/threonine phosphatases, suggesting that its activation is under the control of protein kinases and/or phosphatases (20). Among multiple protein kinases, proteins that belong to the MAPK family could be involved in the regulation of many genes. Because the MAPK signaling pathway is activated by gastrin (21), we first tested whether it contributed to the CCK2 receptor signaling cascade in the InR1G9-CCK2 cells. Results of Western blot analysis verified that gastrin increases ERK1/2 phosphorylation in a time-dependent manner with a peak of stimulation at 5 min in InR1G9-CCK2 cells (Fig. 8A). ERK protein phosphorylation was maintained for at least 1 h of gastrin stimulation and inhibited to nearly basal level with the specific inhibitor of MEK1 signaling pathway, PD98059 (Fig. 8A). The importance of the MEK/ERK cascade in the gastrin-induced glucagon gene transactivation was then investigated. Fig. 8B shows that PD98059 reduced partially but significantly, more than 50% of the gastrin-stimulated glucagon transcriptional activity of the proximal promoter compared with cells not treated with PD98059 (1.69 ± 0.16 and 2.55 ± 0.29-fold basal, respectively).



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  		FIG. 8.
The MAP kinase pathway is critical for Egr-1-dependent gastrin-stimulated glucagon gene expression. A, cells were stimulated with 1 µM gastrin for the indicated time periods in the presence or absence of PD98059. ERK1/2 phosphorylation was then analyzed by Western blot assays (upper panel). As a loading control, the blot was stripped and reprobed with non-phosphospecific anti-ERK1/2 antibody (lower panel). The data shown represent typical results of a series of three independent experiments. B, reporter plasmid containing G1 and G4 elements (-138 bp) of the glucagon gene promoter linked to CAT and plasmid expressing luciferase were cotransfected into InR1G9-CCK2 cells. Cells were pretreated or not with 50 µM MEK1, the specific inhibitor of PD98059, and stimulated (black bars) or not (white bars) with 1 µM gastrin for 24 h. After protein extraction, CAT and luciferase activities were measured. Results represent relative CAT/luciferase activities over nontreated cells. Values are the mean ± S.E. from three separate experiments, each performed twice. *, p < 0.05; **, p < 0.01 compared with control. C, InR1G9-CCK2 nuclear extracts were prepared after stimulation or not with gastrin for 1 h in the presence or absence of 50 µM PD98059 and analyzed in EMSA studies using 120 probe. The data shown represent typical results of three independent experiments. D, nuclear extracts were prepared after stimulation of InR1G9-CCK2 cells with 1 µM gastrin for 1 h, with or without 50 µM PD98059, and Egr-1 nuclear abundance was assayed by Western blot studies. As a loading control, the blot was reprobed with an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody. The data shown represent typical results of three independent experiments.

 
We further explored whether gastrin-induced binding of Egr-1 on the glucagon gene promoter involved MAPK pathway by comparing EMSA analysis with nuclear extracts from gastrin-stimulated InR1G9-CCK2 cells treated or not with PD98059. Binding of Egr-1 was decreased markedly in the presence of the MEK1-specific inhibitor, strongly suggesting that ERK1/2 activation by gastrin contributes to Egr-1 activation (Fig. 8C).

We next investigated whether Egr-1 nuclearization induced by gastrin stimulation was affected by treatment with PD98059. Addition of the MEK1-specific inhibitor dramatically prevented gastrin-induced nuclear accumulation of Egr-1 (Fig. 8D), indicating that ERK1/2 activation is an essential step for nuclearization of the transcription factor. These results were confirmed further by performing an immunocytochemical analysis of gastrin- and PD98059-treated cells. As shown in Fig. 9, Egr-1 nuclear accumulation increased after 1 h of gastrin stimulation in InR1G9-CCK2 cells (Fig. 9, A and B) whereas Egr-1 nuclearization in response to gastrin was decreased strongly in the presence of PD98059 (Fig. 9, C and D).



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  		FIG. 9.
Egr-1 immunocytochemistry. Cells were stimulated (B and D) or not (A and C) with 1 µM gastrin for 1 h in the presence (C and D) or not (A and B) of 50 µM PD98059. Then, immunocytochemical studies were performed to determine Egr-1 localization using an anti-Egr-1 antibody. To visualize nuclei, cells were incubated with DAPI.

 
Taken together, our results demonstrate that gastrin-stimulated glucagon gene expression is dependent on the MAPK signaling pathway in InR1G9-CCK2 cells via nuclearization and DNA binding of Egr-1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the current study highlight a new role for the gastrointestinal regulatory peptide gastrin in the pancreas. Indeed, using a new cell line expressing the CCK2 receptor, we determined that gastrin is an effective stimulant of glucagon RNA expression. This finding provides the first evidence that gastrin directly regulates the expression of a pancreatic endocrine hormone, further supporting a role of gastrin in glucose homeostasis. This together with the previously reported role of gastrin in the regulation of glucagon secretion in vitro from human pancreatic islets and in vivo in gastrin-/- mice (10, 11) implicates gastrin as a determinant of both glucagon secretion and gene expression. Moreover, we identify Egr-1 as a crucial transcription factor for the regulation of basal and gastrin-dependent glucagon gene transactivation.

Our discovery of Egr-1 as an essential transcription factor, interacting with the G4 element of the glucagon gene, settles previous unexplained data that located a cis-regulatory element in the region -120. Indeed, and in agreement with our results, mutation of the intervening sequence between the two E box motifs of the G4 element resulted in more than 60% loss of the transcriptional activity (17). Although the extent of the inhibition reported previously was indicative of an essential role for basal glucagon gene expression, the trans-acting nuclear proteins that bind the intervening sequence remained to be defined. In agreement with the known consensus DNA binding site GCG(G/T)GGGCG (18), we now show that the zinc finger transcription factor Egr-1 recognizes the GC-rich domain contained in the intervening sequence. Egr-1 is encoded by an immediate-early gene and consequently rapidly induced and activated by many environmental signals including growth factors, stress, or hormones (22, 23). Thus activation of Egr-1 may also adapt to rapid elevated glucagon production in stressful conditions such as hypoglycemia. While induction of the Egr-1 gene together with other immediate-early response genes has been linked to long term pleiotropic effects of glucose and secretagogues in pancreatic {beta}-cells (24), this study is the first to report activation of Egr-1 in pancreatic {alpha}-cells.

We also demonstrate that in addition to being a key function in basal glucagon gene expression, Egr-1 binding to the intervening sequence is a critical step in gastrin-dependent regulation of the glucagon gene in pancreatic {alpha}-cells. Furthermore, we show phosphorylation of Egr-1 after gastrin stimulation, consistent with the DNA binding activity of Egr-1 (19). Participation of Egr-1 in gastrin-regulated transactivation of promoter was reported for the chromogranin A gene in gastric epithelial cells (25). Similar to our results, gastrin-dependent binding of Egr-1 to chromogranin A promoter was rapid, transient, disappearing after a maximum at 1 h, and involved the MEK1/ERK1/2 signaling cascade. Although our report provides additional evidence that Egr-1 is one of the gastrin-regulated transcription factors, essential differences exist according to the regulated promoter. First, the fact that Egr-1 controls the basal activity of the glucagon gene although this is not the case for basal transactivation of the chromogranin A gene demonstrates that the functional relevance of the transcription factor is different in the two situations. Moreover, an interplay of Egr-1 and Sp1, resulting in overlapping consensus motifs (26), is crucial for gastrin regulation of the chromogranin A promoter. In the context of the glucagon gene promoter, Sp1 is absent from the DNA-protein complex induced by gastrin, indicating no functional interaction between these two factors.

Finally, in addition to supporting an essential role for gastrin in glucose homeostasis via regulation of islet glucagon cell function, the results of the present study are also of particular interest considering our previous data demonstrating the presence of the CCK2 receptor and of amidated gastrin, its specific ligand in 22-week human fetal pancreas (10). Full understanding of the functions of gastrin in human pancreas during the embryonic period would deserve investigations especially at a period of development earlier than 22 weeks of gestation, but these remain limited because of scarce material. However, the fact that gastrin stimulates glucagon gene expression via activation of its receptor present in glucagon cells opens new interesting prospects concerning its contribution to pancreatic development. Indeed, glucagon is the earliest peptide hormone that is expressed in the developing pancreas, and recent data have proposed a key role for glucagon in the early phase of insulin cell differentiation (27). This suggests that gastrin could actively contribute to the paracrine induction of differentiation of pancreatic cells during fetal life by inducing or maintaining the {alpha}-cell phenotype via induction of Egr-1. Indeed, during mouse embryogenesis, Egr-1 is present in different sites where epithelial mesenchymal interactions are important such as the developing tooth germ and the nasal and salivary glands (28). Induction of its expression after fibroblast growth factor receptor activation has been reported in rat embryonic pancreatic rudiments, suggesting that it may play a role in the promotion of proliferation of embryonic pancreatic epithelial cells (29). Therefore the hypothesis that it is one of the mediators of the developmental effects of gastrin can be raised from now on.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY842856 [GenBank] .

* This work was supported in part by Association pour la Recherche sur le Cancer Grants 4430 and 4514 and by Génopôle of Toulouse Grant ur531-257-8RB06. 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

§ Recipient of support from the Ligue contre le Cancer (région Midi-Pyrénées), University of Toulouse (ATUPS), and the Solvay-Pharma/Club Français du Pancréas. Back

|| Recipient of support from the Ligue contre le Cancer (Tarn et Garonne). Back

** To whom correspondence should be addressed: IFR31, Institut Louis Bugnard, INSERM U531, BP 84225, 31432 Toulouse, Cedex 4, France. Tel.: 33-5-6132-2405; Fax: 33-5-6132-2403; E-mail: dufresne{at}toulouse.inserm.fr.

1 The abbreviations used are: CCK2, cholecystokinin 2; BSA, bovine serum albumin; CAT, chloramphenicol acetyltransferase; Egr-1, early growth response protein 1; EMSA, electrophoretic mobility shift assay; ERK1/2, extracellular signal-regulated kinase 1/2; Luc, luciferase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; RSV, Rous sarcoma virus. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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