Abstract
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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY842856
We demonstrated previously that whereas gastrin expression is extinguished in adult pancreas, cholecystokinin 2 (CCK2) receptors are present in human glucagon-producing cells where their activation stimulates glucagon secretion
We 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
Summary
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 g/ml streptomycin (Invitrogen), and 200 g/ml geneticin (Sigma). Before stimulation with 1 M gastrin for the indicated time periods, cells were cultured overnight in serum-free medium with 0.1% BSA. Cells were washed twice with cold PBS, resuspended carefully in 400 l 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. Binding reactions were performed at room temperature for 20 min in a final volume of 20 l of binding buffer containing 20 mM HEPES, pH 7.9, 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol, 50 mM KCl, 50 g/ml BSA, 10 mM dithiothreitol, 10 M ZnSO4, 1 g of polydeoxyinosinic-deoxycytidylic acid, 10 fmol of oligonucleotide probe, and 10 g of nuclear proteins. 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
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