Interaction of early growth response protein 1 (Egr-1), specificity protein 1 (Sp1), and cyclic adenosine 3'5'-monophosphate response element binding protein (CREB) at a proximal response element is critical for gastrin-dependent activation of the chromogranin A promoter.
Recently, binding of specific protein 1 (Sp1) and cAMP response element binding protein (CREB) to a GC-rich element at -92/-62 has been identified as a critical step in gastrin-dependent regulation of the chromogranin A (CgA) gene in gastric epithelial cells. Here we demonstrate that binding of early growth response protein 1 (Egr-1) to the distal part of the -92/-62 site is also required for gastrin-dependent CgA transactivation. Gastrin elevated cellular and nuclear Egr-1 levels in a time-dependent manner and also increased Egr-1 binding to the CgA -92/-73 region. Disruption of this site reduced gastrin responsiveness without influencing basal promoter activity, while loss of Sp1 and/or CREB binding sites diminished basal and gastrin-stimulated CgA promoter activity. Ectopic Egr-1 overexpression potently stimulated the CgA promoter, whereas coexpression of Egr-1 with Sp1 and/or CREB resulted in additive effects. Functional analysis of Sp1-, Egr-1-, or CREB-specific promoter mutations in transfection studies confirmed the tripartite organization of the CgA -92/-62 element. Signaling studies revealed that MAPK kinase 1 (MEK1)/ERK1/2 cascades are critical for gastrin-dependent Egr-1 protein accumulation as well as Egr-1 binding to the CgA promoter. Our studies for the first time identify Egr-1 as a nuclear target of gastrin and show that functional interplay of Egr-1, Sp1, and CREB is indispensable for gastrin-dependent CgA transactivation in gastric epithelial cells.
Raychowdhury
Raktima
R
Medizinische Klink mit Schwerpunkt Gastroenterologie, Hepatologie, Endokrinologie und Stoffwechsel, Universitätsklinikum Charité, Campus Virchow-Klinikum, Humboldt Universität, 13353 Berlin, Germany.
Schäfer
Georgia
G
Fleming
John
J
Rosewicz
Stefan
S
Wiedenmann
Bertram
B
Wang
Timothy C
TC
Höcker
Michael
M
D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D053379:Chromogranin A; D002864:Chromogranins / Q000235:genetics*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D005755:Gastrins / Q000494:pharmacology*; D006801:Humans; D017874:Immediate-Early Proteins; D015151:Immunoblotting; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020218:Response Elements; D015398:Signal Transduction; D013274:Stomach Neoplasms; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
C506150:CHGA protein, human; D053379:Chromogranin A; D002864:Chromogranins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D004791:Enzyme Inhibitors; D005419:Flavonoids; D005755:Gastrins; D017874:Immediate-Early Proteins; D014157:Transcription Factors; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one