Sp1 and CREB mediate gastrin-dependent regulation of chromogranin A promoter activity in gastric carcinoma cells.
Chromogranin A (CgA) is a multifunctional acidic protein that in the stomach is expressed predominantly in enterochromaffin-like cells (ECL cells) where it is regulated by gastrin. In order to investigate the transcriptional response of the mouse CgA (mCgA) promoter to gastrin stimulation, we studied a 4.8-kilobase mCgA promoter-luciferase reporter gene construct in transiently transfected AGS-B cells. 5'-Deletion analysis and scanning mutagenesis of mCgA 5'-flanking DNA showed that a Sp1/Egr-1 site spanning -88 to -77 base pairs (bp) and a cyclic AMP-responsive element (CRE) at -71 to -64 bp are essential for gastrin-dependent mCgA transactivation. Gastrin stimulation increased cellular Sp1 protein levels and Sp1-binding to the mCgA -88 to -77 bp element, as well as binding of CREB to its consensus motif at -71 to -64 bp. Gastrin also stimulated CREB Ser-133 phosphorylation, and abundance of cellular CREB protein levels. Overexpression of either Sp1 or phosphorylated CREB transactivated the mCgA promoter dose dependently, while coexpression of both transcription factors resulted in an additive mCgA promoter response. mCgA -92 to -64 bp, comprising the Sp1/Egr-1 site and the CRE motif, conferred gastrin responsiveness to a heterologous thymidine kinase promoter system, and therefore functions as a "true" enhancer element. This report demonstrates that Sp1 and CREB mediate CCK-B/gastrin receptor-dependent gene regulation, and that the effect of gastrin on the CgA gene is brought about by cooperative action of both transcription factors.
Höcker
M
M
Medizinische Klink mit Schwerpunkt Gastroenterologie und Hepatologie, Universitätsklinikum Charitè, Campus Virchow-Klinikum, Humboldt Universität Berlin, Germany.
Raychowdhury
R
R
Plath
T
T
Wu
H
H
O'Connor
D T
DT
Wiedenmann
B
B
Rosewicz
S
S
Wang
T C
TC
D000818:Animals; D001483:Base Sequence; D053379:Chromogranin A; D002864:Chromogranins / Q000235:genetics*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D051766:Early Growth Response Protein 1; D005755:Gastrins / Q000494:pharmacology / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D017874:Immediate-Early Proteins; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; 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; C486109:Egr1 protein, mouse; D005755:Gastrins; D017874:Immediate-Early Proteins; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C506155:chromogranin A, mouse; D008156:Luciferases; D013937:Thymidine Kinase