Abstract
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.
Highlights
IntroductionIn a previous study we identified a downstream (ϩ2 to ϩ24) cis-acting element in the human histidine decarboxylase (HDC) (hHDC) promoter through which gastrin exerts its transactivating effect (15)
In order to investigate the transcriptional response of the mouse Chromogranin A (CgA) 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 and a cyclic AMP-responsive element (CRE) at ؊71 to ؊64 bp are essential for gastrin-dependent mCgA transactivation
The purpose of this study was to characterize the regulatory effect of gastrin on the mCgA promoter, along with the cisregulatory elements and transcription factors mediating the actions of gastrin
Summary
In a previous study we identified a downstream (ϩ2 to ϩ24) cis-acting element in the human HDC (hHDC) promoter through which gastrin exerts its transactivating effect (15). This HDC element shows no homology to any previously reported consensus motif, and apparently binds (a) novel transcription factor(s) (15). While previous studies analyzed the structural basis for nicotinestimulated mCgA transcriptional activity in pheocromocytoma cells (18, 19), the molecular mechanisms of gastrin-stimulated CgA expression are still unclear. Our study demonstrates that the mCgA gene is regulated by gastrin on a transcriptional level through binding of two separate transcription factors to their consensus cis-acting elements
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