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J Biol Chem, Vol. 273, Issue 51, 34000-34007, December 18, 1998


Sp1 and CREB Mediate Gastrin-dependent Regulation of Chromogranin A Promoter Activity in Gastric Carcinoma Cells*

Michael HöckerDagger , Raktima Raychowdhury§, Thomas Plath, Hongjang Wuparallel , Daniel T. O'Connorparallel , Bertram WiedenmannDagger **, Stefan RosewiczDagger Dagger , and Timothy C. Wang§§§

From the Dagger  Medizinische Klink mit Schwerpunkt Gastroenterologie und Hepatologie, Universitätsklinikum Charitè, Campus Virchow-Klinikum, Humboldt Universität Berlin, Germany, the  Innere Medizin I, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Germany, the parallel  Department of Medicine and Center for Molecular Genetics, University of California San Diego, San Diego, California 92161, and the § Gastrointestinal Unit and Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Chromogranin A (CgA)1 is a 48-kDa acidic protein which was initially identified as the major soluble protein co-stored and released with neurotransmitters and secretory peptides in the neuroendocrine system (1-3). In neuroendocrine cells, CgA stabilizes secretory granules, influences pro-hormone processing, and regulates peptide sorting into the regulated secretory pathway (1-3). Cleavage products of CgA are able to regulate endocrine and exocrine secretory functions after release into the bloodstream (1-3). In the stomach, enterochromaffin-like cells (ECL cells) of the corpus mucosa, which are responsible for synthesis, storage, and release of histamine controlling gastric acid secretion, have been identified as the main source for CgA expression (4, 5). In vivo experiments in rodents as well as observations made in patients with gastrinoma have shown that hypergastrinemia-induced degranulation of ECL cells is accompanied by a parallel secretion of histamine and CgA, followed by enhanced production of both molecules (4-9). Furthermore, CgA up-regulation is also accompanied by increased synthesis of histidine decarboxylase (HDC), the key enzyme for gastric histamine synthesis (10). The functional importance of gastrin for the parallel expression pattern of HDC and CgA genes was supported by observations in homozygous CCK-B/gastrin receptor- and gastrin-deficient mice, as these knockout mice showed dramatic reductions in HDC and CgA mRNA expression in comparison to wild type littermates (11-13). These observations supported the concept that replenishment of ECL cell histamine stores after a secretory challenge requires the parallel de novo synthesis of the granule matrix protein CgA in order to provide the basis for appropriate packaging and release of histamine.

Histamine production in ECL cells depends on the activity of the rate-limiting enzyme of histamine synthesis, HDC (14, 15). In a previous study we identified a downstream (+2 to +24) 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). Analysis of signal transduction pathways revealed that MAP kinase/ERK signaling pathways are crucial for the regulation of the hHDC promoter (16, 17). While previous studies analyzed the structural basis for nicotine-stimulated mCgA transcriptional activity in pheocromocytoma cells (18, 19), the molecular mechanisms of gastrin-stimulated CgA expression are still unclear. Therefore, to better understand the molecular mechanisms involved in gastrin-dependent CgA gene expression, we have analyzed the regulatory elements and transcription factors involved in the control of the CgA promoter by gastrin. We find that gastrin transactivates the mCgA promoter dose and time dependently in AGS-B gastric carcinoma cells and GH3 pituitary cells. Conventional 5'-deletion analysis, in combination with scanning mutagenesis of the mCgA core promoter region, demonstrate that a DNA-stretch spanning mCgA -93 to -62 bp represents the gastrin-responsive region of the mCgA promoter. Furthermore, we show that two elements located in this region are necessary for full gastrin responsiveness: an Sp1/Egr-1 motif located at -88 to -77 bp and a CRE-like element at -71 to -64 bp. 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Reagents-- Oligonucleotides were provided by the Center for the Study of Inflammatory Bowel Diseases, Massachusetts General Hospital, Boston. Sp1 and CREB consensus oligonucleotides as well as anti-Sp1, anti-CREB/ATF1 antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Anti-phospho-CREB antibody was obtained from Upstate Biotechnology. The Gal4/CREB transactivator plasmid and the protein kinase A expression construct were kindly provided by Dr. J. Habener (20, 21). The Gal4/CREB/A construct contains a mutation at Ser-133 so that it cannot be transactivated (20, 21). The Gal4/Sp1 transactivator plasmid and the Sp1 overexpression construct were kindly provided by Dr. G. Suske (Marburg, Germany) (22, 23). The 5xGal4/Luciferase reporter construct was provided by Dr. Anil Rustgi, Massachusetts General Hospital, Boston, MA. The Sp1 overexpression plasmid has been obtained from Dr. J. Merchant (24). Phorbol ester 12-myristate 13-acetate (PMA) was purchased from Biomol, Plymouth Meeting, PA.

Cell Culture and Transfection Studies-- AGS-B gastric cancer and GH3-B neuroendocrine cells were derived from parent cells (AGS and GH3, ATCC) through stable transfection of the expression vector CCKB-pcDNAINeo, containing the full-length coding region of the human CCK-B/gastrin receptor and the neomycin gene, and have been previously described (25). AGS-B cells were grown in Dulbecco's modified Eagle's medium containing 10% bovine calf serum, 100 IU/ml penicillin, and 100 IU/ml streptomycin in a humidified atmosphere (5% CO2/95% air). Transient transfections of cultured AGS-B and GH3-B cells were carried out using the calcium-phosphate precipitation technique (DNA Transfection Kit, 5 Prime-3 Prime Inc.). Cells were plated at a density of 1 × 106 cells/35-mm well and transfected the next day. In general, cells were harvested and luciferase assays done at 48 h. Luciferase (Luc) assays were performed using luciferin, ATP, and coenzyme A (Promega system) with a Monolight Luminometer (Analytic Luminescence Laboratory) as described previously (16, 17, 25). Incubations were performed in triplicates or quadruplicates and results calculated as mean ± S.E. Values for mCgA-Luc activity were expressed as fold increase in luciferase activity compared with untreated controls. The empty pTK-Luc construct served as an additional control (16, 17, 25). Activities varied less than 15% between transfection experiments. Expression of human growth hormone from the plasmid vector pXGH5, containing the human growth hormone gene under the control of the metallothionein-I promoter, was used as an internal control for transfection efficiency (16, 17, 25).

DNA Constructs and Reporter Plasmids-- The mCgA 5'-deletion constructs, which are all based on the promoterless luciferase reporter gene vector pXP1, have been previously reported (26). A series of scanning mutants between mCgA -100 and -49 bp were prepared by polymerase chain reaction amplification using the pXP100 construct as a template, employing different mutated 5'-oligonucleotide primers and a common 3' primer (26). The mutant fragments were subcloned into XhoI and HindIII sites of pXP1 and mutant sequences were confirmed by dideoxy sequencing (26). In order to study the characteristics of potential mCgA regulatory elements in a heterologous promoter system, the region mCgA -93 to -62 bp was subcloned either adjacent or 1.2 kb away from the enhancerless herpes simplex thymidine kinase (TK) viral promoter into the plasmid pTK-Luc (26).

Electrophoretic Mobility Shift Assays-- Nuclear extracts from AGS-B cells were prepared as described (15). In brief, double-stranded oligonucleotides were radiolabeled with [alpha -32P]dCTP and EMSAs were performed with 5 µg of nuclear extracts in a final volume of 20 µl of binding buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 1 µg of poly(dA-dT), and 10% glycerol. Mixtures were incubated with 10 fmol of double stranded oligonucleotide probes for 20 min at room temperature. DNA-protein complexes were electrophoresed on a 6% nondenaturing polyacrylamide gel containing 0.25 × TBE at a constant current of 15 mA. Gels were dried and exposed to Kodak X-AR films at room temperature. Gel shift assays required 10 µM ZnSO4 for complete Sp1 binding. For competition experiments, nuclear extracts were incubated with a 100-fold excess of double-stranded competitor oligos at room temperature for 10 min before addition of radiolabeled probes. For supershift experiments, nuclear extracts were incubated with 1 µl of anti-Sp1 or anti-CREB antibodies for 10 min at room temperature, followed by an incubation period of 20 min at 4 °C prior to addition of radiolabeled probes.

Western Blotting-- Preparation of cell lysates and SDS-polyacrylamide gel electrophoresis analysis was performed as described previously (16, 17). In brief, AGS-B cells were washed with ice-cold phosphate-buffered saline twice and lysed with ice-cold lysis buffer containing 10 mM HEPES (pH 7.4), 30 mM NaCl, 2% glycerol, 0.2% Triton X-100, 0.3 mM MgCl2, 0.2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 mM sodium orthovanadate, 1 mM sodium fluoride. 50 µg of the lysates were electrophoresed on 8 or 10% SDS-polyacrylamide gels for detection of Sp1 or CREB proteins, respectively. After gel separation, proteins were blotted on nitrocellulose membranes and visualized by the enhanced chemiluminescence (ECL, Amersham) method according to the instructions of the manufacturer using a 1:1000 dilution of antibodies.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Gastrin Stimulation of CgA Promoter Activity-- In AGS-B cells, gastrin stimulated mCgA promoter activity time and dose dependently (Fig. 1), whereas activity of pTK-Luc was not significantly influenced (not shown). Essentially identical kinetics were found for gastrin activation of the mCgA4.8kb-Luc construct in pituitary GH3-B cells (data not shown). Since activation of the mCgA promoter by cAMP-mediated pathways has been established in bovine medullary cells, we investigated the interaction of this signaling route with gastrin- and PMA-activated pathways. In AGS-B cells, dibutyryl-cAMP stimulated mCgA promoter activity 2-3-fold, and co-stimulation with gastrin or PMA and db-cAMP showed additive responses (Fig. 2). 5'-Deletion analysis of mCgA 5'-flanking DNA revealed that 100 bp upstream of the mCgA cap site (+1) are able to confer full responsiveness to gastrin, and that further 5'-deletion to mCgA -77 abrogated gastrin responsiveness (Fig. 3A). To further characterize the region downstream of mCgA -77 functionally, a dense series of scanning mutants was employed in transient transfection experiments. Analysis of scanning mutants demonstrated that two mCgA promoter regions (mutants M6 and M13) are essential for gastrin-dependent transactivation: a DNA-stretch spanning -88 to -77 bp, comprising putative binding sites for Sp1 and Egr-1, and the region between -71 and -64, bp which represents a consensus CRE element (Fig. 3B).


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  		Fig. 1.   Influence of gastrin on the transcriptional activity of the mCgA4.8kb-Luc reporter construct in AGS-B cells. A, AGS-B cells transiently transfected with the mCgA4.8kb-Luc construct (0.5 µg/well) were incubated with or without gastrin under serum-free conditions. After 24 h cells were harvested and analyzed for luciferase activity. B, for time course studies, AGS-B cells were transiently transfected with mCgA4.8kb-Luc and stimulated with gastrin (10-8 M) for various time periods. Luciferase activity is expressed as a fold increase relative to unstimulated controls and represents the mean ± of four separate experiments.


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  		Fig. 2.   mCgA promoter activity after stimulation of different intracellular signaling pathways. AGS-B cells transiently transfected with the mCgA4.8kb-Luc construct (0.5 µg/well) were incubated with or without 10-8 M forskolin (Fors.), 10-5 M dibutyryl (db)-cAMP, 10-8 M gastrin, 10-8 M PMA, or a combination of these compounds. Luciferase activity is expressed as a fold increase relative to unstimulated controls and results represent the mean ± of four separate experiments. All stimulated groups showed a statistically significant difference (p < 0.05) compared with unstimulated controls using Student's t test.


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  		Fig. 3.   Analysis of mCgA 5'-deletion mutants and "scannining mutagenesis" of the mCgA core promoter in AGS-B cells. A, AGS-B cells were transiently transfected with mCgA 5'-deletion mutants of different length (0.5-1.0 µg/well) and incubated with or without gastrin (10-8 M) for 24 h. B, a dense series of scanning mutants of the core promoter region (-100 to -49 bp) was generated by an polymerase chain reaction-based approach (using the construct PxP100-Luc as a template) and transiently transfected into AGS-B cells (0.5-1.0 µg/well). After transfection, gastrin stimulation was performed for 24 h and cell lysates were subsequently analyzed for luciferase activity. Luciferase activity is expressed as a fold increase relative to unstimulated controls and represents the mean ± of four separate experiments. Please note: the mutation in mCgA-M14 converts the mCgA-CRE element to a consensus CRE site.

Subcloning of mCgA -93 to -62 bp Confers Gastrin Responsiveness to a Heterologous Promoter-- To investigate whether the region mCgA -93 to -62 bp is able to confer gastrin responsiveness to a gastrin-insensitive heterologous promoter system, we employed the pTK-Luc construct. In this construct, expression of the luciferase reporter gene is under control of the enhancerless herpes simplex virus thymidine kinase minimal promoter. When constructs in which the mCgA -93 to -62 bp region was subcloned either adjacent or 1.2 kb away from the TK promoter were transfected into AGS-B cells, gastrin stimulation elicited a 2.5-4.5-fold increase in reporter gene activity (Fig. 4).


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  		Fig. 4.   Gastrin stimulates the activity of the mCgA -92 to -64 bp element in a heterologous promoter system. AGS-B cells were transiently transfected with TK-luciferase reporter constructs (1.0 µg/well) into which a single copy of the mCgA -92 to -64 bp element was subcloned either adjacent to, or 1.2 kb upstream of the enhancerless TK promoter. After transfection gastrin stimulation (10-8 M) was performed under serum-free conditions for 24 h and cell lysates were subsequently analyzed for luciferase activity. Luciferase activity is expressed as a fold increase relative to unstimulated controls and represents the mean ± of four separate experiments. The asterisk (*) indicates a statistically significant difference (p < 0.05) compared with unstimulated controls using Student's t test.

CREB and Sp1 Bind to the Gastrin-responsive Region of the mCgA Promoter-- EMSA analysis revealed that in AGS-B cells two nuclear complexes appear to bind to mCgA -93 to -62 bp, the complete element able to confer gastrin responsiveness to a heterologous promoter system. The "A" probe, which represents the complete sequence of this element, is bound by 2 major complexes (Fig. 5B). In experiments using an upstream ("B" probe, mCgA -93 to -73 bp) or downstream probe("C" probe, mCgA -76 to -62 bp), the transcription factor in complex A was identified as Sp1 (Fig. 5C), whereas in complex B the probe is bound by CREB (Fig. 5D). EMSA analysis of nuclear extracts obtained from AGS-B cells after 10 min of gastrin or PMA stimulation revealed that this treatment increased the binding of Sp1 to its upstream binding site (Fig. 5C). Since CREB-dependent transactivation is generally brought about by phosphorylation of the transcription factor at residue serine 133, we performed EMSA supershift experiments employing an anti-phospho-CREB antibody, which is directed against the serine 133 epitope and exclusively recognizes phosphorylated CREB. We found enhanced binding of phosphorylated CREB in response to gastrin and PMA after 10 min of treatment (Fig. 5D). A similar picture emerged when supershift experiments were performed with an antibody identifying CREB proteins also in the unphosphorylated state (Fig. 5D).


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  		Fig. 5.   EMSA of the mCgA -92 to -64 bp region. A, shows the different EMSA probes used: probe A comprised nucleotides mCgA -92 to -62 bp, probe B the region mCgA -92 to -73 bp, and probe C mCgA -76 to -62 bp. For EMSA experiments double-stranded oligonucleotides were synthesized and 32P-end-labeled. Crude AGS-B cell extracts were incubated with double-stranded, labeled probes in the presence or absence of unlabeled (cold) oligonucleotides or antibodies as indicated. For supershift assays, AGS-B nuclear extracts were preincubated with an anti-Sp1 or anti-CREB antibody prior to addition of the appropriate probe. To determine binding of phosphorylated CREB (p-CREB), we used an antibody which selectively recognizes the CREB phosphorylation site at the serine 133 residue. B, EMSA with probe A as a labeled probe, and A, B, or C as a cold competitor. C, EMSA with probe B as a labeled probe, showing competition with consensus Sp1-binding sites and supershift with anti-Sp1 antibody. D, EMSA with probe C as a labeled probe showing supershifts with both anti-CREB and anti-p-CREB.

Gastrin Stimulates CREB Phosphorylation and Sp1 Expression in AGS-B Cells-- To investigate whether increased binding of Sp1 and CREB to the CgA promoter is accompanied by elevated cellular levels of both factors, we analyzed basal and gastrin-stimulated CREB and Sp1 abundance in AGS-B cells by Western blot analysis. We found that gastrin dose dependently stimulated Sp1 abundance, having a pronounced effect as early as 5-10 min after stimulation (Fig. 6A). Similar results were obtained when total lysates from gastrin-stimulated AGS-B cells were analyzed for the abundance of phophorylated CREB (Fig. 6B). Maximal elevation in the abundance of phophorylated CREB was observed after 10 min of gastrin stimulation.


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  		Fig. 6.   Western blot analysis of Sp1 expression and CREB phosphorylation in AGS-B cells. AGS-B cells were stimulated with 10-8 M gastrin for time periods as indicated and total cell lysates were prepared. After resolution of proteins by SDS-polyacrylamide gel electrophoresis, blots were probed with an antibody directed against Sp1 (A) or phosphorylated CREB (B). The anti-phospho-CREB antibody selectively recognizes the phosphorylated serine 133 epitope of human CREB. The blots shown are representative for three independent experiments.

Overexpression of CREB and Sp1 Stimulates mCgA Promoter Activity-- To demonstrate that elevation of cellular Sp1 and CREB levels has functional impact on the transcriptional activity of the CgA promoter, we transiently overexpressed Sp1 or phosphorylated CREB by transfection of appropriate expression vectors into AGS-B cells. Phosphorylation of transfected CREB was achieved by co-transfection of pCMV-CREB with an expression construct for the catalytic subunit (C) of protein kinase A. This strategy has been shown to result in effective phosphorylation of recombinant CREB proteins encoded by the CREB expression construct (20, 21). When increasing amounts of these constructs were co-transfected with mCgA100-Luc into AGS-B cells, reporter gene activity was dose dependently stimulated (Fig. 7B). Similarily, mCgA reporter gene activity was dose dependently stimulated by transfecting increasing amounts of a full-length human Sp1 expression construct (23) together with mCgA4100-Luc (Fig. 7A). When Sp1 and CREB overexpression was performed in cells transfected with the mCgA -93 to -62 bp TK-Luc construct, coexpression of both factors was more effective than expression of either factor alone, indicating positive cooperative action of Sp1 and CREB in the regulation of the mCgA promoter (Fig. 7C).


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  		Fig. 7.   Overexpression of Sp1 and phosphorylated CREB stimulates mCgA promoter activity. AGS-B cells were transiently co-transfected with increasing amounts of expression constructs for Sp1 (A) or CREB (B) and 0.5 µg of the mCgA100-Luc reporter construct. Phosphorylation of recombinant CREB was achieved through co-transfection of a construct encoding the catalytic domain C of protein kinase A (B). In C, AGS-B cells were co-transfected with the mCgA -93 to -62 bp-Luc construct and either the Sp1 or CREB expression construct or a combination of both. Luciferase activity is expressed as a fold increase relative to untransfected controls and represents the mean ± of four separate experiments. The asterisk (*) indicates a statistically significant difference (p < 0.05) compared with unstimulated controls using Student's t test.

Gastrin Stimulates CREB- and Sp1-dependent Transactivation in AGS-B Cells-- After EMSA and Western blot analysis demonstrated that gastrin stimulation of AGS-B cells results in increased abundance of Sp1 and phosphorylated CREB, we next investigated the effect of gastrin on CREB- or Sp1-dependent transactivation in the Gal4/Gal4-5xLuciferase system. The Gal4-CREB transactivator plasmid contains the regulatory sequence of CREB, which comprises the serine 133 residue, critical for its phosphorylation. In the Gal4-CREB/A construct, the serine residue at position 133 is replaced by an arginine which cannot be phosphorylated. The Gal4/Sp1 transactivator plasmid has previously been described (20, 21). After co-transfection of Gal4-transactivator and Gal4-reporter plasmids, binding of Gal4-CREB or Gal4-Sp1 fusion proteins encoded by the transactivator plasmids to the Gal4-binding domain of the reporter plasmid depends on the phosphorylation state of subcloned CREB or Sp1 sequences. In the reporter plasmid 5xGal4-Luciferase, expression of the luciferase reporter gene is under control of a multimer (five copies) of the Gal4-binding domain. Stimulation of AGS-B cells transfected with Gal4-CREB or Gal4-Sp1 transactivator constructs together with the reporter construct Gal4/5xGal4-Luc resulted in a 2.5- or 1.6-fold increase in reporter gene activity, respectively (Fig. 8). Gastrin had no effect on Gal4-CREB/A-dependent transactivation. These results demonstrate that in AGS-B cells, gastrin stimulates CREB-dependent transactivation through enhanced phosphorylation of CREB. Using the Gal4-Sp1 system, we found that gastrin also had a modest stimulatory effect on Sp1-dependent transactivation, raising the possibility of post-translational modification of Sp1 in response to gastrin.


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  		Fig. 8.   Gastrin stimulates CREB- and Sp1-dependent transactivation in AGS-B cells. AGS-B cells were co-transfected with 1.0 µg of Gal4-CREB, Gal4-CREB/A, or Gal4-Sp1 transactivator plasmids along with 1.0 µg of the 5xGal4-Luciferase reporter plasmid and incubated with or without 10-7 M gastrin for 24 h. While Gal4-CREB encodes a fusion protein consisting of the Gal4-binding domain and wild type CREB, in Gal4-CREB/A the serine residue at 133 has been mutated and cannot be phosphorylated anymore. Luciferase activity is expressed as a fold increase relative to unstimulated controls and represents the mean ± of four separate experiments. The asterisk (*) indicates a statistically significant difference (p < 0.05) compared with unstimulated controls using Student's t test.


    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The purpose of this study was to characterize the regulatory effect of gastrin on the mCgA promoter, along with the cis-regulatory elements and transcription factors mediating the actions of gastrin. We found that gastrin transactivates the mCgA promoter dose and time dependently in both AGS-B gastric carcinoma cells and GH3 pituitary cells. Conventional 5'-deletion analysis, in combination with scanning mutagenesis of the mCgA proximal core promoter region, demonstrated that a DNA-stretch spanning -93 to -62 bp represents the gastrin-responsive region of the mCgA promoter, and that two regulatory elements located in this region were necessary for full gastrin responsiveness: an Sp1/Egr-1 motif located at -88 to -77 bp and a CRE-like element at -71 to -64 bp. In the context of the heterologous TK-minimal promoter, the complete element (-93 to -62 bp) was able to confer gastrin-responsiveness independent of distance relative to the TK-minimal promoter, confirming that it displays characteristics of a "true" enhancer element. Selective mutation of either the Sp1/Egr-1 site or the CRE motif completely abrogated the effect of gastrin, demonstrating that integrity of both elements is necessary for gastrin responsiveness. The importance of this functional domain for CgA gene expression is also supported by the observation that both regulatory elements are found evolutionary conserved in tandem upstream of the TATA box in CgA genes of human, murine, bovine, and rat origin (3, 26). EMSA studies showed that Sp1 and CREB specifically bound the CgA promoter element, and transfection studies demonstrated that overexpression of Sp1 and phosphorylated CREB led to increased mCgA promoter activity.

Previous studies examining mCgA promoter regulation in PC12 and AtT20 cells demonstrated that the 5'-flanking region of the mCgA gene contains multiple positive and negative regulatory elements involved in regulation of CgA gene expression (17, 26, 27). These studies identified the proximal mCgA CRE site at -71 to -64 bp as essential for cell type-specific promoter activity, as well as the response to nicotine, a major stimulator of catecholamine biosynthesis in chromaffin cells (17, 27). Our study differs in a number of ways from the findings of these previous reports. In contrast to its essential role for the transmission of gastrin's effects, the Egr-1/Sp1 motif at mCgA -88 to -77 is not involved in response of the mCgA promoter to nicotine (17). In addition, we found using EMSA supershift analysis that Sp1 and CREB are the nuclear factors present in AGS-B cells which bind to the gastrin-responsive region of the mCgA promoter, and that they are induced by gastrin (Fig. 5).

Gastrin stimulation led to an increase in the amount of CREB binding to the mCgA CRE at -71 to 64 bp, but also stimulated time dependently phosphorylation of CREB, as shown by using an antibody which specifically recognizes the CREB Ser-133 epitope in its phosphorylated state. CREB, a member of the "leucine-zipper" family of transcription factors, represents the predominant factor binding to the palindromic CRE-consensus sequence 5'-TGACGTCA-3' present in the 5'-flanking region of a number genes (28, 29). Stimulation of CREB-dependent transcriptional activity is generally achieved by phosphorylation of the transcription factor at a serine residue at position 133 (28). Classically, cAMP-mediated phosphorylation of CREB in response to extracellular stimuli is mediated through direct phosphorylation of the transcription factor by protein kinase A (28-30). In addition, CREB phosphorylation through activation of Ca2+-calmodulin- or MAP kinase-dependent pathways has been reported (31-33). Furthermore, several studies have provided evidence for a model in which the coactivator CBP/p300, a protein that physically interacts with CREB at its transactivation domain dependent on the phosphorylation state, can connect CREB to different intracellular signaling pathways, such as MAP kinase/ERK-cascades and related transcription factors (34-37).

In addition to the mCgA CRE site, an Sp1/Egr-1 motif located directly upstream of the CRE appeared to be indispensible for gastrin responsiveness of the mCgA promoter. EMSA analysis using AGS-B nuclear extracts demonstrated that Sp1 interacted specifically with this element, and that gastrin stimulation of AGS-B cells increased the amount of Sp1 bound to the mCgA Sp1 site. Additionally, Western blot analysis of AGS-B cellular lysates showed a time-dependent increase in Sp1 protein abundance in response to gastrin stimulation. In contrast to our results with Sp1, no binding by Egr-1 to the -88 to -77 bp element could be detected in AGS-B cells under gastrin-stimulated or unstimulated conditions. Previous studies have shown that Sp1 regulates transcriptional activity of target genes by binding to GC-box elements (5'-GGGCGG-3') which can be found in a variety of viral and cellular promoters (38). Functional analysis of the mCgA Sp1 site using scanning mutants showed that in the mutant construct M6, in which the upstream half-site of the overlapping Egr-1 site but only the most upstream base (G at mCgA -83 bp) of the Sp1 consensus motif is mutated, results in a complete loss of gastrin responsiveness (Fig. 3B), whereas construct M7 in which four bases (5'-GtcGacG-3') of the 5'-GGGGCGG-3' Sp1 site are mutated is fully responsive to gastrin (Fig. 3B). Since EMSA analysis using the B-probe (mCgA -93 to -73) identified Sp1 as the nuclear factor binding to Sp1/Egr-1 site, the GC-rich region of the Egr-1 upstream half-site is obviously important for binding of Sp1 to the overlapping Sp1/Egr-1 element of the mCgA gene (Fig. 5C).

While gastrin stimulation led to increased Sp1 protein abundance, it also was able to transactivate a GAL4/Sp1 fusion protein, suggesting the additional possibility of post-translational modification of Sp1 in response to gastrin. While in the past Sp1 was considered a ubiquitiously expressed nuclear factor involved in the transcription of constitutively expressed "housekeeping genes," a number of recent studies have suggested that Sp1 can regulate gene expression in response to activation of diverse signaling pathways (23, 39-42). Functional regulation of Sp1 by post-translational modification has also been reported (43, 44), including both O-glycosylation (23, 44) and phosphorylation (42, 45). Recent studies found that O-glycosylation of Sp1 is critical for its proteasome susceptibility and that modification of the glycosylation state of the factor represents a mechanism for regulation of Sp1 abundance (23, 44, 45). The signaling pathways regulating the expression of Sp1 have not been clearly defined, but several previous reports are consistent with the notion that Sp1 can be functionally controlled by PKC-related pathways (39, 40).

Interestingly, mutation of either the upstream Sp1 site or the downstream CRE site completely abrogated gastrin responsiveness, suggesting the possibility of some sort of cooperative interaction between Sp1 and CREB. Previous studies have reported cooperative action between Sp1 and other transcription factors, including GATA-1 (45), YY-1 (46), NF-kappa B (47), or AP-1 (48), resulting in inhibitory or stimulatory transcriptional effects. Interactions with CREB have not been previously described, although a number of genes have been reported to contain Sp1-binding sites located close to or within a CRE site (41, 49-52). This report represents the first description of a positive interaction between Sp1 and CREB-binding sites to directly stimulate promoter activity in response to activation of a G-protein-coupled, heptahelical receptor. It is unclear at present as to the exact nature of interaction of Sp1 and CREB, and whether it involves changes in DNA conformation or interaction with the basal transcription factor machinery. Our EMSA studies demonstrate that binding of CREB to the mCgA CRE does not depend on the presence of an Sp1 site and vice versa, indicating that the presence of one factor is not a prerequisite for the interaction of the other factor with its recognition motif (Fig. 5, C and D).

Taken together, we found that both Sp1 and CREB are essential for gastrin-dependent regulation of the CgA promoter. In addition, this study for the first time demonstrates that Sp1 and CREB represent nuclear targets of the heptahelical CCK-B/gastrin receptor. Further analysis of gastrin-dependent control of the CgA gene, Sp1, and CREB, as well as associated signal transduction pathways, will provide additional insight into the molecular mechanisms regulating neuroendocrine gene expression in response to the peptide hormone gastrin.

    ACKNOWLEDGEMENT

We acknowledge the technical assistance by Kerstin Waigand.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant RO I DK 48077 (to T. C. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** Supported by Deutsche Forschungsgemeinschaft, the Verum Stiftung, and the Mildred Scheel Stiftung.

Dagger Dagger Supported by a grant of the German Research Council (Deutsche Forschungsgemeinschaft), Bonn.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§§ To whom correspondence should be addressed: Gastrointestinal Unit and Dept. of Medicine, Massachusetts General Hospital, 30 Fruit St., Boston, MA 02114. Tel.: 617-726-9228; Fax: 617-726-3673; E-mail: Wang{at}helix.mgh.harvard.edu.

The abbreviations used are: CgA, chromogranin A; ECL cells, enterochromaffin-like cells; HDC, histidine decarboxylase; ERK, extracellular-signal regulated kinase; CRE, cAMP-responsive element; CREB, CRE-binding protein; bp, base pair(s); PMA, phorbol 12-myristate 13-acetate; kb, kilobase pair(s); TK, thymidine kinase; EMSA, electrophoretic mobility shift assay; MAP, mitogen-activated protein.
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Abstract
Introduction
Procedures
Results
Discussion
References

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