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
Raktima Raychowdhury1,
Georgia Schäfer1,
John Fleming,
Stefan Rosewicz,
Bertram Wiedenmann,
Timothy C. Wang and
Michael Höcker
Medizinische Klink mit Schwerpunkt Gastroenterologie, Hepatologie, Endokrinologie und Stoffwechsel (G.S., S.R., B.W., M.H.), Universitätsklinikum Charité, Campus Virchow-Klinikum, Humboldt Universität, 13353 Berlin, Germany; and Gastrointestinal Unit and Department of Medicine (R.R., J.F., T.C.W.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Priv.-Doz. Dr. Michael Höcker, Gastroenterologie, Hepatologie, Endokrinologie und Stoffwechsel, Universitätsklinikum Charité, Campus Virchow-Klinikum, Humboldt Universität Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: michael.hoecker{at}charite.de.
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ABSTRACT
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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.
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INTRODUCTION
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CHROMOGRANIN A (CgA) represents a multifunctional, secretory granule matrix protein, which is specifically expressed in the neuroendocrine system (1, 2, 3). CgA was initially identified as the major soluble matrix protein of secretory vesicles formed in neuroendocrine cells. Its functions include modulation of secretory granule stability, prohormone processing, and regulation of peptide sorting into secretory pathways (1, 2, 3). Cleavage products of CgA are able to influence endocrine and exocrine secretory functions as well as cellular adhesion and vascular tension through paracrine and/or autocrine pathways (4, 5, 6). In the stomach, enterochromaffin-like cells (ECL cells) of the corpus mucosa, which represent the central regulatory unit for the regulation of gastric acid secretion, have been identified as the main source for CgA expression and secretion (7, 8). Functional in vivo studies in human subjects and rodents demonstrated that CgA synthesis in the ECL cell compartment is under tight control of circulating gastrin blood levels (8, 9, 10, 11, 12, 13). Furthermore, transcriptional activation of the CgA gene promoter appears to be a major mechanism through which the peptide hormone controls gastric CgA expression (8, 9, 10, 11, 12, 13, 14). This concept was confirmed by a recent in vivo study from our group, which showed that hypergastrinemia up-regulates the activity of a 4.8-kb CgA promoter fragment in ECL cells of transgenic mice (15).
In an effort to elucidate the molecular mechanisms mediating gastrin-triggered CgA gene activation, we have recently mapped the CgA gastrin responsive element (Gas-RE) to -92/-62 of the proximal CgA core promoter (14). Within this element, functional integrity of a GC-rich specificity protein 1 (Sp1)/early growth response 1 (Egr-1) motif at -88/-77 and a cAMP response element (CRE)-like binding site at -71/-64 turned out to be indispensable for gastrin stimulation of the CgA gene. Furthermore, an interplay of the transcription factors Sp1 and CRE-binding protein (CREB) at these sites is required for full gastrin responsiveness of the CgA Gas-RE (14). These findings were in contrast to previous studies performed in adrenal and neuronal epithelial cell lines, which showed that basal and agonist-dependent expression of the CgA gene was controlled almost exclusively through binding of CREB to the proximal CRE site, which is highly conserved among mammalian CgA genes (16, 17, 18, 19, 20).
GC-rich cis-acting elements that are commonly regulated through transcription factors of the Sp/XKLF family have been identified and functionally analyzed in numerous mammalian and viral genes, and it was found that they are often critically involved in regulation of fundamental biological processes including developmental patterning, cellular proliferation, and differentiation (21, 22). Furthermore, in a number of genes, consensus Sp1 sites overlapping with recognition motifs for the zinc-finger protein Egr-1 have been identified as important regulatory elements (23, 24, 25, 26, 27). Usually, Egr-1 specifically recognizes promoter regions bearing the DNA sequence 5'-GCG(C/G)GGGCG-3' and cannot bind to GC-rich Sp1 consensus sites, while Sp1 is not able to compete Egr-1 from its DNA binding motifs (28). In contrast, overlapping Sp1/Egr-1 elements allow both factors to bind and interact functionally to direct gene transcription (23, 24, 25, 26, 27). Egr-1 can be activated by mitogens and cytokines including phorbol esters, TGF
, and insulin (23, 24, 25) and, depending on the genetic and/or cellular context, its overall transcriptional effects can be either stimulatory or inhibitory (23, 24, 25, 26, 27, 28, 29). However, a functional role of Egr-1 in gastrin-dependent gene regulation has not yet been established. Therefore, this study was undertaken to clarify the functional role of the overlapping GC-rich Sp1/Egr-1 element at CgA -88/-77 and its potential interaction with the proximal CRE site during gastrin-triggered CgA promoter regulation in gastric epithelial cells.
Our study shows, for the first time, that functional interplay of Sp1, CREB, and Egr-1 is required for full gastrin-dependent CgA transactivation, whereas basal CgA gene promoter activity in gastric epithelial cells is primarily determined through binding of Sp1 and CREB to the Gas-RE. Furthermore, our results link the zinc-finger protein Egr-1 to intracellular pathways regulated through activation of the cholecystokinin B (CCK-B)/gastrin receptor comprising protein kinase C (PKC)-/MAPK kinase 1 (MEK1)-/ERK1/2- dependent signaling cascades as well as de novo protein synthesis.
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RESULTS
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The CgA Gas-RE at -92/-62 Is Bound by Sp1 and CREB
In our initial study, we found that in gastric cancer cells gastrin-stimulated transactivation of the CgA promoter is mediated through a proximal promoter element at CgA -92/-62, which comprises an overlapping Sp1/Egr-1 site and a CRE element (14). EMSA analysis of AGS-B nuclear proteins demonstrated that use of the complete CgA -92/-62 region as a probe (Fig. 1
, CgA "A" probe) did not allow proper analysis of the binding behavior of nuclear proteins at this element (14). Therefore, the CgA -92/-62 sequence was divided into an upstream oligonucleotide representing CgA -92/-73 (CgA B probe) and a downstream oligonucleotide spanning CgA -76/-62 (CgA C probe) (Fig. 1B
). These oligos were [
32P]dCTP end labeled and used as probes in EMSA studies. EMSA analysis of AGS-B nuclear extracts revealed that under resting conditions and after short-term exposure of AGS-B cells (
20 min) to gastrin or the phorbol ester PMA (phorbol 12-myristate-13-acetate), the Sp1/Egr-1 motif at CgA -88/-77 is primarily bound by Sp1 (Ref. 14 and Fig. 2A
). In addition, CREB was identified as the dominant nuclear protein binding to the CRE site at CgA -71/-64 (Ref. 14 and Fig. 2B
). Stimulation of gastric epithelial cells with gastrin or PMA enhanced binding of both Sp1 and CREB, to the -92/-62 region, while mutation within the Sp1/Egr-1 motif or deletion of the CRE element dramatically diminished basal as well as gastrin- and PMA-stimulated CgA promoter activity (14). While this initial investigation demonstrated that Sp1 and CREB are main transcription factors controlling the CgA promoter in gastric epithelial cells (Ref. 14 and Fig. 2
), extended EMSA studies suggested that under conditions of prolonged gastrin/PMA stimulation (
4060 min) additional nuclear protein(s) bind(s) to the -92/-73 bp element in AGS-B cells. Similarly, binding of an additional complex after long-term stimulation was also observed when the A probe, which comprises the entire -92/-62 sequence, was used in EMSAs (not shown), but resolution of the novel complex was complicated by the methodological limitations experienced with this probe (see above and Ref. 14). Because the gastrin-responsive region of the CgA promoter plays a crucial role for the regulation of the CgA gene in gastric cells, we aimed to identify the molecular nature of this additional nuclear factor(s) and analyzed the functional role of this factor(s) for regulation of the CgA promoter.

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Figure 1. Schematic Diagram of the Gastrin-Responsive Region of the CgA Promoter
In previous experiments, the sequence -92/-62 of the 5'-flanking region of the CgA gene has been identified to confer gastrin responsiveness in gastric cancer cells. The influence of gastrin on this element is mediated via activation of the transcription factors Sp1 and CREB (A). The lower panel shows the DNA probes used for basic characterization of the nuclear proteins binding to the CgA Gas-RE sequence (B).
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Figure 2. The CgA Gas-RE at -92/-62 Is Bound by Sp1 and CREB
Nuclear extracts were prepared from AGS-B cells after stimulation with gastrin (10 nM) for 10 min and incubated with [ -32P]dCTP end-labeled oligonucleotide probes as indicated. In mutant probe M13, the sequence -76-TCCTATGACGTAATT-62 was changed to -76-TCCTAcatcaccATT-62. This mutant has previously been shown to abolish CREB binding and agonist-stimulated activity of the CgA CRE (14 16 17 ). Competition studies were carried out using 100-fold molar excess of unlabeled oligonucleotides as indicated. For supershift assays, nuclear extracts were preincubated with anti-Sp1, anti-CREB, or anti-Egr-1 antibodies before addition of radiolabeled probes. The data shown represent typical results of a series of three independent experiments.
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Prolonged Gastrin and PMA Stimulation Results in Binding of Egr-1 to the Overlapping Sp1/Egr-1 Site at CgA -88/-77
Extended time-course analysis of nuclear proteins binding to the Sp1/Egr-1 element was performed in EMSA studies using nuclear extracts prepared from AGS-B cells after gastrin or PMA stimulation for up to 24 h. When the CgA B probe (-92/-73) was incubated with nuclear extracts prepared from AGS-B cells exposed to gastrin or PMA for up to 24 h, we found formation of a new complex (complex II) in response to both stimuli (Fig. 3
, AD). Gastrin-dependent formation of this complex (Fig. 3A
, complex II) was transient, showing peak binding after 1 h of agonist stimulation, whereas the complex disappeared after 15 h of gastrin treatment (Fig. 3B
). Under conditions of PMA stimulation, this complex first appeared after 4060 min (Fig. 3C
, lanes 56), peaked at 13 h, and progressively disappeared at later time points (Fig. 3D
, lanes 46). It is important to note that the appearance of complex II was not associated with any changes in the formation of other EMSA complexes obtained with the -92/-73 probe (Fig. 3
, AD). Furthermore, in contrast to the results obtained with the -92/-73 probe, no changes in the binding patterns of nuclear proteins to the -76/-62 probe comprising the CgA CRE was found during extended stimulation with gastrin or PMA (data not shown). To identify the molecular nature of the nuclear protein(s) contained in the newly formed complex II, supershift assays and competition experiments using excess concentrations of unlabeled oligonucleotides representing consensus or mutant sequences of the Egr-1 binding motif were performed (Fig. 4
). Application of an Egr-1-specific antibody entirely abrogated formation of complex II and led to a supershifted complex (Fig. 4A
, lane 8; Fig. 4B
, lane 3). Similarly, the gastrin- and PMA-induced complex II was competed out by excess concentrations of unlabeled consensus Egr-1 oligo, whereas mutant Egr-1 oligo had no effect on this complex (Fig. 4A
, lanes 6 and 7; Fig. 4B
, lanes 1 and 2). These studies demonstrate that extended stimulation with gastrin or PMA induces the binding of Egr-1 to the GC-rich Sp1/Egr-1 motif, whereas the binding behavior of Sp1 and CREB to their recognition motifs remained unchanged (Figs. 3
and 4
, A and B, for Egr-1; Fig. 4C
for Sp1; data for CREB not shown). In addition to interaction of Egr-1 with the CgA -92/-73 element, we investigated the influence of gastrin and PMA on nuclear accumulation of Egr-1 protein in AGS-B cells. We found that both gastrin and PMA stimulation led to nuclear accumulation of Egr-1 (Fig. 4D
). Highest levels of Egr-1 were observed after 60 min of gastrin stimulation (10-7 M), whereas nuclear Egr-1 abundance appeared to decrease after 90 and 120 min of gastrin exposure (Fig. 4D
). Interestingly, the effect of gastrin on nuclear Egr-1 accumulation was more pronounced than the effect of PMA, whereas in EMSA studies both agents showed similar effectiveness regarding stimulation of Egr-1 binding to the CgA -92/-73 element (compare Fig. 4D
vs. Fig. 3
, AD). To explore the influence of gastrin on Egr-1-dependent transactivation in gastric cells, we used a Gal4-Egr-1/Gal4-luciferase cotransfection system in transient transfection assays (30). In this system the transactivator plasmid, Gal4-Egr-1, encodes a fusion protein comprising the binding domain of the yeast transcription factor Gal4 fused to a regulatory region of Egr-1. Binding of this fusion protein to the Gal4 recognition motif present in the reporter gene construct Gal4-luc depends on modification(s) of the Egr-1 protein sequence and therefore allows determination of the transactivation potency of Egr-1 in response to external stimulation. These studies demonstrated that gastrin treatment increased the transcription-stimulating activity of Egr-1, suggesting that in addition to enhanced nuclear abundance and increased binding to the CgA promoter, posttranslational modification(s) of Egr-1 could contribute to the overall effect of gastrin on this transcription factor in gastric epithelial cells (Fig. 4E
).

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Figure 3. Prolonged Gastrin and PMA Treatment Stimulates Binding of an Additional Nuclear Protein(s) to the CgA Promoter
To determine the binding kinetics of nuclear proteins to the CgA Gas-RE under conditions of prolonged agonist stimulation, AGS-B nuclear extracts were prepared after stimulation with gastrin (10 nM) or PMA (10 nM) for up to 24 h and analyzed in EMSA studies. The EMSA probe used was an [ -32P]dCTP end-labeled oligonucleotide representing the CgA -92 to -73 sequence (B-probe). The time course for binding of nuclear proteins in response to gastrin is given in panels A and B. Panels C and D show time course results for PMA stimulation. The data shown represent typical results of a series of three experiments.
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Figure 4. Gastrin Stimulates Nuclear Accumulation, Transcription-Stimulating Activity, and Binding of Egr-1 to the CgA -92/-73 in AGS-B Cells
To identify the protein(s) present in the new complex II, which appeared after prolonged stimulation with gastrin and PMA, supershift studies using an Egr-1-specific antibody were performed. Nuclear extracts used for these experiments were prepared after stimulation of AGS-B cells with gastrin (panel A) or PMA (panel B) for 60 min. In addition, unlabeled oligonucleotides representing consensus or mutant Egr-1 binding motifs were employed in competition experiments (100-fold excess). Supershift and competitor studies demonstrate that Egr-1 is the other major factor binding to the CgA -92/-73 sequence under conditions of prolonged agonist exposure (panels AC). D, Nuclear extracts were prepared after stimulation of AGS-B cells with 10-7 M gastrin or 10-8 M PMA for the indicated time periods, and Western blot studies were performed using Egr-1 antibody. Equal amounts of proteins (20 µg) were separated by SDS-PAGE and blotted onto membranes. Data shown represent typical results of a series of three independent experiments. E, To investigate the influence of gastrin on Egr-1 transcription-stimulating activity, AGS-B cells were transiently transfected with 1 µg/well of either Gal4-luc alone (negative control) or Gal4-luc along with 2 µg/well of the expression construct Gal4-Egr-13. In the plasmid Gal4-luc, the firefly luciferase reporter gene is under control of a multimer of the Gal4 yeast transcription factor binding element, whereas the construct Gal4-Egr13 encodes a fusion protein comprising the DNA-binding domain of the yeast transcription factor Gal4 and the Egr-1 transactivation domain (amino acids 16335). Cells transfected with the Gal4-Egr-13/Gal4-luc system were treated with 10-7 M gastrin or left untreated, harvested after 8 h, and assayed for luciferase activities. Data shown represent normalized luciferase activity and represent mean ± SEM of three independent transfection experiments.
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Sp1 and Egr-1 Interact with the CgA -88/-77 Element Through Different Binding Regions
To determine the region(s) responsible for binding of Egr-1 to the overlapping Sp1/Egr-1 element in the gastrin-responsive region of the CgA promoter, different mutations were introduced into the CgA -92/-73 sequence (Fig. 5A
). Double-stranded, synthetic oligonucleotides representing wild-type and mutant sequences were [
-32P]dCTP end labeled and used as probes in EMSA studies together with nuclear extracts prepared from AGS-B cells after 60 min of stimulation with gastrin (10 nM) or PMA (10 nM). Analysis of the binding patterns of these mutations revealed that mutant M2 selectively inhibited Sp1 binding. Similarly, mutant M7 abrogated binding of Egr-1 without having substantial effects on Sp1 binding (Fig. 5
, B and C), while mutants M5 and M6 abrogated the binding of both Sp1 and Egr-1 to the CgA -92/-73 sequence. In contrast to mutations M2, M5, M6, and M7, introduction of mutations M1, M3, M4, or M8 into the -92/-73 sequence did not lead to selective inhibition of Egr-1 and/or Sp1 binding, although factor binding was influenced to some degree (Fig. 5B
). Mutant M4 showed altered Sp1 binding, while its binding of Egr-1 was not affected; however, mutant M2 did not display residual Sp1 binding (as M4 did), and therefore, the M2 sequence was chosen for further experiments. Since mutations M5 and M6 both led to interruption of Sp1 and Egr-1 binding, we concentrated in our further functional analysis only on one of these two mutations, namely mutant M6. Figure 5C
shows a direct comparison of the effect of mutants M2, M6, and M7 on the binding of nuclear proteins prepared after stimulation with gastrin or PMA: compared with the wild-type CgA B probe (Fig. 5C
, lanes 13), binding of Sp1 proteins to the CgA -92/-73 mutant M2 was abrogated, whereas binding of Egr-1 to this mutant was not altered (compare Fig. 5C
, lanes 13 vs. lanes 46). When mutant oligo M7 was used as a probe, no binding of Egr-1 could be detected, whereas this mutation did not alter Sp1 binding substantially (Fig. 5C
, lanes 1012). Although mutations M2 and M7 resulted in selective disruption of either Sp1 or Egr-1 binding, conversion of the overlapping Sp1/Egr-1 region from 5'-GGGG-3' to 5'-TCGA-3' resulted in complete loss of both Sp1 and Egr-1 binding (Fig. 5C
, lanes 79). To confirm the studies in which mutant oligos were used as radiolabeled probes, we performed additional EMSAs in which the -92/-73 sequence was used as a probe and competition experiments were carried out using unlabeled oligonucleotides representing mutant M2, M6, and M7 (Fig. 5D
). Additionally, on the same gel Sp1 and Egr-1 were identified by competition experiments employing Sp1 or Egr-1 consensus sequences as competitors (Fig. 5D
). These experiments revealed that CgA -92/-73 mutant M2 is capable of competing away the Egr-1 complex but has no influence on the binding of Sp1 (lane 2), indicating that this mutant has lost its ability to bind Sp1 whereas it is still able to bind Egr-1. These data are in agreement with the findings shown in Fig. 5
, B (lane 4) and C (lanes 46), where the mutant sequence has been used as a probe. Similarly, competition experiments with an excess of CgA -92/-73 mutant M6 oligo confirmed the findings obtained with the radiolabeled mutant oligo: the M6 mutant has lost its ability to bind Sp1 and Egr-1 and therefore is not able to compete away Egr-1 or Sp1 from the CgA -92/-73 sequence (Fig. 5D
, lane 3). Regarding CgA -92/-73 mutant M7, we found that this mutant was not able to influence Egr-1 binding to the CgA -92/-73 probe, whereas the binding of Sp1 was largely reduced (Fig. 5D
, lane 4), suggesting that the M7 mutant is not able to bind Egr-1, but has retained most of its ability to bind Sp1. However, the fact that application of the CgA -92/-73 mutant M7 did not result in complete interruption of Sp1 binding to the CgA -92/-73 element indicates that this mutation may also alter the binding of Sp1 to some extent. However, this feature was infrequently observed when the CgA M7 sequence was used as a probe in EMSAs (Fig. 5
, B and C). The observation that the M7 mutant did not result in significant reduction of basal CgA promoter activity in transfection assays (Fig. 6
, A and B), whereas the Sp1-specific mutant M2, which abolishes Sp1 binding to the CgA -92/-73 element, showed a robust reduction of its basal transcriptional activity, indicates that the alteration of Sp1 binding caused by the M7 mutation has no relevant functional consequences on Sp1-regulated basal expression.

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Figure 5. Selective Mutation of the CgA -92/-73 Region Abrogates the Binding of Either Sp1 or Egr-1 or Both Transcription Factors
To determine the region(s) responsible for binding of Sp1 and Egr-1, different mutations were introduced into the CgA -92/-73 region (A). Double-stranded, synthetic oligonucleotides representing these mutants were end labeled with [ -32P]dCTP and used as probes in EMSA studies with nuclear extracts prepared from AGS-B cells after 60-min exposure to gastrin (10 nM) or PMA (10 nM). Panel B shows a comparison of the binding pattern of AGS-B nuclear extracts to the different mutants used in the study. Panel C displays a direct comparison of the mutants M2 (lanes 46), M6 (lanes 79), and M7 (lanes 1012) after gastrin and PMA stimulation. As a control, stimulated and unstimulated AGS-B extracts were used for EMSA analysis using the wild-type CgA B probe (lanes 13). All other mutations that were introduced into the -92/-73 sequence had no substantial effect on the binding of AGS-B nuclear extracts (data not shown). In panel D, nonradiolabeled oligonucleotides representing the CgA -92/-73 mutants M2, M6, or M7 were used in competition experiments together with the radiolabeled wild-type CgA B probe (lanes 14). In addition, competition studies using unlabeled oligonucleotides representing Egr-1 or Sp1 consensus sequences were performed (lanes 5 and 6). The data shown represent typical results of a series of three experiments.
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Figure 6. Loss of Egr-1, Sp1, and/or CREB Binding Sites Differentially Influence Basal and Gastrin-Dependent CgA Promoter Activity
After analysis of CgA Egr-1/Sp1 mutants in EMSAs, corresponding sequences were subcloned into the enhancerless heterologous reporter vector pT81, in which the firefly luciferase gene is driven by the enhancerless TK promoter, and used in transient transfection studies. CgA Egr-1/Sp1 mutations were analyzed either in the context of the complete CgA Gas-RE sequence [CgA -92/-62 (panel A)], or the upstream CgA -92/-73 fragment (panel B). Gastrin (10 nM) or PMA (10 nM) stimulation was performed for 6 h, and transfected AGS-B cells were subsequently analyzed for luciferase activity. Results for reporter gene activity are expressed as relative light units and represent the mean ± SD of four separate experiments. C, To investigate the individual contribution of CREB, Sp1, or Egr-1 to gastrin-dependent CgA transactivation, appropriate expression constructs (2 µg/well) encoding dominant-negative mutants of CREB or Sp1 or the Egr-1 inhibitor NAB1 were transiently transfected along with 500 ng/well of the CgA-258-luciferase (CgA-258-luc) reporter construct. Cells were stimulated with 10-7 M gastrin or left untreated, harvested after 8 h, and assayed for luciferase activity. Data were calculated as percentage of control transfections in which the corresponding empty vectors were used and represent the mean ± SEM of three independent transfection experiments. Statistical analysis was performed by using Students t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
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Loss of Sp1, Egr-1, or CREB Binding Differentially Influences Basal and Gastrin-Dependent CgA Promoter Activity
Because mutations M2, M6, and M7 selectively abrogated interaction of either Egr-1 or Sp1 or binding of both transcription factors, oligonucleotides containing wild-type and mutant sequences were subcloned into the enhancerless luciferase reporter vector pT81 and used in transfection studies. In this vector, the firefly luciferase reporter gene is under control of the enhancerless herpes simplex thymidine kinase (TK) promoter and therefore represents a suitable heterologous reporter system with which to investigate the enhancer properties of subcloned sequences (14). To investigate manipulations in the presence or absence of the proximal CRE element, mutations were investigated in context of the CgA sequence -92/-62 (containing the CRE) or -92/-73 (lacking the CRE). We found that mutation M2 (which abrogated Sp1 binding in EMSA studies without altering Egr-1 binding) in the context of a complete CgA Gas-RE sequence (-92/-62) reduced basal promoter activity by approximately 80%, whereas gastrin-dependent transactivation was reduced by 5060% (Fig. 6A
). In contrast, the Egr-1-targeting mutant M7 (which abolishes Egr-1 binding to the CgA Gas-RE) had no significant effect on basal promoter activity, but diminished gastrin-dependent CgA transactivation in the context of the -92/-62 sequence to a similar degree as the M2 mutant did (5060% reduction, Fig. 6A
). When the Sp1-targeting M2 mutation was studied in the absence of an intact CRE site using -92/-73-based constructs, basal promoter activity was reduced to background levels, whereas gastrin responsiveness of the construct was abolished (Fig. 6B
). Similar to the observations made with the -92/-62 element, in the -92/-73 context Egr-1 mutant M7 had no significant effect on basal promoter activity (Fig. 6B
). In contrast, regarding gastrin-dependent CgA transactivation, the Egr-1-targeting mutation displayed effects similar to those of the Sp1-targeting mutation M2 [reduction to background levels (Fig. 6B
)]. Simultaneous mutation of Sp1 and Egr-1 binding sites in mutant M6 (which abrogated binding of both, Sp1 and Egr-1, in EMSA studies) completely inhibited basal and stimulated CgA activity in the -92/-73 context (Fig. 6B
). This finding contrasts the results obtained with the M6 mutation in the -92/-62 context (which comprises an intact CRE site at -71/-64), where basal activity was moderately reduced (by 3040%) and gastrin-stimulated CgA activity was diminished by 5060% (compare Fig. 6
, A vs. B). When the same mutant constructs were used under conditions of PMA stimulation, we obtained results that were essentially superimposable to the results obtained with gastrin stimulation (data not shown). To obtain additional insights into the functional importance of Sp1, CREB, and Egr-1 for gastrin-dependent regulation of the CgA gene promoter, we performed transient cotransfection studies using the CgA-258-luc promoter construct (14, 17) or the pT81-based construct CgA -92/-62-luc along with overexpression constructs encoding dominant-negative mutants of CREB (A-CREB) (31) or Sp1 (32) as well as a construct encoding the corepressor nuclear growth factor I-A binding protein 1 (NAB1), which has been characterized as a potent inhibitor of Egr-1-dependent transcription (30). The CgA-258-luc reporter gene construct, which contains 258 nucleotides of the 5'-flanking and 42 nucleotides of the 3'-flanking region of the CgA gene and the firefly luciferase gene, has previously been described (14, 16, 17) and was used to investigate the effects of inhibitory constructs in a more extended promoter CgA context. In CgA -92/-62-luc, the CgA -92/-62 sequence has been inserted into the polylinker of the heterologous reporter gene construct pT81. Expression of A-CREB, dominant-negative Sp1, or NGFI-A binding protein (NAB1) reduced the stimulatory effect of gastrin on CgA-258-luc (Fig. 6C
) and CgA -92/-62-luc (data not shown) in a very similar fashion. Furthermore, inhibitory effects obtained with combinations of inhibitory constructs were not more pronounced than the effects achieved by expression of individual constructs (data not shown). These data confirm a requirement for Sp1, CREB, and Egr-1 for gastrin-dependent CgA transactivation; however, inhibitory effects obtained with these overexpression constructs were smaller than what was found in functional transfection studies with CgA -92/-62 or CgA -92/-73 promoter mutants. Furthermore, these results suggest that functional loss of one or two factor(s) could be compensated for by the unaltered factor(s) and are in agreement with the results obtained with the CgA -92/-62 and CgA -92/-73 constructs and their mutants, showing that the CgA CRE was able to compensate for mutation of Sp1 and/or Egr-1 binding region(s) (Fig. 6A
). Interestingly, whereas mutagenesis experiments indicated a prominent role of the CRE site and its binding factor CREB for gastrin-triggered CgA regulation, dominant-negative inhibition of CREB, even at high concentrations of cotransfected A-CREB plasmid, reduced gastrin- dependent CgA transactivation only by approximately 25% (Fig. 6C
). In contrast, the A-CREB plasmid was able to abrogate the effect of PMA on the cyclooxygenase 2 (cox-2) promoter in AGS-B cells at concentrations identical to those used for cotransfection experiments with the CgA-258 luc reporter gene construct (data not shown). These findings are in agreement with the view that in gastric epithelial cells, the CRE site present in the proximal CRE promoter displays a less dominant role in CgA gene regulation compared with its functional importance in adrenal cells (16, 17). In summary, our data demonstrate that the basal activity of the CgA Gas-RE in AGS-B gastric epithelial cells is controlled through binding of Sp1 and CREB, whereas an interplay of all three factors, Sp1, Egr-1, and CREB, is required for full gastrin- and PMA-dependent transactivation of the CgA gene.
Egr-1, Sp1, and CREB Stimulate the CgA Promoter through a Tripartite Proximal Gastrin Response Element
To explore the functional influence of Egr-1 on the CgA promoter and its interplay with Sp1 and CREB, AGS-B cells were transiently transfected with expression vectors encoding human cDNAs encoding full-length Egr-1 (33), Sp1 (34), and/or CREB (35) along with a reporter construct in which the luciferase reporter gene is controlled by a single copy of the CgA Gas-RE (-92/-62). To explore the transactivating effect of gastrin under conditions of ectopic transcription factor overexpression, transfections were carried out in the presence or absence of 10 nM gastrin or 10 nM PMA for 8 h (Fig. 7A
). Egr-1 overexpression potently transactivated the CgA promoter, and cotransfection of Egr-1 together with an Sp1 overexpression construct resulted in additive effects (Fig. 7A
). As previously shown, the transactivating effect of CREB was strongly enhanced by cotransfection of a construct encoding a constitutively active mutant of the
- subunit of protein kinase A (PKA) (Fig. 7A
; Refs. 14 and 35). Coexpression of all three factors tended to be more effective compared with the expressions of two factors alone. Gastrin stimulation of AGS-B cells transfected with individual expression vectors or combinations of them led to additive transactivating effects on the CgA -92/-62 promoter element (data not shown). To confirm the selectivity of Gas-RE mutations on a functional level, overexpression constructs encoding Egr-1, Sp1, or CREB were cotransfected with reporter constructs containing mutant M7 (Fig. 7B
), mutant M2 (Fig. 7C
), mutant M6 (Fig. 7D
), or the CRE-specific mutation M13 (Fig. 7E
; Ref. 14). In each set of transfections gastrin and PMA stimulations were included. We found that mutation of the Sp1 site (mutant M2) altered Sp1-dependent CgA transactivation, but had virtually no influence on Egr-1 or CREB-dependent activation of CgA Gas-RE (CgA -92/-62) (compare Fig. 7
, A vs. C). Conversely, mutation of the Egr-1 binding region (mutant M7) resulted in diminished responsiveness of the CgA -92/-62 element toward Egr-1 overexpression, but had no influence on the effect of CREB and Sp1 overexpression (Fig. 7
, A vs. B). Simultaneous mutation of both Sp1 and Egr-1 site (mutant M6) abrogated the effect of Egr-1 and Sp1 overexpression, but had no influence on CREB- dependent transactivation (Fig. 7D
). In contrast, mutation of the CRE site (mutant M13, which has no ability to bind CREB, Fig. 2B
) abolished the transactivating effect of CREB, whereas the effectiveness of Sp1 and Egr-1 to transactivate the CgA Gas-RE was not substantially influenced by this modification (Fig. 7E
).

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Figure 7. Functional Dissection of Egr-1, Sp1, and CREB Binding Sites Located within the CgA Gastrin Response Element through Transcription Factor Overexpression Studies
To investigate the influence of mutations targeting the Egr-1, Sp1, or CRE elements in the context of the complete CgA gastrin responsive element, AGS-B cells were transiently transfected with pT81-based reporter constructs containing the wild-type -92/-62 sequence (panel A), mutant M2 (panel C), mutant M7 (panel B), mutant M6 (panel D), or a CRE-specific mutation (panel E) together with expression constructs for Egr-1, Sp1, and/or PKA/CREB. As a control, in each set of transfections gastrin- (10 nM) or PMA- (10 nM) stimulated cells were included. Reporter gene activity is expressed as an increase relative to control transfectants (appropriate empty vectors) and represents the mean ± SEM of four separate experiments.
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The MEK-1/ERK1/2 Pathway Is Critical for Gastrin-Dependent Egr-1 Regulation
Previous studies demonstrated that activation of signaling pathways comprising MEK-1 and ERKs is crucial for gastrin-dependent regulation of the CgA gene by gastrin (Höcker, M., manuscript in preparation). To investigate the importance of this signaling cascade for Egr-1 regulation in AGS-B cells, we analyzed the effect of gastrin on total cellular Egr-1 abundance in the presence or absence of the MEK-1 inhibitor PD98059 by Western blot techniques. In addition, we analyzed the influence of gastrin and PD98059 on ERK1/2 phosphorylation. Our data demonstrate that gastrin exposure stimulates the cellular abundance of Egr-1 in a time-dependent fashion, having a maximum after 1 h. Similarly, PMA stimulation of AGS-B cells potently elevated cellular Egr-1 levels with a maximal effect after 11.5 h (Fig. 8A
, upper panel). The time course of cellular Egr-1 abundance in response to gastrin and PMA was very similar to the kinetics with which both stimuli stimulated nuclear accumulation of Egr-1 (see Fig. 4D
). However, while gastrin was clearly more potent than PMA regarding nuclear Egr-1 accumulation, PMA stimulation led to higher cellular Egr-1 levels compared with gastrin (Fig. 8A
). Use of phospho-specific ERK1/2 antibody revealed that the effects of both stimuli on Egr-1 protein abundance were paralleled by an increase in ERK1/2 phosphorylation (Fig. 8A
, middle panel). Interestingly, the maximal effect of gastrin on ERK1/2 phosphorylation preceded the gastrin-triggered maximum in Egr-1 protein abundance and is therefore in keeping with the concept that activation of this cascade is a prerequisite for gastrin-triggered Egr-1 activation in AGS-B cells. Moreover, application of PD98059 resulted in complete inhibition of gastrin-stimulated ERK1/2 phosphorylation and also abolished the stimulatory effect of gastrin on cellular Egr-1 levels (Fig. 8A
, middle panel: compare lane 2 vs. lane 5). To explore the importance of PKC- and MEK1-dependent signal transduction pathways as well as the role of de novo protein synthesis for gastrin- and PMA-stimulated binding of Egr-1 to the CgA Gas-RE, the influence of the PKC inhibitor H7, the specific MEK1 inhibitor PD98059, and the translation blocker cycloheximide on binding of gastrin- and PMA-stimulated nuclear proteins to the radiolabeled -92/-73 sequence was investigated in EMSAs. We found that PKC inhibition as well as interruption of MEK1-dependent signaling cascades almost completely inhibited gastrin- and PMA-dependent formation of the Egr-1 complex. (Fig. 8B
). Similarly, inhibition of de novo protein synthesis achieved through treatment of AGS-B cells with cycloheximide abrogated formation of the Egr-1 complex (Fig. 8B
, lanes 2 and 8). To explore the functional relevance of the findings in Western blot and EMSA studies, PKC inhibitor H7 and MEK1 inhibitor PD98059 were used in transactivation experiments using the pT81-based construct CgA -92/-73-luc. Both inhibitors elicited a pronounced inhibitory effect on gastrin and PMA-dependent transactivation of the CgA -92/-73 element, indicating that PKC-/MEK1-dependent signaling pathways are required for full transactivation of the CgA promoter in response to PMA and gastrin (Fig. 8C
). To exclude the possibility that the inhibitory action of PD98059 on gastrin-stimulated CgA -92/-73 activity was due to affection of c-jun amino-terminal kinase (JNK)-related pathways, we explored the relevance of JNK cascades to gastrin-dependent CgA promoter activation by cotransfecting a dominant-negative MKK4 mutant (DN-MKK4) (36) along with the CgA -92/-73 reporter construct. Expression of DN-MKK4 had no influence on gastrin-/or PMA-triggered CgA -92/-73 activity but potently inhibited the effect of PMA on an activator protein 1-driven luciferase reporter gene construct (AP-1-luc) (Fig. 8C
). Therefore, it can be concluded that JNK-related pathways appear not to be involved in regulation of gastrin/PMA-dependent activation of the CgA promoter. Taken together, our data demonstrate that gastrin and PMA are capable of stimulating Egr-1 protein abundance in AGS-B cells and strongly indicate that the effect of gastrin on Egr-1 expression as well as Egr-1 binding to the CgA promoter is mediated via activation of MEK1/ERK1/2 signaling pathways. Similarly, transfection assays revealed that the MEK1/ERK1/2 cascade is a critical signaling pathway mediating the transactivating effects of gastrin on the CgA -92/-73 element.

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Figure 8. The MEK1/ERK1/2 Pathway Is Critical for Gastrin-Dependent Egr-1 Regulation in Gastric AGS-B Cells
A, To elucidate the effect of gastrin and PMA on Egr-1 accumulation and to determine the role of the MEK1/ERK1/2 cascade in this context, AGS-B cells were stimulated with 10-7 M gastrin or 10-8 M PMA for the indicated time periods in the presence or absence of the MEK1 inhibitor PD98059. After SDS-PAGE, total cellular proteins were blotted onto nylon membranes and Egr-1 abundance, as well as ERK1/2 phosphorylation, was detected using appropriate antibodies in Western blot assays (upper panels as indicated). As a loading control, the blot was stripped and reprobed with non-phospho-specific anti-ERK1/2 antibody (lower panel). Experiments were performed in triplicate and data shown represent a typical result. B, To explore the importance of PKC/MEK1-dependent signal transduction pathways and de novo protein synthesis for gastrin-/PMA-stimulated binding of Egr-1 to the CgA promoter, the influence of the PKC inhibitor H7 (50 nM), the specific MEK1-inhibitor PD98059 (50 µM), and the translation blocker cycloheximide (25 µg/ml) was investigated. Preincubation with these agents was performed for 1 h, followed by stimulation of AGS-B cells with gastrin (10 nM) or PMA (10 nM) for 60 min. The probe used for these studies was a double-stranded, [ -32P]dCTP end-labeled oligonucleotide representing the CgA -92/-73 bp (CgA B probe) sequence. Positions of Sp1- and Egr-1-containing complexes are indicated by arrows. Data shown represent typical results obtained in three independent experiments. C, To explore the functional relevance of the findings made in Western blot and EMSA studies (A and B), PKC inhibitor H7 as well as MEK1 inhibitor PD98059 were applied in transactivation experiments with the pT81-based construct CgA -92/-73 Luc. Before stimulation with gastrin or PMA, pretreatment of AGS-B cells with the compounds was performed for 1 h. Reporter gene activity is expressed as increase relative to control transfectants and represents the mean ± SEM of four separate experiments.
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DISCUSSION
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In a previous study we demonstrated that in gastric epithelial cells a proximal promoter region spanning CgA -92/-62 functions as a gastrin-responsive element (Gas-RE) of the CgA gene (14). Furthermore, we found that binding of the transcription factors Sp1 and CREB to this element is crucial for regulation of basal and gastrin-dependent CgA promoter activity (14). To extend our initial studies we performed a detailed functional analysis of the CgA Gas-RE region. Our current investigation revealed that in addition to Sp1 and CREB, the transcription factor Egr-1 binds to the overlapping Sp1/Egr-1 element at CgA -88/-77 and critically participates in agonist-regulated transactivation of the CgA promoter. Furthermore, our data show that an interplay of Egr-1, Sp1, and CREB is required for full gastrin/PMA responsiveness of the CgA gene, whereas regulation of basal CgA promoter activity in gastric epithelial cells requires Sp1 and CREB, but not Egr-1.
Functional interaction between Egr-1 and Sp1 transcription factors has been described for a number of human gene promoters and, depending on the genetic and/or cellular context, binding of Egr-1 can result in either stimulation or inhibition of transcription (23, 24, 25, 26, 27, 28, 29). The cis-regulatory sequence required for Egr-1/Sp1 binding is usually represented by GC-rich promoter regions, which comprise overlapping Egr-1 and Sp1 consensus motifs (27). In general, Egr-1 does not bind to Sp1 consensus sites and conversly, Sp1 is not able to compete Egr-1 out from its recognition motifs, but the presence of overlapping Egr-1/Sp1 sites allows binding and functional interaction of both factors (23). For example, activation of the human PDGF (platelet-derived growth factor)-
chain gene by PMA or mechanical stress depends on enhanced binding of Egr-1 to an overlapping Sp1/Egr-1 binding site present in the proximal PDGF-
promoter (26, 27). Upon stimulation, Egr-1 displaces constitutively bound Sp1 and stimulates the transcriptional activity of the PDGF-
promoter (26, 27). In contrast to this stimulatory effect of Egr-1, enhanced binding of Egr-1 at the malic enzyme (ME) gene promoter triggered by insulin leads to displacement of Sp1 from a proximal Sp1/Egr-1 element and results in inhibition of promoter activity (25). Similarly, the transcriptional activity of the P-glycoprotein1/multidrug resistance gene 1 b (Pgp2/mdr1b) promoter is also suppressed by Egr-1, which competes with Sp1 for binding to an overlapping Sp1/Egr-1 site (29).
Our current observations show that an interplay of Sp1 and Egr-1 at the proximal overlapping Sp1/Egr-1 element spanning -88 to -77 is crucial for gastrin- and PMA-dependent regulation of the CgA promoter. However, our results differ significantly in several aspects from previous studies showing an interaction of Egr-1 and Sp1 at target promoters. At the PDGF-
promoter (26, 27), the human copper-zinc superoxide dismutase (SOD1) gene promoter (34), or the cyclin D1 promoter (24), binding of Egr-1 stimulates transcription, whereas Sp1 had no impact on agonist-dependent transactivation and/or undefined regulatory function(s) regarding basal promoter activity. In contrast, our study clearly demonstrates that at the CgA gene promoter both transcription factors, Sp1 and Egr-1, have stimulatory effects and that their binding is required for full gastrin- and PMA-triggered transactivation. This conclusion is supported by the finding that selective interruption of either Sp1 or Egr-1 binding by introduction of mutations into the overlapping Egr-1/Sp1 site was accompanied by inhibition of CgA promoter activity. Furthermore, overexpression of either Egr-1 or Sp1 potently transactivated the CgA promoter, whereas coexpression of both factors led to additive transactivating effects. In addition to establishing the functional importance of Egr-1 and Sp1 for gastrin- and PMA-triggered CgA promoter activation, we found that both factors differentially contribute to regulation of basal CgA promoter activity in gastric epithelial cells. Although selective inhibition of Egr-1 binding reduced gastrin and PMA responsiveness of the CgA promoter by 4050%, independently of the presence of the downstream CRE at -71/-64 (Fig. 6
), this modification had no influence on basal promoter activity. In contrast, selective inhibition of Sp1 binding diminished basal as well as gastrin- and PMA-stimulated promoter activity by approximately 6080% (Fig. 6
). Simultaneous interruption of Sp1 and Egr-1 binding led to a drastic reduction in basal and stimulated CgA promoter activity. Therefore, it can be concluded that binding of Egr-1 is critical for gastrin- and PMA-stimulated CgA transactivation, whereas it has no functional relevance for regulation of basal CgA promoter activity. However, in contrast to the findings for Egr-1, binding of Sp1 to the CgA promoter is indispensable for both basal and agonist-regulated activation of the CgA promoter in gastric epithelial cells (Fig. 9
). Interplay of Sp1 and Egr-1 with high similarity to our findings in context with CgA promoter regulation in gastric epithelial cells has been described for regulation of the tissue factor (TF) gene promoter in HeLa cells: whereas basal TF gene expression requires Sp1 binding to a cluster of three overlapping Sp1/Egr-1 sites in the proximal TF promoter, PMA- and serum- dependent TF gene activation involves both Sp1 and Egr-1 binding to these sites (38).

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Figure 9. Schematic Diagram of the Nuclear Proteins Mediating Gastrin- and PMA-Dependent CgA Transactivation
In gastric epithelial cells, basal CgA gene promoter activity is regulated through Sp1 and CREB binding to the CgA -92/-62 bp element, whereas Egr-1 is not involved. Activation of the CgA promoter in response to gastrin or the phorbol ester PMA requires enhanced binding of all three factors, Egr-1, Sp1, and CREB.
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Displacement of constitutively bound Sp1 protein by Egr-1 has been described as a common mechanism of how both transcription factors interact with overlapping Sp1/Egr-1 sites to influence transcriptional activity of target promoters (21, 22, 23, 24, 25, 26, 27). Our studies provided no clear evidence that such a mechanism contributes to gastrin- and PMA-dependent CgA transactivation. EMSA studies showed that interruption of Sp1 binding to the CgA Gas-RE by either application of a Sp1-specific antibody, use of excess competitor oligonucleotides, or selective mutation of the respective binding element, did not lead to increased binding of Egr-1 to the CgA promoter, a finding that should be expected if the identical sequence would be recognized by both factors. Similarly, interruption of Egr-1 binding to the CgA Gas-RE had no influence on the binding pattern of Sp1. In concert with the concept of Sp1 and Egr-1 binding to individual parts of the overlapping Sp1/Egr-1 site at -88/-77 of the CgA promoter, we were able to define mutations within the Sp1/Egr-1 element that selectively interrupted binding of either Egr-1 or Sp1 without substantially altering the binding pattern of the nontargeted factor. This view was further substantiated by the observation that coexpression of Sp1 and Egr-1 was clearly more effective to transactivate the CgA-Gas-RE compared with individual overexpression of the transcription factors, whereas inhibitory effects were not detected when Egr-1 and Sp1 were coexpressed. Therefore, interaction of Sp1 and Egr-1 with the overlapping GC-rich element in the proximal CgA promoter appears to occur through noncompetitive binding to distinct areas of this element, allowing both factors to exert their stimulatory effects in response to gastrin and PMA treatment.
In addition to demonstrating the crucial functional importance of Egr-1 and Sp1 binding to the CgA promoter, our results also show that in gastric epithelial cells both factors show an interplay with the leucine zipper transcription factor CREB, which binds to an CgA CRE element at -71/-64. The more complex regulation of the CgA promoter in gastric epithelial cells involving Egr-1, Sp1, and CREB is clearly in contrast to findings made in neuronal and adrenal cells, where basal and agonist-dependent transcriptional activation of the CgA gene is almost entirely attributable to binding of CREB to the proximal CRE site (16, 17). Whereas interaction of Egr-1 and Sp1 at overlapping Sp1/Egr-1 elements has been described for several gene promoters (23, 24, 25, 26, 27, 28, 29), a functional interplay of the zinc finger proteins Sp1 and Egr-1 with the leucine zipper factor CREB has not been reported so far, although some human genes contain binding sites for all three nuclear proteins (39, 40). With regard to gastrin-dependent gene regulation, several transcription factors have been identified as nuclear mediators of pathways activated by the heptahelical CCK-B/gastrin receptor. Gastrin-dependent activation of the vesicular monoamine transporter 2 promoter in gastric epithelial cells is mediated via activation of CREB bound to a CRE site at -33/-26 of the vesicular monoamine transporter 2 promoter and an as yet uncharacterized nuclear factor, which binds to an overlapping AP2/Sp1 site at -61 to -48 (38, 39). At the human histidine decarboxylase (HDC) promoter, which is also potently up-regulated by gastrin in gastric epithelial cells, two novel and so far uncharacterized transcription factors, termed GAS-REBP 1 and 2 (gastrin response element binding protein 1 and 2), respectively, were identified to mediate the transactivating effects of gastrin (43). Although these studies clearly added to our understanding of the molecular mechanisms underlying gastrin-dependent gene regulation in gastric cells, none of these investigations uncovered participation of Egr-1 in gastrin-dependent regulation of target genes. Therefore, our report for the first time provides evidence that Egr-1 should be added to the list of gastrin-regulated transcription factors.
In a variety of cells, agonist-induced activation of the immediate-early gene product Egr-1 usually follows a delayed and transient pattern, which frequently depends on de novo protein synthesis (25, 26, 27, 37). Similarly, our studies revealed that gastrin or PMA treatment of AGS-B gastric epithelial cells leads to a transient increase in Egr-1 binding to the CgA Gas-RE, which becomes evident after 40 min and disappears after having a peak at 1 h (Figs. 3
and 4
). In agreement with these data, gastrin treatment also stimulated cellular as well as nuclear accumulation of Egr-1 with maximal effects after 1 h. Probing of underlying signaling pathways suggests that the gastrin effect on Egr-1 is mediated via activation of PKC/MEK1/ERK1/2 pathways. Similarly, our previous signaling studies, analyzing the pathways through which gastrin controls Sp1 and CREB activity in AGS-B cells, demonstrated that a signaling cascade comprising PKCs
Raf-1
MEK1
ERK is crucial for regulation of both factors, and that gastrin responsiveness of Sp1 and CREB depends, to a large extent, on direct phosphorylation of both transcription factors (Ref. 14 and Höcker, M., manuscript in preparation). However, compared with the delayed pattern of Egr-1 stimulation, activation of Sp1 and CREB by gastrin and PMA in AGS-B cells occurs much faster, showing a maximum after 10 min of agonist exposure (14). These results suggest that although Egr-1, Sp1, and CREB are all linked to the same signaling cascade, gastrin- and PMA-dependent Egr-1 activation most likely involves a different mechanism(s) requiring de novo protein synthesis. However, studies employing a Gal4Egr-1/Gal4-luc cotransfection system indicate that posttranslational modification(s) of Egr-1, possibly involving phosphorylation of the transcription factor, may contribute to the overall effect of gastrin on Egr-1 (Fig. 4E
). Based on these observations, a model for gastrin- and PMA-dependent transactivation of the CgA gene in gastric epithelial cells can be postulated according to which immediate transcriptional activation of the CgA gene in gastric epithelial cells is achieved by elevation/phosphorylation of CREB and Sp1 proteins, whereas more delayed induction of the CgA gene involves activation of Egr-1. A mechanism that is very similar to these observations has been described for PMA-dependent tissue factor (TF) gene activation in HeLa cells: whereas Egr-1 displayed a delayed pattern of activation in response to PMA, Sp1 activation appears to be responsible for immediate activation of the TF gene in response to PKC activation (38).
Taken together our results demonstrate that enhanced binding of Egr-1 to the overlapping, GC-rich Sp1/Egr-1 site located within the CgA Gas-RE substantially contributes to gastrin- and PMA-dependent transactivation of the CgA gene in gastric epithelial cells, whereas it is not involved in regulation of basal CgA promoter activity. In contrast, binding of Sp1 and CREB to the CgA Gas-RE is crucial for both agonist-stimulated and basal CgA promoter activity (Fig. 9
). Furthermore, we show that functional interplay of Sp1, CREB, and Egr-1 is indispensable for full gastrin and PMA responsiveness of the CgA promoter. Finally, our results demonstrate, for the first time, that the transcription factor Egr-1 can act as a nuclear effector of pathways activated by the G protein-coupled CCK-B/gastrin receptor. Further analysis of CCK-B/gastrin receptor-dependent regulation of Egr-1 can contribute to a better understanding of the molecular mechanisms underlying the transactivating effects of gastrin on its target genes in gastric epithelial cells.
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MATERIALS AND METHODS
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Reagents
Consensus oligonucleotides representing consensus binding sites for CREB, Egr-1, and Sp1 as well as specific antibodies directed against these transcription factors were purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA). Antibodies recognizing phosphorylated or unphosphorylated ERK1/2 were obtained from New England Biolabs, Inc.(Beverly, MA). PMA, human gastrin-17, and the PKC inhibitor H7 were from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). The MEK1 inhibitor PD98059 and cycloheximide were obtained from Calbiochem(San Diego, CA).
Cell Culture and Transfection Studies
AGS-B gastric cancer cells were derived from plain AGS cells (ATCC, Manassas, VA) through stable transfection of the expression vector CCKB-pcDNA 3.1/Neo, containing the full-length coding region of the human CCK-B/gastrin receptor and the neomycin gene, and have been previously described (30, 44, 45). AGS-B cells express approximately 20,800 receptor molecules per cell, and receptors display characteristics that are typical for the human CCK-B/gastrin receptor (e.g. IC50 for 125I-CCK8 displacement by gastrin: 0.6 nM) (44). Furthermore, analysis of signaling pathways activated by AGS-B CCK-B/gastrin receptors in response to gastrin revealed appropriate coupling to Ca2+/IP3 and Raf-1/MEK1/ERK1/2 cascades (Refs. 44 and 45 and Höcker, M., manuscript in preparation). AGS-B cells were grown in DMEM 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 cells were performed using the calcium-phosphate precipitation technique (DNA Transfection Kit, 5 Prime-3 Prime, Boulder, CO). Cells were plated at a density of 1 x 106 cells/35-mm well and transfected the next day with 0.51 µg of plasmid DNA per well. To correct for transfection efficiency, cotransfection with 50 ng of the renilla luciferase expression construct pRL-TK (Promega Corp., Mannheim, Germany) were performed. For stimulation experiments, cells were incubated in the presence of 10-8 M PMA or 10-7 to 10-8 M gastrin for 8 h, harvested, and assayed for firefly luciferase and renilla luciferase activities in a monolight Luminometer (EG Berthold, Bad Wildbach, Germany) using a dual luciferase reporter assay (Promega Corp.) according to the manufacturers instructions. In transfection studies, determinations were performed in triplicate and results calculated as mean ± SEM. Values for CgA-luc activity were expressed as fold increase in luciferase activity compared with untreated controls.
DNA Constructs and Reporter Plasmids
CgA 5'-deletion constructs and mutant promoter constructs, which are all based on the promoterless luciferase reporter gene vector pXP1, have been previously reported (14, 17). To study the characteristics of potential CgA regulatory elements in a heterologous promoter system, the region CgA -92 to -62 was subcloned adjacent to the enhancerless herpes simplex TK viral promoter into the plasmid pTK-luc (14). In this vector the luciferase reporter gene is under control of the enhancerless herpes simplex TK promoter, and therefore represents a suitable heterologous reporter system to investigate the enhancer properties of subcloned sequences (14). In addition, a series of mutant constructs was generated by subcloning oligonucleotides representing mutated CgA -92/-62 sequences (Fig. 5
) into pT81 polylinker at BamHI (5') and XhoI (3') restriction sites. Before use, all constructs were sequence confirmed by dideoxy sequencing. Furthermore, reporter gene constructs were used in which the Egr-1/Sp1 binding region or the CRE element were deleted in the context of 100 bp of 5'-flanking DNA of the CgA gene employing site-directed mutagenesis. These constructs have been described and used in previous studies (14, 17). To study the effect of transcription factor overexpression on CgA promoter activity, AGS-B cells were transfected with the CgA -92/-62-luc construct along with 2 µg of plasmids containing cDNAs encoding human CREB, human Egr-1, or human Sp1, respectively. To enhance the transactivating effects of CREB, the CREB expression construct was cotransfected with an expression construct encoding a constitutively active mutant of the PKA subunit
, which represents a potent upstream kinase of CREB (14, 35). For interruption of Sp1 effects, we employed the construct pEBGSp1, which has been previously shown to act as dominant-negative inhibitor of Sp1 (32). This plasmid encodes a fusion protein consisting of Schistosoma japonaicum glutathione S-transferase and amino acids 592758 of human Sp1. The plasmid pCMVNAB1 has been used to functionally impair the effects of Egr-1. This construct encodes the corepressor NAB1, which has been shown to act as a specific inhibitor of Egr-1 effects (30). The construct pCMV500-A-CREB encodes a dominant-negative mutant of CREB and has previously been described (31). In experiments with dominant-negative constructs, the plasmid CgA-258-luc, which contains 258 nucleotides of the 5'-flanking and 42 bp of the 3'-flanking region of the CgA gene and the firefly luciferase gene in the reporter gene vector pXP1, and the TK-luciferase construct CgA -92/-62-luc were used. To study the effects of gastrin on the transcription-stimulating potency of Egr-1, cotransfections using 1 µg/well of the reporter plasmid Gal4-luc, in which the luciferase reporter gene is driven by a multimer of the Gal4 yeast transcription factor binding element (30), along with 2 µg/well of the transactivator construct pGal4Egr13, were performed. pGal4Egr13 encodes a fusion protein comprising the DNA-binding domain of the yeast transcription factor Gal4 and the Egr-1 transactivation domain (amino acids 16335) (30). After transfection, cells were incubated in the presence of 10-7 M gastrin for 8 h, harvested, and assayed for luciferase activities as outlined above. The reporter gene plasmid AP1-luc, which served as positive control for the inhibitory effects of a dominant negative MKK4 construct, has been described previously (46).
EMSAs
Crude nuclear extracts were prepared from AGS-B cells and analyzed in EMSAs as previously described (14). In brief, double-stranded, synthetic oligonucleotides were radiolabeled with [
-32P]dCTP and incubated with 5 µg of nuclear extracts in a final volume of 20 µl 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 for 20 min at room temperature. To ensure optimal Sp1 binding, 10 µM ZnSO4 was also added to the incubation mixture. DNA-protein complexes were separated on a 6% nondenaturing polyacrylamide gel containing 0.25x Tris- borate EDTA at a constant current of 15 mA at 4 C. Gels were dried and exposed to X-AR film (Eastman Kodak Co., Rochester, NY) at room temperature. For competition experiments nuclear extracts were incubated with 100-fold excess of double-stranded competitor oligonucleotides at room temperature for 10 min before addition of radiolabeled probes. In supershift experiments, nuclear extracts were incubated with 1 µl of anti-Sp1, anti-CREB, or anti-Egr-1 antibodies for 10 min at room temperature, followed by an incubation period of 20 min at 4 C before addition of radiolabeled probes. For experiments exploring the signaling pathways involved in Egr-1 regulation, serum-starved AGS-B cells were incubated with specific MEK1 inhibitor PD98059 (50 µM) or PKC inhibitor H7 (50 nM) before stimulation with gastrin or PMA. Inhibition of cellular protein synthesis was achieved by preincubation of AGS cells with the translational inhibitor cycloheximide (25 µg/ml) for 1 h followed by stimulation with gastrin or PMA for 60 min.
Immunoblotting
Whole-cell lysates were prepared in 20 mM Tris (pH 7.9), 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 10 mM K2HPO4, 1 mM Na3VO4, 10 mM NaF, 1.25% Nonidet P-40, and 10% glycerol. To study nuclear accumulation of Egr-1, AGS-B nuclear extracts were prepared using the identical protocol after separation of nuclei according to the procedure outlined in the description of EMSA procedures. Equal amounts of protein extracts were separated by SDS-PAGE and blotted on membranes, which were subsequently checked for equal protein loading by transient staining with Ponceau solution. Western blot analysis of nuclear extracts and whole-cell lysates was performed using Egr-1 antibody as well as phospho-specific and non-phospho-specific antibodies recognizing ERK1/2 MAPKs (whole-cell lysates only). To demonstrate equivalent protein loading, membranes were reprobed with corresponding non-phospho-specific anti-ERK1/2 antibodies.
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ACKNOWLEDGMENTS
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We express our gratitude to the following persons for generously providing plasmid DNA: J. Habener (CREB and PKA expression constructs), G. Suske (Sp1 expression construct), R. Maurer (Egr-1 expression construct), G. Thiel (Gal4-Egr-1, pCMV-NAB1, and pEBG-Sp1), M. Naumann (MKK4 expression construct), and C. Vinson (pCMV500-A-CREB). We are also grateful to Dan OConnor for contributing plasmid CgA-258-luc.
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FOOTNOTES
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This study was supported in part by Deutsche Forschungsgemeinschaft Grant HO1288/6-1 (to M.H.) and NIH Grant RO I DK-48077 (to T.C.W.).
1 R.R. and G.S. contributed equally to this study. 
Abbreviations: CCK, Cholecystokinin; CgA, chromogranin A; CgA Gas-RE, chromogranin A gastrin-responsive element; CRE, cAMP responsive element; CREB, CRE binding protein; ECL cells, enterochromaffin-like cells; Egr, early growth response; Gas-RE, gastrin-responsive element; JNK, c-jun amino-terminal kinase; MEK1, MAPK kinase 1; NAB1, nuclear growth factor I-A binding protein; PDGF, platelet- derived growth factor; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate-13-acetate; Sp1, specificity protein 1; TF, tissue factor; TK, thymidine kinase.
Received for publication October 29, 2001.
Accepted for publication August 15, 2002.
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