The human histidine decarboxylase promoter is regulated by gastrin and phorbol 12-myristate 13-acetate through a downstream cis-acting element.

Transcriptional regulation of the human histidine decarboxylase (HDC) gene by gastrin and the phorbol ester phorbol 12-myristate 13-acetate (PMA) was studied using transient transfection of human HDC promoter-luciferase constructs in a human gastric carcinoma cell line (AGS-B) that expresses the human cholecystokinin-B/gastrin receptor. The transcriptional activity of the human HDC promoter was stimulated 3-4-fold by gastrin and 13-fold by PMA, effects that could be blocked by down-regulation or antagonism of protein kinase C. 5'- and 3'-deletion analysis demonstrated that the sequence responsible for gastrin- and PMA-stimulated transactivation (gastrin response element (GAS-RE)) was located in a region (+2 to +24) downstream of the transcriptional start site (+1) in the human HDC promoter and contained a palindrome (5'-CCCTTTAAATAAAGGG-3'). When ligated upstream of the herpes simplex virus 1 thymidine kinase promoter, a single copy of the GAS-RE was sufficient to confer responsiveness to gastrin and PMA. Electrophoretic mobility shift assays with specific competitors and factor-specific antibody supershifts showed that the labeled GAS-RE bound a novel nuclear factor(s). In addition, both gastrin and PMA increased binding of this factor to the GAS-RE. Hence, the palindromic GAS-RE site is sufficient to explain the gastrin/PMA responsiveness of the human HDC promoter and appears to bind a novel transcription factor.

Transcriptional regulation of the human histidine decarboxylase (HDC) gene by gastrin and the phorbol ester phorbol 12-myristate 13-acetate (PMA) was studied using transient transfection of human HDC promoterluciferase constructs in a human gastric carcinoma cell line (AGS-B) that expresses the human cholecystokinin-B/gastrin receptor. The transcriptional activity of the human HDC promoter was stimulated 3-4-fold by gastrin and 13-fold by PMA, effects that could be blocked by down-regulation or antagonism of protein kinase C. 5and 3-deletion analysis demonstrated that the sequence responsible for gastrin-and PMA-stimulated transactivation (gastrin response element (GAS-RE)) was located in a region (؉2 to ؉24) downstream of the transcriptional start site (؉1) in the human HDC promoter and contained a palindrome (5-CCCTTTAAATAAAGGG-3). When ligated upstream of the herpes simplex virus 1 thymidine kinase promoter, a single copy of the GAS-RE was sufficient to confer responsiveness to gastrin and PMA. Electrophoretic mobility shift assays with specific competitors and factor-specific antibody supershifts showed that the labeled GAS-RE bound a novel nuclear factor(s). In addition, both gastrin and PMA increased binding of this factor to the GAS-RE. Hence, the palindromic GAS-RE site is sufficient to explain the gastrin/ PMA responsiveness of the human HDC promoter and appears to bind a novel transcription factor.
Histamine is a chemical mediator that serves a variety of biologic roles, including allergic responses, inflammation, neurotransmission in the central nervous system, and gastric acid secretion (1). The formation of histamine from its precursor, L-histidine, is catalyzed in a single step by the enzyme histidine decarboxylase (HDC) 1 (2). HDC is expressed in a variety of adult cell types, including mast cells/basophils, skin, platelets, histaminergic neurons in the brain, and enterochromaffin-like cells of the gastric corpus (1). In addition to its role in allergic responses, HDC and histamine have been implicated in the modulation of cell growth and/or differentiation. Kahlson et al. (3) initially showed high levels of HDC activity and histamine formation in the rat during late fetal life. Inhibition of HDC at this time leads to arrest of fetal growth (4), suggesting that HDC is essential for fetal development. In addition, HDC activity is significantly elevated during liver regeneration after partial hepatectomy (5,6), in the tissues of healing skin wounds (5), in experimental tumors (7)(8)(9), and in the repair process after reperfusion injury of the ischemic bowel (10). The activation of HDC during stages of growth and regeneration suggested that HDC gene expression may be regulated through growth factor pathways.
The possible regulation of HDC through a protein kinase C (PKC) pathway was initially raised by studies showing that a single application to mouse skin of the phorbol ester 12-Otetradecanoylphorbol-13-acetate (TPA), a known activator of PKC, could result in a rapid increase in HDC activity (11). HDC mRNA was also shown to be increased in rat basophilic leukemia (RBL-2H3) (12), mouse mastocytoma (P-815) (13), and human basophilic leukemia (KU-812-F) (14) cells after TPA treatment. These latter studies suggested that the TPA response was due to changes in the rate of transcription rather than in HDC mRNA stability, but they did not address the mechanism of transcriptional control.
In the gastrointestinal tract, HDC is present at high levels in the enterochromaffin-like cells of the corpus of the stomach, where HDC activity is closely correlated with the level of the circulating peptide hormone gastrin (15). In the rat stomach, gastrin appears to regulate acid secretion through stimulation of histamine release and HDC enzymatic activity. In most species, including humans, gastrin-induced acid secretion can be completely inhibited by histamine-2 receptor antagonists such as cimetidine and ranitidine, indicating that histamine represents the final common pathway for stimulated acid secretion (16). Gastrin also appears to regulate growth in the gastric corpus. Studies in both rats and humans have shown that conditions that lead to higher levels of gastrin in the circulation are associated with hypertrophy of the gastric mucosa and hyperplasia of enterochromaffin-like cells (17). In addition, long-standing hypergastrinemia in rats can lead to the development of gastric carcinoids. Gastrin has also been shown to play a role in the growth of a number of tumors and tumor cell lines (18), although the mechanism for growth stimulation by gastrin in this setting is unclear.
Studies from several groups, utilizing Northern blot and in situ hybridization analyses, have now clearly shown that hypergastrinemic states are associated with elevated HDC mRNA levels in rat gastric enterochromaffin-like cells (19 -21). Gastrin activates HDC gene expression through binding to the CCK-B/gastrin receptor, a member of the superfamily of G protein-coupled receptors with seven transmembrane domains. The cloned human CCK-B/gastrin receptor has been shown to transduce cellular signals in response to both gastrin-17 and CCK-8, with an increase in free cytosolic calcium and in the level of inositol 1,4,5-triphosphate, suggesting that the CCK-B/gastrin receptor is coupled with a signaling pathway activating phosphoinositol hydrolysis (22,23). The activation of PKCrelated pathways by gastrin has been less well studied, although one report demonstrated induction of PKC translocation and increased DNA synthesis by gastrin (24).
To study transcriptional regulation of HDC by gastrin, we recently developed the AGS-B cell line, a human gastric cancer cell line expressing the recombinant human CCK-B/gastrin receptor (21). Initial studies from our laboratory demonstrated that gastrin stimulates the rat HDC promoter in transiently transfected AGS-B cells through a PKC-related pathway (21). In addition, these studies showed that the rat HDC promoter is strongly activated in AGS-B cells by phorbol 12-myristate 13acetate (PMA), a known activator of PKC. The goal of the present study was to extend these initial observations and to define the cis-regulatory elements of the human HDC promoter critical for responsiveness to gastrin and PMA. Through deletion analysis of the HDC promoter, we have identified a 23nucleotide cis-acting DNA element downstream of the transcriptional start site that is sufficient to mediate gastrin and PMA responsiveness. The response element contains a palindrome and appears to bind a novel transcription factor(s) that can be induced by gastrin and PMA.

EXPERIMENTAL PROCEDURES
Cloning of the Human HDC 5Ј-Flanking Region and Construction of Reporter Genes-A human genomic library (human placenta; CLON-TECH, Palo Alto, CA) was screened with a synthetic 50-mer oligonucleotide derived from the proximal 5Ј-noncoding region of the human HDC cDNA (25,26). Of 500,000 plaques examined, three positive recombinants were identified and plaque-purified. A 6-kilobase BamHI restriction fragment positive on Southern blot analysis was isolated from plaque-purified phage DNA, subcloned into plasmid Bluescript II KS(ϩ) (Stratagene), and sequenced by the chain termination method (Sequenase, U. S. Biochemical Corp.).
A human HDC promoter fragment, containing ϳ1.8 kb of 5Ј-flanking DNA and 126 nucleotides of the noncoding first exon, was amplified by polymerase chain reaction (PCR) using primers containing BamHI restriction sites and ligated into the promoterless pXP2 vector containing the firefly luciferase gene (27). A series of human HDC promoter 5Јdeletion constructs (hHDC-luciferase) containing 125, 73, 59, and 47 nucleotides upstream of the start site as well as 126 nucleotides of the noncoding first exon were made by PCR amplification of segments from the human HDC promoter utilizing the 1800-nucleotide promoter template. The 5Ј-deletion PCR primers were designed with a series of sense primers at different positions of the promoter and an antisense primer from the ϩ126 region of exon I (see Table I). Each pair of primers contained a HindIII site at the 5Ј-end of the sense primer and an XhoI site at the 5Ј-end of the antisense primer. After PCR amplification, the products were digested with HindIII and XhoI, gel-isolated, and ligated into the promoterless luciferase vector pXP2.
The 3Ј-deletion constructs were designed using a sense primer from the Ϫ125 region of the promoter and a series of antisense primers at different position(s) in the noncoding portion of exon I (see Table I). Additional pT81 (27) constructs containing various amounts of the human HDC promoter and noncoding first exon ligated upstream of the herpes simplex virus 1 thymidine kinase promoter were also generated by PCR using primers containing HindIII sites in the sense primers and XhoI sites in the antisense primers or using synthetic complementary oligonucleotides with a BamHI site at each end. All pXP2 and pT81 constructs were checked by restriction digests for the correct length of promoter segments and then confirmed by sequencing.
Cell Culture and Transfection Studies-AGS-B cells were derived from plain AGS cells (American Type Culture Collection) through stable transfection of the expression vector CCK-B-pcDNA I-Neo, containing the full-length coding region of the human CCK-B/gastrin receptor and the neomycin resistance gene, and have been previously described (21). AGS-B cells were grown in Dulbecco's modified Eagle's medium (Bio-Whittaker, Inc., Walkersville, MD) containing 10% bovine calf serum, 100 IU/ml penicillin, and 100 g/ml streptomycin in a humidified atmosphere (5% CO 2 , 95% air). Transient transfections of cultured AGS-B cells were carried out using the calcium phosphate precipitation technique (DNA transfection kit, 5 Prime 3 3 Prime, Inc.). AGS-B cells were plated at a density of 1 ϫ 10 6 cells/35-mm well and transfected the next day with 0.5 g of plasmid/well. Sulfated gastrin-17 and CCK-8 (Peninsula Laboratories, Inc., San Diego, CA) and PMA (BIOMOL Research Laboratories Inc., Plymouth Meeting, PA) were added at the appropriate concentrations 12-14 h after transfection, and incubation was continued. The CCK-B/gastrin receptor antagonist L 365,260 and the CCK-A receptor antagonist L 364,718 were generously provided by Merck. In general, cells were harvested, and luciferase assays were done at 48 h.
Luciferase assays were performed using luciferin, ATP, and coenzyme A (Promega system) with a Monolight Luminometer (Analytical Luminescence Laboratory) as described previously (21). Incubations were performed in triplicates or quadruplicates, and results were calculated as the mean Ϯ S.E. Values for HDC-luciferase activity were expressed as -fold increase in luciferase activity compared with untreated controls. The pT81-luciferase construct, in which the luciferase gene is driven by the enhancerless herpes simplex thymidine kinase promoter, served as an additional control (27). Activities in all transfection experiments represent the mean Ϯ S.E. of at least four to six independent transfections. Activities varied Ͻ10% 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.
Primer Extension Analysis and Anchored Polymerase Chain Reaction-The transcriptional initiation site was mapped using a 32 P-endlabeled (1 ϫ 10 7 cpm/pmol) 26-base oligonucleotide probe (5Ј-CTG-GCTCCTTCTCACAGATGGACACG-3Ј) that was designed from the 5Јnoncoding region of the hHDC cDNA counting from Ϫ13 to Ϫ38 upstream of the translational start site. The primer was annealed to poly(A) ϩ RNA (5 g) isolated from plain AGS-B cells, AGS-B cells transfected with the 1.8-kb hHDC-luciferase construct, and human corpus for 12 h at 30°C in 1 M NaCl, 167 mM Hepes (pH 7.5), and 0.33 mM EDTA (pH 8.0). Transcripts were extended for 2 h at 37°C using avian myeloblastosis virus reverse transcriptase (Perkin-Elmer) as described previously (21,28). The extended product was subjected to electrophoresis on a 7 M urea, 6% polyacrylamide gel, and the sizes of the extended products were determined by comparison with the sequencing ladder derived from the same oligonucleotide used for primer extension.
To confirm the results obtained by primer extension analysis, anchored polymerase chain reaction was carried out as described previously (28). Briefly, first-strand cDNA was synthesized from 1 g of mRNA from different sources as described above using the cDNA Cyclekit (Invitrogen) containing a specific primer (5Ј-CTCTCCCTCTCTC-TCGTAC-3Ј) that is complementary to nucleotides ϩ175 to ϩ156 downstream of the transcriptional start site. After removing the excess primer with a Centricon 100 spin filter (Amicon, Inc.), the 3Ј-end of the first-strand cDNA was tailed in a 20-l reaction mixture containing 1 ϫ tailing buffer, 1 mM dATP, and 15 units of terminal deoxynucleotidyltransferase (Life Technologies, Inc.) for 10 min at 37°C and then heated for 15 min at 65°C. 1 l of the reaction mixture was used to synthesize the second-strand cDNA with 10 pmol of anchored oligo(dT) 16 using 2.5 units of Taq DNA polymerase (Perkin-Elmer) at 72°C for 7 min and then subjected to PCR with a nested primer that was the same as that used for primer extension analysis. PCR was performed in a thermal cycler (Perkin-Elmer) as described previously (21). The anchored PCR products were resolved by electrophoresis on a 2.5% agarose gel with X174 DNA/HinfI size markers. The products were then analyzed by Southern blot analysis with 32 P-end-labeled oligonucleotides (5Ј-GGGC-CCACACTGGCTGCCA-3Ј) representing internal sequences to verify the identity of the PCR products.
Electrophoretic Mobility Shift Assays (EMSAs)-Nuclear extracts from AGS-B cells were prepared by Nonidet P-40 detergent lysis and 0.5 M NaCl extraction as described by Schreiber et al. (29). Nuclear extracts from different cell lines for EMSA tissue distribution studies were a kind gift of Dr. Anil K. Rustgi. Protein concentrations were determined by a colorimetric method (Bio-Rad protein assay). EMSAs were performed by incubating the extracts with 4 fmol of double-stranded oligonucleotide probe (40,000 cpm) end-labeled with [␣-32 P]dCTP (Amersham Corp.) by Klenow DNA polymerase (New England Biolabs Inc.) in a 20-l binding reaction mixture containing 25 mM Hepes (pH 7.9), 100 mM KCl, 5 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 0.1 g of poly(dI-dC), and 10 g of nuclear extracts. The wild-type, double-stranded synthetic probe used in this study was ϩ2 CCCTTTA-AATAAAGGGCCCACAC ϩ24 and was polyacrylamide gel-purified.
Competition experiments were carried out by preincubating the nuclear extracts with a 100-fold excess of unlabeled competitor oligonucleotide prior to addition of the probe. After incubation at room temperature for 10 min, samples were loaded onto a 4% nondenaturing polyacrylamide gel and electrophoresed in 0.5 ϫ Tris borate/EDTA at 10 V/cm. The gels were dried and exposed to Kodak X-AR film for 2-12 h at room temperature. Where appropriate, autoradiographs were analyzed by scanning densitometry.
Nuclear extracts and antibodies were incubated for 10 min at room temperature and then for 30 min at 4°C prior to EMSAs. Anti-c-Fos, anti-c-Jun, and anti-CREB antisera were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-c-Fos antiserum has specificity for c-Fos, FosB, Fra-1, and Fra-2 proteins. The anti-c-Jun antiserum has specificity for c-Jun, JunB, and JunD proteins. The anti-CREB antiserum has specificity for CREB-1, CREM-1, and ATF-1. The SP1 antibody was a generous gift of Anil K. Rustgi.

Creation of Human HDC-Luciferase Constructs and Analysis of the Transcriptional Start
Site-The 5Ј-flanking sequence of the human HDC gene was isolated from a human EMBL-3 T7/SP6 genomic library using a synthetic 50-mer oligonucleotide probe corresponding to the proximal 5Ј-noncoding region of the human HDC cDNA. Three genomic DNA clones were isolated after screening 500,000 separate phage clones. A 6-kb BamHI restriction fragment was subcloned from one of the phage isolates and sequenced. 1258 nucleotides of the 5Ј-flanking sequence and 150 nucleotides of exon I from the human HDC gene are shown in Fig. 1. The sequence of our clone is similar to that previously reported (36), except for three nucleotide differences: C instead of G at Ϫ916, two C nucleotides instead of three at Ϫ820 and Ϫ821, and six C nucleotides instead of five at Ϫ558 to Ϫ563. 1800 base pairs of the human HDC promoter were amplified using polymerase chain reaction and ligated into pXP2, a promoterless vector containing the firefly luciferase gene.
The human HDC-luciferase construct was transiently transfected into a human gastric cancer cell line (AGS-B), and the start site was analyzed from poly(A) ϩ RNA prepared from these transfected AGS-B cells. Primer extension was performed using a 26-mer oligonucleotide complementary to the human HDC noncoding sequence in exon I (5Ј-CTGGCTCCTTCTCA-CAGATGGACACG-3Ј) and poly(A) ϩ RNA from human corpus, transfected AGS-B cells, and untransfected AGS-B cells. Ex-tension products of the same size were detected in poly(A) ϩ RNA from transfected AGS-B cells and human corpus, but not in untransfected AGS-B cells (Fig. 2), indicating that the labeled products were derived from the transfected plasmid and that transcription was being initiated from the authentic human HDC promoter. Among the extension products, the predominant band of 126 nucleotides indicated that the major transcriptional start site was located 139 bases upstream of the cap site.
To confirm the results obtained by primer extension analysis, anchored PCR was carried out using poly(A) ϩ RNA from KU-812-F cells, human corpus, and untransfected AGS-B cells using the strategy outlined in Fig. 3A. PCR products of the expected size (142 bp) were seen in KU-812-F human basophilic leukemia cells and human corpus, but not in untrans- The nucleotide sequence of 1258 bp of upstream DNA, as well as some downstream sequence from exons I and II, derived from the 6-kilobase BamHI restriction fragment of the human HDC gene is shown. The numbering refers to the position of each nucleotide relative to the major start site (ϩ1) mapped by primer extension analysis, which is shown in boldface. The locations of the 26-mer oligonucleotide primer used in the primer extension assay (long arrow) and of the initial antisense oligonucleotide primer (ϩ37 to ϩ18) used in anchored PCR (underlined) are indicated. In addition, the general locations of the primers used for PCR generation of the human HDC promoter 5Ј-and 3Ј-deletion mutants are shown (short arrows). The upstream TATA-like sequence (Ϫ47 to Ϫ58) is boxed.
fected AGS-B cells (Fig. 3B). The identity of the PCR bands was confirmed by Southern blot analysis using an internal oligonucleotide probe (Fig. 3B).
Based on these results, the transcriptional start site was identified as the adenine nucleotide located 20 bases upstream of the 5Ј-end of the previously reported cDNA (25), which differs somewhat from a previous report (36). No definitive TATA box was observed in the Ϫ25 to Ϫ30 region of the human HDC promoter. A TATA-like sequence was present farther upstream in the Ϫ48 to Ϫ54 region (Fig. 1). Additional 5Ј-and 3Ј-deletion studies suggested that this TATA-like region of the gene may be functioning as a promoter element (see below and Fig. 6).
The Human HDC Promoter Is Responsive to Gastrin and Phorbol Esters in AGS-B Cells-To study the effect of gastrin stimulation on the activity of the human HDC promoter, the 1.8-kb hHDC-luciferase construct was employed in transient transfection studies with the AGS-B gastric cancer cell line. The AGS-B cell line has been stably transfected with the human CCK-B/gastrin receptor cDNA and expresses functional receptors as previously assessed by Northern blot analysis, binding studies, and measurements of intracellular calcium (21). AGS-B cells were transiently transfected with the 1.8-kb hHDC-luciferase construct and stimulated with various concentrations of gastrin for 24 h. This dose-response analysis showed that a slight increase in luciferase activity could be detected first at 10 Ϫ9 M, while maximal (3-4-fold) stimulation occurred at 10 Ϫ7 M, with a slight decrease seen at higher concentrations (Fig. 4A). An identical dose-response relationship was observed with CCK-8 (data not shown). Time course studies with continuous gastrin stimulation showed that a 2-fold increase in promoter activity could be observed after 4 h, and maximal elevation was achieved after 12-24 h of gastrin exposure (Fig. 4B). No stimulation by gastrin was observed with an enhancerless heterologous promoter (pT81-luciferase) or with the hHDC-luciferase construct in plain (lacking the CCK-B/gastrin receptor) AGS cells (data not shown). In addition, the effects of gastrin could be blocked by the CCK-B/ gastrin receptor antagonists L365,260 and L364,718, with IC 50 values of 5 ϫ 10 Ϫ9 and 5 ϫ 10 Ϫ8 M, respectively (data not shown). These studies indicate that the transcriptional responses seen were due to specific effects of gastrin and were mediated through the CCK-B/gastrin receptor. The 1.8-kb hHDC-luciferase construct could also be stimulated in AGS-B cells by the phorbol ester PMA. Dose-response studies indicated that a slight increase in hHDC promoter activity could be seen with 10 Ϫ10 M PMA, with a maximal 13-fold increase seen with 10 Ϫ7 M PMA (Fig. 4C). Time course studies using a submaximal dose (10 Ϫ9 M) of PMA also showed that the first increase (2.5-fold) occurred after 2 h, with a maximal increase seen at 8 -10 h, followed by a slight decrease at 10 h and then a gradual plateau (Fig. 4D).
The relationship of the protein kinase C-related pathway to the gastrin and PMA responses was studied further through down-regulation of PKC using prolonged PMA pretreatment or antagonism of PKC by application of a specific serine/threonine protein kinase inhibitor (H-7). Transiently transfected AGS-B cells were stimulated with maximally effective concentrations of PMA or gastrin, with or without pretreatment with 10 Ϫ6 M PMA or treatment with the PKC inhibitor H-7. Gastrin-and PMA-induced transcriptional activation of the human HDC promoter was blocked both by prolonged PMA stimulation (Fig.  5A) and by using the specific PKC antagonist, H-7 (Fig. 5B).
Deletion Analysis of Human HDC Promoter Sequences Required for Response to Gastrin and PMA-To identify the cisacting DNA sequences mediating the transcriptional response to gastrin and PMA, deletion analysis of the human HDC promoter was carried out. Initially, 5Ј-deletion analysis was carried out on the original 1.8-kb hHDC-luciferase construct; constructs containing 125, 73, 59, and 47 bp of 5Ј-flanking DNA plus 126 bp of noncoding first exon were generated using synthetic oligonucleotide primers ( Fig. 1 and Table I) and polymerase chain reaction. Transfection studies showed that using constructs as small as Ϫ59 hHDC-luciferase resulted in essentially full basal luciferase activity, along with intact gastrin and PMA responses (Fig. 6A). However, when 12 additional nucleotides were removed, resulting in the Ϫ47 hHDC-luciferase construct, basal promoter activity was completely destroyed, and thus, gastrin and PMA responses could not be assessed (Fig. 6A).
Deletions from the 3Ј-end of the human HDC promoter were then carried out, starting with the Ϫ125 hHDC-luciferase construct, which contained 126 bp of noncoding first exon. Deletion of nucleotides from ϩ126 to ϩ20 reduced basal luciferase activity by 60%, but did not impair gastrin or PMA responsiveness (Fig. 6B). However, further deletions down to ϩ14 had a minimal effect on basal promoter activity, but essentially destroyed the gastrin response. This deletion (ϩ20 to ϩ14) also reduced the PMA response by 50%; interestingly, the Ϫ125 to ϩ3 promoter fragment was quite active (indicating that the AT-rich sequence between ϩ3 and ϩ20 was not essential for basal promoter activity) and retained a significant response to PMA (Fig. 6B), suggesting that there may be an additional PMA response element(s) in this region of the promoter. Thus, taken together, the 5Ј-and 3Ј-deletion studies indicate that the gastrin response element (GAS-RE) is located between Ϫ58 and ϩ20 within the human HDC 5Ј-flanking region.
The ϩ2 to ϩ24 Region of the Human HDC Gene Is Sufficient to Permit Response to Gastrin and PMA-To further localize the cis-acting DNA element mediating responsiveness to gastrin and PMA and to demonstrate that the element acted as an enhancer, a heterologous (thymidine kinase) promoter system was utilized. Various fragments of human HDC 5Ј-flanking DNA were ligated upstream of the enhancerless thymidine kinase promoter in the luciferase-containing plasmid pT81. These pT81 constructs were then used in transfection studies of AGS-B cells to assess responsiveness to gastrin and PMA stimulation. Initially, a large segment (Ϫ47 to ϩ126) of 5Ј-flanking and untranslated DNA from the human HDC promoter was ligated into pT81. This Ϫ47 to ϩ126 sequence resulted in a 10-fold increase in basal luciferase activity as well as significant gastrin (2-fold) and PMA (8-fold) responses (Fig. 7A). Removal of 90 nucleotides from the 3Ј-end and 22 nucleotides from the 5Ј-end to yield the Ϫ25 to ϩ36 hHDC-pT81 construct resulted in no loss of basal activity or of gastrin and PMA responsiveness. An additional deletion from Ϫ25 to Ϫ6, which removed a putative (GGGCGG) SP1 site, resulted in a significant reduction of basal activity, but again, no change in gastrin or PMA responsiveness. Further deletions in pT81 resulted in the identification of a minimal 23-nucleotide element (ϩ2 to ϩ24) that increased basal thymidine kinase promoter activity 4-fold and was sufficient to confer essentially full gastrin and PMA responses, and this element was labeled the GAS-RE. The GAS-RE sequence includes a palindrome ( ϩ2 CCCTTTAAATA-AAGGG ϩ17 ) at the 5Ј-end. Removal of a single nucleotide (C) from the 5Ј-end of the GAS-RE to yield the ϩ3 to ϩ36 hHDC-pT81-luciferase construct resulted in a significant (40%) decrease in gastrin and PMA responsiveness. Removal of seven nucleotides from the 3Ј-end of the GAS-RE to yield the ϩ2 to ϩ17 hHDC-pT81 construct resulted in the complete loss of gastrin and PMA responsiveness (Fig. 7A). Hence, these studies defined the GAS-RE as the minimal sequence (ϩ2 to ϩ24) required to mediate both gastrin and PMA responses.
A Distinct Nuclear Factor Binds to the ϩ2 to ϩ24 Sequence and Is Induced by Gastrin and Phorbol Ester-To characterize the nuclear factors that bind to the GAS-RE and that may therefore mediate gastrin and PMA transcriptional responses, we performed EMSAs. These assays utilized as probe ␣-32 Plabeled, double-stranded oligonucleotides that represented the sequence (ϩ2 to ϩ24) of the GAS-RE shown in Fig. 7B and nuclear extracts prepared from AGS-B cells as described under "Experimental Procedures". Fig. 8 demonstrates that the GAS-RE probe in EMSAs resulted in a specific complex that could be competed away completely by an excess (100-fold) of unlabeled GAS-RE sequences. A computer search revealed no significant similarity between the sequence and previously published transcription factor-binding sites. However, to examine the possibility that the complex consisted of previously identified nuclear factors, we performed additional competition experiments using a 100-fold molar excess of oligonucleotides corresponding to binding sites for known transcription factors, many of which have been shown to be involved in mediating PMA-induced transcription. Fig. 8 shows that complex formation could not be competed away by an excess (100-fold) of AP1, AP2, AP3, CRE, NF-B, SP1, or SRE sequences. Fig. 9A shows the result of EMSAs using antisera specific for some of these known transcription factors. The antibodies to c-Fos, c-Jun, and CREB have broad specificity toward members of the AP1/ CREB family, as detailed under "Experimental Procedures." The antibody to SP1 was used because of the GC-rich nature of the 3Ј-half of the GAS-RE. Antibodies to c-Fos, c-Jun, CREB, and SP1 had no effect on complex formation (Fig. 9A), while these same antibodies either blocked binding (anti-c-Fos and anti-c-Jun) or generated supershifts (anti-CREB and anti-SP1) when using labeled oligonucleotides probes representing known AP1-, CREB-, and SP1-binding sites (Fig. 9B). We sought to identify qualitative and/or quantitative differences in DNA-protein complex formation in response to gastrin or PMA stimulation. Nuclear extracts were prepared prior to and after addition of gastrin (10 Ϫ8 M) or PMA (10 Ϫ8 M) to AGS-B cells cultivated in serum-free medium (Ultraculture, BioWhittaker, Inc.) for 48 h. A significant increase in complex formation was observed 30 and 120 min after addition of gastrin or PMA compared with that observed with nuclear extracts from unstimulated AGS-B cells (Fig. 10A). Scanning densitometry revealed that gastrin stimulation increased binding to the GAS-RE by ϳ3-fold, while PMA stimulation resulted in an ϳ4-fold increase in binding (Fig. 10B). Finally, to characterize the tissue distribution of the GAS-RE-binding protein, we carried out EMSAs using nuclear extracts made from cancer cell lines derived from a variety of human tissues and cell types. As shown in Fig. 11, the GAS-RE-binding protein appeared to be a widely distributed transcription factor that was present in most cell types. DISCUSSION This study demonstrates that the 5Ј-flanking region of the human HDC gene is active and can be regulated by gastrin and the phorbol ester PMA in AGS-B cells. In a previous study, we showed that the rat HDC promoter is regulated by gastrin primarily through a PKC-related pathway (21). In addition, the effect of gastrin on the rat HDC promoter is additive when combined with cAMP, but not additive when combined with PMA (21). In the present study, which represents the first functional characterization of the human HDC promoter, we provide further evidence that gastrin and PMA work through a single mechanism to regulate HDC transcription. In our AGS-B cell culture system, both gastrin and PMA were able to stimulate the human HDC promoter in a similar fashion, although PMA gave somewhat stronger (Ͼ13-fold) and faster (ϳ2 h) responses compared with gastrin (3-4-fold and ϳ4 h, respectively). Both gastrin and PMA responses by the human HDC promoter could be blocked completely by PMA depletion and H-7 blockade, again implicating a protein kinase C-related pathway. Finally, deletions of the human HDC promoter sequence that affected responsiveness to gastrin tended to affect responses to phorbol ester in a similar fashion. The PMA response appeared slightly more complex since only part of the PMA response could be reconstituted by the GAS-RE. In addition, our 3Ј-deletion studies clearly indicated that additional PMA response element(s) were located farther upstream   (Fig. 6B).
Overall, our deletion analysis demonstrates that the predominant gastrin and PMA response elements map to the same region of the promoter. In the AGS-B cell culture system, we have shown initially that gastrin and PMA responses could be mapped by 5Ј-and 3Ј-deletions to a region containing the basal promoter elements, spanning 58 nucleotides of 5Ј-flanking DNA and 36 nucleotides of the untranslated first exon. The human HDC promoter does not contain a canonical TATA box around the Ϫ30 region, as has previously been noted (36); however, it does contain a proximal SP1 site ( Ϫ25 GGGCGG Ϫ20 ) as well as a TATA-like element farther upstream ( Ϫ55 TTAAT-TAAA Ϫ47 ). Deletion of this upstream TATA-like element resulted in the complete loss of promoter activity, suggesting that it may function in some fashion in the assembly of basal transcription factors. Further mapping of the gastrin and PMA response elements, carried out in the heterologous thymidine kinase promoter system, revealed that the responsible element is located downstream of the transcriptional start site of the human HDC gene. This element was further localized to a  7. Localization of the GAS-RE using a heterologous (thymidine kinase) promoter system. A, a series of human HDC promoter fragments were ligated upstream of the enhancerless thymidine kinase (TK) promoter in the construct pT81 to generate a series of hHDC-pT81 constructs for transfection. Transfected AGS-B cells were treated with gastrin (5 ϫ 10 Ϫ8 M) or PMA (10 Ϫ8 M) for 24 h and harvested, and luciferase activity (LUC) measured. Results represent the mean Ϯ S.E. of five independent experiments. Gastrin (GAS) and PMA responses represent the -fold increases over unstimulated controls. TK LUC represents the activity of pT81. B, shown is the sequence of the GAS-RE. The palindromic region is underlined by arrows.
23-bp region, spanning ϩ2 to ϩ24 and including a palindromic sequence ( ϩ2 CCCTTTAAATAAAGGG ϩ17 ), that we have designated the GAS-RE. Our 3Ј-deletion studies (Fig. 6B) also suggested that the 3Ј-end of the GAS-RE may actually be closer to ϩ20 since deletion to ϩ20 did not markedly impair gastrin or PMA responses.
Two pieces of evidence strongly suggest that the GAS-RE functions as an enhancer-like response element rather than as a basal promoter element. First, our 3Ј-deletion studies showed that the Ϫ125 to ϩ3 hHDC-luciferase construct, which lacks the GAS-RE, still has significant basal activity (Fig. 6B), indicating that the GAS-RE is not essential for promoter activity. Second, placement of the ϩ2 to ϩ24 sequence upstream of a heterologous minimal promoter increased basal activity 4-fold and was also sufficient to confer responsiveness to gastrin and PMA, consistent with an enhancer-like response element. In addition, the GAS-RE appears sufficient to act as a binding site for a novel nuclear factor(s).
The activation of HDC by both gastrin and PMA suggests a general pathway for growth stimulation. The phorbol ester FIG. 8. EMSA competition studies for characterization of nuclear factors that bind to the GAS-RE. The double-stranded GAS-RE sequence (ϩ2 to ϩ24) was end-labeled with [␣-32 P]dCTP and used as a probe in EMSAs. Crude nuclear extracts were prepared from unstimulated AGS-B cells. Competition studies were performed using sequence-specific competitors. 100-Fold excesses of unlabeled, doublestranded oligonucleotides representing the GAS-RE, AP1, AP2, AP3, CRE, NF-B, SP1, and SRE were used in this experiment. The arrow indicates free unbound probe. FIG. 9. EMSA studies using factor-specific antibodies. A, the double-stranded GAS-RE sequence was used as a probe, along with antibodies to c-Fos, c-Jun, CREB, and SP1. No supershift or effect on binding was observed. The arrow indicates free unbound GAS-RE probe. B, shown is the effect of the same factor-specific antibodies on AP1, SP1, and CREB. ␣-32 P-Labeled, double-stranded oligonucleotides representing binding sites for AP1, SP1, and CREB (see "Experimental Procedures" for details) were used as probes in EMSAs with (ϩ) or without (Ϫ) factor-specific antibodies. Supershifts (indicated by arrows) were observed with anti-SP1 and anti-CREB, and decreased binding was observed with anti-c-Fos and anti-c-Jun. PMA, a known activator of PKC, exerts its biologic effects at least in part by inducing expression of a number of genes involved in the growth program, including cellular proto-oncogenes such as c-fos, c-myc, and c-sis (37). The peptide hormone gastrin is known to regulate the growth and proliferation of the corpus of the stomach as well as other portions of the gastrointestinal tract (38). Although several studies have now indicated a link between CCK-B/gastrin receptor activation and PKC-related signaling pathways, the mechanism of growth stimulation and possible downstream targets of gastrin have not been explored. Nevertheless, in view of the increasing and widespread use of powerful acid-suppressing medications leading to hypergastrinemic states, these intracellular pathways may be of some clinical relevance. Thus, the study of gastrinresponsive factors regulating genes such as HDC may contribute to our overall understanding of the mechanism of growth control by gastrin. In addition, the possibility has been raised that HDC activation and histamine production may be more directly linked to tissue regeneration (5, 6, 10) and tumor growth (7)(8)(9).
Previous studies suggested that TPA treatment led to increased HDC transcription in KU-812-F cells (12) and increased binding to the TPA response element (TRE) (TGACTCA) of a nuclear factor (presumably AP1) in mouse mastocytoma P-815 cells (39), but the absence of an obvious TRE site in the human HDC 5Ј-flanking region raised the possibility that the HDC promoter may respond to phorbol esters through other mechanisms (36). The most common phorbol ester response element or TRE is the binding site for AP1, but the TRE is only one of several cis-acting elements that mediate induction responses to TPA and other activators of PKC. Other PKC response elements include the SRE of the c-fos gene, the NF-B-binding site, and the AP2 and AP3 recognition sites (40). However, the CRE has been shown to bind AP1 (41) as well as members of the CREM/CREB family that can also mediate transcriptional responses to PKC activation (42). Although these PKC response elements are often located upstream of the basal promoter in eukaryotic genes, several previous studies have identified TPA response elements that have been located downstream of the transcriptional start site. Lee et al. (43) reported that the response element(s) for TPA inducibility of the human vacuolar H ϩ -ATPase B2 subunit gene could be localized to a 178-bp region within the first exon, a region that contained several potential SP1-and AP2-binding sequences. Roebuck et al. (44) identified several c-Fos-responsive elements, which appeared to be TRE/CRE-like sites, that were located downstream of TAR in the transcribed 5Ј-noncoding 5Ј-leader sequence of human immunodeficiency virus type-1.
However, the sequence of our GAS-RE is clearly distinct from known TPA response elements as demonstrated by the lack of competition using these oligonucleotides in gel shift experiments. In addition, our EMSA antibody experiments indicate that the nuclear protein complex binding to the GAS-RE does not contain members of the AP1/CREB family, such as has been described with the NF-AT complex (45). The dyad symmetry or palindromic nature of our GAS-RE sequence is consistent with its being a transcription factor-binding site. A palindromic sequence often implies a binding site for a transcription factor dimer (e.g. CREB and the steroid hormone receptor family) (46). Finally, increased binding activity in EMSAs to the GAS-RE was induced within 30 min by both gastrin and PMA treatment of AGS-B cells, consistent with a role for this factor(s) in HDC transcription. At present, it is unclear if this increased binding activity represents increased synthesis of this factor(s) or modification of an existing factor(s), possibly by phosphorylation. In addition, further studies will be required to characterize the intracellular signal transduction pathways downstream of the CCK-B/gastrin receptor and PKC that are regulating this factor. In conclusion, we believe that the GAS-RE-binding protein(s) represents a novel transcription factor, which is part of a network whose activity is stimulated after activation of PKC.