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Transcriptional Activation of the Rat Vesicular Monoamine Transporter 2 Promoter in Gastric Epithelial Cells

2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês

10.1074/jbc.m006697200

1083-351X

Fiona L. Watson, Rachel S. Kiernan, Damian G. Deavall, Andrea Varró, R. Dimaline,

Neuroscience and Neuropharmacology Research

Vesicular monoamine transporter 2 is important for the accumulation of monoamine neurotransmitters into synaptic vesicles and histamine transport into secretory vesicles of the enterochromaffin-like cell of the gastric corpus. In this study we have investigated the mechanisms regulating the transcriptional activation of the rat vesicular monoamine transporter 2 (VMAT2) promoter in gastric epithelial cells. Maintenance of basal levels of transcription was dependent on the presence of SP1, cAMP-response element (CRE), and overlapping AP2/SP1 consensus sequences within the region of promoter from −86 to +1 base pairs (bp). Gastrin stimulation increased transcriptional activity, and responsiveness was shown to be dependent on the CRE (−33 to −26 bp) and AP2/SP1 (−61 to −48 bp) consensus sites but independent of the SP1 site at −86 to −81 bp. Gastrin-induced transcription was dependent on the cooperative interaction of an uncharacterized nuclear factor of ∼23.3 kDa that bound to the putative AP2/SP1 site, CRE-binding protein (CREB), and CREB-binding protein/p300. Gastrin stimulation resulted in the increased binding of phosphorylated CREB to the promoter, but it did not result in the increased binding of the AP2/SP1-binding protein. The gastrin responsiveness of the promoter was shown to be dependent on both the protein kinase C and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-signaling pathways, which may converge on the AP2/SP1-binding protein. Vesicular monoamine transporter 2 is important for the accumulation of monoamine neurotransmitters into synaptic vesicles and histamine transport into secretory vesicles of the enterochromaffin-like cell of the gastric corpus. In this study we have investigated the mechanisms regulating the transcriptional activation of the rat vesicular monoamine transporter 2 (VMAT2) promoter in gastric epithelial cells. Maintenance of basal levels of transcription was dependent on the presence of SP1, cAMP-response element (CRE), and overlapping AP2/SP1 consensus sequences within the region of promoter from −86 to +1 base pairs (bp). Gastrin stimulation increased transcriptional activity, and responsiveness was shown to be dependent on the CRE (−33 to −26 bp) and AP2/SP1 (−61 to −48 bp) consensus sites but independent of the SP1 site at −86 to −81 bp. Gastrin-induced transcription was dependent on the cooperative interaction of an uncharacterized nuclear factor of ∼23.3 kDa that bound to the putative AP2/SP1 site, CRE-binding protein (CREB), and CREB-binding protein/p300. Gastrin stimulation resulted in the increased binding of phosphorylated CREB to the promoter, but it did not result in the increased binding of the AP2/SP1-binding protein. The gastrin responsiveness of the promoter was shown to be dependent on both the protein kinase C and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-signaling pathways, which may converge on the AP2/SP1-binding protein. vesicular monoamine transporter 2 enterochromaffin-like cell l-histidine decarboxylase chromogranin A mouse CgA mitogen-activated protein MAP kinase extracellular signal-regulated kinase base pair(s) cAMP-response element CRE-binding protein CREB-binding protein electrophoretic mobility shift assay cholecystokinin The transport protein vesicular monoamine transporter 2 (VMAT2)1 plays a central role in cellular physiology. It is responsible for the accumulation of monoamine neurotransmitters into synaptic vesicles and has been implicated in the synthesis and storage of histamine in the interleukin-3-dependent cell line Ea3.123 (1Liu Y. Edwards R.H. Annu. Rev. Neurosci. 1997; 20: 125-156Crossref PubMed Scopus (237) Google Scholar, 2Merickel A. Edwards R.H. Neuropharmacology. 1995; 34: 1534-1547Crossref Scopus (83) Google Scholar, 3Takahashi N. Uhl G. Mol. Brain Res. 1997; 49: 7-14Crossref PubMed Scopus (25) Google Scholar, 4Dimaline R. Struthers J. J. Physiol. 1996; 490: 249-256Crossref PubMed Scopus (68) Google Scholar, 5Watson F. Deavall D.G. Macro J.A. Kiernan R. Dimaline R. Biochem. J. 1999; 337: 193-199Crossref PubMed Scopus (20) Google Scholar). VMAT2 may play a significant role in locomotor stimulation and/or the behavioral reward produced by amphetamines, and malfunctions of monoamine transport have been implicated in psychiatric disease (6Takahashi N. Miner L.L. Sora I. Ujike H. Revay R.R. Kostic V. Jackson, Lewis V. Przedborski S. Uhl G.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9938-9943Crossref PubMed Scopus (339) Google Scholar, 7Liu Y. Peter D. Roghani A. Schuldiner S. Prive G.G. Eisenberg D. Brecha N. Edwards R.H. Cell. 1992; 70: 539-551Abstract Full Text PDF PubMed Scopus (524) Google Scholar). Studies on VMAT2 knockout mice support the idea that vesicular monoamine transport is important in maintaining neuronal function with heterozygote VMAT2+/− mice having dysfunctional monoamine storage and release parameters (6Takahashi N. Miner L.L. Sora I. Ujike H. Revay R.R. Kostic V. Jackson, Lewis V. Przedborski S. Uhl G.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9938-9943Crossref PubMed Scopus (339) Google Scholar, 8Gainetdinov R.R. Fumagalli F. Wang Y-M. Jones S.R. Levey A.I. Miller G.W. Caron M.G. J. Neurochem. 1998; 70: 1973-1978Crossref PubMed Scopus (143) Google Scholar). VMAT2 is also likely to mediate histamine transport into the secretory vesicles of the enterochromaffin-like cell (ECL) of the gastric corpus (2, 4, 9- 11). In the rat stomach the ECL cell is tightly regulated by the peptide hormone gastrin, with gastrin stimulation leading to the release of histamine and the resultant production of gastric acid from the parietal cell (12Prinz C. Kajimura M. Scott D.R. Mercier F. Helander H.F. Sachs G. Gastroenterology. 1993; 105: 449-461Abstract Full Text PDF PubMed Scopus (279) Google Scholar, 13Prinz C. Zanner R. Gerhard M. Mahir S. Neumayer N. Hohne-Zell B. Gratzl M. Am. J. Physiol. 1999; 277: C845-C855Crossref PubMed Google Scholar, 14Black J.W. Shankley N.P. Trends Pharmacol. Sci. 1987; 8: 486-490Abstract Full Text PDF Scopus (73) Google Scholar). Histamine is synthesized within the cytosol of the ECL cell froml-histidine by the action of the enzymel-histidine decarboxylase (HDC) (15Beaven M.A. Ganellin C.R. Parsons M.E. Pharmacology of Histamine Receptors. Wright, Bristol, UK1982: 101-105Google Scholar) and is then sequestered by VMAT2 into secretory vesicles that are stabilized by chromogranin A (CgA), a multi-functional acidic protein expressed both in neuroendocrine cells and in ECL cells (16O'Connor D.T. Wu H. Gill B.M. Rozansky D.J. Tang K. Mahata S.K. Mahata M. Eskeland N.L. Videen J.S. Zhang X. Takiyuddin M.A. Parmer R.J. Ann. N. Y. Acad. Sci. 1993; 729: 36-45Google Scholar, 17Bauerfeind R. Ohashi M. Huttner W.B. Ann. N. Y. Acad. Sci. 1994; 733: 385-390Crossref Scopus (21) Google Scholar, 18Iacangelo A. Eiden L. Regul. Pept. 1995; 58: 65-88Crossref PubMed Scopus (155) Google Scholar, 19Stabile B.E. Howard T.J. Passaro E. O'Connor D.T. Arch. Surg. 1990; 125: 451-453Crossref PubMed Scopus (49) Google Scholar, 20Cetin Y. Grube D. Cell Tissue Res. 1991; 264: 231-241Crossref PubMed Scopus (41) Google Scholar, 21Syversen U. Mignon M. Bonfils S. Kristensen A. Waldum H.L. Acta Oncol. ( Madr. ). 1993; 32: 161-165Crossref PubMed Scopus (67) Google Scholar). Evidence suggests that VMAT2 may be up-regulated to accommodate the increased histamine biosynthesis and secretion that accompanies ECL stimulation. Hypergastrinaemia-induced degranulation of the ECL cell is accompanied by a parallel secretion of histamine and CgA followed by enhanced production of both molecules, and in the pre-B cell line Ea3.123, the mRNA abundance for both VMAT2 and HDC is increased in a parallel fashion after mobilization of their intracellular calcium levels or by activation of protein kinase C (4Dimaline R. Struthers J. J. Physiol. 1996; 490: 249-256Crossref PubMed Scopus (68) Google Scholar, 5Watson F. Deavall D.G. Macro J.A. Kiernan R. Dimaline R. Biochem. J. 1999; 337: 193-199Crossref PubMed Scopus (20) Google Scholar, 20Cetin Y. Grube D. Cell Tissue Res. 1991; 264: 231-241Crossref PubMed Scopus (41) Google Scholar, 21Syversen U. Mignon M. Bonfils S. Kristensen A. Waldum H.L. Acta Oncol. ( Madr. ). 1993; 32: 161-165Crossref PubMed Scopus (67) Google Scholar, 22Watkinson A. Dockray G.J. Regul. Pept. 1992; 40: 51-61Crossref PubMed Scopus (36) Google Scholar, 23Hakanson R. Ding X-Q. Norlen P. Chen D. Gastroenterology. 1995; 108: 1445-1452Abstract Full Text PDF PubMed Scopus (96) Google Scholar). In addition, increases in the abundance of mRNA for HDC, VMAT2, and CgA were observed in animal models of hypergastrinaemia, suggesting that the transcriptional activity of these genes could be regulated by gastrin in vivo (24Dimaline R. Sandvik A.K. FEBS Lett. 1991; 281: 20-22Crossref PubMed Scopus (91) Google Scholar, 25Dimaline R. Evans D. Forster E.R. Sandvik A.K. Am. J. Physiol. 1993; 264: G583-G588Crossref PubMed Google Scholar, 26Dimaline R. Sandvik A.K. Evans D. Forster E.R. Dockray G.J. J. Physiol. 1993; 465: 449-458Crossref PubMed Scopus (49) Google Scholar). Indeed, gastrin has been shown to transcriptionally activate both the HDC and CgA promoters (27Hocker M. Zhang Z. Fenstermacher D.A. Tagerud S. Chulak M. Joseph D. Wang T.C. Am. J. Physiol. 1996; 270: G619-G633Crossref PubMed Google Scholar, 28Zhang Z. Hocker M. Koh T.J. Wang T.C. J. Biol. Chem. 1996; 271: 14188-14197Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 29Hocker M. Raychowdhury R. Plath T. Wu H. O'Conner D.T. Wiedenmann B. Rosewicz S. Wang T.C. J. Biol. Chem. 1998; 273: 34000-34007Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Regulation of the human HDC promoter by gastrin involves a protein kinase C-dependent, MAP kinase/ERK-dependent, and AP1-dependent pathway and involves the binding of distinct nuclear factors to twocis-acting overlapping binding sites (GAS-RE1, +1 to +19; and GAS-RE2 +11 to +27) (30Hocker M. Henihan R.J. Rosewicz S. Riecken E. Zhang Z. Koh T.J. Wang T.C. J. Biol. Chem. 1997; 272: 27015-27024Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 31Hocker M. Zhang Z. Merchant J.L. Wang T.C. Am. J. Physiol. 1997; 272: G822-G830PubMed Google Scholar, 32Raychowdhury R. Zhang Z. Hocker M. Wang T.C. J. Biol. Chem. 1999; 274: 20961-20969Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Gastrin transactivation of the mouse CgA promoter was found to be dependent on the binding of Sp1 to an Sp1/Egr motif located at −88 to −77 bp and a CRE-like element at −71 to −64 bp of the mCgA promoter (29Hocker M. Raychowdhury R. Plath T. Wu H. O'Conner D.T. Wiedenmann B. Rosewicz S. Wang T.C. J. Biol. Chem. 1998; 273: 34000-34007Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Gastrin stimulation resulted in an increased binding of both SP1 and CREB to their consensus sequences within the mCgA promoter, and overexpression of either SP1 or phosphorylated CREB transactivated the promoter. Coexpression of both transcription factors resulted in an additive mCgA promoter response, suggesting that the effect of gastrin was brought about by their cooperative action. In the present study we have investigated the transcriptional regulation of VMAT2 in the gastric epithelial cell line AGS-GR. This cell line has been permanently transfected with the CCKB-gastrin receptor (33Varro A. Wroblewski L. Noble P.J. Bishop L. Ashcroft F. Varro J. Thompson C. Dimaline R. Dockray G.J. Gut. 2000; 46 (abstr.): 56Google Scholar). It has been shown to express a functional CCKβ receptor and utilizes signaling pathways that are common to the ECL cell (33Varro A. Wroblewski L. Noble P.J. Bishop L. Ashcroft F. Varro J. Thompson C. Dimaline R. Dockray G.J. Gut. 2000; 46 (abstr.): 56Google Scholar, 13Prinz C. Zanner R. Gerhard M. Mahir S. Neumayer N. Hohne-Zell B. Gratzl M. Am. J. Physiol. 1999; 277: C845-C855Crossref PubMed Google Scholar). This has allowed us to examine the controls of both basal and gastrin-stimulated VMAT2 transcriptional activity in these gastric epithelial cells. Maintenance of the basal level of transcription was dependent on the presence of SP1, CRE, and overlapping AP2/SP1 consensus sequences within the region of promoter from −86 to +1 bp, whereas full gastrin responsiveness of the promoter depended on the presence of the intact CRE and AP2/SP1 consensus sequences. Regulation of the rat VMAT2 promoter was found to involve the protein kinase C-dependent and MAP kinase/ERK-dependent pathway, binding of a distinct nuclear factor of estimated molecular mass 23.3 kDa to the putative AP2/SP1 consensus site and the binding and phosphorylation of CREB to the CRE site within the promoter. Thus, our study demonstrates that the rat VMAT2 promoter can, like the HDC and mCgA promoters, be transcriptionally regulated by gastrin, but the mechanism of this regulation is distinct from that observed for these other gastrin-sensitive promoters. Ham's F-12, fetal bovine serum, and penicillin/streptomycin were obtained from Life Technologies, Inc. Gastrin I (human) was obtained from Peninsula Laboratories Europe Ltd., and BIOTAQ DNA polymerase was obtained from Bioline, London, UK. A gel shift assay system containing HeLa and AP2 nuclear extracts and consensus oligos for AP1, OCT1, CREB, NF-Kβ, and TFIID was obtained from Promega. Additional oligonucleotides were synthesized by Sigma- Genosys, and all antibodies were purchased from Santa Cruz Biotechnology, Inc. apart from the phospho-CREB antibody, which was from Upstate Biotechnology and peroxidase-conjugated anti-goat IgG from Sigma. Complete, mini-protease inhibitor mixture tablets were from Roche Molecular Biochemicals. The inhibitors GF109303X and PD98059 were obtained from Calbiochem. All other reagents were obtained from Sigma. The promoter region of the rat VMAT2 gene had been previously cloned, and a subclone pV2404-8 was generated in the pGEM-TEasy vector (Promega, Southampton, UK) as described previously (5Watson F. Deavall D.G. Macro J.A. Kiernan R. Dimaline R. Biochem. J. 1999; 337: 193-199Crossref PubMed Scopus (20) Google Scholar). The subclone pV2404-8 was sequenced in both directions by an automated dideoxy method and used to make a series of VMAT2 promoter 5′-deletional constructs in the pGL3- Basic vector (Promega) that contained 1632, 1289, 943, 609, 223, 86, and 36 nucleotides upstream of the start site together with 55 bp of exon 1. Polymerase chain reaction products from pV2404-8 were directionally cloned between the SacI and XhoI sites of pGL3-Basic, and the resultant constructs were sequenced in both directions to confirm their integrity. In addition, polymerase chain reaction products from pV2404-8 that contained 86 and 36 nucleotides upstream of the transcriptional start site together with 55 bp of exon 1 were directionally cloned between the BamHI andXhoI sites of the promoterless luciferase-reporter vector PXP2. Again, the integrity of these constructs and the sequences of mutated constructs generated by polymerase chain reaction were confirmed before their use in experiments. The mutations generated in these constructs are indicated in the appropriate figures and their legends. AGS-GRcells, which were permanently transfected with the human CCKB-gastrin receptor (33Varro A. Wroblewski L. Noble P.J. Bishop L. Ashcroft F. Varro J. Thompson C. Dimaline R. Dockray G.J. Gut. 2000; 46 (abstr.): 56Google Scholar) driven by the EF1-α promoter under puromycin selection, were grown in Ham's F-12 medium supplemented with 10% (fetal bovine serum) and penicillin/streptomycin (100 IU/ml) at 37 °C in 5% CO2. Transient transfections were carried out using TransfastTM transfection reagent from Promega. Cells were plated out at a density of 5 × 105 cells/65-mm well 24 h before transfection. During transfection, cells were incubated for 1.5 h in 2 ml of transfection mix in serum-free media. The transfection mix consisted typically of 3.4 μl of TransfastTM,1.5 μg of firefly luciferase reporter construct, and 0.1 μg of Renillaluciferase (pRL-TK) control vector (Promega) per well. The quantity of firefly luciferase reporter was reduced to 1 μg/ml when additional expression constructs were added. These were typically added at 0.5 μg/well. Cells were stimulated 24 h post-transfection as described in the figure legends and harvested at appropriate time points after stimulation. Luciferase activity was determined using the dual luciferase reporter system (Promega), and each individual transfection was assayed in duplicate. Nuclear extracts from AGS-GR cells and rat gastric corpus epithelium were prepared using the methodology described in Schreiber et al.(34Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3917) Google Scholar). Double-stranded oligonucleotides were radiolabeled with [α-32P]dATP, and EMSAs were performed with 10 μg of nuclear extracts in a final volume of 20 μl of binding buffer containing 10 mm Tris-HCl (pH 7.5), 50 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, 1 μg of poly(dA-dT), 10% glycerol, and 10 μm Zn2+. Mixtures were incubated with 10 fmol of double-stranded oligonucleotide probes for 20 min at room temperature (29Hocker M. Raychowdhury R. Plath T. Wu H. O'Conner D.T. Wiedenmann B. Rosewicz S. Wang T.C. J. Biol. Chem. 1998; 273: 34000-34007Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 32Raychowdhury R. Zhang Z. Hocker M. Wang T.C. J. Biol. Chem. 1999; 274: 20961-20969Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). For supershift assays, nuclear extracts were incubated with 1 μl of anti-phospho-CREB, anti-ATF, and anti-AP2 antibodies but 2 μl of anti-Sp family antibodies. In addition, the Sp supershifts required the addition of 4 μg of bovine serum albumin to the binding reactions. DNA-protein complexes were electrophoresed on a 6% nondenaturing polyacrylamide gel. The dried gel was exposed to a phosphor storage screen, and the image was revealed with a PhosphorImager (Molecular Dynamics, Sevenoaks, Kent, UK). 20-μg aliquots of AGS-GRnuclear extract were incubated with 10 fmol of Klenow [α-32P]dATP end-labeled probes (A,B, and E, as described in the legend to Fig. 6) in EMSA binding buffer for 30 min on ice. The samples were exposed to UV light for 60 min before the addition of an equal volume of 2× SDS sample buffer and heating to 99 °C for 5 min. 10 μg of each sample and prestained molecular weight markers were electrophoresed on a 12% SDS-polyacrylamide gel in Tris/glycine electrophoresis buffer. The gels were dried, and radioactivity associated with the proteins was visualized using a PhosphorImager. 5 × 105AGS-GR cells were plated in 65-mm culture dishes containing 5 ml of complete medium. 18 h later, the medium was removed and replaced with 5 ml of serum-free medium, and the cells were incubated in the absence or presence of gastrin (5 × 10−8m) for 3, 6, 12, and 24 h before preparation of a total cell lysate. To prepare the cellular lysate AGS-GR, cells were washed twice with ice-cold phosphate-buffered saline and resuspended in 250 μl of lysis buffer containing 20 mm Tris (pH 7.8), 150 mm NaCl, 2 mm EDTA, 0.5% Nonidet P-40, 10 mm NaF, 15 mg/ml benzamide, 8.8 mg/ml sodium orthovanadate, 0.5 mmdithiothreitol, 10 mg/ml phenylmethylsulfonyl fluoride, and one protease inhibitor mixture tablet/10 ml of lysis buffer. 40 μg of the lysates were electrophoresed on a 12% SDS-polyacrylamide gel. After electrophoresis, the proteins were blotted on nitrocellulose membranes, and immunodetection of the proteins was performed. The primary antibody used was obtained from Santa Cruz Biotechnology and is specific for mouse, rat, and human VMAT2 but is noncross-reactive with VMAT1. The secondary antibody was a peroxidase-conjugated anti-goat IgG. Enhanced chemiluminescence (SuperSignal® West Pico chemiluminescent substrate; Pierce) was used to identify the VMAT2 protein. Results were analyzed for statistical significance using the Student's t test for independent samples. The basal transcriptional activity of the rat VMAT2 promoter in the gastric environment was characterized by ligating polymerase chain reaction-generated segments of the 5′-flanking region of the VMAT2 gene upstream of the gene for firefly luciferase in the reporter vector pGL3-Basic and transfection of the resultant constructs together with a Renilla luciferase vector into AGS-GR cells. The AGS-GR cell line has been permanently transfected with the CCKB-gastrin receptor and has been demonstrated to show specific activation by gastrin (33Varro A. Wroblewski L. Noble P.J. Bishop L. Ashcroft F. Varro J. Thompson C. Dimaline R. Dockray G.J. Gut. 2000; 46 (abstr.): 56Google Scholar). As shown in Fig. 1, transfection of all the constructs resulted in significant luciferase activity with the largest construct v1632pg (encompassing nucleotides −1632 to +55 bp) showing an approximate 10.3-fold increase in luciferase activity over that seen in the promoterless vector. Activity was maximal in the construct v223pg (−223 to +55 bp), which showed an average 44.7-fold increase in luciferase activity over promoterless vector. This level of expression was maintained in the construct v86pg (−86 to +55 bp). The increase in luciferase activity observed in the v223pg construct as compared with v609pg (−609 to +55 bp) suggests the presence of at least one negative regulatory element in the region from −609 to −223 bp. The schematic illustrated in Fig.2 a shows the sequence of the 5′-flanking region of the rat VMAT2 gene from −86 to +55 bp, with bases numbered relative to the transcriptional start site. Within this region there is a SP1 consensus sequence spanning from −86 to −81 bp, a putative AP2/SP1 site spanning from −61 to −48 bp, and a CRE consensus sequence spanning from −33 to −26 bp. To assess the contribution of the cis-regulatory elements contained in this region to maintenance of basal transcriptional activity, constructs were made in which the SP1, AP2/SP1, or CRE consensus sequences were mutated. A schematic representation of these and other constructs used in this study is shown in Fig. 2 b. As Fig.3 shows, when the intact SP1 site starting at −86 bp was mutated, basal activity of the v86pg construct was reduced to 36.61 ± 5.91% (mean ± S.E.,n = 17) wild type. When the AP2 region of the AP2/SP1 composite site was mutated (Fig. 3), there was no effect on basal activity of the construct v86px. However mutation of the SP1 part of the site reduced basal activity to 37.4 ± 0.04% (mean ± S.E., n = 12) of the control level (Fig. 3). Mutation of the CRE also reduced the basal transcriptional activity of the construct v36pg to 39.9 ± 4.8% (mean ± S.E.,n = 18) of the control level (Fig. 3). Thus the SP1, putative AP2/SP1, and CRE consensus sequences are all important determinants of basal transcriptional activation in the AGS-GR cell line.Figure 2a, sequence of the 5′-flanking region of the rat VMAT2 gene. Bases are numbered relative to the transcriptional start site (+1), which was determined previously by primer extension analysis (5Watson F. Deavall D.G. Macro J.A. Kiernan R. Dimaline R. Biochem. J. 1999; 337: 193-199Crossref PubMed Scopus (20) Google Scholar). Putative cis-regulatory elements (SP1, AP2/SP1, and CRE) are underlined; the 55 bp of exon 1 included in the VMAT2 promoter/luciferase constructs aredouble-underlined. The schematic in b illustrates the deletional and mutated VMAT2 promoter/luciferase constructs used in this study. v, VMAT2; 86, 48, or36 is the number of bases of promoter in the construct;px, PXP2; pg, pGL3b. The sites mutated are indicated both in the construct name and in their associated line diagrams and are as follows: v86sp1pg, mutation of authentic SP1 site GGGCGG to GAGCTC (−86 to −81 bp); v86ap2sp1px, mutation throughout putative AP2/SP1 site CCCCTCCGCCC to AACGTCAGAAC (−61 to −51 bp); v86ap2px, mutation of AP2 part of AP2/SP1 site CCCCT to AACGG (−61 to −57 bp); v86sp1/2px, mutation of SP1 part of AP2/SP1 site CCGCCC to CCGAAT (−56 to −51 bp); v86crepx, mutation of CRE site TGACGT to TGTAGA (−33 to −28 bp); v36mpx/pg, mutation of CRE site TGACGT to TGTAGA (−33 to −28 bp).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Contribution of cis-regulatory elements SP1, AP2/SP1, and CRE to maintenance of basal transcriptional activity of the rat VMAT2 promoter. AGS-GR cells were transiently transfected with mutated VMAT2 promoter/luciferase constructs (v86sp1pg, v86sp1/2px, v86ap2px, and v36 mpg), and their luciferase activity was compared with that found for their parental controls. The luciferase activity of each mutant construct is expressed as the percentage of its parental wild type construct (=100%). Results are the mean ± S.E. and are representative of a minimum of six separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) AGS-GR cells were transiently transfected with the VMAT2-promoter luciferase constructs before stimulation with gastrin. Constructs that contained from 1632 to 86 bp of VMAT2 promoter in the vector pGL3b all showed significant increases in luciferase activity after stimulation with gastrin (Fig.4 a) compared with their basal levels of activity. The luciferase activity of the largest construct, v1632pg, was increased 8.2 ± 0.9-fold (mean ± S.E.,n = 9) over basal activity after stimulation with gastrin (5 × 10−8m) for a 24-h period. Truncation of the promoter region of this construct down to 86 bp did not lead to significant reductions in response. Gastrin stimulation led to a 7.1 ± 1.5-fold (mean ± S.E.,n = 9) increase in the luciferase activity of the construct v609pg, which was not significantly different from that of construct v86pg, which was on average increased 5.2 ± 0.4-fold (mean ± S.E., n = 9). Stimulation of the v86px construct with 5 × 10−8mgastrin increased the transcriptional activity of the promoter within 4 h of stimulation and was maximal at 24 h post-stimulation. Maximal responses were seen with 5 × 10−8m gastrin, but significant stimulation was seen with 5 × 10−9m gastrin (data not shown). Gastrin responsiveness was inhibited by the gastrin antagonist L740093, and Gly-extended gastrin was unable to increase the transcription of the v86px construct (data not shown). Western blot analysis demonstrated VMAT2 protein in the AGS-GR cell line, but its abundance was not significantly elevated after gastrin stimulation of the cells (data not shown). The luciferase activity of the promoterless pGL3b-empty vector was modestly increased after stimulation with 5 × 10−8m gastrin (2.67 ± 0.6-fold over basal levels, mean ± S.E., n = 9; Fig. 4 a). Although this increase was not comparable with those observed with constructs containing the rat VMAT2 promoter segments, new constructs were made in the PXP2 luciferase vector to confirm and extend the data on gastrin responsiveness of the promoter. Stimulation with gastrin did not increase the intrinsic luciferase activity of the promoterless PXP2 vector (Fig. 4 b). However the construct v86px showed a 4.44 ± 0.8 (mean ± S.E.,n = 11) fold increase in luciferase activity over basal levels after gastrin stimulation (Fig. 4 b), confirming our findings originally made using the pGL3b vector that the region of rat VMAT2 promoter from −86 to +55 bp was capable both of sustaining basal transcription and of increasing its transcriptional activity in response to gastrin. The gastrin responsiveness of constructs v48px (48 to +55 bp) and v36px was significantly reduced when compared with that of v86px (p < 0.05) but was significantly greater than that of the promoterless PXP2 empty vector (Fig. 4 b,p < 0.005). The region of promoter (from −86 to 49 bp) contains both the SP1 and AP2/SP1 composite sites, and gastrin responsiveness was reduced on its deletion. As shown by the similar responsiveness of the v48px and v36px constructs, the region from −48 to −37 bp did not appear to contribute to the gastrin responsiveness of the promoter. However, a significant level of gastrin responsiveness was maintained in the smallest construct v36px, which contains the CRE. These data therefore suggested the possibility that all three consensus sites, SP1, AP2/SP1, and CRE, might contribute to the gastrin responsiveness of the promoter. To dissect the gastrin responsiveness further, comprehensive mutagenesis of the constructs v36px and v86px was performed. When the intact SP1 site at position −86 to −81 bp was mutated in construct v86sp1px, the gastrin responsiveness of this construct was not different from its parental construct, v86px (101.9 ± 17.03% gastrin response; mean ± S.E., n = 9). This indicated that this site was not contributing to the gastrin responsiveness of the promoter (Fig. 5 a), and experiments performed with the construct v86pg and the mutated construct v86sp1pg confirmed this finding (data not shown). In contrast, when the CRE site was mutated in the construct v36mpx, the gastrin responsiveness of this construct was reduced to 51 ± 10% (mean ± S.E.,n = 7) of its parental construct, v36px, indicating the importance of the CRE consensus sequence. The importance of this CRE consensus sequence for gastrin responsiveness of the rat VMAT2 promoter was confirmed through its mutation from within the construct v86px (Fig. 5 b). Gastrin increased the luciferase activity of the resultant vcrepx construct by only 1.3 ± 0.2-fold (mean ± S.E., n = 14) over basa