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II Medizinische Klinik (M.G., N.N., E.P.-S., R.Z., C.P.) and Department of Obstetrics and Gynecology (E.L.), Technical University, D-81675 Munich, Germany; and Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Universitätsklinikum Charite, Campus Virchow-Klinikum (T.C., M.H.), D-13353 Berlin, Germany
Address all correspondence and requests for reprints to: Dr. Christian Prinz, II Medizinische Klinik, Technische Universität München, Ismaninger Strasse 22, D-81675 Munich, Germany. E-mail: christian. prinz{at}lrz.tum.de
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Introduction |
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Two VMAT subtypes have been characterized to date. Liu et al. (7, 8, 9, 10, 11, 12) showed that adrenal chromaffin, intestinal enterochromaffin, and neuronal cells store biogenic amines via activation of the VMAT-1. Erikson et al. (13, 14, 15, 16) isolated a different clone from the rat, termed VMAT-2, which was detected in basophilic leukocytes and gastric ECL cells. The rat and human VMAT-2 promoters have been cloned in previous studies (17, 18), but no physiological regulation of VMAT-2 expression by gastrointestinal hormones has been reported. Also, no direct evidence exists about the influence of gastric hormones on VMAT-2 promoter activity.
Gastrin, as a physiological stimulant of ECL cell function, may be a key hormone for the coordinated activation of histamine synthesis and storage. It has already been shown that gastrin induces histamine synthesis by stimulating HDC enzyme activity (4) and HDC mRNA expression (19). Gastrin increases the activity of the HDC promoter (20) and has also been shown to strongly induce the expression of the matrix protein CgA by activation of the CgA promoter (21). Therefore, it appears likely that gastrin exerts a functional synergy in ECL cells by controlling transcriptional regulation not only of HDC and CgA, but also of the VMAT-2 promoter.
Indeed, rats undergoing hypergastrinemia showed increased expression of VMAT-2 in the gastric mucosa, as determined by Northern blot reaction (22). These experiments also revealed an accompanying increase in HDC and CgA mRNA abundance during conditions of hypergastrinemia (22), suggesting a central role of gastrin for the transcriptional regulation of these genes. In the present study, we sought to determine whether this association reflects a direct effect of gastrin on VMAT-2 RNA and protein expression in ECL cells. Further, the effect of gastrin on the activity of the VMAT-2 promoter was investigated in PC-12 cells stably transfected with the gastrin/cholecystokinin B (CCK-B) receptor. Our current experiments yielded clear evidence that the promoter is responsive to gastrin stimulation, indicating the functional importance of this hormone for the coordinated regulation of histamine uptake in secretory vesicles.
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Materials and Methods |
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Stimulation of VMAT-2 mRNA expression in isolated ECL
cells
Total RNA was isolated from enriched ECL cells using
peqGOLDTriFast reagent (Peqlab, Erlangen, Germany). Primers were
designed according to published sequences of the rat VMAT-2 mRNA
(accession no. L00603). Sequences were: VMAT-2 sense,
5'-CTCTGTCTTCACTGGGACAGTCCG-3' (nucleotides 272–294); and VMAT-2
antisense, 5'-ACGGAAATCCAGACCACCAGACC-3' (nucleotides 447–470),
yielding a product of 198 bp. PCR was performed using the
TaqPCR Master Mix (QIAGEN, Hilden, Germany) and
the following temperature cycle profile: 5 min at 94 C, followed by 30
cycles of 45 sec at 94 C, 55 sec at 58 C, 1 min at 72 C, and 10 min at
72 C. PCR products were subjected to horizontal agarose gel
electrophoresis. Amplification products were eluted, subcloned into
TOPO pCR2.1, and sequenced for verification. As control, PCR was
performed simultaneously with primers specific for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), yielding a product of
950 bp. Primers were: GAPDH sense, 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3';
and GAPDH antisense, 5'-CATGTAGGCCATGAGGTCCACCAC-3'.
Western blot analysis
Isolated ECL cells were stimulated with vehicle or the indicated
stimulant. After rinsing cells in ice-cold PBS, cells were lysed in
L-CAM lysis buffer (0.5 M NaCl, 0.1 M
KCl, 0.1 M MgSo4, 0.1 M
CaCl1, 0.1 M Triton X-100, 1
mM phenylmethylsulfonylfluoride) and sonicated. Protein
amounts were determined by bicinchoninic acid kit (Pierce Chemical Co., Rockford, IL). Twenty micrograms of protein from
each sample were boiled for 3 min in 4 x SDS gel loading buffer
(1.5 M dithiothreitol, 6% SDS, 0.1% bromophenol blue,
40% glycerol, and 125 mM Tris, pH 6.8) and loaded.
Proteins were separated by 12% SDS-PAGE and transferred to
polyvinylidene difluoride membranes. The membranes were incubated with
an anti-VMAT-2 antibody (1:500; Santa Cruz Biotechnology Inc., Heidelberg, Germany) overnight at 4 C. After four washes,
membranes were overlaid with 1 µg/ml horseradish-peroxidase-labeled
antirabbit IgG antibody (Amersham Pharmacia Biotech,
Freiburg, Germany) for 1 h at room temperature, and the VMAT-2
band was detected at 50 kDa using the enhanced chemiluminescence system
(Amersham Pharmacia Biotech). Densitometric analysis was
performed using one-dimensional scan software.
Activation of Sp1 and cAMP response element (CRE)-binding protein (CREB) was investigated by preparing nuclear extracts from PC12-G cells after stimulation with gastrin. Antibodies against Sp1 (Santa Cruz Biotechnology Inc.; 1 µg/ml, recognizing p95 and p106) and the antiphospho-CREB (BIOMOL Research Laboratories, Heidelberg, Germany; 1:1000, recognizing Ser133) were applied overnight at 4 C, and detection was performed as described above.
Primer extension analysis
The transcriptional start site of the rat VMAT2 gene was
determined by primer extension of VMAT2 mRNA isolated from PC12 cells
stimulated with forskolin
(10-5 M) or
vehicle. To show that the same transcriptional start site is
functionally important in our promoter constructs, cells were also
transiently transfected with the plasmid containing the rat promoter
cloned in-frame in pGL3 vector (rVMAT2) and a CREB expression plasmid
(provided by Gerald Thiel, University of Erlangen, Erlangen, Germany).
The 32P-labeled oligonucleotide (labeled with T4
polynucleotide kinase, New England Biolabs, Schwalbach,
Germany; 3.55 x 106 cpm/pmol) used
for mapping the rat VMAT2 promoter was the 25-mer
5'-CTCGCCTGTAACTGCGCGGTTATAG-3', complementary to the region +88 to +64
of the gene. Annealing was performed with 2–3 µg mRNA in 16
mM Tris (pH 8.3), 25 mM NaCl, and 1
mM MgCl2 [in the RT first strand
buffer 5x (Life Technologies, Inc., Gaithersburg, MD)
diluted 15 times] by incubation for 25 min at 65 C and subsequent
cooling the mixture to 37 C before chilling them on ice. The extension
reaction was performed in 1 x RT first strand buffer for 75 min
at 42 C. To each annealing mixture the following reagents were added: 3
µl 0.1 M dithiothreitol, 1 µl 10 mM
deoxy-NTP, and 0.5 µl RT Superscript II (Life Technologies, Inc.) in a final volume of 30 µl. After inactivation (10 min
at 70 C), the excess RNA was digested with ribonuclease for 15 min at
37 C. Sequencing dyes and probes were denatured for 4 min at 70 C
before being loaded on a 6% polyacrylamide/7 M urea
gel.
Genomic cloning and construction of reporter constructs
Human VMAT2 promoter.A genomic human (h) VMAT2 fragment
(4.1 kb) cloned in pGFP-1 vector was a gift from Dr. W. Xu (University
College, London, UK). It contained 2.95 kb of 5'-flanking region of the
hVMAT2 gene, exon 1 (100 bp), intron 1 (450 bp), and about 700 bp of
exon 2. A 1.5-kb fragment was amplified by PCR and subcloned into pGL3
basic reporter vector yielding pGL-hVMAT-2-luc. Deletion mutants were
generated by digestion with appropriate single cutting restriction
enzymes giving fragments of -1115, -830, -309, -206, -120, -73,
and +78 bp (numbers referring to the transcriptional start site).
Rat VMAT2 promoter.The rat promoter was cloned using the Genome Walker Kit (CLONTECH Laboratories, Inc., Heidelberg, Germany). Gene-specific primers (GSP) were located in exon II: GSP-1, 5'-TGTCCAGCAGCAGCGCAAGGAACACG-3'; and GSP-2,5'-GCACCAGATCGCTCAGGGCC-3'. Genomic PCR products were subcloned and sequenced. Putative recognition sequences for transcriptional start sites were identified using the TESS program. A 1125-bp fragment was subcloned in-frame into pGL3, yielding pGL-rVMAT-2-luc. 5'-Deletion constructs were generated by digestion with restriction enzymes, yielding fragments -808, -504, -177, -48, and +232 bp (numbers referring to the transcriptional start site). These fragments were further subjected to exonuclease III digestion (see below).
HDC and CgA promoter.The reporter constructs hHDC1.8kB-Luc and mCgA4.8kB-Luc containing the 1.8- or 4.8-kb sequences of human HDC and mouse CgA promoters were provided by Drs. T. C. Wang and D. T. O’Connor, respectively, and have been characterized previously (19, 21).
Dense scanning analysis
Exonuclease III digestion reporter constructs.pGL-rVMAT-2-luc was subjected to progressive unidirectional
(5'
3') nested deletions by digestion with exonuclease III
(Erase-a-Base System, Promega Corp., Heidelberg,
Germany) according to the manufacturer’s instructions using the
following restriction enzymes: for rVMAT2 -800,
SacI-NheI, for the hVMAT2 -309,
SacI-NheI and SacI-MluNI;
for hVMAT2 -1115, KpnI-NheI and
KpnI-PmlI (Roche Molecular Biochemicals, Mannheim, Germany).
PC12 culture and transfection studies.PC12 cells (European Collection of Cell Cultures, London, UK) were grown in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% horse serum, 5% FBS, and 1 x antibiotic solution (100 U/ml penicillin and 0.1 mg/ml streptomycin, Sigma, Munich, Germany). PC12 cells were transfected with a plasmid containing the entire coding sequence for the CCK-B/gastrin receptor (provided by Dr. Martin Beinborn, Massachusetts General Hospital, Boston, MA). For transfection experiments, PC12 cells were plated at a density of 1 x 106 cells/35-mm well and transfected using Cellfectin (QIAGEN, Hilden, Germany) and 1 µg/well pSV-ß-galactosidase control plasmid or 2 µg/well of reporter constructs. Selection media containing G418 were added after 3 days of incubation. Monoclonal cell lines were established by expanding single colonies. Stimulation with gastrin 10-7 M was performed overnight in serum-free medium.
Statistical analysis
Results are expressed as the mean ± SEM. Data
were analyzed by t test or one-way ANOVA and subsequently
with Newman-Keuls test. P < 0.05 was considered
significant.
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Results |
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represents a section of the gastric
mucosa stained with a primary antibody specific against HDC. Numerous
HDC-positive cells were detected at the basis of the gastric glands,
corresponding to the location of ECL cells. Fig. 1B demonstrates a
section of the mucosa stained with an antibody against VMAT-1, but only
a few positive cells were detected. Thus, VMAT-1-positive cells appear
to be rare in the rat gastric mucosa. In contrast, staining of sections
with anti-VMAT-2 antibody (Fig. 1C) revealed numerous small cells at
the basis of the glands, corresponding to the results with the HDC
antibody and the location of ECL cells. As serial sections could not
prove the colocalization of HDC and VMAT-2 in identical ECL cells,
isolated and highly enriched ECL cells were prepared and stained.
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, D and E, highly enriched ECL cells prepared as
cytospins were used for immunocytochemistry. In Fig. 1D, a total of 23
cells can be seen, and 21 of them stain positively using an antibody
against HDC (Fig. 1E). This finding was representative of the whole
preparation. Thus, more than 90% of the cells were HDC positive and
can therefore be regarded as ECL cells. Moreover, isolated and 48-h
cultured cells were stained simultaneously with antibodies specific for
HDC (Fig. 1F) and VMAT-2 (Fig. 1G). As demonstrated, all isolated ECL
cells (100%) were found to express VMAT-2 simultaneously with HDC.
Staining for VMAT-1 was not detected (not shown).
Stimulation of VMAT-2 mRNA and protein expression in ECL cells
Expression of VMAT-2 mRNA was determined using semiquantitative
RT-PCR (Fig. 2A). Gastrin is known to
exert its effect via calcium-signaling and plasmalemmal calcium entry.
Therefore, gastrin and ionomycin were used as stimulants of VMAT-2 mRNA
expression. A single PCR product of 198 bp was amplified and confirmed
by sequencing. Gastrin
(10-7 M; Fig. 2, left lanes 1–6) stimulated mRNA expression after 2–4 h
of incubation, and the signal decreased after 5 h. Similarly,
VMAT-2 mRNA expression was stimulated by ionomycin
(10-5
M; Fig. 2, right lanes 1–6) after
3–5 h of incubation (Fig. 2A). Simultaneously (shown in the
lower panel), PCR was also performed for GAPDH, yielding
bands of identical intensities.
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). A total of 20 µg protein was loaded
onto each lane. Stimulation of VMAT-2 protein expression was observed
with gastrin (10-7
M), ionomycin
(10-5 M), and
forskolin (10-5
M). As a control, actin expression was determined by
Western blot from identical protein samples. Although the intensity of
actin remained unchanged (98–104%), the intensity of VMAT-2 was
stimulated by gastrin 2.5- and 4-fold after 5 and 7 h of
incubation, respectively, which was a statistically significant
difference (by unpaired t test, P < 0.05).
Similarly, forskolin and ionomycin induced a statistically significant
stimulation after 5 h of incubation (P <
0.05).
Determination of the transcriptional initiation site
The transcriptional start site of rat VMAT-2 mRNA was determined
by primer extension analysis (Fig. 3).
Before RNA extraction, PC12 cells were stimulated with forskolin
(10-5M) or
transfected with CREB expression plasmid and rVMAT-2. The
transcriptional start site (+1) was located 8 bp further downstream as
described in previous studies (17). One minor additional
band was detected 18 bp further downstream, which might indicate a
second transcriptional start site.
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b sites located in intron I, and two Sp1
sites within -120 bp upstream of the transcription start site. The
organization of these recognition motifs upstream is illustrated in
Fig. 4A. After transfection into PC12
cells, promoter activity of different 5'-deletion mutants was measured
by luciferase assay. As our initial studies (
Figs. 1–3) were performed
with rat ECL cells, we used the rat promoter constructs for further
detailed analysis. Deletional scanning of the rat VMAT-2 promoter was
performed by repeated exonuclease III digestion. Fig. 4B illustrates
the activity of these elements, defining the -121 bp fragment as the
minimal sequence of the rVMAT-2 promoter exhibiting full basal
activity. This region contains the CRE, AP2, and two Sp1 sites, as
shown in Fig. 4A. To elucidate the importance of these elements for
basal activity of the complete rat promoter (rVMAT-800), mutations were
introduced into the CRE as well as the two Sp1 sites located upstream
(as indicated in Fig. 4C, bottom line). Mutation of either
Sp1 site reduced the promoter activity by approximately 50–60%,
whereas mutation of the CRE reduced activity by 80%. Combined
mutations of the CRE and either one of the adjacent Sp1 sites lead to a
complete derogation of the basal promoter activity.
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, gastrin induced phosphorylation of ERK1/2 within 2.5 min of incubation,
and the signal was stronger than the positive control (PMA). Further,
activation of Sp1 and p-CREB was demonstrated by Western blotting of
nuclear extracts using Abs against Sp1 p95 and p106 and pCREB. Gastrin
stimulation led to an increase in activated transcription factors after
5 min, with a maximum after 10 min (Fig. 5B).
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). Transfection of the complete rat
VMAT-2 promoter (rVMAT-800) into PC12-G cells and consecutive
stimulation with gastrin revealed a 3-fold induction of promoter
activity. The minimal sequence necessary for complete stimulation of
promoter activity by gastrin was found to contain the Sp1 and CRE site
within 121 bp of the 5'-flanking region adjacent to exon 1. The -27
fragment failed to respond to gastrin treatment. To investigate the
role of the transcription factor recognition sites present within the
-121 bp fragment, we introduced mutations into both Sp1 sites and the
CRE. As shown in Fig. 6B, mutation of either Sp1 site decreased gastrin
responsiveness significantly. Mutation of the CRE led to a complete
derogation of the gastrin effect.
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) and stimulated
(Fig. 7, B and C) conditions. As these promoters have been cloned into
different reporter vectors, we normalized luciferase activity to the
activity obtained with empty vectors. Basal promoter activity
(unstimulated) was observed in PC12-G cells transfected with rVMAT-800,
being 2- to 3-fold higher than cells transfected with the HDC or CgA
promoter. Gastrin stimulated transcriptional activity of all three
promoters, being highest in cells with CgA and 2- to 3-fold for VMAT-2
transfections.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using highly enriched ECL cells, we determined that gastrin is an effective stimulant of VMAT-2 mRNA and protein expression. This finding is new and provides the first evidence that this hormone directly regulates the expression of a monoamine transporter. Previous studies in rats described a stimulatory gastrin effect on VMAT-2 expression in vivo over several days (22). These studies, however, could not determine a direct effect of gastrin on ECL cell gene expression, nor was the early time course investigated (22). Thereby, our experiments underline that gastrin is a rather quick stimulus of VMAT-2 mRNA and protein expression within 3–7 h of incubation, enabling a prompt adaptation to increase transport capacities during conditions of hypergastrinemia.
These same findings were observed in part by Watson et al. (17), who found up-regulation of mRNA for VMAT2 and HDC in the pre-B cell line Ea3.123 after stimulation with ionomycin. As this cell line is not of neuroendocrine origin, we investigated the regulation of VMAT-2 promoter in PC12/pheochromocytoma cells. This cell line shows high endogenous expression of VMAT, providing the transcriptional machinery necessary for promoter activation.
The rat and human promoters were cloned, and sequences (17) exhibited a homology of 35%, which is relatively high for noncoding regions. A high similarity was also observed with the mouse VMAT-2 promoter (29). All promoters contain a conserved CRE, multiple Sp1 sites, and one AP2 recognition site positioned within the 140-bp 5'-flanking region of the transcriptional start site.
Using 5'-deletional scanning analysis, we found that a -121 bp rat
fragment upstream of the transcriptional start site was able to confer
full basal promoter activity (
10-fold activity compared with
promoterless vector) in rats. The function of this minimal necessary
element was characterized using dense scanning and mutational analysis
of the rat promoter. This species was chosen for further investigation,
because our physiological data were obtained in a rat model of ECL
cells. As illustrated in Fig. 4, the two Sp1 sites (located -97 to
-88 bp and -68 to -59 bp) and a CRE (-44 to -35 bp) were
indispensable for full basal activity of the rVMAT-2 promoter. This was
confirmed by mutation of each of these recognition sites, which led to
a significant loss of the basal promoter activity, whereas combined
mutation of CRE and the Sp1 completely abolished the basal promoter
activity. Thus, a combined activation of CRE and Sp1 sites may be
responsible for full constitutive activity of the VMAT-2 promoter. A
high constitutive expression of VMAT-2 ensures a continuous
availability of amine transporters in this cell type. Basal promoter
activity appears to be maintained by activation of Sp1 and CRE
recognition motifs adjacent to exon 1, which are highly evolutionary
conserved in tandem in the rat, human, and mouse promoter (17, 18, 29).
Sp1 and CREB have also been identified as growth factor-inducible transcription factors. For example, PC12 cells treated with nerve growth factor react with phosphorylation of CREB at Ser133 (30, 31). Sp1 is activated by diverse signaling pathways after stimulation with growth factors in several tissues, especially by PKC-related pathways (32, 33, 34). This led us to the assumption that Sp1 and CREB are also gastrin-inducible transcription factors that could mediate a stimulatory effect on VMAT-2 expression. We therefore generated and characterized a gastrin-responsive cell line, PC12-G, to investigate the gastrin effect in detail. Indeed, incubation of PC12-G cells with gastrin induced ERK1/2 phosphorylation and Sp1 activation and activated the VMAT-2 promoter. The gastrin effect was obtained with the identical 121-bp fragment responsible for full basal activity. Mutation of the Sp1 sites or the CRE recognition motif decreased gastrin-stimulated promoter activity significantly, demonstrating that gastrin controls transcriptional regulation of VMAT-2 via Sp1 and CREB.
Transcriptional activation of the VMAT-2 and CgA promoter by gastrin might share several similarities. Previous studies reported that gastrin stimulated the CgA promoter in an Sp1- and CRE-dependent process (21). Although gastrin-induced transcriptional activation of CgA appears to be similar to transcriptional stimulation of VMAT-2, the gastrin effect on the HDC promoter is mediated by a distinct gastrin-responsive element within 27 bp upstream of the translational start site (35, 36).
Therefore, the complete promoters of HDC, CgA, or VMAT-2 were directly compared in PC12-G cells. Interestingly, significant differences regarding basal and stimulated promoter activities became evident; the VMAT-2 promoter had the highest activity under basal conditions and was 2.5-fold inducible by gastrin. This observation may reflect physiological conditions. Although there is a low abundance of histamine under basal conditions, stimulation with gastrin leads to increased histamine synthesis within 1–2 h of stimulation. Therefore, a high amount of basal VMAT-2 protein expression is required for the immediate transport of the amine into these vesicles. During hypergastrinemia, expression of this protein is adjusted to the increased cellular histamine content after 5–7 h of gastrin exposure, and therefore, expression of VMAT-2 is increased through a direct effect of gastrin.
In summary, VMAT-2 expression is clearly induced by gastrin in gastric ECL cells. Using a gastrin-responsive cell line as a model, we show that this induction is regulated on the transcriptional level, mainly involving Sp1 and CRE. Our results thereby underline that this hormone has pleiotropic effects, especially on ECL cells, controlling not only histamine release and synthesis, but also histamine storage. Gastrin acts as coordinating hormone, leading to a functional synergy of these enzymes, matrix proteins, and transporters, thereby adapting cellular functions to the differential physiological demands.
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Acknowledgments |
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Footnotes |
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Abbreviations: CgA, Chromogranin A; CCK-B, cholecystokinin-B; CRE, cAMP-responsive element; CREB, CRE binding protein; ECL, enterochromaffin-like; ERK, extracellular signal related kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSP, gene-specific primer; HDC, histidine decarboxylase; h, human; r, rat; VMAT, vesicular monoamine transporter.
Received January 4, 2001.
Accepted for publication April 9, 2001.
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