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

Vesicular monoamine transporter type 2 (VMAT2) is crucial for accumulation of monoamine neurotranmitters into neuronal secretory vesicles and histamine into secretory granules of the enterochromaffin-like cell in the acid-secreting gastric mucosa. Gastric VMAT2 expression is regulated by the antral hormone gastrin acting at the CCK 2 receptor. We demonstrate a gastrin response element ؊56 ccgccccctc ؊47 in the proximal VMAT2 promoter that binds in a gastrin-sensitive manner to nuclear proteins from gastric epithelial cell lines. Mutations within this sequence prevented nuclear protein binding and significantly reduced gastrin-stimulated expression of VMAT2 promoter-reporter constructs in gastric epithelial cells. In a yeast one-hybrid screen of an AR42J cell cDNA library, using the gastrin response element as bait, we identified a beta subunit of the 20 S proteasome, PSMB1, as a potential binding partner. In supershift assays, antibodies to PSMB1 and other proteasome beta subunits disrupted gastrin sensitive nuclear protein binding to the VMAT2 promoter. Moreover, RNA interference of PSMB1 significantly inhibited gastrin-mediated VMAT2 transcription. These data suggest that elements of the 20 S proteasome interact with the VMAT2 promoter to enhance G-proteincoupled receptor-mediated transcription.The vesicular monoamine transporters (VMATs) 2 are responsible for the energy-dependent accumulation of biogenic amines into secretory vesicles of amine producing cells and thus play central roles in cellular physiology. The two members of this family, VMATs 1 and 2, have similar structures and mechanisms of action, but differ in their cellular localization and substrate specificity (1-4). VMAT2 (SLC18A2) is expressed in all monoaminergic neurones of the central nervous system, notably e.g. in dopaminergic neurones of the substantia nigra and histaminergic posterior hypothalamic neurones. Support for the idea that VMAT2 is important for neuronal function comes from studies in VMAT2 knock-out mice. Homozygotes have seriously disrupted patterns of feeding and locomotion and are poorly viable. Heterozygotes are grossly normal but have reduced central monoamine levels and are hypersensitive to the locomotor effects of cocaine and amphetamine and are more sensitive to the neurotoxin 1-methyl-(4-phenyl-1,2,3,6-tetrahydropyridine) (MPTP) and to 3,4-dihydroxyphenylalanine (L-DOPA) (5-7). VMAT2 is able to sequester from cytoplasm, toxins, and transmitters that cause Parkinson-like syndromes, and gain of function haplotypes have been reported to confer protection against Parkinson's disease (8). Transporter affinities for serotonin and the catecholamines are in the low micromolar range and are broadly similar for both VMATs. However, only VMAT2 has micromolar affinity for histamine (3). The ability of VMAT2 to accumulate histamine in secretory vesicles is consistent with its localization in central histaminergic neurones and in enterochromaffin-like (ECL) cells of the gastric epithelium, its only non-neuronal location (4). Hist...

Section: Discussionmentioning

confidence: 99%

Identification of a Gastrin Response Element in the Vesicular Monoamine Transporter Type 2 Promoter and Requirement of 20 S Proteasome Subunits for Transcriptional Activity

Catlow

Ashurst

Varró

et al. 2007

“…For example, gastrin-induced activation of the chromogranin A promoter was mediated by a Sp1/Egr1 binding site (21). In addition, gastrin has been shown to up-regulate the vesicular monoamine transporter-2 promoter through a AP-2/Sp-1 binding site (22).…”

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confidence: 99%

Kruppel-like Factor 4 (KLF4) Represses Histidine Decarboxylase Gene Expression through an Upstream Sp1 Site and Downstream Gastrin Responsive Elements

Liu

Langlois

et al. 2004

Self Cite

Histidine decarboxylase (HDC) is the enzyme that catalyzes the conversion of histidine to histamine, a bioamine that plays an important role in allergic responses, inflammation, neurotransmission, and gastric acid secretion. Previously, we demonstrated that gastrin activates HDC promoter activity in a gastric cancer (AGS-E) cell line through three overlapping downstream promoter elements. In the current study, we used the yeast one-hybrid strategy to identify nuclear factors that bind to these three elements. Among eight positives from the one-hybrid screen, we identified Kruppel-like factor 4 (KLF4) (previously known as gut-enriched Kruppel-like factor (GKLF)) as one factor that binds to the gastrin responsive elements in the HDC promoter. Electrophoretic mobility shift assays confirmed that KLF4 is able to bind all three gastrin responsive elements. In addition, transient cotransfection experiments showed that overexpression of KLF4 dose dependently and specifically inhibited HDC promoter activity. Regulation of HDC transcription by KLF4 was confirmed by changes in the endogenous HDC messenger RNA by KLF4 small interfering RNA and KLF4 overexpression. We further showed that KLF4 inhibits HDC promoter activity by competing with Sp1 at the upstream GC box and also independently by binding the three downstream gastrin responsive elements. Taken together, these results indicate that KLF4 can act to repress HDC gene expression by Sp1-dependent and -independent mechanisms.Histamine is a bioamine that plays an important role in many physiological processes, including allergy, inflammation, neurotransmission, and gastric acid secretion (1-3). Histidine decarboxylase (HDC) 1 is the single enzyme that converts histidine to histamine (4). HDC is expressed in many different cell types, including mast cells, skin cells, platelets, and basophils. However, in the adult mammals, HDC is highly expressed in enterochromaffin-like cells, where the HDC activity is tightly regulated by a gut peptide hormone, gastrin (5). HDC regulation occurs at both the transcriptional and post-translational levels, the latter by proteolytic processing through the ubiquitin-proteasome pathway (6, 7).HDC promoter activity is up-regulated by several different stimuli, including gastrin (8), phorbol ester phorbol 12-myristate 13-acetate (8 -11), oxidative stress (12), thrombopointin (13), and Helicobacter pylori infection (14, 15). Whereas not all of the cis-acting DNA elements or the transcriptional factors involved in regulation of HDC transcription have been identified, three GC-rich gastrin responsive elements located downstream of the transcription initiation site have been characterized in the human HDC promoter region (16,17). Through the use of Southwestern blot and UV cross-linking, the sizes of the three gastrin responsive element-binding factors have previously been assessed at 53, 33, and 110 kDa of apparent molecular mass, respectively (16,17). Recently, a neural peptide pituitary adenylate cyclase-activating polypeptide has been reporte...

“…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 (4, 5, 20 -23). 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 (24 -26).Indeed, gastrin has been shown to transcriptionally activate both the HDC and CgA promoters (27)(28)(29). 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 two cis-acting overlapping binding sites (GAS-RE1, ϩ1 to ϩ19; and GAS-RE2 ϩ11 to ϩ27) (30 -32).…”

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confidence: 99%

“…Indeed, gastrin has been shown to transcriptionally activate both the HDC and CgA promoters (27)(28)(29). 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 two cis-acting overlapping binding sites (GAS-RE1, ϩ1 to ϩ19; and GAS-RE2 ϩ11 to ϩ27) (30 -32).…”

mentioning

confidence: 99%

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

Watson

Kiernan

Deavall

et al. 2001

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. Gastrininduced transcription was dependent on the cooperative interaction of an uncharacterized nuclear factor of ϳ23.3 kDa that bound to the putative AP2/SP1 site, CREbinding 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.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 (1-5). 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 (6, 7).Studies on VMAT2 knockout mice support the idea that vesicular monoamine transport is important in maintaining neuronal function with heterozygote VMAT2ϩ/Ϫ mice having dysfunctional monoamine storage and release parameters (6,8). 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 (12-14). Histamine is synthesized within the cytosol of the ECL cell from L-histidine by the action of the enzyme L-histidine decarboxylase (HDC) (15) 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 (16 -21). Evidence suggests that VMAT2 may be up-regulated to accommodate the increased histamin...