[VEGF-C, VEGF-D and VEGF-receptor 3: novel key regulators of lymphangiogenesis and cancer metastasis].
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Animals↗
Biomedical subjects
M Höcker
Publications and source records attributed to M Höcker.
[Helicobacter pylori and distal gastric adenocarcinoma: Did we underestimate the pathogenic importance of the bacterium?].
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Adenocarcinoma↗
Neuroendocrine-specific and gastrin-dependent expression of a chromogranin A-luciferase fusion gene in transgenic mice.
BACKGROUND AND AIMS: Chromogranin A (CgA) is a multifunctional acidic protein specifically expressed in neuroendocrine cells. In the stomach, CgA is found predominantly in enterochromaffin-like (ECL) cells, where it is regulated by gastrin. We investigated the ability of a promoter fragment comprising 4.8 kb of 5'-flanking DNA of the mouse CgA (mCgA) gene to direct cell-specific expression as well as gastrin responsiveness in the gastroenteropancreatic neuroendocrine system. METHODS: Two independent lines of mCgA 4.8 kb-luc transgenic mice were created. Transgene expression was assessed by determination of luciferase activity and reverse-transcription polymerase chain reaction analysis of luciferase messenger RNA. Cell specificity of transgene expression was investigated by immunohistochemical analysis. The influence of hypergastrinemia on transgene expression was determined after repeated omeprazole injections. RESULTS: In both transgenic lines, mCgA 4.8 kb-luc expression paralleled the expression pattern of the endogenous CgA gene. ECL cells were identified as the major gastric cell population expressing the transgene. Omeprazole treatment stimulated expression of the transgene and the endogenous CgA gene selectivity in the gastric corpus (3-4-fold). CONCLUSIONS: mCgA 5'-flanking DNA (4.8 kb) contain the major cis-regulatory element(s) required for cell-specific CgA expression in the neuroendocrine system and gastrin-responsiveness in the gastric corpus. Further analysis of the CgA promoter in transgenic studies may elucidate the general molecular mechanisms underlying cell-specific gene expression in the gastroenteropancreatic neuroendocrine system.
Animals↗
Isolation, maintenance, and characterization of human pancreatic islet tumor cells expressing vasoactive intestinal peptide.
Tissue from a vasoactive intestinal peptide (VIP)-secreting human tumor has been used to establish and characterize human neuroendocrine primary cell cultures from which permanent, clone-derived cell lines have been established. Viable cells were obtained by enzymatic and mechanical dissociation of freshly resected pancreatic islet tumor and hepatic metastatic tumor tissues. Aliquots of tumor cells were established ex vivo under culture conditions including porous substrata coated with type IV collagen and laminin and a low serum, hormonally defined culture medium. The small (<10 microm) rounded, grape-like cells had a very slow growth rate of doubling times estimated at several weeks or more. After several passages, morphologically uniform cells were derived that strongly expressed neuroendocrine markers of synaptophysin and synaptobrevin. Although chromogranin A and VIP had somewhat weaker expression, both demonstrated phorbol ester-stimulated secretion. The morphologic and secretory properties were maintained by the cells for nearly 2 years in culture. The establishment of this novel VIP-secreting human neuroendocrine cell line (HuNET) makes available a culture model with which to study a transformed version of this pancreatic islet cell type and offers approaches by which to establish islet tumor cell lines.
Adult↗
Dual mechanism of vascular endothelial growth factor upregulation by hypoxia in human hepatocellular carcinoma.
BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) plays a key role in regulation of tumour associated angiogenesis. In the current study we analysed expression of VEGF and its receptors in human hepatocellular carcinoma (HCC) and investigated the molecular mechanisms of VEGF regulation by hypoxia. METHODS: VEGF, kinase domain region (KDR)/fetal liver kinase 1 (flk-1), and flt-1 expression were examined by immunohistochemistry and in situ hybridisation in 15 human HCC tissues. Expression of VEGF and regulation by hypoxia were assessed in three human HCC cell lines using a quantitative competitive reverse transcription-polymerase chain reaction, ELISA, and a series of 5' deletion reporter gene constructs of the human VEGF promoter in transient transfection assays. RESULTS: We observed over expression of VEGF mRNA and protein in HCC compared with cirrhosis or normal liver. Expression of VEGF in tumour cells was strongly increased in areas directly adjacent to necrotic/hypoxic regions. Both VEGF receptors were detected in vascular endothelia of HCC while only KDR/flk-1 receptors were detected in endothelial cells of cirrhotic livers. Expression of VEGF was observed in all human HCC cell lines examined. Hypoxia (1% oxygen) resulted in profound upregulation of VEGF mRNA and protein levels. Furthermore, hypoxia treatment resulted in a doubling of VEGF mRNA stability. Deletion analysis of the human VEGF 5' flanking region -2018 and +50 demonstrated induction of VEGF promoter activity under hypoxic conditions which was significantly decreased following deletion of the region -1286 and -789 suggesting a substantial contribution of the -975 putative hypoxia inducible factor 1 binding site to hypoxia mediated transcriptional activation of the VEGF gene. CONCLUSION: These data suggest hypoxia as a central stimulus of angiogenesis in human HCC through upregulation of VEGF gene expression by at least two distinct molecular mechanisms: activation of VEGF gene transcription and an increase in VEGF mRNA stability.
Analysis of Variance↗
Gastrin induces expression and promoter activity of the vesicular monoamine transporter subtype 2.
Gastric enterochromaffin-like cells produce histamine in response to the antral hormone gastrin and accumulate the biogenic amine in secretory organelles via vesicular monoamine transporter subtype 2. The putative effects of gastrin on vesicular monoamine transporter subtype 2 expression and promoter activity are poorly understood. In the present study we used highly enriched rat enterochromaffin-like cells (purity, >90%) and rat pheochromocytoma cells stably transfected with a gastrin/cholecystokinin B receptor to investigate the expression and transcriptional regulation of vesicular monoamine transporter subtype 2. Stimulation of vesicular monoamine transporter subtype 2 mRNA and protein expression was observed in isolated enterochromaffin-like cells after 3- to 7-h incubation with gastrin (10(-7) M), forskolin (10(-5) M), or ionomycin (10(-5) M). Deletion analysis of the rat vesicular monoamine transporter subtype 2 promoter defined the minimal promoter sequence necessary for full basal activity as a -121 bp segment upstream of exon 1 containing two Sp1 sites (-97 to -88 bp and -68 to -59 bp) and a cAMP-responsive element (-44 to -35 bp). Gastrin (10(-7) M) stimulated extracellular signal related kinase1/2 phosphorylation, activated Sp1 and cAMP-responsive element-binding protein, and further induced activity of the complete rat vesicular monoamine transporter subtype 2 promoter (-800 bp) in gastrin/cholecystokinin B receptor cells. The -121-bp fragment was able to confer full gastrin responsiveness, and site-directed mutagenesis of the Sp1 and cAMP-responsive element motifs demonstrated their crucial importance for basal and inducible activities. Comparison of promoter activity of histidine decarboxylase, chromogranin A, or vesicular monoamine transporter subtype 2 in transfected cell lines revealed significant differences in basal and gastrin-stimulated activities. Our current study provides the first evidence that gastrin directly stimulates the expression and promoter activity of vesicular monoamine transporter subtype 2. Sp1 and cAMP-responsive element-binding protein recognition motifs located within 121 bp upstream of exon 1 appear to be indispensable for full basal and inducible promoter activities. Diverging effects of gastrin on histidine decarboxylase, chromogranin A, and vesicular monoamine transporter subtype 2 promoter may account for the coordinated synthesis and storage of histamine in this neuroendocrine cell type.
Animals↗
Helicobacter pylori activates the histidine decarboxylase promoter through a mitogen-activated protein kinase pathway independent of pathogenicity island-encoded virulence factors.
Helicobacter pylori infection of the gastric mucosa is accompanied by an activated histamine metabolism. Histamine plays a central role in the regulation of gastric acid secretion and is involved in the pathogenesis of gastroduodenal ulcerations. Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine production, and its activity is regulated through transcriptional mechanisms. The present study investigated the effect of H. pylori infection on the transcriptional activity of the human HDC (hHDC) promoter in a gastric epithelial cell line (AGS) and analyzed the underlying molecular mechanisms. Our studies demonstrate that H. pylori infection potently transactivated the hHDC promoter. The H. pylori-responsive element of the hHDC gene was mapped to the sequence +1 to +27 base pairs, which shows no homology to known cis-acting elements and also functions as a gastrin-responsive element. H. pylori regulates the activity of this element via a Raf-1/MEK/ERK pathway, which was activated in a Ras-independent manner. Furthermore, we found that H. pylori-induced transactivation of the hHDC promoter was independent of the cag pathogenicity island and the vacuolating cytotoxin A gene and therefore may be exerted through (a) new virulence factor(s). A better understanding of H. pylori-directed hHDC transcription can provide novel insights into the molecular mechanisms of H. pylori-dependent gene regulation in gastric epithelial cells and may lead to new therapeutic approaches.
Helicobacter pylori↗
New molecular aspects in the diagnosis and therapy of neuroendocrine tumors of the gastroenteropancreatic system.
The nature and biology of neuroendocrine cells and of tumors derived therefrom have been the subject of intense research using cell biological and molecular approaches. Diagnostic procedures for establishing the diagnosis of a neuroendocrine tumor have been improved through the development of new serological markers and imaging procedures. Histopathological diagnosis has been refined by the introduction of a broad spectrum of marker proteins for different subtypes of neuroendocrine neoplasms. The high receptor specificity of somatostatin analogues such as octreotide or lanreotide has made these drugs valuable tools in diagnosis and therapy, and some of the achievements made as well as future directions are reviewed in this article. Another substance in use for therapy of neuroendocrine tumors is interferon-a, whose signal transduction mechanism has been investigated considerably during the past several years. In addition to biotherapy with somatostatin analogues and/or interferon-a, chemotherapy is an accepted strategy in the treatment of advanced neuroendocrine tumor disease derived from the foregut. In this context, streptozotocin has caught some attention due to its somewhat selective toxicity against neuroendocrine tumor cells. Some recent studies on the role of the glucose transporter isoform GLUT2 may provide insight into streptozotocin's action. The multiple endocrine neoplasia type-1 gene has recently been cloned, sequenced and identified as a gene potentially involved in the development of the familial cancer syndrome of multiple endocrine neoplasia type 1 (MEN-1). Mutations of this putative tumor suppressor gene have been described, and the abundance of mutations in MEN-1-related tumors as well as sporadic neuroendocrine tumors at MEN-1 locations have been demonstrated. Whether determination of MEN-1 mutations will be valuable for clinical routine is under investigation.
Animals↗
De novo expression of vascular endothelial growth factor in human pancreatic cancer: evidence for an autocrine mitogenic loop.
BACKGROUND & AIMS: The role of vascular endothelial growth factor (VEGF) and its receptors in tumor angiogenesis has been well established. We analyzed the expression pattern and biologic significance of VEGF and its receptors in human pancreatic cancer. METHODS: VEGF, KDR/flk-1, and flt-1 expression were examined by immunohistochemistry, in situ hybridization, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and receptor phosphorylation. VEGF-stimulated mitogenesis was investigated by mitogen-activated protein kinase (MAPK) phosphorylation, transactivation of a c-fos promoter reporter construct, DNA synthesis assays, and stable transfection of a dominant-negative flk-1 complementary DNA (cDNA) construct. RESULTS: Compared with normal pancreas and chronic pancreatitis, VEGF and its receptors were overexpressed in pancreatic cancer. KDR and flt-1 were detected not only in endothelial cells but also in tumor cells. VEGF expression was observed in all human pancreatic tumor cell lines examined, and the KDR/flk-1 and flt-1 receptor was detected in 2 cell lines. VEGF treatment results in phosphorylation of MAPKs, transactivation of a c-fos promoter construct, and growth stimulation in KDR/flk-1-expressing cell lines, which could be blocked by VEGF antagonists. Furthermore, stable transfection of a dominant-negative flk-1 cDNA significantly inhibited tumor cell growth. CONCLUSIONS: These results not only support the important role of the VEGF/VEGF receptor system in pancreatic tumor biology but also suggest the existence of an autocrine/paracrine mitogenic loop for pancreatic cancer cells.
Adenocarcinoma↗
Interferon-alpha inhibits chromogranin A promoter activity in neuroendocrine pancreatic cancer cells.
Interferon-alpha (IFN-alpha) treatment can suppress the hypersecretion syndrome associated with functional neuroendocrine tumors. Chromogranin A (CgA) is a matrix protein of neuroendocrine secretory vesicles and appears to be essential for an appropriate neuroendocrine secretory function. To test the hypothesis that IFN-alpha can directly interfere with CgA gene transcription, we performed transient transfection studies in pancreatic neuroendocrine tumor cells employing CgA-luciferase reporter gene constructs showing that IFN-alpha inhibited basal and protein kinase C-dependent CgA promoter activity. Using 5'-deletion constructs in combination with mutational analysis of the proximal CgA core promoter, a cyclic AMP response element (CRE) at -71 to -64 bp was identified as the IFN-alpha response element of the CgA gene. Furthermore, functional studies indicated that IFN-alpha exerts its effect on the CgA promoter via interference with CRE binding protein (CREB)/CREB binding protein (CBP)-dependent transactivation of the CgA-CRE.
Animals↗
Activation of human histidine decarboxylase gene promoter activity by gastrin is mediated by two distinct nuclear factors.
The human histidine decarboxylase gene is regulated by gastrin through a cis-acting element known as the gastrin response element (GAS-RE) that was initially localized to a site (+2 to +24) downstream of the transcriptional start site. Electrophoretic mobility shift assays using sequentially deleted DNA probes and nuclear extracts from AGS-B gastric cancer cells showed that the GAS-RE is actually composed of two overlapping binding sites (GAS-RE1, +1 to +19; and GAS-RE2, +11 to +27) that bind distinct nuclear factors. Reporter gene assays demonstrated that each element alone could confer gastrin responsiveness, but the presence of both elements was required for complete gastrin response. Stimulation of AGS-B cells with gastrin for 10-20 min resulted in a >2-fold increase in factor binding. The binding was inhibited by pretreatment of AGS-B cells with cycloheximide and the MEK1 inhibitor PD98059, indicating a requirement for protein synthesis and also indicating that activation occurs through the MEK/mitogen-activated protein kinase pathway. UV cross-linking and Southwestern blot analysis showed that GAS-RE1 bound a 52-kDa protein, whereas GAS-RE2 bound a 35-kDa protein. Hence, activation of histidine decarboxylase gene promoter activity by gastrin is most likely mediated by two separate nuclear factors.
Base Sequence↗
Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells: an immunohistochemical analysis.
Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs; islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and synaptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation.
Aspartic Acid Endopeptidases↗
Therapeutic and diagnostic implications of the somatostatin system in gastroenteropancreatic neuroendocrine tumour disease.
Somatostatin exerts its actions through interaction with specific heptahelical G-protein coupled plasma membrane receptors. Five different somatostatin receptor subtypes have been cloned in man. Different receptor subtypes are coupled to different intracellular transmission cascades in a cell type-dependent manner. In general, somatostatin affects cell proliferation either directly: reducing mitogen-activated protein kinase cascade, activating phosphoproteinphosphatase, stimulating EGF-receptors and adenylate cyclase activity; or indirectly reducing the release of autocrine- and/or paracrine-acting growth factors. Somatostatin can exert cytotoxic (G1 phase cell arrest) or cytostatic (apoptosis induction) effects, also depending on the receptor subtype expressed on the target cell. In gastroenteropancreatic neuroendocrine tumours predominance of sst1 and sst2 with a lesser extent of sst3 and sst5 subtype receptors have been demonstrated using sensitive methods. Synthetic analogues with specific decreasing affinity for sst2 > sst5 > sst3 receptor subtypes have been used as antiproliferative drug in the treatment of gastroenteropancreatic tumours. These compounds (octreotide, lanreotide) resulted in a modest growth-inhibition activity either in functioning or in non-functioning tumours. Combination of somatostatin analogues with alpha-interferon produced a more pronounced antiproliferative effect overcoming therapy resistance developed to either single drug. Finally, the development of radio-labelled somatostatin analogue scintigraphy has contributed to gastroenteropancreatic-tumours lesion localization and future more detailed knowledge of somatostatin receptor mechanisms could improve both the diagnostic and therapeutic application of somatostatin analogues.
Female↗
Sp1 and CREB mediate gastrin-dependent regulation of chromogranin A promoter activity in gastric carcinoma cells.
Chromogranin A (CgA) is a multifunctional acidic protein that in the stomach is expressed predominantly in enterochromaffin-like cells (ECL cells) where it is regulated by gastrin. In order to investigate the transcriptional response of the mouse CgA (mCgA) promoter to gastrin stimulation, we studied a 4.8-kilobase mCgA promoter-luciferase reporter gene construct in transiently transfected AGS-B cells. 5'-Deletion analysis and scanning mutagenesis of mCgA 5'-flanking DNA showed that a Sp1/Egr-1 site spanning -88 to -77 base pairs (bp) and a cyclic AMP-responsive element (CRE) at -71 to -64 bp are essential for gastrin-dependent mCgA transactivation. Gastrin stimulation increased cellular Sp1 protein levels and Sp1-binding to the mCgA -88 to -77 bp element, as well as binding of CREB to its consensus motif at -71 to -64 bp. Gastrin also stimulated CREB Ser-133 phosphorylation, and abundance of cellular CREB protein levels. Overexpression of either Sp1 or phosphorylated CREB transactivated the mCgA promoter dose dependently, while coexpression of both transcription factors resulted in an additive mCgA promoter response. mCgA -92 to -64 bp, comprising the Sp1/Egr-1 site and the CRE motif, conferred gastrin responsiveness to a heterologous thymidine kinase promoter system, and therefore functions as a "true" enhancer element. This report demonstrates that Sp1 and CREB mediate CCK-B/gastrin receptor-dependent gene regulation, and that the effect of gastrin on the CgA gene is brought about by cooperative action of both transcription factors.
Animals↗
Molecular mechanisms of enteroendocrine differentiation.
Passing through a complex series of developmental steps, the visceral endoderm differentiates into four intestinal epithelial lineages comprising enterocytes, goblet cells, paneth cells, and enteroendocrine cells. The intestinal enteroendocrine system consists of at least 15 different cell types, which can be classified on the basis of morphological criteria, expression of secretory products, and abundance of specific marker molecules. During intestinal development and in the adult gut, neuroendocrine subpopulations display strictly controlled differences in their geographical distribution that go along with dramatic differences in cell type-specific gene expression. Identification to transcription factors and regulatory DNA elements responsible for cell-specific gene expression in different neuroendocrine cell types as well as various transgenic and "knock-out" mouse models have largely added to our understanding of mechanisms controlling appropriate special and temporal activation of enteroendocrine differentiation programs. This article reviews current in vitro and in vivo studies analyzing different molecular aspects of enteroendocrine differentiation. In addition, the influence of intestinal diseases including malignant transformation on enteroendocrine differentiation and the underlying mechanisms will be discussed.
Animals↗
Oxidative stress activates the human histidine decarboxylase promoter in AGS gastric cancer cells.
Oxidant stress is thought to play a role in the pathogenesis of many gastric disorders. We have recently reported that histidine decarboxylase (HDC) promoter activity is stimulated by gastrin through a protein kinase C- and extracellular signal-regulating kinase (ERK)-dependent pathway in gastric cancer (AGS-B) cells, and this transcriptional response is mediated by a downstream cis-acting element, the gastrin response element (GAS-RE). To study the mechanism through which oxidant stress affects gastric cells, we examined the effects of hydrogen peroxide (H2O2) on HDC promoter activity and intracellular signaling in AGS-B cells. H2O2 (10 mM) specifically activated the HDC promoter 10-12-fold, and this activation was blocked by both mannitol and N-acetylcysteine. Hydrogen peroxide treatment of AGS-B cells increased the phosphorylation and kinase activity of ERK-1 and ERK-2, but did not affect Jun kinase tyrosine phosphorylation or kinase activity. In addition, treatment of AGS-B cells with H2O2 resulted in increased c-fos/c-jun mRNA expression and AP-1 activity, and also led to increased phosphorylation of epidermal growth factor receptor (EGFR) and Shc. H2O2-dependent stimulation of HDC promoter activity was completely inhibited by kinase-deficient ERKs, dominant-negative (N17 and N15) Ras, and dominant-negative Raf, and partially blocked by a dominant-negative EGFR mutant. In contrast, protein kinase C blockade did not inhibit H2O2-dependent induction of the HDC promoter. Finally, deletion analysis demonstrated that the H2O2 response element could be mapped to the GAS-RE (nucleotides 2 to 24) of the basal HDC promoter. Overall, these studies suggest that oxidant stress activates the HDC promoter through the GAS-RE, and through an Ras-, Raf-, and ERK-dependent pathway at least partially involving the EGFR.
Adaptor Proteins, Signal Transducing↗
Gastrin and phorbol 12-myristate 13-acetate regulate the human histidine decarboxylase promoter through Raf-dependent activation of extracellular signal-regulated kinase-related signaling pathways in gastric cancer cells.
Gastrin stimulates transcription of the human histidine decarboxylase (HDC) gene through binding to the G-protein-coupled cholecystokinin-B/gastrin receptor. We have explored the possibility that mitogen-activated protein kinase cascades play a role in mediating the effects of gastrin on transcription in a gastric cancer (AGS-B) cell line. Gastrin and phorbol 12-myristate 13-acetate (PMA) treatment of AGS-B cells was found to increase the phosphorylation of tyrosine residues of extracellular signal-regulated kinases (ERKs) 1 and 2 and increase ERK activity as determined by the in vitro phosphorylation of myelin basic protein. Reporter gene assays also demonstrated that gastrin and PMA stimulated Elk-1- and c-Myc-dependent transactivation, consistent with gastrin- and PMA-induced activation of ERKs. Overexpression of wild type ERK-1 and ERK-2 or activation of endogenous ERKs using activated MEK-1 (mitogen-activated protein kinase kinase or ERK kinase) overexpression stimulated HDC promoter activity in a dose-dependent fashion. Interruption of the ERK-related pathway using expression vectors for kinase-deficient ERKs or an ERK-specific phosphatase (PAC-1) blocked gastrin- and PMA-stimulated HDC promoter activity. In contrast, inhibition of the Jun kinase pathway using an interfering dominant negative SEK-1 (stress-activated protein kinase/ERK-1) mutant did not inhibit HDC promoter activity. Furthermore, whereas gastrin stimulated phosphorylation of Shc proteins and association with Grb2, activation of the HDC promoter was not influenced by expression of dominant negative Ras (N15 or N17) proteins. However, gastrin stimulated Raf-1 kinase activity, and activation of the HDC promoter was blocked by coexpression of a dominant negative Raf-1 construct. Overall, these data demonstrate that gastrin regulates HDC transcription in a Rafdependent, Ras-independent fashion predominantly through activation of the ERK-related pathway.
Base Sequence↗
Gastrin regulates the human histidine decarboxylase promoter through an AP-1-dependent mechanism.
The histidine decarboxylase (HDC) gene is regulated transcriptionally by gastrin and phorbol 12-myristate 13-acetate (PMA) through a protein kinase C (PKC)-related pathway. To determine the role of AP-1 (fos/jun) in the regulation of the HDC promoter, gastric cancer (AGS-B) cells stably expressing the cholecystokinin-B/ gastrin receptor and the 1.8-kb human (h) HDC-luciferase (luc) construct were cotransfected with constructs expressing c-fos and c-jun. Overexpression of c-fos and c-jun activated the HDC promoter in a dose-dependent fashion in 1.8-kb hHDC-luc/AGS-B cells as well as in transfected F9 embryonal carcinoma cells, which lack endogenous AP-1 activity. PMA was unable to activate the HDC promoter in F9 cells, which were not transfected with c-fos and c-jun. Gastrin stimulation increased c-fos and c-jun mRNA abundance and AP-1-dependent transcriptional activity, as assessed by a reporter construct in which the CAT reporter gene is under the control of a 12-O-tetradecanoylphorbol-13-acetate response element multimer. Gastrin-stimulated HDC promoter activity was blocked by transfection of c-fos antisense and dominant negative c-jun expression constructs. Finally, overexpression of c-fos and c-jun activated the hHDC promoter through a downstream cis-acting element (gastrin response element), which does not bind AP-1. In conclusion, activation of AP-1 is essential for gastrin-stimulated HDC transcription, but the mechanism appears to be indirect.
Antisense Elements (Genetics)↗