Human Vasoactive Intestinal Peptide Gene Research Articles

A restrictive element 1 (RE-1) in the VIP gene modulates transcription in neuronal and non-neuronal cells in collaboration with an upstream tissue specifier element.

The vasoactive intestinal peptide (VIP) gene has been studied extensively as a prototype neuronal gene containing multiple cis-active elements that confer responsiveness to cell lineage, neurotrophic, and activity-dependent intrinsic and extrinsic cues. However, reporter genes containing the presumptive complete regulatory region 5' to the start of transcription do not confer tissue-specific gene expression in vivo. We therefore sought cis-regulatory elements downstream of the transcriptional start that might confer additional tissue-specific and tissue-restrictive properties to the VIP transcriptional unit. We report here a repressor element, similar to the canonical restrictive element-1 (RE-1), located within the first non-coding exon of the human VIP gene. The ability of this element to regulate VIP reporter gene expression in neuroblastoma and fibroblastic cells was examined. Endogenous VIP expression is high in SH-EP neuroblastoma cells, low but inducible in SH-SY5Y cells, and absent in HeLa cells. Endogenous RE-1 silencer factor (REST) expression was highest in SH-EP and HeLa cells, and significantly lower in SH-SY5Y cells. Transient transfection of a VIP reporter gene containing a mutated RE-1 sequence revealed an RE-1-dependent regulation of VIP gene expression in all three cell types, with regulation greatest in cells (SH-EP, HeLa) with highest levels of REST expression. Serial truncation of the VIP reporter gene further revealed a specific interaction between the RE-1 and a tissue-specifier element located 5 kb upstream in the VIP gene. Thus, REST can regulate VIP gene expression in both neuroblastic and non-neuronal cells, but requires coupling to the upstream tissue specifier element.

VIP Gene Transcription Is Regulated by Far Upstream Enhancer and Repressor Elements

SK-N-SH human neuroblastoma subclones differ widely in basal and second messenger induction of the gene encoding the neuropeptide vasoactive intestinal peptide (VIP). These differences were recapitulated by a chimeric gene which consisted of 5.2 kb of the human VIP gene 5′ flanking sequence fused to a reporter. Subsequent gene deletion experiments revealed several regulatory regions on the gene, including a 645-bp sequence located approximately 4.0 upstream from the transcription start site. Here we examined this upstream region in detail. Inhibitory sequences were found to be present on each end of the 645-bp fragment. When removed, basal transcription increased more than 50-fold. Subsequent deletion/mutation analysis showed that the 213-bp fragment contained at least two enhancer elements. One of these was localized to an AT-rich 42-bp sequence shown by others to bind Oct proteins in neuroblastoma cells, while the other corresponded to a composite AP-1/ets element. In addition to these enhancers, a 28-bp sequence on the 213-bp fragment with no apparent homology to known silencers inhibited transcription. The studies provide molecular details of a complex regulatory region on the VIP gene that is likely to be used to finely tune the level of gene transcription in vivo.

Targeting of embryonic and postnatal autonomic and enteric neurons with a vasoactive intestinal peptide transgene.

The neuropeptide vasoactive intestinal peptide (VIP) is expressed in several distinct sites in the CNS, in cholinergic and enteric ganglia, and in a small subpopulation of neurons within sympathetic ganglia. Previous studies on the human VIP gene indicate that transcription in neural crest-derived neuroblastoma and pheochromocytoma cell lines is controlled in part by multiple regulatory elements located along 4.5 kb of upstream 5' flanking sequence. In the current studies, transgenic mice were created with a chimeric gene consisting of 16.5 kb of the mouse VIP gene fused to the beta-galactosidase reporter. In situ hybridization analysis in adult mice indicated that reporter gene expression was correctly targeted to neurons in the esophagus, stomach, small intestine, and colon. No expression was observed in the brain, including regions that contain abundant VIP-expressing cells, such as the thalamus, amygdala, cerebral cortex, hippocampus, and suprachiasmatic nucleus. Analysis of transgene expression in neonatal and embryonic day 13.5 mice revealed a near perfect correlation between VIP and beta-galactosidase gene expression in cranial cholinergic ganglia and the superior cervical ganglia, and lack of transgene expression in sensory ganglia and in nonneuronal tissue. Potential ectopic transgene expression was observed in neonates, in the cerebellar external granule layer and in a small subpopulation of neurons in the olfactory epithelium. We conclude that the 16.5 kb of VIP gene used in these studies contains sequences sufficient for directing expression specifically to VIP neurons in the PNS, and that sequences located elsewhere on the gene are required for proper CNS expression. The VIP gene sequences used here should be capable of targeting other gene products to specific populations of embryonic and adult peripheral neurons without causing significant expression in the CNS.

Region-specific central nervous system expression and axotomy-induced regulation in sympathetic neurons of a VIP-β-galactosidase fusion gene in transgenic mice

To assess the activity of cis-acting elements that direct human vasoactive intestinal peptide (VIP) expression in vivo, two independent transgenic mouse lines were created using a transgene comprised of 1.9 kb of 5′-flanking sequence of the human VIP gene joined to the Escherichia coli β-galactosidase reporter gene. Transgene expression in brain was assessed using β-galactosidase histochemistry and compared to the distribution of endogenous VIP expression. Transgene expression was observed in most central and peripheral nervous system sites in which endogenous VIP is expressed. We investigated whether the VIP-β-galactosidase transgene was regulated in sympathetic neurons in experimental paradigms in which VIP regulation is dependent on the release of leukemia inhibitory factor (LIF). After dissociation in vitro and postganglionic axotomy in vivo there were parallel increases in endogenous VIP and transgene expression in superior cervical ganglia. These results indicate that the 1.9 kb region of 5′-flanking sequence of the human VIP gene includes genomic elements important for cell-specific expression and LIF-dependent regulation in neurons.

Vasoactive intestinal peptide gene expression in the rat pheochromocytoma cell line PC12

Vasoactive intestinal peptide (VIP) gene expression was analyzed in PC12 cells. VIP mRNA was detected in PC12 cells treated with VIP or forskolin whereas no VIP mRNA was detected in the untreated cells. The induction of the VIP mRNA was enhanced by the simultaneous treatment with 12- O-tetradecanoylphorbol-13-acetate (TPA). PC12 cells stimulated with forskolin plus TPA released immunoreactive VIP. Sephadex G-50 column chromatography revealed that the immunoreactive VIP secreted from PC12 cells is comprised of multiple forms, one of which was indistinguishable from the authentic VIP. PC12 cells supported an efficient transcription from the human VIP gene promoter in a cell-specific as well as cAMP-dependent manner. These results definitely demonstrated the expression of the VIP gene in PC12 cells. VIP biosynthesis may be positively regulated by VIP in an autocrine fashion in PC12 cells.

Cyclic AMP- and phorbol ester-induced transcriptional activation are mediated by the same enhancer element in the human vasoactive intestinal peptide gene

Transcription of the human vasoactive intestinal peptide (VIP) gene is regulated by both cyclic AMP and phorbol esters. A 17-nucleotide enhancer element within the human VIP gene mediates transcriptional activation by both phorbol esters and forskolin. Mutations of this element decrease responses to both agents, suggesting that the trans-acting proteins that mediate both modes of transcriptional regulation have similar DNA-binding characteristics. The response of the VIP enhancer element to forskolin, but not to 12-O-tetradecanoylphorbol-13-acetate, was attenuated by treatment with a recombinant inhibitor of the cAMP-dependent protein kinase, suggesting that the cAMP-dependent protein kinase and protein kinase C second messenger pathways that converge on this single enhancer element are distinct. The transcriptional activator cAMP-responsive element-binding (CREB) proteins and the c-fos.c-Jun complex interact with the VIP enhancer. The dual second messenger responses of the VIP gene may result from the interaction of this second messenger enhancer with different transcriptional activator proteins.

VIP: molecular biology and neurobiological function.

In the mammalian brain, a major regulatory peptide is vasoactive intestinal peptide (VIP). This 28 amino acid peptide, originally isolated from the porcine duodenum, was later found in the central and peripheral nervous systems and in endocrine cells, where it exhibits neurotransmitter and hormonal roles. Increasing evidence points to VIP's importance as a mediator or a modulator of several basic functions. Thus, VIP is a major factor in brain activity, neuroendocrine functions, cardiac activity, respiration, digestion, and sexual potency. In view of this peptide's importance, the mechanisms controlling its production and the pathways regulating its functions have been reviewed. VIP is a member of a peptide family, including peptides such as glucagon, secretin, and growth hormone releasing hormone. These peptides may have evolved by exon duplication coupled with gene duplication. The human VIP gene contains seven exons, each encoding a distinct functional domain on the protein precursor or the mRNA. VIP gene transcripts are mainly found in neurons or neuron-related cells. VIP gene expression is regulated by neuronal and endocrine signals that contribute to its developmental control. VIP exerts its function via receptor-mediated systems, activating signal transduction pathways, including cAMP. It can act as a neurotransmitter, neuromodulator, and a secretagog. As a growth and developmental regulator, VIP may have a crucial effect as a neuronal survival factor. We shall proceed from the gene to its multiple functions.

The CGTCA sequence motif is essential for biological activity of the vasoactive intestinal peptide gene cAMP-regulated enhancer.

cAMP-regulated transcription of the human vasoactive intestinal peptide gene is dependent upon a 17-base-pair DNA element located 70 base pairs upstream from the transcriptional initiation site. This element is similar to sequences in other genes known to be regulated by cAMP and to sequences in several viral enhancers. We have demonstrated that the vasoactive intestinal peptide regulatory element is an enhancer that depends upon the integrity of two CGTCA sequence motifs for biological activity. Mutations in either of the CGTCA motifs diminish the ability of the element to respond to cAMP. Enhancers containing the CGTCA motif from the somatostatin and adenovirus genes compete for binding of nuclear proteins from C6 glioma and PC12 cells to the vasoactive intestinal peptide enhancer, suggesting that CGTCA-containing enhancers interact with similar transacting factors.

Sequential expression in the nervous system of c-myb and VIP genes, located in human chromosomal region 6q24.

Vasoactive intestinal peptide (VIP) is a major neuropeptide involved in multiple functions such as vasodilation, smooth-muscle relaxation, sweat secretion, gastrointestinal peristalsis, pancreatic function, and brain activity. In view of the multiple roles associated with VIP, it is important to understand its gene regulation. We have recently isolated the human VIP gene and determined its structure. By in situ hybridization techniques we have now localized this gene to the long arm of chromosome 6, 6q24, a chromosomal region that has been shown previously to contain the coding sequences for the nuclear protooncogene c-myb. Genes found in close proximity to each other on the chromosome are often functionally related and, as VIP is primarily expressed in the nervous system, we investigated the possible correlation of c-myb to VIP in neuronal tissue. A sharp peak of c-myb mRNA was observed in the hippocampus of 3-day-old rats, preceding the peak of VIP mRNA that occurs in this brain area at 8 days of age. Thus, the protooncogene c-myb may be associated with events in brain development occurring prior to the appearance of elevated concentrations of VIP.

Structure and expression of the vasoactive intestinal peptide (VIP) gene in a human tumor

To identify the VIP biosynthetic pathways, we have isolated the human VIP gene, using synthetic oligodeoxynucleotides. These specific hybridization probes were constructed according to the neuroblastoma VIP-cDNA sequence and contained up to 39 bases. The gene structure was deduced by direct chemical nucleotide sequencing. Six exons were thus far discovered; among them two short exons, one encoding VIP and the second encoding PHM-27 (a peptide having a N-terminal histidine and C-terminal methionine amide, closely related in sequence and activity to VIP). As a model system for VIP gene expression, we used a human buccal tumor producing elevated amounts of VIP. In these cells, a major transcript of the VIP-gene was identified as a long RNA containing intron sequences. The occurrence of elevated quantities of a high molecular weight, intron containing, gene transcript which is not processed directly into mature RNA suggests that VIP gene expression may be regulated at the RNA processing level.