Pituitary adenylate cyclase-activating polypeptide regulation of vasoactive intestinal polypeptide transcription requires Ca2+ influx and activation of the serine/threonine phosphatase calcineurin.

A >15-fold increase in vasoactive intestinal polypeptide (VIP) mRNA and VIP peptide levels occurred in primary chromaffin cells following exposure to the neurotrophic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)-27 with an EC50 of approximately 2 nM. PACAP induction of VIP expression was blocked by methoxyverapamil or by a combination of nimodipine and omega-conotoxin MVIIC, indicating a requirement for PACAP-initiated calcium entry through voltage-dependent calcium channels for regulation of VIP biosynthesis. Ascomycin, which inhibits calcineurin through formation of an ascomycin/FKBP12/calcineurin ternary complex, abolished the PACAP-evoked increase in VIP expression, whereas rapamycin, which also binds to FKBP12 but does not cause inhibition of calcineurin, did not. Cyclosporin A, which inhibits calcineurin through formation of a cyclosporin A/cyclophilin/calcineurin complex, also abolished PACAP-evoked VIP biosynthesis. These data indicate that PACAP regulates the expression of VIP via a signaling pathway that requires calcium influx and activation of calcineurin.

Two separate cis-active elements of the vasoactive intestinal peptide gene mediate constitutive and inducible transcription by binding different sets of AP-1 proteins.

Vasoactive intestinal peptide (VIP) gene expression is highly restricted throughout the neuroaxis and regulated by extracellular factors that activate tyrosine- or serine/threonine-directed protein kinase pathways. Cytokine, cyclic AMP, and tissue-specific response elements on the VIP gene have been characterized. Those mediating responsiveness to protein kinase C have not. The endogenous VIP gene and a 5.2-kilobase pair (kb) VIP-luciferase reporter gene, are up-regulated by phorbol 12-myristate 13-acetate (PMA) in SK-N-SH neuroblastoma cells. PMA stimulation was abolished by deletion of sequences at -1.37 to -1.28 or -1.28 to -0.904 kb, but not by removal of the single phorbol ester response element (TRE; TGACTCA) located at -2.25 kb. Mutation of sites at -1.32 or -1.20 that mediate neurotrophin responsiveness of the VIP gene (Symes, A., Lewis, S., Corpus, L., Rajan, P., Hyman, S. E., and Fink, J. S. (1994) Mol. Endocrinol. 8, 1750-1763) each reduced PMA induction in SK-N-SH cells by >50%, and double mutation abolished it. The two mutations also reduced basal VIP reporter gene transcription in SH-EP neuroblastoma cells expressing VIP constitutively. Both cis-active elements bound pre-existing AP-1 proteins in SH-EP- or PMA-stimulated SK-N-SH cell nuclear extracts. The AP-1 complex at both sites contained a Fos-related protein with c-Jun in SH-EP cells and c-Fos with a Jun-related protein in SK-N-SH cells. Recruitment of combinatorially distinct AP-1 complexes to these elements may underlie cell type-specific regulation of the VIP gene.

PACAP activates calcium influx-dependent and -independent pathways to couple met-enkephalin secretion and biosynthesis in chromaffin cells.

Pituitary adenylate cyclase activating polypeptide-27 (PACAP-27) caused a dose-dependent increase in met-enkephalin secretion and increased production of met-enkephalin peptide and proenkephalin A (PEnk) mRNA in bovine chromaffin cells, at concentrations as low as 300 pM. PACAP-38 was less potent than PACAP-27, but had similar effects. Vasoactive intestinal polypeptide (VIP) (1-100 nM) was without appreciable effect on either enkephalin secretion or biosynthesis, implicating PACAP type I receptors in PACAP-stimulated enkephalin secretion and synthesis. PACAP type I receptors can activate adenylate cyclase and stimulate phospholipase C through heterotrimeric G protein interactions, leading to increased intracellular cyclic AMP (cAMP), inositol triphosphate (IP3)-mediated calcium mobilization, and calcium- and diacylglycerol (DAG)-mediated protein kinase C (PKC) activation. Enkephalin secretion evoked by 10-100 nM PACAP-27 was not inhibited by 1 microM (-)-202-791, an L-type specific dihydropyridine calcium channel blocker, but was inhibited 65-80% by the arylalkylamine calcium channel blocker D600. Forty mM potassium-evoked secretion was inhibited > 90% by both D600 and (-)-202-791, 25 microM forskolin-induced secretion was blocked < 50% by D600 and was unaffected by (-)-202-791, and 100 nM phorbol myristate acetate (PMA)-induced secretion was unaffected by either D600 or (-)-202-791. Enkephalin biosynthesis was increased by 10 nM PACAP-27, as measured by increased met-enkephalin pentapeptide content and PEnk A mRNA levels. PACAP-, forskolin-, and PMA-stimulated enkephalin synthesis were not blocked by D600 or (-)-202-791. Elevated potassium-induced enkephalin biosynthesis upregulation was completely blocked by either D600 or (-)-202-791 at the same concentrations. PACAP acting through type I PACAP receptors couples calcium influx-dependent enkephalin secretion and calcium influx-independent enkephalin biosynthesis in chromaffin cells. Restriction of the effects of enhanced calcium influx to stimulation of secretion, but not of biosynthesis, is unique to PACAP. By contrast, potassium-induced enkephalin biosynthesis upregulation is completely calcium influx dependent, specifically via calcium influx through L-type calcium channels. We propose that subpopulations of voltage-dependent calcium channels are differentially linked to intracellular signal transduction pathways that control neuropeptide gene expression and secretion in chromaffin cells.

Five discrete cis-active domains direct cell type-specific transcription of the vasoactive intestinal peptide (VIP) gene.

Vasoactive intestinal peptide (VIP) is a neuromodulator expressed with great anatomical specificity throughout the nervous system. Cell-specific expression of the VIP gene is mediated by a tissue specifier element (TSE) located within a 2.7-kilobase (kb) region between -5.2 and -2.5 kb upstream from the transcription start site, and requires an intact promoter proximal VIP-CRE (cyclic AMP-responsive element) (Hahm, S. H., and Eiden, L. E. (1997) J. Neurochem. 67, 1872-1881). We now report that the TSE comprises a 425-base pair domain located between -4.7 and -4.2 kb containing two AT-rich octamer-like sequences. The 425-base pair TSE is sufficient to provide full cell-specific regulation of the VIP gene, when fused to the 5' proximal 1.55 kb of the VIP gene. Mutational analysis and gel shift assays of these octamer-like sequences indicate that the binding of proteins related to the ubiquitously expressed POU-homeodomain proteins Oct-1 and/or Oct-2 to these octamer-like sequences plays a central role for the function of the TSE. The TSE interacts with three additional discrete domains besides the cAMP response element, which are located within the proximal 1.55 kb of the VIP gene, to provide cell-specific expression. An upstream domain from -1.55 to -1.37 kb contains E-boxes and MEF2-like motifs, and deletion of this domain results in complete abrogation of cell-specific transcriptional activity. The region from -1.37 to -1. 28 kb contains a STAT motif, and further removal of this domain allows the upstream TSE to act as an enhancer in both SH-EP and HeLa cells. The sequence from -1.28 to -0.9 kb containing a non-canonical AP-1 binding sequence (Symes, A., Gearan, T., Eby, J., and Fink, J. S. (1997) J. Biol. Chem. 272, 9648-9654), is absolutely required for TSE-dependent cellspecific expression of the VIP gene. Thus, five discrete domains of the VIP gene provide a combination of enhancer and repressor activities, each completely contingent on VIP gene context, that together result in cell-specific transcription of the VIP gene.

Cis-regulatory elements controlling basal and inducible VIP gene transcription.

The cis-acting elements of the VIP gene important for basal and stimulated transcription have been studied by transfection of VIP-reporter gene constructs into distinct human neuroblastoma cell lines in which VIP transcription is constitutively high, or can be induced to high levels by protein kinase stimulation. The 5.2 kb flanking sequence of the VIP gene conferring correct basal and inducible VIP gene expression onto a reporter gene in these cell lines was systematically deleted to define its minimal components. A 425-bp fragment (-4656 to -4231) fused to the proximal 1.55 kb of the VIP promoter-enhancer was absolutely required for cell-specific basal and inducible transcription. Four additional components of the VIP gene were required for full cell-specific expression driven by the 425 bp TSE (region A). Sequences from -1.55 to -1.37 (region B), -1.37 to -1.28 (region C), -1.28 to -.094 (region D), and the CRE-containing proximal 94 bp (region E) were deleted in various combinations to demonstrate the specific contributions of each region to correct basal and inducible VIP gene expression. Deletion of region B, or mutational inactivation of the CRE in region E, resulted in constructs with low transcriptional activity in VIP-expressing cell lines. Deletion of regions B and C together resulted in a gain of transcriptional activity, but without cell specificity. All five domains of the VIP gene were also required for cell-specific induction of VIP gene expression with phorbol ester. Gelshift analysis of putative regulatory sequences in regions A-D suggests that both ubiquitous and neuron-specific trans-acting proteins participate in VIP gene regulation.

Upstream sequencing and functional characterization of the human cholinergic gene locus.

The 5' flanking region of the human VAChT gene was sequenced to approx 5350 bases upstream of the initiating methionine codon of the VAChT open reading frame (orf). The 5' flanks of the human and rat cholinergic gene loci were compared to identify regions of local sequence conservation, and therefore of potential regulatory importance. Several discrete domains of high homology, including a cluster of far-upstream cis-active consensus motifs, a neuronally restrictive silencer element consensus sequence, and additional conserved sequences within the putative nerve growth factor response domain of the locus, were identified. The probable start of transcription of the VAChT gene was deduced from mapping of sequences of rat and human VAChT cDNAs onto the 5' flanking regions of the human and rat cholinergic gene loci. The actual utilization of a putative 5' VAChT exon in rat central nervous system (CNS) tissue was assessed by in situ hybridization histochemistry. RNA transcripts containing both VAChT and ChAT protein-coding sequences were abundant in spinal cord motoneurons, sympathetic preganglionic cells, basal forebrain, striatum, and cranial motor nuclei. R-exon-containing transcripts could be detected only at low levels in these cell groups, implying that most transcription of VAChT proceeds from a promoter downstream of the R-exon. To assess the structural requirements for expression of the VAChT gene without bias regarding the actual start of transcription, a 5' fragment of the human gene corresponding to approximately 3 kb of sequence extending upstream from within the presumed 5' untranslated region of VAChT itself was fused to a luciferase-encoding reporter and transfected into VAChT-expressing and nonexpressing human and rat cell lines. This portion of the VAChT gene provided strong promoter expression in both cholinergic and noncholinergic cell lines. Deletion of the putative neuronally restrictive silencer element (NRSE) resulted in enhanced transcription in all cell lines. Lack of differential expression of VAChT transcription in VAChT-expressing vs non-VAChT-expressing cell lines suggested that additional enhancer elements controlling cell-specific expression of the VAChT gene exist further upstream in the cholinergic locus 5' flank. Conservation of potential cis-active elements within a 1.4 kb sequence immediately upstream of the NRSE in both rat and human cholinergic gene loci suggests that this domain is required for cholinergic-specific regulation of VAChT and ChAT gene transcription.

Tissue-specific expression of the vasoactive intestinal peptide gene requires both an upstream tissue specifier element and the 5' proximal cyclic AMP-responsive element.

An upstream enhancer element [tissue specifier element (TSE)] located between 4.66 and 4.02 kb from the transcription start site is important for cell type-specific expression and phorbol ester induction of the vasoactive intestinal peptide (VIP) gene. An element located within 100 bases of the VIP promoter [the VIP cyclic AMP-responsive element (VIP-CRE)] confers cyclic AMP and phorbol ester responsiveness to heterologous promoters. The possibility that these two regions of the VIP gene function cooperatively to determine tissue-specific and second messenger-dependent expression of the VIP gene was addressed by assaying transcription from a VIP-luciferase reporter gene with progressive deletions from the 5' flanking sequence of the gene, with or without inactivation of the proximal VIP-CRE. Basal expression of the reporter gene in both SH-EP and SK-N-SH human neuroblastoma cells, which express endogenous VIP mRNA, was absolutely dependent on the presence of the upstream TSE. Full constitutive expression was also dependent on the intact VIP-CRE. Forskolin-mediated induction of the reporter gene in SH-EP and SK-N-SH cells was completely abolished by mutations in the VIP-CRE but not by deletion of the upstream sequence, indicating that the VIP-CRE alone determines cyclic AMP responsiveness. In contrast to reports that the VIP-CRE imparts 12-O-tetradecanoylphorbol 13-acetate (phorbol 12-myristate 13-acetate; PMA) responsiveness to heterologous promoters, PMA stimulation in SK-N-SH cells was independent of an intact VIP-CRE but dependent on a region between -2.5 kb and the VIP-CRE. Sequencing of the entire 5.2-kb VIP 5' flank revealed a consensus PMA-responsive element (TGACTCA) 2.25 kb upstream of the transcription start site that may represent the site imparting PMA responsiveness to the VIP gene.

Transforming growth factor-beta 1 rapidly activates phosphorylase in a calcium-dependent manner in rat hepatocytes.

Transforming growth factor-beta 1 (TGF-beta 1) rapidly activated phosphorylase in isolated rat hepatocytes (half-maximal rate of activation with approximately 0.1 ng/ml). Removal of Ca2+ from the external medium just before TGF-beta 1 addition markedly attenuated phosphorylase activation. TGF-beta 1 (1 ng/ml) produced a small increase in [Ca2+]i (approximately 10% increase after 30 s), which appears sufficient to account for phosphorylase activation. These observations indicate that activation of the TGF-beta 1 signal transduction system in hepatocytes is linked with a small increase in [Ca2+]i, and external Ca2+ may contribute in part to this increase.

Cytokinin increases intracellular Ca2+ in Funaria: detection with Indo-1.

Changes in intracellular [Ca2+] ([Ca2+]i) after cytokinin-treatment in protonema cells of the moss Funaria hygrometrica have been measured using the pentapotassium salt of Indo-1. The extent of dye loading strongly depended on lowering the pH of the incubation medium to 5.0. Exposing dye-loaded cells briefly with Mn2+ did not quench fluorescence suggesting that the source of fluorescence is from the cytoplasm and not from the cell wall. Indo-1 remains responsive to changes in [Ca2+]i in Funaria cells. The [Ca2+]i in quiescent cells (with and without extracellular Ca2+) is 250 nM, which is within the range of reported [Ca2+]i of other plant cells. Treatment of cells with extracellular cytokinin in 4 mM Ca2+ induced a three-fold increase in [Ca2+]i to 750 nM in target caulonema cells. This increase was not observed in Ca(2+)-free medium. These target cells respond to cytokinin treatment by an asymmetrical division, while non-target chloronema cells do not divide. Cytokinin appears to increase [Ca2+]i by extracellular Ca2+ uptake. However, non-target chloronema cells and tip cells also respond to cytokinin treatment by increasing [Ca2+]i. The differential physiological response of these cell types to hormonal stimulation must lie further down the signal transduction chain.