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J A Waschek

Publications and source records attributed to J A Waschek.

At least 37 records · Page 2Linked to original sources
PubMed1999

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.

Aging↗
PubMed1998

Transcriptional control of the galanin gene. Tissue-specific expression and induction by NGF, protein kinase C, and estrogen.

Galanin is a neuropeptide widely expressed in the central and peripheral nervous system where it acts as a neurotransmitter/neuromodulator and possibly an immunoregulator and growth factor. Galanin gene expression is highly regulated during development and by certain hormones and injury situations. We have examined transcriptional control mechanisms for this gene using chimeric bovine galanin/luciferase reporter genes. These were analyzed in cultured cells and in transgenic mice. The studies reveal that enhancer and silencer sequences are involved in conferring cell- and tissue-specific expression, and that specific elements close to the promoter are responsible for nerve growth factor and protein kinase C induction. So far, the studies have not revealed sequences on the bovine gene that mediate the action of estrogen.

Animals↗
PubMed1998

Tissue-specific enhancement and restriction of galanin gene expression in transgenic mice by 5' flanking sequences.

Galanin (GAL) is a 29/30 amino acid residue neuropeptide that regulates a wide variety of neuroendocrine functions. Galanin is expressed in specific populations of neurons in the hypothalamus and other regions of the brain and in numerous peripheral sites. Previous studies in which galanin-reporter genes were transfected into neural crest-derived neuroblastoma and other tumor cells indicated that cell-specific galanin expression is controlled by gene elements on the 5' flanking sequence which enhance and restrict transcriptional activity. To determine how the gene sequences act in vivo, we first determined the distribution of endogenous galanin gene expression in normal mice. Galanin mRNA was detected in several parts of the central nervous system (CNS), and in several peripheral organs, including the pituitary, pancreas, small and large intestine, adrenal gland, lung, tongue, testes, ovary-fallopian tubes, and uterus, but not at detectable levels in the heart, liver, kidney, urinary bladder or skeletal muscle. We then created several lines of transgenic mice which contained either 5 or 0.131 kilobases (kb) of the bovine galanin gene 5' flanking sequence fused to the luciferase (luc) reporter gene (5GAL-luc vs. 0.1GAL-luc mice, respectively) and compared luciferase activity in these and other organs. In some regions of the CNS that expressed high amounts of galanin mRNA, such as the spinal cord, hypothalamus, thalamus, and medulla, transgene expression was significantly higher in 5GAL-luc vs. 0.1GAL-luc mice, whereas in certain other regions of the brain and in all peripheral organs, the ratio was strikingly reversed. It is concluded that 5 kb of flanking sequence contains elements that mediate basal transcriptional activity in certain parts of the CNS, but also contains sequences that restrict expression in many tissues. However, because the larger transgene was expressed at very low levels in some peripheral sites of high galanin expression such as the pituitary, pancreas, adrenal gland, and intestine, it is concluded that sequences on the 5 kb transgene are not sufficient to direct expression to these peripheral tissues in mice.

Animals↗
PubMed1998

Neural tube expression of pituitary adenylate cyclase-activating peptide (PACAP) and receptor: potential role in patterning and neurogenesis.

Neural tube patterning in vertebrates is controlled in part by locally secreted factors that act in a paracrine manner on nearby cells to regulate proliferation and gene expression. We show here by in situ hybridization that genes for the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and one of its high-affinity receptors (PAC1) are widely expressed in the mouse neural tube on embryonic day (E) 10.5. Transcripts for the ligand are present in differentiating neurons in much of the neural tube, whereas the receptor gene is expressed in the underlying ventricular zone, most prominently in the alar region and floor plate. PACAP potently increased cAMP levels more than 20-fold in cultured E10.5 hindbrain neuroepithelial cells, suggesting that PACAP activates protein kinase A (PKA) in the neural tube and might act in the process of patterning. Consistent with this possibility, PACAP down-regulated expression of the sonic hedgehog- and PKA-dependent target gene gli-1 in cultured neuroepithelial cells, concomitant with a decrease in DNA synthesis. PACAP is thus an early inducer of cAMP levels in the embryo and may act in the neural tube during patterning to control cell proliferation and gene expression.

Animals↗
PubMed1998

Differential effects of peptide histidine isoleucine (PHI) and related peptides on stimulation and suppression of neuroblastoma cell proliferation. A novel VIP-independent action of PHI via MAP kinase.

The growth rate of rodent embryonic neuroblasts and human neuroblastoma cell lines is regulated in part by autocrine or paracrine actions of neuropeptides of the family that includes vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and pituitary adenylate cyclase-activating peptide (PACAP). These peptides act via seven transmembrane G-protein-linked receptors coupled to cAMP elevation, phospholipase C activation, intracellular Ca2+ release, and/or of mitogen-activated protein (MAP) kinase activation. Here we investigated the action of these peptides on the mouse neuroblastoma cell line Neuro2a. PHI and VIP inhibited proliferation at concentrations as low as 10(-13) M and 10(-10) M, respectively. In contrast, PACAP action was biphasic, with stimulation occurring at subnanomolar doses and inhibition at higher doses. Peptide actions were studied further by measuring cAMP and ERK1/2 MAP kinase activity and by assessing 3H-thymidine incorporation in conjunction with a panel of signal transduction pathways inhibitors. The data obtained indicated that the PHI-inhibitory and PACAP-stimulatory activities were mediated by corresponding changes in activity of the MAP kinase pathway and independent of protein kinase A (PKA) or protein kinase C (PKC). In contrast, the inhibitory actions of VIP and PACAP were specifically blocked by antagonists of PKA. Northern blot analysis revealed gene expression for only the PACAP-preferring (PAC1) receptor. However, binding experiments using 125I-labeled PACAP27, PHI, and VIP, demonstrated the presence of PACAP-preferring sites, bivalent VIP/PACAP sites, and PHI-binding sites that did not interact with VIP. The studies demonstrate potent regulatory actions of PACAP, PHI, and VIP on neuroblastoma cell proliferation which appear to be mediated by multiple subsets of receptors which differentially couple to MAP kinase and PKA signaling pathways.

Animals↗
PubMed1997

Retinoic acid regulation of the VIP and PACAP autocrine ligand and receptor system in human neuroblastoma cell lines.

Neuroendocrine tumors, neuroblastoma in particular, commonly express the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) and their receptors. Retinoic acid (RA) has been shown to induce differentiation of neuroblastoma cell lines, possibly by augmenting or interfering with neuropeptide autocrine loops. We sought to determine which receptor gene subtypes are expressed in selected human neuroblastoma cell lines (SH-SY5Y, IMR-32, and LA-N-5), and the effect of RA on the VIP/PACAP ligand/receptor system. Expression of both PACAP1 and VIP1/PACAP2 receptor genes was detected by Northern analysis, which characteristically encode Type I (PACAP-preferring), and Type II (bivalent VIP/PACAP) receptors, respectively. Binding experiments carried out on IMR-32 cells, using 125I VIP and 125I PACAP-27 as tracers, corroborated that both receptor subtypes were expressed. In contrast to RA upregulation of VIP binding (confirmed here in IMR-32 cells), levels of both receptor mRNAs were reduced after RA treatment. VIP mRNA in each cell line was increased by RA, whereas PACAP mRNA, detected in IMR-32 cells only, was reduced. The studies indicate that several components of the VIP/PACAP autocrine system are regulated in neuroblastoma cell lines during RA differentiation.

Autocrine Communication↗
PubMed1996

Vasoactive intestinal peptide induces S(alpha)/S(mu) switch circular DNA in human B cells.

Vasoactive intestinal peptide (VIP), a major neurotransmitter of peripheral nerves, has been suggested to function in host defense by regulating local human immune function. Indirect evidence has been marshaled that VIP can function as a switch factor for IgA in human Ig isotype recombination. In this study we directly tested the ability of VIP to function as a factor driving human B cells into IgA producing cells by assessing its ability to induce switch circular DNA representing direct mu to alpha switching. In addition we determined the generation of alpha germ-line transcripts and measured the level of IgA protein produced. Stimulation with VIP and CD40 mAb induced IgA production by human IgD+ B cells while VIP or CD40 alone failed to do so. Stimulation of purified IgD+ B cells with VIP plus CD40 mAb induced generation of switch circular DNA representing in vitro driven isotype switching from mu to alpha. CD40 mAb alone induced alpha germ-line transcripts but not IgA switch circles. Thus VIP, a neurogenic factor, can induce alpha-specific switching in CD40-activated human B cells and may thereby play an important role in directing the humoral immune response at mucosal surfaces.

B-Lymphocytes↗
PubMed1996

Embryonic expression of vasoactive intestinal peptide (VIP) and VIP receptor genes.

Vasoactive intestinal peptide (VIP) exhibits pronounced effects on the growth rate of cultured mouse embryonic day (E) 9.5 embryos and acts in tissue culture as a potent glial mitogen and neuron survival factor. However, previous studies using immunohistochemistry or in situ hybridization in the rat have not revealed the presence and location of VIP or VIP mRNA in the early developing embryo CNS. Using a sensitive in situ hybridization assay with a 33P-labeled riboprobe, we show here that the VIP gene is expressed at least as early as E11 in the mouse hindbrain. Northern blot analysis on RNA from brain dissected from mouse embryos beginning at E14 confirmed that a correct-size mRNA for VIP was present by E14 and at later time points. Expression of the VIP2 receptor gene was also detected by northern analysis in E14 mouse brains. These studies support the hypothesis that VIP produced by the embryo exerts important effects on embryonic nervous system development.

Animals↗
PubMed1995

High levels of vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor mRNA expression in primary and tumor lymphoid cells.

Neuropeptides exert a variety of putative immunomodulatory actions. Despite the molecular cloning of multiple forms of receptors for several neuropeptides with putative immunomodulatory effects, including vasoactive intestinal peptide (VIP), the related peptide pituitary adenylate cyclase-activating peptide (PACAP), the opiate peptides, tachykinins, somatostatin and corticotropin-releasing factor, it has not been reported that any of the receptor genes are expressed at significant levels in cells of the immune system. The low level of expression of these receptors and lack of knowledge concerning receptor subtype has impeded progress in understanding how neuropeptides regulate immune function. For example, it is not understood why VIP produces immunomodulatory effects at concentrations far below its receptor-binding affinity. Receptors for VIP and PACAP have recently been cloned. We show here by Northern blot analysis that the VIP/PACAP1 receptor mRNA is present in total RNA prepared from mouse spleen B- and T-lymphocytes. The VIP/PACAP1 receptor mRNA was also present in human peripheral blood lymphocytes, and in a B-lymphocyte and a myelocytic cell line. The mRNA for a second form of the receptor, the VIP/PACAP2 receptor, was not expressed at detectable levels in normal cells, but was detected in several human T-cell lines and a murine mast cell line. The results indicate that VIP/PACAP1 and perhaps VIP/PACAP2 receptors mediate the diverse effects of VIP and PACAP on immune cells.

Animals↗
PubMed1995

Functional analysis of the 5' flanking sequence in the ovine beta 1-adrenergic receptor gene.

Functional data for the promoter of the beta 1-adrenergic receptor (beta 1AR) gene are lacking. We previously cloned the ovine beta 1AR gene and mapped the transcription start sites. We now report data on ovine beta 1AR gene expression obtained by transient transfection. Progressive deletion of upstream 5' flanking region moderately increased transcription activity in three cell lines compared to the full-length promoter. Deletion of sequences between -1530 and -953 produced the greatest increase in transcriptional activity. This region encompassed a putative GRE and an AP1 site. Deletion of the transcription start sites eliminated nearly all of the activity. Dexamethasone significantly increased activity of each of the promoter constructs tested in C6 glioma cells and an embryonic myocardial cell line, W1 cells. T3 alone had no effect and cotreatment with T3 did not augment the effects of dexamethasone. We conclude, basal transcription activity is repressed by a mechanism which operates through element(s) in the proximal promoter. Glucocorticoids increase transcription through mechanism(s) within the same region. We speculate that this region in the ovine beta 1AR promoter may be responsible for its unique transcription regulation.

Animals↗
PubMed1995

Differential expression of VIP/PACAP receptor genes in breast, intestinal, and pancreatic cell lines.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are structurally-related neuropeptides that function as trophic factors in addition to their more classical roles as neurotransmitters. Binding and molecular cloning studies have shown that their actions are mediated by receptors encoded by at least three different genes. VIP binding has been demonstrated on many tumor types, and radiolabeled VIP has recently been used as a novel method to localize intestinal tumors in humans and their sites of metastasis. To determine the receptor subtype and level of gene expression, we screened breast, intestinal, and pancreatic, cell lines by Northern blot analysis. Breast lines expressed VIP/PACAP1 receptor mRNA levels comparable to intestinal lines, in agreement with the studies showing particularly high VIP binding in these tumors and their derived cell lines. Pancreatic cell lines expressed mRNA for several receptor types. This extends the potential utility of VIP and PACAP in the localization of tumors, and because VIP and PACAP may regulate the growth rate of some tumors by autocrine or other mechanisms, the identification of receptor subtypes on these lines sets the stage for studies in which the activity of these individual receptors in growth and other processes can be investigated.

Animals↗
PubMed1995

Transcription initiation is localized to a TATAless region in the ovine beta 1 adrenergic receptor gene.

We cloned and sequenced the ovine beta 1 adrenergic receptor (beta 1AR) gene and promoter region. The transcription start site was localized by RNase protection and primer extension to a GC-rich region. The predominant initiation sequence did not resemble an initiator element and there was no upstream TATA box. Sequence analysis revealed several potential thyroid hormone and glucocorticoid regulatory elements. Identification of the promoter structure of this important gene will help define its novel regulation during development.

Amino Acid Sequence↗
PubMed1995

Regulation of beta 1-adrenoceptors by glucocorticoids and thyroid hormones in fetal sheep.

The effects of betamethasone alone or in combination with thyroxine (T4) on ovine fetal beta-adrenoceptors were investigated at the molecular level. Ovine fetuses (126 days gestation; term = 150 days) were treated with a single ultrasound-guided intramuscular injection of 0.5 mg/kg betamethasone, betamethasone + 50 micrograms/kg T4, or saline. Forty-eight h after injection, lambs were delivered by cesarean section and evaluated three h for postnatal adaptation. Myocardial beta-adrenoceptor equilibrium dissociation constant (Kd) and maximal receptor density (Bmax), as assessed by [3H]dihydroalprenolol binding, were not significantly different in drug-treated groups compared to the control group. Northern hybridization and RNase protection assays of myocardial total RNA probed with a sheep beta 1-adrenoceptor riboprobe confirmed no changes in expression at the level of the gene. Levels of beta 1-adrenoceptor mRNA in the lung and brain were also unaffected by the treatments. Because other genes are responsive to glucocorticoids and thyroid hormones at this stage, the absence of up-regulation of beta-adrenoceptor number and steady-state levels of mRNA coding for beta 1-adrenoceptor following fetal corticosteroid and thyroid hormone treatment may indicate a specific, developmentally regulated repressor mechanism.

Animals↗
PubMed1995

A cloning strategy for G-protein-coupled hormone receptors: the ovine beta 1-adrenergic receptor.

Regulation of beta 1-adrenergic receptors is unusual in developing animals. For example, glucocorticoid-and thyroid hormone-responsiveness for several genes is seen in animals treated during fetal life but beta 1-responsiveness is not seen until after birth. In order to investigate this at the transcriptional level, the ovine beta 1 receptor gene was cloned from a sheep genomic library. An approach using high-stringency screening with cDNA probes and oligonucleotides from regions of human and rat genes conserved but unique to the beta 1 receptor but not to other seven transmembrane, G-protein-coupled receptors. Over 800,000 clones were screened from which 40-50 positive clones were identified by each of the probes. There was, however, only a single clone which was recognized by each of the probes. A 5-kb insert was subcloned and shown to contain sequences which hybridized to each of the probes. Using the restriction map of the rat beta 1 receptor, a 1.0-kb Pst1 internal fragment was further subcloned for sequence identification. Confirmation of this fragment as the ovine beta 1 receptor was based on homology of the beta 1 receptor from other species and tissue distribution of mRNA. Nucleotide sequence homology was 93% with the human beta 1 receptor and 84% with rat. Amino acid sequence homology was > 75% and approached 100% in the transmembrane regions. The approach described represents a practical approach to cloning and identification of hormone receptors from the highly homologous members of the seven-transmembrane, G-protein-coupled receptors.

Amino Acid Sequence↗
PubMed1995

Vasoactive intestinal peptide: an important trophic factor and developmental regulator?

It has been proposed that vasoactive intestinal peptide (VIP) or a very closely related peptide has important actions very early in embryonic development. Recent data supporting this hypothesis are that subnanomolar concentrations of VIP significantly increased the growth rate of cultured embryonic day-9.5 (E9.5) mouse embryos, and that embryos at this and later stages exhibit a high degree of VIP binding in the brain stem and spinal cord. It is not known whether VIP is derived from the fetus, placenta, or mother at these early stages, or whether VIP acts in this culture system in place of a related peptide. The earliest reported expression of VIP in rat embryos is at E13.5, when the peptide and mRNA are expressed transiently in a high percentage of cells in the rat stellate ganglia. The time course of events mapped in other sympathetic ganglia at this stage suggest that transient expression of VIP in the ganglia might function to regulate neuroblast and/or glial cell proliferation, maturation or survival. Tissue culture studies indicate that VIP can support many of these trophic functions at concentrations that are the same or lower than that necessary to increase cAMP levels by way of classical VIP receptors. For example, VIP at 10(-10) M stimulates the release of neurotrophic factors from glial cells and maximally stimulates the proliferation of astrocytes. Two VIP receptors encoded on different genes have now been cloned. Both are members of the seven transmembrane G-protein-coupled receptor family and, when expressed in mammalian cells, mediate an increase in cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Primary sequence and functional analysis of the bovine galanin gene promoter in human neuroblastoma cells.

Galanin (GAL) is a biologically active neuropeptide that has been suggested to play a role in stress-induced inhibition of insulin secretion, in dementia of the Alzheimer's type, and in the regulation of growth hormone secretion. We report here the isolation of a bovine genomic clone containing more than 5-kb 5'-flanking sequences. Partial sequence analysis of the genomic clone revealed an atypical TATA-box in the promoter (ATAAATA) and several consensus sequences that typically bind transcription factors, including those that bind NF kappa B, Sp1, and AP-2. Primer extension and RNase protection analyses revealed that transcription is initiated at two sites, 28 and 31 bp, respectively, downstream from the TATA-box. To locate functionally active regulatory elements on the GAL gene, we first identified a neural crest-derived human neuroblastoma cell line, SK-N-SH subclone SH-SY5Y, that expressed easily detectable levels of endogenous GAL mRNA. We then constructed plasmids containing various lengths of bovine GAL 5'-flanking sequences and the first exon fused to a reporter plasmid encoding luciferase. Transfection of these plasmids into the SH-SY5Y cells and analysis by transient expression indicated that 131 bp of 5' gene sequence was sufficient to obtain maximal basal expression. Further, expression was suppressed 16-fold when 5 kb were included, suggesting the presence of a distal repressor element(s). In another set of experiments, we found that GAL mRNA levels could be induced more than 10-fold by 20-hr treatment with phorbol 12-myristate 13-acetate (PMA). In cells transfected with the same plasmids, luciferase activity was also induced by PMA, but the degree of induction did not significantly differ among the deletion constructions (varying from six- to eight-fold), suggesting that elements conferring PMA induction and/or RNA stabilization may be located within 131 bp of the transcriptional start site, in the first exon, or on gene sequences not studied here.

Animals↗