Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC(2) receptor for vasoactive intestinal peptide.

Vasoactive intestinal peptide (VIP) and its G protein-coupled receptors, VPAC(1)R and VPAC(2)R, are prominent in the immune system and regulate many aspects of T cell-dependent immunity. In mouse T cells, VPAC(1)R is expressed constitutively, whereas VPAC(2)R is induced by immune stimuli. VPAC(2)R-null (VPAC(2)R(-/-)) mice on a C57BL/6 background are shown here to have normal basic immune characteristics, including serum Ig concentrations, blood levels of all leukocytes, and spleen number of total T cells (CD3(+)) and T cells bearing CD4, CD8, and CD28. Hapten-evoked cutaneous delayed-type hypersensitivity (DTH) was significantly enhanced in VPAC(2)R-null mice compared with age- and sex-matched wild-type mice. In contrast, generation of IgE anti-hapten antibodies and active cutaneous anaphylaxis were > or =70% lower in VPAC(2)R-null mice than in wild-type controls. Cytokine production by splenic CD4(+) T cells, stimulated with adherent anti-CD3 plus anti-CD28 antibodies, revealed higher levels of IL-2 (mean = 3-fold) and IFN-gamma (mean = 3-fold), and lower levels of IL-4 (mean = one-fifth) in VPAC(2)R-null mice than wild-type controls. Loss of VIP-VPAC(2)R maintenance of the normal ratio of Th2/Th1 cytokines thus leads to a state of enhanced DTH and depressed immediate-type hypersensitivity, which may alter both host defense and susceptibility to immune-mediated diseases.

Overexpression of the human VPAC2 receptor in the suprachiasmatic nucleus alters the circadian phenotype of mice.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) belong to a superfamily of structurally related peptide hormones that includes glucagon, glucagon-like peptides, secretin, and growth hormone-releasing hormone. Microinjection of VIP or PACAP into the rodent suprachiasmatic nucleus (SCN) phase shifts the circadian pacemaker and VIP antagonists, and antisense oligodeoxynucleotides have been shown to disrupt circadian function. VIP and PACAP have equal potency as agonists of the VPAC(2) receptor (VPAC(2)R), which is expressed abundantly in the SCN, in a circadian manner. To determine whether manipulating the level of expression of the VPAC(2)R can influence the control of the circadian clock, we have created transgenic mice overexpressing the human VPAC(2)R gene from a yeast artificial chromosome (YAC) construct. The YAC was modified by a strategy using homologous recombination to introduce (i) the HA epitope tag sequence (from influenza virus hemagglutinin) at the carboxyl terminus of the VPAC(2)R protein, (ii) the lacZ reporter gene, and (iii) a conditional centromere, enabling YAC DNA to be amplified in culture in the presence of galactose. High levels of lacZ expression were detected in the SCN, habenula, pancreas, and testis of the transgenic mice, with lower levels in the olfactory bulb and various hypothalamic areas. Transgenic mice resynchronized more quickly than wild-type controls to an advance of 8 h in the light-dark (LD) cycle and exhibited a significantly shorter circadian period in constant darkness (DD). These data suggest that the VPAC(2)R can influence the rhythmicity and photic entrainment of the circadian clock.

Desensitization of the human vasoactive intestinal peptide receptor (hVIP2/PACAP R): evidence for agonist-induced receptor phosphorylation and internalization.

To investigate the role of phosphorylation and internalization in the desensitization of the hVIP2/PACAP receptor, we expressed a C-terminal epitope-tagged (hemagglutinin; YPYDVPDYASL) receptor in COS7 and HEK293 cell lines. Radiolabeling experiments demonstrated that exposure to agonist induced receptor phosphorylation significantly above basal levels. This receptor phosphorylation was greater than that induced by receptor-independent activation of PKA with forskolin and that induced by co-application of forskolin and agonist. This suggests that receptor occupancy promotes phosphorylation and also that receptor phosphorylation may involve a specific G protein-coupled receptor kinase in addition to PKA. Immunocytochemical analysis showed that the receptor was internalized in response to agonist to a single site of accumulation within the cell and this was dependent on temperature, agonist concentration, and time. Further studies will focus on identifying phosphorylation sites and endocytic signals within the hVIP2/PACAP R.

An upstream stimulatory factor (USF) binding motif is critical for rat preprotachykinin-A promoter activity in PC12 cells.

We demonstrate the presence of a functional E box motif in the proximal rat preprotachykinin-A (rPPT) promoter. This element (spanning nucleotides -67 to -47) exhibits the sequence 5'-CACGTG-3' which is recognized and bound by the basic helix-loop-helix family of regulatory proteins. We also show that at least one of the factors bound to this rPPT promoter element in both HeLa and PC12 nuclear extract is the ubiquitously expressed transcription factor, the upstream stimulatory factor (USF). Mutation of this element by insertion of a 10 bp linker into the E box motif, in an rPPT promoter fragment spanning -865 to +92, destroys the ability of this promoter fragment to support reporter gene expression in a PC12 cell model of rPPT promoter activity. The data indicate that this rPPT E box element is likely to function as an important cis-regulatory domain in the rPPT promoter.

Transcriptional control of neuropeptide gene expression in sensory neurons, using the preprotachykinin-A gene as a model.

Control of neuropeptide gene expression in sensory neurons is determined in part by a variety of tissue-specific, developmental, and stimulus-induced transcription factors that interact with the promoters of these genes. We have analysed the regulation of the rat preprotachykinin-A (rPPT) gene, which is expressed in a subset of dorsal root ganglia neurons. A region of the promoter encompassing approximately 1300 base pairs spanning the transcriptional start site has been analysed in detail both by functional analysis of promoter activity in clonal cell lines and dorsal root ganglia neurons grown in culture and by in vitro characterisation of transcription factor interaction with this region. Interestingly our analysis indicates an important role in rPPT gene expression for the E box transcription factor family. This class of transcription factor has been demonstrated to be a major determinant of calcitonin gene related peptide (CGRP) expression, which is also expressed in dorsal root ganglia neurons often under similar conditions as rPPT. In addition, multiple regulatory domains have been identified in the rPPT promoter, which act as activators in a variety of cell types. These elements are silenced in the context of the rPPT promoter in many non-neuronal cells. Therefore, tissue-specific expression of reporter genes directed by the rPPT promoter in transient transfection is determined in part by a variety of silencer elements, which act to repress the function of several domains that act as constitutive enhancers of expression in a wide range of cells. Removal or modulation of silencer elements in the rPPT promoter allows activity in a wider variety of cell types. We postulate that control of rPPT gene expression is the results of dynamic interplay of both positive and negative regulatory elements, a phenomenon observed in several other neuronal-specific genes, including that encoding CGRP.

Three immediate early gene response elements in the proximal preprotachykinin-A promoter in two functionally distinct domains.

The preprotachykinin-A promoter contains two blocks of DNA sequence, with a high degree of homology to one another, both containing activator protein 1/cAMP response element-like elements which constitute cis-acting regulatory domains. These two domains are differentially regulated in HeLa cells and primary cultures of dorsal root ganglion neurons when they are placed in the context of a reporter gene driven by the c-fos minimum promoter. One of the domains, corresponding to a region of the preprotachykinin promoter spanning nucleotides -345 to -308, contains two activator protein 1 elements adjacent to an E-box binding protein consensus sequence. Both of the activator protein 1 elements can bind a complex containing c-fos/c-fos related antigen proteins and the adjacent E-box element is specifically recognized by proteins present in HeLa nuclear extract. This domain requires the synergistic action of both activator protein 1 elements to drive expression of the reporter gene in both HeLa and dorsal root ganglion cells. The second or proximal domain spans nucleotides -198 to -155 and contains a previously characterized activator protein 1/cAMP response element/ATF enhancer element which, in contrast to the activator protein 1 elements in the distal domain, functions in both HeLa and dorsal root ganglion cells as one copy. This domain is differentially regulated in HeLa and dorsal root ganglia. The previously characterized enhancer activity is repressed in the context of the extended cis-acting domain in HeLa cells but remains active in dorsal root ganglion, although no further enhancement of activity supported by the single enhancer is observed when in the context of the extended sequence. This proximal domain, in addition to binding the enhancer complex, can be bound by at least two other complexes, one of which binds to an E-box consensus sequence. As the elements corresponding to the E-box consensus in both domains cross-compete for binding of specific complex(es) it would appear that repression of the activity of the proximal domain is correlated with a specific protein complex binding adjacent to the characterized enhancer in the region spanning nucleotides -198 to -155. The preprotachykinin-A proximal promoter is therefore bound by multiple activator protein I complexes, which in the context of the cis-acting domains in which they are present can be differentially regulated. In the proximal domain their function may also be regulated in a tissue-specific manner by other proteins which bind to adjacent regulatory elements.

Characterisation of a functional E box motif in the proximal rat preprotachykinin-A promoter.

Three E box motifs, which are upstream of the major transcriptional start site, have previously been characterised in the rat preprotachykinin-A (rPPT) promoter. Only one of these, in the proximal promoter spanning nucleotides -67 to -47, has been demonstrated to support reporter gene expression in clonal cell lines under basal growth conditions. Here we demonstrate that the reporter gene expression can be further induced by the action of phorbol 12-myristate 13-acetate (TPA) and nerve growth factor (NGF), respectively, in both HeLa and the neuronally derived PC12 cells. This response is due to the E box motif and not an overlapping consensus sequence for a putative AP1 element, a class of element previously demonstrated to respond to both TPA and NGF in these cell lines. Finally, we demonstrate that this E box motif can support similar levels of reporter gene expression in primary cultures of dorsal root ganglion neurons as observed in clonal cell lines, demonstrating that E box binding complexes can (1) function as a transcriptional regulator in dorsal root ganglion neurons and (2) bind to and therefore presumably regulate rPPT promoter activity.

Repression of preprotachykinin-A promoter activity is mediated by a proximal promoter element.

The rat preprotachykinin-A promoter, which is able to direct reporter gene expression in adult dorsal root ganglia neurons grown in culture, has no detectable activity in HeLa and PC12 cells. DNAase 1 footprinting and electrophoretic mobility shift analyses with HeLa nuclear extract indicated the presence of a protein complex binding to a region of the rat preprotachykinn-A gene promoter between the TATA box and the major transcriptional start site. We demonstrate that the sequence of the preprotachykinin-A promoter spanning nucleotides -47 to +92 functions to repress reporter gene expression in HeLa and PC12 cells but not in adult rat dorsal root ganglia grown in culture, and that this repression is correlated with a protein(s) binding to the element between the TATA box and major transcription initiation site. These results indicate that the tissue-specific expression of the preprotachykinin-A gene could require the interaction of both positive and negative regulatory DNA elements.

The rat preprotachykinin-A promoter is regulated in PC12 cells by the synergistic action of multiple stimuli.

We demonstrate in PC12 cells that although nerve growth factor, forskolin or potassium-evoked depolarisation independently induced minimal or no expression from the rat preprotachykinin-A gene (rPPT) promoter linked to a reporter gene, exposure of the cells to various combinations of these stimuli specifically activated the rPPT promoter in transient transfection assays.

An activator element within the preprotachykinin-A promoter.

The rat Preprotachykinin-A promoter (PPT) directs high levels of expression in dorsal root ganglia (DRG) neurons in culture either endogenously or when linked to a receptor construct. It is not active in any of the established tissue culture cell lines which we have analyzed. To search for transcriptional regulators within this promoter we have started to dissect the promoter into individual elements to determine their function. A DNA element which had previously been suggested to regulate transcription from DNA sequence analysis of the rat PPT promoter occurs at position -200 relative to the major start of transcription within the PPT promoter. The equivalent element from the bovine PPT promoter had previously been proposed to be a cAMP responsive element (CRE). The sequence of this enhancer has similarities with both the AP1 and CRE DNA consensus sequences. We have demonstrated that one copy of this rat PPT element linked to a heterologous basal promoter will enhance transcription in HeLa and PC12 cell lines as well as adult rat DRG neurons grown in culture. It is also demonstrated that the rat PPT element will bind proteins in HeLa nuclear extract distinct from those binding to the well-characterized Gibbon Ape Leukemia Virus (GALV) AP1 or somatostatin CRE sites by gel retardation analysis. This PPT element, when cloned in a heterologous reporter construct, although showing properties of both AP1 and CRE elements, was functionally distinguished from both the somatostatin CRE element and the GALV AP1 enhancer when these elements were tested in the same reporter construct. This PPT element has a constitutive level of activity in adult rat DRG neurons, which is fivefold higher than that driven by the reporter construct promoter. It is also significantly different from the same reporter construct linked to the somatostatin CRE and analyzed in DRG neurons.

A single-stranded DNA binding protein which interacts with sequences within the bovine preprotachykinin promoter: regulation by nerve growth factor.

The production of substance P (SP) and the mRNA encoding its precursor preprotachykinin (PPT) is regulated by nerve growth factor (NGF) in dorsal root ganglion neurons. We have shown previously that two regions of the bovine PPT promoter are capable of mediating the induction by NGF of the downstream structural gene in transfected PC12 cells. Both regions contained a sequence element, similar to a known transcription factor binding site, which is present in several other NGF-regulated genes. We show here that PC12 cells contain a single-stranded DNA binding protein (SSBP-PC12) which can interact specifically with this site. Binding activity was increased by NGF treatment of PC12 cells.

Identification of nerve growth factor-responsive sequences within the 5' region of the bovine preprotachykinin gene.

The production of substance P and the mRNA encoding its precursor (preprotachykinin, PPT) is regulated by nerve growth factor (NGF) in dorsal root ganglion (drg) neurons. To explore the mechanism by which NGF regulates the production of PPT mRNA, we have transfected PC12 cells and F11 cells with plasmids containing the bovine PPT promoter linked to the reporter gene chloramphenicol acetyltransferase (CAT). We have identified (i) functional elements within the PPT promoter which are necessary for expression in the absence of NGF and (ii) two separate regions, each of approximately 250 bp, which confer NGF responsiveness. Both regions contained a sequence element, similar to a known transcription factor binding site, which is present in several other NGF-regulated genes.

A possible role for the pituitary-adrenal system in the effects of nicotine on avoidance behaviour.

Rats were trained on a Sidman lever-pressing avoidance task. Half the animals received 0.4 mg nicotine/kg before each avoidance session. Rats which were successful performers had heavier adrenal glands. Nicotine facillitated avoidance and increased adrenal weight. Successful avoiders also tended to have higher hippocampal 5-HT levels. Hypothalamic 5-HT was elevated in animals which had just received many shocks during the final avoidance session but not in rats which had received a comparable number of unavoidable shocks. No effects of nicotine on noradrenaline or corticosterone were detected nor were these measures related to avoidance performance.

The occurrence of tolerance to a central depressant effect of nicotine.

1. Rats were injected with 0.8 mg nicotine/kg, 0.8 mg amphetamine/kg or with saline immediately before being tested for 30 min in activity boxes.2. During the first 3 trials the nicotine group were less active than the controls but from trial 5 onwards nicotine had a stimulant effect. The stimulant effect of the amphetamine did not alter with repeated injection.