Christine F. Morrison

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, VPAC1R and VPAC2R, are prominent in the immune system and regulate many aspects of T cell-dependent immunity. In mouse T cells, VPAC1R is expressed constitutively, whereas VPAC2R is induced by immune stimuli. VPAC2R-null (VPAC2R−/−) 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 VPAC2R-null mice compared with age- and sex-matched wild-type mice. In contrast, generation of IgE anti-hapten antibodies and active cutaneous anaphylaxis were ≥70% lower in VPAC2R-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-γ (mean = 3-fold), and lower levels of IL-4 (mean = one-fifth) in VPAC2R-null mice than wild-type controls. Loss of VIP-VPAC2R 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.

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.

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.

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.

Desensitization of the Human Vasoactive Intestinal Peptide Receptor (hVIP2/PACAP R): Evidence for Agonist-Induced Receptor Phosphorylation and Internalizationa

Abstract: 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.

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.

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.

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.