VIP receptor 1 (VPAC1) promoter targets the expression of a reporter gene to cerebellum and adrenal medulla in transgenic mice

Vasoactive intestinal peptide (VIP) is a neurotransmitter with neurotropic effects. VIP functions through two distinct G-protein-coupled receptor subtypes (VPAC1 and VPAC2). We have demonstrated expression of VPAC1 in pediatric nervous system tumors, including medulloblastoma arising in the cerebellum and neuroblastoma arising in the adrenal medulla. More recently, we have reported the differentiation of neuroblastoma cells by upregulation of VIP type 1 receptor suggesting a role for VPAC1 in neuronal development.To understand the molecular mechanisms regulating VPAC1 expression in both cerebellum and adrenal medulla, we have cloned the human VPAC1 gene and sequenced 2.6-kb of the 5'-flanking sequence. Expression of the luciferase reporter gene under the control of this 2.6-kb human VPAC1 promoter was induced 35-fold in a human medulloblastoma cell line (DAOY) and 36-fold in a human neuroblastoma cell line (SKNSH). Analysis of 5'-unidirectional deletion derivatives of the 2.6-kb fragment demonstrated that a 241-bp sequence immediately upstream of the VPAC1 coding region retains high activity, suggesting that it contains the core promoter region. Quantitative RT-PCR analysis demonstrated that VPAC1 is expressed in mouse cerebellar and adrenal tissues. The VPAC1 promoter also directed expression of a reporter gene in cerebellum and adrenal medulla in transgenic mice. Along with our previous findings, these results suggest that VPAC1 may play a functional role in development of both cerebellum and adrenal medulla.     

Vasoactive intestinal peptide: autocrine growth factor in neuroblastoma.

Neuroblastoma is the most common solid tumor of children less than 5 years of age; yet the biology of this tumor is poorly understood. Neuroblastoma tumors are derived from neural crest precursors; they synthesize both adrenergic and peptidergic neurotransmitters. This study determined VIP receptor expression in primary neuroblastoma tumors prior to chemotherapy. The VIP receptor was expressed in 12 of 15 neuroblastoma tumors as determined by direct binding studies (KD = 1.3-12.4 nM) and VIP-mediated stimulation of adenylate cyclase. The VIP stimulation index for adenylate cyclase in the primary tumor was inversely correlated with the VIP content of the tumor, suggesting that VIP regulates its own receptor expression. Similar observations were made in vitro by comparison of two human neuroblastoma cell lines, IMR32 and SKNSH. Both cell lines were demonstrated to express specific, high affinity VIP receptors (KD = 4 nM and 2.5 nM for IMR32 and SKNSH, respectively). IMR32 cells contained very low levels of VIP (0.6 pg VIP/10(6) cells). Exogenous VIP stimulated adenylate cyclase 22-fold over basal activity and VIP inhibited proliferation of IMR32 cells by 49% in 6-day cultures. On the other hand, SKNSH cells synthesized high levels of VIP (6.3 pg/10(6) cells), metabolized VIP rapidly and demonstrated a low level of VIP-mediated stimulation of adenylate cyclase; their proliferation rate was minimally inhibited by exogenous VIP. These observations help validate the hypothesis that VIP serves as an autocrine growth factor in neuroblastoma.     

Somatostatin receptor gene expression in neuroblastoma

Somatostatin receptor expression is a favorable prognostic factor in human neuroblastoma. Somatostatin receptors have been demonstrated in vitro by pharmacologic analysis of tumor tissue and in vivo by diagnostic radioreceptor scintigraphy. However, which receptor subtypes (sst(1), sst(2), sst(3), sst(4), and sst(5)) are expressed in these tumors has not yet been delineated. We used RT-PCR to analyze expression of the five somatostatin receptor genes in 32 neuroblastoma tumor specimens. All 32 tumor specimens expressed mRNA for c-abl and sst(1); sst(2) mRNA was detected in 27/32 samples and somatostatin mRNA was detected in 30/32 tumor specimens. The remaining receptor subtypes, sst(3), sst(4), and sst(5) were variably expressed. Receptor protein for sst(1) and sst(2) was visualized in tumor neuroblasts as well as in endothelial cells of tumor vessels using immunostaining with specific anti-receptor antibodies. The effect of high expression of somatostatin receptors on cell proliferation was examined in SKNSH neuroblastoma cells transfected with sst(1) and sst(2). SS(14) binding to wild-type SKNSH cells was undetectable; but the native peptide bound with high affinity to the SKNSH/sst(1) and SKNSH/sst(2) neuroblastoma cell lines. Pharmacologic analysis of binding with two long-acting analogues, CH275 and octreotide, confirmed selective expression of sst(1) and sst(2) in stably transfected SKNSH cells. Formation of neuroblastoma xenograft tumors in nude mice was significantly delayed for both SKNSH/sst(1) (P<0.001) and SKNSH/sst(2) (P<0.05) cells compared to wild-type SKNSH. We conclude that: (1) Somatostatin receptors, sst(1) and sst(2), are expressed in the majority of neuroblastomas at diagnosis; and (2) upregulation of functional sst(1) or sst(2) in neuroblastoma cell lines suppresses tumorigenicity in a xenograft model. These observations suggest that somatostatin receptors may be a useful therapeutic target in neuroblastoma.     

Selective gene expression and activation-dependent regulation of vasoactive intestinal peptide receptor type 1 and type 2 in human T cells

Vasoactive intestinal peptide (VIP) has potent antiproliferative and anti-inflammatory functions in the immune system. Two structurally distinct G-protein-associated receptors, VIP receptor type 1 (VPAC1) and VIP receptor type 2 (VPAC2), mediate the biological effects of VIP. The regulation of VIP receptor gene expression and the distribution of these receptors in different compartments of the human immune systems are unknown. This study reports, for the first time, a quantitative analysis of VPAC1 and VPAC2 mRNA expression in resting and activated T cells as well as in resting monocytes. Purified human peripheral blood CD4(+) T cells and CD8(+) T cells were stimulated via the TCR/CD3 receptor complex. Using the novel fluorometric-based kinetic (real-time) RT-PCR, we determined that VPAC1 is constitutively expressed in resting T cells and monocytes; the levels of expression were significantly higher in monocytes and CD4(+) T cells than in CD8(+) T cells. VPAC1 mRNA expression is significantly higher relative to VPAC2 in resting CD4(+) T cells and CD8(+) T cells. VPAC2 is expressed at very low levels in resting T cells but is not detectable in resting monocytes. In vitro stimulation of Th cells with soluble anti-CD3 plus PMA induced a T cell activation-dependent down-regulation of VPAC1. VPAC1 is down-regulated under conditions of optimal T cell stimulation. Our results suggest that selective VIP effects on T cell function may be mediated via selective expression of VPAC1 and VPAC2 on T cells and monocytes. Furthermore, down-regulation of VPAC1 in CD4(+) T cell subpopulations is highly correlated with T cell activation.     

Differential expression of mRNAs for PACAP and its receptors during neural differentiation of embryonic stem cells

The expressions of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and their receptors (PAC1, VPAC1 and VPAC2) were examined in the five steps of the in vitro neuronal culture model of embryonic stem (ES) cell differentiation. mRNAs for PACAP, VIP, PAC1 receptor, and VPAC2 receptor were moderately expressed in neural stem cell-enriched cultures, while VPAC1 receptor mRNA was most prominently expressed in embryoid bodies (EBs). The expression of PAC1 receptor mRNA was further upregulated after terminal differentiation into neurons. In contrast, the expressions of PAC1 receptor and PACAP mRNAs were markedly decreased after glial differentiation. These results suggest that this in vitro neuronal culture system will be a useful model for future studies on the functional role of the PACAPergic system during different stages of neuronal development.     

Vasoactive intestinal peptide (VIP) stimulates [Ca2+]i and cyclic AMPin CHO cells expressing Galpha16

The stimulatory effect of vasoactive intestinal peptide (VIP) and analogues on [Ca2+]i has been investigated in chinese hamster ovary (CHO) cells stably transfected with the reporter gene aequorin, and expressing either the human VPAC1or VPAC2 receptor in absence or in presence of the Galpha16. In cells that were not transfected with Galpha16 and expressed a similar density of receptors, the VIP induced [Ca2+]i ncrease was higher in VPAC1 than in VPAC2 receptor expressing cells. In aequorin/Galpha16 cotransfected cells, the VIP-induced response was higher, reaching 70 to 80% of the maximal calcium response, obtained after digitonin treatment, in response to both VPAC1 and VPAC2 receptor stimulation.The results suggest that in hematopoietic cells, which express both VIP receptors and Galpha16, the signalling pathway of VIP could be mediated through both cyclic AMP and [Ca2+]i increase.     

Identification of G-proteins coupling to the vasoactive intestinal peptide receptor VPAC(1) using immunoaffinity chromatography: evidence for precoupling.

VPAC(1) receptor subtype-specific G-protein interactions were identified using a strategy that exploits an essential initial signaling event, namely the functional and physical association of the receptor with G-protein. An immunoaffinity purification column was constructed using a previously characterized antibody that had been raised against the first extracellular loop of the VPAC(1) receptor. VPAC(1)/G-protein complexes were solubilized from membranes and copurified. Receptor and Galpha-proteins were detected in eluates using (125)I-VIP labeling and immunoblotting, respectively. Human VPAC(1) transfected in HEK293 cells couples to Gs but not Gi3, Gi1/2, or Gq. Rat VPAC(1) in brain membranes is coupled to Gs and Gi3. Rat VPAC(1) in lung membranes couples to Gs, Gi3, and Gq. Pretreatment of membranes with VIP increased the level of all G-proteins copurifying with VPAC(1). Immunoaffinity chromatography also revealed VPAC(1) receptor precoupling to G-protein in the absence of VIP pretreatment. This was confirmed using a cross-linking procedure to capture VIP receptor/G-protein complexes in the native membrane milieu prior to solubilization. Precoupling suggests that there is a significant basal level of VPAC(1) receptor activity especially in cells, such as some human malignant tumor cells, that express high levels of receptor.     

VIP activates G(s) and G(i3) in rat alveolar macrophages and G(s) in HEK293 cells transfected with the human VPAC(1) receptor

We have characterized vasoactive intestinal peptide (VIP) receptor/G-protein coupling in rat alveolar macrophage (AM) membranes and find that pertussis toxin treatment and antisera against G(alphai3) and G(alphas) reduce high-affinity (125)I-VIP binding, indicating that both G(alphas) and G(alphai3) couple to the VIP-receptor. The predominant VIP-receptor subtype in AM is VPAC(1) and we examined the G-protein interactions of the human VPAC(1) that had been transfected into HEK293 cells. VPAC(1) has a molecular mass of 56 kDa; GTP analogs reduced (125)I-VIP binding to this protein demonstrating that high-affinity binding of VIP to the receptor requires coupling to G-protein. Functional VIP/VPAC(1)/G-protein complexes were captured by covalent cross-linking and analyzed by Western blotting. The transfected human VPAC(1) receptor in HEK293 was found to be coupled to G(alphas) but not G(alphai) or G(alphaq). Furthermore, pertussis toxin treatment had no effect on VPAC(1)/G-protein coupling in these cells. These observations suggest that the G-proteins activated by VPAC(1) may be dependent upon species and cell type.     

VIP provides cellular protection through a specific splice variant of the PACAP receptor: a new neuroprotection target

Vasoactive intestinal peptide (VIP) was known to provide neuroprotection. Three VIP receptors have been cloned: VPAC1, VPAC2 and PAC1. A specific splice variant of PAC1 in the third cytoplasmatic loop, hop2, was implicated in VIP-related neuroprotection. We aimed to clone the hop2 splice variant, examine its affinity to VIP and investigate whether it mediates the VIP-related neuroprotective activity. The PAC1 cDNA was cloned from rat cerebral astrocytes. Using genetic manipulation the hop2 splice variant was obtained, then inserted into an expression vector and transfected into COS-7 cells that were used for binding assays. Results showed that VIP bound the cloned hop2 splice variant. Stearyl-neurotensin(6-11) VIP(7-28) (SNH), an antagonist for VIP, was also found to bind hop2. In addition, VIP protected COS-7 cells expressing hop2 from oxidative stress. Parallel assays demonstrated that VIP increased cAMP accumulation in COS-7 cells expressing hop2. These results support the hypothesis that hop2 mediates the cytoprotective effects attributed to VIP.     

Hypothesis: VPAC G protein-coupled receptors for vasoactive intestinal peptide constitute a dynamic system for signaling T cells from plasma membrane and nuclear membrane complexes

The vasoactive intestinal peptide (VIP)-VPAC(1) and VPAC(2) G protein-coupled receptor (GPCR) systems are autocrine and paracrine regulators of diverse T cell functions. It has been recognized that VIP evokes two types of T cell responses. The first are rapid in onset and brief in duration, such as altered traffic in blood, lymphoid corridors, and tissues. The second are slow in onset and sustained in duration, such as enhanced helper T cell (Th) differentiation in the thymus and increased survival in lymphoid tissues with biases favoring the Th2-type effector and memory subsets. Investigations of some other sets of GPCRs for peptide and lipid mediators have demonstrated expression both in nuclear membranes and plasma membranes with respective linkages to responses that are slow in onset and sustained, and those that are rapid in onset and brief in duration. The hypothesis presented in this paper suggests that plasma membrane VPAC receptors transduce short-term effects of exogenous VIP on T cell effector functions, whereas nuclear VPAC receptors mediate endogenous VIP alterations in differentiation, proliferation, and survival. The types of substantial additional proof needed to support this hypothesis are described, as are its advantages for more selective VIP-directed therapies.     

Molecular cloning and functional expression of a VIP-specific receptor

Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: PAC(1), with high affinity for PACAP, and VPAC(1) and VPAC(2) with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor (VIP(s)) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial NH(2)-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a VPAC(2) receptor from GP gastric smooth muscle and VIP(s) from GP teniae coli smooth muscle. The cDNA sequence of the VIP(s) encodes a 437-amino acid protein (M(r) 49,560) that possesses 87% similarity to VPAC(2) receptors in rat and mouse and differs from the VPAC(2) receptor in GP gastric smooth muscle by only two amino-acid residues, F(40)F(41) in lieu of L(40)L(41). In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity (IC(50) 1.4 nM) and stimulated cAMP formation with high potency (EC(50) 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity (IC(50) 2.3 nM) and stimulated cAMP with equally high potency (EC(50) 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a VIP(s) distinct from VPAC(1) and VPAC(2) receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues (L(40)L(41)) in the NH(2)-terminal ligand-binding domain.     

PACAP-38 induces neuronal differentiation of human SH-SY5Y neuroblastoma cells via cAMP-mediated activation of ERK and p38 MAP kinases

The intracellular signaling pathways mediating the neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP) were investigated in human neuroblastoma SH-SY5Y cells. Previously, we showed that SH-SY5Y cells express the PAC(1) and VIP/PACAP receptor type 2 (VPAC(2)) receptors, and that the robust cAMP production in response to PACAP and vasoactive intestinal peptide (VIP) was mediated by PAC(1) receptors (Lutz et al. 2006). Here, we investigated the ability of PACAP-38 to differentiate SH-SY5Y cells by measuring morphological changes and the expression of neuronal markers. PACAP-38 caused a concentration-dependent increase in the number of neurite-bearing cells and an up-regulation in the expression of the neuronal proteins Bcl-2, growth-associated protein-43 (GAP-43) and choline acetyltransferase: VIP was less effective than PACAP-38 and the VPAC(2) receptor-specific agonist, Ro 25-1553, had no effect. The effects of PACAP-38 and VIP were blocked by the PAC(1) receptor antagonist, PACAP6-38. As observed with PACAP-38, the adenylyl cyclase activator, forskolin, also induced an increase in the number of neurite-bearing cells and an up-regulation in the expression of Bcl-2 and GAP-43. PACAP-induced differentiation was prevented by the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine (DDA), but not the protein kinase A (PKA) inhibitor, H89, or by siRNA-mediated knock-down of the PKA catalytic subunit. PACAP-38 and forskolin stimulated the activation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAP; p38 MAP kinase) and c-Jun N-terminal kinase (JNK). PACAP-induced neuritogenesis was blocked by the MEK1 inhibitor PD98059 and partially by the p38 MAP kinase inhibitor SB203580. Activation of exchange protein directly activated by cAMP (Epac) partially mimicked the effects of PACAP-38, and led to the phosphorylation of ERK but not p38 MAP kinase. These results provide evidence that the neurotrophic effects of PACAP-38 on human SH-SY5Y neuroblastoma cells are mediated by the PAC(1) receptor through a cAMP-dependent but PKA-independent mechanism, and furthermore suggest that this involves Epac-dependent activation of ERK as well as activation of the p38 MAP kinase signaling pathway.     

Inhibition of neuropathy target esterase expressing by antisense RNA does not affect neural differentiation in human neuroblastoma (SK-N-SH) cell line

Neuropathy target esterase (NTE) is phosphorylated and aged by oraganophosphorus compounds (OP) that induce delayed neuropathy in human and some animals. NTE has been proposed to play a role in neurite outgrowth and process elongation during neural differentiation. However, to date, there is no direct evidence of the relevance of NTE in neural differentiation under physiological conditions. In this study we have investigated a possible role for NTE in the all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells by antisense RNA. A NTE antisense RNA construct was generated and then transfected into human neuroblastoma SK-N-SH cells. A positive cell clone that can stably express NTE antisense RNA was obtained by G418 selection and then identified by western blotting. NTE activity was depressed in the transfected cells with only about 50% activity of the enzyme in the control cells. ATRA-induced differentiation of the neuroblastoma cells with lowered NTE activity revealed that inhibition of NTE expression does not affect neural differentiation in SK-N-SH cells. The result suggested that organophosphates may inhibit neural differentiation by initially acting on other targets other than NTE.     

Intermittent hypoxia regulates stem-like characteristics and differentiation of neuroblastoma cells

Background

Neuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas are derived from immature cells of the sympathetic nervous system and are characterized by clinical and biological heterogeneity. Hypoxia has been linked to tumor progression and increased malignancy. Intermittent hypoxia or repeated episodes of hypoxia followed by re-oxygenation is a common phenomenon in solid tumors including neuroblastoma and it has a significant influence on the outcome of therapies. The present study focuses on how intermittent hypoxia modulates the stem-like properties and differentiation in neuroblastoma cells.

Methods and Findings

Cell survival was assessed by clonogenic assay and cell differentiation was determined by morphological characterization. Hypoxia-inducible genes were analyzed by real-time PCR and Western blotting. Immunofluorescence, real-time PCR and Western blotting were utilized to study stem cell markers. Analysis of neural crest / sympathetic nervous system (SNS) markers and neuronal differentiation markers were done by real-time PCR and Western blotting, respectively. Intermittent hypoxia stimulated the levels of HIF-1α and HIF-2 α proteins and enhanced stem-like properties of neuroblastoma cells. In intermittent hypoxia-conditioned cells, downregulation of SNS marker genes and upregulation of genes expressed in the neural crest were observed. Intermittent hypoxia suppressed the retinoic acid-induced differentiation of neuroblastoma cells.

Conclusions

Our results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses differentiation propensities in neuroblastoma cells.     

Cloning of HumanENC-1and Evaluation of Its Expression and Regulation in Nervous System Tumors

We recently identified and characterized a novel murine gene,ENC-1,that is expressed primarily in the nervous system and encodes an actin-binding protein. To gain insight into a potential role forENC-1gene in the processes of cell differentiation and malignant transformation in the human nervous system, we first cloned and characterized the human homologue ofENC-1.The humanENC-1gene appeared to be highly expressed in adult brain and spinal cord, and in a number of cell lines derived from nervous system tumors we detected low steady-state levels ofENC-1mRNA. We used a neuroblastoma differentiation model, the retinoic acid-induced neuronal differentiation of SMS-KCNR cells, to study the regulation of theENC-1gene during neural crest cell differentiation. We found that the expression ofENC-1increased dramatically in the differentiated SMS-KCNR cells as compared to control undifferentiated cells. These results suggest thatENC-1expression plays a role during differentiation of neural crest cells and may be down regulated in neuroblastoma tumors.     

Differential regulation of activity-dependent neuroprotective protein in rat astrocytes by VIP and PACAP

Activity-dependent neuroprotective protein (ADNP) was shown to be a vasoactive intestinal peptide (VIP) responsive gene in astrocytes derived from the cerebral cortex of newborn rats. The present study was set out to identify VIP receptors that are associated with increases in ADNP expression in developing astrocytes. Using VIP analogues specific for the VPAC1 and the VPAC2 receptors, it was discovered that VIP induced changes in ADNP expression in astrocytes via the VPAC2 receptor. The constitutive synthesis of ADNP and VPAC2 was shown to be age-dependent and increased as the astrocyte culture developed. Pituitary adenylate cyclase-activating polypeptide (PACAP) also induced changes in ADNP expression. The apparent changes induced by VIP and PACAP on ADNP expression were developmentally dependent, and while stimulating expression in young astrocytes, an inhibition was demonstrated in older cultures. In conclusion, VIP, PACAP and the VPAC2 receptor may all contribute to the regulation of ADNP gene expression in the developing astrocyte.