preproenkephalin and Neuroblastoma

Ethanol alters neural activity through interaction with multiple neurotransmitters and neuromodulators. The endogenous opioid system seems to play a key role, since the opioid receptor antagonist naltrexone (ReVia®) attenuates craving for alcohol. We recently reported that ethanol and acetaldehyde, the first product of ethanol metabolism, affect transcription of opioid system genes in human SH-SY5Y neuroblastoma cells. In the current study, potential epigenetic mechanisms were investigated to clarify these effects on prodynorphin gene expression. DNA methylation was analyzed by bisulfite pyrosequencing, and chromatin immunoprecipitation was used to assess putative specific histone modifications at the prodynorphin gene promoter. The results demonstrated a temporal relationship between selective chromatin modifications induced by ethanol and acetaldehyde and changes in prodynorphin gene expression quantitated by real-time qPCR. DNA methylation was not altered in any of the experimental conditions used. The epigenetic changes may precede gene transcription, and histone modifications might keep the prodynorphin gene in a poised state for later reactivation. A link has been observed between gene expression alterations and selective epigenetic modulation in the prodynorphin promoter region, demonstrating a specificity of the changes induced by ethanol and acetaldehyde. The latter may be mediating ethanol effects at the genomic level.

Topics: Acetaldehyde; Cell Line, Tumor; Central Nervous System Depressants; Chromatin; DNA Methylation; Enkephalins; Epigenomics; Ethanol; Gene Expression Regulation; Humans; Neuroblastoma; Promoter Regions, Genetic; Protein Precursors

Prodynorphin transcription has been postulated as an important molecular mechanism involved in adaptation/repair processes. Expression of prodynorphin is modulated according to the levels of the second messengers cAMP and Ca(2+). In the neuroblastoma cell lines, the increase of prodynorphin mRNA levels is coupled to an elevation of intracellular cAMP levels. Promoter analyses have revealed that the DRE site, a silencer element present in the prodynorphin promoter, is involved in PKA-dependent prodynorphin derepression. In this way, DREAM, a calcium-dependent repressor, plays an outstanding role. In this study, Ca(2+) release from internal stores has been found to promote an increase of prodynorphin mRNA levels in NB69 cells. Surprisingly, Ca(2+)-dependent prodynorphin gene transcription was insensitive to the broad-spectrum kinase inhibitors and sensitive to agents that alter internal Ca(2+) accumulation. Moreover, we demonstrate that in NB69 cells, the Ca(2+) signaling pathway uses DREAM as an effector to evoke prodynorphin transcription derepression in a kinase-independent manner.

Topics: Animals; Caffeine; Calcium; Calcium Signaling; Calcium-Binding Proteins; Central Nervous System; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enkephalins; Enzyme Inhibitors; Gene Expression Regulation; Genes, Regulator; Humans; Kv Channel-Interacting Proteins; Mutation; Neuroblastoma; Neurons; Protein Biosynthesis; Protein Precursors; Repressor Proteins; RNA, Messenger; Tumor Cells, Cultured

Whereas the role of dopaminergic tone in the cortico-striatal-thalamic system is well-established, the role of endogenous opioids in the function of this system is less understood. We show that Borna disease virus infection of adult rats results in an increase in preproenkephalin transcripts in the striatum of Borna-infected rats, a region important for forming coordinated sequential motor actions and in developing programmes of thought and motivation. Stereotypic behaviours and dyskinesias, the clinical hallmarks of infection in adult Lewis rats (BD rats), are accompanied by a disrupted pattern of immediate early gene c-fos activation in the motor thalamus, with significance for the breakdown in coordinated sequential motor actions. We also find increased preproenkephalin in infected cultured neuroblastoma and rat foetal glial cells. The expression pattern of enkephalin mRNA in vivo and in vitro suggest that increased enkephalin function is one of the neuropharmacological means by which Borna disease virus causes motor disease of animals and possibly cognitive and affective disease in man, and further suggest that enkephalins play a critical role in the maintenance of a balanced tone of activity in the cortico-basal ganglia-thalamo-cortical loops.

Topics: Animals; Astrocytes; Borna Disease; Borna disease virus; Cells, Cultured; Dynorphins; Enkephalins; Gene Expression; Genes, fos; Humans; Male; Neuroblastoma; Protein Precursors; Putamen; Rats; Rats, Inbred Lew; Receptors, Dopamine D2; Tumor Cells, Cultured

Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, alphaCREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of alphaCREM is not required for the interaction, but phospho-alphaCREM shows higher affinity for DREAM. The interaction with alphaCREM is independent of the Ca(2+)-binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both alphaCREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca(2+).

Topics: Amino Acid Motifs; Amino Acid Sequence; Calcium; Calcium-Binding Proteins; Cell Line; Colforsin; Cyclic AMP Response Element Modulator; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; DNA-Binding Proteins; Enkephalins; Gene Expression Regulation; Genes, Regulator; Genes, Reporter; Humans; Kv Channel-Interacting Proteins; Molecular Sequence Data; Mutation; Neuroblastoma; Phosphorylation; Protein Precursors; Recombinant Proteins; Repressor Proteins; RNA, Messenger; Sequence Alignment; Transfection; Tumor Cells, Cultured

Recombinant herpes simplex virus-1 encoding the rat preproenkephalin A (HSVLatEnk1) was generated for driving the expression of preproenkephalin A-derived peptides in dorsal root ganglia of rats in vivo. Three weeks after infection via the hind footpads, quantitative RT-PCR and in situ hybridization experiments showed a strong expression of preproenkephalin A mRNA in lumbar dorsal root ganglia. In addition, a 40-160% increase in radioimmunoassayable Met-enkephalin-like material concentrations was found in the dorsal spinal cord and dorsal root ganglia, respectively, at the lumbar level in HSVLatEnk1-infected rats as compared with animals infected with beta-galactosidase-encoding recombinant herpes simplex virus-1 or control rats. These data demonstrate the efficacy of the preproenkephalin A encoding vector and suggest that it should help in elucidating the role of Met-enkephalin-containing primary afferent fibers in pain transmission and/or control.

Topics: Animals; beta-Galactosidase; Enkephalins; Ganglia, Spinal; Gene Expression Regulation, Viral; Gene Transfer Techniques; Genes, Reporter; Herpesvirus 1, Human; Humans; Male; Neuroblastoma; Pain; Promoter Regions, Genetic; Protein Precursors; Rats; Rats, Sprague-Dawley; Recombinant Proteins; RNA, Messenger; Tumor Cells, Cultured

Induction of the prodynorphin gene has been implicated in medium and long-term adaptation during memory acquisition and pain. By 5' deletion mapping and site-directed mutagenesis of the human prodynorphin promoter, we demonstrate that both basal transcription and protein kinase A (PKA)-induced transcription in NB69 and SK-N-MC human neuroblastoma cells are regulated by the GAGTCAAGG sequence centered at position +40 in the 5' untranslated region of the gene (named the DRE, for downstream regulatory element). The DRE repressed basal transcription in an orientation-independent and cell-specific manner when placed downstream from the heterologous thymidine kinase promoter. Southwestern blotting and UV cross-linking experiments with nuclear extracts from human neuroblastoma cells or human brain revealed a protein complex of approximately 110 kDa that specifically bound to the DRE. Forskolin treatment reduced binding to the DRE, and the time course paralleled that for an increase in prodynorphin gene expression. Our results suggest that under basal conditions, expression of the prodynorphin gene is repressed by occupancy of the DRE site. Upon PKA stimulation, binding to the DRE is reduced and transcription increases. We propose a model for human prodynorphin activation through PKA-dependent derepression at the DRE site.

Topics: 5' Untranslated Regions; Cyclic AMP-Dependent Protein Kinases; DNA-Binding Proteins; Enkephalins; Enzyme Activation; Gene Expression Regulation; Genes, Regulator; Humans; Mutagenesis, Site-Directed; Neuroblastoma; Nuclear Proteins; Promoter Regions, Genetic; Protein Precursors; Transcription, Genetic; Tumor Cells, Cultured

The prodynorphin gene contains several kappa B motifs, suggesting that kappa B-specific DNA-binding factors may regulate its expression. Prodynorphin is known to be expressed in human tumor cell lines [Geiger et al., Regul. Peptides, 34 (1991) 181-188] and we report here that several DNA-binding factors of the NF-kappa B/c-Rel-family are present in the same cells. Three main kappa B-specific factors, presumably a p50 homodimer, NF-kappa B which is a p50/p65 heterodimer and a p65/c-Rel heterodimer were identified using an electromobility shift assay (EMSA), immunoabsorption and UV cross-linking experiments. Minor factors consisting of a novel kappa B-specific protein of about 125 kDa (p125) or being hetero-oligomeric, composed of p125 and either of three other subunits, namely p50, p65 and c-Rel, were also identified. The homo-oligomer of p125 may be identical to the kappa B-specific factor BETA, previously found only in brain [Korner et al., Neuron, 3 (1989) 563-572]. Comparison of prodynorphin mRNA levels with levels of the kappa B-specific DNA-binding factors revealed a negative correlation with the level of p50 homodimer, and a positive correlation with the ratio of the levels of p65/c-Rel to NF-kappa B. No association was found with proenkephalin mRNA levels which were significant in only one cell line. The p50 homodimer, but not p65/c-Rel and NF-kappa B, bound specifically to a DNA-motif within the dynorphin A-encoding gene sequence. This sequence is located in exon 4 and similar to the consensus kappa B-sequence. The dynorphin A-encoding sequence may represent an intragenic target for the p50 homodimer, which when bound to the sequence suppresses transcription.

Topics: Animals; Base Sequence; Binding Sites; Cell Line; Choriocarcinoma; Enkephalins; Gene Expression; Humans; Lung Neoplasms; Molecular Sequence Data; Neuroblastoma; NF-kappa B; Oligodeoxyribonucleotides; Oligonucleotide Probes; Protein Precursors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-rel; Rats; Sequence Homology, Nucleic Acid; Swine; Tumor Cells, Cultured

Regulation of preproenkephalin gene expression was studied in NG108-15 neuroblastoma-glioma hybrid cells. Untreated cells contain 20-120 fg preproenkephalin mRNA per microgram cellular RNA. Treatment of cells with a glucocorticoid (e.g. dexamethasone) for 24 hr or 8 days elevated the abundance of this mRNA to 3 or 9 times the control, respectively. Treatment with 8-bromo-cyclic AMP or an adenylate cyclase activator such as prostaglandin E1 or forskolin elevated preproenkephalin mRNA to twice the control or less. Treatment with both glucocorticoid and forskolin for 24 hr or 8 days markedly increased preproenkephalin mRNA to 5-8 and 30 times the control, respectively. Intracellular Met-enkephalin immunoreactivity was increased in parallel with the mRNA abundance. The results demonstrate that preproenkephalin gene expression is synergistically regulated by glucocorticoids and cAMP.

Topics: Base Sequence; Cells, Cultured; Colforsin; Cyclic AMP; Drug Synergism; Enkephalin, Methionine; Enkephalins; Gene Expression Regulation; Glioma; Glucocorticoids; Hybrid Cells; Neuroblastoma; Protein Precursors; RNA, Messenger