Interplay of the E box, the cyclic AMP response element, and HTF4/HEB in transcriptional regulation of the neurospecific, neurotrophin-inducible vgf gene.

vgf is a neurotrophin response-specific, developmentally regulated gene that codes for a neurosecretory polypeptide. Its transcription in neuronal cells is selectively activated by the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3, which induce survival and differentiation, and not by epidermal growth factor. We studied a short region of the rat vgf promoter which is essential for its regulated expression. A cyclic AMP response element (CRE) within this region is necessary for NGF induction of vgf transcription. Two sites upstream of CRE, an E box and a CCAAT sequence, bind nuclear protein complexes and are involved in transcriptional control. The E box has a dual role. It acts as an inhibitor in NIH 3T3 fibroblasts, together with a second E box located downstream, and as a stimulator in the NGF-responsive cell line PC12. By expression screening, we have isolated the cDNA for a basic helix-loop-helix transcription factor, a homolog of the HTF4/HEB E protein, that specifically binds the vgf promoter E box. The E protein was present in various cell lines, including PC12 cells, and was a component of a multiprotein nuclear complex that binds the promoter in vitro. The E box and CRE cooperate in binding to this complex, which may be an important determinant for neural cell-specific expression.

Expression, processing, and secretion of the neuroendocrine VGF peptides by INS-1 cells.

The neurotropin-inducible gene vgf is expressed in neuronal and endocrine tissues. It encodes a secretory protein that is proteolytically processed in neuronal cells to low molecular mass polypeptides. In the present report, we show that vgf is expressed in different insulinoma cell lines and in normal rat pancreatic islets. In the insulinoma-derived beta-cell line INS-1, vgf messenger RNA was transcriptionally up-regulated by increased levels ofintracellular cAMP, but not by the addition of glucose (20 mM) or phorbol 12-myristate 13-acetate (100 nM). Furthermore, nerve growth factor failed to stimulate vgf gene expression. In INS-1 cells, the VGF protein was shown to be processed in a post endoplasmic reticulum compartment to produce a peptide profile similar to that seen in neurons. The release of such VGF peptides occurred at a low rate in the absence of secretory stimuli (<2%/h). A 3-fold increase in the rate of release was seen after the addition of glucose (15 mM), a 4-fold increase was seen after (Bu)2cAMP (1 mM), and a 6-fold increase was seen after phorbol 12-myristate 13-acetate (100 nM). These results indicated that insulin-containing cells produce VGF-derived peptides that are released via a regulated pathway in response to insulin secretagogues.

Interindividual variability of enkephalin-degrading enzymes in human plasma.

The interindividual variability of the hydrolysis of leucine enkephalin, and of the formation of its hydrolysis by-products has been studied in human plasma. In agreement with known data, the data obtained indicate that Leu-enkephalin is degraded by several enzymes, belonging to three classes: aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases. The relative ratio of the substrate degraded by each enzyme class-as well as the expression of the single enzyme species within each class-appears to be individually determined. Interindividual variability observed seems controlled by two main factors: the pattern of enkephalin-degrading enzymes and, more notably, the low molecular weight plasma inhibitors. Both these factors appear to be partially specific of each donor. Possibly because of the composition of these factors, the hydrolysis pattern of the substrate is characteristic of each donor, and constant in blood obtained from successive drawings, at least within a relatively short period of time.

Molecular cloning and characterization of the human VGF promoter region.

The VGF gene encodes a secretory protein that is expressed in a cell type-restricted pattern in neuroendocrine cells and is up-regulated by nerve growth factor (NGF) in the rat pheochromocytoma PC12 cell line. Here we report the isolation and characterization of the 5'-terminal region of the human VGF gene. In addition to a TATA box and a CCAAT box located at canonical distances from the transcription start site, the human VGF promoter contains several consensus sequences for different transcription factors, including a cyclic AMP response element and an AP-1 element, several GC boxes, and sequences homologous to other neuronal promoters. Transient transfection analysis demonstrates that 2.3 kb of the 5'-flanking sequence acts as a tissue-specific promoter, efficiently used only by neuronal cells that express endogenous VGF. Deletion analysis reveals that a positive regulatory region is located between nucleotides -458 to -204. Negative cis-acting elements that repress promoter activity in cell lines that do not normally express VGF are located between nucleotides -2,305 and -573 and between -458 and -204. The 5'-flanking region of the human VGF gene confers responsiveness to NGF, cyclic AMP, and phorbol ester treatment.

Positive and negative immunomodulation by opioid peptides.

The data that follow review part of the existing evidence concerning the neuroimmune functions mediated by opioid peptides, with particular regard to dual immunomodulatory effects. Limited references to substances other than opioid peptides are included, mainly to emphasize the possible similarities in the mediation of neuroimmune interactions by different informational substances, while the interactions directed from the immune to the nervous system have deliberately been omitted.

Effect of differentiation on the leucine enkephalin-degrading soluble enzymes released by the K562(S) cell line.

Leu-enkephalin hydrolysis kinetics were measured in the presence of soluble supernatants obtained from cultures of the K562(S) leukaemic cell line. Under these conditions, the substrate is degraded with formation of two distinct patterns of the hydrolysis by-products: in one pattern, similar amounts of Tyr and Tyr-Gly are formed; in the other, only Tyr-Gly can be measured. Kinetic data suggest that soluble proteolyses are released by these cells, and that either dipeptidylaminopeptidases alone, or both aminopeptidases and dipeptidylaminopeptidases are involved in substrate hydrolysis. This alternation of hydrolysis patterns appears consistent with existing data on the heterogeneity of K562 cells. In contrast with these results, chromatographic separation of the soluble enzymes indicates the release of all three classes of proteolyses known to hydrolyze enkephalins: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. In cells induced to differentiate by treatment with butyric acid, substrate hydrolysis is increased, and the pattern of the enzymes released is modified. In these cells, variations in both total proteolytic activity, and ratio between the three enzyme classes mentioned above are only minor, while the ratio between the different enzyme species within each class is greatly modified. Data obtained suggest that the expression of soluble enzymes is modified by differentiation. These data may also be interpreted as stressing the role of competition in controlling substrate hydrolysis by the multiple enzymes co-released by K562(S) cells.

Tissue-specific processing of the neuroendocrine protein VGF.

VGF is a neuroendocrine-specific gene product that is up-regulated by nerve growth factor in the PC12 cell line. In rat neuroendocrine tissues two polypeptides of 90 and 80 kDa were detected by an antiserum to an N-terminal domain of VGF (from residues 4 to 240). In parallel, an antiserum directed against the C-terminal nonapeptide of VGF (from residues 609 to 617) revealed several additional posttranslational products. Peptides of apparent molecular sizes of 20, 18, and 10 kDa were prominent in nerve tissues and the hypophysis but absent in the adrenal medulla, and their relative abundance varied in distinct regions of the CNS. In PC12 cells VGF was proteolytically processed only after nerve growth factor treatment, and primary cultures of rat cerebellar granule cells accumulated the low-molecular-weight forms of VGF during in vitro maturation. In these cells the specific cleavages of VGF occurred in a postendoplasmic reticulum compartment; the processed forms were enriched in the secretory vesicles and were preferentially secreted upon cell membrane depolarization. Distinct differential distribution in the CNS and in vitro release of such posttranslational products indicate that these species may represent biologically relevant forms of VGF that play a role in neuronal communication.

Isolation and characterization of VGF peptides in rat brain. Role of PC1/3 and PC2 in the maturation of VGF precursor.

The neurotrophin responsive gene vgf is widely expressed in central and peripheral neurones, and in certain neuroendocrine cell populations. Its encoded VGF precursor protein (proVGF1: 617 amino acids in rat, 615 in man, > 85% homology) gives rise to several low molecular weight species. We studied a range of neuroendocrine and neuronal cells, in which VGF-processing products were prominent with an apparent molecular weight of 20 and 10 kDa (VGF20 and VGF10, respectively). Such peptides were recognized by antibodies specific for the C-terminal rat VGF nonapeptide, thus indicating that they included the C-terminus of proVGF. Ectopic expression of the neuroendocrine-specific prohormone convertases PC1/3 or PC2 in GH3 cells showed that both could generate VGF20, while VGF10 was preferentially produced by PC1/3. Site-directed mutagenesis was used to identify the KRKRKK(488) motif as the target within VGF sequence which leads to the production of VGF20. Molecular characterization of rat VGF10, on the other hand, revealed that this peptide is produced by cleavage at the RPR(555) site. By the combined use of high-resolution separation techniques, matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectrometry and manual Edman degradation we identified in rat brain a VGF fragment analogous to bovine peptide V and two novel peptides also derived from the C-terminal region of proVGF.