Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells.

Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.     

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.     

Nucleotide Sequence and Regulatory Studies of VGF, a Nervous System-Specific mRNA That Is Rapidly and Relatively Selectively Induced by Nerve Growth Factor

A nervous system-specific mRNA that is rapidly induced in PC12 cells to a greater extent by nerve growth factor (NGF) than by epidermal growth factor treatment has been cloned. The polypeptide deduced from the nucleic acid sequence of the NGF33.1 cDNA clone contains regions of amino acid sequence identity with that predicted by the cDNA clone VGF, and further analysis suggests that both NGF33.1 and VGF cDNA clones very likely correspond to the same mRNA (VGF). In this report both the nucleic acid sequence that corresponds to VGF mRNA and the polypeptide predicted by the NGF33.1 cDNA clone are presented. Genomic Southern analysis and database comparison did not detect additional sequences with high homology to the VGF gene. Induction of VGF mRNA by depolarization and phorbol 12-myristate 13-acetate treatment was greater than by serum stimulation or protein kinase A pathway activation. These studies suggest that VGF mRNA is induced to the greatest extent by NGF treatment and that VGF is one of the most rapidly regulated neuronal mRNAs identified in PC12 cells.     

Characterization of vaccinia virus growth factor biosynthetic pathway with an antipeptide antiserum.

A synthetic peptide derived from vaccinia virus growth factor (VGF) was used as an immunogen to prepare antiserum able to immunoprecipitate native VGF from both vaccinia virus-infected cell lysate and cell-free medium. Pulse-chase, tunicamycin treatment, and carbohydrate trimming experiments revealed that VGF is synthesized as a 19-kilodalton (kDa) precursor which is rapidly modified to a high-mannose-type 22-kDa protein. This cell-associated form is further processed into a 25-kDa polypeptide which, after proteolytic cleavage, releases the mature VGF into the medium as a 22-kDa glycoprotein.     

A protein induced by NGF in PC12 cells is stored in secretory vesicles and released through the regulated pathway.

We have previously described the isolation of a cDNA clone corresponding to an mRNA rapidly induced to high levels in PC12 cells by treatment with NGF. We report here the complete amino acid sequence of the protein (named VGF8a) as deduced by nucleotide sequencing of overlapping cDNA clones. VGF8a is particularly rich in proline residues and has a conspicuous number of short stretches of basic amino acid residues which may represent potential targets for proteolytic cleavage. Antibodies directed against recombinant VGF8a-beta-galactosidase fusion proteins were used for immunofluorescent staining of the protein in PC12 cells as well as for its localization, by Western blot analysis, in subfractions of cell homogenates. We demonstrate that in PC12 cells, VGF8a protein is stored in secretory vesicles and is released in response to a variety of stimuli that are known to induce the regulated secretion of neurotransmitters.     

Affinity labeling of a nerve growth factor receptor component on rat pheochromocytoma (PC12) cells

Clonal PC12 rat pheochromocytoma cells were sequentially incubated with 125I-labeled nerve growth factor and the photoreactive bifunctional agent hydroxysuccinimidyl-p-azidobenzoate. This treatment effected the crosslinking of 125I nerve growth factor to a PC12 cell component that exhibits an apparent Mr = 148 000-158 000, and consists of a single polypeptide chain with internal disulfide bonds. The amount of label associated with this Mr = 148 000-158 000 species was proportional to the degree of occupancy of nerve growth factor receptors by 125I-labeled nerve growth factor. Affinity labeling of this species was inhibited by the presence of 0.2 microM unlabeled nerve growth factor during incubation of PC12 cells with 125I nerve growth factor. In membranes prepared from PC12 cells hydroxysuccinimidyl-p-azidobenzoate effected the crosslinking of 125I-labeled nerve growth factor to an Mr = 120 000-130 000 species but not to the Mr = 148 000-158 000 component observed in intact cells. The kinetics of 125I nerve growth factor affinity labeling of the Mr = 148 000-158 000 species closely paralleled the time-course of 125I nerve growth factor association to two kinetically distinct forms of nerve growth factor receptors in PC12 cells. The data indicate that the Mr = 148 000-158 000 species affinity-labeled by 125I nerve growth factor is the native form of a component associated with kinetically different nerve growth factor receptors in PC12 cells.     

Three forms of rat basic fibroblast growth factor are made from a single mRNA and localize to the nucleus

The molecular weight of rat basic fibroblast growth factor is predicted to be 18 kDa when the amino acid sequence is read from the single AUG initiation codon found in the cDNA. DNA sequencing upstream of this AUG codon indicated, however, that there was an extended open reading frame. In vitro translation of the rat cDNA for basic FGF gave three proteins of 18.0, 21.5, and 22.0 kDa in equal abundance. The same proteins were produced in vivo by COS cells transfected with the rat cDNA. Deletion of 81 base pairs from the reading frame upstream of the AUG codon resulted in the expression of only one protein observed at 18.0 kDa. These results indicated that the 22.0 and 21.5 kDa forms of rat basic FGF were formed when translation initiates at the alternative upstream non-AUG codons. Rat cell lines and tissues were found to express all three forms of basic FGF protein. The cDNA was used to analyze the subcellular distribution of the different forms of rat basic FGF. Subcellular fractionation and immunofluorescence of transfected COS cells showed that all three forms of the protein localized preferentially in the nucleus. Expression of a truncated cDNA from which 81 base pairs (27 amino acids) of the upstream reading frame had been deleted, showed localization of the smaller form of bFGF alone in the nucleus. These results demonstrated that although the amino acids that were deleted from the N-terminus of rat basic fibroblast growth factor have a sequence characteristic of nuclear localization motifs, they are not obligatory for the transport of the growth factor into the nucleus. Nuclear extracts taken from transfected cells also contained two smaller proteins of 16 and 12 kDa that were detected by Western blot analysis. It is possible that these are proteolytic products of bFGF.     

Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation

To investigate the possible role of ras proteins in the differentiation process signaled by nerve growth factor, we have microinjected the proto-oncogenic and oncogenic (T24) forms of the human H-ras protein into living rat pheochromocytoma cells (PC12). PC12 cells, which have the phenotype of replicating chromaffin-like cells under normal growth conditions, respond to nerve growth factor by differentiating into nonreplicating sympathetic neuron-like cells. Microinjection of the ras oncogene protein promoted the morphological differentiation of PC12 cells into neuron-like cells. In contrast, microinjection of similar amounts of the proto-oncogene form of the ras protein had no apparent effect on PC12 cells. The induction of morphological differentiation by the ras oncogene protein occurred in the absence of nerve growth factor, was dependent on protein synthesis, and was accompanied by cessation of cell division. Treatment of PC12 cells with nerve growth factor or cAMP analogue prior to injection did not alter the phenotypic changes induced by the ras oncogene protein.     

VGF metabolic-related gene: distribution of its derived peptides in mammalian pancreatic islets.

The vgf gene has been shown to be involved in several metabolic pathways. Because the pancreas is crucial to metabolism and food intake, we studied the VGF peptides in bovine, rat, and pig Langherans islets using antisera raised against specific sites along the primary sequence of the rat/mouse and human VGF protein precursor. Whereas almost all of the pancreatic endocrine cells expressed vgf mRNA, when using the VGF antisera a different staining pattern became apparent. VGF(556-565) and VGF(282-291) immunoreactivity were exclusively found in delta somatostatin-producing cells, whereas the human C-terminus antiserum selectively immunolabeled alpha glucagon and pancreatic polypeptide cells. The same cells were decorated with the VGF(443-588) antiserum, which also weakly labeled beta insulin-secreting cells. Finally, the VGF(298-306) peptide and the rat C terminus were found in virtually all pancreatic endocrine cells. Using bovine, swine, and rat pancreatic extracts, data from chromatography and ELISA assay showed the presence of a high molecular mass form compatible with the proVGF and lower molecular mass fractions corresponding to short VGF peptides. In conclusion, selective VGF distribution may suggest a multifaceted cell type-specific processing of proVGF, resulting in different peptides probably involved in neuroendocrine regulatory metabolic mechanisms.     

Growth inhibition by vaccinia virus growth factor

Vaccinia virus growth factor (VGF), a highly glycosylated 77-residue epidermal growth factor (EGF)-like polypeptide encoded in vaccinia poxvirus, is reported to play an important role in stimulating growth of uninfected cells to facilitate virus infection. We have chemically synthesized the unglycosylated forms of VGF and VGF19-69, a shortened VGF analog consisting of 51 residues and comprising the EGF-homologous region (position 19-69) of VGF. Both synthetic forms of VGFs were purified to homogeneity and vigorously characterized by various criteria, including the Cf-252 ion fission fragment mass spectrometry, amino acid sequencing, and enzymatic digestion to confirm the disulfide linkages. Synthetic VGFs exhibited high affinity binding to the EGF receptors in A431, NRK 49F, NRK clone 3, and NIH 3T3 cells, but, unlike the glycosylated form, showed contrasting mitogenic activities in various cells in vitro. Synthetic VGFs showed low levels of mitogenic and colonogenic activities in NRK clone 49F cells and NIH 3T3 cells, full agonist activities in human keratinocytes and Swiss 3T3 cells, and partial agonist activities in NRK clone 3 cells. Our results suggest that the unglycosylated form of VGF is an EGF antagonist to selected cells and that the production of unglycosylated form of VGF by the cytolytic vaccinia virus may serve as a mechanism whereby inhibition of growth and metabolism of selected host cells may be used to facilitate the propagation of the virus infection.     

Nerve Growth Factor-Induced Changes in the Structure of Sulfated Proteoglycans in PC 12 Pheochromocytoma Cells

Structural changes in proteoglycans (PGs) were examined during the neuritogenesis of PC12 cells induced by nerve growth factor (NGF). (1) A heparan sulfate (HS) PG and a chondroitin sulfate (CS) PG were synthesized by PC12 cells, irrespective of the presence of NGF or the duration of culture. PGs released from PC12 cells into the culture medium were mostly CSPGs. (2) In the absence of NGF, the apparent molecular mass of HSPG prepared from PC12 cells after 3 days of culture was in the range of 90-190 kDa for the intact form (Kav = 0.38 on Sepharose CL-6B), 12 kDa for HS, and 61 kDa for the core protein. In the presence of NGF, these values were 90-190 kDa, 10 kDa, and 51 kDa and 61 kDa, respectively. The intact forms of cell-associated CSPG had apparent molecular mass ranges of 120-150 kDa and 120-190 kDa (Kav = 0.38 and 0.34), with CSs of 15 kDa and 20 kDa in the presence and absence of NGF, respectively. The apparent molecular mass of the core protein of cell-associated CSPG was 92 kDa, irrespective of the presence of NGF. The molecular sizes of cell-associated PGs and their glycosaminoglycans remained unchanged during culture. (3) CSPGs released by PC12 cells into the culture medium were separated into two peaks (I and II) by column chromatography on DEAE-cellulose. The peak II fraction prepared from the medium with NGF after 3 days of culture consisted of CSPG with Kav = 0.22 on Sephacryl S-300 [40-84 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Peripherin Is Tyrosine-Phosphorylated at Its Carboxyl-Terminal Tyrosine

Abstract: Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf-9 cells and SW-13 cl. 2 vim⁻ cells. The identity of peripherin as a tyrosine-phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW-13 cl. 2 vim⁻ cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy-terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild-type peripherin and peripherin Y474F form a filamentous network in SW-13 cl. 2 vim⁻ cells. This indicates that tyrosine phosphorylation of the peripherin C-terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.     

Characterization of an endooligopeptidase A-like protein in PC12 cells: Activity modulation by cAMP but not by basic fibroblast growth factor

Endooligopeptidase A is a putative neuropeptide-metabolizing enzyme. It converts small enkephalin-containing peptides into the corresponding enkephalins and inactivates biopeptides such as bradykinin and neurotensin in vitro. We investigated the presence of endooligopeptidase A in PC12 cells. This cell line was derived from a rat pheochromocytoma tumor and resembles fetal chromaffin cell. Depending on the supplements added to the cell culture, this cell line can be differentiated into mature chromaffin cell or sympathetic neuron-like cell. Endooligopeptidase A activity was measured in soluble cellular extracts using a specific fluorogenic substrate QF-ERP7. The PC12 endooligopeptidase A-like activity shared similar but not identical biochemical properties with rabbit brain endooligopeptidase A. Similarly to rabbit brain endooligopeptidase A, the PC12 endooligopeptidase A-like activity was enhanced by DTT, totally inhibited by DTNB and 1-10 Phenanthroline, partially inhibited by cFP-AAF-pAb, and not affected by PMSF. Furthermore, the PC12 endooligopeptidase A-like activity displayed identical elution profile as rabbit brain endooligopeptidase A in gel filtration and anion-exchange chromatography. In addition, an antiserum raised against rabbit brain endooligopeptidase A cross-reacted with a 71 kDa component from PC12 cell extracts in Western blotting and was also able to partially neutralize the PC12 endooligopeptidase A-like activity. Treatment of PC12 cells with basic fibroblast growth factor (bFGF), a neurotrophic factor for this cell line, did not modify the specific activity of this enzyme. However, cAMP analogs decreased the specific activity of the enzyme. These results indicate the presence of an endooligopeptidase A-like activity in PC12 cells which is modulated by cAMP but not by bFGF.     

Oxygen Toxicity Induces Apoptosis in Neuronal Cells

1. A high oxygen atmosphere induced apoptosis in cultured neuronal cells including PC12 cells and rat embryonic cortical, hippocampal, and basal forebrain neurons associated with DNA fragmentation and nuclear condensation.2. The sensitivity of CNS neurons to a high-oxygen atmosphere was the following order; cortex > basal forebrain > hippocampus.3. Cycloheximide and actinomycin-D inhibited the apoptosis, indicating that it depends on new macromolecular synthesis. In contrast, cultured postnatal CNS neurons were resistant to oxidative stress.4. Neurotrophic factors such as nerve growth factor (NGF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) blocked the apoptosis induced by a high-oxygen atmosphere.     

In vitro production and characterisation of low molecular weight forms of calcitonin gene-related peptide immunoreactivity from rat thyroid

Rat tissues contained two forms of calcitonin gene-related peptide (CGRP) immunoreactivity of lower molecular weight than CGRP itself. Two immunoreactive products of in vitro degradation of synthetic CGRP by rat tissue homogenates were purified and shown to be chromatographically identical to these naturally occurring moieties. They reacted only with a carboxy-terminal directed CGRP antiserum indicating that they were carboxy-terminal fragments of CGRP. The larger fragment was found to have a molecular mass corresponding to amino acid residues 19-37 of the CGRP molecule.