Regulatory elements in the promoter region of vgf, a nerve growth factor-inducible gene.

Abstract

ABSTRACTvgf, a gene coding for a protein secreted through the regulated pathway, is rapidly up-modulated by nerve growth factor in PC12 cells and is expressed in vivo only in cell subpopulations of neuronal and endocrine origin. Here we demonstrate the following: (i) the nerve growth factordependent induction of vgfmRNA occurs at the transcriptional level and requires ongoing protein synthesis, (ii) lack of vgf expression in the nonneuronal cell line HTC is in part mediated by the presence of a repressor, (iii) a 1 10-base-pair sequence in the vgf promoter region contains positive and negative regulatory elements that partially account for its regulated expression, and (iv) a 47-base-pair oligonucleotide within this sequence specifically binds nuclear proteins that differ between vgf-expressing and non-expressing cells.Neurotrophins comprise a family of polypeptides that have growth-promoting activity on distinct populations of neurons (1). The best characterized is nerve growth factor (NGF; ref.2), which exerts a number of effects (tropic, trophic, and differentiative) on neuronal and nonneuronal cells. The most extensively studied action of NGF is the induction of a neuronal phenotype in the pheochromocytoma-derived PC12 cell line (3). In response to NGF, PC12 cells stop proliferating and express neuronal-specific markers and functions (4). The biochemical pathways that lead to PC12 differentiation are not fully understood, although the recent finding that the NGF receptor is composed of two different proteins, one of which, the product of the protooncogene trk, has tyrosinekinase activity, has shed new light on the early events elicited by NGF (5, 6). NGF binding to its receptor activates several signal-transducing proteins, which, in turn, lead to the stimulation of protein synthesis-independent transcription of a number of genes collectively known as "immediate early genes" (7). Many of them code for transcription factors, probably involved in the later-induced transcription of neuronal-specific genes. Most of the immediate early genes in PC12 cells are also induced by agents unable to promote neuronal differentiation and are induced, in other cell types, by mitogenic factors. It is conceivable that the cooperation of a particular subset of early genes with the transcription machinery and the modulation of their function by posttranslational modifications are necessary for achieving the specific response to NGF. To gain insight into the mechanisms responsible for transcriptional activation of neuronalspecific genes in NGF-treated PC12 cells, we have studied the regulation of vgf, a gene whose expression is induced in PC12 cells in response to NGF (8, 9). The vgf-encoded protein is stored in secretory vesicles, is released in response to a number of stimuli (10), and is expressed only in cells of neuronal and endocrine origin (11,12), suggesting that the full-length protein, or peptides derived from it, might have neuromodulatory activity. MATERIALS AND METHODSCell Culture. PC12 cells were grown as described...

Dual effect on the RET receptor of MEN 2 mutations affecting specific extracytoplasmic cysteines

“…In this study, we have assayed the respective capacity of the various RET Cys mutants at inducing the transcription of the chloramphenicol acetyl transferase (CAT) reporter gene driven either by the NFGI-A or by the vgf promoter. NGFI-A and vgf are respectively immediate-early and delayed response genes whose expression is rapidly turned on upon treatment of PC12 cells with nerve growth factor (NGF) (JanssenTimmen et al, 1989;Possenti et al, 1992). As ( Figure 6b).…”

“…pvgf-CAT contains the vgf promoter, the 5' noncoding region and the ®rst methionine (from 7803 to +710) fused in-frame with the initiating methionine of the CAT gene (Possenti et al, 1992). The pBLCAT2 and RSV-CAT vectors contain the CAT gene under the control of the HSV thymidine kinase promoter (position 7105 to +51) and of the Rous sarcoma virus promoter, respectively (Lukow and Schutz, 1987).…”

Role of TFG sequences outside the coiled-coil domain in TRK-T3 oncogenic activation

“…To test the effect of TFG deletions on this activity, PC12 cells were cotransfected with the different TRK-T3 constructs and the VGF8-luc reporter plasmid: the latter is made of the luciferase reporter gene driven by the promoter of the vgf, a gene induced by NGF and considered a differentiation marker (Possenti et al, 1992;Canu et al, 1997). All the mutated proteins were able to induce formation of neurites (data not shown).…”

Signal Transduction Pathways Activated by RET Oncoproteins in PC12 Pheochromocytoma Cells

“…gene is specifically expressed in neuroendocrine cells upon NGF stimulation (34). It is known that c-RET is also predominantly expressed in cells or tissues of neuroendocrine origin (35)(36)(37).…”