Correlation between increased AP-1(NGF) binding activity and induction of nerve growth factor transcription by multiple signal transduction pathways in C6-2B glioma cells

Transcription mechanisms regulating nerve growth factor (NGF) gene expression in the CNS are yet to be thoroughly understood. We have used C6-2B rat glioma cells to characterize the signal transduction pathways that contribute to transcriptional and posttranscriptional regulation of NGF mRNA. Because the NGF promoter contains an AP-1 consensus sequence, we have investigated whether increases in AP-1 binding activity correlate with enhanced NGF mRNA expression. Gel mobility shift assays using an oligonucleotide homologous to the AP-1 responsive element of the rat NGF gene (AP-1(NGF)) revealed that 12-O-tetradecanoyl phorbol-13-acetate (TPA) and, to a lesser extent, isoproterenol (ISO) and thapsigargin, a microsomal Ca2+-ATPase inhibitor, stimulated binding to AP-1(NGF) within 2 h. All of these stimuli increased NGF mRNA levels within 3 h. Cycloheximide pretreatment blocked the TPA and ISO-mediated binding to AP-1(NGF) suggesting that de novo synthesis of c-Fos/c-Jun may be required for the transcriptional regulation of NGF gene. Nuclear run-on assays and NGF mRNA decay studies revealed that TPA increases NGF transcription whereas ISO affects both transcription and mRNA stabilization. We propose that (i) different signal transduction mechanisms regulate the expression of the NGF gene in cells derived from the CNS, and (ii) both mRNA transcription and stability account for the cAMP-mediated increase in NGF mRNA levels.