Transcriptional regulation of 2 ',3 '-cyclic nucleotide 3 '-phosphodiesterase gene expression by cyclic AMP in C6 cells

It was recently shown that the two transcripts encoding the isoforms of 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP1 and CNP2) are differentiall y regulated during the process of oligodendrocyte maturation. In oligodendr ocyte precursors, only CNP2 mRNA is present, whereas in differentiating oli godendrocytes, both CNP1 and CNP2 mRNAs are expressed. This pattern of CNP expression is likely due to stage-specific transcriptional regulation of th e two CNP promoters during the process of oligodendrocyte differentiation. Here, we report the influence of increased intracellular cyclic AMP (cAMP) levels on the transcription of both CNP1 and CNP2 mRNAs in rat C6 glioma ce lls. We found that the transcription of CNP1 mRNA was significantly increas ed in comparison with that of CNP2 mRNA in cells treated with cAMP analogue s to elevate intracellular cAMP levels. This up-regulation of CNP1 expressi on (a) is due to an increase of transcription, (b) requires de novo protein synthesis, and (c) requires the activity of protein kinase A. These result s are physiologically significant and support the idea that a cAMP-mediated pathway is part of the molecular mechanisms regulating the expression of C NP1 in oligodendrocytes. The regulation of CNP1 promoter activity by cAMP w as then investigated in stably transfected C6 cell lines containing various deletions of the CNP promoter directing the bacterial chloramphenicol acet yltransferase gene. We showed that the sequence between nucleotides -126 an d -102 was essential for the cAMP-dependent induction of CNP1 expression. G el retardation analysis showed that two protein-DNA complexes are formed be tween this sequence and nuclear factors from C6 cells treated or not treate d with cAMP. This suggests that the induction of CNP1 mRNA transcription is not mediated by changes in binding of nuclear factors that interact direct ly with the -126/-102 sequence. Sequence analysis of this region revealed t he presence of a putative activator protein-2 (AP-2) binding site. It is in teresting that mutagenesis of this region resulted in a significant reducti on in transcriptional responses to cAMP, implying a possible role for the A P-2 factor in the expression of CNP1. In addition, we have shown that putat ive binding sites for activator protein-4 and nuclear factor-1 adjacent to the AP-2 site are required for efficient induction of CNP1 expression by cA MP. Taken together, our results show that the cAMP-dependent accumulation o f CNP1 mRNA appears to depend on the synergistic interaction of several reg ulatory elements.