Gravel M, Gao E, Hervouet-Zeiber C, Parsons V, Braun P E
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
J Neurochem. 2000 Nov;75(5):1940-50. doi: 10.1046/j.1471-4159.2000.0751940.x.
It was recently shown that the two transcripts encoding the isoforms of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP1 and CNP2) are differentially regulated during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only CNP2 mRNA is present, whereas in differentiating oligodendrocytes, both CNP1 and CNP2 mRNAs are expressed. This pattern of CNP expression is likely due to stage-specific transcriptional regulation of the 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 cells. We found that the transcription of CNP1 mRNA was significantly increased in comparison with that of CNP2 mRNA in cells treated with cAMP analogues to elevate intracellular cAMP levels. This up-regulation of CNP1 expression (a) is due to an increase of transcription, (b) requires de novo protein synthesis, and (c) requires the activity of protein kinase A. These results are physiologically significant and support the idea that a cAMP-mediated pathway is part of the molecular mechanisms regulating the expression of CNP1 in oligodendrocytes. The regulation of CNP1 promoter activity by cAMP was then investigated in stably transfected C6 cell lines containing various deletions of the CNP promoter directing the bacterial chloramphenicol acetyltransferase gene. We showed that the sequence between nucleotides -126 and -102 was essential for the cAMP-dependent induction of CNP1 expression. Gel retardation analysis showed that two protein-DNA complexes are formed between this sequence and nuclear factors from C6 cells treated or not treated with cAMP. This suggests that the induction of CNP1 mRNA transcription is not mediated by changes in binding of nuclear factors that interact directly with the -126/-102 sequence. Sequence analysis of this region revealed the presence of a putative activator protein-2 (AP-2) binding site. It is interesting that mutagenesis of this region resulted in a significant reduction in transcriptional responses to cAMP, implying a possible role for the AP-2 factor in the expression of CNP1. In addition, we have shown that putative 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 cAMP. Taken together, our results show that the cAMP-dependent accumulation of CNP1 mRNA appears to depend on the synergistic interaction of several regulatory elements.
最近研究表明，编码2',3'-环核苷酸3'-磷酸二酯酶（CNP1和CNP2）同工型的两种转录本在少突胶质细胞成熟过程中受到不同的调控。在少突胶质细胞前体中，仅存在CNP2 mRNA，而在分化的少突胶质细胞中，CNP1和CNP2 mRNA均有表达。这种CNP表达模式可能是由于在少突胶质细胞分化过程中，两个CNP启动子的阶段特异性转录调控所致。在此，我们报道细胞内环状AMP（cAMP）水平升高对大鼠C6胶质瘤细胞中CNP1和CNP2 mRNA转录的影响。我们发现，在用cAMP类似物处理以提高细胞内cAMP水平的细胞中，与CNP2 mRNA相比，CNP1 mRNA的转录显著增加。CNP1表达的这种上调（a）是由于转录增加，（b）需要从头合成蛋白质，并且（c）需要蛋白激酶A的活性。这些结果具有生理意义，并支持cAMP介导的途径是调节少突胶质细胞中CNP1表达的分子机制的一部分这一观点。然后，在稳定转染的含有指导细菌氯霉素乙酰转移酶基因的CNP启动子各种缺失的C6细胞系中，研究了cAMP对CNP1启动子活性的调节。我们表明，核苷酸-126至-102之间的序列对于cAMP依赖性诱导CNP1表达至关重要。凝胶阻滞分析表明，在该序列与用或不用cAMP处理的C6细胞的核因子之间形成了两种蛋白质-DNA复合物。这表明CNP1 mRNA转录的诱导不是由直接与-126 / -102序列相互作用的核因子结合变化介导的。对该区域的序列分析揭示了一个推定的激活蛋白-2（AP-2）结合位点。有趣的是，该区域的诱变导致对cAMP的转录反应显著降低，这意味着AP-2因子在CNP1表达中可能发挥作用。此外，我们已经表明，AP-2位点附近的激活蛋白-4和核因子-1的推定结合位点是cAMP有效诱导CNP1表达所必需的。综上所述，我们的结果表明，CNP1 mRNA的cAMP依赖性积累似乎取决于几种调控元件的协同相互作用。
