Bishop J F, Joshi G, Mueller G P, Mouradian M M
Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Brain Res Mol Brain Res. 1997 Oct 15;50(1-2):154-64. doi: 10.1016/s0169-328x(97)00180-0.
Brain-derived neurotrophic factor (BDNF) has potent trophic and protective actions on CNS neurons, including mesencephalic dopaminergic neurons, ventral forebrain cholinergic neurons and spinal motor neurons. To evaluate the effects of calcium and other second messengers on BDNF gene transcription, C6 glioma cells were treated for 4 h with the calcium ionophore A23187, forskolin + isobutyl-methyl-xanthine (IBMX), or the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate. Semi-quantitative RT-PCR analysis revealed that A23187 treatment increased BDNF transcripts containing the protein coding exon by 4.4-6.4-fold. Alternate BDNF transcripts were elevated to varying degrees after treatment with this ionophore and a subset of these transcripts was elevated following forskolin + IBMX treatment. When co-incubated with the RNA polymerase inhibitor, actinomycin D, A23187-induced increases were reduced or abolished, suggesting that calcium-mediated regulation of BDNF mRNA expression occurs at transcriptional as well as post-transcriptional levels. Transient transfection experiments employing reporter constructs containing serial 5' deletions of alternate BDNF promoters suggested that A23187-induced elevations in BDNF exon 1b, 1d and 1e containing transcripts are mediated by putative calcium-responsive regions flanking all three of these exons.
脑源性神经营养因子（BDNF）对中枢神经系统神经元具有强大的营养和保护作用，这些神经元包括中脑多巴胺能神经元、腹侧前脑胆碱能神经元和脊髓运动神经元。为了评估钙和其他第二信使对BDNF基因转录的影响，用钙离子载体A23187、福斯高林 + 异丁基甲基黄嘌呤（IBMX）或佛波酯12 - O - 十四酰佛波醇 - 13 - 乙酸酯处理C6胶质瘤细胞4小时。半定量逆转录 - 聚合酶链反应（RT - PCR）分析显示，A23187处理使含有蛋白质编码外显子的BDNF转录本增加了4.4 - 6.4倍。用这种离子载体处理后，其他BDNF转录本有不同程度的升高，并且在福斯高林 + IBMX处理后，这些转录本中的一部分也升高了。当与RNA聚合酶抑制剂放线菌素D共同孵育时，A23187诱导的增加减少或消除，这表明钙介导的BDNF mRNA表达调节发生在转录水平以及转录后水平。采用含有BDNF不同启动子5'端连续缺失的报告基因构建体的瞬时转染实验表明，A23187诱导的含有外显子1b、1d和1e的BDNF转录本升高是由这三个外显子侧翼的假定钙反应区域介导的。
