Coulombe N, Lefebvre A, Lehoux J G
Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, QC, Canada.
DNA Cell Biol. 1997 Aug;16(8):993-1002. doi: 10.1089/dna.1997.16.993.
A CYP11B2 gene encoding cytochrome P450 aldosterone synthase (P450aldo) was isolated from a hamster genomic library. The gene, which contained 9 exons, was composed of 9,045 bp, of which 3,722 bp were located in the 5' untranslated region (5' UTR). A TATA box sequence (gataaa) and other putative cis elements, previously named Ad1 to Ad6, were identified in the 5' UTR of the hamster gene comparable to the CYP11B2 gene of other animal species. Footprint analysis showed protection by nuclear protein extracts from hamster adrenal zona glomerulosa (ZG) in the regions containing the above mentioned cis elements. In addition, a new protected cis element, between -143 and -161 bp, was demonstrated, and gel-shift assays revealed that the sequence of this new cis element was specifically retarded by factors in the nuclear extracts of hamster adrenal ZG. We then examined the transcriptional activity of the 5' UTR of the CYP11B2 gene, using chloramphenicol acyltransferase (CAT) as the reporter gene. Ten deletion plasmids were constructed using a modified pCAT vector. Transient transfections of the chimeric reporter constructs into Y1 cells showed that the highest basal promoter activity was obtained with the construct containing up to -134 bp. Increasing the length of the regulatory region of CYP11B2 gene to -167 bp resulted in less than two-thirds of the maximal activity, indicating the probability of putative inhibitory cis elements in this area of the gene. Forskolin stimulated the expression of the reporter gene of deletion plasmids excepting the construct containing only the TATA box, and the highest activity also occurred with the -134 bp construct. TPA had no stimulatory effects on any of the constructs, and interestingly it slightly inhibited CAT activity. In contrast to TPA, staurosporine, an inhibitor of the PKC pathway, stimulated CAT activity. To conclude, the promoter region of the hamster CYP11B2 gene transfected in Y1 cells is responsive to forskolin, indicating that the gene is controlled by the PKA signaling pathway. Paradoxically, staurosporine, but not TPA, stimulates the promoter activity of the CYP11B2 gene, indicating that PKC might, at least in Y1 cells, act as a negative regulator on the aldosterone synthase promoter. Moreover, a new cis element was shown to exert a negative effect on basal as well as on stimulated activities of the hamster promoter CYP11B2 gene.
从仓鼠基因组文库中分离出一个编码细胞色素P450醛固酮合成酶（P450aldo）的CYP11B2基因。该基因含有9个外显子，由9045个碱基对组成，其中3722个碱基对位于5'非翻译区（5'UTR）。在仓鼠基因的5'UTR中鉴定出一个TATA盒序列（gataaa）和其他推定的顺式元件，以前称为Ad1至Ad6，与其他动物物种的CYP11B2基因类似。足迹分析表明，仓鼠肾上腺球状带（ZG）的核蛋白提取物对含有上述顺式元件的区域有保护作用。此外，还证实了一个新的受保护顺式元件，位于-143至-161碱基对之间，凝胶迁移试验表明，该新顺式元件的序列被仓鼠肾上腺ZG核提取物中的因子特异性阻滞。然后，我们以氯霉素乙酰转移酶（CAT）作为报告基因，检测了CYP11B2基因5'UTR的转录活性。使用改良的pCAT载体构建了10个缺失质粒。将嵌合报告构建体瞬时转染到Y1细胞中，结果表明，含有-134碱基对的构建体具有最高的基础启动子活性。将CYP11B2基因的调控区域长度增加到-167碱基对，导致活性不到最大值的三分之二，表明该基因这一区域可能存在推定的抑制性顺式元件。福斯高林刺激了除仅含TATA盒的构建体之外的缺失质粒报告基因的表达，最高活性也出现在-134碱基对的构建体中。佛波酯对任何构建体均无刺激作用，有趣的是，它略微抑制了CAT活性。与佛波酯相反，蛋白激酶C（PKC）途径的抑制剂星形孢菌素刺激了CAT活性。总之，转染到Y1细胞中的仓鼠CYP11B2基因的启动子区域对福斯高林有反应，表明该基因受蛋白激酶A（PKA）信号通路控制。矛盾的是，星形孢菌素而非佛波酯刺激了CYP11B2基因的启动子活性，表明PKC可能至少在Y1细胞中作为醛固酮合成酶启动子的负调节因子。此外，一个新的顺式元件对仓鼠CYP11B2基因启动子的基础活性和刺激活性均有负面影响。
