Angiotensin II and potassium regulate human CYP11B2 transcription through common cis-elements.
Angiotensin II and potassium regulate human CYP11B2 transcription through common cis-elements.
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DOI：
10.1210/mend.11.5.9920
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发表时间：
1997-05
影响因子：
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通讯作者：
C. Clyne;Y Zhang-;L. Slutsker;J. Mathis;P. White;W. Rainey
中科院分区：
文献类型：
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作者：
C. Clyne;Y Zhang-;L. Slutsker;J. Mathis;P. White;W. Rainey
醛固酮合酶是一种线粒体酶，可催化 11-脱氧皮质酮转化为有效的盐皮质激素醛固酮。编码醛固酮合酶的基因 CYP11B2 在肾上腺皮质的肾小球带中表达。尽管醛固酮产生的主要生理调节剂是血管紧张素 II (ANG II) 和钾 (K+)，但这些化合物调节 CYP11B2 转录的机制尚不清楚。因此，我们使用瞬时转染表达系统在 H295R 人肾上腺皮质细胞系中分析了人 CYP11B2 5' 侧翼区域。 ANG II 和 K+ 增加了含有 2015 bp 人 CYP11B2 5' 侧翼 DNA 的荧光素酶报告基因构建体的表达。通过钙通道激活剂 BAYK8644 的治疗模拟了这种反应，而用 12-o-tetradecanoylphorbol-13-acetate 激活蛋白激酶 C 通路则没有效果。 (Bu)2cAMP 激活 cAMP 依赖性途径后，报告基因活性也增加。对 CYP11B2 5' 侧翼区域的删除、突变和脱氧核糖核酸酶 I 足迹分析确定了位置 -71/-64 (TGACGTGA) 和 -129/-114 (CTCCAGCCTTGACCTT) 处的两个不同元件，这两个元件都是完整的基础报告基因活性和 cAMP 或钙信号通路最大诱导所需的。通过电泳迁移率变动分析确定，-71/-64 元件类似于共有 cAMP 反应元件 (CRE)，与 H295R 细胞核提取物中的 CRE 结合蛋白结合。使用电泳迁移率变动分析对 -129/-114 元件进行的分析表明孤儿核受体类固醇生成因子 1 和鸡卵清蛋白上游启动子转录因子的结合。这些数据表明 ANG II、K+ 和 cAMP 信号通路利用相同的 SF-1 和 CRE 样顺式元件来调节人 CYP11B2 表达。
Aldosterone synthase is a mitochondrial enzyme that catalyzes the conversion of 11-deoxycorticosterone to the potent mineralocorticoid aldosterone. The gene encoding aldosterone synthase, CYP11B2, is expressed in the zona glomerulosa of the adrenal cortex. Although the major physiological regulators of aldosterone production are angiotensin II (ANG II) and potassium (K+), the mechanisms by which these compounds regulate CYP11B2 transcription are unknown. Therefore we analyzed the human CYP11B2 5'-flanking region using a transient transfection expression system in the H295R human adrenocortical cell line. ANG II and K+ increased expression of a luciferase reporter construct containing 2015 bp of human CYP11B2 5'-flanking DNA. This response was mimicked by treatment with the calcium channel activator BAYK8644, whereas activation of the protein kinase C pathway with 12-o-tetradecanoylphorbol-13-acetate had no effect. Reporter gene activity was also increased after activation of cAMP-dependent pathways by (Bu)2cAMP. Deletion, mutation, and deoxyribonuclease I footprinting analyses of the CYP11B2 5'-flanking region identified two distinct elements at positions -71/-64 (TGACGTGA) and -129/-114 (CTCCAGCCTTGACCTT) that were both required for full basal reporter gene activity and for maximal induction by either cAMP or calcium-signaling pathways. The -71/-64 element, which resembles a consensus cAMP response element (CRE), bound CRE-binding proteins from H295R cell nuclear extracts as determined by electrophoretic mobility shift analysis. Analysis of the -129/-114 element using electrophoretic mobility shift analysis demonstrated binding of the orphan nuclear receptors steroidogenic factor 1 and chicken ovalbumin upstream promoter transcription factor. These data demonstrate that ANG II, K+, and cAMP-signaling pathways utilize the same SF-1 and CRE-like cis-elements to regulate human CYP11B2 expression.