Transcriptional regulation of aquaporin-2 water channel gene by cAMP.
Transcriptional regulation of aquaporin-2 water channel gene by cAMP.
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发表时间：
1997-06
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通讯作者：
Y. Matsumura;S. Uchida;T. Rai;S. Sasaki;F. Marumo
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作者：
Y. Matsumura;S. Uchida;T. Rai;S. Sasaki;F. Marumo
水通道蛋白-2（AQP-2）是尿浓缩的关键分子，在体内脱水时其表达增强。为了阐明这种现象在体外的调节机制，建立了小鼠集合管细胞系，从一个转基因小鼠携带的温度敏感的猿猴病毒40大T抗原基因，然后筛选AQP-2的表达，使用核糖核酸酶保护试验。在一个命名为C4的细胞系中，用氯苯硫基-cAMP（cpt-cAMP）（400 μ M）处理后，通过核糖核酸酶保护试验测量的内源性AQP-2 mRNA水平增加了4倍。相反，佛波醇12-肉豆蔻酸酯13-乙酸酯不影响AQP-2 mRNA水平。为了鉴定cAMP诱导的C4细胞中AQP-2 mRNA上调的分子机制，使用人AQP-2基因的各种5 '侧翼区进行荧光素酶测定。用含有约2.8-kbp或224-bp 5 '-侧翼区的构建体转染的C4细胞中的荧光素酶活性显示出cpt-cAMP处理的3.5倍增加，表明224-bp 5'-侧翼区含有cAMP诱导的调节机制所必需的元件。该区域含有cAMP反应元件（CRE），CRE核心序列的缺失（GACGTCA）或CRE的突变（GTGGTCA）完全消除了对cpt-cAMP的反应性，证实了CRE在cAMP诱导的AQP-2基因转录激活中的关键作用。电泳迁移率变动分析表明，C4细胞和大鼠肾脏中存在与CRE结合的蛋白。与对照组相比，在cpt-cAMP处理的C4细胞和脱水大鼠肾的核提取物中CRE蛋白与CRE的结合增加。这些结果表明，CRE在AQP-2基因启动子是一个关键的顺式元件cAMP介导的转录调控该基因，并可能是重要的在体内调节AQP-2的表达在脱水状态。
Aquaporin-2 (AQP-2) water channel is a key molecule for urinary concentration whose expression is augmented by dehydration in vivo. To elucidate the regulatory mechanism of this phenomenon in vitro, mouse collecting duct cell lines were established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T antigen gene and then screened for the AQP-2 expression, using ribonuclease protection assay. In one cell line designated C4, the endogenous AQP-2 mRNA level measured by ribonuclease protection assay increased fourfold after treatment with chlorophenylthio-cAMP (cpt-cAMP) (400 microM). In contrast, phorbol 12-myristate 13-acetate did not affect the AQP-2 mRNA level. To identify the molecular mechanism(s) of cAMP-induced upregulation of AQP-2 mRNA in C4 cells, luciferase assay was performed using various 5'-flanking regions of the human AQP-2 gene. Luciferase activity in C4 cells transfected with constructs containing approximately 2.8-kbp or 224-bp 5'-flanking region showed a 3.5-fold increase by cpt-cAMP treatment, indicating that the 224-bp 5'-flanking region contains the elements necessary for cAMP-induced regulatory mechanisms. This region contains cAMP-responsive element (CRE), and the deletion of the core sequence of CRE (GACGTCA) or introduction of mutation into CRE (GTGGTCA) completely abolished the responsiveness to cpt-cAMP, confirming the key role of CRE in the cAMP-induced transcriptional activation of the AQP-2 gene. Electrophoretic mobility shift assay revealed the existence of proteins binding to CRE in C4 cells and in rat kidney. The binding of CRE proteins to CRE was increased in the nuclear extract from cpt-cAMP-treated C4 cells and dehydrated rat kidney compared with those from controls. These results demonstrated that the CRE in the AQP-2 gene promoter is a key cis-element for cAMP-mediated transcriptional regulation of this gene and may be important for in vivo regulation of AQP-2 expression in a dehydrated state.