Hypertonicity regulates the aquaporin-2 promoter independently of arginine vasopressin
Hypertonicity regulates the aquaporin-2 promoter independently of arginine vasopressin
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DOI：
10.1093/ndt/gfh677
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发表时间：
2005-03-01
影响因子：
6.1
通讯作者：
Ishikawa, SE
中科院分区：
文献类型：
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作者：
Kasono, K;Saito, T;Ishikawa, SE
背景水通道蛋白-2（AQP-2）是肾脏集合管细胞中由精氨酸加压素（AVP）调节的水通道。本研究旨在通过体外实验确定张力的改变是否可以直接调节AQP-2基因。将小鼠AQP-2基因5 '侧翼区约9.5 kb的不同片段克隆到荧光素酶（Luc）报告质粒中，并将其瞬时转染到Madin-Darby犬肾细胞中。高渗显著增加了含有> 6.1kb的AQP-2基因5 '侧翼区的构建体的Luc活性（-6.1AQP 2）。然而，在bp -570至-560处含有张力响应增强子（TonE）的长度<4.3kb的启动子区域不受高渗刺激。含有AQP-2基因的5'端的约9.5至约1.1kb的TonE缺失的构建体8.4AQP 2也被高渗刺激。丝裂原活化蛋白（MAP）激酶抑制剂SB 203580和U 0126不影响高渗诱导的-6.1 AQP 2的Luc活性。此外，表达显性负性TonE结合蛋白（TonEBP）的载体不影响高渗诱导的-6.1AQP 2的Luc活性。TonEBP过表达可刺激-6.1AQP 2的Luc活性。高渗可进一步增加TonEBP过表达下的-6.1AQP2的Luc活性。这些结果表明，高渗通过AVP非依赖性机制调节AQP-2启动子活性，并且张力响应元件位于AQP-2基因的-6.1和-4.3kb 5 '侧翼区域之间，其中对高渗的响应结构和机制可能不同于TonE。
Background. Aquaporin-2 (AQP-2) is an arginine vasopressin (AVP)-regulated water channel in kidney collecting duct cells. The present study was undertaken to determine whether a change in tonicity could directly regulate the AQP-2 gene in an in vitro experiment.Methods. Various fragments of the 5'-flanking region of the murine AQP-2 gene up to -9.5 kb were cloned into a luciferase (Luc) reporter plasmid, and they were transiently transfected into Madin-Darby canine kidney cells.Results. Hypertonicity significantly increased the Luc activity of the constructs containing > 6.1 kb of the 5'-flanking region of the AQP-2 gene (-6.1AQP2). However, promoter regions < 4.3 kb in length containing the tonicity-responsive enhancer (TonE) at bp -570 to -560 were not stimulated by hypertonicity. The TonE-deleted construct which contains -9.5 to -1.1 kb of the 5' side of the AQP-2 gene, 8.4AQP2, was also stimulated by hypertonicity. Mitogen-activated protein (MAP) kinase inhibitors SB203580 and U0126 did not affect the Luc activity of -6.1 AQP2 induced by hypertonicity. In addition, the vector expressing dominant-negative TonE-binding protein (TonEBP) did not affect the hypertonicity-induced Luc activity of -6.1AQP2. The Luc activity of -6.1AQP2 was stimulated by the overexpression of TonEBP. Hypertonicity further increased the Luc activity of -6.1AQP2 under the overexpression of TonEBP.Conclusion. These findings indicate that hypertonicity regulates AQP-2 promoter activity via an AVP-independent mechanism, and that the tonicity-responsive element resides between the -6.1 and -4.3 kb 5'-flanking region of the AQP-2 gene, in which the structure and mechanism of response to hypertonicity could be distinct from those of TonE.