Hypotonicity reduces the activity of murine aquaporin-2 promoter induced by dibutyryl cAMP

• Published in: Experimental physiology 2008-10, Vol.93 (10), p.1147-1156
• Main Authors: Saito, Takako, Saito, Tomoyuki, Kasono, Keizo, Tamemoto, Hiroyuki, Kawakami, Masanobu, Sasaki, Sei, Ishikawa, San‐e
• Format: Article
• Language: English
• Subjects: Animals; Aquaporin 2 - genetics; Aquaporin 2 - metabolism; Bucladesine - pharmacology; Cell Size - drug effects; Cell Survival - drug effects; Cells, Cultured; Hypotonic Solutions - pharmacology; Kidney Tubules, Collecting - cytology; Kidney Tubules, Collecting - drug effects; Kidney Tubules, Collecting - metabolism; Luciferases; Mice; Plasmids; Promoter Regions, Genetic - genetics; RNA, Messenger - metabolism; Transfection
• ISSN: 0958-0670; 1469-445X
• DOI: 10.1113/expphysiol.2008.042663

The present study was undertaken to determine whether hypotonicity regulates the aquaporin-2 (AQP-2) gene in vitro . The 5′-flanking region of the AQP-2 gene contains the tonicity-response enhancer (TonE) promoter located between −570 and −560 bp, and another distinct hypertonicity-responsive region between −6.1 and −4.3 kb of the AQP-2 gene. The 5′-flanking region of murine AQP-2 gene up to −9.5 kb was cloned into a luciferase (Luc) reporter plasmid. The constructs, which have TonE and/or the hypertonicity-responsive region, together with the murine AQP-2 gene, were co-transfected into murine IMCD 3 cells. When the cells were co-transfected with the construct containing more than 1.1 kb of the 5′-flanking region of murine AQP-2 gene (–9.5AQP2, −6.1AQP2 and −1.1AQP2) and the AQP-2 gene, 24 h exposure to 5 μmol l −1 dibutyryl cAMP (DBcAMP) significantly increased the Luc activity by 2.3-fold in the isotonic medium (300 mosmol kg −1 ). In the hypotonic medium (225 mosmol kg −1 ), basal activity was not altered, and the response of Luc activity to 24 h exposure to 5 μmol l −1 DBcAMP was abolished. Similar findings were obtained in isosmotic, urea-supplemented medium (estimated tonicity, 225 mosmol kg −1 ). The response of Luc activity to 5 μmol l −1 DBcAMP in the hypotonic medium was not affected in cells either transfected with 0.36 kb of the 5′-flanking region of AQP-2 or co-transfected with −1.1AQP2 and a dominant-negative TonE binding protein (pDNTonEBP). Pre-incubation of cells with 1 μmol l −1 SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), restored the response of Luc activity to 5 μmol l −1 DBcAMP under hypotonic conditions. These findings may indicate that hypotonicity reduces the cAMP-induced AQP-2 promoter activity mediated via TonE by activating JNK kinase.