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Hypotonicity reduces the activity of murine aquaporin‐2 promoter induced by dibutyryl cAMP
Author(s) -
Saito Takako,
Saito Tomoyuki,
Kasono Keizo,
Tamemoto Hiroyuki,
Kawakami Masanobu,
Sasaki Sei,
Ishikawa Sane
Publication year - 2008
Publication title -
experimental physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.925
H-Index - 101
eISSN - 1469-445X
pISSN - 0958-0670
DOI - 10.1113/expphysiol.2008.042663
Subject(s) - transfection , microbiology and biotechnology , tonicity , reporter gene , biology , gene , enhancer , in vitro , chemistry , gene expression , endocrinology , biochemistry
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.