Hypotonicity reduces the activity of murine aquaporin-2 promoter induced by dibutyryl cAMP.
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 micromol 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 micromol 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 micromol 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 micromol l(-1) SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), restored the response of Luc activity to 5 micromol 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.
Department of Medicine, Jichi Medical University, Saitama Medical Center, 1-847 Amanuma Omiya-ku, Saitama, Saitama 330-8503, Japan.
Takako
T
Saito
Tomoyuki
T
Saito
Keizo
K
Kasono
Hiroyuki
H
Tamemoto
Masanobu
M
Kawakami
Sei
S
Sasaki
San-e
SE
Ishikawa
D000818:Animals; D051399:Aquaporin 2 / Q000235:genetics* / Q000378:metabolism*; D003994:Bucladesine / Q000494:pharmacology*; D048429:Cell Size / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D007038:Hypotonic Solutions / Q000494:pharmacology; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008156:Luciferases; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
D051399:Aquaporin 2; D007038:Hypotonic Solutions; D012333:RNA, Messenger; D003994:Bucladesine; D008156:Luciferases