Korean J Physiol Pharmacol.  1999 Apr;3(2):157-164.

Molecular analysis of AQP2 promoter. I. cAMP-dependent regulation of mouse AQP2 gene

Affiliations
  • 1Department of Physiology, College of Medicine, Pusan National University, Pusan 602-739, Korea.
  • 2Department of Physiology, Dong-A University College of Medicine, Pusan 602-715, Korea.

Abstract

To determine molecular mechanisms of Aquaporin-CD (AQP2) gene regulation, the promoter region of the AQP2 gene was examined by transiently transfecting a promoter-luciferase reporter fusion gene into mouse renal collecting duct cell lines such as mIMCD-3, mIMCD-K2, and M-1 cells, and NIH3T3 mouse embryo fibroblast cells. PCR-Southern analysis reveals that mIMCD-3 and mIMCD-K2 cells express AQP2, but M-1 and NIH3T3 cells do not, and that the treatment with cpt-cAMP (400 muM) or forskolin/isobutylmethylxanthine (IBMX) increased the AQP2 expression in IMCD cells. In both IMCD and NIH3T3 cells, the constructs containing the promoter of AQP2 gene showed promoter activities, indicating lack of tissue-specific element in the 1.4 kb 5'-flanking region of the mouse AQP2 gene. Luciferase activity in the IMCD cells transfected with the construct containing 5-flanking region showed responsiveness to cpt-cAMP, indicating that the 1.4 kb 5'-flanking region contains the element necessary for the regulatory mechanism by cAMP. The promoter-luciferase constructs which do not have a cAMP-responsible element (CRE) still showed the cAMP responsiveness in IMCD cells, but not in NIH3T3 cells. Increase in medium osmolarity did not affect AQP2 promoter activity in mIMCD-K2 cells. These results demonstrate that AQP2 gene transcription is increased with cAMP treatment through multiple motifs including CRE in the 5'-flanking region of the gene in vitro, and the regulatory mechanism may be important for in vivo regulation of AQP2 expression.

Keyword

AQP2 gene; cAMP; Collecting duct; Regulation

MeSH Terms

Animals
Aquaporin 2
Cell Line
Embryonic Structures
Fibroblasts
Luciferases
Mice*
Osmolar Concentration
Promoter Regions, Genetic
Aquaporin 2
Luciferases
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