cAMP motifs regulating transcription in the aquaporin 2 gene

Am J Physiol. 1996 Jun;270(6 Pt 1):C1695-702. doi: 10.1152/ajpcell.1996.270.6.C1695.

Abstract

Genomic clones including the 5' flanking regions of the AQP2 (aquaporin 2) gene were isolated, and the promoter region was examined by transiently transfecting a promoter-luciferase reporter fusion gene into renal cultured epithelial cells. An orientation specific promoter for the AQP2 gene was found within the proximal 3 kb of 5'-flanking region. Minimal basal promoter activity of the AQP2 gene was found within 198 bp upstream from the transcription start site by deletion analysis. Sequencing the transcriptionally active region revealed a typical TATA box, adenosine 3',5'-cyclic monophosphate (cAMP) responsive element (CRE) and three putative CCAAT boxes in the proximal 1.2-kb region. Significantly, a GATA motif, AP1, AP2, and SP1 transcriptional factor consensus sites were also found in this region. Exposure to cAMP-enhancing agents (1 nM vasopressin or 20 mM forskolin and 250 mM 3-isobutyl-1-methylxanthine) showed that these agents increased luciferase activity in a parallel fashion, suggesting that vasopressin-induced AQP2 gene transcription is mediated through increases in intracellular cAMP in at least one renal cell type, the LLC-PK1 cells. The mechanism of cAMP responsiveness of AQP2 gene transcription was further studied using a series of deletion mutants in renal epithelial cells and other cell types. The cAMP regulatory motifs were shown to exist in a 50-bp sequence between -340 and -290 (containing CRE) and a 65-bp sequence (containing an AP2 site) between -150 and the ATG start site in LLC-PK1 cells. In rat inner medullary collecting duct (IMCD) cells, the cAMP regulatory motifs also exist in a 50-bp sequence between -340 and -290 (containing CRE) and in a 10-bp sequence between -160 and -150 (containing an SP1 site). These separate regions may cooperate to confer full cAMP inducibility to the AQP2 gene in a cell-specific manner.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Aquaporin 2
  • Aquaporin 6
  • Aquaporins*
  • Arginine Vasopressin / pharmacology
  • Base Sequence
  • Cell Line
  • Cyclic AMP / pharmacology
  • Cyclic AMP / physiology*
  • Epithelial Cells
  • Epithelium / physiology
  • Genes, Reporter
  • Genome
  • Humans
  • Ion Channels / genetics*
  • Kidney / cytology
  • Kidney / physiology
  • LLC-PK1 Cells / physiology
  • Molecular Probes
  • Molecular Sequence Data
  • Promoter Regions, Genetic
  • Rats
  • Swine
  • Transcription, Genetic / drug effects
  • Transcription, Genetic / physiology*

Substances

  • AQP2 protein, human
  • Aqp2 protein, rat
  • Aquaporin 2
  • Aquaporin 6
  • Aquaporins
  • Ion Channels
  • Molecular Probes
  • Arginine Vasopressin
  • Cyclic AMP

Associated data

  • GENBANK/U30469