cAMP motifs regulating transcription in the aquaporin 2 gene.
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.
Hozawa
S
S
Renal Unit, Massachusetts General Hospital, Boston 02114, USA.
Holtzman
E J
EJ
Ausiello
D A
DA
D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D001127:Arginine Vasopressin / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology / Q000502:physiology*; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology; D017930:Genes, Reporter; D016678:Genome; D006801:Humans; D007473:Ion Channels / Q000235:genetics*; D007668:Kidney / Q000166:cytology / Q000502:physiology; D018374:LLC-PK1 Cells / Q000502:physiology; D015335:Molecular Probes; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D013552:Swine; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*
C496023:AQP2 protein, human; C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D007473:Ion Channels; D015335:Molecular Probes; D001127:Arginine Vasopressin; D000242:Cyclic AMP