cAMP motifs regulating transcription in the aquaporin 2 gene

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.

Osmolality and solute composition are strong regulators of AQP2 expression in renal principal cells

“…The promoter region of the AQP2 gene contains a CRE, known to be involved in the AVP-induced AQP2 expression (13,25). CREB, once phosphorylated at Ser 133 (pCREB) by PKA, binds to the CRE and, thereby, presumably stimulates AQP2 transcription.…”

“…The subsequent activation of protein kinase A (PKA) initiates the translocation of AQP2-bearing vesicles from the cytosol to the plasma membrane, in which AQP2 is inserted by an exocytosis-like process (short-term regulation) (D. Lorenz, A. Krylov, V. Hagen, J. Zipper, W. Rosenthal, P. Pohl, and K. Maric, unpublished observations; 30). In addition, the signaling cascade described above governs the expression of AQP2 (long-term regulation) by PKA-mediated phosphorylation of the transcription factor cAMP-responsive element (CRE)-binding protein (CREB) (13,16). Given the fact that AQP2 biosynthesis is usually shut off shortly after IMCD cells are established in primary culture (10), most studies on AQP2 long-term regulation have been performed using animal models (29, 37).…”

Hyponatremia in cirrhosis: From pathogenesis to treatment

“…73 In fact, the promoters of rat, mouse, and human AQP2 gene have been cloned, 74,75 and they contain a consensus sequence of cAMP-responsive element, which regulates the transcription of AQP-2. 75,76 In LLC-PK1 cells transfected with the fragment of the human promoter of AQP-2, VP has been demonstrated to regulate via V 2 receptors the activation of the FIG. 2.…”

Mild Nephrogenic Diabetes Insipidus Caused by Foxa1 Deficiency

“…Over the longer term, elevated cAMP levels lead to activation of protein kinase A and the phosphorylation of the transcription factor CREB (cyclic AMP-response element-binding protein) (6). Thus activated, CREB increases transcription of the aquaporin 2 message and ultimately increases aquaporin 2 protein levels (7).…”

“…Inherited nephrogenic DI can be caused by mutations in the genes encoding either the vasopressin 2 receptor or aquaporin 2, highlighting the central role of these two proteins in the pathway (7)(8)(9)(10)(11). The ability of the kidney to concentrate urine also depends on the interstitial osmotic gradient, as illustrated by the molecular defects in Bartter's syndrome.…”