Suppression of ANP gene transcription by liganded vitamin D receptor: involvement of specific receptor domains.
We showed previously that liganded vitamin D receptor (VDR) effects a suppression of human atrial natriuretic peptide (hANP) gene-promoter activity in cultured neonatal rat atrial myocytes. In the present study, we have attempted to identify the structural domains of the VDR that are involved in mediating this suppression. We examined the effects of a series of VDR mutants on a cotransfected hANP promoter-driven chloramphenicol acetyltransferase (CAT) reporter. Neither the native VDR nor any of the mutants tested displayed inhibitory activity in the absence of the 1,25-dihydroxyvitamin D3 (VD3) ligand. Delta134, a deletant harboring solely the DNA binding region of the VDR, and L254G, a mutant shown to be defective in retinoid X receptor (RXR) heterodimer formation in other systems, were as effective as the native VDR in reducing promoter activity. HBD, a deletant containing only the hormone-binding domain of the VDR, and K246G, a point mutant that is defective in the activation function of the receptor, did not attenuate reporter activity. A similar activity profile was displayed when a positively regulated promoter containing a direct-repeat vitamin D responsive element (DR3-CAT) was examined in these cells. Liganded VDR, the delta134 mutant, and liganded L254G effected increases in DR3-CAT activity of 2.5-, 2-, and 4-fold, respectively. Two nonhypercalcemic analogues of VD3 (RO 23-7553 and RO 25-6760) displayed the same inhibitory activity as VD3. These studies suggest that the inhibition of hANP promoter activity requires both the DNA binding and activation functions of the receptor but does not appear to require formation of a classic RXR alpha-VDR heterodimer.
Chen
S
S
Metabolic Research Unit, University of California at San Francisco, 94143, USA.
Wu
J
J
Hsieh
J C
JC
Whitfield
G K
GK
Jurutka
P W
PW
Haussler
M R
MR
Gardner
D G
DG
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D009320:Atrial Natriuretic Factor / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002117:Calcitriol / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D017930:Genes, Reporter; D006325:Heart Atria / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D008024:Ligands; D018167:Receptors, Calcitriol; D009320:Atrial Natriuretic Factor; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D002117:Calcitriol