Analysis of the promoter of the NAD+ dependent 11 beta-hydroxysteroid dehydrogenase (HSD11K) gene in JEG-3 human choriocarcinoma cells.
The NAD+ dependent (K or type 2) isozyme of 11 beta-hydroxysteroid dehydrogenase oxidizes glucocorticoids and thus prevents them from occupying mineralocorticoid receptors. Mutations in the HSD11K (HSD11B2) gene encoding this isozyme cause a genetic form of hypertension, the syndrome of apparent mineralocorticoid excess (AME). This isozyme is expressed at high levels in placenta and kidney but is undetectable in liver. We have now analyzed the proximal 1788 nucleotides (nt) of the 5' flanking region of the HSD11K gene to identify transcriptional regulatory elements that are active in JEG-3 human choriocarcinoma cells. Using luciferase reporter constructs, the region from -2 to -330 nt relative to the initial ATG codon was identified as an essential region for basal transcription of the HSD11K gene. Two segments in this region, -278 to -257 and -215 to -194. were protected in DNase 1 footprinting analysis. Both segments have consensus binding sites for the Spl transcription factor. Gel shift assays of these segments show several DNA-protein complexes using JEG-3 nuclear extract. Only the slowest migrating complex was competed by an antiserum to Spl. These results suggest that the two Spl sites, either alone or in combination, are essential for transcription of the HSD11K gene in JEG-3 cells.
Agarwal
A K
AK
Department of Pediatrics, University of Texas Southwestern Medical Centre, Dallas 75235-9063, USA.
White
P C
PC
D042842:11-beta-Hydroxysteroid Dehydrogenases; D001483:Base Sequence; D001665:Binding Sites; D002822:Choriocarcinoma / Q000473:pathology; D004247:DNA; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D009243:NAD; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D009687:Nuclear Proteins; D009243:NAD; D004247:DNA; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D003850:Deoxyribonuclease I