Cloning of the 11β-hydroxysteroid dehydrogenase (11β-HSD)-2 gene in the baboon:: effects of estradiol on promoter activity of 11β-HSD-1 and -2 in placental JEG-3 cells

In the baboon, estrogen regulated 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyzed metabolism of cortisol and cortisone by the placenta is an important component in the sequence of events regulating the fetal pituitary-adrenocortical axis. The present study was designed to isolate and sequence the promoter region of the baboon 11 beta-HSD-2 gene and to produce constructs of this gene and the 1.7 kb fragment of 5'-flanking region of baboon 11 beta-HSD-1 isolated previously in order to determine whether the promoters of these two genes were activated in human placental JEG-3 cells and whether expression could be modulated by estradiol. The 11 beta-HSD-2 genomic DNA was isolated from a baboon kidney genomic library using a human 11 beta-HSD-2 cDNA as a probe. The sequence of a 1.2 kb fragment of the 5'-flanking region showed extensive homology with that published by others for human 11 beta-HSD-2, particularly in exon 1 (> 95%) and in the proximal promoter (> 90%), Primer extension confirmed that the baboon 11 beta-HSD-2 gene has multiple transcriptional start sites which are preceded by a GC box. To determine promoter activity of 11 beta-HSD-2 and -1, the 5'-flanking regions of these genes were subcloned into luciferase reporter pGL3 vectors, transiently transfected into human placental JEG-3 cells, and then incubated for 16-18 h in the presence or absence of 10(-8) M 17 beta-estradiol or 17 alpha-estradiol, To augment the low level of estrogen receptor (ER) in JEG cells, promoter activity studies were also performed in JEG cells co-transfected with an expression vector containing the human ER cDNA. The promoters of both 11 beta-HSD-1 and -2 were activated following transient transfection into JEG-3 cells although basal activity of 11 beta-HSD-2 (87 +/- 21 RLU/mu g protein) always exceeded (P < 0.05) that of 11 beta-HSD-1 (37 +/- 7). In the absence of co-transfected ER, basal promoter activities of both 11 beta-HSD genes were not altered by 17 beta-estradiol. In contrast, in cells co-transfected with ER, 17 beta-estradiol but not 17 alpha-estradiol increased (P < 0.05) basal promoter activities of 11 beta-HSD-1 and -2 by 8.1 +/- 1.5 and 8.3 +/- 2.0 fold, respectively. Collectively, these findings indicate that the promoter region of the baboon 11 beta-HSD-2 gene is comparable to that in the human and that the 5'-flanking region of both the baboon 11 beta-HSD-1 and -2 genes were active when transiently transfected into JEG-3 cells and that activation could be enhanced by estradiol in the presence of an estrogen receptor. (C) 1999 Elsevier Science B.V. All rights reserved.