Cloning of the 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD)-2 gene in the baboon: effects of estradiol on promoter activity of 11 beta-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 pitu itary-adrenocortical axis. The present study was designed to isolate and se quence 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 ba boon 11 beta-HSD-1 isolated previously in order to determine whether the pr omoters of these two genes were activated in human placental JEG-3 cells an d whether expression could be modulated by estradiol. The 11 beta-HSD-2 gen omic 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'-fl anking region showed extensive homology with that published by others for h uman 11 beta-HSD-2, particularly in exon 1 (> 95%) and in the proximal prom oter (> 90%), Primer extension confirmed that the baboon 11 beta-HSD-2 gene has multiple transcriptional start sites which are preceded by a GC box. T o determine promoter activity of 11 beta-HSD-2 and -1, the 5'-flanking regi ons of these genes were subcloned into luciferase reporter pGL3 vectors, tr ansiently 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-transf ected with an expression vector containing the human ER cDNA. The promoters of both 11 beta-HSD-1 and -2 were activated following transient transfecti on 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 incre ased (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 indi cate that the promoter region of the baboon 11 beta-HSD-2 gene is comparabl e to that in the human and that the 5'-flanking region of both the baboon 1 1 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 reserve d.