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  • Title: Cloning of the 11beta-hydroxysteroid dehydrogenase (11beta-HSD)-2 gene in the baboon: effects of estradiol on promoter activity of 11beta-HSD-1 and -2 in placental JEG-3 cells.
    Author: Pepe GJ, Davies WA, Dong KW, Luo H, Albrecht ED.
    Journal: Biochim Biophys Acta; 1999 Jan 18; 1444(1):101-10. PubMed ID: 9931459.
    Abstract:
    In the baboon, estrogen regulated 11beta-hydroxysteroid dehydrogenase (11beta-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 11beta-HSD-2 gene and to produce constructs of this gene and the 1.7 kb fragment of 5'-flanking region of baboon 11beta-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 11beta-HSD-2 genomic DNA was isolated from a baboon kidney genomic library using a human 11beta-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 11beta-HSD-2, particularly in exon 1 (>95%) and in the proximal promoter (>90%). Primer extension confirmed that the baboon 11beta-HSD-2 gene has multiple transcriptional start sites which are preceded by a GC box. To determine promoter activity of 11beta-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 17beta-estradiol or 17alpha-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 11beta-HSD-1 and -2 were activated following transient transfection into JEG-3 cells although basal activity of 11beta-HSD-2 (87+/-21 RLU/microg protein) always exceeded (P<0.05) that of 11beta-HSD-1 (37+/-7). In the absence of co-transfected ER, basal promoter activities of both 11beta-HSD genes were not altered by 17beta-estradiol. In contrast, in cells co-transfected with ER, 17beta-estradiol but not 17alpha-estradiol increased (P<0.05) basal promoter activities of 11beta-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 11beta-HSD-2 gene is comparable to that in the human and that the 5'-flanking region of both the baboon 11beta-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.
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