Ke-Wen Dong

cDNA cloning and expression of a novel estrogen receptor β-subtype in goldfish (Carassius auratus)

Abstract We have isolated a second goldfish estrogen receptor (ER) β-subtype (gfER-β2) cDNA which is distinct from the liver-derived ER-β (gfER-β1) cDNA reported previously. The 2650-bp cDNA, isolated from a goldfish pituitary and brain cDNA library, encodes a 610 amino acid (aa) protein which shows only a 53% aa sequence identity with gfER-β1 in overall structure. RT-PCR analysis showed that mRNA of gfER-β2, in contrast to that of gfER-β1, was predominantly expressed in pituitary, telencephalon and hypothalamus as well as in liver of female goldfish. The existence of a second distinct ER-β subtype opens new dimensions for studying tissue-specific regulation of gene expression by estrogen in the tetraploid goldfish.

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 11β-hydroxysteroid dehydrogenase (11β-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 11β-HSD-2 gene and to produce constructs of this gene and the 1.7 kb fragment of 5′-flanking region of baboon 11β-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 11β-HSD-2 genomic DNA was isolated from a baboon kidney genomic library using a human 11β-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 11β-HSD-2, particularly in exon 1 (>95%) and in the proximal promoter (>90%). Primer extension confirmed that the baboon 11β-HSD-2 gene has multiple transcriptional start sites which are preceded by a GC box. To determine promoter activity of 11β-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 17β-estradiol or 17α-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 11β-HSD-1 and -2 were activated following transient transfection into JEG-3 cells although basal activity of 11β-HSD-2 (87±21 RLU/μg protein) always exceeded (P<0.05) that of 11β-HSD-1 (37±7). In the absence of co-transfected ER, basal promoter activities of both 11β-HSD genes were not altered by 17β-estradiol. In contrast, in cells co-transfected with ER, 17β-estradiol but not 17α-estradiol increased (P<0.05) basal promoter activities of 11β-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 11β-HSD-2 gene is comparable to that in the human and that the 5′-flanking region of both the baboon 11β-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.

Identification of negative and positive estrogen response elements in human GnRH upstream promoter in the placental JEG-3 cells

Results from our previous studies have demonstrated regulatory effects of estradiol on human gonadotropin-releasing hormone (GnRH) gene expression in human placental cells. The present study was designed to determine the molecular mechanisms whereby estrogens regulate the human GnRH gene expression in the placenta. The effects of estradiol on human GnRH upstream promoter activity in JEG-3 cells depends on the amounts of estrogen receptor (ER) alpha expression vector co-transfected, with the maximal effect obtained at the amount of 1.0 microg of ER expression vector cotransfected. Estriol, an isoform of estradiol, also possesses a regulatory effect on the upstream promoter activity, while estrone, another isoform, does not. Serial deletion studies revealed two estrogen responsive elements in the GnRH upstream promoter region. One element (-987 to -968 bp, E4 element) confers a negative estradiol response, while another one (-827 to -730 bp) is responsible for a positive estradiol effect. Replacement of these two elements with unrelated DNA sequences could abolish the responsiveness to estradiol treatment. Furthermore, footprinting and gel shift assays demonstrated that nuclear protein from estradiol-treated JEG-3 cells, but not from control cells, could bind to a 41 bp DNA fragment (-824 to -784 bp) within the estrogen positive responsive element. Results of gel-shift assay demonstrated that other protein(s) might also be involved in interacting the E4 element to mediate the negative effect of estradiol on the hGnRH upstream promoter activity in JEG-3 cells.

Estrogen down-regulates GnRH gene expression in human placental cytotrophoblast cells

This study aims at investigating and characterizing GnRH gene expression and regulation in human placental cytotrophoblasts by estradiol. RT-PCR analysis demonstrates that the cytotrophoblasts expressed the pro-GnRH gene. PCR with two pairs of oligonucleotide primers generated doublets 870 bases apart at the expected molecular weights, indicating that the upstream and downstream transcriptional start sites of the GnRH gene were utilized and that transcripts with and without intron I were both produced. In human placental cytotrophoblasts, the upstream transcription start site of GnRH gene was the major one and gave rise to an mRNA level three times higher than the downstream start site. RT-PCR assay also revealed that estradiol downregulated the expression of GnRH gene in human cytotrophoblasts. Estradiol at the concentration of 10nM led to a significant (50%) reduction in the mRNA from the upstream transcription start site. Such downregulation was dose-dependent and abolished by the addition of an estrogen antagonist, ICI, indicating that the gene expression was mediated by estrogen receptors. Furthermore, downregulation of GnRH gene expression by estradiol has also been confirmed by luciferase assay.