Estrogen receptor-alpha (ERalpha), but not ERbeta,modulates estrogen stimulation of the ERalpha-truncated variant,TERP-1

Estrogens regulate pituitary gene expression through two nuclear receptors (ERs), ERalpha and ERbeta. Rodent pituitary also expresses high levels of the pituitary-specific ERalpha isoform, truncated ER product-1 (TERP-1), which modulates the response of both ER forms to 17beta-estradiol (E2). Under physiological conditions, E2 stimulates TERP-1 expression from an ERalpha intronic promoter containing several potential binding sites for ERs. To evaluate the role of intact ER proteins on TERP-1 expression, we measured basal expression and steroid stimulation of TERP-1 in wild-type (WT) mice and mice in which either the ERalpha (ERalphaKO) or the ERbeta (ERbetaKO) gene was disrupted. TERP-1 mRNA expression was assessed by semiquantitative RT-PCR, and protein expression was evaluated by immunoblots. Both TERP-1 mRNA and protein were expressed in pituitaries from castrate WT, ERalphaKO, and ERbetaKO male and female mice. E2 stimulated TERP-1 mRNA expression in WT and ERbetaKO mice of both sexes, but had no effect on TERP-1 mRNA in either male or female ERalphaKO mice. Testosterone treatment also stimulated TERP-1 in WT, ERalphaKO, and ERbetaKO male mice. We conclude that ERalpha is critical for E2 stimulation, but not basal expression, of the TERP promoter, and that testosterone may act through the androgen receptor to stimulate the TERP-1 promoter in males.     

A mathematical approach to predict the affinity of estrogen receptors alpha and beta binding to DNA

Estrogen receptors alpha and beta (ERalpha and ERbeta) bind to specific DNA sequences, estrogen response elements (EREs), usually located in the promoters of estrogen-regulated genes. The consensus ERE contains two inverted repeats of the 5'-AGGTCA-3' half-site (1/2 ERE) separated by three base pairs (bp). Many estrogen-responsive gene promoters contain one or more direct repeats (DR) of 1/2 ERE. Here, we examined the affinity of ERalpha and ERbeta binding and estradiol (E(2))-induced transactivation from select EREs and DRs. The affinity of ERalpha and ERbeta binding to imperfect EREs in vitro can be predicted from equations using the number of 1/2 EREs and the number of (AT)-(GC) bp substitutions within the 15-bp candidate ERE sequence as independent variables. Transactivation by ERalpha and ERbeta correlates with the affinity of ER-ERE binding with the exception of ERalpha from two low-affinity EREs. The equations developed here can be used to screen the promoters of estrogen-responsive genes for candidate ERE sequences.     

Transcriptional activation of transforming growth factor alpha by estradiol: requirement for both a GC-rich site and an estrogen response element half-site

17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (-252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGFalpha gene promoter in this study identified a second upstream region of the promoter (-623 to -549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans-acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE(1/2) motif identified in the TGFalpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.     

Differential and opposing regulation of PAI-1 promoter activity by estrogen receptor alpha and estrogen receptor beta in endothelial cells

To investigate the molecular mechanisms involved in the estrogen-dependent control of plasminogen activator inhibitor-1 (PAI-1) gene expression in vascular cells, we compared the transactivation properties of estrogen receptors (ERalpha and ERbeta) in regulating the activity of a human PAI-1 promoter reporter construct in transfected bovine aortic endothelial cells (BAECs). ERalpha increased PAI-1 promoter activity in BAECs by an estrogen-dependent mechanism, whereas ERbeta suppressed PAI-1 promoter activity by an estrogen-independent mechanism. The suppressive activity of ERbeta was dominant over the inductive activity of ERalpha. Mutation of a putative estrogen response element (ERE) located at position -427 in the proximal promoter abolished the ERalpha action without influencing the suppressive effects of ERbeta. Mutation of either AP1-like site did not eliminate the ERalpha or ERbeta actions at the PAI-1 promoter, suggesting that other promoter elements are involved in these responses. These mutations significantly reduced the -3.4kbp PAI-1 promoter response to serum. We concluded that ERalpha and ERbeta exert differential effects on the PAI-1 promoter activity in transfected BAECs. ERalpha activated the PAI-1 promoter through a proximal ERE (-427) and possibly additional EREs located within the PAI-1 promoter, whereas ERbeta suppressed the promoter construct via an unidentified mechanism. This is the first demonstration of the differential regulation of a vascular gene promoter by ERalpha and ERbeta.     

The aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT) heterodimer interacts with naturally occurring estrogen response elements

To determine the molecular mechanisms underlying the "cross talk" between the activity of 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), which binds to arylhydrocarbon receptor (AHR) and estradiol (E2)-liganded estrogen receptor (ER), we first examined the initial step of estrogen action, ligand binding to ER. None of the AHR ligands tested, i.e. TCDD, benzo[a]pyrene, 3,3',4,4',5-pentachlorobiphenyl, beta-naphthoflavone, or alpha-naphthoflavone, bound to ER alpha. We report the first examination of TCDD interaction with ER beta: TCDD did not displace E2 from ER beta. We then examined a second possible mechanism, i.e. direct inhibition of ER alpha binding to estrogen response elements (EREs) by the AHR/AHR nuclear translocator (ARNT) complex. The AHR/ARNT heterodimer did not bind either a full or half-site ERE. However, AHR/ARNT bound specifically to oligomers containing naturally occurring EREs derived from the human c-fos, pS2, and progesterone receptor (PR) gene promoters that include xenobiotic response element (XRE)-like sequences. In contrast, neither purified E2-liganded-ER from calf uterus or recombinant human ER alpha bound a consensus XRE. TCDD inhibited E2-activated reporter gene activity from a consensus ERE and from EREs in the pS2, PR, and Fos genes in transiently transfected MCF-7 human breast cancer cells. However, this inhibition was not reciprocal since E2 did not inhibit TCDD-stimulated luciferase activity from the CYP1A1 promoter in transiently transfected MCF-7 or human endometrial carcinoma HEC-1A cells. We propose that at least part of the mechanism by which the AHR/ARNT complex inhibits estrogen action is by competitively inhibiting ER alpha binding to imperfect ERE sites, adjacent to or overlapping XREs.