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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Estradiol (E2)-liganded estrogen receptor (ER) bound to three or four tandem copies of a consensus ERE (EREc38) in a cooperative manner. E2-ER binding to one or two EREs was non-cooperative. When ER was liganded by the antiestrogen 4-hydroxytamoxifen (4-OHT), ER-ERE binding was not cooperative, regardless of the number of EREs. Here we evaluated how binding to EREc38 affects ER conformation in the ligand binding domain (LBD) as reflected in the dissociation kinetics of [3H]ligand from the ER. Binding of ER to EREc38 slowed the rate of dissociation of either E2 or 4-OHT, indicating that DNA allosterically modulates the LBD conformation creating a tighter fit between the ligand and the ER. Conformational differences in ER induced by E2 versus antiestrogen were not reflected in differences in E2 or 4-OHT dissociation parameters under these conditions. No difference in the association rate of E2- versus 4-OHT-liganded ER binding to EREc38 was detected in electrophoretic mobility shift assay (EMSA). Synergistic, E2-dependent activation of a reporter gene was detected from three and four, but not one or two, tandem copies of EREc38. These observations suggest that cooperative binding of E2-ER to multiple copies of EREc38 is likely responsible for transcriptional synergy and that cooperativity may not involve direct interaction between the LBDs of ERE-bound ER. Since the number of copies of EREc38 did not alter E2 dissociation kinetics, functional synergy must involve cellular factors in addition to the ER ligand.
Mol Cell Endocrinol 1999 Apr 25
PMID:Estrogen receptor binding to estrogen response elements slows ligand dissociation and synergistically activates reporter gene expression. 1041 4

Estrogen-responsive genes are regulated by altering the balance of estrogen receptor (ER) interaction with transcription activators and inhibitors. Here we examined the role of ER ligand on ER interaction with the Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) orphan nuclear receptor. COUP-TF binding to half-site estrogen response elements (EREs) was increased by the addition of estradiol (E2) -liganded ER (E2-ER), but not by ER liganded with the antiestrogens 4-hydroxytamoxifen (4-OHT-ER) or tamoxifen aziridine (TAz-ER). ER did not bind to single half-sites. Conversely, COUP-TF enhanced the ERE binding of purified E2-ER, but did not affect TAz-ER-ERE binding. In contrast, only antiestrogens enhanced direct interaction between ER and COUP-TF as assessed by GST pull-down assays. Identical results were obtained using either purified bovine or recombinant human ERalpha. Co-immunoprecipitation assays showed that ER and COUP-TF interact in extracts from MCF-7 and ERalpha-transfected MDA-MB-231 cells. Here we document that ER ligand impacts COUP-TF-ER interaction. COUP-TF interaction is mediated by the DNA binding and ligand-binding domains of ER. We suggest that changes in ER conformation induced by DNA binding reduce ER-COUP-TF interaction. Transient transfection of human MCF-7 breast cancer cells with a COUP-TFI expression vector repressed E2-induced luciferase reporter gene expression from single or multiple tandem copies of a consensus ERE. COUP-TFI stimulated 4-OHT-induced luciferase activity from a minimal ERE. Alone, COUP-TFI increased transcription from ERE half-sites or a single ERE in a sequence-dependent manner. These data provide evidence that the ERE sequence and its immediate flanking regions influence whether COUP-TF enhances, inhibits, or has no effect on ER ligand-induced ERE reporter gene expression and that COUP-TFI activates gene transcription from ERE half-sites. We suggest that COUP-TFI plays a role in mitigating estrogen-responsive gene expression.
J Steroid Biochem Mol Biol 1999 Nov
PMID:Role of estrogen receptor ligand and estrogen response element sequence on interaction with chicken ovalbumin upstream promoter transcription factor (COUP-TF). 1061 53

Three breast carcinoma cell lines were tested for 17beta-estradiol (E(2)) mediated regulation of vasoactive intestinal polypeptide receptor type-1 (VPAC(1)) expression. In all three, E(2) was found to down-regulate the mRNA level. We studied T47D cells in more details and found a 25 and 70% decrease in the VPAC(1) mRNA level upon 7 and 48 h of E(2) treatment, respectively. The number of vasoactive intestinal polypeptide (VIP) binding sites was reduced 66% upon treatment with E(2) for 72 h. After cycloheximide pretreatment, the E(2) mediated mRNA reduction was attenuated from 50% to 25% after 24 h suggesting the effect to be at least partly independent of protein synthesis. Experiments with the transcriptional inhibitor actinomycin D showed that E(2) did not influence the VPAC(1) mRNA half-life while nuclear run-on experiments indicated that E(2) decreased the VPAC(1) transcription rate. Two antiestrogens: ICI 182780 (ICI) and 4-hydroxy-tamoxifen (4-OHT) mediated a concentration dependent inhibition of E(2)'s effect on the mRNA level. Transient transfection with reporter-gene constructs containing various portions of the VPAC(1) 5'-flanking sequence revealed the most proximal 100 bp to be essential for the basal transcriptional activity. However, E(2) did not influence the expression of the reporter gene using up to 3250 bp of the VPAC(1) 5'-flariking region.
Mol Cell Endocrinol 2001 Feb 14
PMID:Estradiol down regulates expression of vasoactive intestinal polypeptide receptor type-1 in breast cancer cell lines. 1116 54

We have previously shown that estradiol (E2) increases the growth of normal human breast epithelial (HBE) cells and the antiestrogen 4-hydroxytamoxifen (4-OHT) inhibits estrogen-induced proliferation. These effects of estrogens and antiestrogens on proliferation have also been well documented in breast cancer cells. One mechanism for the antiproliferative effects of antiestrogens is the stimulation of TGFbeta in hormone-dependent MCF-7 and T47D cells. The role of this inhibitory growth factor in normal human breast cells has not been well studied. Accordingly, we measured the amounts of total and active TGFbeta1 and TGFbeta2 by specific E(max) immunoassay (EIA) in culture medium from normal breast cells (epithelial and fibroblasts) and from various ER- and ER+ breast cancer cell lines. We established that HBE cells are sensitive to the antiproliferative effect of TGFbetas, and studied the effect of E2 and 4-OHT, alone or in combination, on the secretion and activation of TGFbetas by HBE cells. HBE cells secrete TGFbeta1 and even more TGFbeta2, and are sensitive to these factors. However, in contrast to MCF-7 cells, TGFbeta secretion in normal breast cells is not regulated by E2 and 4-OHT.
Mol Cell Endocrinol 2001 Mar 28
PMID:Estrogen and antiestrogen actions on transforming growth factorbeta (TGFbeta) in normal human breast epithelial (HBE) cells. 1130 68

Estrogens play a critical role in mammary gland development, bone homeostasis, reproduction, and the pathogenesis of breast cancer by activating estrogen receptors (ERs) alpha and beta. Ligand-activated ER stimulates the expression of target proteins by interacting with specific DNA sequences: estrogen response elements (EREs). We have demonstrated that the ERE sequence and the nucleotide sequences flanking the ERE impact ERalpha binding affinity and transcriptional activation. Here, we examined whether the sequence of the ERE modulates ERalpha conformation by measuring changes in sensitivity to protease digestion. ERalpha, occupied by estradiol (E2) or 4-hydroxytamoxifen (4-OHT), was incubated with select EREs and digested by chymotrypsin followed by a Western analysis with antibodies to ERalpha. ERE binding increased the sensitivity of ERalpha to chymotrypsin digestion. We found both ligand-specific and ERE-specific differences in ERalpha sensitivity to chymotrypsin digestion. The ERE-mediated increase in ERalpha sensitivity to chymotrypsin digestion correlates with E2-stimulated transcriptional activity from the same EREs in transiently transfected cells. Transcriptional activity also correlates with the affinity of ERalpha-ERE binding in vitro. Our results support the hypothesis that the ERE sequence acts as an allosteric effector, altering ER conformation. We speculate that ERE-induced alterations in ERalpha conformation modulate interaction with co-regulatory proteins.
Mol Cell Endocrinol 2001 Mar 28
PMID:Estrogen response element sequence impacts the conformation and transcriptional activity of estrogen receptor alpha. 1130 82

The selective estrogen receptor modulator, 4-hydroxytamoxifen (4-OHT) is a full agonist at the transforming growth factor (TGF) alpha gene in ER negative breast cancer cells stably transfected with ER alpha cDNA (Levenson et al., Br. J. Cancer 77 (1998) 1812-1819). E(2) and 4-OHT increase TGF alpha mRNA and protein in a concentration dependent manner. The responses to E(2) and 4-OHT are blocked by the pure antiestrogen ICI 182,780, which does not induce TGF alpha. Transfected MDA-MB-231 cells contain functional ER alpha but no ER beta function was detected. Neo transfected cells that did not express ER alpha or cells stably transfected with the DNA binding domain mutant C202R/E203V which prevents gene activation did not induce TGF alpha mRNA after either E(2) or 4-OHT treatment. An examination of the time course for either 10 nM E(2) or 1 microM 4-OHT for MDA-MB-231 cells stably transfected with cDNA for ER alpha showed increases in TGF alpha mRNA within 2 or 3 h respectively. Cells pretreated with cycloheximide (1 microg/ml) showed induced TGF alpha mRNA in response to E(2) or 4-OHT but TGF alpha mRNA induction was blocked by actinomycin D (1 microg/ml). We conclude that both E(2) and 4-OHT induce TGF alpha by direct interaction of ER alpha with DNA and that ER beta is not involved in the estrogen-like response to 4-OHT in the MDA-MB-231 cells.
J Steroid Biochem Mol Biol 2001 Jul
PMID:Estrogen receptor alpha mediated induction of the transforming growth factor alpha gene by estradiol and 4-hydroxytamoxifen in MDA-MB-231 breast cancer cells. 1153 Feb 83

Genes whose expression is highly induced by estradiol (E(2)) contain multiple estrogen response elements (EREs) in their promoters. Previously we reported that estrogen receptor-alpha (ERalpha) binds cooperatively to and E(2) synergistically activates reporter gene expression from three or four tandem copies of a consensus ERE (EREc38). Here we evaluated how ERalpha binding to one, two, three or four tandem copies of EREc38 affects ERalpha conformation as detected by altered ERalpha trypsin digestion patterns in Western blots. E(2)- or 4hydroxytamoxifen (4-OHT)-occupied ERalpha bound to the pS2 ERE or to a single copy of EREc38 showed enhanced susceptibility to trypsin digestion compared to E(2)- or 4-OHT-ERalpha incubated with DNA lacking an ERE. ERalpha binding to multiple tandem copies of EREc38 further increased sensitivity to trypsin digestion. These results correlate with synergistic transcription and cooperativity of ERalpha binding to multiple tandem copies of EREc38. These observations suggest that EREc38 binding alters the overall conformation of ERalpha and that multiple tandem copies of EREc38 enhance these conformational changes. We hypothesize that ERE-induced alterations in ERalpha conformation modulate interaction with coregulatory proteins, resulting in synergistic transcriptional activation.
J Mol Endocrinol 2001 Dec
PMID:Estrogen response element binding induces alterations in estrogen receptor-alpha conformation as revealed by susceptibility to partial proteolysis. 1171 81

Diseases requiring frequent and lifelong injections of recombinant proteins would be more efficaciously treated by intramuscular delivery of genes encoding secretable proteins. However, the success of this approach largely depends on our capability to temporally regulate transcription of delivered genes. Therefore, we sought to generate a humanized transcription factor to regulate transgene expression in muscle. A novel 4-hydroxytamoxifen (4-OHT)-dependent transcriptional regulator (called HEA-3) was constructed by fusing in-frame the DNA binding domain of the human hepatocyte nuclear factor-1alpha (HNF1alpha), which is not expressed in muscle cells, a G(521)R mutant of the ligand binding domain of human estrogen receptor-alpha (ERalpha), and the activation domain derived from human nuclear factor-kappaB p65 subunit (NF-kappaB p65). We demonstrate that an artificial promoter containing multimeric HNF1alpha binding sites is silent in muscles and in cell lines that lack endogenous HNF1alpha. HEA-3 stimulated transcription from this target promoter in a stringent 4-OHT-dependent manner. The dynamic range of transgene regulation was high, because of the low basal activity and high inducibility of the system. Ex vivo, HEA-3 increased expression of the transfected reporter gene by more than 1000-fold in a ligand-dependent manner. In vivo, HEA-3 stimulated by more than 100-fold, the expression of secreted alkaline phosphatase after delivery as plasmid DNA into mouse muscles. Moreover, long-term modulation of the expression of intramuscularly delivered mouse erythropoietin was achieved in immunocompetent mice.
Mol Ther 2002 Nov
PMID:Long-term and tight control of gene expression in mouse skeletal muscle by a new hybrid human transcription factor. 1240 64

The pattern of transcriptional activation by 17beta-estradiol (E2) and 4-hydroxytamoxifen (4-OHT) was determined in ZR-75 and MDA-MB-231 breast, ECC1 and HEC1A endometrial and HepG2 liver cancer cell lines cotransfected with E2-responsive constructs and wild-type estrogen receptor alpha (ER alpha) or ER beta (ER beta) or variant forms of ER alpha expressing activation function 1, AF1 (ER alpha-AF1) or activation function 2, AF2 (ER alpha-AF2). The E2-responsive constructs contained promoter inserts from the human complement C3 (pC3), human cathepsin D (pCD) and rat creatine kinase B (pCKB) genes. Minimal ER beta-dependent transactivation (<2.5-fold induction) was observed for E2 only in ECC1 and MDA-MB-231 cells transfected with pCKB or pC3, whereas 4-OHT was inactive as an ER beta agonist for all promoters in the four cell lines. The ER alpha agonist and/or antagonist activities for E2 and 4-OHT were highly variable and the transactivation was dependent on ER subtype, ER alpha variant expressed, gene promoter, and cell context. For example, E2 did not activate pCD in HepG2 cells transfected with wild-type or variant ER alpha, whereas E2 activated reporter gene activity in the four endometrial and breast cancer cell lines transfected with ER alpha and pCD, pCKB or pC3. Hormone activation of these constructs by ER alpha-AF1 or ER alpha-AF2 was highly variable among the different cell lines and even in the same cell line transfected with the three E2-responsive constructs. Similar variability was observed for 4-OHT. For example, 4-OHT activates pC3 in HepG2 cells transfected with ER alpha or ER alpha-AF1, and pCKB in HEC1A cells. However, AF1-dependent activation by 4-OHT is not observed for pCKB in ECC1 cells or for pC3 and pCD in HEC1A or ECC1 endometrial cancer cells. The results of this study suggest that transcriptional activation by E2 and 4-OHT induces recruitment of different transcription factor complexes that are dependent on the cell type and also the gene promoter.
J Steroid Biochem Mol Biol 2003 Jan
PMID:17 beta-estradiol- and 4-hydroxytamoxifen-induced transactivation in breast, endometrial and liver cancer cells is dependent on ER-subtype, cell and promoter context. 1264 21

Most cultured cell lines require addition of serum to the medium to maintain their proliferative capacity. For studies examining the cellular effects of estrogens serum is charcoal-stripped to remove steroids. Nonetheless, addition of the selective estrogen receptor modulator (SERM) 4-hydroxytamoxifen (4-OHT) inhibits the basal transcriptional activity of estrogen receptors alpha or beta (ERalpha or ERbeta) in transfected cells. We tested the hypothesis that elimination of serum from the culture medium will block 4-OHT's repression of basal activity. Chinese hamster ovary (CHO-K1) cells adapted to serum-free medium exhibited estrogen responsiveness that was identical with that of the cells grown in serum-containing media. 4-OHT-suppressed basal transcription of an estrogen response element (ERE)-reporter in ERalpha-transfected cells even in the absence of serum, indicating that the 4-OHT suppressive activity is not mediated by blocking ER interaction with serum estrogens. We speculate that 4-OHT-ER recruits co-repressors to suppress basal transcription. We discovered that CHO-K1 cells express ERalpha and ERbeta mRNA. However only ERbeta protein was expressed and use of ERbeta-selective 2,3-bis(4-hydroxy-phenyl)propionitrile (DPN) and ERalpha-selective 4-propyl-1,3,5-tris(4-hydroxy-phenyl)pyrazole) (PPT) revealed that only ERbeta was transcriptionally active. In conclusion, growing CHO-K1 in serum-free medium does not impact the estrogen responsiveness and this cell line expresses functional ERbeta.
J Steroid Biochem Mol Biol 2003 Jul
PMID:Identification of estrogen receptor beta expression in Chinese hamster ovary (CHO) cells and comparison of estrogen-responsive gene transcription in cells adapted to serum-free media. 1294 44


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