Covalently grafted VEGF(165) in hydrogel models upregulates the cellular pathways associated with angiogenesis.

Angiogenesis is an important biological response known to be involved in many physiological and pathophysiological situations. Cellular responses involved in the formation of new blood vessels, such as increases in endothelial cell proliferation, cell migration, and the survival of apoptosis-inducing events, have been associated with vascular endothelial growth factor isoform 165 (VEGF(165)). Current research in the areas of bioengineering and biomedical science has focused on developing polyethylene glycol (PEG)-based systems capable of initiating and sustaining angiogenesis in vitro. However, a thorough understanding of how endothelial cells respond at the molecular level to VEGF(165) incorporated into these systems has not yet been established in the literature. The goal of the current study was to compare the upregulation of key intracellular proteins involved in angiogenesis in human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC) seeded on PEG hydrogels containing grafted VEGF(165) and adhesion peptides Arg-Gly-Asp-Ser (RGDS). Our data suggest that the covalent incorporation of VEGF(165) into PEG hydrogels encourages the upregulation of signaling proteins responsible for increases in endothelial cell proliferation, cell migration, and the survival after apoptosis-inducing events.

Estrogen receptor interaction with estrogen response elements.

The estrogen receptor (ER) is a ligand-activated enhancer protein that is a member of the steroid/nuclear receptor superfamily. Two genes encode mammalian ER: ERalpha and ERbeta. ER binds to specific DNA sequences called estrogen response elements (EREs) with high affinity and transactivates gene expression in response to estradiol (E(2)). The purpose of this review is to summarize how natural and synthetic variations in the ERE sequence impact the affinity of ER-ERE binding and E(2)-induced transcriptional activity. Surprisingly, although the consensus ERE sequence was delineated in 1989, there are only seven natural EREs for which both ERalpha binding affinity and transcriptional activation have been examined. Even less information is available regarding how variations in ERE sequence impact ERbeta binding and transcriptional activity. Review of data from our own laboratory and those in the literature indicate that ERalpha binding affinity does not relate linearly with E(2)-induced transcriptional activation. We suggest that the reasons for this discord include cellular amounts of coactivators and adaptor proteins that play roles both in ER binding and transcriptional activation; phosphorylation of ER and other proteins involved in transcriptional activation; and sequence-specific and protein-induced alterations in chromatin architecture.

Effects of host hormonal status on binding of activated estrogen receptor to nuclei from R3230AC and 7,12-dimethylbenz[a]anthracene-induced mammary tumors.

The effects of various hormonal perturbations that alter growth of two different rat mammary tumors in vivo were investigated by study of the interactions of [3H]estradiol-charged estrogen receptors ([3H]ER) with tumor nuclei in vitro. Nuclei from the transplantable R3230AC adenocarcinoma were isolated after ovariectomy, estrogen treatment, or progesterone treatment. Saturable specific binding of [3H]ER to nuclei was assayed in this in vivo-like system. Scatchard analysis of [3H]ER-nuclear binding data indicated that these perturbations did not affect affinity, which ranged from Kd 1.0 to 2.4 nM. However, the number of [3H]ER-binding sites/nucleus was altered according to the treatment: intact rats, 94,500 +/- 4,200; ovariectomy, 70,400 +/- 3,200; ovariectomy plus estradiol, 82,100 +/- 5,800; and ovariectomy plus progesterone, 73,900 +/- 2,500. Nuclei from primary tumors induced by 7,12-dimethylbenz(a)anthracene displayed similar affinities for [3H]ER, although these tumors had fewer binding sites per nucleus. Animals bearing 7,12-dimethylbenz(a)-anthracene-induced tumors were either ovariectomized or made diabetic by administration of streptozotocin, perturbations that cause regression of the majority of tumors. The number of [3H]ER binding sites per nucleus, in tumors classified according to growth characteristics in host animals subsequent to hormonal perturbation, was: intact growing 36,300 +/- 3,400; ovex regressing, 15,400 +/- 3,400; ovex, estrogen-treated growing, 28,100 +/- 2,700; diabetic regressing, 19,500 +/- 2,400; diabetic static, 32,100 and diabetic growing, 42,000 +/- 7,100. These results indicate that (a) the number of nuclear ER-binding sites can be reduced by hormonal interventions that cause tumor regression and (b) endogenous ovarian hormones may play a role in regulating nuclear ER binding.

Estrogen receptor binding to nuclei from normal and neoplastic rat mammary tissues in vitro.

To investigate the role of hormones in regulating growth of neoplastic mammary cells, we established a heterologous assay for studying interactions of partially purified calf uterine [3H]estradiol-charged estrogen receptor ([3H]ER) with rat tumor nuclei in vitro. This system displays saturable high affinity binding of [3H]ER which is time and salt dependent. Optimal assay conditions required for the heterologous system were identical to those we reported for the homologous calf nuclear binding system. Specificity of [3H]ER binding was demonstrated; 10-fold excess unlabeled estrogen-charged ER (EcR) competed for greater than 90% of the [3H]ER binding sites and binding of [3H]estradiol (not complexed with ER) was less than 1% of [3H]ER binding. Binding of [3H]ER displayed tissue specificity in decreasing order: R3230AC mammary tumor greater than lactating mammary gland = liver greater than kidney greater than lung. Scatchard analysis of saturation data provided estimates of binding affinity to nuclei from R3230AC mammary tumors [Kd, 2.0 +/- 0.3 (SE) nM); the number of binding sites per nucleus for R3230AC tumors was 95,000 +/- 13,800. [3H]ER binding to nuclei isolated from R3230AC rat mammary tumors grown in intact rats was 40% higher than that observed in tumors from ovariectomized animals. Results of administration of individual pharmacological doses of either progesterone or an estrogen to ovariectomized rats did not restore nuclear ER binding levels in R3230AC tumors to those detected in tumors from intact rats. These results suggest that the physiological levels of endogenous hormones produced by the ovaries are important in regulating the number of ER binding sites in nuclei from these mammary tumors.

Cooperative estrogen receptor interaction with consensus or variant estrogen responsive elements in vitro.

Specific binding of estradiol-liganded, partially purified calf uterine estrogen receptor (ER) to a 38-base pair estrogen responsive element (ERE) consensus sequence, containing the inverted repeat 5'-GGTCAnnnTGACC-3', was measured in vitro. The ERE sites were inserted as single or multiple tandem copies in a plasmid vector [p GEM-7Zf(+)]. Results showed that one dimeric ER can interact with one ERE, and steric constraints do not inhibit binding of ER to adjacent EREs. Molybdate-stabilized monomeric (4S) ER did not bind to EREs. ER bound to single and tandem double EREs with Kd values of 0.24 and 0.23 nM, respectively. When the plasmid contained three or more tandem copies of the ERE, ER bound in a cooperative manner, as indicated by convex Scatchard plots and Hill coefficients greater than 1.5. To determine those characteristics of the consensus sequence that are important for maximal high-affinity ER binding, ten variant ERE oligomer sequences were synthesized and cloned into pGEM-7Zf(+) as single copies or as four copies in tandem. ER binding affinity was maximal for the consensus ERE and was reduced for variants containing one or two nucleotide changes in the inverted repeat. The number of nucleotides separating the inverted repeat in the ERE was critical for high-affinity ER binding. Certain sequence-variant EREs when cloned as single copies bound less ER compared to the consensus ERE, yet when cloned as four tandem copies, ER binding displayed cooperativity by Scatchard and Hill analyses. Results demonstrate that cooperative interactions noted in vivo by others are present when measured in vitro. Results strongly imply that the number, spacing, and nucleotide sequence of EREs could precisely control the amount of ER binding to estrogen-responsive genes.

Regulation of estrogenic and nuclear factor kappa B functions by polyamines and their role in polyamine analog-induced apoptosis of breast cancer cells.

The natural polyamines -putrescine, spermidine, and spermine- are essential for cell growth and differentiation. Polyamines are involved in several gene regulatory functions, although their mechanism(s) of action has not been elucidated. We investigated the role of polyamines in the function of NF-kappa B and estrogen receptor-alpha (ER alpha), two transcription factors implicated in breast cancer cell proliferation and cell survival, using MCF-7 breast cancer cells. We found that spermine facilitated the binding of ER alpha and NF-kappa B to estrogen response element (ERE)- and NF-kappa B response element (NRE), respectively, and enhanced ER alpha-mediated transcriptional activation in transient transfection experiments. We also found that the association of the co-regulatory protein CBP/p300 with ER alpha and NF-kappa B was increased by spermine treatment of MCF-7 cells. Spermine also increased the nuclear translocation of NF-kappa B compared to the control. In contrast, treatment of MCF-7 cells with polyamine analogs, BE-3-4-3 and BE-3-3-3, resulted in transcriptional inhibition of both ERE- and NRE-driven reporter plasmids. In addition, polyamine analogs inhibited the association of ER alpha and NF-kappa B with CBP/p300 and were unable to facilitate nuclear translocation of NF-kappa B. APO-BRDU assay demonstrated that polyamine analogs induced apoptosis, with a loss of the anti-apoptotic protein Bcl-2. These data show a gene regulatory function of polyamines involving transcriptional activation of ER alpha and NF-kappa B, potentially leading to the up-regulation of genes involved in breast cancer cell proliferation. Our results with BE-3-4-3 and BE-3-3-3 suggest that down-regulation of ER alpha- and NF-kappa B-regulated genes is a possible mechanism for the action of polyamine analogs in inducing apoptosis of breast cancer cells.

Intranuclear dynamics of DNA polymerase alpha differs between the transplanted R3230AC mammary adenocarcinomas and the host mammary gland depending on lactation cycle.

DNA polymerase alpha activity was markedly higher in all nuclear subfractions, including nuclear matrix, from transplanted R3230AC mammary adenocarcinomas than in the analogous fractions from mammary gland of same tumor-bearing pregnant or lactating rats. Changes in host lactational status had no significant effect on subnuclear distribution of tumor DNA polymerase alpha activity, with the majority (60-75%) localized in soluble nucleoplasm and a significant amount (13-20%) retained in the nuclear matrix. In the host mammary gland, nuclear matrix-bound DNA polymerase alpha was highest, accounting for 48% of total nuclear activity, during late pregnancy when mammary cells undergo rapid raplication. During lactation, when cells in mammary gland cease to divide, only 8% of enzyme activity was in the nuclear matrix, while the majority (60-80%) of DNA polymerase alpha activity was localized in nucleoplasm. In both R3230AC tumor and mammary gland regardless of host's lactational status, the majority (60-80%) of DNA polymerase beta activity was localized in the high salt-soluble chromatin. These present data thus suggest that, regardless of host lactational status, R3230AC tumor has many cycling cells, each with a large pool of DNA polymerase alpha molecules maintaining maximal and constant replicative activity, while normal mammary gland cells have a smaller pool of DNA polymerase alpha which become primarily matrix-bound only during active cell replication during late pregnancy. A constant localization of nuclear DNA polymerase beta in chromatin in both mammary gland and the tumor suggest it is not important in mammary cell proliferation.

A microtiter well assay for quantitative measurement of estrogen receptor binding to estrogen-responsive elements.

Reproducible, rapid measurement of estrogen receptor (ER) binding to DNA was accomplished in microtiter wells treated so that ER-DNA complexes or DNA bound in preference to free ER. Mixtures of 35S-labeled DNA and [3H]estrogen-charged ER ([3H]ER), incubated to equilibrium in microfuge tubes, were transferred to microtiter wells previously treated with histone followed by gelatin. After binding of the DNA or ER-DNA complex to the treated wells, free ER was removed by washing. Radioactivity retained in each well was measured by placing individual wells from snap-apart microtiter plates directly in scintillation fluid. Binding of DNA was saturable, and ER-DNA complex binding was complete within 2 h at 4 C. The use of 35S-labeled DNA and [3H]ER allowed stoichiometric determination of ER bound to DNA. The amount of ER specifically bound to a consensus estrogen-responsive element (ERE) containing the inverted repeat GGTCAgagTGACC was determined by comparing ER bound to plasmid containing or lacking the ERE. At saturating concentrations of ER, plasmids bearing one, two, and four EREs in tandem bound approximately one, two, and four dimeric ER molecules, respectively. Scatchard analysis of saturation binding data revealed a Kd of 0.15 nM for specific ER binding to a single ERE site. Thus, the assay detects ER retaining both DNA-binding and estrogen-binding functions. ER complexed with DNA in the well was also detected using a monoclonal antibody specific for the receptor. Simple modifications of this method would allow study of other DNA-protein interactions.

Effects of multiple estrogen responsive elements, their spacing, and location on estrogen response of reporter genes.

Most highly estrogen-responsive genes possess multiple estrogen-responsive elements (EREs) that act synergistically to activate expression. Synergism between EREs appears to depend on structural features of the EREs and the promoter. To examine the activation process, we cloned single or multiple tandem copies of the consensus ERE into reporter plasmids. These plasmids contained either a chloramphenicol acetyl transferase reporter gene driven by a minimal promoter or a luciferase reporter gene driven by the Simian virus 40 (SV40) promoter. Using MCF-7 human breast cancer cells, we demonstrate that synergism among EREs depends on the number of EREs, their spacing, and the distance of the EREs from the promoter. The induction capacity of EREs falls off slowly with distance from the promoter. Remarkably, multiple EREs can induce effectively and synergize even when they are located more than 2000 nucleotides from the promoter. For EREs located immediately upstream of the promoter, both the distance separating the EREs and the distance to the promoter have to be optimal for synergy. Altering either distance changes the response from synergistic to additive. For distant EREs, presumed to interact by a looping mechanism at the promoter, the length of DNA between the EREs and the promoter is not critical. Synergy among closely spaced EREs that are far from the promoter only requires an optimal distance separating the ERE centers of symmetry. Interestingly, very widely separated EREs can also synergize, presumably also because of their ability to interact by looping. The estrogen response from single or multiple tandem copies of ERE half-palindromes near the SV40 promoter was also tested. The negligible induction capacity of a single half-site was not significantly increased in multiple sites. The biological role of half-EREs is not apparent in the system employed here.

Resveratrol acts as a mixed agonist/antagonist for estrogen receptors alpha and beta.

Epidemiological evidence indicates that phytoestrogens inhibit cancer formation and growth, reduce cholesterol levels, and show benefits in treating osteoporosis. At least some of these activities are mediated through the interaction of phytoestrogens with estrogen receptors alpha and beta (ERalpha and ERbeta). Resveratrol, trans-3,5,4'-trihydroxystilbene, is a phytoestrogen in grapes that is present in red wine. Resveratrol was shown to bind ER in cytosolic extracts from MCF-7 and rat uteri. However, the contribution of ERalpha vs. ERbeta in this binding is unknown. Here we report that resveratrol binds ERbeta and ERalpha with comparable affinity, but with 7,000-fold lower affinity than estradiol (E2). Thus, resveratrol differs from other phytoestrogens that bind ERbeta with higher affinity than ERalpha. Resveratrol acts as an estrogen agonist and stimulates ERE-driven reporter gene activity in CHO-K1 cells expressing either ERalpha or ERbeta. The estrogen agonist activity of resveratrol depends on the ERE sequence and the type of ER. Resveratrol-liganded ERbeta has higher transcriptional activity than E2-liganded ERbeta at a single palindromic ERE. This indicates that those tissues that uniquely express ERbeta or that express higher levels of ERbeta than ERalpha may be more sensitive to resveratrol's estrogen agonist activity. For the natural, imperfect EREs from the human c-fos, pS2, and progesterone receptor (PR) genes, resveratrol shows activity comparable to that induced by E2. We report that resveratrol exhibits E2 antagonist activity for ERalpha with select EREs. In contrast, resveratrol shows no E2 antagonist activity with ERbeta. These data indicate that resveratrol differentially affects the transcriptional activity of ERalpha and ERbeta in an ERE sequence-dependent manner.

Characterization of a cytosolic inhibitor of calf estrogen receptor binding to nuclei.

An inhibitory component that diminishes estrogen receptor (ER) binding to nuclei in vitro is present in cytosol prepared from calf uterus. The inhibitor is heat stable and resistant to enzymatic treatment with trypsin, chymotrypsin, proteinase K, deoxyribonuclease I, or ribonucleases A, T1, and U2. Results of chromatography on DEAE-cellulose and Sephadex G-150 indicate that the factor is a negatively charged macromolecule. Inhibitory activity is sensitive to sequential digestion with chondroitinase ABC, hyaluronidase, and heparinase. Approximately 70% of the inhibitory activity is destroyed by treatment with heparinase alone. Heparitinase destroys only 30% of this activity. Furthermore, the addition of pure hyaluronic acid or chondroitin sulfate to the ER-nuclei binding assay results in little inhibition, whereas addition of heparin inhibits 75% of receptor binding. Overall, these results indicate that glycosaminoglycans, present in bovine uterine cytosol, are capable of inhibiting ER-nuclei interactions. The most potent inhibitory glycosaminoglycan displays heparin-like characteristics.

Estrogen response element binding induces alterations in estrogen receptor-alpha conformation as revealed by susceptibility to partial proteolysis.

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.

Estrogen response element-dependent regulation of transcriptional activation of estrogen receptors alpha and beta by coactivators and corepressors.

One mechanism by which ligand-activated estrogen receptors alpha and beta (ERalpha and ERbeta) stimulate gene transcription is through direct ER interaction with specific DNA sequences, estrogen response elements (EREs). ERE-bound ER recruits coactivators that stimulate gene transcription. Binding of ER to natural and synthetic EREs with different nucleotide sequences alters ER binding affinity, conformation, and transcriptional activity, indicating that the ERE sequence is an allosteric effector of ER action. Here we tested the hypothesis that alterations in ER conformation induced by binding to different ERE sequences modulates ER interaction with coactivators and corepressors. CHO-K1 cells transfected with ERalpha or ERbeta show ERE sequence-dependent differences in the functional interaction of ERalpha and ERbeta with coactivators steroid receptor coactivator 1 (SRC-1), SRC-2 (glucocorticoid receptor interacting protein 1 (GRIP1)), SRC-3 amplified in breast cancer 1 (AIB1) and ACTR, cyclic AMP binding protein (CBP), and steroid receptor RNA activator (SRA), corepressors nuclear receptor co-repressor (NCoR) and silencing mediator for retinoid and thyroid hormone receptors (SMRT), and secondary coactivators coactivator associated arginine methyltransferase 1 (CARM1) and protein arginine methyltransferase 1 (PRMT1). We note both ligand-independent as well estradiol- and 4-hydroxytamoxifen-dependent differences in ER-coregulator activity. In vitro ER-ERE binding assays using receptor interaction domains of these coregulators failed to recapitulate the cell-based results, substantiating the importance of the full-length proteins in regulating ER activity. These data demonstrated that the ERE sequence impacts estradiol-and 4-hydroxytamoxifen-occupied ERalpha and ERbeta interaction with coregulators as measured by transcriptional activity in mammalian cells.

Response element sequence modulates estrogen receptor alpha and beta affinity and activity.

The relationship between estrogen receptor (ER)-estrogen response element (ERE) binding affinity and estradiol (E(2))-induced transcription has not been systematically or quantitatively tested. We examined the influence of ERE palindrome length and the 3' ERE flanking sequence on ERalpha and ERbeta affinity binding in vitro and on the induction of reporter gene activity in transfected cells. The addition of one nucleotide in each arm of the 13 bp ERE palindrome, forming a 15 bp ERE palindrome, increased ERalpha and ERbeta affinity and transcription. In contrast, the addition of an AT-rich flanking sequence from genes highly stimulated by E(2) had little effect on affinity or reporter gene activity. Notable differences between ERalpha and ERbeta include: both K(d) and transcriptional induction were generally higher for ERalpha than ERbeta, better correlation between ERE palindrome length and transcriptional induction for ERalpha than ERbeta, and a better correlation between (ER-ERE)K(d) and transcriptional induction for ERalpha than for ERbeta.

Regulation of cell cycle and cyclins by 16alpha-hydroxyestrone in MCF-7 breast cancer cells.

It has been suggested that alterations in estradiol (E(2)) metabolism, resulting in increased production of 16alpha-hydroxyestrone (16alpha-OHE(1)), is associated with an increased risk of breast cancer. In the present study, we examined the effects of 16alpha-OHE(1)on DNA synthesis, cell cycle progression, and the expression of cell cycle regulatory genes in MCF-7 breast cancer cells. G(1) synchronized cells were treated with 1 to 25 nM 16alpha-OHE(1) for 24 and 48 h. [(3)H]Thymidine incorporation assay showed that 16alpha-OHE(1) caused an 8-fold increase in DNA synthesis compared with that of control cells, whereas E(2) caused a 4-fold increase. Flow cytometric analysis of cell cycle progression also demonstrated the potency of 16alpha-OHE(1) in stimulating cell growth. When G(1) synchronized cells were treated with 10 nM 16alpha-OHE(1) for 24 h, 62+/-3% of cells were in S phase compared with 14+/-3% and 52+/-2% of cells in the control and E(2)-treated groups respectively. In order to explore the role of 16alpha-OHE(1) in cell cycle regulation, we examined its effects on cyclins (D1, E, A, B1), cyclin dependent kinases (Cdk4, Cdk2), and retinoblastoma protein (pRB) using Western and Northern blot analysis. Treatment of cells with 10 nM 16alpha-OHE(1) resulted in 4- and 3-fold increases in cyclin D1 and cyclin A, respectively, at the protein level. There was also a significant increase in pRB phosphorylation and Cdk2 activation. In addition, transient transfection assay using an estrogen response element-driven luciferase reporter vector showed a 15-fold increase in estrogen receptor-mediated transactivation compared with control. These results show that 16alpha-OHE(1) is a potent estrogen capable of accelerating cell cycle kinetics and stimulating the expression of cell cycle regulatory proteins.

Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells.

We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 microM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells.

Estrogen receptor binding to estrogen response elements slows ligand dissociation and synergistically activates reporter gene expression.

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.

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.

Comparison of transcriptional synergy of estrogen receptors alpha and beta from multiple tandem estrogen response elements.

Estrogen receptors alpha and beta (ERalpha and ERbeta) act as ligand-dependent transcriptional enhancers. We reported that ERalpha induces synergistic activation of luciferase reporter gene activity in response to E(2) from three or four tandem copies of a consensus estrogen response element (ERE) in transiently transfected MCF-7 cells. Here we addressed three questions: (1) is the synergistic activation of reporter gene activity from multiple tandem EREs by ERalpha restricted to MCF-7 cells?; (2) does ERbeta induce synergistic activation of reporter activity from multiple tandem EREs?; and (3) does ERbeta bind cooperatively to multiple tandem EREs? To address the first two questions, ER-negative CHO-K1 cells were co-transfected with ERalpha or ERbeta and ERE-driven reporter plasmids. Both ERalpha and ERbeta activated ERE-driven luciferase gene activity in an estradiol-dependent manner. Induction by ERbeta was lower than ERalpha from each ERE. We demonstrate that both ERalpha and ERbeta induce transcriptional synergy with three or four, but not two, tandem copies of an ERE. Electrophoretic mobility shift assays (EMSA) indicated an increase in ER-ERE binding affinity associated with cooperative binding of ERalpha and ERbeta to multiple EREs that may be responsible for transcriptional synergy in transiently transfected cells. We also postulate that interaction of ERalpha and ERbeta with coactivators may also play a role in transcriptional synergy.

Comparison of tamoxifen ligands on estrogen receptor interaction with estrogen response elements.

The estrogen receptor (ER) is a ligand-activated transcription factor that binds to specific DNA sequences, estrogen response elements (EREs). Estradiol-liganded ER (E2-ER) binds cooperatively to stereoaligned EREs that are surrounded by naturally-occurring AT-rich sequences with a stoichiometry of one E2-ER dimer per ERE. When ER is bound by 4-hydroxytamoxifen (4-OHT), the active metabolite of the widely used therapeutic antiestrogen tamoxifen (TAM), the receptor binds to EREs with high affinity. However, one molecule of 4-OHT ligand dissociates from the ER dimer apparently during the process of binding to DNA, yielding a stoichiometry of one [3H]4-OHT molecule per ERE. To determine whether DNA-binding induced ligand dissociation is a general property of type I antiestrogens that are not covalently attached to the ER, we examined the interaction of ER liganded by tamoxifen (TAM) with EREs. We demonstrate that TAM-ER binds EREs with lower affinity than E2-ER, 4-OHT-ER, or ER liganded by the covalent antiestrogen tamoxifen aziridine. Unlike E2-ER, both TAM and 4-OHT-ER bind EREs non-cooperatively. Like 4-OHT, TAM appears to dissociate from the liganded ER as the receptor binds EREs. Additionally, partial proteolysis of ERE-bound ER by trypsin revealed different cleavage patterns for E2 versus 4-OHT and TAM. These findings indicate that the behavior of the ER liganded by TAM is generally similar to that of the antiestrogen 4-OHT.