Estrogen-responsive Promoter Research Articles

The genomic regulatory elements for estrogen receptor alpha transactivation-function-1 regulated genes.

Estrogen receptor alpha (ERα) is a ligand-dependent transcription regulator, containing two transactivation functional domains, AF-1 and AF-2. The selective estrogen receptor modulators (SERMs), including 4-hydroxytamoxifen (4OHT), activate AF-1 preferentially rather than AF-2. However, it is unclear whether this specific function is related to the tissue-selective functionality of SERMs. Moreover, there is no information determining AF-1-dependent estrogenic-genes existing in tissues. We sought to identify AF-1-dependent estrogenic-genes using the AF-2 mutated knock-in (KI) mouse model, AF2ERKI. AF2ER is an AF-2 disrupted estradiol (E2)-insensitive mutant ERα, but AF-1-dependent transcription can be activated by the estrogen-antagonists, fulvestrant (ICI) and 4OHT. Gene profiling and ChIP-Seq analysis identified Klk1b21 as an ICI-inducible gene in AF2ERKI uterus. The regulatory activity was analyzed further using a cell-based reporter assay. The 5'-flanking 0.4kbp region of Klk1b21 gene responded as an ERα AF-1-dependent estrogen-responsive promoter. The 150bp minimum ERα binding element (EBE) consists of three direct repeats. These three half-site sequences were essential for the ERα-dependent transactivation and were differentially recognized by E2 and 4OHT for the gene activation. This response was impaired when the minimum EBE was fused with a thymidine-kinase promoter but could be restored by fusion with the 100bp minimum transcription initiation element (TIE) of Klk1b21, suggesting that the cooperative function of EBE and TIE is essential for mediating AF-1-dependent transactivation. These findings provide the first in vivo evidence that endogenous ERα AF-1 dominant estrogenic-genes exist in estrogen-responsive organs. Such findings will aid in understanding the mechanism of ERα-dependent tissue-selective activity of SERMs.

A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning

Programmable transcriptional regulation is a powerful tool to study gene functions. Current methods to selectively regulate target genes are mainly based on promoter exchange or on overexpressing transcriptional activators. To expand the discovery toolbox, we designed a dCas9-based RNA-guided synthetic transcription activation system for Aspergillus nidulans that uses enzymatically disabled “dead” Cas9 fused to three consecutive activation domains (VPR-dCas9). The dCas9-encoding gene is under the control of an estrogen-responsive promoter to allow induction timing and to avoid possible negative effects by strong constitutive expression of the highly active VPR domains. Especially in silent genomic regions, facultative heterochromatin and strictly positioned nucleosomes can constitute a relevant obstacle to the transcriptional machinery. To avoid this negative impact and to facilitate optimal positioning of RNA-guided VPR-dCas9 to targeted promoters, we have created a genome-wide nucleosome map from actively growing cells and stationary cultures to identify the cognate nucleosome-free regions (NFRs). Based on these maps, different single-guide RNAs (sgRNAs) were designed and tested for their targeting and activation potential. Our results demonstrate that the system can be used to regulate several genes in parallel and, depending on the VPR-dCas9 positioning, expression can be pushed to very high levels. We have used the system to turn on individual genes within two different biosynthetic gene clusters (BGCs) which are silent under normal growth conditions. This method also opens opportunities to stepwise activate individual genes in a cluster to decipher the correlated biosynthetic pathway.Graphical abstractKeypoints• An inducible RNA-guided transcriptional regulator based on VPR-dCas9 was established in Aspergillus nidulans.• Genome-wide nucleosome positioning maps were created that facilitate sgRNA positioning.• The system was successfully applied to activate genes within two silent biosynthetic gene clusters.

Mitochondria-targeted triphenylphosphonium-based compounds do not affect estrogen receptor α.

BackgroundTargeting negatively charged mitochondria is often achieved using triphenylphosphonium (TPP) cations. These cationic vehicles may possess biological activity, and a docking study indicates that TPP-moieties may act as modulators of signaling through the estrogen receptor α (ERα). Moreover, in vivo and in vitro experiments revealed the estrogen-like effects of TPP-based compounds. Here, we tested the hypothesis that TPP-based compounds regulate the activity of ERα.MethodsWe used ERa-positive and ERα-negative human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231, respectively). Cell proliferation was measured using a resazurin cell growth assay and a real-time cell analyzer assay. Cell cycle progression was analyzed using flow cytometry. Real-time PCR was used to assess mRNA expression of endogenous estrogen-responsive genes. Luciferase activity was measured to evaluate transcription driven by estrogen-responsive promoters in cells transfected with an estrogen response element (ERE)3-luciferase expression vector.ResultsThe TPP-based molecules SkQ1 and C12TPP, as well as the rhodamine-based SkQR1, did not increase the proliferation or alter the cell cycle progression of MCF-7 cells. In contrast, 17β estradiol increased the proliferation of MCF-7 cells and the proportion of cells in the S/G2/M-phases of the cell cycle. TPP-based compounds did not affect the induction of transcription of an ERE-luciferase expression vector in vitro, and SkQ1 did not alter the levels of expression of estrogen-dependent genes encoding GREB1, TFF1, COX6, and IGFBP4.ConclusionTPP-based compounds do not possess properties typical of ERα agonists.

ERβ compensates for the absence of ERα function to promote osteoblast viability by inhibition of SOST signaling.

Estrogen receptors α and β (ERα and ERβ) serve key functions in bone development and maintenance, and in the metabolism of bone mineral. ERβ and ERα form heterodimers, and ERβ negatively regulates the transactivation of ERα. ERβ also inhibits recruitment of ERα to the estrogen-responsive promoters. However, the relationship of ERα and ERβ in the regulation of osteoblast viability and differentiation remains unclear. The present study aimed to investigate whether ERβ plays a role in balancing ERα activity in osteoblast cells. Downregulation of ERα by short hairpin RNA (shRNA) was found to significantly increase cell cycle arrest at G1 phase (P<0.01). In addition, this effect was found to be significantly enhanced by downregulation of ERβ (P<0.05). Inversely, ERα-knocked down osteoblasts were treated with ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) to activate ERβ. It was found that activation of ERβ significantly rescued the arrest of cell cycle induced by the downregulation of ERα (P<0.05). Furthermore, downregulation of ERα was found to significantly inhibit cell viability (P<0.01), and knockdown of ERβ was found to have a significant synergic effect with ERα downregulation on the inhibition of cell viability (P<0.01). Treatment with ERβ agonist DPN significantly rescued the effects of downregulation of ERα on cell viability (P<0.01). It was also demonstrated that the synergic effects of ERα and ERβ deletion was via upregulation of SOST gene expression, and the subsequent inhibition of OPG and Runx2 gene expression. Thus, ERβ may serve a function in balancing osteoblast viability and differentiation induced by ERα.

Adaptive Significance of ERα Splice Variants in Killifish (Fundulus heteroclitus) Resident in an Estrogenic Environment.

The possibility that chronic, multigenerational exposure to environmental estrogens selects for adaptive hormone-response phenotypes is a critical unanswered question. Embryos/larvae of killifish from an estrogenic-polluted environment (New Bedford Harbor, MA [NBH]) compared with those from a reference site overexpress estrogen receptor alpha (ERα) mRNA but are hyporesponsive to estradiol. Analysis of ERα mRNAs in the two populations revealed differences in splicing of the gene encoding ERα (esr1). Here we tested the transactivation functions of four differentially expressed ERα mRNAs and tracked their association with the hyporesponsive phenotype for three generations after transfer of NBH parents to a clean environment. Deletion variants ERαΔ6 and ERαΔ6-8 were specific to NBH killifish, had dominant negative functions in an in vitro reporter assay, and were heritable. Morpholino-mediated induction of ERαΔ6 mRNA in zebrafish embryos verified its role as a dominant negative ER on natural estrogen-responsive promoters. Alternate long (ERαL) and short (ERαS) 5'-variants were similar transcriptionally but differed in estrogen responsiveness (ERαS ≫ ERαL). ERαS accounted for high total ERα expression in first generation (F1) NBH embryos/larvae but this trait was abolished by transfer to clean water. By contrast, the hyporesponsive phenotype of F1 NBH embryos/larvae persisted after long-term laboratory holding but reverted to a normal or hyper-responsive phenotype after two or three generations, suggesting the acquisition of physiological or biochemical traits that compensate for ongoing expression of negative-acting ERαΔ6 and ERαΔ6-8 isoforms. We conclude that a heritable change in the pattern of alternative splicing of ERα pre-mRNA is part of a genetic adaptive response to estrogens in a polluted environment.

Abstract P3-05-11: The evolution of the estrogen receptor (ER) complex conformation governs estrogen-induced apoptosis in antihormone-resistant breast cancer cells

Abstract Over the past decade new insights have been gained into the acquired resistance to tamoxifen and the Aromatase Inhibitors (AIs) with the discovery of the new biology of estrogen-induced apoptosis. However, it has also been learned that estrogens can be classified into planar class I and angular class II estrogens. Using model systems of long-term estrogen-deprived breast cancer cells in vitro (MCF-7:5C), it was previously shown that class I estrogens cause immediate apoptosis over a 3-4 day period. Paradoxically, class II estrogens actually block apoptosis caused by planar class I estrogens. To gain insight into this paradox we have successfully crystallized an angular class II triphenylethylene (TPE) estrogen bound to the ligand binding domain (LBD) of the ER and derived a new conformation for the TPE:ER complex (code 3Q97 in the PDB). Surprisingly, Helix 12 seals the LBD with the class II estrogen, but not the same conformation is observed with the planar class I estrogen 17β-estradiol (E2). There would seem to be no reason why the 3Q97 complex would not cause immediate apoptosis. To address this issue we have used Western blot analysis for protein and qRT-PCR for mRNA levels for the ER. ER parameters were monitored for up to 72 hours and results compared and contrasted between E2, the Class II estrogens, 4-hydroxytamoxifen (4OHT) and endoxifen (Endox). ER protein and mRNA levels with 4OHT or Endox accumulated and remained high throughout the study period. In contrast, the planar estrogen E2 produced a rapid decline in the protein and mRNA levels for the ER complex. The angular class II estrogens initially produced an accumulation of the ER protein complex, which decreased with time. Using a chromatin immunoprecipitation (ChIP) technique we demonstrated that the class II estrogens recruit only half of the ER to the estrogen-responsive genes promoters (TFF1 And BREB1) and less than half co-activator binding compared to E2. The TPEs were only partial agonists compared to planar estrogen. These results explain why the Class II estrogens induce delayed apoptosis. We conclude that, for the first time, we have observed the binding of a ligand to a receptor that changes conformation against time and evolves from an antagonist to an agonist conformation to trigger apoptosis. These observations have current relevance to the decryption of the protective effects of estrogen alone therapy against breast cancer incidence in the Women's Health Initiative (WHI) trial. This work was supported by the Susan G. Komen for the Cure Foundation award SAC100009. Citation Format: Maximov PY, Sengupta S, Fernandes DJ, Fan P, Curpan RF, Rajan SS, Greene GL, Jordan VC. The evolution of the estrogen receptor (ER) complex conformation governs estrogen-induced apoptosis in antihormone-resistant breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-11.

Human C-terminally truncated ERα variants resulting from the use of alternative exons in the ligand-binding domain

The nuclear receptor genes contain alternative internal and terminal exons, with alternative exon incorporation yielding mRNA variants that encode various receptor types, including some with C-terminal truncation that exhibit constitutive activation or dominant-negative transcriptional transactivation. However, C-terminally truncated estrogen receptor α (ERα) variants with alternative sequences have rarely been reported in humans. Therefore, we assessed human ERα genomic organization and alternative splicing profiles, and identified both alternative exons and C-terminally truncated ERα variants. These naturally occurring C-terminally truncated ERα proteins were localized in the nuclei of transfected cells. In addition, ERαi45c and ERαΔ5 variants exhibited constitutive transactivation of an estrogen responsive element-driven promoter in transfected cells. We manufactured expression vectors encoding artificially truncated ERα constructs and evaluated their transactivation abilities to establish mechanisms determining the constitutive activity and dominant-negative properties of truncated variants. Lack of the region encoded in exon 8 eliminated basal and ligand-induced transcriptional transactivation. The C-terminally truncated ERα variants/constructs containing the helices 5 in their ligand-binding domains did not exhibit constitutive transactivation. Furthermore, we demonstrated that truncation from C-termini to helices 5 in the variant ligand-binding domains was required for constitutive activation and found that the remnant regions of the ligand-binding domains and variant-specific sequences influenced transcriptional transactivation efficiency. In conclusion, we elucidated the structural and functional features of novel C-terminally truncated ERα variants and revealed the mechanisms underlying constitutive transactivation by C-terminally truncated nuclear receptor variants.

Oncogenic MUC1-C Promotes Tamoxifen Resistance in Human Breast Cancer

Tamoxifen resistance of estrogen receptor-positive (ER+) breast cancer cells has been linked in part to activation of receptor tyrosine kinases, such as HER2, and the PI3K-AKT pathway. Mucin 1 (MUC1) is aberrantly overexpressed in about 90% of human breast cancers, and the oncogenic MUC1-C subunit is associated with ERα. The present studies using HER2 overexpressing BT-474 breast cancer cells, which are constitutively resistant to tamoxifen, demonstrate that silencing MUC1-C is associated with (i) downregulation of p-HER2 and (ii) sensitivity to tamoxifen-induced growth inhibition and loss of clonogenic survival. In contrast, overexpression of MUC1-C in tamoxifen-sensitive MCF-7 breast cancer cells resulted in upregulation of p-AKT and tamoxifen resistance. We show that MUC1-C forms complexes with ERα on the estrogen-responsive promoter of Rab31 and that MUC1-C blocks tamoxifen-induced decreases in ERα occupancy. MUC1-C also attenuated tamoxifen-induced decreases in (i) recruitment of the coactivator CREB binding protein, (ii) Rab31 promoter activation, and (iii) Rab31 mRNA and protein levels. The importance of MUC1-C is further supported by the demonstration that targeting MUC1-C with the cell-penetrating peptide inhibitor, GO-203, sensitized tamoxifen-resistant cells to tamoxifen treatment. Moreover, we show that targeting MUC1-C in combination with tamoxifen is highly synergistic in the treatment of tamoxifen-resistant breast cancer cells. Combined, these findings indicate that MUC1-C contributes to tamoxifen resistance.

Abstract B66: Characterization of bis-phenazine compounds that inhibit estrogen signaling through direct interaction with estrogen response elements

Abstract Hormone therapies interfere with estrogen signaling by targeting Estrogen Receptors (ERs) and are routinely used for the prevention and treatment of ER-expressing breast cancers. However, current therapies have significant limitations related to inherent or acquired resistance. Thus, there is a need to develop therapeutic compounds that do not depend on competitive binding to the ER or on depleting cells of endogenous estrogen. Previous data suggested that certain bis-phenazines, such as our lead compound XR5944, possess potent antiestrogenic properties through a novel mechanism of action: they inhibit gene transcription via direct intercalation with DNA at estrogen response elements (EREs) which are located in estrogen-responsive gene promoters. While these studies have demonstrated the potent antiestrogenic activity of XR5944, they have also demonstrated its capacity to bind to alternative DNA motifs. In order to improve upon ERE target specificity, we have identified XR5944 derivatives that retain potent antiestrogenic properties but may have greater specificity for ERE sequences, to be developed as new therapies for the treatment of estrogen-driven cancers. Such compounds may also be effective in overcoming resistance to current antiestrogen therapies. Here, we characterize DNA sequence interactions and transcriptional effects of five XR5944 derivatives that differ from the parent compound in the composition of their linker chains (compounds 26357 and 26373), cyclic side rings (26576), or both (26536 and 27402). We utilized 1D 1H NMR titration experiments to monitor compound:DNA binding properties to a canonical ERE (AGGTCAcggTGACCT) containing a CGG trinucleotide spacer (ERECGG), electrophoretic mobility shift assays (EMSA) to assess the ability of the derivatives to disrupt ER:ERECGG interactions, and luciferase reporter assays to evaluate their potency in blocking estrogen-dependent transactivation through ERECGG. Our NMR results indicated that alteration of either linker chain or side ring structures of XR5944 reduced the affinity and specificity of compound:ERECGG interactions. These findings correlated with the ability of the compounds to inhibit ER:ERECGG binding in the order of XR5944&amp;gt;26576&amp;gt;26536&amp;gt;26373&amp;gt;26357&amp;gt;27402, as evaluated by EMSA. To further determine the effects of the compounds on ERECGG-driven transactivation, we performed luciferase reporter assays utilizing ERα-expressing MCF-7 breast cancer cells transfected with an ERECGG-luciferase reporter construct. Unlike the EMSA results, there was not a strict concordance between the compound-dependent potency in inhibiting estrogen-driven transcriptional responses and the specificity of interaction with DNA as determined by NMR. Specifically, inhibition of reporter activity was more potent with compounds that had the same cyclic side rings but altered linker chains as XR5944 (26373 and 26357) and was less effective with compounds possessing altered side rings (26576, 26536, and 27402). Together, our findings indicate that the cyclic side chain structures appear to be the most important components of bis-phenazines in disrupting the transcriptional potential of ER:ERE interactions. NMR can be useful for predicting the relative potency of XR5944 derivatives in blocking ER binding to EREs in vitro, but fails to predict their efficacy in inhibiting ERE-mediated transactivation. This information should prove valuable for the rational drug design of new antiestrogenic compounds with improved specificity for ERE sequences. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B66.

Differential Sensitivity to Prepubertal Xenoestrogen Disruption in C57 and CD10 Mice.

Mice are routinely used as model organisms to study the effects of endocrine disrupting chemicals. Different strains of mice differ in their sensitivity to endocrine disruption and the difference in sensitivity may be life stage and or tissue dependent. Previous work has shown that C57BL/6 (C57) and CD-1 mouse strains have differential sensitivity to prepubertal xenoestrogen disruption of the testis. In order to identify mechanisms of differential strain sensitivity to prepubertal xenoestrogen exposure C57 or CD10 mice were backcrossed > seven times to Estrogen Receptor action INdicator (ERIN) mice that contain a transgene with an estrogen responsive promoter linked to the reporter gene beta-galactosidase. At weaning mice were group housed and exposed to ethinyl estradiol [(EE2) 1, 10, or 100 micro-g/kg body weight)] via their drinking water (vehicle control = 0.1% ethanol). Mice were maintained on treated drinking water until collection at 48 days of age. Body, testes, preputial gland, seminal vesicle, and coagulating gland weights were obtained at necropsy. The presence of preputial separation, which is a marker of puberty, and semen in the seminal vesicles was also noted at necropsy. We confirmed that the backcrossed ERIN mice were very similar to their respective wildtype parent strains and exhibited the same differential sensitivity to prepubertal xenoestrogen exposure. Overall, the C57 mice were more sensitive to prepubertal xenoestrogen disruption of the male reproductive tract. The effect on testis weight and preputial separation were the most striking, where C57 mice were over 33-times more sensitive than CD10 mice (IC50 was not reached in CD10 mice). Testes from C57 mice exposed to 1, 10, or 100 micro-g/kg EE2 were 12%, 79%, and 89% smaller, respectively, than those from vehicle exposed mice, whereas testes from CD10 mice were 3%, 11% and 13% smaller than those from vehicle exposed mice. The differential sensitivity of preputial separation was striking at the dose of 10 micro-g/kg EE2, where none of the C57 mice displayed preputial separation while all of the CD10 mice did. Even at the highest EE2 dose, 80% of the CD10 mice still showed preputial separation. This indicates that C57 mice were over 33-times more sensitive to inhibition of puberty than CD10 mice. The preputial glands, seminal vesicles, and coagulating glands decreased in weight following prepubertal xenoestrogen exposure. These tissues were 8-11 times more sensitive to disruption by EE2 in C57 mice than CD10 mice. Preputial gland weight was the most sensitive marker for prepubertal xenoestrogen disruption (IC50 of 1 and 10 micro-g/kg for C57 and CD10 mice, respectively). In summary, C57 mice were much more sensitive to testis disruption by prepubertal xenoestrogen exposure than CD10 mice. The differential sensitivity to estrogen disruption between C57 and CD10 mice, appears to be life stage dependent, however, as we have not found differences in sensitivity to disruption from adult xenoestrogen exposures. Furthermore, the ERIN transgenic mice backcrosses responded very similarly to wildtype mice following prepubertal EE2 exposure and will be used to examine the mechanisms of differential prepubertal disruption by xenoestrogens. (poster)

E6-AP facilitates efficient transcription at estrogen responsive promoters through recruitment of chromatin modifiers

E6-AP is a known coactivator of the estrogen receptor alpha (ERα), however the coactivation mechanism of E6-AP is not clear. This work was undertaken to elucidate the coactivation mechanism of E6-AP. In order to examine the role of E6-AP in ERα signaling, we knocked-down the expression of E6-AP and examined the transactivation functions of ERα. Knockdown of E6-AP showed reduced mRNA production of the ERα target genes pS2 and GREB1 suggesting that E6-AP is required for their proper transcription facilitated by ERα. In order to study the mechanism(s) by which E6-AP regulates the transcriptional functions of ERα, we performed chromatin immunoprecipitation (ChIP) assays under E6-AP knockdown conditions. Our ChIP data suggest that knockdown of E6-AP leads to decreased recruitment of the histone acetylase p300 to the ERα target gene pS2 promoter as well as reduced histone modifications at the promoter. Although there was reduced p300 recruitment to the pS2 promoter, loss of p300 did not account fully for the loss of histone acetylation. Taken together our data suggest that E6-AP regulates the transactivation functions of ERα in part by complexing with p300 and other chromatin modifying enzymes at target gene promoters to create a transcriptionally active promoter environment.

The Role of Calcium in the Activation of Estrogen Receptor-Alpha

Environmental estrogen mimics, including metalloestrogens that can activate estrogen receptor-alpha (ERα), may contribute to breast cancer risk. However, the underlying mechanisms through which these molecular mimics activate the ERα are generally poorly understood. With concern to this important question, we investigated whether intracellular calcium may mediate the cross-talk between signaling pathways that activate ERα and the ligand-binding domain of ERα. MCF-7 cells treated with EGF, ATP, extracellular calcium, or caffeine to increase intracellular calcium triggered a rapid recruitment of ERα to estrogen-responsive promoters and stimulated expression of estrogen-responsive genes including pS2, complement C3, and progesterone receptor. Induction was blocked by an antiestrogen but also by the chelation of intracellular calcium. Treatment with extracellular calcium also increased the growth of MCF-7 cells through an ER-dependent mechanism. We found that EGF and extracellular calcium activated the C-terminus of ERα and the activation was blocked by the antiestrogen. Mechanistic investigations identified four potential sites on the solvent-accessible surface of the ERα ligand-binding domain as important for calcium activation of the receptor. Taken together, our results suggest that calcium mediates the cross-talk between ERα-activating signaling pathways and the ligand-binding domain of ERα providing a potential explanation for the ability of certain environmental metalloestrogens to activate the receptor.

TOX3 is a neuronal survival factor that induces transcription depending on the presence of CITED1 or phosphorylated CREB in the transcriptionally active complex

TOX3 is a nuclear protein containing a high mobility group (HMG)-box domain, which regulates Ca(2+)-dependent transcription in neurons through interaction with the cAMP-response-element-binding protein (CREB). TOX3 appears to be associated with breast cancer susceptibility and was previously shown to be expressed downstream of a cytoprotective cascade together with CITED1, a transcriptional regulator that does not bind directly to DNA. In the present study we show that TOX3 is predominantly expressed in the brain, forms homodimers and interacts with CITED1. TOX3 overexpression protects neuronal cells from cell death caused by endoplasmic reticulum stress or BAX overexpression through the induction of anti-apoptotic transcripts and repression of pro-apoptotic transcripts, which correlates with enhanced transcription involving isolated estrogen-responsive elements and estrogen-responsive promoters. However, both functions cannot be inhibited with the anti-estrogen fulvestrant and are only attenuated by mutation of estrogen-responsive elements. TOX3 also interacts with native CREB and induces the CREB-responsive BCL-2 promoter, which can be inhibited by coexpression of CITED1. Coexpression of CREB, by contrast, abolishes TOX3-mediated transcription from the estrogen-responsive complement C3 promoter. Our results suggest that TOX3 can enhance transcriptional activation from different cytoprotective promoters and that this is dependent on the predominance of either phosphorylated CREB or CITED1 within the transcriptionally active complex.

Estrogen Inhibits Transforming Growth Factor β Signaling by Promoting Smad2/3 Degradation

Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERalpha and ERbeta, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-beta acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERalpha and TGF-beta/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERalpha inhibits TGF-beta signaling by decreasing Smad protein levels. ERalpha-mediated reductions in Smad levels did not require the DNA binding ability of ERalpha, implying that ERalpha opposes the effects of TGF-beta via a novel non-genomic mechanism. Our analysis revealed that ERalpha formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERalpha.

Recruitment of the SWI/SNF Chromatin Remodeling Complex to Steroid Hormone-regulated Promoters by Nuclear Receptor Coactivator Flightless-I

ATP-dependent chromatin remodeling complexes, such as SWI/SNF, are required for transcriptional activation of specific genes and are believed to be recruited to gene promoters by direct interaction with DNA binding transcription factors. However, we report here that recruitment of SWI/SNF to target genes of estrogen receptor alpha (ERalpha) requires the previously described nuclear receptor coactivator protein Flightless-I (Fli-I). Fli-I can bind directly to both ER and BAF53, an actin-related component of the SWI/SNF complex, suggesting that Fli-I may recruit SWI/SNF to ER target genes via interaction with BAF53. Point mutations in Fli-I that disrupt binding to ER or BAF53 compromised the ability of Fli-I to enhance ER-mediated activation of a transiently transfected reporter gene. Depletion of endogenous Fli-I or BAF53 inhibited estrogen-responsive expression of endogenous target genes of ER, indicating a critical role for Fli-I and BAF53. Moreover, depletion of endogenous Fli-I or BAF53 specifically eliminated part of the complex cyclical pattern of recruitment of SWI/SNF to estrogen-responsive promoters in a way that indicates multiple roles and multiple mechanisms of recruitment for SWI/SNF in estrogen-dependent target gene expression. These results begin to establish the functional relationships and interdependencies that coordinate the actions of the many coactivators participating in the transcriptional activation process.

Reciprocal regulation of 17β-estradiol, interleukin-6 and interleukin-8 during growth and progression of epithelial ovarian cancer

Estrogens have been associated with risk for epithelial ovarian cancer (OVCA). Both IL-6 and IL-8 are also likely involved in the progression of OVCA. In order to discover the underline molecular mechanism, we investigated the modulation of estrogen and two cytokines in the growth and progression of epithelial OVCA. In these studies, the effect of 17β-estradiol (E 2) on the expression levels of IL-6, IL-8 and their receptors was investigated. The effect of IL-6 and IL-8 on activation of estrogen-responsive promoter as well as estrogen receptor (ER)α and ERβ expression was also analyzed. Gene expression profile analysis revealed that CAOV-3 and OVCAR-3 cells, which express ER, IL-6 and IL-8 receptors, are suitable model for this study. We found that E 2 not only enhanced IL-6 and IL-8 production via NF-κB signaling pathway, but also modulated their respective receptor expression. Tamoxifen (Txf), an ER antagonist, completely abolished E 2-stimulated cell growth and the expression of IL-6 and IL-8. IL-6/IL-8-induced cell proliferation was completely blocked by their specific neutralizing antibodies, which partially inhibited E 2-induced cell growth. In the absence of estrogen, both cytokines activated estrogen-responsive promoter, which was completely blocked by Txf, and caused a dose-dependent ERα increase and ERβ decrease. Pretreatment of OVCAR-3 with p38 MAPK, MEK1/2 or ErbB2 MAPK inhibitors, respectively, blocked IL-6-mediated induction of estrogen-responsive promoter while Src inhibitor blocked IL-8-induced activation of estrogen-responsive promoter. These results provide a novel mechanism that estrogens, IL-6 and IL-8 may form a common amplifying signaling cascade to modulate OVCA growth and progression. Estrogen-induced OVCA proliferation is partially occurring via enhanced IL-6 and IL-8 production and modulated their receptors, and IL-6/IL-8 could also promote OVCA growth through an ERα pathway.

Regulation of Estrogen-Dependent Transcription by the LIM Cofactors CLIM and RLIM in Breast Cancer

Mammary oncogenesis is profoundly influenced by signaling pathways controlled by estrogen receptor alpha (ERalpha). Although it is known that ERalpha exerts its oncogenic effect by stimulating the proliferation of many human breast cancers through the activation of target genes, our knowledge of the underlying transcriptional mechanisms remains limited. Our published work has shown that the in vivo activity of LIM homeodomain transcription factors (LIM-HD) is critically regulated by cofactors of LIM-HD proteins (CLIM) and the ubiquitin ligase RING finger LIM domain-interacting protein (RLIM). Here, we identify CLIM and RLIM as novel ERalpha cofactors that colocalize and interact with ERalpha in primary human breast tumors. We show that both cofactors associate with estrogen-responsive promoters and regulate the expression of endogenous ERalpha target genes in breast cancer cells. Surprisingly, our results indicate opposing functions of LIM cofactors for ERalpha and LIM-HDs: whereas CLIM enhances transcriptional activity of LIM-HDs, it inhibits transcriptional activation mediated by ERalpha on most target genes in vivo. In turn, the ubiquitin ligase RLIM inhibits transcriptional activity of LIM-HDs but enhances transcriptional activation of endogenous ERalpha target genes. Results from a human breast cancer tissue microarray of 1,335 patients revealed a highly significant correlation of elevated CLIM levels to ER/progesterone receptor positivity and poor differentiation of tumors. Combined, these results indicate that LIM cofactors CLIM and RLIM regulate the biological activity of ERalpha during the development of human breast cancer.

Targeting sodium/iodide symporter gene expression for estrogen-regulated imaging and therapy in breast cancer

Expression of the sodium iodide symporter (hNIS) has been detected in breast cancer tissue, but frequently, not at the levels necessary to mediate (131)I accumulation. Transducing the hNIS gene into breast cancer cells with adenovirus could be a tractable strategy to render breast cancer susceptible to radioiodide therapy. We constructed the replication-incompetent virus, AdSERE, in which an estrogen-responsive promoter directs the expression of hNIS. In vitro, we demonstrate that AdSERE mediates hNIS expression and iodide uptake in ER+ breast cancer cells. In vivo, we show that AdSERE-infected ER+ tumors can be imaged due to tracer accumulation; in addition, AdSERE in combination with therapeutic doses of (131)I suppresses tumor growth.

The Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor (SMRT) Corepressor Is Required for Full Estrogen Receptor α Transcriptional Activity

Multiple factors influence estrogen receptor alpha (ERalpha) transcriptional activity. Current models suggest that the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor functions within a histone deactylase-containing protein complex that binds to antiestrogen-bound ERalpha and contributes to negative regulation of gene expression. In this report, we demonstrate that SMRT is required for full agonist-dependent ERalpha activation. Chromatin immunoprecipitation assays demonstrate that SMRT, like ERalpha and the SRC-3 coactivator, is recruited to an estrogen-responsive promoter in estrogen-treated MCF-7 cells. Depletion of SMRT, but not histone deacetylases 1 or 3, negatively impacts estradiol-stimulated ERalpha transcriptional activity, while exogenous expression of SMRT's receptor interaction domains blocks ERalpha activity, indicating a functional interaction between this corepressor and agonist-bound ERalpha. Stimulation of estradiol-induced ERalpha activity by SMRT overexpression occurred in HeLa and MCF-7 cells, but not HepG2 cells, indicating that these positive effects are cell type specific. Similarly, the ability of SMRT depletion to promote the agonist activity of tamoxifen was observed for HeLa but not MCF-7 cells. Furthermore, impairment of agonist-stimulated activity by SMRT depletion is specific to ERalpha and not observed for receptors for vitamin D, androgen, or thyroid hormone. Nuclear receptor corepressor (N-CoR) depletion increased the transcriptional activity of all four tested receptors. SMRT is required for full expression of the ERalpha target genes cyclin D1, BCL-2, and progesterone receptor but not pS2, and its depletion significantly attenuated estrogen-dependent proliferation of MCF-7 cells. Taken together, these data indicate that SMRT, in conjunction with gene-specific and cell-dependent factors, is required for positively regulating agonist-dependent ERalpha transcriptional activity.

Dynamic Cycling of the O‐GlcNAc Transferase on the Estrogen Responsive pS2 Promoter During the Transcription Cycle.

Dynamic Cycling of the O‐GlcNAc Transferase on the Estrogen Responsive pS2 Promoter During the Transcription Cycle.