ERalpha Transcription Research Articles

Abstract P2-05-02: Functional genomic screening identifies ubiquitin-specific protease 11 (USP11) as a novel regulator of ER-alpha transcription in breast cancer

Abstract Approximately 70% of breast cancers overexpress the estrogen receptor α (ERα) and depend on this key transcriptional regulator for growth and differentiation. The discovery of novel mechanisms controlling ERα function represent major advances in our understanding of breast cancer progression and potentially offer attractive new therapeutic opportunities. Here, we investigated the role of deubiquitinating enzymes (DUBs), which act to remove ubiquitin moieties from proteins, in regulating transcriptional activity of ERα in breast cancer. To identify DUBs involved in the regulation of ERα transcriptional activity, we performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all human DUB genes. The DUB library consisted of pools of four non-overlapping shRNAs targeting all 108 known or putative DUBs (432 shRNAs in total). We found that suppression of a number of DUBs markedly repressed or enhanced the activity of an estrogen-response-element (ERE) luciferase reporter following estradiol (E2) stimulation. Of particular interest, suppression of the BRCA2-associated DUB, USP11, was found to down-regulate ERα transcriptional activity. Subsequent validation using two individual siRNAs targeted to USP11 revealed a notable reduction in expression of endogenous ERα target genes in the ZR-75-1 cell line, as quantified using qRT-PCR. Further validation was carried out in a HEK293T USP11 knockout cell line, where reduced activity of an ERE-luciferase reporter was detected when compared to wild-type cells. This phenotype was rescued with a USP11 overexpression vector, both in the presence and absence of E2. Furthermore, USP11 expression was found to be upregulated in the estrogen-independent cell line LCC1 when compared to their parental MCF7 cells. Knockdown of USP11 in LCC1 cells resulted in decreased mRNA expression of a panel of ERα target genes, while RNA-seq revealed a downregulation of several putative ERα target genes and a downregulation of many cell cycle-associated proteins. To support the prognostic relevance of USP11, immunohistochemical staining of a breast cancer tissue microarray (103 ER+ patients available for final analysis) was performed. Kaplan-Meier analysis of this cohort revealed a highly significant association between high USP11 expression and poor overall (p=0.030) and breast cancer-specific survival (p=0.041). In silico analysis of publically available breast cancer gene expression datasets further supported an association between high USP11 mRNA levels and poor prognosis. We observed a significant correlation between high expression of USP11 mRNA in ER-positive patients and poor distant metastasis-free survival (HR 2, CI 1.37-2.91, p=0.00023). This correlation was also significant in ER-positive patients who had received tamoxifen only (HR 2.9, CI 1.63-5.15, p=0.00015). These results suggest a role for USP11 in driving cellular growth and identify USP11 as novel therapeutic target in breast cancer. Citation Format: Dwane L, Das S, Moran B, O'Connor AE, Mulrane L, Dirac AM, Jirstrom K, Crown JP, Bernards R, Gallagher WM, Ní Chonghaile T, O'Connor DP. Functional genomic screening identifies ubiquitin-specific protease 11 (USP11) as a novel regulator of ER-alpha transcription in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-05-02.

Effects of Hypoglycaemia on Neurotransmitter and Hormone Receptor Gene Expression in Laser‐Dissected Arcuate Neuropeptide Y/Agouti‐Related Peptide Neurones

Arcuate neuropeptide Y (NPY)/agouti-related pepide (AgRP) neurones regulate energy homeostasis, and express the putative glucosensor, glucokinase (GCK). The present study performed multi-transcriptional profiling of these neurones to characterise NPY, AgRP and GCK gene expression during intermediate insulin-induced hypoglycaemia, and to determine whether these transcriptional responses acclimate to repeated insulin dosing. We also examined whether these neurones express insulin, glucocorticoid and oestrogen receptor gene transcripts, and whether the levels of these receptor mRNAs are modified by insulin-induced hypoglycaemia. Individual NPY-immunoreactive neurones were laser-microdissected from the caudal arcuate nucleus after single or serial dosing with neutral protamine Hagedorn insulin (NPH), and evaluated by quantitative real-time reverse transcriptase-polymerase chain reaction for the assessment of neurotransmitter and receptor gene expression. Mean NPY and AgRP mRNA in harvested NPY neurones was unchanged or augmented, respectively, by one NPH dose, although repeated NPH administration up-regulated NPY, whereas AgRP gene transcripts were down-regulated. NPH elicited divergent modifications in the ERalpha and ERbeta mRNA content of sampled neurones. ERalpha transcripts were amplified by both acute and chronic NPH-induced hypoglycaemia, whereas ERbeta gene expression was unaltered during a single bout, but suppressed during recurring hypoglycaemia. Glucocorticoid receptor (GR) mRNA levels were increased by a single insulin dose, but unaffected by serial NPH dosing. Insulin receptor-beta chain (InsRb) gene transcripts were insensitive to acute NPH-induced hypoglycaemia, but repeated NPH inhibited this gene transcript. Neither acute nor recurring hypoglycaemia modified GCK mRNA levels in caudal hypothalamic arcuate nucleus (ARH) NPY/AgRP neurones, but baseline GCK transcription was suppressed by the latter. This evidence for the habituation of hypoglycaemic patterns of InsRb, GR and ERbeta gene transcription to serial NPH dosing implies that such treatment may alter reactivity of caudal ARH NPY/AgRP neurones to receptor ligands, and supports the need to determine whether adaptive changes in neuronal sensitivity to insulin, corticosterone and/or oestrogen cause up- versus down-regulation of NPY and AgRP neurotransmission, respectively, by this caudal ARH subpopulation during chronic hypoglycaemia.

Adaptation of Arcuate Insulin Receptor, Estrogen Receptor-Alpha, Estrogen Receptor-Beta, and Type-II Glucocorticoid Receptor Gene Profiles to Chronic Intermediate Insulin-Induced Hypoglycemia in Estrogen-Treated Ovariectomized Female Rats

Insulin and corticosterone signal energy surfeit and deficiency, respectively, to metabolic structures in the brain, including the hypothalamic arcuate nucleus (ARH). This peripheral input may be subject to ovarian control, since ovariectomy (OVX) increases insulin receptor transcripts and decreases glucocorticoid receptor protein in the hypothalamus. The present studies examined the hypothesis that estradiol regulates basal and hypoglycemic patterns of ARH insulin and glucocorticoid receptor mRNA expression, and governs habituation of these gene profiles to recurring intermediate insulin administration. The premise that estrogen receptor-alpha (ERalpha) and beta (ERbeta) gene profiles may be regulated differentially during acute and chronic hypoglycemia in the presence of estradiol was also evaluated. Insulin receptor-beta chain (InsRb), type-II glucocorticoid receptor (GR), ERalpha, and ERbeta mRNA levels in ARH tissue microdissected from estradiol benzoate (EB)- and oil-implanted OVX rats after single or serial sc neutral protamine Hagedorn insulin (NPH) injection were measured by quantitative real-time RT-PCR. ARH InsRb gene profiles were decreased, relative to baseline, after either one or four NPH injections in OVX + EB rats; mean mRNA levels were significantly lower after serial dosing since basal InsRb transcripts were diminished by precedent NPH treatment. InsRb transcription rates did not differ among OVX + oil treatment groups. Acute insulin elevated ARH GR mRNA relative to baseline in both EB- and oil-implanted rats. Prior NPH injections increased basal GR gene expression and suppressed transcriptional reactivity to a fourth dose of NPH in OVX + EB, but not OVX + oil animals. ARH ERalpha and ERbeta mRNA levels were increased or decreased, respectively, after one insulin dose in OVX + EB rats. Baseline expression of these genes was correspondingly augmented or suppressed after precedent NPH treatment, but ERalpha and ERbeta transcripts were not modified relative to these adjusted baselines after a fourth NPH dose. In the presence of estradiol, ARH InsRb and GR gene profiles exhibit divergent modifications during acute NPH-induced hypoglycemia, as well as opposite adjustments in baseline expression after serial NPH dosing. GR transcriptional acclimation to recurring NPH administration was also estrogen-dependent. Further research is needed to characterize potential effects of adjustments in ARH neuronal sensitivity to insulin and corticosterone on ARH metabolic neurotransmitter release during and after intermediate insulin-induced hypoglycemia in females. The current evidence for converse effects of NPH on ARH ERalpha and ERbeta gene profiles in the presence of estradiol supports the need to identify ARH-directed metabolic activities governed by each ER subtype, including metabolic hormone receptor expression, and to assess the impact of NPH-induced habituation of ER gene profiles on those functions.

Breast cancer-derived M543V mutation in helix 12 of estrogen receptor α inverts response to estrogen and SERMs

We have isolated from human breast cancers several mutations in the Helix 12 component of activation function 2 (AF-2) in the estrogen receptor alpha (ERalpha). We used a novel approach to detect changes in the hormone-binding domain of ERalpha, based on the evidence that antiestrogens, such as 4-hydroxytamoxifen (ZOHT) and ICI 182,780, block the function of ERalpha by binding and folding the AF-2 transcriptional domain in a way that inhibits its association with coactivator proteins. We have identified a Helix 12 mutation, M543V, which leads to greater ERalpha transcription with ZOHT and other antiestrogens (including 1,1-dichloro-2,2,3-triarylcyclopropanes, DTACs) than with 17-beta estradiol (E2). We also found an independent mutation at the same position, M543I, which did not show this inverted ligand phenotype. In comparison to further Helix 12 mutations made in vitro, it appears that relative hydrophobicity of the amino acid side chains on the inner face of Helix 12 is key to maintaining the transcriptionally active, agonist conformation with bound E2. This active conformation can be induced, resulting in increased transcription, by adding excess p160 coactivator AIB1 in transcriptional assays with E2-bound receptors, while the ZOHT-bound receptors were not further activated by AIB1. Other experiments show that the cross talk between ERalpha and AP-1 protein from AP-1-binding sites is not dependent on Helix 12 integrity. We show that two alleles containing a proline substitution in Helix 12 that inactivate AF-2 function of ERalpha at EREs have little negative effect on function through AP-1 elements, supporting a prominent role for the N-terminal AF-1 of ERalpha in AP-1/ERalpha transcriptional cross talk.

Resistance to Antiestrogen Arzoxifene Is Mediated by Overexpression of Cyclin D1

Resistance to tamoxifen treatment occurs in approximately 50% of the estrogen receptor (ER)alpha-positive breast cancer patients. Resistant patients would benefit from treatment with other available antiestrogens. Arzoxifene is an effective growth inhibitor of ERalpha-positive breast cancer cells, including tamoxifen-resistant tumors. In this study, we show that overexpression of a regular component of the ERalpha transcription factor complex, cyclin D1, which occurs in approximately 40% of breast cancer patients, renders cells resistant to the new promising antiestrogen, arzoxifene. Overexpression of cyclin D1 alters the conformation of ERalpha in the presence of arzoxifene. In this altered conformation, ERalpha still recruits RNA polymerase II to an estrogen response element-containing promoter, inducing transcription of an ERalpha-dependent reporter gene and of endogenous pS2, and promoting arzoxifene-stimulated growth of MCF-7 cells. Arzoxifene is then converted from an ERalpha antagonist into an agonist. This can be explained by a stabilization of the ERalpha/steroid receptor coactivator-1 complex in the presence of arzoxifene, only when cyclin D1 is overexpressed. These results indicate that subtle changes in the conformation of ERalpha upon binding to antiestrogen are at the basis of resistance to antiestrogens.

Generation and characterization of a complete null estrogen receptor α mouse using Cre/LoxP technology

Conventional estrogen receptor alpha knockout (neo-ERalphaKO, neo-ERalpha(-/-)) mice contain a truncated and chimeric ERalpha fusion protein that retains 35% estrogen-dependent transactivation activity, and therefore the in vivo ERalpha function is difficult to study thoroughly. Furthermore, these neo-ERalpha(-/-) mice cannot be used for tissue and temporal specific ERalpha deletion. Therefore, there is a clear need to establish a floxed ERalpha mouse line that can knockout ERalpha specifically and completely in each selected cell type. Here we generated floxed ERalpha mice using a self-excising ACN (tACE-Cre/Neo) cassette. Mating the floxed ERalpha mice with ACTB-Cre mice produces a deletion of the floxed allele disrupting the reading frame of the ERalpha transcript so that no ERalpha protein is detected in the ACTB-Cre/ERalpha(-/-) mice. Expression of ERalpha target genes, such as G-6-PD and lactoferrin, is diminished by over 90% in the ACTB-Cre/ERalpha(-/-) uterus, but not in the neo-ERalpha(-/-) uterus. Furthermore, we also validated that ACTB-Cre/ERalpha(-/-) females have a hypoplastic internal genital tract, polycystic ovaries with hemorrhagic follicles, infertility, and higher body weight. Together, our data clearly demonstrate that the newly established floxed ERalpha mouse is a reliable mouse model for future studies of ERalpha roles in vivo in the selective estrogen target tissues. The complete knockout of ERalpha in the ACTB-Cre/ERalpha(-/-) mice will also provide an improved mouse model to study the role of ERalpha in vivo.

Differential regulation of dehydroepiandrosterone and estrogen on bone and uterus in ovariectomized mice

Dehydroepiandrosterone (DHEA) may be useful in the treatment of postmenopausal osteoporosis (PMO). Our present study has found the preferable stimulatory effect of DHEA on bone, in contrast to the proliferative effects of estradiol (E2) on the endometrium and the uterus, which suggests that DHEA has greater potential clinical value than estrogens in prophylaxis and therapeutics for PMO. A series of findings raise the possibility that DHEA may be useful in the treatment of PMO. Our present study thus aimed at the differential effects of DHEA and E2 on bone and the uterus in ovariectomized mice as well as the involvement of aromatase, ERalpha, ERbeta, and AR in the effects. Ovariectomized and sham BALB/c mice were given daily treatment with either DHEA or E2 for three months, respectively. Bone mineral density was determined by DEXA after the last treatment. Mice were necropsied in 3 months after the treatment to analyze the ultrastructure of their femur osteoblasts (OBs) with a transmission electron microscope (TEM); DHEA, DHEA sulfate (DHEAS) and E2 levels were assayed by EIA; production in vitro of E2 in the uterus or tibia was assayed to evaluate the profile of P450arom activity; ERalpha and ERbeta mRNA levels in the uterus and tibia were determined by real-time PCR. The primary murine OBs were treated with DHEA and E2, respectively for 72 h. Real-time polymerase chain reaction (PCR) and western blot were carried out to evaluate aromatase, ERalpha, ERbeta and AR expression in OBs. Both DHEA and E2 significantly improved BMD and OB ultrastructure; E2 but not DHEA has significantly increased uterus wet weight, endometrium epithelial and gland thickness. Dehydroepiandrosterone not only increased serum, femoral DHEA, DHEAS and E2 concentration, but also increased uterine DHEA and DHEAS other than E2 concentration in site, while E2 only increased serum, uterine and femoral E2 concentration, but failed to alter the concentrations of DHEA and DHEAS. Moreover, DHEA significantly increased tibia P450arom enzyme activity, while E2 increased uterine and tibia aromatase activity. Furthermore, DHEA increased uterine ERbeta and ERalpha, and ERbeta transcription in the tibia, while E2 increased ERalpha transcription in the uterus and tibia. Dehydroepiandrosterone increased aromatase, ERalpha, ERbeta and AR expression in OBs, and increased significantly, but E2 apparently decreased the ratio of ERbeta/ERalpha. Although both DHEA and E2 augment BMD, the proliferative effects of E2 on the endometrium and uterus reflect the different modes of action on bone and the uterus, indicating that the preferable stimulatory effect of DHEA on bone appears to the more potential clinical values than estrogens in prophylaxis and therapeutics for PMO. But applicability of the findings from rodents in humans needs further study.

Differing responses of human follicular and nonfollicular scalp cells in an in vitro wound healing assay: Effects of estrogen on vascular endothelial growth factor secretion

Improved wound healing of hairy skin may involve mesenchymal hair follicle cells with stem cell potential and enhancement by estrogen therapy. How estrogen affects follicular dermal papilla (DP) and dermal sheath (DS) cells in wound healing is unknown. Therefore, a comparison of estradiol action on DP, DS, and corresponding interfollicular dermal fibroblasts (DF) in a scratch-wound assay was performed using matching primary cultures established from female temporo-occipital scalp. All three cell types expressed mRNA transcripts and protein for estrogen receptors alpha (ERalpha) and beta (ERbeta). DF ERalpha transcripts were half that of DP and one-third of DS cells, while DF ERbeta transcripts were two-thirds of DP and DS cells. In the scratch-wound assay all three cells types migrated at similar rates, but only the rate of DF was enhanced by estradiol. Mechanical wounding increased DNA synthesis rates of all three cell types and increased the secretion of collagen by DF and DS cells. All three secreted similar basal levels of vascular endothelial growth factor (VEGF), which was increased by wounding DF and DS cells, but not DP cells. DP cells required estradiol to increase VEGF secretion; by contrast VEGF secretion was decreased by estradiol in wounded DS cells. These results highlight differences in the responses of DF, DP, and DS cells to estradiol in a scratch-wound assay, providing further support for the dichotomy of cellular functions in the hair follicle. Further understanding of the role of estrogen in cutaneous wound healing may have important implications for the management of chronic wounds and scarring.

Specific adaptations of estrogen receptor α and β transcripts in liver and heart after endurance training in rats

Estrogens exert their biological roles mainly through estrogen receptors (ER) that function as ligand-activated transcription factors. ER content in a cell is regulated by many factors and is decisive for estrogen action. The purpose of the present study was to investigate the influence of an 8-wk endurance training program on ER expression in the liver, right atrium (RA), and left ventricle (LV) of intact and ovariectomized (Ovx) rats. We measured ERalpha and ERbeta mRNA content by reverse transcription-polymerase chain reaction (RT-PCR). We found an important increase in ERalpha mRNA levels in the liver (300%; P < 0.01) and in ERbeta mRNA levels in the RA (200%; P < 0.05), and a marked decrease in ERalpha (80%; P < 0.01) and ERbeta (40%; P < 0.05) transcripts content in the LV of intact rats after endurance training. On the other hand, ERalpha mRNA levels were depressed by 50% (P < 0.01) in the liver, and increased by 60% (P < 0.01) in LV of Ovx rats after exercise training. These results first indicate that endurance training is associated with modifications of ER transcripts levels in the liver, LV, and RA of female rats. More specifically, these effects are tissue and isoform-specific and the direction of the response (increase or decrease) is different in intact and Ovx rats. It is suggested that some of the adaptations to endurance training in liver and heart may be mediated by an ER-dependent mechanism.

Polyamine Analogues Down-regulate Estrogen Receptor α Expression in Human Breast Cancer Cells

The critical role of polyamines in cell growth has led to the development of a number of agents that interfere with polyamine metabolism including a novel class of polyamine analogues, oligoamines. Here we demonstrate that oligoamines specifically suppress the mRNA and protein expression of estrogen receptor alpha (ERalpha) and ERalpha target genes in ER-positive human breast cancer cell lines, whereas neither ERbeta nor other steroid hormonal receptors are affected by oligoamines. The constitutive expression of a cytomegalovirus promoter-driven exogenous ERalpha in ER-negative MDA-MB-231 human breast cancer cells was not altered by oligoamines, suggesting that oligoamines specifically suppress ERalpha transcription rather than affect mRNA or protein stability. Further analysis demonstrated that oligoamines disrupted the DNA binding activity of Sp1 transcription factor family members to an ERalpha minimal promoter element containing GC/CA-rich boxes. Treatment of MDA-MB-231 cells with the JNK-specific inhibitor SP600125 or expression of the c-Jun dominant negative inhibitor TAM67 blocked the oligoamine-activated JNK/c-Jun pathway and enhanced oligoamine-inhibited ERalpha expression, suggesting that AP-1 is a positive regulator of ERalpha expression and that oligoamine-activated JNK/AP-1 activity may antagonize the down-regulation of ERalpha induced by oligoamines. Taken together, these results suggest a novel antiestrogenic mechanism for specific polyamine analogues in human breast cancer cells.

Identification of the MLL2 Complex as a Coactivator for Estrogen Receptor α

A novel estrogen receptor (ER)alpha coactivator complex, the MLL2 complex, which consists of MLL2, ASH2, RBQ3, and WDR5, was identified. ERalpha directly binds to the MLL2 complex through two LXXLL motifs in a region of MLL2 near the C terminus in a ligand-dependent manner. Disrupting the interaction between ERalpha and the MLL2 complex with small interfering RNAs specific against MLL2 or an MLL2 fragment representing the interacting region with ERalpha significantly inhibited the ERalpha transcription activity. The MLL2 complex was recruited on promoters of ERalpha target genes along with ERalpha upon estrogen stimulation. Inhibition of MLL2 expression decreased the estrogen-induced expression of ERalpha target genes cathepsin D and to a lesser extent pS2. In addition, MCF-7 cell growth was also inhibited by the depletion of MLL2. These results demonstrate that the ERalpha signaling pathway is critically dependent on its direct interaction with the MLL2 complex and suggest a central role for the MLL2 complex in the growth of ERalpha-positive cancer cells.

Paracrine signaling through the epithelial estrogen receptor alpha is required for proliferation and morphogenesis in the mammary gland.

Estradiol is a major regulator of postnatal mammary gland development and thought to exert its effects through estrogen receptor alpha (ERalpha) expressed in the mammary gland stroma and epithelium. Previous studies, however, were confounded by the use of an ERalpha mutant strain that retains some of the protein with transactivation activity. Here, we use an ERalpha-/- mouse strain in which no ERalpha transcript can be detected to analyze mammary gland development in the complete absence of ERalpha signaling. The ERalpha-/- females show no development beyond a rudimentary ductal system. By grafting ERalpha-/- epithelium or stroma in combination with ERalpha WT stroma or epithelium, we show that the primary target for estradiol is the mammary epithelium, whereas a direct response of the mammary stroma is not required for mammary gland development to proceed normally. Mammary glands reconstituted with ERalpha-/- mammary epithelium exposed to pregnancy hormones show increased transcription of milk protein genes, indicating that ERalpha signaling is not an absolute requirement for a transcriptional response to pregnancy hormones. When ERalpha-/- mammary epithelial cells are in close vicinity to ERalpha WT cells, they proliferate and contribute to all aspects of mammary gland development, indicating that estradiol, like progesterone, orchestrates proliferation and morphogenesis by a paracrine mechanism, affecting nearby cells in the mammary epithelium.

Expression and Functional Study of Estrogen Receptor-Related Receptors in Human Prostatic Cells and Tissues

Estrogen receptor-related receptors (ERRs; alpha, beta, gamma) are orphan nuclear receptors and constitutively active without binding to estrogen. Like estrogen receptors (ERs), ERRs bind to estrogen receptor elements and estrogen receptor element-related repeats. Growing evidence suggests that ERRs can cross-talk with ERs in different cell types via competition for DNA sites and coactivators. We hypothesize that ERRs might play regulatory roles in normal and neoplastic prostatic cells by sharing similar ER-mediated pathways or acting independently. In this study, we investigated mRNA and protein expression patterns of three ERR members in normal human prostate epithelial cells, established cell lines, cancer xenografts, and prostatic tissues. Additionally, effects of transient transfection of ERRs on prostatic cell proliferation and ER expression were also examined. RT-PCR showed that ERRalpha and ERRgamma transcripts were detected in most cell lines and xenografts, whereas ERRbeta was detected in normal epithelial cells and few immortalized cell lines but not in most cancer lines. Similar results were demonstrated in clinical prostatic specimens. Western blottings and immunohistochemistry confirmed similar expression patterns that ERR proteins were detected as nuclear proteins in epithelial cells, whereas their expressions became reduced or undetected in neoplastic prostatic cells. Transient transfection confirmed that ERRs were expressed in prostatic cells as nuclear proteins and transcriptionally active in the absence of estradiol. Transfection results showed that overexpression of ERRs inhibited cell proliferation and repressed ERalpha transcription in PC-3 cells. Our study shows that ERRs, which are coexpressed with ERs in prostatic cells, could regulate cell growth and modulate ER-mediated pathways via interference on ERalpha transcription in prostatic cells.

Inhibiting proteasomal proteolysis sustains estrogen receptor-alpha activation.

Estrogen receptor-alpha (ER alpha) is a ligand-dependent transcription factor that mediates physiological responses to 17 beta-estradiol (E2). Ligand binding rapidly down-regulates ER alpha levels through proteasomal proteolysis, but the functional impact of receptor degradation on cellular responses to E2 has not been fully established. In this study, we investigated the effect of blocking the ubiquitin-proteasome pathway on ER alpha-mediated transcriptional responses. In HeLa cells transfected with ER alpha, blocking either ubiquitination or proteasomal degradation markedly increased E2-induced expression of an ER-responsive reporter. Time course studies further demonstrated that blocking ligand-induced degradation of ER alpha resulted in prolonged stimulation of ER-responsive gene transcription. In breast cancer MCF7 cells containing endogenous ER alpha, proteasome inhibition enhanced E2-induced expression of endogenous pS2 and cathepsin D. However, inhibiting the proteasome decreased expression of progesterone receptor (PR), presumably due to the heterogeneity of the PR promoter, which contains multiple regulatory elements. In addition, in endometrial cancer Ishikawa cells overexpressing steroid receptor coactivator 1, 4-hydroxytamoxifen displayed full agonist activity and stimulated ER alpha-mediated transcription without inducing receptor degradation. Collectively, these results demonstrate that proteasomal degradation is not essential for ER alpha transcriptional activity and functions to limit E2-induced transcriptional output. The results further indicate that promoter context must be considered when evaluating the relationship between ER alpha transcription and proteasome inhibition. We suggest that the transcription of a gene driven predominantly by an estrogen-responsive element, such as pS2, is a more reliable indicator of ER alpha transcription activity than a gene like PR, which contains a complex promoter requiring cooperation between ER alpha and other transcription factors.

Protein Kinase A Activation of Estrogen Receptor α Transcription Does Not Require Proteasome Activity and Protects the Receptor from Ligand-Mediated Degradation

17beta-Estradiol (E2)-stimulated estrogen receptor (ERalpha) transcription is accompanied by protein degradation via the 26S-proteasome pathway. Inhibition of proteasome activity stabilizes ERalpha protein and abolishes E2-activated transcription, suggesting functional linkages between transcription and degradation. It is not known whether ligand-independent ERalpha activation is coupled to proteolysis. In pituitary cells, forskolin (FSK) stimulates ERalpha transcription through the protein kinase A (PKA) pathway. This study examined interactions between E2-dependent and PKA-stimulated pathways in GH(3) cells by measuring transcription of a transfected reporter gene and endogenous ERalpha levels. E2 stimulated estrogen response element-mediated transcription 2- to 3-fold and decreased ERalpha protein levels to 40%. In contrast, FSK stimulated ERalpha transcription without decreasing ERalpha protein. Treatment with FSK plus E2 resulted in synergistic ERalpha transactivation, and FSK specifically prevented E2-induced ERalpha degradation. PKA is required for protection and was prevented by H89 (a PKA inhibitor), but not PD98059 (a MAPK kinase inhibitor). Propyl-pyrazole-triol and R,R-diethyl-tetrahydrochrysene, selective ERalpha agonists, reduced ERalpha protein by 50% while stimulating ERalpha transcriptional activity 4- to 8-fold. The antagonist ICI 182,780 similarly decreased ERalpha levels, but prevented ER activation. FSK prevented all ligand-induced ERalpha degradation. Lactacystin, a proteasome inhibitor, abolished E2-stimulated, but not FSK-stimulated, ERalpha transcription. Thus, stimulation of ERalpha transcription by the PKA-dependent pathway is dissociated from receptor degradation and proteasome activity. These data suggest a mechanism of ERalpha transcriptional activation by PKA that is distinct from E2 activation and that may contribute to the synergistic transcriptional activation of ERalpha by ligand-dependent and PKA-dependent pathways.

Truncated Estrogen Receptor Product-1 Stimulates Estrogen Receptor α Transcriptional Activity by Titration of Repressor Proteins

The truncated estrogen receptor product-1 (TERP-1, or TERP) is a pituitary-specific isoform of estrogen receptor alpha (ERalpha), and its expression is regulated by estrogen. TERP modulates the transcriptional activity of ERalpha but has no independent effect on transcription of estrogen-response element-containing promoters. At low concentrations, TERP stimulates ERalpha transcriptional activity in transient transfection assays. At TERP concentrations equal to or greater than full-length ERalpha, TERP forms dimers with ERalpha and reduces both ligand-dependent and -independent transcription. A dimerization mutant of TERP, TERP L509R, stimulated ERalpha transcription at all concentrations. We hypothesized that TERP stimulates ERalpha transcriptional activity by titrating suppressors of ERalpha activity. We found that repressor of estrogen receptor activity (REA), originally isolated from human breast cancer cells, is present in mouse pituitary gonadotrope cell lines. Levels of REA vary slightly throughout the rat reproductive cycle, but TERP mRNA and protein vary much more dramatically. In transfection experiments, REA suppressed ERalpha transcriptional activity, and TERP L509R was able to alleviate transcriptional suppression by REA. In glutathione S-transferase pull-down assays, TERP bound to REA more efficiently than did ERalpha at equivalent concentrations, suggesting that REA will preferentially bind to TERP. Our findings suggest that the stimulation of pituitary ERalpha activity by low concentrations of TERP can occur by titration of corepressors such as REA.

Overexpression of histone deacetylase HDAC1 modulates breast cancer progression by negative regulation of estrogen receptor alpha.

The interaction between 17beta-estradiol and estrogen receptor alpha (ER-alpha) plays an important role in breast carcinogenesis and breast cancer treatment. ER-alpha is a critical growth regulatory gene in breast cancer and its expression level is tightly linked to the prognosis and treatment outcomes of breast cancer patients. Loss of ER-alpha expression in breast epithelial cells is critical for breast cancer progression. The underlying molecular mechanisms for this loss, however, are poorly defined. Histone deacetylases (HDACs) are implicated in the alteration of chromatin assembly and tumorigenesis. We show that histone deacetylase 1 (HDAC1) interacts with ER-alpha in vitro and in vivo and suppresses ER-alpha transcription activity. The interaction of HDAC1 with ER-alpha was mediated by the AF-2 and DBD domains of ER-alpha. We observed an endogenous interaction of HDAC1 with ER-alpha in breast cancer cells, which was decreased in the presence of estrogen. Interestingly, overexpression of HDAC1 in stable transfected MCF-7 clones induced loss of ER-alpha and significantly increased cell proliferation and colony formation, as compared to the control MCF-7 cells, whereas treatment of stable MCF-7 clones with the HDAC specific inhibitor trichostatin A (TSA) induced re-expression of ER-alpha mRNA and protein. Our findings strongly suggest that HDAC1 affects breast cancer progression by promoting cellular proliferation in association with a reduction in both ER-alpha protein expression and transcriptional activity. Thus, HDAC1 may be a potential target for therapeutic intervention in the treatment of a subset of ER-negative breast cancers.

PRb2/p130-E2F4/5-HDAC1-SUV39H1-p300 and pRb2/p130-E2F4/5-HDAC1-SUV39H1-DNMT1 multimolecular complexes mediate the transcription of estrogen receptor-alpha in breast cancer.

The estrogen receptor-alpha (ER) plays a crucial role in normal breast development and is also linked to development and progression of mammary carcinoma. The transcriptional repression of ER-alpha gene in breast cancer is an area of active investigation with potential clinical significance. However, the molecular mechanisms that regulate the ER-alpha gene expression are not fully understood. Here we show a new molecular mechanism of ER-alpha gene inactivation mediated by pRb2/p130 in ER-negative breast cancer cells. We investigated in vivo occupancy of ER-alpha promoter by pRb2/p130-E2F4/5-HDAC1-SUV39 H1-p300 and pRb2/p130-E2F4/5-HDAC1-SUV39H1-DNMT1 complexes, and provided a link between pRb2/p130 and chromatin-modifying enzymes in the regulation of ER-alpha transcription in a physiological setting. These findings suggest that pRb2/p130-multimolecular complexes can be key elements in the regulation of ER-alpha gene expression and may be viewed as promising targets for the development of novel therapeutic strategies in the treatment of breast cancer, especially for those tumors that are ER negative.

Differential messenger RNA transcription of androgen receptor and estrogen receptor in gonad in relation to the sex change in protandrous black porgy, Acanthopagrus schlegeli.

Black porgy, Acanthopagrus schlegeli Bleeker, is a marine protandrous hermaphrodite fish. All are functional males at 1-2 yr of age and then become either males or females at 3 yr of age. To study the process of sex change in this species, mRNA transcripts of two estrogen receptors (ERalpha and ERbeta) and an androgen receptor (AR) were monitored. An AR cDNA was cloned and characterized. ERalpha, ERbeta, and AR were differentially transcribed in bisexual testicular and ovarian tissue according to reverse transcription polymerase chain reaction (RT-PCR) and Southern analysis. A real-time quantification PCR analysis was further developed for the measurement of AR, ERalpha, and ERbeta transcripts. ERalpha and AR transcripts were significantly more plentiful in bisexual testis than in bisexual ovary in 1(+)- and 2(+)-yr-old fish. ERalpha, ERbeta, and AR transcripts decreased in the functional testis of 3-yr-old fish. Similar levels of ERbeta and AR were detected in the ovary of sex-changed females and in functional testis of 3-yr-old males. Significantly decreased AR transcripts were found in testicular tissue of bisexual and functional male and female gonads in 3-yr-old fish as compared with 1- and 2-yr-old fish. In contrast, increased ERalpha transcripts were detected in the bisexual ovary and sex-changed ovary of 3-yr-old fish as compared with the bisexual ovary of 1- and 2-yr-old fish. The data suggest a differential sensitivity in the bisexual testicular and ovarian tissue of black porgy.

Neuroprotective effects of estrogen and tamoxifen in vitro: a facilitative role for glia?

Selective estrogen receptor modulators (SERMs) are steroidal or nonsteroidal compounds that can exhibit either estrogen-like agonistic effects or estrogen-antagonistic effects depending on the target tissue. While SERM actions in the breast, bone, and uterus have been well characterized, their effects in the brain are considerably less well understood. Previous work by our laboratory has demonstrated a beneficial effect of tamoxifen in the reduction of ischemic stroke damage in ovariectomized female rats. The present study utilized neuronal cell culture models to attempt to understand the mechanisms of tamoxifen-mediated neuroprotection. Neither physiologic doses of 17beta-E2 nor clinically therapeutic doses of tamoxifen directly protected GT1-7 neurons or purified cultures of rat cerebrocortical neurons from several forms of cell death. Reverse transcriptase polymerase chain reaction and Western blot analysis revealed that GT1-7 neurons possessed both estrogen receptor-alpha (ERalpha) and ERbeta mRNA and protein, whereas purified embryonic rat cortical neurons only expressed appreciable levels of ERalpha transcript and protein, with little to no expression of ERbeta. In contrast to the lack of protection in the purified neuronal cultures, both 17beta- E2 and tamoxifen significantly protected mixed glial/ neuronal cortical cultures from cell death, suggesting that glia may facilitate 17beta-E2-and tamoxifen-mediated neuroprotection. Furthermore, astrocyte-conditioned media and exogenous transforming growth factor-beta1, a documented astrocyte-derived cytokine, were shown to rescue purified cortical neurons from cell death. Together, these findings support a role for astrocytes in neuroprotection and raise the intriguing possibility that astrocytes may help mediate the neuroprotective effect of 17beta-E2 and tamoxifen.