Human estrogen receptor alpha gene is a target of Runx2 transcription factor in osteoblasts

Several studies into the mechanisms involved in control of osteoblast-specific gene expression have identified Runx2 and ERalpha (estrogen receptor alpha) as essential regulators of osteoblast differentiation. Recently, interactions between Runx2 and ERalpha have been described. Here, we investigate the role of Runx2 on the regulation of ERalpha expression by determining its interaction with the F promoter, one of the multiple promoters of the human ERalpha gene and the only one active in bone. We found that, in this promoter, three Runx2-like sites are present. By electrophoretic mobility shift assay in combination with supershift and ChIP experiments, we demonstrated that Runx2 preferentially binds one of the Runx2 motifs of the F promoter. To understand whether or not they are involved in influencing F promoter activity, different promoter-reporter deletion and mutation constructs were transiently transfected into human osteoblastic cells. Comparison of luciferase activities allowed the identification of a prevalent negative role of a sequence context, within the -117,877/-117,426 region, which may be under the control of Runx2 (a) site. Finally, silencing and overexpression of endogenous Runx2 provided evidence that Runx2 has a more complex role than initially expected. In fact, Runx2 (a) and Runx2 (b) sites carried out opposite roles which are conditioned by Runx2 levels in bone cells. Therefore, the resulting F promoter activity may be tightly regulated by a dynamic interplay between these two Runx2 sites, with a predominance of negative effect of the Runx2 (a) site.     

Polymorphisms of estrogen receptor alpha gene in endometrial cancer

It is hypothesized that polymorphisms of estrogen receptor-alpha (ERalpha) gene are involved in endometrial cancer. To test this hypothesis, the genotype distributions of six different loci (codon 10 T-->C, codon 87 G-->C, codon 243 C-->T, codon 325 C-->G, codon 594 G-->A, and intron 1 C-->G) of the ERalpha gene were investigated and their association with endometrial cancer was determined. The DNA from 113 cases of human endometrial cancer was analyzed by sequence-specific polymerase chain reaction. The relative risk of variant genotype was calculated by comparison with 200 healthy controls. The frequency of variant genotype on codon 10 was significantly lower in endometrial cancer patients as compared to controls. Nine of 113 endometrial cancer patients (8.0%) showed genotype 10C/C compared to 27 of 200 healthy controls (13.5%). The relative risk of genotype 10C/C was calculated as 0.44, compared to wild-type. Forty-five of 113 endometrial cancer patients (39.8%) showed genotype T/C on codon 10 compared to 111 of 200 healthy controls (55.5%). The relative risk of genotype 10T/C was calculated as 0.67, compared to wild-type. The polymorphism on codon 87 was not detected both in endometrial cancer patients and in healthy control. Other loci, intron 1, and codons 243, 325, and 594, did not show a correlation with endometrial cancer. The frequency of alleles on codon 10 was also significantly lower in endometrial cancer patients as compared to controls. Sixty-three of 226 alleles (27.9%) of endometrial cancer patients showed allele C compared to 165 of 400 (41.2%) of healthy controls. The relative risk of allele 10C was calculated as 0.67, compared to wild-type. Other loci, intron 1, and codons 243, 325, and 594, did not show a difference between cancer patients and controls. All genotype and allelic distributions were in accordance with the Hardy-Weinberg equilibrium. The present study demonstrates for the first time a protective effect of 10C allele against endometrial cancer. Thus, inherited alterations in ERalpha may be associated with changes in estrogen metabolism and thereby may possibly explain inter-individual differences in disease incidences of endometrial cancer.     

Structural insight into the antagonistic action of diarylheptanoid on human estrogen receptor alpha

Estrogen receptor α (ER α) is an important therapeutic target in the regulation of ligand dependent signaling in breast cancer. The current study investigates the anti-estrogenic potential of the Diarylheptanoid, 5-hydroxy-7-(4-hydroxy-3 methoxyphenyl)-1-phenyl-3-heptanone (DAH) in silico. Rigid Docking analysis of DAH at the ligand binding domain (LBD) of ER α showed hydrogen bond interactions with Arg394 and Glu353 at the active site, similar to the positive controls 4-Hydroxy Tamoxifen (4-OHT) and Fulvestrant (FUL). The protein and the protein–DAH complexes were further analyzed using molecular dynamics simulations for a time scale of 50 ns using GROMACS. Root mean square fluctuation (RMSF) analysis showed large fluctuations at the N-terminal region of Helices (H) 3, 9 and at the C-terminal region of H11, which could be involved in the antagonistic conformational change. Interestingly, H12 appeared to move away from the ligand binding pocket and occupy the co-activator binding groove at the LBD of ER α. Secondary structure analysis of the protein upon binding of DAH and CUR showed structural change from α-helix to Turn conformation at H4. We hypothesize that this structural change at H4, similar to the positive control, could hinder the activity of AF-2 by blocking the binding of co-activator. These conformational changes in ER α indicate an anti-estrogenic and therapeutic potential of the DAH.     

Estrogen regulation of trefoil factor 1 expression by estrogen receptor alpha and Sp proteins

Estrogen-responsive genes in human breast cancer cells often have an estrogen response element (ERE) positioned next to an Sp1 binding site. In chromatin immunoprecipitation (ChIP) assays, we investigated the binding of estrogen receptor alpha (ER), Sp1, and Sp3 to the episomal and native estrogen-responsive trefoil factor 1 (TFF1; formerly pS2) promoter in MCF-7 breast cancer cells. Mutation of the Sp site upstream of the ERE reduced estrogen responsiveness and prevented binding of Sp1 and Sp3, but not ER to the episomal promoter. In the absence of estradiol (E2), Sp1, Sp3, histone deacetylase 1 (HDAC), and HDAC2, and low levels of acetylated H3 and H4 are associated with the native promoter, with the histones being engaged in dynamic reversible acetylation. Following E2 addition, levels of ER and acetylated H3 and H4 bound to the native promoter increases. There is clearance of Sp1, but not of Sp3, from the promoter while HDAC1 and HDAC2 remain bound. These data are consistent with a model in which Sp1 or Sp3 aid in recruitment of HDACs and histone acetyltransferases (HATs) to mediate dynamic acetylation of histones associated with the TFF1 promoter, which is in a state of readiness to respond to events occurring following the addition of estrogen.     

Estradiol and a selective estrogen receptor modulator affect steroid hormone receptor messenger RNA levels and turnover in explant cultures of sheep endometrium

Estrogens upregulate estrogen receptor (ER) and progesterone receptor (PR) gene expression in endometrium immediately before ovulation to prepare it for nurturing embryos. Most in vitro model systems have lost the ability to upregulate expression of the ER gene in response to estradiol (E2) or the ability to express the ER gene at all. Here, we used explant cultures from control and E2-treated ewes and assessed expression of four genes (ER, PR, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], and cyclophilin [CYC] genes) that are upregulated by E2 in vivo on Northern blots. In cultures from control and E2-treated ewes, ER and PR messenger ribonucleic acid (mRNA) levels dropped significantly during 24 h of culture in the absence of E2. Glyceraldehyde 3-phosphate dehydrogenase mRNA levels increased 300% in explants from control ewes to match the higher levels in the endometrium of the E2-treated ewe (in vivo and in explant culture). The only effect of E2 in the explant cultures was to prevent the decrease in PR mRNA. The new selective ER modulator, EM-800 (EM), decreased ER and PR mRNA levels in explants from control ewes but upregulated GAPDH and CYC mRNA levels. The EM treatment in vitro mimicked that of E2 by increasing the half-life of ER mRNA in endometrial explants. These data illustrate distinct, gene-specific effects of the explant culture process, E2, and EM on the expression of endometrial genes.     

Endometrial effects of selective estrogen receptor modulators (SERMs) on estradiol-responsive gene expression are gene and cell-specific

Three selective estrogen receptor modulator (SERM) drugs which included 4-OH-tamoxifen (Tam), EM-800 (EM) and GW 5638 (GW) were investigated to determine their ability to inhibit estradiol-responsive gene expression in sheep endometrium. The uteri of ovariectomized ewes (10 ewes per SERM group) were infused with 10⁻⁷ M SERMs for 24 h prior to hysterectomy. Five ewes from each group received 50 μg 17β-estradiol (E2) and the remaining five ewes received vehicle 18 h prior to hysterectomy. Northern blot analyses and in situ hybridization demonstrated that E2 treatment increased estrogen receptor (ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and cyclophilin (CYC) mRNA levels in most endometrial cells examined. Tam and GW exhibited characteristics similar to E2 by increasing ER gene expression, but they antagonized the E2-induced increases in PR and CYC mRNA levels. EM acted as an E2-agonist of GAPDH gene expression, but antagonized the E2 up-regulation of ER, PR and CYC gene expression in most endometrial cells. Immunohistochemistry determined that EM decreased ER protein levels in the glandular epithelium, and the SERMs investigated antagonized increases in PR protein levels in endometrium. In conclusion, GW and EM exhibit fewer agonist effects than Tam on endometrial gene expression. EM demonstrated the greatest antagonism of E2-enhanced levels of ER, PR and CYC, likely due to the inhibition of ER gene expression at both mRNA and protein levels.     

二恶英对雌激素受体的干扰作用

核受体相关的肿瘤,如乳腺癌和前列腺癌的发生、发展主要依赖于性激素的分泌。环境内分泌干扰物如Ⅲ二恶英,可作为雌激素受体的诱导配体,直接或间接地与转录因子相互作用,从而干扰基因的转录。此外,二恶英还能使雌激素受体发生泛素化修饰,促进它的降解,并使雌激素依赖性的基因转录受到抑制,成为内分泌干扰因子。本文综述了二恶英的类雌激素效应、致癌性以及激素信号干扰的最新研究进展。     

Estrogen receptor alpha/beta isoforms, but not betacx, modulate unique patterns of gene expression and cell proliferation in Hs578T cells

The actions of 17beta-estradiol (E2) and selective estrogen receptor modulators (SERMs) have been extensively investigated regarding their ability to act through estrogen receptor-alpha (ERalpha) to perturb estrogen receptor positive (ER+) breast cancer (BC) growth. However, many BCs also express ERbeta, along with multiple estrogen receptor (ER) splice variants such as ERbetacx, an ERbeta splice variant incapable of binding ligand. To gain a more comprehensive understanding of ER action in BC cells, we stably expressed ERalpha, ERbeta, or ERbetacx under doxycycline (Dox) control in Hs578T cells. Microarrays performed on E2 or 4OH-tamoxifen (4HT) treated Hs578T ERalpha and ERbeta cells revealed distinct ligand and receptor-dependent patterns of gene regulation, while the induction of ERbetacx did not alter gene expression patterns. E2 stimulation of Hs578T ERbeta cells resulted in a 27% decrease in cellular proliferation, however, no significant change in proliferation was observed following the exposure of Hs578T ERalpha or ERbeta cells to 4HT. Expression of ERbetacx in Hs578T cells did not effect cellular proliferation. Flow cytometry assays revealed a 50% decrease in E2-stimulated Hs578T ERbeta cells entering S-phase, along with a 17% increase in G0/G1 cell-cycle arrest. We demonstrate here that ERalpha and ERbeta regulate unique gene expression patterns in Hs578T cells, and such regulation likely is responsible for the observed isoform-specific changes in cell proliferation. Hs578T ER expressing cell-lines provide a unique BC model system, permitting the comparison of ERalpha, ERbeta, and ERbetacx actions in the same cell-line.     

Myometrial effects of selective estrogen receptor modulators on estradiol-responsive gene expression are gene and cell-specific

We examined in vivo effects of selective estrogen receptor modulators (SERMs) 4-OH-tamoxifen (Tam), GW 5638 (GW) and EM-800 (EM) on myometrial gene expression. The uteri of ovariectomized ewes were infused with 10⁻⁷ M of one SERM via indwelling catheters for 24 h preceding hysterectomy. Half of the ewes in each SERM group received an intramuscular injection of 50 μg 17β-estradiol (E2) 18 h prior to hysterectomy. Northern blot analysis and in situ hybridization demonstrated that E2 increased estrogen receptor (ER), progesterone receptor (PR) and cyclophilin (CYC) gene expression in the cells of both inner layer of myometrium (IM) and outer layer of myometrium (OM) as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in OM. Tam also increased ER mRNA levels in OM. EM appeared to increase ER gene expression, but antagonized E2’s up-regulation of PR and CYC gene expression in both IM and OM. Tam and GW also antagonized E2 up-regulation of PR gene expression in OM but not IM. No SERM affected GAPDH gene expression with or without E2. Immunohistochemistry indicated that E2 increased nuclear ER and PR protein levels in both IM and OM. EM was unique in up-regulating ER protein levels, opposite to its effects in endometrial cells. All SERMs tested antagonized this increase in PR immunostaining preferentially in OM compared to the IM layer. These results illustrate gene and cell layer-specific effects of SERMs in sheep myometrium.     

Estrogen-mediated neuroprotection against beta-amyloid toxicity requires expression of estrogen receptor alpha or beta and activation of the MAPK pathway

It is well documented that estrogen can activate rapid signaling pathways in a variety of cell types. These non-classical effects of estrogen have been reported to be important for cell survival after exposure to a variety of neurotoxic insults. Since direct evidence of the ability of the estrogen receptors (ERs) alpha and/or beta to mediate such responses is lacking, the hippocampal-derived cell line HT22 was stably transfected with either ERalpha (HTERalpha) or ERbeta (HTERbeta). In HTERalpha and HTERbeta cells, but not untransfected cells, an increase in ERK2 phosphorylation was measured within 15 min of 17beta-estradiol treatment. The ER antagonist ICI 182, 780 (1 microm) and the MEK inhibitor, PD98059 (50 microm) blocked this increase in ERK2 phosphorylation. Treatment of HT22, HTERalpha and HTERbeta cells with the beta-amyloid peptide (25-35) (10 micro m) resulted in a significant decrease in cell viability. Pre-treatment for 15 min with 10 nm 17beta-estradiol resulted in a 50% increase in the number of living cells in HTERalpha and HTERbeta cells, but not in HT22 cells. Finally, ICI 182, 780 and PD98059 prevented 17beta-estradiol-mediated protection. This study demonstrates that both ERalpha and ERbeta can couple to rapid signaling events that mediate estrogen-elicited neuroprotection.     

Genotype/age interactions on aggressive behavior in gonadally intact estrogen receptor beta knockout (betaERKO) male mice

Estrogen, as an aromatized metabolite of testosterone, has a facilitatory effect on male aggressive behavior in mice. Two subtypes of estrogen receptors, alpha (ER-alpha) and beta (ER-beta), in the brain are known to bind estrogen. Previous studies revealed that the lack of ER-alpha gene severely reduced the induction of male aggressive behavior. In contrast, mice that lacked the ER-beta gene tended to be more aggressive than wild type (WT) control mice, although the behavioral effects of ER-beta gene disruption were dependent on their social experience. These findings lead us to hypothesize that estrogen may facilitate aggression via ER-alpha whereas it may inhibit aggression via ER-beta. In the present study, we further investigated the role of ER-beta in the regulation of aggressive behavior by examining developmental changes starting at the time of first onset, around the age of puberty. Aggressive behaviors of ER-beta gene knockout (betaERKO) mice were examined in three different age groups, puberty, young-adult, and adult. Each mouse was tested every other day for three times in a resident-intruder paradigm against olfactory bulbectomized intruder mice and their trunk blood was collected for measurements of serum testosterone after the completion of the study. Overall, betaERKO mice were significantly more aggressive than WT. These genotype differences were more pronounced in puberty and young adult age groups, but not apparent in the adult age group, in which betaERKO mice were less aggressive than those in two younger age groups. Serum testosterone levels of betaERKO mice were significantly higher than those of WT mice only in the pubertal age group, but not in young adult (when betaERKO mice were still significantly more aggressive than WT mice) and adult (when no genotype differences in aggression were found) age groups. These results suggest that ER-beta mediated actions of gonadal steroids may more profoundly be involved in the inhibitory regulation of aggressive behavior in pubertal and young adult mice.