Estrogen receptor-Beta activated apoptosis in benign hyperplasia and cancer of the prostate is androgen independent and TNF-alpha mediated

Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are androgen-dependent diseases commonly treated by inhibiting androgen action. However, androgen ablation or castration fail to target androgen-independent cells implicated in disease etiology and recurrence. Mechanistically different to castration, this study shows beneficial proapoptotic actions of estrogen receptor–β (ERβ) in BPH and PCa. ERβ agonist induces apoptosis in prostatic stromal, luminal and castrate-resistant basal epithelial cells of estrogen-deficient aromatase knock-out mice. This occurs via extrinsic (caspase-8) pathways, without reducing serum hormones, and perturbs the regenerative capacity of the epithelium. TNFα knock-out mice fail to respond to ERβ agonist, demonstrating the requirement for TNFα signaling. In human tissues, ERβ agonist induces apoptosis in stroma and epithelium of xenografted BPH specimens, including in the CD133+ enriched putative stem/progenitor cells isolated from BPH-1 cells in vitro. In PCa, ERβ causes apoptosis in Gleason Grade 7 xenografted tissues and androgen-independent cells lines (PC3 and DU145) via caspase-8. These data provide evidence of the beneficial effects of ERβ agonist on epithelium and stroma of BPH, as well as androgen-independent tumor cells implicated in recurrent disease. Our data are indicative of the therapeutic potential of ERβ agonist for treatment of PCa and/or BPH with or without androgen withdrawal.

Estrogen Receptor-beta Affects the Prognosis of Human Malignant Mesothelioma

Malignant pleural mesothelioma is an asbestos-related neoplasm with poor prognosis, refractory to current therapies, the incidence of which is expected to increase in the next decades. Female gender was identified as a positive prognostic factor among other clinical and biological prognostic markers for malignant mesothelioma, yet a role of estrogen receptors (ERs) has not been studied. Our goal was to investigate ERs expression in malignant mesothelioma and to assess whether their expression correlates with prognosis. Immunohistochemical analysis revealed intense nuclear ER beta staining in normal pleura that was reduced in tumor tissues. Conversely, neither tumors nor normal pleura stained positive for ER alpha. Multivariate analysis of 78 malignant mesothelioma patients with pathologic stage, histologic type, therapy, sex, and age at diagnosis indicated that FRO expression is an independent prognostic factor of better survival. Moreover, studies in vitro confirmed that treatment with 17 beta-estradiol led to an ER beta-mediated inhibition of malignant mesothelioma cell proliferation as well as p21(CIP1) and p27(KIP1) up-regulation. Consistently cell growth was suppressed by ER beta overexpression, causing a G(2)-M-phase cell cycle arrest, paralleled by cyclin B1 and survivin down-regulation. Our data support the notion that ER beta acting as a tumor suppressor is of high potential relevance to prediction of disease progression and to therapeutic response of malignant mesothelioma patients. [Cancer Res 2009;69(11):4598-604]

Evidence for associations between common polymorphisms of estrogen receptor beta gene with homocysteine and nitric oxide

Background Homocysteine and asymmetric dimethylarginine (ADMA) affect nitric oxide (NO) concentration, thereby contributing to cardiovascular disease (CVD). Both amino acids can be reduced in vivo by estrogen. Variation in the estrogen receptor (ER) may influence homocysteine and ADMA, yet no information is available on associations with single nucleotide polymorphisms in the estrogen receptor genes ER alpha (PvuII and XbaI) and ER beta (1730G -> A and cx+56 G -> A). Objective To find relationships between common polymorphisms associated with cardiovascular disease and cardiovascular risk factors homocysteine and ADMA. Methods In a cross-sectional study with healthy postmenopausal women (n = 89), homocysteine, ADMA, nitric oxide metabolites (NOx), plasma folate and ER alpha and beta polymorphisms ER alpha PvuII, ER alpha XbaI; ER beta 1730G -> A (AluI), ER beta cx+56 G -> A (Tsp5091) were analyzed. Results Women who are homozygotic for ER beta cx+56 G -> A A/A exhibited higher homocysteine (p = 0.012) and NOx (p = 0.056) levels than wildtype or heterozygotes. NOx concentration was also significantly affected by ER beta 1730 G -> A polymorphism (p = 0.025). The ER beta (p < 0.001) and ER alpha (p < 0.001) polymorphisms were in linkage disequilibrium. Conclusions Women who are homozygotic for ER beta cx+S6 G -> A A/A may be at increased risk for cardiovascular disease due to higher homocysteine levels.

Immunohistochemical detection of estrogen receptor beta in alveolar bone cells of estradiol-treated female rats: possible direct action of estrogen on osteoclast life span

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estrogen receptor β activation impairs mitochondrial oxidative metabolism and affects malignant mesothelioma cell growth in vitro and in vivo

Estrogen receptor (ER)-β has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERβ coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERβ, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERβ agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERβ-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.

High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

In this study, we evaluated the effects of obesity and insulin resistance induced by a high-fat diet on prostate morphophysiology, focusing on cell proliferation, expression of androgen (AR) and estrogen receptors (ER) and proteins of the insulin signaling pathway. Adult male Wistar rats were fed a high-fat diet (20% fat) for 15 weeks, whereas control animals received a balanced diet (4% fat). Both groups were then divided and treated for 2 weeks with 1 mg/kg body weight/day of the aromatase inhibitor letrozole or vehicle only. The ventral prostate was analyzed with immunohistochemical, histopathological, stereological, and Western blotting methods. Obese rats showed insulin resistance, hyperinsulinemia, and reduced plasma testosterone levels. The incidence of prostatic intraepithelial neoplasia (PIN) was 2.7 times higher in obese rats and affected 0.4% of the gland compared with 0.1% PIN areas found in control rats. Obesity doubled cell proliferation in both prostate epithelium and stroma. AR content decreased in the prostate of obese rats and estrogen receptor beta (ER beta) increased in this group. Protein levels of insulin receptor substrate 1 and protein kinase B diminished in the obese group, whereas phosphatidylinositol 3-kinase (PI3K) increased significantly. Most structural changes observed in the prostate of obese rats normalized after letrozole treatment, except for increased stromal cell proliferation and ER beta expression, which might be associated with insulin resistance. This experimental model of obesity and insulin resistance induced by a high-fat diet increases cell proliferation in rat prostate. Such alterations are associated with decreased levels of AR and increased ER beta and PI3K proteins. This change can facilitate the establishment of proliferative lesions in rat prostate.

Estrogen receptor subtype beta2 is involved in neuromast development in zebrafish (Danio rerio) larvae

Estrogens are known to play a role in both reproductive and non-reproductive functions in mammals. Estrogens and their receptors are involved in the development of the central nervous system (brain development, neuronal survival and differentiation) as well as in the development of the peripheral nervous system (sensory-motor behaviors). In order to decipher possible functions of estrogens in early development of the zebrafish sensory system, we investigated the role of estrogen receptor beta(2) (ERbeta(2)) by using a morpholino (MO) approach blocking erbeta(2) RNA translation. We further investigated the development of lateral line organs by cell-specific labeling, which revealed a disrupted development of neuromasts in morphants. The supporting cells developed and migrated normally. Sensory hair cells, however, were absent in morphants' neuromasts. Microarray analysis and subsequent in situ hybridizations indicated an aberrant activation of the Notch signaling pathway in ERbeta(2) morphants. We conclude that signaling via ERbeta(2) is essential for hair cell development and may involve an interaction with the Notch signaling pathway during cell fate decision in the neuromast maturation process.

Differential crosstalk between estrogen receptor (ER) alpha and ER beta and the thyroid hormone receptor isoforms results in flexible regulation of the consensus ERE

Crosstalk between nuclear receptors is important for conversion of external and internal stimuli to a physiologically meaningful response by cells. Previous studies from this laboratory have demonstrated crosstalk between the estrogen (ER) and thyroid hormone receptors (TR) on two estrogen responsive physiological promoters, the preproenkephalin and oxytocin receptor gene promoter. Since ERa and ERb are isoforms possessing overlapping and distinct transactivation properties, we hypothesized that the interaction of ERa and b with the various TR isoforms would not be equivalent. To explore this hypothesis, the consensus estrogen response element (ERE)derived from the Xenopus vitellogenin gene is used to investigate the differences in interaction between ERa and b isoforms and the different TR isoforms in fibroblast cells. Both the ER isoforms transactivate from the consensus ERE, though ERa transactivates to a greater extent than ERb. Although neither of the TRb isoforms have an effect on ERa transactivation from the consensus ERE, the liganded TRa1 inhibits the ERa transactivation from the consensus ERE. In contrast, the liganded TRa1 facilitates ERb-mediated transactivation. The crosstalk between the TRb isoforms with the ERa isoform, on the consensus ERE, is different from that with the ERb isoform. The use of a TRa1 mutant, which is unable to bind DNA, abolishes the ability of the TRa1 isoform to interact with either of the ER isoforms. These differences in nuclear receptor crosstalk reveal an important functional difference between isoforms, which provides a novel mechanism for neuroendocrine integration.

Evaluation of estrogen receptor alpha and beta and progesterone receptor expression and correlation with clinicopathologic factors and proliferative marker Ki-67 in breast cancers

To elucidate the molecular profile of hormonal steroid receptor status, we analyzed ER-alpha, ER-beta, and PGR mRNA and protein expression in 80 breast carcinomas using reverse transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR, and immunohistochemical analysis. Qualitative analysis revealed positive expression of ER-alpha, ER-beta, and PGR mRNA in 48%, 59%, and 48% of the breast carcinomas, respectively. ER-alpha, ER-beta, and PGR transcript overexpression was observed in 51%, 0%, and 12% of the cases, respectively, whereas moderate or strong protein expression was detected in 68%, 78%, and 49% of the cases, respectively. Tumor grade was negatively correlated with transcript and protein levels of ER-alpha (P = .0169 and P = .0006, respectively) and PGR (P = .0034 and P = .0005, respectively). Similarly, proliferative index Ki-67 was negatively associated with transcript and protein levels of ER-alpha (P = .0006 and P < .0001, respectively) and PGR (P = .0258 and P =. 0005, respectively). These findings suggest that ER-alpha and PGR expression are associated with well-differentiated breast tumors and less directly related to cell proliferation. A significant statistical difference was observed between lymph node status and ER-beta protein expression (P = .0208). In ER-alpha-negative tumors, we detected a correlation between ER-beta protein expression and high levels of Ki-67. These data suggest that ER-beta could be a prognostic marker in human breast cancer. (C) 2008 Elsevier B.V. All rights reserved.

Differential expression of aromatase, estrogen receptor alpha and 17 beta-HSD associated with the processes of total testicular regression and recrudescence in the bat Myotis nigricans (Chiroptera: Vespertilionidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The measurement of adenosine and estrogen receptor expression in rat brains following ovariectomy using quantitative PCR analysis

In our laboratory we have developed a quantitative-polymerase chain reaction (Q-PCR) strategy to examine the differential expression of adenosine receptor (ADOR), A(1), A(2A), A(2B) and A(3), and estrogen receptors (ER) alpha and beta. Brain and uterine mRNA were first used to optimise specific amplification conditions prior to SYBR Green I real time analysis of receptor subtype expression. SYBR Green I provided a convenient and sensitive means of examining specific PCR amplification product in real time, and allowed the generation of standard curves from which relative receptor abundance could be determined. Real time Q-PCR analysis was then performed, to examine changes in receptor expression levels in brains of adult female Wistar rats 3-month post ovariectomy. Comparison with sham-operated age-matched control rats demonstrated both comparative and absolute-copy number changes in receptor levels. Evaluation of both analytical methods investigated 18S rRNA as an internal reference for comparative gene expression analysis in the brain. The results of this study revealed preferential repression of ADORA(2A) (>4-fold down) and consistent (>2-fold) down-regulation of ADORA(1), ADORA(3), and ER-beta, following ovariectomy. No change was found in ADORA(2B) or ER-alpha. Analysis of absolute copy number in this study revealed a correlation between receptor expression in response to ovariectomy, and relative receptor subtype abundance in the brain.

Differential interaction of estrogen receptor and thyroid hormone receptor isoforms on the rat oxytocin receptor promoter leads to differences in transcriptional regulation

Both the estrogen receptor (ER) and thyroid hormone receptor (TR) are members of the nuclear receptor superfamily. Two isoforms of the ER, alpha and beta, exist. The TRalpha and beta isoforms are products of two distinct genes that are further differentially spliced to give TRalpha1 and alpha2, TRbeta1 and beta2. The TRs have been shown to interfere with ER-mediated transcription from both the consensus estrogen response element (ERE) and the rat preproenkephalin (PPE) promoter, possibly by competing with ER binding to the ERE or by squelching coactivators essential for ER-mediated transcription. The rat oxytocin receptor (OTR) gene is thought to be involved in several facets of reproductive and affiliative behaviors. 17beta-Estradiol-bound ERs upregulate the OTR gene in the ventromedial hypothalamus, a region critical for the induction of lordosis behavior in several species. We investigated the effects of the ligand-binding TR isoforms on the ER-mediated transcription from a physiological promoter of a behaviorally relevant gene such as the OTR. Only ERalpha could induce the OTR gene in two cell lines tested, the CV-1 and the SK-N-BE2C neuroblastoma cell lines. ERbeta was incapable of inducing the gene in either cell line. ERalpha is therefore not equivalent to ERbeta on this physiological promoter. Indeed, in the neural cell line, ERbeta can inhibit ERalpha-mediated induction from the OTR promoter. While the TRalpha1 isoform inhibited ERalpha-mediated induction in the neural cell line, the TRbeta1 isoform stimulated induction, thus demonstrating isoform specificity in the interaction. The use of a DNA-binding mutant, the TR P box mutant, showed that inhibition of ERalpha-mediated induction of the rat OTR gene promoter by the TRalpha1 isoform does not require DNA-binding ability. SRC-1 overexpression relieved TRalpha1-mediated inhibition in both cell lines, suggesting that squelching for coactivators is an important molecular mechanism in TRalpha-mediated inhibition. Such interactions between TR and ER isoforms on the rat OTR promoter provide a mechanism to achieve neuroendocrine integration.