Ovarian ERß cistrome and transcriptome reveal chromatin interaction with LRH-1.

BACKGROUND: Estrogen receptor beta (ERß, Esr2) plays a pivotal role in folliculogenesis and ovulation, yet its exact mechanism of action is mainly uncharacterized. RESULTS: We here performed ERß ChIP-sequencing of mouse ovaries followed by complementary RNA-sequencing of wild-type and ERß knockout ovaries. By integrating the ERß cistrome and transcriptome, we identified its direct target genes and enriched biological functions in the ovary. This demonstrated its strong impact on genes regulating organism development, cell migration, lipid metabolism, response to hypoxia, and response to estrogen. Cell-type deconvolution analysis of the bulk RNA-seq data revealed a decrease in luteal cells and an increased proportion of theca cells and a specific type of cumulus cells upon ERß loss. Moreover, we identified a significant overlap with the gene regulatory network of liver receptor homolog 1 (LRH-1, Nr5a2) and showed that ERß and LRH-1 extensively bound to the same chromatin locations in granulosa cells. Using ChIP-reChIP, we corroborated simultaneous ERß and LRH-1 co-binding at the ERß-repressed gene Greb1 but not at the ERß-upregulated genes Cyp11a1 and Fkbp5. Transactivation assay experimentation further showed that ERß and LRH-1 can inhibit their respective transcriptional activity at classical response elements. CONCLUSIONS: By characterizing the genome-wide endogenous ERß chromatin binding, gene regulations, and extensive crosstalk between ERß and LRH-1, along with experimental corroborations, our data offer genome-wide mechanistic underpinnings of ovarian physiology and fertility.

Regulation of Follicular Development in Chickens: WIF1 Modulates Granulosa Cell Proliferation and Progesterone Synthesis via Wnt/ß-Catenin Signaling Pathway.

Proliferation, apoptosis, and steroid hormone secretion by granulosa cells (GCs) and theca cells (TCs) are essential for maintaining the fate of chicken follicles. Our previous study showed that the Wnt inhibitor factor 1 (WIF1) plays a role in follicle selection. However, the significance of WIF1 in GC- and TC-associated follicular development was not explicitly investigated. This study found that WIF1 expression was strongly downregulated during follicle selection (p < 0.05) and was significantly higher in GCs than in TCs (p < 0.05). WIF1 inhibits proliferation and promotes apoptosis in GCs. Additionally, it promotes progesterone secretion in prehierarchal GCs (pre-GCs, 1.16 ± 0.05 ng/mg vs. 1.58 ng/mg ± 0.12, p < 0.05) and hierarchal GCs (hie-GCs, 395.00 ng/mg ± 34.73 vs. 527.77 ng/mg ± 27.19, p < 0.05) with the participation of the follicle-stimulating hormone (FSH). WIF1 affected canonical Wnt pathways and phosphorylated ß-catenin expression in GCs. Furthermore, 604 upregulated differentially expressed genes (DEGs) and 360 downregulated DEGs in WIF1-overexpressed GCs were found through RNA-seq analysis (criteria: |log2⁡(FoldChange)| > 1 and p_adj < 0.05). Cytokine-cytokine receptor interaction and the steroid hormone biosynthesis pathway were identified. In addition, the transcript of estrogen receptor 2 (ESR2) increased significantly (log2⁡(FoldChange) = 1.27, p_adj < 0.05). Furthermore, we found that WIF1 regulated progesterone synthesis by upregulating ESR2 expression in GCs. Additionally, WIF1 suppressed proliferation and promoted apoptosis in TCs. Taken together, these results reveal that WIF1 stimulates follicle development by promoting GC differentiation and progesterone synthesis, which provides an insight into the molecular mechanism of follicle selection and egg-laying performance in poultry.

Bisphenol A and Di(2-Ethylhexyl) Phthalate promote pulmonary carcinoma in female rats via estrogen receptor beta: In vivo and in silico analysis.

The prevalence of lung cancer in women currently merits our attentions. However, cigarette exposure alone does not tell the whole story that lung cancer is more prevalent among non-smoking women. Since female lung cancer is closely linked to estrogen levels, many of endocrine disrupting chemicals (EDCs), as the substances similar to estrogen, affect hormone levels and become a potential risk of female lung cancer. Additionally, the combined toxicity of EDCs in daily environment has only been discussed on a limited scale. Consequently, this study explored the cancer-promoting effect of two representative substances of EDCs namely Bisphenol A (BPA) and Di(2-Ethylhexyl) Phthalate (DEHP) after their exposure alone or in combination, using a rat pulmonary tumor model published previously, combining bioinformatics analysis based on The Comparative Toxicogenomics Database (CTD) and The Cancer Genome Atlas (TCGA) databases. It demonstrated that BPA and DEHP enhanced the promotion of pulmonary tumor in female rats, either alone or in combination. Mechanistically, BPA and DEHP mainly directly bound and activated ESR2 protein, phosphorylated CREB protein, activated HDAC6 transcriptionally, induced the production of the proto-oncogene c-MYC, and accelerated the formation of pulmonary tumor in female rats. Remarkably, BPA, rather than DEHP, exhibited a much more critical effect in female lung cancer. Additionally, the transcription factor ESR2 was most affected in carcinogenesis, causing genetic disruption. Furthermore, the TCGA database revealed that ESR2 could enhance the promotion and progression of non-small cell lung cancer in females via activating the WNT/ß-catenin pathway. Finally, our findings demonstrated that BPA and DEHP could enhance the promotion of pulmonary carcinoma via ESR2 in female rats and provide a potential and valuable insight into the causes and prevention of lung cancer in non-smoking women due to EDCs exposure.

Estrogen Receptor-ß Gene Cytosine-Adenine (ESR2-CA) Repeat Polymorphism in Postmenopausal Colon Cancer.

The pathobiological role of estrogen is controversial in colorectal cancer. Cytosine-adenine (CA) repeat in the estrogen receptor (ER)-ß gene (ESR2-CA) is a microsatellite, as well as representative of ESR2 polymorphism. Though its function is unknown, we previously showed that a shorter allele (germline) increased the risk of colon cancer in older women, whereas it decreased it in younger postmenopausal women. ESR2-CA and ER-ß expressions were examined in cancerous (Ca) and non-cancerous (NonCa) tissue pairs from 114 postmenopausal women, and comparisons were made considering tissue types, age/locus, and the mismatch repair protein (MMR) status. ESR2-CA repeats <22/≥22 were designated as 'S'/'L', respectively, resulting in genotypes SS/nSS (=SL&LL). In NonCa, the rate of the SS genotype and ER-ß expression level were significantly higher in right-sided cases of women ≥70 (≥70Rt) than in those in the others. A decreased ER-ß expression in Ca compared with NonCa was observed in proficient-MMR, but not in deficient-MMR. In NonCa, but not in Ca, ER-ß expression was significantly higher in SS than in nSS. ≥70Rt cases were characterized by NonCa with a high rate of SS genotype or high ER-ß expression. The germline ESR2-CA genotype and resulting ER-ß expression were considered to affect the clinical characteristics (age/locus/MMR status) of colon cancer, supporting our previous findings.

Genomic exploration of the targets of FOXL2 and ESR2 unveils their implication in cell migration, invasion, and adhesion.

FOXL2 and ESR2 are key transcriptional regulators in ovarian granulosa cells. To explore their transcriptional roles and their interplay, we have depleted Foxl2 and Esr2 in mouse primary granulosa cells to assess their ability to bind their targets and/or to modulate gene expression and cellular functions. We show that FOXL2 is involved in a large number of regulatory actions essential for the maintenance of granulosa cell fate. A parallel ChIP-seq analysis showed that FOXL2 mainly binds to sites located in intergenic regions quite far from its targets. A bioinformatic analysis demonstrated that FOXL2-activated genes were enriched in peaks associated with the H3K27ac mark, whereas FOXL2-repressed genes were not, suggesting that FOXL2 can activate transcription through binding to enhancer sites. We also identified about 500 deregulated genes upon Esr2 silencing, of which one third are also targets of FOXL2. We provide evidence showing that both factors modulate, through a coherent feed-forward loop, a number of common targets. Many of the FOXL2/ESR2 targets are involved in cell motility and, consistently, granulosa cells depleted for either Foxl2 or Esr2 exhibit decreased migration, invasion and adhesion. This effect is paralleled by the depletion of their target Phactr1, involved in actin cytoskeleton dynamics. Our analysis expands the number of direct and indirect transcriptional targets of both FOXL2 and ESR2, which deserve investigation in the context of adult-type granulosa cell tumors whose molecular diagnostic hallmark is the presence of the C134W FOXL2 pathogenic variant.

Association of ESR1 and ESR2 Polymorphisms with Osteoporosis: A Meta-Analysis from 36 Studies.

BACKGROUND: Recently, the roles of ESR1 and ESR2 polymorphisms in osteoporosis have been extensively reported, with conflicting findings. Therefore, we performed this present study to evaluate the potential associations between ESR1 and ESR2 polymorphisms and osteoporosis risk. METHODOLOGY: All included literatures published up to April 2021 were identified by searching Pubmed, Embase, Web of Science, Cochrane Library, Chinese National Knowledge Infrastructure (CNKI) and Wanfang databases. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated the associations using a fixed or random effects model. RESULTS: 36 observational studies involving five gene polymorphisms (ESR1 PvuII, ESR1 XbaI, ESR1 G2014A, ESR2 AluI and ESR2 RsaI) covering 12507 cases and 18487 controls were included. The results of our meta-analysis demonstrated the variant A allele of ESR2 RsaI polymorphism might play a remarkable protective role in developing osteoporosis under all genetic models. However, no associations were observed between ESR1 PvuII, ESR1 XbaI, ESR1 G2014A and ESR2 AluI polymorphisms with the risk of osteoporosis under all genetic models. CONCLUSIONS: Our meta-analysis suggests that genetic polymorphism in ESR2 RsaI may lead to decreased risk for osteoporosis. Further larger studies are needed to confirm this conclusion.

The masculinization steroid milieu caused by fluorene-9-bisphenol disrupts sex-typical courtship behavior in female zebrafish (Danio rerio).

In vertebrates, the behavior of congenital sex differences between males and females is highly dependent on steroid signals and hormonal milieu. The presence of endocrine disrupting chemicals (EDCs) in the environment generally plays a similar role to sex hormones, so its interference with aquatic organism population stability can not be ignored and is worth studying. Fluorene-9-bisphenol (BHPF) has been clarified as an endocrine disruptor on organisms by several studies but its mechanism in perturbation of courtship behavior of female zebrafish is not clear. Here, we proposed an automated multi-zebrafish tracking method quantifying the courtship process and reported that zebrafish females exposed to BHPF, are not receptive to males but rather court females, and lose normal ovarian function with an altered sex steroid milieu. Our results showed that BHPF damaged 17ß-estradiol synthesis by down-regulation of sox3 and cyp19a1a, linking apoptosis with ovary development and female fecundity. The down-regulated expression of estrogen signaling through an estrogen receptor, esr2b, caused the induction of masculinization of female courtship behavior and sexual preference in zebrafish females after BHPF treatment. This process might be mediated by inhibiting the transcription of a neuropeptide B (npb) in the brain. Our study reveals that the estrogen signaling pathway may play an important role in classical courtship behavior and sexual preference of zebrafish. This study provided evidence that anti-estrogenic chemical exposure caused adverse effects on the regulation of the brain-gonad-estrogen axis of aquatic organisms, which should be of concern and highlighted the importance of controlling environmental contamination.

The role of CYP19A1 and ESR2 gene polymorphisms in female androgenetic alopecia in the Polish population.

Introduction: Androgenetic alopecia is the most common type of non-cicatricial alopecia both in male and female patients. The mechanism that leads to hair loss is similar in both sexes, but the underlying cause, and especially the role of genes and sex hormones in the pathogenesis of the disease in women has not fully been explained as of yet. So far, a few attempts have been made to assess selected SNPs for CYP19A1 and ESR2 genes, but their results are not unequivocal and fully reproducible. Aim: To investigate the association of 13 CYP19A1 and 11 ESR2 gene SNPs with female androgenetic alopecia (FAGA) in a population of Polish patients, including some already genotyped SNPs of possible importance for FAGA pathophysiology in other populations. Material and methods: Twenty-four genetic polymorphisms were analysed for the ESR2 and CYP19A1 genes in 117 patients with FAGA and 128 healthy subjects treated at the Department of Dermatology in Krakow. Results: In the studied Polish population, none of the selected SNPs, frequently detected in the Caucasian population and linked with the transformation pathway of sex hormones, showed a significant association with FAGA. Conclusions: Further studies into the genetic background of androgenetic alopecia are needed. Ethnic differences as well as the size of the studied population may be of great significance for the obtained results.

ESR2 regulates PINK1-mediated mitophagy via transcriptional repression of microRNA-423 expression to promote asthma development.

Asthma represents an inflammatory airway disease related to the induction of airway eosinophilia, mucus overproduction, and bronchial hyperresponsiveness. This study explored the effects of microRNA-423 (miR-423) on mitophagy and inflammation in asthmatic mice challenged with house dust mites (HDMs) and rhinovirus (RV). By searching for differentially expressed miRNAs in the GSE25230 microarray, miR-423 was identified as our target. Moreover, miR-423 was expressed at low levels in the lung tissues from patients with asthma, and agomiR-423 significantly inhibited RV-induced inflammatory injury and activation of inflammasome signaling in mouse lung tissues. Additionally, miR-423 downregulated the expression of IL-1ß/NLRP3/Caspase-1 inflammasome signaling by targeting phosphatase and tensin homolog-induced putative kinase 1 (PINK1). Furthermore, luciferase reporter experiments and ChIP-qPCR assays revealed that estrogen receptor 2 (ESR2) transcriptionally repressed miR-423 expression by coordinating with H3K9me2 modification of the miR-423 promoter histone. Overall, ESR2 synergized with the H3K9me2 modification of the miR-423 promoter histone to transcriptionally repress miR-423 expression and increase PINK1 expression in lung tissues, resulting in asthma exacerbation.

Association between single nucleotide polymorphism of the CYP19A1 and ESR2 genes and endometriosis.

PURPOSE: Endometriosis is a frequent gynaecological condition, both in Poland and in the world. The development of this disease is supported by hormonal, immunological and environmental factors. During the recent years, a particular attention has been focused on the genetic polymorphisms which may be of particular significance for the increased incidence rates of endometriosis. According to literature data, Oestrogen Receptor 2 (ESR2) and Cytochrome P450 Family 19 Subfamily A Member 1 (CYP19A1) genes may be accounted to the potential risk factors of infertility associated with endometriosis. The reported research was aimed to evaluate the association between single nucleotide polymorphisms (SNPs) rs17179740 of ESR2 and rs2899470 of CYP19A1 genes and the incidence of endometriosis. METHODS: The study material included blood specimens, collected from patients (n = 200) with endometriosis. Blood samples from age-matched, endometriosis-free women (n = 200) served as control. The High-Resolution Melter (HRM) technique was applied for polymorphism analysis. RESULTS: Regarding rs2899470 polymorphism TT homozygotes was significantly more prevalent among the patients with endometriosis than in the controls (OR 2.19; p = 0.04). In case of rs17179740, GG homozygotes, as well as AG-AA genotypes, were significantly more prevalent among the endometriosis patients (OR 2.48, p = 0.04 and OR 2.36, p = 0.04, respectively). CONCLUSION: Summing up, the investigated polymorphisms of ESR2 and CYP19A1 gene are associated with the observed incidence of endometriosis.

Altered Expression of ESR1, ESR2, PELP1 and c-SRC Genes Is Associated with Ovarian Cancer Manifestation.

Ovarian cancer remains the leading cause of death due to gynecologic malignancy. Estrogen-related pathways genes, such as estrogen receptors (ESR1 and ESR2) and their coregulators, proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), and proto-oncogene tyrosine-protein kinase c-Src (SRC) are involved in ovarian cancer induction and development, still they require in-depth study. In our study, tissue samples were obtained from 52 females of Caucasian descent (control group without cancerous evidence (n = 27), including noncancerous benign changes (n = 15), and the ovarian carcinoma (n = 25)). Using quantitative analyses, we investigated ESRs, PELP1, and SRC mRNA expression association with ovarian tumorigenesis. Proteins' presence and their location were determined by Western blot and immunohistochemistry. Results showed that PELP1 and SRC expression levels were found to differ in tissues of different sample types. The expression patterns were complex and differed in the case of ovarian cancer patients compared to controls. The most robust protein immunoreactivity was observed for PELP1 and the weakest for ESR1. The expression patterns of analyzed genes represent a potentially interesting target in ovarian cancer biology, especially PELP1. This study suggests that specific estrogen-mediated functions in the ovary and ovary-derived cancer might result from different local interactions of estrogen with their receptors and coregulators.

Generation of an all-exon Esr2 deleted mouse line: Effects on fertility.

Estrogen receptor beta (ERß), encoded by the Esr2 gene, is one of two nuclear receptors that mediate the functions of the steroid hormone estradiol. The binding of estradiol to the receptor results in enhanced transcription of many genes that have estrogen response elements in promoter or enhancer regions. Several genetically modified mouse lines with mutations or deletions of exons in the Esr2 gene have been developed and results from analysis of these are not completely consistent, especially regarding ERß's role in fertility. To address these controversies, we have used the CRISPR/Cas9 genome editing system to make a deletion of the entire Esr2 gene in the mouse genome and determined the effect of this mutation on fertility. We show that female Esr2 deleted mice, Esr2ΔE1-10, are subfertile at young age, with fewer litters and smaller litter size, and that they become infertile/have severely reduced fertility at around six months of age, while the male Esr2ΔE1-10 mice are fertile. Ovaries from Esr2ΔE1-10 mice are smaller than those from wild-type littermates and the morphology of the ovary displays very few corpora lutea, indicating a defect in ovulation. We also show that the estradiol levels are reduced at diestrus, the phase in the estrous cycle when levels are expected to start to increase before ovulation. Our results verify that ERß has an important function in female reproduction, likely as a regulator of serum estradiol levels, and that its loss does not affect male reproductive function.

Transcriptomic insight into salinomycin mechanisms in breast cancer cell lines: synergistic effects with dasatinib and induction of estrogen receptor ß.

BACKGROUND: Tumors are heterogeneous in nature, composed of different cell populations with various mutations and/or phenotypes. Using a single drug to encounter cancer progression is generally ineffective. To improve the treatment outcome, multiple drugs of distinctive mechanisms but complementary anticancer activities (combination therapy) are often used to enhance antitumor efficacy and minimize the risk of acquiring drug resistance. We report here the synergistic effects of salinomycin (a polyether antibiotic) and dasatinib (a Src kinase inhibitor). METHODS: Functionally, both drugs induce cell cycle arrest, intracellular reactive oxygen species (iROS) production, and apoptosis. We rationalized that an overlapping of the drug activities should offer an enhanced anticancer effect, either through vertical inhibition of the Src-STAT3 axis or horizontal suppression of multiple pathways. We determined the toxicity induced by the drug combination and studied the kinetics of iROS production by fluorescence imaging and flow cytometry. Using genomic and proteomic techniques, including RNA-sequencing (RNA-seq), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Western Blot, we subsequently identified the responsible pathways that contributed to the synergistic effects of the drug combination. RESULTS: Compared to either drug alone, the drug combination showed enhanced potency against MDA-MB-468, MDA-MB-231, and MCF-7 human breast cancer (BC) cell lines and tumor spheroids. The drug combination induces both iROS generation and apoptosis in a time-dependent manner, following a 2-step kinetic profile. RNA-seq data revealed that the drug combination exhibited synergism through horizontal suppression of multiple pathways, possibly through a promotion of cell cycle arrest at the G1/S phase via the estrogen-mediated S-phase entry pathway, and partially via the BRCA1 and DNA damage response pathway. CONCLUSION: Transcriptomic analyses revealed for the first time, that the estrogen-mediated S-phase entry pathway partially contributed to the synergistic effect of the drug combination. More importantly, our studies led to the discoveries of new potential therapeutic targets, such as E2F2, as well as a novel drug-induced targeting of estrogen receptor ß (ESR2) approach for triple-negative breast cancer treatment, currently lacking of targeted therapies.

Association of the polymorphisms of the genes APOC3 (rs2854116), ESR2 (rs3020450), HFE (rs1799945), MMP1 (rs1799750) and PPARG (rs1801282) with lipodystrophy in people living with HIV on antiretroviral therapy: a systematic review.

The aim of this study was to perform a systematic review to identify data reported in the literature concerning the association of APOC3 (rs2854116), ESR2 (rs3020450), HFE (rs1799945), MMP1 (rs1799750) and PPARG (rs1801282) polymorphisms with lipodystrophy in people living with HIV (PLWHIV) on antirretroviral therapy. The research was conducted in six databases and the studies were selected in two steps. First, a search was undertaken in the following electronic databases: PubMed, Science Direct, Medline, World Wide Science, Directory of Open Access Journals, Scielo, Lilacs and Medcarib. The titles and abstracts of 24,859 articles were read to select those that match the elegibilty criteria. Five papers that addressed the association of HAART, lipodystrophy and polymorphisms were selected for the review. There was no association between the polymorphisms of the genes APOC3 and PPARG and lipodystrophy. Another study described an association between the variant allele (G) of HFE and protection concerning the development of lipoatrophy (0.02) when compared with the reference allele (C). On the other hand, the variant allele (T) of the ESR2 gene was associated with the development of lipoatrophy (p = 0.007) when compared with the reference allele (C). In addition, the genotype and the variant allele of the gene MMP1 (2G) were associated with lipodystrophy in PLWHIV on HAART (p = 0.0002 and p = 0.0008, respectively). Therefore, further studies with other populations, involving PLWHIV on HAART are necessary to better understand the role of genetic markers, which may be involved in a predisposition to lipodystrophy.

The Genetic Background of Endometriosis: Can ESR2 and CYP19A1 Genes Be a Potential Risk Factor for Its Development?

Endometriosis is defined as the presence of endometrial foci, localized beyond their primary site, i.e., the uterine cavity. The etiology of this disease is rather complex. Its development is supported by hormonal, immunological, and environmental factors. During recent years, particular attention has been focused on the genetic mechanisms that may be of particular significance for the increased incidence rates of endometriosis. According to most recent studies, ESR2 and CYP19A1 genes may account for the potential risk factors of infertility associated with endometriosis. The paper presents a thorough review of the latest reports and data concerning the genetic background of the risk for endometriosis development.

The direct effects of gonadotropin-releasing hormone on proliferation of granulosa cells and development of follicles in goose.

1. The study objectives were to determine the direct effects of gonadotropin-releasing hormone (GnRH) on the proliferation of ovarian granulosa cells (GCs) and the development of follicles in geese (Anser cygnoides) by colorimetry and ethynyl-2'-deoxyuridine (EdU) cell proliferation assays, in which primary GCs were treated with different concentrations of GnRH agonist (alarelin acetate) and an antagonist (cetrorelix acetate). Differently expressed genes (DEGs) were identified by RNA-sequencing and validated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting. 2. The EdU assays showed that the proliferation of GCs was affected by the GnRH agonist and antagonist in a dose-dependent manner. The effect of treatment on cell proliferation was statistically significant at the concentrations of 10-5 mol/l alarelin and 1 mg/l cetrorelix acetate. A total of 134 DEGs (76 downregulated and 58 upregulated for alarelin treatment) and 226 DEGs (90 downregulated and 136 upregulated for cetrorelix) were identified by RNA-sequencing analysis, respectively. Enrichment analysis indicated that DEGs were enriched in the GO terms of cell-cell signalling and cell junctions. The pathways that regulate the development of follicles were identified, including the biological progress of cAMP accumulation, ovulation cycle and vasculature that are essential to follicular selection. 3. The results suggested that GnRH might directly regulate GC proliferation via autocrine or paracrine pathways related to cell junctions. In particular, it was confirmed that the mRNA and protein expression levels of the oestrogen receptor 2 (ESR2) gene, a negative transcription factor involved in follicular maturation and ovulation, were affected by GnRH agonist or antagonist in GCs. 4. In conclusion, GnRH might play an important role in follicular development by changing the expression of genes that participate in cAMP accumulation, ovulation cycle and cell junctions in ovarian GCs.

Structural analysis of estrogen receptors: interaction between estrogen receptors and cav-1 within the caveolae†.

Pregnancy is a physiologic state of substantially elevated estrogen biosynthesis that maintains vasodilator production by uterine artery endothelial cells (P-UAECs) and thus uterine perfusion. Estrogen receptors (ER-α and ER-ß; ESR1 and ESR2) stimulate nongenomic rapid vasodilatory responses partly through activation of endothelial nitric oxide synthase (eNOS). Rapid estrogenic responses are initiated by the ∼4% ESRs localized to the plasmalemma of endothelial cells. Caveolin-1 (Cav-1) interactions within the caveolae are theorized to influence estrogenic effects mediated by both ESRs. Hypothesis: Both ESR1 and ESR2 display similar spatial partitioning between the plasmalemma and nucleus of UAECs and have similar interactions with Cav-1 at the plasmalemma. Using transmission electron microscopy, we observed numerous caveolae structures in UAECs, while immunogold labeling and subcellular fractionations identified ESR1 and ESR2 in three subcellular locations: membrane, cytosol, and nucleus. Bioinformatics approaches to analyze ESR1 and ESR2 transmembrane domains identified no regions that facilitate ESR interaction with plasmalemma. However, sucrose density centrifugation and Cav-1 immunoisolation columns uniquely demonstrated very high protein-protein association only between ESR1, but not ESR2, with Cav-1. These data demonstrate (1) both ESRs localize to the plasmalemma, cytosol and nucleus; (2) neither ESR1 nor ESR2 contain a classic region that crosses the plasmalemma to facilitate attachment; and (3) ESR1, but not ESR2, can be detected in the caveolar subcellular domain demonstrating ESR1 is the only ESR bound in close proximity to Cav-1 and eNOS within this microdomain. Lack of protein-protein interaction between Cav-1 and ESR2 demonstrates a novel independent association of these proteins at the plasmalemma.

Pregnenolone as a potential candidate for hormone therapy for female reproductive disorders targeting ERß.

Many steroid hormones such as estrogen (E2) bind to their receptors for the regulation of biological processes. Pregnenolone (P5) is the precursor form of almost all steroid hormones and is often used to treat skin disorders and neurological complications. However, the mechanism and physiological function of P5 in reproductive organs are not well established. In this study, we investigated the effects of P5 on activation and expression of E2 receptor (ER) in the uteri and ovaries. To study the mechanism of P5 directly, Ishikawa cells were transfected with E2 response element (ERE)-luciferase plasmid and isoforms of ER. ERE-luciferase activity induced by P5 was similar to that induced by E2, and P5 showed high activity for ERß without any relevance to P5-metabolizing hormones such as progesterone (P4) and E2. In an animal study, immature female rats treated with P5 showed upregulation of ERα and downregulation of ERß in the uteri, which is the main organ expressing ERα. In ERß-expressing organ ovaries, estrogen receptor 1, estrogen receptor 2, and P4 receptor were all downregulated by P5 and E2. Also, a decrease of ovarian cell proliferation and viability was observed in response to P5 relative to the control, suggesting that P5 may be a candidate for antiproliferative hormone of ovarian cancer. These findings suggest that P5 stimulates ERE promoter by ERß-mediated signaling in the uteri and ovaries. Activation of ERß by P5 may help in understanding the mechanism of ER-related female reproductive diseases such as endometriosis and ovarian cancer.

Applicability of Anti-Human Estrogen Receptor ß Antibody PPZ0506 for the Immunodetection of Rodent Estrogen Receptor ß Proteins.

Several lines of controversial evidence concerning estrogen receptor ß (ERß) remain to be solved because of the unavailability of specific antibodies against ERß. The recent validation analysis identified a monoclonal antibody (PPZ0506) with sufficient specificity against human ERß. However, the specificity and cross-reactivity of PPZ0506 antibody against ERß proteins from laboratory animals have not been confirmed. In the present study, we aimed to validate the applicability of PPZ0506 to rodent studies. The antibody exhibited specific cross-reactivity against mouse and rat ERß proteins in immunoblot and immunocytochemical experiments using transfected cells. In immunohistochemistry for rat tissue sections, PPZ0506 showed immunoreactive signals in the ovary, prostate, and brain. These immunohistochemical profiles of rat ERß proteins in rat tissues accord well with its mRNA expression patterns. Although the antibody was reported to show the moderate signals in human testis, no immunoreactive signals were observed in rat testis. Subsequent RT-PCR analysis revealed that this species difference in ERß expression resulted from different expression profiles related to the alternative promoter usage between humans and rats. In conclusion, we confirmed applicability of PPZ0506 for rodent ERß studies, and our results provide a fundamental basis for further examination of ERß functions.

Neurochemical Characterization of Neurons Expressing Estrogen Receptor ß in the Hypothalamic Nuclei of Rats Using in Situ Hybridization and Immunofluorescence.

Estrogens play an essential role in multiple physiological functions in the brain, including reproductive neuroendocrine, learning and memory, and anxiety-related behaviors. To determine these estrogen functions, many studies have tried to characterize neurons expressing estrogen receptors known as ERα and ERß. However, the characteristics of ERß-expressing neurons in the rat brain still remain poorly understood compared to that of ERα-expressing neurons. The main aim of this study is to determine the neurochemical characteristics of ERß-expressing neurons in the rat hypothalamus using RNAscope in situ hybridization (ISH) combined with immunofluorescence. Strong Esr2 signals were observed especially in the anteroventral periventricular nucleus (AVPV), bed nucleus of stria terminalis, hypothalamic paraventricular nucleus (PVN), supraoptic nucleus, and medial amygdala, as previously reported. RNAscope ISH with immunofluorescence revealed that more than half of kisspeptin neurons in female AVPV expressed Esr2, whereas few kisspeptin neurons were found to co-express Esr2 in the arcuate nucleus. In the PVN, we observed a high ratio of Esr2 co-expression in arginine-vasopressin neurons and a low ratio in oxytocin and corticotropin-releasing factor neurons. The detailed neurochemical characteristics of ERß-expressing neurons identified in the current study can be very essential to understand the estrogen signaling via ERß.