Nuclear oestrogen receptors in rat liver. Development of assay conditions, characterization and the translocation of oestrogens in vivo.
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ABSTRACT:
SUBMITTER: Tamulevicius P
PROVIDER: S-EPMC1158583 | biostudies-other | 1982 Aug
REPOSITORIES: biostudies-other
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Project description:The administration of oestradiol-17 beta or ethynyloestradiol as well as the synthetic progestogen norethisterone acetate resulted in translocation of the oestrogen receptor. Progesterone and the synthetic progestogen (+)-norgestrel were ineffective. The increases in nuclear oestrogen receptor content 1 h after injection of each steroid were similar but different subsequently. The increase with oestradiol-17 beta extended for 3--6 h and for at least 9 h with ethynyloestradiol. With norethisterone acetate, nuclear content was still increased after 24 h. Oestrogen injection resulted in cytosol receptor depletion and a 'deficit' in receptor content extending for 6 h, whereas norethisterone acetate-induced translocation was quantitative. With injections of norethisterone acetate + ethynyloestradiol the increase at 1 h and retention of the nuclear receptors were similar to that with norethisterone acetate alone. In contrast, the depletion of cytosol receptor and its restoration were similar to that seen with ethynyloestradiol alone, suggesting that norethisterone acetate did not interfere with the oestrogen receptor replenishment. Specific binding in vitro of [3H]oestradiol-17 beta in liver cytosols was inhibited by (+)-norgestrel and norethisterone acetate, but not progesterone, at concentrations of 10--100 microM. Nuclear receptors present after norethisterone acetate injection bound oestrogen with high affinity (Kd = 1.52 nM), similar to receptors of oestrogen-injected animals. In the uterus, differential retention of nuclear receptors in response to oestrogens is associated with different cellular responses. The differences in the response of the receptor system in liver to the various steroids suggests that the corresponding tissue responses may also be dissimilar. These results are discussed in relation to the problems of liver dysfunction in oral-contraceptive users.
| S-EPMC1162133 | biostudies-other
Project description:BackgroundOestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein).ResultsThe response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1-2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2), the synthetic oestrogen 17α- ethinyloestradiol (EE2), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures). For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish). We also demonstrate that our construct works in medaka, another model fish test species, suggesting the transient assay is applicable for testing oestrogenic chemicals in fish generally.ConclusionOur results indicate that the transient expression assay system can be used as a rapid integrated testing system for environmental oestrogens and to detect the oestrogenic target sites in developing fish embryos.
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Project description:Oestrogens influence the pathology and development of hormone-sensitive breast cancers. Tissue factor pathway inhibitor (TFPI) has been shown to be associated with breast cancer pathogenesis. Recently, we found TFPI mRNA levels to be significantly reduced by oestrogens in a breast cancer cell line (MCF7), a process mediated through the oestrogen receptor alpha (ER?). The aim of the present study was to investigate the mechanism(s) by which oestrogens may regulate TFPI at the transcriptional level. The TFPI 5'-flanking region contains three oestrogen response element (ERE) half-sites at positions -845, -769 and -50. Constructs containing the wild type or mutated ERE half-sites of the TFPI 5'-flanking region were generated in a luciferase reporter gene vector and transiently co-transfected with an ER? expression vector into HEK293 cells and subsequently treated with oestrogens. We found that luciferase activity was significantly downregulated after oestrogen stimulation in cells transfected with the wild type construct, an effect that was abolished by mutating either ERE half-sites. Electrophoretic mobility shift assay suggested direct and specific interaction of ER? with the ERE half-sites in the TFPI 5'-flanking region. Chromatin immunoprecipitation showed that ER? was recruited to the region -899 to -578 of the TFPI 5'-flanking region in vivo, where the ERE half-sites -845 and -769 are located. Our results indicate that ER? can interact with all three ERE half-sites in the TFPI 5'-flanking region and thus participate in the repression of oestrogen mediated TFPI transcription in breast cancer cells.
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Project description:Oestrogens are well-known proliferation and differentiation factors that play an essential role in the correct development of sex-related organs and behaviour in mammals. With the use of the ERE-Luc reporter mouse model, we show herein that throughout mouse development, oestrogen receptors (ERs) are active starting from day 12 post conception. Most interestingly, we show that prenatal luciferase expression in each organ is proportionally different in relation to the germ layer of the origin. The luciferase content is highest in ectoderm-derived organs (such as brain and skin) and is lowest in endoderm-derived organs (such as liver, lung, thymus and intestine). Consistent with the testosterone surge occurring in male mice at the end of pregnancy, in the first 2 days after birth, we observed a significant increase in the luciferase content in several organs, including the liver, bone, gonads and hindbrain. The results of the present study show a widespread transcriptional activity of ERs in developing embryos, pointing to the potential contribution of these receptors in the development of non-reproductive as well as reproductive organs. Consequently, the findings reported here might be relevant in explaining the significant differences in male and female physiopathology reported by a growing number of studies and may underline the necessity for more systematic analyses aimed at the identification of the prenatal effects of drugs interfering with ER signalling, such as aromatase inhibitors or endocrine disrupter chemicals.
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Project description:As regulators of steroidogenesis, development, and metabolism, the nuclear receptor 5A (NR5A) subfamily members steroidogenic factor 1 (SF-1) and liver receptor homologue 1 (LRH-1) are important pharmacological targets for cancers and metabolic diseases. Evaluation of small molecule modulators and candidate endogenous ligands for these orphan receptors has been hindered by the lack of accessible, robust direct-binding assays. Here, we leverage the potency of our new NR5A agonist (6N) to create a high-affinity probe for fluorescence polarization competition assays by conjugating 6N to fluorescein (FAM). The 6N-FAM probe tightly binds the NR5A receptors and detects direct binding of synthetic and phospholipid ligands. For 25 LRH-1 agonists, affinity predicts potency in cellular activation assays, demonstrating the potential for this assay in drug discovery. Moreover, phospholipids dilauroylphosphatidylcholine and phosphatidylinositol(4,5)phosphate bind with high affinity, demonstrating this assay is robust for evaluation of candidate endogenous ligands for human NR5A receptors.
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Project description:A major proportion of the hypothalamic nuclear oestrogen receptors were available for complexing with radioactive oestradiol in vitro at 4 degrees C and were apparently unoccupied . At 6 h after oestradiol administration the content of unoccupied nuclear receptors had increased 2.5-fold and represented 71% of the total nuclear receptor content. These results suggest that unoccupied receptors may be active elements in the 'long-term' receptor population of the hypothalamus. Androgenized females had lower contents of these receptors.
| S-EPMC1162165 | biostudies-other
Project description:Nuclear receptors are transcription factors which sense changing environmental or hormonal signals and effect transcriptional changes to regulate core life functions including growth, development, and reproduction. To support this function, following ligand-activation by xenobiotics, members of subfamily 1 nuclear receptors (NR1s) may heterodimerize with the retinoid X receptor (RXR) to regulate transcription of genes involved in energy and xenobiotic metabolism and inflammation. Several of these receptors including the peroxisome proliferator-activated receptors (PPARs), the pregnane and xenobiotic receptor (PXR), the constitutive androstane receptor (CAR), the liver X receptor (LXR) and the farnesoid X receptor (FXR) are key regulators of the gut:liver:adipose axis and serve to coordinate metabolic responses across organ systems between the fed and fasting states. Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and may progress to cirrhosis and even hepatocellular carcinoma. NAFLD is associated with inappropriate nuclear receptor function and perturbations along the gut:liver:adipose axis including obesity, increased intestinal permeability with systemic inflammation, abnormal hepatic lipid metabolism, and insulin resistance. Environmental chemicals may compound the problem by directly interacting with nuclear receptors leading to metabolic confusion and the inability to differentiate fed from fasting conditions. This review focuses on the impact of nuclear receptors in the pathogenesis and treatment of NAFLD. Clinical trials including PIVENS and FLINT demonstrate that nuclear receptor targeted therapies may lead to the paradoxical dissociation of steatosis, inflammation, fibrosis, insulin resistance, dyslipidemia and obesity. Novel strategies currently under development (including tissue-specific ligands and dual receptor agonists) may be required to separate the beneficial effects of nuclear receptor activation from unwanted metabolic side effects. The impact of nuclear receptor crosstalk in NAFLD is likely to be profound, but requires further elucidation. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.
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Project description:Binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to the plasma membrane TRAIL-R1/-R2 selectively kills tumor cells. This discovery led to evaluation of TRAIL-R1/-R2 as targets for anti-cancer therapy, yet the corresponding clinical trials were disappointing. Meanwhile, it emerged that many cancer cells are TRAIL-resistant and that TRAIL-R1/-R2-triggering may lead to tumor-promoting effects. Intriguingly, recent studies uncovered specific functions of long ignored intracellular TRAIL-R1/-R2, with tumor-promoting functions of nuclear (n)TRAIL-R2 as the regulator of let-7-maturation. As nuclear trafficking of TRAIL-Rs is not well understood, we addressed this issue in our present study. Cell surface biotinylation and tracking of biotinylated proteins in intracellular compartments revealed that nTRAIL-Rs originate from the plasma membrane. Nuclear TRAIL-Rs-trafficking is a fast process, requiring clathrin-dependent endocytosis and it is TRAIL-dependent. Immunoprecipitation and immunofluorescence approaches revealed an interaction of nTRAIL-R2 with the nucleo-cytoplasmic shuttle protein Exportin-1/CRM-1. Mutation of a putative nuclear export sequence (NES) in TRAIL-R2 or the inhibition of CRM-1 by Leptomycin-B resulted in the nuclear accumulation of TRAIL-R2. In addition, TRAIL-R1 and TRAIL-R2 constitutively localize to chromatin, which is strongly enhanced by TRAIL-treatment. Our data highlight the novel role for surface-activated TRAIL-Rs by direct trafficking and signaling into the nucleus, a previously unknown signaling principle for cell surface receptors that belong to the TNF-superfamily.
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Project description:Tamoxifen is a potent inhibitor of specific oestrogen-induced yolk protein synthesis by chicken liver. The oestradiol receptor in salt extracts of liver nuclei from oestrogen-treated chicks has a K(d) for oestradiol of 0.7+/-0.2nm. Tamoxifen and its metabolite, monohydroxytamoxifen, compete for binding to the salt-soluble nuclear receptor with K(i) values of 2.6 and 0.1nm respectively. The anti-oestrogens show much less inhibition of [(3)H]oestradiol binding when assays are carried out using intact nuclei. The competition by unlabelled oestradiol for [(3)H]oestradiol binding to receptor is identical in both salt extracts and intact nuclei. This suggests that intact nuclei contain components which bind anti-oestrogens, but not oestradiol. While tamoxifen and desmethyltamoxifen will readily dissociate from the salt-soluble nuclear oestrogen receptor, monohydroxytamoxifen does not dissociate under the conditions generally used for exchange assays. A modified assay was developed in which 60-70% of monohydroxytamoxifen-bound sites were shown to be exchangeable for [(3)H]oestradiol. Soluble receptor preparations were first incubated in a 1.7% charcoal suspension at 37 degrees C for 15min before assay of specific oestradiol binding. This technique was used in examining the effects of tamoxifen and monohydroxytamoxifen given in vivo on the nuclear oestrogen receptor concentration. Despite their 30-fold difference in binding affinity for the receptor, both anti-oestrogens increase nuclear receptor levels to about the same degree. When given with oestradiol, both compounds have the same apparent partial inhibitory effect on the oestrogen-induced increase in nuclear receptor. These data are consistent with the metabolic hydroxylation of tamoxifen before binding to the hepatic oestrogen receptor.
| S-EPMC1158596 | biostudies-other
Project description:Prostate cancer (PCa) incidence has been dramatically increasing these last years in westernized countries. Though localized PCa is usually treated by radical prostatectomy, androgen deprivation therapy is preferred in locally advanced disease in combination with chemotherapy. Unfortunately, PCa goes into a castration-resistant state in the vast majority of the cases, leading to questions about the molecular mechanisms involving the steroids and their respective nuclear receptors in this relapse. Interestingly, liver X receptors (LXRα/NR1H3 and LXRβ/NR1H2) have emerged as new actors in prostate physiology, beyond their historical roles of cholesterol sensors. More importantly LXRs have been proposed to be good pharmacological targets in PCa. This rational has been based on numerous experiments performed in PCa cell lines and genetic animal models pointing out that using selective liver X receptor modulators (SLiMs) could actually be a good complementary therapy in patients with a castration resistant PCa. Hence, this review is focused on the interaction among the androgen receptors (AR/NR3C4), estrogen receptors (ERα/NR3A1 and ERβ/NR3A2), and LXRs in prostate homeostasis and their putative pharmacological modulations in parallel to the patients' support.
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