A Bipartite Recombinant Yeast System for the Identification of Subtype-Selective Estrogen Receptor Ligands

Since the estrogen receptor α (ERα) and β (ERβ) are thought to mediate different biological effects, there is intense interest in designing or screening subtype-selective ER ligands. Here, we constructed a biosensor identified as bipartite recombinant yeast system (BRYS) to screen and evaluate subtype-selective ER ligands. Uniform design and immunoblotting was used to determine an optimal dose of copper which control the expression of ERs through a copper inducible metallothionine promoter (CUP1). Some chemicals including natural estrogen (17β-estradiol), specific estrogen receptor agonist (PPT and DPN), and phytoestrogens (genistein) were tested to validate this system. There was obvious and anticipative discrimination between the agonistic activities when these chemicals were identified and characterized. Furthermore, antagonist (ICI 182,780), which could antagonize the transactivation of estrogen, and chromatin immunoprecipitation (ChIP) were used to confirm that the agonistic effects were mediated through ERs. Comparative studies showed that this system was reproducible and sensitive. 4.7 pM (1.3 ng/l) estrogen was the lowest concentration that could be detected to ERα and 0.12 nM (33.5 ng/l) for ERβ. The results from our study showed that in vitro screening for subtype-selective ER ligands could be conducted in a simple yeast system that is rapid, sensitive, reliable, and quantitative. An erratum to this article can be found at     

Estrogen receptors,antiestrogens, and non-small cell lung cancer

This review considers data on expression of different types of estrogen receptors (ERα and ERβ) in in vitro cultured cells of non-small cell lung cancer and also in human and animal lung tumors. Estrogens are shown to play an important role in genesis and development of non-small cell lung cancer because the estrogen-stimulated cell proliferation as well as antiestrogen-caused inhibition of proliferation occurred only in the cells expressing different types of estrogen receptors. In general, the situation is similar to that observed in breast cancer, but in the cells of non-small cell lung cancer not ERα are expressed in more than half of cases but ERβ. Just estrogen receptors β play the crucial role in inducing cell proliferation in response to estrogens, and ERβ is a prognostic marker of a favorable course of non-small cell lung cancer. Data on the interactions between ER and EGFR signaling pathways, as well as on the additive antitumor effect of antiestrogens (tamoxifen and fulvestrant) combined with tyrosine kinase inhibitors (gefitinib, erlotinib, and vandetanib) are considered. The review also includes data on the influence of estrogens on genesis and development of lung cancer in humans and animals and the frequency of ERα and ERβ expression in non-small cell lung cancer in tissues from patients of the two sexes. Problems of quantitative determination of α and β estrogen receptors in the tumor cells are also discussed.     

Elevated β1,4-galactosyltransferase-I induced by the intraspinal injection of lipopolysaccharide

β1,4-Galactosyltransferase-I (β1,4-GalT-I) is one of the best studied glycosyltransferases. Previous studies demonstrated that β1,4-GalT-I was a major galactosyltransferase responsible for selectin-ligand biosynthesis and that inflammatory responses of β1,4-GalT-I deficient mice were impaired. In this study, we investigate the expression of β1,4-GalT-I in lipopolysaccharide (LPS)-induced neuroinflammatory processes. The results of this study demonstrated that β1,4-GalT-I was strongly induced by intraspinal administration of LPS. More than 90% galactose-containing glycans and β1,4-GalT-I were expressed in immune cells. The ELISA assay shows focal injection LPS also induces TNF-α alteration. Double staining indicated β1,4-GalT-I overlapped with TNF-α. Moreover, RT-PCR for β1,4-GalT-I mRNA showed that β1,4-GalT-I mRNA in microglia in vitro was affected in a dose- and time dependent manner in response to LPS or TNF-α stimulation. All these results indicated that the increase of β1,4-GalT-I might attribute to the effect of TNF-α excreting during inflammation. E-selectin, which ligand was modified by β1,4-GalT-I, was correlated with galactose-containing glycans following injecting LPS into spinal cord. We therefore suggest that β1,4-GalT-I may play an important role in regulating immune cell migration into the inflammatory site. Aiguo Shen and Jianping Chen contributed equally to this work.     

The Effects of Estradiol on Estrogen Receptor and Glutamate Transporter Expression in Organotypic Hippocampal Cultures Exposed to Oxygen--Glucose Deprivation

The molecular basis of estrogen-mediated neuroprotection against brain ischemia remains unclear. In the present study, we investigated changes in expression of estrogen receptors (ERs) α and β and excitatory amino acid transporters (EAAT) 1 and 2 in rat organotypic hippocampal slice cultures treated with estradiol and subsequently exposed to oxygen--glucose deprivation (OGD). Pretreatment with 17β-estradiol (10 nM) for 7 days protected the CA1 area of hippocampus against OGD (60 min), reducing cellular injury by 46% compared to the vehicle control group. Levels of ERα protein were significantly reduced by 20% after OGD in both vehicle- and estradiol-treated cultures, whereas ERβ was significantly up-regulated by 25% in the estradiol-treated cultures. In contrast, EAAT1 and EAAT2 levels were unchanged in response to estradiol treatment in this model of OGD. These findings suggest that estrogen-induced neuroprotection against ischemia might involve regulation of ERβ and, consequently, of the genes influenced by this receptor.Helena Cimarosti and Ross D. O’Shea, equal first authors.     

Phylogenetic Analyses Reveal Ancient Duplication of Estrogen Receptor Isoforms

To determine the origin and evolutionary significance of a recently discovered isoform of the estrogen receptor (ERβ), we examined the phylogenetic relationship of ERβ to the well-known α isoform (ERα) and other steroid receptors. Our phylogenetic analyses traced the origin of ERβ to a single duplication event at least 450 million years ago. Since this duplication, the evolution of both ER isoforms has apparently been constrained such that 80% of the amino acid positions in the DNA binding domain (DBD) and 53% of the ligand binding domain (LBD) have remained unchanged. Using the phylogenetic tree, we determined the amount of evolutionary change that had occurred in two ER isoforms. The DBD and the LBD had lower rates of evolutionary change compared to the NH₂ terminal domain. However, even with strong selective constraints on the DBD and LBD, our phylogenetic analyses demonstrate two clearly separate phylogenetic histories for ERα and ERβ dating back several hundred million years. The ancient duplication of ER and the parallel evolution of the two ER isoforms suggest that, although ERα and ERβ share a substantial degree of sequence identity, they play unique roles in vertebrate physiology and reproduction. Received: 19 January 1999 / Accepted: 26 May 1999     

The Expression of Tumor Necrosis Factor-α (TNF-α) by the Intrathecal Injection of Lipopolysaccharide in the Rat Spinal Cord

The proinflammatory and lipopolysaccharide (LPS)-inducible cytokine tumor necrosis factor α (TNF-α) has been shown to enhance primary sensory nociceptive signaling. However, the precise cellular site of TNF-α synthesis is still a matter of controversy. Therefore, we focused our study on TNF-α protein synthesis and expression patterns in spinal dorsal horn of naives and rats under intrathecal challenge with LPS. The enzyme-linked immunosorbent (ELISA) assay showed that the protein level of TNF-α reached peak at 8 h. Double immunofluorescence revealed that LPS-induced expression of TNF-α exclusively located in a subpopulation of microglia, which increased at 8 h in the rat spinal dorsal horn (the injected side). Positive staining of TNF receptor 1 (TNFR1) were also found in microglia. These observations have demonstrated the production of this proinflammatory cytokine by central nerve glia especially microglia. Synthesized TNF-α might directly act on microglia via TNFR1, but the inherent mechanisms remain unknown. Further studies are needed to confirm the pathogenic role of tumor necrosis factor in the early stage of inflammation. Aiguo Shen and Dan Zhou contributed equally to this work.     

Estrogen receptor-beta agonist diarylpropionitrile counteracts the estrogenic activity of estrogen receptor-alpha agonist propylpyrazole-triol in the mammary gland of ovariectomized Sprague Dawley rats

Although estrogen can bind both types of estrogen receptors, estrogen receptor-alpha (ERα) is dominant in mediating estrogenic activity in the mammary gland and uterus. Excessive estrogenic activity such as estrogen-based postmenopausal hormone replacement therapy increases the risk for breast and endometrial cancers. The adverse effect of estrogen on uterine endometrium can be opposed by progestins; however, estrogen-plus-progestin regimen imposes substantially greater risk for breast cancer than estrogen alone. In this study, we used ERα-selective agonist propylpyrazole-triol (PPT) and ERβ-selective agonist diarylpropionitrile (DPN) to activate ERα and estrogen receptor-beta (ERβ) separately in an ovariectomized rat model and determined whether PPT-activated ERα function in the mammary gland can be suppressed by DPN activated ERβ. Ovariectomized rats were randomly divided into six groups and treated with DMSO (control), DPN, PPT, PPT/DPN, PPT/Progesterone, and PPT/Progesterone/DPN, respectively. In the mammary gland, PPT but not DPN increased cell proliferation and amphiregulin gene expression; importantly, the stimulatory effect of PPT on mammary cell proliferation and amphiregulin gene expression can be suppressed by DPN. In the uterus, the effect of PPT on uterine weight and endometrial cell proliferation was not inhibited by DPN but can be inhibited by progesterone. These data provide in vivo evidence that PPT activated ERα activity in the mammary gland can be opposed by ERβ-selective agonist DPN, which may be explored for the development of better hormone replacement therapy regimen with less risk for breast cancer.     

Induction of G protein-coupled estrogen receptor (GPER) and nuclear steroid hormone receptors by gonadotropins in human granulosa cells

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation. The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH. Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized granulosa cells.     

Cellular localization of estrogen receptor-alpha (ERα) and -beta (ERβ) mRNA in the boar testis

Boar testes synthesize high amounts of estrogens which are known to stimulate several male sexual functions in a variety of extragonadal target tissues. Possible effects within the testis depend on the existence of the estrogen receptor subtypes α and β (ERα, ERβ). The precise cellular localization of these subtypes within the testis was, so far, based mainly on protein expression studies using different antibodies in several species including boars shows contradictory results. Therefore, we investigated the ERα and ERβ gene expression using RT-PCR of testis homogenates and RT-PCR after UV-single cell microdissection combined with in-situ hybridization of four fertile boars with an average age of 32 weeks. Both ERα and ERβ mRNA were found in testis homogenates. Using in-situ hybridization and UV-single cell microdissection ERα mRNA was present in type A and type B spermatogonia up to mid-pachytene primary spermatocytes in stage V–VIII and stage I of the seminiferous epithelial cycle, but not in other cells. ERβ mRNA was found only in Sertoli cells. Interstitial Leydig cells revealed neither ERα nor ERβ mRNA. The data suggest a direct impact of estrogen in the boar on Sertoli cell function via ERβ and germ cell formation via ERα.     

Estradiol's salutary effects on keratinocytes following trauma-hemorrhage are mediated by estrogen receptor (ER)-alpha and ER-beta

Although administration of 17beta-estradiol (estrogen) following trauma-hemorrhage attenuates the elevation of cytokine production and mitogen-activated protein kinase (MAPK) activation in epidermal keratinocytes, whether the salutary effects of estrogen are mediated by estrogen receptor (ER)-alpha or ER-beta is not known. To determine which estrogen receptor is the mediator, we subjected C3H/HeN male mice to trauma-hemorrhage (2-cm midline laparotomy and bleeding of the animals to a mean blood pressure of 35 mmHg and maintaining that pressure for 90 min) followed by resuscitation with Ringer's lactate (four times the shed blood volume). At the middle of resuscitation we subcutaneously injected ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), ER-beta agonist diarylpropionitrile (DPN; 5 microg/kg), estrogen (50 microg/kg), or ER antagonist ICI 182,780 (150 microg/kg). Two hours after resuscitation, we isolated keratinocytes, stimulated them with lipopolysaccharide for 24 h (5 microg/mL for maximum cytokine production), and measured the production of interleukin (IL)-6, IL-10, IL-12, and TNF-alpha and the activation of MAPK. Keratinocyte cytokine production markedly increased and MAPK activation occurred following trauma-hemorrhage but were normalized by administration of estrogen, PPT, and DPN. PPT and DPN administration were equally effective in normalizing the inflammatory response of keratinocytes, indicating that both ER-alpha and ER-beta mediate the salutary effects of estrogen on keratinocytes after trauma-hemorrhage.     

Estrogen-dependent regulation of sodium/hydrogen exchanger-3 (NHE3) expression via estrogen receptor β in proximal colon of pregnant mice

Although constipation is very common during pregnancy, the exact mechanism is unknown. We hypothesized that the involvement of estrogen receptor (ER) in the regulation of electrolyte transporter in the colon leads to constipation. In this study, the intestines of normal female ICR mouse and pregnant mice were examined for the expression of ERα and ERβ by immunohistochemistry and in situ hybridization. ERβ, but not ERα, was expressed in surface epithelial cells of the proximal, but not distal, colon on pregnancy days 10, 15, and 18, but not day 5, and the number of ERβ-positive cells increased significantly during pregnancy. Expression of NHE3, the gene that harbors estrogen response element, examined by immunohistochemistry and western blotting, was localized in the surface epithelial cells of the proximal colon and increased in parallel with ERβ expression. In ovariectomized mice, NHE3 expression was only marginal and was up-regulated after treatment with 17β-estradiol (E₂), but not E₂ + ICI 182,780 (estrogen receptor antagonist). Moreover, knock-down of ERβ expression by electroporetically transfected siRNA resulted in a significant reduction of NHE3 expression. These results indicate that ERβ regulates the expression of NHE3 in the proximal colon of pregnant mice through estrogen action, suggesting the involvement of increased sodium absorption by up-regulated NHE3 in constipation during pregnancy.     

Ontogeny of estrogen receptor alpha,estrogen receptor beta and androgen receptor,and their co-localization with Islet-1 in the dorsal root ganglia of sheep fetuses during gestation

The aims of the present study were to detect the ontogeny of estrogen receptor (ERα and ERβ) and androgen receptor (AR) expressions and their co-localization with Islet-1 in the developing dorsal root ganglia (DRG) of sheep fetuses by immunohistochemistry. From the single staining results, the ERα immunoreactivity (ERα-ir), ERβ immunoreactivity (ERβ-ir) and AR immunoreactivity (AR-ir) was first detected at days 90, 120 and 90 of gestation, respectively. From days 90 to 120, ERα and AR were consistently detected in the nuclei of DRG neurons and the relative percentage (approximately 60%) of ERα-ir or AR-ir cells did not change significantly. Moreover, there was no change in ERα expression, while a dramatic loss of AR expression was observed at birth. From day 120 of gestation to birth, very few neurons (approximately 8%) showed nuclear ERβ immunoreactivity. The dual staining results showed that Islet-1 was co-localized with ERα, ERβ or AR in the nuclei of DRG neurons with various frequencies, and over 70% ERα-ir, ERβ-ir or AR-ir cells contained Islet-1. These results imply that ERs, AR and Islet-1 may be important in regulating the differentiation and functional maintenance of some phenotypes of DRG neurons after mid-gestation in the sheep fetus.     

Aging of Brain: Role of Estrogen

The brain undergoes many structural and functional changes during aging. Some of these changes are regulated by estrogens which act mainly through their intracellular receptors, estrogen receptor ERα and ERβ. The expression of these receptors is regulated by several factors including their own ligand estrogen, and others such as growth hormone and thyroid hormone. The levels of these factors decrease during aging which in turn influence estrogen signaling leading to alterations in brain functions. In the present paper, we review the effects of aging on brain structure and function, and estrogen action and signaling during brain aging. The findings suggest key role of estrogen in the maintenance of brain functions during aging.     

The Role of TNF-<Emphasis Type="Italic">α</Emphasis> and its Receptors in the Production of <Emphasis Type="Italic">β</Emphasis>-1,4-galactosyltransferase I mRNA by Rat Primary Type-2 Astrocytes

β-1,4-galactosyltransferase I (β-1,4-GalT I) plays an important role in the synthesis of the backbone structure of adhesion molecules involved in leukocyte–endothelial cell interaction. The expression of β-1,4-GalT I mRNA increased in primary human endothelial cells after exposure to tumor necrosis factor-α (TNF-α). In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-α when stimulated with Lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of β-1,4-GalT I mRNA in type-2 astrocytes after exposure to TNF-α and LPS. Real-time PCR showed that TNF-α or LPS affected β-1,4-GalT I mRNA expression in a time- and dose-dependent manner. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes, and that TNF-α, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-α or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes, and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes, and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed β-1,4-GalT I mRNA expression induced by TNF-α or LPS. From these results, we conclude that TNF-α signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce β-1,4-GalT I mRNA. In addition, we observed that not only exogenous TNF-α but also TNF-α produced by type-2 astrocytes affected β-1,4-GalT I mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-α contributes to the production of β-1,4-GalT I mRNA in response to inflammation. Chunlin Xia is the co-first author.