ABSTRACT
Endothelial cells express multiple receptors mediating estrogen responses; including the G protein-coupled estrogen receptor (GPER). Past studies on nitric oxide (NO) production elicited by estrogens raised the question whether 17-ß-estradiol (E2) and natural phytoestrogens activate equivalent mechanisms. We hypothesized that E2 and phytoestrogens elicit NO production via coupling to distinct intracellular pathways signalling. To this aim, perfusion of E2 and phytoestrogens to the precontracted rat mesentery bed examined vasorelaxation, while fluorescence microscopy on primary endothelial cells cultures quantified single cell NO production determined following 4-amino-5-methylamino-2',7'-difluoroescein diacetate (DAF) incubation. Daidzein (DAI) and genistein (GEN) induced rapid vasodilatation associated to NO production. Multiple estrogen receptor activity was inferred based on the reduction of DAF-NO signals; G-36 (GPER antagonist) reduced 75 % of all estrogen responses, while fulvestrant (selective nuclear receptor antagonist) reduced significantly more the phytoestrogens responses than E2. The joint application of both antagonists abolished the E2 response but not the phytoestrogen-induced DAF-NO signals. Wortmannin or LY-294002 (PI3K inhibitors), reduced by 90% the E2-evoked signal while altering significantly less the DAI-induced response. In contrast, H-89 (PKA inhibitor), elicited a 23% reduction of the E2-induced signal while blocking 80% of the DAI-induced response. Desmethylxestospongin-B (IP3 receptor antagonist), decreased to equal extent the E2 or the DAI-induced signal. Epidermal growth factor (EGF) induced NO production, cell treatment with AG-1478, an EGF receptor kinase inhibitor reduced 90% DAI-induced response while only 53% the E2-induced signals; highlighting GPER induced EGF receptor trans-modulation. Receptor functional selectivity may explain distinct signalling pathways mediated by E2 and phytoestrogens.
Subject(s)
Cyclic AMP-Dependent Protein Kinases , ErbB Receptors , Estradiol , Nitric Oxide , Phosphatidylinositol 3-Kinases , Phytoestrogens , Signal Transduction , Vasodilation , Animals , Phytoestrogens/pharmacology , Estradiol/pharmacology , Nitric Oxide/metabolism , Rats , Signal Transduction/drug effects , Vasodilation/drug effects , Cyclic AMP-Dependent Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , ErbB Receptors/metabolism , Male , Isoflavones/pharmacology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Genistein/pharmacology , Receptors, Estrogen/metabolism , Rats, WistarABSTRACT
INTRODUCTION: The prevalence of female infertility in Pakistan is currently estimated at 22%, and emerging research suggests that vitamin D (VD) deficiency (VDD) may play a significant role in influencing female fertility. The focus of this study was to investigate the single nucleotide polymorphism (SNP) patterns within the VD binding protein (VDBP). The study aimed to explore dysregulated pathways and gene enrichment through an interaction network analysis, specifically focusing on the interplay between the VD receptor (VDR) and VDBP in females experiencing unexplained infertility (UI) coupled with VDD. METHODS: A cross-sectional study was conducted on VD-deficient, fertile, and UI female subjects. VDBP and VDR were assessed by enzyme-linked immunoassay and genotyping performed. FunRich (version 3.1.3; http://funrich.org/index.html) was employed for analysis of the identified proteins: VDR and VDBP and with their mapped gene datasets, gene enrichment, and protein-protein interaction (PPI) network. RESULTS: The mean VD and VDR values of infertile females were significantly lower than those of fertile females. VDBP in infertile females (median (IQR)): 296.05 (232.58-420.23)) was lower than that of fertile females (469.9 (269.57-875.55), (p=0.01)). On sequence analysis, a mutation rs 4588 SNP (Thr 436 Lys) was found in exon 11 of the VDBP gene of UI females, but no mutation in exons 8 and 9 of the VDR gene, with some insignificant intronic variants, was observed. The proteins such as plasma membrane estrogen receptor signaling pathway (p < 0.001), VDR, SMAD3, NCOR1, CREBBP, NCOA1, STAT1, GRB2, PPP2CA, TP53, and NCOA2 were enriched after biological pathway grouping when VDR was made the focused gene and directly interacting with VDBP. CONCLUSION: The females with UI exhibited significantly low VD, VDBP, and VDR. The plasma membrane estrogen receptor signaling pathway was enriched in VDD infertile females.
ABSTRACT
There is a need to fully know the physiology of Eurasian beaver due to its essential role in environmental homeostasis. However, a "human factor" impacts this, including stress conditions and environmental pollution. Adrenal glands protect these all. The regulation of endocrine processes by nonclassical androgen and estrogen signaling, the first and fastest control, is still a matter of research. The specific analyses performed here in mature female and male beaver adrenals contained: anatomical and histological examinations, expression and localization of membrane androgen receptor (zinc transporter, Zinc- and Iron-like protein 9; ZIP9) and membrane estrogen receptor coupled with G protein (GPER), and measurement of zinc (Zn2+) and copper (Ca2+) ion levels and corticosterone levels. We revealed normal anatomical localization, size, and tissue histology in female and male beavers, respectively. Equally, ZIP9 and GPER were localized in the membrane of all adrenal cortex cells. The protein expression of these receptors was higher (p < 0.001) in male than female adrenal cortex cells. Similarly, Zn2+ and Ca2+ ion levels were higher (p < 0.05, p < 0.01) in male than female adrenal cortex. The increased corticosterone levels (p < 0.001) were detected in the adrenal cortex of females when compared to males. The present study is the first to report the presence of nonclassical androgen and estrogen signaling and its possible regulatory function in the adrenal cortex of Eurasian beavers. We assume that this first-activated and fast-transmitted regulation can be important in the context of the effect of environmental physical and chemical stressors especially on adrenal cortex cells. The beaver adrenals may constitute an additional supplementary model for searching for universal mechanisms of adrenal cortex physiology and diseases.
Subject(s)
Adrenal Cortex , Receptors, Androgen , Receptors, Estrogen , Rodentia , Signal Transduction , Animals , Female , Male , Receptors, Estrogen/metabolism , Receptors, Androgen/metabolism , Adrenal Cortex/metabolism , Signal Transduction/physiology , Rodentia/physiology , Corticosterone/blood , Corticosterone/metabolism , Zinc/metabolism , Copper/metabolismABSTRACT
The phenomenon of remaining paramesonephric ducts (uterus masculinus) in males of some animal species concerning its role is still an unresolved issue. Now it is well-recognized that sex hormonal regulation of reproductive physiology involves also fast nongenomic control of cellular processes through noncanonical signaling. Herein, in the uterus masculinus of Eurasian beaver membrane androgen receptor (metal ion transporter Zrt- and Irt-like protein 9; ZIP9) and membrane estrogen receptor (G protein-coupled estrogen receptor; GPER) were studied. Scanning electron microscopy together with anatomical analysis revealed that Eurasian male beavers possess one double uterus (uterus duplex). Two odd parts open into the vagina but do not form a common lumen. The length of the horns is the most differential feature of this organ in studied animals. Uterus masculinus is not a tightly closed tubular structure. Histological analysis showed an analogy to the female uterus structure however no glands but gland-like structures were observed. The presence and abundant localization of ZIP9 and GPER proteins in cells of uterus masculinus was confirmed by immunohistochemistry while their expression was measured by western blotting. GPER expression in remnants was lower (P < 0.001) than those in the female uterus. Parallelly, the concentration of progesterone and estradiol but not testosterone was lower (P < 0.05 and P < 0.01, respectively) in comparison to the female uterus. Our study, for the first time, reports the involvement of fast hormonal regulation in the uterus masculinus of Eurasian beavers reflecting the participation of this organ in the creation local hormonal environment. Moreover, the uterus masculinus seems to be a useful research model for understanding and resolving urgent biological problems such as gender identities and having children by women with a lack of uterus or anatomical barriers on this level.
Subject(s)
Androgens , Receptors, Estrogen , Animals , Child , Female , Male , Humans , Receptors, Estrogen/metabolism , Androgens/metabolism , Rodentia , Estrogens/metabolism , Estradiol/metabolism , Uterus/metabolism , Receptors, Androgen/metabolismABSTRACT
Estrogen receptors were initially identified as intracellular, ligand-regulated transcription factors that result in genomic change upon ligand binding. However, rapid estrogen receptor signaling initiated outside of the nucleus was also known to occur via mechanisms that were less clear. Recent studies indicate that these traditional receptors, estrogen receptor α and estrogen receptor ß, can also be trafficked to act at the surface membrane. Signaling cascades from these membrane-bound estrogen receptors (mERs) can rapidly alter cellular excitability and gene expression, particularly through the phosphorylation of CREB. A principal mechanism of neuronal mER action has been shown to occur through glutamate-independent transactivation of metabotropic glutamate receptors (mGlu), which elicits multiple signaling outcomes. The interaction of mERs with mGlu has been shown to be important in many diverse functions in females, including driving motivated behaviors. Experimental evidence suggests that a large part of estradiol-induced neuroplasticity and motivated behaviors, both adaptive and maladaptive, occurs through estradiol-dependent mER activation of mGlu. Herein we will review signaling through estrogen receptors, both "classical" nuclear receptors and membrane-bound receptors, as well as estradiol signaling through mGlu. We will focus on how the interactions of these receptors and their downstream signaling cascades are involved in driving motivated behaviors in females, discussing a representative adaptive motivated behavior (reproduction) and maladaptive motivated behavior (addiction).
Subject(s)
Receptors, Estradiol , Receptors, Metabotropic Glutamate , Humans , Female , Estradiol , Receptors, Estrogen , LigandsABSTRACT
Multiplex immunohistochemistry (mIHC) use markers staining different cell populations applying widefield optical microscopy. Resolution is low not resolving subcellular co-localization. We sought to colocalize markers at subcellular level with antibodies validated for clinical diagnosis, including the single secondary antibody (combination of anti-rabbit/mouse-antibodies) used for diagnostic IHC with any primary antibody, and confocal microscopy. We explore colocalization in the nucleus (ColNu) of nuclear hormone receptors (ERa, PR, and AR) along with the baseline marker p63 in paired samples of breast and prostate tissues. We established ColNu mIHCF as a reliable technique easily implemented in a hospital setting. In ERa+ breast cancer, we identified different colocalization patterns (nuclear or cytoplasmatic) with PR and AR on the luminal epithelium. A triple-negative breast-cancer case expressed membrane-only ERa. A PR-only case was double positive PR/p63. In normal prostate, we identified an ERa+/p63+/AR-negative distinct population. All prostate cancer cases characteristically expressed ERa on the apical membrane of the AR+ epithelium. We confirmed this using ERa IHC and needle-core biopsies. ColNu mIHCF is feasible and already revealed a new marker for prostate cancer and identified sub-patterns in breast cancer. It could be useful for pathology as well as for functional studies in normal prostate and breast tissues.
ABSTRACT
BACKGROUND: Classic nuclear-initiated estrogen signaling stimulates corticotropin-releasing hormone (CRH) gene expression as a transcription factor. However, the possible mechanism by which membrane-initiated estrogen signaling (MIES) influences CRH expression remains unclear. There are indications that MIES may upregulate nitric oxide (NO) production through the phosphatidylinositol 3-hydroxy kinase (PI3K) and potentially through the mitogen-activated protein kinase (MAPK) pathway. OBJECTIVES: We investigated the effect of MIES-mediated kinase pathways on CRH expression with or without NO synthesis. METHOD: In SK-N-SH cell culture, estradiol-bovine serum albumin (E2-BSA) was used as the specific membrane estrogen receptor activator, with a specific NO donor, and/or inhibitors for NO synthase (NOS), PI3K, MAPK, protein kinase A (PKA), and protein kinase C (PKC). RESULTS: E2-BSA significantly increased NO and CRH levels in the medium and NOS1-mRNA levels in the cells. In addition, NO donor up-regulated CRH expression, while NOS-inhibitor down-regulated it. When the inhibitor of MAPK and/or the inhibitor of PI3K was added to the medium, only the latter appeared to significantly block the stimulating effect of E2-BSA on NO synthesis, and this was accompanied by an increased CRH expression in the medium. We further studied the effect of the MIES-PKC-mediated pathway on CRH expression, with or without NOS-inhibitor, while the MIES-PKA(-PI3K) pathway served as a control. We found that MIES-PKC upregulated CRH expression independent of NO synthesis. CONCLUSION: MIES can efficiently upregulate CRH expression via various intracellular kinase pathways and may thus be a crucial component in the stress response.
Subject(s)
Corticotropin-Releasing Hormone/metabolism , Estradiol/pharmacology , Estrogens/metabolism , Gene Expression Regulation/physiology , Nitric Oxide/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase C/metabolism , Receptors, Estrogen/metabolism , Serum Albumin, Bovine/pharmacology , Signal Transduction/physiology , Cells, Cultured , HumansABSTRACT
BACKGROUND: Dipsaci Radix has been clinically used for thousands of years in China for strengthening muscles and bones. Sweroside is the major active iridoid glycoside isolated from Dipsaci Radix. It has been reported that sweroside can promote alkaline phosphatase (ALP) activity in both the human osteosarcoma cell line MG-63 and rat osteoblasts. However, the underlying mechanism involved in these osteoblastic processes is poorly understood. PURPOSE: This study aimed to characterize the bone protective effects of sweroside and to investigate the signaling pathway that is involved in its actions in MC3T3-E1 cells. METHODS: Cell proliferation, differentiation and mineralization were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, ALP test and Alizarin Red S staining, respectively. The concentration of sweroside in intracellular and extracellular fluids was determined by ultra-performance liquid chromatography coupled to triple quadrupole xevo-mass spectrometry (UPLC/TQ-XS-MS). Proteins associated with the osteoblastic signaling pathway were analysed by western blot and immunofluorescence methods. RESULTS: Sweroside did not obviously affect the proliferation but significantly promoted the ALP activity and mineralization of MC3T3-E1 cells. The maximal absorption amount 0.465 ng/ml (1.3 × 10-9 M) of sweroside was extremely lower than the tested concentration of 358.340 ng/ml (10-6 M), indicating an extremely low absorption rate by MC3T3-E1 cells. Moreover, the ALP activity, the protein expression of ER-α and G protein-coupled receptor 30 (GPR30) induced by sweroside were markedly blocked by both the ER antagonist ICI 182780 and the GPR30 antagonist G15. In addition, sweroside also activated the phosphorylation of p38 kinase (p-p38), while the phosphorylation effects together with ALP and mineralization activities were completely blocked by a p38 antagonist, SB203580. Additionally, the phosphorylation of p38 induced by sweroside were markedly blocked by both the ER antagonist ICI 182780 and the GPR30 antagonist G15. CONCLUSIONS: The present study indicated that sweroside, as a potential agent in treatment of osteoporosis, might exert beneficial effects on MC3T3-E1 cells by interaction with the membrane estrogen receptor-α and GPR30 that then activates the p38 signaling pathway. This is the first study to report the specific mechanism of the effects of sweroside on osteoblastic differentiation and mineralization of MC3T3-E1 cells.
Subject(s)
Estrogen Receptor alpha/metabolism , Iridoid Glucosides/pharmacology , Osteoblasts/drug effects , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Alkaline Phosphatase/metabolism , Animals , Calcification, Physiologic/drug effects , Cell Differentiation/drug effects , Cell Line , Cell Proliferation/drug effects , Mice , Osteoblasts/metabolism , Phosphorylation/drug effectsABSTRACT
Organotypic culture of testicular fragments from 7-day-old male pigs (Polish White Large) was used. Tissues were treated with an antagonist of G-protein coupled estrogen receptor (GPER) (G-15; 10 nM), and bisphenol A (BPA), and its analogs (TBBPA, TCBPA; 10 nM) alone or in combination and analyzed using electron and light (stainings for collagen fibers, lipid droplet and autophagy markers) microscopes. In addition, mRNA and protein abundances and localization of molecules required for miRNA biogenesis and function (Drosha, Exportin 5; EXPO5, Dicer, and Argonaute 2; AGO2) were assessed together with calcium ion (Ca2+) and estradiol concentrations. Regardless of GPER blockade and/or treatment with BPA, TBBPA and TCBPA, there were no changes in Leydig cell morphology. Also, there were no changes in lipid droplet content and distribution but there were changes in lipid and autophagy protein abundance. In the interstitial tissue, there was an increase of collagen content, especially after treatment with BPA analogs and G-15 + BPA. Independent of the treatment, there was downregulation of EXPO5 and Dicer genes but the Drosha and AGO2 genes were markedly upregulated as a result of treatment with G-15 + BPA and TCBPA, respectively. There was always a lesser abundance of EXPO5 and AGO2 proteins regardless of treatment. There was markedly greater abundances of Drosha after G-15 + BPA treatment, and this also occurred for Dicer after treatment with G-15 + TCBPA. Immunolocalization of miRNA proteins indicated there was a cytoplasmic-nuclear pattern in control and treated cells. There was an increase of Ca2+ concentrations after treatment with G-15 and BPA analogs. Estradiol secretion decreased after antagonist and chemical treatments when these were administered alone, however, there was an increase in estradiol secretion after treatment with combinations of these compounds.
Subject(s)
Benzhydryl Compounds/pharmacology , Epigenesis, Genetic/drug effects , Leydig Cells/drug effects , Phenols/pharmacology , Receptors, Estrogen/physiology , Receptors, G-Protein-Coupled/physiology , Testis/drug effects , Animals , Gene Expression Regulation, Developmental/drug effects , Gene-Environment Interaction , Leydig Cells/metabolism , Male , MicroRNAs/drug effects , MicroRNAs/genetics , MicroRNAs/metabolism , Receptors, Estrogen/genetics , Receptors, G-Protein-Coupled/genetics , Sexual Maturation/drug effects , Sexual Maturation/genetics , Swine , Testis/metabolismABSTRACT
In estradiol-primed nonreceptive ovariectomized rats, activation of G protein-coupled estrogen receptor 1 (GPER) in the arcuate nucleus of the hypothalamus (ARH) rapidly facilitates sexual receptivity (lordosis). Estradiol priming activates ARH ß-endorphin (ß-END) neurons that then activate medial preoptic (MPN) µ-opioid receptors (MOP) to inhibit lordosis. ARH infusion of non-esterified 17ß-estradiol (E2) 47.5â¯h after 17ß-estradiol benzoate (2⯵gâ¯EB) priming deactivates MPN MOP and rapidly facilitates lordosis within 30â¯min via activation of GPER. Since it was unclear where GPERs were located in the neuron, we tested the hypothesis that GPER signaling is initiated at the plasma membrane. Membrane impermeable estradiol (17ß-estradiol conjugated to biotin; E-Biotin) infused into the ARH of EB primed rats facilitated lordosis within 30â¯min, and MPN MOP was deactivated. These actions were blocked by pretreating with GPER antagonist, G-15. Further, we used cell fractionation and western blot techniques to demonstrate that GPER is expressed both in plasma membrane and cytosolic ARH fractions. In previous studies, the orphanin FQ/nociceptin-opioid receptor-like receptor-1 (OFQ/N-ORL-1) system mediated estradiol-only facilitation of lordosis. Therefore, we tested whether the OFQ/N-ORL-1 system mediates E-Biotin-GPER facilitation of lordosis. Pretreatment of UFP-101, an ORL-1 selective antagonist, blocked the facilitation of lordosis and deactivation of MPN MOP by ARH infusion of E-Biotin. Double-label immunohistochemistry revealed that GPER is expressed within approximately 70% of OFQ/N neurons. These data indicate that membrane GPER mediates the E2/E-Biotin facilitation of lordosis by inducing OFQ/N neurotransmission, which inhibits ß-END neurotransmission to reduce MPN MOP activation.
Subject(s)
Estradiol/pharmacology , Opioid Peptides/metabolism , Posture/physiology , Receptors, G-Protein-Coupled/physiology , Receptors, Opioid/metabolism , Sexual Behavior, Animal/drug effects , Animals , Estradiol/metabolism , Female , Hypothalamus/drug effects , Hypothalamus/metabolism , Male , Neurons/drug effects , Neurons/metabolism , Rats , Rats, Long-Evans , Sexual Behavior, Animal/physiology , Signal Transduction/drug effects , Nociceptin Receptor , NociceptinABSTRACT
AIMS: The G-protein-coupled estrogen receptor GPR30 (also referred to as GPER) has been implicated in the estrogenic regulation of hippocampal plasticity and spatial memory; however, the molecular mechanisms are largely unclear. METHODS: In this study, we initially examined the levels of GPR30 in the hippocampus of postnatal, ovariectomy (OVX)- and letrozole (LET)-treated female mice. Under G1, G15, and/or OVX treatment, the spatial memory, spine density, levels of ERα, ERß, and SRC-1, selected synaptic proteins, mTORC2 signals (Rictor and p-AKT Ser473), and actin polymerization dynamics were subsequently evaluated. Furthermore, G1, G15, and/or E2 combined with SRC-1 and/or PI3K inhibitors, actin cytoskeleton polymerization modulator JPK, and CytoD treatments were used to address the mechanisms that underlie GPR30 regulation in vitro. Finally, mTORC2 activator A-443654 (A4) was used to explore the role of mTORC2 in GPR30 regulation of spatial memory. RESULTS: The results showed that high levels of GPR30 were detected in the adult hippocampus and the levels were downregulated by OVX and LET. OVX induced an impairment of spatial memory, and changes in other parameters previously described were reversed by G1 and mimicked by G15. Furthermore, the E2 effects on SRC-1 and mTORC2 signals, synaptic proteins, and actin polymerization were inhibited by G15, whereas G1 effects on these parameters were inhibited by the blockade of SRC-1 or PI3K; the levels of synaptic proteins were regulated by JPK and CytoD. Importantly, G15-induced actin depolymerization and spatial memory impairment were rescued by mTORC2 activation with A4. CONCLUSIONS: Taking together, these results demonstrated that decreased GPR30 induces actin depolymerization through SRC-1 and PI3K/mTORC2 pathways and ultimately impairs learning and memory, indicating its potential role as a therapeutic target against hippocampus-based, E2-related memory impairments.
Subject(s)
Actins/metabolism , Estrogens/metabolism , Hippocampus/metabolism , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/metabolism , Spatial Memory/physiology , Animals , Cell Line , Dendritic Spines/metabolism , Female , Hippocampus/growth & development , Maze Learning/physiology , Mechanistic Target of Rapamycin Complex 2/metabolism , Mice, Inbred C57BL , Nuclear Receptor Coactivator 1/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Estrogen/antagonists & inhibitors , Receptors, G-Protein-Coupled/antagonists & inhibitorsABSTRACT
The hypothalamus is most often associated with innate behaviors such as is hunger, thirst and sex. While the expression of these behaviors important for survival of the individual or the species is nested within the hypothalamus, the desire (i.e., motivation) for them is centered within the mesolimbic reward circuitry. In this review, we will use female sexual behavior as a model to examine the interaction of these circuits. We will examine the evidence for a hypothalamic circuit that regulates consummatory aspects of reproductive behavior, i.e., lordosis behavior, a measure of sexual receptivity that involves estradiol membrane-initiated signaling in the arcuate nucleus (ARH), activating ß-endorphin projections to the medial preoptic nucleus (MPN), which in turn modulate ventromedial hypothalamic nucleus (VMH) activity-the common output from the hypothalamus. Estradiol modulates not only a series of neuropeptides, transmitters and receptors but induces dendritic spines that are for estrogenic induction of lordosis behavior. Simultaneously, in the nucleus accumbens of the mesolimbic system, the mating experience produces long term changes in dopamine signaling and structure. Sexual experience sensitizes the response of nucleus accumbens neurons to dopamine signaling through the induction of a long lasting early immediate gene. While estrogen alone increases spines in the ARH, sexual experience increases dendritic spine density in the nucleus accumbens. These two circuits appear to converge onto the medial preoptic area where there is a reciprocal influence of motivational circuits on consummatory behavior and vice versa. While it has not been formally demonstrated in the human, such circuitry is generally highly conserved and thus, understanding the anatomy, neurochemistry and physiology can provide useful insight into the motivation for sexual behavior and other innate behaviors in humans.
ABSTRACT
Estrogens affect dopamine transmission in the striatum, increasing dopamine availability, maintaining D2 receptor density, and reducing the availability of the dopamine transporter. Some of these effects of estrogens are rapid, suggesting that they are mediated by membrane associated receptors. Recently our group demonstrated that there is extra-nuclear labeling for ERα, ERß, and GPER1 in the striatum, but that ERα and GPER1 are not localized to dopaminergic neurons in this region. GABAergic neurons are the most common type of neuron in the striatum, and changes in GABA transmission affect dopamine transmission. Thus, to determine whether ERα or GPER1 are localized to GABAergic neurons, we double labeled the striatum with antibodies for ERα or GPER1 and GABA and examined them using electron microscopy. Ultrastructural analysis revealed that ERα and GPER1 are localized exclusively to extranuclear sites in the striatum, and â¼35% of the dendrites and axon terminals labeled for these receptors contain GABA immunoreactivity. Binding at membrane-associated ERα and GPER1 could account for rapid estrogen-induced decreases in GABA transmission in the striatum, which, in turn, could affect dopamine transmission in this region.
Subject(s)
Corpus Striatum/metabolism , Estrogen Receptor alpha/metabolism , GABAergic Neurons/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Female , Rats, Sprague-DawleyABSTRACT
Tamoxifen is a selective estrogen receptor modulator that competitively binds the ligand-binding domain of estrogen receptors. Binding of tamoxifen displaces its cognate ligand, 17ß-estradiol, thereby hampering the activation of estrogen receptors. Cellular labeling of ER is typically carried out using specific antibodies which require permeabilization of cells, incubation with secondary antibodies, and are expensive and time consuming. In this article, we describe the usefulness of FLTX1, a novel fluorescent tamoxifen derivative, which allows the labeling of estrogen receptors in immunocytochemistry and immunohistochemistry studies, both under permeabilized and non-permeabilized conditions. Further, besides labeling canonical estrogen receptors, this novel fluorescent probe is also suitable for the identification of unconventional targets such membrane estrogen receptors as well as other noncanonical targets, some of which are likely responsible for the number of undesired side effects reported during long-term tamoxifen treatments.
Subject(s)
Fluorescent Dyes/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Oxadiazoles/metabolism , Receptors, Estrogen/metabolism , Tamoxifen/analogs & derivatives , Uterus/metabolism , Animals , Female , Humans , MCF-7 Cells , Mice , Protein Binding , Tamoxifen/metabolism , WorkflowABSTRACT
Trafficking studies of plasma membrane-localized intracellular estrogen receptors have mainly relied on biochemical and histological techniques to locate the receptor before and after estradiol stimulation. More often than not these experiments were performed using postmortem, lysed, or fixed tissue samples, whose tissue or cellular structure is typically severely altered or at times completely lost, making the definitive localization of estrogen receptors difficult to ascertain. To overcome this limitation we began using total internal reflection fluorescence microscopy (TIRFM) to study the trafficking of plasma membrane estrogen receptors. This real-time imaging approach, described in this chapter, permits observation of live, intact cells while allowing visualization of the steps (in time and spatial distribution) involved in receptor activation by estradiol and movements on and near the membrane. TIRFM yields high-contrast real-time images of fluorescently labeled E6BSA molecules on and just below the cell surface and is ideal for studying estrogen receptor trafficking in living cells.
Subject(s)
Cell Membrane/metabolism , Estrogen Receptor alpha/metabolism , Microscopy, Fluorescence/methods , Neurons/metabolism , Animals , Cell Line , Cell Membrane/drug effects , Estradiol/pharmacology , Estrogen Receptor alpha/agonists , Estrogens/pharmacology , Image Interpretation, Computer-Assisted , Mice , Neurons/drug effects , Protein Transport , Time Factors , WorkflowABSTRACT
Breast cancer is a leading killer of women worldwide. Cyclodextrin-based estrogen receptor-targeting drug-delivery systems represent a promising direction in cancer therapy but have rarely been investigated. To seek new targeting therapies for membrane estrogen receptor-positive breast cancer, an estrogen-anchored cyclodextrin encapsulating a doxorubicin derivative Ada-DOX (CDE1-Ada-DOX) has been synthesized and evaluated in human breast cancer MCF-7 cells. First, we synthesized estrone-conjugated cyclodextrin (CDE1), which formed the complex CDE1-Ada-DOX via molecular recognition with the derivative adamantane-doxorubicin (Ada-DOX) (Kd =1,617 M(-1)). The structure of the targeting vector CDE1 was fully characterized using (1)H- and (13)C-nuclear magnetic resonance, mass spectrometry, and electron microscopy. CDE1-Ada-DOX showed two-phase drug-release kinetics with much slower release than Ada-DOX. The fluorescence polarization analysis reveals that CDE1-Ada-DOX binds to recombinant human estrogen receptor α fragments with a Kd of 0.027 µM. Competition assay of the drug complex with estrogen ligands demonstrated that estrone and tamoxifen competed with CDE1-Ada-DOX for membrane estrogen receptor binding in MCF-7 cells. Intermolecular self-assembly of CDE1 molecules were observed, showing tail-in-bucket and wire-like structures confirmed by transmission electronic microscopy. CDE1-Ada-DOX had an unexpected lower drug uptake (when the host-guest ratio was >1) than non-targeting drugs in MCF-7 cells due to ensconced ligands in cyclodextrins cavities resulting from the intermolecular self-assembly. The uptake of CDE1-Ada-DOX was significantly increased when the host-guest ratio was adjusted to be less than half at the concentration of CDE1 over 5 µM due to the release of the estrone residues. CDE1 elicited rapid activation of mitogen-activated protein kinases (p44/42 MAPK, Erk1/2) in minutes through phosphorylation of Thr202/Tyr204 in MCF-7 cells. These results demonstrate a targeted therapeutics delivery of CDE1-Ada-DOX to breast cancer cells in a controlled manner and that the drug vector CDE1 can potentially be employed as a molecular tool to differentiate nongenomic from genomic mechanism.
Subject(s)
Antineoplastic Agents , Breast Neoplasms/metabolism , Cyclodextrins , Drug Delivery Systems , Estrogens , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Cyclodextrins/chemistry , Cyclodextrins/pharmacokinetics , Estrogens/chemistry , Estrogens/pharmacokinetics , Female , HumansABSTRACT
GPER possesses structural and functional characteristics shared by members of the G-protein-coupled receptor (GPCR) superfamily, the largest class of plasma membrane receptors. This newly appreciated estrogen receptor is localized predominately within intracellular membranes in most, but not all, cell types and its surface expression is modulated by steroid hormones and during tissue injury. An intracellular staining pattern is not unique among GPCRs, which employ a diverse array of molecular mechanisms that restrict cell surface expression and effectively regulating receptor binding and activation. The finding that GPER displays an intracellular predisposition has created some confusion as the estrogen-inducible transcription factors, ERα and ERß, also reside intracellularly, and has led to complex suggestions of receptor interaction. GPER undergoes constitutive retrograde trafficking from the plasma membrane to the endoplasmic reticulum and recent studies indicate its interaction with PDZ binding proteins that sort transmembrane receptors to synaptosomes and endosomes. Genetic targeting and selective ligand approaches as well as cell models that express GPER in the absence of ERs clearly supports GPER as a bonafide "stand alone" receptor. Here, the molecular details that regulate GPER action, its cell biological activities and its implicated roles in physiological and pathological processes are reviewed.
Subject(s)
Receptors, Estrogen/chemistry , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Animals , Endosomes/metabolism , Humans , Intracellular Membranes/metabolism , PDZ Domains , Protein Binding , Protein Transport , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Signal TransductionABSTRACT
Steroid receptors exist and function in multiple compartments of cells in most organs. Although the functions and nature of some of these receptors is being defined, important aspects of receptor localization and signaling to physiology and pathophysiology have been identified. In particular, extranuclear sex steroid receptors have been found in many normal cells and in epithelial tumors, where they enact signal transduction that impacts both nongenomic and genomic functions. Here, I focus on the progress made in understanding the roles of extranuclear estrogen receptors (ER) in physiology and pathophysiology. Extranuclear ER serve as a model to selectively intervene with novel receptor reagents to prevent or limit disease progression. Recent novel mouse models and membrane ER-selective agonists also provide a better understanding of receptor pool cross-talk that results in the overall integrative actions of sex steroids.
Subject(s)
Cell Membrane/metabolism , Receptors, Estrogen/physiology , Animal Structures/physiology , Animals , Humans , Mice , Models, Animal , Organogenesis/genetics , Protein TransportABSTRACT
The GPR30, a former orphan GPCR, is a putative membrane estrogen receptor that can activate rapid signaling pathways such as extracellular regulated kinase (ERK) in a variety of cells and may contribute to estrogen's effects in the central nervous system. The distribution of GPR30 in the limbic system predicts a role for this receptor in the regulation of learning and memory and anxiety by estrogens. Though acute G-1 treatment is reported to be anxiogenic in ovariectomised female mice and in gonadally intact male mice, the effect of GPR30 activation is unknown in gonadectomised male mice. In this study, we show that an acute administration of G-1 to gonadectomised male mice, but not female mice, was anxiolytic on an elevated plus maze task, without affecting locomotor activity. In addition, though G-1 treatment did not regulate ERK, it was associated with increased estrogen receptor (ER)α phosphorylation in the ventral, but not dorsal, hippocampus of males. In the female, G-1 increased the ERK activation solely in the dorsal hippocampus, independent of state anxiety. This is the first study to report an anxiolytic effect of GPR30 activation in male mice, in a rapid time frame that is commensurate with non-genomic signaling by estrogen.
Subject(s)
Anxiety/drug therapy , Anxiety/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Corticosterone/metabolism , Cyclopentanes/pharmacology , Estradiol/pharmacology , Estrogen Receptor alpha/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Hippocampus/drug effects , Hippocampus/metabolism , Male , Mice , Mice, Inbred C57BL , Orchiectomy , Ovariectomy , Phosphorylation/drug effects , Quinolines/pharmacology , Receptors, Estrogen , Receptors, G-Protein-Coupled/agonists , Sex Factors , Signal TransductionABSTRACT
In the present work, we investigated the effect of 17ß-estradiol (E2) and basic fibroblast growth factor 2 (FGF2) on the lactotroph cell-proliferative response and the related membrane-initiated signaling pathway. Anterior pituitary mixed-cell cultures of random, cycling 3-mo-old female rats were treated with 10 nM E2, E2 membrane-impermeable conjugated BSA (E2-BSA), PPT (ERα agonist), and DPN (ERß agonist) alone or combined with FGF2 (10 ng/ml) for 30 min or 4 h. Although our results showed that the uptake of BrdU into the nucleus of lactotrophs was not modified by E2 or FGF2 alone, a significant increase in the lactotroph uptake of BrdU was observed after E2/FGF2 coincubation, with this effect being mimicked by PPT/FGF2. These proliferative effects were blocked by ICI 182,780 or PD-98059. The involvement of membrane ER in the proliferative response of prolactin cells induced by the steroid and FGF2 coincubation was confirmed using E2-BSA, and the association between ERα and FGF receptor was observed after E2/FGF2 treatment by immunoprecipitation. A significant increase in the ERK1/2 expression was noted after E2, E2-BSA, PPT, and FGF2 alone, which was more noticeable after E2-BSA/FGF2, E2/FGF2, or PPT/FGF2 treatments. This study provides evidence that E2 and FGF2 exert a cooperative effect on the lactotroph proliferation principally by signaling initiated at the plasma membrane triggering a genomic effect mediated by MEK/ERK1/2, a common signaling pathway, that finally regulates the lactotroph population, thus contributing to pituitary plasticity.