Nongenomic Actions Of Estrogens Research Articles

The G-Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Proliferation and Causes Apoptosis in Leukemia Cell Lines of T Lineage.

The G-protein–coupled estrogen receptor (GPER) mediates non-genomic action of estrogen. Due to its differential expression in some tumors as compared to the original healthy tissues, the GPER has been proposed as a therapeutic target. Accordingly, the non-steroidal GPER agonist G-1, which has often demonstrated marked cytotoxicity in experimental models, has been suggested as a novel anticancer agent for several sensitive tumors. We recently revealed that cell lines derived from acute T-cell (query) lymphoblastic leukemia (T-ALL) express the GPER. Here, we address the question whether G-1 is cytotoxic to T-ALL. We have shown that G-1 causes an early rise of intracellular Ca2+, arrests the cell cycle in G2/M, reduces viability, and provokes apoptosis in T-ALL cell lines. Importantly, G-1 caused destabilization and depolymerization of microtubules. We assume that it is a disturbance of the cytoskeleton that causes G-1 cytotoxic and cytostatic effects in our model. The observed cytotoxic effects, apparently, were not triggered by the interaction of G-1 with the GPER as pre-incubation with the highly selective GPER antagonist G-36 was ineffective in preventing the cytotoxicity of G-1. However, G-36 prevented the intracellular Ca2+ rise provoked by G-1. Finally, G-1 showed only a moderate negative effect on the activation of non-leukemic CD4+ lymphocytes. We suggest G-1 as a potential antileukemic drug.

Non-Genomic Actions of Estrogens on the DNA Repair Pathways Are Associated With Chemotherapy Resistance in Breast Cancer.

Estrogens have been implicated in the etiology of breast cancer for a long time. It has been stated that long-term exposure to estrogens is associated with a higher incidence of breast cancer, since estradiol (E2) stimulates breast cell growth; however, its effect on DNA damage/repair is only starting to be investigated. Recent studies have documented that estrogens are able to modify the DNA damage response (DDR) and DNA repair mechanisms. On the other hand, it has been proposed that DDR machinery can be altered by estrogen signaling pathways, that can be related to cancer progression and chemoresistance. We have demonstrated that E2 promotes c-Src activation and breast cancer cell motility, through a non-genomic pathway. This review discusses scientific evidence supporting this non-genomic mechanism where estrogen modifies the DNA repair pathways, and its relationship to potential causes of chemoresistance.

Characterization of G protein-coupled estrogen receptors in Japanese medaka, Oryzias latipes.

The G protein-coupled estrogen receptor 1 (Gper1) is a membrane-bound estrogen receptor that mediates non-genomic action of estrogens. A Gper1-mediating pathway has been implicated in reproductive activities in fish, including oocyte growth, but Gper1 has been characterized in only a very limited number of fish species. In this study, we cloned and characterized two genes encoding medaka (Oryzias latipes) Gper1s, namely, Gper1a and Gper1b, and phylogenic and synteny analyses suggest that these genes originate through a teleost-specific whole genome duplication event. We found that Gper1a induced phosphorylation of mitogen-activated protein kinase (MAPK) in 293T cells transfected with medaka Gper1s on exposure to the natural estrogen, 17β-estradiol (E2) and a synthetic Gper1 agonist (G-1), and treatment with both E2 and G-1 also decreased the rate of spontaneous maturation in medaka oocytes. These findings show that the processes for oocyte growth and maturation are sensitive to estrogens and are possibly mediated through Gper1a in medaka. We also show that 17α-ethinylestradiol (EE2), one of the most potent estrogenic endocrine-disrupting chemicals, and bisphenol A (BPA, a weak environmental estrogen) augmented phosphorylation of MAPK through medaka Gper1s in 293T cells. Interestingly, however, treatment with EE2 or BPA did not attenuate maturation of medaka oocytes. Our findings support that Gper1-mediated effects on oocytes are conserved among fish species, but effects of estrogenic endocrine-disrupting chemicals on oocytes acting through Gper1 may be divergent among fish species.

Role of GPER-Mediated Signaling in Testicular Functions and Tumorigenesis.

Estrogen signaling plays important roles in testicular functions and tumorigenesis. Fifteen years ago, it was discovered that a member of the G protein-coupled receptor family, GPR30, which binds also with high affinity to estradiol and is responsible, in part, for the rapid non-genomic actions of estrogens. GPR30, renamed as GPER, was detected in several tissues including germ cells (spermatogonia, spermatocytes, spermatids) and somatic cells (Sertoli and Leydig cells). In our previous review published in 2014, we summarized studies that evidenced a role of GPER signaling in mediating estrogen action during spermatogenesis and testis development. In addition, we evidenced that GPER seems to be involved in modulating estrogen-dependent testicular cancer cell growth; however, the effects on cell survival and proliferation depend on specific cell type. In this review, we update the knowledge obtained in the last years on GPER roles in regulating physiological functions of testicular cells and its involvement in neoplastic transformation of both germ and somatic cells. In particular, we will focus our attention on crosstalk among GPER signaling, classical estrogen receptors and other nuclear receptors involved in testis physiology regulation.

Impact of Estrogens on the Regulation of White, Beige, and Brown Adipose Tissue Depots.

As adipose tissue depots are active endocrine organs, they secrete a variety of hormones (including estrogens from white adipose) and inflammatory mediators, which have important implications in numerous obesity-associated diseases. Adipose tissues are broadly characterized as consisting of white, beige, and brown depot types. The endocrine, metabolic, and inflammatory profiles of adipose are depot dependent and influenced by the estrogenic and androgenic status of the adipose tissue. Estrogen receptors mediate both the genomic and nongenomic actions of estrogens and are expressed in the brain, heart, and other peripheral tissues. All three known estrogen receptor α (ERα) and estrogen receptor β (ERβ), and the G-protein coupled estrogen receptor (GPER/GPR30) are expressed in white adipose and can modulate adipose mass. Expression of each receptor is dependent on depot location, adipose cell type, and estrogen levels. Estrogen receptor expression profiles in beige and brown adipocytes are less well established. This review will discuss the effects of estrogens on the differential deposition of the major adipose tissues and the impact of estrogens within white adipose depots. © 2019 American Physiological Society. Compr Physiol 9:457-475, 2019.

A theory of eu-estrogenemia: a unifying concept.

Objective:The aim of the study was to propose a unifying theory for the role of estrogen in postmenopausal women through examples in basic science, randomized controlled trials, observational studies, and clinical practice.Methods:Review and evaluation of the literature relating to estrogen.Discussion:The role of hormone therapy and ubiquitous estrogen receptors after reproductive senescence gains insight from basic science models. Observational studies and individualized patient care in clinical practice may show outcomes that are not reproduced in randomized clinical trials. The understanding gained from the timing hypothesis for atherosclerosis, the critical window theory in neurosciences, randomized controlled trials, and numerous genomic and nongenomic actions of estrogen discovered in basic science provides new explanations to clinical challenges that practitioners face. Consequences of a hypo-estrogenemic duration in women's lives are poorly understood. The Study of Women Across the Nation suggests its magnitude is greater than was previously acknowledged. We propose that the healthy user bias was the result of surgical treatment (hysterectomy with oophorectomy) for many gynecological maladies followed by pharmacological and physiological doses of estrogen to optimize patient quality of life. The past decade of research has begun to demonstrate the role of estrogen in homeostasis.Conclusions:The theory of eu-estrogenemia provides a robust framework to unify the timing hypothesis, critical window theory, randomized controlled trials, the basic science of estrogen receptors, and clinical observations of patients over the past five decades.

GPER Mediates Non-Genomic Effects of Estrogen.

Estrogens are important modulators of a broad spectrum of physiological functions in humans. However, despite their beneficial actions, a number of lines of evidence correlate the sustained exposure to exogenous estrogen with increased risk of the onset of various cancers. Mainly these steroid hormones induce their effects by binding and activating estrogen receptors (ERα and ERβ). These receptors belong to the family of ligand-regulated transcription factors, and upon activation they regulate the expression of different target genes by binding directly to specific DNA sequences. On the other hand, in recent years it has become clear that the G protein-coupled estrogen receptor 30 (GPR30/GPER) is able to mediate non-genomic action of estrogens in different cell contexts. In particular, GPER has been shown to specifically bind estrogens, and in turn to functionally cross-react with diverse cell signaling systems such as the epidermal growth factor receptor (EGFR) pathway, the Notch signaling pathway and the mitogen-activated protein kinases (MAPK) pathway. In this chapter we will present some of the different experimental techniques currently used to demonstrate the functional role of GPER in mediating non-genomic actions of estrogens, such as the dual luciferase assay, assessment of the involvement of GPER in the stimulation of cell migration in breast cancer cell lines and in cancer-associated fibroblasts, and chromatin immunoprecipitation assay. Overall, the experimental procedures described herein represent key instruments for assessing the biological role of GPER in mediating non-genomic signals of estrogen.

Ligand Accessibility and Bioactivity of a Hormone–Dendrimer Conjugate Depend on pH and pH History

Estrogen conjugates with a polyamidoamine (PAMAM) dendrimer have shown remarkably selective regulation of the nongenomic actions of estrogens in target cells. In response to pH changes, however, these estrogen-dendrimer conjugates (EDCs) display a major morphological transition that alters the accessibility of the estrogen ligands that compromises the bioactivity of the EDC. A sharp break in dynamic behavior near pH 7 occurs for three different ligands on the surface of a PAMAM-G6 dendrimer: a fluorophore (tetramethylrhodamine [TMR]) and two estrogens (17α-ethynylestradiol and diphenolic acid). Collisional quenching and time-resolved fluorescence anisotropy experiments with TMR-PAMAM revealed high ligand shielding above pH 7 and low shielding below pH 7. Furthermore, when the pH was cycled from 8.5 (conditions of ligand-PAMAM conjugation) to 4.5 (e.g., endosome/lysosome) and through 6.5 (e.g., hypoxic environment) back to pH 8.5, the 17α-ethynylestradiol- and diphenolic acid-PAMAM conjugates experienced a dramatic, irreversible loss in cell stimulatory activity; dynamic NMR studies indicated that the hormonal ligands had become occluded within the more hydrophobic core of the PAMAM dendrimer. Thus, the active state of these estrogen-dendrimer conjugates appears to be metastable. This pH-dependent irreversible masking of activity is of considerable relevance to the design of drug conjugates with amine-bearing PAMAM dendrimers.

Neuronal differentiation dictates estrogen-dependent survival and ERK1/2 kinetic by means of caveolin-1.

Estrogens promote a plethora of effects in the CNS that profoundly affect both its development and mature functions and are able to influence proliferation, differentiation, survival and neurotransmission. The biological effects of estrogens are cell-context specific and also depend on differentiation and/or proliferation status in a given cell type. Furthermore, estrogens activate ERK1/2 in a variety of cellular types. Here, we investigated whether ERK1/2 activation might be influenced by estrogens stimulation according to the differentiation status and the molecular mechanisms underling this phenomenon. ERK1/2 exert an opposing role on survival and death, as well as on proliferation and differentiation depending on different kinetics of phosphorylation. Hence we report that mesencephalic primary cultures and the immortalized cell line mes-c-myc A1 express estrogen receptor α and activate ERK1/2 upon E2 stimulation. Interestingly, following the arrest of proliferation and the onset of differentiation, we observe a change in the kinetic of ERKs phosphorylation induced by estrogens stimulation. Moreover, caveolin-1, a main constituent of caveolae, endogenously expressed and co-localized with ER-α on plasma membrane, is consistently up-regulated following differentiation and cell growth arrest. In addition, we demonstrate that siRNA-induced caveolin-1 down-regulation or disruption by means of ß-cyclodextrin treatment changes ERK1/2 phosphorylation in response to estrogens stimulation. Finally, caveolin-1 down-regulation abolishes estrogens-dependent survival of neurons. Thus, caveolin-1 appears to be an important player in mediating, at least, some of the non-genomic action of estrogens in neurons, in particular ERK1/2 kinetics of activation and survival.

The many faces of estrogen signaling.

Estrogens have long been known as important regulators of the female reproductive functions; however, our understanding of the role estrogens play in the human body has changed significantly over the past years. It is now commonly accepted that estrogens and androgens have important functions in both female and male physiology and pathology. This is in part due to the local synthesis and action of estrogens that broadens the role of estrogen signaling beyond that of the endocrine system. Furthermore, there are several different mechanisms through which the three estrogen receptors (ERs), ERα, ERβ and G protein-coupled estrogen receptor 1 (GPER1) are able to regulate target gene transcription. ERα and ERβ are mostly associated with the direct and indirect genomic signaling pathways that result in target gene expression. Membrane-bound GPER1 is on the other hand responsible for the rapid non-genomic actions of estrogens that activate various protein-kinase cascades. Estrogen signaling is also tightly connected with another important regulatory entity, i.e. epigenetic mechanisms. Posttranslational histone modifications, microRNAs (miRNAs) and DNA methylation have been shown to influence gene expression of ERs as well as being regulated by estrogen signaling. Moreover, several coregulators of estrogen signaling also exhibit chromatin-modifying activities further underlining the importance of epigenetic mechanisms in estrogen signaling. This review wishes to highlight the newer aspects of estrogen signaling that exceed its classical endocrine regulatory role, especially emphasizing its tight intertwinement with epigenetic mechanisms.

Abstract B82: G-protein coupled estrogen receptor (GPER) agonist G-1 inhibits growth of human granulosa cell tumor cells via blocking microtubule assembly

Abstract Accumulating evidence indicates that the G-protein coupled estrogen receptor (GPER) mediates the non-genomic actions of estrogen in many different cells and tissues. The selective GPER agonist G-1 (1-[4-(6-bromobenzo[1,3]dioxol-5yl)-3a,4,5,9b-tetrahydro- 3H-cyclopenta[c]quinolin-8-yl]-ethanone) was developed to distinguish estrogen actions mediated by GPER from those mediated by the traditional nuclear estrogen receptors. Our recent data showed that G-1 suppresses the growth of breast and ovarian cancer cells in a GPER-independent manner. However, the mechanisms underlying the suppressive actions of G-1 on cancer cell growth are still unclear. The aim of the present study was to explore potential mechanisms of G-1 action in human ovarian granulosa tumor cells. The KGN cell line, a cell line derived from a metastatic adult ovarian granulosa cell tumor (aGCT), and the COV434 cell line, a cell line derived from a juvenile granulosa cell tumor (jGCT), were used as cellular models. Cell proliferation and viability were determined by cell number counting and MTT assay, respectively. Cell cycle analysis was monitored by flow cytometry. Key proteins associated with cell cycle progression were analyzed with Western blot. Cell apoptosis was detected by analysis of caspase activity and DNA integrity. Cellular cytoskeleton changes were monitored by fluorescent immunocytochemistry, confocal microscopy and in vitro microtubule assembly assays. Consistent with our previous results, the putative GPER agonist G-1 suppressed proliferation in a concentration- and time-dependent manner in both KGN and COV434 cells. G-1 inhibited GCT cell growth via suppressing cell viability, which was indicated by a significant decrease in the MTT metabolism, an increase in DNA fragmentation, and an increase in PARP cleavage. G-1 treatment did not affect the expression of cyclin D1, cyclin D2 and cyclin E, suggesting that G-1 treatment had no effect on the early stage cell cycle progression, a result further confirmed by flow cytometry. However, flow cytometry showed that G-1 treatment arrested the cell cycle in the G2/M phase. G2/M phase arrest of KGN cells was confirmed by a significant increase in the phosphorylation of histone H3 after G-1 treatment. Surprisingly, the Western blot analysis showed that G-1 treatment did not affect the activation of mitotic promoting factors (MPF) and had no effect on G2/M entry checkpoint proteins and kinases. Fluorescent immunocytochemistry and confocal microscopy showed that G-1 treatment disrupted the segregation of chromosomes during mitosis, an effect that may be attributed to the abnormal assembly of microtubules. In vitro microtubule assembly assays demonstrated that G-1 treatment indeed suppressed tubulin polymerization and thereby inhibited the assembly of the microtubules. In conclusion, our results from the present study demonstrate that G-1, the putative GPER agonist, suppresses GCT cell proliferation and induces tumor cell death via blocking microtubule assembly. The ability of G-1 to suppress tumor cell proliferation and induce tumor cell apoptosis by targeting microtubules makes it a promising candidate drug for the treatment of GCT. Citation Format: Xiangmin Lv, Guohua Hua, Chunbo He, John S Davis, Cheng Wang. G-protein coupled estrogen receptor (GPER) agonist G-1 inhibits growth of human granulosa cell tumor cells via blocking microtubule assembly. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B82.

The G-protein-coupled estrogen receptor agonist G-1 suppresses proliferation of ovarian cancer cells by blocking tubulin polymerization

The G-protein-coupled estrogen receptor 1 (GPER) has recently been reported to mediate the non-genomic action of estrogen in different types of cells and tissues. G-1 (1-[4-(6-bromobenzo[1,3] dioxol-5yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone) was developed as a potent and selective agonist for GPER. G-1 has been shown to induce the expression of genes and activate pathways that facilitate cancer cell proliferation by activating GPER. Here we demonstrate that G-1 has an anticancer potential with a mechanism similar to vinca alkaloids, the commonly used chemotherapy drugs. We found that G-1 blocks tubulin polymerization and thereby interrupts microtubule assembly in ovarian cancer cells leading to the arrest of cell cycle in the prophase of mitosis and the suppression of ovarian cancer cell proliferation. G-1 treatment also induces apoptosis of ovarian cancer cells. The ability of G-1 to target microtubules to suppress ovarian cancer cell proliferation makes it a promising candidate drug for treatment of ovarian cancer.

Genomic and non-genomic actions of estrogen: recent developments.

Abstract Estrogen affects transcriptional status by activating its corresponding nuclear receptor, the estrogen receptor (ER). It can also induce rapid cellular reactions within a few minutes, and this feature cannot be explained by the transcription-mediated effects of estrogen. The latter mechanisms are called 'non-genomic actions' of estrogen. In contrast, the former classic modes of action came to be called 'genomic actions'. One of the recent developments of research on estrogen was the substantiation of the non-genomic actions of estrogen; these were initially observed and reported as intriguing phenomena more than 40 years ago. The interacting molecules as well as the biological significance of non-genomic actions have now been shown. In the field of genomic actions, invention and spread of new technologies, including high-throughput sequencers, promoted a comprehensive view of estrogen-mediated transcriptional regulation.

Cytoplasmic estrogen receptor β as a potential marker in human non-small cell lung carcinoma

Objectives:Estrogen has been reported to promote an increased susceptibility to lung cancer development. This study focusses on the role of cytoplasmic estrogen receptor β (c-ERβ) in NSCLC.Methods:NSCLC (n = 162) cases were analyzed using immunohistochemistry (IHC) for c-ERβ expression and its association with clinicopathological variables. Significance of c-ERβ expression was further examined using in vitro studies in NSCLC cell lines.Results:Among ERβ and aromatase positive NSCLC females, c-ERβ was significantly associated with greater tumor diameter and tended to be associated with worse overall survival. A549 and LCAM1 cells expressed aromatase, as well as c-ERβ and nuclear ERβ (n-ERβ). U0126 (MAPK/extracellular-signal-regulated kinase (ERK) inhibitor) abrogated MAPK phosphorylation, caused by estradiol via c-ERβ, more effectively than ICI 182780 (ER blocker) in either cell line. However, ICI 182780 completely abrogated the estrogen responsive elements (ERE)-luciferase activity caused by estradiol. Combination therapy with ICI 182780 and U0126 turned out to be far more effective than either treatment alone in either A549 or LCAM1 cells.Conclusion:The results indicated that ERβ may contribute to NSCLC via non-genomic action of estrogen through its cytoplasmic form, in addition to the genomic actions via n-ERβ. These actions of estrogen in NSCLCs may be abrogated by combination therapy with ICI 182780 and U0126.

Estrogen Receptor β-Selective Agonists Stimulate Calcium Oscillations in Human and Mouse Embryonic Stem Cell-Derived Neurons

Estrogens are used extensively to treat hot flashes in menopausal women. Some of the beneficial effects of estrogens in hormone therapy on the brain might be due to nongenomic effects in neurons such as the rapid stimulation of calcium oscillations. Most studies have examined the nongenomic effects of estrogen receptors (ER) in primary neurons or brain slices from the rodent brain. However, these cells can not be maintained continuously in culture because neurons are post-mitotic. Neurons derived from embryonic stem cells could be a potential continuous, cell-based model to study nongenomic actions of estrogens in neurons if they are responsive to estrogens after differentiation. In this study ER-subtype specific estrogens were used to examine the role of ERα and ERβ on calcium oscillations in neurons derived from human (hES) and mouse embryonic stem cells. Unlike the undifferentiated hES cells the differentiated cells expressed neuronal markers, ERβ, but not ERα. The non-selective ER agonist 17β-estradiol (E2) rapidly increased [Ca2+]i oscillations and synchronizations within a few minutes. No change in calcium oscillations was observed with the selective ERα agonist 4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT). In contrast, the selective ERβ agonists, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), MF101, and 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3 benzoxazol-5-ol (ERB-041; WAY-202041) stimulated calcium oscillations similar to E2. The ERβ agonists also increased calcium oscillations and phosphorylated PKC, AKT and ERK1/2 in neurons derived from mouse ES cells, which was inhibited by nifedipine demonstrating that ERβ activates L-type voltage gated calcium channels to regulate neuronal activity. Our results demonstrate that ERβ signaling regulates nongenomic pathways in neurons derived from ES cells, and suggest that these cells might be useful to study the nongenomic mechanisms of estrogenic compounds.

Rapid estrogen signalling in mouse primordial germ cells

We report here that in the mouse embryonic gonads in addition to gonadal somatic cells, primordial germ cells (PGCs) the precursors of adult gametes, express estrogen receptor α (ERα) and that through this receptor, 17-β-estradiol (E2) is able to modulate in such cells molecular signalling known to be crucial for their development. We demonstrated that PGCs from 11.5 to 12.5 days post coitum (dpc) mouse embryos express ERα transcripts and protein and that at concentrations of 10 − 8 M E2 stimulates rapid (within 20 min) about 4-fold AKT (Ser473) and 3-fold ERK2 (Thr202/Tyr204) and SRC (Tyr418) phosphorylation. In addition, the E2 stimulatory effects were associated with increased phosphorylation of the KIT receptor (Tyr568/570). While the ER antagonist ICI182780 was able to abolish these effects, AKT phosphorylation induced by E2 was also inhibited by the PI3K inhibitor LY294002 and the SRC family inhibitor PP2. This latter was also able to abolish the increased phosphorylation of KIT and ERKs caused by E2. Taken together these results suggest that E2 may modulate via ERα non-genomic signalling/phosphorylation cascade in mouse PGCs. This was also supported by the finding that PGCs express MNAR, a scaffold protein that regulate ER activation in other cell types. Finally, we found that when PGCs were cultured in the presence of 10 − 8 M E2 a significant ICI inhibitable increase of their number occurred. The present study provides evidence for novel direct non-genomic actions of estrogens on PGCs and suggests that these cells can represent a potential target for estrogens and estrogenic compounds during the early stages of embryo development in mammals.

Genomic and non-genomic actions of estrogen on synaptic plasticity in SH-SY5Y cells

Estrogen modulates synaptic plasticity, an important mechanism of memory storage. Previously, we have reported that estrogen rapidly increases the expression of Arc (activity-regulated cytoskeleton associated protein), a key protein for synaptic plasticity, via non-genomic phosphoinositide-3 kinase (PI-3K)-, mitogen-activated protein kinase (MAPK)-, and estrogen receptor (ER)-dependent pathways in SH-SY5Y cells. The present study aimed to investigate the role of each ER subtype, α and β, in synaptic plasticity in SH-SY5Y cells. The specific agonist of ERβ (DPN) markedly induced Arc expression that mimics treatment with estrogen, but not ERα (PTT). Determination of subcellular localization of ERβ using immunocytochemistry shows that ERβ was retained in the cytoplasm of the untreated cells. In estrogen-treated cells, the membrane and cytosolic ERβ gradually decreased, while nuclear ERβ progressively increased in time-dependent manner, suggesting estrogen-dependent nuclear translocation of ERβ. Nuclear accumulation of ERβ at 6–12 h post-estrogen treatment, leads to increased PSD-95 and SYP mRNA expression, indicating the classical genomic estrogenic action on synaptic plasticity. However, the block of PI-3K signaling by Wortmannin partially suppressed estrogen (48 h)-induced PSD-95 and SYP expression, suggesting a crosstalk mechanism between genomic and non-genomic actions of estrogen on synaptic plasticity. Therefore, the estrogen-enhanced synaptic plasticity is ERβ-dependent and involves the crosstalk mechanism of non-genomic and genomic estrogenic actions.

Neuroprotective actions of sex steroids in Parkinson’s disease

The sex difference in Parkinson’s disease, with a higher susceptibility in men, suggests a modulatory effect of sex steroids in the brain. Numerous studies highlight that sex steroids have neuroprotective properties against various brain injuries. This paper reviews the protective effects of sex hormones, particularly estradiol, progesterone and androgens, in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of Parkinson’s disease as compared to methamphetamine toxicity. The molecular mechanisms underlying beneficial actions of sex steroids on the brain have been investigated showing steroid, dose, timing and duration specificities and presently focus is on the dopamine signaling pathways, the next frontier. Both genomic and non-genomic actions of estrogen converge to promote survival factors and show sex differences. Neuroprotection by estrogen involves activation of signaling molecules such as the phosphatidylinositol-3 kinase/Akt and the mitogen-activated protein kinase pathways. Interaction with growth factors, such as insulin-like growth factor 1, also contributes to protective actions of estrogen.

Association of Estrogen Receptor α and Histone Deacetylase 6 Causes Rapid Deacetylation of Tubulin in Breast Cancer Cells

Estrogen receptor alpha (ERalpha) is a nuclear receptor that functions as a ligand-activated transcription factor. Besides its genomic action in nuclei, ERalpha could exert nongenomic actions at the plasma membrane. To investigate the mechanism underlying the nongenomic action of ERalpha in breast cancer cells, we generated a construct of membrane-targeted ERalpha (memER), an expression vector of ERalpha without the nuclear localizing signal and including instead the membrane-targeting sequence of Src kinase. MemER was stably expressed in human breast cancer MCF-7 cells. Cell migration test and tumorigenic assay in nude mice revealed that the in vitro motility and the in vivo proliferation activity of MCF-7 cells expressing memER were significantly enhanced compared with those of vector-transfected cells. Interestingly, the acetylation level of tubulin in memER-overexpressing cells was lower than that in control cells. We found that histone deacetylase (HDAC) 6 translocated to the plasma membrane shortly after estrogen stimulation, and rapid tubulin deacetylation subsequently occurred. We also showed that memER associated with HDAC6 in a ligand-dependent manner. Although tamoxifen is known for its antagonistic role in the ERalpha genomic action in MCF-7 cells, the agent showed an agonistic function in the memER-HDAC6 association and tubulin deacetylation. These findings suggest that ERalpha ligand dependently forms a complex with HDAC6 and tubulin at the plasma membrane. Estrogen-dependent tubulin deacetylation could provide new evidence for the nongenomic action of estrogen, which potentially contributes to the aggressiveness of ERalpha-positive breast cancer cells.

Estrogen Facilitates both Phosphatidylinositol 3-Kinase/Akt and ERK1/2 Mitogen-Activated Protein Kinase Membrane Signaling Required for Long-Term Neuropeptide Y Transcriptional Regulation in Clonal, Immortalized Neurons

It is established that increases in neuropeptide Y (NPY) expression are associated with hyperphagia and obesity. These effects can be reversed by estrogen, a recognized anorexigen. We found that 17beta-estradiol (E(2)) regulates biphasic NPY gene expression in a clonal, immortalized hypothalamic cell line, N-38, through estrogen receptor (ER) action at the level of the NPY promoter. However, rapid, nongenomic actions of estrogen, linked to the phosphatidylinositol 3-kinase (PI3-K)/Akt and ERK1/2 mitogen-activated protein kinase (MAPK) pathways, may also play a role. We therefore examined the changes in the phosphorylation status of Akt, ERK1/2, and cAMP response element-binding protein (CREB) after treatment with 10 nm E(2) in the N-38 neurons and found activation of these signaling proteins within 5-30 min. We also demonstrated possible cross talk between the estrogen-activated PI3-K/Akt and MAPK/extracellular signal-regulated kinase pathways using pharmacological inhibitors. We find that only ERalpha is involved in the early signaling events using the ERalpha agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol and the ERbeta agonist 2,3-bis(4-hydroxyphenyl)-propionitrile. Furthermore, we can detect colocalization of ERalpha and caveolin-1, a membrane-associated signaling protein. Remarkably, we find that the membrane-mediated events are critical for the long-term estrogen-mediated repression of NPY gene expression that can be mapped to within -97 bp of the NPY promoter. To link the early signaling events to downstream effectors, we detected induction of c-fos and inactivation of MSK-1 by estrogen and binding of CREB to this minimal promoter region. These observations suggest that rapid estrogen-mediated signaling is mediated by ERalpha, and the signal transduction events potentiate the genomic actions of estrogen on NPY gene expression in the N-38 NPY neurons.