Nongenomic Actions Of Estrogens Research Articles

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.

Relationship Between Non-Genomic Actions of Estrogens and Insulin Resistace

Numerous experimental and clinical data show that the physiological actions of insulin and sexual steroids interact in target tissues for these hormones. In the other hand, sexual steroids has effects on peripheral tissues, and since the skeletal muscle is the main responsible for peripheral glucose uptake, it would be possible that the sexual steroids induce directly in the muscle a decrease of the sensibility of this tissue to insulin action. Some of the biological actions of the estrogens are too fast like to be compatible with this classical mechanism of action, and this mechanism has been called not classical, non-genomic or rapid actions of the estrogens. Moreover, some experiments have shown that low concentrations of estradiol, induce an increase in the rate of IRS-1 phosphorylation, promotes the association between IRS-1 and the subunit of PI3-k, p85alpha, causes a decrease in the rate of IRS-1 serine phosphorylation and increases the rate of Akt phosphorylation. Therefore, the evidences suggest the existence of a narrow interrelation between the estrogens and insulin sensitivity, but relatively few studies have tried to resolve the molecular base of this relation in insulin-dependent tissues. The resolution of these unknown questions would be able to have a great long-term therapeutic repercussion. In this sense, we should not forget that insulin resistance is the underlying cause of several associated pathologies to the female aging, as Type 2 diabetes, cardio-circulatory pathology or neurodegenerative disease.

MNAR plays an important role in ERa activation of Src/MAPK and PI3K/Akt signaling pathways

Estrogens play a critical role in the regulation of cellular proliferation, differentiation, and apoptosis. Evidence indicates that this regulation is mediated by a complex interface of direct control of gene expression (so-called “genomic action”) and by regulation of cell-signaling/phosphorylation cascades (referred to as the “non-genomic”, or “extranuclear” action). However, the mechanisms of the non-genomic action of estrogens are not well defined. We have recently described the identification of a novel scaffold protein termed MNAR (modulator of non-genomic action of estrogen receptor), that couples conventional steroid receptors with extranuclear signal transduction pathways, thus potentially providing additional and tissue- or cell-specific level of steroid hormone regulation of cell functions. We have demonstrated that the MNAR is required for ER alpha (ERa) interaction with p60 src (Src), which leads to activation of Src/MAPK pathway. Our new data also suggest that activation of cSrc in response to E2 leads to MNAR phosphorylation, interaction with p85, and activation of the PI3 and Akt kinases. These data therefore suggest that MNAR acts as an important scaffold that integrates ERa action in regulation of important signaling pathways. ERa non-genomic action has been suggested to play a key role in estrogen-induced cardio-, neuro-, and osteo-protection. Therefore, evaluation of the molecular crosstalk between MNAR and ERa may lead to development of functionally selective ER modulators that can separate between beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS and the “detrimental”, proliferative effects in reproductive tissues and organs.

Estrogen, inflammation, and platelet phenotype

Although exogenous estrogenic therapies increase the risk of thrombosis, the effects of estrogen on formed elements of blood are uncertain. This article examines the genomic and nongenomic actions of estrogen on platelet phenotype that may contribute to increased thrombotic risk. To determine aggregation, secretion, protein expression, and thrombin generation, platelets were collected from experimental animals of varying hormonal status and from women enrolled in the Kronos Early Estrogen Prevention Study. Estrogen receptor beta predominates in circulating platelets. Estrogenic treatment in ovariectomized animals decreased platelet aggregation and adenosine triphosphate (ATP) secretion. However, acute exposure to 17beta-estradiol did not reverse decreases in platelet ATP secretion invoked by lipopolysaccharide. Thrombin generation was positively correlated to the number of circulating microvesicles expressing phosphatidylserine. Assessing the effect of estrogen treatments on blood platelets may lead to new ways of identifying women at risk for adverse thrombotic events with such therapies.

Genomic and non-genomic action of estrogen in the mouse skeletal muscle cells.

Genomic and non-genomic action of estrogen in the mouse skeletal muscle cells.

Modulation of Aβ-induced neurotoxicity by estrogen receptor alpha and other associated proteins in lipid rafts

Some evidences have demonstrated the participation of estrogen receptors (ERs) in rapid, non-genomic actions of estrogen to promote neuroprotection against different toxic agents. However, there is still very little information about the structural nature of these receptors and the manner these proteins may be integrated into the plasma membrane. One of the plausible possibilities is that they may be localized in lipid rafts microstructures where they would be associated with other, still unknown, molecules which may modulate their physiological activities related to cell survival. In this work, we have identified in caveolar fractions of murine septal and hippocampal neurons a membrane-related ER shown to physically interact with, both, a voltage-dependent anion channel and scaffold protein caveolin-1.

Signaling by estrogens

By regulating activities and expression levels of key signaling molecules, estrogens control mechanisms that are responsible for crucial cellular functions. Ligand binding to estrogen receptor (ER) leads to conformational changes that regulate the receptor activity, its interaction with other proteins and DNA. In the cytoplasm, receptor interactions with kinases and scaffolding molecules regulate cell signaling cascades (extranuclear/nongenomic action). In the nucleus, estrogens control a repertoire of coregulators and other auxiliary proteins that are associated with ER, which in turn determines the nature of regulated genes and level of their expression (genomic action). The combination of genomic and nongenomic actions of estrogens ultimately confers the cell-type and tissue-type selectivity. Recent studies have revealed some important new insights into the molecular mechanisms underlying ER action, which may help to explain the functional basis of existing selective ER modulators (SERMs) and provide evidence into how ER might be selectively targeted to achieve specific therapeutic goals. In this review, we will summarize some new molecular details that relate to estrogen signaling. We will also discuss some new strategies that may potentially lead to the development of functionally selective ER modulators that can separate between the beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS as well as the "detrimental," proliferative effects in reproductive tissues and organs.

Short Days and Estrogen Make for Short Tempers

The nuclear hormone estrogen is reported to increase aggressive behavior in most birds and domesticated mice but to reduce aggression in Bluebanded gobies, California mice, and humans. Trainor et al . present data showing that in their preferred model, male beach mice, estrogen can produce both effects, depending on the season (or, more precisely, the photoperiod to which the animal is exposed). Short, winterlike days cause testicular regression and aggressive behavior in these mice. In castrated mice (in which constant testosterone concentrations were provided by an implant) exposed to short days (8 hours of light and 16 hours of darkness), activation of estrogen receptors increased aggression (measured as the time elapsed before they attacked a male intruder in their home cage). But when the mice were maintained on long days (16 hours of light and 8 of darkness), estrogen treatment caused a decrease in aggression. Microarray analysis of gene expression in an area of the brain associated with aggression showed that the decrease in aggression produced in animals exposed to long days was associated with increased estrogen-dependent gene expression. The opposite effects of estrogen to increase aggression in animals exposed to short days could be detected within 15 minutes, a time frame likely to be too short for transcriptionally mediated effects. Thus, the authors propose that the increased aggression is influenced by nongenomic actions of estrogens. Together, the findings show that the effects of these nuclear receptors on aggressive behavior are highly dependent on environmental cues. B. C. Trainor, S. Lin, M. S. Finy, M. R. Rowland, R. J. Nelson, Photoperiod reverses the effects of estrogens on male aggression via genomic and nongenomic pathways. Proc. Natl. Acad. Sci. U.S.A. 104 , 9840-9845 (2007). [Abstract] [Full Text]

Nongenomic Estrogen Effects on Nitric Oxide Synthase Activity in Rat Adipocytes

Estrogens exert multiple genomic effects on adipose tissue through binding to nuclear estrogen receptors. However, there is evidence for additional nongenomic mechanisms whereby estrogens may exert their control on adipose tissue metabolism through rapid activation of various membrane-initiated kinase cascades. Here, we tested rapid effects of estrogens on nitric oxide production in white adipose tissue using 17-beta estradiol (E2) and its membrane impermeant albumin conjugated form (17-beta estradiol hemisuccinate BSA, E2-BSA). We found that both E2 and E2-BSA stimulate nitric oxide synthase (NOS) activity in adipocytes. These effects were abolished by 1) ICI 182-780, a selective estrogen receptor antagonist; 2) wortmannin, an inhibitor of phosphatidylinositol 3-kinase; and 3) N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89) an inhibitor of protein kinase A. In contrast to NOS activation by E2, E2-BSA-induced NOS activity was abolished by UO126, an inhibitor of MAPK kinase/ERK (p42/p44 MAPKs). Immunoblotting studies have shown that both estrogens phosphorylate endothelial NOS (NOS III) on Ser(1179), an effect that is prevented by wortmannin and H89, suggesting that NOS III is the target for estrogen-induced NOS activity. Furthermore, only the E2-BSA-induced NOS III phosphorylation on Ser(1179) was totally abolished by UO126. These results indicate that the signaling cascades involved in adipocyte NOS stimulation by estrogens are different depending on whether estrogens are free or conjugated to albumin and therefore underline the importance of estrogen receptor locations in the nongenomic actions of estrogens in these cells.

Nongenomic actions of estrogens: Exciting opportunities for pharmacology

Estrogens, like other steroids, elicit a variety of rapid effects in many tissues in addition to their delayed action on gene expression in the cell nucleus. The rapid responses occur without participation of the genome, and are therefore termed nongenomic. Some of the estrogen induced effects acutely modulate vascular function and may contribute to the gender difference in cardiovascular susceptibility. While some actions may be mediated by novel, nonclassic receptors, the classic estrogen receptor has been shown to also act on signalling cascades. There are sparse examples for compounds structurally related to the endogenous hormone estradiol that bind to the estrogen receptor but may selectively elicit nongenomic responses. The further development of such selectively acting drugs holds much promise for better therapies with fewer side effects, e.g. for vascular malfunction, but also for estrogen-dependent cancer.

Estrogen Receptor α Interacts with Gα13 to Drive Actin Remodeling and Endothelial Cell Migration via the RhoA/Rho Kinase/Moesin Pathway

Sex steroids control cell movement and tissue organization; however, little is known of the involved mechanisms. This report describes the ongoing dynamic regulation by estrogen of the actin cytoskeleton and cell movement in human vascular endothelial cells that depends on rapid activation of the actin-regulatory protein moesin. Moesin activation is triggered by the interaction of the C-terminal portion of cell membrane estrogen receptor alpha with the G protein Galpha(13), leading to activation of the small GTPase RhoA and of the downstream effector Rho-associated kinase. The resulting phosphorylation of moesin on Thr(558) is the means of moesin's binding to actin and the remodeling of the actin cytoskeleton. This cascade of events ensues within minutes of estradiol administration and results in changes in cell morphology and to the development of specialized cell membrane structures such as ruffles and pseudopodia that are necessary for cell movement. These findings expand our knowledge of the basis of estrogen's effects on human cells, including the regulation of actin assembly, cell movement and migration. They highlight novel pathways of signal transduction of estrogen receptor alpha through nontranscriptional mechanisms. Furthermore, exposure of this estrogen receptor-dependent, nongenomic action of estrogen on human vascular endothelial cells is especially relevant to the present interest in the role of estrogen in cardiovascular protection.

Estrogen-Mediated Regulation of p38 Mitogen-Activated Protein Kinase in Human Endometrium

Estrogen predominantly exerts its biological effects through the genomic pathway by binding to its intracellular receptors, interacting with the estrogen response element located in the promoter region of the target gene, and thus regulating gene transcription. There has been an increasing body of evidence regarding nongenomic actions of estrogen. Estrogen activates multiple signaling cascades, including the MAPK pathway. p38 MAPK plays key roles in mediating apoptosis, proliferation, and inflammation, which all take place in the endometrium during cyclical changes under the influence of estrogen. We hypothesized that estrogen might activate the p38 MAPK pathway in endometrial cells and exert some of its actions through this pathway in the endometrium. p38 MAPK phosphorylation was analyzed using in vivo and in vitro techniques. Total and phosphorylated p38 MAPK immunostainings were more intense in epithelial cells compared with stromal cells, and the phosphorylated/total p38 MAPK ratio was significantly higher in the functional endometrial layer compared with the basal layer (P < 0.05). Estradiol significantly increased p38 MAPK phosphorylation in endometrial stromal cells in culture within 2 min (P < 0.05), and this phosphorylation was blocked by a specific p38 MAPK inhibitor. Moreover, tamoxifen and raloxifene also increased phosphorylation of p38 MAPK. The estrogen receptor antagonist ICI 182,780 reversed the estrogen-induced p38 MAPK phosphorylation in endometrial stromal and epithelial cells, suggesting involvement of the estrogen receptor. Our results indicate the involvement of estrogen in regulating p38 MAPK activity in endometrial cells, suggesting a nongenomic action of estrogen through this MAPK in the endometrium.

Estrogen rapid action via protein complex formation involving ERα and Src

This review provides insight into biomolecular knowledge regarding the non-genomic actions of estrogen in hormone-dependent breast cancer, particularly its role in the rapid stimulation of pathways that transmit signals to increase cell division or decrease programmed cell death. Until recently, attention to estrogenic effects focused primarily on events in the nucleus, where most estrogen receptors (ERalpha and beta) reside. However, a fraction of ERalpha associated with the cell membrane also participates in rapid estrogen-induced cell membrane-mediated events via formation of a protein complex with many signaling molecules, leading to activation of the mitogen-activated protein kinase and Akt signaling pathways. Understanding the mechanisms underlying these relationships, with the aim of abrogating specific steps, should lead to more targeted strategies to treat hormone-dependent breast cancer.

Integration of steroid hormone initiated membrane action to genomic function in the brain

Estrogen is a ligand for the estrogen receptor (ER), which on binding 17β-estradiol, functions as a ligand-activated transcription factor and regulates the transcription of target genes. This is the slow genomic mode of action. However, rapid non-genomic actions of estrogen also exist at the cell membrane. Using a novel two-pulse paradigm in which the first pulse rapidly initiates non-genomic actions using a membrane-limited estrogen conjugate (E-BSA), while the second pulse promotes genomic transcription from a consensus estrogen response element (ERE), we have demonstrated that rapid actions of estrogen potentiate the slower transcriptional response from an ERE-reporter in neuroblastoma cells. Since rapid actions of estrogen activate kinases, we used selective inhibitors in the two-pulse paradigm to determine the intracellular signaling cascades important in such potentiation. Inhibition of protein kinase A (PKA), PKC, mitogen activated protein kinase (MAPK) or phosphatidylinositol 3-OH kinase (PI-3K) in the first pulse decreases potentiation of transcription. Also, our data with both dominant negative and constitutive mutants of Gα subunits show that Gα q initiates the rapid signaling cascade at the membrane in SK-N-BE(2)C neuroblastoma cells. We discuss two models of multiple kinase activation at the membrane Pulses of estrogen induce lordosis behavior in female rats. Infusion of E-BSA into the ventromedial hypothalamus followed by 17β-estradiol in the second pulse could induce lordosis behavior, demonstrating the applicability of this paradigm in vivo. A model where non-genomic actions of estrogen couple to genomic actions unites both aspects of hormone action.

Mechanisms of Estrogen Receptor Signaling: Convergence of Genomic and Nongenomic Actions on Target Genes

Estrogen receptors (ERs) act by regulating transcriptional processes. The classical mechanism of ER action involves estrogen binding to receptors in the nucleus, after which the receptors dimerize and bind to specific response elements known as estrogen response elements (EREs) located in the promoters of target genes. However, ERs can also regulate gene expression without directly binding to DNA. This occurs through protein-protein interactions with other DNA-binding transcription factors in the nucleus. In addition, membrane-associated ERs mediate nongenomic actions of estrogens, which can lead both to altered functions of proteins in the cytoplasm and to regulation of gene expression. The latter two mechanisms of ER action enable a broader range of genes to be regulated than the range that can be regulated by the classical mechanism of ER action alone. This review surveys our knowledge about the molecular mechanism by which ERs regulate the expression of genes that do not contain EREs, and it gives examples of the ways in which the genomic and nongenomic actions of ERs on target genes converge. Genomic and nongenomic actions of ERs that do not depend on EREs influence the physiology of many target tissues, and thus, increasing our understanding of the molecular mechanisms behind these actions is highly relevant for the development of novel drugs that target specific receptor actions.

17beta-estradiol enhances the production of granulocyte-macrophage colony-stimulating factor in human keratinocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is effective for impaired wound repair. Estrogen is known to enhance wound repair. We examined if 17beta-estradiol (E2) may in vitro enhance GM-CSF production in human keratinocytes. E2 and membrane-impermeable bovine serum albumin-conjugated E2 increased GM-CSF secretion, mRNA stability, and promoter activity. The element homologous to activator protein-1 (AP-1) on the promoter was responsible for the activation. E2 enhanced transcriptional activity and DNA binding of AP-1. E2 transiently generated c-Fos protein, and shifted AP-1 composition from c-Jun homodimers to c-Fos/c-Jun heterodimers in keratinocytes. E2-induced enhancement of GM-CSF secretion, mRNA stability, and promoter activity were not suppressed by estrogen receptor antagonist ICI 182,780, however, suppressed by conventional protein kinase C inhibitor Gö6976 and PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK). Gö6976 and PD98059 suppressed E2-induced c-Fos expression and enhancement of DNA-binding and transcriptional activity at AP-1. E2 induced membrane translocation of protein kinase Calpha, which was suppressed by phosphatidylinositol (PI)-specific phospholipase C (PLC) inhibitor U73122. E2 stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), which was suppressed by PD98059, Gö6976, and U73122. E2 transiently generated inositol 1,4,5-triphosphate in keratinocytes, which was suppressed by U73122 and guanine nucleotide-binding protein inhibitor. These results suggest that E2 may enhance GM-CSF production via guanine nucleotide-binding protein-coupled membrane receptors and signaling cascade of PI-specific PLC/protein kinase Calpha/MEK/ERK.

Role of estrogen in avian osteoporosis

One of the difficulties associated with commercial layer production is the development of osteoporosis in hens late in the production cycle. In light of this fact and because of hens′ unique requirements for Ca, many studies have focused on the regulation of Ca and the role of estrogen in this process. The time course of estrogen synthesis over the productive life of hens has been well documented; increased circulating estrogen accompanies the onset of sexual maturity while decreases signal a decline in egg production prior to a molt. Numbers of estrogen receptors decrease with age in numerous tissues. The parallel changes in calcium-regulating proteins, primarily Calbindin D28K, and in the ability of duodenal cells to transport Ca, are thought to occur as a result of the changes in estrogen, and are also reversible by the molt process. In addition to the traditional model of estrogen action, evidence now exists for a possible nongenomic action of estrogen via membrane-bound receptors, demonstrated by extremely rapid surges of ionized Ca in chicken granulosa cells in response to 17β-estradiol. Estrogen receptors have also been discovered in duodenal tissue, and tamoxifen, which binds to the estrogen receptor, has been shown to cause a rapid increase in Ca transport in the duodenum. In addition, recent evidence also suggests that mineralization of bone per se may not explain entirely the etiology of osteoporosis in the hen but that changes in the collagen matrix may contribute through decreases in bone elasticity. Taken together, these studies suggest that changes in estrogen synthesis and estrogen receptor populations may underlie the age-related changes in avian bone. As with postmenopausal women, dietary Ca and vitamin D are of limited benefit as remedies for osteoporosis in the hen.

A case for estrogen receptors on cell membranes and nongenomic actions of estrogen.

A case for estrogen receptors on cell membranes and nongenomic actions of estrogen.

Evidence for genomic and nongenomic actions of estrogen in growth plate regulation in female and male rats at the onset of sexual maturation.

Recently, both estrogen receptor (ER) alpha and beta were detected in growth plate chondrocytes of rats before sexual maturation, implying a role for estrogen at this stage. In this study, therefore, we investigated the effects of ovariectomy (OVX) or estrogen supplementation on parameters of longitudinal growth in 26-day-old rats, which were sexually immature at the start of the experiment. OVX caused an increase in body weight gain, tibial length and growth plate width due to an increased proliferating zone. This increase correlated with an increase in cell number, with a decrease in cell diameter and with increased proliferating cell nuclear antigen (PCNA) immunostaining compared with sham. Interestingly, the increase in proliferation was not caused by an increase in insulin-like growth factor-I (IGF-I) mRNA expression in the growth plate as assessed by real-time PCR. In contrast to OVX, 17beta-estradiol (E(2)) supplementation (0.5 mg/21 days) of 26-day-old female rats caused a strong decrease in body weight gain, tibial length and growth plate width. The latter was explained by a reduction of the proliferating zone width, which correlated with a reduced number of PCNA-positive cells (not significant) and by a reduction of the hypertrophic zone width. In male rats supplemented with E(2), similar effects were observed compared with the females. ERalpha and beta immunostaining was found predominantly in late proliferating and early hypertrophic chondrocytes. OVX did not affect ER expression but E(2) supplementation strongly decreased immunostaining for both ERalpha and beta in both sexes. Besides E(2), desoxyestrone (DE), an activator of nongenomic estrogen-like signaling (ANGEL) and 2-methoxyestradiol (2-MeO-E(2)), a tissue-selective naturally occurring metabolite of E(2), were administered to female and male rats of the same age. Compared with E(2), these compounds had less pronounced, though significant, effects on some parameters of longitudinal growth in both sexes, especially on growth plate characteristics. In conclusion, E(2) may exert effects on longitudinal growth before and at the onset of sexual maturation, despite very low endogenous serum levels at these stages. There may be a role for nongenomic signaling in body weight gain, tibial length and growth plate width but genomic signaling prevails.

A novel nongenomic action of estrogens: the regulation of exocytotic kinetics.

A novel nongenomic action of estrogens: the regulation of exocytotic kinetics.