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Endocrinology Vol. 147, No. 4 1796-1804
Copyright © 2006 by The Endocrine Society

Estradiol Reduces Nonclassical Transcription at Cyclic Adenosine 3',5'-Monophosphate Response Elements in Glioma Cells Expressing Estrogen Receptor Alpha

Andrew J. Mhyre, Robert A. Shapiro and Daniel M. Dorsa

Department of Pharmacology (A.J.M.), University of Washington School of Medicine, Seattle, Washington 98195; and Department of Physiology and Pharmacology (R.A.S., D.M.D.), Oregon Health & Science University, Portland, Oregon 97239

Address all correspondence to: Dr. Andrew J. Mhyre, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, D1-100, P.O. Box 19024, Seattle, Washington 98109-1024. E-mail: amhyre{at}fhcrc.org. Address reprint requests to: Dr. Daniel Dorsa, Research Development and Administration, Oregon Health & Science University, L335, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estradiol can protect the brain from a variety of insults by activating membrane-initiated signaling pathways, and thereby modulate gene expression and lead to functional changes in neurons. These direct neuronal effects of the hormone have been well documented; however, it is less understood what effects estradiol may have on nonneuronal cells of the central nervous system. There is evidence that estradiol levels can induce the release of glial-derived growth factors and other cytokines, suggesting that estradiol may both directly and indirectly protect neurons. To determine whether 17ß-estradiol (E2) can activate rapid signaling and modulate nonclassical transcription in astrocytes, we stably transfected the C6 rat glioblastoma cell line with human estrogen receptor (ER) {alpha} (C6ER{alpha}) or rat ERß (C6ERß). Introduction of a cAMP response element-luciferase reporter gene into C6, C6ER{alpha}, and C6ERß cells leads to the observation that E2 treatment reduced isoproterenol-stimulated luciferase activity by 35% in C6ER{alpha} but had no effect on reporter gene expression in C6ERß or untransfected C6 cells. A similar effect was seen with a membrane-impermeable estrogen (E2-BSA), suggesting the modulation of nonclassical transcription by estradiol treatment is mediated by the activation of a membrane-initiated signaling pathway. Furthermore, pretreatment with wortmannin (phosphatidylinsositol 3-kinase) or U73122 (phospholipase C) attenuated the E2-induced reduction in nonclassical transcription. We conclude that E2 treatment reduces cAMP response element-mediated transcription in glioma cells expressing ER{alpha} and that this reduction is dependent on the activation of membrane-initiated signaling. These findings suggest a novel model of estrogen rapid signaling in astrocytes that leads to modulation of nonclassical transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN is a class of gonadal sex hormones that plays a multifaceted role throughout the body and in the central nervous system (CNS). Local levels of estradiol, the predominant form of estrogen, can mediate reproductive behavior, but also influence cognition (1) and neuronal survival (2). During the natural aging process in women, menopause is marked by a dramatic decline in circulating estrogen levels, leaving the body susceptible to a number of different diseases. Hormone therapy has been prescribed to counteract the decline in hormone levels and alleviate some of the more troublesome symptoms of menopause such as hot flashes and disruption of sleep patterns. Epidemiological evidence suggests that the use of hormone therapy correlates with an increase in cognitive abilities and a decrease in the incidence of Alzheimer’s disease in postmenopausal women (3, 4). Estradiol directly protects primary neurons in culture (5), but the activation of nonneuronal cells of the CNS may augment these beneficial direct effects of estradiol on cognition and neuronal survival.

In the classical model of steroid receptor function, the ligand-bound estrogen receptors (ERs) form homo- and/or heterodimers and modulate transcription of genes containing estrogen response elements (EREs). However, this single pathway model does not account for the complexity of effects seen in response to changes in estradiol levels. In the presence of ligand, the ER monomer can complex with fos and jun and regulate transcription at activator protein-1 sites (6, 7). In addition to directly stimulating numerous genomic targets, estradiol activates membrane-initiated signaling pathways, resulting in changes in intracellular calcium (8), increases in cAMP (9), and activation of signaling pathway kinases such as Akt (10), p42/44 MAPK (11), phospholipase C (PLC) (12), and cAMP response element binding protein (CREB) (13). Activation of these membrane-initiated signaling pathways after estradiol treatment can alter gene expression independent of ER-DNA interactions (14). For example, the promoter region of the neurotensin/neuromedin gene lacks sequences that mimic an ERE-like element, but estradiol can activate the protein kinase A/CREB pathway, leading to transcription at cAMP response elements (CREs), which drives the expression of the neurotensin/neuromedin gene (15). The diversity in the various membrane-initiated signaling pathways and the resulting transcriptional events adds to the complexity of the functional changes in response to estradiol levels.

Although considerable efforts have been devoted to understanding how estradiol directly effects neurons in both in vitro (5, 16) and in vivo models (2, 17), the effects of estradiol on nonneuronal cells of the CNS remain largely unclear. ERs have been detected in astrocytes, the predominant glial cell type, in both in vitro (18, 19, 20) and in vivo (21, 22, 23) studies, suggesting that estradiol may modulate the role glial cells play in maintaining normal homeostasis and/or modulating inflammatory signals in the brain. It has recently been shown that 17ß-estradiol (E2) treatment of cortical astrocytes increases the secretion of TGF-ß, a neurotrophic growth factor (24, 25), suggesting that estradiol treatment may enhance neuronal viability via a noncell autonomous mechanism. In neurons, it has been demonstrated that E2 treatment activates the p42/44 MAPK signaling pathway, which partially mediates protection against glutamate excitotoxicity (26). It remains unclear whether estradiol stimulates similar rapid signaling events in astrocytes to indirectly modulate neuronal function and survival. To test the hypothesis that estradiol treatment activates membrane-initiated signaling pathways leading to modulation of nonclassical transcription in astrocytes, we developed an astrocyte cell model by stably transfecting the C6 rat glioblastoma with either ER{alpha} or ERß. In this study, we examined whether E2 modulates transcription at CREs in ER{alpha}- and/or ERß-expressing glioma cells and if so, what membrane-initiated signaling pathways are responsible for these alterations in gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
E2 and E2-BSA (17ß-estradiol-6-CMO:BSA) were purchased from Steraloids, Inc. (Newport, RI) and ICI 182,780 (ER antagonist) was obtained from Tocris Cookson (Ellisville, MO). Akt Inhibitor IV in solution (dimethylsulfoxide), calphostin C [protein kinase C (PKC) inhibitor], isoproterenol (ß-adrenergic receptor agonist), pertussis toxin (inactivates Gi/o proteins), U0126 [MAPK kinase (MEK) 1/2 inhibitor], U73122 (PLC inhibitor), and wortmannin [phosphatidylinositol 3-kinase (PI3K) inhibitor] were purchased from EMD Biosciences, Inc. (La Jolla, CA). All treatments were prepared as 1000x stocks in appropriate vehicles and diluted into cell culture media. Isoproterenol was dissolved directly in media immediately before adding it to the cells.

Cell culture
The C6 rat glioblastoma cell line was obtained from ATCC (Manassas, VA) and grown on tissue culture-treated dishes in media (10%FM) consisting of phenol red-free DMEM (Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin (100 U/ml), streptomycin (100 µg/ml), and 2 mM L-glutamine (Invitrogen Corp., Carlsbad, CA). When the cultures reached 95% confluency, cells were subcultured with TrypLE (Invitrogen Corp.). The cells were maintained at 37 C and 5% CO2 atmosphere and were used for experiments between passage 5 and 20.

Generation of transfected cell lines
For transfection, C6 cells were grown in transfection media (10%CSM) consisting of phenol red-free DMEM supplemented only with 10% charcoal-stripped fetal bovine serum (Hyclone) and 2 mM L-glutamine. Once the cells reached 85% confluency, a 100-mm tissue culture dish was transfected with a mixture of Lipofectamine 2000 (Invitrogen Corp.) and pcDNA3.1 hygromycin (15 µg) containing cDNA encoding the appropriate gene. The ERs expressed included full-length human ER{alpha} (hER{alpha}) (27) (a kind gift from Dr. Pierre Chambon, Strasbourg, France), full-length rat ERß (rERß) (28) (a kind gift from Dr. George Kuiper, Karolinska Institute, Sweden) and the hER{alpha} gene with two point mutations in the DNA binding domain (C202H and C205H), making it transcriptionally inactive at ERE sites (29, 30). Lipofectamine 2000 was premixed with the various plasmids at a ratio of 1 µg DNA to 2.5 µl transfectant for 30 min before the mixture was added to the cells. After 48 h, the media were replaced and the cells were treated with Hygromycin B (Invitrogen Corp.; 400 µg/ml) to select for incorporation of the plasmid. Single colonies were isolated after 10 d of growth in selection media and tested for receptor expression by Western blot and functional assays. The monoclonal ER-expressing cell lines were maintained in 10%FM containing Hygromycin B (100 µg/ml) to suppress the loss of plasmid expression.

Immunoblotting
Each monoclonal cell line was tested for ER expression by Western blot techniques. Cells, grown to 80% confluency, were rinsed with ice-cold PBS, and lysed with immunoprecipitation buffer [25 mM HEPES (pH 7.5), 5 mM EDTA, 5 mM EGTA, 100 mM sodium pyrophosphate, 50 mM NaF, 100 µM NaVO4, and 150 mM NaCl] containing 1% Triton X-100 and Proteinase Inhibitor Cocktail (EMD Biosciences). The lysates were sonicated for 2 min and cleared by centrifugation at 20,000 x g for 10 min. Protein concentrations were quantified using the bicinchoninic acid assay (Pierce Biotechnology Inc., Rockford, IL) and equal amounts of protein (50 µg) from each lysate were diluted in Laemmli sodium dodecyl sulfate sample buffer, resolved by electrophoresis on 4–12% Bis-Tris precast NuPage gels (Invitrogen Corp.) in running buffer [50 mM 2-(N-morpholino)ethane sulfonic acid, 50 mM Tris base, 0.1% sodium dodecyl sulfate, and 1 mM EDTA] as described by the manufacturer, and transferred to polyvinylidene difluoride. Recombinant hER{alpha} (10 pg) and hERß (100 pg) proteins (Affinity Bioreagents, Golden, CO) were loaded as positive controls. The membranes were blocked in 5% nonfat dry milk diluted in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature and then incubated overnight at 4 C in 5% nonfat dry milk/TBS-T containing either a mouse anti-ER{alpha} antibody (1:500; 6F11 from Novocostra Laboratories, Newcastle upon Tyne, UK) or a rabbit anti-ERß antibody (1:200; 51–7700 from Zymed Laboratories Inc., San Francisco, CA). Secondary goat antimouse or antirabbit antibodies (1:2000; Santa Cruz Biotechnology Inc., Santa Cruz, CA) conjugated to horseradish peroxidase were used for enhanced chemiluminescence (Pierce Biotechnology, Inc.) and photographed in an Epi Chemi II Darkroom (UVP Inc., Upland, CA).

Immunocytochemistry
Immunocytochemistry was used to determine the subcellular localization of the ERs in the stably transfected cell lines. C6ER{alpha}, C6ERß, or untransfected C6 cells were plated on 0.001% poly-D-ornithine-coated glass coverslips and allowed to attach overnight. The cultured cells were fixed with acetone:methanol (50:50; 10 min), blocked with 10% BSA/1% Cold Water Fish Skin Gelatin in PBS (1 h) at room temperature and incubated overnight at 4 C in 1% BSA/0.1% Gelatin (PBG) containing the appropriate antibody (1:200): rabbit anti-ER{alpha} antibody (AB-16; Lab Vision Corp., Fremont, CA) or rabbit anti-ERß antibody (06-629; Upstate, Charlottesville, VA). After four washes in PBG, fluorescently tagged secondary antibodies (Molecular Probes, Inc., Eugene, OR) were used to visualize the primary antibodies bound to the ER in each cell line (1 h at room temperature). The coverslips were washed with PBG four times, mounted with Vectasheild containing 4'-6-diamidino-2-phenylindole (DAPI) (Vector, Burlingame, CA), and sealed. Images were acquired using a fluorescent microscope.

Activation of luciferase (luc) reporter genes
The ability of E2 to modulate gene expression at either CRE or ERE sites was assessed using reporter gene assays. Cell lines were plated in 12-well tissue culture-treated plates at a density of 8 x 104 cells/well in 10%CSM and grown overnight. Lipofectamine 2000 (Invitrogen Corp.) was premixed for 30 min with the various reporter gene constructs at a ratio of 1 µg DNA to 2 µl transfection reagent before the mixture was added to the cells. Each well was transfected with a luc reporter gene (750 ng), tkERE-luc (a kind gift from Dr. Peter Burbach, Rudolf Magnus Institute, University of Utrecht, Utrecht, The Netherlands) or CRE-luc (a kind gift from Dr. Daniel Storm, University of Washington, Seattle, WA). In addition, each well was cotransfected with pCH110 (500 ng), a ß-galactosidase (ß-gal) reporter (Amersham Pharmacia Biotech, Uppsala, Sweden), to normalize for transfection efficiency. E2-BSA was dissolved in Tris-HCl and filtered with a 3-kDa cut-off filter as previously described (31). After 6 h of transfection, the media were replaced with 10%CSM containing either E2 or conjugated E2-BSA. For CRE experiments, the cells were treated with E2 or vehicle (0.095% ethanol) for 30 min before isoproterenol (10 µM final) was added. The cells were incubated for 24 h and then lysed in Glo Lysis Buffer (Promega Corp., Madison, WI). Each lysate was assayed for both luc and ß-gal activity using the Steady Glo Assay and Beta Glo Assay, respectively, as described by the manufacturer (Promega Corp.). The luminescence was measured on a Fushion plate reader (PerkinElmer, Inc., Boston, MA).

Cell treatments with pharmacological inhibitors
The effect of E2 on CRE-luc reporter gene expression was quantified in the presence of specific pharmacological inhibitors. C6ER{alpha} cells were plated in 12-well plates and transfected with the CRE-luc and pCH110 constructs as described above. To test the involvement of Gi/o proteins, the cells were pretreated with pertussis toxin (300 ng/ml) for 18 h before and during the transfection with the reporter constructs. The endotoxin catalyzes ADP-ribosylation of Gi and Go inactivating the G proteins. After 6 h of transfection with the reporter constructs, the media were replaced with fresh 10%CSM containing the appropriate pharmacological inhibitor: Akt Inhibitor IV, calphostin C (PKC inhibitor), ICI 182,780 (ER antagonist), pertussis toxin (inactivates Gi/o), U0126 (MEK1/2 inhibitor), U73122 (PLC inhibitor), and wortmannin (PI3K inhibitor). Thirty minutes after changing the media, the cells were preexposed to E2 (10 nM) or vehicle (0.095% ethanol) for 30 min before the addition of isoproterenol (10 µM final). The cells were exposed to all three treatments (inhibitor, E2, and isoproterenol) for 24 h and then lysed and assayed as described above.

Statistical analysis
For each experiment, the significance of differences among groups was determined by one-way ANOVA followed by a Tukey’s multiple comparison test (Prism 4.0; GraphPad, San Diego, CA). P < 0.05 was considered significant. Each treatment was performed in quadruplicate, and the experiments were repeated at least three times. All values are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transfected glioma cells express functional ERs
We developed an astrocyte cell model using the C6 rat glioblastoma cell line to examine the role of ER{alpha} or ERß in modulating glioma cell function. After transfection with cDNAs encoding either hER{alpha} or rERß, monoclonal cell lines were screened for receptor expression by Western blot analysis, and a single clone was selected for each receptor transfected (C6ER{alpha} and C6ERß). We detected immunoreactive bands in whole cell lysates from these cell lines that were the same size as those detected in the positive controls, recombinant hER{alpha} and hERß proteins (Fig. 1AGo). As expected, immunoreactivity for ER{alpha} and ERß were not detected in untransfected C6 cells. To characterize the localization of the receptors in the transfected cell lines, immunocytochemistry was performed on C6ER{alpha}, C6ERß, and untransfected C6 cells. Using immunofluorescence, we observed that ER{alpha} and ERß localized predominantly to the DAPI-stained nucleus, although we also observed cytoplasmic and plasma membrane immunoreactivity (Fig. 1BGo).


Figure 1
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  		FIG. 1. Generation of stable C6 glioblastoma cell lines expressing ER{alpha} or ERß. A, Western blot analysis of transfected cell lines expressing immunoreactive proteins of the expected size for ER{alpha} (67 kDa) and ERß (54 kDa). Protein lysates were separated on 4–12% Bis-Tris gels and immunoblotted with a monoclonal mouse anti-ER{alpha} antibody (6F11) or a polyclonal rabbit anti-ERß antibody (51–7700). Ten picograms of recombinant hER{alpha} or 100 pg of hERß were loaded as positive controls. B, Fluorescent microscopy of C6, C6ER{alpha}, and C6ERß cell lines after immunocytochemistry using rabbit polyclonal anti-ER{alpha} antibody (AB-16) or anti-ERß antibody (06-629). Cells were counterstained with DAPI.

 
To confirm the functional activity of ER{alpha} and ERß in the transfected glioma cell lines, we introduced a tkERE-luc reporter gene into C6, C6ER{alpha}, and C6ERß cells along with a control plasmid (pCH110) that expresses ß-gal to control for transfection efficiency. We then exposed the cells to increasing concentrations of E2 (100 fM to 10 nM) and found that in C6ER{alpha} and C6ERß cells, E2 increased luc activity in a dose-dependent manner (Fig. 2Go). In C6ER{alpha} cells E2 treatment resulted in a maximal 8-fold increase in luc activity with an EC50 of 32.8 pM, whereas in C6ERß cells treatment increased luc activity by 6-fold with an EC50 of 143.3 pM. No change in activity was detected in untransfected cells confirming the lack of ER expression in untransfected C6 cells. Pretreatment with the ER antagonist, ICI 182,780 (1 µM), abolished the ERE-mediated response to 1 nM E2 in C6ER{alpha} and C6ERß cells (data not shown). Together, these data demonstrate that ER{alpha} and ERß expressed in the stable glioma cell lines are functionally active in modulating classical ERE-mediated transcription.


Figure 2
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  		FIG. 2. Dose response of E2-stimulating ERE-mediated transcription in cells stably transfected with ER{alpha} or ERß. Each well was transfected with a tkERE-luc reporter gene (750 ng) and pCH110 (500 ng) and treated with increasing concentrations of E2 (100 fM to 10 nM). After 24 h treatment, the cells were lysed, and both luc and ß-gal expression were assayed for luminescence activity. Data are represented as fold increase of relative ERE-luc activity (ratio of luc to ß-gal activity) compared with vehicle-treated controls ± SEM. The experiment was repeated four times in quadruplicate.

 
E2 reduces CRE-mediated transcription
Previous findings in in vivo (13) and in vitro models (32) demonstrated that estradiol treatment can activate CREB signaling in neurons. To test this effect in glioma cells, we used a CRE-luc reporter gene, which drives luc expression in response to CREB activation. C6ER{alpha}, C6ERß, and untransfected C6 cells were transiently transfected with the CRE-luc and pCH110 constructs, exposed to various treatments for 24 h and assayed for luc and ß-gal activity. In contrast to its effect in the neuronal cell model, E2 treatment alone failed to increase luc activity in transfected glioma cells (Fig. 3AGo). Stimulation with isoproterenol (10 µM) increased CRE-luc reporter gene activity in all three cell lines by activating the Gs-coupled ß-adrenergic receptor. Surprisingly, we discovered that treatment of C6ER{alpha} cells with E2 reduced isoproterenol-stimulated CRE-mediated luc activity by 35% with an EC50 of 285.1 pM (Fig. 3BGo). No effect of E2 treatment was observed in either C6ERß or untransfected C6 cells. To confirm that the estradiol-induced modulation of nonclassical transcription at CRE sites was mediated by the ER, C6ER{alpha} cells were pretreated with ICI 182,780 (1 µM). Although the antagonist had no effect on its own, it blocked the effect of E2 on isoproterenol-stimulated luc activity (Fig. 4Go). These data demonstrate that the estradiol-induced reduction in CRE-mediated transcription in glioma cells is dependent on the expression and activation of ER{alpha}, a nuclear ER.


Figure 3
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  		FIG. 3. E2 treatment reduces CRE-mediated transcription in glioma cells. C6, C6ER{alpha}, and C6ERß cell lines, transiently transfected with CRE-luc (750 ng) and pCH110 (500 ng) in 12-well plates, were pretreated with vehicle or E2 for 30 min, followed by addition of isoproterenol (10 µM). After 24 h, luc and ß-gal expression was assessed by luminescence activity, and the data are represented as percent of relative CRE-luc activity (ratio of luc to ß-gal activity) compared with isoproterenol stimulation ± SEM. A, E2 reduces CRE-mediated transcription in C6ER{alpha} cells, but not in C6ERß or untransfected C6 cells. Each cell line was treated with 10 nM E2 or vehicle in the absence or presence of 10 µM isoproterenol. B, C6ER{alpha} and C6ERß cells treated with increasing doses of E2 (1 pM to 100 nM) followed by isoproterenol (10 µM) stimulation. All experiments were performed at least three times in quadruplicate. Asterisks (*) indicate a P value of 0.001 or less when compared with vehicle-treated controls, as determined by one-way ANOVA. **, Statistically different from cells treated with either vehicle or isoproterenol (P ≤ 0.001).

 

Figure 4
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  		FIG. 4. Reduction in CRE-mediated transcription is sensitive to ICI 182,780. After transfection with CRE-luc (750 ng/well) and pCH110 (500 ng/well), C6ER{alpha} cells were pretreated with 10 nM E2 for 30 min in the presence and absence of ICI 182,780 (1 µM), followed by stimulation with 10 µM isoproterenol. After 24 h of treatment, luc and ß-gal expression were quantified by luminescence activity. Data are represented as percent inhibition of relative CRE-luc activity (ratio of luc to ß-gal activity) compared with isoproterenol stimulation ± SEM. {psi}, P value of 0.001 or less when compared with isoproterenol-stimulated controls, as determined by one-way ANOVA. {Omega}, Significantly different from cells treated with both E2 and isoproterenol (P ≤ 0.001).

 
Modulation of nonclassical transcription is dependent on initiation of signaling at the plasma membrane
As our initial test to assess the mechanism by which estradiol modulates CRE-mediated transcription in these glioma cells, we examined whether a membrane-impermeable estrogen, E2-BSA, could modulate CRE-luc reporter gene expression. When we treated C6ER{alpha} cells with E2-BSA, we found that isoproterenol-stimulated luc activity was reduced by 24%, similar to the results with unconjugated estradiol, whereas C6ERß cells were unresponsive to E2-BSA (Fig. 5AGo) as were untransfected C6 cells (data not shown). This conjugated estrogen had no effect on the expression of the tkERE-luc reporter gene activity in either C6ER{alpha} (Fig. 5BGo) or C6ERß (data not shown) cells, demonstrating that this preparation of E2-BSA was free of unconjugated estradiol. These data suggest that E2 binds ER{alpha} at or near the plasma membrane, which in glioma cells leads to activation of a signal transduction cascade that results in a reduction in isoproterenol-stimulated transcription at CRE sites.


Figure 5
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  		FIG. 5. Reduction in CRE-mediated transcription is mediated by membrane-initiated signaling pathways. After transfection with the reporter genes, cells were treated with either E2 or E2-BSA, a membrane-impermeable estrogen. A, Treatment with E2-BSA reduces CRE-mediated transcription similar to free E2. C6ER{alpha} and C6ERß cells were transfected with the CRE-luc and pCH110 reporter genes, treated with either E2 or E2-BSA followed by isoproterenol (10 µM) and assayed as described and presented in Fig. 4Go. B, E2-BSA is unable to activate classical ERE-mediated transcription. C6ER{alpha} cells were transfected with the tkERE-luc and pCH110 reporter genes, treated with 10 nM E2 or conjugated E2-BSA and assayed as described and presented in Fig. 2Go. {Delta}, P value of 0.001 or less when compared with vehicle controls, as determined by one-way ANOVA.

 
Inhibition of transcription at CRE sites requires activation of PI3K and PLC
To determine which membrane-initiated signaling pathway leads to inhibition of CRE-mediated transcription, we quantified the E2-induced reduction in isoproterenol-stimulated CRE-luc reporter gene activity in the presence of pathway-specific inhibitors. We found that wortmannin (100 nM) abolished the ability of E2 (10 nM) to reduce CRE-mediated luc activity (Fig. 6Go), implicating the PI3K pathway. No effect was observed with either ADP-ribosylation of Gi and Go proteins with pertussis toxin (300 ng/ml) or pharmacological inhibition of the p42/44 MAPK pathway with U0126 (1 µM). Activation of PI3K can lead to a number of signaling cascades including Akt, PKC, and PLC. Estradiol treatment has been previously shown to activate Akt in primary cortical neurons (33), PKC in pituitary cells (34), and PLC in fibroblasts (12). Although pretreatment with either Akt Inhibitor IV (1 µM) or calphostin C (500 nM: PKC inhibitor) had no effect, we observed that exposure to U73122 (10 µM) attenuated the estradiol-induced reduction in isoproterenol-stimulated luc expression (Fig. 7Go). These data suggest that estradiol binds ER{alpha} and activates the PI3K and PLC signal transduction pathway leading to inhibition of CRE-mediated transcription.


Figure 6
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  		FIG. 6. Sensitivity to wortmannin in reducing CRE-mediated transcription suggests role for PI3K pathway. After transfection with the CRE-luc pCH110 reporter genes, C6ER{alpha} cells were treated with 10 nM E2 followed by 10 µM isoproterenol in the presence and absence of pathway-specific pharmacological inhibitors: pertussis toxin (300 ng/ml; Inactivates Gi/o proteins), U0126 (1 µM; Inhibits MEK1/2), and wortmannin (100 nM; Inhibits PI3K). After 24 h of treatment, luc and ß-gal expression were assessed as described and presented in Fig. 4Go.

 

Figure 7
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  		FIG. 7. PLC, but not Akt or PKC, is implicated in attenuation of CRE-mediated transcription. After transfection with the CRE-luc and pCH110 reporter genes, C6ER{alpha} cells were treated with 10 nM E2 followed by 10 µM isoproterenol in the presence and absence of pathway-specific pharmacological inhibitors: calphostin C (500 nM; Inhibits PKC), U73122 (10 µM; Inhibits PLC) and Akt Inhibitor IV (1 µM). After 24 h of treatment, luc and ß-gal expression were assessed as described and presented in Fig. 4Go.

 
Modulation of transcription at CRE sites is independent of classical effects at ERE sites
To determine the contribution of classical ERE-mediated genomic effects in modulating gene expression at CRE sites, ER{alpha} mutated in the DNA binding domain (C202H;C205H: HE27) (29) was stably expressed in C6 cells (C6ER{alpha}HE27). Using the tkERE-luc reporter gene, we found that this mutated receptor was transcriptionally inactive at ERE sites when expressed in the C6ER{alpha}HE27 cell line (Fig. 8AGo) consistent with previous findings in HT-22, a hippocampal-derived neuronal cell line (30). To test whether this mutated receptor can mediate the effects of E2 on CRE-mediated transcription, we introduced the CRE-luc reporter gene into C6ER{alpha}HE27 cells and assessed the modulation of luc activity in response to E2 treatment. When C6ER{alpha}HE27 cells were pretreated with E2 (10 nM), we detected a 35% reduction in isoproterenol-stimulated CRE-mediated luc reporter gene activity comparable with the estradiol-induced reduction in C6ER{alpha} cells expressing wild-type receptor (Fig. 8BGo). Treatment of C6ER{alpha}HE27 cells with the membrane-impermeable E2-BSA also reduced isoproterenol stimulated CRE-luc reporter gene expression by 29% similar to free E2 (Fig. 8CGo). These data demonstrate that estradiol reduces transcription at CRE sites independent of its effects on classical ERE-mediated gene expression and that this effect on nonclassical transcription is mediated by a membrane-initiated signaling pathway.


Figure 8
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  		FIG. 8. Reduction in CRE-mediated transcription is independent of classical genomic effects at ERE sites. A, Mutation of ER{alpha} in the DNA binding domain (ER{alpha}HE27) makes the receptor transcriptionally inactive at classical ERE sites. C6ER{alpha} and C6ER{alpha}HE27 cells were transfected with the tkERE-luc and pCH110 reporter genes, treated with 1 nM E2 or vehicle and assayed as described and presented in Fig. 2Go. B, Estradiol binding to either ER{alpha} or ER{alpha}HE27 reduces transcription at CRE sites in glioma cells. C6ER{alpha} and C6ER{alpha}HE27 cells were transfected with the CRE-luc and pCH110 reporter genes and then treated with 10 nM E2 followed by 10 µM isoproterenol. After 24 h, luc and ß-gal expression were assessed as described and presented in Fig. 4Go. C, Membrane-impermeable estrogen reduces nonclassical transcription similar to free E2. C6ER{alpha}HE27 cells transfected with the CRE-luc and pCH110 reporter genes were treated with 10 nM E2 or E2-BSA and followed by stimulation with 10 µM isoproterenol. The cells were treated for 24 h and assayed for both luc and ß-gal expression as described and presented in Fig. 4Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The classical effects of estradiol have been attributed solely to modulation of gene expression at ERE sites. Recent observations have begun to characterize activation of membrane-initiated signaling events leading to modulation of ERE-independent gene expression (14). In this study, we provide functional evidence that E2 treatment attenuates nonclassical transcription at CRE sites in glioma cells expressing ER{alpha}, but not ERß. This estradiol-induced reduction in CRE-mediated gene expression is sensitive to the pharmacological inhibitors, wortmannin and U73122, strongly suggesting the activation of the PI3K/PLC signaling pathway.

Three observations provide evidence that estradiol modulates nonclassical CRE-mediated transcription in glioma cells by activating ER{alpha}. First, E2 treatment of transfected C6 cells inhibited isoproterenol-stimulated CRE-luc reporter gene expression. This reduction in CRE-mediated transcription required the expression of ER{alpha}, whereas untransfected and ERß-expressing C6 cells were unresponsive to E2 treatment. Second, pretreatment with the ER antagonist, ICI 182,780, blocked the reduction in reporter gene activity. This sensitivity to the ER antagonist demonstrated that the estradiol-induced reduction in transcription at CRE sites is mediated by the activation of the ER and not another putative receptor (35) or GPR30 (36). Third, the mutated receptor, ER{alpha}HE27, which is transcriptionally inactive at ERE sites, was also able to mediate the effect of E2 on transcription at CRE sites similar to wild-type ER{alpha}. Together, these results demonstrate that E2, through activation of ER{alpha}, inhibits transcription at CRE sites in glioma cells independent of classical ERE-mediated transcription.

A possible mechanism for the E2-induced reduction in nonclassical transcription is the interaction between the ER and CREB-binding protein (CBP), a cofactor involved in transcription at various response elements including CRE sites (37). E2 treatment has been shown to increase the interaction between ER{alpha} and CBP (38). Furthermore, in 293A cells, expression of ER{alpha} negatively modulates transcription at nonclassical response elements, which is augmented with E2 treatment (39). Kamei et al. (40) have suggested that the activation of ERE-mediated transcription by estradiol may sequester CBP at ERE sites, and therefore, decrease activity of CBP at other transcriptional sites such as CREs. This study presents evidence that, in glioma cells, E2 is not modulating the transcriptional machinery, but instead activating a rapid signaling event. This is indicated by the observations that treatment with either E2 or ICI 182,780 alone had no effect on the basal level of CRE-mediated transcription, but rather CRE-luc reporter gene expression was only modulated by E2 when the CREB signaling pathway was activated, such as when the cells were stimulated with isoproterenol. Furthermore, mutation in the DNA binding domain (ER{alpha}HE27), making it transcriptionally inactive at ERE sites, had no effect on the receptor’s ability to mediate the reduction in transcription at CRE sites in response to E2 treatment. These data suggest that estradiol modulates CRE-mediated transcription in glioma cells independent of CBP and its classical genomic effects at ERE sites.

In addition to our finding that E2 treatment leads to modulation of nonclassical transcription at CRE sites in glioma cells, two independent approaches provide evidence that the reduction in transcription is mediated by the activation of membrane-initiated signaling pathways. First, treating C6ER{alpha} or C6ER{alpha}HE27 cells with E2-BSA, a membrane-impermeable estrogen, showed that the estradiol-induced reduction in CRE-mediated transcription is initiated by the receptor at or near the plasma membrane. Similar to the results with free E2, C6ERß and untransfected C6 cells were unresponsive to E2-BSA, further suggesting that the estradiol-induced reduction in transcription at CRE sites requires the expression and activation of ER{alpha} at the plasma membrane. Pawlak et al. (41) have recently demonstrated that, in primary cultures of midbrain astrocytes, ER{alpha} localized to the plasma membrane and along the astrocyte processes. Localization of ER{alpha} has been shown to be dependent on palmitoylation of the receptor (42) in addition to an interaction with Caveolin-1 (43) that transiently anchors the receptor to the membrane. In the transfected glioma cells, we detected ER{alpha} immunoreactivity outside of the DAPI-stained nucleus, suggesting that at least some of the ER{alpha} expressed in these cells localizes to the plasma membrane where it can bind to free or BSA-conjugated estradiol and activate rapid signaling pathways. A second approach, blocking signaling molecules with specific pharmacological inhibitors, suggests that the modulation of CRE-mediated transcription in glioma cells requires the activation of a rapid signaling pathway. The estradiol-induced reduction in CRE-mediated transcription was sensitive to wortmannin (PI3K) and U73122 (PLC), suggesting that E2 treatment of glioma cells activates the PI3K/PLC pathway leading to modulation of transcription at CRE sites. This sensitivity to wortmannin and U73122 was unexpected as previous findings from our laboratory demonstrated that E2 treatment of transfected hippocampal-derived neuronal cell lines (HTER{alpha} or HTERß) activates p42/44 MAPK and CREB, resulting in an increase in CRE-mediated transcription (16, 32). However, treatment of C6ER{alpha} and C6ERß cells with physiological concentrations of E2 failed to affect the phosphorylation state of either CREB or p42/44 MAPK (44). Together, these data demonstrate that E2 treatment activates a membrane-initiated signaling pathway in glioma cells leading to a reduction in CRE-mediated transcription, which is dissimilar to its effects in neuronal cells.

Although treating C6ER{alpha} cells with E2 caused a reduction in CRE-luc reporter gene expression, the ligand failed to affect CRE-mediated transcription in either C6ERß or untransfected C6 cells. C6 cells have been used in the past to model the antioxidant properties of estradiol in glial cells, but this effect required supraphysiological doses of E2 (45, 46), consistent with reports that C6 cells do not express either of the ERs (47). Although recent reports have shown detectable levels of mRNA for ER{alpha} in C6 cells by RT-PCR (44, 48), under the culture conditions used in this study, we were unable to detect ER{alpha} protein in untransfected C6 cells by immunoblotting or immunocytochemistry. Moreover, the ERE-luc reporter gene assay confirmed the lack of functional receptors in untransfected C6 cells because E2 had no effect on luc expression in these cells. Transfection with either hER{alpha} or rERß resulted in a significant increase in ERE-mediated transcription in response to physiological concentrations of E2. This observation further demonstrates that the estradiol-induced reduction in transcription at CRE sites is dependent on the activation of ER{alpha}, and not another endogenously expressed receptor.

The functional evidence that E2 treatment reduces CRE-mediated transcription in ER{alpha}-expressing glioma cells suggests a novel estrogen-activated rapid signaling pathway in astrocytes. This new model is based on the results that E2 induces a membrane-initiated signaling pathway that is sensitive to wortmannin and U73122. In other cell lines, estradiol treatment has been shown to activate the p42/44 MAPK pathway and CREB (9, 11) and increase CRE-mediated transcription (32). CREB, a transcription factor that modulates gene expression at CRE sites, is a coincidence detector that is responsive to several different signaling pathways including cAMP/protein kinase A, p42/44 MAPK, and PI3K pathways. This study suggests that E2 binds ER{alpha} and activates PI3K/PLC at or near the plasma membrane of glioma cells, which results in attenuation of the transcriptional activity at CRE sites, independent of the classical effects of estradiol on ERE-mediated gene expression.

An important prediction of this rapid signaling model is that estradiol levels may reduce neuroinflammation by attenuating the increase in CRE-mediated transcription in response to proinflammatory signals. There is a growing body of evidence suggesting that estradiol can modulate the functional activity of astrocytes and modify their ability to protect neurons from different insults (24). After brain injury, both ER{alpha} and ERß are up-regulated in reactive astrocytes suggesting that these cells are a direct target of estradiol stimulation (49, 50). Furthermore, astrocytes release growth factors such as TGF-ß in response to E2 (24, 25), and also respond to and modulate the immune response by releasing both pro- and antiinflammatory cytokines. Inhibition of CRE-mediated transcription may provide a mechanism for estradiol to shift the response in astrocytes to antiinflammation. Lipopolysaccharides increase cyclooxygenase-2 expression through p42/44 MAPK and PKC signaling cascades leading to an increase in prostaglandins and inflammation (51). Treatment of primary astrocyte cultures with pigment epithelium-derived factor increases proinflammatory cytokines like IL-1ß, IL-6, and TNF-{alpha} (52). Both of these inflammatory signals activate CREB and nuclear factor-{kappa}B, which drive the expression of cyclooxygenase-2 and proinflammatory cytokines. In this study, we provide functional evidence that E2 treatment reduces CRE-mediated transcription and therefore, may suppress proinflammatory signals from reactive astrocytes leading to neuroprotection from degenerative insults that stimulate inflammation.

In summary, we have found that E2 treatment of ER{alpha}-expressing glioma cells reduces nonclassical transcription at CRE sites. The inhibitory effect on nonclassical transcription appears to be mediated by a signal transduction cascade initiated with the activation of PI3K and PLC at or near the plasma membrane. These results suggest a novel nonclassical signaling model for estrogen rapid signaling in astrocytes. If true, this model has important implications in how estradiol can modulate the responses to inflammation in astrocytes and other nonneuronal cells of the CNS. A deeper understanding of estrogen rapid signaling in the various cell-types of the brain may prove important in treating neuroinflammation and preventing neurodegenerative disorders.


   Footnotes
 
This work was supported by National Institutes of Health, National Institute of Neurological Disorders and Stroke, NS20311-23 and the Alzheimer’s Disease Research Center of the University of Washington.

A.J.M., R.A.S., and D.M.D. have nothing to declare.

First Published Online January 26, 2006

Abbreviations: ß-gal, ß-Galactosidase; CBP, CREB-binding protein; CNS, central nervous system; CRE, cAMP response element; CREB, CRE binding protein; 10%CSM, transfection media consisting of phenol red-free DMEM supplemented with 10% charcoal-stripped fetal bovine serum and L-glutamine; DAPI, 4'-6-diamidino-2-phenylindole; E2, 17ß-estradiol; E2-BSA, 17ß-estradiol-6-CMO:BSA, a membrane-impermeable estrogen; ER, estrogen receptor; ERE, estrogen response element; 10%FM, growth media consisting of phenol red-free DMEM supplemented with 10% fetal bovine serum, penicillin, streptomycin and L-glutamine; hER{alpha}, human ER{alpha}; hERß, human ERß; luc, luciferase; MEK, MAPK kinase; PBG, 1% BSA/0.1% fish skin gelatin in PBS; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; rERß, rat ERß; TBS-T, Tris-buffered saline containing 0.1% Tween 20.

Received October 17, 2005.

Accepted for publication January 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sherwin BB 2002 Estrogen and cognitive aging in women. Trends Pharmacol Sci 23:527–534[CrossRef][Medline]
  2. Dubal DB, Kashon ML, Pettigrew LC, Ren JM, Finklestein SP, Rau SW, Wise PM 1998 Estradiol protects against ischemic injury. J Cereb Blood Flow Metab 18:1253–1258[CrossRef][Medline]
  3. Sherwin BB 1994 Estrogenic effects on memory in women. Ann NY Acad Sci 743:213–230[Abstract]
  4. Paganini-Hill A, Henderson VW 1994 Estrogen deficiency and risk of Alzheimer’s disease in women. Am J Epidemiol 140:256–261[Abstract/Free Full Text]
  5. Singer CA, Rogers KL, Strickland TM, Dorsa DM 1996 Estrogen protects primary cortical neurons from glutamate toxicity. Neurosci Lett 212:13–16[CrossRef][Medline]
  6. Weisz A, Rosales R 1990 Identification of an estrogen response element upstream of the human c-fos gene that binds the estrogen receptor and the AP-1 transcription factor. Nucleic Acids Res 18:5097–5106[Abstract/Free Full Text]
  7. Webb P, Lopez GN, Uht RM, Kushner PJ 1995 Tamoxifen activation of the estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like effects of antiestrogens. Mol Endocrinol 9:443–456[Abstract]
  8. Morley P, Whitfield JF, Vanderhyden BC, Tsang BK, Schwartz JL 1992 A new, nongenomic estrogen action: the rapid release of intracellular calcium. Endocrinology 131:1305–1312[Abstract]
  9. Aronica SM, Kraus WL, Katzenellenbogen BS 1994 Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription. Proc Natl Acad Sci USA 91:8517–8521[Abstract/Free Full Text]
  10. Ahmad S, Singh N, Glazer RI 1999 Role of AKT1 in 17ß-estradiol- and insulin-like growth factor I (IGF-I)-dependent proliferation and prevention of apoptosis in MCF-7 breast carcinoma cells. Biochem Pharmacol 58:425–430[CrossRef][Medline]
  11. Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E, Auricchio F 1996 Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO J 15:1292–1300[Medline]
  12. Imai A, Iida K, Tamaya T 1990 Phospholipase C in human endometrial fibroblasts and its regulation by estrogens. Comp Biochem Physiol B 97:617–621[CrossRef][Medline]
  13. Zhou Y, Watters JJ, Dorsa DM 1996 Estrogen rapidly induces the phosphorylation of the cAMP response element binding protein in rat brain. Endocrinology 137:2163–2166[Abstract]
  14. Bjornstrom L, Sjoberg M 2005 Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Mol Endocrinol 19:833–842[Abstract/Free Full Text]
  15. Watters JJ, Dorsa DM 1998 Transcriptional effects of estrogen on neuronal neurotensin gene expression involve cAMP/protein kinase A-dependent signaling mechanisms. J Neurosci 18:6672–6680[Abstract/Free Full Text]
  16. Fitzpatrick JL, Mize AL, Wade CB, Harris JA, Shapiro RA, Dorsa DM 2002 Estrogen-mediated neuroprotection against ß-amyloid toxicity requires expression of estrogen receptor {alpha} or ß and activation of the MAPK pathway. J Neurochem 82:674–682[CrossRef][Medline]
  17. Simpkins JW, Rajakumar G, Zhang YQ, Simpkins CE, Greenwald D, Yu CJ, Bodor N, Day AL 1997 Estrogens may reduce mortality and ischemic damage caused by middle cerebral artery occlusion in the female rat. J Neurosurg 87:724–730[Medline]
  18. Jung-Testas I, Renoir M, Bugnard H, Greene GL, Baulieu EE 1992 Demonstration of steroid hormone receptors and steroid action in primary cultures of rat glial cells. J Steroid Biochem Mol Biol 41:621–631[CrossRef][Medline]
  19. Santagati S, Melcangi RC, Celotti F, Martini L, Maggi A 1994 Estrogen receptor is expressed in different types of glial cells in culture. J Neurochem 63:2058–2064[Medline]
  20. Hosli E, Jurasin K, Ruhl W, Luthy R, Hosli L 2001 Colocalization of androgen, estrogen and cholinergic receptors on cultured astrocytes of rat central nervous system. Int J Dev Neurosci 19:11–19[CrossRef][Medline]
  21. McEwen B, Akama K, Alves S, Brake WG, Bulloch K, Lee S, Li C, Yuen G, Milner TA 2001 Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation. Proc Natl Acad Sci USA 98:7093–7100[Abstract/Free Full Text]
  22. Milner TA, McEwen BS, Hayashi S, Li CJ, Reagan LP, Alves SE 2001 Ultrastructural evidence that hippocampal {alpha} estrogen receptors are located at extranuclear sites. J Comp Neurol 429:355–371[CrossRef][Medline]
  23. Platania P, Laureanti F, Bellomo M, Giuffrida R, Giuffrida-Stella AM, Catania MV, Sortino MA 2003 Differential expression of estrogen receptors {alpha} and ß in the spinal cord during postnatal development: localization in glial cells. Neuroendocrinology 77:334–340[CrossRef][Medline]
  24. Sortino MA, Chisari M, Merlo S, Vancheri C, Caruso M, Nicoletti F, Canonico PL, Copani A 2004 Glia mediates the neuroprotective action of estradiol on ß-amyloid-induced neuronal death. Endocrinology 145:5080–5086[Abstract/Free Full Text]
  25. Dhandapani KM, Wade FM, Mahesh VB, Brann DW 2005 Astrocyte-derived transforming growth factor-ß mediates the neuroprotective effects of 17ß-estradiol: involvement of nonclassical genomic signaling pathways. Endocrinology 146:2749–2759[Abstract/Free Full Text]
  26. Singer CA, Figueroa-Masot XA, Batchelor RH, Dorsa DM 1999 The mitogen-activated protein kinase pathway mediates estrogen neuroprotection after glutamate toxicity in primary cortical neurons. J Neurosci 19:2455–2463[Abstract/Free Full Text]
  27. Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941–951[CrossRef][Medline]
  28. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930[Abstract/Free Full Text]
  29. Kumar V, Green S, Staub A, Chambon P 1986 Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J 5:2231–2236[Medline]
  30. Mize AL, Shapiro RA, Dorsa DM 2003 Estrogen receptor-mediated neuroprotection from oxidative stress requires activation of the mitogen-activated protein kinase pathway. Endocrinology 144:306–312[Abstract/Free Full Text]
  31. Stevis PE, Deecher DC, Suhadolnik L, Mallis LM, Frail DE 1999 Differential effects of estradiol and estradiol-BSA conjugates. Endocrinology 140:5455–5458[Abstract/Free Full Text]
  32. Wade CB, Dorsa DM 2003 Estrogen activation of cyclic adenosine 5'-monophosphate response element-mediated transcription requires the extracellularly regulated kinase/mitogen-activated protein kinase pathway. Endocrinology 144:832–838[Abstract/Free Full Text]
  33. Honda K, Sawada H, Kihara T, Urushitani M, Nakamizo T, Akaike A, Shimohama S 2000 Phosphatidylinositol 3-kinase mediates neuroprotection by estrogen in cultured cortical neurons. J Neurosci Res 60:321–327[CrossRef][Medline]
  34. Drouva SV, Gorenne I, Laplante E, Rerat E, Enjalbert A, Kordon C 1990 Estradiol modulates protein kinase C activity in the rat pituitary in vivo and in vitro. Endocrinology 126:536–544[Abstract]
  35. Toran-Allerand CD, Guan X, MacLusky NJ, Horvath TL, Diano S, Singh M, Connolly ESJ, Nethrapalli IS, Tinnikov AA 2002 ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. J Neurosci 22:8391–8401[Abstract/Free Full Text]
  36. Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER 2005 A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307:1625–1630[Abstract/Free Full Text]
  37. Mulholland DJ, Dedhar S, Coetzee GA, Nelson CC 2005 Interaction of nuclear receptors with the Wnt/ß-catenin/Tcf signaling axis: Wnt you like to know? Endocr Rev 26:898–915[Abstract/Free Full Text]
  38. Hanstein B, Eckner R, DiRenzo J, Halachmi S, Liu H, Searcy B, Kurokawa R, Brown M 1996 p300 Is a component of an estrogen receptor coactivator complex. Proc Natl Acad Sci USA 93:11540–11545[Abstract/Free Full Text]
  39. Wang MM, Traystman RJ, Hurn PD, Liu T 2004 Non-classical regulation of estrogen receptor-[alpha] by ICI182,780. J Steroid Biochem Mol Biol 92:51–62[CrossRef][Medline]
  40. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414[CrossRef][Medline]
  41. Pawlak J, Karolczak M, Krust A, Chambon P, Beyer C 2005 Estrogen receptor-{alpha} is associated with the plasma membrane of astrocytes and coupled to the MAP/Src-kinase pathway. Glia 50:270–275[CrossRef][Medline]
  42. Acconcia F, Ascenzi P, Bocedi A, Spisni E, Tomasi V, Trentalance A, Visca P, Marino M 2005 Palmitoylation-dependent estrogen receptor {alpha} membrane localization: regulation by 17ß-estradiol. Mol Biol Cell 16:231–237[Abstract/Free Full Text]
  43. Razandi M, Alton G, Pedram A, Ghonshani S, Webb P, Levin ER 2003 Identification of a structural determinant necessary for the localization and function of estrogen receptor {alpha} at the plasma membrane. Mol Cell Biol 23:1633–1646[Abstract/Free Full Text]
  44. Mhyre AJ, Dorsa DM 9 December 2005 Estrogen activates rapid signaling in the brain: role of ER{alpha} and ERß in neurons and glia. Neuroscience 10.1016/j.neuroscience.2005.10.019
  45. Bishop J, Simpkins JW 1994 Estradiol treatment increases viability of glioma and neuroblastoma cells in vitro. Mol Cell Neurosci 5:303–308[CrossRef][Medline]
  46. Haghighat N, Oblinger MM, McCandless DW 2004 Cytoprotective effect of estrogen on ammonium chloride-treated C6-glioma cells. Neurochem Res 29:1359–1364[CrossRef][Medline]
  47. Jacobsson SOP, Rongard E, Stridh M, Tiger G, Fowler CJ 2000 Serum-dependent effects of tamoxifen and cannabinoids upon C6 glioma cell viability. Biochem Pharmacol 60:1807–1813[CrossRef][Medline]
  48. Yague JG, Lavaque E, Carretero J, Azcoitia I, Garcia-Segura LM 2004 Aromatase, the enzyme responsible for estrogen biosynthesis, is expressed by human and rat glioblastomas. Neurosci Lett 368:279–284[CrossRef][Medline]
  49. Garcia-Ovejero D, Veiga S, Garcia-Segura LM, Doncarlos LL 2002 Glial expression of estrogen and androgen receptors after rat brain injury. J Comp Neurol 450:256–271[CrossRef][Medline]
  50. Takahashi N, Tonchev AB, Koike K, Murakami K, Yamada K, Yamashima T, Inoue M 2004 Expression of estrogen receptor-[beta] in the postischemic monkey hippocampus. Neurosci Lett 369:9–13[CrossRef][Medline]
  51. Mestre JR, Mackrell PJ, Rivadeneira DE, Stapleton PP, Tanabe T, Daly JM 2001 Redundancy in the signaling pathways and promoter elements regulating cyclooxygenase-2 gene expression in endotoxin-treated macrophage/monocytic cells. J Biol Chem 276:3977–3982[Abstract/Free Full Text]
  52. Yabe T, Sanagi T, Schwartz JP, Yamada H 2005 Pigment epithelium-derived factor induces pro-inflammatory genes in neonatal astrocytes through activation of NF-{kappa}B and CREB. Glia 50:223–234[CrossRef][Medline]



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