The present study sought to determine the characteristics of ICI 182,780 (Faslodex) action in rat primary hippocampal neurons. We first investigated the neuroprotective efficacy of ICI 182,780 against neurodegenerative insults associated with Alzheimer's disease and related disorders. Dose-response analyses revealed that ICI 182,780, in a concentration-dependent manner, significantly promoted neuron survival following exposure to either excitotoxic glutamate (200 μM)- or β-amyloid1–42 (1.5 μM)-induced neurodegeneration of hippocampal neurons. At a clinically relevant concentration of 50 ng/ml, ICI 182,780 exerted nearly maximal neuroprotection against both insults with efficacy comparable with that induced by the endogenous estrogen 17β-estradiol. Thereafter, we investigated the impact of 50 ng/ml ICI 182,780 on mechanisms of 17β-estradiol-inducible neuronal plasticity and neuroprotection. Results of these analyses demonstrated that ICI 182,780 directly induced a series of rapid intracellular Ca2+ concentration ([Ca2+]i) oscillations in a pattern comparable with that of 17β-estradiol. In addition, ICI 182,780 exerted dual regulation of the glutamate-induced rise in [Ca2+]i identical to that induced by 17β-estradiol. Further analyses demonstrated that ICI 182,780 induced significant activation of extracellular signal-regulated kinase 1/2 and Akt (protein kinase B) and significantly increased expression of spinophilin and Bcl-2, with efficacy comparable with neurons treated with 17β-estradiol. Taken together, results of these in vitro analyses of ICI 182,780 provide direct evidence of an estrogenic agonist profile of ICI 182,780 action in rat hippocampal neurons. Therapeutic development of neuroselective estrogen receptor modulators that mimic ICI 182,780 is discussed with respect to the potential of safe and efficacious alternatives to estrogen therapy for the prevention of postmenopausal cognitive decline and late-onset Alzheimer's disease.
ICI 182,780 (Faslodex) is a 7α-alkylsulfinyl analog of the endogenous estrogen 17β-estradiol and binds to both estrogen receptor (ER) subtypes with a comparable affinity to 17β-estradiol (Wakeling et al., 1991). ICI 182,780 is an efficacious antagonist of the ER-dependent proliferative actions of estrogen in reproductive organs such as the breast and uterus (Howell et al., 2000). Moreover, it is a “pure” antiestrogen lacking estrogen-like agonist activity in these organs, as seen with tamoxifen (TMX) (Howell et al., 2000). Partial agonist activity of TMX is believed to be the major cause of the development of resistance to TMX therapy for the treatment of estrogen-dependent breast cancers (Howell et al., 2000). The absence of estrogenic activity and the observations that TMX-resistant tumors remain sensitive to ICI 182,780 treatment led to Food and Drug Administration approval for the use of ICI 182,780 as an adjuvant endocrine therapy to treat ER-positive metastatic breast cancers in postmenopausal women with disease progression following the first line antiestrogen therapy, i.e., TMX-resistant breast cancers (Bross et al., 2002).
The ER antagonism of ICI 182,780 in the breast and uterus has been associated with its “down-regulation” of ER, resulting from its disruption of the shuttling of ER between the cytoplasm and the nucleus, a constitutively active process involving ER in both the absence and presence of 17β-estradiol (Dauvois et al., 1993). The accumulated ER presence in the cytoplasm undergoes elevated receptor turnover and degradation (Linstedt et al., 1986). Inhibition of nuclear uptake of ER induced by ICI 182,780 and its structural analogs has been proposed to be related to the terminal portion of the 7α side chains of these compounds, which protrude out of the ligand binding pocket and interfere with ER dimerization, a structural prerequisite for the translocation of ER into the nucleus (Fawell et al., 1990). In addition, ICI 182,780 has been demonstrated to fully abolish ER transcription regulated by both activation function AF-1 and AF-2 domains, which is distinct from the ER partial agonist/antagonist TMX or its active metabolite, 4-hydroxyl-tamoxifen, where only the AF-2 domain is blocked, and the estrogenic agonist activity of these selective estrogen receptor modulators (SERMs) can be achieved through the activation of the other ligand-independent AF-1 domain (Wakeling et al., 1991).
Although a large number of studies demonstrated that a high concentration of ICI 182,780 was an antiestrogen in brain (Purves-Tyson and Keast, 2004; Miller et al., 2005), there is a lack of research on ICI 182,780 action at a relatively low pharmacological and clinically relevant concentration. Several reports indicated estrogenic activity of ICI 182,780 in select brain tissue/cells. For example, in vitro ICI 182,780 induced a significant increase in phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in neonatal rat primary cerebellar neurons (Wong et al., 2003). Another in vitro study demonstrated that ICI 182,780 failed to block the activation of ERK1/2 induced by 17β-estradiol in basal forebrain cholinergic neurons (Dominguez et al., 2004). The ineffectiveness of ICI 182,780 in inhibiting 17β-estradiol-induced agonistic effects in the brain tissue was also indicated in studies on hypothalamic neurons and our own studies in hippocampal neurons (Cambiasso and Carrer, 2001). These novel findings of “deviated” properties from the conventional notion of ICI 182,780 acting as a “full” estrogen antagonist led us to pursue in more detail the pharmacological characteristics of ICI 182,780 action in neurons vulnerable to neurodegeneration associated with Alzheimer's disease and related disorders. In the present study, we sought to determine the estrogen agonist and/or antagonist properties of ICI 182,780 action on indicators widely accepted as key outcomes and mechanisms of estrogenic action in hippocampal neurons. Results of our in vitro analyses indicate that ICI 182,780 effectively and significantly induced estrogenic outcomes and activated underlying biochemical mechanisms consistent with estrogen actions, supporting its estrogenic agonist activity in these neurons.
Materials and Methods
Chemicals. 17β-Estradiol was purchased from Steraloids (Newport, RI). ICI 182,780 was purchased from Tocris Cookson (Ellisville, MO). The sources of other materials are indicated in the experimental methods described below.
Neuronal Cultures. The use of animals was approved by the Institutional Animal Care and Use Committee at the University of Southern California. Primary cultures of rat hippocampal neurons were prepared according to the method described previously (Zhao et al., 2004). In brief, hippocampi were dissected from the brains of embryonic day 18 Sprague-Dawley rat fetuses, treated with 0.02% trypsin in Hanks' balanced salt solution (137 mM NaCl, 5.4 mM KCl, 0.4 mM KH2PO4, 0.34 mM Na2HPO4·7H2O, 10.0 mM glucose, and 10.0 mM HEPES) at 37°C for 5 min and dissociated by repeated passage through a series of fire-polished constricted Pasteur pipettes. For intracellular Ca2+ imaging analyses, approximately 104 cells were seeded onto poly-d-lysine (10 μg/ml)-coated 22-mm coverslips in covered 35-mm Petri dishes. For neuroprotection and Western immunoblotting analyses, approximately 106 cells/ml were seeded onto poly-d-lysine-coated solid black and clear bottom 96-well culture plates and 60-mm Petri dishes, respectively. Cells were grown in phenol-red free neurobasal medium (NBM; Invitrogen, Carlsbad, CA) supplemented with B27, 5 U/ml penicillin, 5 μg/ml streptomycin, 0.5 mM glutamine, and 25 μM glutamate at 37°C in 10% CO2 for the first 3 days and NBM without glutamate afterward. Cultures grown in serum-free NBM yields approximately 99.5% neurons and 0.5% glial cells.
Glutamate Exposure. Primary hippocampal neurons grown on solid black and clear bottom 96-well culture plates for 7 days in vitro (DIV) were pretreated with vehicle alone or steroids for 48 h, followed by exposure to 200 μM glutamate at room temperature for 5 min in HEPES-buffered saline (HBS) solution (containing 100 mM NaCl, 2.0 mM KCl, 2.5 mM CaCl2, 1.0 mM MgSO4, 1.0 mM NaH2PO4, 4.2 mM NaHCO3, 10.0 mM glucose, and 12.5 mM HEPES). Immediately following glutamate exposure, cultures were washed once with HBS and replaced with fresh NBM containing test steroids. Cultures were returned to the culture incubator and allowed to incubate for an additional 24 h before neuronal viability measurements on the following day.
β-Amyloid1–42 Exposure. β-Amyloid1–42 (American Peptide Company, Sunnyvale, CA) was dissolved in 10 mM HCl at 1 mM and stored at –20°C as a stock solution. Aggregation of β-amyloid1–42 was prepared by incubating the stock solution in 0.1 M phosphate-buffered saline (PBS) solution (3.42 ml of 1 M Na2HPO4 and 1.58 ml of 1 M NaH2PO4 in 50 ml of double-distilled H2O) at room temperature for 3 days before use, which yielded a concentration of β-amyloid1–42 at 100 μM. Primary hippocampal neurons grown on solid black and clear-bottom 96-well culture plates for 7 DIV were pretreated with vehicle alone or steroids for 48 h, followed by exposure to freshly prepared 1.5 μM β-amyloid1–42 in NBM in the presence of vehicle alone or test steroids at 37°C for 3 days before neuronal viability measurement.
Neuronal Viability. Neuronal viability was determined with calcein acetoxymethyl ester (AM; Molecular Probes, Eugene, OR) staining, which indicates the metabolically live cells in the cultures (Zhao et al., 2004). Following glutamate or β-amyloid1–42 exposure, cultures were rinsed with warm PBS twice and incubated with 1 μM calcein AM in PBS at room temperature for 30 min. For cultures exposed to glutamate, the amount of fluorescence generated by calcein, a green fluorescent product derived from enzymatic esteration of calcein AM by esterases in live cells, was measured on a Spectra-Max microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA) at excitation/emission of 485/530 nm. For cultures exposed to β-amyloid1–42, fluorescent micrographs were captured with a Marianas digital microscopy workstation (Intelligent Imaging Innovations, Denver, CO), and the number of live cells in the cultures indicated by the green fluorescence staining was counted blind to the experimental condition with the image analysis program SlideBook 4.0 (Intelligent Imaging Innovations).
Intracellular Ca2+Imaging. The intracellular Ca2+ concentration ([Ca2+]i) in hippocampal neurons was measured by ratiometric Ca2+ imaging with the Ca2+-sensitive fluorescent dye, fura-2 AM (Molecular Probes), on an InCyt2 fluorescence imaging system (Intracellular Imaging, Cincinnati, OH). Before imaging, hippocampal neurons grown on poly-d-lysine-coated coverslips for 7 DIV were loaded with 2 μM fura-2 AM in HBS (containing 100 mM NaCl, 2.0 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 1.0 mM NaH2PO4, 4.2 mM NaHCO3, 10.0 mM glucose, and 12.5 mM HEPES) at 37°C for 40 min. Excess fura-2 AM dye was removed by washing with HBS twice, and then the cultures were incubated in HBS at 37°C for 30 min to equilibrate. The coverslip with fura-2 AM-loaded neurons was mounted in a perfusion chamber placed on an inverted microscope (IMT-2; Olympus Optical, Melville, NY) equipped with epifluorescence optics (20×; Nikon, Kanagawa, Japan). The perfusion system connected to the perfusion chamber was balanced using two variable speed pumps. The cultures were perfused with HBS at a flow rate of 2 ml/min. Fura-2 AM was sequentially excited by a xenon light source at 340 and 380 nm by means of two narrow beam bandpass filters selected by a computer-controlled filter wheel. The emitted fluorescence was filtered through a 520-nm filter. A pair of images, one at 340-nm excitation and one at 380 nm, were captured with an intensified charge-coupled device camera (COHU, San Diego, CA) and analyzed with the program InCyt Im2 (Intracellular Imaging). As the [Ca2+]i rises, the fluorescence intensity generated at 340-nm excitation increases, whereas the intensity at 380-nm excitation decreases. The relative [Ca2+]i was determined by taking the ratio of emission intensity at 340- to 380-nm excitation.
Western Immunoblotting. Primary hippocampal neurons grown on poly-d-lysine-coated culture dishes for 7 DIV were treated with vehicle alone or steroids for 25 min [for analyses on ERK1/2 and protein kinase B (Akt) expression] or 48 h (for analyses on spinophilin and Bcl-2 expression). Cultures were washed with ice-cold PBS once and scraped off the dish in 1 ml of PBS. Cells were then centrifuged at 5000 rpm for 5 min, and the pellets were dissolved in the radioimmunoprecipitation assay lysis buffer (PBS, 1% Triton, 0.2% SDS, and protease and phosphatase inhibitor cocktail set I; Calbiochem, San Diego, CA) and suspended by passage through a 200-μl pipette tip. Following incubation at 4°C for 45 min, the samples were centrifuged at 12,000 rpm for 10 min, and the supernatants were collected as the whole-cell protein extracts. Protein concentration was determined by the bicinchoninic acid method. Twenty-microgram protein samples were diluted in 15 μl of 2× SDS containing sample buffer, and the final volume was 30 μl with water. After denaturation on a hot plate at 95 to 100°C for 5 min, 20 μl of the mixture was loaded per lane on 10% SDS-polyacrylamide minigels followed by electrophoresis at 90 V. A high-range Precision Protein Standard (Bio-Rad Laboratories, Hercules, CA) was used to determine the protein size. The proteins were then electrotransferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) from the gels. Nonspecific binding sites were blocked with 5% nonfat dry milk in PBS containing 0.05% Tween 20.
For detection of pERK1/2 expression, membranes were incubated with the primary antibody against phospho-ERK1/2 (pTpY185/187, polyclonal; Biosource International, Camarillo, CA) at 1:760 dilution in PBS-Tween at 4°C overnight and then incubated with the HRP-conjugated secondary antibody (1:3300 dilution; Vector Laboratories, Burlingame, CA) at room temperature for 1 h. After scanning, membranes were stripped and reprobed with the primary antibody against ERK2 (polyclonal, 1:5000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight followed by incubation with the HRP-conjugated secondary antibody (1:5000 dilution; Vector Laboratories) at room temperature for 1 h.
For detection of pAkt expression, membranes were incubated with the primary antibody against phospho-Akt (pS473, monoclonal; Cell Signaling Technology, Beverly, MA) at 1:2000 dilution in PBS-Tween at 4°C overnight and then incubated with the HRP-conjugated secondary antibody (1:5000 dilution; Vector Laboratories). After scanning, membranes were stripped and reprobed with the primary antibody against Akt (polyclonal, 1:1000 dilution; Cell Signaling Technology) at 4°C overnight followed by incubation with the HRP-conjugated secondary antibody (1:5000 dilution; Vector Laboratories) at room temperature for 1 h.
For detection of spinophilin expression, membranes were incubated with the primary antibody against spinophilin (polyclonal; Upstate, Charlottesville, VA) at 1:750 dilution in PBS-Tween at 4°C overnight and then incubated with the anti-rabbit HRP-conjugated secondary antibody (1:10,000 dilution; Vector Laboratories) at room temperature for 1 h. For detection of Bcl-2 expression, membranes were incubated with the primary antibody against Bcl-2 (monoclonal; BD Bioscience, San Jose, CA) at 1:250 dilution in PBS-Tween at 4°C overnight and then incubated with the HRP-conjugated secondary antibody (1:5000 dilution; Vector Laboratories) at room temperature for 2 h. After scanning, membranes were stripped and reprobed with the primary antibody against β-actin (monoclonal, 1:250 dilution; Santa Cruz Biotechnology) at room temperature for 2 h followed by incubation with the anti-mouse HRP-conjugated secondary antibody (1:10,000 dilution; Vector Laboratories) at room temperature for 1 h.
All the membranes were developed with a 3,3′,5,5′-tetramethylbenzidine peroxidase substrate kit (Vector Laboratories). Relative intensities of the immunoreactive bands were quantified by optical density analysis using an image digitizing software, Un-Scan-It version 5.1 (Silk Scientific, Orem, UT).
Statistical Analyses. Data are presented as group means ± S.E.M. Statistically significant differences were determined by a one-way analysis of variance followed by a Newman-Keuls post hoc analysis.
ICI 182,780 Promoted Neuronal Survival against Neurodegenerative Insults in Rat Primary Hippocampal Neurons. Multiple in vitro and in vivo studies indicate that 17β-estradiol is a highly efficacious neuroprotective agent against a wide variety of neurodegenerative insults when administered prior to the insults (Brinton, 2001). In the course of attempts to use ICI 182,780 as an estrogen antagonist in our previous experiments, we discovered that ICI 182,780 did not function as an antagonist in neurons (L. Zhao and R. D. Brinton, unpublished data). To determine in detail the estrogen agonist and/or antagonist properties of ICI 182,780 action in neurons, we first investigated the impact of ICI 182,780 on neuronal viability when challenged by neurodegenerative insults, glutamate and β-amyloid1–42, in a prevention model paradigm using rat primary hippocampal neurons. Hippocampal neurons grown on poly-d-lysine-coated 96-well culture plates for 7 DIV were pretreated with vehicle alone or ICI 182,780 at multiple concentrations (1, 10, 50, 100, 500, and 1000 ng/ml) for 48 h before exposure to 200 μM glutamate for 5 min followed by recovery for 24 h (Fig. 1A) or 1.5 μM β-amyloid1–42 for 3 days (Fig. 1B). 17β-Estradiol (10 ng/ml; 36.7 nM) was used as a positive control and analyzed in parallel. Neuronal viability was determined by calcein AM staining, which is indicative of metabolically active cells. Results are presented as neuroprotective efficacy (NE), which is defined as the percentage of the neurotoxin-induced cell death prevented by experimental treatment and quantitated by the equation: NE = (Vtreatment – Vneurotoxin)/(Vcontrol – Vneurotoxin) × 100%, where Vtreatment is the individual value from experimental-treated cultures, Vneurotoxin is the mean value from neurotoxin alone-treated cultures, Vcontrol is the mean value from vehicle-treated control cultures, and neurotoxin refers to glutamate or β-amyloid1–42. Exposure of neurons to either 200 μM glutamate or 1.5 μM β-amyloid1–42 induced a significant decline in neuronal viability as demonstrated by reduced fluorescence intensity or reduced number of live cell staining in the cultures, respectively (Fig. 1, **, P < 0.01 compared with vehicle alone-treated control cultures). Pretreatment with ICI 182,780 significantly increased neuron survival against both excitotoxic glutamateand β-amyloid1–42-induced neurotoxicity. The neuroprotective potency of ICI 182,780 against both insults was comparable, and 10 ng/ml was the minimally effective concentration at which near 60% of neurons survived from both insults. Against excitotoxic glutamate, 10 ng/ml ICI 182,780 exerted the greatest neuroprotection (Fig. 1A, 51.4 ± 7.8% increase in neuronal viability compared with glutamate alone-treated cultures; **, P < 0.01), whereas 100 ng/ml was the EC100 against β-amyloid1–42-induced cell death (Fig. 1B, 84.8 ± 12.3% increase in neuronal viability compared with β-amyloid1–42 alone-treated cultures; **, P < 0.01). There was no statistically significant difference in neuroprotective efficacy among 10, 50, and 100 ng/ml ICI 182,780. Based on the equal efficacy of 10, 50, and 100 ng/ml, we selected 50 ng/ml as the optimal dose for further analyses because ICI 182,780 50 ng/ml exhibited nearly maximal neuroprotection against both glutamate and β-amyloid1–42-induced neurotoxicity (Fig. 1A, 39.9 ± 5.2% increase in neuronal viability compared with glutamate alone-treated cultures; **, P < 0.01; Fig. 1B, 73.6 ± 7.3% increase in neuronal viability compared with β-amyloid1–42 alone-treated cultures; **, P < 0.01). Moreover, 50 ng/ml ICI 182,780 induced a neuroprotective efficacy comparable with that induced by 17β-estradiol (Fig. 1A, 33.1 ± 4.2% increase in neuronal viability compared with glutamate alone-treated cultures; **, P < 0.01; Fig. 1B, 85.9 ± 8.6% increase in neuronal viability compared with β-amyloid1–42 alone-treated cultures; **, P < 0.01). Based on these analyses, we selected 50 ng/ml (82.4 nM), a relatively low and clinically relevant concentration of ICI 182,780 (Howell et al., 1996; Robertson et al., 2004), for subsequent comparative analyses of the impact of ICI 182,780 on the molecular mechanisms underlying estrogen-inducible neurotrophism and neuroprotection (Zhao et al., 2005).
ICI 182,780 Directly Induced a Series of Rapid But Irregular [Ca2+]i Oscillations in a Manner Comparable with 17β-Estradiol in Rat Primary Hippocampal Neurons. Although the full spectrum of molecular mechanisms that underlie estrogen promotion of neuronal plasticity and survival remains to be elucidated, a number of cellular responses have been well documented (Zhao et al., 2005). Key among these is 17β-estradiol regulation of intracellular Ca2+ signaling (Zhao et al., 2005). Our previous studies demonstrated that 17β-estradiol induces intracellular Ca2+ influx through voltage-dependent L-type Ca2+ channels, which is required for downstream activation of Src and mitogen-activated protein kinase signaling pathways. This signaling pathway is required for estrogen induction of neurotrophic and neuroprotective outcomes in neurons (Wu et al., 2005; Zhao et al., 2005).
In this experiment, we determined whether ICI 182,780 would directly induce a rapid rise in [Ca2+]i in neurons comparable with that induced by 17β-estradiol. Primary hippocampal neurons grown on poly-d-lysine-coated coverslips for 7 DIV were loaded with the Ca2+-sensitive dye, fura-2 AM, followed by ratiometric intracellular Ca2+ imaging analyses. The relative change in [Ca2+]i in neurons was determined by monitoring the change in the ratio of emission fluorescence intensity at 340- to 380-nm excitation. Results of these analyses indicated that similar to with 17β-estradiol (10 ng/ml, Fig. 2A), direct perfusion of neurons with ICI 182,780 (50 ng/ml, Fig. 2B) induced a series of rapid [Ca2+]i oscillations in approximately 40 to 50% of the neurons within the 30-min observation period. Eight responsive neurons are shown to provide a representative profile of the heterogeneity of the [Ca2+]i change in both the temporal characteristics and the magnitude of response (Fig. 2). Approximately 10 to 20% of the neurons responded at an intermediate level within the first 15 min (Fig. 2). At 15 to 30 min, both 17β-estradiol and ICI 182,780-perfused cultures exhibited a greater number of responding neurons with a greater magnitude of response (Fig. 2). Overall, ICI 182,780 induced a comparable average change (0.9) in the ratio of fluorescence intensities generated at 340- and 380-nm excitation, respectively, in responding neurons, to that (1.2) induced by 17β-estradiol (Fig. 2). A change in [Ca2+]i was not observed in neurons perfused with vehicle alone within the same observation period (30 min, data not shown).
ICI 182,780 Differentially Regulated [Ca2+]i Rise in Response to Glutamate in a Manner Comparable with 17β-Estradiol in Rat Primary Hippocampal Neurons. Previous findings from our group demonstrated that estrogens, including 17β-estradiol and conjugated equine estrogens, exerted a dual regulation of the intracellular Ca2+ dynamics in the presence of exogenous glutamate in neurons, depending on whether the concentration of glutamate is in the tolerable physiological range or in the excitotoxic range (Nilsen et al., 2002). At a physiological concentration of glutamate (25 μM) (Nilsen et al., 2002), estrogens potentiated the glutamate-induced [Ca2+]i rise, a cellular response leading to estrogen promotion of neuronal morphogenesis and new synapse formation (Nilsen et al., 2002). In contrast, at an excitotoxic level of glutamate (200 μM), estrogens attenuated the glutamate-induced [Ca2+]i rise in neurons, a cellular response considered as a fundamental event leading to estrogen promotion of neuronal defense and survival (Nilsen et al., 2002). Therefore, we pursued whether ICI 182,780 would regulate hippocampal neuron intracellular Ca2+ dynamics in a manner comparable with estrogens. Primary hippocampal neurons grown on poly-d-lysine-coated coverslips for 7 DIV were pretreated with vehicle alone, 17β-estradiol (10 ng/ml), or ICI 182,780 (50 ng/ml) for 48 h prior to ratiometric intracellular Ca2+ imaging analyses by fura-2 AM. 17β-Estradiol induced an average of 34.3% potentiation and an average 32.5% attenuation of the [Ca2+]i rise induced by 25 and 200 μM glutamate, respectively (Fig. 3A, 34.3 ± 3.1% increase; Fig. 3B, 32.5 ± 3.2% reduction compared with vehicle alone-pretreated control cultures; *, P < 0.05; **, P < 0.01). ICI 182,780 induced a comparable direction and magnitude of the [Ca2+]i change with those induced by 17β-estradiol. Neurons pretreated with ICI 182,780 and subsequently exposed to 25 μM glutamate exhibited an average 39.1% potentiation of the [Ca2+]i rise induced by glutamate (Fig. 3A; 39.1 ± 2.8% increase compared with vehicle alone-pretreated control cultures; *, P < 0.05). Consistent with neuronal response to 17β-estradiol, ICI 182,780 protected neurons from excess intracellular Ca2+ with an average 32.9% attenuation of the [Ca2+]i rise induced by 200 μM excitotoxic glutamate (Fig. 3B; 32.9 ± 3.9% reduction compared with vehicle alone-pretreated control cultures; **, P < 0.01).
ICI 182,780 Rapidly Increased ERK1/2 and Akt Phosphorylation with Efficacy Comparable with 17β-Estradiol in Rat Primary Hippocampal Neurons. One of the key downstream signaling events initiated by estrogen-induced intracellular Ca2+ rise and required for activation of the transcription factor, the cyclic AMP response element-binding protein (CREB), which results in increased transcription of various neurotrophic and neuroprotective genes, is the rapid activation of Src and ERK1/2 (Wu et al., 2005; Zhao et al., 2005). In parallel, through a unified upstream mechanism, estrogen binding to ER and interaction with phosphatidylinositol-3-kinase activates the Akt signaling pathway, which plays a pivotal role in altering the expression and function of the Bcl-2 family proteins (Znamensky et al., 2003; Mannella and Brinton, 2006). Based on these findings, we determined whether ICI 182,780 would activate these same signaling mechanisms in rat primary hippocampal neurons. Neurons grown on poly-d-lysine-coated culture dishes for 7 DIV were B27 supplement-deprived for 45 min prior to incubation with vehicle alone, 17β-estradiol (10 ng/ml), ICI 182,780 (50 ng/ml), or 17β-estradiol (10 ng/ml) plus ICI 182,780 (50 ng/ml) for 25 min prior to harvesting of proteins for detection of phosphorylated ERK and Akt expression by Western immunoblotting analyses. Total ERK and Akt expression levels in the same protein samples were also detected and used as the loading controls. Results of these analyses indicate that exposure of neurons to ICI 182,780 rapidly induced a significant increase in phosphorylation of both ERK2 and Akt (Fig. 4A, 28.6 ± 5.6%; Fig. 4B, 33.1 ± 5.0% increase compared with vehicle alone-treated control cultures, respectively; **, P < 0.01), with efficacy comparable with that induced by 17β-estradiol (Fig. 4A, 55.0 ± 13.6%; Fig. 4B, 25.8 ± 2.3% increase compared with vehicle alone-treated control cultures, respectively; **, P < 0.01). The presence of ICI 182,780 failed to block the effect of 17β-estradiol and combined use of both steroids exerted a comparable impact on both pERK1/2 and pAkt (Fig. 4A, 43.0 ± 9.6%; Fig. 4B, 30.9 ± 6.9% increase compared with vehicle alone-treated control cultures, respectively; **, P < 0.01). There were no statistically significant differences among the 17β-estradiol, ICI 182,780, and 17β-estradiol plus ICI 182,780 treatment groups.
ICI 182,780 Increased Spinophilin and Bcl-2 Expression with Efficacy Comparable with 17β-Estradiol in Rat Primary Hippocampal Neurons. Spinophilin, a protein that is enriched in the heads of neuronal dendritic spines, has been demonstrated to play a significant role in modulating both dendritic morphology and glutamatergic synaptic activity (Feng et al., 2000; Lee et al., 2004). We have shown previously that activation of CREB initiated by estrogen activation of the intracellular Ca2+/Src/ERK signaling cascade led to increased expression of spinophilin in hippocampal neurons, which is consistent with estrogen-inducible promotion of neuronal morphogenesis and synaptoplasticity (Zhao et al., 2005).
In light of these findings, we conducted Western immunoblotting analyses to determine the impact of ICI 182,780 on the expression of spinophilin in rat primary hippocampal neurons. We used β-actin as an internal loading control in our analyses because we determined that β-actin does not change in response to either 17β-estradiol or progesterone even though β-actin is enriched in spines and interacts with spinophilin. Hippocampal neurons grown for 7 DIV and treated with 50 ng/ml ICI 182,780 for 48 h had a significant increase in spinophilin expression (Fig. 5A, 44.8 ± 8.9% increase compared with vehicle alone-treated control cultures; **, P < 0.01), which was statistically comparable with that induced by 10 ng/ml 17β-estradiol (Fig. 5A, 29.5 ± 7.4% increase compared with vehicle alone-treated control cultures; *, P < 0.05). Coadministration of 17β-estradiol (10 ng/ml) and ICI 182,780 (50 ng/ml) resulted in a comparable effect with that induced by either 17β-estradiol or ICI 182,780 alone (Fig. 5A, 36.0 ± 8.1% increase compared with vehicle alone-treated control cultures; *, P < 0.05).
Estrogen activation of both intracellular Ca2+/Src/ERK/CREB and Akt signaling cascades leads to up-regulation of the Bcl-2 family antiapoptotic proteins, such as Bcl-2 and Bcl-xl, has been proposed as a critical component underlying estrogen promotion of neuronal survival (Nilsen and Brinton, 2003). Estrogen-inducible elevation in Bcl-2 and Bcl-xl expression in neurons enhances mitochondrial Ca2+ load tolerability induced by neurotoxic insults, leading to estrogen neuroprotection through sustained mitochondrial function (Nilsen and Brinton, 2003).
Based on these earlier findings, we evaluated the impact of ICI 182,780 on expression of Bcl-2 protein in rat primary hippocampal neurons. Neurons grown for 7 DIV were treated with vehicle alone, 17β-estradiol (10 ng/ml), ICI 182,780 (50 ng/ml), or 17β-estradiol (10 ng/ml) plus ICI 182,780 (50 ng/ml) for 48 h followed by Western immunoblotting analyses. Results of these analyses indicated that both 17β-estradiol and ICI 182,780 significantly and comparably increased Bcl-2 expression in neurons (Fig. 5B; 44.6 ± 8.0% and 40.4 ± 4.1% increase compared with vehicle alone-treated control cultures, respectively; **, P < 0.01). The presence of ICI 182,780 not only failed to block the effect of 17β-estradiol, but the combined use of 17β-estradiol and ICI 182,780 induced a comparable increase in Bcl-2 expression with that induced by either 17β-estradiol or ICI 182,780 alone (Fig. 5B; 38.0 ± 7.2% increase compared with vehicle alone-treated control cultures; **, P < 0.01). There were no statistically significant differences among the 17β-estradiol, ICI 182,780, and 17β-estradiol plus ICI 182,780 treatment groups.
Data presented herein provide in vitro evidence indicating an estrogenic agonist profile of ICI 182,780 action, at a clinically relevant concentration, in rat primary hippocampal neurons. First, the data demonstrate that ICI 182,780 was an efficacious neuroprotective agent against neurodegenerative insults associated with Alzheimer's disease and related disorders. Second, ICI 182,780 significantly activated biochemical mechanisms required for estrogen promotion of neuronal plasticity and neuroprotection. The data further demonstrate that ICI 182,780-induced neuronal responses were of comparable magnitude with those induced by 17β-estradiol. Moreover, the presence of ICI 182,780 did not inhibit 17β-estradiol-induced neural responses.
Our findings are in contrast to the conventional classification of ICI 182,780 as a “full” antiestrogen in ER-positive tissues. In addition to our data derived from hippocampal neurons, studies by the Simpkins group demonstrated that ICI 182,780 was protective against β-amyloid25–35 or H2O2 toxicity in SK-N-SH and human lens cells (Wang et al., 2003, 2006; Kumar et al., 2005). Estrogen agonist properties of ICI 182,780 have also been observed in bone where ICI 182,780 promoted bone growth (Sibonga et al., 1998). Taken together, these earlier investigations along with our present analyses suggest that as with other SERMs, exemplified by TMX and raloxifene (RAL), ICI 182,780 should also be classified as a mixed estrogen agonist/antagonist.
We propose that one factor to account for the “inversed” pharmacological properties exhibited by ICI 182,780 in distinct tissues/cells could be the nature of an estrogen-binding receptor involved. Estrogen antagonism of ICI 182,780 in reproductive tissues has been associated with the conformational remodeling of the classical ER induced by its bulky side chain substituted on the 7α position of the 17β-estradiol core structure. The terminal portion of this long tail structure, present as an additional structure absent in both TMX and RAL, binds along the coactivator recruitment site in the AF-2 cleft, which in turn precludes helix 12 from adopting either the characteristic agonist orientation, as seen with 17β-estradiol, in which helix 12 is aligned over the ligand-binding pocket (Brzozowski et al., 1997), or the AF-2 antagonist orientation, as seen with TMX and RAL, in which helix 12 orients along the coactivator binding surface in AF-2 (Brzozowski et al., 1997; Shiau et al., 1998). One functional consequence derived from this conformational remodeling of ER induced by ICI 182,780 is the impaired dimerization of ER, a structural prerequisite for the translocation of ER into the nucleus, resulting in the inhibition of the ER-DNA binding (Fawell et al., 1990; Brzozowski et al., 1997). The estrogenic agonist activity of ICI 182,780 in parallel to 17β-estradiol in hippocampal neurons suggests that these two ER ligands, although they exhibit opposite pharmacological profiles in some ER-positive tissues, in particular reproductive tissues, are able to activate the same molecular target and the downstream signaling cascades, leading to their estrogen agonist actions in neurons. In view of the unique characteristics of ICI 182,780 interaction with ER, these analyses may suggest that the parallel estrogenic agonist activity of 17β-estradiol and ICI 182,780 in hippocampal neurons is likely mediated by a nonclassical signaling pathway separate from proliferative actions of estrogen in reproductive tissues.
Increasing evidence indicates that there is a plethora of structural variants of ERα and ERβ enriched in the brain (Brinton and Nilsen, 2001; Toran-Allerand, 2004), which may carry out distinct functions from the classical nuclear receptors and serve as the molecular basis to confer ICI 182,780-induced agonist activity in neurons. In support of this hypothesis, specific mutations in the ER could reverse the antiestrogenic property of an ER antagonist to that representing an estrogen agonist. For instance, a study conducted by Mahfoudi et al. (1995) demonstrated that both TMX and ICI 164,384 acted as potent agonists in HeLa cells expressing ER mutants introduced in a conserved hydrophobic region of AF-2. Such mutations also promoted the stability and nuclear localization of the receptor compared with the wild-type ER following ICI 164,384 treatment (Mahfoudi et al., 1995). Based on this observation, it is conceivable that there might exist ER variants (e.g., with the structural alteration in AF-2) in neurons; binding of ICI compounds could induce the repositioning of helix 12 distinct from that observed in the full-length receptor and lead to exposure of the AF-2 without affecting the dimerization and nuclear uptake capability of the receptors.
Alternatively, studies from multiple laboratories, including our own, indicate that a membrane-associated estrogen-binding receptor induces the rapid actions of 17β-estradiol and ICI 182,780 in neurons (Brinton, 2001; McEwen, 2002). Based on our recent observations that both ERα-selective agonist PPT and ERβ-selective agonist DPN were effective in activating the neurotrophic and neuroprotective outcomes and the same underlying mechanisms, we propose that this membrane-associated estrogen-binding receptor could be ERα or ERβ or their structural analogs (Zhao et al., 2004). This hypothesis is partially supported by increasing evidence indicating the subcellular localization of ERα and ERβ in non-nuclear compartments including membranes in rat and mouse cortical and hippocampal neurons (Milner et al., 2001; Nishio et al., 2004; Kalita et al., 2005). In addition, our observation that PPT and DPN compete for the same membrane binding site as the BSA-17β-estradiol conjugate (BSA-17β-estradiol-fluorescein isothiocyanate) in rat hippocampal neurons provides indirect evidence for membrane localization of ERα and ERβ or at least ERα- and ERβ-related receptors (Wu et al., 2005). Functionally, it is likely that these membrane-associated classical ER-related receptors could associate and act with other membrane structures, leading to initiation of signaling cascades (Norman et al., 2004; Mannella and Brinton, 2006).
An alternative hypothesis for a membrane-associated estrogen-binding receptor in neurons could be a structurally distinct class of receptors independent of the classical ER. In support of this hypothesis, recent discoveries demonstrated that a transmembrane G protein-coupled receptor, GPR30, although it remains controversial whether it localizes to the plasma membrane or exclusively to the endoplasmic reticulum, can be activated by a number of the same ER ligands and responsible for estrogen-induced rapid signaling independently of classical ER in some cell types, including ER-negative cell types (Revankar et al., 2005; Thomas et al., 2005). ICI 182,780 exhibits high binding affinity to GPR30 and acts as an agonist of GPR30 (Thomas et al., 2005), which has important implications for a membrane-associated estrogen-binding receptor in mediating the consistent agonistic effects of 17β-estradiol and ICI 182,780 in hippocampal neurons.
From a therapeutic development perspective, our findings that ICI 182,780 acts as an estrogen agonist in neurons provides a translational opportunity for development of an ideal brain-selective ER modulator that mimics the beneficial effects of estrogen agonists in brain while lacking or antagonizing activation of estrogenic proliferative responses in reproductive organs (neuro-SERMs) (Zhao et al., 2005). Because ICI 182,780 does not readily cross the blood-brain barrier (Howell et al., 2000), design and development of a brain-accessible ICI-like molecule that is structurally and functionally analogous to ICI 182,780 while possessing improved chemical features that promote blood-brain barrier penetration is required. The availability of such a molecule will provide a valuable pharmacological tool to further decipher the in vivo activity of this category of ER ligands in the brain. Proof of principle of the in vivo efficacy of ICI to mimic neuro-SERMs is anticipated to provide a dual therapeutic profile to treat ER-positive breast cancers while simultaneously preventing estrogen deficiency-associated cognitive decline and neurodegeneration in postmenopausal women.
We gratefully thank Sean Iwamoto for providing excellent technical support.
This work was supported by grants from the National Institutes of Mental Health, the National Institutes of Aging, the K. T. and E. L. Norris Foundation, the L. K. Whittier Foundation, and the Stanley Family Trust (to R.D.B.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ICI, ICI 182,780 (Faslodex); ER, estrogen receptor; TMX, tamoxifen; AF, activation function; SERM, selective estrogen receptor modulator; ERK, extracellular signal-regulated kinase; NBM, neurobasal medium; DIV, day(s) in vitro; HBS, HEPES-buffered saline; Akt, protein kinase B; PBS, phosphate-buffered saline; AM, acetoxymethyl ester; [Ca2+]i, intracellular Ca2+ concentration; HRP, horseradish peroxidase; NE, neuroprotective efficacy; CREB, cyclic AMP response element-binding protein; RAL, raloxifene; ICI 164,384, (N-n-butyl-N-methyl-11-[3,17β-dihydroxyestra-1,3,5(10)-trien-7α-yl]-undecanamide; E2, 17β-estradiol.
- Received June 21, 2006.
- Accepted August 31, 2006.
- The American Society for Pharmacology and Experimental Therapeutics