Estrogen Receptor-Mediated Neuroprotection from Oxidative Stress Requires Activation of the Mitogen-Activated Protein Kinase Pathway
Abstractu.*w|0, 百拇医药
It is well documented that estrogen mediates responses by both genomic and nongenomic mechanisms, both of which are important for cell survival. Because direct evidence showing that the estrogen receptors (ERs) {alpha} and/or ß can activate rapid signaling that may mediate neuroprotection is lacking, the hippocampal-derived cell line, HT22, was stably transfected with ER{alpha} (HTER{alpha} ), ERß (HTERß), or a mutated form of ER{alpha} (HTER{alpha} HE27), which lacks the ability to mediate ER element-mediated transcription. Treatment of HT22, HTER{alpha} , HTERß, and HTER{alpha} HE27 cells with glutamate (5 mM) resulted in a significant decrease in cell viability. Pretreatment for 15 min with 10 nM 17ß-estradiol resulted in a 50% increase in the number of living cells in HTER{alpha} and HTERß cells but not in HT22 cells. The ER antagonist ICI 182,780 and the MEK inhibitor PD98059 prevented 17ß-estradiol-mediated protection. In HTER{alpha} HE27 cells, 17ß-estradiol rapidly phosphorylated ERK2 (within 15 min), in the absence of estrogen response element-mediated transcription. Treatment of HTER{alpha} HE27 cells with 10 nM 17ß-estradiol partially reversed the cell death produced by glutamate treatment. This study demonstrates that activation of either ER{alpha} or ERß can result in neuroprotection and that activation of the MAPK pathway is an important part of the neuroprotective mechanism.
Introductionp{n@?, 百拇医药
THE GONADAL SEX steroid hormone estradiol serves many important functions. In addition to its classical reproductive role, numerous studies demonstrate that it plays an important trophic and protective role in the brain. Epidemiological evidence suggests that estrogen replacement therapy for postmenopausal women is associated with an improvement of some measures of cognitive performance, protection against cognitive deterioration, and decreased incidence of Alzheimer’s disease (1, 2, 3, 4, 5). In addition, beneficial effects of estrogen on the mortality and morbidity associated with cerebral stroke have also been demonstrated (6, 7, 8).p{n@?, 百拇医药
At the cellular level, estrogen is known to exert neuroprotective effects in various model systems. In vitro studies have shown that 17ß-estradiol reduces neuronal damage caused by serum deprivation (9, 10), ß-amyloid treatment (11, 12), and exposure to glutamate (12, 13). Although activation of estrogen receptors has been suggested in mediating neuroprotection, the complex mechanisms by which estrogen protects neurons against injury are not completely understood.
Although estrogen is generally thought to mediate its effects by activation of transcription via nuclear receptors, increasing evidence suggest that estrogen may also cause rapid activation of signal transduction pathways. As examples, estrogen is known to produce mobilization of intracellular calcium (14) production of cAMP (15, 16), activation of Akt (17) as well as MAPK, ERK1, and ERK2 (11, 18, 19, 20). Several lines of evidence suggest estrogen neuroprotection may be mediated by rapid intracellular signaling events rather than estrogen response element (ERE)-mediated gene transcription. For example, activation of protein kinase A, protein kinase C, and MAPK have been linked to neuroprotection in various cellular model systems (19, 21, 22). In addition, recent reports from our laboratory have shown that estrogen receptor (ER) activation of MAPK is important to protect neuronal cells from ß-amyloid toxicity (11). However, it is not clear whether nuclear estrogen receptor activation of rapid signaling pathways is sufficient to elicit neuroprotection in the absence of ERE-mediated transcription.
Our study was conducted to examine whether expression of ER{alpha} and ERß are required for low doses of estrogen to elicit neuroprotection against glutamate toxicity in a neuronal cell line. In addition, we have examined the relative role of MAPK activation vs. classical ERE-mediated transcription in eliciting estrogen-mediated neuroprotection.{ovr, 百拇医药
Materials and Methods{ovr, 百拇医药
Chemicals{ovr, 百拇医药
17ß-Estradiol, glutamate, and PD98059 were purchased from Sigma (St. Louis, MO). ICI 182,780 was purchased from Tocris Cookson (Ballwin, MO).{ovr, 百拇医药
Cell culture{ovr, 百拇医药
HT22 cells were given as a kind gift from Dr. Pamela Maher (The Scripps Research Institute, La Jolla, CA). These cells were grown on 100-mm tissue culture dishes and maintained in DMEM (Sigma) media supplemented with 5% fetal bovine serum and 1% Pen-Strep (Gem Cell, Woodland, CA) at 37 C in a 5% CO2 atmosphere. Cell density was maintained 70% or less confluency as described previously (23).
Plasmids and oligonucleotide mutagenesisq, 百拇医药
The zinc finger mutation (C202H;C205H) corresponding to HE27, described previously (23), was introduced into ER{alpha} HEGO/pcDNA3.1 hygromycin by a sequence overlap extension procedure. Overlapping PCR fragments were first generated in separate reactions and then reannealed and extended in a second PCR. In the first step, two oligonucleotides (complement and reverse complement) containing the desired nucleotides were synthesized for each mutation. Two fragments were then synthesized by PCR using a T7 primer and the reverse complement (5'-GAATACTTCTCTTGAAGAAGGCCTTGTGGCCCTCATGACACCAGACTCCATAATGG-3') to generate the 5' fragment and with the complement (5'-CCATTATGGAGTCTGGTCTCATGAGGGCCACAAGGCCTTCTTCAAGAGA-AGTATTC3') and a 3' oligo (5'TGTACACTCCAGAATTAAGC-3') to generate the 3' fragments. Altered nucleotides are indicated in bold type. A novel BspH1 site is underlined. After gel purification, 10 ng of each fragment were mixed and used as a template in a second PCR with the two outer oligonucleotides. The resulting 1400-bp fragment was digested with HindIII and a 1020-bp fragment used to replace the corresponding fragment in ER{alpha} HEGO/pcDNA3.1 hygromycin. All nucleotide changes were confirmed by DNA sequencing.
Generation of HT22 stable transfectants*i!}l*, 百拇医药
Stable transfectants were generated as described previously (11). Briefly, HT22 cells were grown to approximately 60% confluency before being transfected with the lipid transfection reagent, TransFast (Promega Corp., Madison, WI). The pCDNA3.1 hygromycin (7.3 µg) containing either the full-length human ER{alpha} cDNA (24), a gift from Dr. Pierre Chambon (Strasbourg, France), or rat ERß cDNA (25), a gift from George Kuiper (Karolinska Institute, Sweden), was added at a 1:1 ratio with Transfast per 100-mm plate. Medium was changed 24 h later, and hygromycin (125 µg/ml) was added 72 h after transfection for selection of ER-expressing clones. Single colonies were isolated after the 10th day of growth in selective conditioned media and tested for receptor expression by immunoblotting.*i!}l*, 百拇医药
Activation of ERK2*i!}l*, 百拇医药
ER-positive cells were grown to 70–80% confluency on 100-mm plates. Eighteen hours before treatment, the medium was replaced with phenol red-free DMEM supplemented with 1% charcoal-stripped serum. Cells were treated with ethanol vehicle (0.1% final concentration) or 17ß-estradiol (10 nM) for the indicated times. The medium was removed, and the cells were washed in ice-cold PBS. Cells were rinsed with ice-cold PBS buffer, scraped into immunoprecipitation buffer (1 M HEPES, 0.1 M EGTA, 0.5 M EDTA, 0.5 M Na+ pyrophosphate, 1 M NaF, 1 mM NaVO4, and 9 mM NaCl) and incubated on ice for at least 5 min. The samples were then sonicated for 2 min followed by centrifugation at 15,000 rpm for 10 min. The supernatant was transferred to a new tube and protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce Chemical Co., Rockford, IL). The proteins samples were diluted in Laemmeli sodium dodecyl sulfate sample buffer and equal volumes of cell lysate were loaded and resolved by electrophoresis on 4–12% Bis-Tris precast gels (Invitrogen, Carlsbad, CA) in running buffer [50 mM 2-(N-morpholine)ethane sulfonic acid, 50 mM Tris base, 0.1% sodium dodecyl sulfate, and 1 mM EDTA] as described by the manufacturer. The proteins were transferred to polyvinylidene diflouride membranes and blocked in 5% nonfat dry milk in Tris-buffered saline containing 0.2% Tween 20 for 1 h at room temperature. ERK2 phosphorylation was detected using mouse antiphospho-p44/42 MAPK (1:2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) that recognizes phospho-THR202 and THR204 forms of ERK1 and 2. Phosphorylation at these sites has been correlated with increased activity (26, 27, 28). Total ERK2 was detected using rabbit anti-ERK2 (1:10,000, Santa Cruz Biotechnology, Inc.).
Secondary goat antimouse antibodies (1:5000, Santa Cruz Biotechnology, Inc.) or goat antirabbit antibodies (1:5000) conjugated to horseradish peroxidase were used for detection by enhanced chemiluminescence (NEN Life Science Products, Boston, MA) on film. The resulting film samples were scanned and analyzed with an image analysis program (NIH Image, Scion Corp., Frederick, MD). Data are presented as a ratio of phospho-ERK2/total ERK2 in the sample, normalized to vehicle-treated samples.a, http://www.100md.com
Transient transfectionsa, http://www.100md.com
HT22 cells (~ 5 x 105 cells/well) were plated into 24-well plates and transfected with 1.0 µg/well tk-ERE-luciferase (kindly provided by Dr. Peter Burbach, Rudolf Magnus Institute, University of Utrecht) using the TransFast protocol. One microgram pCH110, a ß-galactosidase reporter (Amersham Pharmacia, Uppsala, Sweden) was added to each well to normalize for transfection efficiency. After 24 h in DMEM, cells were transfected at 37 C in Transfast reagent in media supplemented with 10% charcoal-stripped calf serum for 1 h. Media was then replaced, and cells were allowed to recover for 24 h before pharmacological manipulation.
Cell treatments[\, 百拇医药
Cells were grown to 70–80% confluency in 12-well plates. Twenty-four hours before treatment the medium was replaced with phenol red free DMEM supplemented with charcoal-stripped fetal bovine serum (1%). Glutamate was diluted to a final concentration of 5 mM in culture medium and cells were exposed for 24 h. 17ß-Estradiol was initially dissolved in 95% ethanol at a concentration of 1 mM and diluted to the appropriate concentration (10 nM) in culture medium. Exposure to 17ß-estradiol was initiated 15 min before glutamate addition. Ethanol was used at a final concentration of 0.1% as a vehicle control. This concentration of ethanol had no effect on cell viability or glutamate toxicity. ICI 182,780 and PD98059 were made as 1000x stocks in 100% dimethylsulfoxide and were added to cells 15 min before 17ß-estradiol exposure.[\, 百拇医药
Dimethylthiazoldiphenyltetra-zoliumbromide (MTT) assay[\, 百拇医药
The MTT assay measures cellular viability by assessing mitochrondrial activity in the cells. After completion of incubation, 200 µl of a 5 mg/ml MTT stock in PBS was added to each well, and the incubation continued for 4 h. After removal of the MTT solution, 1 ml solubilization solution containing 50% dimethylformamide, 20% sodium dodecyl sulfate (pH 4.8) was added. Absorption values at 570 nM were read on a Packard Spectra spectrophotometer.
Calcein acetomethyl ester (AM)+5ae3x, 百拇医药
Following incubation of cells in glutamate, cells were rinsed once with PBS and incubated with 1 µM calcein AM dye (Molecular Probes, Inc., Eugene, OR) at 37 C for 15 min, washed twice with PBS, and coverslips applied. Green fluorescent cells are the product of mitochondrial cleavage of calcein AM and were observed using a Optiphot 2 microscope (Nikon) with the EF-D fluorescence attachment and G-1B and DM510 filters and counted as living cells.+5ae3x, 百拇医药
Statistical analysis+5ae3x, 百拇医药
The significance of differences among groups was determined by one-way ANOVA followed by a Tukey’s multiple comparison test. P < 0.05 was considered significant, and each group consisted of 6–12 wells or plates. All values are expressed as mean ± SEM.+5ae3x, 百拇医药
Results+5ae3x, 百拇医药
Figure 1 demonstrates that HT22, HTER{alpha} , and HTERß cells are sensitive to glutamate exposure, in which greater than 50% of cells were killed after a 24-h treatment. To further characterize the steps leading to cell death, various compounds were tested to determine their ability to block glutamate-induced cell death. As suggested by previous reports, both N-methyl-D-aspartate and kainate glutamate receptor antagonists failed to prevent glutamate toxicity in HTER{alpha} and HTERß cells (Table 1). However, cystine was able to completely reverse the cellular death.
fig.ommitteed25t, http://www.100md.com
Figure 1. Glutamate significantly decreases cell survival in HT22, HTER, and HTERß cells. HT22, HTER, and HTERß cells were treated with glutamate (5 mM) for 24 h. Cellular viability was assessed using the MTT assay. Data are expressed as the percentage of the maximal number of living cells in a vehicle-treated control. All results represent the mean ± SEM from three to four separate platings. *, Statistically different from vehicle-treated cells, P < 0.05.25t, http://www.100md.com
fig.ommitteed25t, http://www.100md.com
Table 1. Toxicity of glutamate and its analogs25t, http://www.100md.com
HT22, HTER{alpha} , and HTERß cells were treated with either 1 µM or 10 nM 17ß-estradiol for 15 min before glutamate exposure to determine whether a short pretreatment with the hormone would elicit neuroprotection. In HTER{alpha} and HTERß cells, a 15-min pretreatment with a near physiological dose of 17ß-estradiol (10 nM) significantly increased cellular viability as evidenced by calcein AM staining (Fig. 2, A and B). By contrast, at this dose of estrogen HT22 cells were not protected from glutamate toxicity. A high dose of estrogen (1 µM) protected all three cell lines from glutamate toxicity, irrespective of ER expression. Figure 2B is a representative experiment depicting the abundance of living cells treated with glutamate after a 15-min pretreatment with 17ß-estradiol or vehicle.
fig.ommitteedz]f, http://www.100md.com
Figure 2. Fifteen-minute pretreatment with low-dose 17ß-estradiol protects HTER{alpha} and HTERß cells from glutamate toxicity. HT22, HTER{alpha} , and HTERß cells were treated with 17ß-estradiol (1 µM or 10 nM) for 15 min, followed by glutamate (5 mM) for 24 h. Cellular viability was assessed by calcein AM staining. The number of living cells was assessed by fluorescence with calcein AM. A, Numbers of living cells are expressed as percent of vehicle-treated controls. All results represent the mean ± SEM from three to four separate platings. *, Statistically different from glutamate-treated cells, P < 0.05. B, Representative micrograph showing the presence of living cells, compared with vehicle-treated controls.z]f, http://www.100md.com
To confirm that the neuroprotective effect of 17ß-estradiol was dependent on ERs, cells were pretreated with ICI 182,780. Although ICI 182,780 (1 µM) had no cytotoxic or neuroprotective effects on its own (data not shown), it blocked the ability of 17ß-estradiol to increase living cell number (Fig. 3, A and B), demonstrating that the protective effects of low-dose estrogen occurred in an ER-dependent manner. Figure 3B is a representative experiment depicting the abundance of living cells treated with glutamate in the presence and absence of 17ß-estradiol and the reduction of living cells observed after glutamate treatment in the presence of estrogen and ICI 182,780.
fig.ommitteed^%, http://www.100md.com
Figure 3. ICI 182,780 blocks 17ß-estradiol-mediated neuroprotection against glutamate-induced toxicity in HTER{alpha} and HTERß cells. HTER{alpha} and HTERß cells were treated with 17ß-estradiol (10 nM) for 15 min in the presence and absence of ICI 182,780 (1 µM), followed by glutamate (5 mM) for 24 h. Cellular viability was assessed as described and presented in Fig. 2.^%, http://www.100md.com
To determine whether 17ß-estradiol-mediated neuroprotection was dependent on activation of MAPK, cellular cytotoxicity was determined in the presence and absence of PD98059. Although PD98059 (50 µM) had no effect on cytotoxicity alone, it abolished the ability of 17ß-estradiol to increase living cell number (Fig. 4, A and B). This datum suggests that the protective effects of estrogen are occurring through a MAPK-dependent pathway. Figure 4B is a representative experiment depicting the abundance of living cells treated with glutamate in the presence and absence of 17ß-estradiol and the reduction of living cells observed after glutamate treatment in the presence of estrogen and PD98059.
fig.ommitteed'], 百拇医药
Figure 4. PD98059 blocks 17ß-estradiol-mediated neuroprotection against glutamate induced toxicity in HTER{alpha} and HTERß cells. HTER{alpha} and HTERß cells were treated with 17ß-estradiol (10 nM) for 15 min in the presence and absence of PD98059 (50 µM), followed by glutamate (5 mM) for 24 h. Cellular viability was assessed as described in Fig. 2.'], 百拇医药
To evaluate the relative contribution of rapid signaling vs. genomic mechanisms in mediating neuroprotection, an ER{alpha} mutated to conform with that reported by Kumar et al. (29) was stably expressed into HT22 cells (HE27: C202H;C205H;HTER{alpha} HE27). First, it was confirmed that in HTER{alpha} HE27 cells, estrogen was still able to mediate rapid activation of the MAPK pathway. Figure 5A shows that ERK2 phosphorylation is increased within 15 min and returns to basal levels by 30 min in cells expressing the mutated ER{alpha} . Under these conditions, no ERK2 phosphorylation was observed in untransfected HT22 cells (11) (data not shown). To ensure that the mutated ER cell line was unable to elicit ERE-mediated transcription, HT22 cells were transfected with either full-length ER{alpha} or HE27 and an ERE-luciferase reporter construct and the transcriptional activity was measured (Fig. 5B). In cells transfected with ER{alpha} , addition of 17ß-estradiol resulted in a 2.5-fold increase in luciferase activity. No activity was observed in untransfected cells or cells transfected with HTER{alpha} HE27.
fig.ommitteed5r, 百拇医药
Figure 5. 17ß-Estradiol treatment increases ERK2 phosphorylation in HTER{alpha} HE27 cells but does not activate an ERE reporter gene construct. A, Lysates from HTER{alpha} HE27 cells treated with 10 nM 17ß-estradiol for 5, 15, or 30 min were analyzed by immunoblotting for changes in phosphorylation of ERK2. Lysates were probed with an antiphospho-specific p42/44 MAPK (ERK1/ERK2) antibody. Total ERK2 was detected using an anti-ERK2 antibody that recognizes both phosphorylated and unphosphorylated ERK2. Relative amounts of phosphorylated ERK2 were determined from densitometric scanning of ECL-exposed film. Data are represented as fold increase in relative phosphorylated ERK2, compared with vehicle treatment ± SEM. All experiments were performed three times in triplicate, and asterisks (*) indicate a P value of 0.05 or less, as determine by one-way ANOVA. B, Transiently transfected HTER{alpha} HE27 cells were treated with vehicle or 50 nM 17ß-estradiol for 24 h and assayed for luciferase and ß-galactosidase activity. Data are represented as the ratio of luciferase/ß-galactosidase activity. All experiments were performed three times in triplicate, and asterisks (*) indicate a P value of 0.05 or less, as determined by one-way ANOVA.
To determine the contribution of MAPK activation vs. ERE-mediated transcription in eliciting neuroprotection, HTER{alpha} HE27 cells were treated with glutamate in the presence and absence of 17ß-estradiol. Figure 6A shows that HTER{alpha} HE27 cells are similarly sensitive to glutamate exposure in which greater than 50% of cells were killed after a 24-h treatment. Following a 15-min pretreatment with 10 nM 17ß-estradiol, significant neuroprotection was observed. The magnitude of this neuroprotection was in between that of wild-type ER{alpha} and untransfected HT22 cells. Cells expressing the mutated form of ER{alpha} were only partially protected from glutamate toxicity, compared with the full neuroprotection observed in cells expressing a nonmutated form of the receptor. Figure 6B is a representative experiment depicting the abundance of living cells treated with glutamate after a 15-min pretreatment with 17ß-estradiol or vehicle.*y, 百拇医药
fig.ommitteed
Figure 6. Ffiteen-minute pretreatment with low-dose 17ß-estradiol partially protects HTER{alpha} HE27 cells from glutamate toxicity. HTER{alpha} HE27 and HTER{alpha} cells were treated with 17ß-estradiol (10 nM) for 15 min, followed by glutamate (5 mM) for 24 h. Cellular viability was assessed as described in Fig. 2. *, Significantly different from vehicle-treated controls (P 0.05). **, Significantly different from cells treated with vehicle or glutamate (P 0.05).k, 百拇医药
Discussionk, 百拇医药
In this study, the contribution of ER{alpha} and ERß in mediating neuroprotection against glutamate toxicity was examined. In addition, the contribution of estrogen rapid signaling vs. ERE-mediated transcription in the neuroprotection pathway was determined. Cells were pretreated with 17ß-estradiol followed by glutamate for 24 h. Physiological concentrations of 17ß-estradiol (10 nM) were protective against glutamate in HTER{alpha} and HTERß cells but not in HT22 cells. The 10-nM dose of estrogen is identical with that required to elicit protection from glutamate in primary cortical neurons in culture (13). This suggests that under the culture conditions used, ER expression is required for low concentrations of estrogen to be neuroprotective against glutamate toxicity. A supraphysiological concentration of 17ß-estradiol (1 µM) was neuroprotective in HT22, HTER{alpha} , and HTERß cells, an effect previously attributed to antioxidant properties of the molecule (23). Neuroprotection produced by low-dose 17ß-estradiol was blocked by ICI 182,780, further demonstrating that ER{alpha} or ERß is required for 17ß-estradiol to elicit neuroprotection at physiological concentrations. Activation of the MAPK pathway was found to be necessary for estrogen to mediate neuroprotection because blockade of this pathway with the mitogen/extracellular-signal regulated kinase kinase-1 (MEK-1) inhibitor, PD98059, prevented estrogen from being neuroprotective. To confirm that estrogen rapid signaling is important for neuroprotection, a mutated form of the ER{alpha} , HE27, was stably transfected into cells for comparison (HTER{alpha} HE27). Estrogen still activated MAPK in HTER{alpha} HE27 cells, although ERE-mediated transcription was not observed, suggesting that the zinc-finger of the ER is not required for cultivating rapid effects. In the HTER{alpha} HE27 cell line, estrogen still had a marked effect on cell survival following glutamate toxicity. These studies indicated that estrogen-mediated neuroprotection against glutamate toxicity requires the presence of an ER and activation of the MAPK pathway.
Glutamate is thought to be a major cause of neuronal cell death in a number of different neurodegenerative diseases (30, 31, 32). Two pathways for glutamate toxicity have been described; excitotoxicity (33), which occurs through the activation of glutamatergic receptors (34, 35), and oxidative glutamate toxicity, which is mediated via a series of disturbances to the redox homeostasis of the cell (36). These pathways are incompletely characterized, but both result in the production of free radicals (31, 36). Our results are in accordance with others (23), showing that HT22 cells die via the oxidative pathway from glutamate toxicity, as glutamatergic receptor antagonists fail to block glutamate induced toxicity. It has been reported that exposure of HT22 cells to glutamate results in an inhibition of cystine transport into the cell (36), leading to an inability to maintain intracellular glutathione levels. The low level of intracellular glutathione reduces the cell’s ability to counteract oxidative reactions within the cell and, ultimately, cell death. Our results support this notion because a high dose of cystine is able to protect the cells from glutamate toxicity.
There is significant debate over whether estrogen’s neuroprotective actions are mediated by one of the known ERs or nonspecific antioxidant mechanisms. The theory that at supraphysiological doses estrogen acts through antioxidant mechanism to prevent cell death has been clearly established. In doses greater than 10 µM 17ß-estradiol can reduce phospholipid oxidation in a cell-free system (37). More recently it has been shown that micromolar doses of estrogen are able to protect estrogen receptor-negative neuronal cell lines form cytotoxicity (12, 38). There is, however, also a body of evidence showing that exposure to lower, more physiological doses of estrogen is possible to achieve neuroprotection in cells containing ERs. For example, Gollapudi and Oblinger (39) found that estradiol exposure attenuates serum deprivation toxicity in PC12 cells transfected with ER{alpha} but not those transfected with a control plasmid. In addition, in an in vivo model of ischemia, ER{alpha} was found to be the critical link in mediating the protective effects of physiological concentrations of estradiol in brain injury (40). Furthermore, we have previously reported that a short pretreatment with 17ß-estradiol protects cells from ß-amyloid toxicity, and this protection is dependent on ER expression (11).
The role of ERß in mediating neuroprotection is more controversial. For example, in studies by Dubal et al. (40), ERß was not sufficient for protection against ischemia in an in vivo model. Kim et al. (41) did not observe neuroprotection against ß-amyloid peptide toxicity in HT22 cells stably transfected with ERß. However, we observed pronounced neuroprotection in HTERß cells. In support of our findings, studies by Sawada and Shimohama (42) demonstrated estrogen to be neuroprotective in mesencephalon dopaminergic neurons, which exclusively express ERß. In addition, Wang et al. (43) demonstrated the importance of ERß in the survival of neurons throughout the brain by measuring a neuronal deficit in ERß knockout mice. In their studies, the neuronal loss was increased with age, suggesting a role for ERß in the prevention of neurodegenerative diseases.76ptwn:, http://www.100md.com
The time frame of MAPK activation in HTER{alpha} and HTERß cells (11) correlates to the pretreatment time required to see neuroprotection. Indeed, PD98059 blocked neuroprotection, suggesting that transient phosphorylation of MAPK results in estrogen’s ability to be neuroprotective. It is possible that estrogen is eventually activating transcription of genes important for neuroprotection. This has been shown to occur in SK-N-BE2C cells transfected with ER{alpha} . In these cells, the membrane-impermeable E2-BSA acted through nongenomic mechanisms at the cell membrane to facilitate the ability of estrogen to activate an ERE-luciferase reporter. The events were proven to act through the classical ER at the cell membrane because ICI 182,780 blocked the transcriptional effects (44). Alternatively, genes responding to activation of the MAPK pathway, independent of ERE activity, may also be induced. For example, BCL2 is induced by estrogen in primary neuron cultures, and it has been recently reported that cAMP response element activation in the BCL promoter may be critical for this effect (45). Therefore, it is possible that a 15-min pretreatment with estrogen is neuroprotective because of the subsequent transcription of multiple genes (i.e. a nonclassical pathway for estrogen activation of transcription).
The generation of a cell line expressing a mutated form of ER{alpha} allowed us to demonstrate more clearly that rapid signaling was indeed important for neuroprotection. This mutated receptor contains two point mutations in the zinc finger domain, rendering the receptor unable to bind the palindromic ERE on DNA to activate ERE-mediated transcription (29). We clearly showed that HTER{alpha} HE27 is incapable of inducing luciferase activity from an ERE, verifying the critical role of these cystine residues in interacting with this element. Although the receptor was unable to activate an ERE reporter gene, HTER{alpha} HE27 was fully capable of rapidly activating MAPK in a time frame similar to what was seen in cells expressing ER{alpha} or ERß (11). In addition, 10 nM 17ß-estradiol phosphorylated MAPK approximately 5-fold, which is the same fold activation observed in HT22 cells expressing the wild-type ER{alpha} (11). When examining the neuroprotective capabilities of HTER{alpha} HE27, significant neuroprotection was seen (approximately two thirds of neuroprotection observed in wild-type ER{alpha} cells). This further suggests that estrogen’s ability to activate the MAPK pathway is important for neuroprotection against glutamate toxicity. These results are consistent with our earlier reports, demonstrating that estrogen-mediated neuroprotection against glutamate toxicity required activation of the MAPK pathway in primary cortical neurons (21). The involvement of the MAPK pathway in the protection of neurons from excitotoxicity has been further confirmed by the ability of PD98059 to block estrogen’s effects on protection from N-methyl-D-aspartate and kainate induced toxicity in hippocampal slice cultures (46). In addition, estrogen activation of MAPK through the ERs provides neuroprotection in primary neuronal cultures against a variety of toxic insults including quinolinic acid toxicity (47) and glutamate excitotoxicity (21).
Although clear and significant neuroprotection was found in the HTER{alpha} HE27 cell line, estrogen was not able to block 100% of the cell death brought about by glutamate exposure. This suggests that multiple pathways may be involved in the complex mechanism of neuroprotection. It is possible that acutely, estrogen activates MAPK to establish one pathway of neuroprotection, although longer treatments with the hormone work through the classical ERE-mediated transcriptional pathway to up-regulate protective genes within the cell. Further studies need to be conducted to delineate more clearly how the different pathways work together to mediate the neuroprotection pathway.y33, 百拇医药
Our studies clearly show that ER{alpha} and ERß expression is necessary for estrogen-mediated neuroprotection against glutamate toxicity. In HTER{alpha} and HTERß cells, 17ß-estradiol provided neuroprotection following a 15-min pretreatment. Neuroprotection was blocked by ICI 182,780 and PD98059, demonstrating that estrogen activation of MAPK via the ER is necessary for 17ß-estradiol to be neuroprotective. In addition, in cells expressing the mutated form of ER{alpha} , HE27, moderate neuroprotection was still observed. These data suggest that either ER{alpha} or ERß can mediate estrogen neuroprotection in a neuronal cell model and that rapid activation of the MAPK pathway is indeed important in the mechanism of neuroprotection. Therefore, a further understanding of estrogen rapid signaling may be important to increase neuroprotection and prevent neurodegenerative diseases.
Received July 9, 2002.v.u[j|j, 百拇医药
Accepted for publication September 23, 2002.v.u[j|j, 百拇医药
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It is well documented that estrogen mediates responses by both genomic and nongenomic mechanisms, both of which are important for cell survival. Because direct evidence showing that the estrogen receptors (ERs) {alpha} and/or ß can activate rapid signaling that may mediate neuroprotection is lacking, the hippocampal-derived cell line, HT22, was stably transfected with ER{alpha} (HTER{alpha} ), ERß (HTERß), or a mutated form of ER{alpha} (HTER{alpha} HE27), which lacks the ability to mediate ER element-mediated transcription. Treatment of HT22, HTER{alpha} , HTERß, and HTER{alpha} HE27 cells with glutamate (5 mM) resulted in a significant decrease in cell viability. Pretreatment for 15 min with 10 nM 17ß-estradiol resulted in a 50% increase in the number of living cells in HTER{alpha} and HTERß cells but not in HT22 cells. The ER antagonist ICI 182,780 and the MEK inhibitor PD98059 prevented 17ß-estradiol-mediated protection. In HTER{alpha} HE27 cells, 17ß-estradiol rapidly phosphorylated ERK2 (within 15 min), in the absence of estrogen response element-mediated transcription. Treatment of HTER{alpha} HE27 cells with 10 nM 17ß-estradiol partially reversed the cell death produced by glutamate treatment. This study demonstrates that activation of either ER{alpha} or ERß can result in neuroprotection and that activation of the MAPK pathway is an important part of the neuroprotective mechanism.
Introductionp{n@?, 百拇医药
THE GONADAL SEX steroid hormone estradiol serves many important functions. In addition to its classical reproductive role, numerous studies demonstrate that it plays an important trophic and protective role in the brain. Epidemiological evidence suggests that estrogen replacement therapy for postmenopausal women is associated with an improvement of some measures of cognitive performance, protection against cognitive deterioration, and decreased incidence of Alzheimer’s disease (1, 2, 3, 4, 5). In addition, beneficial effects of estrogen on the mortality and morbidity associated with cerebral stroke have also been demonstrated (6, 7, 8).p{n@?, 百拇医药
At the cellular level, estrogen is known to exert neuroprotective effects in various model systems. In vitro studies have shown that 17ß-estradiol reduces neuronal damage caused by serum deprivation (9, 10), ß-amyloid treatment (11, 12), and exposure to glutamate (12, 13). Although activation of estrogen receptors has been suggested in mediating neuroprotection, the complex mechanisms by which estrogen protects neurons against injury are not completely understood.
Although estrogen is generally thought to mediate its effects by activation of transcription via nuclear receptors, increasing evidence suggest that estrogen may also cause rapid activation of signal transduction pathways. As examples, estrogen is known to produce mobilization of intracellular calcium (14) production of cAMP (15, 16), activation of Akt (17) as well as MAPK, ERK1, and ERK2 (11, 18, 19, 20). Several lines of evidence suggest estrogen neuroprotection may be mediated by rapid intracellular signaling events rather than estrogen response element (ERE)-mediated gene transcription. For example, activation of protein kinase A, protein kinase C, and MAPK have been linked to neuroprotection in various cellular model systems (19, 21, 22). In addition, recent reports from our laboratory have shown that estrogen receptor (ER) activation of MAPK is important to protect neuronal cells from ß-amyloid toxicity (11). However, it is not clear whether nuclear estrogen receptor activation of rapid signaling pathways is sufficient to elicit neuroprotection in the absence of ERE-mediated transcription.
Our study was conducted to examine whether expression of ER{alpha} and ERß are required for low doses of estrogen to elicit neuroprotection against glutamate toxicity in a neuronal cell line. In addition, we have examined the relative role of MAPK activation vs. classical ERE-mediated transcription in eliciting estrogen-mediated neuroprotection.{ovr, 百拇医药
Materials and Methods{ovr, 百拇医药
Chemicals{ovr, 百拇医药
17ß-Estradiol, glutamate, and PD98059 were purchased from Sigma (St. Louis, MO). ICI 182,780 was purchased from Tocris Cookson (Ballwin, MO).{ovr, 百拇医药
Cell culture{ovr, 百拇医药
HT22 cells were given as a kind gift from Dr. Pamela Maher (The Scripps Research Institute, La Jolla, CA). These cells were grown on 100-mm tissue culture dishes and maintained in DMEM (Sigma) media supplemented with 5% fetal bovine serum and 1% Pen-Strep (Gem Cell, Woodland, CA) at 37 C in a 5% CO2 atmosphere. Cell density was maintained 70% or less confluency as described previously (23).
Plasmids and oligonucleotide mutagenesisq, 百拇医药
The zinc finger mutation (C202H;C205H) corresponding to HE27, described previously (23), was introduced into ER{alpha} HEGO/pcDNA3.1 hygromycin by a sequence overlap extension procedure. Overlapping PCR fragments were first generated in separate reactions and then reannealed and extended in a second PCR. In the first step, two oligonucleotides (complement and reverse complement) containing the desired nucleotides were synthesized for each mutation. Two fragments were then synthesized by PCR using a T7 primer and the reverse complement (5'-GAATACTTCTCTTGAAGAAGGCCTTGTGGCCCTCATGACACCAGACTCCATAATGG-3') to generate the 5' fragment and with the complement (5'-CCATTATGGAGTCTGGTCTCATGAGGGCCACAAGGCCTTCTTCAAGAGA-AGTATTC3') and a 3' oligo (5'TGTACACTCCAGAATTAAGC-3') to generate the 3' fragments. Altered nucleotides are indicated in bold type. A novel BspH1 site is underlined. After gel purification, 10 ng of each fragment were mixed and used as a template in a second PCR with the two outer oligonucleotides. The resulting 1400-bp fragment was digested with HindIII and a 1020-bp fragment used to replace the corresponding fragment in ER{alpha} HEGO/pcDNA3.1 hygromycin. All nucleotide changes were confirmed by DNA sequencing.
Generation of HT22 stable transfectants*i!}l*, 百拇医药
Stable transfectants were generated as described previously (11). Briefly, HT22 cells were grown to approximately 60% confluency before being transfected with the lipid transfection reagent, TransFast (Promega Corp., Madison, WI). The pCDNA3.1 hygromycin (7.3 µg) containing either the full-length human ER{alpha} cDNA (24), a gift from Dr. Pierre Chambon (Strasbourg, France), or rat ERß cDNA (25), a gift from George Kuiper (Karolinska Institute, Sweden), was added at a 1:1 ratio with Transfast per 100-mm plate. Medium was changed 24 h later, and hygromycin (125 µg/ml) was added 72 h after transfection for selection of ER-expressing clones. Single colonies were isolated after the 10th day of growth in selective conditioned media and tested for receptor expression by immunoblotting.*i!}l*, 百拇医药
Activation of ERK2*i!}l*, 百拇医药
ER-positive cells were grown to 70–80% confluency on 100-mm plates. Eighteen hours before treatment, the medium was replaced with phenol red-free DMEM supplemented with 1% charcoal-stripped serum. Cells were treated with ethanol vehicle (0.1% final concentration) or 17ß-estradiol (10 nM) for the indicated times. The medium was removed, and the cells were washed in ice-cold PBS. Cells were rinsed with ice-cold PBS buffer, scraped into immunoprecipitation buffer (1 M HEPES, 0.1 M EGTA, 0.5 M EDTA, 0.5 M Na+ pyrophosphate, 1 M NaF, 1 mM NaVO4, and 9 mM NaCl) and incubated on ice for at least 5 min. The samples were then sonicated for 2 min followed by centrifugation at 15,000 rpm for 10 min. The supernatant was transferred to a new tube and protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce Chemical Co., Rockford, IL). The proteins samples were diluted in Laemmeli sodium dodecyl sulfate sample buffer and equal volumes of cell lysate were loaded and resolved by electrophoresis on 4–12% Bis-Tris precast gels (Invitrogen, Carlsbad, CA) in running buffer [50 mM 2-(N-morpholine)ethane sulfonic acid, 50 mM Tris base, 0.1% sodium dodecyl sulfate, and 1 mM EDTA] as described by the manufacturer. The proteins were transferred to polyvinylidene diflouride membranes and blocked in 5% nonfat dry milk in Tris-buffered saline containing 0.2% Tween 20 for 1 h at room temperature. ERK2 phosphorylation was detected using mouse antiphospho-p44/42 MAPK (1:2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) that recognizes phospho-THR202 and THR204 forms of ERK1 and 2. Phosphorylation at these sites has been correlated with increased activity (26, 27, 28). Total ERK2 was detected using rabbit anti-ERK2 (1:10,000, Santa Cruz Biotechnology, Inc.).
Secondary goat antimouse antibodies (1:5000, Santa Cruz Biotechnology, Inc.) or goat antirabbit antibodies (1:5000) conjugated to horseradish peroxidase were used for detection by enhanced chemiluminescence (NEN Life Science Products, Boston, MA) on film. The resulting film samples were scanned and analyzed with an image analysis program (NIH Image, Scion Corp., Frederick, MD). Data are presented as a ratio of phospho-ERK2/total ERK2 in the sample, normalized to vehicle-treated samples.a, http://www.100md.com
Transient transfectionsa, http://www.100md.com
HT22 cells (~ 5 x 105 cells/well) were plated into 24-well plates and transfected with 1.0 µg/well tk-ERE-luciferase (kindly provided by Dr. Peter Burbach, Rudolf Magnus Institute, University of Utrecht) using the TransFast protocol. One microgram pCH110, a ß-galactosidase reporter (Amersham Pharmacia, Uppsala, Sweden) was added to each well to normalize for transfection efficiency. After 24 h in DMEM, cells were transfected at 37 C in Transfast reagent in media supplemented with 10% charcoal-stripped calf serum for 1 h. Media was then replaced, and cells were allowed to recover for 24 h before pharmacological manipulation.
Cell treatments[\, 百拇医药
Cells were grown to 70–80% confluency in 12-well plates. Twenty-four hours before treatment the medium was replaced with phenol red free DMEM supplemented with charcoal-stripped fetal bovine serum (1%). Glutamate was diluted to a final concentration of 5 mM in culture medium and cells were exposed for 24 h. 17ß-Estradiol was initially dissolved in 95% ethanol at a concentration of 1 mM and diluted to the appropriate concentration (10 nM) in culture medium. Exposure to 17ß-estradiol was initiated 15 min before glutamate addition. Ethanol was used at a final concentration of 0.1% as a vehicle control. This concentration of ethanol had no effect on cell viability or glutamate toxicity. ICI 182,780 and PD98059 were made as 1000x stocks in 100% dimethylsulfoxide and were added to cells 15 min before 17ß-estradiol exposure.[\, 百拇医药
Dimethylthiazoldiphenyltetra-zoliumbromide (MTT) assay[\, 百拇医药
The MTT assay measures cellular viability by assessing mitochrondrial activity in the cells. After completion of incubation, 200 µl of a 5 mg/ml MTT stock in PBS was added to each well, and the incubation continued for 4 h. After removal of the MTT solution, 1 ml solubilization solution containing 50% dimethylformamide, 20% sodium dodecyl sulfate (pH 4.8) was added. Absorption values at 570 nM were read on a Packard Spectra spectrophotometer.
Calcein acetomethyl ester (AM)+5ae3x, 百拇医药
Following incubation of cells in glutamate, cells were rinsed once with PBS and incubated with 1 µM calcein AM dye (Molecular Probes, Inc., Eugene, OR) at 37 C for 15 min, washed twice with PBS, and coverslips applied. Green fluorescent cells are the product of mitochondrial cleavage of calcein AM and were observed using a Optiphot 2 microscope (Nikon) with the EF-D fluorescence attachment and G-1B and DM510 filters and counted as living cells.+5ae3x, 百拇医药
Statistical analysis+5ae3x, 百拇医药
The significance of differences among groups was determined by one-way ANOVA followed by a Tukey’s multiple comparison test. P < 0.05 was considered significant, and each group consisted of 6–12 wells or plates. All values are expressed as mean ± SEM.+5ae3x, 百拇医药
Results+5ae3x, 百拇医药
Figure 1 demonstrates that HT22, HTER{alpha} , and HTERß cells are sensitive to glutamate exposure, in which greater than 50% of cells were killed after a 24-h treatment. To further characterize the steps leading to cell death, various compounds were tested to determine their ability to block glutamate-induced cell death. As suggested by previous reports, both N-methyl-D-aspartate and kainate glutamate receptor antagonists failed to prevent glutamate toxicity in HTER{alpha} and HTERß cells (Table 1). However, cystine was able to completely reverse the cellular death.
fig.ommitteed25t, http://www.100md.com
Figure 1. Glutamate significantly decreases cell survival in HT22, HTER, and HTERß cells. HT22, HTER, and HTERß cells were treated with glutamate (5 mM) for 24 h. Cellular viability was assessed using the MTT assay. Data are expressed as the percentage of the maximal number of living cells in a vehicle-treated control. All results represent the mean ± SEM from three to four separate platings. *, Statistically different from vehicle-treated cells, P < 0.05.25t, http://www.100md.com
fig.ommitteed25t, http://www.100md.com
Table 1. Toxicity of glutamate and its analogs25t, http://www.100md.com
HT22, HTER{alpha} , and HTERß cells were treated with either 1 µM or 10 nM 17ß-estradiol for 15 min before glutamate exposure to determine whether a short pretreatment with the hormone would elicit neuroprotection. In HTER{alpha} and HTERß cells, a 15-min pretreatment with a near physiological dose of 17ß-estradiol (10 nM) significantly increased cellular viability as evidenced by calcein AM staining (Fig. 2, A and B). By contrast, at this dose of estrogen HT22 cells were not protected from glutamate toxicity. A high dose of estrogen (1 µM) protected all three cell lines from glutamate toxicity, irrespective of ER expression. Figure 2B is a representative experiment depicting the abundance of living cells treated with glutamate after a 15-min pretreatment with 17ß-estradiol or vehicle.
fig.ommitteedz]f, http://www.100md.com
Figure 2. Fifteen-minute pretreatment with low-dose 17ß-estradiol protects HTER{alpha} and HTERß cells from glutamate toxicity. HT22, HTER{alpha} , and HTERß cells were treated with 17ß-estradiol (1 µM or 10 nM) for 15 min, followed by glutamate (5 mM) for 24 h. Cellular viability was assessed by calcein AM staining. The number of living cells was assessed by fluorescence with calcein AM. A, Numbers of living cells are expressed as percent of vehicle-treated controls. All results represent the mean ± SEM from three to four separate platings. *, Statistically different from glutamate-treated cells, P < 0.05. B, Representative micrograph showing the presence of living cells, compared with vehicle-treated controls.z]f, http://www.100md.com
To confirm that the neuroprotective effect of 17ß-estradiol was dependent on ERs, cells were pretreated with ICI 182,780. Although ICI 182,780 (1 µM) had no cytotoxic or neuroprotective effects on its own (data not shown), it blocked the ability of 17ß-estradiol to increase living cell number (Fig. 3, A and B), demonstrating that the protective effects of low-dose estrogen occurred in an ER-dependent manner. Figure 3B is a representative experiment depicting the abundance of living cells treated with glutamate in the presence and absence of 17ß-estradiol and the reduction of living cells observed after glutamate treatment in the presence of estrogen and ICI 182,780.
fig.ommitteed^%, http://www.100md.com
Figure 3. ICI 182,780 blocks 17ß-estradiol-mediated neuroprotection against glutamate-induced toxicity in HTER{alpha} and HTERß cells. HTER{alpha} and HTERß cells were treated with 17ß-estradiol (10 nM) for 15 min in the presence and absence of ICI 182,780 (1 µM), followed by glutamate (5 mM) for 24 h. Cellular viability was assessed as described and presented in Fig. 2.^%, http://www.100md.com
To determine whether 17ß-estradiol-mediated neuroprotection was dependent on activation of MAPK, cellular cytotoxicity was determined in the presence and absence of PD98059. Although PD98059 (50 µM) had no effect on cytotoxicity alone, it abolished the ability of 17ß-estradiol to increase living cell number (Fig. 4, A and B). This datum suggests that the protective effects of estrogen are occurring through a MAPK-dependent pathway. Figure 4B is a representative experiment depicting the abundance of living cells treated with glutamate in the presence and absence of 17ß-estradiol and the reduction of living cells observed after glutamate treatment in the presence of estrogen and PD98059.
fig.ommitteed'], 百拇医药
Figure 4. PD98059 blocks 17ß-estradiol-mediated neuroprotection against glutamate induced toxicity in HTER{alpha} and HTERß cells. HTER{alpha} and HTERß cells were treated with 17ß-estradiol (10 nM) for 15 min in the presence and absence of PD98059 (50 µM), followed by glutamate (5 mM) for 24 h. Cellular viability was assessed as described in Fig. 2.'], 百拇医药
To evaluate the relative contribution of rapid signaling vs. genomic mechanisms in mediating neuroprotection, an ER{alpha} mutated to conform with that reported by Kumar et al. (29) was stably expressed into HT22 cells (HE27: C202H;C205H;HTER{alpha} HE27). First, it was confirmed that in HTER{alpha} HE27 cells, estrogen was still able to mediate rapid activation of the MAPK pathway. Figure 5A shows that ERK2 phosphorylation is increased within 15 min and returns to basal levels by 30 min in cells expressing the mutated ER{alpha} . Under these conditions, no ERK2 phosphorylation was observed in untransfected HT22 cells (11) (data not shown). To ensure that the mutated ER cell line was unable to elicit ERE-mediated transcription, HT22 cells were transfected with either full-length ER{alpha} or HE27 and an ERE-luciferase reporter construct and the transcriptional activity was measured (Fig. 5B). In cells transfected with ER{alpha} , addition of 17ß-estradiol resulted in a 2.5-fold increase in luciferase activity. No activity was observed in untransfected cells or cells transfected with HTER{alpha} HE27.
fig.ommitteed5r, 百拇医药
Figure 5. 17ß-Estradiol treatment increases ERK2 phosphorylation in HTER{alpha} HE27 cells but does not activate an ERE reporter gene construct. A, Lysates from HTER{alpha} HE27 cells treated with 10 nM 17ß-estradiol for 5, 15, or 30 min were analyzed by immunoblotting for changes in phosphorylation of ERK2. Lysates were probed with an antiphospho-specific p42/44 MAPK (ERK1/ERK2) antibody. Total ERK2 was detected using an anti-ERK2 antibody that recognizes both phosphorylated and unphosphorylated ERK2. Relative amounts of phosphorylated ERK2 were determined from densitometric scanning of ECL-exposed film. Data are represented as fold increase in relative phosphorylated ERK2, compared with vehicle treatment ± SEM. All experiments were performed three times in triplicate, and asterisks (*) indicate a P value of 0.05 or less, as determine by one-way ANOVA. B, Transiently transfected HTER{alpha} HE27 cells were treated with vehicle or 50 nM 17ß-estradiol for 24 h and assayed for luciferase and ß-galactosidase activity. Data are represented as the ratio of luciferase/ß-galactosidase activity. All experiments were performed three times in triplicate, and asterisks (*) indicate a P value of 0.05 or less, as determined by one-way ANOVA.
To determine the contribution of MAPK activation vs. ERE-mediated transcription in eliciting neuroprotection, HTER{alpha} HE27 cells were treated with glutamate in the presence and absence of 17ß-estradiol. Figure 6A shows that HTER{alpha} HE27 cells are similarly sensitive to glutamate exposure in which greater than 50% of cells were killed after a 24-h treatment. Following a 15-min pretreatment with 10 nM 17ß-estradiol, significant neuroprotection was observed. The magnitude of this neuroprotection was in between that of wild-type ER{alpha} and untransfected HT22 cells. Cells expressing the mutated form of ER{alpha} were only partially protected from glutamate toxicity, compared with the full neuroprotection observed in cells expressing a nonmutated form of the receptor. Figure 6B is a representative experiment depicting the abundance of living cells treated with glutamate after a 15-min pretreatment with 17ß-estradiol or vehicle.*y, 百拇医药
fig.ommitteed
Figure 6. Ffiteen-minute pretreatment with low-dose 17ß-estradiol partially protects HTER{alpha} HE27 cells from glutamate toxicity. HTER{alpha} HE27 and HTER{alpha} cells were treated with 17ß-estradiol (10 nM) for 15 min, followed by glutamate (5 mM) for 24 h. Cellular viability was assessed as described in Fig. 2. *, Significantly different from vehicle-treated controls (P 0.05). **, Significantly different from cells treated with vehicle or glutamate (P 0.05).k, 百拇医药
Discussionk, 百拇医药
In this study, the contribution of ER{alpha} and ERß in mediating neuroprotection against glutamate toxicity was examined. In addition, the contribution of estrogen rapid signaling vs. ERE-mediated transcription in the neuroprotection pathway was determined. Cells were pretreated with 17ß-estradiol followed by glutamate for 24 h. Physiological concentrations of 17ß-estradiol (10 nM) were protective against glutamate in HTER{alpha} and HTERß cells but not in HT22 cells. The 10-nM dose of estrogen is identical with that required to elicit protection from glutamate in primary cortical neurons in culture (13). This suggests that under the culture conditions used, ER expression is required for low concentrations of estrogen to be neuroprotective against glutamate toxicity. A supraphysiological concentration of 17ß-estradiol (1 µM) was neuroprotective in HT22, HTER{alpha} , and HTERß cells, an effect previously attributed to antioxidant properties of the molecule (23). Neuroprotection produced by low-dose 17ß-estradiol was blocked by ICI 182,780, further demonstrating that ER{alpha} or ERß is required for 17ß-estradiol to elicit neuroprotection at physiological concentrations. Activation of the MAPK pathway was found to be necessary for estrogen to mediate neuroprotection because blockade of this pathway with the mitogen/extracellular-signal regulated kinase kinase-1 (MEK-1) inhibitor, PD98059, prevented estrogen from being neuroprotective. To confirm that estrogen rapid signaling is important for neuroprotection, a mutated form of the ER{alpha} , HE27, was stably transfected into cells for comparison (HTER{alpha} HE27). Estrogen still activated MAPK in HTER{alpha} HE27 cells, although ERE-mediated transcription was not observed, suggesting that the zinc-finger of the ER is not required for cultivating rapid effects. In the HTER{alpha} HE27 cell line, estrogen still had a marked effect on cell survival following glutamate toxicity. These studies indicated that estrogen-mediated neuroprotection against glutamate toxicity requires the presence of an ER and activation of the MAPK pathway.
Glutamate is thought to be a major cause of neuronal cell death in a number of different neurodegenerative diseases (30, 31, 32). Two pathways for glutamate toxicity have been described; excitotoxicity (33), which occurs through the activation of glutamatergic receptors (34, 35), and oxidative glutamate toxicity, which is mediated via a series of disturbances to the redox homeostasis of the cell (36). These pathways are incompletely characterized, but both result in the production of free radicals (31, 36). Our results are in accordance with others (23), showing that HT22 cells die via the oxidative pathway from glutamate toxicity, as glutamatergic receptor antagonists fail to block glutamate induced toxicity. It has been reported that exposure of HT22 cells to glutamate results in an inhibition of cystine transport into the cell (36), leading to an inability to maintain intracellular glutathione levels. The low level of intracellular glutathione reduces the cell’s ability to counteract oxidative reactions within the cell and, ultimately, cell death. Our results support this notion because a high dose of cystine is able to protect the cells from glutamate toxicity.
There is significant debate over whether estrogen’s neuroprotective actions are mediated by one of the known ERs or nonspecific antioxidant mechanisms. The theory that at supraphysiological doses estrogen acts through antioxidant mechanism to prevent cell death has been clearly established. In doses greater than 10 µM 17ß-estradiol can reduce phospholipid oxidation in a cell-free system (37). More recently it has been shown that micromolar doses of estrogen are able to protect estrogen receptor-negative neuronal cell lines form cytotoxicity (12, 38). There is, however, also a body of evidence showing that exposure to lower, more physiological doses of estrogen is possible to achieve neuroprotection in cells containing ERs. For example, Gollapudi and Oblinger (39) found that estradiol exposure attenuates serum deprivation toxicity in PC12 cells transfected with ER{alpha} but not those transfected with a control plasmid. In addition, in an in vivo model of ischemia, ER{alpha} was found to be the critical link in mediating the protective effects of physiological concentrations of estradiol in brain injury (40). Furthermore, we have previously reported that a short pretreatment with 17ß-estradiol protects cells from ß-amyloid toxicity, and this protection is dependent on ER expression (11).
The role of ERß in mediating neuroprotection is more controversial. For example, in studies by Dubal et al. (40), ERß was not sufficient for protection against ischemia in an in vivo model. Kim et al. (41) did not observe neuroprotection against ß-amyloid peptide toxicity in HT22 cells stably transfected with ERß. However, we observed pronounced neuroprotection in HTERß cells. In support of our findings, studies by Sawada and Shimohama (42) demonstrated estrogen to be neuroprotective in mesencephalon dopaminergic neurons, which exclusively express ERß. In addition, Wang et al. (43) demonstrated the importance of ERß in the survival of neurons throughout the brain by measuring a neuronal deficit in ERß knockout mice. In their studies, the neuronal loss was increased with age, suggesting a role for ERß in the prevention of neurodegenerative diseases.76ptwn:, http://www.100md.com
The time frame of MAPK activation in HTER{alpha} and HTERß cells (11) correlates to the pretreatment time required to see neuroprotection. Indeed, PD98059 blocked neuroprotection, suggesting that transient phosphorylation of MAPK results in estrogen’s ability to be neuroprotective. It is possible that estrogen is eventually activating transcription of genes important for neuroprotection. This has been shown to occur in SK-N-BE2C cells transfected with ER{alpha} . In these cells, the membrane-impermeable E2-BSA acted through nongenomic mechanisms at the cell membrane to facilitate the ability of estrogen to activate an ERE-luciferase reporter. The events were proven to act through the classical ER at the cell membrane because ICI 182,780 blocked the transcriptional effects (44). Alternatively, genes responding to activation of the MAPK pathway, independent of ERE activity, may also be induced. For example, BCL2 is induced by estrogen in primary neuron cultures, and it has been recently reported that cAMP response element activation in the BCL promoter may be critical for this effect (45). Therefore, it is possible that a 15-min pretreatment with estrogen is neuroprotective because of the subsequent transcription of multiple genes (i.e. a nonclassical pathway for estrogen activation of transcription).
The generation of a cell line expressing a mutated form of ER{alpha} allowed us to demonstrate more clearly that rapid signaling was indeed important for neuroprotection. This mutated receptor contains two point mutations in the zinc finger domain, rendering the receptor unable to bind the palindromic ERE on DNA to activate ERE-mediated transcription (29). We clearly showed that HTER{alpha} HE27 is incapable of inducing luciferase activity from an ERE, verifying the critical role of these cystine residues in interacting with this element. Although the receptor was unable to activate an ERE reporter gene, HTER{alpha} HE27 was fully capable of rapidly activating MAPK in a time frame similar to what was seen in cells expressing ER{alpha} or ERß (11). In addition, 10 nM 17ß-estradiol phosphorylated MAPK approximately 5-fold, which is the same fold activation observed in HT22 cells expressing the wild-type ER{alpha} (11). When examining the neuroprotective capabilities of HTER{alpha} HE27, significant neuroprotection was seen (approximately two thirds of neuroprotection observed in wild-type ER{alpha} cells). This further suggests that estrogen’s ability to activate the MAPK pathway is important for neuroprotection against glutamate toxicity. These results are consistent with our earlier reports, demonstrating that estrogen-mediated neuroprotection against glutamate toxicity required activation of the MAPK pathway in primary cortical neurons (21). The involvement of the MAPK pathway in the protection of neurons from excitotoxicity has been further confirmed by the ability of PD98059 to block estrogen’s effects on protection from N-methyl-D-aspartate and kainate induced toxicity in hippocampal slice cultures (46). In addition, estrogen activation of MAPK through the ERs provides neuroprotection in primary neuronal cultures against a variety of toxic insults including quinolinic acid toxicity (47) and glutamate excitotoxicity (21).
Although clear and significant neuroprotection was found in the HTER{alpha} HE27 cell line, estrogen was not able to block 100% of the cell death brought about by glutamate exposure. This suggests that multiple pathways may be involved in the complex mechanism of neuroprotection. It is possible that acutely, estrogen activates MAPK to establish one pathway of neuroprotection, although longer treatments with the hormone work through the classical ERE-mediated transcriptional pathway to up-regulate protective genes within the cell. Further studies need to be conducted to delineate more clearly how the different pathways work together to mediate the neuroprotection pathway.y33, 百拇医药
Our studies clearly show that ER{alpha} and ERß expression is necessary for estrogen-mediated neuroprotection against glutamate toxicity. In HTER{alpha} and HTERß cells, 17ß-estradiol provided neuroprotection following a 15-min pretreatment. Neuroprotection was blocked by ICI 182,780 and PD98059, demonstrating that estrogen activation of MAPK via the ER is necessary for 17ß-estradiol to be neuroprotective. In addition, in cells expressing the mutated form of ER{alpha} , HE27, moderate neuroprotection was still observed. These data suggest that either ER{alpha} or ERß can mediate estrogen neuroprotection in a neuronal cell model and that rapid activation of the MAPK pathway is indeed important in the mechanism of neuroprotection. Therefore, a further understanding of estrogen rapid signaling may be important to increase neuroprotection and prevent neurodegenerative diseases.
Received July 9, 2002.v.u[j|j, 百拇医药
Accepted for publication September 23, 2002.v.u[j|j, 百拇医药
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