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Endocrinology Vol. 146, No. 12 5397-5406
Copyright © 2005 by The Endocrine Society

Rapid Estrogenic Regulation of Extracellular Signal- Regulated Kinase 1/2 Signaling in Cerebellar Granule Cells Involves a G Protein- and Protein Kinase A-Dependent Mechanism and Intracellular Activation of Protein Phosphatase 2A

Scott M. Belcher, Hoa H. Le, Lynda Spurling and Jeremy K. Wong

Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: Scott M. Belcher, Ph.D., Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert Sabin Way, P.O. Box 670575, Cincinnati, Ohio 45267-0575. E-mail: scott.belcher{at}uc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In neonatal rat cerebellar neurons, 17ß-estradiol (E2) rapidly stimulates ERK1/2 phosphorylation through a membrane-associated receptor. Here the mechanism of rapid E2-induced ERK1/2 signaling in primary cultured granule cells was investigated in more detail. The results of these studies show that E2 and ICI182,780, a steroidal antagonist of estrogen receptor transactivation, rapidly increased ERK signaling with a time course similar to the transient activation induced by epidermal growth factor (EGF). However, EGF receptor (EGFR) autophosphorylation was not increased by E2, and blockade of EGFR tyrosine kinase activity did not abrogate the rapid actions of E2. The involvement of Src-tyrosine kinase activity was demonstrated by detection of increased c-Src phosphorylation in response to E2 and by blockade of E2-induced ERK1/2 activation by inhibition of Src-family tyrosine kinase activity. Inhibition of G{alpha}i signaling or protein kinase A (PKA) activity blocked the ability of ICI182,780 to rapidly stimulate ERK signaling. Under those conditions, E2 treatment induced a rapid and transient suppression of basal ERK1/2 phosphorylation. Protein phosphatase 2A (PP2A) activity was rapidly increased by E2 but not by E2 covalently linked to BSA. Rapid E2-induced increases in PP2A activity were insensitive to pertussis toxin. The presented evidence indicates that the rapid effects of estrogens on ERK signaling in cerebellar granule cells are induced through a novel G protein-coupled receptor mechanism that requires PKA and Src-kinase activity to link E2 to the ERK/MAPK signaling module. Along with stimulating ERK signaling, E2 rapidly activates PP2A via an independent signaling mechanism that may serve as a cell-specific regulator of signal duration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS MEDIATE MANY of their effects through binding at cognate ligand-activated receptors. These estrogen receptors (ERs) are members of the steroid/thyroid superfamily of nuclear receptors and act to regulate the expression of estrogen-responsive genes (1). However, the molecular mechanisms, through which the diverse and cell-specific physiological effects of the major endogenous estrogen 17ß-estradiol (E2) are regulated, are not fully understood. In addition to regulating estrogen-responsive gene expression, E2 rapidly affects a variety of signal transduction pathways. These signaling effects occur within minutes, are not blocked by transcriptional and translational inhibitors, and can be activated in many different types of cells by membrane-impermeable protein-conjugated ligands (2). Those properties suggest that rapid estrogen-induced signaling operates through mechanisms distinct from those mediated by the intracellular ER’s association with coregulators to influence expression of E2-responsive genes.

Accumulating evidence suggests that the details of rapid E2-induced signaling may vary greatly from cell type to cell type and may even vary during ontogeny in the same cell. Of the several signaling pathways that can be activated by E2, the ERK/MAPK signaling pathway is commonly responsive to estrogens in many types of cells. Rapid E2-induced ERK activation has been described in various tumor cell lines (3, 4, 5), vascular endothelium (6, 7), osteocytes (8, 9), and neurons (10, 11, 12). The ability of membrane-impermeable E2 to rapidly activate ERK signaling in these cell types indicates that rapid estrogenic actions are initiated at the plasma membrane (13). In some cells, including neurons, a small fraction of the classical ERs (ER{alpha} or ERß) is associated with the plasma membrane (14, 15, 16). The membrane-associated ERs may influence ERK signaling through direct association with nonreceptor tyrosine kinases (4) or by interacting with the adaptor proteins Shc (17) or MNAR (modulator of nongenomic activity of ER) (18), which link the activated receptor to the growth factor-associated signaling pathways. In some neurons and breast cancer cells, heterotrimeric G protein-coupled mechanisms are also involved in rapid estrogenic signaling (19, 20, 21, 22). The role of ERß in rapid ERK signaling is less well studied; however, results from recombinant cell models indicate that ERß can function to activate rapid ERK1/2 signaling with unique temporal and pharmacological properties (23).

Previous studies from our laboratory have demonstrated that ER expression in the rat cortex and cerebellum was regulated during critical periods of development (24, 25, 26). In cerebellar granule cell neurons, where only ERß is expressed at appreciable levels, low physiological concentrations of E2 and ICI182,780 (a steroidal ER antagonist of coactivator association) rapidly activate ERK1/2 signaling via a membrane-initiated mechanism that differentially regulates cell death and mitosis of immature granule cell precursors (12). In contrast to the sustained ERK activation induced by E2 in cultured cortical neurons, ERK signaling was transiently increased in cerebellar granule cells (10, 12, 27). The unique temporal nature of E2’s effect on ERK1/2 signaling in these different neuronal populations point to important differences in the mechanism regulating rapid E2-induced ERK signaling.

In this study, pharmacological approaches were used to investigate the mechanism of rapid ERK1/2 activation by E2 and ICI182,780 in primary cultures of cerebellar granule cell neurons. The presented results reveal that E2 and ICI182,780 activate ERK1/2 signaling through a G protein-dependent mechanism that is sensitive to pertussis toxin (PTX) and inhibitors of protein kinase A (PKA) or Src kinase activity. Unlike cell types where the membrane ER is coupled to G{alpha}s and activates ERK signaling by transactivation of epidermal growth factor receptor (EGFR), in the ERß-expressing granule cells, rapid activation of ERK signaling involves a PTX-sensitive G protein-coupled mechanism. Pharmacologically, this mechanism resembles the Gs/Gi switching mechanism initially described for ß-adrenergic receptor activation of ERK signaling (28). Additional evidence is presented that reveals an independent intracellular signaling pathway that results in protein phosphatase 2A (PP2A) activation and ERK dephosphorylation and is also rapidly responsive to E2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Timed pregnant Sprague-Dawley rats were obtained from the supplier (Charles River Laboratories, Wilmington, MA) at least 48 h before giving birth. The litter size was adjusted to a total of 10 pups on the day pups first appeared, designated postnatal d 0 (P0). When possible, each litter contained an equal number of male and female pups. On P7, the sex and weight of each animal was determined and recorded before granule cell preparation. At this age, the mean weight of the pups was 16.53 ± 1.23 g (mean ± SD; n = 39); any pups weighing significantly less than the average were not used for study. All animal procedures were done in accordance with protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee and followed National Institutes of Health guidelines.

Preparation of primary cultures of cerebellar neurons
Primary cerebellar cultures were prepared and maintained under serum- and steroid-free conditions as previously described (12, 29). Cerebella were isolated from P7–P9 male or female Sprague-Dawley rat pups. After rapid dissection, the cerebellum was immediately immersed in ice-cold culture medium, and meninges were gently removed. The cerebellum was transferred to 2–5 ml of fresh medium and chopped finely with a sterile scalpel blade. Cerebellar cells were then dissociated without enzymatic treatment by repeated trituration of the tissue. Dissociated cells were filtered through a 40-µm nylon cell strainer (Falcon, Franklin Lakes, NJ) to remove any remaining clumps of cells. The final volume of the resulting cell suspension was adjusted to 10 ml and the number of viable cells determined by counting trypan blue dye-excluding cells using a Neubauer hemacytometer. Based on the calculated cell numbers, cerebellar cells were serially diluted in an appropriate volume of culture medium and seeded at an initial density of 1.5 x 105 granule cells/cm2. Cultures were maintained in a humidified incubator in 5% CO2 at 37 C. After 24 h in culture, a final concentration of 10 µM cytosine ß-D-arabinofuranoside (Sigma Chemical Co., St. Louis, MO) was added to inhibit proliferation of nonneuronal cells. Treatments were carried out after 7 d in culture. Serum-free granule cell culture medium lacking phenol red was composed of 1x DMEM, 25 mM glucose, 0.5 mM L-glutamine, 26 mM NaHCO3, 0.23 mM sodium pyruvate (Invitrogen, Carlsbad, CA), 25 mM KCl, 10 mM HEPES (pH 7.2) (Sigma), 1.5 mg/ml BSA (Sigma), 5 µg/ml insulin, 5 µg/ml transferrin, 5 pg/ml selenium (BioWhittaker, Bedford, MA), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen).

Drug treatments
Cerebellar cultures were exposed for various times to a final concentration of 10–11, 10–10, or 10–8 M 1,3,5(10)-estratrien-3,17ß-diol (E2) (Sigma or Steraloids, Newport, RI; no difference in E2 actions between vendors was observed) or 10–8 M ICI182,780 (Tocris Cookson Inc., Ellisville, MO) that was serially diluted into dimethylsulfoxide/PBS vehicle (final dimethylsulfoxide concentration, 0.001%). Ether-extracted E2-hemisuccinate-BSA (E2-BSA) (molecular ratio of E2 to BSA was 35, and a final concentration of 10–10 M E2 was used; Steraloids) and BSA (fraction V) was reconstituted and diluted in PBS (pH 7.4) with potentially contaminating free estradiol removed by microfiltration (30-kDA cutoff) (Micron YM-30; Millipore, Bedford, MA). Positive controls for ERK1/2 activation included treatment with brain-derived neurotrophic factor (BDNF) (100 ng/ml; Promega, Madison, WI) or EGF (100 ng/ml; Invitrogen). For selective inhibitor experiments, cultures were pretreated for 30 min with the selective PKA antagonist H89 (10 µM; Calbiochem, San Diego, CA), the EGFR tyrosine kinase inhibitor tyrphostin AG 1478 (1 µM; Tocris Cookson), the Src-family tyrosine kinase inhibitor PP2 (10 µM; Tocris Cookson), or PTX (100 ng/ml; Sigma) for 16–18 h before treatment. In experiments using antagonists, the duration of antagonist-vehicle exposure was identical to the time cultures were exposed to antagonist. Thus, vehicle-treated negative controls were exposed to vehicle that was identical in composition and concentration to that received by the experimental cultures.

Cerebellar cell lysates and Western blot analysis
After treatment, media were aspirated and attached cells washed with ice-cold Hanks’ balanced salt solution. Cells were detached on ice with cold 2 mM EDTA in PBS (pH 7.4) and collected at 2 C by centrifugation at 1000 x g for 2 min. Pelleted cells were resuspended on ice in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml Pefabloc, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 µg/ml aprotinin, 1 mM EDTA, 1 mM EGTA, 0.05% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerol phosphate, and 1 mM Na3VO4. Cellular homogenates were lysed with three freeze/thaw cycles in liquid N2 and then cleared by centrifugation at 14,000 x g for 10 min at 2 C. Total protein present in each lysate was quantified using a modified Lowry assay (DC protein assay; Bio-Rad, Hercules, CA).

SDS-PAGE, Western blotting, and densitometric analysis were done using standard protocols (12, 26). The following primary antibodies were purchased from Cell Signaling Technologies (Beverly, MA) and used for immunoblotting at 1:1000 dilution: phospho-(thr202/tyr204) p44/42 MAPK (pERK1/2; no. 9101), phospho-(ser473) AKT (no. 9271), AKT (no. 9272), phospho-(tyr1068) EGFR (no. 2234), and phospho-(tyr416) Src (no. 2101). The anti-ERK antibody K-23 was used at a 1:30,000 dilution, and MAPK phosphatase 1 (MKP1), MKP2, and MKP3 antisera were diluted 1:100 (sc1119, sc1120, and sc8598; Santa Cruz Biotechnology, Santa Cruz, CA).

For ERK1/2 immunoblot analysis, 5–10 µg/lane of each protein lysate was fractionated on 10% gels by SDS-PAGE and then electrotransferred to nitrocellulose or polyvinylidene difluoride membranes. For immunodetection of EGFR, Akt, and Src, 30 µg/lane was used. Membranes were blocked for 1 h in Tris-buffered saline, 0.1% Tween 20 (TBS-T) with 5% nonfat dry milk or 5% BSA, washed in TBS-T, and incubated with primary antibodies overnight at 4 C. Antigens bound by primary antibodies were detected with appropriate horseradish peroxidase-conjugated anti-IgG secondary antibodies (1:30,000 dilution; Kirkegaard and Perry Laboratories, Gaithersburg, MD). Immunoreactive bands were visualized onto preflashed x-ray film by enhanced chemiluminescence using the SuperSignal West Pico Substrate (Pierce, Rockford, IL). Multiple exposures of each blot were collected, and those determined in the linear range of the films response were used for densitometric analysis. Digital images of appropriate films were captured with the EDAS290 imaging system (Kodak, New Haven, CT), and the optical density of each immunoreactive band was determined with KODAK 1D image analysis software. OD were calculated as arbitrary units after local area background subtraction, normalized to the density of the phospho-independent ERK immunoreactivity, and reported as fold change relative to control.

Phosphatase activity analysis
Phosphatase activity was calculated by determining the amount of free phosphate released from a phosphopeptide substrate by measuring the absorbance of a molybdate-malachite green-phosphate complex (nonradioactive serine/threonine phosphatase assay system; Promega). Granule cell cultures were generated and treated as described above. Cell lysates were prepared in a phosphate-free buffer in the absence of phosphatase inhibitors [20 mM Tris (pH 8.0), 0.1 mM EDTA, 0.1% Triton X-100, and Complete protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN)]. Particulate material was pelleted by centrifugation at 100,000 x g for 60 min. Free phosphate was removed from cell lysates by centrifugation through Sephadex-25 at 400 x g for 10 min. The protein concentration for each lysate was determined as above. In a final reaction volume of 50 µl, 15–25 µg protein was incubated for 20 min at room temperature in PP2A-specific buffer composed of 50 mM imidazole (pH 7.2), 200 µM EGTA, 0.02% ß-mercaptoethanol, 0.1 mg/ml BSA, plus or minus serine/threonine phosphopeptide substrate (100 µM; RRApTVA, where pT represents phosphothreonine). The reaction was terminated by the addition of 50 µl molybdate dye additive. After incubation at room temperature for an additional 30 min to allow full color development, the absorbance was measured at 590 nm. Phosphatase activity was calculated using a standard curve generated from standards of known free phosphate concentrations and expressed as picomoles of free phosphate per microgram protein per minute. Presented results are from three independent experiments in which three to four independent cultures/samples were analyzed for each treatment or control group.

Statistical analysis
Unless noted otherwise, all data presented are representative of at least three experiments or quantitative assessments of mean values ± SEM. Statistical analysis was conducted using a one-way ANOVA with posttest comparison between treatment groups using Tukey-Kramer multiple comparison test. The level of statistical significance between absolute values for each treatment group is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data were analyzed with Excel (Microsoft) and GraphPad Prism version 4.0 (GraphPad Software Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time dependency of rapid activation of ERK signaling by E2
Using the low concentrations of E2 (10–11 M) and ICI182,780 (10–8 M) previously found efficacious for inducing rapid ERK1/2 activation in cerebellar granule cell neurons (12), we qualitatively compared the time course of estradiol-mediated ERK1/2 activation with activation induced by the EGF/EGFR and the tropomyosin-related kinase B (trkB)/BDNF signaling pathways. In E2- or ICI182,780-treated cultures, a rapid and transient increase in the level of activated phospho-ERK1/2 (pERK) was observed (Fig. 1Go, A and B). Maximal increases above control levels peaked at the 10- and 15-min time points and returned to baseline by 30 min of exposure (Fig. 1Go, A and B). Exposure of granule cells to EGF produced a rapid activation of ERK1/2, with an increase in pERK1/2 observed as early as 2 min after treatment. Similar to E2-induced ERK signaling, the levels of pERK1/2 were much reduced by 30 min of exposure (Fig. 1CGo). In contrast, BDNF produced a rapid and sustained increase in pERK1/2 that was maintained for at least 60 min (Fig. 1DGo).



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  		FIG. 1. Qualitative comparison of the time course of E2-induced and growth factor-induced ERK1/2 stimulation. Shown are representative results of time course studies evaluating the temporal profile of ERK1/2 activation by 10–11 M E2 (A), 10–8 M ICI182,780 (B), 100 ng/ml EGF (C), and 100 ng/ml BDNF (D) in cerebellar granule cell cultures. pERK immunoreactivity was detected in granule cell protein lysates (~5 µg/lane) with antiserum specific for the dually phosphorylated active form of ERK1/2 (pERK) (top). To confirm equal loading of total ERK1/2 proteins, the blots were reanalyzed using phosphorylation-state-independent ERK1/2 (ERK1/2) (bottom) antiserum. In EGF, E2, or ICI182,780-treated cultures, rapid and transient profiles of increased ERK1/2 phosphorylation were detectable by the 10-min time point. Exposure to BDNF induced a sustained stimulation of pERK levels. Treatments and treatment times are indicated above each pair of panels. Vehicle-treated control groups (V10') were treated for 10 min.

 
Assessment of rapid estrogen-signaling desensitization
Because ERK signaling rapidly decreases after E2-induced stimulation, we investigated whether the receptor/signaling system responsible for E2 signaling becomes desensitized after E2 exposure (Fig. 2Go). To test this possibility and determine the availability of spare receptors in the presence of E2, granule cell cultures were pretreated with 10–11 M E2 for 30 min, a time when E2-mediated ERK1/2 phosphorylation has returned to baseline levels; without removal of E2, a second dose of 10–11 M E2 (Fig. 2Go, A and C) or 10–8 M ICI182,780 (Fig. 2Go, B and C) was added to the E2-treated cultures for 10, 15, or 30 min. Comparison of pERK levels at different times after this second exposure, with pERK levels from E2-treated control cultures (a total treatment time of 40 min), revealed that E2 or ICI182,780 induced a second stimulation of ERK signaling. Although modestly diminished (2.4-fold vs. 3.6-fold), the second E2- and ICI182,780-induced (not shown) increase in pERK levels was significant and again decreased after 30 min of exposure (Fig. 2CGo). Those results reveal that the rapidly induced receptor/signaling system in granule cells was not saturated or fully desensitized.



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  		FIG. 2. E2 and ICI182,780 can stimulate reactivation of ERK1/2. Shown are representative Western blots demonstrating that after a 30-min pretreatment with 10–11 M E2, an additional bolus of 10–11 M E2 (A) or 10–8 M ICI182,780 (ICI*) (B) to primary cultures of cerebellar granule cells resulted in a second significant increase in pERK levels. C, Densitometric analysis of E2- and ICI182,780-induced activation of ERK1/2 after10–11 M E2 pretreatment. Levels of E2-induced ERK1 activation, expressed as fold increase above vehicle-treated control were determined by densitometric analysis of pERK1 immunoreactivity. Vehicle control groups (V10') were treated for 10 min. Groups pretreated with E2 are indicated with a bar below each lane; experimental treatments and times are indicated above each panel pair. Secondary treatments are indicated by asterisks. Results are expressed as means ± SEM (n = 4) of the fold induction compared to vehicle control with levels of significance between values for treatment and control groups indicated above the bars (F =74.6; df = 31).

 
Analysis of PTX sensitivity of rapid E2 effects
In breast cancer cell lines, there is evidence that supports E2 rapidly activating ERK signaling through EGFR transactivation via an orphan seven-transmembrane G{alpha}i-coupled receptor (20), or through a mechanism where a membrane-associated form of the classical ER functions as a G{alpha}i-coupled receptor (22). To investigate the potential involvement of G proteins in E2- or ICI182,780-mediated ERK1/2 activation in granule cells, cultures were pretreated with PTX before treatment. In PTX-treated granule cell cultures, neither E2 (Fig. 3AGo) nor ICI182,780 (Fig. 3BGo) stimulated ERK1/2 phosphorylation. Quantitative assessments revealed that in control cultures, E2 and ICI182,780 stimulated pERK levels 5.7- and 4.2-fold above baseline (Fig. 3DGo). Pertussis toxin treatment completely blocked the response to ICI182,780, whereas E2 treatment reduced pERK1 levels 6.1-fold below the baseline levels of PTX-treated vehicle controls. In contrast, ERK signaling mediated through the G protein-independent EGF/EGFR and BDNF/TrkB signaling pathways was not influenced by PTX (Fig. 3CGo).



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  		FIG. 3. Pretreatment with the G{alpha}i/o inhibitor PTX blocks E2-induced ERK1/2 activation. Shown are representative Western blotting results characterizing the E2 responsiveness of ERK1/2 in primary cultures of cerebellar granule cells that were treated with 100 ng/ml PTX or vehicle control cultures. After overnight pretreatment, pERK levels were assessed at different times after addition of 10–11 M E2 (A), 10–8 M ICI182,780 (B), 100 ng/ml EGF (C), or 100 ng/ml BDNF (C). In PTX-treated cultures, rapid induction of pERK in response to E2 or ICI182,780 was completely blocked, suggesting the involvement of a G protein-dependent mechanism. Growth factor-mediated activation of ERK1/2 was not influenced by PTX. Groups pretreated with PTX are indicated with a bar below each set of panels; experimental treatments and times are indicated above each panel pair. D, E2 or ICI182,780 changes in pERK1 levels were quantified by densitometric analysis and expressed as fold change from vehicle-treated control levels. Results are expressed as means ± SEM (n = 3 except for PTX-ICI 15' where n = 4) of the fold change compared with vehicle control indicated with dashed lines. Levels of significance between values for treatment and control groups are indicated above the bars (F = 39.87; df = 28).

 
Analysis of PKA sensitivity of rapid E2 effects
The potential for PKA activation to influence rapid E2-induced ERK1/2 phosphorylation was assessed using the PKA inhibitor H89 (Fig. 4Go). In contrast to breast cancer cell lines where PKA inhibition potentiated E2-induced pERK activation (31), in cerebellar neurons, H89 blockade of PKA resulted in a depression of E2-induced ERK1/2 signaling (Fig. 4Go). In H89-treated cultures, a 10-min E2 exposure decreased significantly the basal pERK1 levels to approximately 30% of control (Fig. 4Go, A and B). Although the pERK stimulatory effects of ICI182,780 were blocked, only an insignificant decrease in basal pERK levels was observed. Recovery from E2-induced ERK dephosphorylation was evident by 15 min, with pERK levels in E2- and ICI182,780-treated cultures at approximately 50 and 94% of control, respectively (Fig. 4Go, A and B). In control cultures not exposed to H89, a 10-min exposure to E2 increased pERK1 by 2.6-fold (Fig. 4CGo). In the presence of H89, a 10-min E2 treatment inhibited baseline ERK1 phosphorylation by 2.9-fold. By the 15-min time point, pERK levels had recovered to vehicle control baseline (Fig. 4CGo).



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  		FIG. 4. Pretreatment with the PKA inhibitor H89 blocks E2-induced ERK1/2 activation. Shown is a representative Western blot characterizing E2 responsiveness of ERK1/2 in primary cultures of cerebellar granule cells that were treated with 10 µM H89 or untreated control cultures. A, After 30 min of H89 pretreatment, pERK levels were assessed at 10 and 15 min after addition of E2 or ICI182,780. In the presence of H89, rapid induction of pERK in response to E2 and ICI182,780 was completely blocked. Groups pretreated with H89 are indicated with a bar below each lane; experimental treatments and times are indicated above each panel pair. B, Shown are the results of densitometric analysis of the blot in A represented as fold induction of pERK1 levels compared with vehicle control. C, Results from experiments comparing changes in pERK1 levels after E2 exposure in the absence or presence of H89 were quantified and expressed as fold change from vehicle-treated control level. Results are means of the fold change compared with vehicle control indicated with a dashed line (± SEM) (for E2, n = 5; for H89-E2 10 min and H89-E2 15 min, n = 6). Levels of significance between values for treatment and control groups are indicated above the bars (F = 30.54; df = 29).

 
Analysis of rapid E2-induced phosphatase activity
The rapid decrease in pERK levels after transient estrogenic stimulation, or in response to E2 in the presence of PTX and PKA inhibitors, suggested that a pERK-specific phosphatase was rapidly activated in response to E2. One possible mechanism through which levels of active ERKs can be regulated is by the rapid increase in expression of the MKP family of dual-specificity phosphatases (DSPs). The activity of MKPs is transcriptionally regulated; in some cell types, increased growth factor signaling can induce very rapid stimulation of MKP expression and activity (32, 33). In cultured granule cells, an increase in MKP1, MKP2, or MKP3 immunoreactivity was not detected at 10 or 30 min after E2 or serum exposure (Fig. 5AGo). These results suggest that transcriptional regulation of MKPs in granule cells is too slow to account for the rapid inactivation of E2-mediated ERK signaling. In contrast, PP2A activity was found to be significantly increased in response to 10–10 M E2, an effect that was insensitive to PTX (Fig. 5BGo). In response to E2, significant increases in PP2B or PP2C were not detected (not shown). Membrane-impermeable E2-BSA was unable to stimulate PP2A (Fig. 5BGo), suggesting that the concerted activation of PP2A is initiated through intracellular E2 binding.



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  		FIG. 5. Analysis of changes in MKP expression and PP2A activity in response to E2. A, Representative Western blotting results of proteins (50 µg/lane) isolated from cerebellar granule cell cultures that were treated for 10 min with vehicle (V10'), 10% fetal bovine serum, or 10–11 M E2 for 10 or 30 min. HeLa cell lysates were used for positive blotting controls. No detectable increase in MKP1, MKP2, or MKP3 expression that parallels rapid pERK1/2 inactivation after E2 or in response to growth factor-like stimulation of ERK signaling by serum was detectable. B, Analysis of PP2A activity in response to E2. Shown are results from experiments where PP2A activity was determined after exposure to 10–8 M E2 (E2 10–8) or 10–10 M E2 (E2 10–10) in the presence or absence of PTX. About a 2-fold increase in PP2A activity that was insensitive to PTX was detected after a 10-min exposure to 10–10 M E2. Increases in PP2A activity were not detected in response to E2-BSA (E2 concentration of 10–10 M). Results are presented as mean percent ± SEM of vehicle-treated control activity. Levels of significance between values for treatment and control groups are indicated above the bars (F = 4.803 df = 41; vehicle and E2 10–10, n = 7; E2 10–8, vehicle PTX, and E2 10–10 PTX, n = 6; E2 10–8 PTX, n = 4; vehicle BSA and E2-BSA 10–10, n = 3).

 
Analysis of the intracellular pathway mediating rapid E2 effects
To investigate whether E2 activates ERK signaling in granule cell neurons through G protein-coupled receptor (GPCR)-mediated EGFR transactivation, protein lysates from E2- and EGF-treated cultures were analyzed with phosphospecific antisera targeting activation of the AKT kinase in the phosphatidylinositol-3-kinase signaling pathway, autophosphorylation of EGFR at tyrosine 1068, and activation of c-Src at tyrosine 416. Protein lysates from granule cell cultures treated for 10 min with vehicle, E2, ICI182,780 (not shown), or EGF were collected and analyzed by Western blotting. Shown in Fig. 6Go, A–E, are the results of a representative immunoblot experiment in which the same lysates were analyzed for increased phosphorylation of ERK1/2, AKT, EGFR, or c-Src (Fig. 6Go, A–D). Treatment with E2 induced a 2.5-fold increase in pERK1 levels; however, unlike EGF/EGFR-mediated signaling, E2-induced increases in pAKT or autophosphorylation of EGFR were not detected (Fig. 6Go, A–E).



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  		FIG. 6. Analysis of the role of EGFR and c-Src in mediating rapid actions of E2 in granule cells. Shown are results of Western blotting analysis of the signaling intermediates associated with E2-induced ERK1/2 activation. The same samples of representative granule cell lysates from cultures treated for 10 min with 10–11 M E2 (E210'), 100 ng/ml EGF (EGF10') or vehicle (V10') were probed with antiserum specific for pERK1/2 or total ERK1/2 (A), phosphotyrosine 1068 EGFR (pEGFR) or total EGF (B), phosphoserine 473 AKT (pAKT) or total AKT (C), or phosphotyrosine 416 c-Src (D). E, Densitometric analysis of the results in A–D showed that E2 and EGF stimulate pERK1/2 levels; in response to EGF but not E2, pAKT levels and EGFR autophosphorylation were increased; and in the same lysates, E2 caused a modest increase in c-Src activation. F, Densitometric quantification of experiments investigating E2’s ability to increase phospho-c-Src levels (n = 4) revealed a significant 1.8-fold increase compared with control (F = 27.52; df = 12). Results are normalized to total ERK1 of the same sample and expressed as mean ± SEM fold induction compared with vehicle control. Normalization results to total ERK2, total AKT, or EGFR gave results similar to those normalized to total ERK1. Levels of significance between values for treatment and control groups are indicated above the bars.

 
Quantification of Western blot analysis for phosphorylation at tyrosine 416 of c-Src revealed a significant 1.8-fold increase in phospho-Src levels after 10 min of E2 treatment (Fig. 6Go, D–F). Involvement of a Src protein kinase was further demonstrated by the ability of the Src-family tyrosine kinase inhibitor PP2 to completely abolish the ability of E2 or ICI182,780 to rapidly stimulate ERK signaling (Fig. 7AGo). In contrast, the highly potent and specific inhibitor of EGFR kinase activity AG 1478 (Fig. 7Go, B and C) did not influence E2-induced ERK activation. In the presence of AG 1478, E2 stimulated a 2.6- and 3.7-fold increase at the 10- and 15-min time points, respectively (Fig. 7CGo). These results suggest further that E2-induced ERK1/2 activation in granule cells involves Src tyrosine kinase activity but is not mediated by a mechanism requiring EGFR activation.



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  		FIG. 7. Rapid E2-induced stimulation of ERK1/2 signaling is dependent on Src tyrosine kinase activity and independent of EGFR tyrosine kinase activity. A, Results of representative Western blot analysis evaluating the ability of 10–11 M E2 and 10–8 M ICI182,780 to activate ERK1/2 in the presence the Src family tyrosine kinase inhibitor PP2. Inhibition of Src-tyrosine kinase activity blocked the ability of either estrogen to rapidly increase pERK levels. Vehicle treatment for ICI182,780 (V) was for 15 min. B, Representative Western blot results of E2-induced ERK activation in the presence of the specific EGFR tyrosine kinase antagonist AG 1478. C, Results of densitometry analysis quantifying E2-induced ERK activation in the presence of AG 1478. Results are expressed as means ± SEM (n = 6) of the fold induction compared with vehicle control. Levels of significance between values for treatment and control groups are indicted above the bars (F = 9.588; df = 17).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Evidence from many different tissues and cell types suggests that there are multiple mechanisms through which estradiol acts to stimulate rapid intracellular signaling (2, 34, 35). Previous results from our laboratory described important differences in the physiological consequences of rapid E2-induced ERK signaling between immature and mature cerebellar neurons (12). The rapid and transient ERK activation induced by E2 in granule cells is also different from the more sustained activation that occurs in cultured cortical neurons (10, 27). Dose-dependency analysis of E2-induced ERK1/2 activation in primary cultured cerebellar granule cells revealed that 10–11 to 10–10 M E2 was more effective at activating the ERK1/2 MAPK signaling pathway in cultured granule cells than the 10–8 M concentration that activates ERK signaling in immature neurons from the cerebral cortex and midbrain (10, 14, 27). Those differences suggest that rapid E2 signaling can operate with differences in mechanistic detail during ontogeny and in different populations of neurons.

Here we have provided evidence that in granule cell neurons, E2’s rapid effects are mediated via the ERK1/2 MAPK cascade through a novel G protein-dependent mechanism that temporally resembles EGFR-mediated signaling but does not involve transactivation of the EGFR. Importantly, the effects of E2 and the estrogen-like steroidal antagonist of ER transcriptional activity ICI182,780 were nearly identical, supporting our previous conclusion that ICI182,780 was a full agonist of rapid E2-mediated ERK signaling (12). The temporal character of ERK1/2 activation induced by E2 or ICI182,780 was compared with growth factor signaling induced through the EGFR by EGF or by BDNF through trkB. Reminiscent of the response induced by EGF, through the granule cell EGFR system, E2 stimulation of ERK signaling was transient with pERK1/2 levels returning to, and often falling below, baseline by 30 min of exposure.

In MCF7 breast cancer cells, where E2 can rapidly activate c-Src to stimulate ERK activation, less than 10–20% receptor occupancy was reported sufficient to stimulate maximal c-Src activation by E2 (4). Those results were interpreted as indicative of a membrane-associated subpopulation of ERs. However, an alternative interpretation of those results, more consistent with known hormone receptor behavior, would be that maximal membrane-initiated actions of E2 are achieved at low receptor occupancy. The finding that E2 can stimulate maximal ERK activity in granule cells at concentrations 10 times lower than the Kd of known ERs is consistent with this possibility. Using a two-pulse E2-treatment protocol, the functional nature of the ER/signaling system responsible for rapid ERK signaling in granule cells was explored in more detail. In the presence of a maximally efficacious (10–11 M) dose of E2, increases in pERK levels were observed upon retreatment with either 10–11 M E2 or 10–8 M ICI182,780. Compared with the initial response, the magnitude of ERK stimulation in response to the second E2 pulse was reduced. The results of the two-pulse analysis demonstrate that the responsiveness of ERK signaling to E2 was not saturated by the most efficacious concentration of ligand and that spare populations of receptors are available for reactivation of the ERK signaling cascade. The diminution of the initial response may have resulted in a partial desensitization to additional stimulation, possibly through receptor internalization.

The vast majority of ERs expressed in granule cells are extranuclear localized ERß1 (25, 26). Based on current understanding, it is reasonable to assume that ERß1, through a mechanism that is unique from its activity as a nuclear ER, could act to mediate rapid E2 signaling in granule cells. The Kd (50% receptor occupancy) for E2 binding at ERß1 is approximately 0.1 nM (36); therefore, in the presence of 0.01 nM E2, less than 10% of the receptors are expected to be occupied by ligand. Based on the ability of an additional pulse of E2 or ICI182,780 to stimulate ERK signaling in the presence of a fully efficacious ligand, the remaining unoccupied receptors must be available to bind ligand, reconstitute the activated E2/ER-signaling complexes, and stimulate ERK signaling. Physiologically, the ability of a signaling system to induce maximal responses well below the Kd of the receptor affords sensitivity to small changes in hormone concentrations. Such sensitive responsiveness is not possible if occupancy of a large percentage of the available receptors is required to elicit maximal responsiveness. Like the classical nuclear ER system, the pharmacological and physiological properties of rapid E2-mediated ERK signaling indicate an estrogen receptor system tuned to respond fully to small variations in low concentrations of the biologically active (free) fraction of circulating E2.

In some neurons, endothelial cells, pancreatic cells, and tumor cell lines, heterotrimeric G protein-coupled mechanisms appear involved in rapid estrogenic signaling (19, 20, 21, 22, 37, 38). The similarities between the time courses of EGF- and rapid E2-induced ERK activation were suggestive of a mechanism involving cross-talk between rapid estrogenic signaling and the EGFR signaling pathways. Therefore, we investigated whether E2 was mediating cross-talk between the EGFR via a GPCR mechanism previously described as operative in other cell systems (2, 13, 39). As expected, the G{alpha}i/o inhibitor PTX did not inhibit G protein-independent ERK1/2 activation by EGF and BDNF. However, in granule cells pretreated with PTX, the response to E2 and ICI182,780 was fully blocked. Additionally, in PTX-treated cultures, a rapid reduction of basal pERK levels was observed in response to E2 but not ICI182,780. The inhibitory actions of E2 on ERK phosphorylation were also observed in granule cells after blockade of PKA activity with H89. Together, those experiments indicated that E2- and ICI182,780-induced ERK1/2 activation operates via a PKA-dependent and PTX-sensitive G{alpha}i/o or Gß{gamma}-dependent mechanism. Furthermore, the inversion of the E2-induced effects suggests that an additional PTX- and PKA-insensitive signaling pathway that increases protein phosphatase activity was concomitantly activated by E2.

DSPs regulate MAPK activity by selective dephosphorylation of phosphotyrosine and phosphothreonine residues of specific MAPK isoforms (32, 33). The activity of DSPs is transcriptionally regulated and, in response to growth factors, DSP gene expression is rapidly up-regulated, causing a parallel dephosphorylation of MAPKs. The potential role of dual-specificity MKPs in rapid down-regulation of E2-induced ERK activity was previously unknown. The prototypical DSP gene family member MKP1 specifically acts on the p38 and stress-activated protein kinase/c-Jun terminal kinase MAPKs, signaling pathways that are not rapidly influenced by E2 in granule cells (12). The MKP2 and MKP3 phosphatases are expressed in the brain, specifically inactive pERK, and in some cases, their expression is robustly stimulated by serum or growth factors in less than 15–30 min (40, 41). No rapid increases in MKP1–3 expression paralleling the decrease in the pERK levels during 30-min exposures to E2 were observed, suggesting that MKPs do not regulate rapid inactivation of E2-stimulated ERK signaling.

PP2A is also an important regulator of ERK/MAPK signaling that acts at multiple levels of the signaling cascade (42, 43, 44). Interestingly, PP2A indirectly regulates ERK-induced transcriptional activity of the ER (45). In contrast to the MKPs, E2 significantly increased PP2A activity after a 10-min exposure, an effect that was not influenced by PTX or mimicked by E2-BSA. Those results, combined with the finding that ICI182,780 did not stimulate ERK dephosphorylation in the presence of PTX or H89, suggest that rapid E2-induced ERK signaling and PP2A activation, although temporally linked, are mediated through different signaling mechanisms involving unique membrane-associated and intracellular E2 binding sites, respectively. Thus, the results presented here implicate PP2A as regulator of the rapid membrane-mediated actions of E2 that may play a pivotal role in determining the context-specific responsiveness of different cell types to estrogens.

Cross-talk between G protein-dependent mechanisms and the EGFR are frequently coupled via the nonreceptor tyrosine kinase c-Src, which may serve as the bridging molecule between the two signaling pathways. Similar to what was observed in neurons from the mouse neocortex (46), blockade of the Src-family kinase antagonist activity completely abrogated E2-induced ERK activation in cerebellar neurons. An increase in c-Src phosphorylation after brief E2 exposure was also detected. Those results raised the possibility that the rapid actions of E2 were mediated via transactivation of the EGFR. Rapid E2-induced transactivation was first proposed in breast cancer cells to involve activation of an orphan GPCR (GPR30) to mediate E2-dependent liberation of heparin-binding EGF (HB-EGF) (20). Subsequent studies in breast cancer and endothelial cell lines suggested a G protein-dependent requirement for ER{alpha} to induce transactivation of EGFR (22). The results of the later studies suggested that the rapid mechanism of E2-induced ERK activation involved Src-kinase-dependent activation of matrix metalloproteinases (MMPs) to liberate HB-EGF, which acts as a ligand to stimulate autophosphorylation of the EGFR and initiate the signaling events required for ERK1/2 activation. Thus, ER{alpha} appears to act as a GPCR that usurps the prototypical mechanism of EGFR transactivation (47). Yet, as initially proposed in the same MCF7 cells, there is strong evidence in support of a direct interaction between ER{alpha} and the Src homology 2 domain of c-Src, which links estrogen signaling directly to the ERK1/2 MAPK signaling complex and does not require EGFR activation (4, 48).

Because of the similarity between E2- and EGF-induced ERK signaling and the similar dependence on G proteins and Src-tyrosine kinase activity for rapid ERK activation in granule cells and MCF7 cells, we initially hypothesized that in granule cells rapid actions of E2 were mediated through ERß-dependent transactivation of EGFR. Consistent with this hypothesis, it was found that that rapid E2-mediated ERK activation in granule cells is G protein and Src-kinase activity dependent. However, after E2 treatment, EGFR autophosphorylation was not detected, and blockade of EGFR tyrosine kinase activity did not influence rapid ERK activation. Although transactivation of other receptor tyrosine kinases linked to the ERK signaling cascade have not been ruled out, transactivation of the EGFR is not critically involved in the mechanism of rapid E2-induced ERK stimulation in granule cells. The finding that E2 exposure did not rapidly increase pAKT levels in granule cells further supports a lack of EGFR involvement in rapid E2-induced ERK signaling.

The finding that transactivation mechanisms requiring highly regulated shedding of HB-EGF to stimulate the regulation of E2 signaling are not operative is teleologically consistent with granule cell physiology. Unlike MCF7 cells, in vivo granule cell neurons are a highly migratory cell type that normally express high levels of active MMPs to regulate process extension, synaptic plasticity, and migration (30, 49). We have compared the level of MMP-2 and MMP-9 activity in primary cultures of granule cells with activity in MCF7 cultures and found that MMP activity is at least 20-fold greater in primary cultures of granule cells than in MCF7 cells (unpublished observation). As a sensitive mechanism to regulate rapid actions of E2, the high levels of endogenous MMP activity that are a normal aspect of granule cell physiology would likely preclude sensitive exploitation of MMP/HB-EGF transactivation in response to fluctuations in free concentrations of E2. In contrast, our current results extend the mechanistic diversity of rapid E2 signaling, suggesting that rapid E2-induced regulation of ERK signaling in granule cell neurons may be mediated through a mechanism (Fig. 8Go) that closely resembles ERK signaling through a ß-adrenergic receptor-like pathway involving PKA-dependent Gs/Gi switching (28).



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  		FIG. 8. A model for rapid and transient E2-induced G protein-dependent activation of ERK signaling in granule cells. Shown is a schematic model to describe the signaling mechanism of rapid E2-induced ERK activation. The presented results suggest a model similar to that of ß-adrenergic receptor activation of ERK signaling (28 ). E2-induced increases in ERK1/2 phosphorylation are mediated through a membrane-associated receptor (12 ) that is coupled to Gs. E2-induced activation of Gs results in activation of PKA, which subsequently mediates receptor coupling to Gi, presumably through phosphorylation of the membrane-associated receptor or components of the signaling complex, which leads to ß{gamma}-mediated ERK activation. Concerted activation of PP2A, via intracellular binding of E2 and activation of a PTX- and PKA-independent pathway, functions to rapidly inactivate increased ERK signaling. AC, Adenylate cyclase; MEK, mitogen-activated ERK-activating kinase/MAPK kinase.

 
Activation of the ERK signaling cascade is the result of a complex set of signaling inputs from different receptor tyrosine kinases, GPCRs, and other sources such as integrins and ion channels that converge to influence the ERK1/2 MAPK module. The precise nature of these signaling inputs will vary in different cells and even at different times in the same cell. Of the many different extracellular stimuli active during neuronal development, the actions of the steroid hormone estrogen are particularly potent and surprisingly varied. This variability speaks directly to the importance of understanding the effects of E2 in the context of individual cell types. Because of the requirement for a fine-tuned system to respond to small changes in E2 concentration, it appears that the mechanisms through which E2 acts to modulate rapid signaling are highly adaptable in their ability to exploit signaling cascades in different cell types. Thus, the rapid signaling mechanisms of estrogen, and potentially those of other hormones, display a high degree of variability based on the physiological context of a given cell type. This plasticity suggests that these rapidly acting mechanisms can vary from cell to cell, in a single cell during development in response to environmental changes, and potentially in different subcellular localizations within the same cell.


   Footnotes
 
These studies were supported by National Institutes of Health Grant R01-NS42798, a basic research grant from the Pediatric Brain Tumor Foundation of the United States, and by National Institute of Environmental Health Sciences through the University of Cincinnati Center for Environmental Genetics (P30-ES06096).

First Published Online August 25, 2005

Abbreviations: BDNF, Brain-derived neurotrophic factor; DSP, dual-specificity phosphatase; E2, 17ß-estradiol; E2-BSA, E2-hemisuccinate-BSA; EGFR, epidermal growth factor receptor; ER, estrogen receptor; GPCR, G protein-coupled receptor; HB-EGF, heparin-binding EGF; MKP, MAPK phosphatase; MMP, matrix metalloproteinase; P0, postnatal d 0; pERK, phospho-ERK; PKA, protein kinase A; PP2A, protein phosphatase 2A; trk, tropomyosin-related kinase.

Received May 10, 2005.

Accepted for publication August 16, 2005.


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