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The Journal of Neuroscience, February 15, 1999, 19(4):1179-1188
Estrogen-Induced Activation of Mitogen-Activated Protein Kinase
in Cerebral Cortical Explants: Convergence of Estrogen and Neurotrophin
Signaling Pathways
Meharvan
Singh1,
György
Sétáló Jr1, 2,
Xiaoping
Guan1,
Matthew
Warren1, and
C. Dominique
Toran-Allerand1
1 Departments of Anatomy and Cell Biology and Neurology
and Centers for Neurobiology and Behavior and Reproductive
Sciences, Columbia University College of Physicians and
Surgeons, New York, New York 10032, and 2 Department of
Biology, University Medical School of Pécs, Pécs H-7643,
Hungary
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ABSTRACT |
We have shown that estrogen elicits a selective enhancement of the
growth and differentiation of axons and dendrites (neurites) in the
developing CNS. We subsequently demonstrated widespread colocalization
of estrogen and neurotrophin receptors (trk) within developing forebrain neurons and reciprocal transcriptional regulation of these receptors by their ligands. Using organotypic explants of the
cerebral cortex, we tested the hypothesis that estrogen/neurotrophin receptor coexpression also may result in convergence or
cross-coupling of their signaling pathways. Estradiol elicited rapid
(within 5-15 min) tyrosine phosphorylation/activation of the
mitogen-activated protein (MAP) kinases, ERK1 and ERK2, that persisted
for at least 2 hr. This extracellular signal-regulated protein kinase
(ERK) activation was inhibited successfully by the MEK1 inhibitor
PD98059, but not by the estrogen receptor (ER) antagonist ICI 182,780, and did not appear to result from estradiol-induced activation of
trk. Furthermore, we also found that estradiol elicited
an increase in B-Raf kinase activity. The latter and subsequent
downstream events leading to ERK activation may be a consequence of our
documentation of a multimeric complex consisting of, at least, the ER,
hsp90, and B-Raf. These novel findings provide an alternative mechanism for some of the estrogen actions in the developing CNS and could explain not only some of the very rapid effects of estrogen but also
the ability of estrogen and neurotrophins to regulate the same broad
array of cytoskeletal and growth-associated genes involved in neurite
growth and differentiation.
Key words:
estradiol; estrogen receptor; ERK; neurotrophins; signal
transduction; cross-coupling; brain; cerebral cortex
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INTRODUCTION |
Estrogen and numerous growth
factors, including the neurotrophins (NGF, BDNF, NT-3, NT-4/5), play
important roles associated with neuronal differentiation and survival
(Toran-Allerand, 1996a ,b). We previously showed that estrogen elicits
significant enhancement of neurite growth and differentiation within
organotypic explant cultures of the hypothalamus, preoptic area, and
cerebral cortex (Toran-Allerand, 1976, 1980, 1984) and that estrogen
and neurotrophin receptors colocalize in developing forebrain neurons
(Toran-Allerand et al., 1992; Miranda et al., 1993). This pattern of
coexpression suggested the potential for interaction between these two
receptor systems. In fact, subsequent studies demonstrated differential and reciprocal regulation of estrogen and nerve growth factor (NGF)
receptor mRNAs by their ligands, along with the regulation of estrogen
receptor (ER) binding, in adult female rat sensory neurons (Sohrabji et
al., 1994b), PC12 cells (Sohrabji et al., 1994a) and
cerebral cortical explants (Miranda et al., 1996). In addition,
estrogen also has been shown to regulate the levels of NGF and
brain-derived neurotrophic factor (BDNF) mRNA expression in the adult
rat cerebral cortex (Singh et al., 1995; Sohrabji et al., 1995),
olfactory bulb (Sohrabji et al., 1995), and hippocampus (Singh et al.,
1995). A critical and, as yet, unanswered question is whether the
developmental actions of estrogen on neurite growth and differentiation
are mediated directly or result from intermediate steps via modulatory
interactions with endogenous growth factors and their signaling pathways.
The classical mechanism of estrogen action requires the binding of
intranuclear receptors to specific estrogen response elements (EREs)
within DNA, resulting in the regulation of gene expression. This
concept, however, inadequately explains the complete and extensive
range of actions of estrogen, which include the ability of estrogen to
regulate non-ERE-containing genes (Sukovich et al., 1994) and the very
rapid (seconds to minutes) effects of estrogen (Chiaia et al., 1983;
Garcia-Segura et al., 1987; Migliaccio et al., 1993), collectively
suggesting alternative mechanisms of action.
In extraneural targets, interactions between the ER and growth factor
signaling pathways (e.g., EGF; TGF- /TGF- ; IGF-I, insulin) have
been implicated in the mediation of an increasing number of
estrogen-induced differentiative processes (Read et al., 1989; Kato et
al., 1995). For example, the uterotrophic effects of epidermal growth
factor (EGF) (Ignar-Trowbridge et al., 1992) and the differentiative effects of insulin on neuroblastoma cells (Patrone et al., 1996) were
both found to be estrogen receptor-dependent. Furthermore, the effect
of insulin required the presence of p21Ras, an early signaling
component of the mitogen-activated protein (MAP) kinase cascade also
used by the neurotrophins. As a result of these observations, taken
collectively with the knowledge that estrogen and the neurotrophins can
regulate the same cytoskeletal and growth-associated genes [such as
MAP-2 (Black et al., 1986; Fischer et al., 1991; Lorenzo et al., 1992),
tau microtubule-associated protein (Drubin et al., 1985; Matsuno et
al., 1997), -tubulin (Guo and Gorski, 1988), and GAP-43 (Costello et
al., 1990; Lustig et al., 1991)], we hypothesized that the MAP kinase
cascade also may be used by estrogen in its differentiative actions on
the CNS.
Here, we report for the first time in the developing brain, which
normally expresses wild-type ERs, the rapid activation of the MAP
kinase isoforms, extracellular signal-regulated protein kinase-1 (ERK1)
and ERK2 by estradiol, an action blocked by the mitogen-activated
protein kinase kinase (MEK1) inhibitor PD98059, but not by the ER
antagonist ICI 182,780. Moreover, estradiol-induced ERK phosphorylation
did not result from trk activation but is likely a
consequence of our observed estradiol-induced activation of B-Raf. A
possible mechanism for estrogen action is suggested by the work of
Jaiswal et al. (1996), which demonstrated an intracellular association
between the heat shock protein, hsp90, and B-Raf in PC12 cells. In view
of the known association of hsp90 with the ER (Segnitz and Gehring,
1995), we postulated that the ER also may be a component of this
putative complex and could be associated with one or more of members of
the MAP kinase cascade. We confirmed this in explants of the cerebral
cortex, where the ER coimmunoprecipitated with hsp90 and B-Raf, but not
with MEK1. These novel findings suggest that the estrogen-induced
activation of ERK may occur via direct interaction of the ER in a
complex with members of the MAP kinase cascade. Such multimeric
complexes would provide intracellular convergence points for the rapid
activation of ERK by estrogen and, conversely, support the previously
reported regulation of the ER by neurotrophins (Miranda et al.,
1996).
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MATERIALS AND METHODS |
Tissue culture. Organotypic explants were derived
from ~360-µm-thick hemicoronal slices of postnatal day 2 (P2)
frontal and cingulate cerebral cortex (day of birth = P1) or from
parasagittal slices of P9 cerebellum, obtained from pups born of
timed-pregnant Sprague Dawley rats (Zivic-Miller, Allison Park, PA, and
Taconic Farms, Germantown, NY) as previously described (Miranda et al., 1996). Explant slices were maintained as roller tube cultures on
rat-tail collagen-coated/poly-L-lysine-precoated glass
coverslips for 6 d to allow them to reach the stage of maximal
cortical ER expression (Friedman et al., 1983; Gerlach et al., 1983).
The steroid-deficient and phenol red-free maintenance nutrient medium (25% gelding serum, 22.5% HBSS, 50% BME, 7.5 mg/ml glucose, 2 mM L-glutamine, and 50 µg/ml ascorbic acid)
was supplemented with a cocktail of human recombinant neurotrophins
[50 ng/ml each of NGF, BDNF, NT-3, and NT-4/5 (Genentech, San
Francisco, CA, and Intergen, Purchase, NY)] and 17- estradiol (10 nM; Sigma, St. Louis, MO). Antibiotics were never used.
Treatment of cultures. After 6 d in culture a 24 hr
washout period was performed, consisting of both omitting exogenously added estradiol and neurotrophins and adding blocking antibodies to
rodent NGF (Sigma), BDNF, and NT-3 (gift of R. A. Rush, Flinders University, Adelaide, Australia) (Zhou et al., 1996). For the experiment identifying the effect of the ER antagonist, 1 µM ICI 182,780 (gift of A. E. Wakeling, Zeneca
Pharmaceuticals, Cheshire, England) or 0.1% DMSO (vehicle control) was
added to the appropriate tubes at this time as well. The next day the
explants were pulsed with 10 nM estradiol for 5, 15, and 30 min and 1, 2, and 4 hr or with 100 ng/ml of each individual
neurotrophin (NGF, BDNF, NT-3, and NT-4/5) administered together as a
cocktail for a single 30 min time point. For the MEK1 inhibition
experiment the cultures were pretreated with either the MEK1 inhibitor
PD98059 (100 µM; New England Biolabs, Beverly, MA) or
0.1% DMSO for 4-5 hr before the cultures were pulsed with the
appropriate estradiol or neurotrophin treatments. We also evaluated the
effect of estradiol on cerebellar cultures for which the ER levels
(estrogen binding) relative to the cerebral cortex are considerably
lower at the postnatal age investigated (C. D. Toran-Allerand and
N. J. MacLusky, unpublished observation). Cerebellar cultures were
treated either with 10 nM estradiol for 30 or 60 min or
with the neurotrophin cocktail for 30 min.
Tissue processing. Harvested cultures (two to four
hemicoronal sections per coverslip) were washed with ice-cold PBS and
immersed into lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM
Na3VO4, and 100 mM NaF plus
5 µM ZnCl2, 10% glycerol, and 1%
Triton X-100 and the following protease inhibitors: 10 µg/ml
aprotinin, 1 µg/ml leupeptin, 1 mM PMSF, and 0.5 µM okadaic acid. After homogenization and centrifugation
at 100,000 × g for 15 min at 4°C, the resulting
supernatants (lysates) were normalized for protein content (Lowry
Assay, Bio-Rad Protein Assay Kit, Hercules, CA).
Western blot analysis (ERK phosphorylation). After
electrophoretic separation the polyacrylamide gels were transferred
onto polyvinylidene difluoride (PVDF) membranes (0.22 µm pore size, Bio-Rad), blocked overnight with 3% BSA (Fraction V, Sigma) in Tris-buffered saline containing 0.2% Tween 20 (TBS-T), and probed with
the following antibodies: for ERK phosphorylation, rabbit anti-phospho-MAPK [dual phosphospecific (Thr202/Tyr204), 1:1000; New
England Biolabs]; for ERK protein assessment, rabbit anti-ERK1 (1:1000) and rabbit anti-ERK2 (1:1000; Santa Cruz Biotechnologies, Santa Cruz, CA). Binding of the antibody to the membrane was detected by a secondary antibody (either goat anti-rabbit or goat anti-mouse) conjugated to horseradish peroxidase (HRP; 1:40,000; Pierce, Rockford, IL) and visualized on autoradiographic film, using enzyme-linked chemiluminescence (ECL; Amersham, Arlington Heights, IL). All blots
were stripped and reprobed with the appropriate antibody to verify the
equal loading of protein across lanes.
In-gel kinase assay (ERK activation). Nondenatured lysates
were separated on a 10% polyacrylamide gel containing 0.6 mg/ml myelin
basic protein (MBP; Upstate Biotechnology, Lake Placid, NY). Then the
gel was washed with a 50 mM Tris, pH 8, and 20% isopropanol solution and subsequently was denatured in Buffer A
(consisting of 50 mM Tris, pH 8, and 5 mM
2-mercaptoethanol), followed by treatment with 6 M
guanidinium-HCl in Buffer A. After renaturation in Buffer A containing
0.04% Tween 40, the gel was exposed to the ATP mixture [containing 40 mM HEPES, pH 8, 1 mM MnCl2,
2 mM DTT, 0.1 mM EGTA, pH 7, 5 mM
MgCl2, and 0.025 mM cold ATP plus 250 µCi of [ -32P]ATP (New England Nuclear, Wilmington,
DE) per 10 ml of ATP mixture] for 1 hr at room temperature and washed
extensively in 5% TCA/1% sodium pyrophosphate solution. After the gel
was dried onto Whatman filter paper, exposure to autoradiographic film
allowed for the visualization of 32P-incorporated bands.
Immunoprecipitation of trk. Cerebral cortical explants were
lysed as describe above. Lysates were immunoprecipitated with the
rabbit anti-pan-trk (203) antibody (gift of D. R. Kaplan, Montreal Neurological Institute, Canada) overnight and
precipitated with pre-BSA-blocked, protein A-Sepharose beads (Pharmacia
Biotech, Uppsala, Sweden). After overnight incubation with protein
A-Sepharose, the beads were washed four times with lysis buffer and
subsequently were reconstituted in Laemmli buffer, boiled for 5 min,
and separated on a 7.5% SDS-PAGE gel. The separated proteins were
transferred onto PVDF membranes and probed with the mouse
anti-phosphotyrosine (4G10) antibody (Upstate Biotechnology). Untreated
PC12 cells and the trkA-deficient NNR-5 cell line served as
negative controls, whereas NGF-treated PC12 cells served as the
positive methodological control. Neurotrophin-treated cerebral cortical
explants served as the internal experimental control.
B-Raf kinase assay. Cerebral cortical explants were lysed
and immunoprecipitated with a rabbit anti-B-Raf antibody (Santa Cruz)
and precipitated with anti-rabbit IgG-coated magnetic beads (Dynabeads,
Dynal A. S., Oslo, Norway). After four washes with lysis buffer,
the beads were used as the starting material for the B-Raf kinase
assay. The assay procedure was done according to the protocol provided
in the B-Raf kinase assay kit (Upstate Biotechnology) and is based on
the phosphorylation of MBP by a B-Raf-activated kinase cascade that
used radioactive ATP as the final phosphate donor. Briefly, assay
dilution buffer [containing (in mM) 20 MOPS, pH 7.2, 25 -glycerol phosphate, 5 EGTA, 1 Na3VO4, and 1 dithiothreitol] and the
magnesium/cold ATP cocktail were added in conjunction with 0.4 µg of
inactive MEK1 and 1 µg of inactive GST-p42 MAP kinase. Then this
mixture was incubated for 30 min at 30°C. Subsequently, additional
assay dilution buffer, MBP, and [-32P]ATP were added
and incubated for another 10 min at 30°C while being thoroughly
shaken. After the samples were boiled for 5 min, 25 µl of the
supernatant was spotted onto P81 phosphocellulose paper, which exhibits
differential binding of the phosphorylated MBP from unincorporated
32P. Radioactivity incorporated into the P81 paper then was
counted with a scintillation counter (Model Delta 300, Searle, Skokie, IL).
Coimmunoprecipitation of the ER with B-Raf, MEK1, and hsp90.
Lysates obtained from explants were immunoprecipitated with either anti-ER antibody [ER715; National Hormone and Pituitary Program (NHPP), National Institute of Diabetes and Digestive and Kidney Diseases] or ER Ab C1355 (Friend et al., 1997) (gift of M. Shupnik, University of Virginia) or anti-MEK1 (Transduction Laboratories, Lexington, KY). Then the immunoprecipitates were separated and probed
for various members of the putative complex. Lysates immunoprecipitated with the ER antibody were probed for MEK1, B-Raf, and hsp90. Negative controls were run in parallel, where a control (preimmune) IgG was used
to immunoprecipitate the sample lysate, and subsequently probed with
the appropriate antibody. Additionally, a control peptide or lysate was
always used as a positive control to verify the identity of the band in
the experimental lanes. In evaluating the presence of B-Raf kinase
activity in the ER immunoprecipitates, we immunoprecipitated
cerebral cortical lysates with either ER antibodies or preimmune IgG,
followed by precipitation with the Dynabeads. After four washes of the
Dynabeads the resulting pellet was used as the starting material for
the B-Raf kinase assay. In all coprecipitation experiments a
detergent-free lysis buffer was used to wash the precipitates.
Densitometric analysis of ERK phosphorylation/activation time
course. Autoradiograms were scanned with an Epson ActionScanner II
(Epson America, Torrance, CA) and analyzed by Kodak 1D Image Analysis
software (Eastman Kodak, Rochester, NY). Net intensity values were
calculated by subtracting the background within the area measured for
each band from the total intensity within this same measured area to
account for any variation in background intensity across the film.
Statistical analysis. Resulting counts per minute (cpm)
values obtained from the B-Raf kinase assay were analyzed by using a
one way ANOVA, followed by Scheffé's post hoc
analysis for group differences. Values obtained in comparing B-Raf
kinase activity in control IgG-immunoprecipitated lysates versus
ER-immunoprecipitated lysates were analyzed by using a two-tailed
Student's t test. All statistical analyses were performed
by using the SPSS software (SPSS, Chicago, IL).
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RESULTS |
Estradiol-induced phosphorylation and activation of ERK1
and ERK2
To test whether estrogen signaling uses pathways also activated by
neurotrophins in the developing brain, we investigated whether estrogen
could elicit tyrosine phosphorylation and activation of ERK, a
component of the MAP kinase cascade. 17- estradiol rapidly induced
tyrosine phosphorylation of both ERK1 and ERK2. The responses were seen
between 5 and 15 min, became maximal at 1 hr, and declined to control
levels by 4 hr (Fig. 1). In addition, when explant cultures were treated with control media that did not
contain estrogen or exogenously added neurotrophins, no increase in ERK
phosphorylation was observed (data not shown), suggesting that the
effect of estradiol on ERK phosphorylation was specific and not a
consequence of procedural manipulation of the cultures. Furthermore,
when cerebellar explants, a region of the brain that expresses
considerably lower levels of estrogen binding, were treated for the
same length of time that elicited maximal ERK phosphorylation in the
cerebral cortex, little to no phosphorylation of ERK was detected (Fig.
2). Neurotrophin treatment, however, did result in ERK phosphorylation in the cerebellar explants (Fig. 2).
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Figure 1.
Estradiol-induced ERK phosphorylation. Lysates
derived from cerebral cortical explants were probed with a
phosphospecific ERK1/ERK2 antibody. Shown is a time course for
estradiol-induced ERK phosphorylation (top panel)
and the reprobed blot for ERK1 and ERK2 protein (middle
panel). Explants also were treated with a 100 ng/ml
neurotrophin cocktail (NGF, BDNF, NT-3, and NT-4/5) for a single 30 min
time point that served as the experimental positive control. Note the
similarity in the intensity of the response to estrogen and the
neurotrophins in the cortical explants. Untreated PC12 and NGF-treated
PC12 cells served as negative and positive methodological controls,
respectively. Densitometric representations of the relative intensities
of the phosphorylated ERK1 and ERK2 bands are provided also
(bottom panel).
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Figure 2.
Effect of estradiol on ERK phosphorylation in
cerebellar cultures. Cerebellar explants were treated with 10 nM estradiol for 30 min or for 1 hr. Only marginal
phosphorylation of ERK was observed, whereas the neurotrophin cocktail
treatment (positive control) resulted in robust phosphorylation of ERK.
The bottom panel represents the same blot, which was
stripped and reprobed for ERK1 and ERK2 protein to verify equal loading
of protein across lanes.
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As expected, the neurotrophins also increased tyrosine phosphorylation
of ERK1 and ERK2 in the cerebral cortical explants (see Fig. 1). The
in-gel kinase assay (Fig. 3) demonstrates
that phosphorylation of the ERKs correlated with an increase in ERK activity, because estradiol activation of ERK followed a similar time
course observed in the ERK phosphorylation study (see Fig. 1).
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Figure 3.
Temporal pattern of ERK activation after estradiol
exposure. Shown is an in-gel kinase assay, using MBP as a substrate,
for the temporal pattern of ERK1 and ERK2 activation in response to 10 nM estradiol. The effects of 100 ng/ml of NGF, BDNF, or
NT-3 on ERK activation were used as controls to document the action of
all of these neurotrophins on ERK activation in cerebral cortical
explants. Untreated PC12 cells and NGF-treated PC12 cells served as
methodological controls. A densitometric representation of the relative
intensities of ERK2 activation is provided also (bottom
panel).
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Pharmacological blockade of estradiol-induced
ERK phosphorylation
The effect of estradiol on ERK phosphorylation was not affected by
the ER antagonists ICI 182,780 (Fig.
4A) or ICI 164,384 (data not shown). However, the MEK1 inhibitor PD98059 did inhibit the
effect of both estradiol and the neurotrophins on ERK phosphorylation (Fig. 5). Furthermore, treatment with the
MEK inhibitor alone also inhibited baseline phosphorylation of ERK.
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Figure 4.
Effect of ICI 182,780 on estradiol-induced ERK
phosphorylation in rat cerebral cortical explants. Cultures were
pretreated with either vehicle (DMSO, 0.1%) or 1 µM ICI
182,780 for 24 hr before being pulsed with either estradiol in
combination with ICI 182,780 or estradiol with vehicle.
A, Shown is the phospho-ERK blot documenting the
inability of ICI 182,780 to block either estradiol- or
neurotrophin-induced ERK phosphorylation. B, Documented
is the positive control for the ICI 182,780 compound performed on the
mammary tumor cell line, MCF-7, treated with either 10 nM
estradiol or estradiol in combination with 10 µm of ICI 182,780. The
bottom panels in both A and
B represent the same blot, which was stripped and
reprobed for ERK1 and ERK2 protein to verify equal loading of protein
across lanes.
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Figure 5.
Effect of the MEK inhibitor PD98059 on estrogen-
and neurotrophin-induced ERK phosphorylation. After a 5 hr
preincubation with 100 µM PD98059 the effects of both
estradiol and the neurotrophins on ERK phosphorylation were abolished
in the continued presence of the MEK inhibitor. The bottom
panel represents the same blot, which was stripped and reprobed
for ERK1 and ERK2 protein to verify equal loading of protein across
lanes.
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Estradiol-induced increase in B-Raf kinase activity
The activity of the signaling protein B-Raf, which is immediately
upstream of MEK, also was evaluated for responsiveness to estrogen
exposure in cerebral cortical explants. Both the 30 and 60 min
estradiol treatments resulted in significant increases in B-Raf
activity relative to untreated controls (Fig.
6). In addition, the activation seen
after 60 min of estradiol treatment was statistically equivalent to the
activation of B-Raf observed after 30 min of neurotrophin
treatment.
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Figure 6.
Effect of estradiol on B-Raf activity. The amount
of 32P incorporated into MBP was evaluated from lysates of
cerebral cortical explants immunoprecipitated with the B-Raf antibody
(clone C-19). Both 30 and 60 min estradiol treatment significantly
increased the kinase activity of B-Raf relative to untreated control
explants. Treatment of the explants with the neurotrophin cocktail for
30 min served as the experimental positive control and also revealed a
significant increase in B-Raf kinase activity. Values represent the
average of four samples and were calculated by subtracting the activity
level obtained from an IgG-immunoprecipitated control (background) from
the values obtained in the experimental samples. The difference (in
cpm) obtained after a control (untreated) sample was immunoprecipitated
with preimmune IgG versus being immunoprecipitated with the B-Raf
antibody is presented as the inset. Statistical analysis
was performed by using a one-way ANOVA, followed by Scheffé's
analysis for group differences (#, different from all
treated groups, p < 0.05; *difference between 30'
E2 group and 30' NT group, with p < 0.05).
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Estradiol does not induce tyrosine phosphorylation of
trk receptors
To assess whether estradiol elicits the activation of ERK
indirectly, i.e., via the release of endogenous neurotrophins that could result in the activation of trk, we investigated the
ability of estradiol to induce tyrosine phosphorylation of the cortical trk receptors. After 30 min of estradiol treatment, a time
point that elicited significant ERK phosphorylation, no increase in phosphorylation of trk receptors was observed. In contrast,
a 30 min treatment with the neurotrophin cocktail (consisting of NGF,
BDNF, NT-3, and NT-4/5) resulted in robust phosphorylation of
trk (Fig. 7).
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Figure 7.
Effect of estradiol on trk
phosphorylation. Lysates of cerebral cortical explants were
immunoprecipitated with the pan-trk (203) antibody
before Western analysis and probed with an anti-phosphotyrosine (4G10)
antibody. Shown are the effects of a 30 min pulse of 10 nM
estradiol and treatment with a 100 ng/ml neurotrophin cocktail for 30 min. Estradiol did not elicit tyrosine phosphorylation of
trk receptors, whereas the neurotrophins did. Untreated
PC12 cells and the trkA-deficient NNR-5 cell line served
as methodological negative controls, whereas NGF-treated PC12 cells
served as the prototypical positive control.
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Characterization of a putative complex containing the ER
Western analysis of cerebral cortical lysates that used a
B-Raf-specific antibody revealed significant levels of B-Raf (Fig. 8A). The intensity of
the 95 kDa band observed in the cerebral cortex was approximately
one-half that observed in PC12 cells. Interestingly, a lower molecular
weight isoform of B-Raf (68 kDa), known to be recognized by our B-Raf
antibody (clone C-19; Santa Cruz), was seen only in the cerebral cortex
and not in PC12 lysates (Fig. 8A). On the basis of
the observation by Jaiswal and colleagues (1996), which documented an
association between B-Raf and the heat shock protein hsp90 along with
the well described association between hsp90 and the ER, we evaluated
the potential association of the ER with B-Raf. Coprecipitation
experiments in which cerebral cortical lysates were immunoprecipitated
with anti-ER antibodies and subsequently probed with antibodies to
B-Raf, in fact, revealed an association between the ER and B-Raf (Fig.
8B). A band of ~95 kDa was evident in the
ER-immunoprecipitated lane and was conspicuously absent in the
IgG-immunoprecipitated lane. Although the slightly higher molecular
weight band seen in the cerebral cortex relative to the PC12 cell band
easily could be explained by the known existence of multiple B-Raf
isoforms that are present in the brain (Barnier et al., 1995), we
evaluated B-Raf kinase activity in the ER-immunoprecipitated explants
to verify that the coprecipitated band observed was, in fact,
kinase-active Raf. We found that lysates precipitated for ER did
contain kinase-active Raf for which the activity was statistically
higher than that observed in lysates immunoprecipitated with preimmune
rabbit IgG (Fig. 8C). In additional experiments we evaluated
the ER/B-Raf association in lysates from cerebral cortices of P10 rat
pups. As was seen with the explants, after immunoprecipitation with the
ER antibody, probing for B-Raf revealed a specific band (~95 kDa)
that was not observed in the IgG-immunoprecipitated control lane (Fig.
9A). Subsequent reprobing for
the ER revealed ER immunoreactivity (67 kDa) in the
ER-immunoprecipitated lane that was not observed in the IgG lane (Fig.
9B).
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Figure 8.
Coprecipitation of the ER with B-Raf in cerebral
cortical explants. A, Evaluation of B-Raf levels in the
cerebral cortical explants by Western analysis revealed significant
levels of a 95 kDa B-Raf band that migrated to a similar
position as that observed in PC12 cells. An additional band of ~68
kDa also was observed in the cerebral cortical lysates that was absent
in PC12 cell lysates. B, After immunoprecipitation with
an anti-ER antibody (ER715), probing with an anti-B-Raf antibody
revealed an association between the ER and the 95 kDa isoform of B-Raf
that was conspicuously absent in the IgG-immunoprecipitated lane.
C, Confirmation of the identity of this specific B-Raf
band as kinase-active Raf was performed by evaluating the amount of Raf
activity present in cerebral cortical explants immunoprecipitated with
the ER and comparing it with samples immunoprecipitated with preimmune
IgG. Statistical analysis that used a two-tailed Student's
t test revealed that the level of Raf activity in the
ER-immunoprecipitated samples was significantly higher than that
observed in the IgG-immunoprecipitated control
(*p < 0.05).
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Figure 9.
Coprecipitation of the ER and B-Raf in P10
cerebral cortical tissue. A, Immunoprecipitation of
cerebral cortical lysates derived from P10 rat pups with the ER
antibody, followed by Western analysis for B-Raf, revealed a similar
association of B-Raf with the ER (as seen in explants of similar age),
which was not found in the IgG-immunoprecipitated control.
B, Subsequent reprobing of the blot for the ER revealed
a specific ER (67 kDa) band that was not present in the
IgG-immunoprecipitated control lane.
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Immunoprecipitation with the anti-ER antibody, followed by probing with
anti-hsp90, also revealed the expected association of hsp90 with the ER
(Fig. 10A). The hsp90
band corresponded to the observed 90 kDa band from the positive control
(Fig. 10A) and was markedly less in the negative
control. The presence of a faint band in the negative control was
attributed to the likely nonspecific association of hsp90 with protein
A-Sepharose because of its sheer abundance in cells (1-2% of total
cellular protein). In contrast, immunoprecipitation of cerebral
cortical lysates with anti-MEK1, followed by probing with the anti-ER
antibody, failed to reveal an association between the ER and MEK1 (Fig.
10B). Similarly, reversal of the order of
immunoprecipitation and probing antibodies also revealed a lack in
association of the ER with MEK1 (Fig. 10C).
View larger version (18K):
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Figure 10.
Coimmunoprecipitation of the ER with hsp90, but
not with MEK1. A, Immunoprecipitation with the ER and
probing for hsp90 revealed an association in the ER with hsp90. The
HeLa lysate served as a positive control for the detection of hsp90.
Immunoprecipitation with either anti-MEK1 (B) or
the ER (C) failed to reveal an association
between the ER and MEK1. Recombinant estrogen receptor protein
(C) and A-431 lysate
(B) served as positive controls for the
detection of the ER and MEK1, respectively.
|
|
| |
DISCUSSION |
NGF, the prototypical neurotrophin, activates multiple signal
transduction pathways, including the MAP kinase cascade, which serve to
amplify and propagate signals generated at the cell surface into
complex biological responses. Briefly, the MAP kinase cascade is
propagated by Ras activation of Raf, followed by sequential phosphorylation and the activation of MEK and a member of the MAP
kinase family, ERK. Activated ERK then can translocate to the nucleus
to regulate transcription factors and immediate early and late response
genes (Marshall, 1995).
In the present study we sought to identify an alternative estrogen
signaling pathway that displayed similarities to the one used by growth
factors, including the neurotrophins, and specifically the MAP kinase
cascade. We document here for the first time in developing brain
tissue, which expresses wild-type (normal) ERs, the estrogen-induced
tyrosine phosphorylation of both ERK1 and ERK2. In contrast to the
recent report by Migliaccio et al. (1996), which demonstrated transient
estrogen-induced ERK activation in MCF-7 mammary carcinoma cells, the
pattern of activation observed in the cerebral cortex in our study,
although rapid in onset, was longer-lasting (up to at least 2 hr).
Because sustained activation of ERK in PC12 cells has been associated
with neuronal differentiation (Qui and Green, 1992; Marshall, 1995),
the persistent ERK phosphorylation that followed estrogen exposure
would appear to be consistent with the observed differentiative actions
of estrogen on neurites (Toran-Allerand, 1976, 1980, 1984) in this
culture system. Not all studies, however, support the sufficiency of
ERK activation for neurite outgrowth. Some studies support the
importance of ERK activation for neurite outgrowth (Qui and Green,
1992; Pang et al., 1995), whereas others oppose it (Kuo et al., 1996),
citing the importance of Raf activation instead. Because estrogen
elicits both B-Raf and ERK activation in the cerebral cortex, studies are currently underway to investigate whether estrogen activation of
these specific nodes within the MAP kinase pathway could play a role in
estrogen enhancement of neurite growth, using transfection strategies
with dominant MEK, Raf, and Ras constructs.
Sustained activation of ERK has been reported to result in nuclear
translocation of ERK (Traverse et al., 1992, 1994; Nguyen et al.,
1993), which is then capable of regulating gene transcription itself
(Davis, 1995). In contrast, mitogenic peptides (such as EGF in PC12
cells) elicit rapid but transient activation of ERK (Nguyen et al.,
1993) unaccompanied by nuclear translocation. By acting via its own
receptor, estrogen thus not only could elicit direct transcriptional
effects (the classical mechanism) but also could regulate gene
transcription indirectly via ERK activation. This alternative route of
estrogen signaling may explain the ability of estrogen to regulate
genes that do not exhibit an apparent canonical ERE (Sukovich et al.,
1994).
The lack of inducible ERK phosphorylation by estradiol in the
cerebellar explants also suggested that the estradiol-induced stimulation of ERK phosphorylation in the cerebral cortex may be a
direct consequence of the presence of adequate ER levels and that the
significantly lower levels of ER binding in the developing cerebellum
(C. D. Toran-Allerand and N. J. MacLusky, unpublished observations) were insufficient for estrogen to elicit ERK
phosphorylation. Interestingly, neither the ER antagonist ICI 182,780 nor ICI 164,384 was able to block estradiol-induced phosphorylation of
ERK in the cerebral cortical explants. Because the efficacy of this and other ER antagonists has been characterized principally on the basis of
their ability to prevent the transcriptional activation of
ERE-containing genes, it is possible that this "nongenomic" effect
of estradiol, by being upstream of any potential interaction with an
ERE, may not be influenced by these antagonists. In agreement with this
idea is the apparent lack of effect of either tamoxifen or ICI 182,780, two ER antagonists, on the effect of estrogen on MAPK in a human
neuroblastoma tumor cell line (Watters et al., 1997). In the
non-neuronal mammary tumor cell line, MCF-7, however, the ICI compound
blocked the effect of estrogen on ERK activation (Migliaccio et al.,
1996), which suggests that the effect of estrogen receptor antagonists
also may be cell type-specific, possibly a consequence of differences
in the ratios of ER subtypes within the cells.
To explore the mechanism and pathway(s) by which estradiol activated
ERK, we first addressed the possibility that exposure of the cerebral
cortical explants to estradiol elicited trk phosphorylation. Because estrogen has been reported to elicit tyrosine
phosphorylation of the EGF receptor (Reddy et al., 1992), we first
addressed the possibility that exposure of the cortical explants to
estradiol may have first elicited tyrosine phosphorylation of
trk, either directly or as a result of estrogen-induced
endogenous neurotrophin release. As such, this would initiate the
cascade of events that follows trk autophosphorylation,
eventually leading to ERK activation. Estradiol was without effect in
this regard, whereas neurotrophin exposure did elicit trk
phosphorylation, supporting the hypothesis that the effect of estradiol
is not simply attributable to the release of endogenous neurotrophins
or to trk activation. Second, the ability of the MEK1
inhibitor PD98059 to inhibit estrogen-induced phosphorylation of ERK
suggests the involvement of MEK1 in the estrogen signaling pathway.
Finally, our observation of estradiol-induced activation of B-Raf also
implicates B-Raf in the cross-coupling of the ER and neurotrophin
signaling pathways. Together, these observations support the hypothesis
that convergence of the ER and neurotrophin receptor signaling pathways
occurs downstream of trk and begins at least at the level of
B-Raf, or perhaps further upstream.
Recent studies by Jaiswal et al. (1996) have documented an
intracellular complex formed by the association of hsp90 with a member
of the trk signaling cascade, B-Raf. This intracellular complex had a molecular weight that was >300 kDa, suggesting the possibility of additional components within this complex. Because the
ER has been well documented to associate with hsp90 (Ratajczak et al.,
1990; Segnitz and Gehring, 1995), we addressed the possibility that the
ER may be part of a multimeric complex consisting of at least B-Raf,
MEK1, and hsp90. The results showed a clear association of the ER with
B-Raf and hsp90, although not with MEK. The B-Raf band observed in the
cerebral cortical explant sample seemed to migrate to a slightly higher
molecular weight (~95-96 kDa) than that observed in PC12 cells
(~94 kDa). Although multiple isoforms of B-Raf have been
documented in the brain, ranging from 86 to 99 kDa (Barnier et al.,
1995), it was necessary to ensure that the band was, in fact,
kinase-active Raf. As such, we found that ER immunoprecipitates did
contain kinase-active Raf, for which the activity was
significantly higher than that observed in IgG-immunoprecipitated cerebral cortical lysates. In addition, coimmunoprecipitation also
was performed with cerebral cortical tissue derived from P10 rats and
revealed a similar association that was absent in controls (see Fig.
9A), further supporting that the association of the ER with
B-Raf is real and not simply a consequence of the culture system.
Thus, the initial route taken to elicit rapid activation of ERK may
involve direct activation of this putative multimeric complex via dissociation and conformational changes
consequent to phosphorylation of one or more of the already identified
ER/signaling proteins associated within the complex. Alternatively,
estradiol may induce a greater association of B-Raf into
this complex. This recruitment could then instruct, via
phosphorylation, further downstream members of the cascade, such as MEK
and ERK, to become rapidly activated. Such a putative multimeric
complex or complexes, consisting of the ER and members of the MAP
kinase cascade, could serve as an intracellular junction linking the
estrogen and neurotrophin signaling pathways. Thus, cross-coupling of
the ER with growth factor signaling pathways, which appears to be a
general property of the ER (Kato et al., 1995; Bunone et al., 1996),
may argue for a mutual requirement of both the estrogen and the
neurotrophin systems to elicit their effects efficiently, if not completely.
Finally, with the discovery of ER- (Kuiper et al., 1996) we cannot
exclude the possibility that the effect of estrogen on ERK activation
may occur either via this novel receptor, a proposed membrane-associated estrogen receptor (Pietras and Szego, 1977; Pappas
et al., 1995; Karthikeyan and Thampan, 1996), or even an as yet
unidentified ER. Thus, it is possible that more than one subtype of the
ER could interact with this putative complex. The sum of these findings
suggests that, depending on the ER subtype (for example, vs ) or
cellular location (membrane vs cytosolic vs nuclear) or even on
hormonal or growth factor milieu, the ER and various signaling proteins
may coexist in a variety of multimeric associations or complexes.
Experiments directed to understand the nature of the complex(es) after
various treatments are currently underway.
Although our results document the involvement of ERK in estrogen
signaling, they do not rule out the involvement of other substrates
such as adenylate cyclase/cAMP (Aronica et al., 1994) or
Ca2+ channels (Morley et al.,
1992) in the mechanism of estrogen action. These substrates also may
act, either in parallel or by converging into the MAP kinase pathway,
to elicit estrogen-induced differentiation of neurons. Nevertheless,
the existence of ERs in multimeric complexes with growth factor
signaling proteins creates the potential for a broader scope of the
actions of estrogen and provides support for alternative signal
transduction pathways underlying the differentiative effects of
estrogen on CNS tissue. Although the traditional view of estrogen
action has been that the ligand-activated ER binds directly to EREs,
our findings could explain how estrogen and the neurotrophins, via the
intermediary of ERK translocation into the nucleus, could regulate the
same broad array of ERE-containing genes such as GAP-43 (Costello et
al., 1990; Lustig et al., 1991) and tau microtubule-associated protein
(Drubin et al., 1985; Matsuno et al., 1997) and non-ERE-containing
genes such as -tubulin (Guo and Gorski, 1988) and MAP-2 (Black et
al., 1986; Fischer et al., 1991; Lorenzo et al., 1992) involved in
neuronal differentiation and neurite growth.
| |
FOOTNOTES |
Received March 13, 1998; revised Nov. 6, 1998; accepted Nov. 25, 1998.
This work was supported in part by grants from National Institutes of
Health (National Institute on Aging, National Institute of Mental
Health, and National Science Foundation), the Alzheimer's Association/Burks B. Lapham grant, the Bader Foundation, and an Alcohol, Drug Abuse, and Mental Health Administration Research Scientist Award to C.D.T.-A. We thank Hae-Jung Chung, Cynthia Leung,
and Dr. Elisabetta Mauri for their expert technical assistance. We are
indebted to Dr. Lloyd A. Greene (Columbia University, NY) for his
technical advice, for critically reviewing this manuscript, and for
providing valuable discussions; Dr. David R. Kaplan (McGill University,
Montréal, Québec, Canada) for valuable discussions and for
the gift of the anti-pan trk 203 antibody; Dr. Robert A. Rush (Flinders University, Adelaide, Australia) for the anti-rat blocking antibodies to BDNF and NT-3; Dr. Margaret Shupnik (University of Virginia, Charlottesville, VA) for the anti-estrogen receptor antibody (C1355); and Dr. Alan E. Wakeling (Zeneca Pharmaceuticals, Cheshire, UK) for the gift of ICI 182,780 and ICI 164,384. We also
thank Drs. James Angelastro and Matthew Cunningham (Columbia University) for their technical advice.
Parts of this paper have been published previously at the 26th and 27th
Annual Meetings of the Society for Neuroscience, Washington, DC,
November 16-21, 1996, and New Orleans, LA, October 25-30, 1997, respectively.
Correspondence should be addressed to Dr. Dominique Toran-Allerand,
Department of Anatomy and Cell Biology, Columbia University College of
Physicians and Surgeons, 650 West 168th Street, BB 1615, New York, NY 10032.
| |
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[Abstract]
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K. N. Schultz, S. A. von Esenwein, M. Hu, A. L. Bennett, R. T. Kennedy, S. Musatov, C. D. Toran-Allerand, M. G. Kaplitt, L. J. Young, and J. B. Becker
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Protein Kinase C Signaling in the Hypothalamic Arcuate Nucleus Regulates Sexual Receptivity in Female Rats
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S. Bake, L. Ma, and F. Sohrabji
Estrogen Receptor-{alpha} Overexpression Suppresses 17{beta}-Estradiol-Mediated Vascular Endothelial Growth Factor Expression and Activation of Survival Kinases
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K. Vagnerova, I. P. Koerner, and P. D. Hurn
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H. Abe, K. L. Keen, and E. Terasawa
Rapid Action of Estrogens on Intracellular Calcium Oscillations in Primate Luteinizing Hormone-Releasing Hormone-1 Neurons
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P. Dewing, M. I. Boulware, K. Sinchak, A. Christensen, P. G. Mermelstein, and P. Micevych
Membrane Estrogen Receptor-{alpha} Interactions with Metabotropic Glutamate Receptor 1a Modulate Female Sexual Receptivity in Rats
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J. W. Gatson and M. Singh
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N. Vasudevan and D. W. Pfaff
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M. Singh, J. A. Dykens, and J. W. Simpkins
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I. S. Nethrapalli, A. A. Tinnikov, V. Krishnan, C. D. Lei, and C. D. Toran-Allerand
Estrogen Activates Mitogen-Activated Protein Kinase in Native, Nontransfected CHO-K1, COS-7, and RAT2 Fibroblast Cell Lines
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A. M. Kennedy, K. L. Shogren, M. Zhang, R. T. Turner, T. C. Spelsberg, and A. Maran
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M. M. McCarthy
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R. I. Fernando and J. Wimalasena
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M. K. Jezierski and F. Sohrabji
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J. Nilsen and R. D. Brinton
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A. M. Etgen and M. Acosta-Martinez
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L. Zhao, S. Chen, and R. D. Brinton
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J. Nilsen and R. D. Brinton
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Estradiol (E2) Elicits Src Phosphorylation in the Mouse Neocortex: The Initial Event in E2 Activation of the MAPK Cascade?
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R. D. Brinton
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P. M. Wise and D. B. Dubal
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K. Takeda, T. Ichiki, T. Tokunou, N. Iino, and A. Takeshita
15-Deoxy-Delta 12,14-prostaglandin J2 and Thiazolidinediones Activate the MEK/ERK Pathway through Phosphatidylinositol 3-Kinase in Vascular Smooth Muscle Cells
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D. B. Dubal, H. Zhu, J. Yu, S. W. Rau, P. J. Shughrue, I. Merchenthaler, M. S. Kindy, and P. M. Wise
Estrogen receptor alpha , not beta , is a critical link in estradiol-mediated protection against brain injury
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R. Bi, G. Broutman, M. R. Foy, R. F. Thompson, and M. Baudry
The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus
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Top
Abstract
Introduction
