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The Journal of Neuroscience, March 1, 2000, 20(5):1694-1700
Estrogen-Induced Activation of the Mitogen-Activated
Protein Kinase Cascade in the Cerebral Cortex of Estrogen
Receptor- Knock-Out Mice
Meharvan
Singh1,
György
Sétáló Jr1, 3,
Xiaoping
Guan1,
Donald E.
Frail4, and
C. Dominique
Toran-Allerand1, 2
Departments of 1 Anatomy and Cell Biology, and Centers
for Neurobiology and Behavior and Reproductive Sciences, and
2 Neurology, Columbia University College of Physicians and
Surgeons, New York, New York 10032, 3 Department of
Biology, University Medical School of Pécs, Pécs H-7643,
Hungary, and 4 Womens Health Research Institute,
Wyeth-Ayerst Research, Radnor, Pennsylvania 19087
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ABSTRACT |
We have shown previously in the developing cerebral cortex that
estrogen elicits the rapid and sustained activation of multiple signaling proteins within the mitogen-activated protein (MAP) kinase
cascade, including B-Raf and extracellular signal-regulated kinase
(ERK). Using estrogen receptor (ER)- gene-disrupted (ERKO) mice, we addressed the role of ER- in mediating this action of estrogen in the brain. 17 -Estradiol increased B-Raf activity and MEK
(MAP kinase/ERK kinase)-dependent ERK phosphorylation in
cerebral cortical explants derived from both ERKO and their wild-type
littermates. The ERK response was stronger in ERKO-derived cultures
but, unlike that of wild-type cultures, was not blocked by the estrogen
receptor antagonist ICI 182,780. Surprisingly, both the ER-
selective ligand 16-iodo-17-estradiol and the ER- selective
ligand genistein failed to elicit ERK phosphorylation, suggesting that
a different mechanism or receptor may mediate estrogen-induced ERK
phosphorylation in the cerebral cortex. Interestingly, the
transcriptionally inactive stereoisomer 17-estradiol did elicit a
strong induction of ERK phosphorylation, which, together with the
inability of the ER-- and ER--selective ligands to elicit ERK
phosphorylation, and of ICI 182,780 to block the actions of estradiol
in ERKO cultures, supports the hypothesis that a novel,
estradiol-sensitive and ICI-insensitive estrogen receptor may mediate
17-estradiol-induced activation of ERK in the brain.
Key words:
estradiol; estrogen receptor; ERK; ERKO; signal
transduction; brain; cerebral cortex
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INTRODUCTION |
The traditional view of estrogen
action is that the estrogen receptor (ER) acts as a transcriptional
modulator, requiring direct interaction with DNA. This mechanism,
however, is neither sufficient to explain the rapidity of the effects
of estrogen nor adequate to account for its wide range of
actions in the brain, which include such responses as the enhancement
of axon and dendrite (neurite) growth and differentiation
(Toran-Allerand, 1976 , 1984), neuroprotection (via multiple mechanisms)
(Behl et al., 1995; Green et al., 1996; Simpkins et al., 1997), and
influences on cognitive behaviors and functions (Singh et al., 1994;
Luine et al., 1998). We have proposed an alternative hypothesis for
estrogen action in the brain, involving interactions of the estrogen
receptor system with various growth factors, including the
neurotrophins, a class of molecules also known to be important for the
development and survival of neurons (Toran-Allerand, 1996). We
demonstrated previously colocalization of the estrogen receptor with
the neurotrophin ligands and their receptor systems
(p75NTR and the trks) in neurons of the
developing CNS (Toran-Allerand et al., 1992; Miranda et al.,
1993), providing an anatomical substrate and the potential for
interactions of estrogen with the neurotrophins. In fact, not only has
reciprocal regulation of these two receptor systems by their ligands
been described previously (Sohrabji et al., 1994), but convergence, or
cross-coupling, of their signal transduction pathways has also been
documented in the developing cerebral cortex in which estradiol
elicited the rapid and sustained activation of multiple members of the
mitogen-activated protein (MAP) kinase cascade, including B-Raf and the
extracellular signal-regulated kinases ERK1 and ERK2 (Singh et al.,
1999). Through the intermediary of ERK activation, the very rapid
effects of estrogen (Chiaia et al., 1983; Garcia-Segura et al., 1987;
Migliaccio et al., 1993), as well as the influence of estrogen on genes
that do not contain a canonical estrogen response element (ERE)
(Sukovich et al., 1994), may be better explained.
With the recent cloning of a second estrogen receptor, ER- (in which
the classical ER is now termed ER-), it became important to identify
the estrogen receptor subtype that mediates estrogen activation of ERK.
We studied the effects of 17-estradiol on cerebral cortical explants
derived from the ER- gene-disrupted (ERKO) mouse (Lubahn et al.,
1993) and show here that 17-estradiol activates B-Raf and ERK in the
cerebral cortex derived from both ERKO and their wild-type littermates,
with the ERK response being stronger in ERKO-derived cultures.
Moreover, neither the ER--selective ligand
16-iodo-17-estradiol (Shughrue et al., 1999) nor the ER--selective ligand genistein (Witkowska et al., 1997) were capable
of eliciting ERK phosphorylation. Together with the inability of the
estrogen receptor antagonist ICI 182,780 to block this action of
estradiol in ERKO tissue and the ability of the transcriptionally inactive stereoisomer 17-estradiol to elicit strong ERK
phosphorylation, our data support the hypothesis that a novel,
estradiol-sensitive and ICI-insensitive estrogen receptor may mediate
the stimulatory effect of estradiol on ERK activation in the developing
cerebral cortex.
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MATERIALS AND METHODS |
Mice. Six breeding pairs of C57BL/6J mice
heterozygous for the ER- gene disruption (Lubahn et al., 1993) were
mated for each experiment.
Genotyping. On the day of birth [postnatal day 1 (P1)],
tail snips were obtained from the pups and used for genotyping.
Briefly, tissues were digested with Proteinase K at 56°C for 90 min,
followed by a 99°C incubation for 10 min. The samples were then
vortexed vigorously, and insoluble material was pelleted in a
microfuge. Supernatants were used in a PCR reaction that used one
primer pair (primer 1, 5'-CGG TCT ACG GCC AGT CGG GCA TC-3'; primer 2, 5'-GTA GAA GGC GGG AGG GCC GGT GTC-3') for the ER- gene product (product size of 239 bp) and one primer pair (primer 2 from above with
NEO primer 5'-GCT GAC CGC TTC CTC GTG CTT TAC-3') for the neomycin
insert-containing gene product (product size of 790 bp). The PCR
program was performed as follows: 1 cycle at 94°C for 3 min, and 30 cycles of 94°C for 45 sec, 62°C for 1 min, and 72°C for 1 min 40 sec, followed by a final extension cycle of 72°C for 7 min. Products
were analyzed by agarose gel electrophoresis. Wild-type animals
revealed the smaller, 239 bp band, homozygous knock-outs showed the
larger 790 bp band, and heterozygotes displayed both bands.
Each breeding resulted in the generation of approximately six to eight
homozygous ERKO pups, approximately the same number of wild-type
animals, and almost double the number of heterozygotes. Pups homozygous
for the ERKO and wild-type littermates, but not heterozygotes, were
used to prepare cultures on P3.
Tissue culture. Hemicoronal slices (360 µm thick)
of P3 frontal and cingulate cerebral cortex derived from either
wild-type or ERKO pups were prepared and maintained by the roller tube
culture technique in steroid-deficient and phenol red-free nutrient
medium, supplemented with a cocktail of human recombinant neurotrophins (NGF, BDNF, NT-3, NT-4/5; 50 ng/ml each; Intergen, Purchase, NY) and
17-estradiol (2 nM; Sigma, St. Louis, MO), as
described previously (Singh et al., 1999). Previous observations in our
laboratory have established that the addition of estrogen and the
neurotrophins to the nutrient media optimizes the development of the
cultures (C. D. Toran-Allerand, unpublished observations).
Treatment of cultures. After 6 d in vitro, a
24 hr washout period was performed, consisting of both omitting
exogenously added 17-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 and
Rush, 1995)]. The following day, explants were pulsed with 10 nM 17-estradiol or 100 ng/ml each individual
neurotrophin (NGF, BDNF, NT-3, and NT-4/5) administered together as a
cocktail for a single 30 min time point. The neurotrophin-treated cultures served as positive controls. For the evaluation of the effect
of the transcriptionally inactive stereoisomer of 17-estradiol, 17-estradiol, cultures were pulsed with 10 nM
17-estradiol for 0, 15, 30, or 60 min.
The individual contributions of ER- and ER- to estrogen-induced
ERK phosphorylation were evaluated using the selective ligands 16-iodo-17-estradiol and genistein, respectively.
16-Iodo-17-estradiol (Hochberg and Rosner, 1980) (gift of R. B. Hochberg, Yale University, New Haven, CT), which is selective for
ER- at concentrations below 10 nM (Shughrue et al.,
1999), was administered for 30 min to the cerebral cortical explants.
Genistein (Upstate Biotechnology, Lake Placid, NY), an ER--selective
ligand (Witkowska et al., 1997), was also studied with respect to its
ability to phosphorylate ERK. Displacement curves evaluating the
displacement of 3H-estradiol from either
the ER- or ER- ligand binding domains have revealed that
genistein binds preferentially to ER- in the low to mid nanomolar
range (Witkowska et al., 1997). As such, genistein was administered to
cerebral cortical explants for 30 min at concentrations of 0.1, 1, 10, and 100 nM. However, in the presence of 10 nM
17-estradiol, 100 µM genistein, a concentration at
which genistein acts as a tyrosine kinase inhibitor (Akiyama et al.,
1987), served as a positive control, verifying the activity of the
genistein used.
To identify the effect of the estrogen receptor antagonist ICI 182,780 (gift of A. E. Wakeling, Zeneca Pharmaceuticals, Macclesfield, Cheshire, UK) on 17-estradiol-induced ERK phosphorylation, cultures were pretreated with either 1 µM ICI 182,780 or 0.1%
DMSO (vehicle control) 24 hr before pulsing the explants. After 24 hr,
cultures were treated with 10 nM 17-estradiol in the
continued presence of either the antagonist or the vehicle control. For
MEK (MAP kinase/ERK kinase) inhibition, the cultures were pretreated
with either the MEK1/2 inhibitor PD98059 (100 µM; New
England Biolabs, Beverly, MA) or 0.1% DMSO for 4-5 hr before pulsing
the cultures with the appropriate 17-estradiol or neurotrophin treatments.
Western blot analysis (ERK phosphorylation). Cultures were
harvested into protease inhibitor- and phosphatase inhibitor-containing lysis buffer and prepared for PAGE, as described previously
(Singh et al., 1999). After electrophoretic separation, proteins were transferred onto polyvinylidene difluoride membranes (0.22 µm pore size; Bio-Rad, Hercules, CA), blocked overnight with 3% BSA (Fraction V; Sigma) in Tris-buffered saline containing 0.2%
Tween-20, and probed with the following antibodies: for ERK
phosphorylation, rabbit anti-phosphoMAPK (dual phospho-specific
(Thr202/Tyr204), 1:1000; New England Biolabs); for ERK protein
determination, goat or rabbit anti-ERK1 (1:1000), or goat or rabbit
anti-ERK2 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). Antibody
binding to the membrane was detected using a secondary antibody (either
goat anti-rabbit or donkey anti-goat) conjugated to horseradish
peroxidase (1:40,000; Pierce, Rockford, IL) and visualized on
autoradiographic film using enzyme-linked chemiluminescence
(Amersham, Arlington Heights, IL). All blots depicting phosphoERK
levels were reprobed with the anti-ERK1 and ERK2 antibodies to verify
equal loading of protein across lanes, and where necessary,
densitometric analysis of the ERK protein levels were performed to
ensure similar levels of protein loaded across lanes.
B-Raf kinase assay. Cerebral cortical explants were lysed
and incubated with a rabbit anti-B-Raf antibody (Santa Cruz
Biotechnology) for 3 hr and precipitated using anti-rabbit IgG-coated
magnetic beads (Dynabeads; Dynal, Oslo, Norway). As a negative control, a preimmune IgG-immunoprecipitated cortical lysate was always run in
parallel with the experimental samples. After four washes with lysis
buffer, the B-Raf-bound beads were used as the starting material for
the B-Raf kinase assay. The assay procedure was performed according to
the protocol provided in the B-Raf kinase assay kit (Upstate
Biotechnology) and is based on the phosphorylation of myelin basic
protein (MBP) by a B-Raf-activated kinase cascade using radioactive ATP
as the final phosphate donor. Briefly, assay dilution buffer [20
mM 3-(N-morpholino) propane sulfonic acid (MOPS), pH 7.2, 25 mM -glycerol phosphate, 5 mM EGTA, 1 mM
Na3VO4, and 1 mM dithiothreitol]
and the magnesium-cold ATP cocktail were added in conjunction with 0.4 µg of inactive MEK1 and 1 µg of inactive glutathione
S-transferase-p42 MAP kinase. This mixture was then
incubated for 30 min at 30°C. Additional assay dilution buffer, MBP,
and [ -32P] ATP were added
subsequently and incubated for another 10 min at 30°C while shaking
thoroughly. After boiling the samples 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 was then counted using a scintillation counter (Searle model
Delta 300).
Statistical analysis. Resulting counts per minute
values obtained from the B-Raf kinase assay were analyzed using a
one-way ANOVA, followed by Scheffe's post hoc
analysis for group differences. Statistical analyses were performed
using the SPSS software (SPSS Inc., Chicago, IL).
Densitometric analyses. Autoradiograms were scanned using an
HP Scanjet 6200C (Hewlett Packard Company, Greeley, CO) and analyzed using 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.
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RESULTS |
A single 30 min treatment of 17-estradiol, a time point shown
previously to elicit significant ERK1 and ERK2 phosphorylation and
activation (Singh et al., 1999), was administered to cerebral cortical
explants derived from ERKO and wild-type mice. Whereas 17-estradiol
elicited ERK phosphorylation in both ERKO and wild-type cerebral cortex
(Fig. 1), ERKO cultures showed a more
robust response. ERK phosphorylation levels in ERKO tissue were
increased 29-fold in response to 17-estradiol, whereas wild-type
cultures exhibited only a fourfold increase. Interestingly, the effect
of the neurotrophins was also augmented in ERKO tissue.
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Figure 1.
17-Estradiol-induced ERK phosphorylation in
wild-type and ERKO cerebral cortex. Cerebral cortical explants derived
from either wild-type or ERKO mice were treated for a single 30 min
time period with either 10 nM 17-estradiol or a
neurotrophin cocktail (of NGF, BDNF, NT-3, and NT-4/5 at 100 ng/ml
each). Cortical lysates were immunoprecipitated for ERK protein and
subsequently probed for phosphotyrosine using the 4G10
anti-phosphotyrosine antibody (Upstate Biotechnologies). ERK
phosphorylation was seen both in wild-type and ERKO cultures, although
the extent of phosphorylation was stronger in ERKO cultures. The
bottom panel represents reprobing of the phosphotyrosine
blot for ERK protein to verify equal loading of protein across lanes.
Densitometric representation of the relative intensities of ERK
phosphorylation is also provided, revealing the significant enhancement
of 17-estradiol-induced ERK phosphorylation in ERKO cultures. Data
shown are representative of three independent experiments.
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To further evaluate the individual contributions of ER- and ER-
on ERK phosphorylation in wild-type cultures, we analyzed the effects
of the halogenated estrogen 16-iodo-17-estradiol (Hochberg and
Rosner, 1980), which has a selective affinity for mouse ER- at
concentrations below 10 nM (Shughrue et al., 1999), and of
the phytoestrogen genistein, which has a selective affinity for ER-
(Witkowska et al., 1997). 16-Iodo-17-estradiol at concentrations of 0.1, 1, and 10 nM not only failed to elicit ERK
phosphorylation but actually resulted in an ~70% reduction of
baseline ERK phosphorylation (Fig. 2).
The effect of the ER--selective ligand genistein (Witkowska et al.,
1997) was also evaluated and found to be without influence on ERK
phosphorylation at concentrations ranging from 0.1 to 100 nM (Fig. 3). However, at 100 µM, a concentration at which genistein acts as a tyrosine
kinase inhibitor (Akiyama et al., 1987), 17-estradiol-induced ERK
phosphorylation was successfully inhibited, verifying that the
genistein used was indeed biologically active. Because both 16-iodo-17-estradiol and 17-estradiol have similar affinities for ER- (Hochberg and Rosner, 1980), the inability of
16-iodo-17-estradiol to stimulate ERK phosphorylation is probably
not attributable to inadequate binding to the estrogen receptor,
particularly because a concentration as low as 0.1 nM
17-estradiol was sufficient to activate ERK (data not shown).
Interestingly, the transcriptionally inactive stereoisomer for
17-estradiol, 17-estradiol, was equally capable of eliciting
phosphorylation of ERK (Fig. 4).
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Figure 2.
Effect of 16-iodo-17-estradiol on ERK
phosphorylation in wild-type cerebral cortex. The ER--selective
ligand 16-iodo-17-estradiol was evaluated for its ability to
phosphorylate ERK [using the dual phospho-specific ERK antibody (New
England Biolabs)]. At concentrations known to selectively bind ER-,
this ligand failed to elicit ERK phosphorylation. The bottom
panel represents reprobing of the phosphoERK blot for ERK
protein to verify equal loading of protein across lanes. Densitometric
representation of the relative intensities of ERK1 phosphorylation is
also provided, demonstrating significant inhibition of ERK1
phosphorylation in response to 16-iodo-17-estradiol. Data are
representative of three independent experiments.
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Figure 3.
The effect of genistein on ERK phosphorylation in
wild-type cerebral cortical explants. Concentrations of the
ER--selective ligand genistein, ranging from 0.1 to 100 nM, were evaluated for ERK phosphorylation. Genistein
failed to elicit ERK phosphorylation at any concentration tested. As a
control, we evaluated the efficacy of the compound at a higher (100 µM) concentration at which inhibition of tyrosine kinase
activity is expected and found that genistein successfully inhibited
estradiol-induced ERK phosphorylation to below baseline levels. The
bottom panel represents reprobing of the phosphoERK blot
for ERK protein to verify equal loading of protein across lanes.
Densitometric representation of the relative intensities of ERK1
phosphorylation is also provided, documenting the lack of effect of
genistein at the nanomolar concentrations on ERK phosphorylation. Data
are representative of two independent experiments.
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Figure 4.
The effect of 17-estradiol on ERK
phosphorylation in cerebral cortical cultures. Cultures were treated
with 10 nM 17-estradiol for 15, 30, and 60 min. At each
time point evaluated, 17-estradiol elicited ERK phosphorylation. The
bottom panel represents reprobing of the phosphoERK blot
for ERK protein to verify equal loading of protein across lanes.
Densitometric analysis of the relative intensities of ERK1
phosphorylation is also provided.
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The ability of the estrogen receptor antagonist ICI 182,780 to inhibit
17-estradiol induction of ERK phosphorylation was also evaluated in
the cerebral cortex derived from both genotypes. ICI 182,780 blocked
the effect of 17-estradiol on ERK phosphorylation in wild-type
cortical cultures (Fig. 5A)
but not in those derived from ERKO mice (Fig. 5B).
Neurotrophin induction of cortical ERK phosphorylation was unaffected
by the ICI compound. The MEK1/2 inhibitor (PD98059), on the other hand,
inhibited 17-estradiol- and neurotrophin-induced ERK phosphorylation
in both wild-type and ERKO cultures (Fig.
6).
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Figure 5.
The effect of ICI 182,780 on ERK phosphorylation
in wild-type and ERKO cerebral cortex. Cultures were pretreated with
either vehicle (DMSO, 0.1%) or the ER antagonist ICI 182,780 at a
concentration of 1 µM for 24 hr before pulsing. Cultures
were pulsed with either 17-estradiol (or neurotrophins) in
combination with ICI 182,780 or 17-estradiol (or neurotrophins) with
vehicle. ICI 182,780 successfully inhibited the phosphorylation of ERK
only in the wild-type cultures (A), but no effect
was observed in ERKO cultures (B). Parallel
controls evaluating the effect of either the vehicle (0.1% DMSO) or
ICI 182,780 alone revealed no contribution to the observed effect of
either 17-estradiol or the estrogen antagonist. The bottom
panels represent reprobing of the phosphoERK blots for ERK
protein to verify equal loading of protein across lanes. Densitometric
representation of the relative intensities of ERK1
phosphorylation is also provided, revealing the significant inhibition
of 17-estradiol-induced ERK phosphorylation by ICI 182,870 in
wild-type cultures. Data are representative of two independent
experiments.
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Figure 6.
Effect of PD98059 on ERK phosphorylation in
wild-type and ERKO cerebral cortex. The MEK1/2 inhibitor PD98059 was
used to evaluate the involvement of the upstream signaling protein MEK
in the 17-estradiol-induced ERK phosphorylation seen in both
wild-type and ERKO cultures. PD98059 (100 µM)
successfully inhibited both 17-estradiol- and neurotrophin-induced
ERK phosphorylation in the cultures of both genotypes. The
bottom panels represent reprobing of the phosphoERK blot
for ERK protein to verify equal loading of protein across lanes.
Densitometric representation of the relative intensities of ERK1
phosphorylation is also provided, revealing the significant inhibition
of both 17-estradiol- and neurotrophin-induced ERK phosphorylation
with PD98059 in both wild-type and ERKO cultures. Data are
representative of three independent experiments.
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To determine whether the activation of elements upstream of ERK was
still possible after the disruption of cortical ER- expression, we
studied the effect of 17-estradiol on B-Raf activity in both genotypes. In keeping with our earlier observation that cortical B-Raf
activity in the rat was strongest in response to a 1 hr exposure of
17-estradiol (Singh et al., 1999), we found that a 1 hr treatment
with 17-estradiol similarly increased B-Raf activity in cerebral
cortical explants derived from both wild-type and ERKO mice (Fig.
7).
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Figure 7.
Effect of 17-estradiol on B-Raf kinase activity
in both wild-type and ERKO cerebral cortex. The amount of
32P incorporated into MBP was evaluated from lysates of
cerebral cortical explants immunoprecipitated with the B-Raf antibody.
A single 60 min 17-estradiol treatment significantly increased the
kinase activity of B-Raf relative to untreated control in cerebral
cortical explants derived from both wild-type and ERKO explants.
Treatment of the explants with the neurotrophin cocktail for 30 min
served as the positive experimental 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 in an IgG-immunoprecipitated control (background) from
the values obtained in the experimental samples
[*p  0.05 vs untreated control;
 p 0.05 vs estrogen
(E2)].
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DISCUSSION |
We have proposed recently a novel and alternative mechanism for
estrogen action in the developing brain that involves the activation of
multiple signaling intermediates within the MAP kinase cascade through
convergence or cross-coupling of the estrogen and neurotrophin signal
transduction pathways (Singh et al., 1999). The identification of a
second estrogen receptor, ER- (Kuiper et al., 1996; Mosselman et
al., 1996; Ogawa et al., 1998), which is expressed with ER- in
explants of the developing cerebral cortex (Toran-Allerand, unpublished
observations), raised questions regarding the identity of the estrogen
receptor subtype(s) that mediates estrogen activation of ERK. We
therefore evaluated the role of ER- and ER- in estrogen-induced
activation of the MAP kinase cascade using both the ER-
gene-disrupted mouse model (ERKO) and pharmacological agonists
previously documented to have a selective affinity for either ER- or
ER-.
The present study revealed that the disruption of ER- gene
expression in the ERKO cerebral cortex did not prevent 17-estradiol from activating members of the MAP kinase cascade, including B-Raf and
MEK-dependent ERK phosphorylation. In fact, ERK phosphorylation was
significantly stronger in ERKO cortical cultures (Fig. 1). Because the
level of ER- mRNA is approximately the same in wild-type as in ERKO
animals (Couse et al., 1997) and assuming that the level of translation
to functional ER- protein is similar, it is unlikely that the
enhanced response to estrogen is caused by a compensatory upregulation
of ER-. To further evaluate the role of ER- in the regulation of
ERK by estrogen, we analyzed the effect of the ER--selective ligand
16-iodo-17-estradiol on ERK phosphorylation. At concentrations of
10 nM and below [concentrations that bind preferentially
to mouse ER- (Shughrue et al., 1999)], this ligand failed to elicit
the phosphorylation of ERK. In fact, a significant inhibition (70%
below baseline) was observed, suggesting that ER- may serve to
mediate inhibition of ERK phosphorylation. The enhanced
response of ERK to estrogen in ERKO tissue, seen in Figure 1, may also
be consistent with this proposed inhibitory role of ER- in which, in
the absence of such inhibitory control, 17-estradiol elicits a more
robust stimulation of ERK phosphorylation.
We also found that neurotrophin-induced ERK phosphorylation was
similarly augmented in the ERKO tissue. Because certain upstream elements of the MAP kinase pathway that are activated by the
neurotrophins, such as B-Raf, exist in a multimolecular complex with
ER- (Singh et al., 1999), the disruption of ER- expression could
have consequences for neurotrophin signaling as well. We are currently
exploring the mechanisms underlying the enhanced responsiveness of ERK
to the neurotrophins in ERKO tissue.
Interestingly, the ER--selective ligand genistein (Witkowska et al.,
1997) also failed to elicit ERK phosphorylation, suggesting that ER-
may not be involved in this estrogen action either. Because no specific
end point that can be ascribed to selective ER- activation is
currently known, we were not able to verify whether, in fact, the
concentrations of genistein used in our experimental system interact
with ER- exclusively. However, in vitro binding studies
have demonstrated preferential binding of genistein to the rodent
ER- ligand binding domain (Witkowska et al., 1997) at concentrations
similar to those used in our study, providing support for its
selectivity in our rodent cerebral cortical explant system. In
contrast, others (Kuiper et al., 1998) have documented that, in a
transient gene expression assay in which both ER- and ER- were
cotransfected with an estrogen-dependent reporter system, low to mid
nanomolar concentrations of genistein could induce transcriptional
activity of both ER- and ER-. However, the inability of genistein
to elicit ERK phosphorylation despite its apparent ability to activate
both ER- and ER- only further supports our hypothesis that
neither of these known receptors are involved in the ability of
estradiol to elicit activation of the MAP kinase cascade. Moreover, we
found that the estrogen receptor antagonist ICI 182,780, which binds to
both ER- and ER- (Kuiper et al., 1997), did not disrupt
17-estradiol-induced ERK phosphorylation in the ERKO cultures.
We have also observed that 17-estradiol, a transcriptionally
inactive stereoisomer of 17-estradiol in adult reproductive tissues
with 100-fold lower affinity for the estrogen receptor (Ginsburg et
al., 1977; Merriam et al., 1980), is equally capable of eliciting
robust ERK phosphorylation in wild-type cultures of the cerebral cortex
(Fig. 4). 17-Estradiol, like its stereoisomer, has also been shown
to be a potent neuroprotective agent, with only moderate sensitivity to
estrogen receptor antagonists (Green et al., 1997) and is postulated to
act independently of currently known estrogen receptors (Behl et al.,
1997). Thus, it is possible that the ability of 17-estradiol to
phosphorylate ERK may occur via a similar alternative pathway, perhaps
involving a novel, antagonist-insensitive estrogen receptor. Although
these data suggest that a novel estrogen receptor may exist, further
analysis in mice lacking the ER- receptor (Krege et al., 1998) will
undoubtedly help test this hypothesis.
The existence of novel estrogen receptors has, in fact, been proposed
previously. These include the identification of a 112 kDa estrogen
receptor in the adult rat cerebral cortex (Asaithambi et al., 1997) and
the characterization of membrane estrogen receptors (estrogen binding
sites) in extraneural estrogen targets (Pietras and Szego, 1977; Pappas
et al., 1995; Karthikeyan and Thampan, 1996). The latter may either
represent a subpopulation of the known ER- or ER- (Pappas et al.,
1995; Razandi et al., 1999) or may be a unique, structurally distinct
estrogen receptor (Anuradha et al., 1994).
Alternatively, the ability of estradiol to elicit ERK
phosphorylation in ERKO tissue could also result from the existence of
a functional splice variant of ER- in the ERKO model (Moffatt et
al., 1998). For example, the identification of residual ER- mRNA and
immunoreactivity in ERKO mice was used to support the hypothesis that
this residual estrogen receptor could mediate the continued ability of
17-estradiol to regulate progesterone receptor expression in
specific hypothalamic regions (Moffatt et al., 1998).
Our data describe the ability of ICI 182,780 to inhibit
estradiol-induced ERK phosphorylation only in wild-type but not ERKO cerebral cortex. These findings suggest that ER- may be required to
mediate inhibitory modulation of ERK phosphorylation. Support for this
hypothesis comes from experiments in which selective activation of
ER- with 16-iodo-17-estradiol resulted in as much as a 70%
inhibition of baseline ERK phosphorylation (Fig. 2). Moreover, the role
of ER- as an inhibitory regulator could explain the enhanced
phosphorylation of ERK seen after 17-estradiol treatment in the ERKO
cerebral cortex (Fig. 1). Thus, after disruption of ER- expression
(ERKO), removal of such inhibitory control would enable estradiol to
elicit an unopposed, and hence more robust, stimulation of ERK phosphorylation.
Differences in the responsiveness of wild-type and ERKO tissue to the
estrogen receptor antagonist ICI 182,780 are not without precedent. Das
et al. (1997), in fact, showed that the effect of the catecholestrogen
4-hydroxy estradiol on uterine lactoferrin expression was not only
mediated by a potentially novel estrogen receptor but that ICI 182,780 inhibited this effect in wild-type, but not ERKO, tissue (Das et al.,
1997). Additional studies also support the existence of novel,
ICI-insensitive estrogen receptors in the rapid and nongenomic actions
of estrogens (Das et al., 1997; Gu et al., 1999). For example,
17-estradiol-induced potentiation of kainate-induced currents was
not blocked by ICI 182,780 in isolated hippocampal CA1 neurons of both
wild-type and ERKO mice (Gu et al., 1999).
Because the data described here argue against a direct role for ER-
in the mediation of estrogen-induced ERK phosphorylation, ER- must
modulate the inhibition of ERK phosphorylation by influencing the
estrogen receptor specifically responsible for the induction. Such an
influence of one receptor over another could be achieved through an
interaction (heterodimerization) between ER- and other receptor
subtypes. Heterodimerization of estrogen receptor isoforms (ER- and
ER-) has, in fact, been demonstrated (Pace et al., 1997; Pettersson
et al., 1997; Ogawa et al., 1998). We propose that inhibitory
modulation of estrogen-induced ERK phosphorylation may occur through
heterodimerization of ER- with a novel, ICI-insensitive estrogen
receptor. However, after disruption of ER- expression (as in ERKO),
no ER--containing heterodimers would exist, and this mediator of
inhibitory regulation would be absent. As a result, ICI 182,780 would
become incapable of influencing estradiol-induced phosphorylation of
ERK in ERKO cerebral cortex, a hypothesis consistent with our findings.
Convergence of estrogen- and growth factor-signaling pathways in the
brain provides a novel mechanism that may explain how estrogen and the
neurotrophins can each regulate the same growth- and neurite-related
genes, such as -tubulin (Guo and Gorski, 1988), MAP2 (Black et al.,
1986; Lorenzo et al., 1992), tau microtubule-associated protein (Drubin
et al., 1985; Ferreira and Caceres, 1991), and growth-associated
protein 43 (Federoff et al., 1988; Lustig et al., 1991).
Estrogen activation of the MAP kinase cascade may also be particularly
relevant for cognition and its associated disorders, including such
neurodegenerative diseases as Alzheimer's disease (AD). Both estrogen
exposure (Jaffe et al., 1994; Xu et al., 1998) and activation of the
MAP kinase cascade (Mills et al., 1997; Desdouits-Magnen et al., 1998)
have been shown to reduce the potential of forming -amyloid, a
protein of considerable focus in the pathophysiology of AD (Selkoe,
1994), by upregulating -secretase activity. Thus, estrogen
activation of the MAP kinase cascade may be very important not only for
neuronal differentiation but may also explain how, through regulation
of amyloid metabolism, estrogen could exert some of its known
neuroprotective effects in neurodegenerative disorders such as AD
(Fillit et al., 1986; Paganini-Hill and Henderson, 1994; Tang et al.,
1996; Simpkins et al., 1997).
| |
FOOTNOTES |
Received Sept. 27, 1999; revised Nov. 30, 1999; accepted Dec. 13, 1999.
This work was supported in part by grants from National Institutes of
Health (National Institute on Aging), National Institute of Mental
Health, National Science Foundation, the Alzheimer's Association/Burks
B. Lapham Grant, an Alzheimer's Association/T.L.L. Temple Foundation
Discovery Award, the Bader Foundation, and an Alcohol, Drug Abuse, and
Mental Health Administration Research Scientist Award, all to
C.D.T.-A. We thank H.-J. Chung and M. F. Warren for their
expert technical assistance. We also thank our colleagues at Columbia
University: L. A. Greene for his technical advice and for
providing valuable discussions, N. J. MacLusky for his valuable
suggestions and critically reviewing our manuscript, and J. Angelastro
and M. Cunningham for their technical advice. We also thank R. A. Rush (Flinders University, Adelaide, Australia) for the anti-rat
blocking antibodies to BDNF and NT-3, A. E. Wakeling (Zeneca
Pharmaceuticals, Macclesfield, Cheshire, UK) for the gift of ICI
182,780, R. B. Hochberg (Yale University, New Haven, CT) for the
gift of 16-iodo 17-estradiol, and the Bioresource staff at
Wyeth-Ayerst for providing the ERKO animals.
Correspondence should be addressed to Dr. Dominique Toran-Allerand,
Department of Anatomy and Cell Biology, 650 West 168th Street, BB 1615, New York, NY 10032. E-mail: cdt2{at}columbia.edu.
| |
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[Abstract]
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J. H. Morrison, R. D. Brinton, P. J. Schmidt, and A. C. Gore
Estrogen, Menopause, and the Aging Brain: How Basic Neuroscience Can Inform Hormone Therapy in Women
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J. L. Turgeon, M. C. Carr, P. M. Maki, M. E. Mendelsohn, and P. M. Wise
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S. Musatov, W. Chen, D. W. Pfaff, M. G. Kaplitt, and S. Ogawa
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M. Singh, J. A. Dykens, and J. W. Simpkins
Novel Mechanisms for Estrogen-Induced Neuroprotection.
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M. M. Khan, M. Hadman, C. Wakade, L. M. De Sevilla, K. M. Dhandapani, V. B. Mahesh, R. K. Vadlamudi, and D. W. Brann
Cloning, Expression, and Localization of MNAR/PELP1 in Rodent Brain: Colocalization in Estrogen Receptor-{alpha}- But Not in Gonadotropin-Releasing Hormone-Positive Neurons
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[Abstract]
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C. D. Toran-Allerand, A. A. Tinnikov, R. J. Singh, and I. S. Nethrapalli
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[Abstract]
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K. D. Yi, J. Chung, P. Pang, and J. W. Simpkins
Role of Protein Phosphatases in Estrogen-Mediated Neuroprotection
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J. A. Arreguin-Arevalo and T. M. Nett
A Nongenomic Action of 17{beta}-Estradiol as the Mechanism Underlying the Acute Suppression of Secretion of Luteinizing Hormone
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A. Hasbi, B. F. O'Dowd, and S. R. George
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P. Mendez, I. Azcoitia, and L. M. Garcia-Segura
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M. Karl, M. Potier, I. H. Schulman, A. Rivera, H. Werner, A. Fornoni, and S. J. Elliot
Autocrine Activation of the Local Insulin-Like Growth Factor I System Is Up-Regulated by Estrogen Receptor (ER)-Independent Estrogen Actions and Accounts for Decreased ER Expression in Type 2 Diabetic Mesangial Cells
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S. V. Vaidya, S. E. Stepp, M. E. McNerney, J.-K. Lee, M. Bennett, K.-M. Lee, C. L. Stewart, V. Kumar, and P. A. Mathew
Targeted Disruption of the 2B4 Gene in Mice Reveals an In Vivo Role of 2B4 (CD244) in the Rejection of B16 Melanoma Cells
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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
Endocrinology,
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[Abstract]
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M. R. I. Williams, T. Dawood, S. Ling, A. Dai, R. Lew, K. Myles, J. W. Funder, K. Sudhir, and P. A. Komesaroff
Dehydroepiandrosterone Increases Endothelial Cell Proliferation in Vitro and Improves Endothelial Function in Vivo by Mechanisms Independent of Androgen and Estrogen Receptors
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A. K. Murashov, R. R. Islamov, R. J. McMurray, E. S. Pak, and D. A. Weidner
Estrogen increases retrograde labeling of motoneurons: evidence of a nongenomic mechanism
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I. M. Abraham, M. G. Todman, K. S. Korach, and A. E. Herbison
Critical in Vivo Roles for Classical Estrogen Receptors in Rapid Estrogen Actions on Intracellular Signaling in Mouse Brain
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R. I. Fernando and J. Wimalasena
Estradiol Abrogates Apoptosis in Breast Cancer Cells through Inactivation of BAD: Ras-dependent Nongenomic Pathways Requiring Signaling through ERK and Akt
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C. D. Toran-Allerand
Minireview: A Plethora of Estrogen Receptors in the Brain: Where Will It End?
Endocrinology,
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R. Dominguez, C. Jalali, and S. de Lacalle
Morphological Effects of Estrogen on Cholinergic Neurons In Vitro Involves Activation of Extracellular Signal-Regulated Kinases
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C. L. Koski, S. Hila, and G. E. Hoffman
Regulation of Cytokine-Induced Neuron Death by Ovarian Hormones: Involvement of Antiapoptotic Protein Expression and c-JUN N-Terminal Kinase-Mediated Proapoptotic Signaling
Endocrinology,
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[Abstract]
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J. Qiu, M. A. Bosch, S. C. Tobias, D. K. Grandy, T. S. Scanlan, O. K. Ronnekleiv, and M. J. Kelly
Rapid Signaling of Estrogen in Hypothalamic Neurons Involves a Novel G-Protein-Coupled Estrogen Receptor that Activates Protein Kinase C
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I. M. Abraham, S.-K. Han, M. G. Todman, K. S. Korach, and A. E. Herbison
Estrogen Receptor {beta} Mediates Rapid Estrogen Actions on Gonadotropin-Releasing Hormone Neurons In Vivo
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D. Herring, R. Huang, M. Singh, L. C. Robinson, G. H. Dillon, and N. J. Leidenheimer
Constitutive GABAA Receptor Endocytosis Is Dynamin-mediated and Dependent on a Dileucine AP2 Adaptin-binding Motif within the {beta}2 Subunit of the Receptor
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J. K. Wong, H. H. Le, A. Zsarnovszky, and S. M. Belcher
Estrogens and ICI182,780 (Faslodex) Modulate Mitosis and Cell Death in Immature Cerebellar Neurons via Rapid Activation of p44/p42 Mitogen-Activated Protein Kinase
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K. J. Ho and J. K. Liao
Nonnuclear Actions of Estrogen
Arterioscler Thromb Vasc Biol,
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[Abstract]
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K. M. Dhandapani and D. W. Brann
Protective Effects of Estrogen and Selective Estrogen Receptor Modulators in the Brain
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C. D. Toran-Allerand, X. Guan, N. J. MacLusky, T. L. Horvath, S. Diano, M. Singh, E. S. Connolly Jr, I. S. Nethrapalli, and A. A. Tinnikov
ER-X: A Novel, Plasma Membrane-Associated, Putative Estrogen Receptor That Is Regulated during Development and after Ischemic Brain Injury
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K. J. Ho and J. K. Liao
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P. J. Shughrue, G. R. Askew, T. L. Dellovade, and I. Merchenthaler
Estrogen-Binding Sites and Their Functional Capacity in Estrogen Receptor Double Knockout Mouse Brain
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E. F. Rissman, A. L. Heck, J. E. Leonard, M. A. Shupnik, and J.-A. Gustafsson
Disruption of estrogen receptor beta gene impairs spatial learning in female mice
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M. R. I. Williams, S. Ling, T. Dawood, K. Hashimura, A. Dai, H. Li, J.-P. Liu, J. W. Funder, K. Sudhir, and P. A. Komesaroff
Dehydroepiandrosterone Inhibits Human Vascular Smooth Muscle Cell Proliferation Independent of ARs and ERs
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R. X.-D. Song, R. A. McPherson, L. Adam, Y. Bao, M. Shupnik, R. Kumar, and R. J. Santen
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Mol. Endocrinol.,
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I. S. Nethrapalli, M. Singh, X. Guan, Q. Guo, D. B. Lubahn, K. S. Korach, and C. D. Toran-Allerand
Estradiol (E2) Elicits Src Phosphorylation in the Mouse Neocortex: The Initial Event in E2 Activation of the MAPK Cascade?
Endocrinology,
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E.-M. Tsai, S.-C. Wang, J.-N. Lee, and M.-C. Hung
Akt Activation by Estrogen in Estrogen Receptor-negative Breast Cancer Cells
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S. M. Belcher and A. Zsarnovszky
Estrogenic Actions in the Brain: Estrogen, Phytoestrogens, and Rapid Intracellular Signaling Mechanisms
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R. Bi, M. R. Foy, R.-M. Vouimba, R. F. Thompson, and M. Baudry
Cyclic changes in estradiol regulate synaptic plasticity through the MAP kinase pathway
PNAS,
October 25, 2001;
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P. S. Green, S.-H. Yang, K. R. Nilsson, A. S. Kumar, D. F. Covey, and J. W. Simpkins
The Nonfeminizing Enantiomer of 17{beta}-Estradiol Exerts Protective Effects in Neuronal Cultures and a Rat Model of Cerebral Ischemia
Endocrinology,
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J. M. Hall, J. F. Couse, and K. S. Korach
The Multifaceted Mechanisms of Estradiol and Estrogen Receptor Signaling
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R. Bi, M. R. Foy, R.-M. Vouimba, R. F. Thompson, and M. Baudry
Cyclic changes in estradiol regulate synaptic plasticity through the MAP kinase pathway
PNAS,
November 6, 2001;
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TOP
ABSTRACT
INTRODUCTION
