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and ERß Exhibit Unique Pharmacologic Properties When Coupled to Activation of the Mitogen-Activated Protein Kinase Pathway1
Departments of Pharmacology (C.B.W., D.M.D.) and Psychiatry and Behavioral Sciences (R.A.S., D.M.D.), and Graduate Program in Neurobiology and Behavior (S.R.), University of Washington, School of Medicine, Seattle, Washington 98195
Address all correspondence and requests for reprints to: Christian B. Wade, University of Washington, Department of Pharmacology, Box 357280, 1959 Northeast Pacific Street, Seattle, Washington 98195. E-mail: cwade{at}u.washington.edu
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Abstract |
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or rat ERß and examined their ability to couple to activation of MAPK
in response to 17ß-estradiol (17ß-E2) and other
ligands. For both receptors, addition of E2 resulted
in a rapid phosphorylation of MAPK. Activation of MAPK in
ER-transfected cells was partially and completely blocked by the
antiestrogens tamoxifen and ICI 182,780, respectively. In
ERß-transfected cells, MAPK activation was less sensitive to
inhibition by tamoxifen and ICI 182,780. We have also observed that, in
this model system, a membrane-impermeable estrogen
(BSA-E2) and 17-E2 were both able
to activate MAPK in a manner similar to E2 alone. Here
also, ICI 182,780 blocked the ability of BSA-E2 to
activate MAPK through ER, but failed to block ERß-mediated
effects. BSA-E2 treatment, however, failed to activate
nuclear estrogen-response-element-mediated gene transcription. These
data show that these nuclear ERs are necessary for estrogen’s effects
at the membrane. This model system will be useful in identifying
molecular interactions involved in the rapid effects mediated by the
ERs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß) are
members of the superfamily of nuclear steroid hormone receptors, which
are able to regulate the transcriptional activity of target genes by
interacting with different DNA response elements. Classically, ERs are
thought to bind ligand, dimerize, and activate transcription at
estrogen response elements (EREs). It has also been shown that both ERs
can interact with fos/jun to form a complex that augments transcription
at activator protein-1 (AP-1) sites (1). In
addition to its ability to mediate gene transcription, estrogen also
elicits rapid, nontranscriptional effects (2), including
changes in intracellular calcium (3), increases in cAMP
(4, 5), modulation of neuronal calcium channels
(6) and potassium channels (7),
phosphorylation of cAMP response element binding protein (CREB)
(8, 9), and activation of mitogen-activated protein kinase
(MAPK) (10, 11, 12). The rapidity of these responses and the
ability of membrane-impermeable estrogens to activate MAPK
(11) have led to the proposal that these actions may
involve a novel, membrane-bound ER (13). Alternatively,
recent evidence has shown that the known ERs may mediate many of these
rapid effects. Transient transfection of ER into COS cells results
in estrogen-induced interaction of receptor with the nonreceptor
tyrosine kinase, src, and activation of MAPK
(10). A recent report (14) has demonstrated
that ER or ERß, transiently transfected into Chinese
hamster ovary (CHO) cells, can localize to the membrane and mediate
several of the previously reported rapid effects of estradiol
(E2), including MAPK activation, increases in cAMP,
and phosphatidylinositol hydrolysis.
This laboratory has previously reported that a 24-h pretreatment with
E2 can protect neurons from glutamate toxicity
(15). More recently, we have shown that this protection
may involve activation of the MAPK pathway via activation of
src (12). The MAPK pathway is further
implicated as a crucial player in estrogen-dependent neuroprotection,
by the finding that tyrosine phosphorylation of sos1, grb2, and
p21ras-GTP activating protein all increase within
minutes of estrogen exposure. Neuroprotection can be blocked by the
addition of the MEK1 inhibitor, PD98059, as well as ICI 182,780,
suggesting an ER-dependent mechanism. To further characterize the
kinetics and ligand specificity of ER-mediated MAPK activation, we have
transfected Rat-2 fibroblasts and selected stable cell lines expressing
either ER or ERß. This model system will be useful in dissecting
the molecular mechanisms underlying the rapid actions of estrogen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-E2, BSA-E2, and tamoxifen) were
obtained from Sigma. BSA- conjugated
E2 was purified following the protocol described by
Stevis et al. (1999), using a Microcon cartridge with a
3-kDa cut-off (Amicon, Beverly, MA). ICI 182,780 was obtained from
Zeneca Pharmaceuticals (Wilmington, DE).
Vector constructs
The complementary DNAs (cDNAs) encoding both human ER
(16) and rat ERß (17) were subcloned into
the R1 site of the expression vector, pcDNA3 (Invitrogen,
Carlsbad, CA), which contains the Neo resistance gene for selection in
G418.
Generation of stable ER and ERß cell lines
Plates (100 mm) of Rat-2 cells (50–60% confluent) were
transfected with 5 µg ER/pcDNA3 or ERß/pcDNA3 using standard
calcium phosphate procedures described elsewhere (Promega Corp., Madison, WI). After 48 h, cells were split 1:10 into
media containing 500 µg/ml G418 (Life Technologies, Inc., Gaithersburg, MD). Single G418-resistant colonies were
picked after 10 days, and assayed by Western blotting for
E2-mediated MAPK activation using an antiactive MAPK
antibody (Phospho-p44/p42 MAPK; New England Biolabs, Inc.,
Beverly, MA).
MAPK phosphorylation
A monoclonal antibody that recognizes the dual phosphorylation
state of MAPK on Thr202 and Tyr204 (Phospho-p44/42 MAPK; New England Biolabs, Inc.) was used to detect activation of MAPK.
Whole-cell lysates were prepared in immunoprecipitation buffer (2.5
mM HEPES, pH 7.5, 10% glycerol, 5 mM EDTA, 5
mM EGTA, 100 mM NaCl, 100 mM Na
pyrophosphate, 50 mM NaF, 0.1 mM
NaVO4, 1% Triton X-100, 1 mM
benzamidine, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, 2 µg/ml pepstatin) and separated,
under denaturing conditions, by SDS-PAGE on 10–20% gradient
polyacrylamide Tris-glycine gels (Novex Corp., San Diego,
CA). After transfer to polyvinylidene difluoride (Immobilon-P;
Millipore Corp., Bedford, MA), the membrane was blocked
with 5% nonfat dry milk in 0.2% Tween-20 in Tris-buffered
saline (T-TBS) for 1 h at room temperature. The membrane was then
placed in a 1:2500 dilution of the antiactive MAPK antibody in 5%
nonfat dry milk/T-TBS overnight, at 4 C, with mild agitation. Membranes
were then washed 4 x 5 min in T-TBS before incubation in a 1:2500
dilution of horseradish peroxidase-conjugated rabbit antimouse IgG
diluted in 1% milk/T-TBS. The membranes were then washed again before
proceeding to visualization by enhanced chemiluminescence (ECL)
(Renaissance; NEN Life Science Products, Boston, MA).
Membranes were stripped (Re-blot Western blot Recycling Kit; Chemicon,
Temecula, CA) and reblotted with a polyclonal antibody against
extracellular-regulated kinase 2 (ERK2) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:5000, washed, and
incubated in horseradish peroxidase-conjugated goat antirabbit IgG
diluted in 1% milk/T-TBS (Santa Cruz Biotechnology, Inc.), followed by enhanced chemiluminescence detection. The
resulting film samples were scanned and analyzed with an image analysis
program (NIH Image; Scion Corporation, Frederick, MD). Data are
presented as a ratio of phospho-ERK2/total ERK2 in the sample,
normalized to control.
Western blot for ER and ERß
A mouse monoclonal antibody (Clone TE111.5D11: Neomarkers,
Fremont, CA) was used to detect ER at a final concentration of 1
µg/ml and incubated for 2 h at room temperature. A rabbit
polyclonal antibody (Upstate Biotechnology, Lake Placid,
NY) was used to detect ERß at a final concentration of 1 µg/ml.
Each membrane was exposed to film for 5 min. All other procedures were
performed similarly to the Western blot protocol above.
Transient transfections
Rat-2 cells (
5 x 105 cells/well)
were plated into 6-well plates and transfected with 1.5 µg/well
tk-ERE-luciferase (kindly provided by Dr. Peter Burbach, Rudolf Magnus
Institute, University of Utrecht) and 1.0 µg/well of either human
ER or rat ERß, or the expression vector pcDNA3 using the
diethylaminoethyl/Dextran protocol (Promega Corp.), with
slight modifications. One microgram of pCH110, a ß- galactosidase
reporter (Amersham Pharmacia Biotech, Uppsala, Sweden),
was added to each well to normalize for transfection efficiency. After
24 h in DMEM, cells (70% confluent) were transfected at 37 C
in diethylaminoethyl/Dextran and 50 µM chloroquine
phosphate (Sigma) in media supplemented with 10%
charcoal-stripped calf serum (Sigma), for 4 h. Media
was then replaced, and cells were allowed to recover for 24 h
before pharmacological manipulation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or rat ERß, and stable cell lines were selected
for G418 resistance. A series of clonal populations were isolated and
screened for estrogen-sensitive MAPK activation. A single
G418-resistant clone was then selected for each receptor transfected
(ER and ERß). We first examined the cells for the presence of
ER and ERß, by Western blot. As expected, untransfected Rat-2
cells do not express either ER or ERß; whereas the appropriate
receptor is detected, using an equivalent exposure time, in ER- or
ERß-transfected cells (Fig. 1
).
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and ERß in activating MAPK,
transfected Rat-2 cells were treated with 10 nM
17ß-E2 for 15 min and assayed for estrogen-dependent
MAPK activation by immunoblotting with an antiactive MAPK antibody that
detects only the dual-phosphorylation state of ERK1/2 (Fig. 2, upper bands). Blots were
then stripped and incubated with an anti-ERK2 antibody to control for
the total amount of protein in each lane (Fig. 2, lower
bands). Both ER and ERß were able to mediate
estrogen-sensitive MAPK activation in Rat-2 cells (Fig. 2). No
activation was observed in untransfected Rat-2 cells, demonstrating
that MAPK activation is dependent on the transfected receptors. The
magnitude of the response (4- to 6-fold) is consistent with that
previously observed in primary neuronal cultures (12). We
chose a dose of E2 that we found to be saturating in
this cell type, namely 10 nM, as determined by
the dose response profile (Fig. 3).
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). MAPK activation by ER peaked at 15
min but returned to near-control levels by 30 min. ERß activation of
MAPK displayed slightly different kinetics, reaching a maximum near 15
min, with substantial activation still detectable after 1 h. Both
ER- and ERß-transfected cells displayed a 4- to 6-fold increase in
MAPK activation when treated with E2.
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and ERß (19) and in complex
promoters (20). To compare the effects of various SERMs,
we next examined their ability to modulate E2-mediated
MAPK activation (Fig. 5). Tamoxifen is a
partial agonist and is able to reduce E2-induced MAPK
activation mediated by ER. In contrast to the effects seen in
Rat-2/ER cells, tamoxifen seems to potentiate ERß-mediated MAPK
activation when combined with E2. The dose response
profile of tamoxifen-mediated MAPK activation differed significantly
between the two receptors (Fig. 6A).
Doses as high as 1 µM only weakly activated MAPK in
ER-transfected cells, whereas ERß more robustly increased MAPK
phosphorylation in response to this same dose. We also determined the
effect of ICI 182,780 on membrane- localized ERs. ICI 182,780
blocks ER but fails to block ERß-mediated effects, even at doses
as high as 1 µM (Fig. 6B). These data suggest that the
nuclear ERs are able to function at the cell membrane.
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-E2 has been shown to be
neuroprotective in SK-N-SH cells (21), but it only weakly
activates transcription. To determine whether 17-E2
is able to activate MAPK, we treated ER- and ERß-transfected cells
with increasing doses of 17-E2 for 15 min (Fig. 7). After treatment with
17-E2, ER-transfected cells displayed increased
MAPK phosphorylation, compared with cells treated with vehicle
alone.
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or ERß (Fig. 8A), at a dose similar to that of
E2 treatment alone (10 nM). Similarly to
free estrogen, this increase in MAPK phosphorylation is blocked by ICI
182,780 in ER-transfected cells but not in those stably expressing
ERß.
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). To
confirm the absence of free E2 in our filtered
BSA-E2, we examined its ability to activate
ERE-mediated transcription using an ERE-luciferase reporter assay in
transiently transfected Rat-2 cells (Fig. 9). In accordance with our previous
observations in SK-N-SH cells (11), BSA-conjugated
E2 was unable to activate an ERE reporter construct.
Thus, if there is any unbound estrogen in this filtered
BSA-E2 preparation, the concentration of free estrogen
is insufficient to activate nuclear receptor mediated
transcription.
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-E2 to weakly activate ER-mediated
transcription at EREs has been demonstrated in some cell types. Because
17-E2 was able to activate MAPK for both ER- and
ERß-expressing Rat-2 cells, we measured its transcriptional activity
in transiently transfected Rat-2 cells (Fig. 9). In these cells,
17-E2 weakly activated ERE-mediated transcription,
compared with 17ß-E2. The transcriptional activity
of both 17-E2 and 17ß-E2 was
blocked by ICI 182,780. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism by which the ERs mediate the rapid cytoplasmic effects of estrogen is not well understood. E2 can elicit a rapid and sustained activation of MAPK in various cells that seems to be mediated by the nonreceptor tyrosine kinase, src (10, 12). In addition to src, a number of nonreceptor tyrosine kinases of the src family might be involved in mediating MAPK activation. The ER has also been shown to interact with B-raf and heat shock protein 90 (27), suggesting the possibility of an ER-containing complex involving multiple intracellular proteins.
ER and ERß are differentially expressed in a variety of tissues.
High levels of ER are found in pituitary, kidney, epididymis, and
adrenal; whereas ERß is found in prostate, lung, and bladder.
Expression of both subtypes overlaps in the brain, ovary, testis, and
uterus (28). Much of the work that has been done on MAPK
activation by ERs has been observed in tissues containing both ER
and ERß. In addition, ER and ERß can heterodimerize in
vitro (29), thus complicating an analysis of the
molecular mechanisms of estrogen action. To examine each receptor
individually, we have stably transfected Rat-2 cells, which lack ERs,
with cDNA clones encoding either ER or ERß. We found that
transfection of either receptor is sufficient to impart
estrogen-sensitivity for MAPK phosphorylation. Though the dual
phosphorylation of MAPK has been shown to be essential for MAPK
activity, we did not directly measure this in the present study. The
magnitude of MAPK activation did not differ significantly between the
two receptors.
The temporal aspects of activation of MAPK differed between ER and
ERß. ER displayed maximal MAPK activation at 15 min, but this
activation dropped to control levels shortly thereafter.
ERß-transfected cells also reached a peak at 15 min, but significant
activation was still detectable for at least 1 h after estrogen
treatment. Though the kinetics of the two receptors seem to differ
slightly, this is in accordance with our earlier observations in
primary cortical neuronal cultures, where maximal MAPK activation is
seen near 30 min and is sustained for at least 1 h. Furthermore,
the duration and timing of MAPK activity have been recently reported as
being important to NGF-induced cellular differentiation
(18). The rapid and sustained activation of MAPK by ERß
is similar to the kinetics observed in primary neuronal cultures
(12), a finding that is consistent with ERß being the
predominant ER in the adult cortex (30). Perhaps this
sustained pattern of MAPK activation by ERß could be important in
determining how estrogen will influence cellular function and/or
differentiation.
The molecular mechanisms of action of many antiestrogens, such as
tamoxifen and ICI 182,780, are not completely understood. Tamoxifen is
used clinically in the treatment of breast cancer and is presumed to
exert its therapeutic benefits by acting as an ER antagonist. It has
been demonstrated in vivo that tamoxifen functions as an
agonist of ERß-mediated effects at AP-1 elements (19).
Because estrogen would be expected to reduce tamoxifen’s effects on
ERß AP-1-mediated gene transcription, the pharmacology of
tamoxifen’s effects on MAPK activation might play a role in this mode
of transcription. Our results show that, with respect to MAPK
activation, tamoxifen seems to function as an agonist for ERß, even
slightly potentiating ERß-mediated effects of E2.
Tamoxifen does not seem to be an agonist for ER-mediated MAPK
phosphorylation.
The antiestrogen ICI 182,780 and the other ICI compounds were once
thought to be pure antagonists of estrogen action for both ER and
ERß. ICI 182,780 is thought to inhibit receptor dimerization
(31) and decrease the half-life of the ER proteins. More
recent evidence suggests that the ICI compounds, like tamoxifen, can
behave as agonists of ERß-mediated AP-1 gene transcription (1, 19). We have found that whereas ICI is able to antagonize
E2-induced MAPK activation by ER, ERß is
relatively insensitive to this same compound. This result is consistent
with observations in primary cortical explants (27), which
express predominantly ERß in the adult brain (30).
We also treated transfected Rat-2 cells with 17-E2,
a stereoisomer of E2 that has been shown to be
neuroprotective in SK-N-SH cells (21). We found that
17-E2 was able to activate MAPK in a manner similar
to that of 17ß-E2 for both ER and ERß.
17-E2 has approximately one third of the binding
affinity of E2 for ER. It is also a weak agonist
for ERß at ERE sites (32). In our cells,
17-E2 can weakly activate ER-mediated transcription
at ERE elements, and this activation is susceptible to blockade by ICI
182,780. It is possible that membrane-associated ERs might display
similar affinities for both 17-E2 and
17ß-E2. The neuroprotective actions of
17-E2 are not thought to be transcriptional in
nature (21). However, its ability to activate MAPK might
contribute to this protection in certain cell types.
There has been considerable speculation on the existence of a novel,
membrane-associated ER. Previous results from our laboratory have shown
that MAPK activation in SK-N-SH cells can occur after treatment with
the membrane-impermeable estrogen, BSA-E2
(11). When we treated transfected Rat-2 cells with
BSA-E2, we were able to show an increase in MAPK
activation similar to that with E2 treatment for both
ER and ERß. This is in addition to the activation we previously
observed with E2 alone. BSA-E2 is
unable to activate ERE-mediated transcription, thereby demonstrating
the membrane-specific nature of this compound. Thus, our results agree
with those of Razandi et al. (14), who reported that
introduction of ER into cultured cells imparts sensitivity to the
membrane-based actions of estrogen.
There has also been recent interest in the ability of ICI 182,780 to
modulate membrane-based ERs differently than for the nuclear population
of receptors. Here, ICI might be acting to potentiate
membrane-initiated events. It has been demonstrated that ICI 182,780
elicits an increase in cAMP production (4), an effect that
also occurs after BSA-E2 treatment (33).
We found that ICI blocks BSA-E2-induced MAPK signaling
in ER-, but not ERß-, transfected cells. This pharmacological
pattern is identical for both BSA-E2 and free estrogen
treatment.
It has recently been demonstrated that many commercially available
BSA-E2 preparations contain substantial quantities of
free, unconjugated E2. Purification of the BSA
conjugate, using a method previously documented to eliminate free
estrogen (25), did not alter its ability to activate MAPK.
Though it is apparent that BSA-E2 is not the ideal
reagent for studies of membrane-based ERs, our results demonstrate that
it is able to activate rapid signaling pathways similarly to that of
free estrogen. Furthermore, BSA-E2 treatment of
ER-transfected cells was blocked by ICI 182,780, whereas
ERß-transfected cells remained insensitive to ICI blockade.
The downstream targets of estrogen-mediated MAPK activation are just
beginning to be explored. This effect on cell signaling is likely to
interact with other rapid actions of estrogen, such as PKA activation,
as well as coupling to the classical genomic effects of the hormone.
MAPK has been shown to directly phosphorylate the ER (34),
which modulates its transcriptional activity. Phosphorylation of the
receptor via src is also involved in regulating receptor
dimerization and transcription at ERE elements. MAPK activation may
also contribute to the ability of E2 to increase CREB
phosphorylation (9), thus enhancing cAMP response
element-mediated gene transcription. In summary, ER and ERß are
sufficient to mediate estrogen-dependent MAPK activation. The effect
examined here is too rapid to evoke gene transcription as a possible
mechanism of MAPK activation. The unique pharmacology of ER and
ERß for rapid signaling of this type presents an intriguing new
molecular target for future drug development.
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Acknowledgments |
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Footnotes |
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Received August 23, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -ß mRNA in the
rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
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