From the Division of Endocrinology, Veterans Affairs Medical
Center, Long Beach, Long Beach, California 90822 and the
Departments of Medicine and § Pharmacology,
University of California, Irvine, California 92717
Received for publication, June 8, 2002, and in revised form, October 11, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Estradiol (E2) rapidly stimulates signal
transduction from plasma membrane estrogen receptors (ER) that are G
protein-coupled. This is reported to occur through the transactivation
of the epidermal growth factor receptor (EGFR) or insulin-like growth
factor-1 receptor, similar to other G protein-coupled receptors. Here, we define the signaling events that result in EGFR and ERK activation. E2-stimulated ERK required ER in breast cancer and endothelial cells
and was substantially prevented by expression of a dominant negative
EGFR or by tyrphostin AG1478, a specific inhibitor for EGFR
tyrosine kinase activity. Transactivation/phosphorylation of EGFR by E2
was dependent on the rapid liberation of heparin-binding EGF (HB-EGF)
from cultured MCF-7 cells and was blocked by antibodies to this ligand
for EGFR. Expression of dominant negative mini-genes for
G Steroid hormones such as estrogen are essential to the development
and reproductive functions of prokaryotic and eukaryotic organisms.
Traditionally, steroid hormone action was exclusively attributed to the
binding of nuclear receptors and the subsequent transactivation of
target genes that led to cell biological effects (1). More recently, it
has become clear that steroids rapidly act on cells, in seconds to
minutes, effects that are classified as "nongenomic" (reviewed in
Ref. 2). For estrogen, this has been attributed in most cells to
binding a population of receptors that exists within caveolar rafts and
other domains in the plasma membrane (3-5). It is at the plasma
membrane that estradiol (E2)1-liganded estrogen receptors
(ER) physically associate with the scaffold protein, caveolin-1 (5), and a variety of proximal signaling
molecules, including G proteins (6, 7), Src and Ras (8, 9), and
B-Raf (10). This results in the activation of cascades of signal
transduction, mainly evolving from G protein activation. Comparable
with many other G protein coupled receptors (GPCR), G protein
activation by ER (6, 7) leads to the stimulation of phospholipase C
(11), protein kinase C (12), ERK (9), and phosphatidylinositol 3-kinase
and nitric-oxide synthase (13). These positive signaling effects are
cell context-specific, and in some cells, estrogen inhibits
cytokine-related signal transduction to cell differentiation,
proliferation, migration, or cell death (14-17).
What is the nature of the membrane ER, and how does it enact signal
transduction? Current evidence favors the idea that the membrane and
nuclear ER are the same protein. Antibodies directed against many
epitopes of the classical ER As a GPCR, the membrane ER associates with and activates several G
proteins. In transfected CHO cells, membrane ER The utilization of EGFR by E2/ER to signal results from a linked series
of events involving multiple upstream molecules, only some of which
have been defined. For instance, we do not know the range of G
proteins that can be activated to cross-talk to EGFR activation, and it
is not clear what signals immediately downstream of G proteins are
important. Src participates in the transactivation of EGFR in response
to other GPCR ligands and is probably upstream of HB-EGF shedding (29),
but its exact role and requirement for ER signaling is unclear.
Furthermore, although matrix metalloproteinase (MMP) activation is
required for HB-EGF liberation (and subsequent EGFR activation), the
identity of the required MMP(s) is mainly undefined, especially as
regards ER signaling. These issues are addressed in the studies
described here. Finally, much of the interaction between GPCRs and EGFR has examined ERK activation. Thus, we sought additional signaling molecules in several cell types and the structural requirements within
ER that utilize this interactive mechanism following endogenous ER
ligation by E2.
Materials--
Antibodies and substrate for kinase
activation/activity were from Santa Cruz Biotechnology (Santa Cruz,
CA). PD 98059 was a generous gift from Dr. Alan Saltiel (Parke-Davis).
LipofectAMINE was from Invitrogen. Primary cultures of bovine aortic EC
were prepared and used as previously described (30). In transfection studies, EC were generally used in passages 4 and 5, based upon the
previous observation that this greatly increases the transfection efficiency of these cells. Breast cancer cell lines were obtained from
ATCC. The cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 or RPMI 1640 with serum until 48 h prior to experimentation, when they were placed in serum-free conditions and in
medium without phenol red. Gelatin was from Sigma, and kinase
substrates were from Upstate Biotechnology, Inc. (Lake Placid, NY) or
Santa Cruz Biotechnology. PP2, Src family kinase inhibitor, and GM6001,
a matrix metalloproteinase (MMP) inhibitor, were from Calbiochem (San
Diego, CA).
Kinase Activity Assays--
For ERK or p38 Transient Transfections--
MCF-7, HCC-1569, ZR-75-1, or
bovine aortic endothelial cells (passages 4 and 5) were grown to
40-50% confluence and then transiently transfected with 1.5 µg
(each well of 6-well plates) or 10 µg of fusion plasmid DNA (100-mm
dishes). Plasmids included wild type mouse ER Gelatin Zymography Substrate Cleavage and Antisense Studies for
MMP Activity--
MMP activity, as secreted into the medium of
cultured MCF-7 cells, was analyzed by substrate gel electrophoresis
(zymography). The cells were synchronized in serum-free medium for
24 h and then incubated in medium with or without 10 nM estrogen for 2 min at 37 °C in a CO2
incubator. The cell medium was removed, concentrated 20-fold by
ultrafiltration, and mixed with native gel sample buffer
(Bio-Rad), and the proteins were separated by electrophoresis on an 8%
gel co-polymerized with 1 mg/ml gelatin (Sigma). Active MMP-2 and MMP-9
(Calbiochem) was loaded into additional lanes on the gel. After
electrophoresis, the gels were washed in 2.5% Triton X-100 at room
temperature for 1 h and incubated for16 h at 37 °C (in 0.05 M Tris, pH 7.5, 5 mM CaCl2, 0.02%
NaN3). The gel was stained with 0.5% Coomassie Blue and
destained in 10% acetic acid, 10% propanol. The study was repeated
twice. Gelatinolytic activity appears as a clear band on a blue
background. For the fluorescent substrate assay, MCF-7 cells were
synchronized for 24 h and then incubated without or with 10 nM estrogen for 2 min. The incubation medium was
concentrated 10-fold, and 1 ml of assay buffer (100 mM
Tris, pH 7.5, 100 nM NaCl2) containing 5 µM of the Mca-Pro-Leu-Dpa-Ala-Arg-NH2
substrate for MMP-2/MMP-9 was added and then incubated at 37 °C for
3 h. Excitation at 328 nm and emission at 393 nm were determined
in a fluorimeter. To implicate MMP-2 and MMP-9 in the shedding of
HB-EGF, the cells were incubated with antisense (ASO) or scrambled
antisense (MSO) with the same base composition for each of the two
MMPs. The oligonucleotides were: MMP-2, ASO, CCGGGCCATTAGCGCCTCCAT, and
MSO, TCACCGCGGTACGCATGCCCT; and MMP-9, ASO, CAGGGGCTGCCAGAGGCTCAT, and
MSO, GCGAGCTAGGACTGTGCAGCC. The oligonucleotides were added with
LipofectAMINE for 5 h, and the cells were recovered overnight and
synchronized in the absence of serum for 12 h. Transfection
efficiency exceeded 60%, based upon co-expression of PEGFPc2. Western
blot studies were carried out to confirm the efficacy of the ASO but
not the MSO to inhibit specific protein production. Studies of
E2-induced signaling were then carried out in cells expressing the
various oligonucleotides.
Western Blot for HB-EGF and EGFR
Phosphorylation--
Subconfluent, transfected, or nontransfected
cultured bovine aortic endothelial cells were serum-deprived for
24 h and then incubated under various conditions for 10 min with
inhibitors followed by 10 min of treatment with stimulants. This
included several 17- Activation of ERK by E2 Requires an ER and the Activation of EGFR
by HB-EGF--
We first established that E2 required both the presence
of an ER and the activation of EGFR to signal to ERK. HCC-1569 cells lack ER, and the cells did not respond to E2 with ERK activation (Fig.
1A, lanes
1 and 2). When ER
What ligand for EGFR is involved in the transactivation of this
receptor by E2? Although there are many members of the EGF family that
can bind the EGFR, HB-EGF has often been implicated in the setting of
GPCR signaling via this receptor (36). To examine this, we first
determined whether E2 could stimulate the secretion of HB-EGF,
determined by Western blot. As seen in Fig. 2A, E2
dose-responsively induced a significant enhancement of HB-EGF
shedding/secretion from the MCF-7 cells after 3 min of incubation. This
was prevented by ICI182780 and by GM6001, an MMP inhibitor. To
determine that HB-EGF was the important ligand for EGFR signaling to
ERK, we incubated the MCF-7 cells with 10 nM E2, in the
presence or absence of antibody to HB-EGF. In the setting of this added
antibody, E2 could not significantly activate ERK (Fig. 2B).
In contrast, antibody to TGF Matrix Metalloproteinases 2 and 9 Are Activated and Are Necessary
for Signaling by E2--
Current evidence supports the idea that GPCRs
activate MMP activity, thereby liberating HB-EGF from the cell matrix,
leading to the transactivation of the EGFR (36, 37). Therefore, MMP activation represents the step immediately upstream of HB-EGF liberation. In many cell paradigms, including E2 action, the precise MMP(s) activated by GPCR signaling are unknown. We therefore showed that E2 activates MMP activity by demonstrating that the incubation medium from MCF-7 cells treated with E2 for 2 min induces the cleavage
of substrate specific for MMP-2 and MMP-9 (Fig.
3A). In contrast,
substrate specific for MMP-13 or MMP-3 was not cleaved by the
E2-treated cell medium (data not shown), even though breast cancer
cells produce these proteolytic enzymes. We then sought to further
identify the MMPs by carrying out gelatin zymography. E2 treatment of
the cultured MCF-7 cells for 2 min led to the increased secretion and
activation of MMP-2 and -9 (Fig. 3B, first and
second lanes). To corroborate the identify of the digested gelatin band activities, active MMPs (Calbiochem) were also run in
parallel on a separate gel (data not shown). Functionally, activation
of MMP activity was necessary for E2-induced ERK. This was shown in
that the MMP inhibitor completely reversed the ability of E2 to
activate ERK in both MCF-7 and ZR-75-1 cells (Fig. 3C, left and right panels). This compound did not
affect EGF-induced ERK activation, supporting the idea that MMP-related
events occur upstream to EGFR activation in this pathway.
Although E2 activates these two MMPs, it is not clear that they are
responsible for E2-induced HB-EGF shedding. We therefore used ASO or
MSO, with the latter comprised of the same base composition as the ASO
for MMP-2 and MMP-9, and expressed them in MCF-7 cells. First, we
validated the constructs by showing that the ASO (but not the MSO) for
MMP-2 or MMP-9 inhibited the respective protein production in a
dose-related manner (Fig. 4A,
left panel). Similarly, we validated the function of the
MMP-2 or MMP-9 to specifically inhibit only the intended protein target
(Fig. 4A, right panel). Using these ASO and MSO,
we next determined whether MMP-2 and MMP-9 each contributed to HB-EGF
shedding and ERK activation (Fig. 4, B and C).
Each ASO significantly down-regulated E2-induced HB-EGF liberation, and
expressing the ASO to both MMPs completely blocked this E2 action. The
ASO to MMP-2 almost completely prevented the ability of E2 to activate
ERK in MCF-7 cells, whereas the ASO to MMP-9 was also substantially
able to prevent this signaling; neither MSO had any effect, and the
results were similar to those in EC. E2/ER stimulation of MMP-2 and
MMP-9 may therefore underlie several important actions in breast
cancer, including signaling through ERK to cell proliferation and
survival (5, 9). Metalloproteinase activation also contributes to the
disengagement of cells from matrix, a necessary initial step preceding
invasion and migration behaviors (38). MMP-2 and MMP-9 are well
recognized to contribute to these events in various contexts (38,
39).
Specific G Proteins Are Involved in E2-induced Transactivation of
EGFR--
It has previously been established that E2 can activate
G Calcium, PLC, and PKC Activities Mediate E2-induced MMP
Activation--
The signaling through the identified G proteins
potentially leads to the activation of MMP activity and the subsequent
downstream signaling through EGFR. We examined which signal pathways
immediately downstream of G protein activation that we
identified here could mediate MMP activation. E2-induced MMP activity
was significantly inhibited by EDTA, an extracellular calcium chelator,
but was not affected by BAPTA-AM (Fig. 2A). This
indicates that calcium entry through surface channels, but not the
mobilization of intracellular calcium, contributes to E2-induced MMP
activation. It has previously been shown that E2 activates several
calcium channels that lead to an influx of calcium into the cell (40,
41), and this can result from G
We also found that soluble inhibitors of PLC and PKC (calphostin C and
U-73122, respectively) significantly prevented E2 activation of MMP
activity (Fig. 3A). This is consistent with our
identification here of G Role of Src in Shedding of HB-EGF--
It has been documented that
E2-liganded ER complex with and activate the Src tyrosine kinase, and
this is necessary for E2 stimulation of ERK (8, 17). Src could
potentially play a role both upstream and downstream of EGFR
activation. We therefore determined where Src activation is required
for the proximal signaling induced by E2, leading to EGFR
transactivation. As shown in Fig. 3B, E2-induced MMP-2 and
MMP-9 activation and secretion at 2 min (first lane versus second
lane). This was substantially prevented by the Src family kinase
inhibitor, PP2 (third lane), or by expressing a specific
dominant negative Src construct, pRC-csrc-K298M (26) (fifth
and sixth lanes compared with first and
second lanes). Thus, these results define a novel role for
Src in E2-induced signaling from the membrane, and we suggest that this
molecule may play a similar role in other GPCR-induced activation of
EGFR through this mechanism.
ER Is Required for Proximal Signaling Events--
We earlier
showed that E2 requires an ER to activate ERK (Fig. 1). To further
support the idea of the necessity of ER presence for E2 action, we
expressed ER The Role of EGFR in E2 Activation of Multiple Signaling
Pathways--
Most studies invoking the role of EGFR in GPCR signaling
have examined ERK activation. Regarding E2 signaling, ERK and cAMP generation are the two pathways that have been identified to require EGFR activation (24, 25), but this has only been established in breast
cancer cells. To further define the role of EGFR in E2-induced
signaling from membrane ER, we examined breast cancer cells and EC,
cells that express endogenous ER (9, 20). In MCF-7 cells, we found that
E2 activated protein kinase B (AKT) (Fig.
7A). This was
substantially prevented by the soluble inhibitor of MMP activity and by
tyrphostin AG1478, implicating the EGFR. These two compounds had no
effects by themselves (data not shown). In EC, we previously showed
that E2 activates the p38 E Domain Activation of Signaling--
What structural aspect of
the membrane ER is necessary for activation of the signal cascade that
results in EGFR activation and ERK up-regulation? This is an important
issue, and assuming that the membrane and nuclear proteins are the same
(6), there is no typical catalytic or kinase domain sequence present in
ER The ability of E2 to signal from membrane ER is increasingly
appreciated as being important to the effects of this sex steroid. E2
triggers calcium increases in seconds and rapidly activates PKC and
adenylate cyclase. Downstream activation of several kinases then leads
to cell biological effects in a variety of cell types (44). Ischemia
reperfusion injury of muscle in rats is limited by E2, and this occurs
through the physical association of ER with phosphatidylinositol
3-kinase, the subsequent up-regulation of kinase activity, and the
generation of NO (13). E2 acts as a survival factor for neurons (27),
breast cancer (16, 45), and osteoblasts (17) while suppressing
osteoclast differentiation (14). The sex steroid also acts as a
survival and angiogenesis-promoting factor for EC (33). These effects
are related to the modulation of ERK, JNK, and p38 MAP kinases,
regulated through membrane ER. Recently, it was shown that the
administration of antibodies to ER Now established for a variety of GPCRs, membrane ER localize
substantially to caveolae (3-5). Here, they can physically complex with or activate signal molecules, including G proteins, receptor tyrosine kinases (insulin-like growth factor-1 receptor and EGFR), nonreceptor tyrosine kinases (Src family), and a variety of adapter and
threonine/serine kinase proteins. This probably occurs on a scaffold
platform provided by caveolin-1 (47, 48) and in part related to
tyrosine 14 phosphorylation of this protein (49). Interestingly, this
tyrosine is the principal substrate for Src kinase action (49), and the
ability of E2 to activate Src at the membrane (8, 17) may therefore
contribute to assembling the mature signalsome upon ER ligation by E2.
Caveolin-1 can bind to and promote the assemblage of G proteins, Src,
Grb7, Raf, Ras, MEK, and the EGFR at the plasma membrane (48).
Caveolin-1 also facilitates ER translocation to the plasma
membrane and localization within the caveolae microdomain (5).
In this way, localizing ER and the signaling molecules to a confined
area could augment the ability of E2/ER to transactivate EGFR,
resulting in the stimulation of ERK activity (this work and Ref. 25).
However, upon GPCR ligation, caveolin probably dissociates from binding
to the EGFR, leading to the activation of this receptor tyrosine kinase
(50).
Although some details of the mechanisms of EGFR transactivation by ER
(or any GPCR) are known, there are several aspects that are not clear.
We found that the ability of membrane ER to activate Gq and G
i blocked E2-induced,
EGFR-dependent ERK activation, and G
also
contributed. G protein activation led to activation of matrix
metalloproteinases (MMP)-2 and -9. This resulted from Src-induced MMP
activation, implicated using PP2 (Src family kinase inhibitor) or the
expression of a dominant negative Src protein. Antisense
oligonucleotides to MMP-2 and MMP-9 or ICI 182780 (ER antagonist) each
prevented E2-induced HB-EGF liberation and ERK activation. E2 also
induced AKT up-regulation in MCF-7 cells and p38
MAP kinase
activity in endothelial cells, blocked by an MMP inhibitor, GM6001, and
tyrphostin AG1478. Targeting of only the E domain of ER
to the
plasma membrane resulted in MMP activation and EGFR transactivation.
Thus, specific G proteins mediate the ability of E2 to activate MMP-2
and MMP-9 via Src. This leads to HB-EGF transactivation of EGFR and
signaling to multiple kinase cascades in several target cells for E2.
The E domain is sufficient to enact these events, defining additional
details of the important cross-talk between membrane ER and EGFR in
breast cancer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor identify membrane ER by
immunocytochemistry (18). Expression of antisense DNA to the
"nuclear" ER also abrogates the detectable expression of membrane
ER in cells containing endogenous receptor (19). In CHO cells,
expression of a single cDNA for either ER
or ER
produces both
membrane and nuclear receptor populations and results in E2 activation
of signal transduction from the membrane (6). In many cell types,
endogenous membrane ER have been identified (15, 18, 20) and appear to
reflect the localization of receptors that also have the capacity to
translocate to the nucleus. The structural aspects of the membrane ER
that allow it to activate signaling molecules are not well defined.
Assuming that the sequence of the nuclear receptor is the same as the
membrane ER, there is no catalytic or kinase sequence inherent to the
structure. Recent evidence favors the idea that the E domain of the
membrane ER is essential (and perhaps sufficient) for activation of the ERK cascade (5), leading to cell survival (17). Additionally, the AF-1
domain of ER
has been identified to interact with the adapter
protein, Shc, in whole cell homogenates (21). Thus, the membrane ER
acts similarly to many other GPCR that also lack catalytic or kinase
domains yet signal to important events in cell biology.
or ER
co-precipitates with and activates G
s and
G
q proteins (6). This leads to the expected downstream
signaling to cAMP and inositol 1,4,5-trisphosphate generation,
signaling that has been shown in cells expressing endogenous ER (22,
23). In EC, endogenous membrane ER physically associates with
G
i and activate endothelial nitric-oxide
synthase; this probably takes place within caveolae (7). Additionally,
it has been proposed in breast cancer cells that E2/ER transactivates
the epidermal growth factor receptor (EGFR), leading to the downstream
signaling to ERK activation (24, 25). This occurs through the
activation of G
, the liberation of heparin-binding EGF (HB-EGF),
which results in the binding and activation of the EGFR, and the
subsequent stimulation of the ERK signaling cascade. In some of these
respects, the membrane ER acts similarly to a wide range of GPCR (26).
However, it was further proposed in breast cancer cells that E2 in some
undefined way activates the orphan GPCR, GPR30, to stimulate signaling, and this interaction does not require ER (25). These latter data are
not in concert with many studies from other laboratories, indicating
that E2 requires an ER for signaling from the membrane in various cell
types (5, 6, 8, 20, 27, 28).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
activity assays,
the cells were synchronized for 24 h in serum- and growth
factor-free medium. The cells were then exposed to E2 for 8 (ERK) or 15 (p38) minutes, with or without additional substances, as previously
described (30, 33). The cells were lysed, and lysate was
immunoprecipitated with protein A-Sepharose conjugated to antiserum for
p38 or ERK. Immunoprecipitated kinases were washed and then added to
the proteins ATF-2 (for p38) or myelin basic protein (for ERK)
for in vitro kinase assays. This was followed by SDS-PAGE
separation and autoradiography/laser densitometry. In addition, the
E2-induced phosphorylation of AKT kinases at 10 min was
determined to assess activation. Cultured cell lysates were pelleted
and dissolved in SDS sample buffer, boiled, separated, and then
transferred to nitrocellulose. Phosphorylated kinase proteins were
detected using phospho-specific monoclonal antibodies (Santa Cruz) and
the ECL Western blot kit (Amersham Biosciences). Equal samples from the
cells were also immunoprecipitated, and immunoblots of the precipitated
kinase protein from each experimental condition were determined to show
equal gel loading. All of the experiments were repeated two or three times.
(31) (kindly provided
by Dr. Ken Korach) PRK5-HER, a dominant negative EGF receptor construct
(kindly provided by Dr. A. Ullrich (32), a dominant negative Src
construct, pRC-csrc-K298M (kindly provided by Drs. Louis Luttrell and
Robert Lefkowitz (26), a dominant negative, truncated
-adrenergic
receptor kinase plasmid (BARK1-CT pRK5) from Dr. Walter Koch (34), and
truncated G
subunit plasmids, serving as specific dominant negative
constructs for Gs, Gq, Gi,
G12, and G13 (35). Transfection was carried out
using LipofectAMINE (Invitrogen). The cells were incubated with
liposome-DNA complexes at 37 °C for 5 h, followed by overnight recovery in culture medium containing 10% fetal bovine serum, 24 h of synchronization in serum-free medium, and then treatment with E2
with or without other substances.
-E2 concentrations, ICI 182780 (1 µM), and 100 nM GM6001, a broad MMP
inhibitor. The cells were lysed, and antibodies to HB-EGF or EGFR
(tyrosine 1138) (1:50 dilution) were conjugated to Sepharose beads and
then added to the cell lysate for 2 h at 4 °C. After pelleting
and washing, the samples were electrophoretically separated on a 7%
SDS gel, transferred to nitrocellulose, and immunoblotted. Detection
utilized the ECL kit (Amersham Biosciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was expressed in these cells, 17-
-E2 (lane 4), but not 17-
-E2 (lane 8),
was capable of activating ERK, and this was substantially blocked by
the ER antagonist, ICI182780 (lane 5). As a positive
control, these cells express the EGFR and appropriately respond to EGF
(lane 7). The requirement of ER is similar to our previous
findings in CHO-K1 cells (6). We then asked whether E2 activation of
ERK depends upon EGFR tyrosine kinase activity. We examined this in
MCF-7 and ZR-75-1 breast cancer cells and EC (all with ER).
Tyrphostin AG1478, specifically directed against the EGFR tyrosine
kinase function, prevented EGFR-induced ERK activation in both MCF-7
and ZR-75-1 cells (Fig. 1B, left and
center panels). Importantly, tyrphostin AG1478 also substantially prevented the ability of E2 to activate ERK in the three
cell types (Fig. 1B, all panels, lanes 2 versus lanes 6). To corroborate this finding, we expressed a
dominant negative EGFR (31) in MCF-7 cells, and E2 was much less
effective in stimulating this MAP kinase, compared with cells
expressing the empty vector (control) (Fig. 1C).
View larger version (26K):
[in a new window]
Fig. 1.
E2 activates ERK via ER and EGFR.
A, 17- -E2 activates ERK only when ER is present. HCC-1569
cells (ER negative) were incubated with 10 nM
17-
-E2 or were transfected to express wild type mouse ER
(mERa) and then incubated with 17-
-E2 or 17-
-E2, and
ERK activity (against myelin basic protein) was determined after
8 min in an in vitro tube assay as described under
"Experimental Procedures." Immunoblots of total ERK protein are
shown below the activity. The bar graph represents three
combined experiments. *, p < 0.05 for control
versus E2 or EGF; +, p < 0.05 for E2
versus E2 with ICI182780 (ER antagonist). B,
inhibition of EGFR tyrosine kinase function with tyrphostin AG1478
prevents E2-induced ERK activation in MCF-7 cells (left
panel), in ZR-75-1 cells (center panel), or in
endothelial cells (right panel). The cells were incubated as
described above with 17-
-E2 with or without a specific EGFR
tyrosine kinase inhibitor, and ERK activity was determined. Each
bar graph represents three combined experiments. *,
p < 0.05 for control versus E2 or EGF; +,
p < 0.05 for E2 versus E2 with ICI182780
(ER antagonist) or E2 or EGF versus either E2 or EGF with
AG1478 (tyrphostin). C, expression of a dominant negative
EGFR (EGFR (DN)) prevents E2-induced ERK activation in MCF-7
cells. The cells were transfected to transiently express PRK5-HER
dominant negative EGFR, recovered overnight in 10% serum, and 24 h after cell recovery, E2 activation of ERK was determined after 8 min
of incubation. *, p < 0.05 for control
versus E2; +, p < 0.05 for E2
versus PRK5-HER transfected cells incubated with E2.
-1, another ligand for the EGFR, had no
effect on E2-induced ERK, and the antibodies by themselves did not
affect basal ERK activity. Similarly, antibody to HB-EGF (but not to
TGF
-1) prevented E2-induced phosphorylation of the EGFR (Fig.
2C). Identical findings were determined from EC incubated
with E2 (data not shown). These results support the interactions of
secreted HB-EGF with EGFR, leading to ERK activation in breast cancer
and vascular cells. The data also support ER-mediated, MMP-dependent release of HB-EGF.
View larger version (15K):
[in a new window]
Fig. 2.
E2 rapidly stimulates HB-EGF release via
matrix metalloproteinase action and ER. A, MCF-7 cells
were incubated with E2, 0.1-10 nM with or without
ICI182780, or 100 nM GM6001, an MMP inhibitor, for 3 min.
The HB-EGF shed into the medium was determined by Western blot. The bar graph is three
experiments combined. *, p < 0.05 for control
versus E2; +, p < 0.05 for E2.
B, antibody to HB-EGF but not TGF blocks E2-induced ERK
activation. MCF-7 were incubated with 10 nM E2 with or
without 10 µg/ml antibody to HB-EGF or TGF
, and ERK activity was
determined after 8 min. The antibodies alone had no effect on ERK
activity. C, HB-EGF but not TGF
antibody blocks the
E2-induced transactivation/phosphorylation of EGFR. The cells were
incubated with 10 nM E2 with or without antibodies for 8 min, and lysate was subjected to SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with an antibody to tyrosine 1173 of
the EGF receptor. *, p < 0.05 for control
versus E2 or E2 plus TGF
antibody; +, p < 0.05 for E2 versus E2 with HB-EGF antibody.
View larger version (35K):
[in a new window]
Fig. 3.
E2 activates matrix metalloproteinase 2 and 9 secretion and activity. A, cells were incubated with or
without E2 with or without BAPTA (intracellular calcium
inhibitor), EDTA (chelates extracellular calcium), a specific PLC
inhibitor, U73122, or a specific PKC inhibitor, calphostin C for 2 min.
Cleavage of substrate for MMP-2/MMP-9 by the medium from MCF-7 cells
incubated with 10 nM E2 for 2 min was determined by
spectroflurometry. The data are from triplicate determinations in a
representative experiment, repeated twice. B, MCF-7 cells
were incubated with E2 with or without PP2 (soluble Src inhibitor) or
with E2 in cells transfected to express a dominant negative Src
(pRC-csrc-K298M). By gelatin zymography (see "Experimental
Procedures"), active (act) MMP-9 and MMP-2 are shown,
along with the inactive (pro) MMP-2 protein.
M.W., molecular weight. C, matrix
metalloproteinase or ER inhibition prevents E2-induced ERK activation.
MCF-7 cells (left panel) and ZR-75-1 cells (right
panel) were incubated with E2 ± ICI182780 or GM6001
(MMPI), and ERK activity was determined. EGF was
also added as a control. The bar graph is three experiments
combined. *, p < 0.05 for control versus E2
or EGF; +, p < 0.05 for E2 versus E2 with
MMPI. MMPI, matrix metalloproteinase inhibitor;
MBP, myelin basic protein.
View larger version (27K):
[in a new window]
Fig. 4.
Matrix metalloproteinases 2 and 9 mediate
E2/ER effects. A, validation of ASO to MMP-2 and MMP-9.
MCF-7 cells were incubated with ASO or MSO with LipofectAMINE, and
MMP-2 or MMP-9 protein was detected by Western blot, 24 h after
transfection. The left panel shows the dose-responsive
effects of the ASO to MMP-2 (top panel) or to MMP-9
(bottom panel) to inhibit the intended protein production,
but no effect of MSO was found. The right panel shows the
specificity of an ASO for only its target MMP. Control is in the
presence of E2. B and C, HB-EGF shedding
(B) or ERK activation (C) in response to E2 is
prevented by an MMP-2 or MMP-9 ASO but not MSO. HB-EGF shedding was
carried out in MCF-7 cells, whereas ERK activation was determined in
MCF-7 (left panel) or EC (right panel). The data
are representative of three experiments, except for the bar
graph, where three experiments are combined. *, p < 0.05 for control versus E2; +, p < 0.05 for E2 versus E2 with MMP-2 or MMP-9 ASO.
s and G
q, as well as G
i
in several cell models (6, 7). Therefore, one or more G proteins
activated by E2 could ultimately result in EGFR signaling to ERK. To
examine this issue, we expressed mini-genes for G
subunits of
Gs, Gi, G12, G13, and
Gq, constructs that have been shown to act as dominant
negatives for specific endogenous G protein subunit activation (35). As
seen in Fig. 5A, ERK
activation in response to E2 in cells expressing the control plasmid,
G
ir (lane 3), was substantially
prevented after expressing the inhibitory mini-genes for
G
i and G
q (lanes 4 and 5).
However, dominant negative constructs for the
subunits of
Gs, G12, and G13 had insignificant
effects on this signaling. We also expressed a C-terminal truncated
-adrenergic receptor kinase, pRK5-BARK1-(495-689), that inhibits
G
signaling (33). Expression of this construct significantly but
incompletely prevented the ability of E2 to activate ERK and HB-EGF
liberation (Fig. 5B). Upon expressing ER
in HCC-1569
cells, E2 could now activate ERK in a G
i,
G
q, and G
-dependent fashion (Fig.
5C). Therefore, both G
and G
subunits contribute to
the ability of E2/ER to activate the signaling pathway that ultimately
results in EGFR transactivation.
View larger version (22K):
[in a new window]
Fig. 5.
G subunit protein
activation is required for E2-induced ERK activation in MCF-7
cells. A, expression of dominant negative mini-genes
for G
q and G
i but not G
s,
G12, or G13 prevents E2-induced ERK. The cells
were transfected to express truncated G
subunits, serving as
dominant negatives, then recovered, and exposed to E2 10 nM
for 8 min. ERK activity was determined. Lanes 1 and
2 are nontransfected cells; lane 3 is
E2-stimulated ERK after control plasmid transfection. The bar
graph represents three experiments combined. *, p < 0.05 for control versus E2, or control versus
E2-incubated, G
ir-transfected cells. +,
p < 0.05 for E2-incubated,
G
ir-transfected cells versus E2 in
G
i or G
q mini-gene expressing cells.
B, G
contributes to E2-induced ERK activation. The
cells were transiently transfected to express a dominant negative,
C-terminally truncated
-adrenergic receptor kinase (BARK1-CT), the
cells were recovered for 24 h, and then ERK activation by E2 was
determined. A representative experiment, repeated twice, is
shown.
q or G
activation.
q and G
as mediating
E2-induced ERK activation, because PLC and PKC up-regulation results
from the activation of these G protein subunits. We previously showed
that E2 can activate G
q, PLC, and inositol
1,4,5-trisphosphate generation via membrane ER (6), and E2 has been
described to stimulate PKC activity in several cell types (reviewed in
Ref. 42). PKC-dependent signaling in growth plate
chondrocytes mediates E2-induced regulation of these cells, and
originates from membrane action of the steroid (43). These findings
link the most proximal signaling events to later events (MMP activation
and HB-EGF shedding), mediating EGFR transactivation.
in HCC-1569 cells and determined the proximal signaling
events implicated. We first demonstrated that expression of the
dominant negative G
i and G
q mini-genes substantially blocked E2-induced ERK, compared with kinase activity in
the presence of the control (inactive) construct, G
ir
(Fig. 6A).
Similarly, expression of C-terminal truncated
-adrenergic receptor
kinase (BARK1) also down-regulated E2/ER-induced ERK, whereas the
truncated mini-gene for G
13 was without effect (similar to control). We also examined HB-EGF secretion and found that E2
stimulated the secretion of this receptor ligand only when ER was
expressed (Fig. 6B, lanes 1 and 2 versus lanes 3 and 4). ICI182780 and
MMP inhibition significantly prevented the stimulation of HB-EGF
secretion. Finally, we found that in the presence (but not in the
absence) of ER
, E2 stimulated the activation of MMP-2 and MMP-9
(Fig. 6C). This was partially dependent upon extracellular calcium, PLC, and PKC signaling. These data strongly support the idea
that the classical ER
is required for E2 to activate rapid signaling
in breast cancer.
View larger version (38K):
[in a new window]
Fig. 6.
ER is necessary for E2-induced proximal
signaling. A, specific G protein subunits are required
for ER-induced ERK. HCC-1569 cells were co-transfected to express ER
or pcDNA3 and truncated G
subunits or the C-terminal truncated
-adrenergic receptor kinase (BARK1-CT). The cells were recovered and
then incubated with 10nME2 for 10 min, and ERK activity was determined.
The bar graph represents two experiments combined.
B, HB-EGF secretion in response to E2. ER
or pcDNA3
was expressed, and the cells were incubated with E2 with or without
MMPI or ICI182780 for 3 min. Electrophoresed proteins were then
subjected to Western blot. The bar graph is three
experiments combined. C, MMPI activation by E2 requires ER.
ER
-transfected HCC-1569 cells were incubated with E2 for 2 min, and
the cell lysate was used to determine MMP activity by
spectroflurometry. EDTA is a calcium chelator, U-73122 is a PLC
inhibitor, and calphostin C is a PKC inhibitor.
member of the MAP kinase family, and this
was essential for E2 to act as a cell survival factor during hypoxia,
to preserve EC morphology after metabolic insult, and to stimulate EC
migration and primitive capillary formation (33). Here, we show that
MMP inhibition or EGFR tyrosine kinase inhibition significantly
prevents E2 signaling to the activation of p38
(Fig. 7B).
Thus, additional signal transduction pathways are rapidly triggered by
E2 in several cell types, and these pathways require EGFR
transactivation via the linked events we show here.
View larger version (13K):
[in a new window]
Fig. 7.
Additional signaling pathways that depend
upon ER to EGFR cross-talk. A, E2-induces AKT
activation in MCF-7 cells, dependent upon MMP activation and EGFR
tyrosine kinase function. MCF-7 cells were incubated with E2 with or
without GM6001 or tyrphostin AG1478 for 10 min, and AKT phosphorylation
at serine 473 was determined. B, p38 activation in
endothelial cells by E2 is significantly prevented by inhibition of MMP
activity or the EGFR tyrosine kinase. EC were incubated with 10 nM E2 for 15 min, and the p38 immunoprecipitated from the cell lysate(s) was used for in
vitro kinase activity assays, with ATF-1 as substrate. The
bar graphs are from three experiments combined.
or ER
. It has recently been shown in CHO cells lacking
endogenous ER that targeting of the E domain of ER
to the plasma
membrane is sufficient to allow strong activation of ERK by E2 (5). This same, localized construct rescues HeLa cells from apoptotic cell
death in response to etoposide (17), and in both situations, targeting
of the E domain to the nucleus had no effect. We therefore asked
whether targeting the E domain of ER
to the plasma membrane was sufficient to activate the signal pathway that we define here. This
was accomplished in the HCC-1569 breast cancer cells that do not
express ER. Targeting the E domain to the plasma membrane resulted in
MMP activation (Fig. 8A) and
EGFR activation (Fig. 8B), leading to ERK up-regulation (5).
In the absence of the expressed E domain, E2 was unable to stimulate
any of these events. Targeting the E domain to the nucleus also did not
result in activation of this pathway. Therefore, the E domain appears
to be sufficient for the complex interactions at the plasma
membrane that allow assembly and activation of the signalsome in
response to E2.
View larger version (32K):
[in a new window]
Fig. 8.
Structure/function relationship between ER
and signaling. A, targeting the E domain of ER to the
membrane of breast cancer cells augments MMP activity. HCC-1569 cells
were transiently transfected to express the E domain targeted to the
nucleus (lanes 1 and 2) or to the plasma membrane
(lanes 3 and 4), followed by 2 min of treatment
with E2 and determination of MMP activity. A representative study is
shown, repeated once. B, expressing the E domain in the
membrane leads to the transactivation of EGFR by E2. The transfected
cells were assayed for EGFR phosphorylation by Western Blot, using an
antibody against tyrosine 1138 (lane 3 versus lane
4). EGF-induced transactivation of its receptor serves as a
positive control. The study was repeated twice.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in nude mice blocked the growth
of human breast cancer xenografts. This probably resulted from the
antibodies inhibiting membrane ER signaling to ERK and
phosphatidylinositol 3-kinase (46). Therefore, it is important to
understand how E2 acts through membrane ER to trigger signal transduction.
q
and G
i, but not the
subunits of Gs,
G12, and G13, was important to the subsequent
(but still rapid) signaling events upstream of EGFR activation (Fig.
9). G
inhibition also prevented the full transactivation of EGFR and ERK up-regulation in response to E2.
This underscores the ideas that E2 activates several G proteins (6) and
that there are specific functions for each but with some degree of
redundancy. The partial redundancy we demonstrate may be related to the
requirement that full signaling by E2 requires multiple G protein
activations. Supporting this idea, we found that co-expressing dominant
negative mini-genes for G
q and G
i (but
not co-expression with G
S) added to the inhibition of E2/ER
signaling to MMP activation and EGFR activation, compared with the
inhibition of single G
subunits.2 This may be
related to the necessity for complete activation of Src and Src-induced
signaling to MMP activation (see below).
View larger version (13K):
[in a new window]
Fig. 9.
E2-induced proximal signaling to the
transactivation of EGFR, leading to ERK
activation.
We also found that MMP-2 and MMP-9 were necessary for E2 to stimulate
the secretion of HB-EGF and the transactivation of EGFR. First, E2
activated these two enzymes, as determined by gelatin zymography and substrate cleavage studies. However, E2 did not up-regulate MMP-3 and MMP-13 activity, thus showing the specificity of
our results. Second, E2 induced the release of HB-EGF into the cell
culture medium after only 3 min of incubation, and this was
substantially prevented by the specific antisense (but not missense)
constructs for MMP-2 and MMP-9, with the effects being additive.
Shedding of HB-EGF is a complicated process, and involvement of
Ras-Raf-Mek (51), Rac (52), or PKC and the
metalloprotease-disintegrin, MDC9 (37), has been proposed. In some
cellular contexts, unknown metalloproteinase(s) mediates this shedding
(53). Recently, TACE/ADAM17 has been shown to cleave expressed HB-EGF
at 24 h in fibroblasts (54). However, our results suggest that
MMP-2 and MMP-9 are sufficient.
The specific signal from GPCRs that leads to MMP activation is not well understood. In this regard, we report the novel finding that Src is necessary for E2 to activate MMP-2 and MMP-9 and subsequent HB-EGF shedding. A previous study implicated Src as upstream from HB-EGF, but its role was undetermined (29). The precise mechanisms by which Src accomplishes MMP secretion and activation is unknown but is under investigation. It should be appreciated that this kinase is also downstream of EGFR, either through Src binding this receptor or through potential cross-talk of EGFR to G protein-coupled receptors, leading to Src activation (55, 56).
Our novel identification of MMP-2 and MMP-9 secretion and
activation as being involved in estrogen signaling underlies the overall contribution of these MMPs to breast cancer biology. MMP-2 and
MMP-9 have been implicated in the aggressive behavior of breast cancer
(57, 58). The ability of breast cancer cells to migrate or
invade/metastasize is importantly dependent on the degradation of cell
matrix by MMPs. However, these proteases also play additional important
roles to mediate cell survival, differentiation, and angiogenesis
(reviewed in Ref. 59). Recently, MMP-2 production in response to E2 was
found to be dependent on ERK signaling to the up-regulated activity of
the AP-2 transcription factor in mesangial cells (60). In our model,
MMP activation is necessary for E2 to stimulate HB-EGF secretion into
the culture media, and HB-EGF but not TGF transactivates the EGFR to
signal to ERK.
The ability of EGFR to underlie E2/ER-induced ERK may
represent only a single example of the wider signaling interactions of
these two growth modulatory systems. We therefore asked whether other
important signaling pathways that originate from membrane ER are also
dependent on EGFR. We report here that E2 activates protein kinase B in
breast cancer cells and p38 MAP kinase in EC and that both pathways
are also dependent upon transactivation of the EGFR. Utilization of
EGFR to activate ERK is relatively common for a variety of GPCRs (38,
61); however, GPCRs can also activate ERK by pathways apart from EGFR
(62). In this respect, we previously showed that E2 stimulates
G
s and cAMP, as well as ERK in CHO cells that are
transiently transfected to express ER but that lack endogenous EGFR
(6). Other EGF receptor family members might facilitate GPCR signaling
to distinct pathways and thereby contribute to the specificity of
signaling. For instance, the ability of muscarinic receptors to
activate AKT is dependent upon the transactivation of the ErbB3 member
of the EGFR family (63). In other situations, platelet-derived growth
factor or insulin-like growth factor-1 receptors may be necessary for
GPCR effects (64-66). Thus, the tyrosine kinase receptor milieu in a particular cell may specifically control the panoply of signaling typically enacted by a GPCR ligand.
These interactions extend to cross-talk in both directions, including from the growth factor receptor tyrosine kinase to ER (67). Insulin-like growth factor-1 and EGF can signal to transcription via ER, independently of E2 (68, 69). This occurs through growth factor receptor-induced phosphorylation of the nuclear sex steroid receptor (70) or co-accessory proteins (71), and the induction of several kinase cascades is important in this regard (70, 72). These complex interactions are important in that they may contribute to the ability of breast cancer cells to proliferate or survive via ER, even when circulating levels of E2 are low, as in the post-menopausal woman.
An additional important issue is whether ER is required for E2 to
activate signaling pathways from the membrane. It has previously been
shown that E2 can transactivate the EGFR and signal in breast cancer
cells that do not express ER (24). This purportedly occurs through a
nondefined interaction with the orphan GPCR, GPR 30, and can
nonspecifically be activated by estrogen receptor antagonists (ICI182780) and relatively inactive steroisomers (17--E2). We report
here that in the absence of ER, E2 can not activate ERK in HCC-1569
cells that lack this receptor. Expressing ER (or the E domain targeted
to the cell membrane) allows E2 to signal through specific
G proteins, MMP activation, and HB-EGF secretion that activates EGFR. Although some cells have been reported to respond rapidly to E2 in a nontraditional ER-related or ER-independent fashion
(10, 73), the mechanisms underlying these reports remain unknown.
Furthermore, the majority of studies indicate the requirement of ER for
E2 action (5, 6, 8, 20, 27, 28), and these studies identify typical
receptor pharmacology for the nongenomic actions of this sex steroid
(reviewed in Ref. 43).
What part of ER is necessary for signal transduction at the membrane?
Tyrosine 537 in the AF-2 portion of the E domain is essential for the
interaction of ER with Src and functional up-regulation of ERK in MCF-7
cells (8). Recently, an interaction between the AF-1 domain of ER
and the phosphotyrosine binding and SH2 domains of the adapter
protein Shc was postulated to mediate ERK activation in MCF-7 cells
(20). However, Razandi et al. (5) recently
showed that targeting the E domain of ER
to the plasma membrane of
CHO cells is sufficient for robust ERK activation by E2, and Kousteni
et al. (17) showed that this was sufficient to rescue HeLa
cells from apoptosis. Here, we show that targeting the E domain to the
membrane (and not to the nucleus) of HCC-1569 cells results in MMP-2
activation, EGFR transactivation, and ERK up-regulation. Thus, it is
not clear what the significance of the AF-1 region of ER might be for
signaling from the membrane. We propose that elements in the E domain,
such as AF-2, allows for the complex interactions with G proteins,
caveolin, Src, and other signaling molecules.
In summary, E2 activation of ERK is dependent on several
G and G
subunits of small GTP-binding proteins.
Src-dependent stimulation of MMP-2 and MMP-9 activity in
response to E2/ER releases HB-EGF, leading to EGFR transactivation, and
signaling to MAP kinase. The E domain of ER
appears to be sufficient
to activate these mechanisms. The assemblage of signal transduction
complexes probably platformed on caveolin or growth factor receptor
tyrosine kinase proteins (EGFR and insulin-like growth factor receptor) accounts for much of the ability of E2 to signal through
membrane-localized ER to different pathways. This mechanism is
increasingly appreciated to play important roles in the cellular
biology of E2 actions, and manipulation of these pathways could
therapeutically modulate the effects of this sex steroid.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Research Service of the Department of Veteran's Affairs, a grant from the Avon Products Breast Cancer Research Foundation, Department of Defense Breast Cancer Research Program Grant BC990915, and National Institutes of Health Grant HL-59890 (to E. R. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Medical Service (111-I), Long Beach VA Medical Center/UC-Irvine, 5901 E. 7th St., Long Beach, CA 90822. Tel.: 562-826-5748; Fax: 562-826-5515; E-mail: ellis.levin@med.va.gov.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M205692200
2 M. Razandi, A. Pedram, and E. R. Levin, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: E2, estradiol; ER, estrogen receptor(s); EGF, epidermal growth factor; EGFR, EGF receptor(s); GPCR, G protein-coupled receptor(s); MMP, matrix metalloproteinase(s); PLC, phospholipase C; PKC, protein kinase C; HB-EGF, heparin-binding epidermal growth factor; TGF, transforming growth factor; EC, endothelial cell(s); CHO, Chinese hamster ovary; ASO, antisense oligonucleotide(s); MSO, scrambled antisense oligonucleotide(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Tsai, M.-J., and O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486[CrossRef][Medline] [Order article via Infotrieve] |
2. | Falkenstein, E., and Wehling, M. (2000) Eur. J. Clin. Invest. 3 (suppl.), 51-54 |
3. | Kim, H. P., Lee, J. Y., Jeong, J. K., Bae, S. W., Lee, H. K., and Jo, I. (1999) Biochem. Biophys. Res. Commun. 263, 257-262[CrossRef][Medline] [Order article via Infotrieve] |
4. | Chambliss, K. L., Yuhanna, I. S., Mineo, C., Liu, P., German, Z., Sherman, T. S., Mendelsohn, M. E., Anderson, R. G. W., and Shaul, P. W. (2000) Circ. Res. 87, e44-e52[Medline] [Order article via Infotrieve] |
5. |
Razandi, M., Oh, P.,
Pedram, A.,
Schnitzer, J.,
and Levin, E. R.
(2002)
Mol. Endocrinol.
16,
100-115 |
6. |
Razandi, M.,
Pedram, A.,
Greene, G. L.,
and Levin, E. R.
(1999)
Mol. Endocrinol.
13,
307-319 |
7. |
Wyckoff, M. H.,
Chambliss, K. L.,
Mineo, C.,
Yuhanna, I. S.,
Mendelsohn, M. E.,
Mumby, S. M.,
and Shaul, P. W.
(2001)
J. Biol. Chem.
276,
27071-27076 |
8. |
Migliaccio, A.,
Castoria, G., Di,
Domenico, M.,
de Falco, A.,
Bilancio, A.,
Lombardi, M.,
Vitorria Barone, M.,
Ametrano, D.,
Zannini, M. S.,
Abbondanza, C.,
Bontempo, P.,
and Auricchio, F.
(2000)
EMBO J.
19,
5406-5417 |
9. | Migliaccio, A., Di, Domenico, M., Castoria, G., de Falco, A., Bontempo, P., Nola, E., and Auricchio, F. (1996) EMBO J. 15, 1292-1300[Abstract] |
10. |
Singh, M.,
Setalo, G., Jr.,
Guan, X.,
Warren, M.,
and Toran-Allerand, C. D.
(1999)
J. Neurosci.
19,
1179-1188 |
11. |
Le Mellay, V.,
Grosse, B.,
and Lieberherr, M.
(1997)
J. Biol. Chem.
272,
11902-11907 |
12. |
Ansonoff, M. A.,
and Etgen, A. M.
(1998)
Endocrinology
139,
3050-3056 |
13. | Simoncini, T., Hafezi-Moghadam, A., Brazil, D. P., Ley, K., Chin, W. W., and Liao, J. K. (2000) Nature 407, 538-541[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Shevde, N. K.,
Bendixen, A. C.,
Dienger, K. M.,
and Pike, J. W.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
7829-7834 |
15. |
Morey, A. K.,
Pedram, A.,
Razandi, M.,
Prins, B. A., Hu, R.-M.,
Biesiada, E.,
and Levin, E. R.
(1997)
Endocrinology
138,
3330-3339 |
16. |
Razandi, M.,
Pedram, A.,
and Levin, E. R.
(2000)
Mol. Endocrinol.
14,
1434-1447 |
17. | Kousteni, S., Bellido, T., Plotkin, L. I., O'Brien, C. A., Bodenner, D. L., Han, L., Han, K., DiGregorio, G. B., Katzenellenbogen, J. A., Katzenellenbogen, B. S., Roberson, P. K., Weinstein, R. S., Jilka, R. L., and Manolagas, S. C. (2001) Cell 104, 719-730[Medline] [Order article via Infotrieve] |
18. |
Pappas, T. C.,
Gametchu, B.,
Yannariello-Brown, J.,
Collins, T. J.,
and Watson, C. S.
(1995)
FASEB J.
9,
404-410 |
19. |
Norfleet, A. M.,
Thomas, M. L.,
Gametchu, B.,
and Watson, C. S.
(1999)
Endocrinology
140,
3805-3814 |
20. |
Russell, K. S.,
Haynes, M. P.,
Sinha, D.,
Clerisme, E.,
and Bender, J. R.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5930-5935 |
21. |
Song, R. X.,
McPherson, R. A.,
Adam, L.,
Bao, Y.,
Shupnik, M.,
Kumar, R.,
and Santen, R. J.
(2002)
Mol. Endocrinol.
16,
116-127 |
22. | Aronica, S. M., Kraus, W. L., and Katzenellenbogen, B. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8517-8522[Abstract] |
23. | Lieberherr, M., Grosse, B., Kachkache, M., and Balsan, S. (1993) J. Bone Min. Res. 8, 1365-1376[Medline] [Order article via Infotrieve] |
24. |
Filardo, E. J.,
Quinn, J. A.,
Frackelton, A. R., Jr.,
and Bland, K. I.
(2002)
Mol. Endocrinol.
16,
70-84 |
25. |
Filardo, E. J.,
Quinn, J. A.,
Bland, K. I.,
and Frackelton, A. R.
(2000)
Mol. Endocrinol.
14,
1649-1660 |
26. |
Luttrell, L. M.,
Hawes, B. E.,
van Biesen, T.,
Luttrell, D. K.,
Lansing, T. J.,
and Lefkowitz, R. J.
(1996)
J. Biol. Chem.
271,
19443-19450 |
27. |
Singer, C. A.,
Figueroa-Masot, X. A.,
Batchelor, R. H.,
and Dorsa, D. M.
(1999)
J. Neurosci.
19,
2455-2463 |
28. |
Zhu, Y.,
Bian, Z., Lu, P.,
Karas, R. H.,
Bao, L.,
Cox, D.,
Hodgin, J.,
Shaul, P. W.,
Thoren, P.,
Smithies, O.,
Gustafsson, J. A.,
and Mendelsohn, M. E.
(2002)
Science
295,
505-508 |
29. | Pierce, K. L., Tohgo, A., Ahn, S., Field, M. E., Luttrell, L. M., and Lefkowitz, R. J. (2001) J. Biol. Chem. 22, 23155-23160[CrossRef] |
30. |
Pedram, A.,
Razandi, M.,
and Levin, E. R.
(1998)
J. Biol. Chem.
273,
26722-26728 |
31. | Couse, J. F., Curtis, S. W., Washburn, T. F., Lindzey, J., Golding, T. S., Lubahn, D. B., Smithies, O., and Korach, K. S. (1995) Mol. Endocrinol. 9, 1441-1454[Abstract] |
32. | Redemann, A., Holzmann, B., von Ruden, T., Wagner, E. F., Schlessinger, J., and Ullrich, A. (1992) Mol. Cell. Biol. 12, 491-498[Abstract] |
33. |
Razandi, M.,
Pedram, A.,
and Levin, E. R.
(2000)
J. Biol. Chem.
275,
38540-38546 |
34. |
Koch, W. J.,
Hawes, B. E.,
Inglese, J.,
Luttrell, L. M.,
and Lefkowitz, R. J.
(1994)
J. Biol. Chem.
269,
6193-6197 |
35. | Gilchrist, A., Li, A., and Hamm, H. E. (2002) Methods Enzymol. 344, 58-69[Medline] [Order article via Infotrieve] |
36. | Prenzel, N., Zwick, E., Daub, H., Leserer, M., Abraham, R., Wallasch, C., and Ullrich, A. (1999) Nature 402, 884-888[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Izumi, Y.,
Hirata, M.,
Hasuwa, H.,
Iwamoto, R.,
Umata, T.,
Miyado, K.,
Tamai, Y.,
Kurisaki, T.,
Sehara-Fujisawa, A.,
Ohno, S.,
and Mekada, E.
(1998)
EMBO J.
17,
7260-7272 |
38. | Duffy, M. J., Maguire, T. M, Hill, A., McDermott, E., and O'Higgins, N. (2000) Breast Cancer Res. 2, 252-257[CrossRef][Medline] [Order article via Infotrieve] |
39. | Hua, J., and Muschel, R. J. (1996) Cancer Res. 56, 5279-5284[Abstract] |
40. | Perret, S., Dockery, P., and Harvey, B. J. (2001) Mol. Cell. Endocrinol. 176, 77-84[CrossRef][Medline] [Order article via Infotrieve] |
41. | Rubio-Gayosso, I., Sierra-Ramirez, A., Garcia-Vazquez, A., Martinez- Martinez, A., Munoz-Garcia, O., Morato, T., and Ceballos-Reyes, G. (2000) J. Cardiovasc. Pharmacol. 36, 196-202[CrossRef][Medline] [Order article via Infotrieve] |
42. | Kelly, M. J, and Wagner, E. J. (1999) Trends Endocrinol. Metab. 10, 369-374[CrossRef][Medline] [Order article via Infotrieve] |
43. | Sylvia, V. L., Walton, J., Lopez, D., Dean, D. D., Boyan, B. D., and Schwartz, Z. (2001) J. Cell. Biochem. 81, 413-429[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Levin, E. R.
(2001)
J. Applied Physiol.
91,
1860-1867 |
45. | Teixeira, C., Reed, J. C., and Pratt, M. A. (1995) Cancer Res. 55, 3902-3907[Abstract] |
46. | Marquez, D. C., and Pietras, R. J. (2001) Oncogene 20, 5420-5430[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Liu, J., Oh, P.,
Horner, T.,
Rogers, R. A.,
and Schnitzer, J. E.
(1997)
J. Biol. Chem.
272,
7211-7222 |
48. |
Okamoto, T.,
Schlegel, A.,
Scherer, P. E.,
and Lisanti, M. P.
(1998)
J. Biol. Chem.
273,
5419-5422 |
49. |
Lee, H.,
Volonte, D.,
Galbiati, F.,
Iyengar, P.,
Lublin, D. M.,
Bregman, D. B.,
Wilson, M. T.,
Campos-Gonzalez, R.,
Bouzahzah, B.,
Pestell, R. G.,
Scherer, P. E.,
and Lisanti, M. P.
(2000)
Mol. Endocrinol.
14,
1750-1775 |
50. |
Ushio-Fukaki, M.,
Hilenski, L.,
Santanam, N.,
Becker, P. L., Ma, Y.,
Griendling, K.,
and Alexander, R. W.
(2001)
J. Biol. Chem.
276,
48269-48275 |
51. | Gechtman, Z., Alonso, J. L., Raab, G., Ingber, D. E., and Klagsbrun, M. (1999) J. Biol. Chem. 274, 18828-18835 |
52. |
Umata, T.,
Hirata, M.,
Takahashi, T.,
Ryu, F.,
Shida, S.,
Takahashi, Y.,
Tsuneoka, M.,
Miura, Y.,
Masuda, M.,
Horiguchi, Y.,
and Medada, E.
(2001)
J. Biol. Chem.
276,
30475-30482 |
53. |
Dong, J.,
Opresko, L. K.,
Dempsey, P. J.,
Lauffenburger, D. A.,
Coffey, R. J.,
and Wiley, H. S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6235-6240 |
54. |
Sunnarborg, S. W.,
Hinkle, C. L.,
Stevenson, M.,
Russell, W. E.,
Raska, C. S.,
Peschon, J. J.,
Castner, B. J.,
Gerhart, M. J.,
Paxton, R. J.,
Black, R. A.,
and Lee, D. C.
(2002)
J. Biol. Chem.
277,
12838-12845 |
55. |
Seta, K.,
Nanamori, M.,
Modrall, J. G.,
Neubig, R. R.,
and Sadoshima, J.
(2002)
J. Biol. Chem.
277,
9268-9277 |
56. | Luttrell, L. M., Daaka, Y., and Lefkowitz, R. J. (1999) Curr. Opin. Cell Biol. 11, 177-183[CrossRef][Medline] [Order article via Infotrieve] |
57. |
Westermarck, J.,
and Kahari, V.-M.
(1999)
FASEB J.
13,
781-792 |
58. |
Ray, J. M.,
and Stetler-Stevenson, W. G.
(1994)
Eur. Respir. J.
7,
2062-2072 |
59. |
Coussens, L. M.,
Fingleton, B.,
and Matrisian, L. M.
(2002)
Science
295,
2387-2392 |
60. | Guccione, M., Silberger, S., Lei, J., and Nuegarten, J. (2002) Am. J. Physiol. 282, F164-F169[CrossRef][Medline] [Order article via Infotrieve] |
61. | Hackel, P. O., Zwick, E., Prenzel, N., and Ullrich, A. (1999) Curr. Opin. Cell Biol. 11, 184-189[CrossRef][Medline] [Order article via Infotrieve] |
62. |
Andreev, J.,
Galisteo, M. L.,
Kranenburg, O.,
Logan, S. K.,
Chiu, E. S.,
Okigaki, M.,
Cary, L. A.,
Moolenaar, W. H.,
and Schlessinger, J.
(2001)
J. Biol. Chem.
276,
20130-20135 |
63. |
Tang, X.,
Batty, I. H.,
and Downes, C. P.
(2002)
J. Biol. Chem.
277,
338-344 |
64. |
Schmidt, M.,
Frings, M.,
Mono, M. L.,
Guo, Y.,
Weernink, P. A.,
Evellin, S.,
Han, L.,
and Jakobs, K. H.
(2000)
J. Biol. Chem.
275,
32603-32610 |
65. |
Kahlert, S.,
Nuedling, S.,
van Eickels, M.,
Vetter, H.,
Meyer, R.,
and Grohe, C.
(2000)
J. Biol. Chem.
275,
18447-18453 |
66. |
Sumitomo, M.,
Milowsky, M. I.,
Shen, R.,
Navarro, D.,
Dai, J.,
Asano, T.,
Hayakawa, M.,
and Nanus, D. M.
(2001)
Cancer Res.
61,
3294-3298 |
67. | Yee, D., and Lee, A. V. (2000) J. Mammary Gland Biol. Neoplasia 5, 107-115[CrossRef][Medline] [Order article via Infotrieve] |
68. |
Klotz, D. M.,
Curtis Hewitt, S.,
Ciana, P.,
Raviscioni, M.,
Lindzey, J. K.,
Foley, J.,
Maggi, A.,
DiAugustine, R. P.,
and Korach, K. S.
(2002)
J. Biol. Chem.
277,
8531-8537 |
69. |
Curtis, S. W.,
Washburn, T.,
Sewall, C.,
DiAugustine, R.,
Lindzey, J.,
Couse, J. F.,
and Korach, K. S.
(1996)
Proc. Natl. Acad. Sci.
93,
12626-12630 |
70. | Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., Metzger, D., and Chambon, P. (1995) Science 270, 1491-1494[Abstract] |
71. |
Lopez, G. N.,
Turck, C. W.,
Schaufele, F.,
Stallcup, M. R.,
and Kushner, P. J.
(2001)
J. Biol. Chem.
276,
22177-22182 |
72. |
Martin, M. B.,
Franke, T. F.,
Stoica, G. E.,
Chambon, P.,
Katzenellenbogen, B. S.,
Stoica, B. A.,
McLemore, M. S.,
Olivo, S. E.,
and Stoica, A.
(2000)
Endocrinology
141,
4503-4511 |
73. |
Guo, Z.,
Krucken, J.,
Benten, W. P.,
and Wunderlich, F.
(2002)
J. Biol. Chem.
277,
7044-7050 |