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Departments of Surgery (E.J.F., K.I.B.) and Medicine (E.J.F., J.A.Q.), Rhode Island Hospital (E.J.F., J.A.Q., K.I.B.) and Roger Williams Hospital (A.R.F.) and Brown University, Providence, Rhode Island 02903
Address all correspondence and requests for reprints to: Edward J. Filardo, Rhode Island Hospital, Department of Medicine, Division of Clinical Pharmacology, Aldrich Building, Room 712, 593 Eddy Street, Providence, Rhode Island 02903. E-mail: Edward_Filardo{at}brown.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit protein-dependent
transactivation of the epidermal growth factor (EGF) receptor through
the release of pro-heparan-bound EGF from the cell surface. Here we
investigate the mechanism by which Erk-1/-2 activity is rapidly
restored to basal levels after estrogen stimulation. Evidence is
provided that attenuation of Erk-1/-2 activity by estrogen occurs via
GPR30-dependent stimulation of adenylyl cyclase and cAMP-dependent
signaling that results in Raf-1 inactivation. We show that 17ß-E2
represses EGF-induced activation of the Raf-to-Erk pathway in human
breast carcinoma cells that express GPR30, including MCF-7 and SKBR3
cells which express both or neither, ER, respectively. MDA-MB-231
cells, which express ERß, but not ER
, and low levels of GPR30
protein, are unable to stimulate adenylyl cyclase or promote
estrogen-mediated blockade of EGF-induced activation of Erk-1/-2.
Pretreatment of MDA-MB-231 cells with cholera toxin, which
ADP-ribosylates and activates Gs subunit proteins, results in G
protein-coupled receptor (GPCR)-independent adenylyl cyclase activity
and suppression of EGF-induced Erk-1/-2 activity. Transfection of GPR30
into MDA-MB-231 cells restores their ability to stimulate adenylyl
cyclase and attenuate EGF-induced activation of Erk-1/-2 by estrogen.
Moreover, GPR30-dependent, cAMP-mediated attenuation of EGF-induced
Erk-1/-2 activity was achieved by ER antagonists such as tamoxifen or
ICI 182, 780; yet not by 17-E2 or progesterone. Thus, our
data delineate a novel mechanism, requiring GPR30 and estrogen, that
acts to regulate Erk-1/-2 activity via an inhibitory signal mediated by
cAMP. Coupled with our prior findings, these current data imply that
estrogen balances Erk-1/-2 activity through a single GPCR via two
distinct G protein-dependent signaling pathways that have opposing
effects on the EGF receptor-to-MAPK pathway. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Stimulation of the EGFR-to-MAPK pathway is initiated by the specific
binding of cognate ligands, such as EGF, TGF, heregulin, and
heparan-bound EGF (HB-EGF), to specific EGFRs. This interaction results
in the formation of EGFR homo- and heterodimers and autophosphorylation
of tyrosyl residues within their cytoplasmic domains. Specific
recognition of these phosphotyrosines by the adapter proteins Grb-2
and/or Shc, and guanine nucleotide exchange factors, such as Sos,
serves to link-activated EGFR to MAPK via the monomeric GTPase, p21Ras.
Thus activated, Ras is capable of recruiting the serine-threonine
kinase Raf-1, which in turn promotes cascade phosphorylation and
activation of Mek-1 and its dedicated substrates Erk-1 and Erk-2
(3). Under conditions of normal growth and behavior,
activation of the EGFR to MAPK pathway is transient and attenuated by a
variety of control mechanisms, which prevent downstream
activation of Erk-1/-2 (4), as well as by phosphatases,
which dephosphorylate, and thereby inactivate, Erk-1/-2
(5). In contrast, constitutive activation of the EGFR to
MAPK pathway results in dysregulated cellular behaviors associated with
carcinogenesis (6, 7).
Several lines of evidence suggest that dysregulation of the EGFR to MAPK pathway may have particular significance for breast carcinogenesis. First, overexpression of the EGFR family member, HER2, is a common event in breast tumors (8), an event that is known to increase both the amplitude and duration of EGF-stimulated Erk-1/-2 activation (9). Second, Erk-1-mediated phosphorylation of serine residue 118 of the ER enhances its gene activation function (10, 11). Third, estrogen stimulates activation of Erk-1/-2 (12, 13, 14, 15). In this regard, constitutive Erk-1/-2 may initiate dysregulated cellular behaviors exhibited by estrogen-independent tumors; additionally, Erk-1/-2 may also provide a mechanism whereby hyperactive growth factor signaling may activate estrogen-dependent tumor growth. The association of increased Erk-1/-2 activity with invasive breast cancer suggests this hypothesis (16).
Aside from receptor tyrosine kinases, as represented by the EGFRs, G protein-coupled receptors (GPCRs) comprise a second major class of transmembrane receptors that signal via Erk-1/-2. Unlike EGFRs, GPCRs activate Erk-1/-2 through several distinct mechanisms, some of which couple via the monomeric GTPases, Ras or Rap; others activate Raf or Mek directly (17). In some instances, GPCR stimulation leads to the activation of Src-related tyrosine kinases and the assembly of Grb-2/Sos/Shc complexes on the cytoplasmic domain of EGFRs (18). In conjunction with the finding that Src can directly phosphorylate the EGFR (19), these observations suggest that GPCRs may activate EGFRs via Src-mediated phosphorylation of the EGFR cytoplasmic tail. More recently, ligands for some GPCRs, including endothelin, bombesin, and lysophosphatidic acid have been shown to transactivate the EGFR through their ability to cleave and release surface-associated precursors of EGF-related polypeptides (20). These findings parallel observations that other receptors that lack intrinsic enzymatic function, such as integrins (21) and cytokine receptors (22), also transactivate the EGFR. The fact that may different receptors transactivate the EGFR to MAPK pathway suggests that coordinated signaling is required to regulate the activity of this commonly used signaling axis.
Recently, we have shown that GPR30 is required for
estrogen-induced activation of the MAPKs, Erk-1 and Erk-2
(23). This activation response is rapid and occurs via
Gß-subunit protein-dependent release of surface-associated HB-EGF
and transactivation of the EGF receptor. GPR30-dependent,
estrogen-mediated Erk-1/-2 activation is transient, rapidly returning
to basal levels 10–15 min after initial exposure to estrogen. This
rapid inactivation of Erk-1/-2 implies the existence of a tightly
controlled regulatory mechanism. Others have shown that estrogen
(24, 25, 26) also promotes stimulation of adenylyl cyclase
activity and production of intracellular cAMP. In some cell
settings, cAMP acts as a potent inhibitor of Erk-1/-2 activity
(27, 28). In other cell types, Erk-1/-2 are activated by
cAMP via its ability to promote B-raf-mediated stimulation of Mek-1
(29). Because adenylyl cyclases are commonly linked to
GPCRs (30, 31) we investigated whether GPR30
participates in estrogen-mediated stimulation of adenylyl cyclase.
Here, we show that GPR30 is required for estrogen-induced stimulation
of adenylyl cyclase and cAMP-mediated inhibition of Erk-1/-2.
Moreover, we demonstrate that ER antagonists, including the
antiestrogens tamoxifen and
7-[9-[(4,4,5,5,5,-pentafluoropentyl)sulphinyl]nonyl]estra-1,3,5(10)-triene-3,17ß-diol
(ICI 182,780), can also induce these same GPR30-dependent
rapid signaling events. Our results suggest that estrogens and
antiestrogens signal via GPR30-mediated stimulation of adenylyl
cyclase to inhibit the EGFR to MAPK pathway.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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s-proteins are required in these cells for
estrogen-mediated stimulation of adenylyl cyclase
(32). Traditionally, adenylyl cyclase activity is known to
be regulated by receptors that couple to heterotrimeric G proteins
(30). Although the ER has been shown to exist in the
plasma membrane (33, 34), there are no known functional
motifs within the structure of the ER that permit Gs protein
coupling or activation (35). Because we have shown that
GPR30 is required for transactivation of the EGFR by estrogen
(23), we queried whether this receptor, or the known ERs,
promote estrogen-mediated stimulation of adenylyl cyclase.
To discriminate between these possibilities, we measured the ability of
estrogen to stimulate cAMP production in membranes isolated from human
SKBR3 breast cancer cells that express neither ER nor ERß
(36) yet express GPR30 protein (23). SKBR3
membranes exposed to 17ß-E2 produced substantial levels of cAMP (Fig. 1A
). This activity was not promoted by
the isomer, 17-E2. In agreement with the observations of Aronica and
colleagues (24), demonstrating that ER antagonists can
stimulate adenylyl cyclase activity in MCF-7 membranes, the
antiestrogen ICI 182, 780, also stimulated cAMP production in membranes
from SKBR3 cells (Fig. 1A). In contrast, as had been previously noted
by others (24), we found that membranes from MDA-MB-231
cells that express ERß but not ER protein (37) did
not generate cAMP upon exposure to either 17ß-E2 or ER antagonists
(Fig. 1B). Nevertheless, cholera toxin, an agonist that ADP-ribosylates
and directly activates Gs subunit proteins, stimulated a 15-fold
increase in cAMP in MDA-MB-231 membranes, indicating that the
MDA-MB-231 membrane preparations retained Gs proteins capable of
activating adenylyl cyclase (Fig. 1B).
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). The saturation dose for 17ß-E2-mediated
stimulation of adenylyl cyclase activity was near 1 µM,
whereas approximately 10 nM of 17ß-E2 showed a
half-maximal response. Half-maximal stimulation was achieved with 0.2
µM ICI 182, 780, a concentration that closely
approximates the half-maximal dose for a response of similar amplitude
in SKBR3 cells (Fig. 1A). No increases in cAMP production were observed
in MDA-MB-231 (GPR30) membranes treated with 17-E2, an isomer of
17ß-E2 that is unable to support ER function. Similarly, the sex
steroid hormone progesterone failed to elicit cAMP production from
MDA-MB-231 (GPR30) membranes. Collectively, these results indicate that
GPR30 acts independently of the known ERs to promote estrogen-mediated
stimulation of adenylyl cyclase.
Inhibition of PKA Prolongs Estrogen-Induced Erk-1 and Erk-2
Activity
Agents that elevate intracellular cAMP possess either
stimulate or inhibit Erk-1/-2 activity in different cell types
(27, 28, 29). The ability of cAMP to activate Erk-1/-2 has
been attributed to the cellular expression of the 95-kDa isoforms of
B-Raf (29). Therefore, we measured B-Raf expression in
MDA-MB-231 (GPR30) cells by Western blotting (Fig. 2A). Simian SV40-transformed COS-7 kidney
epithelial cells, which undergo Erk-1/-2 activation in response to
cAMP, expressed elevated levels of both the 95-kDa and 68-kDa isoforms
of B-Raf. In contrast, MDA-MB-231 (GPR30) breast cancer cells grown in
serum expressed the 95-kDa isoform of B-Raf, and little, if any,
detectable 68-kDa B-Raf (Fig. 2A). However, we found that when these
cells were serum-starved they failed to express detectable levels of
95-kDa B-Raf. This finding is consistent with a prior report noting
that the 95-kDa isoform of B-Raf is inhibited in serum-starved cells
(38) and suggests that serum-starved MDA-MB-231(GPR30)
cells may be refractory to cAMP-dependent activation of Erk. To
directly test this hypothesis, Erk-1/-2 phosphorylation was
measured after exposure of MDA-MB-231 (GPR30) cells to either cholera
toxin or dibutyryl cAMP, a membrane-permeable cAMP congener. Although
either estrogen or EGF induced Erk-1/-2 activation in MDA-MB-231
(GPR30) cells, neither cholera toxin nor dibutyryl cAMP promoted
Erk-1/-2 stimulation (Fig. 2B). However, MDA-MB-231 (GPR30) cells
exposed to either cholera toxin or dibutyrl cAMP were able to blunt
EGF-induced Erk-1/-2 activity, suggesting that cAMP antagonizes
Erk in these cells (Fig. 2C). This finding implies that via its ability
to stimulate adenylyl cyclase, estrogen may transmit a cAMP inhibitory
signal that acts to attenuate estrogen-mediated, transactivation of the
EGFR-to-Erk signaling axis.
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, the duration of this
response is transient. Increases in Erk-1/-2 phosphorylation were
detected as early as 1 min after exposure to 17ß-E2. Peak Erk-1/-2
phosphorylation levels occurred at 5 min (3- to 4-fold increase) with
Erk-1/-2 activity returning to baseline levels by 30–60 min. Cells
exposed to KT5720 for 2 h exhibited reduced basal levels of
Erk-1/-2 activity relative to untreated control cells. However, after
estrogen stimulation, the rate and amplitude of the Erk-1/-2 activation
response in KT5720 pretreated cells was similar to that observed in
control cells with peak activity observed within 5 min. In contrast to
untreated control cells, KT5720-treated cells maintained elevated
levels of Erk-1/-2 activity for an extended period of time (greater
than 1 h) after estrogen stimulation (Fig. 3). This observation
suggests that activation of cAMP-dependent PKA is required to restore
estrogen-induced Erk-1/-2 activity to basal levels.
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, stimulation of quiescent MCF-7 cells (ER+, ERß+, GPR30+) with EGF
induces substantial (5- to 10-fold) increases in the phosphorylation
state, or activity, of Erk-1/-2 within 15 min. Pretreatment of MCF-7
cells with 17ß-E2 for 30 min significantly inhibited EGF-induced
Erk-1/-2 phosphorylation or activity (Fig. 4A). This state of
E2-induced suppression of EGF-induced Erk-1/-2 phosphorylation could be
measured in cells maintained in 17ß-E2 for as long as 120 min prior
to EGF stimulation. Reprobing these filters with phosphorylation
state-independent Erk-2 antibodies verified that these changes in
Erk-1/-2 phosphorylation were not due to changes in Erk-2 protein
expression. To address whether the suppressive effect of estrogen on
EGF-stimulated Erk-1/-2 activity might be due to a delay of the onset
of EGF-induced Erk-1/-2 activation, Erk-1/-2 phosphorylation was
measured in MCF-7 cells that were pretreated with estrogen and then
stimulated with EGF for various lengths of time. Basal Erk-1/-2
phosphorylation levels were observed in cells that had been pretreated
with 17ß-E2 and subsequently challenged with EGF for any of the time
intervals tested (Fig. 4B), indicating that 17ß-E2 did not delay the
onset of EGF-induced Erk-1/-2 activity in these cells. ER-antagonists,
4- hydroxytamoxifen or ICI 182, 780 behaved similarly to 17ß-E2
with regards to their ability to attenuate EGF-induced Erk-1/-2
phosphorylation (data not shown). To determine whether the
estrogen-induced suppressive effect on EGF-induced Erk-1/-2 activity
also occurs via activation of cAMP-dependent PKA, MCF-7 cells were
incubated with KT5720 before exposure to tamoxifen and then stimulated
with EGF. KT5720-treatment completely abrogated tamoxifen-mediated
attenuation of EGF-induced phosphorylation of Erk-1/-2 in
these cells (Fig. 5A). No changes were
observed in the expression of total Erk-2 protein in response to
KT5720, whereas this treatment abolished tamoxifen-mediated
repression of EGF-induced Erk-1/-2 activity (Fig. 5A). We
found that estrogen suppression of EGF-induced Erk-1/-2 was also
observed in ER-negative SKBR3 cells (Fig. 5B). Repression of
EGF-induced Erk-1/-2 activity in these cells was achieved by not only
17ß-E2 but also the ER-antagonists tamoxifen and ICI 182, 780 (Fig. 5B). As was the case for MCF-7 cells (Fig. 5A), estrogen-mediated
repression of EGF-induced Erk-1/-2 activation in SKBR3 cells was
similarly sensitive to the cAMP congener, KT5720 (Fig. 5B).
Because KT5720 functions as an inhibitor of cAMP-dependent PKA
(39), our data suggests that PKA-mediated, cAMP-dependent
signaling is necessary for repression of Erk-1/2 activity by estrogens
and antiestrogens. These findings indicate that the ER is not required
for this estrogen suppressor activity.
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).
Prior exposure to tamoxifen (Fig. 5A) or 17ß-E2 (data not shown) did
not inhibit EGF-induced stimulation of Erk-1/-2 phosphorylation in
these cells. However, exposure of these parental MDA-MB-231 cells to
either dibutyrl cAMP or the potent cAMP agonist, cholera toxin resulted
in a dramatic reduction of EGF-stimulated Erk-1/-2 activity and
phosphorylation (Fig. 6A). In contrast, GPR30-transfected MDA-MB-231
cells expressed the estrogen suppressor phenotype. These cells
exhibited 20-fold less EGF-induced Erk-1/-2 phosphorylation after
tamoxifen treatment than mock-transfected MDA-MB-231 cells (Fig. 7). A similar inhibition of EGF-induced
Erk-1/-2 phosphorylation was observed for MDA-MB-231(GPR30) cells
treated with 17ß-E2 (data not shown). However, attenuation of
EGF-induced Erk-1/-2 phosphorylation was not inhibited in MDA-MB-231
(GPR30) cells exposed to 500 nM of either the inactive
17-E2 isomer or progesterone (Fig. 8).
No differences were observed between vector- and GPR30-transfected
MDA-MB-231 cells in total Erk-2 protein expression under any of these
conditions (Figs. 7 and 8). Thus, collectively these data suggest that
the cAMP-signaling pathway promoting estrogen-mediated repression of
Erk-1/-2 is intact in MDA-MB-231 cells, and that these cells are unable
to potentiate estrogen suppressor activity due to a defect in the
pathway leading to Gs-subunit protein activation. Overexpression of
GPR30 protein reconstitutes the estrogen suppressor phenotype
suggesting that GPR30 is required for estrogen-mediated suppression of
the EGFR-to-MAPK signaling axis. Moreover, these data provide
specificity for the GPR30-dependent responses measured here, and
suggest a novel mechanism by which estrogenic hormones can regulate
growth factor signaling.
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). Comparable amounts of tyrosine
phosphorylated erbB1/EGFR was observed at 60 min after estrogen
stimulation (Fig. 9A), even though basal levels of phosphorylated
Erk-1/-2 are present at these later time points (Fig. 3). To further
investigate whether restoration of Erk-1/-2 to basal levels of activity
after estrogen stimulation may be the consequence of EGF receptor
down-modulation, surface expression of erbB1/EGFR was measured after
estrogen stimulation (Fig. 9B). MDA-MB-231(GPR30) cells were treated
with 17ß-E2 or EGF, or pretreated with 17ß-E2 for 30 min and then
exposed to EGF. After stimulation at 37 C, cells were fixed in
paraformaldehyde, immunostained with Ab-1, an ErbB1/EGFR- specific
monoclonal antibody directed against an epitope that maps outside the
EGF-binding pocket of the receptor, and analyzed by flow cytometry. As
observed in Fig. 9B, exposure of cells to EGF (100 ng/ml) for 15 min
resulted in a 50% decrease in surface EGFR. In contrast, less than a
5% decrease in surface EGFR was observed in cells exposed to estrogen
for 3, 10, 30, or 60 min. Yet, cells which were pre-exposed to
17ß-E2, internalized 50% of their surface EGFR within 15 min
subsequent to stimulation with EGF (Fig. 9B). Taken together, these
data imply that the restoration of estrogen-induced Erk-1/-2 activity
to basal levels observed by 30 min following exposure to estrogen is
not due to a decrease in EGFR activity or expression and suggests that
the estrogen-induced blockade of Erk-1/-2 activity occurs downstream of
the EGFR.
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) and Raf-1 activity (Fig. 9D). 17ß-E2 stimulation of these cells
also induced rapid, yet transient, Mek-1 and Raf-1 phosphorylation and
activity with a kinetic response that paralleled the activation
response observed for estrogen-induced Erk-1/-2 phosphorylation
observed in Fig. 3. Both Raf-1 and Mek-1 activation by 17ß-E2 in this
cell background is dependent on GPR30 expression (data not shown).
Pretreatment with 17ß-E2 abrogated both EGF-induced Mek-1
phosphorylation (Fig. 9C) and Raf-1 activation (Fig. 9D), suggesting
that estrogen-mediated repression of EGF-induced Erk-1/-2 activity
occurs at, or upstream of, Raf-1.
To further investigate the inhibitory effect of estrogen on the
EGFR-to-Erk signaling pathway, we employed a Ras affinity assay to
measure the ability of endogenous Ras-1 to couple to a GST fusion
protein containing the Ras-binding domain of Raf-1. Raf-1/Ras complexes
were detected as early as 3 min following exposure to estrogen or EGF
(Fig. 9E). However, these complexes were transient and were no longer
detected after 30 min of exposure to either stimulant. Cells pretreated
with a diphtheria toxin mutant, CRM-197, that sequesters HB-EGF from
the cell surface (40), abrogated estrogen-mediated
activation of Ras, demonstrating that extracellular release of HB-EGF
is necessary for estrogen-induced Ras activity. In contrast, cells
exposed to estrogen for 30 min, a time interval sufficient to stimulate
cAMP (Fig. 1) and restore Erk to baseline (Fig. 9E), did not block
EGF-induced Ras activation yet did blunt EGF-induced stimulation of Erk
(Fig. 9E).
Thus, together these data suggest that restoration of Erk-1/-2 activity to basal levels in breast carcinoma cells stimulated by estrogen or growth factor is achieved through GPR30-mediated stimulation of adenylyl cyclase, which suppresses the EGFR-to-Erk pathway through PKA-dependent inhibition of Raf-1 activity. Furthermore, these data imply that breast tumors that fail to express GPR30, or produce mutant variants of this GPCR that are unable to couple to adenylyl cyclase, may no longer be able to effectively regulate the EGFR-to-Erk pathway in response to estrogens or antiestrogens.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß. It has long been
appreciated that these ERs belong to the steroid hormone receptor
superfamily and function as ligand-activated transcription factors
(41). Over the past decade, a number of investigators have
reported that estrogen (12, 15, 24, 42, 43, 44, 45), and other
steroid hormones (46, 47, 48, 49) trigger rapid intracellular
signaling events typically associated with membrane receptors that
possess intrinsic tyrosine kinase activity or couple to heterotrimeric
G proteins. Previously, we have demonstrated that estrogen acts via the
GPCR, GPR30, to promote rapid transactivation of the EGFR to MAPK
pathway through the release of pro-HB-EGF (23). Here, we
show that through GPR30, estrogen stimulates adenylyl cyclase and
inhibits Erk-1/-2 activity via a cAMP-dependent mechanism. Together
these data demonstrate that estrogen signals via GPR30 to trigger
opposing G protein- dependent signaling mechanisms that act to
balance Erk-1/-2 activity. This mechanism of GPCR-Erk-1/-2 regulation
is consistent with prior data showing a dual regulatory effect on MAPK
by a single ß-adrenergic receptor (50).
Here we provide several lines of evidence suggesting that
estrogen-mediated activation of adenylyl cyclase occurs independently
of known ERs but rather requires GPR30 protein. First, the
antiestrogens, tamoxifen and ICI 182, 780, do not antagonize
estrogen-induced activation of adenylyl cyclase but rather act as
agonists capable of stimulating adenylyl cyclase activity (Fig. 1).
Second, we show that either antiestrogens or 17ß-E2 are able to
promote activation of adenylyl cyclase activity in MCF-7 and
SKBR3 human breast cancer cell lines that express both
(37) or neither (23, 36) ER and ERß,
respectively, but do express elevated levels of GPR30 protein.
Conversely, we find that MDA-MB-231 cells that express ERß, but not
ER and express only low levels of GPR30 protein are unable to
stimulate adenylyl cyclase activity (Fig. 1B) or mediate cAMP-dependent
suppression of the EGFR to MAPK pathway (Fig. 6). However, we do show
that MDA-MB-231 cells forced to overexpress GPR30 are able to regulate
these activities (Figs. 1C and 7) in response to estrogen.
A requirement for GPR30 in stimulation of adenylyl cyclase by estrogen
is consistent with studies that have implicated GPCRs and G proteins in
rapid membrane signaling events mediated by estrogen (32, 33, 45) and other steroid hormones (47, 48, 49). Our
finding that antiestrogens also promote adenylyl cyclase stimulation
has previously been reported by others who demonstrated that ER
antagonists, namely tamoxifen and ICI 164, 384, could stimulate this
activity and generate intracellular cAMP in human MCF-7 breast cells
(24). These investigators also found increased levels of
cAMP in the uterus of rats injected with either estrogen or the
aforementioned antiestrogens. In this regard, it is noteworthy that
prolonged tamoxifen use in women has been associated with endometrial
hyperplasia (51) and that intrauterine injection of
cholera toxin has been induces estrogen-like growth in the uterus of
rats (52). Others have provided evidence that estrogen
induced stimulation of adenylyl cyclase may occur via a GPCR-dependent
mechanism (26, 32). These investigators have shown that
SHBG, a serum protein that binds circulating estrogen and androgens
with high affinity, when unliganded, specifically interacts with a
membrane receptor on breast and prostate cancer cells, termed SHBGR.
Upon exposure to estrogen or androgens, these preformed SHBG/SHBGR
complexes bind hormone and stimulate adenylyl cyclase activity
(32). Although the molecular nature of the SHBG
receptor remains unknown, recent data demonstrating that: 1)
nonhydrolyzable GTP analogs inhibit SHBG binding and 2) a dominant
negative Gs-subunit protein decreases estrogen-induced,
SHBG-dependent cAMP signaling, indicates that this receptor may belong
to the GPCR superfamily (53). Although it is possible that
GPR30 may serve as a receptor for SHBG, in our experiments, as well as
those conducted by others (24), no exogenous factors are
required to initiate estrogen-induced activation of adenylyl cyclase.
Furthermore, in contrast to the findings reported for SHBG-mediated
estrogen action (25), we find that GPR30-dependent
activation of adenylyl cyclase can also be promoted by the
antiestrogens, tamoxifen, and ICI 182, 780 (Fig. 1C).
In other cell types, cAMP agonists are known to promote
stimulation of MAPK activity via activation of the monomeric GTPase,
Rap-1, which in turn, promotes B-Raf-mediated activation of Mek-1 and
Erk-1/-2 (29). A similar Rap-1 dependent mechanism is
activated in LNCaP prostatic carcinoma cells in response to agents that
elevate cAMP (54). We have found that neither dibutyrl
cAMP or cholera toxin are capable of inducing rapid activation of
Erk-1/-2 in MDA-MB-231 (GPR30) cells (Fig. 2B), an effect that we show
is likely due to the fact that these cells down-modulate the 95-kDa
B-Raf isoform upon serum starvation (Fig. 2A). We show that
estrogen-mediated repression of EGF-induced activation of the
Raf-to-Erk cascade can be reversed by the cell permeant cAMP
congener, KT5720 (Fig. 3). Because this analog irreversibly binds to
the regulatory subunits of PKA, and thereby prevents its catalytic
activation, our data indicate that estrogen mediated suppression of the
EGFR-to-MAPK cascade via GPR30 occurs via PKA-dependent signaling.
Other hormones and agonists that elevate cAMP are known to oppose
activation of the EGFR-to-MAPK cascade in many other cell types.
Several distinct PKA-dependent inhibitory mechanisms have been shown to
operate. Direct phosphorylation of Raf-1 by PKA at serine residues 43
(28, 55) and 621 (56, 57) have been proposed
to be responsible for this inhibitory effect. Still others have
provided evidence that PKA may act upstream of Raf-1 (58).
Here we show that estrogen promotes Raf-1 inactivation (Fig. 9D),
which, in turn, is associated with decreased activity of Erk-1/-2 and
its activating kinase Mek-1 (Fig. 9C). Our data indicate that this
estrogen action does not interfere with the ability of Ras to couple to
Raf-1 in vitro (Fig. 9E). However, we did not explore the
possibility that estrogen promotes cAMP-dependent signals via GPR30
that prevent in vivo coupling of Ras to Raf-1. This
mechanism of GPCR- dependent inhibition of Erk has been associated
with Rap-1-dependent sequestration of Raf-1 in HEK293 cells
(59).
Estrogen-responsive cells employ both serum growth factors and estrogen for their growth and survival. Coordinated signaling between growth factor receptors and estrogen receptors is required for controlled growth and behavior of normal mammary epithelium. The discovery that these distinct extracellular stimuli utilize common intracellular signaling pathways, as exemplified by the EGFR-to-MAPK signaling axis, further emphasizes this concept. Several lines of evidence support the concept that the EGFR-MAPK signaling axis is a common pathway that is regulated by estrogen. EGF-related ligands enhance ER transcriptional activity (60), and this has been shown to result from MAPK-mediated phosphorylation of serine 118 within the activation function II (ATF-II) domain of the ER (10, 11). In this regard, these studies indicate that the ER lies downstream of the EGFR-MAPK signaling axis and may enhance ER-dependent cellular growth. Conversely, estrogen has been shown to increase EGFR expression and activity in the uterus (61, 62). However, it is important to note that this response is transient, and ultimately, results in the restoration of EGFR expression to levels observed before estrogen stimulation (63). Studies designed to investigate the refractoriness of ER-transfected cells to undergo estrogen-dependent proliferation have demonstrated that EGFR signaling must be silenced for estrogen-dependent proliferation to occur in these cells (64). Others have shown that estrogen can inhibit serum-mediated, MAPK-dependent growth of vascular smooth muscle cells (65).
Although our studies indicate that GPR30 may affect estrogen-mediated
regulation of the EGFR-MAPK axis, others have also indicated that the
ER may promote activation of MAPK (12, 13, 14, 15). A novel
functional role for the ER in rapid estrogen has also been suggested
from studies that have indicated that the ER can engage and promote
activation of phosphatidylinositol 3'OH kinase (66) and
PKB/AKT (67). It is noteworthy that these downstream
signaling effectors lie downstream of receptor tyrosine kinases,
including the EGFR. Although the data presented here and previously
(23) strongly suggest that GPR30 participates in the
regulation of the EGFR-to-MAPK signaling axis, whether or not GPR30
acts alone or functions as part of a receptor complex remains to be
determined. However, it is worth reiterating that we have demonstrated
that estrogen is capable of regulating the EGFR-to-MAPK signaling axis
in SKBR3 breast cancer cells that lack ER as well as ERß, but
express GPR30 (data presented here and in Ref. 23). It is
possible, however, in other cell types, GPR30 may form a signaling
complex with the ER, or communicate with the ER to promote rapid
nongenomic estrogen signaling.
A schematic diagram depicting a likely mechanism by which GPR30
may regulate growth factor receptor and ER signal transduction pathways
is shown in Fig. 10. We have previously
shown that estrogenic hormones and GPR30 act to stimulate
Gß-subunit protein dependent transactivation of the EGFR-to-Erk
signaling axis through the release of proHB-EGF (23).
Here, we demonstrate that estrogen also stimulates adenylyl cyclase
activity and cAMP-dependent PKA-mediated suppression of the EGFR-Erk
pathway. Our model outlines a regulatory loop comprised of
opposing signals, triggered by estrogen and requiring GPR30, that
serve to balance the EGFR-to-Erk pathway. Although our experiments
indicate that these opposing mechanisms can be activated by estrogen
in vitro, our results raise an interesting question
regarding which one of these opposing estrogen-induced signals prevails
in breast tumors in vivo. Amplification of EGFRs is the
most common genetic alteration associated with breast cancer and
is detected in 30% of all breast tumors and primarily among those
tumors that fail to express ER (8). Likewise, dysregulated
expression of MAPK has been reported to be a frequent event in breast
cancer (16). However, mutations in Ras genes are
rarely observed (less than 5% of all breast cancer cases) even though
they occur frequently in other carcinomas (68). These data
suggest that intermediate components of the EGFR-to-Erk cascade are
tightly regulated in normal breast epithelial cells. In this regard,
genetic alterations that affect signaling pathways that attenuate the
EGFR-MAPK signaling cascade, including loss or mutation of GPR30, may
be a common occurrence in breast cancer.
|
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
+, ERß+), MDA-MB-231 (ER-, ERß+),
and SKBR3 (ER-, ERß-) breast carcinoma cell lines were
obtained from the American Tissue Culture Collection
(Manassas, VA). MDA-MB-231 (GPR30) cells are stable transfectants
expressing GPR30 protein and have been described previously
(23). Both MCF-7 and SKBR3 cells express elevated levels
of GPR30 protein relative to MDA-MB-231 cells (23). All
cultures were grown in phenol red-free DMEM/Ham’s F12 media (1:1)
supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin.
MDA-MB-231 (GPR30) cells were maintained in the same medium
supplemented with 500 µg/ml geneticin (Sigma, St. Louis,
MO).
Growth Factors, Estrogens, and Antiestrogens, cAMP Agonists, and
Congeners
Recombinant human EGF was purchased from the Upstate Biotechnology, Inc. (Lake Placid, NY). Water-soluble 17ß-E2;
its inactive isomer, 17-E2; progesterone; 4-hydroxytamoxifen; and
cholera toxin were purchased from Sigma. The pure ER
antagonist, ICI 182, 780 was obtained from Tocris Chemicals (Ballwin,
MN). Dibutyrl-cAMP was obtained from Roche Molecular Biochemicals (Indianapolis, IN) and the cell permeant cAMP
congener, KT5720 from Calbiochem (La Jolla, CA). The
diphtheria toxin mutant, CRM-197, was purchased from Berna Products
(Coral Gables, FL).
Antibodies
The p42/44 MAPK antibody that recognizes total Erk-1 and Erk-2
protein (phosphorylation state-independent) and phospho-specific
antibodies that recognize either phosphorylated Erk-1 and -2
(phospho-Erk), or phosphorylated Mek-1 (phospho-Mek) were purchased
from New England Biolabs, Inc., now Cell Signaling
Technologies, Inc. (Beverly, MA). The Erk-2 antibodies were also
purchased from the same vendor and are also known to cross react with
Erk-1. Monoclonal antibodies Ab-1 (Calbiochem) and 29.1
(Sigma) recognize the ErbB1/EGFR receptor and do not
cross-react with ErbB2 (Her-2/Neu), ErbB3, or ErbB4. Monoclonal
antibody 29.1 recognizes an epitope external to the ligand binding
domain of the EGFR and does not interfere with EGF binding. The
phosphotyrosine-specific monoclonal antibody, PY20, was purchased from
Transduction Laboratories, Inc., Lexington, KY). Raf-1
(C-12) antibodies raised against a peptide from the carboxyl terminus
of the human Raf-1 protein were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies that recognize
the 95- and 68-kDa isoforms of B-Raf (C-19) were purchased from the
same vendor. Ras monoclonal antibody (clone RAS10) recognizes both the
Ha- and Ki-Ras isoforms at 21 kDa and was obtained from
Upstate Biotechnology, Inc.
Conditions for Cell Stimulation
Breast carcinoma cells were seeded onto 90-mm Falcon tissue
culture dishes in phenol-red free DMEM/F12 medium containing 10% FCS.
The following day, the cell monolayers were washed three times with
phenol-red free, serum-free DMEM/F12, and exchanged for fresh
phenol-red free, serum-free media on each of the following 3 d.
Stimulations of quiescent cells were carried out at 37 in serum-free
medium as described in the figure legends. After stimulation,
monolayers were washed twice with ice-old PBS, and lysed in ice-cold
RIPA buffer (150 mM NaCl, 100 mM Tris, pH 7.5,
1% deoxycholate, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 3.5
mM NaVO4, 2 mM
phenylmethylsulfonylfluoride, 50 mM NaF, 100 mM
sodium pyrophosphate plus a protease inhibitor cocktail; Complete,
Roche Molecular Biochemicals). Crude lysates were
clarified by centrifugation and cellular protein concentration was
determined using the bichichoninic acid method according to
manufacturer’s suggestions (Pierce Chemical Co.,
Rockford, IL). Detergent lysates were stores at -70 C until use.
Western Blotting
Total cellular protein (50 µg) was boiled in standard Laemmli
buffer with reducing agents and resolved by SDS-PAGE. Proteins were
electrotransferred onto nitrocellulose membranes (0.45 µM
pore size; Schleicher & Schuell, Inc., Keene, NH) using a
semi-dry transfer cell (CBS Scientific Co., Del Mar, CA) at 1
mA/cm2 for 4 h. Phospho-Erk was detected by
probing membranes, which were preblocked in Tris-buffered saline
containing 0.1% Tween-20 and 2% BSA (TBST-BSA), with
phospho-Erk-specific antibodies diluted 1:1,000 in TBST-BSA for 1
h at room temperature. Rabbit antibody-antigen complexes were detected
with horseradish peroxidase-coupled goat antibodies to rabbit IgG
diluted 1:5,000 in TBST-BSA and visualized by enhanced
chemiluminescence (Amersham Pharmacia Biotech,
Arlington Heights, IL). Relative levels of total Erk-2 protein in each
sample were determined by stripping the phospho-specific Erk
rabbit antibodies from the nitrocellulose membrane and reprobing
with antibodies to Erk-2. Phosphorylated Mek-1 protein was detected in
much the same manner, except that filters to be probed with phospho-Mek
antibodies were blocked in TBST containing 5% nonfat dry milk and
antibodies were delivered overnight in TBST-BSA. Apparent molecular
weights were determined from Rainbow molecular weight standards
(Amersham Pharmacia Biotech).
Adenylyl Cyclase Activity
Cells (50 x 106) were homogenized in
20 ml of 10 mM Tris-HCl (pH 7.4), 5 mM EDTA
samples were sonicated, and sedimented twice (1,000 x
g for 5 min and 40,000 x g for 20 min). The
membrane pellet was resuspended at a final concentration of 3–5 mg/ml
in 75 mM Tris-HCl, pH 7.4, 2
mM EDTA, 5 mM
MgCl2 and stored at - 80 C. Ten micrograms
of membrane protein were added to reactions containing 1
mM ATP, 50 nM GTP, 0.2 IU
pyruvate kinase, 0.1 IU myokinase, 2.5 mM
phosphoenolpyruvate, and 1.0 mM
isobutylmethylxanthine, and treated with 17ß-E2, 17-E2,
progesterone, 4- hydroxytamoxifen, or cholera toxin for 20 min at
37 C. Reactions were terminated by precipitating the samples with
ice-cold ethanol. Supernatants were dried and cAMP was measured in a
competitive ELISA using rabbit cAMP-specific antisera (Cayman
Biochemicals, Ann Arbor, MI).
Detection of Erk-1/-2 and Raf-1 Activity
Erk-1/-2 activity was measured by standard immune complex assay
utilizing myelin basic protein (MBP) as a substrate. Erk-1 and -2 were
immunopurified from 500 µg of lysate using 2 µg/sample of p42/44
MAPK antibody plus 50 µl of a 50% slurry of protein G-agarose
(Pierce Chemical Co.). Erk immunoprecipitates were washed
twice in 50 mM HEPES (pH 7.9), 100 mM NaCl and
then resuspended in immune complex kinase buffer: 25 mM
HEPES, pH 7.9, 1 mM DTT, 10 mM cold ATP, 50
µM 32P-ATP (0.25 µCi), and 8
µg MBP (Upstate Biotechnology, Inc.). After a 30-min
incubation at 30 C, samples were boiled in standard Laemmli buffer
and subjected to SDS-PAGE. Gels were dried and exposed to
Kodak XAR film for autoradiography. Raf-1 activity using a
kinase cascade assay kit, essentially as described by the manufacturer
(Upstate Biotechnology, Inc.). Raf-1 was
immunoprecipitated from 500 µg of lysate using 2 µg/sample of Raf-1
antibody plus 50 µl of a 50% slurry of protein G-agarose. Raf-1
immunoprecipitates were washed three times in assay dilution buffer (20
mM MOPS, pH 7.2; 25 mM ß-glycerol phosphate,
5 mM EGTA, 1 mM sodium orthovanadate, and 1
mM dithiothreitol) and then resuspended in the same buffer
containing 1 mM ATP, 75 mM
MgCl2 and 0.4 µg of unactivated
(unphosphorylated) Gst-Mek1 protein. After a 30-min incubation at 30 C,
1.0 µg of unactivated (unphosphorylated) Gst-Erk2 was added to
this kinase reaction and incubated an additional 30 min at the
same temperature. The reaction was terminated by the addition of
boiling standard Laemmli buffer. Products of the reaction were
separated by SDS-PAGE and phosphorylated GST-Erk2 was detected by
immunoblotting sing phospho-Erk-specific antibodies as described
above.
Affinity Assay for Ras Activation
Serum-starved cells were stimulated at 37 C for indicated times
and then immediately lysed in ice-cold MLB lysis buffer (25
mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal
CA-630, 0.25% sodium deoxycholate, 10% glycerol, 25 mM
NaF, 10 mM MgCl2, 1 mM
EDTA, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 10
µg/ml aprotinin). Per the manufacturer’s specifications, activated
Ras was isolated from these lysates using GST-Raf1BD coupled to
glutathione agarose beads (Upstate Biotechnology, Inc.).
Proteins were eluted from the beads by boiling in 2x Laemmli buffer,
resolved through 12% SDS-polyacrylamide gels and transferred to
nitrocellulose. Membranes were then blocked in PBS containing 0.05%
Tween 20 and 5% nonfat dried milk and probed with a Ras monoclonal
antibody (clone RAS10) overnight at 4 C. Ras antibodies were detected
using horseradish peroxidase- coupled antimouse secondary
antibodies and a chemiluminescent substrate.
Detection of Phosphotyrosyl Residues on the EGFR
Tyrosine phosphorylation of the EGFR was assessed by
immunoblotting EGFR immunoprecipitates with phosphotyrosine-specific
antibodies. EGFR was immunoprecipitated from 250 µg of total cell
protein, extracted in RIPA buffer using 2 µg/sample of the
ErbB1-specific monoclonal antibody, Ab-1. EGFR-Ab-1 complexes were
precipitated with 50 µl of a 50% slurry of protein G-agarose
(Pierce Chemical Co.). EGFR immunoprecipitates were
washed, resuspended in standard Laemmli buffer containing reducing
agents, and subjected to SDS-PAGE. After electrophoresis, the
immunoprecipitated material was then transferred to nitrocellulose
membranes, blocked with TBST-BSA, and then immunoblotted with the
phosphotyrosine-specific monoclonal antibody, PY20.
EGFR Internalization
Serum-deprived MDA-MB-231 (GPR30) cells were detached in
HEPES-buffered saline containing 5 mM EDTA, washed twice in
phenol red-free DMEM/F12 containing 0.5% BSA and resuspended at a
concentration of 106/ml in the same buffer in the
absence of BSA. One million cells were aliquoted into flow cytometry
tubes and allowed to equilibrate to 37 C in a water bath for 15 min.
Samples were either untreated or exposed to 1 nM 17ß-E2
or 10 ng/ml of EGF for various lengths of time at 37 C. After
stimulation, cells were fixed by adding an equal volume of 8%
paraformaldehyde to each sample. Cells were collected by
centrifugation, washed twice in PBS-containing 0.5% BSA (PBS-BSA) and
resuspended in the same. Fixed cells were incubated with 5 µg/ml EGFR
mAB 29.1 for 30 min at room temperature. Cells were then washed twice
in PBS-BSA, resuspended in the same buffer containing a 1:250 dilution
of fluorescein isothiocyanate- conjugated antimouse IgG antibodies,
and incubated for 30 min at room temperature. Cells were then
centrifuged, washed, and surface expression was assessed by flow
cytometry using a FACScan instrument.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Abbreviations: EGF, Epidermal growth factor; EGFR, EGF
receptor; Erk, extracellular signal-regulated kinase; HB-EGF,
heparan-bound EGF; GPCR, G protein-coupled receptor; ICI 182,
780,
(7-[9-[(4,4,5,5,5,-pentafluoropentyl)sulphinyl]nonyl]estra-1,3,5(10 )-triene-3,17ß-diol),
a high affinity ER antagonist; MBP, myelin basic protein; Mek, MAPK/ERK
kinase (same as MAP kinase kinase); SHBGR, a membrane receptor on
breast and prostate cancer cells.
Received for publication May 29, 2001. Accepted for publication September 21, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
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phosphorylation of the epidermal growth factor receptor. J Biol
Chem 273:4637–4644
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Mol Endocrinol 13:307–319 and ß: two receptors of a kind? Curr Med Chem 7:561–576[Medline]
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C. Wang, E. R. Prossnitz, and S. K. Roy Expression of G Protein-Coupled Receptor 30 in the Hamster Ovary: Differential Regulation by Gonadotropins and Steroid Hormones Endocrinology, October 1, 2007; 148(10): 4853 - 4864. [Abstract] [Full Text] [PDF]
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G. W. Woodfield, A. D. Horan, Y. Chen, and R. J. Weigel TFAP2C Controls Hormone Response in Breast Cancer Cells through Multiple Pathways of Estrogen Signaling Cancer Res., September 15, 2007; 67(18): 8439 - 8443. [Abstract] [Full Text] [PDF]
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A. H. Eid, K. Maiti, S. Mitra, M. A. Chotani, S. Flavahan, S. R. Bailey, C. S. Thompson-Torgerson, and N. A. Flavahan Estrogen increases smooth muscle expression of {alpha}2C-adrenoceptors and cold-induced constriction of cutaneous arteries Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1955 - H1961. [Abstract] [Full Text] [PDF]
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 P. Dewing, M. I. Boulware, K. Sinchak, A. Christensen, P. G. Mermelstein, and P. Micevych Membrane Estrogen Receptor-{alpha} Interactions with Metabotropic Glutamate Receptor 1a Modulate Female Sexual Receptivity in Rats J. Neurosci., August 29, 2007; 27(35): 9294 - 9300. [Abstract] [Full Text] [PDF]
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A. Morales, M. Gonzalez, R. Marin, M. Diaz, and R. Alonso Estrogen inhibition of norepinephrine responsiveness is initiated at the plasma membrane of GnRH-producing GT1-7 cells J. Endocrinol., July 1, 2007; 194(1): 193 - 200. [Abstract] [Full Text] [PDF]
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E. Filardo, J. Quinn, Y. Pang, C. Graeber, S. Shaw, J. Dong, and P. Thomas Activation of the Novel Estrogen Receptor G Protein-Coupled Receptor 30 (GPR30) at the Plasma Membrane Endocrinology, July 1, 2007; 148(7): 3236 - 3245. [Abstract] [Full Text] [PDF]
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 L. A. Gilad and B. Schwartz Association of estrogen receptor {beta} with plasma-membrane caveola components: implication in control of vitamin D receptor J. Mol. Endocrinol., June 1, 2007; 38(6): 603 - 618. [Abstract] [Full Text] [PDF]
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A. Zheng, A. Kallio, and P. Harkonen Tamoxifen-Induced Rapid Death of MCF-7 Breast Cancer Cells Is Mediated via Extracellularly Signal-Regulated Kinase Signaling and Can Be Abrogated by Estrogen Endocrinology, June 1, 2007; 148(6): 2764 - 2777. [Abstract] [Full Text] [PDF]
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J. Cheng, S. C. Watkins, and W. H. Walker Testosterone Activates Mitogen-Activated Protein Kinase via Src Kinase and the Epidermal Growth Factor Receptor in Sertoli Cells Endocrinology, May 1, 2007; 148(5): 2066 - 2074. [Abstract] [Full Text] [PDF]
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 Y.-C. Hsieh, H.-P. Yu, M. Frink, T. Suzuki, M. A. Choudhry, M. G. Schwacha, and I. H. Chaudry G Protein-Coupled Receptor 30-Dependent Protein Kinase A Pathway Is Critical in Nongenomic Effects of Estrogen in Attenuating Liver Injury after Trauma-Hemorrhage Am. J. Pathol., April 1, 2007; 170(4): 1210 - 1218. [Abstract] [Full Text] [PDF]
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 J. Veliskova Estrogens and Epilepsy: Why Are We So Excited? Neuroscientist, February 1, 2007; 13(1): 77 - 88. [Abstract] [PDF]
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 E. J. Filardo, C. T. Graeber, J. A. Quinn, M. B. Resnick, D. Giri, R. A. DeLellis, M. M. Steinhoff, and E. Sabo Distribution of GPR30, a Seven Membrane-Spanning Estrogen Receptor, in Primary Breast Cancer and its Association with Clinicopathologic Determinants of Tumor Progression. Clin. Cancer Res., November 1, 2006; 12(21): 6359 - 6366. [Abstract] [Full Text] [PDF]
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 A. Howell Pure oestrogen antagonists for the treatment of advanced breast cancer. Endocr. Relat. Cancer, September 1, 2006; 13(3): 689 - 706. [Abstract] [Full Text] [PDF]
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A. Pedram, M. Razandi, and E. R. Levin Nature of Functional Estrogen Receptors at the Plasma Membrane Mol. Endocrinol., September 1, 2006; 20(9): 1996 - 2009. [Abstract] [Full Text] [PDF]
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L. F. Macedo, Z. Guo, S. L. Tilghman, G. J. Sabnis, Y. Qiu, and A. Brodie Role of androgens on mcf-7 breast cancer cell growth and on the inhibitory effect of letrozole. Cancer Res., August 1, 2006; 66(15): 7775 - 7782. [Abstract] [Full Text] [PDF]
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J. E Sanchez-Criado, J. C Garrido-Gracia, C. Bellido, R. Aguilar, P. Guelmes, P. Abreu, R. Alonso, I. Barranco, Y. Millan, and J. M. de las Mulas Oestradiol-17{beta} inhibits tamoxifen-induced LHRH self-priming blocking hormone-dependent and ligand-independent activation of the gonadotrope progesterone receptor in the rat. J. Endocrinol., July 1, 2006; 190(1): 73 - 84. [Abstract] [Full Text] [PDF]
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 M. R. Meyer, E. Haas, and M. Barton Gender Differences of Cardiovascular Disease: New Perspectives for Estrogen Receptor Signaling Hypertension, June 1, 2006; 47(6): 1019 - 1026. [Full Text] [PDF]
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J. Qiu, M. A. Bosch, S. C. Tobias, A. Krust, S. M. Graham, S. J. Murphy, K. S. Korach, P. Chambon, T. S. Scanlan, O. K. Ronnekleiv, et al. A G-Protein-Coupled Estrogen Receptor Is Involved in Hypothalamic Control of Energy Homeostasis J. Neurosci., May 24, 2006; 26(21): 5649 - 5655. [Abstract] [Full Text] [PDF]
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 A W Horne, E-N Lalani, R A Margara, and J O White The effects of sex steroid hormones and interleukin-1-beta on MUC1 expression in endometrial epithelial cell lines. Reproduction, April 1, 2006; 131(4): 733 - 742. [Abstract] [Full Text] [PDF]
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V. Vijayanathan, S. Venkiteswaran, S. K. Nair, A. Verma, T.J. Thomas, B. T. Zhu, and T. Thomas Physiologic levels of 2-methoxyestradiol interfere with nongenomic signaling of 17beta-estradiol in human breast cancer cells. Clin. Cancer Res., April 1, 2006; 12(7 Pt 1): 2038 - 2048. [Abstract] [Full Text] [PDF]
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S. M. Belcher, H. H. Le, L. Spurling, and J. K. Wong Rapid Estrogenic Regulation of Extracellular Signal- Regulated Kinase 1/2 Signaling in Cerebellar Granule Cells Involves a G Protein- and Protein Kinase A-Dependent Mechanism and Intracellular Activation of Protein Phosphatase 2A Endocrinology, December 1, 2005; 146(12): 5397 - 5406. [Abstract] [Full Text] [PDF]
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E. R. Levin Integration of the Extranuclear and Nuclear Actions of Estrogen Mol. Endocrinol., August 1, 2005; 19(8): 1951 - 1959. [Abstract] [Full Text] [PDF]
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C. Grossmann, A. Benesic, A. W. Krug, R. Freudinger, S. Mildenberger, B. Gassner, and M. Gekle Human Mineralocorticoid Receptor Expression Renders Cells Responsive for Nongenotropic Aldosterone Actions Mol. Endocrinol., July 1, 2005; 19(7): 1697 - 1710. [Abstract] [Full Text] [PDF]
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 A. Hasbi, B. F. O'Dowd, and S. R. George A G Protein-Coupled Receptor For Estrogen: The End Of The Search? Mol. Interv., June 1, 2005; 5(3): 158 - 161. [Abstract] [Full Text] [PDF]
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J.-R. Chen, L. I. Plotkin, J. I. Aguirre, L. Han, R. L. Jilka, S. Kousteni, T. Bellido, and S. C. Manolagas Transient Versus Sustained Phosphorylation and Nuclear Accumulation of ERKs Underlie Anti-Versus Pro-apoptotic Effects of Estrogens J. Biol. Chem., February 11, 2005; 280(6): 4632 - 4638. [Abstract] [Full Text] [PDF]
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P. Thomas, Y. Pang, E. J. Filardo, and J. Dong Identity of an Estrogen Membrane Receptor Coupled to a G Protein in Human Breast Cancer Cells Endocrinology, February 1, 2005; 146(2): 624 - 632. [Abstract] [Full Text] [PDF]
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B. Schafer, B. Marg, A. Gschwind, and A. Ullrich Distinct ADAM Metalloproteinases Regulate G Protein-coupled Receptor-induced Cell Proliferation and Survival J. Biol. Chem., November 12, 2004; 279(46): 47929 - 47938. [Abstract] [Full Text] [PDF]
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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 Mol. Biol. Cell, July 1, 2004; 15(7): 3266 - 3284. [Abstract] [Full Text] [PDF]
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H. E. Cunliffe, M. Ringner, S. Bilke, R. L. Walker, J. M. Cheung, Y. Chen, and P. S. Meltzer The Gene Expression Response of Breast Cancer to Growth Regulators: Patterns and Correlation with Tumor Expression Profiles Cancer Res., November 1, 2003; 63(21): 7158 - 7166. [Abstract] [Full Text] [PDF]
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L. B. Lutz, M. Jamnongjit, W.-H. Yang, D. Jahani, A. Gill, and S. R. Hammes Selective Modulation of Genomic and Nongenomic Androgen Responses by Androgen Receptor Ligands Mol. Endocrinol., June 1, 2003; 17(6): 1106 - 1116. [Abstract] [Full Text] [PDF]
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S. R. Hammes The further redefining of steroid-mediated signaling PNAS, March 4, 2003; 100(5): 2168 - 2170. [Full Text] [PDF]
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E. R. Levin Bidirectional Signaling between the Estrogen Receptor and the Epidermal Growth Factor Receptor Mol. Endocrinol., March 1, 2003; 17(3): 309 - 317. [Abstract] [Full Text] [PDF]
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M. Razandi, A. Pedram, S. T. Park, and E. R. Levin Proximal Events in Signaling by Plasma Membrane Estrogen Receptors J. Biol. Chem., January 17, 2003; 278(4): 2701 - 2712. [Abstract] [Full Text] [PDF]
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T. M. Ahola, N. Alkio, T. Manninen, and T. Ylikomi Progestin and G Protein-Coupled Receptor 30 Inhibit Mitogen-Activated Protein Kinase Activity in MCF-7 Breast Cancer Cells Endocrinology, December 1, 2002; 143(12): 4620 - 4626. [Abstract] [Full Text] [PDF]
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D. Rueda, B. Navarro, A. Martinez-Serrano, M. Guzman, and I. Galve-Roperh The Endocannabinoid Anandamide Inhibits Neuronal Progenitor Cell Differentiation through Attenuation of the Rap1/B-Raf/ERK Pathway J. Biol. Chem., November 22, 2002; 277(48): 46645 - 46650. [Abstract] [Full Text] [PDF]
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J. Duchene, J. P. Schanstra, C. Pecher, A. Pizard, C. Susini, J.-P. Esteve, J.-L. Bascands, and J.-P. Girolami A Novel Protein-Protein Interaction between a G Protein-coupled Receptor and the Phosphatase SHP-2 Is Involved in Bradykinin-induced Inhibition of Cell Proliferation J. Biol. Chem., October 18, 2002; 277(43): 40375 - 40383. [Abstract] [Full Text] [PDF]
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T. M. Ahola, T. Manninen, N. Alkio, and T. Ylikomi G Protein-Coupled Receptor 30 Is Critical for a Progestin-Induced Growth Inhibition in MCF-7 Breast Cancer Cells Endocrinology, September 1, 2002; 143(9): 3376 - 3384. [Abstract] [Full Text] [PDF]
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Z. Guo, W. P. M. Benten, J. Krucken, and F. Wunderlich Nongenomic Testosterone Calcium Signaling. GENOTROPIC ACTIONS IN ANDROGEN RECEPTOR-FREE MACROPHAGES J. Biol. Chem., August 9, 2002; 277(33): 29600 - 29607. [Abstract] [Full Text] [PDF]
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 A. C. B. Cato, A. Nestl, and S. Mink Rapid Actions of Steroid Receptors in Cellular Signaling Pathways Sci. Signal., June 25, 2002; 2002(138): re9 - re9. [Abstract] [Full Text] [PDF]
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