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and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells
Department of Pathology (H.L., F.J., Q.W., S.V.N., W.B.)
University of South Florida College of Medicine and H. Lee Moffitt
Cancer Center Tampa, Florida 33612-4799
Department of
Immunology (J.Y., B.S.) M. D. Anderson Cancer Center
Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,
and the recently cloned ERß (3, 4, 5). Both ERs contain activation
domains located in the A/B region at the amino terminus and the E
region at the carboxyl terminus, known as activation function 1 and 2
(AF-1 and AF-2), respectively. While AF-1 is active to a certain extent
in the absence of ligands, the activity of AF-2 is highly hormone
dependent. The AFs mediate the transcriptional activation or repression
of target genes independently and/or synergistically in a cell- and
promoter-specific manner (6, 7). So far, the most notable difference
between ER and ERß is that they are expressed in a
tissue-specific manner. For example, in females, ER is the
predominant receptor expressed in uterus while ERß is the predominant
type in ovary (8, 9).
In addition to steroids, molecules such as kinase activators,
phosphatase inhibitors, neurotransmitters, cell cycle regulators, and
growth factors also regulate the activity of steroid receptors
including ERs (10, 11, 12). The non-ligand-induced ER activation has
profound clinical implications. First, it may contribute to the
hormone-independent growth of human cancers. Second, it may contribute
to the tissue-specific agonistic activity of synthetic antiestrogens
such as tamoxifen, the prototype of selective ER modulators (SERMs).
Furthermore, the physiological relevance of the non-ligand-induced ER
activation has been demonstrated in whole animal experiments. For
example, epidermal growth factor (EGF) and insulin-like growth factor I
(IGF-I) increase progesterone receptor synthesis in fetal uterus and
antiestrogens block this effect (13, 14, 15). In the mouse uterus, EGF
promotes growth, increases lactoferrin production, and induces
biochemical changes of ERs such as increased DNA binding and production
of heterogenous forms of the receptor (11, 16). These effects of EGF,
which mimic those of estrogens, do not occur in ER-deficient mice
and thus are ER mediated (17). Other studies suggest that ER
activation by EGF in COS1 (18) and Hela (19) cells is mediated through
the direct phosphorylation of Ser118 in the AF-1
region by the external signal-regulated kinases (ERKs), which are the
prototype of mitogen-activated protein kinases (MAPKs).
The MAPK family includes not only the ERKs but also the more recently identified c-Jun N-terminal kinases (JNKs), also referred as stress-activated protein kinases (SAPKs) and p38/Hog-1. MAPKs are phosphorylated and activated by MAPK kinases (MAPKKs; e.g. MEKs and JNKKs), which in turn are phosphorylated and activated by MAPK kinase kinases (MAPKKKs; e.g. RAF, Mos, and MEKKs). Whereas RAF and Mos primarily activate the ERKs, MEKKs are known to preferentially regulate JNK pathways.
To assess the involvement of the different MAPK pathways in ER
activation, we examined the transcriptional activity of ER in
endometrial and ovarian cancer cells transfected with constitutively
active forms of three different MAPKKKs, MEKK1, MEKK2, and RAF. Our
studies demonstrated that the transcriptional activity of the ER was
enhanced by the expression of full-length or constitutively active
MEKK1 but not by its relatives, MEKK2 or RAF. We also found that
MEKK1-induced ER activation was mediated through both JNK and p38
and that MEKK1 activation fully blocked the antagonistic activity of
4-hydroxytamoxifen, a metabolite of tamoxifen that is a well
established estrogenic mitogen for endometrial cancers.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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in
Endometrial Cancer Cells. Because
Ishikawa cells are heterogeneous with respect to endogenous ER
expression (20), we first assessed the effect of endogenous as well as
transfected ER on the activity of an ER reporter gene, EREe1bCAT, in
which synthetic estrogen response elements were placed in front of the
simple promoter of adenovirus E1b gene and chloramphenicol acetyl
transferase (CAT). As shown in Fig. 1A
, 17ß-estradiol did not activate the ER reporter gene in the absence of
the ER expression vector. In contrast, when the cells were
cotransfected with the ER expression vector and treated with
17ß-estradiol, reporter activity increased about 5 fold. Ectopic
ER expression had little effect on reporter activity in the absence
of 17ß-estradiol. These findings demonstrate that endogenous ER, if
present, lacks detectable transcriptional activity. However, the
transcriptional machinery required for estrogen-induced ER activation
is intact in these cells.
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in Ishikawa cells. As determined by
reporter gene assays, the ER activity induced by
10-10 M 17ß-estradiol was further
enhanced by coexpression of constitutively active MEKK1, but not by
active MEKK2 or RAF (Fig. 1B
). The extent of enhancement was comparable
to that caused by a 100-fold increase in estrogen concentration, and
thus apparently is significant. MEKK1 did not activate the reporter
gene in the absence of exogenous ER expression, confirming that
activation of the reporter gene by MEKK1 is ER-dependent. This
conclusion is consistent with the capacity of ER antagonist ICI 182,780
to block the MEKK1 activation (Fig. 1C) and the promoter independency
of the activation, which was similarly observed on both EREe1bCAT and
EREtkCAT (Fig. 1D). EREtkCAT is a more complex ER reporter in which
CAT expression is controlled by synthetic estrogen response elements
linked to a portion of thymidine kinase (tk) promoter which, in
contrast to E1b promoter, contains binding sites for multiple
sequence-specific transcription factors.
As determined by Western blotting, expression of active MEKK2 or RAF
increased ER levels in the absence of estrogen, and estrogen
treatment decreased the ER level as expected (Fig. 1E). However,
constitutive active MEKK1 did not increase ER expression in either
the absence or presence of estrogen, indicating that the increase in
ER activity induced by MEKK1 was not due to increased expression of
the transfected ER. In the presence of estrogen, the ER level in
MEKK1-transfected cells is much lower than the level in cells
transfected with control vector. Since it is known that ER activation
is associated with increased ER degradation (21), the data further
indicate that MEKK1 activated ER. Consistent with transcriptional
data in Fig. 1A, little endogenous ER protein was detected in cells
transfected with SR3 but without ER expression vector and treated
with or without estrogen (Fig. 1E). The ER antibody detected ER
in MCF-7 cells but did not detect any signal at the predicted size in
ER-negative COS cells (data not shown). These findings indicate that
this antibody is ER-specific.
To determine dosage effects of the different MAPKKKs on ER
activation, various amounts of active MEKK1, MEKK2, and RAF were
cotransfected with the ER expression plasmid and EREe1bCAT reporter
into Ishikawa cells, and the cultures were treated with
10-8 M 17ß-estradiol. For
comparative purposes, replicate cultures were transfected with similar
amounts of the MAPKKKs and an AP1 reporter, AP-1-CAT, to determine the
effect of the MAPKKKs on endogenous AP1. As shown in Fig. 2A, MEKK1 activated ER at all three
tested dosages, but the activation was highest at an intermediate dose
of 0.2 µg DNA. A similar profile was observed for AP1 activity (Fig. 2B), suggesting that MEKK1 at 1 µg may result in a level of MEKK1
expression that is toxic to the cell. Although neither MEKK2 nor RAF
activated ER at any of the dosages examined, these kinases activated
AP1 as effectively as did MEKK1. The finding indicates that the lack of
ER activation by MEKK2 or RAF was not due to lower kinase activity
or level of protein expression in the transfected cells.
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activation by the active MEKK1 was due to artifacts resulting
from truncation of the kinase, the effect of full-length MEKK1 on ER
activity was examined. As shown in Fig. 3A, the full-length MEKK1 activated the
ER in a dose- dependent manner, albeit to a lesser extent than did
active MEKK1 (compare with Figs. 1B and 2A). This is not surprising
because full-length MEKK1 in its basal state is less active and depends
on upstream activators such as Rac1 to induce its activity to a higher
level.
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, expression of
active Rac1 efficiently activated ER. Cotransfection of a dominant
negative MEKK1 inhibited the effect of Rac1 in a dose-dependent manner,
thus indicating that Rac1 enhanced ER activity via activation of
endogenous MEKK1. As a negative control, we also show that a dominant
negative MEKK2 did not affect the Rac1-mediated ER activation.
Activation of ER by MEKK1 Was Mediated through JNK and p38
To elucidate the mechanism of ER activation by MEKK1, the
involvement of MAPKKs downstream of MEKK1 in ER activation was
examined. Since some published studies (22) suggested that MEKK1 could
also activate MEK in addition to JNKKs, the involvement of MEK and
JNKK1 in MEKK1-induced ER activation was investigated. As shown in
Fig. 4A, coexpression of a dominant
negative JNKK1 blocked ER activation by MEKK1. On the other hand,
the specific MEK inhibitor PD98059 did little to affect ER
activation by MEKK1 (Fig. 4B), although, as expected, it inhibited the
ERK activation by RAF (Fig. 4C). These data suggest that MEKK1
activation of ER is mediated through JNKK instead of MEK.
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activation by
MEKK1, the effects of MEKK1 on different MAPKs, JNK1, ERK2, and p38
were first examined. These proteins were expressed in Ishikawa
cells and JNK, ERK, and p38 activities were determined by in
vitro kinase assay using appropriate substrates. In agreement with
previous findings, MEKK1 activated JNK1 (Fig. 5A). In contrast to other studies which
reported that MEKK1 activated ERK but not p38 (23), we found that MEKK1
did not activate ERK (Fig. 5B) but significantly increased the activity
of p38 (Fig. 5C) in Ishikawa cells. As shown in Fig. 4C, ERK was,
however, effectively activated by RAF, thus indicating that the lack of
effect of MEKK1 on ERK activity was not artifactual. The capacity of
MEKK1 to activate JNK1 and p38 suggests that both kinases may mediate
the effect of MEKK1 on ER activation.
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activation by
MEKK1, we transfected cells with a dominant negative JNK1 together with
active MEKK1, and the ER activity was determined. As shown in Fig. 6A, expression of dominant negative JNK1
decreased the MEKK1-induced ER activation in a dose-dependent
manner. More interestingly, dominant negative JNK1 also prevented the
estrogen-induced ER activation in the absence of ectopic MEKK1
expression (Fig. 6B). As expected, dominant negative JNK1 inhibited Jun
activation by MEKK1 (Fig. 6C). In this study, Jun activation was
determined by measuring the activity of transfected Gal-Jun fusion
protein on a Gal4 reporter, 3x17 mer-Gal-CAT. In the Gal-Jun, Gal4 DNA
binding domain was fused to c-Jun transcriptional activation domain of
which the activity is known to depend on the phosphorylation of
Ser63 and Ser73 by JNKs.
The lack of MEKK1 effect on the Gal-Jun
(Ala63/73) mutant confirmed that the
MEKK1-induced increase in the Gal4 reporter activity accurately
measured MEKK1 activation of JNK1. The dominant negative JNK1 did not
affect the activation of endogenous AP1 by RAF (Fig. 6D), a process
that is mediated through ERK pathway. Thus, dominant negative JNK1
specifically targeted the JNK pathway. These findings demonstrate that
JNK1 activity is necessary for both MEKK1-induced and
17ß-estradiol-induced ER activation.
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activation. In these
experiments, Ishikawa cells transfected with active MEKK1 were treated
with p38 inhibitor SB203580. As shown in Fig. 7, SB203580 inhibited MEKK1-induced ER
activation in a dose-dependent manner (Fig. 7A) and partially inhibited
the estrogen-induced activation in the absence of ectopic MEKK1
expression (Fig. 7B), suggesting that p38 is also involved in the ER
activation. As expected, SB203580 inhibited MEKK1-induced p38
activation in a dose-dependent manner (Fig. 7C) but had no effect on
MEKK1-induced activation of endogenous AP1 (Fig. 7D), a process that is
mainly mediated through JNK pathways. The data demonstrate that the
effect of the inhibitor on MEKK1 activation of the ER was indeed due to
the specific repression of p38 and that the inhibitor did not affect
transcription in a nonspecific manner in the cells. These findings,
coupled with those presented above, implicate both JNK1 and p38 in the
activation of ER by MEKK1 and estrogen.
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contains five known phosphorylation sites, some of which are
Ser-Pro motifs that are part of the consensus sequence for
phosphorylation by MAPKs. To test the requirement for each of these
sites for MEKK1-induced ER activation, we transfected into Ishikawa
cells ER mutants in which one or two of the known phosphorylation
sites were changed to alanines together with active MEKK1, and reporter
activity was determined. Although a difference in fold increase was
observed, all of the ER mutants were substantially activated by
MEKK1 (Fig. 8). This finding indicates
that MEKK1 activation either does not depend on phosphorylation of the
receptor at any of the known phosphorylation sites or is mediated
through redundant receptor phosphorylation of these sites.
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phosphorylation sites did not
eliminate ER activation by MEKK1, it is possible that p38 and/or JNK
phosphorylates ER at a yet unidentified site. To determine whether
p38 phosphorylates ER, we transfected Flag-tagged p38 into
Ishikawa cells with or without active MEKK1. p38 was then
immunoprecipitated from the cell extracts, and in vitro
immunocomplex kinase assays were performed using full-length
biologically active human ER
protein as a substrate. As shown
in Fig. 9A, ER was phosphorylated by
p38 immunocomplex prepared from cells both containing and lacking
active MEKK1. Cotransfection of active MEKK1, however, increased ER
phosphorylation about 3-fold, an amount that correlates well with fold
increase in ER transcriptional activity that is typically induced by
MEKK1. No phosphorylation of the ER was detected in the
immunoprecipitates prepared from the control vector-transfected cells,
suggesting that the kinase that phosphorylated ER in the
immunoprecipitates is indeed Flag-p38. For comparative purposes, we
also examined the phosphorylation of GST-ATF2 by p38 (Fig. 9B). Taking
into consideration the fact that the mol wt of ER is more than twice
that of GST-ATF2 and that the amount of ER used in the kinase assays
was one-sixth that of GST-ATF2, p38 phosphorylated ER at least
20-fold better than GST-ATF2 (Fig. 9B). Therefore, ER is an
excellent substrate for p38.
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was then determined by the
same protocol used in the p38 studies described above. In contrast to
p38, however, hemagglutinin (HA)-tagged JNK1 did not detectably
phosphorylate ER regardless of whether or not active MEKK1 was
cotransfected (Fig. 9C). As shown in Fig. 9C, ER was basally
phosphorylated in immunoprecipitates lacking HA-JNK1, and the presence
of MEKK1-activated HA-JNK1 in immunoprecipitates did not increase the
ER phosphorylation above this basal level. In contrast,
MEKK1-activated HA-JNK1 efficiently phosphorylated GST-c-Jun.
MEKK1-activated HA-JNK1 also phosphorylated GST-c-Jun in assays in
which ER protein was added to the reaction mixture, eliminating the
possibility that the lack of ER phosphorylation was due to any
inhibition of activated JNK1 activity by buffer components used to
dissolve ER. The basal level of ER phosphorylation detected in
these kinase assays presumably reflects the presence of a
contaminating kinase in the immunoprecipitates.
These studies suggest that JNK and p38 may have different roles in
ER activation.
MEKK1 Activates ER in Endometrioid Ovarian Cancer Cells
To determine whether the ER activation by MEKK1 is limited to
endometrial cancer cells, ER was transfected into epithelial ovarian
cancer cells containing no endogenous ER, and its activation by
MEKK1 was examined. As determined by ER reporter assay, active MEKK1
increased ER activity in the endometrioid ovarian cancer cell line,
MDAH 2774 (Fig. 10A). The activation
was inhibited by ICI 182,780, and neither MEKK2 nor RAF increased the
ER activity. On the other hand, none of these kinases, even at high
doses, activated the ER in the serous ovarian cancer cell, OV1063
(Fig. 10B). These data suggest that ER activation by MEKK1 may
be restricted to endometrial cancer and endometrioid ovarian cancer
cells, which are morphologically and characteristically
indistinguishable.
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is the major ER subtype in the uterus, and tamoxifen is
known to act as an ER agonist in the development of endometrial tumors.
Thus, the effect of MEKK1 on the agonistic activity of tamoxifen was
examined in Ishikawa cells. We found that in the absence of active
MEKK1, 4-hydroxytamoxifen only slightly activated ER (Fig. 11) and thus is a poor agonist.
Expression of active MEKK1 resulted in a significant increase in
4-hydroxytamofen-induced ER activity, the level of which was
comparable to that of cells stimulated with 10-8
M 17ß-estradiol. These findings show that MEKK1
activation allows 4-hydroxtamoxifen to function as a potent ER
agonist.
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activity when added to Ishikawa cells in combination with
17ß-estradiol (Fig. 11). In cells not expressing active MEKK1,
4-hydroxytamoxifen inhibited the estrogen-induced ER activation as
efficiently as did ICI 182,780. In contrast, in cells expressing active
MEKK1, 4-hydroxytamoxifen did not inhibit the estrogen-induced ER
activation, and in fact, slightly enhanced the activation. These
results demonstrate that 4-hydroxytamoxifen acts as an ER antagonist in
the absence of MEKK1 and an agonist in the presence of MEKK1. ICI
182,780, on the other hand, ablated ER activation by 17ß-estradiol
and thus functions as an ER antagonist irrespective of whether or not
active MEKK1 is expressed. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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expression plasmid,
and thus is mediated through ER. Since MEKK1 did not enhance the
level of ER expression, it must increase the reporter activity by
enhancing the specific transcriptional activity of ER. Although
MEKK1 consistently enhanced ER activity in cells treated with
different concentrations of 17ß-estradiol, its effects on the ER
in the absence of ligands were variable. Thus, more studies are
required to determine whether MEKK1 participates in the
ligand-independent activation of ER.
Our data in endometrial cancer cells show that MEKK1 activated both
JNK1 and p38, and that ER activation by MEKK1 was mediated through
JNKK-JNK/p38 pathways. These results differ from those of previous
studies which implicate the RAF-ERK pathway in ER activation in Hela
(19) and COS (18) cells. In these studies, the ER activation resulted
from ERK-mediated phosphorylation of ER at
Ser118. In our studies, however,
Ala118 ER mutant was still activated by MEKK1.
In addition, MEKK1 did not activate ERK in our system, and active RAF
did not activate ER. These results strongly argue against the
involvement of the RAF-ERK pathway in MEKK1-induced ER activation.
It is important to note that ER activation by various signals is
both tissue- and promoter specific, and thus our results and others are
not necessarily contradictory. In fact, they raise the interesting
possibility that different cell types use different MAPK pathways to
activate ER.
Our studies suggest that none of the known phosphorylation sites on
ER, including Ser118, is absolutely required
for ER activation by MEKK1. This finding suggests that MEKK1-induced
ER activation may involve the phosphorylation of ER at a novel
site. p38 activity was required for ER activation by MEKK1, and we
found that p38 efficiently phosphorylated ER in a MEKK1-regulated
manner. p38 phosphorylated ER to a greater extent than it
phosphorylated GST-ATF2, a physiological substrate for p38; thus, ER
is an excellent substrate for p38. Although JNK activity was also
needed for MEKK1-induced ER activation, it did not detectably
phosphorylate ER. Overall, these data suggest that ER activation
by MEKK1 is mediated through two independently acting downstream
kinases: p38, which directly phosphorylates ER, and JNK1,
which indirectly promotes ER activation, perhaps by
phosphorylating proteins interacting with ER.
Our previous studies on the progesterone receptor (PR) showed that PR-A and PR-B are differentially activated by MEKK1 and MEKK2 in Hela cells (24). However, in contrast to the findings in the current study, PR activation by MEKKs was independent of JNK and p38. In the case of glucocorticoid receptor, JNK activation repressed receptor activity, and the repression was relieved by mutation of JNK sites (25). During the preparation of this manuscript, it was reported that reporter genes for androgen receptors were activated by MEKK1 in prostate and breast cancer cells (26). However, in this instance, it was not clear whether the observed activation resulted from increases in the expression level or the transcriptional capacity of the receptor. Moreover, the involvement of JNK and p38 in MEKK1-induced androgen receptor activation was not examined. We believe our data are the first to demonstrate that JNK and p38 signaling pathways activate steroid receptors.
As mentioned in the Introduction, the
uterus exemplifies a system in which both steroid and nonsteroid
signals impinge on the ER in a physiologically relevant manner and
tamoxifen stimulates tumorigenesis. ER activation by MEKK1 and the
consequent increase in the agonistic activity of tamoxifen compounds
suggest that MEKK1-JNK/p38 may be involved in mediating the stimulatory
effect of estrogen and tamoxifen in tumorigenesis of endometrial
epithelium. The MEKK1-induced activation of ER transfected into MDAH
2774 cells suggests that MEKK1 may also play a similar positive role in
the formation of endometrioid ovarian cancers, which are
morphologically and characteristically indistinguishable from
endometrial cancers and may have a similar sensitivity to estrogen and
tamoxifen. Although some studies have implicated MEKK1 in apoptosis
(27), a positive role for MEKK1-JNK pathways in tumorigenesis has been
suggested by studies showing that cellular transformation by oncogenes
such as Met (28), Abl (29) and certain mutant EGF receptors (30)
depends on JNK activity and that the genetic deletion of MEKK1 from
mice impaired the survival of murine embryonic fibroblasts to stress
signals (23).
To our knowledge, our data showing that MEKK1 abrogates the antagonistic activity of 4-hydroxytamoxifen is the first demonstration of such an effect by an active kinase. Targeting the MEKK1-JNK/p38 pathway, therefore, represents a potential means of eliminating the undesired stimulatory effects of tamoxifen in endometrial tumorigenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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protein was from Affinity BioReagents, Inc.
(Golden, CO). GST-ATF2 and GST-c-Jun proteins were from New England Biolabs, Inc. (Beverly, MA). The ECL reagents for
immunoblotting were from Amersham Pharmacia Biotech Inc.
(Piscataway, NJ). Antibody for ER (F-10) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). 12CA5 and protease
inhibitor cocktail tablets were from Roche Molecular Biochemicals (Indianapolis, IN). ICI 182,780 was from Tocris
(Baldwin, MO). All other reagents were reagent grade.
Plasmids
The pLEN-hER, EREe1bCAT, and EREtkCAT were kindly provided by
Dr. Carolyn L. Smith (31). pLENßgal (24), pGal-ATF2 (32), HA-JNK1
(32), HA-ERK2 (32), Flag-p38 (32), AP-1-CAT (33), pGal-Jun(1–223)
(32), pGal-Jun(1–223)(Ala63/73) (32), and 3x17
mer-Gal-CAT (34) have been described in previous studies. Dominant
negative MEKK2 is an unpublished construct (B. Su, unpublished).
Constitutively active MEKK1 [SRMEKK1(CT)] (35), MEKK2
[SRHAMEKK2(CT)] (36), RAF1 [SRRAF(BxB)] (37), Rac1 (37) as
well as dominant negative JNK1 [SRHAJNK1(APF)] (38), MEKK1 (35),
JNKK1 [SRHAJNKK1(AL)] (36), and full-length MEKK1
(SR51p1) (39) are all in the SR3 vector and
have also been described previously.
The receptor phosphorylation site mutants in pCMV5 were kindly provided by Dr. Benita Katzenellenbogan (40). Due to our unpublished observation that the cytomegalovirus (CMV) promoter was strongly activated by constitutively active MEKK1, which interferes with our transcriptional assays, all mutants were subcloned into the same pLEN vector used to express the wild-type receptor as well as ß-galactosidase, the internal standard for normalization of the reporter activity in all of our transcription assays. The Ala537 mutant was removed from pCMV5 by BamHI and ligated into the BamHI site in pLEN. pCMV5 expressing the remainder of the mutants was first digested with EcoRI at the 5'-end of the mutant ER cDNA and a BamHI-EcoRI linker was then ligated to it. The plasmids were further cut by BamHI at the 3'-end, and the mutant cDNAs were ligated into the BamHI site of the pLEN vector.
Transfections
Ishikawa cells were grown in DMEM containing 10% FBS. One day
after plating, cells were transfected by a lipofectamine-mediated
transfection procedure following the protocol from Life Technologies, Inc. In brief, the medium was removed, and phenol
red-free DMEM was added to the cells. The DNA-lipofectamine complexes
were prepared and added dropwise to the cells. After 5 h
incubation, cells were washed with HBSS, and phenol red-free DMEM
containing 1% charcoal-stripped FBS was added to the cells. On the
following day, hormones and inhibitors were added to the cells. After
24 h, cells were harvested and assayed for CAT and
ß-galactosidase (ß-gal) activity as indicated below. For in
vitro kinase assays and Western blots, cells were treated on the
third day after transfection with hormone or kinase inhibitors for 30
min and lysed.
Transcriptional Assays
The transfected cells were harvested by scraping into TEN buffer
(40 mM Tris, 1 mM EDTA, 150 mM
NaCl, pH 8.0), collected by centrifugation, resuspended in 0.25
M Tris, pH 7.5, and lysed by freeze-thawing. CAT activity
(41, 42) and ß-gal activity (41) were assayed as previously
described. CAT activity was normalized to ß-gal activity. Duplicate
samples were analyzed for each data point.
In Vitro Immunocomplex Kinase Assays
Transfected cells were washed with ice-cold PBS and lysed in
buffer containing 20 mM Tris (pH. 7.6), 250 mM
NaCl, 3 mM EDTA, 3 mM EGTA, 5 mM
ß-glycerophosphate, 100 µM
Na3VO4, 0.5% NP-40, and
protease inhibitor cocktail (1 tablet/10 ml) for JNK1 or ERK2 kinase
assays. For p38 kinase assays, the transfected cells were lysed in
buffer containing 10 mM HEPES (pH 7.6), 250 mM
NaCl, 3 mM EDTA, 100 mM
Na3VO4, 1% Triton X-100,
and protease cocktail (1 tablet/10 ml). After centrifugation, half of
the supernatant was immunoprecipitated with 12CA5 antibody for HA-ERK2
and HA-JNK1 or M2 antibody for Flag-p38. Kinase reactions were
performed at 30 C in 20 µl buffer containing 20 mM HEPES
(pH 7.6), 10 mM PNPP, 20 mM
MgCl2, 2 mM EDTA, 2 mM
EGTA, 100 µM
Na3VO4, 10 µM
ATP, 10 µCi 
-32P-ATP with the appropriate
substrate. For MBP, GST-c-Jun, and GST-ATF2, 3 µg were used and, for
the purified ER protein, 0.5 µg was used. Reactions were
terminated by adding 2x SDS-PAGE sample buffer, analyzed by SDS-PAGE,
and visualized by autoradiography. For each kinase assay, duplicate
samples were analyzed.
Immunoblotting Analysis
To determine ER expression, transfected cells were
washed with PBS and lysed by adding 0.5 ml hot 2x SDS-PAGE sample
buffer directly to the plate. The cells were scraped into 1.5-ml
microcentrifuge tubes, heated at 100 C for 10 min, and spun for 10 min
at room temperature in a microcentrifuge set at maximum speed.
Supernatants (50 µl) was separated on a 7.5% SDS gel.
To determine the expression of HA-ERK2, HA-JNK1, and Flag-p38, half of the cell lysate prepared for kinase assays was mixed with one sixth of 6x SDS-PAGE sample buffer, heated for 10 min at 100 C, and separated on a 10% SDS gel.
After separation on SDS-PAGE, all samples were transferred to nitrocellulose membrane, probed with the corresponding antibodies, and visualized using an ECL kit following the instructions of the manufacture. For each Western blot, duplicate samples were analyzed.
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ACKNOWLEDGMENTS |
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and Drs. Doug Cress, Nancy Olashaw, and
Warren J. Pledger for critical reading of the manuscript. |
FOOTNOTES |
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This work was supported by NIH Grant R29 CA-79530 to W.B.
Received for publication April 12, 2000. Revision received August 1, 2000. Accepted for publication August 7, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 Q.-P. Li, X. Qi, R. Pramanik, N. M. Pohl, M. Loesch, and G. Chen Stress-induced c-Jun-dependent Vitamin D Receptor (VDR) Activation Dissects the Non-classical VDR Pathway from the Classical VDR Activity J. Biol. Chem., January 19, 2007; 282(3): 1544 - 1551. [Abstract] [Full Text] [PDF]
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B. Liu, J.-f. Wu, Y.-y. Zhan, H.-z. Chen, X.-y. Zhang, and Q. Wu Regulation of the Orphan Receptor TR3 Nuclear Functions by c-Jun N Terminal Kinase Phosphorylation Endocrinology, January 1, 2007; 148(1): 34 - 44. [Abstract] [Full Text] [PDF]
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 T M Sadler, M Gavriil, T Annable, P Frost, L M Greenberger, and Y Zhang Combination therapy for treating breast cancer using antiestrogen, ERA-923, and the mammalian target of rapamycin inhibitor, temsirolimus. Endocr. Relat. Cancer, September 1, 2006; 13(3): 863 - 873. [Abstract] [Full Text] [PDF]
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D. V. Henley and K. S. Korach Endocrine-Disrupting Chemicals Use Distinct Mechanisms of Action to Modulate Endocrine System Function Endocrinology, June 1, 2006; 147(6): s25 - s32. [Abstract] [Full Text] [PDF]
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 D. E. Frigo, A. Basu, E. N. Nierth-Simpson, C. B. Weldon, C. M. Dugan, S. Elliott, B. M. Collins-Burow, V. A. Salvo, Y. Zhu, L. I. Melnik, et al. p38 Mitogen-Activated Protein Kinase Stimulates Estrogen-Mediated Transcription and Proliferation through the Phosphorylation and Potentiation of the p160 Coactivator Glucocorticoid Receptor-Interacting Protein 1 Mol. Endocrinol., May 1, 2006; 20(5): 971 - 983. [Abstract] [Full Text] [PDF]
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A. Vivacqua, D. Bonofiglio, A. G. Recchia, A. M. Musti, D. Picard, S. Ando, and M. Maggiolini The G Protein-Coupled Receptor GPR30 Mediates the Proliferative Effects Induced by 17{beta}-Estradiol and Hydroxytamoxifen in Endometrial Cancer Cells Mol. Endocrinol., March 1, 2006; 20(3): 631 - 646. [Abstract] [Full Text] [PDF]
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 X. Cui, R. Schiff, G. Arpino, C. K. Osborne, and A. V. Lee Biology of Progesterone Receptor Loss in Breast Cancer and Its Implications for Endocrine Therapy J. Clin. Oncol., October 20, 2005; 23(30): 7721 - 7735. [Abstract] [Full Text] [PDF]
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M. C. Gutierrez, S. Detre, S. Johnston, S. K. Mohsin, J. Shou, D. C. Allred, R. Schiff, C. K. Osborne, and M. Dowsett Molecular Changes in Tamoxifen-Resistant Breast Cancer: Relationship Between Estrogen Receptor, HER-2, and p38 Mitogen-Activated Protein Kinase J. Clin. Oncol., April 10, 2005; 23(11): 2469 - 2476. [Abstract] [Full Text] [PDF]
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Y. M. Shah and B. G. Rowan The Src Kinase Pathway Promotes Tamoxifen Agonist Action in Ishikawa Endometrial Cells through Phosphorylation-Dependent Stabilization of Estrogen Receptor {alpha} Promoter Interaction and Elevated Steroid Receptor Coactivator 1 Activity Mol. Endocrinol., March 1, 2005; 19(3): 732 - 748. [Abstract] [Full Text] [PDF]
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 M. S. Jansen, S. C. Nagel, P. J. Miranda, E. K. Lobenhofer, C. A. Afshari, and D. P. McDonnell Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition PNAS, May 4, 2004; 101(18): 7199 - 7204. [Abstract] [Full Text] [PDF]
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T. Watanabe, H. Jono, J. Han, D. J. Lim, and J.-D. Li Synergistic activation of NF-{kappa}B by nontypeable Haemophilus influenzae and tumor necrosis factor {alpha} PNAS, March 9, 2004; 101(10): 3563 - 3568. [Abstract] [Full Text] [PDF]
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H. Leong, J. E. Riby, G. L. Firestone, and L. F. Bjeldanes Potent Ligand-Independent Estrogen Receptor Activation by 3,3'-Diindolylmethane Is Mediated by Cross Talk between the Protein Kinase A and Mitogen-Activated Protein Kinase Signaling Pathways Mol. Endocrinol., February 1, 2004; 18(2): 291 - 302. [Abstract] [Full Text] [PDF]
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S. K. Kolluri, N. Bruey-Sedano, X. Cao, B. Lin, F. Lin, Y.-H. Han, M. I. Dawson, and X.-k. Zhang Mitogenic Effect of Orphan Receptor TR3 and Its Regulation by MEKK1 in Lung Cancer Cells Mol. Cell. Biol., December 1, 2003; 23(23): 8651 - 8667. [Abstract] [Full Text] [PDF]
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 A. Jones Combining trastuzumab (Herceptin(R)) with hormonal therapy in breast cancer: what can be expected and why? Ann. Onc., December 1, 2003; 14(12): 1697 - 1704. [Abstract] [Full Text] [PDF]
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F. Jiang, P. Li, A. J. Fornace Jr., S. V. Nicosia, and W. Bai G2/M Arrest by 1,25-Dihydroxyvitamin D3 in Ovarian Cancer Cells Mediated through the Induction of GADD45 via an Exonic Enhancer J. Biol. Chem., November 28, 2003; 278(48): 48030 - 48040. [Abstract] [Full Text] [PDF]
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P. Li, H. Lee, S. Guo, T. G. Unterman, G. Jenster, and W. Bai AKT-Independent Protection of Prostate Cancer Cells from Apoptosis Mediated through Complex Formation between the Androgen Receptor and FKHR Mol. Cell. Biol., January 1, 2003; 23(1): 104 - 118. [Abstract] [Full Text]
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 R. Schiff, S. Massarweh, J. Shou, and C. K. Osborne Breast Cancer Endocrine Resistance: How Growth Factor Signaling and Estrogen Receptor Coregulators Modulate Response Clin. Cancer Res., January 1, 2003; 9(1): 447s - 454s. [Abstract] [Full Text]
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H. Lee and W. Bai Regulation of Estrogen Receptor Nuclear Export by Ligand-Induced and p38-Mediated Receptor Phosphorylation Mol. Cell. Biol., August 15, 2002; 22(16): 5835 - 5845. [Abstract] [Full Text] [PDF]
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P. H. Driggers, J. H. Segars, and D. M. Rubino The Proto-oncoprotein Brx Activates Estrogen Receptor beta by a p38 Mitogen-activated Protein Kinase Pathway J. Biol. Chem., December 7, 2001; 276(50): 46792 - 46797. [Abstract] [Full Text] [PDF]
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M. J. Ellis, A. Coop, B. Singh, L. Mauriac, A. Llombert-Cussac, F. Janicke, W. R. Miller, D. B. Evans, M. Dugan, C. Brady, et al. Letrozole Is More Effective Neoadjuvant Endocrine Therapy Than Tamoxifen for ErbB-1- and/or ErbB-2-Positive, Estrogen Receptor-Positive Primary Breast Cancer: Evidence From a Phase III Randomized Trial J. Clin. Oncol., September 15, 2001; 19(18): 3808 - 3816. [Abstract] [Full Text] [PDF]
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T. Shen, K. B. Horwitz, and C. A. Lange Transcriptional Hyperactivity of Human Progesterone Receptors Is Coupled to Their Ligand-Dependent Down-Regulation by Mitogen-Activated Protein Kinase-Dependent Phosphorylation of Serine 294 Mol. Cell. Biol., September 15, 2001; 21(18): 6122 - 6131. [Abstract] [Full Text] [PDF]
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 M. Sun, J. E. Paciga, R. I. Feldman, Z.-q. Yuan, D. Coppola, Y. Y. Lu, S. A. Shelley, S. V. Nicosia, and J. Q. Cheng Phosphatidylinositol-3-OH Kinase (PI3K)/AKT2, Activated in Breast Cancer, Regulates and Is Induced by Estrogen Receptor {alpha} (ER{alpha}) via Interaction between ER{alpha} and PI3K Cancer Res., August 1, 2001; 61(16): 5985 - 5991. [Abstract] [Full Text] [PDF]
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P. Li, S. V. Nicosia, and W. Bai Antagonism between PTEN/MMAC1/TEP-1 and Androgen Receptor in Growth and Apoptosis of Prostatic Cancer Cells J. Biol. Chem., June 1, 2001; 276(23): 20444 - 20450. [Abstract] [Full Text] [PDF]
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