From the Department of Microbiology and the Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016
Received for publication, June 23, 2000, and in revised form, November 1, 2000
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ABSTRACT |
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The estrogen receptor The estrogen receptor To identify novel proteins that modulate ER transcriptional activation,
we have carried out a genetic screen in the yeast Saccharomyces
cerevisiae. The ability of the human ER to function within yeast
allows a wide variety of genetic approaches to be taken toward further
defining the mechanism of ER transcriptional activation given the ease
of genetic manipulation and simplicity of gene identification in yeast.
In addition, with the large number of orthologous proteins carrying out
the same biological functions in both S. cerevisiae and
metazoans (8-10), it is likely that the yeast factors affecting ER
transactivation will have mammalian counterparts, which can be examined
in vertebrate systems.
The genetic approach we have used to identify factors that affect ER
transcriptional activation is dosage suppression analysis. In this
technique, a mutant ER protein with a reduced ability to activate
transcription is used as a substrate to isolate yeast gene product(s)
that are capable of overcoming this mutant phenotype, thus restoring
receptor transcriptional activity. The mutant ER derivative used in
this screen is defective in transactivation by virtue of serine to
alanine mutations in the three major N-terminal phosphorylation sites,
serines 104, 106, and 118 (ERAAA). This mutant was selected
as the substrate because it has only a modest effect on ER
transactivation and therefore has the potential to isolate a broad
range of factors that affect receptor activity. We expect to isolate
yeast factors that enhance ER transcriptional activity and,
importantly, have human homologues that can then be examined in
mammalian cells for effects on ER transcriptional response. This
approach has proven successful for investigating various aspects of ER
signal transduction (11). Using this system, we have isolated RDI1, the
yeast Rho guanine nucleotide dissociation inhibitor (Rho GDI), as a
gene product that is capable of increasing both ERAAA and
WT ER transcriptional activation when overexpressed. This gene
product is the yeast homologue of the mammalian Rho GDI In this report, we extend the findings of Rubino et al. (26)
and examine the effects of human Rho GDI Plasmids
Yeast--
The reporter plasmid ERE-CYC1-LacZ contains a single
estrogen response element (ERE) upstream of a truncated CYC1
promoter linked to the Mammalian Cells--
The ER reporter plasmid contains one ERE
from the Xenopus vitellogenin A2 gene, upstream of the
herpes simplex virus thymidine kinase promoter ( Yeast Strains, Growth Conditions, and The yeast strain W303a (a ade2 leu2 his3 trp1
ura3) was used to screen for ER activators. Yeast transformation
was performed by the lithium acetate/polyethylene glycol method (40). To assay ER transcriptional activation, cells were cultured overnight in the appropriate selective medium containing 2% glucose and subcultured 1:20 in selective minimal medium containing 2% galactose, 1% raffinose to induce receptor expression and treated with
17 Cell Culture, Transfection, and Luciferase Assays
Human osteosarcoma U2OS (HTB 96) and human breast cancer MCF-7
(HTB-22) cell lines were obtained from the American Type Culture Collection (Manassas, VA), and the Ishikawa human uterine cancer cell
line was obtained from Dr. Seth Guller (NYU School of Medicine). Cells
were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum
(HyClone), 10 units/ml each of penicillin and streptomycin (Cellgro),
and 2 mM L-glutamine (Cellgro). Between 1.2 and
1.3 × 105 cells were seeded onto 35-mm plates in
phenol red-free Dulbecco's modified Eagle's medium (Cellgro)
supplemented with 10% charcoal-stripped fetal bovine serum and 2 mM L-glutamine. Transfections using
LipofectAMINE Plus reagent (Life Technologies) were performed according
to the manufacturer's recommendation. Cells were treated with hormone agonists (100 nM 17 Immunoblotting
To prepare protein extracts from transfected cells, whole cell
extracts prepared for luciferase assay in 1× reporter lysis buffer
were normalized for total protein and boiled for 3 min in SDS sample
buffer. Protein extracts were fractionated by 12% SDS-polyacrylamide
gel electrophoresis, transferred to Immobilon membrane (Millipore
Corp.), and probed with anti-ER A Genetic Screen for Activators of ER
The ERAAA phenotype is most striking at 0.1 nM
17
A search of the yeast genome data base revealed that two of the
candidate suppressors were yeast homologues of mammalian proteins previously shown to affect ER transactivation. These include 1) CKA1 (44), a homologue of the mammalian Rho GDI Expression Increases ER Transactivation--
Among the
human Rho GDIs, RDI1 is most similar to human Rho GDI Rho GDI Specifically Activates Steroid Hormone Receptors--
We
next tested the ability of Rho GDI Rho GTPases Inhibit ER Transactivation--
The GTPases known to
interact with Rho GDI
As an independent means of examining the effect of RhoA inhibition on
ER transcriptional activation, we ectopically expressed the
Clostridium botulinum C3 transferase, a protein toxin that ADP-ribosylates and inhibits RhoA but not Rac1 or Cdc42 (37, 38). As
with Rho GDI, expression of C3 transferase results in an enhancement of
ER transcriptional activity but decreases SRF transcriptional
activation in U2OS cells (Fig.
7A and data not shown).
Inhibition of ER transcriptional activation by activated RhoA, but not
Rac1 or Cdc42, was also relieved by C3 coexpression (not shown).
Ectopic expression of C3 transferase also increased ER transcriptional
activation in MCF-7 and Ishikawa cells (not shown). Likewise,
inhibition of endogenous RhoA by expression of a dominant negative form
of RhoA (RhoA.N19) also results in greater ER transactivation (Fig.
7B). Thus, inhibition of RhoA results in enhanced ER
transcriptional activation, indicating that Rho-mediated signaling
events suppress ER transactivation.
We have demonstrated that Rho GDI What is the mechanism underlying the modulation of ER transactivation
by RhoA? Since Rho GTPases mediate actin cytoskeleton reorganization as
well as the activation of multiple signaling pathways, such as c-Jun
N-terminal kinase (JNK) and p38, Rho-mediated inhibition of ER may
result from either of these events. The ability of the Rho GTPase
family members to repress ER transcriptional activity suggests that
RhoA-, Rac1-, and Cdc42-mediated signaling to ER may converge at some
common point through a shared signaling molecule. An attractive
candidate for such a common regulator is LIM kinase (53). The
GTP-bound forms of RhoA and Rac1/Cdc42 activate LIM kinase via
phosphorylation through effector kinases ROCK and Pak, respectively
(54, 55). The activated LIM kinase phosphorylates cofilin, an
actin-binding protein, thereby inhibiting its actin-depolymerizing
activity and leading to the accumulation of actin filaments. Recently,
it has been shown that changes in the actin cytoskeleton can affect
transcriptional activation by SRF (56). In a model reminiscent of that
proposed for regulation of SRF by actin (56), we speculate that
suppression of ER transactivation could result either from releasing an
ER corepressor that is associated with free G-actin or from binding a
coactivator to actin filaments, thus preventing its interaction with
the ER. One such putative actin-regulated factor is the SWI/SNF
complex, which has previously been shown to be a coactivator for
steroid receptors, including ER, in both yeast and mammalian cells
(57-59) and contains Alternatively, changes in ER transcriptional regulatory properties may
result from the activation of signal transduction pathways by Rho
GTPases. For example, Rac1/Cdc42 activate JNK and p38 mitogen-activated protein kinase pathways, which may affect ER or its coregulatory factors via phosphorylation. Unlike Rac1/Cdc42, RhoA is not thought to
activate the JNK and p38 pathways; therefore, it is unlikely that the
activated Rho GTPases are effecting ER transactivation via these
pathways. Nevertheless, since we have not excluded the possibility that
JNK and p38 are mediating the effect of the Rho GTPases on ER
transcriptional activation, we are currently testing the impact of
activation and inhibition of JNK and p38 on receptor transactivation.
A cellular activity induced by activated RhoA, Rac1, and Cdc42 is
NF- Recently, an ER-interacting protein, termed Brx, was identified and
shown to contain a domain virtually identical to the Rho GEF Lbc,
although its enzymatic activity has not been demonstrated (26).
Overexpression of Brx in Ishikawa cells increases ER transcriptional activation, and a dominant negative form of Cdc42, but not RhoA or
Rac1, reduces its coactivator function (26). Our results differ from
this report in assessing the effect of Rho GTPases on ER
transactivation and showing that RhoA, Rac1, and Cdc42 negatively regulate ER transcriptional activity. This apparent discrepancy between
what would be predicted from Rubino et al. (26), that Cdc42
increases ER transactivation, and our results showing that Cdc42, RhoA,
and Rac1 decreased ER transcriptional activation may be attributed to
methodological or cell-specific differences. Alternatively, since Brx
coactivator function is probably mediated by direct ER binding, the
inhibition of ER activity by the dominant negative form of Cdc42 may
have resulted from competition between ER and dominant negative Cdc42
for Brx binding, rather than from blocking the signaling pathway
downstream of Brx. In contrast, the effect of Rho GDI and the Rho
GTPases on ER appears to be indirect. Localization studies indicate
that ER and Rho GDI are found in distinct subcellular compartments,
with Rho GDI residing in the cytoplasm, whereas ER is confined to the
nucleus.3 In addition, GST-Rho GDI is unable to associate
with estradiol-bound ER, although it is capable of binding Rho A
in vitro.3 While Brx and Rho GTPases may
modulate ER activity through distinct mechanisms, the identification of
different components in the Rho signaling pathway as modulators of ER
transactivation underscores their importance in receptor regulation.
Our findings suggest that the Rho GTPases decrease transcriptional
activation by ER (ER) is a
ligand-dependent transcription factor that plays a critical
role in the development and progression of breast cancer, in part, by
regulating target genes involved in cellular proliferation. To identify
novel components that affect the ER transcriptional response, we
performed a genetic screen in yeast and identified RDI1, a Rho guanine
nucleotide dissociation inhibitor (Rho GDI), as a positive regulator of
ER transactivation. Overexpression of the human homologue of RDI1, Rho
GDI
, increases ER
, ER
, androgen receptor, and
glucocorticoid receptor transcriptional activation in mammalian cells
but not activation by the unrelated transcription factors serum
response factor and Sp1. In contrast, expression of constitutively
active forms of RhoA, Rac1, and Cdc42 decrease ER transcriptional
activity, suggesting that Rho GDI increases ER transactivation by
antagonizing Rho function. Inhibition of RhoA by expression of either
the Clostridium botulinum C3 transferase or a dominant
negative RhoA resulted in enhanced ER transcriptional activation, thus
phenocopying the effect of Rho GDI expression on ER transactivation.
Together, these findings establish the Rho GTPases as important
modulators of ER transcriptional activation. Since Rho GTPases regulate
actin polymerization, our findings suggest a link between the major regulators of cellular architecture and steroid receptor
transcriptional response.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(ER)1 is a
ligand-dependent transcription factor that transduces the
estrogen signal (1). Activation of ER is responsible for female sexual
development and maintenance of bone density (2, 3). In addition, ER
plays a critical role in the development and progression of breast
cancer by regulating genes and signaling pathways involved in cellular
proliferation (4). Regulation of gene expression by the ER requires the
coordinate activity of ligand binding, phosphorylation, and cofactor
interactions, with particular combinations probably resulting in the
tissue-specific responses elicited by the receptor (5-7). However, the
extracellular cues and intracellular signaling pathways modulating
these components and regulating ER transcriptional activation are not
fully understood.
, a
cytoplasmic protein originally identified as a negative regulator of
the Rho family of GTP-binding proteins (12-15). The Rho family of
GTPases, which include RhoA, Rac1, and Cdc42, are best known for their
ability to regulate actin cytoskeletal remodeling in response to
extracellular signals, thereby promoting changes in cell morphology,
adhesion, and motility (16). In addition, by affecting multiple
signaling pathways, Rho family members regulate gene transcription and
cell cycle progression and have been implicated in cellular
transformation and metastasis (17-22). The Rho family members possess
intrinsic GTPase activity and cycle between the inactive cytoplasmic
GDP-bound and the active membrane-associated GTP-bound state. The
exchange of GDP for GTP induces a conformational change in the G
protein that allows effector molecules, such as protein kinases, to
bind and initiate downstream signaling events (23). This GTP/GDP cycle
is tightly regulated in response to extracellular signals by three
different classes of proteins. Guanine nucleotide exchange factors
(GEFs) catalyze the exchange of GDP for GTP, GTPase-activating proteins
(GAPs) accelerate the intrinsic GTPase activity of the Rho GTPases, and
GDIs antagonize their activity by blocking GEFs and GAPs (12,
24). However, since the cytoplasmic GDP-bound Rho GTPases predominate
under physiological conditions, Rho GDI acts as a negative regulator of
Rho GTPases mainly by blocking the dissociation of GDP. In addition,
Rho GDI controls the subcellular localization of the GTPases,
stimulating their release from the plasma membrane (12, 25).
Interestingly, Rubino et al. (26) identified an
ER-interacting protein, termed Brx, which contains a domain virtually
identical to the Rho GEF Lbc and was first to demonstrate a link
between ER signaling and the Rho GTPases.
as well as the Rho GTPases,
RhoA, Rac1, Cdc42, on transcriptional activation by ER and other
members of the steroid receptor family in mammalian cells. Our findings
indicate that Rho GDI
specifically increases the transcriptional
activity of ER
and ER
as well as the glucocorticoid receptor (GR)
and androgen receptor (AR), but not of the unrelated transcription
factors serum response factor (SRF) and Sp1, and that this activation
is mediated via repression of Rho GTPases. These results further
establish the Rho-mediated signaling pathway as an important regulator
of ER, GR, and AR transcriptional activity.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase gene (27). The yeast high copy genomic library was described by Engebrecht et al. (28) and was generated by subcloning Sau3A partially digested yeast
genomic DNA into the BamHI site of the YEP351
plasmid. WT ER and ERAAA were expressed from the Gal1-10
promoter in Trp1, 2 µM plasmid (p2T-GAL) (29).
p2T-GAL-ERAAA was constructed by subcloning the
BamHI fragment containing ERAAA sequence from
pcDNA3 (30) plasmid into p2T-GAL (29).
109) linked to the
firefly luciferase coding sequence (XETL) (31). The GR reporter plasmid
(XG46TL) is identical to XETL, except two consensus GREs
are substituted for the ERE (31). The steroid receptor expression
plasmids are pcDNA3-human ER
, pCMV5-human ER
(32),
pcDNA3-rat GR (33), and pcDNA3-human AR (34). A
BamHI/EcoRI fragment of human Rho GDI
from
pGEX2T (35) was subcloned into pcDNA3 to create a Rho GDI
mammalian expression
construct.2 Expression
plasmids for N-terminally Myc-tagged Rac1.L61 and Cdc42.L61 have been
described previously (36). pRK5-Myc-RhoA.V14 was subcloned as an
EcoRI fragment containing Myc-tagged RhoA.V14 from EXV
plasmid (18). Both EXV.RhoA.V14 and EFC3- expressing Myc-tagged C3
transferase under the EF1
promoter have been described elsewhere
(37, 38). The dominant negative form of RhoA, RhoA.N19, was made by
site-directed mutagenesis using the oligonucleotide 5'-GGAGCCTGTGGAAAGAACTGCTTGCTCATAGTC-3' and the QuickChange
mutagenesis kit (Stratagene) with pRK5-Myc-RhoA as the template. The
entire RhoA.N19 coding region was sequenced to verify the base changed and to ensure that no other mutations were introduced. The Sp1 reporter
contains six Sp1 binding sites upstream of the adenovirus major late
promoter in front of the luciferase gene (39), and SRF reporter
contains a fragment of the c-Fos promoter upstream of luciferase
(18).
-Galactosidase Assay
-estradiol for 12 h. Quantitative liquid
-galactosidase
assays were performed as described previously (11). Plate assays were
performed by replica-plating colonies from glucose plates onto
galactose X-gal indicator plates containing 0.1 nM
17
-estradiol.
-estradiol, 100 nM
dexamethasone, and 100 nM R1881 for ER, GR, and AR,
respectively), the ER antagonist ICI 182,780 (41) (100 nM),
or ethanol vehicle 12 h post-transfection for 24 h.
Transfected cells were washed once in phosphate-buffered saline and
harvested in 1× reporter lysis buffer (Promega) as per the
manufacturer's instructions. Luciferase activity was quantified in a
reaction mixture containing 25 mM glycylglycine, pH 7.8, 15 mM MgSO4, 1 mM ATP, 0.1 mg/ml
bovine serum albumin, 1 mM dithiothreitol, using an LB 9507 luminometer (EG & G Berthold) and 1 mM
D-luciferin as substrate. The steroid receptor
transcriptional activity is normalized to reporter activity in the
absence of transfected steroid receptors and to protein concentration
as determined by the Bradford protein assay (Bio-Rad). Since MCF-7
cells contain endogenous ER, transcriptional activity of the receptor
is normalized to XETL activity in the presence of ER antagonist ICI
182,780. The data presented represent the average of two experimental
values, with error bars representing the range of the data points.
polyclonal antibody (HC-20; Santa
Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Rho GDI polyclonal
antibody (A-20; Santa Cruz Biotechnology), or anti-c-Myc monoclonal
antibody (9E10; Santa Cruz Biotechnology). The blots were developed
using horseradish peroxidase-coupled sheep anti-mouse or goat
anti-rabbit antibodies and the ECL substrate as per the manufacturer's
instructions (Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Transcriptional
Enhancement--
Concomitant serine to alanine mutations at N-terminal
phosphorylation sites 104, 106, and 118 (ERAAA) result in a
~50% reduction in ER transcriptional activity in mammalian cells
(42, 43). To determine whether the transcriptional activity of
ERAAA is also reduced in yeast, strains were constructed
containing a galactose-inducible expression vector encoding either WT
ER or ERAAA and an ER-responsive reporter plasmid. The
transcriptional activities of WT ER and ERAAA were measured
as a function of hormone concentration. Compared with WT ER,
ERAAA exhibited ~40% reduction of transcriptional activity at all hormone concentrations tested, suggesting that the
ERAAA is less efficient at engaging in the interactions
necessary for transcriptional activation (Fig.
1A).
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Fig. 1.
Isolation of yeast factors that increase
transcriptional activation by ER .
A, transcriptional activation of WT ER and ERAAA
as a function of 17
-estradiol concentration. Yeast strains were
transformed with either a galactose-inducible WT ER or
ERAAA, along with an ERE-containing
-galactosidase
reporter plasmid. Transcriptional activation by the WT ER
(dotted line) and ERAAA
(solid line) in response to increasing
17
-estradiol concentration was determined by liquid
-galactosidase assay as described under "Experimental
Procedures." Note that the ERAAA in yeast exhibits
~40% of the WT ER transcriptional activity at each estradiol
concentration tested. The dosage suppression screen was carried out in
the presence of 1 × 10
10 M
17
-estradiol, conditions under which the ERAAA phenotype
is the most pronounced. B, the relative activity of WT ER,
ERAAA, and ERAAA with an ER activator. Three
independent colonies on X-gal indicator plates in the presence
of 1 × 10
10 M
17
-estradiol are shown and represent WT ER with an empty expression
vector (WT ER), ERAAA plus an empty expression
vector (ER (AAA)), and ERAAA plus the RDI1
suppressor plasmid (ER (AAA) + ER activator).
Under these conditions, colonies expressing WT ER are blue,
ERAAA-expressing colonies appear white, and
ERAAA-expressing the ER activator RDI1 are blue.
-estradiol. Under these conditions, yeast colonies expressing WT
ER are blue, while ERAAA-expressing colonies appear white
(Fig. 1B). To screen for ER activators, yeasts expressing
ERAAA, along with an estrogen-responsive reporter gene,
were transformed with a high copy yeast genomic library and assayed for
receptor transcriptional activation on X-gal indicator plates
containing 0.1 nM 17
-estradiol. Candidate high copy
suppressors changing the ERAAA-expressing yeast from white
to blue were selected for further analysis (Fig. 1B). Of the
29,000 colonies screened, which represents approximately 3 times the
size of the yeast genome, we identified six yeast open reading frames
that enhance ER transcriptional activation (Table
I).
Yeast genes that enhance ER transactivation
-subunit
of casein kinase II that phosphorylates ER at serine 167 in
vitro (45) and 2) CAD1 (46), a member of the Jun
transcription factor family that synergizes with ER in mammalian cells
(47). In addition to genes known to regulate ER activity, several genes
not known to affect ER were identified. YAK1 (48), a
serine/threonine kinase with homology to ANPK, a protein kinase that
interacts with the zinc finger region of the AR and increases
AR-dependent transcriptional activation, was isolated once
(49). In addition, MCK1 (50), a protein kinase with homology
to glycogen synthase kinase-3 (51), was identified once. We also
isolated RDI1 (14), the yeast Rho guanine nucleotide
dissociation inhibitor (Rho GDI), three times, and
LRG1 (52), a yeast protein that contains a GTPase-activating protein (GAP) homology domain, once. Although LRG1 is
presently linked to GAP merely through sequence homology, it is
interesting to note that GAP and RDI1 are both negative regulators of
Rho GTPases. The recovery of known ER regulators together with the
repeated isolation of certain genes indicates that the approach was
sound and that the library was probably screened to saturation. Since
Rho GDI negatively regulates Rho GTPases, this result suggests that the
Rho GTPases may modulate ER transcriptional activation and is the focus
of this report.
, which
negatively regulates the best studied Rho GTPases, RhoA, Rac1, and
Cdc42. To examine whether the mammalian Rho GDI affects ER
transcription in mammalian cells, we tested the ability of human Rho
GDI
to enhance ER transcriptional activity in the human osteosarcoma
cell line U2OS. ER-negative U2OS cells were transiently transfected
with ER
, an ER-responsive reporter plasmid, along with increasing
amounts of Rho GDI
. As shown in Fig.
2A, Rho GDI
stimulates ER
transactivation in a dose-dependent manner. Enhancement of
ER transcriptional activation by Rho GDI
was also observed for
ERAAA mutant (not shown). To ensure that this enhanced transcriptional activity was not a result of increased ER protein production, we monitored protein expression in whole cell extracts using Western blot analysis. Fig. 2B illustrates that ER
levels are not increased by Rho GDI expression. Indeed, the steady
state concentration of ER decreased slightly with increasing Rho GDI expression, indicating that the effect of Rho GDI
on ER activity is
greater than that observed. The effect of Rho GDI on ER transactivation is not restricted to single cell type, since Rho GDI
also enhanced ER transactivation in MCF-7 and Ishikawa cells (Fig. 2C).
Thus, Rho GDI
can act as a positive regulator of
ER-dependent transcriptional activation in a variety of
mammalian cell lines.
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Fig. 2.
Enhancement of ER transcriptional
activation by overexpression of Rho GDI
.
A, ER-deficient U2OS cells (1.2 × 105
cells/35-mm dish) were transiently transfected using LipofectAMINE Plus
reagent with 0.1 µg of ER
expression construct or empty vector,
0.2 µg of the ERE-containing reporter gene XETL, and increasing
amounts of Rho GDI
, as indicated. 12 h after the transfection,
cells were treated with 100 nM 17
-estradiol
(E2) (dark bars) or the ethanol
vehicle (light bars) for 24 h, harvested,
and assayed for luciferase activity. ER
transcriptional activity is
normalized to XETL reporter activity in the absence of ER. The data
represent the mean of an experiment done in duplicate, which was
repeated three times. B, expression of ER
does not
increase by Rho GDI
coexpression. Whole cell extracts were prepared
from transfected cells as described under "Experimental
Procedures," and the expression of ER
and Rho GDI
was analyzed
by Western blotting. C, MCF-7 and Ishikawa cells were
transfected as above and assayed for luciferase activity. For Ishikawa
cells, ER
transcriptional activity is normalized to XETL reporter
activity in the absence of ER. For MCF-7 cells that contain endogenous
ER, transcriptional activity of the receptor is normalized to XETL
activity in the presence of ER antagonist ICI 182,780. The data
represents the mean of experiments done in duplicate, which were
repeated two times.
to affect transactivation by
other members of the steroid receptor family, ER
, GR, and AR, using
transient transfection assays. Our results indicate that Rho GDI
also increased the transcriptional activity of ER
, GR, and AR in a
dose-dependent manner (Fig.
3, A-C). To determine whether
Rho GDI-mediated activation is specific to steroid receptors, we tested
the effect of Rho GDI
on Sp1- and SRF-dependent
transactivation. Rho GTPase signaling has been previously shown to
enhance transcriptional activation by SRF (18); thus, we would expect
Rho GDI
, as a negative regulator of Rho GTPases, to decrease SRF
transcriptional activity. Consistent with this idea, Rho GDI
expression decreased SRF activity from a reporter plasmid containing
the c-Fos SRF element (Fig.
4A). Similarly, Sp1
transcriptional activity using an Sp1-responsive reporter also
decreased in response to Rho GDI overexpression (Fig. 4B).
Taken together, these results strongly suggest that Rho GDI
specifically increases transactivation by steroid hormone receptors,
perhaps through a mechanism involving suppression of Rho GTPase
signaling.
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Fig. 3.
Rho GDI enhances the
transcriptional activation by ER
, GR, and
AR. U2OS cells were transfected as described in Fig. 2 with paired
expression and reporter plasmids for ER
+ XETL (A), GR + XG46TL (B), or AR + XG46TL
(C) and, along with the indicated amount of Rho GDI
, were
treated with 100 nM 17
-estradiol (E2),
dexamethasone (Dex), and R1881, respectively, and harvested.
In each case, receptor transcriptional activity shown is normalized to
reporter activity in the absence of the receptor. The data shown
represent experiments done in duplicate that have been repeated at
least twice with similar results.
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Fig. 4.
Rho GDI inhibits transcriptional activation
by SRF and Sp1. U2OS cells were transfected as in Fig. 2 with 0.4 µg of Rho GDI together with 0.2 µg of SRF reporter
(A) or Sp1 reporter (B), harvested after 24 h, and assayed for luciferase activity. Results shown represent an
experiment done in duplicate and repeated twice.
include RhoA, Rac1, and Cdc42. To determine
whether Rho GDI increases ER transactivation by inhibiting the Rho
GTPases, we expressed constitutively active forms of Rho GTPases
(RhoA.V14, Rac1.L61, and Cdc42.L61) in U2OS cells and examined ER
transcriptional activation. As shown in Fig.
5, expression of RhoA.V14, Rac1.L61, and
Cdc42.L61 decreased ER transcriptional enhancement. Active forms of Rho
GTPases also decreased ER transactivation in MCF-7 and Ishikawa cells
(Fig. 6). In all three cell types,
expression of the constitutively active forms of RhoA, Rac1, and Cdc42
resulted in an accumulation of filamentous actin, as determined by
fluorescent phalloidin staining.3 These results are
consistent with the model that Rho GDI activates ER transcriptional
enhancement by antagonizing Rho GTPases.
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Fig. 5.
The Rho GTPases, RhoA, Rac1, and Cdc42,
inhibit ER transcriptional activation. U2OS cells were transfected
as in Fig. 2 with the indicated amount of constitutively active forms
of the Rho GTPases, RhoA.V14, Rac1.L61, and Cdc42.L61, along with 0.1 µg of ER and 0.2 µg of XETL. Cells were treated with 100 nM 17
-estradiol (E2) 12 h post-transfection and
harvested after 24 h of estradiol treatment. ER transcriptional
activity as depicted is normalized to reporter activity in the absence
of ER. Results shown represent an experiment done in duplicate and
repeated twice with similar results.
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Fig. 6.
The Rho GTPases inhibit ER transcriptional
activation in MCF-7 and Ishikawa cells. MCF-7 and Ishikawa cells
were transfected with the indicated amount of the constitutively active
forms of the Rho GTPases, RhoA.V14, Rac1.L61, and Cdc42.L61, and ER
transcriptional activity was measured as described in the legend to
Fig. 2. Shown is a representative experiment performed in duplicate and
repeated three times with similar results.
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Fig. 7.
Inhibition of endogenous RhoA by C3
transferase and dominant negative RhoA potentiates ER
transactivation. U2OS cells were transfected as in Fig. 2 with the
indicated amount of C3 expression vector (A) or dominant
negative form of RhoA (RhoA.N19) (B) along with 0.1 µg of
ER and 0.2 µg of XETL. Cells were treated as described in the
legend to Fig. 2, and ER transcriptional activation was measured. Shown
is a representative experiment performed in duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
enhances the transcriptional
activity of the ER
as well as ER
, GR, and AR but not SRF or Sp1.
We also show that activated mutant forms of RhoA, Rac1, and Cdc42
decrease, whereas inhibition of endogenous RhoA by C3 transferase or
dominant negative RhoA increases ER transcriptional activation. From
these results, we conclude that the enhanced ER transactivation
observed upon Rho GDI
overexpression is mediated by antagonism of
Rho GTPases and implicates the Rho family proteins RhoA, Rac1, and
Cdc42 in signaling to ER.
-actin as well as two actin-related protein
subunits (60-62). We are currently testing whether SWI/SNF
and/or LIM kinase mediate the modulatory effects of Rho GTPases on ER
transactivation. While the effect of actin cytoskeletal changes on ER
remains unknown, actin dynamics may provide a means of modulating ER
transcriptional activity during normal development or in pathological
settings, such as tumor progression, when cells undergo extensive actin reorganization.
B, which has been shown to inhibit steroid receptor transactivation by forming inhibitory heterocomplexes (63, 64). It is
tempting to speculate that the inhibition of ER by the Rho GTPases is
mediated by NF-
B. However, our preliminary findings suggest that
inhibition of NF-
B by overexpressing I
B does not relieve the
repressive effects of Rho GTPases on ER transactivation (not shown),
suggesting that Rho GTPases regulate ER independent of NF-
B.
, thus establishing a novel pathway of cross-talk
between cell surface receptors that regulate Rho GTPase signaling and
steroid receptor transcriptional activation. Another example of
cross-talk between the cell surface and ER is the modulation of ER
ligand-independent transcriptional activation by the epidermal growth
factor/Ras/mitogen-activated protein kinase signaling pathway (65, 66).
It has been shown that treatment of cells with epidermal growth factor
results in ER ligand-independent activation and phosphorylation by the
mitogen-activated protein kinase, Erk1 (31, 67). Although the mechanism
of this increased ER transcriptional activation remains to be
elucidated, it probably involves phosphorylation-dependent cofactor recruitment (68). Thus, Ras acts as a positive regulator of ER
transcriptional enhancement
(67),3 whereas Rho GTPases
suppress receptor transactivation. We speculate that the opposing
actions of Ras and Rho GTPases on ER-mediated transcriptional
activation provide a means of fine tuning the ER transcriptional
response to changes in the extracellular environment.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Richard Treisman,
Alan Hall, and Mark Philips for RhoA, Rac1, Cdc42, C3 transferase, and
Rho GDI plasmids; Dr. Jan-Ake Gustafsson for the ER
expression
construct; Dr. Roger Miesfeld for the AR plasmid; and Dr. Naoko Tanese
for the Sp1 reporter construct. We are grateful to Dr. Seth Guller for
the Ishikawa cells and Dr. Alan Wakeling for the ICI compound. We thank
Dr. Danny Manor for helpful discussion and Drs. Mark Philips, Susan
Logan, and Inez Rogatsky for critically reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Army Breast Cancer Research Fund Career Development Award DAMD17-96-6032 and the Irma T. Hirschl Charitable Trust (to M. J. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Army Breast Cancer Research Fund Predoctoral Grant
DAMD17-97-7275 and National Institutes of Health (NIH) Grant T32 GM07308.
§ Supported by Army Breast Cancer Research Fund Predoctoral Grant DAMD17-98-8134 and NIH Grant T32 GM07308.
¶ To whom correspondence should be addressed: Dept. of Microbiology, NYU School of Medicine, 550 First Ave., New York, NY 10016. Tel.: 212-263-7662; Fax: 212-263-8276; E-mail: garabm01@med.nyu.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M005547200
2 D. Michaelson and M. Philips, unpublished results.
3 L. F. Su and M. J. Garabedian, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ER or ER, estrogen receptor
;
ER
, estrogen receptor
;
GDI, guanine
nucleotide dissociation inhibitor;
WT, wild type;
GEF, guanine
nucleotide exchange factor;
GAP, GTPase-activating protein;
GR, glucocorticoid receptor;
AR, androgen receptor;
SRF, serum response
factor;
ERE, estrogen response element;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
RLU, relative luminescence units.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Parker, M. G. (1998) Biochem. Soc. Symp. 63, 45-50[Medline] [Order article via Infotrieve] |
2. | Warner, M., Nilsson, S., and Gustafsson, J. A. (1999) Curr. Opin. Obstet. Gynecol. 11, 249-54[CrossRef][Medline] [Order article via Infotrieve] |
3. | Bland, R. (2000) Clin. Sci. 98, 217-240[Medline] [Order article via Infotrieve] |
4. | Jordan, V. C. (1999) J. Lab. Clin. Med. 133, 408-414[Medline] [Order article via Infotrieve] |
5. |
Robyr, D.,
Wolffe, A. P.,
and Wahli, W.
(2000)
Mol. Endocrinol.
14,
329-347 |
6. |
Schapira, M.,
Raaka, B. M.,
Samuels, H. H.,
and Abagyan, R.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1008-1013 |
7. | Weigel, N. L. (1996) Biochem. J. 319, 657-667[Medline] [Order article via Infotrieve] |
8. |
Rubin, G. M.,
Yandell, M. D.,
Wortman, J. R.,
Gabor Miklos, G. L.,
Nelson, C. R.,
Hariharan, I. K.,
Fortini, M. E.,
Li, P. W.,
Apweiler, R.,
Fleischmann, W.,
Cherry, J. M.,
Henikoff, S.,
Skupski, M. P.,
Misra, S.,
Ashburner, M.,
Birney, E.,
Boguski, M. S.,
Brody, T.,
Brokstein, P.,
Celniker, S. E.,
Chervitz, S. A.,
Coates, D.,
Cravchik, A.,
Gabrielian, A.,
Galle, R. F.,
Gelbart, W. M.,
George, R. A.,
Goldstein, L. S.,
Gong, F.,
Guan, P.,
Harris, N. L.,
Hay, B. A.,
Hoskins, R. A.,
Li, J.,
Li, Z.,
Hynes, R. O.,
Jones, S. J.,
Kuehl, P. M.,
Lemaitre, B.,
Littleton, J. T.,
Morrison, D. K.,
Mungall, C.,
O'Farrell, P. H.,
Pickeral, O. K.,
Shue, C.,
Vosshall, L. B.,
Zhang, J.,
Zhao, Q.,
Zheng, X. H.,
Zhong, F.,
Zhong, W.,
Gibbs, R.,
Venter, J. C.,
Adams, M. D.,
and Lewis, S.
(2000)
Science
287,
2204-2215 |
9. |
Chervitz, S. A.,
Aravind, L.,
Sherlock, G.,
Ball, C. A.,
Koonin, E. V.,
Dwight, S. S.,
Harris, M. A.,
Dolinski, K.,
Mohr, S.,
Smith, T.,
Weng, S.,
Cherry, J. M.,
and Botstein, D.
(1998)
Science
282,
2022-2028 |
10. |
Botstein, D.,
Chervitz, S. A.,
and Cherry, J. M.
(1997)
Science
277,
1259-1260 |
11. |
Knoblauch, R.,
and Garabedian, M. J.
(1999)
Mol. Cell. Biol.
19,
3748-3759 |
12. | Fukumoto, Y., Kaibuchi, K., Hori, Y., Fujioka, H., Araki, S., Ueda, T., Kikuchi, A., and Takai, Y. (1990) Oncogene 5, 1321-1328[Medline] [Order article via Infotrieve] |
13. |
Leonard, D.,
Hart, M. J.,
Platko, J. V.,
Eva, A.,
Henzel, W.,
Evans, T.,
and Cerione, R. A.
(1992)
J. Biol. Chem.
267,
22860-22868 |
14. |
Masuda, T.,
Tanaka, K.,
Nonaka, H.,
Yamochi, W.,
Maeda, A.,
and Takai, Y.
(1994)
J. Biol. Chem.
269,
19713-19718 |
15. | Koch, G., Tanaka, K., Masuda, T., Yamochi, W., Nonaka, H., and Takai, Y. (1997) Oncogene 15, 417-422[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Hall, A.
(1998)
Science
279,
509-514 |
17. | Narumiya, S. (1996) J. Biochem. (Tokyo) 120, 215-228[Abstract] |
18. | Hill, C. S., Wynne, J., and Treisman, R. (1995) Cell 81, 1159-1170[Medline] [Order article via Infotrieve] |
19. | Coso, O. A., Chiariello, M., Yu, J. C., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J. S. (1995) Cell 81, 1137-1146[Medline] [Order article via Infotrieve] |
20. | Minden, A., Lin, A., Claret, F. X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157[Medline] [Order article via Infotrieve] |
21. | Olson, M. F., Ashworth, A., and Hall, A. (1995) Science 269, 1270-1272[Medline] [Order article via Infotrieve] |
22. | Clark, E. A., Golub, T. R., Lander, E. S., and Hynes, R. O. (2000) Nature 406, 532-535[CrossRef][Medline] [Order article via Infotrieve] |
23. | Bishop, A. L., and Hall, A. (2000) Biochem. J. 348, 241-255[CrossRef][Medline] [Order article via Infotrieve] |
24. | Hart, M. J., Maru, Y., Leonard, D., Witte, O. N., Evans, T., and Cerione, R. A. (1992) Science 258, 812-815[Medline] [Order article via Infotrieve] |
25. | Hoffman, G. R., Nassar, N., and Cerione, R. A. (2000) Cell 100, 345-356[Medline] [Order article via Infotrieve] |
26. | Rubino, D., Driggers, P., Arbit, D., Kemp, L., Miller, B., Coso, O., Pagliai, K., Gray, K., Gutkind, S., and Segars, J. (1998) Oncogene 16, 2513-2526[CrossRef][Medline] [Order article via Infotrieve] |
27. | Picard, D., Khursheed, B., Garabedian, M. J., Fortin, M. G., Lindquist, S., and Yamamoto, K. R. (1990) Nature 348, 166-168[CrossRef][Medline] [Order article via Infotrieve] |
28. | Engebrecht, J., Hirsch, J., and Roeder, G. S. (1990) Cell 62, 927-937[Medline] [Order article via Infotrieve] |
29. | Schena, M., Picard, D., and Yamamoto, K. R. (1991) Methods Enzymol. 194, 389-398[Medline] [Order article via Infotrieve] |
30. |
Rogatsky, I.,
Trowbridge, J. M.,
and Garabedian, M. J.
(1999)
J. Biol. Chem.
274,
22296-22302 |
31. | Bunone, G., Briand, P. A., Miksicek, R. J., and Picard, D. (1996) EMBO J. 15, 2174-2183[Abstract] |
32. |
Kuiper, G. G.,
Carlsson, B.,
Grandien, K.,
Enmark, E.,
Haggblad, J.,
Nilsson, S.,
and Gustafsson, J. A.
(1997)
Endocrinology
138,
863-870 |
33. |
Hittelman, A. B.,
Burakov, D.,
Iniguez-Lluhi, J. A.,
Freedman, L. P.,
and Garabedian, M. J.
(1999)
EMBO J.
18,
5380-5388 |
34. |
Chamberlain, N. L.,
Whitacre, D. C.,
and Miesfeld, R. L.
(1996)
J. Biol. Chem.
271,
26772-26778 |
35. | Hancock, J. F., and Hall, A. (1993) EMBO J. 12, 1915-1921[Abstract] |
36. | Nobes, C. D., and Hall, A. (1995) Cell 81, 53-62[Medline] [Order article via Infotrieve] |
37. |
Narumiya, S.,
Sekine, A.,
and Fujiwara, M.
(1988)
J. Biol. Chem.
263,
17255-17257 |
38. |
Sekine, A.,
Fujiwara, M.,
and Narumiya, S.
(1989)
J. Biol. Chem.
264,
8602-8605 |
39. |
Tanese, N.,
Saluja, D.,
Vassallo, M. F.,
Chen, J. L.,
and Admon, A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
13611-13616 |
40. | Schiestl, R. H., and Gietz, R. D. (1989) Curr. Genet. 16, 339-346[Medline] [Order article via Infotrieve] |
41. |
Dauvois, S.,
White, R.,
and Parker, M. G.
(1993)
J. Cell Sci.
106,
1377-1388 |
42. |
Le Goff, P.,
Montano, M. M.,
Schodin, D. J.,
and Katzenellenbogen, B. S.
(1994)
J. Biol. Chem.
269,
4458-4466 |
43. | Ali, S., Metzger, D., Bornert, J. M., and Chambon, P. (1993) EMBO J. 12, 1153-1160[Abstract] |
44. | Chen-Wu, J. L., Padmanabha, R., and Glover, C. V. (1988) Mol. Cell Biol. 8, 4981-4990[Medline] [Order article via Infotrieve] |
45. | Arnold, S. F., Obourn, J. D., Jaffe, H., and Notides, A. C. (1994) Mol. Endocrinol. 8, 1208-1214[Abstract] |
46. |
Wu, A.,
Wemmie, J. A.,
Edgington, N. P.,
Goebl, M.,
Guevara, J. L.,
and Moye-Rowley, W. S.
(1993)
J. Biol. Chem.
268,
18850-18858 |
47. |
Uht, R. M.,
Anderson, C. M.,
Webb, P.,
and Kushner, P. J.
(1997)
Endocrinology
138,
2900-2908 |
48. | Garrett, S., and Broach, J. (1989) Genes Dev. 3, 1336-1348[Abstract] |
49. |
Moilanen, A. M.,
Karvonen, U.,
Poukka, H.,
Janne, O. A.,
and Palvimo, J. J.
(1998)
Mol. Biol. Cell.
9,
2527-2543 |
50. |
Su, S. S.,
and Mitchell, A. P.
(1993)
Genetics
133,
67-77 |
51. | Bianchi, M. W., Plyte, S. E., Kreis, M., and Woodgett, J. R. (1993) Gene (Amst.) 134, 51-56[CrossRef][Medline] [Order article via Infotrieve] |
52. | Muller, L., Xu, G., Wells, R., Hollenberg, C. P., and Piepersberg, W. (1994) Nucleic Acids Res. 22, 3151-3154[Abstract] |
53. | Lawler, S. (1999) Curr. Biol. 9, 800-802[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Ohashi, K.,
Nagata, K.,
Maekawa, M.,
Ishizaki, T.,
Narumiya, S.,
and Mizuno, K.
(2000)
J. Biol. Chem.
275,
3577-3582 |
55. | Edwards, D. C., Sanders, L. C., Bokoch, G. M., and Gill, G. N. (1999) Nat. Cell Biol. 1, 253-259[CrossRef][Medline] [Order article via Infotrieve] |
56. | Sotiropoulos, A., Gineitis, D., Copeland, J., and Treisman, R. (1999) Cell 98, 159-169[Medline] [Order article via Infotrieve] |
57. | Yoshinaga, S. K., Peterson, C. L., Herskowitz, I., and Yamamoto, K. R. (1992) Science 258, 1598-1604[Medline] [Order article via Infotrieve] |
58. | Chiba, H., Muramatsu, M., Nomoto, A., and Kato, H. (1994) Nucleic Acids Res. 22, 1815-1820[Abstract] |
59. | Ichinose, H., Garnier, J. M., Chambon, P., and Losson, R. (1997) Gene (Amst.) 188, 95-100[CrossRef][Medline] [Order article via Infotrieve] |
60. | Zhao, K., Wang, W., Rando, O. J., Xue, Y., Swiderek, K., Kuo, A., and Crabtree, G. R. (1998) Cell 95, 625-636[Medline] [Order article via Infotrieve] |
61. |
Peterson, C. L.,
Zhao, Y.,
and Chait, B. T.
(1998)
J. Biol. Chem.
273,
23641-23644 |
62. | Cairns, B. R., Erdjument-Bromage, H., Tempst, P., Winston, F., and Kornberg, R. D. (1998) Mol. Cell. 2, 639-651[Medline] [Order article via Infotrieve] |
63. | Perona, R., Montaner, S., Saniger, L., Sanchez-Perez, I., Bravo, R., and Lacal, J. C. (1997) Genes Dev. 11, 463-475[Abstract] |
64. |
McKay, L. I.,
and Cidlowski, J. A.
(1998)
Mol. Endocrinol.
12,
45-56 |
65. | Katzenellenbogen, B. S. (1996) Biol. Reprod. 54, 287-293[Abstract] |
66. | Kato, S., Kitamoto, T., Masuhiro, Y., and Yanagisawa, J. (1998) ONCOLOGY 55 Suppl. 1, 5-10[CrossRef][Medline] [Order article via Infotrieve] |
67. | Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., Metzger, D., and Chambon, P. (1995) Science 270, 1491-1494[Abstract] |
68. |
Endoh, H.,
Maruyama, K.,
Masuhiro, Y.,
Kobayashi, Y.,
Goto, M.,
Tai, H.,
Yanagisawa, J.,
Metzger, D.,
Hashimoto, S.,
and Kato, S.
(1999)
Mol. Cell. Biol.
19,
5363-5372 |