Estrogen Receptor-mediated Activation of the Serum Response
Element in MCF-7 Cells through MAPK-dependent
Phosphorylation of Elk-1*
Renqin
Duan,
Wen
Xie,
Robert C.
Burghardt, and
Stephen
Safe
From the Department of Veterinary Physiology and Pharmacology,
Texas A & M University, College Station, Texas 77843-4466
Received for publication, June 22, 2000, and in revised form, November 13, 2000
 |
ABSTRACT |
17
-Estradiol (E2) induces c-fos
protooncogene expression in MCF-7 human breast cancer cells, and
deletion analysis of the c-fos promoter showed that
the serum response element (SRE) at
325 to
296 was E2-responsive.
The mechanism of ligand-activated estrogen receptor
(ER
)-dependent activation of gene expression through the
SRE was determined by mutational analysis of the promoter, analysis of
mitogen-activated protein kinase (MAPK) pathway activation by
E2, and transforming growth factor
(TGF-
) as a positive control. In addition, ER
-negative MDA-MB-231 breast cancer and Chinese hamster ovary cells were used as reference cell lines. The results showed that transcriptional activation of the SRE by E2 was
due to ER
activation of the MAPK pathway and increased binding of
the serum response factor and Elk-1 to the SRE. Subsequent studies with
dominant negative Elk-1, wild type, and variant GAL4-Elk-1 fusion
proteins confirmed that phosphorylation of Elk-1 at serines 383 and 389 in the C-terminal region of Elk-1 is an important downstream target
associated with activation of an SRE by E2. Both E2
(ER
-dependent) and growth factors (ER
-independent)
activated the SRE in breast cancer cells via the Ras/MAPK
pathway; however, in ER-negative CHO cells that do not express a
receptor for TGF-
, only hormone-induced activation was observed in
cells transfected with ER
.
 |
INTRODUCTION |
c-fos protooncogene plays an important role in
regulation of normal cell growth, differentiation, and cellular
transformation processes (1-4). c-fos is a prototypical
"immediate early" gene that is rapidly induced in response to
diverse extracellular stimuli including various mitogens and the
steroid hormones during the early phases of recruitment of quiescent
(G0) cells in the cell cycle (5-28). Homozygous
c-fos
/
mice, although viable,
are growth-retarded and develop osteopetrosis, with deficiencies in
bone remodeling and tooth eruption (29). The failure to observe a more
compromised phenotype could be due to redundancy and compensation by
other Fos-related proteins. c-Fos, Jun, and related transcription
factors are nuclear phosphoproteins that share common modular
organization consisting of multiple, separable domains each with a
defined structure and function (30, 31). The "leucine zipper" and
an adjacent basic region are required for protein-protein and
region-specific DNA interactions that are required for hetero- and
homodimerization of Ap1 proteins to form the activating protein
(activating protein 1) transcription factor complex that regulates
expression of multiple genes, including those involved in cell growth,
differentiation, and transformation (4).
Transcriptional regulation of c-fos protooncogene is
modulated, in part, by interactions of nuclear proteins with multiple cis-elements in the c-fos gene promoter (30-34).
Proximal to the c-fos TATA box is a cAMP response element
that binds cAMP response element-binding protein or ATF proteins that
mediate c-fos induction in response to neurotransmitters and
polypeptide hormones. This response uses either cAMP or
Ca2+ as second messengers to activate either protein kinase
A- or calmodulin-dependent protein kinases,
respectively (35).
Another cis element that regulates c-fos
transcription is a Sis-inducible enhancer
(SIE)1 that is recognized by
the signal transducers and activators of transcription (STAT) group of
transcription factors (36). STATs are activated and translocated to the
nucleus in response to signals that activate the Janus kinase group of
tyrosine kinases (37). A third cis element, called the serum
response element (SRE), mediates c-fos induction by growth
factors, cytokines, and other extracellular stimuli that activate MAPK
pathways (30-34). SRE is recognized by a dimer of the serum response
factor (SRF), whose binding recruits the monomeric ternary complex
factors (TCFs) that cannot bind SRE alone (30). TCFs have at least
three members, including SAP1 (SRF accessory protein), Elk-1, and SAP2,
that form a subgroup of the Ets family of transcription factors
(30).
The downstream
178 to
144 region of the fos promoter is
also required for induction of fos gene expression by
vitamin D in osteoblastic cells, and this response was associated with
binding of the vitamin D receptor and CTF/NF-1 proteins to a composite response element (38). Several studies have also demonstrated that
another member of the nuclear receptor superfamily, estrogen receptor
(ER
), also mediates 17
-estradiol (E2)-activated expression of
c-fos, which is induced as an immediate early gene in
ER
-positive breast cancer cell lines (22-28, 39). Research in this
laboratory showed that interaction of an ER
-Sp1 complex with
a GC-rich site in the distal region (39) of the c-fos gene
promoter was required for estrogen action in breast cancer cells.
Further analysis of this promoter for elements required for growth
factor and hormone responsiveness showed that transactivation by both
mitogens was observed using constructs containing the SRE. ER
activation through the SRE involved the MAPK signaling pathway and
subsequent phosphorylation of Elk-1 and showed that hormone-induced
c-fos expression in breast cancer cells involves ER
action at two elements (GC-rich and SRE) that do not require direct
receptor/DNA interactions.
 |
EXPERIMENTAL PROCEDURES |
Chemicals, Cells, Antibodies, Oligonucleotides, and
Plasmids--
MCF-7, MDA-MBA-231, and Chinese hamster ovary (CHO) cell
lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were routinely maintained in Dulbecco's modified Eagle's/F-12 medium with phenol red and supplemented with 5% fetal bovine serum plus 10 ml of antibiotic-antimycotic solution (Sigma) in
an air/carbon dioxide (95:5) atmosphere at 37 °C. For transient transfection studies, cells were grown for 1 day in Dulbecco's modified Eagle's/F-12 medium without phenol red and 5% fetal bovine serum treated with dextran-coated charcoal. The wild-type human ER
(hER) expression plasmid was provided by Dr. Ming-Jer Tsai (Baylor
College of Medicine, Houston, TX). the IGF-1 receptor antagonist, H1356
(amino acids CYAAPLKPAKSC) (40) was provided by Sigma-Genosys. Human
recombinant TGF-
, IGF-1, and E2 were purchased from Sigma, and
PD98059 was purchased from Calbiochem-Novabiochem. SRF, SAP1,
Elk-1, and ERK1 antibodies were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Active ERK1 antibody was
purchased from Promega Corp. (Madison, WI). The ER deletion mutants
HE11, HE15, and HE19 were provided by Dr. Pierre Chambon (Institut de
Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France). The dominant negative (dn) Elk, dn Ras N17, and
inhibitory MAPK expression plasmids were kindly provided by Dr. Roger
Davis (University of Massachusetts, Worcester, MA), Dr. Joseph
Baldassare (St. Louis University, St. Louis, MO), and Dr. Arthur
Gutierrez-Hartmann (University of Colorado, Denver, CO), respectively.
The plasmids GAL-ElkC and GAL-ElkC(383/389) were received from
Dr. Roger Treisman (Imperial Cancer Research Center, London, United
Kingdom). pFC2-BLCAT plasmid, which contains the
1400 to +41
5'-flanking sequence from the human c-fos gene linked to a
bacterial CAT reporter gene, was kindly provided by Dr. Alessandro
Weisz (Universita di Napoli, Naples, Italy) (23). ICI 182,780 was
provided by Alan Wakeling (Zeneca Pharmaceuticals, Macclesfield, UK).
Me2SO was used as solvent for E2 and the
antiestrogens. All other chemicals and biochemicals were of the highest
quality available from commercial sources.
Oligonucleotides derived from the c-fos protooncogene
promoter and other oligonucleotides were synthesized by the Gene
Technologies Laboratory, Texas A & M University (College Station,
TX). The complementary strands were annealed, and the 5'-overhangs were used for cloning. The structures of these oligonucleotides are summarized as follows, and the SIE and SRE sites are underlined (mutations incorporated into mutant oligonucleotides are denoted by
asterisks): SS (SIE/SRE) (
354/
296) (sense strand), 5'-AGC TTG AGC
AGT TCC CGT CAA TCC CTC CCC CCT TAC ACA GGA TGT
CCA TAT TAG GAC ATC TGC G-3'; SRE (
325/
296) (sense
strand), 5'-AGC TTA CAC AGG ATG TCC ATA TTA GGA
CAT CTG CG-3'; SRE.m1 (
325/
296) (sense strand), 5'-AGC TTG*
T*AC T*GT* ATG TCC ATA TTA GGA CAT CTG CG-3'; SRE.m2 (
325/
296) (sense strand), 5'-AGC TTA CAC AGG ATG
TCG* C*A*A TC*G* C*GA CAT CTG CG-3'; a perfect palindromic
17-mer GAL4 binding site (17M) (sense strand) (41), 5'-CGG AGG ACT GTC
CTC CG-3'.
SIE/SRE, SRE, SRE.m1, and SRE.m2 oligonucleotides and five copies of
17M were cloned into the pBLTATA-CAT vector at the HindIII and BamHI sites to give the pSS, pSRE, pSRE.m1, pSRE.m2, and
GAL4-CAT constructs, respectively, as previously described.
Transient Transfection and CAT Assays--
MCF-7, MDA-MB-231,
and CHO cells were transiently transfected, utilizing the calcium
phosphate method with 5 µg of c-fos gene promoter-derived
constructs and 2.5 µg (MCF-7 cells) or 5 µg (MDA-MB-231 cells) of
wild-type or variant ER expression plasmids plus 1 µg of
lacZ expression plasmid as an internal control for
transfection efficiency. pCDNA3-Neo (InVitrogen, Inc., Carlsbad,
CA) was utilized as an empty vector (control) and was also added in
some experiments to maintain uniform levels of added DNA. After 18 h, the medium was changed, and cells were treated with
Me2SO (0.2% total volume), 10 nM E2, 10 nM TGF-
, 10 nM IGF-1, 1 µM ICI
182,780, or other compounds or their combinations in Me2SO
for 44 h. Cells were then washed with phosphate-buffered saline
and scraped from the plates. Cell lysates were prepared in 0.15 ml of
0.25 M Tris-HCl (pH 7.5) by three freeze-thaw-sonication
cycles (3 min each). Protein concentrations were determined using
bovine serum albumin as a standard, and analysis for CAT activity in
cell lysates was standardized to
-galactosidase activity. Lysates
were incubated at 56 °C for 7 min to remove endogenous deacetylase
activity. CAT activity was determined by incubating aliquots of the
cell lysates with 0.2 mCi of
D-threo-[dichloroacetyl-1-14C]chloramphenicol
and 4 mM acetyl-CoA. Acetylation was allowed to proceed to
<20-25% completion (linear range), and acetylated metabolites were
analyzed by TLC. Following TLC, acetylated products were visualized and
quantitated using an Instant Imager analyzer. CAT activity was
calculated as fraction of that observed in cells treated with
Me2SO alone (arbitrarily set at 100), and results are
expressed as means ± S.D. The experiments were carried out at
least in triplicate and statistical significance was determined by
analysis of variance and Scheffe's test.
Electrophoretic Mobility Shift Assays--
Synthetic
oligonucleotides were synthesized, purified, annealed, and
32P-labeled as previously described (39). DNA binding was
measured using a gel retardation assay. Nuclear extracts from MCF-7
cells treated with Me2SO, 10 nM E2, or 10 nM TGF-
for 30 min were incubated in HEGD buffer (2.5 mM HEPES, 1.5 mM EDTA, 10% glycerol, 1.0 mM dithiothreitol, pH 7.6) with poly(dI-dC) (200 ng)
for 15 min at 20 °C. The mixture was then incubated for an
additional 15 min (20 °C) after the addition of
32P-labeled DNA. Aliquots from reaction mixtures were then
loaded into a 5% polyacrylamine gel (acrylamide/bisacrylamide, 30:0.8) and electrophoresed at 110 V in 0.9 M Tris borate and 2 mM EDTA, pH 8.0. The gel was dried, and protein/DNA
interactions were determined and quantitated by scanning on a Betascope
603 blot analyzer imaging system and visualized by autoradiography.
Western Immunoblotting--
Nonphosphorylated and phosphorylated
Elk-1 antibodies were purchased from New England Biolabs, and ER
antibodies were obtained from Santa Cruz Biotechnology. The plasmid
expressing Elk-1 (from Dr. R. Ianknecht, The Salk Institute) and hER
was transiently transfected into MCF-7 cells using calcium phosphate;
18 h after transfection, cells were incubated in serum-free medium
for 36 h and were then treated with chemicals and harvested at
designated time points. Cells were washed once in ice-cold
phosphate-buffered saline and collected by scraping in 0.3 ml of
ice-cold lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 µg/ml
aprotinin, 50 mM phenylmethylsulfonyl fluoride, 50 mM sodium orthovanadate). The lysates were incubated on ice
for 1 h with intermittent vortexing followed by centrifugation (15,000 × g, 5 min). Equal amounts of protein from
each treatment group were separated by SDS-polyacrylamide gel
electrophoresis (7.5% gel, 150 V) and electrophoresed (15 V,
overnight) to polyvinylidene difluoride membrane using a Bio-Rad
Trans-Blot Electrophoretic Transfer Cell and transfer buffer (48 mM Tris, 39 mM glysine, 0.025% SDS).
Subsequent procedures followed the manufacturer's recommendation (New
England Biolabs). The lysates were also used for immunoblotting for
ERKs and active ERKs. However, subsequent procedures followed different
manufacturers' recommendations (Promega for active ERKs; Santa Cruz
Biotechnology for ERKs and ER
).
Fluorescence Immunocytochemistry--
CHO-K1 cells were
subcultured in four-well Lab-Tek chambered slides (Nunc Inc.,
Naperville, IL) in Dulbecco's modified Eagle's/F-12 medium without
phenol red 5% fetal bovine serum stripped with dextran-coated
charcoal. After 24 h, cells were transiently transfected with 1 µg of hER or HE 11 expression plasmids. Transfections were done with
SuperFect Transfection Reagent (Qiagen, Inc., Valencia, CA); cells were
incubated with DNA-SuperFect Transfection Reagent complexes at 37 °C
for 3 h, followed by 24 h of recovery in Dulbecco's modified
Eagle's/F-12 medium. Prior to fixation, slides were washed three times
in Dulbecco's phosphate-buffered saline (DPBS) and then fixed for 10 min at room temperature in 2% paraformaldehyde in DPBS and 0.15 M sucrose in the presence or absence of 0.5% Nonidet P-40
for detection of nuclear and membrane ER
, respectively. Slides were
subsequently washed three times in DPBS followed by a 10-min incubation
in 100 mM ammonium chloride before a 1-h blocking step in
3% normal goat serum (G-9023; Sigma). For nuclear localization of
ER
, the rat mAb raised against the ligand binding domain of the
human ER
(H222; a generous gift of Dr. Geoffrey Greene) was diluted
to a final concentration of 3 µg/ml in DPBS containing 0.5% bovine
serum albumin, 0.1% goat serum, and 0.3% Tween 20. Rat IgG at
the same concentration was used as a control. Tween 20 (0.3%) was
included in all antibody, blocking steps, and washes for nuclear
localization of ER
, whereas it was excluded from all incubations for
membrane ER
localization. Following incubation in H222 antibody
overnight, cells were washed three times in DPBS. Cells were then
incubated for 1 h in a 1:200 dilution fluorescein isothiocyanate-conjugated goat-anti-rat IgG (62-9511; Zymed
Laboratories Inc., S. San Francisco, CA) in DPBS containing 0.1%
goat serum. Cells were then washed six times over a period of 2 h
and transferred to distilled water prior to coverslip mounting with
ProLong Antifade mounting reagent (Molecular Probes, Inc., Eugene, OR).
For each treatment, representative fluorescence images were recorded
using a Zeiss Axioplan microscope (Carl Zeiss, Thornwood, NY) equipped with a Hamamatsu chilled 3CCD color camera (Hamamatsu, Japan) using
Adobe Photoshop 5.0 (Adobe Systems, Seattle, WA) image capture software. Images from all treatment groups were captured at the same
time using identical image capture parameters.
 |
RESULTS |
Transactivation of c-fos-derived Constructs by E2 and Growth
Factors in MCF-7 and MDA-MB-231 Cells--
Both E2 and growth factors
induce c-fos protooncogene expression in MCF-7 cells
(22-27, 39), and initial studies showed that in cells transfected with
pFC2 (containing the
1400 to +41 region of the c-fos
promoter), E2 alone (10-100 nM) induced a maximal 90%
increase in CAT activity. 10 nM IGF or TGF-
alone also
significantly increased CAT activity in MCF-7 cells transfected with
pFC2. In cells treated with 10 nM E2, IGF, or TGF-
plus
hER
expression plasmid, the induction was further enhanced compared
with treatment with E2, IGF, or TGF-
alone (Fig.
1A). In ER-negative MDA-MB-231 cells transfected with pFC2, no induction response was observed after
treatment with E2 alone; however, E2 induced CAT activity in this cell
line after cotransfection with ER
expression plasmid (Fig.
1B). Since cotransfection with ER
enhanced E2
responsiveness in transient transfection assays, ER
expression
plasmid was routinely used in studies with the c-fos-derived
constructs. Previous studies identified a distal E2-responsive GC-rich
site (
1168 to
1161) that was activated through ER
/Sp1
interactions (39); however, in MCF-7 cells cotransfected with ER
and
pSS containing a more proximal fos gene promoter insert
(
354 to
296), both E2 and TGF-
induced reporter gene activity
(Fig. 1C). Moreover, in cells cotreated with the ER
antagonist ICI 182,780, only E2-induced transactivation was inhibited.
In the absence of cotransfected ER
, only TGF-
induced CAT
activity, and ICI 182,780 did not inhibit this response (data not
shown). Previous studies (12, 14-20) in other cell lines have
demonstrated that this region of the fos gene promoter is
activated by multiple mitogenic compounds, but this is the first report
of transcriptional activation by E2.

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Fig. 1.
Transactivation of
c-fos-derived constructs by E2 and growth factors in
MCF-7 and MDA-MB-231 cells. Shown are effects of E2 and growth
factors on CAT activity in MCF-7 (A) and MDA-MB-231
(B) cells transiently transfected with pFC2-BLCAT plus or
minus hER expression plasmids. MCF-7 cells were transiently transfected
with pFC2-BLCAT and treated with Me2SO (CTL),
10-100 nM E2, 10 nM IGF-1, or TGF- , and CAT
activity was determined as described under "Experimental
Procedures." Relative intensities of acetylated products in E2 and
TGF- groups are compared with the control group, and relative
intensities that are significantly higher than that in controls are
indicated (*). Results are means ± S.D. for three separate
determinations. C, the effect of ICI 182,780 on E2- and
TGF- -induced CAT activity in MCF-7 cells transiently transfected
with pSS and hER expression plasmids. MCF-7 cells were transiently
transfected with pSS and hER expression plasmid and treated with
Me2SO (DMSO), 10 nM E2, 10 nM TGF- , 1 µM ICI 182,780, or their
combinations, and CAT activity was determined as described under
"Experimental Procedures." Significant (p < 0.05)
induction (*) was observed for E2 and TGF- , but only CAT activity
induced by E2 was inhibited by ICI 182,780 (**). Results were
determined in triplicate (three separate experiments) and expressed as
means ± S.D.
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The
354 to
296 region of the fos gene promoter does not
contain an estrogen-responsive element, and DNA binding of ER
was not observed in a gel mobility shift assay (data not shown). The requirement for the DNA binding domain of ER
for estrogen action was
further investigated in MCF-7 and ER-negative MDA-MB-231 cells transfected with pSS plus wild-type ER
, mutant HE11 (DNA binding domain deletion), HE15 (expressing AF1), or mutant HE19 (expressing AF2) expression plasmids (Fig.
2B). E2-induced reporter gene
activity in both cell lines only after cotransfection of wild-type
ER
or HE11, confirming that DNA binding was not required for
transactivation and that both AF1 and AF2 domains of ER
were
required for an induction response.

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Fig. 2.
Effects of wild-type or variant ER on CAT
activity induced by E2 in MCF-7 (A) or MDA-MB-231
(B) cells cotransfected with pSS or pSRE plus
wild-type or variant ER . The transient
transfection and CAT assays were performed as described under
"Experimental Procedures." Cells were treated with
Me2SO (light bars) or 10 nM E2 (dark bars). *, the relative
induction response was significantly higher (p < 0.001) than in controls. Results were determined in triplicate (three
separate experiments) and expressed as means ± S.D.
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Kinase-dependent Activation of fos-derived Promoter
Constructs pSS (
354/
296) and pSRE (
325/
296)--
Previous
studies have shown that, like growth factors, E2 activates the MAPK and
other kinase-dependent pathways (42-48), and the results
in Fig. 2 suggest a kinase-dependent activation pathway. Fig. 3A summarizes the effects
of the MAPK kinase inhibitor, PD98059, on induction of CAT activity by
10 nM E2, 10 nM IGF, or 10 nM TGF-
in MCF-7 cells transfected with pSS and ER
expression
plasmid. TGF-
, IGF, and E2 induce reporter gene activity, and
PD98059 inhibits this response. In the absence of ER
, E2 is
inactive, whereas TGF-
and IGF induce CAT activity and PD98059
inhibits this response (data not shown). Activation of pSS by E2 was
also inhibited after cotransfection with dominant negative expression plasmids for MAPK or ras (Fig. 3B). The
254 to
296 region of the fos gene promoter contains an
upstream SIE and a downstream SRE that is composed of two binding
sites: an SRF site for binding an SRF dimer and a TCF site that binds
Ets family transcription factors, SAP1, SAP2, and Elk-1 (32). Deletion
analysis of pSS (Fig. 3C) showed that pSRE (
325 to
296
fos gene promoter insert) alone retained E2 responsiveness;
however, mutation of either the TCF or SRF binding sites resulted in
loss of hormone-mediated transactivation. In addition, cotransfection
of pSRE with dominant negative MAPK or ras expression
plasmids resulted in loss of inducibility by E2 (Fig. 3D),
suggesting that the SRE region of the fos gene promoter was
sufficient for E2 action, and inducibility was associated with
activation of the Ras/MAPK pathway. Previous studies in ER-negative CHO
cells showed that transfection with ER
also resulted in activation of the Ras/MAPK pathway via membrane ER
(45); the results in Fig.
3E illustrate that transfection of CHO cells with hER (or HE11) also activates pSRE as noted in Fig. 2 for both MCF-7 and MDA-MB-231 cells.

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Fig. 3.
Activation of c-fos-derived
constructs by E2 and growth factors is dependent on the Ras/MAPK
pathway. A, effect of PD98059 on E2- and growth
factor-induced CAT activity. MCF-7 cells were transiently transfected
with pSS and hER expression plasmid and treated with Me2SO
(DMSO), 10 nM E2, 10 nM IGF-1 or
TGF- , 50 µM PD98059, or their combinations, and CAT
activity was determined as described under "Experimental
Procedures." Significant (p < 0.05) induction (*)
was observed for E2, IGF-1, and TGF- , and PD98059 inhibited both E2-
and growth factor-induced CAT activity (**). All data presented in this
figure were derived from at least three separate experiments and are
expressed as means ± S.D. B, effects of dominant
negative Ras or MAPK on CAT activity induced by E2. MCF-7 cells were
cotransfected with pSS plus dn Ras N-17 and MAPK expression
plasmids, or an empty vector pLNCX (as control) (total amount of DNA
was kept constant). The transient transfection and CAT assays were
performed as described under "Experimental Procedures." Cells were
treated with Me2SO (light bars) or 10 nM E2 (dark bars). *, the relative
intensity was significantly higher (p < 0.001) than in
control. C, effects of E2 on CAT activity in MCF-7 cells
transiently transfected with pSS, pSRE, pSRE.m1, or pSRE.m2. MCF-7
cells were transiently transfected with the various plasmids and
treated with Me2SO or 10 nM E2, and CAT
activity was determined as described under "Experimental
Procedures." Significant (p < 0.05) induction (*)
was observed for E2 with pSS or pSRE but not with pSRE1.m1 or pSRE.m2.
D, effects of dominant negative Ras or MAPK expression on
CAT activity induced by E2 in MCF-7 cells transiently transfected with
pSRE and hER expression plasmid. MCF-7 cells were cotransfected with
pSRE plus dn Ras N-17 and MAPK expression plasmids or an empty
vector pLNCX (as control) (total amount of DNA was kept constant). The
transient transfection and CAT assays were performed as described under
"Experimental Procedures." Cells were treated with
Me2SO (light bars) or 10 nM E2 (dark bars). *, the relative
intensity was significantly higher (p < 0.001) than in
control. E, pSRE activity in CHO cells cotransfected with
wild-type and variant ER . This experiment was carried out as
described in Fig. 2, and E2 induced CAT activity
(p < 0.001) only after cotransfection with wild-type
ER or HE11.
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Protein Interactions with the c-fos SRE in Gel Mobility Shift
Assays--
Protein binding to the SRE was investigated using
[32P]SS containing the
354 to
296 region of the
fos gene promoter. After incubation of the radiolabeled
oligonucleotide with nuclear extracts from untreated MCF-7 cells,
analysis by gel mobility shift assay showed three major bands (C1, C2,
and C3) (Fig. 4A,
lane 1), and the intensity of these bands was
increased after incubation with extracts from MCF-7 cells treated with
10 nM E2 (lane 2) or 10 nM TGF-
(lane 3) for 30 min. In
antibody supershift studies using nuclear extracts from E2-treated
MCF-7 cells (Fig. 4B, lane 1), band C3
was supershifted with SRF and Elk-1 antibodies; bands C1 and C2 were
also supershifted with SRF antibody, whereas SAP1 antibody did
not give a supershifted band. These data are consistent with results of
previous gel shift assays showing that the least mobile C3 band is the
ternary complex composed of SRF dimer and Elk-1, whereas C2 is the SRF
homodimer (49, 50).

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Fig. 4.
Treatment of MCF-7 cells with E2 and
TGF- enhanced formation of ternary complex
composed of SRF and Elk-1. A, treatment of MCF-7 cells
with E2 and TGF- enhanced protein-DNA complex C3. Gel shift analysis
was performed as described under "Experimental Procedures" using 3 µg of nuclear extracts from MCF-7 cells treated with
Me2SO (DMSO), 10 nM E2, or 10 nM TGF- for 30 min. The retarded protein-DNA complex
bands (see arrows) were visualized by autoradiography.
B, protein-DNA complex C3 is a ternary complex composed of
SRF and Elk-1. Gel shift analysis was performed as described for
A, and 3 µg of nuclear extracts from MCF-7 cells treated
with 10 nM for 30 min were used. The retarded protein-DNA
complex bands (see arrows) were visualized by
autoradiography.
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Phosphorylation and Activation of Elk-1 by E2 via
ER-dependent MAPK Pathway--
Previous studies showed
that the C-terminal domain of Elk-1 is phosphorylated by MAPK at
multiple sites, and this C-terminal region (Elk-1, amino acids
307-428) can function as a transactivation domain when fused to the
DNA binding domain of GAL4 (49). Both E2 and TGF-
induced CAT
activity in MCF-7 cells transfected with ER
expression plasmid, the
GAL-ElkC fusion protein, and GAL4-dependent promoter
construct (GAL4-CAT) (Fig.
5A). In contrast, induction was not observed using a reporter without the GAL4 response elements (i.e. TATA-CAT empty vector). Transcriptional activation by
E2 or TGF-
was not observed in the GAL-ElkC fusion protein
containing mutations at serines 383 and 389 that are important for
transactivation (49) (GAL-ElkC(383/389)). Results shown in Fig.
5B also demonstrate that E2 alone (10-100 nM)
induced reporter gene activity in MCF-7 cells transfected with GAL-ElkC
and GAL4-CAT, and this response was enhanced after cotransfection with
ER
expression plasmid. Similar results were obtained using pFC2
(Fig. 1A) in which the activity of E2 alone was enhanced by
transfection with ER
. Transcriptional activation of the GAL-ElkC
fusion protein was further investigated in MCF-7 cells transiently
transfected with GAL-ElkC, GAL4-CAT, and ER
and treated with various
reagents (Fig. 5C). E2, IGF, and TGF-
induced CAT
activity, and these responses were inhibited by PD98059 (MAPK
inhibitor), whereas the antiestrogen ICI 182,780 inhibited E2-induced
but not IGF- or TGF-
-induced CAT activity. Razandi et al.
(45) previously showed that transfection of CHO cells with ER
activated the MAPK pathway, and results in Fig. 5D
demonstrate that the pattern of GAL-ElkC activation by E2 in CHO cells
is similar to that observed in MCF-7 cells (Fig. 5C). E2
induces CAT activity in CHO cells transfected with GAL4-CAT and
GAL-ElkC, and this response is inhibited by both ICI 182,780 and
PD98059. In contrast, TGF-
is inactive in this cell line as
previously reported (45) due to a nonfunctional receptor for this
mitogen.

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Fig. 5.
Activation of Elk-1 by E2 is ER- and
MAPK-dependent. A, E2 and TGF- induced
Elk-1-mediated transcriptional activation. MCF-7 cells were transiently
cotransfected with GAL4-CAT plus GAL-ElkC, GAL-ElkC (383/389), or
GAL4-DBD or with TATA-CAT plus GAL-ElkC and then treated with
Me2SO (CTL), 10 nM E2, or 10 nM TGF- . *, the relative intensity was significantly
higher (p < 0.001) than in control. Results were
determined in triplicate (three separate experiments) and expressed as
means ± S.D. B, effect of ER cotransfection. MCF-7
cells were transfected with GAL4-CAT/GAL-ElkC treated with 10-100
nM E2 in the presence or absence of cotransfected ER
expression plasmid as described under "Experimental Procedures." E2
alone (50 and 100 nM) significantly induced CAT activity,
and this response was enhanced after cotransfection with ER . Results
were determined in triplicate and are presented as means ± S.E.
Effects of ICI 182,780 and PD98059 on E2- or growth factor-induced CAT
activity in MCF-7 (C) or CHO (D) cells
cotransfected with GAL4-CAT and GAL-ElkC plasmids are shown.
Transfection and CAT assays were performed as described under
"Experimental Procedures." *, the relative intensity was
significantly higher (p < 0.001) than in control. **,
the relative intensity was significantly lower (p < 0.001) than in cells treated with E2 or growth factors. Results were
determined in triplicate (three separate experiments) and expressed as
means ± S.D.
|
|
Hormone-induced activation of pSRE via MAPK-dependent
activation of Elk-1 was further confirmed by showing that induction of
CAT activity in MCF-7 cells transfected with pSRE was decreased by
cotransfection with dominant negative Elk-1 expression plasmid (Fig.
6A). The effects of 10 nM E2 and EGF alone and in combination with cotransfected
ER
on ERK1/2 phosphorylation were determined in whole cell extracts
(Fig. 6B), and both E2 and EGF alone induced phosphorylation
(active ERK1/2). ERK1/2 phosphorylation was enhanced 60-100% in
extracts from cells transfected with ER
expression plasmid, and this
was consistent with increased activation of constructs in transient
transfection assays using ER
expression plasmids. Moreover, since
not all cells are transfected, increased phosphorylation of ERK1/2 by
ER
would be higher in transfected cells than observed for the whole
cell extracts (Fig. 6B). The effects of E2, E2 plus ICI
182,780, and E2 plus PD98059 on immunoreactive ERK1 and ERK2 and the
corresponding active proteins (P-ERKs) were also determined (Fig.
6C). E2 (and TGF-
, data not shown) induced immunoreactive
levels of active-ERK1 and active-ERK2, and this response was inhibited
by ICI 182,780 and PD98059, whereas the effects of TGF-
were not
affected by ICI 182,780 (data not shown). These results paralleled
activation of pSRE by E2 and TGF-
and confirmed that both mitogens
activate kinase-dependent signaling that converge on
MAPK-dependent phosphorylation and activation of proteins
binding the SRE in the fos gene promoter. The specific effects of ICI 182,780 on ER
-mediated responses are consistent with
hormone-dependent activation of pSRE and the MAPK pathway through an ER
-dependent pathway (42, 45). The effects of E2 on immunoreactive Elk-1 and phosphorylated Elk-1 proteins were also
investigated (Fig. 6D) in MCF-7 cells; treatment with E2, E2
plus ICI 182,780, E2 plus PD98059, and TGF-
did not affect levels of
immunoreactive Elk-1 protein, whereas E2 induced phosphorylation of
Elk-1 (P-Elk-1), and this response was inhibited by ICI 182,780 and
PD98059. As a positive control, TGF-
also induced P-Elk-1.

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Fig. 6.
Phosphorylation of ERK1/2 and Elk-1 and its
role in E2-induced activation of pSRE. A, effects of
dominant negative Elk on E2-induced CAT activity. MCF-7 cells were
cotransfected with pSRE plus various amounts of dn Elk expression
plasmids or an empty vector pCDNA3 (as control) (total amount of
DNA was kept constant). The transient transfection and CAT assays were
performed as described under "Experimental Procedures." Cells were
treated with Me2SO (DMSO, light
bars) or 10 nM E2 (dark
bars). *, the relative intensity was significantly higher
(p < 0.001) than in control. B, activation
of ERK1/2. Whole cell extracts were obtained from MCF-7 cells treated
with solvent control (CTL), 10 nM E2, and
TGF- in the presence or absence of cotransfected ER , and
immunoblot analysis of activated ERK1/2, ERK1/2, and ER was carried
out as described under "Experimental Procedures." Cotransfection
with ER increased ERK1/2 activation by 60-100%, and levels of
ER protein were also increased; replicate (two) experiments
gave similar results. Shown are effects of PD98059 and ICI 182,780 on
E2-induced active ERKs (C) and phosphorylation of
Elk-1 (D) in MCF-7 cells. Western immunoblottings were
performed as described under "Experimental Procedures." The lysates
were obtained from MCF-7 cells treated with Me2SO, E2, or
TGF- for 15 min or cotreated with ICI 183,780 or PD98059 for 4 h plus E2 or TGF- for 15 min.
|
|
Role of Cytosolic ER
Interaction with the IGF-1 Receptor and
Detection of Membrane ER in Transfected CHO-K1 Cells--
The results
illustrated in Fig. 7, A and
B, demonstrate that E2, IGF, and TGF-
activate pSRE and
GAL-ElkC in ER-positive ZR75 and T47D breast cancer cells. Thus,
E2 and growth factors activate this kinase-dependent
pathway in three ER-positive breast cancer cell lines (MCF-7, ZR75, and
T47D), ER-negative MDA-MB-231 breast cancer cells, and CHO cells,
indicating that this response is not confined to one or two breast
cancer cell lines. A recent study (51) reported that E2 also activated
MAPK signaling in ER-negative COS7 and HEK293 cells transfected with
ER
; however, this response was not linked to membrane ER
but to
direct physical interactions of cytosolic ER
with the membrane-bound
IGF-1 receptor. Moreover, activation of MAPK by E2 was inhibited in
these cells after cotreatment with H1356, a polypeptide that binds the
IGF-1 receptor and blocks IGF-1 action. Therefore, the possible role of
cytosolic ER
/IGF-1 receptor interactions with activation of MAPK was
investigated in MCF-7 cells transfected with pSRE or GAL4-CAT/GAL-ElkC
(Fig. 7, C and D, respectively). 10 nM E2 and IGF-1 induce reporter gene activity in cells
transfected with these constructs, and as a positive control H1356
polypeptide inhibited the IGF-1-induced response. In contrast, H1356
did not affect E2-induced activity in MCF-7 cells transfected with pSRE or GAL4-CAT/GAL-ElkC, indicating that cytosolic ER
interactions with
the IGF-1 receptor in MCF-7 cells were not responsible for activation
of MAPK as previously reported in COS7 and HEK293 cells (51).

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Fig. 7.
E2 and growth factor activation of pSRE and
GAL4-CAT/GAL-ElkC. A and B, ZR-75 and T47D
cells. ER-positive ZR-75 (A) or T47D (B) cells
were transfected with pSRE or GAL4-CAT/GAL-ElkC and treated with 10 nM TGF- , IGF-1, or E2, and reporter gene activity was
determined as described under "Experimental Procedures."
Significant (*, p < 0.05) induction was observed for
E2 and growth factors. C and D, effects of H1356
on E2 and IGF-1-induced activity. MCF-7 cells were transfected with
pSRE (C) or GAL4-CAT/GAL-ElkC (D) and treated
with 10 nM E2 or IGF-1 alone or in combination with 0.1-10
µM H1356 polypeptide, and CAT activity was determined as
described under "Experimental Procedures." Significant (*,
p < 0.05) induction of CAT activity was observed for
E2 and IGF-1, and H1356 significantly (*, p < 0.05)
inhibited only the IGF-1-induced response. Results were determined in
triplicate (three separate determinations) and expressed as means ± S.D.
|
|
Fluorescence immunocytochemistry was used to detect membrane hER and
HE11 ectopically expressed in ER-negative CHO-K1 cells (Fig.
8). For detection of membrane hER and
HE11, cells were fixed with paraformaldehyde to minimize membrane
permeability, and following staining with the H222 antibody increased
membrane staining was observed in cells transfected with hER (Fig.
8B) or HE11 (Fig. 8C), whereas a signal was not
observed in nontransfected cells (Fig. 8A). In cells using
IgG alone or after transfection with hER or HE11, membrane signals were
not detected (Figs. 8, D-F, respectively). Nuclear staining
was observed in cells fixed in the presence of detergent and
transfected with hER or HE11 (Fig. 8, H and I),
whereas no signal was observed in nontransfected cells (Fig.
8G).

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Fig. 8.
Immunofluorescence detection of plasma
membrane (A-C) and nuclear (G-I)
hER or HE11 ectopically expressed in CHO-K1 cells.
A-C, cells were stained with H222 antibody following
fixation without detergent to minimize permeabilization of the plasma
membrane. Nontransfected cells (A) exhibit no nuclear or
plasma membrane staining. After ectopic expression of hER or HE11,
~25% of the cells exhibit a detectable increase in membrane hER
(B) or HE11 (C) signal. Occasional nuclear
staining in less than 1% of the cells was attributed to transfected
cells with plasma membranes that were compromised during staining
(B). Cells subjected to the same treatments with H222
antibody replaced by rat IgG show no plasma membrane or nuclear signal
in nontransfected (D), hER-transfected (E), or
HE11-transfected cells (F). Fixation in the presence of
detergent (G-I) reveals nuclear signal in ~25% of the
cells after transfection with hER (H) or HE11 (I)
in contrast to nontransfected cells (G).
|
|
 |
DISCUSSION |
Interactions between estrogens, growth factors, and other mitogens
in breast cancer cells are complex and play an important role in
activating signaling pathways required for cell cycle progression and
proliferation (52-56). Growth factor activation of ligand-independent
ER
action has been extensively investigated (reviewed in Refs.
57-59) and is primarily associated with phosphorylation of specific
amino acids required for a functional response using constructs
containing an ERE-dependent promoter. For example, growth
factor activation of the MAPK pathway results in
kinase-dependent phosphorylation of Ser118 in
the AF1 domain of ER
and is required for ligand-independent ER
action (60). It has also been reported that
pp90rsk1-dependent phosphorylation of ER
(at
Ser118 and Ser167) enhances
AF1-dependent transcriptional activity of ER
(61). Protein kinase A also increases phosphorylation and ligand-independent ER
action (62-65), and this was associated with
phosphorylation of serine 236 (65).
In addition to growth factor activation of ER
, estrogens also
activate diverse growth factor/mitogen-like signaling, including the
Src/Ras/MAPK and cAMP pathways (42-48), and these responses may be
associated with activation of membrane ER. Transient transfection of
ER
(or ER
) in ER-negative CHO cells results in incorporation of
ER in cell membranes, and E2 activates the MAPK pathway in the
transfected cells (45). A recent study also reported that estrogens
rapidly activated the MAPK pathway in human neuroblastoma cells and
induced reporter gene activity in cells transiently transfected with a
construct containing a mouse c-fos gene promoter insert
(46).
c-fos protooncogene is induced by E2 and growth factors in
human breast cancer cells, and estrogen action is associated with ER
/Sp1 interactions with a GC-rich site in the distal region of the
promoter (39). Since the c-fos gene promoter also contains downstream elements responsive to growth factors, we have used various
fos promoter-derived constructs to study growth factor-ER
cross-talk. Results showed that not only growth factors but E2 also
induced reporter gene activity in MCF-7 cells transfected with
constructs containing
1400 to +41 (pFC2),
354 to
296 (pSS), or
325 to
296 (pSRE) fos gene promoter inserts, and the
former two constructs contained only SIE/SRE that are activated via
kinase-dependent pathways. These results are consistent
with previous studies showing that in MCF-7 cells E2 induces kinase
cascades that activate MAPK (42-45). E2, IGF-1, and TGF-
alone
significantly activated reporter gene activity in MCF-7 cells
transfected with pFC2 (Fig. 1A) and GAL-ElkC/GAL4-CAT (Fig.
5B) constructs, and the induction response was markedly
enhanced after cotransfection with ER
expression plasmid. Similar
results have also been observed for kinase-dependent activation of endothelial nitric-oxide synthase (constructs) by E2 in
pulmonary arterial endothelial cells (66). The role of ER
expression
in mediating increased reporter gene activity was further investigated
and shown to be related to enhanced ERK1/2 activation (60-100%) as
determined by immunoblots of whole cell extracts of MCF-7 cells (Fig.
6B). Increased reporter gene activity associated with ER
cotransfection was higher in transient transfection studies (>3-fold)
than the enhanced ERK1/2 phosphorylation; however, since only a
fraction of the cells are transfected, the overall enhanced kinase
activation in these cells will be higher than observed in whole cell
extracts (Fig. 6B).
Razandi et al. (45) showed that in ER-negative CHO cells,
transfection with ER
resulted in activation of signaling pathways associated with activation of kinase pathways and nuclear ER
. To
confirm that transient transfection of ER
can lead to enhanced kinase activation, we used ER-negative MDA-MB-231 and CHO cells transfected with wild-type and variant ER expression plasmids and
determined activation of pSS or pSRE. The results obtained in
MDA-MB-231 cells (Fig. 2B) showed that in cells transfected with pSS and ER
or HE11 (DNA binding domain deletion), E2 induced CAT activity; similar results were observed for CHO cells using pSRE
(Fig. 3E), suggesting that this kinase-dependent
response is not associated with nuclear ER
action. The fact that a
DNA binding domain-deficient mutant (HE11) also activates pSS or pSRE supports the ER-mediated kinase activation pathway in MDA-MB-231, CHO,
and MCF-7 cells and is consistent with results of previous studies in
ER-negative CHO cells transfected with ER
(45). Moreover, E2 and
growth factors also activate pSRE and GAL4-CAT/GAL-ElkC in ER-positive
ZR75 and T47D breast cancer cells (Fig. 7, A and B). Deletion analysis of the fos gene promoter
showed that the SRE (
325 to
296) was the minimal promoter activated
by both TGF-
and E2, and previous studies have also demonstrated
that other mitogens and growth factors induce c-fos through
kinase activation of the MAPK pathway and phosphorylaton of proteins binding the SRE (32). Results obtained using the MAPK kinase inhibitor
PD98059, dominant negative ras, and MAPK expression plasmids
confirm that both E2 and TGF-
also activate the c-fos SRE
in breast cancer cells through MAPK pathways, and this was also
associated with increased formation of the ternary complex of SRF and
Elk-1 that binds to this element in gel mobility shift assays (Fig.
4).
Activation of fos promoter constructs through the SRE is
dependent on the mitogenic stimuli and cell context. For example, induction of reporter gene activity in HeLa cervical carcinoma cells by
serum and epidermal growth factor required an intact SRF, whereas
mutation of the TCF site did not affect induction by serum, and
induction by epidermal growth factor was decreased by 50% (14). In
contrast, growth hormone activation of the SRE in 3T3-F442A and Chinese
hamster ovary cells stably transfected with the growth hormone receptor
required intact SRF and TCF sites (19, 20). E2 (and growth factor)
activation of pSS/pSRE in breast cancer cells also requires both SRF
and TCF sites, and results obtained with the MAPK kinase inhibitor
PD98059 and dominant negative MAPK, Ras, and Elk-1 expression plasmids
are consistent with previous reports showing E2-dependent
activation of Ras-MAPK signaling in breast cancer cells. Results
obtained using CHO cells confirm that ER
-dependent
activation of MAPK (45) is paralleled by activation of Elk-1 and pSRE
(Figs. 3E, 5D, and 7) as observed in multiple
breast cancer cell lines.
In summary, our results show that hormone-induced activation
(phosphorylation) of the Elk-1 transcription factor is an important downstream target of E2-dependent Ras-MAPK signaling in
breast cancer cells. Although growth factors also induced Ras-MAPK
activation and phosphorylation of Elk-1 in MCF-7 cells, ICI 182,780 did
not inhibit this response, suggesting that there are differences
between growth factor- and E2-mediated activation of the
c-fos pSRE. Results of this study show that E2
activates kinase-dependent phosphorylation of Elk-1, and
this may be due, in part, to a membrane receptor (Fig. 8). However, a
recent study showed that activation of MAPK signaling and IGF-1
receptor phosphorylation by E2 was associated with direct interaction
of ER
with the IGF-1 receptor in COS7 and HEK293 cells (51). In
contrast, our results in MCF-7 cells demonstrate that interaction of
cytosolic ER
with the IGF-1 receptor does not play a role in
activation of MAPK in MCF-7 cells (Fig. 7). Moreover, we have also
shown by fluorescence immunocytochemistry (Fig. 8) that ectopic
expression of hER or HE11 in CHO cells leads to accumulation of
membrane (Fig. 8, B and C) and nuclear (Fig. 8,
H and I) ER
or HE11, and this supports a
pathway involving membrane ER
(Fig.
9). Current studies are focused on
further characterization of the mechanisms of hormone- and growth
factor-induced expression of c-fos and other
SRE-dependent genes/gene promoters in breast cancer cell
lines.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants ES09253 and ES09106 and the Texas Agricultural Experiment Station.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.
A Sid Kyle Professor of Toxicology. To whom correspondence should
be addressed: Dept. of Veterinary Physiology and Pharmacology, Texas
A & M University, College Station, TX 77843-4466. Tel.: 979-845-5988;
Fax: 979-862-4929; E-mail: ssafe@cvm.tamu.edu.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M005492200
 |
ABBREVIATIONS |
The abbreviations used are:
SIE, Sis-inducible
enhancer;
CHO, Chinese hamster ovary;
E2, 17
-estradiol;
ER, estrogen
receptor;
SRE, serum response element;
SRF, serum response factor;
STAT, signal transducers and activators of transcription;
TCF, ternary
complex factor;
TGF-
, transforming growth factor
;
MAPK, mitogen-activated protein kinase;
hER, human ER;
IGF, insulin-like
growth factor;
ERK, extracellular signal-regulated kinase;
dn, dominant
negative;
CAT, chloramphenicol aminotransferase;
DPBS, Dulbecco's
phosphate-buffered saline.
 |
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