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Molecular Endocrinology 22(9):2116–2127
Copyright © 2008 by The Endocrine Society
doi: 10.1210/me.2008-0059
Nuclear and Extranuclear Pathway Inputs in the
Regulation of Global Gene Expression by
Estrogen Receptors
Zeynep Madak-Erdogan, Karen J. Kieser, Sung Hoon Kim, Barry Komm, John A. Katzenellenbogen,
and Benita S. Katzenellenbogen
Department of Cell and Developmental Biology (Z.M.-E., B.S.K.), Department of Chemistry (K.J.K.,
S.H.K., J.A.K.), and Department of Molecular and Integrative Physiology (K.J.K., B.S.K.), University of
Illinois, Urbana, Illinois 61801; and Women’s Health and Musculoskeletal Biology (B.K.), Wyeth
Research, Collegeville, Pennsylvania 19426
Whereas estrogens exert their effects by binding to
nuclear estrogen receptors (ERs) and directly altering target gene transcription, they can also initiate extranuclear signaling through activation of
kinase cascades. We have investigated the impact of estrogen-mediated extranuclear-initiated
pathways on global gene expression by using
estrogen-dendrimer conjugates (EDCs), which
because of their charge and size remain outside
the nucleus and can only initiate extranuclear
signaling. Genome-wide cDNA microarray analysis of MCF-7 breast cancer cells identified a subset of 17␤-estradiol (E2)-regulated genes (⬃25%)
as EDC responsive. The EDC and E2-elicited increases in gene expression were due to increases
in gene transcription, as observed in nuclear run-on
assays and RNA polymerase II recruitment and
phosphorylation. Treatment with antiestrogen or
ER␣ knockdown using small interfering RNA abol-
ished EDC-mediated gene stimulation, whereas
GPR30 knockdown or treatment with a GPR30selective ligand was without effect, indicating ER
as the mediator of these gene regulations. Inhibitors of MAPK kinase and c-Src suppressed both E2
and EDC stimulated gene expression. Of note, in
chromatin immunoprecipitation assays, EDC was
unable to recruit ER␣ to estrogen-responsive regions of regulated genes, whereas ER␣ recruitment by E2 was very effective. These findings suggest that other transcription factors or kinases that
are downstream effectors of EDC-initiated extranuclear signaling cascades are recruited to regulatory regions of EDC-responsive genes in order
to elicit gene stimulation. This study thus highlights
the importance of inputs from both nuclear and
extranuclear ER signaling pathways in regulating
patterns of gene expression in breast cancer cells.
(Molecular Endocrinology 22: 2116–2127, 2008)
E
small population of extranuclear ERs. These extranuclear receptors have been shown to play important
roles in certain rapid signaling events, such as intracellular calcium mobilization, nitric oxide synthesis,
and activation of various kinases (4, 5). We have only
an incomplete understanding, however, of the cross
talk between nuclear and extranuclear ERs in mediating the actions of estrogen in regulation of gene expression. Hence, our aim in this study was to examine
the impact of extranuclear-initiated estrogen action on
gene expression regulation in breast cancer cells.
Based on current thinking, the regulation by 17␤estradiol (E2) of gene expression likely involves both
genomic and nongenomic signaling (1–5). The former,
for which there is much evidence, involves direct action of nuclear-localized ER in its function as a ligandregulated transcription factor or coregulator. By contrast, nongenomic signaling involves extranuclear
events mediated by ER or other estrogen binders;
these can impact gene expression in the nucleus indirectly, by activation through posttranslational modifications of other transcription or chromatin-modifying
factors, or even of ER and its coregulatory partners.
This implies that the regulation of gene expression by
STROGENIC HORMONES are important for the
regulation of many physiological processes in
both reproductive and nonreproductive tissues, and
they impact the phenotypic properties of cancers,
such as breast cancer, that develop in these tissues.
These effects are exerted by binding of estrogens to
their receptors [estrogen receptors (ER␣ and ER␤)],
which are members of the nuclear receptor superfamily of ligand-activated transcription factors (1–3). Although ERs have long been considered to be nuclearlocalized proteins, recent studies have revealed a
First Published Online July 10, 2008
Abbreviations: BMP, Bone morphogenetic protein; ChIP,
chromatin immunoprecipitation; EDC, estrogen dendrimer
conjugate; E2, 17␤-estradiol; ER, estrogen receptor; HSPB8,
heat shock protein B8; JNK, c-Jun N-terminal kinase;
LRRC54, leucine-rich repeat containing 54; MEK, MAPK kinase; PgR, progesterone receptor; PMAIP1, PMA-induced
protein 1; Q-PCR, quantitative PCR; RNA Pol II, RNA polymerase II; siRNA, small interfering RNA; TSS, transcription
start site.
Molecular Endocrinology is published monthly by The
Endocrine Society (http://www.endo-society.org), the
foremost professional society serving the endocrine
community.
2116
Madak-Erdogan et al. • Extranuclear Signaling and Gene Regulation by ER
estrogen has both genomic and nongenomic inputs,
and that the balance of these inputs may vary in a celland gene-specific manner.
To dissect the nuclear/genomic vs. extranuclear/
nongenomic actions of estrogen in the regulation of
gene expression, we have used estrogen-dendrimer
conjugates (EDCs), which because of their charge and
size, remain outside the nucleus. These large, abiotic,
nondegradable polyamidoamine dendrimer macromolecules, which are conjugated to multiple estrogen
molecules through chemically robust linkages, are capable of activating only extranuclear pathways (6). By
comparing the actions of EDC and E2 in genome-wide
gene regulation, we show in this report that extranuclear-initiated pathways of estrogen action can alter
the transcription of a portion of estrogen target genes,
and that they do so in a mechanistically distinct manner that does not result in the recruitment of ER to ER
binding sites of target genes. Moreover, we provide
evidence that extranuclear estrogen-initiated gene
regulation is blocked by some kinase inhibitors and
Mol Endocrinol, September 2008, 22(9):2116–2127 2117
by antiestrogens or knockdown of ER, implying the
requirement for ER and certain protein kinases
in both nuclear-initiated and extranuclear-initiated
gene regulations.
RESULTS
EDCs Regulate the Expression of a Subset of
Estrogen Target Genes in MCF-7 Cells
Extranuclear signaling by estrogen has been shown to
activate signaling pathway components, including kinases, by processes that do not involve gene transcription, but little attention has been focused on the
effect of estrogen-regulated extranuclear pathways on
gene expression. As shown in Fig. 1, we investigated
the impact of estrogen-mediated extranuclear initiated
pathways on global gene expression in MCF-7 breast
cancer cells by using an EDC. MCF-7 cells were
treated with vehicle control, E2, EDC, or empty den-
Fig. 1. cDNA Microarray Analysis of Genes Regulated by E2 and EDC
A, Experimental design. MCF-7 cells were treated with vehicle, 10 nM E2, empty dendrimer, or 10 nM E2 equivalent EDC for
4 h. RNA was then hybridized to Affymetrix Hu-U133 GeneChips, and analysis was performed as described in Materials and
Methods. B, Cluster diagram of EDC- and E2-regulated genes. C, Venn diagram showing the distribution of genes regulated by
E2 only, commonly regulated by E2 and EDC, or regulated by EDC only, and their characterization in functional categories
according to GeneSpring and ErmineJ softwares and web-based DAVID functional annotation tool. Ctrl, Control; Dend, empty
dendrimer; Veh, vehicle.
2118 Mol Endocrinol, September 2008, 22(9):2116–2127Madak-Erdogan et al. • Extranuclear Signaling and Gene Regulation by ER
ulated by E2 only, 373 (⬃47%) of the genes had ER␣
binding sites in a window of 100 kb upstream or downstream of the transcriptional start site (TSS). For E2
and EDC commonly regulated genes, 90 of 243 genes
(⬃37%) had ER␣ binding sites, and for EDC-only
genes, 39 of the 92 genes (⬃42%) had ER␣ binding
sites within 100 kb of the gene TSS.
Regarding the 92 genes categorized as being regulated only by EDC, we found that the level of regulation of these genes was low and close to the cutoff
used to determine regulation. We further examined 15
genes in this category by quantitative PCR (Q-PCR)
and confirmed that all of them were stimulated by both
EDC and E2, but slightly more by EDC. Hence, these
appear to be genes that are preferentially regulated by
EDC. This group of genes is considered further in the
Discussion.
drimer control, and cDNA microarray analyses were
carried out using Affymetrix HG-U133A GeneChips.
We used multivariate analysis (LIMMA), which assigns
statistical significance to contrasts and controls for
multiple testing, to find genes that are differentially
regulated by each ligand (Fig. 1A). In this manner, we
identified a subset of E2-regulated genes that were
also EDC responsive (Fig. 1, B and C). As shown in the
Venn diagram (Fig. 1C), 1036 genes were up- or downregulated by E2 at 4 h, 243 genes were regulated by
both E2 and EDC, and 92 genes were classified as
being regulated by EDC only. Thus, the genes commonly regulated by E2 and EDC represent 23% of the
total E2-regulated genes.
Functional classification of the gene regulations,
using the functional annotation tools DAVID (http://
david.abcc.ncifcrf.gov/), Gene Spring, and ErmineJ all
agreed in showing that genes regulated only by E2
encoded mostly transcription factors, growth factors,
and mitosis-related genes (Fig. 1C; Venn diagram,
left), which is consistent with our previous study in
which we found that EDC did not stimulate the proliferation of MCF-7 cells (6). Interestingly, genes regulated by both E2 and EDC (commonly regulated
genes; Venn diagram, middle) included many genes
involved in RNA metabolism and the cytoskeleton.
Genes regulated by EDC only were enriched in those
associated with RNA metabolism.
Recent reports using chromatin immunoprecipitation (ChIP)-on-chip (7, 8) and ChIP-paired end tag
cloning approaches (9, 10) have determined the location of ER␣ binding sites in MCF-7 cells. Of the 793
genes that we identified by cDNA microarray as reg-
Analysis of the Expression of Target Genes
Regulated by EDC and Estradiol
To examine gene expression regulation by EDC vs. E2
in detail, we selected several genes that were highly
regulated by both E2 and EDC in our cDNA microarray
data sets [leucine-rich repeat containing 54 (LRRC54),
heat shock protein (HSP)B8, and PMA-induced protein 1 (PMAIP1); Fig. 1B]. These genes encode proteins having different cell functions. LRRC54 is a bone
morphogenetic protein (BMP) inhibitor shown to inhibit
BMP-2/4 action upon direct binding to BMPs and
chordin proteins (11). HSPB8 is a small heat shock
protein that appears to play important roles in cardiac
hypertrophy, cell cycle regulation, apoptosis, and
mRNA
(fold change)
LRRC54
7
6
5
4
3
2
1
0
HSPB8
PMAIP1
25
E2
12
20
8
6
10
EDC
EDC
2
0
2
4
6
time (h)
8
0
0
2
4
6
time (h)
8
0
2
4
6
time (h)
8
pS2
PgR
150
200
mRNA
(fold change)
EDC
4
5
0
E2
10
E2
15
E2
E2
100
100
50
EDC
0
0
2
4
6
time (h)
EDC
0
8
0
2
4
6
time (h)
8
Fig. 2. Time Course of Regulation of Gene Expression by E2 or EDC
MCF-7 cells were treated with 0.1% EtOH vehicle, 10 nM E2 (solid line), empty dendrimer or 10 nM estrogen equivalent EDC
(dotted line) for 2, 4, and 8 h, and mRNA levels were monitored by Q-PCR. mRNA levels were normalized relative to 36B4, and
fold change was calculated relative to control. Results are the average ⫾ SD of at least three independent experiments.
Madak-Erdogan et al. • Extranuclear Signaling and Gene Regulation by ER
breast carcinogenesis (12, 13). PMAIP1/NOXA is a
proapoptotic protein that mediates p53-induced apoptosis together with PUMA, p21, and MDM2 (14). Progesterone receptor (PgR) and pS2, which are stimulated by E2 but not EDC at the concentration used for
microarray analysis (10⫺8 M), were included for comparison as E2-only regulated genes.
In time course experiments, we observed a similar
profile of mRNA stimulation by E2 and EDC for the
three genes, LRRC54, HSPB8, and PMAIP1, with
maximum RNA levels generally being reached by 4 h
(Fig. 2). By contrast, pS2 and PgR mRNA levels were
up-regulated by E2 but not by EDC. Empty (unconjugated) dendrimer (data not shown) elicited no change
in expression of any of these genes.
Both E2 and EDC Transcriptionally Increase
Expression of the Commonly Regulated Genes
Because changes in mRNA levels for genes could
reflect either altered rates of gene transcription and/or
changes in mRNA stability, we undertook several experiments to determine whether changes in expression of the E2 and EDC commonly regulated genes
represented primary responses, and whether the increases in mRNA reflected increases in gene transcription or changes in mRNA half-life. Moreover, in
our functional gene category overrepresentation analyses, RNA metabolism-related genes were one of the
highest scoring groups for genes that were regulated
by both E2 and EDC.
As shown in Fig. 3A, treatment with the protein
synthesis inhibitor cycloheximide did not prevent E2or EDC-stimulated expression of these genes, which
suggests that the five selected genes are primary response genes. We also carried out nuclear run-on
experiments in which nuclei were isolated from cells
treated with E2 or EDC, and the incorporation of labeled nucleotides into RNA was assessed. As shown
in Fig. 3B, we observed that both E2 and EDC increased transcription rates for the three commonly
regulated genes (Fig. 3B, left three panels), whereas
only E2 increased transcription of the PgR or pS2
genes (Fig. 3B, right two panels).
Next, to determine whether E2 or EDC altered the
stabilities of these mRNAs, cells were cotreated with
the transcription inhibitor, actinomycin D, and with E2
or EDC, and changes in mRNA levels were monitored
over time. Neither E2 nor EDC altered the mRNA halflife for the three commonly regulated genes tested,
which suggests that E2 and EDC do not alter the
stability of these mRNAs (Fig. 3C). As another indicator
of gene transcription, we used ChIP assays to monitor
recruitment of RNA polymerase II (RNA Pol II) to the
TSS of the genes of interest. These ChIP studies (Fig.
3D) show that E2 and EDC treatment both resulted in
enhanced recruitment of total RNA Pol II and of phosphor-Ser5-Pol II at the three commonly regulated
genes (LRRC54, HSPB8, and PMAIP1; Fig. 3D, left
three panels). By contrast, only E2 increased total RNA
Mol Endocrinol, September 2008, 22(9):2116–2127 2119
Pol II and phosphor-Ser5-RNA Pol II recruitment at the
PgR and pS2 genes (Fig. 3D, right two panels). All of
the data in Fig. 3 suggest that E2 and EDC up-regulate
gene expression by enhancing transcription of these
genes, without requiring new protein synthesis or affecting mRNA stability.
ER␣ Mediates the Actions of EDC and E2 on the
Regulation of Gene Expression
Because recent studies have indicated that an extranuclear form of ER␣ and/or the G protein GPR30
could be responsible for the nongenomic actions of E2
(4, 15), we used several approaches to examine the
involvement of these two proteins in the regulation of
gene expression by E2 and EDC. We first used the
ER␣ antagonist ICI182,780, which we found to completely abrogate EDC- and E2-induced gene regulations (Fig. 4A). ER␣ knockdown (which reduced ER
protein to ⬃20% of control level; see inset of Fig. 4B,
right) also greatly reduced E2- and EDC-induced gene
stimulations (Fig. 4B). We also examined the effect of
a GPR30 agonist ligand, G1, which was previously
shown to bind to GPR30 but not ER (16). G1 did not
alter E2- or EDC-stimulated expression of these genes
(Fig. 4C) or affect expression of any of the genes when
tested alone at a range of G1 concentrations (1–500
nM) (Fig. 4D). We also carried out small interfering RNA
(siRNA) knockdown of GPR30 and found that its
knockdown did not affect gene stimulation by either
EDC or E2 (data not shown). Our findings imply that
ER␣, not GPR30, is responsible for the regulation of
gene expression by EDC and E2.
Inhibiting MAPK Kinase (MEK) and c-Src Kinase
Activity Impairs EDC-Induced Gene Expression
In our previous studies we showed that E2 and EDC
rapidly activated p42/44-MAPK and c-Src kinase (6).
Furthermore, we have found that E2 and EDC treatment increased activated p38-MAPK and stress-activated protein kinase/c-Jun N-terminal kinase (JNK)MAPKs in MCF-7 cells (Madak-Erdogan, Z., and B. S.
Katzenellenbogen, unpublished results). We therefore
investigated whether activation of these kinase pathways is important for EDC- or E2-mediated gene stimulations by using small molecule inhibitors for each
kinase. MCF-7 cells were treated with either the MEK
inhibitor PD98059, c-Src inhibitor PP2, p38 MAPK
inhibitor SB203580, or JNK inhibitor SP600125 in the
presence of EDC or E2, and effects on gene expression were determined (Fig. 5). On the three genes
regulated by EDC (all except pS2 and PgR), EDCstimulated gene expression was significantly dampened by PP2 or PD98059. E2-induced gene expression was also reduced by treatment with PP2 on all
except the PgR gene, and PD98059 also decreased all
gene stimulations by E2. These data imply that both E2
and EDC utilize c-Src and/or MAPK pathways as part
of the stimulatory inputs that control the induction of
2120 Mol Endocrinol, September 2008, 22(9):2116–2127Madak-Erdogan et al. • Extranuclear Signaling and Gene Regulation by ER
LRRC54
mRNA
(fold change)
A
25
HSPB8
25
4
20
20
3
15
15
10
10
5
5
0
transcription
(fold change)
B
E2
EDC
Veh
0
CHX
LRRC54
PgR
400
300
100
2
200
50
1
Veh
0
CHX
Veh
100
0
CHX
Veh
PMAIP1
20
CHX
70
60
40
10
8
Veh
pS2
50
12
15
0
CHX
PgR
14
12
10
pS2
150
HSPB8
14
50
8
30
40
6
20
30
10
6
4
4
5
2
0
Veh E2 Dend EDC
0
0
Veh E2 Dend EDC
mRNA
(fold change)
0
Veh E2 Dend EDC
1.2
1.2
1.0
1.0
1.0
0.8
0.8
0.8
0.6
0.6
0.6
0.4
0.4
0.4
0.2
0.2
0.2
0.0
0.0
1
2
3
4
Actinomycin D (h)
LRRC54
0.8
Veh E2 Dend EDC
0
1
2
3
4
0.0
Actinomycin D (h)
HSPB8
0.4
0.7
0
1 2 3 4
Actinomycin D (h)
pS2
0.2
0.4
E2-Pol II
EDC-Pol II
E2-Ser5
EDC-Ser5
1.2
0.6
0.3
0.3
1.0
0.5
Veh E2 Dend EDC
Veh
E2
Dend
EDC
PgR
PMAIP1
1.4
0
PMAIP1
1.2
0
10
HSPB8
LRRC54
D
20
10
2
C
% input
PMAIP1
0.8
0.4
0.2
0.1
0.2
0.6
0.3
0.2
0.4
0.1
0.1
0.2
0.1
0.0
0.0
0
40
80
time (min)
120
0.0
0
40
80
time (min)
120
0.0
0
40
80
time (min)
120
0.0
0
40
80
time (min)
120
0
40
80
120
time (min)
Fig. 3. EDC and E2 Increase Gene Expression by Increasing Gene Transcription and Not Altering mRNA Stability
A, MCF-7 cells were pretreated with 10 ␮g/ml cycloheximide for 2 h to stop protein translation and were then treated with
10 nM E2 or 10 nM E2 equivalent EDC for 4 h in the continued presence of cycloheximide. Total RNA was isolated and reverse
transcribed. Q-PCR was performed. B, Nuclear run-on assays were performed on nuclei isolated from MCF-7 cells after
treatment with 0.1% ethanol vehicle, 10 nM E2, empty dendrimer (Dend), or 10 nM E2 equivalent EDC for 2 h. Fold change
in transcription is shown. C, MCF-7 cells were treated with 5 ␮g/ml actinomycin D in the presence or absence of 10 nM E2
or 10 nM E2 equivalent EDC for 2 or 4 h. Total RNA was isolated and reverse transcribed. Q-PCR was performed. D, MCF-7
cells were treated with 10 nM E2 or 10 nM E2 equivalent EDC for 15 min, 1 h, and 2 h. Total Pol II/DNA complexes or
phosphor-Ser5-Pol II/DNA complexes were immunoprecipitated overnight using specific anti-Total Pol II antibody, antiphospho-Ser5_Pol II (CTD4H8), or rabbit IgG (Santa Cruz) as negative control. Immunoprecipitated DNA levels were
measured by Q-PCR, and percent input was calculated. Values are the mean ⫾ SD of at least three independent experiments.
CHX, Cycloheximide; Pol II, polymerase II; Veh, vehicle.
Madak-Erdogan et al. • Extranuclear Signaling and Gene Regulation by ER
LRRC54
mRNA
(fold change)
A
HSPB8
5
10
4
8
3
6
2
4
1
2
10
PgR
100
pS2
150
8
5
50
50
E2 EDC E2 EDC
Ctrl
3
0
E2 EDC E2 EDC
ICI 182,780
Ctrl
LRRC54
B
mRNA
(fold chage)
6
10
Ctrl
5
PMAIP1
1
0
E2 EDC E2 EDC
siGL3 siERα
LRRC54
15
E2 EDC E2 EDC
siGL3 siERα
5
40
15
pS2
70
10
5
5
0
0
20
30
0
0
E2 EDC E2 EDC
siGL3 siERα
Ctrl
G1
G1
0
E2 EDC E2 EDC
siGL3
PMAIP1
siERα
E2 EDC E2 EDC
siGL3
8
70
7
60
siERα
pS2
PgR
80
150
100
50
40
4
Ctrl
10
9
5
E2 EDC E2 EDC
β -actin
20
30
3
E2 EDC E2 EDC
ERα
50
40
10
2
20
1
10
0
siCtrl siERα
60
30
1
6
10
ICI 182,780
Ctrl
PgR
50
HSPB8
20
E2 EDC E2 EDC
ICI 182,780
Ctrl
6
2
0
E2 EDC E2 EDC
3
2
D
0
ICI 182,780
4
3
20
E2 EDC E2 EDC
HSPB8
15
4
0
ICI 182,780
5
0
mRNA
(fold change)
PMAIP1
100
0
C
Mol Endocrinol, September 2008, 22(9):2116–2127 2121
E2 EDC E2 EDC
Ctrl
0
G1
50
E2 EDC E2 EDC
Ctrl
G1
0
E2 EDC E2 EDC
Ctrl
G1
mRNA
(fold change)
50
LRRC54
HSPB8
PMAIP1
PgR
30
10
10
5
0
Ctrl
10 nM E2
1 nM G1
10 nM G1
100 nM G1
500 nM G1
Fig. 4. ER␣ Is the Mediator for EDC Regulation of Gene Expression
A, MCF-7 cells were pretreated with 1 ␮M ICI182780 for 2 h and then treated with 10 nM E2 or 10 nM E2 equivalent EDC for
4 h. B, MCF-7 cells were transfected with control GL3 siRNA or ER␣ siRNA for 48 h and then treated with 10 nM E2 or 10 nM E2
equivalent EDC for 4 h. Western blot inset in panel B right shows the extent of ER knockdown with siRNA to be about 80%. C,
MCF-7 cells were cotreated with 10 nM G1 (GPR30 agonist ligand) during 10 nM E2 or 10 nM E2 equivalent EDC treatment. D,
MCF-7 cells were treated with the indicated concentrations of G1 ligand for 4 h. Total RNA was isolated, reverse transcribed, and
analyzed by Q-PCR. Values are the mean ⫾ SD of at least three independent experiments. Ctrl, Control; siER␣, small interfering
siER␣; siGL3, control siRNA.
2122 Mol Endocrinol, September 2008, 22(9):2116–2127Madak-Erdogan et al. • Extranuclear Signaling and Gene Regulation by ER
LRRC54
mRNA
(fold change)
4
HSPB8
10
***
3
8
***
#
2
***
#
***
6
#
#
4
1
#
0
Veh
PP2
PD98059
Veh
PP2
PP2
6
5
4
#
3
2
***
#
PP2
PD98059
Veh
#
PP2
PD98059
EDC
mRNA
(fold change)
125
*
***
100
75
40
*
50
#
PP2
E2
EDC
pS2
60
Veh
PD98059
1
0
PgR
0
PP2
***
E2
20
Veh
PMAIP1
***
Veh
**
PD98059
E2
EDC
mRNA
(fold change)
E2
Veh
PD98059
#
##
2
0
80
***
PD98059
#
25
Veh
PP2
PD98059
EDC
0
Veh
PP2
E2
PD98059
Veh
PP2
PD98059
EDC
Fig. 5. Inhibitors of c-Src Kinase or MEK Dampen E2- and EDC-Induced Gene Regulation
MCF-7 cells were pretreated with 1 ␮M PP2 or 50 ␮M PD98059 for 2 h and then treated with control vehicle, 10 nM E2, empty
dendrimer, or 10 nM E2 equivalent EDC for 4 h. Total RNA was isolated and reverse transcribed. Q-PCR was performed. ***, P ⬍
0.001; or **, P ⬍ 0.01; or *, P ⬍ 0.05, significantly up-regulated by E2 or EDC over control. #, P ⬍ 0.05; or ##, P ⬍ 0.01; significantly
different from no inhibitor control. Values are the mean ⫾ SD of three or more independent experiments. Veh, Vehicle.
these genes. By contrast, we saw no impact of inhibitors of JNK or p38 MAPK on these gene regulations
by E2 or EDC (data not shown).
EDC Is Ineffective in Recruiting ER␣ to Putative
Estrogen-Responsive Regions of Target Genes
Because ER␣ is known to be recruited to ER binding
sites of regulated genes upon exposure to E2, we performed ChIP assays to assess whether ER␣ is recruited
to the putative regulatory regions of the selected genes
upon EDC or E2 treatment of cells. We used ER binding
sites for our genes of interest, based on sites reported in
Refs. 8 and 10 (Fig. 6). ER_7120 is 10 kb upstream of the
LRRC54 TSS. The HSPB8 gene has a strong ER binding
site (MOPET11) 7.5 kb upstream of the HSPB8 TSS (10).
ER_7204 is about 5 kb downstream of the PgR 3⬘-untranslated region, and ER_10218 is about 300 bp upstream of the pS2 TSS. (PMAIP1 is not shown because
no ER binding site is known for this gene.)
Upon E2 treatment, ER␣ was found to be highly recruited to the responsive regions of all of the selected
genes after 45 min, and then ER␣ recruitment decreased
after 2 and 3 h of E2 treatment, except for the PgR gene,
where significant ER presence at the ER binding site was
still observed at the later times (Fig. 6). By contrast, EDC
did not increase ER␣ recruitment to the regulatory regions of any of these genes.
Madak-Erdogan et al. • Extranuclear Signaling and Gene Regulation by ER
A
Mol Endocrinol, September 2008, 22(9):2116–2127 2123
TSS
10 kb
ER_7120
LRRC54
TSS
MOPET11
7.5 kb
TSS
HSPB8
3’-UTR
PgR
ER_7204
5 kb
TSS
ER_10218
B
pS2
300 bp
HSPB8
% input
LRRC54
6
5
5
4
4
3
E2
3
2
1
0
E2
2
1
EDC
0
50
100
EDC
150
200
0
0
50
100
150
200
Time (min)
Time (min)
pS2
PgR
4
6
% input
5
3
4
E2
3
E2
2
2
1
1
0
EDC
0
50
100
150
200
Time (min)
EDC
0
0
50
100
150
200
Time (min)
Fig. 6. EDC Is Ineffective in Recruiting ER␣ to ER Binding Sites of Estrogen Target Genes whereas E2 Elicits a Robust ER␣
Recruitment
A, Schematic showing the location of ER␣ binding sites for selected genes. B, MCF-7 cells were treated with 10 nM E2 or 10 nM E2
equivalent EDC for 45 min, 2 h, and 3 h. ER␣/DNA complexes were immunoprecipitated using specific ER␣ antibody or rabbit IgG as
a negative control. Immunoprecipitated DNA levels were measured by Q-PCR, and percent input was calculated. Values are the
mean ⫾ SD of three independent experiments. 3⬘-UTR, 3⬘-untranslated region, MOPET, maximum overlap paired end tag.
These results indicate that EDC is not effective
in recruiting ER␣ to responsive regions of the
EDC-regulated genes, even though EDC affects the
transcription of these genes. This suggests that
estrogen action through the extranuclear-initiated
pathway may be regulating these genes through
transcription factors other than ER. As expected, we
did not see any ER␣ recruitment by EDC to the
estrogen-responsive region of the pS2 or PgR
genes, which are well-known direct genomic targets
of ER␣, whereas E2 gave robust ER recruitment and
stimulation of gene expression.
DISCUSSION
There is increasing evidence that estrogens utilize extranuclear as well as nuclear signaling in target cells.
2124 Mol Endocrinol, September 2008, 22(9):2116–2127Madak-Erdogan et al. • Extranuclear Signaling and Gene Regulation by ER
Extranuclear signaling has been shown to activate
important signaling pathway components, including
various protein kinases (e.g. c-Src, phosphatidylinositol-3 kinase, MAPK, etc.), that result in Ca2⫹ mobilization from the endoplasmic reticulum, rearrangement
of the cytoskeleton, and induction of nitric oxide production, with specific activations dependent on the
nature of the target cell (4, 5).
Our findings document that estrogen action initiated
outside of the nucleus, which can be achieved selectively by EDC treatment, stimulates the transcription
and expression of a significant portion (⬃25%) of the
total number of estrogen-regulated genes. This extranuclear-initiated gene regulation by EDC requires
ER, but, intriguingly, it seems to occur without direct
ER recruitment to the putative ER binding sites of
these regulated genes. By contrast, E2 stimulation of
these genes is associated with a very robust recruitment of ER to these ER binding sites. Whereas it is
possible that EDC might be promoting a less stable
binding of ER to the ER binding sites so that they might
not be detectable by ChIP assays or that extranuclear
action of EDC might promote the binding of ER to sites
other than those to which it is recruited when occupied
by E2, we believe that it is most likely that EDC regulation of these genes, which does require ER, involves
transcription factors other than ER directly binding to
chromatin in the nucleus. Future experiments will be
needed to distinguish among these possibilities.
Our findings also indicate that protein kinases
are involved in gene regulations by both so-called
genomic (nuclear-initiated) and nongenomic (extranuclear-initiated) pathways. Hence, we found that in
MCF-7 cells, inhibition of the kinases c-Src and/or
MEK suppressed the expression of genes up-regulated by both E2 and EDC (LRRC54, HSPB8, and
PMAIP1), as well as genes regulated by E2 only (PgR
and pS2). This is perhaps to be expected, because the
input of extranuclear signaling pathways on elements
of the estrogen-regulated nuclear pathway is well established, because phosphorylation state is known to
markedly affect the activity and functional properties
of ER itself (17, 18), ER coregulator protein partners
(19), many other DNA-binding transcription factors,
RNA Pol II, histones, and other chromatin-associated
proteins (20). Of note, regulation of the genes we investigated was affected only by certain kinases, c-Src
and MEK in particular, because we found inhibitors of
JNK and p38 MAPK to be without effect. The importance of different kinases is likely to be cell dependent,
because different cells possess different complements of various kinases and phosphatases. We also
observed some differences in efficacies of various kinase inhibitors in suppression of different genes. In our
studies, PD98059 was a generally more complete inhibitor than was PP2, implying the importance of the
MAPK pathway in MCF-7 cells. Several publications
have shown a rapid activation of MAPK signaling in
impacting various aspects of MCF-7 cell functioning
(4, 21, 22). Hence, our findings imply that kinase path-
ways provide important inputs for regulation of both
nuclear and extranuclear gene stimulations, and that
one cannot assign kinase activity uniquely to nuclear
or extranuclear-initiated pathways. Indeed, the observation that MEKK1 colocalizes with progesterone receptor at progesterone receptor binding sites of the
mouse mammary tumor virus promoter (23) suggests
extensive interrelationships between hormone-regulated transcription factors and protein kinases in regulation of gene expression.
We explored the possibility that the c-Src and MEK
inhibitors might suppress transcriptional gene activity
by blocking the recruitment of RNA pol II or ER␣ to the
regulated genes. However, we found that treatment of
cells with these inhibitors had little if any impact on the
recruitment of RNA pol II to the transcription start site
of the gene or of ER␣ to the ER binding site, implying
that the block in gene regulation is not at the level of
ER or pol II recruitment. It is quite possible that these
inhibitors might be affecting the state of phosphorylation and activity of other important components of
transcription complexes, such as coregulators or mediator components. For example, a recent report has
shown that estrogen stimulates ERK phosphorylation
of MED1/thyroid hormone receptor-associated protein
220/vitamin D receptor interacting protein 205, a step
required for its association with the mediator complex
and for its nuclear receptor coactivator activity (24).
Likewise, estrogen-regulated MAPK phosphorylation
of steroid receptor coactivator 3 regulates its association with ER␣ and thereby ER transcriptional activity
(25). Moreover, in different systems, MAPK signaling
has been shown to activate other kinases, such as
MSK1 and RSK2 that would phosphorylate histones
and alter the chromatin environment (20). Hence, the
suppressive effects of protein kinase inhibitors that we
have observed might arise from alterations in the state
of phosphorylation of a number of key components of
the active transcription complex.
In our classification of E2- and/or EDC-regulated
genes, we identified some genes that were regulated
preferentially by EDC. Whereas it might be unexpected that genes would be preferentially regulated by
EDC compared with E2, it is possible that on these
genes the genomic component of E2 action opposes
(down-regulates) the stimulating effect of the nongenomic component, so that the combined genomic
plus nongenomic output by E2 would be less than that
from the nongenomic component stimulated by EDC.
Further analysis of such a mechanism would require
use of either an agonist agent that would only stimulate the genomic component or the use of an antagonist agent that would block the nongenomic
component of E2 action without affecting the genomic component.
Recent studies have also highlighted that ER or
other E2 binding proteins (GPR30) located in or close
to the cell membrane can initiate signaling cascades
from growth factor receptor tyrosine kinases [i.e. epidermal growth factor receptor and HER2/neu], or in-
Madak-Erdogan et al. • Extranuclear Signaling and Gene Regulation by ER
teract with key signal transduction adaptors and kinases such as Shc, phosphatidylinositol-3 kinase, and
c-Src (15, 22, 26–28). Our findings indicate that ER is
the mediator of the extranuclear regulated gene expression we have investigated. This is supported both
by use of the antiestrogen ICI 182,780 and by knockdown of ER, which greatly reduced gene regulations
by EDC. There are several reports addressing the role
of GPR30, a G protein-coupled receptor, which has
been proposed by some to be the nonclassical E2
receptor and the effector of some extranuclear signaling induced by estrogen. Upon E2 treatment, GPR30
was reported to stimulate cAMP-dependent signaling,
which attenuated the MAPK pathway induced by epidermal growth factor receptor (15, 27). However, Ca2⫹
mobilization and MAPK activation upon E2 treatment
were greatly impaired in endothelial cells from ER␣/
ER␤ double knockout mice (22). Although the nature of
the estrogen binding protein, whether ER and/or
GPR30 or other, may depend on the target cell and
responses being monitored, our findings are supportive of those of Pedram et al. (22), who also found ER
to be the mediator of extranuclear signaling in MCF-7
cells.
One of our most interesting observations is that EDC
and E2 both increased gene transcription, but that
only gene transcription induced by E2 was accompanied by the recruitment of ER to putative ER binding
sites of the regulated genes. We selected ER binding
sites to be examined based on datasets from Carroll et
al. (8) and Lin et al. (10) in which these high-affinity ER
binding sites were identified. In addition, in a genomewide study which has used ChIA-PET (whole-genome
chromatin interaction analysis using paired-end ditagging) methodology, the ER␣ binding sites we examined in the LRRC54 and HSPB8 genes were found
to interact through looping events with another site
in the coding sequence (LRRC54) or with the transcription start site (HSPB8), suggesting that these
are true ER␣ binding sites associated with the
LRRC54 and HSPB8 genes, and not other genes
(Cheung, E., Y. Ruan, and E. T. Liu, personal communication; manuscript submitted). Our findings are
consistent with the hypothesis that EDC may elicit
these gene up-regulations by activation of other
transcription factors, perhaps involving phosphorylations that proceed through extranuclear-initiated
signaling cascades. The suppression of gene stimulation by some protein kinase inhibitors would be in
keeping with such a mechanism. Future investigations will be focused on the identification of the
transcription factors or kinases that are involved in
mediating gene regulation through these ER-dependent extranuclear pathways. Our current study highlights the importance of integration of nuclear and
extranuclear ER signaling inputs activated by estrogens in regulating patterns of gene expression in
breast cancer cells.
Mol Endocrinol, September 2008, 22(9):2116–2127 2125
MATERIALS AND METHODS
Compounds and Materials
The EDC was prepared as previously described (6). Because
the EDC contains 20 molecules of estrogen per dendrimer,
we state EDC concentration in terms of E2 equivalents. One
EDC equals 20 E2 molecules; therefore 0.5 nM EDC provides
10 nM E2 (6). In all experiments, E2 and EDC were used at
equivalent estrogen concentrations. E2, cycloheximide, and
actinomycin D were from Sigma Chemical Co. (St. Louis,
MO). GPR30 agonist G1, c-Src inhibitor PP2, and MEK inhibitor PD98059 and other protein kinase inhibitors were from
Calbiochem (La Jolla, CA).
Cell Culture, RNA Extraction, and Real-Time PCR
Analysis of Gene Expression Regulation
MCF-7 human breast cancer cells were maintained in culture
as previously described (29). At 6 d before E2 treatment, cells
were switched to phenol red-free media containing charcoaldextran-treated calf serum. Medium was changed on d 2 and
d 4 of culture, and cells were then treated with compounds as
indicated. After cell treatments, total RNA was isolated, reverse transcribed, and analyzed by real-time PCR exactly as
described previously (29). Primers for the genes studied are
as follows: LRRC54 forward (f), GGGCTACACGACGTTGGCT; LRRC54 reverse (r), GAGGTCAAGCGACTCCAGGTA;
HSPB8f, TGGATACGTGGAGGTGTCTGG; HSPB8r, GATCCACCTCTGCAGGAAGC; PMAIP1f, CGAAAGACCTCAAGCTGCTC; PMAIP1r, CCAATCCATTGCCTTTATGG. Primers
for progesterone receptor and pS2 are published elsewhere (29, 30).
Nuclear Run-On Assays
Nuclear run-on assays were carried out as described previously (31). MCF-7 cells were treated with 10 nM E2 or 10 nM
E2 equivalent EDC for 2 h. Then cells were washed once with
PBS and harvested using HEPES-EDTA. Then 1 ml lysis
buffer [0.5% Nonidet P-40, 10 mM KCl, 10 mM MgCl2, 10 mM
HEPES (pH 7.9), and 0.5 mM ␤-mercaptoethanol] was added
per plate, and cell suspensions were incubated on ice for 5
min. After centrifugation, the nuclei were washed once with
lysis buffer without Nonidet P-40 and then centrifuged again.
Finally nuclei from each treatment were resuspended in 100
␮l of storage buffer (50 mM Tris-HCl, 5 mM MgCl2, 0.5 mM
␤-mercaptoethanol, 40% glycerol) and kept frozen at ⫺80 C.
For transcription, 100 ␮l transcription buffer [10 mM Tris-HCl
(pH 8.0), 0.3 M MgCl2, 5 mM dithiothreitol, 40 U of ribonuclease inhibitor (Roche, Indianapolis, IN), 1⫻ biotin labeling mix
(Roche)] was added to nuclei, and the reaction was incubated
at 30 C for 45 min. Then RNA was isolated using Trizol
Reagent (Invitrogen, Carlsbad, CA). The final RNA was dissolved in 50 ␮l of diethylpyrocarbonate water. Streptavidinconjugated magnetic beads (50 ␮l) (Invitrogen) resuspended
in binding buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and
100 mM NaCl) was added to each sample, and the mix was
incubated at room temperature for 2 h. Beads were washed
twice in 500 ␮l of 2⫻ standard sodium citrate, 15% formamide for 15 min and once in 500 ␮l of 2⫻ standard sodium
citrate for 5 min. Beads were finally dissolved in 12 ␮l diethylpyrocarbonate water. RNA was reverse transcribed, and
quantitative real-time PCRs were carried out. The fold change
in transcription of each gene was calculated as described
previously (29). The primers used for the nuclear run-on assays were the same as those used for gene expression
experiments.
2126
Mol Endocrinol, September 2008, 22(9):2116–2127Madak-Erdogan et al. • Extranuclear Signaling and Gene Regulation by ER
GeneChip Microarrays, Statistical Analysis, and
Functional Categorization of Target Genes
MCF-7 cells were treated with 10 nM E2 or 10 nM E2 equivalent EDC for 4 h in three separate experiments (Fig. 1A), and
total RNA was prepared from each sample, further purified,
and used to generate cRNA, which was labeled with biotin.
cRNAs were then hybridized on Affymetrix human Hu-133A
GeneChips, which contain oligonucleotide probe sets representing approximately 23,000 human genes and expressed
sequence tags. After washing, the chips were scanned and
data were analyzed as described previously (32). Briefly, the
data was analyzed using GeneChip Operating Software (Affymetrix, Santa Clara, CA). CEL files were then analyzed
using affy and gcrma package protocols in R/Bioconductor.
Probesets with consistently low expression values were discarded after which statistical multivariate analysis was done
by the limma package. Next, probesets were also filtered
based on best overall significance by the F-test statistic (32).
The criteria for genes regulated by E2 or EDC were set so that
they have a false discovery rate (FDR) of 1% and a fold
change of 1.5 or greater over vehicle-treated samples. The
entire data set will be available through the National Center
for Biotechnology Information Gene Expression Omnibus.
GeneSpring and ErmineJ software and the web-based DAVID
functional annotation tool from National Institutes of Health
(NIH) were used for functional classification of the genes.
ChIP Assays
Chromatin immunoprecipitation assays were performed as
described elsewhere (32, 33). MCF-7 cells were treated with
10 nM E2 or 10 nM E2 equivalent EDC for 45 min, 2 h, or 3 h
before harvest. The antibodies used, purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA) were ER␣ (HC-20),
total RNA Pol II (N20), phosphor-Ser5 RNA Pol II (CTD4H8).
Controls using rabbit IgG were routinely done in all ChIP
assays, as indicated in the figure legends. We also used a
control non-ER binding region of the pS2/TFF1 gene as an
additional check for specificity of ChIPs (30, 32). Quantitative
real-time PCR was used to calculate recruitment to the regions studied, as described previously (32).
siRNA Transfection and ER␣ Knockdown
siRNA knockdown of ER␣ was done as previously described
(30, 32). MCF-7 cells were transfected with siRNA duplex
against the F-domain of ER␣ or with control GL3 luciferase
(no. D-001400–01), both obtained from Dharmacon (Lafayette, CO). Both siRNAs were transfected into cells at a final
concentration of 20 nM using the DharmaFECT transfection
reagent (Dharmacon) as per the manufacturer’s recommendations at 48 h before ligand treatment. The small interfering
ER␣ forward sequence is UCAUCGCAUUCCUUGCAAAdTdT, and the reverse sequence is UUUGCAAGGAAUGCGAUGAdTdT. The efficiency of ER␣ knockdown was verified
at the RNA level by Q-PCR and at the protein level by Western blot.
Acknowledgments
We thank Edwin Cheung, Yijun Ruan, and Edison Liu of the
Genome Institute of Singapore for sharing their unpublished
data with us.
Received February 19, 2008. Accepted July 3, 2008.
Address all correspondence and requests for reprints to:
Dr. Benita S. Katzenellenbogen, University of Illinois, Department of Molecular and Integrative Physiology, 524 Burrill Hall,
407 South Goodwin Avenue, Urbana, Illinois 61801-3704.
E-mail: [email protected].
This work was supported by grants from the National
Institutes of Health (NIH) [NIH CA 18119 (to B.S.K.), DK 15556
(to J.A.K.), T32 ES07326 (to Z.M.E.)] and a grant from The
Breast Cancer Research Foundation (to B.S.K.).
Disclosure Statement: Z.M.E., K.J.K., S.H.K., J.A.K, and
B.S.K. have nothing to declare. B.K. is employed by Wyeth
Research.
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