REA in Endometriosis_2015

Multiple Beneficial Roles of Repressor of Estrogen
Receptor Activity (REA) in Suppressing the
Progression of Endometriosis
Yuechao Zhao, Yiru Chen, Ye Kuang, Milan K. Bagchi, Robert N. Taylor,
John A. Katzenellenbogen, and Benita S. Katzenellenbogen
Departments of Molecular and Integrative Physiology (Y.Z., Y.C., M.K.B., B.S.K.) and Chemistry (J.A.K.),
University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; Department of Gynecology and
Obstetrics (Y.K.), The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang
150086, China; and Department of Obstetrics and Gynecology (R.N.T.), Wake Forest School of
Medicine, Winston-Salem, North Carolina 27157
Endometriosis is an estrogen-dependent, inflammation-driven gynecologic disorder in which en-
dometrial tissue creates inflammatory lesions at extrauterine sites, leading to pelvic pain and
impaired fertility. Although dysregulated estrogen receptor (ER) signaling has been implicated,
understanding of this disease is incomplete and current therapies are of limited benefit. Using an
immunocompetent syngeneic murine model, we used combinations of donor uterine tissue and/or
recipienthostmicewithpartialgeneticdeletionoftheERcoregulator,repressorofERactivity(REA)
(also known as prohibitin 2), to investigate roles of REA in the contributions of donor uterine tissue
and host cell influences on endometriosis establishment and progression. Ectopic lesions derived
from donor tissue with half the wild-type gene dosage of REA (REAϩ/Ϫ
) grown in REAϩ/Ϫ
hosts
displayed enhanced proliferation, vascularization, and markedly increased neuron innervation
and inflammatory responses, including elevated cytokine production, nuclear factor kappa B ac-
tivation, cyclooxygenase-2 expression, and immune cell infiltration. Although lesion progression
was greatest when REA was reduced in both donor tissue and host animals, other donor/host
combinations indicated that distinct stimulatory inputs were derived from ectopic tissue (prolif-
erative signals) and host cells (inflammatory signals). Importantly, depletion of REA in primary
human endometriotic stromal cells led to elevated proliferation and expression of cell cycle reg-
ulators. Notably, REA was significantly lower in human endometriotic tissue versus normal human
endometrium. Thus, REA modulates cross talk among multiple cell types in the uterine tissue and
host background, serving as a brake on the estradiol-ER axis and restraining multiple aspects that
contribute to the pathologic progression of endometriosis. (Endocrinology 157: 900–912, 2016)
Endometriosis is an estrogen-dependent and inflamma-
tion-driven disorder in which endometrial tissue at-
taches at extrauterine ectopic sites, proliferates, and forms
invasive lesions. It affects 10%–14% of reproductive age
women, with an even higher prevalence of 35%–50%
among patients with pelvic pain and infertility (1–4). The
devastating effects of this disease on millions of women
and the high rate of disease recurrence after treatment
indicate the need for better mechanistic understanding of
this disorder with the ultimate goal of developing more
effective therapies and long-term management of
endometriosis.
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in USA
Copyright © 2016 by the Endocrine Society
Received April 10, 2015. Accepted December 3, 2015.
First Published Online December 14, 2015
Abbreviations: CD3, cluster of differentiation 3 T cell co-receptor; COX2, cyclooxygen-
ase-2; E2, estradiol; EGFP, enhanced green fluorescent protein; ER, estrogen receptor;
F4/80, macrophage marker EGF-like module-containing mucin-like hormone receptor-like
1; GFP, green fluorescent protein; GL3, luciferase reporter vector GL3; IHC, immunohis-
tochemistry; Ki67, proliferation marker nuclear protein Ki67; KO, knockout; PGP9.5, pro-
tein gene product 9.5; PGR, progesterone receptor; p65, transcription factor p65; P-H3,
phosphohistone-H3; qPCR, quantitative polymerase chain reaction; REA, repressor of ER
activity; siGL3, GL3 luciferase control siRNA; siREA, REA siRNA; siRNA, small interfering
RNA; WT, wild type.
O R I G I N A L R E S E A R C H
900 press.endocrine.org/journal/endo Endocrinology, February 2016, 157(2):900–912 doi: 10.1210/en.2015-1324
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 June 2016. at 13:10 For personal use only. No other uses without permission. . All rights reserved.

Endometriosis is complex in that it is promoted by and
dependent upon extensive cross talk among the numerous
cell types that comprise the endometriotic lesions. These
include endometrial cells from the uterus, likely from ret-
rograde menstrual flow, plus immune cells that infiltrate
into the lesions, and vascular endothelial cells, blood ves-
sels and nerves that grow into and support lesion estab-
lishment, survival and progression. Host peritoneal cells
into which the lesion embeds may also contribute by pro-
viding a favorable environment for lesion survival. Many
of these multiple cell types express estrogen receptors
(ERs) and ER coregulators (5–7) that can work together to
enhance or restrain the estrogen signaling that promotes
progression of the disease.
A hallmark of endometriosis is excessive estrogen sig-
naling (8, 9). This is supported by increased local produc-
tion of estrogen driven by the aromatase gene, cytochrome
P450, family 19, subfamily A, polypeptide 1 (2, 10), as
wellasalteredexpressionofER␣andER␤(11,12).There-
fore, current hormonal treatments, including progestins,
androgens, GnRH agonists, and aromatase inhibitors, fo-
cus on reducing systemic levels of estrogens. These treat-
ments, however, are limited by side effects, incomplete
effectiveness, and high rates of disease recurrence after
treatment cessation (2). To interrogate the molecular
eventsunderlyingtheestablishmentandprogressionofthe
uterine tissue at ectopic sites, murine models have been
widely used recently (13). Critical roles of the ERs in en-
dometriosis pathogenesis are also supported by studies
employing ER knockout (KO) mice (14) and preclinical
analyses with ER subtype-selective ligands (15–17). Be-
cause ER coregulators are recruited to chromatin by ste-
roid hormone nuclear receptors in a temporally and spa-
tially specific manner for precise gene regulation, these
components also emerge as contributors to and potential
therapeutic targets for controlling the multiple hyper-
estrogenic stimulatory activities that drive endometriosis
establishment and progression.
The ER coregulator, repressor of ER activity (REA) (also
known as prohibitin 2) (18, 19), has been shown to serve as
a brake on ER activity in estrogen target tissues such as the
uterus (20, 21) and mammary gland (22) and to affect cell
signaling pathways. Homozygous ablation of REA in the
uterus led to infertility due to severely compromised uterine
development and failure of implantation (21). However,
uteri of heterozygous REAϩ/Ϫ
mice, with half the normal
wild-type (WT) level of REA, showed an accelerated and
amplified decidualization process and subfertility, due to hy-
perresponsiveness to estrogen signaling (20, 21, 23). These
studies, documenting REA to be a physiologic, protective
factor against excessive estrogen-driven activity in uterine
tissue, led us to hypothesize that REA might also play a crit-
ical role in modulating or moderating the establishment and
progression of endometriosis.
To explore this hypothesis, we have used an immuno-
competent murine model, in which estrogen hyperstimu-
lation of proliferation and inflammatory signaling in ec-
topic lesions recapitulate the disease in humans (24). Our
findings reported herein using donor tissue and host ani-
mals with reduced levels of REA highlight distinctive roles
that this ER corepressor plays in the ectopic uterine tissue
and host tissues: normal levels of REA in ectopic uterine
tissue restrain estrogen-supported implant growth and
vascularization, whereas normal REA levels in host tissues
suppress inflammatory responses associated with lesion
progression. Elevated proliferative activity of human en-
dometriotic stromal cells upon loss of REA and our com-
parison of human endometriotic tissue from patients vs
normal human endometrium, which revealed significantly
lower REA in endometriotic lesions further support the
clinical relevance of REA and the usefulness of observa-
tions from our animal model. The findings highlight REA
as a protective restraint on the estradiol (E2)-ER-driven
axis in endometriosis that acts as a corepressor of multiple
aspects of the pathologic progression of this disease.
Materials and Methods
Animals and immunocompetent mouse model of
endometriosis
All animals were maintained in accordance with the National
Institutes of Health Guide for Care and Use of Laboratory An-
imals, and all procedures were approved by the University of
Illinois Institutional Animal Care and Use Committee. C57BL/6
mice were purchased from Harlan Laboratories or The Jackson
Laboratory (EGFP, stock number 006567). REA heterozygous
(REAϩ/Ϫ
) mice on the C57BL/6 background and their WT lit-
termatesweremaintainedandgenotypedasdescribedpreviously
(20, 23).
Endometriotic-like lesions were surgically transplanted as de-
scribed before (17, 24). Briefly, female WT, EGFP, or REAϩ/Ϫ
mice (8–10 wk of age) served as either donor or recipient ani-
mals, or both. The uterine horns were removed from donor mice,
opened longitudinally, cut into fragments using a 3-mm dermal
biopsy punch (Miltex) and transplanted onto the peritoneal wall
of recipient mice by suturing. In each experimental group, uter-
ine tissue was collected from at least 6 donor mice and trans-
planted into 6 recipient mice. Ectopic lesion volume was calcu-
lated as before (17).
To examine roles of REA in E2-supported lesion establish-
ment,ovariectomizedrecipientmicewereimplantedwithapellet
of E2 (Innovative Research of America) sc and underwent ecto-
pic tissue transplantation on the same day. The dosage of
0.125-mg E2/pellet was chosen as optimal based on our pre-
vious work (17). At the times indicated, both eutopic uterine
tissue and ectopic endometriotic lesions were collected for
further analysis.
doi: 10.1210/en.2015-1324 press.endocrine.org/journal/endo 901

To interrogate functions of REA during chronic lesion pro-
gression, uterine fragments from donor mice were transplanted
on alternate sides of the peritoneal incision into intact female
recipients without any hormonal administration. The ectopic
tissues were collected at 2, 4, and 8 weeks after transplantation
surgery for further analysis.
In order to minimize hormonal variation in cycling mice, all
donor mice used above, as well as intact recipients, underwent
tissue transplantation at the diestrous stage. In addition, recip-
ient mice were killed at diestrus for collection of tissues as de-
scribed before (17, 24).
Primary human endometrial and endometriotic
stromal cell cultures and small interfering RNA
(siRNA) studies
Our studies involving human eutopic endometrial biopsies,
endometriotic lesion biopsies, and primary cell cultures were
approved by the Institutional Review Boards of the University of
Illinois, Emory University, and Wake Forest University School of
Medicine. All protocols adhere to the regulations set forth for the
protection of human subjects participating in clinical research,
including the establishment of a data and safety monitoring plan.
Isolation and culture of primary human endometriotic stro-
mal cells were conducted as described (23). Cells were cultured
in DMEM/F-12 medium (Invitrogen) containing 5% charcoal-
dextran-treated fetal bovine serum. For siRNA experiments, en-
dometriotic stromal cells were transfected with REA siRNA
(siREA) or GL3 luciferase control siRNA (siGL3) (Dharmacon)
following the Silent-Fect kit protocol (Bio-Rad Laboratories) as
before (23). After 24 hours of transfection, cells were exposed to
20-ng/mL TNF␣ (R&D Systems) and 10nM E2 (Sigma-Aldrich)
for the times indicated.
Histological analyses, immunohistochemistry (IHC),
and immunofluorescence
IHC and immunofluorescence were performed in cultured
cells or paraffin-embedded mouse or human tissue sections as
described (23). Primary antibodies used (Supplemental Table 2)
were: REA (Millipore Co), GFP (Cell Signaling Technology),
Ki67 (Bioss), platelet endothelial cell adhesion molecule (PE-
CAM) (Abcam), IL-6 (Invitrogen), p65 (Cell Signaling Technol-
ogy), CD3 (Abcam), F4/80 (Acris Antibodies), COX2 (Abcam),
protein gene product 9.5 (PGP9.5) (Abcam), progesterone re-
ceptor (PGR) (DAKO), and phosphohistone-H3 (P-H3) (Milli-
pore Co). The stain signal was quantified by monitoring the
average numbers of positively stained cells to the total number of
cells from 6 randomly chosen fields.
RNA isolation and real-time PCR
Total RNA was isolated from eutopic or ectopic tissues or
primary cells using TRIzol Reagent (Life Technologies) to pre-
pare cDNA (17, 23, 24). Real-time PCR was performed to quan-
tifygeneexpressionusingspecifichumanormouseprimers(Sup-
plementalTable1)andSYBRGreenkits(Bio-RadLaboratories).
After analysis by the delta cycle threshold method, data were
normalized to 36B4 gene expression (23).
Statistical analysis
Statistical analyses included paired or unpaired t test, one- or
two-way ANOVA with Bonferroni’s multiple comparison test
and used GraphPad Prism version 5.00 (GraphPad Software).
Data are expressed as mean Ϯ SD, and P Ͻ .05 was assigned as
statistically significant.
Results
Impact of partial depletion of REA on E2-
supported endometriosis-like lesion establishment
We investigated the role of REA in E2-supported lesion
establishment using different combinations of donor uter-
ine tissue and recipient host background in which the level
of REA was WT (REAϩ/ϩ
) or reduced (REAϩ/Ϫ
). We first
compared in parallel donor uterine tissue with different
levels of REA transplanted into the same recipient animal.
(Because REA null uteri displayed a severely atrophic phe-
notype [21], REA heterozygous [REAϩ/Ϫ
] uteri were cho-
sen for comparison with WT donor tissue with the full
complement of REA [REAϩ/ϩ
].) As shown in Figure 1, A
and B, after 2 weeks of E2 supplementation, both WT and
REAϩ/Ϫ
donor (D) uterine fragments, surgically trans-
planted onto the peritoneal surface of ovariectomized WT
recipient (R) mice, were able to form endometriosis-like le-
sions denoted as Dwt
-Rwt
and Dϩ/Ϫ
-Rwt
, respectively. No-
tably, the growth of Dϩ/Ϫ
-Rwt
lesions was significantly
greater than Dwt
-Rwt
(Figure 1B), suggesting a restraining
role for REA present in ectopic endometriosis-like lesion
growth.
AlthoughtheheterozygousREAϩ/Ϫ
transcriptandpro-
tein levels (ϳ50%) were confirmed in REAϩ/Ϫ
donor uter-
ine tissue compared with WT uteri by qPCR (Figure 1C)
and IHC (Figure 1D), it was noteworthy that Dϩ/Ϫ
-Rwt
ectopic lesions contained about 70% of the WT level of
REA mRNA (Figure 1C) and REA protein (Figure 1E),
implying a contribution of REA from infiltrating host
cells. Indeed, as shown in Figure 1F, the presence of REA
in host cells that infiltrate the ectopic lesion was validated
by cellular colocalization of REA and EGFP proteins in
EGFP-transgenic host mice by dual immunofluorescence
analysis when WT donor uterine tissue was transplanted
into EGFP recipients, and these recipients were given 2
weeks of E2 treatment.
In order to evaluate the importance of host cell REA in
ectopiclesionprogression,weexamineddonortissuegrowth
and phenotypic properties in E2-treated REAϩ/Ϫ
recipient
mice(Figure2A).AlthoughDϩ/Ϫ
-Rwt
ectopictissuesformed
larger lesions compared with Dwt
-Rwt
lesions (Figure 1B),
WTlesionsestablishedinREAheterozygousrecipients(Dwt
-
Rϩ/Ϫ
) displayed a growth rate similar to that of Dwt
-Rwt
lesions (Figure 2B), implying that the proliferative signal in
902 Zhao et al REA in Endometriosis Endocrinology, February 2016, 157(2):900–912

lesions is affected largely by the REA content in the donor
tissue.
Because the host environment and cells that infiltrate
into the ectopic lesion from the host animal can contribute
to inflammatory aspects of the disease (17), we next ex-
amined the expression of several cytokines known to be
highly expressed in human endometriotic tissue (25–28)
and regulated by E2 signaling in endometriosis (17). As
Donor Uterine
Tissue
2 weeks of
E2 treatment
REA⁺/
⁺ REA⁺/-
A
Lesionvolume(mm³)
*
B
OVX Recipient (REA⁺/
⁺)
Dwt-Rwt
Dwt-Rwt
Dwt-Rwt
Ectopic
lesion
S
E
S
E
50μm
S
E
D+/--Rwt Dwt-Rwt
IgGREAE
Dwt-Rwt D+/--RwtREA⁺/⁺
REA⁺/-
Ectopic lesion
a
b
a
c
mRNAFoldChange
REA
C
C
C
D⁺/-
-Rwt
D⁺/-
-Rwt
D
0
10
20
30
40
C
LE
S
GE LE
S
GE
LEREA
50μm
REA⁺/
⁺ REA⁺/-
F
REA GFP Merged
100μm
Figure 1. Ectopic lesions with a reduced level of REA show enhanced growth in E2-treated WT recipient mice. A, Endometriosis-like lesions were
established by transplanting donor (denoted by D) uterine fragments from REA WT (REAϩ/ϩ
, n ϭ 6) and REA heterozygous (REAϩ/Ϫ
, n ϭ 6) mice
of diestrous stage into ovariectomized (OVX) REAϩ/ϩ
recipient (denoted by R) mice, as described in Materials and Methods. B, Growth of ectopic
lesions was monitored and lesion volume was quantified after 2 weeks of E2 treatment, P Ͻ .05 (paired t test). C, qPCR analysis of REA mRNA in
donor uterine tissue and ectopic lesions after 2 weeks of E2 treatment. Levels of mRNA are expressed relative to the transcript level in REAϩ/ϩ
donor uterine tissue which is set at 1.0. Different letters indicate P Ͻ .05 by one-way ANOVA with Bonferroni’s multiple comparison test. D, Donor
uterine tissue from REAϩ/ϩ
and REAϩ/Ϫ
mice at diestrous stage was subjected to IHC staining for REA. GE, glandular epithelium; LE, luminal
epithelium; S, stromal tissue. E, IHC staining of REA in ectopic lesions after 2 weeks of E2 treatment. IgG served as negative control. C,
endometriotic cyst; E, epithelial tissue; S, stromal tissue. F, Dual immunofluorescence of REA and GFP in ectopic lesions derived from WT donor
tissue transplanted into WT EGFP recipient mice treated with E2 for 2 weeks. D, donor; R, recipient.

shown in Figure 2D, when compared with Dwt
-Rwt
le-
sions, levels of IL-6 (Il6), chemokine (C-C motif) ligand 2
(Ccl2) and Ccl5 in Dwt
-Rϩ/Ϫ
lesions were increased,
whereas the expression of TNF␣ (Tnf␣) remained
unchanged.
REA expressed in donor uterine tissue suppresses
ectopic cell proliferation and vascularization
To further investigate functions of donor tissue REA in
lesion progression, we used a model in which both REA
donor WT (Dwt
-Rwt
) and REA donor heterozygous (Dϩ/
Ϫ-Rwt
) ectopic tissues were allowed to become established
in the same WT intact recipient mice, and growth was
monitored over 8 weeks. As seen in Figure 3A, Dϩ/Ϫ
-Rwt
ectopic lesions showed an enhanced growth rate after 2
weeks of lesion progression compared with that of Dwt
-
Rwt
lesions. Notably, elevated ectopic lesion cell prolifer-
ation was seen in 4-week Dϩ/Ϫ
-Rwt
ectopic lesions vs Dwt
-
Rwt
lesions by IHC analysis of the proliferation marker
Ki67 (Figure 3, B and C). By contrast, Ki67 staining anal-
ysis of eutopic uterine WT REAϩ/ϩ
or REAϩ/Ϫ
donor tis-
sue indicated no difference in cell proliferation (Supple-
mental Figure 1A), further suggesting the specific roles of
donor REA level in lesion progression. Immunofluores-
cence for the blood vessel marker, PECAM (Figure 3D),
and quantification of PECAM-positive cells as an indica-
tor of vascularization (Figure 3E) also documented that
Dϩ/Ϫ
-Rwt
lesions were more highly vascularized com-
pared with Dwt
-Rwt
ectopic lesions, but remained at a sim-
Ccl2 Ccl5 afnT6LI REA
*
*
*
*
A
mRNAFoldChange
2 weeks of E2 treatment
Dwt
-Rwt
Dwt
-R+/-
Dwt-R+/-
Dwt-Rwt
OVX Recipient (REA⁺/-)
REA⁺/
⁺
OVX Recipient (REA⁺/⁺)
C
Lesionvolume(mm³)
B
Dwt-Rwt
Dwt
-R+/-
0
10
20
30
40
Figure 2. Ectopic lesions from E2-treated recipients with reduced level of REA. A, Donor uterine tissue from REAϩ/ϩ
mice (n ϭ 12) was
transplanted into OVX REAϩ/ϩ
(n ϭ 6) or REAϩ/Ϫ
(n ϭ 6) recipients, and assays were conducted after 2 weeks of E2 treatment in Dwt
-Rwt
and Dwt
-
Rϩ/Ϫ
ectopic lesions. B, Volume of ectopic lesions at 2 weeks of E2 treatment. Lesion volumes were not statistically different (unpaired t test). C,
Il6, Ccl2, Ccl5, Tnf␣, and REA expression levels in Dwt
-Rwt
and Dwt
-Rϩ/Ϫ
ectopic tissues were analyzed by qPCR after 2 weeks of E2 treatment.
mRNA levels are expressed relative to the transcript level in Dwt
-Rwt
lesions, which is set at 1.0. *, P Ͻ .05 (unpaired t test).

ilar level in eutopic tissue (data not shown). By contrast,
cytokine production was similar in Dϩ/Ϫ
-Rwt
lesions
and WT lesions (Figure 3F). Taken together, these find-
ings suggest that growth and inflammatory signals orig-
inate from distinct tissue loci, with the E2-supported
lesion growth and vascularization being largely con-
trolled by REA gene dosage in the donor uterine tissue,
whereas cytokine production in the ectopic lesion is
principally determined by the host level of REA in in-
filtrating cells.
Host REA restrains inflammation and innervation
of ectopic endometriotic lesions
Next, both WT and REAϩ/Ϫ
mice were used as recip-
ients to interrogate the impact of REA in the host tissue on
endometriotic lesions that developed from REA WT uter-
ine tissue. Consistent with our observations for cytokine
expression in WT lesions from E2-treated heterozygous
recipients (Dwt
-Rϩ/Ϫ
) (Figure 2C), higher Il6, Ccl2, Ccl5,
and Tnf␣ mRNA levels were seen by qPCR analysis in WT
lesions established in ovary intact REAϩ/Ϫ
recipients
(Dwt
-Rϩ/Ϫ
) and monitored over 8 weeks of progression
(Figure 4A). Immunostaining also demonstrated a greatly
increased level of IL6 protein and p65 protein in Dwt
-Rϩ/Ϫ
lesions vs Dwt
-Rwt
lesions (Figure 4, B and C). The greater
than 2-fold increase in the number of nuclear p65-positive
cells in Dwt
-Rϩ/Ϫ
ectopic lesions supports stronger nuclear
factor kappa B activity in these lesions (Figure 4C). Im-
munofluorescence for the T-cell marker CD3 and macro-
phage marker F4/80 also revealed more immune cells in
the Dwt
-Rϩ/Ϫ
lesions (Figure 4D). COX2 protein, another
hallmark of endometriotic lesion-associated inflamma-
tion (2), was also present at a higher level in Dwt
-Rϩ/Ϫ
vs
Dwt
-Rwt
lesions (Figure 4E). Also notable was the in-
creased innervation of Dwt
-Rϩ/Ϫ
lesions, observed by
staining of the pan neuron marker, PGP9.5 (Figure 4E).
Taken together, these data support suppressive roles of
REA in host cells and tissues in multiple inflammatory
responses and in lesion innervation that accompanies le-
sion progression. Notably, however, Dwt
-Rϩ/Ϫ
ectopic tis-
sues showed similar lesion growth rate (Figure 4F) and
cell proliferation activity indicated by Ki67 staining
(Supplemental Figure 1B) to that of Dwt
-Rwt
lesions,
which is distinctly different from what was observed in
Dϩ/Ϫ
-Rwt
lesions. These findings highlight the impor-
tance of REA status in donor uterine tissue on prolif-
erative drive, in addition to distinctive contributions
from the host environment in the inflammatory aspects
of endometriosis.
*
*
Dwt
-Rwt
D+/-
-Rwt
Dwt
-Rwt D+/-
-Rwt
A
Lesionvolume(mm³)
D
Weeks
*
*
B C
S
S
BV
BV
BV
BV
50μm
PECAM
Dwt-Rwt
D+/-
-Rwt
Ectopic lesion (4 weeks, intact recipient)
S
E
S
E
50μm
Ki67
D+/--RwtDwt-Rwt
Intact recipient
F
perﬁeld
E
D+/--RwtDwt-Rwt
F
Ccl2 Ccl5IL6 Tnfa
mRNAFoldChange
Dwt-Rwt
D+/--Rwt
Figure 3. Impact of REA level in donor uterine tissue on lesion progression. Donor uterine tissue from REA WT (REAϩ/ϩ
, n ϭ 6) and REA
heterozygous (REAϩ/Ϫ
, n ϭ 6) mice were transplanted into intact WT recipient mice (n ϭ 6) and were followed over 8 weeks for formation of
Dwt
-Rwt
and Dϩ/Ϫ
-Rwt
lesions, respectively. Both donor and recipient mice underwent transplantation surgery at the diestrous stage. A, Growth of
Dwt
-Rwt
and Dϩ/Ϫ
-Rwt
lesion volume over time was quantified as shown. *, P Ͻ .05 (two-way ANOVA with Bonferroni’s multiple comparison test).
B, IHC staining for Ki67 in Dwt
-Rwt
and Dϩ/Ϫ
-Rwt
lesions after 4 weeks of progression in intact WT recipients. C, Quantification of Ki67 staining
signals in lesions. *, P Ͻ .05 (paired t test). D, PECAM staining of vasculature in ectopic lesions at 4 weeks of progression in intact WT recipients. E,
Quantification of PECAM-positive cells at 4 weeks. *, P Ͻ .05 (paired t test). BV, blood vessel; C, endometriotic cyst; E, epithelial tissue;
S, stromal tissue. F, Il6, Ccl2, Ccl5, and Tnf␣ mRNA levels in Dwt
-Rwt
and Dϩ/Ϫ
-Rwt
ectopic tissues were analyzed by qPCR at 4 weeks of lesion
progression. Transcript levels are expressed relative to the transcript level in Dwt
-Rwt
lesions which is set at 1.0. No significant difference was
detected.

Lesion growth and inflammation are most
increased when REA level is reduced in both
donor and recipient host tissues
To further understand the actions of REA in the cross
talk between donor and host tissues, REAϩ/Ϫ
donor uter-
ine tissue was transplanted into heterozygous REA host
mice (Dϩ/Ϫ
-Rϩ/Ϫ
) and lesions were collected after 8
weeks. Quantification of lesion volume (Figure 5A)
showed that the reduced gene dosage of REA in both do-
nor and host tissues (Dϩ/Ϫ
-Rϩ/Ϫ
) resulted in the greatest
*
A
mRNAFoldChange
S
E
SC
E
C
IL6
B
C
CD3
F4/80
S
S
S
S
S
S S
SCOX2
F4/80
PGP9.5
D
E
Dwt-Rwt
Dwt-R+/-
50 μm
100 μm
C
C
E
E
S
S
p65
*
afnT5lcC2lcC6LI
*
*
*
* *
* *
Weeks
0 2 4 6 80 2 4 6 80 2 4 6 80 2 4 6 8
0000
10
20
30
40
10
100
150
100
200
300
400
500
20
40
60
80
100
Weeks
F
Lesionvolume(mm³)
Dwt-Rwt
Dwt-R+/-
Dwt-Rwt Dwt-R+/-
Dwt-Rwt Dwt-R+/-
Dwt-Rwt Dwt-R+/-
Dwt-Rwt
Dwt-R+/-
Dwt-R+/-
Dwt-Rwt
Dwt-Rwt
Dwt-R+/-
IL6
p65
CD3 F4/80
Dwt
-Rwt Dwt
-R+/-
Dwt
-Rwt Dwt
-R+/-
0
10
20
30
40
0
10
20
30
* *
0
5
10
15
20
25
0
5
10
15
20
COX2 PGP9.5
Dwt
-Rwt Dwt
-R+/-
Dwt
-Rwt Dwt
-R+/-
*
*
* *
Figure 4. Impact of reduced host REA level on lesion progression. Donor uterine tissue from REAϩ/ϩ
mice at diestrous stage (n ϭ 12) was
engrafted into intact REAϩ/ϩ
(n ϭ 6) or REAϩ/Ϫ
(n ϭ 6) recipients at diestrus and followed over 8 weeks for lesion progression, forming Dwt
-Rwt
and Dwt
-Rϩ/Ϫ
implants, respectively. A, Il6, Ccl2, Ccl5, and Tnf␣ transcript levels in Dwt
-Rwt
and Dwt
-Rϩ/Ϫ
ectopic tissues were profiled by qPCR
over 8 weeks of lesion progression in intact recipients. mRNA levels are expressed relative to the transcript level in REAϩ/ϩ
eutopic donor tissue,
which is set at 1.0. *, P Ͻ .05 (two-way ANOVA with Bonferroni’s multiple comparison test). IHC staining of (B) IL6 or (C) p65 in Dwt
-Rwt
and Dwt
-
Rϩ/Ϫ
ectopic lesions at 8 weeks. The IHC signals were quantified. *, P Ͻ .05 (unpaired t test). C, endometriotic cyst; E, epithelial tissue; S, stromal
tissue. D, Immunofluorescence of immune cell markers CD3 and F4/80 and of (E) Cox2 as well as the pan-neuron marker PGP9.5 were performed
and quantified (*, P Ͻ .05) (paired t test) in Dwt
-Rwt
and Dwt
-Rϩ/Ϫ
ectopic lesions at 8 weeks. Quantitation of CD3, F4/80, COX2, and PGP9.5 are
shown (at the right). F, Growth of WT lesions in intact WT (Dwt
-Rwt
, n ϭ 6) and heterozygous (Dwt
-Rϩ/Ϫ
, n ϭ 6) recipients was monitored over 8
weeks. No significant difference was detected (two-way ANOVA with Bonferroni’s multiple comparison test).

increase in lesion size, compared with donor REAϩ/Ϫ
im-
plants in recipient WT hosts (Dϩ/Ϫ
-Rwt
), which were
larger than WT donor uterine implants grown in WT hosts
(Dwt
-Rwt
) or heterozygous hosts (Dwt
-Rϩ/Ϫ
). qPCR anal-
ysis (Figure 5B) demonstrated significantly higher tran-
script levels for most of the inflammation-associated cy-
tokines examined in Dϩ/Ϫ
-Rϩ/Ϫ
lesions compared with
Dwt
-Rwt
and Dϩ/Ϫ
-Rwt
lesions. In addition, although
mRNA expression levels of ER␣ or ER␤ were similar in
ectopic tissue (Supplemental Figure 2A), IHC analysis of
PGR, which serves as an indicator of estrogen signaling
activity, showed enhanced PGR with reduced REA level in
the donor tissue (Dϩ/Ϫ
-Rwt
) which
was further elevated in Dϩ/Ϫ
-Rϩ/Ϫ
lesions (Supplemental Figure 2B),
demonstrating the restraining role of
REA in estrogen signaling during le-
sion progression. Therefore, the en-
hanced progression observed in Dϩ/
Ϫ-Rϩ/Ϫ
lesions reveals that REA is
not only able to suppress multiple
characteristic aspects of endometri-
osis, but that it also critically con-
tributes in estrogen signaling, and
the cross talk among the multiple cell
types in the donor uterine tissue and
the host background, which
are important in endometriosis
progression.
REA regulates proliferation of
human endometriotic stromal
cells
To examine the functional signif-
icance of REA in human endometri-
otic cells from patient samples, we
employed siRNA knockdown of
REA in primary human endometri-
otic stromal cells cultured in vitro.
Treatment with siREA resulted in
greatly reduced levels of REA
mRNA (Figure 6A) and protein
(Supplemental Figure 3). When cells
were exposed to TNF␣ and E2 to
mimic the in vivo hyperestrogenic
and inflammatory microenviron-
ment characteristic of endometriosis
(29, 30), the mRNA levels of several
key cell cycle regulators, such as cy-
clin-dependent kinase 2 (CDK2), cy-
clin B2 (CCNB2), cyclin D2
(CCND2), and minichromosome
maintenance complex 2, were more
markedly elevated in siREA treated than in control siGL3-
treated human endometriotic stromal cells, especially in
cells treated with E2 ϩ TNF␣ (Figure 6A). By contrast, the
level of ER␣ was not changed (Figure 6A). Increased cell
proliferation was evident from immunofluorescence as-
says of Ki67, P-H3, and by quantitation of cell numbers in
cells with knockdown of REA (Figure 6B). Thus, REA
normally acts as a suppressor of human endometriotic cell
proliferation, reflected by enhanced expression of key cell
cycle regulators and proliferation markers when REA was
reduced in the cells.
Il6
Ccl5
Ccl2
Tnfa
B
mRNAFoldChange
a a
c
a a
bb
c
b
b
a a
a a
a
b
mRNAFoldChange
Dwt
-Rwt
D+/-
-Rwt Dwt
-R+/-
D+/-
-R+/-
A Lesionvolume(mm³)
Dwt
-Rwt
Dwt
-R+/-
D+/-
-Rwt
D+/-
-R+/-
a a
b
c
Dwt
-Rwt
D+/-
-Rwt Dwt
-R+/-
D+/-
-R+/-
0
20
40
60
80
100
0.0
0.5
1.0
1.5
2.0
0
5
10
15
0
1
2
3
4
0.0
0.5
1.0
1.5
2.0
2.5
Figure 5. Impact of reduced REA levels in both donor uterine tissue and host animals on lesion
progression. Donor tissues from REAϩ/Ϫ
mice at diestrous stage (n ϭ 6) were transplanted into
intact REAϩ/Ϫ
recipients (n ϭ 6) to form Dϩ/Ϫ
-Rϩ/Ϫ
lesions. At 8 weeks after transplantation,
quantification of (A) lesion volume and (B) cytokine mRNA levels by qPCR were monitored in Dwt
-
Rwt
, Dϩ/Ϫ
-Rwt
, Dwt
-Rϩ/Ϫ
, and Dϩ/Ϫ
-Rϩ/Ϫ
lesions. Donors and recipients were intact animals
chosen from the diestrous stage. Different letters indicate P Ͻ .05 by one-way ANOVA with
Bonferroni’s multiple comparison test.

REA is lower in human endometriotic tissue vs
normal human endometrium
As shown in Figure 7, we compared by IHC the presence
of REA protein in normal eutopic endometrium (n ϭ 4
women, ages 25–44) and in ectopic endometriosis samples
from women with the disease (n ϭ 12 patients, ages 22–50).
Quantitation of REA in tissue sections (3 tissue blocks per
patient and 6 fields quantitated per section from each block)
revealed that REA was significantly lower in endometriosis
samples compared with normal eutopic endometrium.
Cellnumber/ﬁeld
E2 + TNFα
siGL3 siREA siGL3 siREA
Veh
a a
b
c
A
B
mRNAFoldChangemRNAFoldChange
siGL3 siREA siGL3 siREA siGL3 siREA siGL3 siREA siGL3 siREA siGL3 siREA
Veh E2+TNF α Veh E2+TNF α Veh E2+TNFα
CDK2
MCM2REA
CCNB2 CCND2
siGL3 siREA siGL3 siREA siGL3 siREA siGL3 siREA siGL3 siREA siGL3 siREA
Veh E2+TNF α Veh E2+TNF α Veh E2+TNFα
siGL3
siREA
αFNT+2EheV
Ki67 + DAPI Ki67 + DAPI
E2 + TNF α
Veh
Ki67
siGL3
siREA
P-H3 + DAPI P-H3 + DAPI
αFNT+2EheV
Phospho-H3
E2 + TNFαVeh
20μm
20μm
a
b b
a
a
b b
c
c
a
b
a,b
a
b b
c
a
b b
c
a a
b
c
a a
b
c
a a
a
a
ERα
Figure 6. Impact of REA knockdown in human endometriotic stromal cells on cell proliferation and expression of cell cycle regulators. 24 hours
after control siGL3 or siREA transfection, human endometriotic stromal cells were treated with E2 (10nM) and TNF␣ (20 ng/mL), or vehicle. A,
qPCR analysis for the indicated RNAs after 24 hours of ligand or vehicle treatment. B, Immunofluorescence for Ki67 and P-H3 at 48 hours after
treatment. DAPI staining indicates nuclei. Quantification of staining signals and measurement of cell numbers are shown (right panels). Different
letters indicate P Ͻ .05 by one-way ANOVA with Bonferroni’s multiple comparison test.

Discussion
Estrogen and inflammatory signaling, which are con-
trolled by nuclear receptors and their coregulators, are
essential for the survival of endometriotic tissue and for
disease progression (31, 32). Endometriotic tissue, like
normal uterine tissue, is reliant on estrogen, but endome-
triosis is unique in that the endocrine milieu and hormone
receptor status of the endometriotic lesions are very dif-
ferent from those in normal reproductive tissues. In par-
ticular, estrogen production and ER regulation are altered
in endometriotic lesions. The ectopic tissue overexpresses
aromatase and COX2 (2, 33), thereby causing continuous
local production of estrogens and prostaglandins. Also
endometriotic lesions have increased levels and increased
activity of ERs which elicit a state of hyperstimulation (11,
12, 34) that drives progression of the disease. Coregula-
tors partner with ERs to control receptor activity and, in
this study, we have found that the corepressor, REA, func-
tions as a restraint on ER to suppress the estrogen-stim-
ulated proliferative drive of endometriotic lesions. Thus,
when REA was reduced, it exacerbated and promoted
pathologic progression of the disease.
Ourobservationshighlightandsupporttheexistenceof
extensive cross talk among various cell types that collab-
orate to support the growth and phenotypic properties of
the endometriotic lesions; these include the ectopic uterine
endometrial cells and supporting cells from the immune,
nervous and vascular systems (17, 35) that are found in-
filtrating the ectopic lesions as they develop (1, 36). Our
studies using host EGFP transgenic mice demonstrated
infiltration of host cells into the ectopic lesion and an en-
hanced macrophage-monocyte complement in heterozy-
gous REAϩ/Ϫ
host animals. Our findings indicate that
REA modulates this cross talk between donor uterine-de-
rived cells and infiltrating host cells, and that reduction of
the REA level in both donor and host tissues most greatly
accelerates the growth and inflammatory signaling in en-
dometriotic lesions.
Notably, as shown in the model in Figure 8, our find-
ingssuggestthatthegrowthandinflammatorysignalsthat
contribute to endometriotic lesion progression originate
principally from distinct tissue loci, with stimulatory in-
puts from the ectopic uterine tissue primarily responsible
for control of lesion proliferation and vascularization, and
host cells and tissues primarily responsible for control of
inflammation and neurogenesis in lesions. Our use of dif-
ferent combinations of donor tissue and host back-
grounds, that allow modulation of REA gene dosage in
each, enabled us to specify distinct stimulatory inputs
from the ectopic uterine donor and host cells. However,
the donor/recipient experimental observations also have
revealed that the donor and recipient tissues impact each
other in ways that influence the progression of endome-
triosis, because lesion growth and inflammatory signaling
were greatest when both the uterine donor tissue and the
recipient host mice were heterozygous for REA.
Of note, our studies in primary human endometrial
stromal cells in which REA levels were experimentally re-
duced revealed that REA normally restrains proliferation
so that its depletion resulted in elevated proliferative ac-
tivity and enhanced expression of cell cycle regulators.
Furthermore, in clinical specimens, REA was found to be
significantly lower in human endometriotic lesions from
patients compared with normal endometrium. The find-
REA-Endometriosis samples
REA-Normal endometrium
50μm
REAposivecells/cm2
Normal
endometrium
Endometriosis
samples
P = 0.0077
0
4000
3000
2000
1000
BA
Control IgG
CG CG CG
CG
SS
S
S
S
S
EG EG EG
Figure 7. REA protein by IHC in clinical human endometriosis samples and in human eutopic normal endometrium. Formalin-fixed and paraffin-
embedded sections of endometriosis tissue or normal eutopic endometrium were examined for REA protein by IHC using REA antibody or IgG. A,
Representative images are shown for REA staining in endometriosis samples from 3 patients and normal eutopic endometrium from 2 women not
affected by endometriosis. CG, cystic gland; EG, endometrial gland; S, stroma. B, Quantitation of REA in tissue samples. P ϭ .0077 by unpaired t
test.

ings with clinical samples support observations made in
our preclinical mouse endometriosis model and suggest a
critical role for REA in the pathologic progression of
endometriosis.
Previous studies have documented that REA represses
ER signaling (21) and exerts modulatory roles on path-
ways controlling cell survival and metabolism consistent
with its name also as prohibitin 2 (18, 19, 37). REA has
been established as a key ER corepressor in the mammary
gland and female reproductive tract, as well as in breast
cancer cells (20–23). In the current study, we have high-
lighted the pleiotropic ability of REA to suppress lesion
progression by modulating multiple aspects of estrogen-
mediated signaling in endometriosis. The findings provide
evidence that this coregulator acts as a restraint on ER
activities, repressing ER signaling that contributes to the
pathologic molecular milieu in endometriotic lesions.
Some proteins that function as coactivators of ER have
also been shown to impact endometriosis. For example,
thecoactivatorsteroidreceptorcoactivator-1iscleavedby
TNF␣-activated matrix metallopeptidase 9 into a cyto-
plasmic 70-kDa shortened isoform, which notably pre-
vents TNF␣-mediated apoptosis in ectopic endometriotic
cells (38). Also of interest, endometrial deficiency of the
transcription factor Krüppel-like factor 9, which acts as a
regulator of ER␣ signaling, promoted endometriotic le-
sion establishment and affected notch-, hedgehog-, and
steroid receptor-regulated pathways (39). Thus, both co-
activators and corepressors appear to regulate key aspects
in the pathogenesis of endometriosis.
Endometriosis is associated with chronic inflammation
(1–4) and in recent results from murine models, Burns et
al reported that compared with WT lesions transplanted
into WT hosts, WT lesions were proliferative in ER␣KO
recipient mice but showed decreased inflammatory re-
sponses upon E2 treatment (14). Consistent with this, the
novel ER ligand, oxabicycloheptene sulfonate, with pref-
erential affinity for ER␣, lost its suppressive effects in WT
ectopic lesion-associated inflammatory responses in
ER␣KO recipients (17) or in WT recipient mice depleted
of macrophages with clodronate liposomes (17), suggest-
ing critical roles of ER␣ and host myeloid responses.
REA(+/+)
REA(+/+)
ERE2
Endometriotic
Cells
Infiltrating
Host Cells
Proliferation
Vascularization
Neurogenesis
Inflammation
Disease
Progression
REA(+/-)
REA(+/-)
ERE2
Endometriotic
Cells
Infiltrating
Host Cells
Proliferation
Vascularization
Neurogenesis
Inflammation
Enhanced
Disease
Progression
Figure 8. Model depicting the cross talk and interrelationships between cells of the endometriotic lesion and host tissues, and the impact of REA.
Our findings suggest that the growth and inflammatory signals that contribute to endometriotic lesion progression originate principally from
distinct tissue loci, with stimulatory inputs from the ectopic uterine tissue primarily responsible for control of lesion proliferation and
vascularization, and host cells and tissues primarily responsible for control of inflammation and neurogenesis in lesions. These influences were
queried using different combinations of donor tissue and host background that were either WT (full complement of REA) or heterozygous
(expressing reduced REA levels). Further, donor and recipient tissues impact each other such that lesion growth and inflammatory signaling were
greatest when both the uterine donor tissue and the recipient host mice were heterozygous for REA.

In the current work, we have used an immune-intact
syngeneic murine model, in which the impacts of donor
and host REA could be clearly distinguished and com-
pared. We found that E2-supported chronic inflammatory
responses in intact animals that mimic clinical findings.
These include cytokine production, nuclear factor kappa
B activation, Cox2 expression and immune cell infiltra-
tion, all of which were elevated upon partial loss of host
but not donor REA. By contrast, donor uterine tissue REA
level was most important in the control of ectopic lesion
proliferation and vascularization. These findings under-
line the cellular and functional complexity of endometri-
osis lesions and support separate contributions of the do-
nor tissue and the host environment in the proliferative
and inflammatory aspects of endometriosis driven by the
estrogen-ER axis.
Lesion innervation is thought to be involved in endo-
metriosis-associated pain (40–42), and we showed pre-
viously that treatment with dual antiestrogenic and anti-
inflammatory compounds suppressed the innervation of
murine endometriotic-like lesions (17). Interestingly, neu-
roangiogenesis, a critical process driving the disease, has
been shown to be regulated by E2 signaling by Greaves et
al (43). Moreover, E2 is not only able to stimulate mac-
rophage infiltration into ectopic lesions but also to acti-
vate interactions of macrophages and nerves, and thus
may exacerbate endometriosis-associated pain (44). The
current findings of reduced REA increasing lesion inflam-
matory signaling and nerve innervation suggest that by
impacting E2-ER signaling, host-derived REA might serve
as a potential regulator of pain in endometriosis.
Current medical management of endometriosis pa-
tients, which is primarily focused on suppressing E2 pro-
duction (2), has not proven to be fully satisfactory. ERs,
which are known to be essential and dysregulated in the
pathogenesis of endometriosis, and their coregulators
emerge as promising therapeutic targets. For example, our
novel ER ligands, oxabicycloheptene sulfonate and
chloroindazole, displayed dual suppression of estrogenic
and inflammatory activities and were effective in prevent-
ing the establishment and progression of endometriotic
lesions in mice (17). The selective ER modulators, baze-
doxifene (15) and ERB-041 (16), have also been shown to
suppress endometriotic lesion growth. Because REA/pro-
hibitin2suppressestheproliferationofhumanendometri-
otic stromal cells and endometriosis-like lesion progres-
sion in the preclinical mouse model, and is reduced in
human endometriotic tissue compared with its level in
normal human endometrium, it appears that maintenance
of adequate levels of REA may be important in preventing
the development of this disease. The clinical relevance of
our study is also highlighted by our observation that neu-
ron innervation, which may be involved in the chronic
pelvic pain of endometriosis (40–42), was also suppressed
by host REA. Therefore, our findings provide new insights
into critical roles of coregulators in endometriosis, and
imply that novel therapeutic approaches based on modu-
lation of such coregulators might hold future potential for
improving medical care of women with this challenging
disease.
Acknowledgments
Address all correspondence and requests for reprints to: Dr
Benita S. Katzenellenbogen, Department of Molecular and In-
tegrative Physiology, University of Illinois at Urbana-Cham-
paign, Urbana, IL 61801. E-mail: katzenel@illinois.edu.
This work was supported by the National Institutes of Health
(NIH) Grant U54 HD055787 as part of the Eunice Kennedy
Shriver National Institute of Child Health and Human Devel-
opment/NIH Centers Program in Reproduction and Infertility
Research (to B.S.K., M.K.B., and R.N.T.) and by the NIH Grant
PHS 5R01DK015556 (to J.A.K.).
Disclosure Summary: The authors have nothing to disclose.
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