Steroids 64 (1999) 648 653

Progesterone and transforming growth factor- coordinately regulate

suppression of endometrial matrix metalloproteinases in a model of
experimental endometriosis
Kaylon L. Brunera, Esther Eisenberga, Fred Gorsteinb, Kevin G. Osteena,*
a
Center for Reproductive Medicine Research, Dept. of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, TN, USA
b
Dept. of Pathology, Jefferson Medical College, Philadelphia, PA, USA

Abstract

Endometriosis is a benign, though aggressive, disease of the female reproductive tract that consists of endometrial stromal and epithelial
cells growing at an extrauterine site. Although it is widely accepted that the majority of cases of endometriosis result from the ectopic
implantation of refluxed menstrual tissue, the precise mechanisms by which this disease becomes established are not well understood. Matrix
metalloproteinases (MMPs), enzymes which are important for extracellular matrix turnover, have recently been implicated in the
development of endometriosis. MMPs appear to be overexpressed in endometriotic lesions, but expression levels decrease following
successful medical therapy. Intriguingly, although transforming growth factor- (TGF-) mediates progesterone suppression of specific
endometrial MMPs, this growth factor is overexpressed in women with endometriosis. In the current study, we used an established
experimental model of endometriosis to explore MMP regulation by TGF-. Our findings indicate that blocking the action of TGF-
opposes progesterone-mediated suppression of MMPs and blocks the ability of this steroid to prevent experimental endometriosis. However,
we also show that the action of TGF- does not lead to a sustained suppression of MMPs as observed following progesterone treatment.
Taken together, our data suggest that in the absence of a normal progesterone response, common in ectopic lesions of endometriosis,
sensitivity to TGF- may be altered, resulting in a failure to regulate MMPs. 1999 Elsevier Science Inc. All rights reserved.

Keywords: Ovarian steroids; Progesterone; Endometriosis; TGF-; MMPs

1. Introduction
The growth and function of human endometrium is inti-
mately associated with fluctuating levels of ovarian steroids.
Under the influence of estradiol, this dynamic tissue is
rapidly renewed following the desquamation which occurs
during menstruation. The post-ovulatory rise in ovarian
progesterone opposes endometrial proliferation and pro-
motes cellular differentiation in preparation for implanta-
tion. In the absence of nidation, steroid hormone levels fall
and menstruation ensues, marking the beginning of a new
cycle. Coordinate with the extensive endometrial remodel-
ing that occurs during the proliferative and menstrual phases
is the expression of matrix metalloproteinases (MMPs),
which mediate extracellular matrix (ECM) turnover [1].
Progesterone effectively suppresses endometrial MMPs of
* Corresponding author. Vanderbilt University School of Medicine,
Dept. of Obstetrics & Gynecology, B-1100 Medical Center North, 1161
21st Ave South, Nashville, TN 37232.
the stromelysin class both in vivo and in vitro [25]. Pro-
gesterone-mediated MMP regulation is associated with an
increase in endometrial expression of transforming growth
factor- (TGF-) [6 8], which plays critical roles in estab-
lishment of pregnancy [6,9]. Outside the reproductive tract,
TGF- acts to suppress MMP-3 in rat fibroblasts [10] and
MMP-7 in mesangial cells of the human kidney [11].
TGF- also induces expression of tissue inhibitors of
MMPs (TIMPs) in numerous tissues [11] and TIMP-1
mRNA correlates with rising levels of TGF- in the human
endometrium [3,6,7].
Maintaining the delicate balance between MMPs and
their inhibitors is critical to prevent excessive matrix de-
struction or deposition; thus, expression of these enzymes is
very tightly regulated in normal tissues. Inappropriate ex-
pression of both MMPs and TIMPs has been implicated in
numerous metastatic disease states [12] as well as the be-
nign disease endometriosis [13,14]. Endometriosis is an
invasive disease requiring MMP expression for establish-
ment of endometrial glands and stroma at ectopic sites.

0039-128X/99/$ see front matter 1999 Elsevier Science Inc. All rights reserved.
PII: S 0 0 3 9 - 1 2 8 X ( 9 9 ) 0 0 0 4 8 - 3
 K.L. Bruner et al. / Steroids 64 (1999) 648 653
Whether the ability of TGF- to regulate MMP and TIMP
expression plays an important role in preventing the exper-
imental disease is not known. However, induction of
TIMP-1 expression appears to be a component of the suc-
cessful treatment of women with endometriosis [15]. Using
a nude mouse model of endometriosis, we have demon-
strated a requirement of MMPs in the establishment of
human endometrial tissue as ectopic lesions [16]. The de-
velopment of experimental endometriosis in nude mice can
be prevented by progesterone, which suppresses MMP ex-
pression, or by TIMP-1, which inhibits MMP activity.
In this report, we examine the interactive role of proges-
terone and TGF- in the regulation of MMP-3 and MMP-7
in human endometrium. Additionally, we determined the
relative abilities of progesterone and TGF- to prevent the
development of endometriotic-like lesions in a nude mouse
model of the disease. Our results indicate that, in our model
of endometriosis, progesterone and TGF- must act in con-
cert to suppress MMPs and prevent ectopic lesion establish-
ment.
2. Experimental
2.1. Acquisition of human tissue
Samples of endometrial tissue were obtained during the
proliferative phase (Days 9 12) of the menstrual cycle from
a donor population of women (ages 21 45) exhibiting reg-
ular menstrual cycles. An endometrial thickness of greater
than 9 mm was confirmed by vaginal ultrasound prior to
each biopsy, and a serum progesterone level of less than 1.0
ng/ml was necessary for inclusion in this study. Biopsies
were obtained from the uterine fundus with a Pipelle
endometrial suction curette (Unimar, Inc.; Wilton, CT,
USA) and immediately washed in prewarmed (37C) phe-
nol red-free Dulbeccos modified Eagles medium
(DMEM)/Hams F-12 Medium (DME/F-12) (Sigma Chem-
ical; St. Louis, MO, USA) to remove blood and mucous.
The use of human tissues was approved by Vanderbilt
Universitys Institutional Review Board and Committee for
the Protection of Human Subjects.
2.2. Organ cultures of endometrium
Endometrial tissues were sectioned into 2 mm3 frag-
ments with the aid of a dissecting microscope. Tissue
fragments were equally divided into groups required for
experimental treatments and suspended as organ cultures
within tissue culture inserts (Millipore; Bedford, MA,
USA) near the air:media interface [16]. Each culture was
maintained under serum-free conditions in DME/F-12
medium supplemented with 1% ITS (Collaborative Bio-
medical; Bedford, MA, USA) and 0.1% Excyte (Miles
Scientific; Kankakee, IL, USA) and incubated at 37C in
a humidified chamber with 95% air:5% CO2. Steroid
649
treatments of organ cultures included estradiol alone
(108 M) or estradiol plus progesterone (E 109 M;
P 5 107 M). Additionally, some cultures received
2 ng/ml TGF-1 or 10 g/ml pan-specific TGF- poly-
clonal antibody (both obtained from R & D Systems,
Minneapolis, MN, USA). The pan-specific antibody was
demonstrated by R & D Systems to neutralize TGF-1,
TGF-1.2, TGF-2, TGF-3, and TGF-5 but does not
crossreact with any other cytokine tested.
2.3. Western analysis of MMP secretion in
endometrial cultures
Western analysis was performed using standard meth-
odology [17,18]. Briefly, secreted proteins were concen-
trated using Centricon-10 filters (Amicon, Beverly, MA,
USA), resuspended in TE Buffer (10 mM Tris-HCl, pH
7.4, 0.1 mM EDTA) and concentration determine using
the Bradford method (Bradford, 1976; Coomassie Plus;
Pierce, Rockford, IL). Proteins, 20 g/lane were sepa-
rated by SDS-PAGE using a 10% polyacrylamide gel and
transferred to a PVDF membrane (Immobilon-P; Milli-
pore). Blots were blocked in 1 PBS/10% non-fat milk/
0.05% Tween-20, incubated overnight at 4C with pri-
mary antibody followed by three washes in 1 PBS/
0.05% Tween-20. Blots were then incubated one hour at
room temperature with peroxidase-conjugated secondary
antibody, washed as before, and immunoreactive bands
visualized using chemiluminescent (Amersham Life Sci-
ences, Arlington Heights, IL, USA). As a negative con-
trol, identical blots were not incubated in primary anti-
body, but remained in blocking solution overnight prior
to incubation with secondary antibody.
MMP-3 antibody (Oncogene Research Products, Cam-
bridge, MA, USA) is a mouse monoclonal antibody ob-
tained by immunizing mice with pro-MMP-3 purified from
rheumatoid synovial fibroblast conditioned media. It recog-
nizes both the precursor (59/56 kDa) and active (45/28 kDa)
forms of MMP-3. Lyophilized antibody was reconstituted in
PBS to a concentration of 100 g/ml and diluted 1:2000 in
1 PBS/5% milk/0.05% Tween 20. Antibody to MMP-7
was a rat monoclonal obtained using full-length human
MMP-7 and recognizes both the active (19 kDa) and pre-
cursor (29 kDa) forms of MMP-7. For western analysis,
antibody was diluted 1:5000 in 1 PBS/5% milk/0.05%
Tween 20. Secondary antibodies for MMP-3 and MMP-7
were a sheep anti-mouse (Amersham) and a goat anti-rat
(Jackson Immuno Research Laboratories), respectively.
Both were diluted in 1 PBS/5% milk/0.05% Tween-20 to
concentrations equal that of the primary antibody.
2.4. Experimental model of endometriosis
Nude mouse experiments were performed as previ-
ously described [16]. Briefly, 5-week-old athymic (nude),
ovariectomized mice were purchased from Taconic Lab-
650 K.L. Bruner et al. / Steroids 64 (1999) 648 653
Fig. 1. Western analysis of expression and regulation of MMP-3 (A) and MMP-7 (B) in proliferative endometrial tissue maintained in organ cultures for 48
h. Tissues were treated with 108 M estradiol (lane 1), 109 M estradiol plus 5 107 M progesterone (lane 2) 109 M estradiol 5 107 M progesterone
plus 10 ng/ml of a pan-specific antibody to TGF- (lane 3) or 109 M estradiol plus 2 ng/ml TGF-1 (lane 4).
oratory Animals (Germantown, NY, USA) and housed on
site in sterilized cages and bedding. The animal room was
maintained at 26.7C with a 12-h light/12-h dark cycle
and mice were provided with sterile Purina Rodent
Chow and water ad libitum. Metafane (Pittman-
Moore, Toledo, OH, USA) was used to anesthetize ani-
mals prior to invasive procedures, which were performed
in a laminar flow hood using sterile instruments. Mice
were randomly divided into groups and sterile 60-day
release capsules containing 1.5 mg 17 estradiol (Inno-
vative Research of America; Sarasota, FL) were inserted
subcutaneously (s.c.) at a site just below the scapula 24 h
prior to tissue injection. Human endometrial tissue, main-
tained for 24 h in culture as described above, was washed
briefly in sterile PBS before injection into recipient nude
mice. Each animal received a single intraperitoneal (i.p.)
injection of 8 10 endometrial tissue fragments in 200 l
sterile PBS using tuberculin syringes and 18-gauge nee-
dles. Some mice received 0.2 g TIMP-1 in 200 l PBS
by i.p. injection at 0, 6, 12, and 18 h after tissue injec-
tions. TIMP-1 was prepared for use in animals by Syn-
ergen, Inc (Boulder, CO, USA) and was a generous gift
from Dr George Stricklin. Ten to twelve days after re-
ceiving human tissue, mice were sacrificed and necrop-
sied for signs of endometriotic-like lesions.
3. Results
We previously demonstrated that TGF- acts in concert
with progesterone to suppress MMP-7 in co-culture exper-
iments of human endometrial epithelial and stromal cells
[7]. To explore the role of TGF- in regulating the expres-
sion of both MMP-7 and MMP-3, we established organ
cultures of proliferative phase human endometrium. As
shown in Fig. 1, endometrial organ cultures treated 48 h
with estradiol express both MMP-3 and MMP-7 (Fig. 1, A
and B, lane 1). Treatment of similar tissues with estradiol
and progesterone results in a suppression of MMP-3 and
MMP-7 (Fig. 1A and B, lane 2) although a pan-specific
antibody to TGF- completely abolishes the suppressive
effect of progesterone on either enzyme (Fig. 1A and B,
lane 3). Similar to results obtained with progesterone treat-
ment, suppression of MMP-3 and MMP-7 expression is
observed following treatment with estradiol plus TGF-
(Fig. 1A and B, lane 4). These results support an important
role for TGF- in mediating progesterone-associated MMP
suppression. Additionally, the results described above sug-
gest TGF- action may be an important component in the
prevention of experimental disease.
To address the role of TGF- in the establishment of
ectopic human endometrial lesions in nude mice, we have
compared the relative ability of progesterone versus
TGF- to prevent lesion development. However, as
shown in Table 1, mice implanted with human endome-
trial tissue treated with estradiol alone or with estradiol
plus TGF- were equally likely to develop disease (91.7
vs. 92.3, respectively). These results demonstrate that
TGF- treatment cannot duplicate the ability of proges-
terone to prevent experimental endometriosis despite the
role of this growth factor in regulating MMPs (Fig. 1). To
further investigate the role of TGF- in establishment of
the experimental disease, human tissues were treated
with estradiol and progesterone with or without a pan-
specific antibody to TGF- prior to injection into nude
mice. As shown in Table 1, only one mouse receiving
human tissue treated with estradiol and progesterone de-
veloped lesions, although 91.7% of animals receiving
similar tissue additionally treated with anti-TGF- anti-
body developed lesions. These results indicate that
TGF- is required for progesterone action in the preven-
tion of experimental endometriosis. Together, the above
studies suggest that progesterone-priming may be neces-
sary for appropriate endometrial response to TGF-. A
requirement of both progesterone and TGF- to prevent
development of human lesions in nude mice is supported
by the lack of lesion development in mice receiving
tissues treated with estradiol, progesterone and TGF-
(data not shown).
Table 1
Effects of TGF- on the establishment of human tissue as lesions in
nude mice
Tissue Animal Number Total Lesions per Percent
treatment treatment w/lesions number diseased animal w/lesions
E E 11 12 2.8 91.7
E TGF- E 12 13 4.7 92.3
EP E 1 13 1.0 7.6
EP TGF- E 11 12 3.2 91.7
 K.L. Bruner et al. / Steroids 64 (1999) 648 653
Fig. 2. Western analysis of MMP-3 (A) and MMP-7 (B) expression in proliferative endometrial tissue maintained in organ cultures. Tissues were treated for
24 h in the presence of 108 M estradiol (lane 1), 109 M estradiol plus 5 107 M progesterone (lane 2), 109 M estradiol plus 2 ng/ml TGF-1 (lane
3) or 109 M estradiol, 5 107 M progesterone and 2 ng/ml TGF-1 (lane 4) followed by 24 h in the absence of steroids or growth factor.
To explore why TGF- treatment fails to prevent
development of endometriotic-like lesions, we examined
endometrial MMP regulation in the absence of proges-
terone. To mimic the manner in which human endome-
trial tissues are treated prior to injection into mice, tissue
fragments were cultured 24 h in experimental medium,
rinsed in PBS and subsequently cultured an additional
24 h in the absence of either steroids or growth factors.
Endometrial organ cultures treated initially with estradiol
maintained expression of MMP-3 and MMP-7 in the
absence of continued steroid treatment (Fig. 2AB, lane
1). Organ cultures treated with estradiol plus progester-
one for 24 h and then transferred to medium containing
no steroids secrete neither MMP-3 or MMP-7 protein
over the next 24 h (Fig. 2A and B, lane 2). However,
tissues treated with estradiol and TGF- for the initial
24-h period, followed by 24 h in steroid and growth
factor-free medium, expressed both MMP-3 and MMP-7
at levels equal to or greater than tissues treated with
estradiol alone (Fig. 2A and B, lane 3). The addition of
TGF- to cultures treated with progesterone did not alter
MMP suppression, even following 24 h without steroids
or growth factor (Fig. 2A and B, lane 4), indicating that
TGF- does not interfere with progesterone regulation of
MMPs.
The results described above suggest that the failure of
TGF- treatment to prevent development of human tissue
lesions in nude mice may be due to a resumption of MMP
secretion. To indirectly address this possibility, a second set
of in vivo experiments was conducted in which mice re-
ceiving tissues treated with estradiol and TGF- were also
given a series of i.p. injections of TIMP-1, an inhibitor of
MMP activity. The majority of mice receiving human tis-
sues treated with estradiol plus TGF- (Table 1) developed
lesions. However, development of the experimental disease
was dramatically reduced in mice receiving tissue fragments
Table 2
Effect of TIMP-1 on TGF- induction of experimental endometriosis
Tissue Animal Number Total Lesions per Percent
treatment treatment w/lesions number diseased animal w/lesions
E E 8 8 2.2 100.0
E TGF- E TIMP 2 8 2.1 25.0
651
treated identically, but also given TIMP-1 injections (Table
2). Taken together, our results support a cooperative effect
of TGF- and progesterone for regulation of endometrial
MMP expression. Additionally, this coordinate action of
progesterone and TGF- appears critical to the prevention
of experimental endometriosis.
4. Discussion
Although it is generally accepted that endometriosis
largely develops as a consequence of ectopic implantation
of refluxed menstrual tissue, the mechanisms by which this
process occurs are unknown. Recently, a number of studies
have provided circumstantial evidence suggesting a role for
MMPs in the pathophysiology of endometriosis [1315,19].
Using the nude mouse as an experimental model, we ob-
tained more direct evidence linking MMP expression and
action to the development of an endometriotic-like disease.
Our studies have shown that progesterone-mediated sup-
pression of MMPs in organ cultures of human endometrium
prevents the subsequent establishment of ectopic lesions by
human tissue in nude mice [16]. Although TGF- can sup-
press MMP expression in a wide variety of tissues [7,10,11]
women with endometriosis show an increased expression of
peritoneal TGF- [20], which may promote the develop-
ment of adhesions [21]. The current study was designed to
investigate the apparent paradoxical relationship between
progesterone and TGF- in the regulation of endometrial
MMPs in a model of endometriosis.
Our results suggest that suppression of endometrial
MMPs requires the action of both progesterone and locally
produced TGF-. Treatments of human tissue with anti-
TGF- antibody blocks the in vitro ability of progesterone
to suppress expression of either MMP-3 or MMP-7. How-
ever, exogenous treatment of human endometrial tissues
with TGF-, in the absence of progesterone, does not main-
tain MMP suppression in vitro, nor does TGF- treatments
of human tissue prevent development of endometriotic-like
lesions in nude mice. Importantly, treatment of human en-
dometrial tissue with anti-TGF- antibody blocks the ability
of progesterone to prevent establishment of human lesions
in this experimental model. These results imply that when
progesterone and TGF- act in concert, endometrial MMP
652 K.L. Bruner et al. / Steroids 64 (1999) 648 653
expression is suppressed and experimental endometriosis is
prevented. However, if this steroid-cytokine relationship is
absent or diminished, MMPs are not suppressed and ectopic
endometrial lesions may be established. Since the effects of
TGF- on cells can be greatly influenced by the local tissue
environment [22], it is possible that progesterone-associated
endometrial changes may alter cellular response to TGF-.
This possibility is supported by the observations of Zhang et
al. [23] who recently described a requirement of both pro-
gesterone and TGF- for induction of platelet-derived en-
dothelial cell growth factor by isolated human endometrial
epithelial cells. Unfortunately, adhesions and fibrosis in
ectopic sites of naturally occurring endometriosis is associ-
ated with diminished progesterone responsiveness [24], per-
haps explaining aberrant expression of both TGF- and
MMPs in this disease [1315,19,20].
In summary, we have described a critical interaction
between progesterone and TGF- for the normal regulation
of endometrial MMP expression, including preventing an
endometriotic-like disease in nude mice. These results are
important to understanding the pathophysiology of endome-
triosis since ectopic endometrial sites show a decrease in
progesterone sensitivity [24 26] coupled with increased
levels of i.p. TGF-1 [20]. Other processes, important to the
development of endometriosis, may also be affected by the
interactions of progesterone and TGF-. For example,
TGF- acts to stimulate both matrix deposition and angio-
genesis [27,28], which could ultimately enhance the devel-
opment and/or progression of endometriosis.
Acknowledgments
The authors wish to express gratitude to all the volun-
teers who provided endometrial biopsies for these studies.
We greatly appreciate the comments and suggestions of Dr
Gary Olson (Vanderbilt) during the preparation of this
manuscript.
This work was supported by grants from the National
Institutes of Health (HD-28128 and HD-30472; KGO) and
a Reproductive Biology, Biochemistry and Endocrinology
training grant (HD-07043; KLB).
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