Stromal PRs Mediate Induction of 17ß-Hydroxysteroid Dehydrogenase Type 2 Expression in Human Endometrial Epithelium: A Paracrine Mechanism for Inactivation Of E2
Sijun Yang,
Zongjuan Fang,
Bilgin Gurates,
Mitsutoshi Tamura,
Josephine Miller,
Karen Ferrer and
Serdar E. Bulun
Division of Reproductive Endocrinology, University of Illinois at
Chicago, Chicago, Illinois 60612
Address all correspondence and requests for reprints to: Serdar E. Bulun, M.D., University of Illinois at Chicago Medical Center, 820 South Wood Street, M/C 808, Chicago, Illinois 60612. E-mail:
bulun{at}uic.edu
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ABSTRACT
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Progesterone stimulates the expression of 17ß-hydroxysteroid
dehydrogenase (HSD) type 2, which catalyzes the conversion of the
potent estrogen, E2, to an inactive form, estrone, in epithelial cells
of human endometrial tissue. Various effects of progesterone on uterine
epithelium have recently been shown to be mediated by stromal PRs in
mice. We describe herein a critical paracrine mechanism whereby
progesterone induction of 17ß-HSD type 2 enzyme activity, transcript
levels, and promoter activity in human endometrial epithelial cells are
mediated primarily by PR in endometrial stromal cells. Medium
conditioned with progestin-pretreated human endometrial stromal cells
robustly increased 17ß-HSD type 2 enzyme activity (2-fold) and mRNA
levels (13.2-fold) in Ishikawa malignant endometrial epithelial cells.
In contrast, direct progestin treatment of Ishikawa epithelial cells
gave rise to much smaller increases in enzyme activity (1.2-fold) and
mRNA levels (4-fold). These results suggest that progesterone-
dependent paracrine factors arising from stromal cells are
primarily responsible for the induction of epithelial 17ß-HSD type 2
expression in the endometrium. We transfected serial deletion mutants
of the -1,244 bp 5'-flanking region of the 17ß-HSD type 2 gene into
Ishikawa cells. No progesterone response elements could be identified
upstream of the 17ß-HSD type 2 promoter. Stromal PR-dependent
induction of the 17ß-HSD type 2 promoter was mediated by a critical
regulatory region mapped to the -200/-100 bp sequence. Direct
treatment of Ishikawa cells with progestin gave rise to a maximal
increase in the activity of -200 bp/Luciferase construct only
by 1.2-fold, whereas medium conditioned by progestin-pretreated
endometrial stromal cells increased promoter activity up to 2.4-fold in
a time- and concentration-dependent manner. The stimulatory effect of
medium conditioned by progestin-pretreated stromal cells was enhanced
strikingly by increasing stromal cell PR levels with the addition of
estrogen. This epithelial-stromal interaction was specific for
endometrial epithelial cells, since 17ß-HSD type 2 could not be
induced in malignant breast epithelial cells by media conditioned with
progestin-treated breast or endometrial stromal cells. In conclusion,
progesterone regulates the conversion of biologically active E2 to
estrone by inducing the 17ß-HSD type 2 enzyme in human endometrial
epithelium primarily via PR in stromal cells, which secrete factors
that induce transcription mediated primarily by the -200/-100 bp
5'-regulatory region of the 17ß-HSD type 2 promoter.
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INTRODUCTION
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ESTROGEN STIMULATES the proliferation of
the inner lining of the uterus, named endometrium, and, at the same
time, prepares this tissue for the action of progesterone by inducing
PRs (1). Progesterone, on the other hand, causes the
differentiation of both endometrial stromal and epithelial cells and
limits cell proliferation. For example, progestins inhibit and even
reverse estrogen-induced growth, hyperplasia, or adenocarcinoma of the
endometrium. This progesterone effect on endometrial epithelial cells
was recently shown to be mediated by PRs in stromal cells using uterine
tissues of PR knockout (PRKO) mice (2). These findings
reinforced the concept of stromal cell-derived factors that act on
epithelial cells to counteract the effects of estrogens. One potential
mechanism is progesterone-induced decrease in the synthesis of ERs
(3). Another striking effect of progesterone on the
endometrium is to stimulate the expression of the enzyme
17ß-hydroxysteroid dehydrogenase (HSD) type 2 in epithelial cells
(4, 5, 6, 7).
17ß-HSD type 2 catalyzes the conversions of E2 to estrone (E1),
T to androstenedione, and 20
- hydroxyprogesterone to
progesterone in specific human tissues including the liver,
endometrium, breast, and placenta (8, 9, 10). The activity of
this enzyme in the liver possibly accounts for the global inactivation
of circulating E2 and T via conversion to estrone and androstenedione.
In an adult nonpregnant woman, endometrial epithelium represents
another specific site of 17ß-HSD type 2 expression (7, 10, 11, 12). In contrast to the liver, the major biological impact
of this enzyme in the endometrium may be at a local level by lowering
tissue E2 concentrations via converting E2 to estrone and by increasing
tissue progesterone levels via converting 20
-hydroxyprogesterone to
progesterone. These local functions of 17ß-HSD type 2 are especially
significant in view of the fact that endometrial growth and
differentiation are regulated primarily by E2 and progesterone
(8).
Extremely high levels of 17ß-HSD type 2 mRNA and protein have been
demonstrated in the glandular epithelial cells (but not stromal cells)
of the human endometrium during the secretory phase, suggesting that
progesterone stimulates the activity of this enzyme (7, 11, 12). This is in agreement with the pioneering studies of Tseng
and Gurpide (5, 6) on endometrial tissue E2 dehydrogenase
activity (oxidation of E2
E1), which was stimulated by progesterone.
The conversion of the potent estrogen E2 to a virtually inactive
steroid E1 during the secretory phase endometrium has been viewed as a
critical protective mechanism against estrogen-induced growth (6, 7). Subsequently, Andersson and co-workers (7, 8)
cloned and characterized a full-length cDNA, which encodes this enzyme.
The 5'-flanking region of the 17ß-HSD type 2 gene has been cloned by
Labrie and co-workers (13).
The cellular and molecular mechanisms responsible for the regulation of
17ß-HSD type 2 in endometrial epithelium by progesterone have
remained elusive to date for the following reasons: 1) Although
17ß-HSD type 2 enzyme activity, protein, and mRNA levels increase
strikingly in intact endometrial tissues in response to progesterone,
direct treatment of endometrial epithelial cells with a progestin gave
rise to only minimal to modest increases of these parameters (4, 6, 7). 2) The -1,245 bp 5'-flanking region of the 17ß-HSD
type 2 gene does not contain any classical progesterone response
elements (13). 3) The use of tissues from transgenic mice
with disrupted PR gene revealed that all studied effects of
progesterone on uterine epithelium were mediated by stromal cell PR in
a paracrine fashion.
In light of these preliminary data, we hypothesized that 17ß-HSD type
2 expression in human endometrial cells is regulated primarily by
stromal factors secreted in response to progesterone, and we uncovered
an epithelial-stromal interaction responsible for the regulation of
17ß-HSD type 2 in endometrial tissue. We used an endometrial stromal
cell line derived from normal tissue to represent stromal cells and
also well differentiated Ishikawa malignant endometrial epithelial cell
line in lieu of epithelial cells (14, 15). Both cells were
responsive to estrogen and progesterone, and Ishikawa cells but not
stromal cells expressed 17ß-HSD type 2. Our findings based on this
model were physiologically relevant, since they could be reproduced
using cultured primary stromal cells isolated from normal endometrium.
17ß-HSD type 2 expression showed minimal to modest increases in
response to direct treatment of Ishikawa endometrial epithelial cells
with a progestin. This minimal induction was possibly mediated by an
autocrine mechanism in response to secretory factors released from
Ishikawa cells in a progestin-dependent manner but not via binding of
ligand-activated PR to 17ß-HSD type 2 promoter. On the other hand,
conditioned medium (CM) from progestin-pretreated endometrial stromal
cells showed a striking induction of 17ß-HSD type 2 enzyme activity,
mRNA levels, and promoter activity. We report herein a paracrine
mechanism in human endometrium whereby stromal PR is primarily
responsible for the induction of an epithelial enzyme, which
inactivates E2.
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RESULTS
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Regulation of 17ß-HSD Oxidase (E2
E1) Activity in Ishikawa
Cells
We used the Ishikawa human endometrial cancer cell line in lieu of
epithelial cells, since these cells are of epithelial origin and
express ER
, PR, and 17ß-HSD type 2 (15, 16) (also see
Figs. 13

). On the other hand, a benign human decidual fibroblast
(HuDF) line was used as the stromal cell component, since these cells
represent differentiated endometrial stromal cells and respond to
progesterone via PR (14).

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Figure 1. 17ß-HSD Oxidase (E2 E1) Activity in Ishikawa
Cells
The conversion rate of radiolabeled E2 to E1 was shown in Ishikawa
epithelial cells. The results are reported as percentages of the enzyme
activity in untreated cells. Direct treatment with PR agonist R5020
(10-7 M) gave rise to a minimal increase
(20%), whereas medium conditioned with R5020-treated HuDF endometrial
stromal cells doubled the baseline value. This stimulatory effect of CM
could be reversed, in part, by cotreatment of HuDF with ZK98299.
Statistically significant differences (P < 0.01)
between treatments are indicated with asterisks. R5020
and/or ZK98299 were routinely removed from CM before its use to treat
Ishikawa cells.
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Figure 2. 17ß-HSD Type 2 mRNA Levels in Ishikawa Cells
17ß-HSD type 2 mRNA levels were determined by RPA. Arbitrary units
were calculated as the ratio of the densitometric value of 17ß-HSD
type 2 to that of the housekeeping gene GAPDH. Direct treatment
Ishikawa cells with R5020 increased 17ß-HSD type 2 mRNA level by
4-fold, whereas medium conditioned (CM) with R5020-treated HuDF stromal
cell line gave rise to a 13.2-fold increase. This effect of CM could be
potentiated by cotreatment of HuDF with E2 and inhibited by cotreatment
of HuDF with progesterone antagonist ZK98299. Luteal phase endometrial
tissue was used as a positive control, whereas an RNA sample from
primary human breast adipose fibroblasts served as a negative control.
R5020, E2, and/or ZK98299 were routinely removed from CM before its
addition to treat Ishikawa cells.
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Figure 3. 17ß-HSD Type 2 Promoter Activity in Ishikawa
Cells
Expression of deletion mutants of 17ß-HSD type 2 promoter fused to
Luciferase reporter gene revealed that a critical region within the
-200/-100 bp sequence is responsible for baseline activity and
stromal PR-mediated (CM from HuDF) induction. Direct treatments of
Ishikawa cells with R5020 gave rise to only minimal inductions (1.1- to
1.3-fold), whereas CM from R5020-treated HuDF stromal cells produced 2-
to 2.4-fold inductions. Differences between direct R5020 (dotted
columns) and CM from R5020-treated HuDF (gray
columns) were statistically significant for -200, -300,
-500, -750, and -1,244 bp constructs. The addition of E2 to R5020
during preparation of CM from HuDF potentiated the induction by
increasing PR in the HuDF stromal cell line. R5020 and E2 were removed
before the incubation of Ishikawa cells with CM. Results were expressed
as multiples of an arbitrary unit of 1 assigned to the pGL3.0 Basic
(empty) vector.
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We initially determined the 17ß-HSD oxidase (E2
E1) and
ketoreductase (E1
E2) activities in Ishikawa cells in the presence or
absence of progesterone agonist R5020 and HuDF-CM (Fig. 1
). None of these treatments altered the
ketoreductase (E1
E2) activity (data not shown). On the other hand,
oxidase activity (E2
E1) is slightly increased in response to R5020.
A robust increase in Ishikawa cell oxidase activity, however, was
observed in the presence of CM from HuDF stromal cells treated with
R5020. This effect could be blocked by the progesterone antagonist
ZK98299. Before the addition of CM from the R5020 and
ZK98299-pretreated HuDF line, all steroids were removed from the CM by
charcoal stripping as a control measure (Fig. 1
). We interpreted these
findings as progesterone induction of epithelial oxidase (E2
E1)
activity via stromal PR by way of a paracrine interaction.
Regulation of 17ß-HSD Type 2 mRNA Levels in Ishikawa Cells
We sought to determine whether stromal PR-mediated induction of
oxidase (E2
E1) activity in Ishikawa cells is accompanied by parallel
changes in the 17ß-HSD type 2 mRNA levels (Fig. 2
). Direct treatment of Ishikawa cells
with R5020 increased 17ß-HSD type 2 mRNA level by 4-fold, whereas CM
from R5020- pretreated HuDF gave rise to a 13.2-fold increase.
(R5020 and other steroids were routinely removed from CM before its
use.) This stimulatory effect of R5020 was potentiated by the addition
of E2, which induces PR expression in both cell types (Fig. 2
).
Treatment with E2 by itself did not induce 17ß-HSD type 2 levels
significantly. Addition of the progesterone antagonist ZK98299 to R5020
treatment during the generation of CM from stromal cells eliminated the
stimulatory effect of CM on 17ß-HSD type 2 mRNA levels in Ishikawa
cells (Fig. 2
). Changes in 17ß-HSD type 2 mRNA levels in response to
these treatments were comparable to those in 17ß-HSD type 2 enzyme
activity (E2
E1) in Ishikawa cells and supported the notion that
progesterone-dependent gene regulation in endometrial epithelium is
mediated primarily by PR in stromal cells via a paracrine
mechanism.
Regulation of 17ß-HSD Type 2 Promoter Activity in Ishikawa
Cells
Next, we determined the regulation of the 17ß-HSD type 2 gene
promoter activity in Ishikawa cells. We initially characterized the
regions that confer maximum baseline activity and stromal PR-mediated
induction of promoter activity. We transferred seven serial deletion
mutants of the 17ß-HSD type 2 gene promoter fused to Luciferase
reporter gene (Fig. 3
). The maximum level
of baseline activity was observed with the use of the -200 bp
Luciferase construct (Fig. 3
). In particular, the cis-acting
elements within the -200/-100 bp region were critical, since baseline
activity of the -200 bp construct was 3 times that of the -100 bp
construct. Stromal PR-mediated induction (by CM from R5020-pretreated
HuDF) increased from 1.2-fold in the -100 bp construct to 2.4-fold in
the -200 bp construct, indicating that stromal PR-mediated induction
is also mediated by regulatory elements within the -200/-100 bp
region. This stimulatory effect of CM from R5020-pretreated stromal
cells (HuDF) could be potentiated by the addition of E2 via increasing
stromal cell PR levels (Fig. 3
). Finally, direct treatments of Ishikawa
cells with R5020 gave rise to relatively small increases of promoter
activity (1.1- to 1.3-fold), whereas CM from R5020-pretreated stromal
cells (HuDF) caused significantly higher inductions for each construct
up to 2.4-fold (Fig. 3
). The differences between treatments with R5020
(direct) and CM from R5020-pretreated HuDF were statistically
significant (largest, P < 0.05) for each construct
except for the -65 bp and -100 bp constructs that contained minimal
portions of the promoter regulatory region. These findings were
consistent with the alterations observed in enzyme activity and mRNA
levels in response to similar treatments (Figs. 1
and 2
).
Figure 4
depicts the sequence of the
5'-regulatory region of 17ß-HSD type 2 promoter. We identified five
potential regulatory elements that may confer stromal PR-mediated
activation of this promoter using the computer-assisted program
TFSEARCH on the internet
(http://molsun1.cbrc.aist.go.jp/research/db/ TFSEARCH.html). These
include binding sites (with at least 85% homology) for heat shock
factor (HSF), activated protein-1 (AP-1), the product of the homebox
gene CdxA, and the product of the sex-determining region Y (SRY) gene.
Additionally, an alcohol dehydrogenase regulatory protein-1 (ADR1)
binding site was identified within the first -100 bp region (Fig. 4
).
A computer-assisted analysis of the entire -1,244 bp flanking region
did not show any palindromic repeats that may represent a steroid
receptor binding site (Fig. 4).

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Figure 4. 5'-Flanking DNA Upstream of the 17ß-HSD Type 2
Gene Promoter (13 )
We identified a number of potential cis-acting elements
in the -200/-100 bp region, which is critical for stromal PR-mediated
induction. These motifs were identified using the TFSEARCH database on
the internet and showed at least 85% homology to originally described
consensus sequences. This program did not identify any PREs even at the
search level of 50% homology.
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Regulation of the -200 bp 5'-Flanking Region of 17ß-HSD Type 2
Promoter in Endometrial Epithelium via Stromal PR
We used the Luciferase construct with the highest levels of
baseline activity and stromal PR-mediated induction (-200/-1 bp) in
the following experiments. Treatment of Ishikawa endometrial epithelial
cells directly with progesterone agonist R5020, E2, forskolin, phorbol
diacetate, dexamethasone, or serum either did not alter the activity of
the -200/-1 bp construct significantly or caused a minimal
stimulation (1.2-fold induction by direct treatment with R5020) (Fig. 5
, part of negative data not shown).

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Figure 5. The Effects of a Number of Treatments on the
17ß-HSD Type 2 Promoter Construct (-200/-1 bp) in Ishikawa Cells
Direct treatment of Ishikawa cells with R5020 increased promoter
activity by 1.2-fold, whereas CM from R5020-treated HuDF stromal cells
gave rise to a 2.4-fold induction that could be blocked by the
progesterone antagonist ZK98299. This stromal PR-mediated effect could
be potentiated by the addition of E2, which increased progesterone
levels in HuDF. Asterisks show statistically significant
differences (P < 0.001) between incubation
conditions. R5020, E2, and/or ZK98299 were removed from CM before its
addition to cultured Ishikawa cells.
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Incubation of Ishikawa cells with medium conditioned with
R5020-pretreated HuDF (endometrial stromal cell line), however, gave
rise to a 2.4-fold induction compared with untreated cells (Fig. 5
).
This induction by CM from R5020-pretreated HuDF stromal cells was
significantly higher than the induction by CM from untreated HuDF. CM
from E2 plus R5020-pretreated HuDF stimulated the promoter activity to
a maximum level (4-fold). This we interpreted as E2-stimulated increase
of PR in HuDF, to enhance this paracrine effect, because all steroids
were routinely removed from CM before treatments of Ishikawa cells, and
direct treatment with only E2 did not stimulate promoter activity. This
stromal PR-mediated induction could be reversed by the progesterone
antagonist ZK98299 used in 100 molar excess (Fig. 5
). Again, the
changes in 17ß-HSD type 2 promoter activity in response to these
treatments were comparable to those in levels of enzyme activity and
mRNA in Ishikawa cells (Figs. 1
and 2
).
Dose- and Time Dependency of the Induction of Epithelial 17ß-HSD
Type 2 Promoter Activity by Stromal Factors
Figure 6A
shows that HuDF
(endometrial stromal cell)-derived factors induce the activity of
17ß-HSD type 2 promoter (-200/-1 bp Luciferase construct) in a
dose-dependent fashion. Confluent HuDF cell line maintained in
serum-free RPMI 1640 had been treated for 48 h by E2
(10-8 M) plus R5020
(10-7 M). E2 and R5020 were
subsequently removed from conditioned RPMI 1640. HuDF-conditioned RPMI
1640 was diluted serially by nonconditioned RPMI 1640 to obtain media
containing 20%, 40%, 60%, and 80% CM. These gave rise to a
dose-dependent stimulation of 17ß-HSD type 2 promoter activity in
Ishikawa cells (Fig. 6A
). A time course experiment (Fig. 6B
) also
showed that the stimulation of this promoter activity steadily
increased until the 48 h time point.

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Figure 6. Dose- and Time-Dependent Inductions of 17ß-HSD
Type 2 Promoter Activity
A, Dose dependency for 17ß-HSD type 2 promoter activity in Ishikawa
cells treated with CM from E2 plus R5020- pretreated HuDF.
17ß-HSD type 2 promoter (-200/-1 bp) activity in Ishikawa cells
increased in response to incubation with CM from E2 plus R5020-treated
HuDF endometrial stromal cell line in a dose-dependent fashion. B, Time
course for 17ß-HSD type 2 promoter activity in Ishikawa cells treated
with CM from E2 plus R5020-pretreated HuDF. Incubations of Ishikawa
cells with CM from E2 plus R5020-pretreated HuDF for various
intervals showed progressive increases in promoter activity up to the
48-h time point. R5020 and E2 were removed from CM before its addition
to cultured Ishikawa cells.
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PRs in HuDF and Ishikawa Cell Lines
We determined the regulation of PR levels in HuDF and Ishikawa
cells. Both E2 and R5020, alone or in combination, stimulated the
levels of both PR-A and PR-B in either cell type (Fig. 7
, A and B). In fact, E2 treatment, alone
or in combination with R5020, gave rise to higher levels of PR compared
with R5020 treatment alone (Fig. 7
, A and B). This may be the basis for
the highest 17ß-HSD type 2 mRNA levels and promoter activity in
response to incubation with CM from HuDF pretreated with E2 plus R5020
(Figs. 2
, 3
, and 5
). We also concluded that the lack of a robust
induction of epithelial 17ß-HSD type 2 in response to direct
treatment with R5020 was not due to the lack of functional PR in
Ishikawa cells, since R5020 alone stimulated PR levels in these cells
(Fig. 7B
).

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Figure 7. Functional PRs Are Present in Ishikawa Cells
A, PRs in HuDF Cells. Western analysis revealed that levels of PR
isoforms A and B in the HuDF stromal cell line increased upon
treatments with E2, R5020, or E2 plus R5020. B, PRs in Ishikawa Cells.
PR isoforms A and B were also present in Ishikawa epithelial cells
(Western analysis). Treatment with E2 plus R5020 gave rise to
strikingly increased PR levels in both cells lines. Arbitrary units
represent mean densitometric values of PR-A and PR-B isoforms for each
treatment. C, Promoter activity of a consensus PRE-PGL3-SV40 construct
in Ishikawa and T47D cells treated with R5020. Ishikawa cells contain
functional PR. R5020 stimulated promoter activity of a Luciferase
construct containing an SV40 promoter and two consensus progesterone
response elements (PRE) comprised of two palindromic repeats. We also
transfected T47D cells with the same progestin-responsive construct as
a positive control. Thus, we showed that the lack of a robust induction
of 17ß-HSD type 2 expression in response to direct treatment with
R5020 in Ishikawa cells was not due to the absence of functional PR in
these cells.
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The presence of functional PR in Ishikawa cells was also demonstrated
by a Luciferase reporter gene with an SV40 promoter fused to a 40-bp
sequence containing two consensus progesterone response elements
(PREs) that consisted of two sets of palindromic repeats (Fig. 7C
). This construct could be stimulated by R5020 in a dose-dependent
manner in Ishikawa cells (Fig. 7C
).
Thus, we demonstrated the presence of functional PR in Ishikawa cells
using two different approaches. Despite the presence of functional PR
in Ishikawa cells, a robust induction of 17ß-HSD type 2 remained
strictly dependent on CM from progestin-pretreated stromal cells.
Cell Specificity of the Stromal PR-Mediated Effect On Epithelial
Cells
We asked the question whether this progesterone-mediated
epithelial-stromal interaction is specific for uterine cells. First, we
used media conditioned with E2 plus a R5020-pretreated murine 3T3-L1
adipose fibroblast cell line, primary human breast adipose fibroblasts,
and primary human endometrial stromal cells (Fig. 8
). Again, R5020 and E2 were removed by
charcoal stripping before the addition of CM on Ishikawa cells. Both
media from murine and human adipose fibroblasts gave rise to
intermediate increases (2.2-fold) in 17ß-HSD type 2 promoter activity
in Ishikawa cells (Fig. 8
). This showed that PR in stromal cell types
other than endometrial fibroblasts might give rise to the production of
similar paracine factors to induce 17ß-HSD type 2 in endometrial
epithelial cells.

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Figure 8. 17ß-HSD Type 2 Promoter Activity (-200/-1
Luciferase) in Ishikawa Cells Treated with CM from Various E2 plus
R5020-Pretreated Cells
Medium conditioned with (E2 plus R5020-treated) human or murine adipose
tissue fibroblasts also induced 17ß-HSD type 2 promoter activity in
Ishikawa cells to intermediate levels. The highest levels, however,
were achieved by treatments with CM from either HuDF endometrial
stromal cell line or primary endometrial stromal cells. The stimulatory
effect of CM from HuDF could be inactivated by heat. Steroids were
removed from CM before its addition to Ishikawa cells.
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On the other hand, CM from primary endometrial stromal cells and the
endometrial stromal cell line HuDF produced the highest levels of
induction (4.7- and 4.4-fold, respectively). Thus, although a variety
of stromal cells are capable of secreting similar paracrine factors in
response to progesterone, endometrial stromal cells have the highest
capacity to induce 17ß-HSD type 2 in epithelial cells in a paracrine
fashion. Moreover, this stimulatory effect could be decreased
significantly by exposing CM from HuDF to heat (95 C for 15 min) before
the addition CM to Ishikawa cells. This indicated that the secretory
products of stromal cells were possibly proteinaceous in nature.
Subsequently, we asked the opposite question: can stromal PR mediate
stimulation of 17ß-HSD type 2 in a nonendometrial epithelial cell
type? We determined 17ß-HSD type 2 mRNA levels and promoter
activity in T47D breast epithelial cell line in response to CM
from primary human breast adipose fibroblasts and HuDF endometrial
stromal cells (Fig. 9
). Readily
detectable baseline levels of 17ß-HSD type 2 mRNA and promoter
activity were present in T47D cells. CM from E2 plus R5020-pretreated
breast or endometrial stromal cells (HuDF) did not stimulate 17ß-HSD
type 2 mRNA levels or promoter activity in T47D breast (epithelial)
cancer cells (Fig. 9
, A and B). Thus, we conclude that endometrial
epithelial cell type is specific for stromal PR-mediated induction of
17ß-HSD type 2.

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Figure 9. 17ß-HSD Type 2 mRNA Levels (Panel A) and
17ß-HSD Type 2 Promoter Activity in T47D Breast Cancer Cells (Panel
B)
CM from endometrial (HuDF) or primary adipose tissue fibroblasts did
not stimulate 17ß-HSD type 2 mRNA levels (panel A, RPA) or promoter
activity (panel B) in T47D breast cancer epithelial cell line. Thus,
although this breast epithelial line expresses readily detectable
baseline levels of 17ß-HSD type 2, a stromal PR-mediated induction
could not be demonstrated. Steroids were routinely removed from CM
before its addition to T47D cells.
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DISCUSSION
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Recently introduced data from uterine tissue recombination
experiments on knockout mice with disrupted ER
and PR genes
demonstrated a number of epithelial-stromal interactions that mediate
the effects of ovarian steroids on endometrial tissue (2, 17, 18). For example, critical mitogenic and differentiative effects
of estrogen and progesterone on epithelial cells were shown to be
mediated by stromal ER
and PR (2, 17, 18). We use
herein an alternative technique to characterize a physiologically
critical epithelial-stromal interaction in the human endometrium.
Stromal cells of the endometrium respond to progesterone by secreting
water-soluble and heat-sensitive factors that act on epithelial cells
to induce the E2-metabolizing enzyme 17ß-HSD type 2. This interaction
is specific for endometrial epithelial cells because breast or
endometrial stromal cells do not induce this enzyme in breast
epithelial cells, which also express readily detectable baseline levels
of 17ß-HSD type 2 mRNA and promoter activity.
In contrast to the robust induction of 17ß-HSD type 2 expression in
Ishikawa epithelial cells by medium conditioned with
progestin-pretreated endometrial stromal cells, direct treatments of
Ishikawa cells with progestin gave rise to only 2025% of the
fold-inductions observed with CM. An autocrine mechanism is likely
reponsible for the induction (albeit minimal) of 17ß-HSD type 2
expression in response to direct treatment of Ishikawa epithelial cells
with R5020. In other words, as in the case of CM from endometrial
stromal cells, activation of PR in Ishikawa cells also gave rise to
secretion of factors into the culture medium, which in turn stimulated
17ß-HSD type 2 expression in the same cell in an autocrine fashion.
The capacity of Ishikawa epithelial cells for the production of these
factors, however, was markedly less compared with that of endometrial
stromal cells.
Although progesterone induction of 17ß-HSD type 2 enzyme activity,
mRNA, and protein in epithelial cells of human endometrial tissue had
been demonstrated using tissue explants or by circumstantial in
vivo evidence, its transcriptional regulation could not be studied
to date because of the lack of a suitable cell culture model
(4, 5, 6, 7, 8, 10, 12). Treatment of epithelial cells with stromal
cell CM has proven to be a useful model that permitted us to ask
functionally relevant questions about endometrial physiology. The
answers, however, gave rise to more questions such as the identity of
stroma-derived factors that induce epithelial 17ß-HSD type 2. Studies
to identify these heat-sensitive factors are under way.
To our knowledge this report represents the first published data on the
transcriptional regulation of 17ß-HSD type 2 gene in any human cell
type. We also uncovered a critical paracrine regulatory mechanism
responsible for the induction of this gene, evidenced by the presence
of readily detectable levels of functional PR in Ishikawa cells, the
lack of a progesterone response element in the 17ß-HSD type 2
promoter, and the dominant stimulatory effect of progesterone-dependent
stromal factors. This paracrine effect is conferred by a critical
region in the -200/-100 bp sequence of the 17ß-HSD type 2 promoter.
Four potential cis-acting elements in this region include
binding sites for heat shock factor, CdxA homebox gene product, sex
determining region Y gene product, and activator protein-1 (Fig. 4
).
Future studies are needed to identify trans-activating
factors and signaling mechanisms that mediate this physiologically
critical effect of stromal factors on epithelial cells in response to
progesterone.
 |
MATERIALS AND METHODS
|
---|
Cultures of Cell Lines and Primary Human Endometrial Stromal
Cells
We used three types of cells of endometrial origin: 1) The human
decidual fibroblast line (HuDF) represents endometrial stromal cells.
This cell line was spontaneously immortalized by serial passages and
was found to express PRL in response to treatment with progesterone.
HuDF cells also contain ER
and PR (14). HuDF cells were
cultured to confluency in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, MD) containing 10% FBS. 2) The human
Ishikawa malignant endometrial epithelial cell line was used in lieu of
endometrial epithelial cells (15). Ishikawa cells were
also cultured to confluency in RPMI 1640 plus 10% FBS. 3) Primary
stromal cells in monolayer culture were prepared from human
endometrium, as previously described (n = 4 patients)
(19). Primary endometrial stromal cells were cultured to
confluency in DMEM/F12 1:1 (Life Technologies, Inc.)
containing 10% FBS. Subsequently, all stromal cells were maintained in
serum-free RPMI 1640 to generate CM. Human tissues were obtained
following protocols approved by the Institutional Review Board of the
University of Illinois at Chicago.
The following cells were used as positive and negative controls: 1)
Mouse 3T3-L1 fibroblast line was cultured to confluency in DMEM/F12 1:1
plus 10% FBS. 2) Human adipose tissue fibroblasts in primary monolayer
cultures were grown to confluency in DMEM/F12 1:1 plus 10% FBS, as
previously described (20). 3) The human T47D malignant
breast epithelial cell line was grown to confluency in RPMI 1640 with
10% FBS.
Generation of CM from Endometrial Stromal Cells and Control
Cells
The HuDF line, primary endometrial stromal cells, primary
adipose fibroblasts, 3T3-L1 cells, and T47D cells were cultured in
appropriate growth media in T75 flasks in a humidified atmosphere with
5% CO2 at 37 C, as described above. Media were
changed at 48-h intervals, until the cells became 95% confluent. Then,
growth media were aspirated and washed out twice with PBS, and cells
were incubated in serum-free RPMI 1640 overnight for further washout.
The next day, serum-free RPMI 1640 was added to all cell types (30
ml/T75 flask). These media were collected at the end of a 48-h period
and used as CM to treat Ishikawa epithelial cells. During the
generation of CM, cells were subjected to combinations of the following
treatments: 1) serum; 2) E2 (10-8
M); 3) progesterone agonist R5020
(10-7 M); and 4) progesterone
antagonist ZK98299 (10-5 M, a
generous gift from Schering AG, Berlin, Germany). At the
end of the 48-h incubation, media conditioned by these various cell
types were harvested. These CM inevitably contained residual hormones
that were initially added to treat cells during the conditioning
process. Therefore, CM were routinely stripped by charcoal to remove
E2, R5020, or ZK98299. Stripped CM were preserved frozen at -80 C
until use. Frozen CM were thawed at 37 C immediately before the
addition to Ishikawa cells.
17ß-HSD Type 2 Enzyme Activity
Ishikawa cells were plated in 35-mm culture dishes until the
cells became 95% confluent. After being washed twice with PBS, the
cells were kept in serum-free medium for 12 h. Ishikawa cells were
then incubated with hormones (R5020, E2) or media conditioned with
hormone-treated HuDF endometrial stromal cell line. Cells were rinsed
twice with PBS and then frozen in liquid nitrogen and kept at -80 C
until assayed. Cell pellets were allowed to thaw on ice and mixed with
homogenization buffer (0.5 ml packed cells/ml buffer) consisting of 10
mM Tris (pH 7.2) 150 mM KCl, 0.3 M
sucrose, 1 mM EDTA, and 1 mM
phenylmethylsulfonyl fluoride. Cells were homogenized with a polytron
using five strokes at setting 5. 17ß-HSD type 2 enzyme assay was
carried out using 50 mg of protein homogenate, in 100 mM
Tris (pH 9.0), [3H]-E2 (0.5 µCi), unlabeled
E2 (5 µM), and NAD+ (1.5
mM) (total volume of 500 µl). Assay was begun by the
addition of the cofactor and carried out for 60 min at 37 C. Assay was
terminated by the addition of 100 µl of 0.1 N NaOH.
Excess unlabeled E2 and E1 were added to aid for the visualization of
steroid products after TLC, and samples were extracted in diethyl
ether-ethyl acetate (9:1). Products were separated using TLC (methylene
chloride-ethyl acetate, 3:1), visualized with iodine vapor, and the
spots were scraped into scintillation vials. Product formation was
calculated as the number of counts in the product spot divided by the
total number of counts in the product plus the substrate spots. No
significant counts were identified in other sites on the plate.
Deletion Mutations and Preparation of Luciferase Fusion
Constructs
The -1,244/-1 bp 5'-flanking region of the 17ß-HSD type 2
gene was amplified by PCR from a sample of human genomic DNA using
single-stranded oligonucleotides complementary to the previously
published sequence of this region (13). These 25 mer
oligonucleotides contained nonannealing ends for the restriction sites
KpnI (5'-primer) and HindIII (3'-primer). This
PCR-amplified product was directly subcloned into PCR 2.1 using the TA
Cloning System as described in the manufacturers protocol
(Invitrogen, Carlsbad, CA). This PCR 2.1 vector
containing the -1,244/-1 bp sequence was used as a template to
amplify deletion fragments containing -750/-1, -500/-1, -300/-1,
-200/-1, -100/-1, and -65/-1 bp sequences of the 17ß-HSD type 2
promoter flanking region. All deletion fragments were subcloned into
PCR 2.1 and sequenced to check their fidelity. These deletion fragments
of 17ß-HSD type 2 promoter were released from the PCR2.1 vector by
restriction digest with KpnI and HindIII and were
subcloned into KpnI and HindIII sites of the
pGL3-Basic vector (Promega Corp., Madison, WI), thereby
generating pGL3 promoter constructs containing -1,244/-1, -750/-1,
-500/-1, -300/-1, -200/-1, -100/-1, and -65/-1 bp deletion
fragments. All vectors were reconfirmed by sequencing. These vectors
were then transfected along with cytomegalovirus-Luciferase internal
control into Ishikawa cells.
We also prepared a generic progesterone-responsive Luciferase construct
to use in control experiments (Fig. 7C
). A 40-bp double-stranded
oligonucleotide (5'-CAAAGA
ACACCCTGTTCTACACAGAACACCCTGTTCTACC-3')
containing two consensus PREs (each represented by two palindromic
repeats) was subcloned into KpnI and HindIII
sites of the pGL3-SV40 vector (Promega Corp.). Thus, we
generated a Luciferase reporter gene construct with an SV40 promoter
downstream of a 40-bp regulatory sequence that contains two consensus
PREs.
Transient Transfections and Luciferase Assays
Transient transfection of Ishikawa cells in culture was carried
out in 35-mm dishes using the LipofectAMINE Plus reagent (Life Technologies, Inc.) with the following plasmids: 1) 1 µg of
the pGL3-Basic Luciferase reporter plasmid that contains serial
deletion mutants of 17ß-HSD type 2 promoter; and 2) 10 ng
of cytomegalovirus-PRL plasmid as an internal control (Promega Corp.). After transfection for 4 h in serum-free medium,
medium was changed to RPMI 1640 with antibiotics, 10
mM HEPES, and 10% FBS. After overnight recovery
in the serum-containing medium, cells were kept in serum-free medium
for 12 h. Thereafter, cells in serum-free medium were treated for
48 h with hormones or media conditioned with hormone-treated cells
(HuDF, primary endometrial cells, primary adipose fibroblasts, and
3T3-L1). We performed a dose-response experiment using dilutions (0%,
20%, 40%, 60%, 80%, 100%) of medium conditioned with E2 plus
R5020-pretreated HuDF stromal cell line. (Steroids were routinely
removed by charcoal stripping before the use of CM.) We also performed
a time-course experiment using HuDF-CM to treat Ishikawa cells for
1 h, 3 h, 6 h, 9 h, 12 h, 24 h, and
48 h. After treatment, transfected Ishikawa cells were washed
twice in PBS and lysed in 250 µl of a 1x lysis buffer (0.1
M potassium phosphate, pH 7.8; 1% Triton X-100;
1 mM dithiothreitol; 2 mM
EDTA). Luciferase assays were performed using 10 µl of cell lysate
employing a Dual-Luciferase Reporter Assay System kit (Promega Corp.). Luminescent activities were measured using LUMAT LB9507
luminometer (EG&G Berthold, Bad Wildbad, Germany) Results were
presented as the average of data from three replicate experiments
± SEM.
Ribonuclease Protection Assay (RPA)
Ishikawa and T47D cells were plated in 100-mm culture dishes
until the cells became 95% confluent, washed with PBS three times, and
kept in serum-free medium for 12 h. Cells were treated directly
with hormones or media conditioned with HuDF or other cells, as
described above. Treatments were continued for 48 h. Treated cells
were then washed with PBS, and total RNA was isolated. Riboprobes were
prepared for the RPA in the following fashion: RT was performed using 3
µg of total RNA from human luteal phase endometrium using SuperScript
II (Life Technologies, Inc.) and random hexamers to
generate a cDNA library. A full- length cDNA for 17ß-HSD type 2 was
generated by PCR and ligated into PCR2.1 vector
(Invitrogen), which was sequenced to confirm its identity
(8). Specific oligodeoxynucleotide primers were
synthesized according to the published information for 17ß-HSD type 2
cDNA (8). A 319-bp cDNA fragment was generated by PCR and
represents the coding sequence from +925 bp to +1,243 bp. Sense primer
was 5'-GGACATTCTGGACCACCTCC-3'; and the antisense primer included a T7
polymerase site
(5'-ATCCTAATACGACTCACTATAGGGAGGAGGCCTTTTTCTTGTAGTTAG-3'). This PCR
product was gel purified and used for riboprobe preparation employing
the Maxiscript T7 polymerase kit (Ambion, Inc., Austin,
TX). During this process, riboprobes were labeled with
[32P]UTP. mRNA levels of the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined as a
control for starting equal amounts of total RNA. The riboprobe for
GAPDH mRNA was prepared from a 195-bp cDNA fragment (from +78 bp to
+272 bp) using total RNA from luteal endometrium.
The RPA was performed according to RPA III kit manual (Ambion, Inc.). The 17ß-HSD type 2 and GAPDH probes were added to total
RNA samples (20 µg each) from treated Ishikawa cells and hybridized
overnight at 56 C. After ribonuclease digestion, the protected
fragments were separated on a 6% polyacrylamide gel. Total RNA from
human luteal phase endometrium and human adipose fibroblasts were used
as positive and negative controls, respectively.
Western Blotting for PRs
Ishikawa cells and HuDF cells were cultured in 100-mm dishes
until 95% confluence as described above and switched to serum-free
media for 12 h. These cells were then incubated under various
conditions, i.e. 1% FBS, E2 (10-8
M), R5020 (10-7
M) for 48 h. Total protein was extracted
from whole cells. The T47D cell line was used as a positive control.
Whole cell extracts (each containing 25 µg of total protein) were
electrophoresed on 7.5% polyacrylamide SDS-gels (5% stacking, 7.5%
separating gels) at 25 mA for 15 min and then at 45 mA for 50 min.
Protein samples were then transferred to a nitrocellulose membrane in
Transblot buffer (25 mM Tris, 192
mM glycine, and 20% methanol) at 4 C for 12
h at 50 V (Bio-Rad Laboratories, Inc. Hercules, CA).
Membranes were then blocked with 2% milk in TBS buffer (20
mM Tris, pH 7.2; 140 mM
NaCl) overnight and incubated with an anti-PR (1294) monoclonal
antibody at 1:500 dilution, for 1 h at room temperature
(21). Membranes were then washed five times for 10 min
with 0.1% Tween-20 in TBS and incubated with a 1:2,500 dilution of
secondary antibody (antimouse IgG-horseradish peroxidase, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at room
temperature. Next, membranes were washed five times (10 min) each with
0.1% Tween-20 in TBS. The signal was detected using chemiluminescence
(SuperSignal Ultra Chemiluminescent Kit, Pierce Chemical Co., Rockford, IL) according to the manufacturers
protocol.
 |
ACKNOWLEDGMENTS
|
---|
Dee Alexander provided skilled editorial assistance. We thank
Dr. Asgerally Fazleabas for providing HuDF and Ishikawa cell lines and
Dr. Dean Edwards for providing the monoclonal anti-PR antibody
1294.
 |
FOOTNOTES
|
---|
This work was supported by NICHD Grant HD-38691 (to S.E.B.).
Abbreviations: CM, Conditioned medium; E1, estrone; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; HSD, hydroxysteroid
dehydrogenase; PRE, progesterone response element; RPA, ribonuclease
protection assay.
Received for publication June 14, 2001.
Accepted for publication August 22, 2001.
 |
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