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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}- 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{alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}, PR, and 17ß-HSD type 2 (15, 16) (also see Figs. 1–3GoGoGo). 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).



View larger version (15K):
[in this window]
[in a new window]
 
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.

 


View larger version (64K):
[in this window]
[in a new window]
 
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.

 


View larger version (19K):
[in this window]
[in a new window]
 
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.

 
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. 1Go). 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. 1Go). 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. 2Go). 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. 2Go). 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. 2Go). 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. 3Go). The maximum level of baseline activity was observed with the use of the -200 bp Luciferase construct (Fig. 3Go). 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. 3Go). 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. 3Go). 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. 1Go and 2Go).

Figure 4Go 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. 4Go). 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).



View larger version (50K):
[in this window]
[in a new window]
 
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.

 
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. 5Go, part of negative data not shown).



View larger version (16K):
[in this window]
[in a new window]
 
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.

 
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. 5Go). 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. 5Go). 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. 1Go and 2Go).

Dose- and Time Dependency of the Induction of Epithelial 17ß-HSD Type 2 Promoter Activity by Stromal Factors
Figure 6AGo 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. 6AGo). A time course experiment (Fig. 6BGo) also showed that the stimulation of this promoter activity steadily increased until the 48 h time point.



View larger version (17K):
[in this window]
[in a new window]
 
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.

 
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. 7Go, 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. 7Go, 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. 2Go, 3Go, and 5Go). 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. 7BGo).



View larger version (34K):
[in this window]
[in a new window]
 
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.

 
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. 7CGo). This construct could be stimulated by R5020 in a dose-dependent manner in Ishikawa cells (Fig. 7CGo).

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. 8Go). 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. 8Go). 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.



View larger version (15K):
[in this window]
[in a new window]
 
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.

 
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. 9Go). 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. 9Go, A and B). Thus, we conclude that endometrial epithelial cell type is specific for stromal PR-mediated induction of 17ß-HSD type 2.



View larger version (29K):
[in this window]
[in a new window]
 
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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recently introduced data from uterine tissue recombination experiments on knockout mice with disrupted ER{alpha} 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{alpha} 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 20–25% 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. 4Go). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} 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 manufacturer’s 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. 7CGo). 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 manufacturer’s 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Eckert R, Katzenellenbogen B 1981 Human endometrial cells in primary tissue culture: modulation of the progesterone receptor level by natural and synthetic estrogens in vitro. J Clin Endocrinol Metab 52:699–708[Medline]
  2. Kurita T, Kurita T, Young P, Brody JR, Lydon JP, O’Malley BW, Cunha GR 1998 Stromal progesterone receptors mediate the inhibitory effects of progesterone on estrogen-induced uterine epithelial cell deoxyribonucleic acid synthesis. Endocrinology 139:4708–4713[Abstract/Free Full Text]
  3. Tseng L, Gurpide E 1975 Effects of progestins on estradiol receptor levels in human endometrium. J Clin Endocrinol Metab 41:402–404[Abstract]
  4. Satyaswaroop PG, Wartell DJ, Mortel R 1982 Distribution of progesterone receptor, estradiol dehydrogenase, and 20{alpha}-dihydroprogesterone dehydrogenase activities in human endometrial glands and stroma: progestin induction of steroid dehydrogenase activities in vitro is restricted to the glandular epithelium. Endocrinology 111:743–749[Abstract]
  5. Tseng L, Gurpide E 1974 Estradiol and 20{alpha}-dihydroprogesterone dehydrogenase activities in human endometrium during the menstrual cycle. Endocrinology 94:419–423[Medline]
  6. Tseng L, Gurpide E 1975 Induction of human endometrial estradiol dehydrogenase by progestins. Endocrinology 97:825–833[Abstract]
  7. Casey ML, MacDonald PC, Andersson S 1994 17ß- Hydroxysteroid dehydrogenase type 2: chromosomal assignment and progestin regulation of gene expression in human endometrium. J Clin Invest 94:2135–2141[Medline]
  8. Wu L, Einstein M, Geissler WM, Chan HK, Elliston KO, Andersson S 1993 Expression cloning and characterization of human 17ß-hydroxysteroid dehydrogenase type 2, a microsomal enzyme possessing 20{alpha}-hydroxysteroid dehydrogenase activity. J Biol Chem 268:12964–12969[Abstract/Free Full Text]
  9. Moghrabi N, Head JR, Andersson S 1997 Cell type-specific expression of 17ß-hydroxysteroid dehydrogenase type 2 in human placenta and fetal liver. J Clin Endocrinol Metab 82:3872–3878[Abstract/Free Full Text]
  10. Vihko P, Isomaa V, Ghosh D 2001 Structure and function of 17ß-hydroxysteroid dehydrogenase type 1 and type 2. Mol Cell Endocrinol 171:71–76[CrossRef][Medline]
  11. Mustonen, MVJ, Isomaa V V, Vaskivuo T, Tapanainen J, Poutanen M, Stenbeck F, Vihko RK, Vihko PT 1998 Human 17ß-hydroxysteroid dehydrogenase type 2 messenger ribonucleic acid expression and localization in term placenta and in endometrium during the menstrual cycle. J Clin Endocrinol Metab 83:1319–1324[Abstract/Free Full Text]
  12. Zeitoun K, Takayama K, Sasano H, Suzuki T, Moghrabi N, Andersson S, Johns A, Meng L, Putman M, Carr B, Bulun SE 1998 Deficient 17ß-hydroxysteroid dehydrogenase type 2 expression in endometriosis: failure to metabolize estradiol-17{alpha}. J Clin Endocrinol Metab 83:4474–4480[Abstract/Free Full Text]
  13. Labrie Y, Durocher F, Lachance Y, Turgeon C, Simard J, Labrie C, Labrie F 1995 The human type II 17ß-hydroxysteroid dehydrogenase gene encodes two alternatively spliced mRNA species. DNA Cell Biol 14:849–861[Medline]
  14. Richards JS, Fitzpatrick SL, Clemens JW, Morris JK, Alliston T, Sirois J 1995 Fibroblast cells from term human decidua closely resemble endometrial stromal cells: induction of prolactin and insulin-like growth factor binding protein-1 expression. Biol Reprod 52:609–615[Abstract]
  15. Ishiwata I, Ishiwata C, Soma M, Arai J, Ishikawa H 1984 Establishment of human endometrial adenocarcinoma cell line containing estradiol-17ß and progesterone receptors. Gynecol Oncol 17:281–290[Medline]
  16. Lessey BA, Ilesanmi AO, Castelbaum AJ, Yaum L, Somkuti SG 1996 Characterization of the functional progesterone receptor in an endometrial adenocarcinoma cell line (Ishikawa): progesterone-induced expression of the {alpha}1 integrin. J Steroid Biochem Mol Biol 59:31–39[CrossRef][Medline]
  17. Kurita T, Lee KJ, Cooke PS, Lydon JP, Cunha GR 2000 Paracrine regulation of epithelial progesterone receptor and lactoferrin by progesterone in the mouse uterus. Biol Reprod 62:831–838[Abstract/Free Full Text]
  18. Cooke PS, Buchanan DL, Young P, Setiawan T, Brody J, Korach KS, Taylor J, Lubahn DB, Cunha GR 1997 Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium. Proc Natl Acad Sci USA 94:6535–6540[Abstract/Free Full Text]
  19. Ryan I, Schriock ED, Taylor R 1994 Isolation, characterization, and comparison of human endometrial and endometriosis cells in vitro. J Clin Endocrinol Metab 78:642–649[Abstract]
  20. Zhao Y, Nichols JE, Bulun SE, Mendelson CR, Simpson ER 1995 Aromatase P450 gene expression in human adipose tissue: role of a Jak/STAT pathway in regulation of the adipose-specific promoter. J Biol Chem 270:16449–16457[Abstract/Free Full Text]
  21. Clemm DL, Sherman L, Boonyaratanakornkit V, Schrader WT, Weigel NL, Edwards DP 2000 Differential hormone-dependent phosphorylation of progesterone receptor A and B forms revealed by a phosphoserine site-specific monoclonal antibody. Mol Endocrinol 14:52–65[Abstract/Free Full Text]