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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-1567
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 12 4825-4835
Copyright © 2006 by The Endocrine Society

Regulation of A Disintegrin And Metalloproteinase with ThromboSpondin Repeats-1 Expression in Human Endometrial Stromal Cells by Gonadal Steroids Involves Progestins, Androgens, and Estrogens

Jiadi Wen, Hua Zhu, Shuko Murakami, Peter C. K. Leung and Colin D. MacCalman

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4

Address all correspondence and requests for reprints to: Colin D. MacCalman, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, Child and Family Research Institute, Room I3091-950, West 28th Avenue, Vancouver, British Columbia, Canada V5Z 4H4. E-mail: cdmaccalman{at}hotmail.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Gonadal steroids are key regulators of the extracellular matrix remodeling events that occur in the human endometrium during each menstrual cycle. The spatiotemporal expression of A Disintegrin And Metalloproteinase with ThromboSpondin repeats (ADAMTS)-1 in human endometrial stroma in vivo suggests that this novel metalloproteinase may contribute to this tightly regulated developmental process.

Objective: The objective of the study was to determine whether progesterone (P4), 17ß-estradiol (E2), or the nonaromatizable androgen dihydrotestosterone (DHT), alone or in combination, is capable of regulating ADAMTS-1 mRNA and protein levels in human endometrial stromal cells in a concentration- and time-dependent manner.

Design: A real-time quantitative PCR strategy and Western blotting were used to examine ADAMTS-1 mRNA and protein expression levels in primary cultures of human endometrial stromal cells.

Results: P4 and DHT but not E2 increased the levels of the ADAMTS-1 mRNA transcript and protein species (110 kDa) present in endometrial stromal cells in vitro in a concentration- and time-dependent manner. A combination of P4 and DHT resulted in an additional increase in stromal ADAMTS-1 expression, whereas E2 attenuated the regulatory effects of P4 and DHT in a concentration-dependent manner. The antisteroidal compounds, mifepristone (RU486) and hydroxyflutamide, were also found to inhibit specifically the P4- and DHT-mediated increase in ADAMTS-1 mRNA and protein expression levels in these primary cell cultures in a concentration-dependent manner, respectively.

Conclusions: These studies demonstrate that progestins, androgens, and estrogens, alone and in combination, have distinct regulatory effects on ADAMTS-1 mRNA and protein expression levels in human endometrial stromal cells in vitro.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GONADAL STEROIDS REGULATE the proliferation, differentiation, and shedding of the human endometrium during each menstrual cycle (1, 2). After menstruation, 17ß-estradiol (E2) coordinates cellular proliferation in the glandular epithelium, vasculature, and stroma of the endometrium. In turn, increasing progesterone (P4) levels after ovulation induce glandular secretion and the terminal differentiation of the vasculature and stroma, which is characterized by the appearance of prominent spiral arterioles surrounded by cuffs of pseudodecidual cells. If fertilization occurs, levels of P4 levels continue to increase, causing further development of the endometrial vasculature and extensive decidualization of the stroma, whereas in the absence of pregnancy, P4 levels decrease and menstruation ensues.

There is increasing evidence to suggest that androgens also play integral roles in the cyclic remodeling events that occur in the endometrium in preparation for pregnancy. In particular, androgen and androgen receptor (AR) expression levels are tightly regulated in the human endometrium during the menstrual cycle (3, 4, 5). Aberrant expression levels of the androgen-AR base system in the endometrium have also been linked with recurrent pregnancy loss and infertility (4, 6). Although less well understood, the direct biological actions of androgens on the human endometrium mimic many of those previously assigned to P4 and include antagonizing the E2-mediated proliferation of endometrial cells and promoting decidualization (7, 8).

Remodeling of the extracellular matrix (ECM) is a hallmark of the steroid-mediated morphological and functional maturation of the endometrium (9, 10). The proteolytic enzymes that are assigned key roles in this highly regulated series of developmental events include urokinase plasminogen activator and matrix metalloproteinases, which act in concert or in cascades to degrade/process specific components of the endometrial ECM (11, 12). However, members of the novel family of metalloproteinases, known as the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats), may also represent a significant molecular mechanism underlying the cyclic proteolysis of the endometrial ECM. Although more than 20 distinct ADAMTS subtypes have been described at the structural level, ADAMTS-1 remains one of the best characterized members of this gene family (13, 14).

ADAMTS-1 was initially identified as an inflammation-associated gene in an experimental colon carcinoma model system (15) but has since been implicated in the ECM remodeling events underlying cancer and osteoarthritis in humans (13, 14) and pre- and postnatal growth and development of tissues in mice (16, 17). ADAMTS-1 has also been found to be spatiotemporally expressed in the human endometrium during the menstrual cycle and in pregnancy (2, 18), with accumulation of this ADAMTS subtype being associated with the onset and progression of decidualization in vivo (18). As this expression pattern strongly suggests that gonadal steroids are key regulators of ADAMTS-1 in the human endometrium, we examined the ability of P4, E2, and the nonaromatizable androgen, dihydrotestosterone (DHT), alone or in combination, to regulate ADAMTS-1 mRNA and protein expression levels in primary cultures of human endometrial stromal cells in a concentration- and time-dependent manner. In addition, we determined whether the antiprogestin, RU486 (mifepristone), and the antiandrogen, hydroxyflutamide, are capable of inhibiting the observed regulatory effects of P4 and DHT on stromal ADAMTS-1 expression levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues

Endometrial tissue samples were obtained from women of reproductive age (range 35–45 yr) undergoing hysterectomy for reasons other than endometrial cancer or hyperplasia in accordance with a protocol for use of human tissues approved by the Committee of Ethical Review of Research Involving Human Subjects, University of British Columbia. All of these women had normal menstrual cycles and did not receive hormonal treatments for 3 months or longer before the time of surgery. The stage of the menstrual cycle was determined by the last menses and confirmed by subsequent histological evaluation (1). Only endometrial tissues (n = 12) that were obtained before the perimenstrual stage of the late secretory phase were used for stromal cell isolation.

Cell isolation and culture

Enriched cultures of stromal cells were isolated from endometrial tissues according to a previously described protocol (19). Briefly, endometrial tissue samples were minced and subjected to 0.1% collagenase (type IV)/hyaluronidase (type I-S; Sigma Aldrich, St. Louis, MO) digestion in a shaking water bath at 37 C for 60 min. The cell digest was then passed through a nylon sieve (38 µm) after which the eluate containing the stromal cells was centrifuged at 800 x g for 10 min at room temperature. The resultant cell pellet was washed once and resuspended in phenol red-free DMEM containing 25 mM glucose, L-glutamine, antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin) and supplemented with 10% charcoal-stripped fetal bovine serum. All of the endometrial stromal cell cultures included in these studies were determined to have a purity of 99% or greater by immunocytochemical analysis for a variety of cellular markers (19, 20).

Experimental culture conditions

Endometrial stromal cells (passage 4–6) were plated in 60-mm2 tissue culture dishes (Becton Dickinson and Co., Franklin Lakes, NJ) at a density of 5 x 106 cells/dish and grown to 80% confluency. The cells were then washed with PBS and cultured in phenol red-free DMEM supplemented with 10% charcoal-stripped fetal bovine serum and containing either increasing concentrations of P4 (10 nM to 5 µM), E2 (0.1–100 nM), or DHT (1–200 nM) for 24 h or a fixed concentration of P4 (1 µM), E2 (30 nM), or DHT (100 nM) for 0–72 h.

Combinatorial effects of gonadal steroids on ADAMTS-1 mRNA and protein expression levels were investigated by culturing stromal cells in the presence of P4 (1 µM) plus DHT (100 nM) for 0–72 h or a combination of P4 (1 µM) or DHT (100 nM) plus either a fixed concentration of E2 (30 nM) for 0–72 h or increasing concentrations of E2 (0.1–100 nM) for 72 h.

To determine whether the observed regulatory effects of P4 and DHT on stromal ADAMTS-1 mRNA and protein expression levels could be inhibited by antisteroidal compounds, endometrial stromal cells were cultured in the presence of increasing concentrations of RU486 (25 nM to 10 µM) or hydroxyflutamide (0.1 nM to 1 µM) alone or in combination with P4 (1 µM) or DHT (100 nM) for 72 h.

Endometrial stromal cells cultured with vehicle (0.1% ethanol) served as controls for these experiments. The concentrations of gonadal steroids and antisteroidal compounds examined in this study are based on previous reports (7, 19, 20, 21).

RNA preparation and synthesis of first-strand cDNA

Total RNA was extracted from the endometrial stromal cell cultures using a RNeasy minikit (QIAGEN, Mississauga, Ontario, Canada). The purity and concentration of total RNA present in each of these extracts were quantified by absorbance (260/280 nm) using a Du-64 UV-spectrophotometer (Beckman Coulter, Mississauga, Ontario, Canada).

Aliquots (~1 µg) of the total RNA extracts prepared from the endometrial stromal cell cultures were subsequently reverse transcribed into cDNA using a first-strand cDNA synthesis kit (Amersham Pharmacia Biotech, Oakville, Ontario, Canada).

Real-time quantitative (q)RT-PCR

The first-strand cDNA generated from the endometrial stromal cell cultures served as a template for qRT-PCR using the ABI PRISM 7000 sequence detection system (PerkinElmer Applied Biosystems, Foster City, CA) equipped with a 96-well optical reaction plate for primers specific for ADAMTS-1 or the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The specific nucleotides sequences for these primers are as follows: ADAMTS-1 forward, 5'-GCTCA TCTGC CAAGC CAAAG-3', reverse, 5'-CTACA ACCTT GGGCT GCAAA A-3'; GAPDH forward, 5'-TGGAA ATCCC ATCAC CATCT T-3', reverse, 5'-CGCCC CACTT GATTT TGG-3'.

Real-time qPCR was performed using 12.5 µl SYBR Green PCR master mix (PerkinElmer Applied Biosystems), 7.5 µl of primer mixture (300 nM), and 5 µl of cDNA template [diluted 1:7 (vol/vol)] under the following optimized conditions: 52 C for 2 min followed by 95 C for 10 min and 40 cycles of 95 C for 15 sec and 60 C for 1 min. All PCRs were performed in duplicate, with the mean being used to determine mRNA levels. A control containing water instead of sample cDNA was included in each plate. Each set of primers generated a single PCR product of the appropriate size when visualized by agarose gel electrophoresis and the melt curve analysis after qRT-PCR. Nucleotide sequences of the resultant PCR products were confirmed by BLAST (http://www.ncbi.nlm.nih.gov). The amplification efficiency was determined by plotting log cDNA dilution against {Delta}CT ({Delta}CT = CT.Target – CT.GAPDH), the slope of which was close to zero, indicating maximal and similar efficiency of the target and reference genes (data not shown). Relative ADAMTS-1 mRNA levels were determined using the formula 2–{Delta}{Delta}CT where {Delta}{Delta}CT = (CT.Target – CT.GAPDH)X – (CT.Target-CT.GAPDH)0. In this formula, X represents any time point or experimental treatment with control cultures being assigned a value of zero (22). Data were analyzed using SDS 2.0 software (PerkinElmer Applied Biosystems). This experimental approach was further validated by the observation that differences between the CT for the target gene and GAPDH remained relatively constant for each amount of cDNA examined.

Western blot analysis

Endometrial stromal cell cultures were washed three times in PBS and incubated in 100 µl of cell extraction buffer (Biosource International, Camarillo, CA) supplemented with 1.0 mM phenylmethylsulfonyl fluoride and protease-inhibitor cocktail for 30 min on a rocking platform. The cell lysates were centrifuged at 10,000 x g for 10 min at 4 C and the supernatants used for Western blot analysis. The concentrations of protein in the cell lysates were determined using a BCA kit (Pierce Chemicals, Rockford, IL). Western blots containing aliquots (~30 µg) of the cell lysates were prepared and immunoblotted using a polyclonal antibody directed against human ADAMTS-1 (Biodesign International, Saco, ME) as previously described (18). To standardize the amounts of protein loaded into each lane, the blots were reprobed with a polyclonal antibody directed against human ß-actin (Sigma-Aldrich). The Amersham enhanced chemiluminescence (ECL) system was used to detect the amount of each antibody bound to antigen and the resultant autoradiograms analyzed by UV densitometry. The absorbance values obtained for ADAMTS-1 were then normalized relative to the corresponding ß-actin absorbance value.

Statistical analysis

The absorbance values obtained by qRT-PCR and Western blotting were subjected to statistical analysis using GraphPad Prism 4 computer software (GraphPad, San Diego, CA). Statistical differences between the absorbance values were assessed by the ANOVA. Differences were considered significant for P ≤ 0.05. Significant differences between the means were determined using Dunnett’s test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time-dependent effects of gonadal steroids on stromal ADAMTS-1 mRNA and protein levels

ADAMTS-1 mRNA transcripts and a single protein species (110 kDa) were detected in all of the endometrial stromal cell cultures (Figs. 1–6GoGoGoGoGoGo). This protein species corresponds to the ADAMTS-1 zymogen (23, 24). The addition of vehicle to the culture medium had no significant effect on stromal ADAMTS-1 mRNA and protein expression levels at any of the time points examined in these studies (data not shown).


Figure 1
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  		FIG. 1. Time-dependent effects of gonadal steroids on stromal ADAMTS-1 mRNA and protein levels. A, qRT-PCR analysis of ADAMTS-1 mRNA levels in human endometrial stromal cells cultured in the presence of a fixed concentration of either P4 (1 µM), DHT (100 nM), or E2 (30 nM) for 0, 6, 12, 24, 48, or 72 h. Values for the levels of the ADAMTS-1 mRNA transcript present in each sample were normalized to the corresponding GAPDH mRNA levels. B–D, Representative autoradiograms of Western blots containing total protein extracted from endometrial cells cultured in P4 (1 µM) (B), DHT (100 nM) (C), or E2 (30 nM) (D) for 0, 6, 12, 24, 48, or 72 (lanes 1–6, respectively) and probed with rabbit polyclonal antibodies directed against ADAMTS-1 or human ß-actin. The Amersham ECL system was used to detect antibody bound to antigen. The resultant autoradiograms were scanned and the values obtained for ADAMTS-1 protein levels normalized to absorbance values obtained for the corresponding ß-actin. The results derived from both these analyses as well as those from at least three other sets of experiments were standardized to the 0-h control and are represented (mean ± SEM; n ≥ 4) in the bar graphs (*, P < 0.05 vs. 0-h control).

 

Figure 2
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  		FIG. 2. Concentration-dependent effects of gonadal steroids on stromal ADAMTS-1 mRNA and protein levels. A, qRT-PCR analysis of ADAMTS-1 mRNA levels in human endometrial stromal cells cultured in the presence of increasing concentrations of P4 (0, 10, or 100 nM; 1 or 5 µM; lanes 1–5, respectively), DHT (0, 1, 10, 50, 100, or 200 nM; lanes 1–6, respectively), or E2 (0, 0.1, 1, 10, 30, or 100 nM; lanes 1–6, respectively) for 72 h. Values for the levels of the ADAMTS-1 mRNA transcript present in each sample were normalized to the corresponding GAPDH mRNA levels. B–D, Representative autoradiograms of Western blots containing total protein extracted from endometrial cell cultured with increasing concentrations of P4 (0, 10, or 100 nM; 1 or 5 µM; lanes 1–5, respectively) (B), DHT (0, 1, 10, 50, 100, or 200 nM; lanes 1–6, respectively) (C), or E2 (0, 0.1, 1, 10, 30, or 100 nM; lanes 1–6, respectively) (D) for 72 h and probed with rabbit polyclonal antibodies directed against human ADAMTS-1 or ß-actin. The Amersham ECL system was used to detect antibody bound to antigen. The resultant autoradiograms were scanned and the values obtained for ADAMTS-1 protein levels normalized to absorbance values obtained for the corresponding ß-actin. The results derived from both analyses as well as those from at least three other sets of experiments were standardized to the untreated control and are represented (mean ± SEM; n ≥ 4) in the bar graphs (*, P < 0.05 vs. untreated control).

 

Figure 3
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  		FIG. 3. Combinatorial effects of gonadal steroids on stromal ADAMTS-1 mRNA and protein expression levels. A, qRT-PCR analysis of ADAMTS-1 mRNA levels in endometrial stromal cells cultured in the presence of P4 (1 µM) plus DHT (100 nM) for 0, 6, 12, 24, 48, or 72 h. A representative autoradiogram of a Western blot containing total protein extracted from the corresponding endometrial cell cultures (lanes 1–6, respectively) and probed for ADAMTS-1 and ß-actin is also shown. B, qRT-PCR analysis of ADAMTS-1 mRNA levels in endometrial stromal cells cultured in the presence of P4 (1 µM) plus E2 (30 nM) for 0, 6, 12, 24, 48, or 72 h. A representative autoradiogram of a Western blot containing total protein extracted from the corresponding endometrial cell cultures (lanes 1–6, respectively) and probed for ADAMTS-1 and human ß-actin is also shown. C, qRT-PCR analysis of ADAMTS-1 mRNA levels in endometrial stromal cells cultured in the presence of DHT (100 nM) plus E2 (30 nM) for 0, 6, 12, 24, 48, or 72 h. A representative autoradiogram of a Western blot containing total protein extracted from the corresponding endometrial cell cultures (lanes 1–6, respectively) and probed for ADAMTS-1 and ß-actin is also shown. Values for the levels of the ADAMTS-1 mRNA transcript present in each sample were normalized to the corresponding GAPDH mRNA levels. Western blotting was performed using the Amersham ECL system to detect antibody bound to antigen. The resultant autoradiograms were scanned and the values obtained for ADAMTS-1 protein levels normalized to absorbance values obtained for the corresponding ß-actin. The results derived from both analyses as well as those from at least three other sets of experiments were standardized and are represented (mean ± SEM; n ≥ 4) in the bar graphs (*, P < 0.05 vs. 0-h control).

 

Figure 4
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  		FIG. 4. Effects of varying concentrations of E2 on the P4- and DHT-mediated increase in stromal ADAMTS-1 mRNA and protein levels. A, qRT-PCR analysis of ADAMTS-1 mRNA levels in endometrial stromal cells cultured in the absence or presence of P4 (1 µM) alone or in combination with increasing concentrations of E2 (0.1, 1, 10, 30, 100 nM) for 72 h. A representative autoradiogram of a Western blot containing total protein extracted from the corresponding cell cultures and probed for ADAMTS-1 and ß-actin is also shown. B, qRT-PCR analysis of ADAMTS-1 mRNA levels in endometrial stromal cells cultured in the absence or presence of DHT (100 nM) alone or in combination with increasing concentrations of E2 (0.1, 1, 10, 30, 100 nM) for 72 h. A representative autoradiogram of a Western blot containing total protein extracted from the corresponding cell cultures and probed for ADAMTS-1 and ß-actin is also shown. Values for the levels of the ADAMTS-1 mRNA transcript present in each sample were normalized to the corresponding GAPDH mRNA levels. Western blotting was performed using the Amersham ECL system to detect antibody bound to antigen. The resultant autoradiograms were scanned and the values obtained for ADAMTS-1 protein levels normalized to absorbance values obtained for the corresponding ß-actin. The results derived from both analyses as well as those from at least three other sets of experiments were standardized and are represented (mean ± SEM; n ≥ 4) in the bar graphs (*, P < 0.05 vs. untreated control; **, P < 0.05 vs. steroid alone).

 

Figure 5
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  		FIG. 5. Regulatory effects of RU486, alone or in combination with P4 or DHT, on stromal ADAMTS-1 mRNA and protein expression levels. A, qRT-PCR analysis of ADAMTS-1 mRNA levels in endometrial stromal cells cultured in the absence or presence of increasing concentrations of RU486 (0, 25, or 250 nM; 2.5, 5, or 10 µM) alone. A representative autoradiogram of a Western blot containing total protein extracted from the corresponding cell cultures (lanes 1–6) and probed for ADAMTS-1 and ß-actin is also shown. B, qRT-PCR analysis of ADAMTS-1 mRNA levels in endometrial stromal cells cultured in the absence or presence of P4 (1 µM) alone or in combination with increasing concentrations of RU486 (25 or 250 nM; 2.5, 5, or 10 µM) for 72 h. A representative autoradiogram of a Western blot containing total protein extracted from the corresponding cell cultures and probed for ADAMTS-1 and ß-actin is also shown. C, qRT-PCR analysis of ADAMTS-1 mRNA levels in endometrial stromal cells cultured in the absence or presence of DHT (100 nM) alone or in combination with increasing concentrations of RU486 (25 or 250 nM; 2.5, 5, or 10 µM) for 72 h. A representative autoradiogram of a Western blot containing total protein extracted from the corresponding cell cultures and probed for ADAMTS-1 and ß-actin is also shown. Values for the levels of the ADAMTS-1 mRNA transcript present in each sample were normalized to the corresponding GAPDH mRNA levels. Western blotting was performed using the Amersham ECL system to detect antibody bound to antigen. The resultant autoradiograms were scanned and the values obtained for ADAMTS-1 protein levels normalized to absorbance values obtained for the corresponding ß-actin. The results derived from both these analyses as well as those from at least three other sets of experiments were standardized and are represented (mean ± SEM; n ≥ 4) in the bar graphs (*, P < 0.05 vs. untreated control; **, P < 0.05 vs. steroid alone).

 

Figure 6
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  		FIG. 6. Regulatory effects of hydroxyflutamide, alone or in combination with DHT or P4, on stromal ADAMTS-1 mRNA and protein expression levels. A, qRT-PCR analysis of ADAMTS-1 mRNA levels in endometrial stromal cells cultured in the absence or presence of increasing concentrations of hydroxyflutamide (0, 0.1, 1, 10, 100, or 500 nM or 1 µM) alone. A representative autoradiogram of a Western blot containing total protein extracted from the corresponding cell cultures (lanes 1–6) and probed for ADAMTS-1 and ß-actin is also shown. B, qRT-PCR analysis of ADAMTS-1 mRNA levels in endometrial stromal cells cultured in the absence or presence of DHT (100 nM) alone or in combination with increasing concentrations of hydroxyflutamide (0.1, 1, 10, 100, or 500 nM or 1 µM) for 72 h. A representative autoradiogram of a Western blot containing total protein extracted from the corresponding cell cultures and probed for ADAMTS-1 and ß-actin is also shown. C, qRT-PCR analysis of ADAMTS-1 mRNA levels in endometrial stromal cells cultured in the absence or presence of P4 (1 µM) alone or in combination with increasing concentrations of hydroxyflutamide (0.1, 1, 10, 100, or 500 nM or 1 µM) for 72 h. A representative autoradiogram of a Western blot containing total protein extracted from the corresponding cell cultures and probed for ADAMTS-1 and ß-actin is also shown. Values for the levels of the ADAMTS-1 mRNA transcript present in each sample were normalized to the corresponding GAPDH mRNA levels. Western blotting was performed using the Amersham ECL system to detect antibody bound to antigen. The resultant autoradiograms were scanned and the values obtained for ADAMTS-1 protein levels normalized to absorbance values obtained for the corresponding ß-actin. The results derived from both these analyses as well as those from at least three other sets of experiments were standardized and are represented (mean ± SEM; n ≥ 4) in the bar graphs (*, P < 0.05 vs. untreated control; **, P < 0.05 vs. steroid alone).

 
P4 and DHT significantly increased ADAMTS-1 mRNA levels in endometrial stromal cells after 24 h of culture (Fig. 1AGo). The levels of the ADAMTS-1 mRNA transcript present in these cell cultures continued to increase until the termination of these studies at 72 h. In contrast, E2 alone did not significantly alter stromal ADAMTS-1 mRNA levels, at least not at any of the time points examined (Fig. 1AGo). Similarly, there was a significant and continuous increase in stromal ADAMTS-1 protein expression after 48 h of culture in the presence of P4 (Fig. 1BGo) and after 24 h of culture in the presence of DHT (Fig. 1CGo), whereas the levels of the ADAMTS protein species present in endometrial cells cultured in E2 remained relatively constant for the duration of these studies (Fig. 1DGo).

Concentration-dependent effects of gonadal steroids on stromal ADAMTS-1 mRNA and protein levels

A significant increase in ADAMTS-1 mRNA and protein expression levels was observed only in endometrial stromal cells cultured in the higher concentrations of P4 (1 or 5 µM; Fig. 2Go, A and B) or DHT (100 or 200 nM; Fig. 2Go, A and C) examined in these studies. In contrast, increasing concentrations of E2 did not significantly alter stromal ADAMTS-1 mRNA and protein expression levels (Fig. 2Go, A and D).

Combinatorial effects of gonadal steroids on stromal ADAMTS-1 mRNA and protein expression levels

A combination of P4 plus DHT caused a significant increase in ADAMTS-1 mRNA levels over time in culture in a similar manner to those observed in endometrial stromal cells cultured in the presence of either steroid alone (Fig. 3AGo). The levels of the ADAMTS-1 protein species present in these cell cultures were, however, greater than those detected in cells cultured in either P4 or DHT alone at all of the time points examined (Fig. 3AGo).

In contrast to endometrial stromal cells cultured in the presence of either P4 or DHT alone, we failed to detect any significant increases in ADAMTS-1 mRNA and protein expression levels in cells cultured in E2 plus P4 (Fig. 3BGo) or E2 plus DHT (Fig. 3CGo).

Effects of varying concentrations of E2 on the P4- and DHT-mediated increase in stromal ADAMTS-1 mRNA and protein levels

In agreement with our preceding findings, P4 alone caused a significant increase in stromal ADAMTS-1 mRNA and protein levels (Fig. 4AGo). There was no significant difference between ADAMTS-1 mRNA and protein levels in these cells and those cultured with P4 plus the lower concentrations of E2 (0.1 or 1 nM) examined. A significant decrease in ADAMTS-1 mRNA and protein levels was first detected in cells cultured in the presence of P4 plus 10 nM of E2. The addition of higher concentrations (30 or 100 nM) of E2 to the culture medium did not result in any further decrease in ADAMTS-1 mRNA and protein levels in these cells. Similarly, only the higher concentrations (30 and 100 nM) of E2 were capable of attenuating the DHT-mediated increase in stromal ADAMTS-1 mRNA and protein expression levels (Fig. 4BGo).

Regulatory effects of antisteroidal compounds on stromal ADAMTS-1 mRNA and protein expression levels

RU486 alone had no significant effect but inhibited the P4-mediated increase in stromal ADAMTS-1 mRNA and protein expression levels in a concentration-dependent manner (Fig. 5Go, A and B, respectively). In contrast, levels of the ADAMTS-1 mRNA transcript and protein species remained elevated in stromal cells cultured in the presence of DHT and increasing concentrations of this progesterone receptor (PR) antagonist (Fig. 5CGo).

Similarly, ADAMTS-1 mRNA and protein levels remained relatively constant in endometrial stromal cells cultured in increasing concentrations of hydroxyflutamide alone (Fig. 6AGo). Hydroxyflutamide, however, inhibited the DHT-mediated increase in stromal ADAMTS-1 mRNA and protein expression levels in a concentration-dependent manner (Fig. 6BGo) but had no significant effect on the levels of the ADAMTS-1 mRNA transcript and protein species present in stromal cells cultured in the presence of P4, at least at the concentrations of this antisteroidal compound examined in these studies (Fig. 6CGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we report that gonadal steroids, alone and in combination, have distinct regulatory effects on ADAMTS-1 mRNA and protein expression levels in human endometrial stromal cells in vitro. In particular, P4 and DHT increased ADAMTS-1 expression, whereas E2 alone had no regulatory effect on the levels of this ADAMTS subtype in these primary cell cultures. A combination of DHT and P4 potentiated the increase in the levels of the ADAMTS-1 protein species present in these cell cultures, whereas E2 was capable of attenuating the stimulatory effects of both P4 and DHT on stromal ADAMTS-1 mRNA and protein levels. In contrast, the antisteroidal compounds, RU486 and hydroxyflutamide, specifically inhibited the increase in ADAMTS-1 expression levels mediated by P4 and DHT, respectively.

ADAMTS-1 is a secreted, multidomain, multifunctional protein (13, 14). Gene knockout studies in mice have been assigned key roles for ADAMTS-1 in folliculogenesis and the ovulatory process and in the development of the urogenital system (17, 25). In contrast, the biological function(s) of ADAMTS-1 in the morphological and functional maturation of the endometrium remains ambiguous. Endometrial tissues of mice null-mutant for this gene have been shown to either develop large cysts (16) or be capable of undergoing normal morphological decidualization (17). However, all ADAMTS-1 gene knockout female mice are reported to have reduced pregnancy rates (16, 17). Taken together, these observations suggest that ADAMTS-1 is neither necessary nor sufficient to mediate decidualization of the endometrial stroma but fulfills a specific role in other aspect(s) of endometrial development. For example, ADAMTS-1, independent of its proteolytic activity, has been shown to have both angioinhibitory and angiogenic properties in vitro and in vivo (14, 16, 25). Thus, ADAMTS-1 has the potential to contribute to the development of a uterine environment capable of supporting a pregnancy via the degradation of the endometrial ECM and/or by modulating, at least in part, the extensive remodeling of the vasculature that occurs in this dynamic tissue during the menstrual cycle and pregnancy (2).

ADAMTS-1 is synthesized as a zymogen (110 kDa), which undergoes two consecutive rounds of posttranslational modification to yield two proteolytic fragments (87 and 67 kDa) (23, 24). All three of these ADAMTS-1 protein species have been detected in cellular extracts prepared from mouse ovaries (26); although similar to our findings, significant levels of only the ADAMTS-1 zymogen are present in primary cultures of human chondrocytes (24) and decidual stromal cells (18). These differences are likely attributable to the loss of the endogenous proteolytic factors responsible for ADAMTS-1 cleavage after the isolation and culture of these enriched populations of cells.

P4 increased ADAMTS-1 mRNA and protein expression in our cultures of endometrial stromal cells in a concentration- and time-dependent manner, the regulatory effects of which were attenuated by RU486. Similarly, RU486 has been shown to inhibit the increase in ADAMTS-1 expression levels in cumulus-oocyte complexes isolated from the porcine ovary after the preovulatory surge of gonadotropins (27). The ability of the progestin synthesis inhibitor, epostane, to abolish the preovulatory increase in ADAMTS-1 mRNA levels in rat follicles (28), which was also not observed in mice null-mutant for the PR gene (29), provides further indirect evidence that P4 is a key regulator of ADAMTS-1 expression levels in mammalian tissues and cells. However, the levels of the mRNA transcript encoding this ADAMTS subtype were found to be higher in early stage corpus luteum of the bovine ovary when P4 levels are low and decline during the midluteal phase of the estrous cycle when the circulating levels of this gonadal steroid are high, suggesting that other factors are involved in the regulation of ADAMTS-1 mRNA levels (30). Computer-based searches of the nucleotide sequence and functional assays subsequently failed to identify a PR response element in the promoter region of the murine ADAMTS-1 gene (31). Instead, P4 is believed to regulate ADAMTS-1 gene expression, at least in the mouse, through an indirect mechanism(s) that involves the DNA binding transcription factors specificity protein (Sp)1/Sp3, CCAAT/enhancer-binding protein ß, and/or neurofibromatosis type 1 (31). In support of this regulatory mechanism being operative in the human endometrium, IL-1ß and TGF-ß1 have also been shown to regulate ADAMTS-1 in primary cultures of decidual stromal cells (19). These two cytokines not only mediate many of the biological actions of P4 on the human endometrium (32) but also regulate gene expression via the Sp1/Sp3 complex (33, 34).

E2 alone had no regulatory effects on ADAMTS-1 mRNA or protein levels in isolated human endometrial stromal cells. These findings are indicative of the low levels of this ADAMTS subtype previously observed in proliferative endometrial tissues of mice and humans (18, 35) and the preovulatory follicles of rodent, equine, and bovine ovaries in vivo (30, 36, 37). Furthermore, our studies demonstrate that E2 inhibits the ability of P4 and DHT to increase ADAMTS-1 expression levels in primary cultures of endometrial stromal cells in a concentration-dependent manner. Because ADAMTS-1 accumulation is first observed in the mammalian ovary and endometrium during the periovulatory period (18, 30, 35, 36, 37), when there is a marked increase in the local and circulating levels of P4 and androgens and a concomitant decrease in E2 (2, 30, 37), it is tempting to speculate that ADAMTS-1 expression in at least these two steroid-responsive tissues is dependent on a threshold balance between the counterregulatory effects of these gonadal steroids. To date, the molecular mechanisms by which E2 modulates the P4- and DHT-mediated increase in ADAMTS-1 expression in human endometrial stromal cells remain to be elucidated but are unlikely to involve any direct actions of E2 on the transcriptional regulation of either PR or AR levels in these cell cultures (4, 8). Instead, these regulatory effects may be mediated by the novel membrane-bound E2 receptor (G protein-coupled receptor 30) recently shown to be biologically active in human endometrial cancer cell lines (38).

Of the gonadal steroids examined in this study, DHT was the most potent regulator of ADAMTS-1 protein expression levels in human endometrial stromal cells in vitro. The noncoordinate regulation of the ADAMTS-1 mRNA transcript and protein species present in these primary cell cultures suggests that DHT is capable of regulating the expression of this stromal ADAMTS subtype at both the transcriptional and translational level. Similarly, DHT and P4 have similar potencies on the decidua of the rodent endometrium, with concurrent treatment of both steroids having an additive stimulatory effect on this cellular compartment (39). Thus, the biological actions of DHT on the endometrium do not simply mimic those of P4 but are instead mediated by independent but cooperative receptor-based signaling pathway(s). The likelihood that ARs mediate the biological actions of DHT on the endometrium in a mutually exclusive manner is supported by the ability of hydroxyflutamide but not the PR antagonist, RU486, to block the regulatory effects of this nonaromatizable androgen on stromal ADAMTS-1 mRNA and protein expression levels. However, although hydroxflutamide is capable of suppressing decidualization of the rodent endometrium, its effects could be rescued by the administration of P4 (40). Furthermore, hydroxyflutamide and RU486 are both capable of partially inhibiting the biological actions of DHT and P4 on the decidua of the rodent endometrium (39), suggesting some degree of cross talk/overlap between these two receptor-mediated pathways during this multistep developmental process in vivo. However, the biological significance of androgen regulation of ADAMTS-1 expression in endometrial stromal cells in vitro remains to be elucidated because the concentrations of DHT used in this study exceed the normal physiological range of this gonadal steroid reported in women throughout the menstrual cycle (3, 6). Furthermore, hydroxyflutamide had no significant effect on the levels of ADAMTS-1 in cultures of porcine cumulus-oocyte complexes, suggesting that these regulatory effects may also be tissue and/or species specific (27).

In summary, our current results demonstrate that gonadal steroids are key regulators of ADAMTS-1 expression levels in primary cultures of human endometrial stromal cells and provide the basis for further studies into the molecular mechanisms underlying the hormonal regulation and biological function(s) of ADAMTS-1 in the human endometrium.


   Acknowledgments
 
The authors thank Dr. D. Miller (Division of Gynecological Oncology, Department of Obstetrics/Gynecology, University of British Columbia) and Dr. B. Gilks (Department of Pathology and Laboratory Medicine, University of British Columbia) for their invaluable contributions to this study.


   Footnotes
 
These studies are supported by an operating grant from the Canadian Institutes of Health Research (to C.D.M. and P.C.K.L.). C.D.M. is a career investigator of the Child and Family Research Institute. P.C.K.L. is the recipient of a Senior Scientist award from the Michael Smith Foundation for Health Research. J.W. is the recipient of a studentship from the Interdisciplinary Training Program in Women’s Reproductive Health, Child and Family Research Institute. S.M. is the recipient of the Hoffmeister Postdoctoral Fellowship, Child and Family Research Institute.

Disclosure statement: The authors have nothing to disclose.

First Published Online October 3, 2006

Abbreviations: ADAMTS, A Disintegrin And Metalloproteinase with ThromboSpondin repeats; AR, androgen receptor; DHT, dihydrotestosterone; E2, 17ß-estradiol; ECL, enhanced chemiluminescence; ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; P4, progesterone; PR, progesterone receptor; q, quantitative; Sp, specificity protein.

Received July 18, 2006.

Accepted September 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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