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Biology of Reproduction 60, 471-478 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Gelatinases A and B and Tissue Inhibitors of Metalloproteinases 1, 2, and 3 during In Vivo and In Vitro Decidualization of Rat Endometrial Stromal Cells1

R.K. Nuttalla, and T.G. Kennedy2,a

a Departments of Physiology and Obstetrics and Gynaecology, The University of Western Ontario, London, Ontario, Canada N6A 5C1


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An important event during decidualization is the remodeling of the extracellular matrix, an event controlled by the balance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). A putative regulator of decidualization is prostaglandin E2 (PGE2). The present study shows that endometrial mRNA levels for TIMPs 1, 2, and 3 were increased while gelatinase A levels remained unchanged and gelatinase B levels decreased during oil-induced decidualization. The production of TIMPs 1, 2, and 3 and gelatinases A and B during in vitro decidualization was examined, as was the role of PGE2 as a regulator. Ovariectomized rats were given a regimen of estrogen and progesterone, which sensitized their uteri for decidualization, at which time endometrial stromal cells were isolated and cultured in serum-free conditions for 72 h. Northern blot analyses indicated the presence of the mRNAs for TIMPs and gelatinases, while reverse zymography and zymography showed the presence of their proteins. PGE2 decreased mRNA levels for TIMP-1 and gelatinase A but had no effect on gelatinase B or TIMPs 2 and 3. Indomethacin had no effect on any of the transcripts. These data indicate that rat endometrial stromal cells undergoing decidualization in vitro secrete gelatinases and TIMPs, and suggest that PGE2 may play a role in regulating tissue remodeling during decidualization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extensive remodeling of the uterine extracellular matrix (ECM) occurs during implantation, due both to its degradation associated with blastocyst invasion [1] and to the collagen turnover accompanying decidualization [2]. Prior to implantation, the ECM surrounding the stromal cells is composed of fibrillar collagens (types I and III), collagens V and VI, and fibronectin [3–5]. During decidualization, the collagen matrix becomes less fibrous [3], collagen VI is removed [5], and the decidual cells produce basement membrane components such as laminin, entactin, and collagen type IV [4, 6]. These same changes in matrix composition occur during artificially induced decidualization [3, 5], indicating that this tissue remodeling is independent of an embryo.

The process of tissue remodeling requires a balance between the activities of matrix metalloproteinases (MMPs), enzymes that degrade components of the ECM, and their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). MMPs include the collagenases, gelatinases, stromelysins, metalloelastases, and membrane-type MMPs (reviewed in [7]), while the TIMPs include TIMPs 1, 2, 3, and 4 (reviewed in [7] and [8]). In addition, remodeling is regulated by serine proteases, particularly the plasminogen activators (PAs), which cleave plasminogen to plasmin. This plasmin can degrade ECM components such as fibronectin and laminin, and also serves as an activator of MMPs. The natural inhibitors to the PAs are the plasminogen activator inhibitors, PAIs [9].

The mouse decidua expresses the mRNA of, and shows activity for, gelatinases A (MMP-2) and B (MMP-9) [10] as well as TIMPs 1, 2 [10, 11], and 3 [12, 13] during the periimplantation period. In situ hybridization studies have also shown the presence of TIMPs 1, 2, and 3 and gelatinase A and B mRNA within the uterus during the periimplantation period [10, 14]. During oil-induced decidualization, TIMP-3 [13, 15] and gelatinase A [15] mRNA are up-regulated within the endometrium. The MMPs and TIMPs are believed to be involved in mediating the remodeling associated with decidualization, while the inhibitors may have the additional function of controlling the extent of trophoblast invasion. Consistent with this notion are the observations that, in the mouse and rat, the blastocyst produces gelatinase B [10, 12] and urokinase-type plasminogen activator (uPA) [16] during implantation.

In the present study, we employed a model of pseudopregnancy to study changes in mRNA levels for gelatinases A and B and TIMPs 1, 2, and 3 during the tissue remodeling that occurs in response to artificially induced decidualization. This model is used to examine the uterine remodeling that occurs independent of embryo invasion. In addition, we employed an in vitro model of decidualization whereby endometrial stromal cells are isolated from hormonally sensitized rats and cultured for 72 h, during which time they undergo decidualization [17].

Decidualization is regulated in part by prostaglandins, particularly PGE2, which has been implicated in regulating the increase in vascular permeability within the uterus as well as the decidual cell reaction itself, as determined by uterine weight and alkaline phosphatase activity [18]. Prostaglandins can regulate MMPs and TIMPs in other cell types [19, 20], while Zhang et al. [21] have shown that endometrial stromal cells of the rat secrete uPA, which is up-regulated by PGE2. To further investigate the possible role of prostaglandins in controlling ECM remodeling, the effects of PGE2 on TIMP and gelatinase expression in cultured rat endometrial stromal cells were determined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

All procedures involving animals were performed in accordance with the guidelines of the Canadian Council on Animal Care and the University Council on Animal Care at the University of Western Ontario. Female Sprague-Dawley rats (200–225 g BW; Harlan Sprague-Dawley Inc., Indianapolis, IN) were housed in temperature- and light-controlled conditions (lights-on from 0500 to 1900 h) with free access to food and water. Two days after arrival, animals were ovariectomized under ether anesthesia and allowed at least four days to recover. To obtain rats with uteri sensitized for decidualization, estradiol and progesterone were administered s.c. in sesame oil as described previously ([17], protocol 2).

Oil-Induced Decidualization

On the equivalent of Day 5 of pseudopregnancy, 0.1 ml of sesame oil was injected bilaterally into the uterine lumen of one group of rats; a second group, to be used as a control, remained unstimulated. Both groups continued to receive daily morning injections of 0.1 µg estradiol/4 mg progesterone s.c. in sesame oil. Rats were killed by decapitation on each day of equivalent pseudopregnancy from Day 2 through Day 10; the uterine horns were removed and cleaned in ice-cold saline, and the endometrium was separated from the myometrium by scraping while on ice [22]. The endometrium was then homogenized in guanidinium isothiocyanate (GITC) solution [23] and frozen at -20°C until the RNA was isolated.

Endometrial Stromal Cell Isolation and Culture

Rats with uteri sensitized for decidualization (the equivalent of Day 5 of pseudopregnancy) were killed by decapitation, and endometrial stromal cells were obtained from uterine horns by enzymatic dispersion as described in detail elsewhere [24]. Stromal cells were suspended in Dulbecco's Modified Eagle's medium:F12 nutrient mixture (DMEM:F12) containing 10% heat-inactivated charcoal-stripped fetal calf serum, penicillin (50 IU/ml), streptomycin (50 µg/ml), and fungizone (1.25 µg/ml; all Gibco-BRL, Burlington, ON, Canada) and plated at 5 x 105 cells per well in 24-well plates (Becton Dickinson, Lincoln Park, NJ). After 2-h incubation at 37°C under 5% CO2:95% air to allow for differential attachment of stromal cells, the medium was removed and replaced with serum-free DMEM:F12; this time period was designated as 0 h. Media were changed every 24 h.

For the 24-h time-course experiments, culture media were removed and cells were harvested in GITC solution at either 0, 6, 12, or 24 h. For the 72-h time-course experiment, media were removed and frozen at -20°C and the cells were harvested in GITC solution at either 24 h, 48 h, or 72 h. To determine the effects of PGE2, cells were assigned to one of four treatment groups: control (1% ethanol), PGE2 (1 µg/ml; Cayman Chemical, Ann Arbor, MI), indomethacin (IM; 10-5 M; Sigma Chemical Co., St. Louis, MO), or PGE2+IM, with the treatments commencing at 0 h and lasting 72 h. At the end of the culture period, the media were removed and frozen at -20°C and the cells were harvested in GITC solution.

Northern Blot Analyses

Total RNA was isolated from the GITC-lysed cells by extraction with phenol/chloroform [23], and its integrity was checked by electrophoresis; it was quantified by absorbance at 260 nm. Total RNA (10 µg) was denatured and subjected to electrophoresis in a denaturing gel as described in Sambrook et al. [25]. RNA within the gel was blotted by capillary transfer onto Hybond-N membranes (Amersham, Oakville, ON, Canada) and then cross-linked to the membrane by exposure to 1.2 x 105 µJ/cm2 of UV energy (Hoefer Pharmacia Biotech, San Francisco, CA).

Northern blot analyses were performed as described by Church and Gilbert [26] with some modifications. Briefly, membranes were prehybridized in Church buffer (7% SDS, 0.25 M Na2HPO4 [pH 7.2], 1 mM EDTA, and 1% BSA) at 60°C for 30 min. Mouse cDNA probes for gelatinase A [27] and TIMP-1 [28] were 435- and 800-base pair (bp) fragments, respectively, from plasmids kindly provided by Dr. R. Khokha. Mouse cDNA probes for gelatinase B [29] and TIMP-3 [30] were 3000- and 1800-bp fragments, respectively, from plasmids kindly provided by Dr. D. Edwards. The mouse cDNA for TIMP-2 [31] was a 700-bp fragment of a plasmid kindly provided by Dr. X. Zhang. Complementary DNA probes (25 ng) were labeled by the random-priming technique in the presence of [{alpha}-32P]dCTP (Amersham) using an oligo-labeling kit (Random Primers Labeling System; Gibco-BRL). Hybridizations were carried out at 65°C for approximately 20 h. Membranes were then washed three times (15 min each) in 20 mM Na2HPO4, pH 7.2, with 4% SDS at 60°C and subjected to autoradiography at -70°C with intensifying screens. Between hybridizations, the blots were stripped in 1 mM Tris, 1 mM EDTA, and 0.1-strength Denhardt's reagent (single-strength Denhardt's: 2% BSA, 2% Ficoll, 2% polyvinylpyrrolidone; pH 8.0) for 2 h at 75°C.

Finally, blots were probed with a radiolabeled cDNA for 18S rRNA to determine the relative amounts of RNA loaded into each lane and transferred onto the membrane [32].

Zymography

To determine whether the cultured stromal cells secreted gelatinases, gelatin zymography was performed [33]. Briefly, conditioned medium (CM, 15 µl) was diluted with 4-strength sample buffer (8 mM urea, 8% SDS in 0.5 M Tris, pH 6.8, containing bromophenol blue without 2-mercaptoethanol) and electrophoresed on a 10% polyacrylamide gel containing 60 µg/ml gelatin; a protein molecular weight marker (Amersham) was also run in each gel. Gels were then rinsed in 2.5% Triton X-100 and incubated for 24 h at 37°C in CAB buffer (50 mM Tris, 0.2 M NaCl, 3 mM CaCl2, 0.5 mg/ml Brij 35, 0.2 mg/ml NaN3, pH 7.2). Gels were stained with 0.1% Brilliant Blue R (Sigma) for 10 min and then destained. Clear bands indicated gelatinolytic activity.

To determine whether the cultured stromal cells secreted TIMPs, reverse zymography was performed [34]. Briefly, 500 µl CM was lyophilized and reconstituted in 50 µl sample buffer (50 mM Tris, 1% SDS, 0.025% bromophenol blue, 10% glycerol, pH 6.8). Reconstituted product (20 µl) was electrophoresed on a 12% polyacrylamide gel containing 1 mg/ml gelatin and 1 ml of CM from a known source of gelatinases (Reverse Zymography Kit; UTI, Calgary, AB, Canada). Gels were then rinsed in 2.5% Triton X-100 and incubated for 24 h at 37°C in 50 mM Tris containing 5 mM CaCl2, pH 7.5. Gels were stained with 0.25% Brilliant Blue R (Sigma) for 30 min and then destained. Dark bands indicated inhibition of gelatinase activity.

Statistical Analysis

In vivo experiments were performed twice on separate groups of ovariectomized, hormone-treated rats. In vitro experiments were performed at least three times with different endometrial cell preparations. The relative intensities of the mRNA signals on the autoradiograms were quantified by densitometry (Image Master VDS; Pharmacia Biotech). The relative densities of each target mRNA were expressed as the ratio to the relative density of the 18S rRNA in the same sample, and are presented as the mean ± SEM relative to control. In vitro time-course experiments were analyzed on the raw data by ANOVA using orthogonal polynomial comparisons. To determine the effects of treatment with or without PGE2 and indomethacin, the data were analyzed by two-way ANOVA within blocks, with individual cell preparations being considered blocks. A p value less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pseudopregnancy and Oil-Induced Decidualization

To determine the temporal pattern of expression for the TIMPs during decidualization, RNA was isolated from the endometrium on the equivalent of Day 2 through Day 10 of pseudopregnancy and subjected to Northern blot hybridization (Fig. 1). Messenger RNA for TIMP-1 was detected as a single band at 0.9 kilobases (kb) as reported by others [28] and was present on Day 2 of pseudopregnancy; its levels declined through to Day 5. However, on Day 6, the levels increased, to peak on Day 8, but only within the endometria that had received a deciduogenic stimulus. TIMP-2 mRNA was detected as two transcripts at 3.5 and 1.0 kb as reported by others [31] and was present on Days 2 and 3; levels of these transcripts declined and remained low until Day 7. By Day 8, mRNA became elevated only within the endometria that had received a deciduogenic stimulus. TIMP-3 transcripts were undetectable until Day 8, when they were detected as two transcripts at 4.5 and 2.8 kb as reported by others [7]; these appeared only within the endometria that had received a deciduogenic stimulus, and the levels increased through to Day 10.



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  		FIG. 1. Northern blot analyses of A) TIMPs 1 and 2 and B) TIMP-3 and gelatinases A and B from rat endometrium isolated on the equivalent of Days 2 through 10 of pseudopregnancy. Beginning on Day 6; N, nonstimulated (no oil injected into lumen); S, stimulated (0.1 ml sesame oil injected into the uterine lumen on the equivalent of Day 5 of pseudopregnancy). Shown are two different blots prepared from RNA from the same experiment. Experiments were performed twice on different groups of ovariectomized, hormone-treated rats.

Messenger RNA for gelatinase A was detected as a single band at 3.1 kb as found by others [35] and was present on each day of pseudopregnancy. There was no difference between values in those uteri that had received a deciduogenic stimulus and those that had not. Gelatinase B was detected as two transcripts, one occurring at 2.9 kb, which has been detected by others [35], and the other at approximately 1.3 kb. The transcripts were detected on the equivalent of Days 2 through 5 of pseudopregnancy; but from the equivalent of Day 6 onward, transcripts were detected only within those uteri that had not received a deciduogenic stimulus.

Effect of Time on mRNA Levels In Vitro

In the first series of experiments, the effects of duration of culture of endometrial stromal cells on transcript levels for the TIMPs were determined. Cells were cultured for 24 h (Fig. 2A) or 72 h (Fig. 2B), and the RNA was analyzed by Northern blot hybridization. Probing the membranes with TIMP-1 cDNA revealed a 0.9-kb signal; TIMP-2 revealed a 3.5-kb and a 1.0-kb signal; and TIMP-3 revealed a 4.5-kb and a weaker 2.8-kb signal. The densitometric analyses are presented in Figure 2, C and D. There was no significant change in TIMP-1 transcript levels throughout the 72 h of culture. ANOVA showed that the effect of time (linear) on TIMP-2 (both 3.5- and 1.0-kb bands) and TIMP-3 (4.5-kb band) transcript levels was significant (p < 0.05), indicating that over the first 24 h, TIMP-2 mRNA increased and TIMP-3 mRNA decreased in a manner that did not depart significantly from linearity. The levels of these transcripts were unchanged during the subsequent 48 h of culture.



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  		FIG. 2. Northern blot analyses of TIMPs 1, 2, and 3 from rat endometrial stromal cells cultured for either 24 h (A) or 72 h (B) in serum-free medium. Mean (± SEM, n = 3) ratios of target mRNA/18S rRNA signals, as determined by image analysis, with the first time point from each experiment (0 h or 24 h) set at 1, are shown in C and D. Shown in B are two different blots prepared from RNA from the same experiment.

In the next series of experiments, the effects of duration of culture on transcript levels for the gelatinases were determined. Cells were cultured for 24 h (Fig. 3A) or 72 h (Fig. 3B), and the RNA was analyzed by Northern blot hybridization. Probing the membrane with gelatinase A cDNA revealed a 3.1-kb signal, while gelatinase B cDNA revealed a 2.9-kb signal. The densitometric analyses are presented in Figure 3, C and D. ANOVA showed that the effect of time (linear) on gelatinase A levels was significant (p < 0.05), indicating that during the first 24 h the mRNA levels increased in a manner that did not depart significantly from linearity; transcript levels were not affected during the subsequent 48 h. Gelatinase B mRNA levels did not change significantly throughout the full 72 h of culture.



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  		FIG. 3. Northern blot analyses of gelatinases A and B from rat endometrial stromal cells cultured for either 24 h (A) or 72 h (B) in serum-free medium. Mean (± SEM, n = 3) ratios of target mRNA/18S rRNA signals, as determined by image analysis, with the first time point from each experiment (0 h or 24 h) set at 1, are shown in C and D.

Effect of PGE2 on mRNA Levels In Vitro

To determine whether PGE2 affects transcript levels, endometrial stromal cells were cultured for 72 h in the presence of vehicle, PGE2, indomethacin (an inhibitor of prostaglandin production [36]), or a combination of PGE2 and indomethacin. Experiments were repeated four times with different endometrial stromal cell preparations. Shown are one representative blot after probing with TIMPs 1, 2, and 3 (Fig. 4A) and the same membrane probed for gelatinases A and B (Fig. 4B). As indicated by ANOVA, the relative abundance of mRNA for TIMP-1 (Fig. 4C) and gelatinase A (Fig. 4D) significantly decreased (p < 0.05) when the cells were treated with PGE2. There was no significant effect of PGE2 on transcript levels of TIMP-2 (either the 3.5-kb band and the 1.0-kb band), TIMP-3 (4.5-kb band), or gelatinase B. Indomethacin had no significant effect (p > 0.05) on any of the transcripts examined.



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  		FIG. 4. Northern blot analyses of gelatinases A and B and TIMPs 1, 2, and 3 from rat endometrial stromal cells cultured for 72 h either under control conditions (CON), with PGE2 (1 µg/ml), with indomethacin (IM, 10-5 M), or with PGE2+IM (P+I). A) Autoradiograph of membrane probed sequentially with 32P-labeled cDNA for gelatinase A, gelatinase B, and 18S rRNA. B) Autoradiograph of the same membrane probed sequentially with 32P-labeled cDNA for TIMP-1, TIMP-2, and TIMP-3. C and D) Mean (± SEM, n = 3) ratios of target mRNA/18S rRNA signals, as determined by image analysis, with control set at 1.

Zymographic Assays

Gelatin zymography of the CM from the 72-h time course (Fig. 5A) and that for the PGE2- and indomethacin-treated cell culture (Fig. 5B) both revealed three major bands of gelatinolytic activity occurring at approximately 90 kDa, 70 kDa, and 66 kDa. The known size of latent gelatinase A is 72 kDA; active gelatinase A is 66 kDa, latent gelatinase B is 92 kDa, and active gelatinase B is 84 kDa [37]. Reverse zymography of lyophilized CM from the 72-h time course revealed numerous bands occurring between 30 and 20 kDa (Fig. 6A). Lyophilized CM from the PGE2- and indomethacin-treated culture (Fig. 6B) revealed the presence of four major bands that showed inhibition of gelatinase activity occurring within the range of 21–28 kDa. The known size of TIMP-1 is 28 kDa; TIMP-2 is 21 kDa, and TIMP-3 is 24 kDa [7]. The time-course results are representative of three zymographic assays, while the PGE2 results are representative of four zymographic assays, each performed on medium from a different endometrial stromal cell preparation.



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  		FIG. 5. CM from endometrial stromal cells was subjected to electrophoresis through a 10% polyacrylamide gel containing gelatin. Clear areas represent gelatinase activity. A) One representative blot (n = 3) showing the gelatinase levels within CM from stromal cells cultured in serum-free conditions for 72 h. Samples were collected at 24 h, 48 h, and 72 h. B) One representative blot (n = 4) showing gelatinase levels within CM from stromal cells cultured either under control conditions (CON), with PGE2 (1 µg/ml), with indomethacin (IM, 10-5 M), or with PGE2+IM (P+I).



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  		FIG. 6. CM from endometrial stromal cells was subjected to electrophoresis through a 12% polyacrylamide gel containing gelatin and gelatinase. Dark areas represent inhibition of gelatinase activity. A) One representative blot (n = 3) showing the gelatinase levels within CM from stromal cells cultured in serum-free conditions for 72 h. Samples were collected at 24 h, 48 h, and 72 h. B) One representative blot (n = 4) showing gelatinase levels within CM from stromal cells cultured either under control conditions (CON), with PGE2 (1 µg/ml), with indomethacin (IM, 10-5 M), or with PGE2+IM (P+I).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, transcripts for gelatinases A and B and for TIMPs 1, 2, and 3 were detected in endometria from ovariectomized rats treated with steroid hormones to mimic pseudopregnancy. Endometrial stromal cells from rats on the equivalent of Day 5 of pseudopregnancy express these genes while undergoing decidualization in vitro. In addition, activities of the gelatinases and TIMPs were detected in the media of the cultured cells by zymography and reverse zymography, respectively.

Transcripts for gelatinase A were present in the endometrium on all days of pseudopregnancy, with no apparent changes in the levels over time or in response to a deciduogenic stimulus. By contrast, Yang and Zhang [15] reported low levels of this transcript in the endometrium of rats until Day 7, when its level increased irrespective of whether an artificial deciduogenic stimulus had been given on Day 5. By Day 9, levels remained elevated in the stimulated endometrium undergoing decidualization, but decreased in nonstimulated endometrium. Others have reported the presence of transcripts for gelatinase A prior to the onset of decidualization in mice, with levels decreasing during decidualization [10, 14]. During decidualization in vitro, gelatinase A transcript levels increased during the first 24 h of culture and remained unchanged thereafter. At present, the basis for these differences is not known.

Gelatinase B transcripts were detected in the endometrium on the equivalent of Days 2, 3, and 5 of pseudopregnancy. From Day 6 of pseudopregnancy, the transcripts were present only within the endometrium that had not received a deciduogenic stimulus. Since the steroid treatment regimen was identical for both groups of rats, the absence of transcripts in the stimulated endometrium is presumably a consequence of decidualization, rather than an effect of duration of hormone treatment. These results are consistent with those of Romagnano et al. [38], who reported the presence of gelatinase B protein activity within the endometrial stroma before implantation and its absence after implantation. By contrast, endometrial stromal cells undergoing decidualization in vitro had detectable mRNA for gelatinase B, the relative abundance of which did not change over the 72 h of culture.

The role of the gelatinases within the endometrium is unknown. Gelatinase A acts on ECM components such as collagen types IV and V and fibronectin [39] and may be involved in the degradation of collagen V and fibronectin that occurs during decidualization [3]. However, if this is its role, the role is apparently accomplished without a change in the levels of its transcripts; regulation could be at the level of translation, activation of the latent enzyme, or changes in the concentration of inhibitors. Gelatinase B also acts on similar substrates [39], but because it is not expressed during decidualization in vivo, it is unlikely that gelatinase B is involved in the tissue remodeling during this process. During blastocyst implantation, gelatinase B mRNA has been detected only within the invading trophoblast cells [10, 13, 14], suggesting that it may be involved in trophoblast invasion. However, targeted disruption of the gelatinase B gene apparently does not affect implantation [40].

The activity of the MMPs is inhibited by TIMPs [8]. Elevated levels of transcripts for TIMP-1 and TIMP-2 were detected in the endometrium immediately following the induction of decidualization in vivo in a pattern similar to that reported for the mouse during pregnancy [11]. Elevated levels for TIMP-3 were not detected until the equivalent of Day 8 of pseudopregnancy, 3 days after the induction of decidualization; this observation is similar to the findings of Yang and Zhang [15]. Because transcript levels for the TIMPs increased only within stimulated uteri, the increases are a consequence of decidualization rather than a response to the duration of hormone treatment. The presence of the TIMPs within the decidua suggests that they have a role in regulating the remodeling associated with decidualization. In support of this notion is the observation that overexpression of TIMP-1 in mice results in reduced decidualization [10]. The TIMPs are able to inhibit a broad array of MMPs, with TIMP-1 being more effective than TIMP-2 against the collagenases while TIMP-2 is more effective at inhibiting the gelatinases [41, 42]. TIMP-3 is an inhibitor of gelatinase B [41], and because its expression has been localized spatially and temporally to the stromal cells adjacent to trophoblast cells during implantation, it is believed to inhibit the proteinase activity of the invading embryo [10, 13, 14]. The presence of transcripts for TIMP-3 in the decidua during artificially induced decidualization indicates that its expression does not require the presence of a conceptus.

Transcripts for the TIMPs were also detected during decidualization in vitro, with TIMP-1 levels remaining constant throughout 72 h of culture and TIMP-2 and TIMP-3 levels changing only during the first 24 h. The proteins for the TIMPs were detected within CM obtained from stromal cells cultured in vitro. The bands detected by reverse zymography are most likely to be TIMPs 1 and 2, but not TIMP-3, which is bound to the ECM and therefore probably not present in the medium [30].

The patterns of expression of the gelatinases and TIMPs during in vivo and in vitro decidualization were not identical. Whereas gelatinase B expression was not detected in the endometrium undergoing decidualization in vivo, it was detected in vitro. The patterns of TIMP-3 expression also differed; transcripts were not detected in vivo until 3 days after uterine stimulation, whereas in vitro their levels decreased during the first 24 h of culture and then remained unchanged. The reason for these differences is unknown. They may be a consequence of differences in the structure of the ECM in vivo and in vitro. During isolation of endometrial stromal cells, the fibrillar collagen of the uterus is enzymatically degraded [43] and the stromal cells, when plated, produce basement membrane components such as fibronectin, laminin, and collagen type IV [4, 44]. MMP and TIMP expression may be dependent, in part, on interactions with the ECM.

Prostaglandins have an obligatory role in decidualization (reviewed in [18]) and are known to affect MMPs and TIMPs in other cell types, including rat lymphocytes [45], rat mesangial cells [19], and human ciliary smooth muscle cells [20]. The current experiments show that PGE2 reduces the transcript levels of gelatinase A and TIMP-1 within endometrial stromal cells, with no effect on gelatinase B or TIMPs 2 and 3. Indomethacin, an inhibitor of prostaglandin synthesis [36], had no effect on any of the transcripts examined, probably because the cells under serum-free conditions produce relatively little endogenous PGE2. Thus one of the effects of PGE2 within the uterus during decidualization could be to facilitate remodeling of the ECM by decreasing TIMP-1 levels, thereby increasing MMP activity. The effect of PGE2 on gelatinase A was surprising in that the decrease was concurrent with the decrease in TIMP-1. Although not found in the present study, others have reported a decrease in gelatinase A levels after the induction of decidualization [10, 14] when PGE2 concentrations in the uterus are elevated [46]. It is possible, therefore, that increased PGE2 concentrations could be responsible for mediating decreases in gelatinase A and TIMP-1, and thus mediate ECM remodeling.

Remodeling of the ECM during decidualization is not dependent only on MMPs and their inhibitors; the PAs and their inhibitors may also be involved. Messenger RNA for uPA is present at low levels within the endometrium, but its levels increase within 2 days following induction of decidualization. The inhibitor, PAI, is also present postimplantation. Transcripts for uPA have also been detected during decidualization in vitro, and PGE2 increases their levels [47]. PAs act by cleaving plasminogen to plasmin, which can not only degrade numerous components of the ECM but also convert the inactive pro-form of MMPs to their active form [9]. Thus, remodeling of the ECM during decidualization is probably mediated through the MMP/TIMP and the PA/PAI systems, as well as the interaction between them. Both systems may be regulated, at least in part, by PGE2.

In addition to their role as inhibitors of MMPs, TIMPs are also believed to act as growth factors. TIMP-1, which is homologous to erythroid-potentiating activity, has mitogenic activity on a wide range of cell types [48], while TIMP-2 also has growth-promoting activity [49]. However, our finding that PGE2 decreases TIMP-1 transcript levels and presumably TIMP-1 protein levels during decidualization in vitro, a period during which the cells are undergoing differentiation and proliferation [43], suggests that TIMP-1 does not function as a growth factor regulating endometrial cell proliferation.

The current study demonstrates that endometrial stromal cells undergoing decidualization in vivo show an up-regulation of TIMPs. There was no change in gelatinase A levels within the endometrium, while gelatinase B was absent in the endometrium during decidualization. During in vitro decidualization, endometrial stromal cells produce the mRNA for gelatinases A and B, and TIMPs 1, 2, and 3, throughout 72 h of culture. The transcript levels of gelatinase A and TIMP-1 are regulated to a small degree by PGE2. This study, in addition to the work done by Zhang and colleagues [21, 47] with the PA/PAI system, suggests that PGE2, in addition to its role in increasing vascular permeability, may also play a role in regulating the tissue remodeling accompanying decidualization. Much work still needs to be done to further elucidate the role of PGE2 in remodeling, as well as the role of MMPs, TIMPs, and members of the PA/PAI system in controlling the changes within the ECM during decidualization and implantation.


   ACKNOWLEDGMENTS
 
The authors wish to thank Liz Ross, Gerry Barbe, and Dawn Bowron for all their technical assistance and Dr. R. Khokha at the Ontario Cancer Institute, Dr. X. Zhang at Northwestern University Medical School, and Dr. D. Edwards at The University of East Anglia for kindly providing the cDNA probes.


   FOOTNOTES
 
1 This work was supported by the Medical Research Council of Canada, Grant #MT-10414. Back

2 Correspondence: T.G. Kennedy, Department of Physiology, The University of Western Ontario, London, ON, Canada N6A 5C1. FAX: 519 661 3827; tkennedy{at}physiology.uwo.ca Back

Accepted: September 25, 1998.

Received: June 23, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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