Effects of Epidermal Growth Factor and Interleukin-1 on Plasminogen Activator Secretion and Decidualization in Rat Endometrial Stromal Cells¹

^a Departments of Physiology and Obstetrics and Gynaecology, University of Western Ontario, London, Ontario, Canada N6A 5C1 ^b Departments of Obstetrics and Gynaecology, The University of Calgary, Calgary, Alberta, Canada T2N 2T9

	ABSTRACT

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Prostaglandin E₂ (PGE₂) increases decidualization and net plasminogen activator (PA) activity in the medium of cultured endometrial stromal cells from ovariectomized rats sensitized for the decidual cell reaction. Because interleukin-1 (IL-1) and epidermal growth factor (EGF) stimulate PGE₂ production by these cells, the present study determined their effects on decidualization and on the levels of PA activity in the medium. Cells were treated with or without IL-1 (20 ng/ml) and EGF (40 ng/ml) for up to 72 h, and net PA activity in the medium and alkaline phosphatase (ALP) activity (a marker for decidualization) in the cells were measured. After 48 and 72 h of treatment with IL-1, net PA activity levels decreased by 60% and 85%, respectively. EGF significantly increased net PA activity at 24, 48, and 72 h. ALP activity in the cells at 24, 48, and 72 h increased in response to IL-1 but not EGF. These results indicate that IL-1, but not EGF, enhances decidualization of the cells as indicated by ALP activity. Moreover, they suggest that net PA activity in the medium is not a useful marker of decidualization.

	INTRODUCTION

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Decidualization is the process by which the fibroblast-like endometrial stromal cells differentiate into large decidual cells that eventually form the maternal component of the placenta. In rodents, decidualization begins in the endometrium in response to an implanting blastocyst or in response to artificial stimuli [1]. It only occurs during a specific period of time during pregnancy and pseudopregnancy or in ovariectomized animals if the uterus has been sensitized by an appropriate regimen of hormones [1–3]. Cultured rat endometrial stromal cells also undergo decidualization [4], providing a good in vitro model for studying endometrial stromal cell function during decidualization.

Decidualization involves substantial tissue remodeling [5, 6]. Since plasminogen activation has a role in tissue remodeling in other tissues [7], it may also have a role in the endometrium during decidualization. The secretion of urokinase-type plasminogen activator (uPA), but not tissue-type plasminogen activator (tPA), by rat endometrial stromal cells dramatically increases during in vitro decidualization [8]. Further, in vivo, endometrial stromal cells undergoing decidualization express uPA during early pregnancy and after artificially induced decidualization [9]. Therefore, it appears that uPA expression increases in rat endometrial stromal cells during decidualization in vivo and in vitro. However, little is known about the control of plasminogen activator (PA) levels in the endometrium during implantation and decidualization.

Epidermal growth factor receptors (EGFR), type 1 interleukin-1 receptors (IL-1R1), and their ligands are present in the rodent uterus during implantation [10–19]. Although they are present in the uterus, the role of the EGFR- and IL-1R1-signaling systems in implantation are not clear. However, one possible role may be to modulate the PA-plasmin system in the endometrium. In support of this possible role are the observations that uPA secretion by rat endometrial stromal cells is stimulated, in part, by prostaglandin (PG) E₂ (PGE₂) [8]. Because epidermal growth factor (EGF) and interleukin-1 (IL-1) increase PGE₂ accumulation in the medium of these rat endometrial cells [20, 21], the first objective of the present study was to determine the effect of EGF and IL-1 on uPA secretion by rat endometrial stromal cells isolated from uteri sensitized for decidualization. Since PGE₂ is also known to modulate the decidualization of cultured rat endometrial stromal cells [22], the second objective of the present study was to determine the effects of EGF and IL-1 on the in vitro decidualization of rat endometrial stromal cells.

	MATERIALS AND METHODS

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Animals and Cell Isolation

Female Harlan Sprague-Dawley rats (200-225 g; Harlan Sprague-Dawley Inc., Indianapolis, IN) were housed under temperature- and light-controlled conditions (lights-on from 0500 to 1900 h) with free access to food and water. The animals were ovariectomized under ether anesthesia and were allowed at least 5 days to recover. To obtain rats with uteri sensitized for decidualization, estradiol and progesterone (Sigma Chemical Company, St. Louis, MO) in sesame oil were administered s.c. as described elsewhere [21].

Endometrial stromal cells were obtained from the sensitized uteri as described elsewhere [21, 22]. The stromal cells were suspended in Dulbecco's Modified Eagle medium: F-12 nutrient mixture (DMEM:F12) containing penicillin (50 U/ml), streptomycin (50 µg/ml), fungizone (1.25 µg/ml), and 10% heat-inactivated charcoal-stripped fetal calf serum (all from Gibco-BRL, Burlington, ON, Canada). The cell suspension was filtered through nylon mesh (70 µm) to remove glands and clumps of cells. The cells were plated at 5 x 10⁵ cells (in 0.5 ml of medium) in 24-well plates (Becton-Dickinson, Lincoln Park, NJ) and incubated at 37°C under 5% CO₂:95% air for 2 h to allow for differential attachment of the stromal cells, after which the medium and free floating cells were removed and replaced with serum-free DMEM:F12 containing antibiotics and fungizone. This was defined as 0 h of Day 1 of culture. The resulting cultures of attached cells were free of contaminating epithelial-type cells as indicated by the absence of positively staining cells for cytokeratin by immunocytochemistry (data not shown).

Cells were incubated with various treatments for 24, 48, or 72 h, with changes of the media every 24 h. Treatments included human recombinant EGF (Gibco-BRL), human recombinant IL-1 (kindly provided by the National Cancer Institute, Frederick, MD), indomethacin (IM; Sigma) and PGE₂ (Cayman Chemical Company, Ann Arbor, MI). Since they have previously been shown to maximally stimulate PGE₂ accumulation in the medium of these cells [20, 21], concentrations of 40 ng/ml for EGF and 20 ng/ml for IL-1 were used. At the termination of treatments, the media were collected and stored at -20°C until PA activity and PGE₂ were measured as described below. The cells were washed with Dulbecco's PBS (Gibco-BRL), solubilized in 0.25% sodium deoxycholate (Sigma; pH 8.0), then stored at -70°C. The amount of cellular protein in each well was determined using a Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories, Mississauga, ON, Canada).

Plasminogen Activator Assay

Net PA activity levels in the medium were measured using the chromogenic assay of Coleman and Green [23] as modified by Zhang et al. [8]. In brief, samples were incubated with 25 ng of plasminogen (Sigma) in 96-well microtiter plates for 30 min to generate plasmin. Chromogenic substrates for plasmin, 5,5'-dithio-bis(2-nitrobenzoic acid) (0.22 mM) and N-carbobenzoxy-L-lysine thiobenzyl ester (0.18 mM) (Sigma), were then added, and after 15–30 min the absorbance at 405 nm was determined using an automated microplate reader. Standard curves were constructed using 0–20 milli-International Units (mIU) of human urokinase (Calbiochem, San Diego, CA). Under these conditions, the assay was able to detect as little as 2 mIU of PA activity. Phenol red dye, at concentrations present in the samples, had no detectable effect on the assay (data not shown). Net plasminogen activator activity in the medium was expressed as mIU of activity accumulated in the medium over the 24 h of incubation per microgram of cellular protein. The level of net PA activity was expressed in this manner to compensate for small changes in cell protein between wells.

Measurement of Alkaline Phosphatase (ALP)

The level of ALP in the cells was determined using the method of Lowry [24]. ALP activity was expressed as nanomoles of substrate (p-nitrophenol phosphate) hydrolyzed per hour per microgram of cellular protein.

PGE₂ RIA

PGE₂ was measured directly in the medium by RIA as described previously [20, 21]. PGE₂ accumulation was expressed as picograms of PGE₂ accumulated in the medium over the 24 h of incubation per microgram of cellular protein.

Statistical Analysis

Heterogeneity of variance was reduced by logarithmically transforming the ALP, PA, and PGE₂ data before statistical analysis. ANOVA was used to determine treatment effects. When significant interactions (p < 0.05) were detected, Duncan's multiple-range tests were used for group comparisons. All statistical analyses were carried out using SAS statistical software (Cary, NC). Data for PGE₂ accumulation, ALP activity, and PA activity are presented as means (± SEM, n = 4–6) from single experiments. Experiments were performed more than once with different endometrial cell preparations. Because of significant differences between experiments, the data from different cell preparations have not been pooled. However, in all experiments, treatment effects were qualitatively similar.

	RESULTS

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Cells were incubated with or without EGF (40 ng/ml) in the presence or absence of IM (10 µM) for 24, 48, or 72 h after the differential attachment period. Compared to results in vehicle-treated controls, EGF caused significant (p < 0.01) increases in net PA activity in the medium at 24, 48, and 72 h (Fig. 1A). There was a significant interaction (p < 0.01) between the effects of EGF and time on net PA activity in the medium, a consequence of a greater effect of EGF at 72 h than that at 24 or 48 h. IM, an inhibitor of PG production, significantly (p < 0.001) reduced net PA activity in the medium. However, EGF significantly increased net PA activity in the medium in the presence of IM at each time. EGF had no significant (p > 0.05) effect on ALP activity in the cells (Fig. 1B). On the other hand, in the presence or absence of EGF, IM significantly (p < 0.001) reduced ALP activity in the cells. Compared to results in vehicle-treated controls, EGF significantly (p < 0.01) increased PGE₂ accumulation in the medium after incubations of 24, 48, and 72 h (Fig. 2A). IM significantly inhibited PGE₂ accumulation in the medium as indicated by the inability to detect it in the medium (< 0.5 ng/mg) of cells incubated with IM or IM plus EGF.

View larger version (20K): [in this window] [in a new window]   FIG. 1. Effects of EGF and IM on net PA activity in the medium (A) and ALP activity in the cells (B) of cultured rat endometrial stromal cells. Cells were incubated with vehicle (serum-free DMEM:F12, 0.5% ethanol), EGF (40 ng/ml), IM (10 µM), or EGF plus IM for 24, 48, or 72 h. Each point represents the mean ± SEM for 6 replicates.

View larger version (17K): [in this window] [in a new window]   FIG. 2. PGE2 accumulation in the medium of cultured rat endometrial stromal cells. A) Effects of EGF and IM: cells were incubated with vehicle (serum-free DMEM:F12, 0.5% ethanol), EGF (40 ng/ml), IM (10 µM), or EGF plus IM for 24, 48, or 72 h. B) Effect of IL-1: cells were incubated with vehicle or IL-1 (20 ng/ml) for 24, 48, or 72 h. Each point represents the mean ± SEM for 6 (A) or 5 (B) replicates.

Preliminary work indicated that IL-1 reduced net PA activity in the medium after treatment for 72 h (data not shown). Because previous work showed that PGE₂ stimulates PA activity in the medium of these cells [8], we sought to determine whether this inhibitory effect of IL-1 could be prevented by PGE₂. Cells were incubated with or without IL-1 (20 ng/ml) in the presence or absence of PGE₂ (1 µg/ml) for 24, 48, or 72 h after the differential attachment period. Compared to results in vehicle-treated controls, PGE₂ significantly (p < 0.05) increased net PA activity in the medium at 48 and 72 h, but not at 24 h (Fig. 3A). Although causing a small but significant (p < 0.05) increase at 24 h, IL-1 significantly (p < 0.05) reduced net PA activity in the medium after 48 and 72 h in the absence or presence of PGE₂. Analysis of variance indicated a significant interaction between the effects of IL-1 and PGE₂ on net PA activity at 72 h, but not at 48 h; the effects of PGE₂ and IL-1 on PA activity were additive and less than additive at 48 and 72 h, respectively. Both PGE₂ and IL-1 caused significant (p < 0.005) increases in ALP activity in the medium (Fig. 3B); these effects were less than additive at 48 and 72 h, as indicated by significant interaction (p < 0.05) upon analysis of variance. As shown in Figure 2B, IL-1 caused significant (p < 0.01) increases in PGE₂ accumulation in the medium at 24, 48, and 72 h, compared to controls. However, there was a significant (p < 0.01) interaction between the effects of IL-1 and time because the magnitude of the IL-1-induced PGE₂ accumulation differed between times, being the greatest at 48 h.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Effects of IL-1 and PGE2 on net PA activity in the medium (A) and ALP activity in the cells (B) of cultured rat endometrial stromal cells. Cells were incubated with vehicle (serum-free DMEM:F12, 0.5% ethanol), IL-1 (20 ng/ml), PGE2 (1 µg/ml), or IL-1 plus IM for 24 h. Each point represents the mean ± SEM for 4 and 5 replicates for PA activity and ALP activity, respectively.

Since IL-1 decreased net PA activity in the medium of the cultured rat endometrial stromal cells after 24 h of treatment, the next set of experiments was carried out to determine the effect of IL-1 on EGF-induced net PA activity. Cells were incubated with or without EGF (40 ng/ml) in the presence or absence of IL-1 (20 ng/ml) for 24, 48, or 72 h. Compared to vehicle treatment of controls, treatment with EGF alone significantly (p < 0.05) increased net PA activity in the medium at 24, 48, and 72 h (Fig. 4A). By contrast, treatment with IL-1 caused a significant (p < 0.05) increase in net PA activity at 24 h but a significant (p < 0.05) decrease at 48 h and 72 h in comparison to results in controls. There was a significant (p < 0.02) interaction between the effects of EGF and IL-1 on net PA activity at 24 h but not at 48 and 72 h. The respective stimulatory and inhibitory effects of EGF and IL-1 at 48 and 72 h were additive whereas the stimulatory effects at 24 h were less than additive. Compared to results in vehicle-treated controls, EGF and IL-1 each caused significant (p < 0.05) increases in PGE₂ accumulation in the medium at each time studied (Fig. 4B). As indicated by a significant (p < 0.001) interaction between the effects of EGF and IL-1, these stimulatory effects were less than additive at each time.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Effects of EGF and IL-1, alone or combined, on net PA activity (A) and PGE2 accumulation (B) in the medium of cultured rat endometrial stromal cells. Cells were incubated with vehicle (serum-free DMEM:F12), EGF (40 ng/ml), IL-1 (20 ng/ml), or EGF plus IL-1 for 24, 48, or 72 h. Each point represents the mean ± SEM for 4 replicates.

	DISCUSSION

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In the present study, incubation of rat endometrial stromal cells isolated from uteri sensitized for the decidual cell reaction with either EGF or IL-1 for up to 72 h increased PGE₂ accumulation in the medium. Because PGE₂ can modulate net PA activity in the medium [8], it was hypothesized that the EGF- and IL-1-induced increases in PGE₂ accumulation observed in the present study should be accompanied by an increase in net PA activity in the medium. EGF increased net PA activity in the medium, an effect not eliminated by inhibition of PG production by treatment with IM. Consequently, the effect of EGF on net PA activity cannot be entirely explained by an increase in PG accumulation. Although IL-1 caused a slight increase after 24 h, it greatly reduced net PA activity in the medium after 48 and 72 h even in the presence of exogenous PGE₂. Therefore, the effect of IL-1 on net PA activity in the medium cannot be predicted by its effect on PG accumulation in the medium.

The chromogenic assay used in this study measures net PA activity. Therefore, the changes in net PA activity in the medium in response to EGF and IL-1 may have been due to changes in the secretion of PAs and/or inhibitors of PAs (PAIs). The increase in PA activity in the medium might have been due to either an increase in PA secretion or a decrease in PAI secretion. Alternatively, the decrease in PA activity in the medium of cells incubated with IL-1 might have been due to decreased PA and/or increased PAI secretion, respectively. Further studies are required to determine the effects of EGF and IL-1 on the expression of PAs and PAIs in rat endometrial stromal cells.

PGE₂ enhances the in vitro decidualization [22] of rat endometrial stromal cells as indicated by cellular ALP activity. Since EGF and IL-1 significantly increased PGE₂ accumulation in the medium, it was hypothesized that incubation of the cells with EGF or IL-1 in the present study would enhance the decidualization of the cells. As expected, IL-1 significantly increased ALP activity in the cells; by contrast, EGF had no effect on ALP activity. Whether or not the effect of IL-1 was a consequence of its effect on PG accumulation was not investigated. However, the results of the present study show that IL-1 but not EGF enhances the decidualization of rat endometrial stromal cells in vitro. These findings differ from those in the human, in which it has been reported that EGF enhances [25] while IL-1 reduces [26] progesterone-dependent decidualization of cultured human endometrial stromal cells as assessed by prolactin secretion. It seems that the effects of ligands of the EGFR and IL-1R1 on the in vitro decidualization of rat and human endometrial stromal cells are not similar. Further studies are required to determine why the EGF-induced PG accumulation did not result in an increase in ALP activity.

Urokinase-type PA is expressed in the rat endometrium during decidualization. Tarachand and Pawse [27] have shown that PA activity in the rat uterus increases shortly after the onset of implantation and peaks at around 4 days later. They found a similar increase after an artificial induction of decidualization in pseudopregnant rats. Correlated with these changes in activity are changes in steady-state uPA mRNA levels in the uterus; the changes have been localized to the endometrial stromal cells undergoing decidualization [9]. Rat endometrial stromal cells secrete uPA while undergoing decidualization in vitro [8]. These data suggested that uPA may be a marker of decidualization. However, the present study shows that IL-1 enhances decidualization while decreasing net PA activity in the medium. Further, EGF increases net PA activity with no effect on decidualization. Therefore, measurement of net PA activity in the medium appears not to be a useful marker of decidualization in vitro. A similar conclusion was reached in studies in which endometrial stromal cells from rat uteri that were differentially sensitized for the decidual cell reaction in vivo and that undergo differing degrees of decidualization in vitro were cultured and PA activity in the medium determined [28].

In summary, the present study shows that IL-1 but not EGF can influence the decidualization of rat endometrial stromal cells in vitro. Further, EGF and IL-1 can each influence net PA activity in the medium. These results suggest that ligands of the EGFR and IL-1R1 may play a role in modulating the PA/plasmin system in the endometrium during decidualization. The PA/plasmin system is probably only a part of the biochemical mechanism involved in the control of tissue remodeling during decidualization. Although there is a decrease in the decidualization of the endometrium in uPA-deficient mice, gene deletion studies in mice show that uPA and tPA are not absolutely required for decidualization and implantation [29, 30]. This may be due to considerable functional redundancy of proteins involved in tissue remodeling [31]. More studies are required to understand how cytokines control the PA/plasmin system in conjunction with other proteins that may play a role in tissue remodeling in the uterus during implantation.

	ACKNOWLEDGMENTS

 
We would like to thank M.A. Shu, H.E. Ross, and G. Barbe for technical assistance.

	FOOTNOTES

 
¹ The Medical Research Council of Canada supported this research by Grant MT-10414 to T.G.K., and Grant MT-12107 to X.Z. who was also the recipient of a MRC Scholarship Award.

² Correspondence: T.G. Kennedy, Department of Physiology, The University of Western Ontario, M223 Medical Sciences Building, London, ON, Canada N6A 5C1. FAX: (519) 661-3827; tkennedy{at}physiology.uwo.ca

³ Current address: Department of Obstetrics and Gynecology, Northwestern University, Chicago, IL 60611.

Accepted: February 27, 1998.

Received: December 5, 1997.

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