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BOR - Papers in Press, published online ahead of print August 31, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.044925
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BIOLOGY OF REPRODUCTION 74, 13–22 (2006)
DOI: 10.1095/biolreprod.105.044925
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Difference in Progesterone-Receptor Isoforms Ratio Between Early and Late First-Trimester Human Trophoblast Is Associated with Differential Cell Invasion and Matrix Metalloproteinase 2 Expression

Shlomit Goldman 2, and Eliezer Shalev 1 23 

Laboratory for Research in Reproductive Sciences,2 Department of Obstetrics and Gynecology, Ha'Emek Medical Center, Afula 18101, Israel Rappaport Faculty of Medicine,3 Technion, Israel Institute of Technology, Haifa 31096, Israel

ABSTRACT

The expression profile of the progesterone-receptor isoforms and progesterone regulation of matrix metalloproteinase 2 (MMP2) were investigated in early and late first-trimester trophoblast cells. Human trophoblast cells were obtained from legal abortions (6–12 wk of gestation). Purity of 95–98% was verified using immunohistochemistry with specific antibodies. Evaluation of cell count was performed with XTT Reagent kit, and invasion was tested using Matrigel invasion assay. Zymography was used to detect proteolytic activity, and Western blot immunoassay was used to study protein concentration. Gene expression of PGRB, PGR, and MMP2 was studied using reverse transcription-polymerase chain reaction with the housekeeping gene GAPDH used for normalization. Promoter activity was determined using luciferase reporter assay. Differential progesterone-receptor profile was documented with the dominance of PGRB in early trophoblast and the dominance of PGRA in late trophoblast. This differential profile is compatible with the inverse effect of progesterone on the two cell populations, decreasing invasion and gelatinase expression in the early first-trimester trophoblast and increasing invasion and gelatinase expression in the late first-trimester trophoblast. A decrease in MMP2 promoter activity in early trophoblast cells exposed to progesterone suggests that MMP2 expression is regulated by progesterone at the transcriptional level as well. Early trophoblast cells transfected with expressing vector for PGR encoding PGRA revealed less MMP2 activity and reversal of its response to progesterone similar to the effect observed in late trophoblast cells.

gene regulation, mechanisms of hormone action, progesterone receptor, steroid hormone receptors, syncytiotrophoblast

INTRODUCTION

Successful implantation depends on the ability of the embryo to degrade the basement membrane of the uterine epithelium and to invade the uterine stroma. Trophoblast invasion is facilitated by degradation of the extracellular matrix of the endometrium/decidua, closely linked to the expression of matrix metalloproteinases (MMPs) [1–4]. In a previous study, we have shown that the gelatinases are differentially secreted by the first-trimester trophoblasts. Whereas in early first-trimester trophoblasts (6–8 wk of gestation) MMP2 is the main secreted gelatinase and the key enzyme in trophoblast invasion, in late first-trimester trophoblasts (9–11 wk of gestation) both MMP2 and MMP9 participate in trophoblast invasion [3]. The regulation of trophoblast invasion is still poorly understood [5, 6].

Progesterone is considered to be an essential hormone for development of endometrial receptivity and maintenance of the pregnancy [6, 7]. The development of a mouse model carrying a null mutation of the progesterone receptor (PGR) gene (Pgr) firmly established the essential role of this receptor in regulating key female reproductive processes. Female Pgr knockout mice, which are infertile, fail to ovulate. Furthermore, the uteri of these mutant mice are hyperplastic [8, 9]. The role of progesterone in the uterus is well documented, but data concerning the role of progesterone and its receptor in trophoblast invasion are very limited. Several studies have demonstrated that progesterone restrains endometrial tissue breakdown by inhibiting the stimulation of MMPs [10, 11]. Secretion of MMP9 from trophoblasts was reduced in the presence of progesterone [12]. The inhibitory effect of progesterone on the MMPs implies that progesterone also impedes the invasion of trophoblast cells into endometrial tissue. This phenomenon apparently contradicts the essential role of the hormone in the establishment and maintenance of pregnancy and deserves further clarifications. Progesterone acts through activation of PGRs A and B (PGRA and PGRB). The receptor isoforms, belonging to the family of nuclear receptors, are identical, except that PGRB has a longer NH2 terminus, consisting of 164 amino acids, not present in PGRA [13–15]. Both isoforms share an identical ligand-binding domain, which includes an activation function. The PGRA and PGRB can be considered as two independent receptors that display different transcriptional activities [16, 17]. It has been described that a different set of genes is regulated by progesterone in cells that express different PGR isoform profiles [16–19]. Only a limited number of publications concern the role of progesterone and its receptors on trophoblast cell invasion and MMP regulation, and the mechanisms of action are largely unknown. The present study was aimed to explore the expression profile of the progesterone-receptor isoforms in early and late first-trimester trophoblast cells and to investigate progesterone regulation of MMP2 in these cells.

MATERIALS AND METHODS

Ethics

The present study was approved by the ethical committee of Ha'Emek Medical Center in compliance with the Helsinki declaration. Consent was obtained from each of the participating patients.

Isolation and Cultivation of Human Cytotrophoblast Cells

First-trimester trophoblasts were divided into two groups: 6–8 wk (early) and 9–12 wk (late) of gestation. Human trophoblast cells were obtained from legal abortions (6–12 wk of gestation). Trophoblast cells were isolated as described previously [3]. Briefly, tissues were digested by 0.25% trypsin (Sigma) and DNase I (Sigma) at 37°C, and then trophoblast cells were separated from blood cells and decidua on a discontinuous Percoll gradient (Sigma) and immunopurified with magnetic antibody CD45RB (DAKO). This method supplies a 95–98% purity of trophoblastic cells, including all trophoblastic subgroups. We verified the purity of trophoblast cells using immunohistochemistry with specific antibodies to cytokeratin 7 (positive) and vimentin (negative), commonly used as an indication of trophoblast purity [20].

Culture Conditions

The cells were plated at 1–2 x 105 cells/well in 24-well plates or in Transwell plates (Corning) with M-199 medium supplemented with 1.5% fetal calf serum and 1% penicillin/streptomycin and kept in 5% CO2 at 37°C in the absence and presence of progesterone (progesterone water soluble p-7556; Sigma). Wide ranges of progesterone concentrations, varying from 10 ng/ml to 100 µg/ml, have been used in experiments with cell culture [12, 21–25]. In two separate studies of trophoblast cells, tested progesterone concentrations varied between 10–5 M to 2 x 10–4 M. Because the latter study [12] examined the effect of progesterone on gelatinase B, we tested the effect of progesterone between 2 x 10–6 M to 2 x 10–4 M

Cell Count Assay

Evaluation of cell proliferation was performed with XTT Reagent kit (cell proliferation kit; XTT) according to manufacturer's protocol. This assay is based on the activity of mitochondrial enzymes in live cells, reducing tetrazolium salts (XTT) into colored formazan compounds that can be detected colorimetrically with a spectrophotometer at 450 nm (ELISA reader). Dye absorbance is proportional to the number of cells in each well.

Substrate Gel Electrophoresis (Zymography)

To detect proteolytic activity in conditioned media collected after 48–72 h of culture, substrate gel electrophoresis (zymography) on gels containing gelatin as the substrate were used as described previously [26]. Briefly, conditioned media were diluted in sample buffer (5% SDS and 20% glycerol in 0.4 mol/L of Tris, pH 6.8, containing 0.02% bromophenol blue without 2-mercaptoethanol) and electrophoresed through a 10% polyacrylamide gel containing 0.5% gelatin (50 mg/ml). Afterward, gels were washed twice in 2.5% Triton X-100 for 15 min and incubated for 24 h at 37°C in 0.2 mol/L of NaCl, 5 mmol/L of CaCl2, 0.2% Brij 35, and 50 mmol/L of Tris (pH 7.5). The buffer was decanted and the gels stained with Coomassie blue G in 30% methanol and 10% acetic acid for 10 min at room temperature on a rotary shaker. Stain was washed out with water until clear bands were seen. Areas in which proteolytic activity degraded the gelatin were seen as an absence of staining. Identification of each gelatinase band was done in accordance to their molecular weight and commercial standards (gelatinize A and B, 7 µl; Oncogene Science; data not shown). These bands (pro-MMP) were quantified using the BioImaging gel documentation system (Dinco & Renum) endowed with TINA software (Raytest). The MMP secretion was expressed as a percentage of the control value.

Matrigel Invasion Assay

Matrigel invasion assay was prepared in our laboratory as described in detail previously [3]. Briefly, Matrigel (diluted 1:10; 1 mg/ml; BD Biosciences) in serum-free cell culture media was added to the upper chamber of a 24-well Transwell plate and incubated at 37°C for 3–4 h for gelling. The JAR cells were harvested from tissue culture by trypsin/EDTA, washed, and resuspended in 1.5% fetal calf serum in M-199 medium and added to the upper wells at a density of 105 cells/well in 200 µl of medium, whereas 500 µl of medium were added to the lower well. First-trimester trophoblasts were cultured in the upper wells at a density of 2 x 105 cells/well in 100 µl of medium. Cells, in the absence or presence of progesterone (2 x 10–4 M), were seeded at the same density in a well without Transwell and counted at time of the invasion assay as a reference of total cells. Preliminary studies found no significant Matrigel-mediated changes in multiplication rates between 6- to 8-wk and 9- to 12-wk trophoblasts, whether seeded on Matrigel or on the plastic bottom of the well (data not shown). Progesterone (2 x 10–4 M) and 2 µg/ml of inhibiting MMP2 or MMP9 antibodies (catalog nos. IM33L and IM09L, respecively, concentrations as recommended by manufacture; Oncogene Science) were added to the medium in the upper and lower wells. Plates were incubated at 37°C for 36–48 hours, and then noninvaded cells on top of the Transwell were scraped off with a cotton swab. The amount of invaded cells in the lower well as a percentage of total seeded cells was evaluated with XTT Reagent kit. The percentage invasion was calculated as (Absorbance of invaded cells/Absorbance of seeded cells) x 100 = Invasion index (%) where invasion was expressed as an Invasion index (% of control).

Preparation of Nuclear and Cytosolic Extracts

Nuclear and cytosolic extract proteins were prepared from the cell culture after the incubation period. Culture were lysed with ice-cold lysis buffer (10 mM Hepes [pH 7.9], 10 mM KCL, 1 mM EDTA, 1 mM dithiotheitol, 1 mM PMSF, 10 µg/ml of leupeptin, and 50 µg/ml of aprotinin). Suspensions were incubated for 15 min in 4°C, and Nonidet P-40 at a 0.4% final concentration was added. The cell suspension was centrifuged for 1 minute at 3000 rpm at 4°C, the supernatant containing the cytosolic fraction was removed; and the pellet was resuspended in the same lysis buffer, which contained 400 mM NaCl instead of KCl. After 15 min of incubation, the pellet suspensions were centrifuged for 5 min at 12000 rpm at 4°C. The nuclear extract was collected and stored at –20°C together with the cytosolic fraction until use.

Western Blot Analysis

Western blot analysis was conducted as described previously [26]. Briefly, to detect tissue inhibitor of metalloproteinase-1 (TIMP1) and PGR, conditioned media, cytosolic extracts, and nuclear extracts, together with mass marker (10 µl), were diluted with 4x sample buffer (5% SDS and 20% glycerol in 0.4 M Tris, pH 6.8, containing 0.02% bromophenol blue) and subjected to 10% polyacrylamide gel electrophoresis. After electrophoresis, the proteins (30 µg/lane) were blotted from the SDS-PAGE onto 0.45-µm nitrocellulose membranes (Scheicher & Schuel). Nonspecific binding sites were blocked by incubating the nitrocellulose membranes overnight with 20% nonfat milk and Tris-buffered saline containing 0.01% Tween-20. The membranes were then washed twice with Tris-buffered saline containing 0.5% Tween-20 and incubated for 1 h with either mouse anti-human TIMP1 antibody (1.0 µg/ml; Oncogene Science) or rabbit anti human PGR polyclonal antibody (1.0 µg/ml; sc-539; Santa Cruz Biotechnology) in 10% nonfat milk and Tris-buffered saline containing 0.01% Tween-20. The membranes were subsequently washed with Tris-buffered saline containing 0.5% Tween-20 and incubated for 1 h with horseradish peroxidase-conjugated anti-mouse rabbit secondary antibody (Jackson ImmunoResearch) in 10% nonfat milk and Tris-buffered saline containing 0.01% Tween-20, then detected by enhanced chemiluminescence (Amersham International) and quantified by densitometry as above.

Semiquantitative RT-PCR

Reverse transcription-polymerase chain reaction was conducted with the following primers: PGRB: forward, 5'-ACACCTTGCCTGAAGTTTCG-3'; reverse, 5'-CTGTCCTTTTCTGGGGGACT-3' (196-bp product); PGRAB: forward, 5'-TGGAAGAAATGACTGCATCG-3'; reverse, 5'-TAGGGCTTGGCTTTCATTTG-3' (196-bp product); MMP2: forward, 5'-ACCTGGATGCCGTCGTGGAC-3'; reverse, 5'-GTGGCAGCACCAGGGCAGC-3' (447-bp product). For normalization, we used the levels of the housekeeping gene GAPDH with the following primers: forward, 5'-TGATGACATCAAGAAGGTGGTGAAG-3'; reverse, 5'-TCCTTGGAGGCCATGTGGGCCAT-3' (240-bp product).

Total RNA was extracted from frozen samples with TRIzol reagent according to the manufacturer's instructions (Life Technologies, Inc.-BRL). The RNA concentrations were determined spectrophotometrically, and all RNA samples were stored at –80°C until used for cDNA synthesis. An RT kit (SuperScript preamplification system; Life Technologies, Inc.-BRL) was used in the synthesis and amplification of cDNA. Total RNA (5 µg) was denatured at 70°C for 10 min and then reverse transcribed in the presence of 25 ng/µl of oligo(deoxythymidine) primer (Life Technologies, Inc.), 2.5 mM MgCl2, 0.5 mM deoxy-NTPs, 10 mM dithiothreitol, and 10 U of ribonuclease H-reverse transcriptase (SuperScript II RT; Life Technologies, Inc.) for 60 min at 42°C and 5 min at 95°C. Subsequently, 10 µl of the resulting cDNA were used as a template for PCR. The PCR was set up using 3 mM MgCl2, 50 pmol of each primer, and 2.5 U of Taq DNA polymerase (Sigma). The PCR conditions were 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec, 60°C for 45 sec, and 72°C for 60 sec, with a 72°C extension for 10 min. After PCR, the products were resolved on a 2.5% agarose ethidium bromide gel. Images were captured with Polaroid film under ultraviolet light. Products were quantified using PhosphorImager and ImageQuant software (Molecular Dynamics, Inc.).

Protein Assay

The total protein content of trophoblast cells was determined using a protein assay kit with BSA as the standard (Bio-Rad Laboratories, Inc.). Five to twenty microliters of sample were used in the assay. The assay is based on the Bradford dye-binding procedure.

Transient Transfection and Luciferase Reporter Assay

The luciferase reporter gene, driven by either human MMP2 or rat MMP2 promoter, was used. The hpGL2-MMP2 (or rpGL2-MMP2) is a pGL2-basic reporter construct containing a full-length firefly luciferase gene under the control of human MMP2 (or rat MMP2) promoter. The hpGL2-MMP2 with the full-length human MMP2 promoter-luciferase (hpGL2-MMP2) was kindly provided by Dr. E.N. Benveniste (Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL) [27], and the rpGL2-MMP2 with the full-length rat MMP2 promoter (–1686 bp to the ATG codon) was kindly provided by Dr. Alan Wolfman (Department of Cell Biology, Cleveland Clinic Lerner College of Medicine, Cleveland, OH) and Dr. David Lovett (Department of Medicine, San Francisco Veterans Affairs Medical Center, University of California, San Francisco, CA) [28, 29]. Cells were transfected as described elsewhere in detail [29]. Briefly, 24 h before transfection, cells were plated in six-well plates at a density of 6 x 105 cells per well. Cells were transfected by LipofectAMINE/Plus Reagent (Invitrogen) with one of the following plasmids (4 µg): hpGL2-MMP2, rpGL2-MMP2, or PGL2-basic. The transfection was performed on primary cultures of human first-trimester trophoblast cells according to the manufacturer's directions (Invitrogen). Progesterone (2 x 10–4 M) was added immediately after transfection. Luc assays of resultant cell lysates were performed 48 h after transfection according to the manufacturer's instructions (Promega Corp.). Luciferase reporter enzyme activity was determined by correcting for ß-galactosidase and cell extract protein content as determined by the Bradford assay. Results from four independent experiments, each with duplicate wells, were averaged and presented as the mean ± SEM.

Transient Transfection of Human Progesterone-Receptor cDNA

Early first-trimester trophoblast cells were transfected as described previously [30] with the PGR expression vector hPGRA (pSG5-PGRA), which was generously provided by Prof. P. Chambon (CNRS, Inserm Universite Louis Pasteur, Strasbourg, France) [31]. Briefly, transient transfection was carried out 10 h after plating cells. Cells were transiently transfected with 0.5 µg of empty vector or expression vector hPGR2, which contained human PGR cDNA encoding PGRA, in pSG5 plasmid. Transfection was carried out overnight using LipofectAMINE/Plus Reagent (Invitrogen) according to the manufacturer's instructions. The next morning, cells were washed and incubated in serum-free M199 medium with and without progesterone (2 x 10–5 M and 2 x 10–4 M). After 24 h, cells were harvested for RNA extract and medium wad collected for MMP analysis. Data for MMP were normalized to the protein concentration in each sample. For normalization of the PGRA:PGRB ratio, we used the levels of the housekeeping gene GAPDH.

Statistical Analysis

Results are expressed as the mean ± SEM of 5–10 independent experiments, and each treatment was performed in duplicate. Statistical analysis was performed using the SPSS statistical software. Student t-test and one-way ANOVA were used when appropriate. A level of P < 0.05 was considered to be significant.

RESULTS

Dose-Dependent Effect of Progesterone on pro-MMP2 and pro-MMP9 Secretion by Early (6–8 wk) and Late (9–12 wk) First-Trimester Trophoblast

First-trimester trophoblast cells (2 x 105) obtained from early (6–8 wk) and late (9–12 wk) gestations were incubated 48 h in the absence or presence of progesterone (2 x 10–6 M to 2 x 10–4 M), and media were analyzed by zymography for gelatinase secretion. Progesterone significantly decreased secretion of pro-MMP2 in a dose-dependent manner in early trophoblast cells (P < 0.05). Incubation with progesterone (2 x 10–5 M) decreased pro-MMP2 secretion by 38% (P < 0.01) (Fig. 1, A and C, lane 3) and by 47% (P<0.01) (Fig. 1, A and C, lane 4) at a concentration of 2 x 10–4 M. To verify the progesterone effect on gelatinase secretion, 10–6 M mifepristone, a progesterone antagonist, was used. Mifepristone abolished the inhibitory effect of progesterone on pro-MMP2 in early trophoblast cells (Fig. 1, A and C). In sharp contrast to the early trophoblast cells, incubation of late first-trimester trophoblast cells with progesterone significantly enhanced both pro-MMP2 and pro-MMP9 secretion (P < 0.01) (Fig. 1, B and D). Incubation with progesterone (2 x 10–5 M) caused a 2.2- and 1.9-fold increase in the secretion of pro-MMP2 and pro-MMP9, respectively, as compared to control (P < 0.05) (Fig. 1, B and D, lane 3). Incubation with progesterone (2 x 10–4 M) caused a 2.3- and 2.1-fold increase in the secretion of pro-MMP2 and pro-MMP9, respectively, as compared to control (P < 0.05) (Fig. 1, B and D, lane 4). In late trophoblast cells, mifepristone at 10–6 M failed to antagonize the stimulatory effect induced by progesterone on gelatinase secretion in trophoblast cell (Fig. 1. B and D).


Figure 1
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  		FIG. 1. Secretion of pro-MMP2 and pro-MMP9 after 48 h of incubation of early and late first-trimester trophoblast (2 x 105) in the presence of progesterone (2 x 10–6 M to 2 x 10–4 M) and progesterone (P4; 2 x 10–4M) with mifepristone (RU 486; 10–6 M). Representative zymography gels are shown for early (6–8 wk) (A) and late (9–12 wk) (B) first-trimester trophoblast cells. Also shown are bar graphs representing the mean ± SEM from 10 independent experiments detecting pro-MMP2 (72 kDa) and pro-MMP9 (92 kDa) in early (C) and late (D) first-trimester trophoblast cells. Lane 1: control (Cont.); lane 2: 2 x 10–6 M progesterone; lane 3: 2 x 10–5 M progesterone; lane 4: 2 x 10–4 M progesterone; lane 5: 10–6 M mifepristone with 2 x 10–4 M P4. White bars represent pro-MMP9 secretion; black bars represent pro-MMP2 secretion. *P < 0.05 vs. control, **P < 0.01 vs. control

Effect of Progesterone on Cell Invasive Properties in Early (6–8 wk) and Late (9–12 wk) First-Trimester Trophoblast

First-trimester trophoblast cells of 6–8 wk and 9–12 wk (2 x 105 cells/well) were incubated for 48 h in the absence or presence of progesterone 2 x 10–4 M on top of a Transwell membrane coated with Matrigel. Progesterone decreased cell invasion in early trophoblast cells by 20% (P < 0.05) (Fig. 2A). In late (9–12 wk) first-trimester trophoblast cells, progesterone caused a 1.58-fold increase in cell invasion (P < 0.05) (Fig. 2A). The addition of inhibitory MMP2 antibody significantly decreased invasion of 6- to 8-wk trophoblast cells by 32% (P < 0.05) but did not affect 9- to 12-wk trophoblasts. No difference was found in the invasive property of early (6–8 wk) trophoblast cells induced with progesterone in the presence or absence of MMP2 antibody (Fig. 2A). A reduction of 15.8% in cell invasion was observed in progesterone-induced late trophoblast cells in the presence of inhibitory MMP2 antibody (P < 0.05). The presence of inhibitory MMP9 antibody in progesterone-induced cells (9–12 wk) decreased invasive property by 11.7% (P < 0.05) (Fig. 2A). To exclude the possible effect of the antibody itself, IgG was added to the culture medium, but IgG did not affect trophoblast invasive property (data not shown). To verify the progesterone effect on trophoblast cell invasion, 10–6 M mifepristone, a progesterone antagonist, was used. Mifepristone abolished the inhibitory effect of progesterone on early trophoblast cell invasive properties (Fig. 2B). In late trophoblast cells, mifepristone failed to antagonize the stimulatory effect induced by progesterone on trophoblast cell invasive properties (Fig. 2B). Based on these results, we speculate that in the early trophoblast cells, progesterone acts via its receptor, whereas in the late trophoblast cells, progesterone also may act via the nongenomic pathway.


Figure 2
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  		FIG. 2. Cell invasive property of early and late first-trimester trophoblast tested with Transwell Invasion Assay. A) Mean ± SEM from 10 independent experiments of cells treated in the absence or presence of progesterone (2 x 10–4 M) alone or in combination with either MMP2 or MMP9 inhibitory antibodies. Black bars represent early (6–8 wk) trophoblast cells. White bars represent late (9–12 wk) trophoblast cells. *P < 0.05 vs. control, ** P < 0.05 vs. late trophoblast in the presence of progesterone. B) Mean ± SEM from 10 independent experiments of cells with progesterone (2 x 10–4 M) alone or in combination with 10–6 M mifepristone. Black bars represent control, white bars cells treated with progesterone, and diagonal line bar cells treated with progesterone and mifepristone. *P < 0.05 vs. control

Time Course of Progesterone-Regulated Effect on MMP2 Expression in Early (6–8 wk) and Late (9–12 wk) First-Trimester Trophoblast

The inhibitory effect on MMP2 by progesterone was the most significant. To validate our results further, MMP2 gene expression was studied by RT-PCR. For normalization, we used the levels of the housekeeping gene GAPDH. Total RNA was extracted from early and late first-trimester trophoblast cells. We next examined the time course of progesterone-dependent regulation of MMP2 gene expression. Early (6–8 wk) and late (9–12 wk) trophoblast cells treated with progesterone for 1, 3, 6, and 24 h are shown in Figures 3 and 4. To exclude the possibility of fluctuation in gene expression during the 24-h period, we studied the basal MMP2 gene expression at 0 and 24 h without progesterone (Figs. 3 and 4). We detected the transcripts of MMP2 by semiquantitative RT-PCR (Figs. 3 and 4 show a representative 447-amplicon human MMP2 cDNA). In the early (6–8 wk) first-trimester trophoblast, progesterone significantly decreased MMP2 transcript expression level in a time-response manner (P < 0.05). To compare MMP2 mRNA relative expression levels between groups, we analyzed the ratio of each independent experiment between the expression level of either MMP2 and GAPDH from the same tissue under the same treatment. Rapid inhibition was observed for MMP2 (beginning after 3 h of incubation) (Fig. 3) in early (6–8 wk) trophoblast cells. After 24 h, MMP2 mRNA relative expression decreased by 85% (ratio: 0.78 ± 0.04 without progesterone vs. 0.0184 ± 0.003 with progesterone) (Fig. 3). Delayed induction pattern was noted for MMP2 in late (9–12 wk) trophoblast cells (Fig. 4). The relative level of MMP2 mRNA was increased after 6 h by 23-fold (P< 0.01; ratio: 0.032 ± 0.041 without progesterone vs. 0.748 ± 0.1 with progesterone) (Fig. 4) and reduced sharply after 24 h. Based on these results, we speculate that MMP2, which is rapidly inhibited, is a primary target of regulation by progesterone-bound PGR. In contrast, MMP2 in the late trophoblast cells, showing delayed induction, likely is a downstream target of early hormone inducible genes.


Figure 3
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  		FIG. 3. Time course for progesterone-dependent repression of MMP2 gene expression in early first-trimester trophoblast cells. A) Representative agarose gel of RT-PCR for MMP2 and GAPDH in the absence or presence of progesterone (2 x 10–4 M). B) Bar graph representing the mean ± SEM of the MMP2:GAPDH ratio from six independent experiments. Product size: MMP2, 447 bp; GAPDH, 240 bp. *P < 0.05. C, control (no progesterone)


Figure 4
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  		FIG. 4. Time course of progesterone-dependent induction of MMP2 gene expression in late first-trimester trophoblast. A) Representative agarose gel of RT-PCR for MMP2 and GAPDH in the absence or presence of progesterone (2 x 10–4 M). B) Bar graph represents the mean ± SEM of the MMP2:GAPDH ratio from six independent experiments. Product size: MMP2, 447 bp; GAPDH, 240 bp. **P < 0.01. C, control (no progesterone)

Transcriptional Regulation of the MMP2 Gene by Progesterone in Early (6–8 wk) and Late (9-12 wk) First-Trimester Trophoblast

To examine the effect of progesterone on MMP2 transcription, the human MMP2 and rat MMP2 promoters linked upstream of a luciferase reporter gene were transfected into early and late first-trimester trophoblast cells, which then were treated with progesterone. Cells were lysed, and luciferase assay was performed to determine the activity of human MMP2 and rat MMP2 promoters. Consistent with decreased MMP2 gelatinase activity together with the decrease of mRNA relative level, a significant decrease was observed in both human (Fig. 5B) and rat (Fig. 5A) MMP2 promoters activity in early (6–8 wk) trophoblast cells after treatment with progesterone. Progesterone significantly decreased human MMP2 promoter activity by 52.7% (P < 0.01) and the activity of the rat MMP2 promoter by 53.75% (P < 0.01). These results suggest that progesterone may directly regulate MMP2 expression at the transcriptional level. In the late trophoblast cells, addition of progesterone did not change human (Fig. 5B) or rat (Fig. 5A) MMP2 promoter activity. A significant difference in promoter activity in the untreated trophoblast of both (human and rat) was observed between early and late gestation. Human MMP2 promoter activity in early trophoblast cells was significantly higher than in late trophoblast cells (912 ± 62.21 vs. 74 ± 6.7 relative luciferase activities, respectively; P < 0.05) (Fig. 5B). Similar results were observed in the activity of rat MMP2 promoter (160.5 ± 8.17 vs. 99.25 ± 11.1 relative luciferase activity, respectively; P < 0.05) (Fig. 5A).


Figure 5
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  		FIG. 5. Promoter activity in early and late first-trimester trophoblast cells transiently transfected with PGL2-basic reporter with empty vector or with PGL2-basic reporter containing firefly luciferase gene under the control of full-length rat (A) or human (B) MMP2 promoters. Luciferase assay was used to measure the promoter activity. Results represent the mean ± SEM from six independent experiments. Black bar represents pGL2-basic without promoter, white bar transfected cells without treatment, and diagonal line bar transfected cells in the presence of progesterone (2 x 10–4 M). **P < 0.01 vs. untreated cells, *P < 0.05 vs. early trophoblast cells

Effect of Progesterone on TIMP1 Secretion by Early (6–8 wk) and Late (9–12 wk) First-Trimester Trophoblast

First-trimester trophoblast cells (2 x 105) obtained from 6–8 wk (early) and 9–12 wk (late) were incubated 48 h in the absence or presence of progesterone, and media were analyzed by Western blot analysis for TIMP1 secretion. Whereas in early trophoblast cells progesterone significantly enhanced TIMP1 secretion by 2.4-fold (P < 0.05) (Fig. 6A), in the late first-trimester trophoblast progesterone significantly reduced TIMP1 secretion by 73% (P < 0.01) (Fig. 6B).


Figure 6
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  		FIG. 6. Expression of TIMP1 in early and late first-trimester trophoblast. Representative Western blots for early (A) and late (B) trophoblast cells. C) Bar graph representing the mean ± SEM from six independent experiments. Black bar represents control, and white bar represents cells treated with progesterone (2 x 10–4 M). *P < 0.05, **P < 0.01

Progesterone-Receptor Expression in Early (6–8 wk) and Late (9–12 wk) First-Trimester Trophoblast

Nuclear and cytosolic extracts (30 µg/lane) from 12 independent experiments obtained from early (six experiments) and late (six experiments) first-trimester trophoblast cells were tested with Western blot analysis. Figure 7 shows both PGR isoforms, with the full-length PGRB of 116 kDa and the N-terminal truncated PGRA of 82 kDa expressed in the cytosol and nuclear fractions. To exclude the possibility of nonequal blot efficiency between lanes, the PGRB:PGRA ratio was calculated for each lane separately. The PGRB:PGRA ratio in the nucleus was significantly higher in early first-trimester trophoblast cells as compared with late first-trimester trophoblast cells (2.2 ± 0.23 vs. 0.48 ± 0.1, respectively; P<0.01) (Fig. 7, A and D). The level of PGRB expression was significantly higher then PRGA (1109 ± 12.6 vs. 504 ± 7.5 in arbitrary units, respectively; P < 0.05) in the early trophoblast. In the late trophoblast cells, the expression level of PGRB was significantly lower than that of PGRA (320 ± 6.7 vs. 666 ± 7.1 in arbitrary units, respectively; P < 0.05). Progesterone significantly increased the PGRB:PGRA ratio by 1.59-fold (P < 0.05) (Fig. 7, A and D) in 6- to 8-wk trophoblast while significantly decreasing the PGRB:PGRA ratio in 9- to 12-wk trophoblast by 43.5% (P < 0.05) (Fig. 7, A and D). A similar ratio was observed in the cytosolic fraction with a high PGRB:PGRA ratio in early first-trimester trophoblast cells as compared to that in late first-trimester trophoblast cells (Fig. 7, B–D). To validate our results further, PGRB gene expression was studied by RT-PCR. For normalization, we used the levels of the housekeeping gene GAPDH.


Figure 7
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  		FIG. 7. Expression of PGR isoforms in early and late first-trimester trophoblast. A) Representative Western blot gel from nuclear extract of early and late trophoblast (C, Control; P, cell treated with progesterone [2 x 10–4 M]). B and C) Cytosol extracts of early (B) and late (C) trophoblast. D) Bar graph showing the mean PGRB:PGRA ratio ± SEM of PGR isoforms from six independent experiments. Black bar represents early trophoblast cells, and white bar represents late trophoblast cells. *P < 0.05, **P < 0.01

Figure 8 shows a significant increase in the relative PGRB mRNA level in the early trophoblast cells in the presence of progesterone. To compare relative PGRB mRNA expression level between groups, we analyzed the ratio of each independent experiment between the expression level of PGRB and GAPDH from the same tissue under the same treatment. The expression level was increased by 1.42-fold without progesterone (ratio: 0.4 ± 0.06 vs. 0.568 ± 0.054; P < 0.001) with progesterone (Fig. 8). The opposite was observed in the late first-trimester trophoblast. The expression level of PGRB was decreased by 26% without progesterone (ratio: 0.7 ± 0.05 vs. 0.518 ± 0.019; P < 0.05) with progesterone (Fig. 8).


Figure 8
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  		FIG. 8. RT-PCR for PGRB in early and late first-trimester trophoblast cells in the absence (c) or presence (p) of progesterone (2 x 10–4 M). A) Representative agarose gel of PGRB and GAPDH. B) Bar graph representing the mean ± SEM of PGRB:GAPDH ratio from six independent experiments. Product size: PGRB, 196 bp; GAPDH, 240 bp. Black bar represents control, and white bar represents cells treated with progesterone. *P < 0.05, **P < 0.01

MMP2 Expression in Transfected Early (6–8 wk) First-Trimester trophoblast

As a complementary approach and a more direct way to assess the effect of PGR on MMP2 expression, we transfected early first-trimester trophoblast with vector hPGR2 containing human PGR cDNA encoding PGRA. Transfected early first-trimester trophoblast cells (1 x 106 transfected cells) revealed a different MMP profile as compared with that of cells transfected with empty vector (control 6- to 8-wk trophoblast cells). In early trophoblast cells transfected with PGRA, MMP2 was reduced, and MMP9 dominated (Fig. 9, C and E). The MMP9:MMP2 ratio in these cells was significantly higher than the ratio in control cells (2.5 ± 0.63 vs. 1.4 ± 0.52, respectively; P < 0.05). The profiles of MMP2 and MMP9 observed in the early trophoblast cells overexpressing PGRA were similar to those of control cells from late (9–12 wk) trophoblasts. Progesterone significantly increased MMP2 secretion by 2.2-fold in the early trophoblast cells overexpressing PGRA (Fig. 9, C and E, lane 3). This effect is similar to that of progesterone on late trophoblast cells.


Figure 9
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  		FIG. 9. A) Representative gel of RT-PCR for PGRB, PGR, and GAPDH in early first-trimester trophoblast cells (1, Control; 2, transfected cells). Product size: PGRAB, 196 bp; PGRB, 196 bp; GAPDH, 240 bp. B) Representative zymography gels of early trophoblast cells transfected with an empty vector. C) Early trophoblast cells transfected with PGRA-expressing vector. Lane 1: control; lane 2 = progesterone (2 x 10–5 M); lane 3, progesterone (2 x 10–4 M). D and E) Bar graphs representing the mean ± SEM from four independent experiments detecting pro-MMP9 (white bars) and pro-MMP2 (black bars) in control (D) and transfected (E) early first-trimester trophoblast cells. Lane 1: control; lane 2: progesterone (2 x 10–5 M); lane 3: progesterone (2 x 10–4 M). *P < 0.05 vs. control

DISCUSSION

Early human trophoblast has dramatic invasive properties [32]. In the present study, incubation of human first-trimester trophoblast with progesterone modified the invasive properties of the trophoblast cells and the expression and secretion of MMP2 and MMP9. These changes were inversely related to the gestational age of the trophoblast cells. In the early (6–8 wk) trophoblast cells, progesterone decreased pro-MMP2 secretion and expression and reduced cell invasive properties. An inverse effect was observed when progesterone was incubated with late (9–12 wk) trophoblast cells. Progesterone increased the secretion and the expression level of both MMPs as well as cell invasion. A similar inhibitory effect on cell invasive property was achieved by progesterone or by inhibitory MMP2 antibodies added to the growth media of the early trophoblast in vitro. No additive inhibitory effect was found on cell invasion following the addition of both progesterone and inhibitory antibodies to the growth media. Inhibitory MMP2 antibodies did abolish part of the invasion-stimulatory effect induced by progesterone on late trophoblast cells. These results further support the pivotal role played by the MMPs in trophoblast invasion [1–3]. In addition, MMP2 was documented, both by ourselves and by others, to be expressed more strongly in extravillous trophoblasts and vascular endothelial cells in the early first-trimester trophoblast [1–3]. It likely is the primary mediator in invasion of the trophoblast into the decidual endometrium as well as in vascular remodeling and angiogenesis.

Trophoblast invasion needs strict regulation, which calls for mechanisms that attenuate its activity. Progesterone has been shown to decrease gelatinase activity in many systems [10, 11]. The hormone reduces collagenolytic activity in uterine cervical fibroblasts by decreasing collagenase production and increasing TIMP activity [33]. The same mechanism seems to operate in controlling the changes that occur in the endometrium during menstruation [34]. A decrease in the progesterone level after regression of the corpus luteum permits collagenase activity, causing the breakdown of the endometrium [35]. The inhibitory effect of progesterone on the MMPs implies that progesterone also impedes the invasion of trophoblast cells into endometrial tissue. This phenomenon deserves clarification, because it contradicts the essential role of the hormone in the establishment and maintenance of a pregnancy. During implantation, the extravillous trophoblasts invade the uterine wall (interstitial invasion) and the spiral arteries (endovascular invasion), replacing the cells of the vessel wall and creating a high-flow, low-resistance vessel. This process involves two invasion waves of the cytotrophoblast inside the uterine wall. The first wave is strictly controlled to the implantation site and is completed at 8 wk of gestation. Following a pause phase, a second wave of invasion begins. This second wave is deep, comprising a third of the uterine wall, and is completed between 14 and 16 wk of gestation [36, 37]. Cytotrophoblast invasion is a unique process under the direction of uterine endo- and myometrium. Following the two cytotrophoblast invasion waves, a permanent uterine-placental blood circulation is formed [36, 37].

It has been suggested that several complications of late pregnancy, such as growth retardation and preeclampsia, have their origins in the earliest weeks of gestation [38–40]. Preeclampsia has been attributed, in part, to the failure of the extravillous cytotrophoblast to invade the maternal uterine spiral arteries to a sufficient depth at the second invasion wave, resulting in a poor vascular exchange between mother and placenta [39, 40]. Ultrasound placental resistance indices were found to be significantly higher from 8 wk of gestation in women that were more prone to develop pregnancy complications [41]. Progesterone is secreted at the beginning of pregnancy from the corpus luteum, followed by its secretion from the developing placenta. At the beginning of pregnancy, its level in the maternal plasma is approximately 20 ng/ml at the fourth week, rising to approximately 130 ng/ml toward the end of gestation [42]. However, the exact progesterone concentration at the fetomaternal interface is not clear. Several studies have found progesterone to be synthesized in large quantities, with its concentration reaching much higher levels (e.g., 1–10 µM, or 3200–32000 ng/ml) in humans [43]. It is tempting to suggest that under paracrine-autocrine regulation, the progesterone secretion level changes at the fetomaternal interface, subsequently regulating the invasive phenotype of the trophoblast cells, most probably via MMP2 expression.

The inhibitory effect of progesterone on MMP2 secretion, relative mRNA expression, and cell invasive properties, with an enhancement effect on TIMP1, are suggestive of an important role for the hormone in endocrine regulation of the first wave of trophoblast invasion. The finding that from the ninth week of gestation progesterone has an inductive effect on the trophoblast is an indication for its possible role in autocrine-paracrine regulation of the second wave of invasion. The antagonistic effect of RU-486 on progesterone in the early trophoblast cells indicates that progesterone most probably acts via its receptor, whereas in the late trophoblast cells, the failure of RU-486 to antagonize progesterone suggests that progesterone also might act via a nonclassical mechanism. Further support for this hypothesis came from the time course of progesterone-dependent regulation of MMP2 gene expression. In early trophoblast cells, MMP2, which is rapidly inhibited, is a primary target of regulation by progesterone-bound PGR. In contrast, in the late trophoblast cells, MMP2, which shows delayed induction, likely is a downstream target of early hormone-inducible genes. These data suggest that in the late trophoblast cells, progesterone also acts via a nongenomic pathway to regulate MMP2 expression. Clearly, transcriptional and signaling stimulatory activities of PGRs are independent [21, 44, 45]. Progesterone receptors are known to shuttle between nuclear and extranuclear compartments, and only a small amount of these receptors is required to stimulate the signaling. However, it is not clear whether, in addition to the shuttling, other mechanisms enhance the interaction of receptors with signaling proteins acting at the level of the cell membrane or cytoplasm [21, 45]. Progesterone can activate mitogen-activated protein kinase (otherwise known as Src-MEK1), the extracellular-regulated kinase pathway, and the phosphoinositol-3-phosphate, protein kinase C, mitogen-activated protein kinase, serine/theronine kinase AKT pathway. Activation of the two pathways is simultaneous, and both of them are required to trigger cell signaling [44, 45]. How a single hormone activates different signaling pathways, however, is still an open question. The hormone-triggered assembly of these macromolecular complexes has multiple effects and implications. It offers the opportunity of interfering specifically at the level of protein-protein interactions associated with the signaling pathway activation without affecting the transcriptional activity of the receptors [21, 44, 45].

Classically, progesterone acts via its PGRs. To our knowledge, the expression of PGR isoforms have not been demonstrated previously in human first-trimester trophoblast cells; the present study is the first to examine the relative expression of the PGRA and PGRB isoforms during early and late first-trimester trophoblast. In the early (6–8 wk) first-trimester trophoblast, PGRB expression dominates over PGRA. After the ninth week, this ratio is the inverse, with PGRA dominating over PGRB. Early first-trimester trophoblast transfected with human PGR cDNA encoding PGRA revealed a different gelatinase profile, similar to that of the late trophoblast cells. The transfected cells differed in their response to progesterone as compared with nontransfected cells. These data might suggest that the differing responsiveness of the cells to the same progesterone concentration related to the status of PGR rather than to progesterone itself. The PGRB:PGRA ratio was found to be clinically important in several tissues and, especially, in malignant processes, such as ductal carcinomas and invasive breast lesions [46, 47]. Loss of one PGR isoform with the expression of only single PGR isoform was more commonly observed in endometrial cancers and was associated with a higher clinical grade [48, 49]. An abnormally low concentration of PGR was found in intermediate trophoblastic cells following spontaneous abortions when compared to concentrations found with elective abortions [50]. Whether this is a primary or secondary event is unknown, but this information may be an important finding in attempting to characterize both the molecular etiology of implantation and the molecular pathophysiology of spontaneous abortion. Alterations in the ratio of PGR isoform expression likely cause disordered regulation of target genes, resulting in altered progestin action in the uterus [51]. Progesterone inhibits human endometrial cancer cell growth and invasiveness [52]. Overexpression of PGRB in endometrial carcinoma cells was found to inhibit invasive activity, suppressing MMPs [53, 54]. These results are consistent with our observations and hypothesis suggesting an inhibitory role for PGRB on cell invasion and MMP expression at the early stage of gestation in trophoblast cells.

The exact mechanism by which progesterone, via its receptor, inhibits MMP expression is poorly understood. Several studies mapping the potential response element on MMP2 promoters suggested the existence of eight major response elements [27, 28, 53]. It appears that the promoter of MMP2 lacks a progesterone-response element (PRE) [27, 28, 48]. Recently, it was suggested that progesterone could regulate several genes through a novel mechanism that involves interactions between PGR and several transcription factors on non-PRE DNA-binding sites [54–56]. In the present study, progesterone inhibited both human and rat MMP2 promoter activity, most probably by transcriptional cross-coupling between PGR and other transcription factors. In part, PGR isoforms may define the pathway by which each isoform interacts with transcriptional components, resulting in the modulation of a distinct repertoire of progesterone-regulated genes.

In late (9–12 wk) trophoblast cells, the basal MMP2 promoter activity is reduced significantly compared with that of early trophoblast cells, and progesterone failed to affect MMP2 promoter activity. The reduced basal activity is consistent with a reduction in MMP2 secretion from the ninth week of gestation. However, we cannot exclude the possibility that the low basal transcription level of MMP2 gene introduces technical difficulties. Because the PCR technique is more sensitive then the luciferase assay, it is possible that the stimulatory transcriptional regulatory effect of progesterone cannot be detected on these cells. The increase in MMP2 secretion observed in the late trophoblast cells after the addition of progesterone can be explained by the shift (decrease) in PGRB:PGRA ratio. It has been documented that PGRA and PGRB inversely regulate the same genes [16, 17]. In endometrial cancer, cells that express only PGRB were found to be the most proliferative and migrative, whereas cells that express only PGRA, or that express no PGR at all, showed minimal growth and spread [46, 47]. The exact mechanism of each receptor on the transcriptional mechanism is not fully understood and needs further investigation. These results open novel approaches by suggesting that progesterone-receptor status, more than progesterone concentration, is related to impaired implantation and several gestational pathologies.

ACKNOWLEDGMENTS

The authors thank Dr. E.N. Benveniste (Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL) for providing the human MMP2 promoter, Dr. Alan Wolfman (Department of Cell Biology, Cleveland Clinic Lerner College of Medicine, Cleveland, OH), Dr. David Lovett (Department of Medicine, San Francisco Veterans Affairs Medical Center, University of California, San Francisco, California) for providing the rat MMP2 promoter, and Prof. Pierre Chambon (Institute of Genetics and Molecular and Cellular Biology, Strasbourg, France) for kindly providing the progesterone-receptor expression vectors hPR1 and hPR2.

FOOTNOTES

1 Correspondence. FAX: 972 4 649 4032; shaleve{at}tx.technion.ac.il Back

Received: 30 June 2005.

First decision: 19 July 2005.

Accepted: 26 August 2005.

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