Mechanisms of matrix metalloproteinase-2 (mmp-2) transcriptional repression by progesterone in jar choriocarcinoma cells
© Goldman et al; licensee BioMed Central Ltd. 2009
Received: 07 April 2009
Accepted: 09 May 2009
Published: 09 May 2009
Although the MMP-2 promoter lacks a canonical progesterone response element (PRE), the hormone inhibits MMP-2 expression and is part of treatment protocols in gynecological invasive pathologies, including endometriosis and endometrial hyperplasia. This study aimed to explore the mechanism by which progesterone inhibits MMP-2 expression.
The effect of progesterone on MMP-2 expression in the JAR human choriocarcinoma cell line was analyzed by gelatin zymography. MMP-2 transcript expression was studied using Northern blot and semi-quantitative RT-PCR. Rat promoter deletion analysis, electrophoretic mobility shift and chromatin immuno-precipitation assays were performed in order to locate the DNA binding site and the transcription factors involved in MMP-2 regulation.
Progesterone significantly decreased secretion of pro-MMP-2 and MMP-2 transcript expression level in a dose-dependent manner. Progesterone (1 microM) significantly decreased both human and rat MMP-2 promoter activity (80.1% +/- 0.3 and 81.3% +/- 0.23, respectively). Progesterone acts through the SP1 family transcription factors-binding site, located between -1433 and -1342 bp region from the transcriptional start site of the rat MMP-2 promoter, which are present in the orthologous human MMP-2 promoter. Progesterone receptor (PR), SP2, SP3 and SP4 proteins are constitutively bound to this consensus sequence.
Progesterone reducesPR and SP4 binding to the MMP-2 promoter, thereby suppressing transcription. Progesterone also promotes SP4 degradation. These novel mechanisms of MMP-2 regulation by progesterone provide the biological rationale for the use of progesterone in clinical settings associated with increased MMP-2 expression.
Matrix metalloproteinase-2 (MMP-2) plays a critical role in invasion, metastasis, angiogenesis and tissue remodelling . The protein is widely expressed by a number of normal and transformed cells . Recent studies have highlighted the important role of MMP-2 in the invasive potential of metastatic endometrial [3, 4], ovarian  and trophoblast cells , as well as invasion of the normal trophoblast . The ability of trophoblasts to infiltrate the uterine wall and to anchor the placenta to it, as well as their ability to infiltrate and adjust utero-placental vessels to pregnancy, is dependent upon MMP-2 secretion . Abnormal MMP-2 secretion can also result in impaired implantation and is closely linked to several gestational pathologies, including repeated abortion , preeclampsia , gestational trophoblastic neoplasia and preterm labor [11, 12]. The activity of MMP-2 is regulated at multiple levels, including gene transcription, translation, proenzyme activation and inhibition by the tissue inhibitors of metalloproteinases . An extensive body of data has demonstrated a complex transcriptional regulatory network operative during both normal development and disease [[14–17], and ]. The human, and orthologous rat and murine MMP-2 proximal promoters lack a canonical TATA box and are composed of a relatively GC-rich region adjoining multiple transcriptional start sites. Functional binding sites for numerous transcription factors, including AP-1, AP-2, p53, nm-23β, and SP1/3 have been reported [14–19].
MMP-2 transcription is also regulated by reproductive hormones. Several studies have demonstrated induction of MMP-2 by estrogen in a variety of tissues, including vascular smooth muscle cells, glomerular mesangial cells and granulosa-lutein cells [20–22]. Estrogen responsiveness of human MMP-2 transcription was mapped to a half-palindromic binding site for the estrogen receptor (ERE), the activity of which was affected by an adjacent single nucleotide polymorphism . In contrast, we, and others, have demonstrated that progesterone (PG) inhibits MM-2 expression in a variety of tissues [7, 24–27]. This inhibitory effect of PG on MMP-2 expression occurs in the absence of a canonical progesterone response element (PRE) in the MMP-2 promoter. Progesterone has been part of the treatment protocol in number of conditions associated with enhanced MMP-2 secretion, including endometrial hyperplasia, imminent abortion and as luteal support in assisted reproduction [28–30]. Progesterone is also used as prophylactic treatment in women with a previous history of preterm labor .
Given the clinical importance of MMP-2 regulation by progesterone, we examined the transcriptional regulation of MMP-2 by progesterone using the model human JAR choriocarcinoma cell line. Our results support a novel mechanism by which progesterone inhibits MMP-2 expression. Within the context of human choriocarcinoma JAR cells basal transcription of MMP-2 is mediated by binding of primarily transcription factor SP4 to the MMP-2 proximal promoter. Progesterone suppresses MMP-2 transcription by reducing progesterone receptor (PR) and SP4 binding to the MMP-2 promoter. The inhibitory effect of progesterone on MMP-2 transcription may also result from enhanced proteasomal degradation of SP4/SP2.
Culture media and fetal calf serum (FCS) were obtained from Biological industries, Beit-Ha'Emek, Israel. Progesterone (P4) and Mifepristone (RU486) were obtained from Sigma (St. Louis, MO, USA); Antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The BCA assay kit was obtained from Bio-Rad Laboratories, Inc, USA. All of the other reagents, unless otherwise specified, were purchased from Sigma.
Human choriocarcinoma cell line (JAR) was obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in M199 medium containing 10% FCS in 60 mm culture plates, 24 wells and 96 wells kept in a humidified (37°C, 5% CO2) chamber. For the experiments described below, cells were trypsinized and harvested on reaching 80% to 90% confluence. The incubation period was 6–48 hours in the absence or presence of 0.1–10 μM progesterone (water soluble p-7556, Sigma, USA), and 10-6 M Mifepristone (RU486, Sigma, USA) in serum free M-199 medium supplemented 1% penicillin/streptomycin and kept in 5% CO2 at 37°C.
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 . 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.
Western blot analysis
Western blot analysis was conducted as described previously . To detect SP1, SP2, SP3, SP4, PR ubiquitin and GAPDH (for normalization), cell extracts (30 μg/lane) were diluted with 4× 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. Transfer and blocking were performed as reported . Blocked membranes were incubated for 1 h with either mouse anti-human SP1, SP2, SP3, SP4, ubiquitin and GAPDH antibody (1.0 μg/ml; Santa Cruz Biotechnology) or rabbit anti-human PR polyclonal antibody (1.0 μg/ml; sc-539; Santa Cruz Biotechnology) in 10% non-fat 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% non-fat milk and Tris-buffered saline containing 0.01% Tween-20, then detected by enhanced chemiluminescence (Amersham International) and quantified by densitometry as above.
The total protein content of trophoblast cells was determined using a protein assay kit with BSA as the standard (Bio-Rad Laboratories, Inc. USA).
JAR cells were washed 3 times with PBS and fixed with 3.7% paraformaldehyde in PBS for 20 minutes at room temperature (RT) and then permeabilized for 3 minutes with 0.1% Triton X-100 in PBS. Incubation for 1 hour with blocking buffer (PBS supplemented with 3% FCS serum) and three washings in PBS. The cells were then incubated with primary antibodies (anti-SP4, sc-645 (3 μl into 700 μl PBS supplemented with 1.5% FCS), for 30 minutes at RT, followed by additional five washings in PBS. Cells were incubated in the dark for 30 minutes at RT with fluorescein-labelled phalloidin (AlexaFluor-488, A-12379, Molecular Probes) for F-actin (3 μl into 700 μl PBS supplemented with 1.5% FCS) and the secondary goat anti-rabbit IgG antibodies were conjugated with Alexa Fluor-546 (1 μl into 700 μl PBS supplemented with 1.5% FCS, A-11035, Molecular Probes) or secondary goat anti-mouse IgG antibodies were conjugated with AlexaFluor-633 (1 μl into 700 μl PBS supplemented with 1.5% FCS, A-21052, Molecular Probes). Following washing in PBS, stained cells were photographed using a confocal microscope. The photos were analyzed by Image Pro software that quantifies density per area.
Reverse transcription-polymerase chain reaction was conducted with the following primers: MMP-2: forward, 5'-ACCTGGATGC-CGTCGTGGAC-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. A 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 and quantified using BioImaging gel documentation system (Dinco & Renum) endowed with TINA software (Raytest).
Firefly luciferase reporter plasmids incorporating either the human or rat MMP-2 promoters were prepared using the promoterless luciferase expression vector, pGL2-Basic (Promega). The human promoter pGL2-MMP-2 construct (denoted HpGL2-MMP2) extended to -1659 bp relative to the transcriptional start site was provided kindly by Dr. E.N. Benveniste . The rat promoter pGL2-MMP-2 construct (denoted RpGL2-MMP-2) extended to -1686 bp relative to the translational start site. Serial deletion constructs of the RpGL2-MMP-2 extended to 1007, 573, 383, 321, 267 bp relative to the translational start site. A second series of serial deletion constructs extended from 1686 bp to 1433 bp, 1345 bp, 1262 bp and 1181 bp.
Transient transfection and luciferase assays
Cells were transfected as described elsewhere in detail . Briefly, 24 h before transfection, cells were plated in 24 or 96-well plates at a density of 6 × 105 cells per well. Cells were transfected by LipofectAMINE/Plus Reagent (Invitrogen) with full length or constructs plasmids (4 μg) or PGL2-basic (empty vector). The transfection was performed on JAR cells according to the manufacturer's directions (Invitrogen). Progesterone (1 μM) was added immediately after transfection. Luciferase assays of resultant cell lysates were performed 6 h after progesterone treatment according to the manufacturer's instructions (Promega Corp.). Luciferase reporter enzyme activity was determined by correcting for β-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.
Nuclear extract preparation
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.
Electrophoretic mobility shift assay
Chromatin immunoprecipitation (ChIP) Assay
Cells were fixed with 1% formaldehyde at 37°C for 10 min. Cells were washed twice with ice-cold PBS with protease inhibitors (1 mM phenylmethylsulphonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml pepstatin A), scraped and pelleted by centrifugation at 4°C. Cells were resuspended in a lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris-HCl, pH 8.1), incubated for 10 min on ice, and sonicated to shear DNA. After sonication, lysate was centrifuged for 10 min at 13,000 rpm at 4°C. The supernatant was diluted in CHIP dilution buffer (0.01% SDS, 1% Triton X-100, 2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, and protease inhibitors). Primary antibodies were added and incubated overnight at 4°C with rotation. The immuno-complex was collected by protein A/G agarose beads and washed with low salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 200 mM Tris-HCl, pH 8.1, and 150 mM NaCl), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 200 mM Tris-HCl, pH 8.1, and 500 mM NaCl), LiCl washing buffer (0.25 M LiCl, 1% NP40, 1% deoxycolate, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.1), and finally 1' TE buffer (10 mM Tris-HCl, and 1 mM EDTA, pH 8.0). After that, the immuno-complex was eluted by the elution buffer (1% SDS, 0.1 M NaHCO3, and 200 mM NaCl) and the cross-links were reversed by heating at 65°C for 6 h. After reaction, the samples were adjusted to 10 mM EDTA, 20 mM Tris-HCl, pH 6.5, and 40 μg/ml proteinase K, and incubated at 45°C for 1 h. DNA was recovered and was subjected to PCR amplification of the rat MMP-2 promoter which contains the SP1 site at the -1433/-1342 bp region compliance with human promoter (Figure 1). The sequences for the primers are: forward 5-ATCACTGGCTCTCCAACTTGG-3,; reverse 5-TTAGCTCGCAAGGAGTCTCTT-3. The predicted size for the PCR product is 250 bp.
Ubiquitinated SP4 protein immuno-precipitation
JAR cells were seeded into 60 mm tissue culture plates in maintenance medium and allowed to grow to approximately 90% confluence. Cells were treated with progesterone 1 μM for 2 h. Whole-cell extracts for the control and treatment group were obtained using RIPA buffer (50 mM Tris-HCL, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) with the addition of protease inhibitor cocktail. Duplicate aliquots of 500 μg were used for the experiments. Cell extracts were diluted in ice-cold PBS containing protease inhibitor cocktail to a final volume of 1 ml, followed by the addition of 30 μl of protein A/G PLUS-agarose beads (Santa Cruz, USA). The reactions were placed on a rocker at 4°C for 3 h, followed by 600 g centrifugation at 4°C for 5 min. A 900-μl aliquot of supernatant from each sample was transferred into a new Eppendurf tube on ice. Rabbit polyclonal anti-Sp4 (1 μg), SP1 (1 μg), SP2 (1 μg), SP3 (1 μg), or normal rabbit IgG (1 μg) was added to either control or treatment set, followed by the addition of 30 μl of protein A/G PLUS-agarose beads. The samples were then placed on a rocker at 4°C overnight, followed by centrifugation at 2500 rpm at 4°C for 5 min. The supernatant was removed by aspiration and the immuno-precipitates were washed with two cycles of 1 ml of ice-cold RIPA buffer followed by 1 ml of ice-cold PBS using centrifugation at 600 g at 4°C for 5 min. The agarose pellet was resuspended in 40 μl of loading buffer, boiled, and centrifuged. The supernatant was separated by SDS-10% PAGE, electrophoresed. Total protein (input) was tested for either, SP1, SP2, SP3, SP4, PR, ubiquitin and GAPDH. The immuno-precipitant was tested for the presence of ubiquitin.
Results are expressed as the mean ± SEM of 3 to 4 independent experiments where each treatment was performed in duplicate. Statistical analysis was performed using the SPSS statistical software, guided by the statistician annalist from our central statistical centre. A level of P < 0.05 was considered to be significant.
Progesterone inhibits MMP-2 expression by JAR choriocarcinoma cells
The time course of progesterone-dependent regulation of MMP-2 gene expression was examined. Transcript expression of MMP-2 in JAR choriocarcinoma trophoblast cells treated with progesterone for several time intervals is presented using Northern blot (Figure 2B) and semi-quantitative RT-PCR (Figure 2C). Progesterone (1 μM) significantly decreased MMP-2 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 the house keeping gene GAPDH from the same tissue under the same treatment. Inhibition was observed after 4 h of incubation.
Progesterone inhibits MMP-2 via transcriptional repression
Progesterone suppresses MMP-2 promoter activity via SP1 site
In MMP-2 5'-deletion construct extending to -1502 bp the inhibitory effect of progesterone was slightly reduced, however in the -1433 bp deletion construct progesterone significantly increased its ability to inhibit luciferase activity (153 ± 10.1 versus 101.2 ± 7.1 percent; P < 0.05), (Figure 4B). This inhibitory effect of progesterone was abolished in the -1342 bp deletion construct (92.8 ± 12 and 119.3 ± 11 percent of luciferase activity; P < 0.05). The progesterone-responsive element should be therefore located between -1432 to -1342 bp region of the rat-MMP-2 promoter. In the -1262 bp and -1181 bp deletion constructs no effect of progesterone was observed.
Transcription factor binding site analysis  localized three potential SP1 binding sites in this sequence that is conserved in the homologous human MMP-2 promoter sequence (Figure 1). The sequence analyses suggest that the inhibitor effect of progesterone on MMP-2 transcription may involve interaction of members of the SP1 transcription factor family with specific binding sites in the 91 bp sequence extending between -1433 and -1342 bp of the rat MMP-2 promoter and the corresponding region of the human MMP-2 promoter.
Progesterone reduces transcription factors binding to the response element on the MMP-2 promoter
In order to study the possible mechanism for the reduction in SP4 binding following progesterone treatment, western blot for the classical SP1 family was performed with and without progesterone. Results (Figure 6B) show that SP1, SP2, SP3 and SP4 are expressed in JAR cells. Following treatment with progesterone only SP4 expression is reduced significantly. These results suggested that progesterone not only affects SP4/PR binding to the intrinsic MMP-2 promoter but may also affect SP4 protein levels, per se.
Role of progesterone on SP4 degradation
The effects of progesterone on Ubiquitin binding capacity to the SP1, SP2, SP3, SP4 and PR proteins were investigated. (Figure 7B). Whole-cell lysates from control and progesterone-treated groups were initially immunoprecipitated with a control IgG or antibodies to SP1, SP2, SP3, SP4 and PR. Immunoprecipitated fractions were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-ubiquitin antibodies. Antibodies to GAPDH were used as an internal control (data not shown). The addition of progesterone induced specific ubiquitin binding only to the SP4 protein.
Synthetic progestational agents have been used extensively in invasive gynecological pathologies such as endometriosis  and endometrial hyperplasia . Progesterone is a paradoxical hormone having either stimulatory or inhibitory effects, depending on the tissue in question and the dose and treatment regimen [34, 35]. Classical P4 activity is mediated by interaction of the hormone with the two classical intracellular progesterone receptor (PR) isoforms PR-A and PR-B [34, 35]. Both isoforms, encoded by a single gene independently regulated by separate promoter, regulate different subsets of genes. PR frequently acts as ligand-inducible transcription factor in the respective target cell by binding to specific progesterone response elements (PRE) in the promoter of target genes . The mechanisms of progesterone are only partial explained by their isoforms profile ratio expression.
Recent reports indicate that progesterone can also regulate promoters lacking a canonical progesterone response element [37–40]. In these cases, progesterone receptors interact indirectly with the promoter through complex formation with various transcription factors, including several members of the SP1 transcription factor family [38, 41, 37].
We have previously reported that progesterone inhibits MMP-2 synthesis in reproductive tissues . JAR choriocarcinoma cells synthesize high levels of MMP-2, which is responsible for the highly invasive nature of these cells. In this report we demonstrate that this inhibitory effect of progesterone occurs at a transcriptional level in the absence of a canonical PRE in the human or rat MMP-2 promoters and this inhibitory effect is reversed by Mifepristone. Using a combination of techniques, we mapped the region responsible for high level MMP-2 transcription in JAR cells to a GC-rich region in the MMP-2 proximal promoter which contained several potential SP1 transcription family binding sites. In the basal state the MMP-2 promoter is occupied by SP2/PR and SP4/PR complexes. Addition of progesterone reduces MMP-2 transcription through a reduction in SP4 and in PR binding to the intrinsic MMP-2 promoter.
In addition to displacement of SP4/PR binding from the MMP-2 promoter, progesterone also affects the net concentration of the SP4 protein. Addition of progesterone to JAR cells specifically promoted degradation of the SP4 protein, while there was no effect on SP1-3 protein degradation. The failure of Mifepristone (progesterone receptor antagonist) to reverse this progesterone effect, together with lack of effect by CHX, might suggest that progesterone is involved, via non classical mechanism, in SP4 protein degradation rather than inhibition of the protein synthesis. The ability of progesterone to enhance specific ubiquitin binding to SP4 protein after four hours incubation might suggest that ubiquitination of the SP4 protein would result in enhanced rates of SP4 protein degradation in the proteasome, thereby reducing SP4 protein levels and the ability to form transactivating SP4/PR complexes. Repression of SP4-mediated transactivation via enhanced proteasomal degradation may be a more common event than previously recognized. Adelrahim and Safe, , recently demonstrated that cyclooxygenase inhibitors decrease vascular endothelial cell growth factor expression via enhanced degradation.
Progesterone reduces PR and SP4 binding to the MMP-2 promoter and promotes SP4 degradation, thereby suppressing MMP-2 promoter transcription. These findings provides further example for the complex nature of progesterone-dependent transcriptional regulation, particularly in the absence of a canonical PRE in the proximal promoter. To our knowledge, the mechanisms of progesterone-mediated MMP-2 transcriptional suppression are unique and provide a firm biological basis for the clinical application of progesterone in the treatment of gynecologic and obstetric disorders associated with increased MMP-2 expression.
The authors thank Dr. E.N. Benveniste (Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL) for providing the human MMP-2 promoter. This work was support by the Israel Ministry of Health Chief Scientist Grant No. 3-3228 (S.G. and E.S.) and the National Institutes of Health RO1 DK 39776 (D.H.L.).
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