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Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5
Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, Room 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca.
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Introduction |
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The urokinase plasminogen activator (uPA) and its endogenous inhibitor, plasminogen activator inhibitor type 1 (PAI-1), play key roles in the highly regulated series of remodeling events that occur in the endometrium in preparation for pregnancy (6, 7, 8). In particular, uPA is believed to mediate, at least in part, the degradation of the endometrial extracellular matrix (ECM) underlying the development of the decidua and the regulated invasion of extravillous cytotrophoblasts. The proteolytic activity of uPA at the maternal-fetal interface is counterbalanced, in both an autocrine and a paracrine manner, by PAI-1 secreted by the decidual cells and the subpopulation(s) of highly invasive extravillous cytotrophoblasts. Although it is recognized that the remodeling of the decidual ECM is a critical step in the establishment of pregnancy, the factors capable of regulating the coordinate expression of uPA and PAI-1 in this dynamic tissue remain poorly characterized.
We have recently determined that both the classical form of GnRH (GnRH I) and the second mammalian form of this hormone (GnRH II) are key regulators of uPA-1 and PAI-1 mRNA and protein expression levels in extravilllous cytotrophoblasts propagated from first trimester placental explants (9). In particular, GnRH I and GnRH II increased uPA and concomitantly decreased PAI-1 expression levels in these primary cell cultures. As GnRH I and GnRH II are secreted by both human placenta and endometrium (10, 11, 12, 13), it is tempting to speculate that these two hormones may be key regulators of the proteolytic degradation of ECM that occurs at the maternal-fetal interface during early pregnancy. To gain a better understanding of the role(s) of GnRH I and GnRH II in this developmental process, we examined the ability of GnRH I and GnRH II to regulate uPA and PAI-1 mRNA and protein expression levels in primary cultures of stromal cells isolated from first trimester decidual tissues in a dose- and time-dependent manner.
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Materials and Methods |
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Tissue samples of first trimester decidua parietalis were obtained from women undergoing elective termination of pregnancy. The use of these tissues was approved by the committee for ethical review of research involving human subjects, University of British Columbia. All patients provided informed written consent.
Cell isolation and culture
Stromal cells were isolated from the decidual tissue samples by enzymatic digestion and mechanical dissociation using a protocol modified from that reported by Shiokawa et al. (14). Briefly, the decidual tissue samples were minced and subjected to 0.1% collagenase (type IV; Sigma-Aldrich, St. Louis, MO) and 0.1% hyaluronidase (type I-S) digestion in a shaking water bath at 37 C for 60 min. The cell digest was then passed through a nylon sieve (38 µm). The isolated glands and any undigested tissue fragments were retained on the sieve, and the eluate containing the stromal cells was collected in a 50-ml tube. The stromal cells were then pelleted by centrifugation at 800 x g for 10 min at room temperature. The cell pellet was washed once, resuspended, and plated in DMEM containing 25 mM glucose, L-glutamine, antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin), and supplemented with 10% fetal bovine serum, 17ß-estradiol (10 nM; Sigma-Aldrich), and progesterone (1 µM; Sigma-Aldrich). The culture medium was replaced 30 min after plating to reduce epithelial cell contamination. The purity of the decidual stromal cell cultures was determined by immunocytochemical staining for vimentin, cytokeratin, muscle actin, and factor VIII (data not shown). These cellular markers have been used previously to determine the purity of human endometrial cell cultures (15). As defined by these criteria, the decidual stromal cell cultures used in these studies contained less than 1% epithelial or vascular cells.
Hormone treatments
Decidual stromal cells (passage 2) were plated in 35-mm2 tissue culture dishes (BD Biosciences, Franklin Lakes, NJ) at a density of 1 x 106 cells/dish and grown to 80% confluence. The cells were then cultured in the presence of increasing concentrations of GnRH I or GnRH II (0, 0.1, 1, 10, or 100 nM) for 24 h or a fixed concentration of GnRH I or GnRH II (100 nM) for 0, 3, 6, 12, 24, or 48 h. In addition, decidual stromal cell cultures were treated with a combination of GnRH I or GnRH II (100 nM) and increasing concentrations (0, 1, 10, or 100 nM) of the GnRH I antagonist, Cetrorelix (AnaSpec, Inc., San Jose, CA), for 24 h. Cells treated with vehicle (0.1% ethanol) alone served as a control for these experiments. The viability of the decidual cell cultures under these experimental conditions was determined by a trypan blue exclusion assay.
Primer design
The design and nucleotide sequences of the primer sets specific for uPA, or PAI-1 used in the quantitative competitive-PCR (QC-PCR) analysis, or the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase, used to quantify and assess the integrity of the total RNA samples, have been described in detail previously (9).
RNA preparation and RT-PCR
Total RNA was prepared from the decidual stromal cell cultures with an RNeasy Mini Kit (Qiagen, Inc., Mississauga, Canada) using a protocol recommended by the manufacturer. The concentration of total RNA present in each of the extracts was quantified by optical densitometry (260/280 nm) using a Du-64 UV spectrophotometer (Beckman Coulter, Fullerton, CA). An aliquot (1 µg) of the total RNA extracts prepared from these cell cultures was reverse transcribed into cDNA using a First Strand cDNA Synthesis Kit according to the manufacturer’s protocol (Amersham Pharmacia Biotech, Oakville, Canada).
PCR was performed using template cDNA generated from the total RNA extracts prepared from cultures of untreated decidual stromal cells and the primer sets specific for glyceraldehyde-3-phosphate dehydrogenase, uPA, or PAI-1. The PCR conditions were as follows: 1 min at 94 C; 1 min at 57.5 or 56 C for PAI-1 and uPA, respectively; and 1.5 min at 72 C, followed by a final extension at 72 C for 15 min. The cycles were repeated 20–35 times. A linear relationship between the yield of the PCR products and the number of cycles performed was observed after 27 cycles for uPA and after 30 cycles for PAI-1 (data not shown).
The resultant PCR products for uPA and PAI-1 were separated using gel electrophoresis and visualized by ethidium bromide staining (data not shown). An aliquot of these uPA and PAI-1 PCR products was subcloned into the PCR II vector (Invitrogen, Carlsbad, CA) and subjected to DNA sequence analysis to confirm the specificity of the primers. These clones were also used to generate target or internal standard uPA and PAI-1 cDNA fragments by standard molecular biology techniques.
QC-PCR
The QC-PCR strategy employed in these studies is based upon the competitive co-amplification of a known amount of an internal standard specific for uPA or PAI-1 added to aliquots of the first strand cDNA prepared from our primary cultures of decidual stromal cells (9, 16, 17).
To determine the optimal amounts of the internal standards to be used in the QC-PCR analysis, PCR reaction mixtures containing a fixed amounts of the target uPAI-1 or PAI-1 cDNAs (1 µl) and decreasing concentrations of the corresponding internal standard cDNAs (8–0.0625 pg/µl for uPA or 8–0.125 pg/µl PAI-1, respectively) were prepared. PCR was then performed using these cDNA mixtures and the distinct uPA or PAI-1 primer sets under the optimized conditions described above.
An aliquot (10 µl) of the resultant uPA and PAI-1 PCR products was separated by electrophoresis in a 1% agarose gel and visualized by ethidium bromide staining (Fig. 1
). The intensity of the ethidium bromide staining of the PCR products was analyzed using UV densitometry (Biometra, Whiteman Co., Gottigen, German). Volume counts (square millimeters) of the PCR products were then determined using Scion Image computer software (Scion Image Co., Frederick, MD). The absorbance values obtained for each of the target and corresponding internal standard were plotted against the amount of internal standard added to the initial reaction mixtures, with the point of interception on these line graphs being taken as the optimal amount of internal standard to be used in the QC-PCR analysis (Fig. 1). Based upon these observations, uPA or PAI-1 internal standard cDNAs were added to aliquots of the first strand cDNA generated from the human decidual cells to be used in the QC-PCR analysis at concentrations of 1 or 0.5 pg/µl, respectively.
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. These standard curves were highly reproducible and linear. The values obtained from these regression curves (y = b + mx) were subsequently used to quantify the levels of uPA and PAI-1 mRNA transcripts present in our decidual stromal cell cultures. QC-PCR was performed using the uPA or PAI-1 primer sets and 1 µl of the first strand cDNA synthesized from each of the treated decidual stromal cell cultures containing the optimized amount of the corresponding internal standard cDNA under the PCR conditions described above. The ratios of the intensity of ethidium bromide staining of the resultant target/internal standard PCR products were logarithmically transformed and compared to the values obtained from the standard curves.
ELISA
The expression levels of uPA and PAI-1 in the conditioned medium collected from the decidual stromal cell cultures were measured using ELISA kits for uPA or PAI-1 (American Diagnostica, Inc., Greenwich, CA). uPA was detected in the conditioned culture medium with mean intra- and interassay coefficients of variation of 4.9% and 8.2%, respectively, whereas PAI-1 was detected in the conditioned medium with mean intra- and interassay coefficients of variation of 6.1% and 8.8%, respectively. All samples were assayed in duplicate.
Statistical analysis
The absorbance values obtained from the ethidium bromide-stained gels were subjected to statistical analysis using GraphPad PRISM 2 computer software (San Diego, CA). Statistical differences between the absorbance values were assessed by ANOVA. Differences were considered significant for P 
0.05. Significant differences between the means were determined using Dunnett’s test. The results are presented as the mean relative absorbance ± SEM obtained from five independent experiments.
Statistical differences between the dose- or time-dependent effects of GnRH I or GnRH II on uPA or PAI-1 protein expression levels detected in the conditioned media of decidual stromal cells were assessed by ANOVA, followed by Dunnett’s test. The results are presented as the mean protein expression levels ± SEM obtained from five independent experiments.
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Results |
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u-PA mRNA transcripts and protein expression were detected in all of the decidual stromal cell cultures. The addition of vehicle to the culture medium had no significant effect on the uPA mRNA and protein expression levels in these primary cell cultures at any of the time points examined in these studies (data not shown). In contrast, GnRH I and GnRH II increased uPA mRNA levels and protein expression in the cultured decidual stromal cells in a dose-dependent manner (Fig. 2).
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). Although there was a slight decline in uPA mRNA levels in decidual cells cultured in the presence of GnRH I for 48 h, the levels of this mRNA transcript were still significantly greater than those detected in the 0 h control. A significant and progressive increase in uPA protein expression levels was detected in these cell cultures until the termination of these studies at 48 h.
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). A coordinate increase in the expression levels of uPA in these decidual stromal cell cultures was also observed at all of the time points examined (Fig. 3). Differential effects of GnRH I and GnRH II on PAI-1 mRNA and protein levels in decidual stromal cells
PAI-1 mRNA transcripts and protein expression were detected in all of the decidual stromal cell cultures. The addition of vehicle to the culture medium had no significant effect on PAI-1 mRNA or protein expression levels in these cells at any of the time points examined in these studies (data not shown).
GnRH I increased, whereas GnRH II decreased, PAI-1 mRNA and protein expression levels in our primary cultures of decidual stromal cells in a dose-dependent manner (Fig. 4).
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). There was a subsequent and progressive decline in PAI-1 mRNA and protein expression levels in these primary cell cultures until the termination of these studies at 48 h.
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). Effects of Cetrorelix on the GnRH I- or GnRH II-mediated regulation of uPA and PAI-1 mRNA and protein expression levels in decidual stromal cells
Cetrorelix inhibited the stimulatory effects of GnRH I on uPA mRNA and protein expression levels in cultured decidual stromal cells in a dose-dependent manner. In contrast, Cetrorelix had no significant effect on the GnRH II-mediated increase in uPA mRNA and protein levels in these primary cell cultures at any of the concentrations examined in these studies (Fig. 6).
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The ratio of uPA/PAI-1 expression levels in the samples of conditioned media obtained from decidual cells cultured in the presence of GnRH II was significantly greater than those obtained from cells treated with GnRH I at all of the hormone concentrations examined in these studies (Fig. 8).
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Discussion |
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GnRH II has been shown to mimic the biological actions of GnRH I (21). Often, the effects of GnRH II on extrapituitary tissues and cells appear to be significantly greater than those observed with native GnRH I and/or its synthetic analogs. For example, the antiproliferative effects of GnRH II on human endometrial and ovarian cancer cells were significantly greater than those observed in cells cultured in the presence of equivalent concentrations of GnRH I or the GnRH I agonist, triptorelin (22). GnRH II has also been shown to have a more potent regulatory effect on the secretion of human chorionic gonadotropin by human term placenta and the steroidogenic capacity of granulosa-lutein cells in vitro (13, 23). However, we determined that GnRH I and GnRH II have differential effects on PAI-1 mRNA and protein expression levels in primary cultures of decidual stromal cells. To our knowledge, these studies are the first to demonstrate that GnRH I and GnRH II are capable of having differential biological actions on mammalian cells and suggest that the regulatory effects of these two hormones may be tissue/cell-specific. Although the molecular mechanisms underlying the differential effects of GnRH I and GnRH II on PAI-1 mRNA and protein expression levels in these cell cultures have yet to be elucidated, our observations suggest that these distinct biological actions may be due to differences in the binding affinity of GnRH I and GnRH II to one or more GnRH receptors (GnRHRs) and/or the activation of distinct intracellular signaling pathways.
Although significant GnRHR mRNA levels have not been detected in total RNA extracts prepared from human endometrial tissues obtained at any stage of the menstrual cycle or in early pregnancy (24), recent studies have demonstrated that this mRNA transcript is present in primary cultures of human endometrial stromal cells (25). In addition, low binding affinity/high capacity binding and high binding affinity/low capacity binding sites for GnRH I have been detected in normal and malignant human endometrial cells, suggesting that two distinct GnRHRs are present in this dynamic tissue (26). Recently, a gene encoding a second receptor for GnRH (GnRHR II) has been identified in the human genome (21, 27). Although a full-length mRNA transcript encoding this second form of human GnRHR has not been isolated, GnRHR II mRNA transcripts have been detected in the human endometrium and placenta (22). The ability of Cetrorelix, an antagonist believed to be specific for the GnRHR I (21), to inhibit the regulatory effects of GnRH I, but not GnRH II, on uPA and PAI mRNA levels in stromal cells isolated from first trimester decidual tissues provides further evidence that the biological actions of these two hormones may be elicited by distinct receptors.
Prolonged exposure to GnRH I resulted in decreases in uPA and PAI-1 mRNA and protein expression levels in the primary cultures of decidual stromal cells. Similarly, the inhibitory effects of GnRH II on PAI-1 mRNA and protein expression levels in decidual stromal cells were reduced with time in culture. This biphasic effect may be attributed to the desensitization of GnRHR-I, a biological phenomenon often observed in GnRH I-stimulated pituitary cells (28). GnRH I binding to its receptor activates several intracellular signaling pathways, including protein kinase A, protein kinase C, and/or MAPK (ERK1/2) cascades (29). Recent studies indicate that the activation of the ERK1/2 signaling cascade by GnRH I involves the trans-activation of the epidermal growth factor receptor (EGFR) (30). Interestingly, there is a marked increase in EGFR expression in the endometrial stroma as it undergoes decidualization, with maximum levels being detected in decidua cells present at the maternal-fetal interface (31). EGFR agonists have also been shown to increase PAI-1 expression levels in human endometrial stromal cells undergoing steroid-mediated decidualization in vitro (31, 32). In contrast, invasive extravillous cytotrophoblasts do not express significant levels of EGFR (33, 34). Thus, the differential effects of GnRH on PAI-1 mRNA and protein expression levels in primary cultures of decidual stromal cells and extravillous cytotrophoblasts may be due to the presence or absence of EGR in these two cell types, respectively.
In summary, we have determined that GnRH I and GnRH II increase uPA mRNA and protein expression levels in primary cultures of stromal cells isolated from first trimester decidual tissues in a dose- and time-dependent manner. In contrast, GnRH I increased, whereas GnRH II decreased, the expression levels of the endogenous inhibitor of uPA, PAI-1, in these cell cultures. These findings strengthen our hypothesis that the two forms of GnRH secreted by the placenta and endometrium play key regulatory roles in the ECM remodeling events that occur at the maternal-fetal interface during pregnancy in humans.
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Footnotes |
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C.D.M. and P.C.K.L. contributed equally to these studies.
C.D.M. is a Career Investigator with the British Columbia Research Institute for Children’s and Women’s Health.
P.C.K.L. is a Recipient of a Senior Investigatorship from the Michael Smith Foundation for Health Research.
Abbreviations: ECM, Extracellular matrix; EGFR, epidermal growth factor receptor; GnRHR, GnRH receptor; PAI-1, plasminogen activator inhibitor type 1; QC-PCR, quantitative competitive-PCR; uPA, urokinase-type plasminogen activator.
Received December 12, 2002.
Accepted May 12, 2003.
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