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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 2 680-688
Copyright © 2003 by The Endocrine Society

Dose-Dependent Effects of Gonadotropin Releasing Hormone on Matrix Metalloproteinase (MMP)-2, and MMP-9 and Tissue Specific Inhibitor of Metalloproteinases-1 Messenger Ribonucleic Acid Levels in Human Decidual Stromal Cells in Vitro

Chun-Shan Chou, Chen-Jei Tai, Colin D. MacCalman and Peter C. K. Leung

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia V6H 3V5, Canada

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 V6H 3V5, Canada. E-mail: peleung{at}interchange.ubc.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue-specific inhibitor of matrix metalloproteinases (TIMPs), play key roles in the cyclic remodeling events that occur in the human endometrium in preparation for pregnancy. To date, the factors capable of regulating the expression of MMPs and TIMPs in the human decidua remain poorly characterized. The spatiotemporal expression of GnRH in the human endometrium during the menstrual cycle and early pregnancy suggests that this hormone may have a regulatory role in the development of this dynamic tissue. In view of these observations, we have examined the ability of GnRH to regulate MMP-2, MMP-9, and TIMP-1 mRNA levels in primary cultures of human decidual stromal cells using a quantitative competitive PCR strategy. GnRH was capable of increasing MMP-2 and MMP-9 mRNA levels in these primary cell cultures in a dose-dependent manner. The GnRH antagonist, antide, was capable of inhibiting the GnRH-mediated increase in the levels of the MMP-2 and MMP-9 mRNA transcripts present in these decidual stromal cells in a dose-dependent manner. In contrast, GnRH or antide did not have a significant effect on TIMP-1 mRNA level in these primary cell cultures at any of the concentrations used in these studies. Taken together, these observations suggest that GnRH plays an integral role in human implantation, by virtue of its ability to regulate the balance between MMP and TIMP expression in decidual cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DECIDUAL CELLS of the endometrium fulfill paracrine, nutritional, immunoregulatory, and embryoregulatory functions throughout pregnancy (1, 2). Decidualization, which begins in the secretory phase and continues into early pregnancy in the human, involves the morphological and biochemical differentiation of the endometrial stroma (3, 4). This highly regulated series of developmental events involves the remodeling of the stromal/decidual cell extracellular matrix (ECM). In particular, the interstitial-type ECM of the proliferative endometrium, which is enriched in fibronectin and collagen types I, III, V, and VI is replaced with a basal lamina composed primarily of residual interstitial proteins, laminin, heparin sulfate proteoglycan and collagen type IV (5, 6).

The spatiotemporal expression of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue-specific inhibitor of matrix metalloproteinases (TIMPs), are believed to mediate, at least in part, the cyclic remodeling of the endometrial ECM (7, 8). In particular, MMP-2 and MMP-9 expression has been associated with the decidualization of human endometrial stromal cells (2, 8, 9). The composition of the decidual ECM is also modulated by MMPs secreted by the trophoblastic cells of the implanting embryo (7, 8). Of the multiple MMP subtypes identified in the human placenta, the expression of MMP-2 and MMP-9 have been assigned key roles in the invasion of trophoblasts into the underlying maternal decidua (10, 11). The production of these MMPs by human trophoblasts is down-regulated during the second trimester of pregnancy, paralleling the decline in the invasive capacity of these cells with gestational age (10). TIMP-1, TIMP-2, and TIMP-3, which are constitutively expressed in the human endometrium and placenta, are believed to counterbalance the MMP-mediated degradation of the decidual ECM in both an autocrine and paracrine manner (11, 12). To date, the factors capable of regulating the balance between MMP/TIMP expression levels at the maternal-fetal interface remain poorly characterized.

GnRH has been detected in the glandular epithelium and stroma of the human endometrium at all stages of the menstrual cycle with maximum levels being expressed in the secretory endometrium and first trimester decidua (13, 14). Furthermore, high levels of GnRH have been detected in first trimester placental tissues and cells (15, 16). To date, the biological functions of GnRH in these tissues remain poorly understood. However, in view of the close correlation between the expression patterns of GnRH and MMP-2/MMP-9 in the human endometrial stroma and placenta, it is tempting to speculate that this hormone plays a key regulatory role in the MMP-mediated remodeling of the endometrial ECM required for the establishment of pregnancy. In these studies, we have examined the ability of GnRH to regulate MMP-2, MMP-9, and TIMP-1 mRNA levels in primary cultures of human decidual stromal cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues

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. (17). Briefly, the decidual tissue samples were minced and subjected to 0.1% collagenase (type IV,Sigma, 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 with DMEM containing 10% fetal bovine serum before being resuspended and plated in DMEM containing 25 mM glucose, L-glutamine, antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin) and supplemented with 10% fetal bovine serum. 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 to determine the purity of human endometrial cell cultures (18). As defined by these criteria, the decidual stromal cell cultures used in these studies contained less than 1% epithelial or vascular cells.

Hormone treatments

The decidual stromal cells were cultured in the presence of increasing concentrations of GnRH (0, 0.1, 1, 10, or 100 nM) for 24 h before being harvested for total RNA extraction. The hormone concentrations used in these experiments were selected on the basis of previous studies (19, 20). In addition, decidual stromal cell cultures were treated with a combination of GnRH (100 nM) and increasing concentrations (0, 1, 10, or 100 nM) of the GnRH antagonist, antide, for 24 h. Cells treated with vehicle (0.1% ethanol) alone served as a control for these experiments.

Generation of first strand cDNA

Total RNA was prepared from the decidual cell cultures using a RNeasy Mini Kit (QIAGEN, Valencia, CA) and 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, Inc., Fullerton, CA).

An aliquot (1 µg) of the total RNA extracts prepared from the decidual stromal 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).

Primer design

Nucleotide sequences specific for MMP-2, MMP-9, or TIMP-1, and which also spanned different exons, were identified in the human mRNA sequences (accession nos. XM_165656, BC006093, and XM_033878, respectively) deposited in GenBank (National Center for Biotechnology Information). Forward and reverse primers corresponding to these DNA sequences were synthesized at the Nucleic Acid and Protein Services Unit, University of British Columbia. Primers specific for the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which have been previously described (21) were used to quantify and assess the integrity of the total RNA samples. To construct a competitive cDNA fragment, floating primers with a sequence complementary to a short nucleotide sequence between the forward- and reverse-primer binding sites were also generated by attaching the complementary sequence of the binding site to the 3'-end of the original reverse primers specific for MMP-2, MMP-9 or TIMP-1 (Fig. 1Go). A similar approach has been used to examine the effects of IL-1 on urokinase plasminogen activator (u-PA), soluble u-PA receptor, and plasminogen activator inhibitor-1 mRNA levels in primary cultures of human endometrial stromal cells (22). The specific sequences of these primer sets and the expected sizes of the resultant PCR products are listed in Table 1Go.



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  		Figure 1. A representative schematic diagram illustrating the construction of an internal standard cDNA for MMP-2. An internal standard fragment was constructed by deletion of 212-bp fragment from the specific target cDNA to be detected.

 

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  		Table 1. Oligonucleotide primers for decidual cell mRNA amplification

 
Semiquantitative PCR

Semiquantitative PCR was performed using template cDNA generated from the total RNA extracts prepared from cultures of untreated decidual stromal cells and the primers specific for MMP-2, MMP-9, or TIMP-1. The PCR conditions were as follows: 1 min at 94 C, 1 min at 57.5 C or 56 C for PAI-1 and u-PA, 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.

The resultant PCR products of 506, 544, and 369 bp for MMP-2, MMP-9, and TIMP-1, respectively, were separated using gel electrophoresis and visualized by ethidium bromide staining (Fig. 2Go). To confirm the specificity of the primers, an aliquot of the MMP-2, MMP-9, and TIMP-1 cDNAs generated using total RNA extracts prepared from the decidual cell cultures were subcloned into the PCR II vector (Invitrogen, Carlsbad, CA) and subjected to DNA sequence analysis.



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  		Figure 2. Semiquantitative PCR analysis of MMP-2 mRNA levels in primary cultures of human decidual cells. Autoradiograms of Southern blots containing PCR products amplified from first strand cDNA synthesized from decidual stromal cells and primers specific for GAPDH (top) or MMP-2 (bottom) using an increasing number of cycles. The autoradiograms were scanned, and the absorbance values obtained for each PCR product were plotted against the corresponding number of cycles. The observed linear relationships between the yield of these distinct PCR products and the number of amplification cycles are shown in the graphs below. A linear relationship between the yield of PCR product and the number of amplification cycles using template cDNA synthesized from decidual stromal cells was also observed for MMP-9 and TIMP-1 (data not shown)

 
Southern blot analysis using an aliquot (10 µl) of the MMP-2, MMP-9, and TIMP-1 cDNAs demonstrated that there was a linear relationship between the yield of the PCR products and number of cycles performed. Consequently, 21 cycles for the amplification of GAPDH, 27 cycles for MMP-2, and 30 cycles for MMP-9 and TIMP-1 from first strand cDNA templates generated from decidual stromal cells were used in these studies.

Similarly, PCR using template cDNA generated from the decidual stromal cell cultures and a combination of the regular forward primer specific for MMP-2, MMP-9, or TIMP-1 and the corresponding reverse, floating primer yielded truncated cDNAs with the expected sizes of 294, 395, and 185 bp, respectively. These PCR products were also subcloned into the PCR II vector and subjected to DNA sequence analysis to confirm the specificity of these primer sets.

Quantitative Competitive-PCR (QC-PCR)

The QC-PCR strategy employed in these studies was based upon the competitive coamplification of known amounts of the truncated MMP-2, MMP-9, or TIMP-1 PCR products added to aliqouts of first strand cDNA prepared from primary cultures of decidual stromal cells.

To determine the ideal amounts of each template to be added to the reaction mixture, PCR was performed using fixed amount of competitive cDNA (15, 0.1, and 1 pg for MMP-2, MMP-9, and TIMP-1, respectively) and decreasing concentrations of target cDNA (51.2-0.1, 6.4-0.003, and 12.8-0.05 pg for MMP-2, MMP-9, and TIMP-1, respectively), obtained by serial dilution as templates and the corresponding sets of primer. The PCR conditions and number of cycles were performed for the MMP and TIMP primer sets as described above.

An aliquot (10 µl) of the PCR products were separated by electrophoresis in a 1% agarose gel and visualized by ethidium bromide staining (Fig. 3Go). The intensity of the ethidium bromide staining of the PCR products was analyzed using UV densitometry (Biometra, Whiteman Co., Gottingen, Germany). Volume counts (mm2) of the PCR products were then determined using the Scion Image computer software (Scion Image Co., Frederick, MD). The intensity of the ethidium bromide staining of an aliquot (10 µl) of a 100-bp DNA ladder (Life Technologies, Inc.) served as an internal standard.



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  		Figure 3. Preparation of a standard curve for the quantitative competitive PCR analysis of MMP-2 mRNA levels in decidual stromal cells. A, Photomicrograph of an ethidium bromide stained gel containing PCR products generated using an increasing amount of native cDNA and the two primer sets specific for MMP-2. The sizes of the resultant PCR products relative to a 100-bp ladder (MW) are indicated. The intensity of the ethidium bromide staining of the target and competitive PCR products was determined by UV densitometry. The observed linear relationship between the logarithmically transformed ratios of target:competitive cDNA and the amount of native cDNA added to each PCR is shown in the graph below. B, Photomicrograph of an ethidium bromide gel containing PCR products generated using a fixed amount of template cDNA (1 µl) synthesized from decidual stromal cells and a decreasing amount of the 294-bp internal standard cDNA for MMP-2 obtained by serial dilutions. The intensity of the ethidium bromide staining of the target and competitive PCR products was determined by UV densitometry. The linear relationship between the logarithmically transformed ratios of target:internal standard cDNAs and the amount of mutant cDNA added to the initial PCR is shown in the graph below. The point at which these two graphs intercept (12.5–25 pg) indicates the amount of MMP-2 cDNA that can be generated from 1 µg of total RNA extracted from isolated human decidual stromal cells. Standard curves for MMP-9 and TIMP-1 were also generated using the same approach (data not shown).

 
A standard curve was generated using the logarithmically transformed ratios of target:competitive cDNA plotted against the log amount of target cDNA initially added to the PCR reaction (Fig. 3Go). This standard curve was highly reproducible and linear. The values obtained from this regression curve (y = b + mx) were then used to determine the amounts of cDNA transcripts present in unknown samples. An aliquot of MMP-2 (15 pg/µl), MMP-9 (0.1 pg/µl) or TIMP-1 (1 pg/µl) competitive cDNA was subsequently added to the aliquots of the first strand cDNA generated from the human decidual cells for QC-PCR.

QC-PCR was performed using the MMP-2, MMP-9, or TIMP-1 primer sets, 1 µl of the first-strand cDNA synthesized from each of the cell cultures, and the PCR conditions described above. The ratios of the intensity of ethidium bromide staining of the target:competitive cDNAs were then normalized to the corresponding GAPDH, logarithmically transformed and compared with the values obtained from the standard curve.

Statistical analysis

The absorbance values obtained from the ethidium bromide-stained gels were subjected to statistical analysis using GraphPad Software, Inc. Prism 2 software (San Diego, CA). Statistical differences between the absorbance values were assessed by the ANOVA. Differences were considered significant for P values no greater than 0.05. Significant differences between the means were determined using Dunnett’s test. The results are presented as the mean relative absorbance (± SEM) obtained using five or more different tissue samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose-dependent effects of GnRH on MMP-2, MMP-9, and TIMP-1 mRNA levels in human decidual stromal cells

MMP-2, MMP-9, and TIMP-1 mRNA transcripts were detected in all of the total RNA extracts prepared from the isolated human decidual stromal cells (Fig. 4Go). The addition of vehicle (0.1% ethanol) had no significant effect on the levels of the MMP-2, MMP-9, or TIMP-1 mRNA transcripts in these primary cell cultures (data not shown). In contrast, GnRH increased MMP-2 and MMP-9 mRNA levels in isolated decidual stromal cells in a dose-dependent manner (Fig. 5Go, A and B). Maximum levels of MMP-2 mRNA levels were observed in decidual cells cultured in the presence of 1 nM GnRH. The addition of higher concentrations of GnRH to the culture medium had no further effect on the levels of the MMP-2 mRNA transcript present in these cells. A significant increase in MMP-9 mRNA levels was only observed in decidual stromal cells cultured in the presence of 100 nM GnRH. In contrast, GnRH had no significant effect on the levels of the mRNA transcripts encoding TIMP-1 present in these primary cell cultures at any of the concentrations used in these studies (Fig. 5CGo).



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  		Figure 4. Photomicrograph of an agarose gel containing PCR products generated using template cDNA synthesized from isolated human decidual stromal cells and primers specific for GAPDH (lane 1), MMP-2 (lane 2), TIMP-1 (lane 3), and MMP-9 (lane 4). The expected sizes of the distinct PCR products; GAPDH (373 bp), MMP-2 (506 bp), MMP-9 (544 bp), and TIMP-1 (379 bp) are indicated relative to a 100-bp DNA ladder (lane MW). Similar results were obtained using different cell cultures on five independent occasions.

 


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  		Figure 5. QC-PCR analysis of total RNA extracts prepared from decidual cells cultured in the presence of increasing concentrations of GnRH. Representative photomicrographs of ethidium bromide-stained gels containing PCR products generated using template cDNA synthesized from decidual cells cultured in the presence of 0, 0.1, 1, 10, or 100 nM (lanes 1–5, respectively) and the primer sets specific for MMP-2 (A), MMP-9 (B), or TIMP-1 (C). Each sample was coamplified in the presence of a defined amount of internal standard cDNA. The sizes of the resultant target and competitive PCR products relative to a 100-bp ladder (lane MW) are marked to the right of the photomicrograph. The intensity of the ethidium bromide staining of the PCR products was determined by UV densitometry and the absorbance values obtained for each PCR normalized to the values obtained for the corresponding GAPDH (lower panels). These normalized values were used to calculate the ratio of target to internal standard cDNA for each QC-PCR. The results derived from this analysis as well as from four other studies (data not shown) are represented (mean ± SEM, n = 5) in the bar graphs below (a, P < 0.05 vs. untreated control).

 
Antide inhibits the stimulatory effects of GnRH on MMP-2 and MMP-9 mRNA levels in primary cultures of human decidual cells

The GnRH antagonist, antide, inhibited the stimulatory effects of GnRH on MMP-2 and MMP-9 mRNA levels in isolated human stromal decidual cells in a dose-dependent manner (Fig. 6Go, A and B). In contrast, antide had no significant effect on TIMP-1 mRNA levels in decidual cells cultured in the presence or absence of GnRH (Fig. 6CGo).



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  		Figure 6. QC-PCR analysis of total RNA extracts prepared from decidual cells cultured in the presence or absence of GnRH and increasing concentrations of antide. Representative photomicrographs of ethidium bromide-stained gels containing PCR products generated using template cDNA synthesized from untreated decidual cells (lane 1) or cells cultured in the presence of 100 nM GnRH and 0, 1, 10, or 100 nM antide (lanes 2–5, respectively) and the primer sets specific for MMP-2 (A), MMP-9 (B), or TIMP-1 (C). Each sample was coamplified in the presence of a defined amount of internal standard cDNA. The sizes of each of the target and competitive PCR products relative to a 100-bp ladder (lane MW) are marked to the right of the photomicrographs. The intensity of the ethidium bromide staining of the PCR products was determined by UV densitometry and the absorbance values obtained for each normalized to the values obtained for the corresponding GAPDH. These normalized values were used to calculate the ratio of target to internal standard cDNA for each QC-PCR. The results derived from this analysis as well as from four other studies (data not shown) are represented (mean ± SEM n = 5) in the bar graphs below (a, P < 0.05 vs. untreated control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MMP-2 and MMP-9 were readily detectable in stromal cells isolated from first trimester human decidual tissues. Similarly, MMP-2 and MMP-9 activity has been detected in human decidual tissues throughout pregnancy (23). The expression of these two MMPs is also highly regulated during the decidualization of the rat uterus (24, 25). Furthermore, the administration of the MMP inhibitor doxycycline to these animals during early pregnancy delayed decidualization but did not inhibit implantation (25). Taken together, these observations suggest that MMP-2 and MMP-9 play key roles in the terminal differentiation of endometrial stomal cells into decidua. These MMPs may mediate decidualization not only via their ability to remodel the endometrial ECM but also in the release and/or activation of growth factors, and vasoactive factors (25).

The spatiotemporal expression and/or activity of MMPs in the human endometrium during the menstrual cycle suggests that gonadal steroids are key regulators of these proteases (7, 9). Progesterone has been shown to be capable of decreasing MMP-2 and MMP-9 expression levels in human endometrial stromal cells in vitro (26, 27). However, as MMP-2 and MMP-9 are readily detectable in the decidua, when progesterone levels are elevated, it is likely in addition to gonadal steroids, MMP expression levels in the endometrium are modulated by localized, regulatory factors. These regulatory factors may act in an autocrine and/or paracrine manner. For example, recent studies indicate that activation of MMP-2 in the endometrial stroma involves as yet unidentified, soluble factor(s) that are secreted by glandular epithelial cells (29). We have determined that GnRH, which is expressed by both the glandular epithelium and stroma of the endometrium (14), is capable of regulating MMP expression levels in decidual stromal cells in vitro. In addition, GnRH secreted by subpopulations of human trophoblasts at the maternal fetal interface is also likely to regulate MMP activity in the decidua.

Increasing concentrations of GnRH had differential effects on MMP-2 and MMP-9 mRNA levels in decidual stromal cells. The decrease in MMP-2 mRNA levels in the cells cultured in the presence of the highest concentrations of GnRH used in these studies may be attributed to desensitization, a biological phenomenon observed in the GnRH-stimulated secretion of gonadotropins by pituitary cells (30). Furthermore, as MMP-9 mRNA levels were increased in these cell cultures, it is tempting to speculate that the regulatory effects of GnRH on the expression of these two MMPs in the human decidua is mediated by distinct intracellular signaling pathways. Recent studies indicate that GnRH is capable of activating the protein kinase A, protein kinase C, and/or MAPK signaling pathways in a wide variety of human cells (31). Although the importance of one or more of these signaling pathways in the GnRH-mediated expression of MMPs in human decidual cells has yet to be elucidated, at least one response element for the downstream effector of the MAPK cascade, activator protein-1, has been detected in the promoter region of the human gene encoding MMP-9 (32). In contrast, the MMP-2 promoter appears to be devoid of such response elements (32).

Human endometrial stromal cells cultured in the presence of gonadal steroids undergo morphological and biochemical differentiation processes that mimic decidualization (18). However, Raga et al. (19) failed to detect MMP-9 mRNA transcripts in these primary cell cultures, a predominant MMP subtype present in human decidual tissues (19). Furthermore, in contrast to our findings, GnRH agonists were shown to increase the levels of TIMP-1 and TIMP-3 but have no effect on MMP-9 mRNA in these primary cell cultures (19). These discrepancies may be attributed to differences between the repertoire of MMP/TIMPs expressed by endometrial stromal cells that have undergone decidualization in vivo or in vitro and/or the culture conditions used in these two studies. In particular, the pharmacological doses of gonadal steroids required to induce decidualization in human endometrial stromal cells in vitro is likely to have profound effects on MMP and TIMP expression levels in these primary cell cultures.

TIMP-1 is constitutively expressed in the human endometrium throughout the menstrual cycle and pregnancy (18). As TIMPs inactivate MMP activity by binding their active forms with a 1:1 stoichiometry (33), the localized remodeling of the endometrial ECM will be dependent on the balance between the expression levels of MMPs and TIMPs in this dynamic tissue. We have determined that GnRH regulates MMP-2/MMP-9 but not TIMP-1 mRNA levels in primary cultures of human decidual stromal cells. Similarly, epidermal growth factor and basic fibroblast growth factor were capable of increasing MMP-3, MMP-9, and MMP-13 but had no significant effect on TIMP-1, TIMP-2, or TIMP-3 in rat endometrial stromal cells undergoing decidualization in vitro (34). In contrast, IL-1 increased MMP-9 and decreased TIMP-1 and TIMP-3 mRNA levels in primary cultures of human endometrial stromal cells, whereas TGF-ß decreased the levels of MMP-9 and increased TIMP-1 and TIMP-2 mRNA levels in these primary cell cultures (35).

Low binding affinity/high capacity binding sites for GnRH have been observed in the endometrium and endometrial carcinomas suggesting that this hormone plays a direct, regulatory role in the remodeling processes that occur in this dynamic tissue under normal and pathological conditions (36). Furthermore, immunization of immature mice with antibodies directed against a peptide corresponding to amino acids 5–17 of the murine GnRH receptor (GnRHR) resulted in the specific regression/inhibition of endometrial cell proliferation (37). Antide was capable of inhibiting the GnRH-mediated increase MMP-2 and MMP-9 mRNA levels in isolated decidual stromal cells in a dose-dependent manner suggesting that these stimulatory effects are mediated by GnRH interacting with its receptor (GnRHR). However, previous studies have failed to detect significant levels of GnRHR in the human endometrium during the secretory phase of the menstrual cycle and decidua of early pregnancy (19, 38). Recently, a gene encoding a second receptor for GnRH (GnRHR II) has been identified in the human genome (39, 40). 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 human uterine tissues and endometrial cancer lines (40, 41).

In summary, we have determined that GnRH is capable of increasing MMP-2 and MMP-9, but not TIMP-1 mRNA levels in primary cultures of stromal cells isolated from first trimester human decidual tissues. The GnRH antagonist, antide, was capable of inhibiting the stimulatory effects of GnRH on MMP-2 and MMP-9 mRNA levels in these primary cell cultures. Collectively, these observations suggest that GnRH is a key regulator of the MMP-mediated ECM remodeling events that occur in the stroma of the human endometrium in preparation for pregnancy.


   Acknowledgments
 


   Footnotes
 
These studies were supported by an operating grant from the Canadian Institutes of Health Research (CIHR) (to P.C.K.L. and C.D.M.). P.C.K.L. is the recipient of a career investigatorship from the Michael Smith Foundation for Health Research. C.D.M. is the recipient of a scholarship from the CIHR.

C.D.M. and P.C.K.L. contributed equally to these studies.

Abbreviations: ECM, Extracellular matrix; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GnRHR, GnRH receptor; MMP, matrix metalloproteinases; QC-PCR, quantitative competitive PCR; TIMP, tissue-specific inhibitor of matrix metalloproteinases; u-PA, urokinase plasminogen activator.

Received August 13, 2002.

Accepted October 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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