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Endocrinology Vol. 142, No. 9 3941-3949
Copyright © 2001 by The Endocrine Society

ARTICLES

Relaxin Increases Secretion of Matrix Metalloproteinase-2 and Matrix Metalloproteinase-9 during Uterine and Cervical Growth and Remodeling in the Pig

Judy A. Lenhart, Peter L. Ryan1, Kathleen M. Ohleth, Stephen S. Palmer2 and Carol A. Bagnell

Department of Animal Sciences (J.A.L., P.L.R., K.M.O., C.A.B.), Rutgers University, New Brunswick, New Jersey 08901; and The R. W. Johnson Pharmaceutical Research Institute (S.S.P.), Raritan, New Jersey 08869

Address all correspondence and requests for reprints to: Carol A. Bagnell, Ph.D., Department of Animal Sciences, 84 Lipman Drive, Rutgers University, New Brunswick, New Jersey 08901.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Matrix metalloproteinases are proteolytic enzymes that degrade the extracellular matrix and are essential for tissue remodeling. Uterine and cervical growth require remodeling of structural barriers to cell invasion and matrix metalloproteinase-2 and -9 degrade type IV collagen, the major component of basement membranes. Relaxin stimulates uterine and cervical growth and remodeling, which includes remodeling of support elements such as basement membranes. The objective of this study was to determine whether relaxin alters the production and/or activity of matrix metalloproteinase-2 and -9 in the uterus or cervix of the pig. The growth-promoting effects of relaxin were elicited by administering relaxin to prepubertal gilts every 6 h for 54 h. The expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 was characterized by gel zymography, and proteins were quantified by immunoblotting. Total enzyme activity was measured using matrix metalloproteinase-specific fluorescent substrate assays. In both uterine and cervical tissues, immunoreactive matrix metalloproteinase-2 and matrix metalloproteinase-9 protein expression was similar in relaxin-treated and control animals. However, tissue-associated gelatinase activity was attenuated by relaxin (P < 0.05). In contrast, relaxin significantly increased the secretion of active matrix metalloproteinase-2 and -9 protein into uterine fluid (P < 0.05). Given the importance of matrix metalloproteinases in extracellular matrix degradation, the observation that relaxin promotes uterine secretion of matrix metalloproteinase-2 and -9 supports the concept that relaxin facilitates the growth and remodeling of reproductive tissues by increasing extracellular proteolysis in the pig reproductive tract.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UTERINE AND CERVICAL growth during pregnancy are the result of cellular proliferation as well as remodeling of the connective tissue framework of these organs to facilitate expansion. This remodeling requires highly coordinated breakdown and rebuilding of the extracellular matrix (1, 2, 3). Matrix metalloproteinases (MMPs) are a family of extracellular proteinases that target the extracellular matrix and are important for tissue remodeling (3). The production of MMPs is under the influence of a diverse array of stimuli, including cytokines, growth factors, and hormones (4), many of which can be synthesized locally by cells of the connective tissue matrix (5). The gelatinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), are the MMP subclass that degrade type IV collagen, the major component of the basement membrane and the scaffold to which other basement membrane components bind (6, 7, 8). Therefore, MMP-2- and MMP-9-mediated degradation of the basement membrane is an early event in the process of cellular invasion and tissue expansion and is thus important for reproductive tissue growth and remodeling (9, 10).

Relaxin stimulates the growth and remodeling of the porcine uterus and cervix (11, 12, 13), and connective tissue is a prime target for relaxin in reproductive tissues. During pregnancy, relaxin promotes uterine growth and increases the distensibility of the collagenous framework of the uterus, which is important for fetal accommodation (14). In preparation for parturition, relaxin plays a major collagenolytic role in the cervix, altering the connective tissue matrix composition (15) and proteolytic enzyme profile (16) to induce cervical ripening and dilation (15, 17, 18). These observations point to the important relationship between relaxin-mediated reproductive tissue reorganization during growth and the production, activation, and/or activity of connective tissue enzymes. Studies from our laboratory show that relaxin-induced uterine and cervical growth in prepubertal gilts is accompanied by increases in serine protease activity and protein (i.e. urokinase-type plasminogen activator) in uterine luminal fluids (19). Of interest is the observation that serine proteases play a role in activating some of the MMPs, including MMP-9 (20).

Given the importance of relaxin in growth and remodeling of the uterus and cervix, we were interested in examining the impact of relaxin on the uterine and cervical MMP system, specifically on the gelatinases, MMP-2 and MMP-9. Thus, the objective of this study was to determine the effect of relaxin on the production and activity of MMP-2 and MMP-9 during growth and remodeling of the pig uterus and cervix. In addition, we describe the use of two novel MMP activity assays in which gelatinase-specific activity was monitored through the degradation of fluorescently labeled gelatinase substrates.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Purified porcine relaxin (CM-A fraction; 3000 U/mg) was prepared at the Department of Biomedical Sciences (University of Guelph, Guelph, Canada) by extraction and purification from ovaries of pregnant sows using the method of Sherwood and O’Byrne (21). Purity was confirmed by SDS-PAGE, which revealed a single band at approximately 6.2 kDa. The biological activity of the relaxin preparation was ascertained by inhibition of spontaneous uterine motility in vitro (22), and immunoreactivity was verified by RIA (23). Electrophoresis gels for gelatin zymography (10% gelatin zymogram) and Western analysis (NuPAGE), electrophoresis buffers, and zymogram renaturing and developing buffers were purchased from Novex (San Diego, CA). Monoclonal antihuman MMP-2 (Ab-3) and antihuman MMP-9 (Ab-1) antibodies were obtained from Oncogene Research Products (Calbiochem, Cambridge, MA). Human MMP-2 enzyme was obtained from PanVera Corp (Madison, WI). Human MMP-3 and MMP-9 enzymes were obtained from Biogenesis (Sandown, NH). Goat antimouse IgG-horseradish peroxidase-conjugated antibody was purchased from Transduction Laboratories, Inc. (Lexington, KY). Renaissance Chemiluminescence Reagent Plus was obtained from NEN Life Science Products (Wilmington, DE). Autoradiographic film (Hyperfilm-ECL) was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). All other chemicals were purchased from Sigma (St. Louis, MO) and Life Technologies, Inc. (Gaithersburg, MD), unless otherwise specified.

Animals
Prepubertal (~115-d-old) Yorkshire-Landrace gilts (Swine Unit of the New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, NJ) were injected with porcine relaxin (0.5 mg/0.5 ml, im) or saline (0.5 ml, im) every 6 h for 54 h (12). Three hours after the last injection, animals were killed by exsanguination after stunning. Uterine and cervical tissues and uterine flushes were collected and processed as described by Wang-Lee et al. (19). The animal experimentation procedures described here were reviewed and approved by the Rutgers University Animal Care Advisory Committee.

The marked trophic effects of relaxin on the uterus (12, 24) and cervix (19) and the systemic and local concentrations of relaxin achieved after in vivo relaxin administration in this animal model have been reported (24). The prepubertal status of the gilts was confirmed by the absence of 17ß-estradiol and progesterone in the plasma and uterine flushes of all animals before and after the treatment regimen (24, 25).

Gelatin zymography
Gelatinases were extracted from uterine and cervical tissues with high calcium and heating to dissociate the enzymes from endogenous substrates and inhibitors as described by Sellers and Woessner (26). Briefly, tissues were homogenized in 0.25% Triton X-100 and 10 mM CaCl2 and centrifuged at 9,000 x g for 30 min at 4 C, and the supernatant was designated the Triton extract. The pellet was then heated in 10 vol buffer containing 50 mM Tris-HCl, 100 mM CaCl2, and 0.15 M NaCl (pH 7.4) at 60 C for 6 min and centrifuged at 27,000 x g for 30 min at 4 C. The resulting supernatant was designated the heat extract. Triton extracts, heat extracts, and uterine flushes were desalted before analysis using Micro Bio-Spin chromatography columns (P-6, Bio-Rad Laboratories, Inc., Hercules, CA). Uterine flushes and tissue extracts (20 µg) were diluted in Tris-glycine-SDS sample buffer, and proteins were separated on a 10% polyacrylamide gel containing 0.1% gelatin. After electrophoresis, SDS was eluted from the gels in renaturing buffer (2.5% Triton X-100 in distilled water) for 30 min at room temperature, then equilibrated in developing buffer (50 mM Tris, 0.2 M NaCl, 5 mM CaCl2, and 0.2% Brij 35) for 30 min at room temperature. Gels were incubated in fresh developing buffer overnight (8–16 h) at 37 C with gentle agitation. Gels were stained with Coomassie blue R-250 for 30 min and then destained. Clear bands indicated gelatinolytic activity.

Immunoblot analysis of MMP-2 and MMP-9
Protein was extracted from tissue samples as described previously (25). Briefly, tissues were homogenized in boiling lysis buffer, sonicated to reduce viscosity, and centrifuged to remove insoluble material. Uterine and cervical proteins (20 µg) were resolved on 10% Bis-Tris-HCl-buffered polyacrylamide electrophoresis gels (NuPage), under either reducing (MMP-2) or nonreducing (MMP-9) conditions according to the manufacturer’s recommendations. Human MMP-2 and MMP-9 (50 ng) served as the positive controls. Proteins were transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). Membranes were blocked in 5% BSA in Tris-buffered saline [TBST; 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween-20] for 1 h at room temperature, then incubated with anti-MMP-2 or anti-MMP-9 antibody (0.4 µg/ml) in TBST/1% BSA overnight at 4 C. Membranes were washed with TBST and incubated with horseradish peroxidase-conjugated goat antimouse IgG (1:5000 in TBST/5% nonfat dry milk) for 1 h at room temperature. After washing in TBST, membrane-bound antibodies were detected by enhanced chemiluminescence.

MMP preparations and activation
Human progelatinase-A (MMP-2) was activated with 4-aminophenyl mercuric acetate in 100% dimethylsulfoxide (final concentration, 2 mM) for 1 h at 37 C. Human progelatinase-B (MMP-9) was activated with stromelysin-1 (MMP-3) at a ratio of 40:1 MMP-9 to MMP-3, for 2 h at 37 C. Enzymes were serially diluted in assay buffer [50 mM tricine (pH 7.4), 200 mM NaCl, 100 mM CaCl2, 2.5 mM ZnSO4, and Brij 35 at 0.05% for the peptide assay and 0.005% for the collagen IV assay] for the fluorescent activity analyses. For negative controls, the progelatinases were inactivated, either by incubation in 100% dimethylsulfoxide without 4-aminophenyl mercuric acetate for 1 h at 37 C or by heating to 100 C for 5 min, with similar results. Gelatinases were extracted from uterine and cervical tissues as described for zymography. To measure total enzyme activity, Triton and heat extracts were combined after desalting. Pooled tissue extracts (20 µg) and uterine flushes (10 µl) were diluted in assay buffer before analysis.

Inhibitor preparation
Metalloproteinase inhibitors included 1,10-phenanthroline (0.5 mM) and EDTA (5 mM). Inhibitors of serine, cysteine, and aspartic proteases included aprotinin (0.05 mg/ml), phenylmethylsufonylfluoride (PMSF; 1 mM), soybean trypsin inhibitor (0.05 mg/ml), leupeptin (1 mM), and pepstatin A (0.1 mM). Inhibitors were diluted to the desired concentrations for fluorescent analyses with assay buffer.

MMP activity assays
Two novel MMP activity assays, the peptide substrate and collagen IV assays, described and validated by Kraft et al. (27) were used to quantify MMP-2 and MMP-9 activities in uterine flush and tissue samples. The peptide substrate assay measures the ability of MMP-2 and MMP-9 to degrade a small, fluorescently labeled, synthetic gelatinase-selective peptide. The collagen IV assay was used to confirm that the change in fluorescence intensity resulted from specific proteolytic activity against a native gelatinase substrate. Although gelatinase-mediated proteolysis also increases fluorescence intensity in this assay, the change in fluorescence polarization of the substrate can also be measured using the fluorescein-labeled collagen IV. Fluorescent polarization changes are proportional to changes in the size of the substrate and the subsequent change in the rate of rotation of the fragments. Thus, the collagen IV activity assay was employed to verify that the observed change in fluorescence intensity was due to a gelatinase-catalyzed change in the size of the substrate and was not the result of nonspecific changes in fluorescence quenching.

Peptide substrate assay
The peptide substrate used in this assay was (Aedens)EAGPRGMAGQFSH(Dabcyl)K-amide, a gelatinase-selective, FRET peptide, developed at R. W. Johnson Pharmaceutical Research Institute (Raritan, NJ). Aedens is a fluorescent donor that excites at 340 nm and emits at 530 nm. Dabcyl quenches the fluorescence emitted from Aedens when the peptide is intact, and the two are in close proximity. Gelatinase-mediated proteolysis of the peptide separates the donor (Aedens) from the quencher (Dabcyl), which allows the fluorescence to be detected. The fluorescent peptide substrate was prepared in 100% dimethylsulfoxide (8 mM) and was diluted to a 1.5-mM working concentration in 0.1 M HEPES. The peptide substrate was further diluted to a final concentration of 20 µM with assay buffer just before use. In the assay, tissue extracts (20 µg) were diluted to a volume of 20 µl in assay buffer. Immediately before analysis, inhibitors in assay buffer or assay buffer alone (5 µl) were added, and the final reaction volume was brought up to 100 µl in assay buffer containing the fluorescently labeled peptide substrate (final substrate concentration, 20 µM). Gelatinase-catalyzed proteolysis of the peptide substrate was quantified by measuring changes in fluorescence intensity at 37 C every 30 min for 8 h using a CytoFluor multiwell plate reader (series 4000, Perseptive Biosystems, Framingham MA). The reference blank for this assay was assay buffer, and the positive control was 0.05 mg/ml trypsin diluted in assay buffer. The negative controls were substrate in assay buffer without enzyme and substrate in the presence of inactive MMP-2 or MMP-9.

Collagen IV assay
Fluorescein-conjugated, human placental DQ type IV collagen (Molecular Probes, Inc., Eugene, OR; 1 mg/ml stock in distilled water) served as the substrate for the collagen IV assay. The multiple fluoresceins bound to collagen IV (15 fluoresceins/molecule collagen IV), excite at 485 nm, and emit at 530 nm, but are quenched in the intact state due to the proximity of each fluorescein residue to another. In this assay, tissue extracts (20 µg) were diluted to a volume of 16 µl in assay buffer. Immediately before analysis, inhibitors in assay buffer or assay buffer alone (4 µl) were added, and the final reaction volume was brought up to 200 µl in assay buffer containing fluorescently labeled collagen IV (final substrate concentration, 0.01 µM). Gelatinase-mediated proteolysis of the collagen IV substrate was analyzed at 37 C using a POLARstar (BMG Instruments, Offenburg, Germany), 96-well spectrofluorometer (in polarization mode), which measures changes in both fluorescence intensity and polarization. Activity was determined at 0, 6, and 21 h. A 0.25-µM fluorescein solution was used to adjust the gain and k-factor at zero time. For this assay the reference blank was substrate assay buffer, the positive control was 0.05 mg/ml trypsin, and the negative control was substrate in assay buffer.

Densitometry and statistical analysis
MMP-2 and MMP-9 were quantified in immunoblots by scanning densitometry (SigmaGel, SPSS, Inc., Chicago, IL). Data for immunoblot analysis and enzyme activity are expressed as the mean ± SEM of samples from control and relaxin-treated gilts using at least three animals per group. Data were analyzed by t test. P < 0.05 was accepted as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gelatin zymography and immunoblot analysis
Gelatin zymography was used to qualitatively identify the type and relative abundance of gelatinases in the uterus, uterine flushes, and cervix of control and relaxin-treated gilts. In uterine tissue Triton extracts (n = 8; Fig. 1AGo), zymographic analysis revealed a prominent doublet band of lysis at 66 and 72 kDa, which is the reported size of MMP-2 (28). In uterine tissue heat extracts (n = 8; Fig. 1BGo), a third band of approximately 68–72 kDa was also evident. Additionally, a faint band of lysis was observed at 92 kDa, as reported for MMP-9 (29), in two of the samples from relaxin-treated animals. Multiple bands of gelatinolytic activity (66–92 kDa) were detected in uterine flushes from control and relaxin-treated animals (n = 6; Fig. 1CGo) corresponding to the different active and proenzyme forms of MMP-2 and MMP-9. In cervical tissues from both control and relaxin-treated animals, zymographic analysis revealed three gelatinolytic bands between 66–72 kDa in both the Triton (n = 8; Fig. 1DGo) and heat (data not shown) extracts.



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  		Figure 1. Effect of relaxin on the expression of MMP-2 and MMP-9 in control and relaxin-treated pigs. Zymographic analysis of MMP-2 and MMP-9 activity in uterine tissue. A, Triton extracts (50 µg); B, heat extracts (20 µg); C, uterine flush samples (10 µl); D, cervical tissue Triton extracts (20 µg). Clear zones against the dark background indicate the presence of MMPs.

 
Immunoblot analysis confirmed that the gelatinolytic activity detected by zymography corresponded in size to immunoreactive MMP-2 and MMP-9 gelatinases. In uterine tissues, a single immunoreactive band of protein at 66 kDa (MMP-2) was identified (Fig. 2AGo, right panel); however, relaxin did not significantly alter the expression of uterine MMP-2 protein compared with the control (Fig. 2AGo, left panel). In contrast, relaxin significantly increased the secretion of immunoreactive MMP-2 (P < 0.05) protein into the uterine lumen (Fig. 2BGo). In cervical tissues, two immunoreactive MMP-2 proteins were identified at 66 and 72 kDa (Fig. 2CGo, right panel). Although the expression of the immunoreactive 66-kDa MMP-2 protein was similar in control and relaxin-treated gilts, the immunoreactive 72-kDa MMP-2 cervical protein was significantly lower (P < 0.05) in gilts after relaxin-induced growth (Fig. 2CGo, left panel). Analysis of MMP-9 expression revealed an immunoreactive band of protein at 92 kDa in uterine and cervical samples (Fig. 3Go, A–C). MMP-9 expression was similar to that of MMP-2, in that immunoreactive protein in uterine and cervical tissue extracts was not different between control and relaxin-treated animals, but was significantly greater in uterine flushes after relaxin administration in vivo (P < 0.05).



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  		Figure 2. Effect of relaxin on MMP-2 expression in the porcine uterus and cervix. Uterine and cervical tissue proteins (20 µg) and uterine flush samples (10 µl) were resolved by SDS-PAGE, and MMP-2 proteins were detected using a monoclonal MMP-2-specific antibody. Positive controls included activated (A) and inactive (I) MMP-2 proteins (+; 50 ng). Left panel, Representative immunoblots; right panel, quantitative analysis of MMP-2 protein expressed as the mean ± SE. A, Uterine tissues; B, uterine flushes; C, cervical tissues from control and relaxin-treated pigs. *, Values significantly different (P < 0.05) from the control.

 


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  		Figure 3. Immunoblot analysis of MMP-9 expression in the porcine uterus and cervix. Uterine and cervical tissue proteins (20 µg) and uterine flush samples (10 µl) were resolved by SDS-PAGE, and MMP-9 proteins were detected using a monoclonal MMP-9-specific antibody. Left panel, Representative immunoblots; right panel, densitometric analysis of MMP-9 protein expressed as the mean ± SE. A, Uterine tissues; B, uterine flushes; C, cervical tissues from control and relaxin-treated pigs. *, Values significantly different (P < 0.05) from the control.

 
Validation of peptide substrate and collagen IV substrate fluorescent assays
A linear concentration-activity relationship was demonstrated for serially diluted MMP-2 and MMP-9 in both the peptide substrate (Fig. 4AGo) and the collagen IV substrate (data not shown) activity assays. Enzyme activity was characterized as metalloproteinase given that the increase in gelatinase-catalyzed fluorescence could be significantly attenuated by metalloproteinase inhibitors (5 mM EDTA or 0.5 mM 1–10 phenanthroline), but not by inhibitors of serine, cysteine, or aspartic proteases (aprotinin, leupeptin, pepstatin, PMSF, or soybean trypsin inhibitor; Fig. 4BGo). In addition, the specificity of these metalloproteinase inhibitors was confirmed by their inability to inhibit nonspecific substrate cleavage by trypsin, a serine protease, which was effectively inhibited by soybean trypsin inhibitor (data not shown).



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  		Figure 4. Characterization of metalloproteinase activity in fluorogenic substrate assays. A, Representative graph of enzyme activity, demonstrating assay linearity for serially diluted MMP-2 and MMP-9. B, Representative graph of enzyme inhibition, using MMP-2 (30 ng) in the collagen IV (0.01 µM) substrate assay, showing inhibition of fluorescence intensity by metalloproteinase inhibitors EDTA (5 mM) and 1,10-phenanthroline (0.5 mM), along with the absence of inhibition in the presence of serine, cysteine, and aspartic proteases. C, Gelatinase activity in tissue extracts of control and relaxin-treated pigs, in the presence or absence of protease inhibitors. Gelatinase-specific cleavage of the peptide substrate (20 µM) by tissue extracts ({blacksquare}) was similar to that of MMP-2, in that activity was not affected by PMSF (1 mM; {square}), but could be attenuated by 1–10 phenanthroline (0.5 mM; ). Nonspecific trypsin (0.05 mg/ml) activity was unaffected by either inhibitor.

 
Tissue extract and uterine flush enzyme activity in these assays were similarly characterized. Serial dilutions of each sample were analyzed to ensure that the maximum change in fluorescence intensity due to substrate hydrolysis was within the linear portion of the established MMP-2 and MMP-9 standard curves. This insured that the amount of gelatinase present in the sample did not exceed the available substrate. For both assays, the specificity of the sample enzyme activity was further characterized as metalloproteinase using nonmetalloproteinase- and metalloproteinase-specific inhibitors, as described for purified MMP-2 and MMP-9 (Fig. 4C).

Gelatinase activity
Compared with controls, relaxin significantly increased the gelatinase-catalyzed fluorescence intensity of the peptide substrate in uterine flushes (P < 0.05; Fig. 5BGo, left panel) while attenuating uterine tissue-associated activity (P < 0.05; Fig. 5AGo, left panel). Cervical tissue gelatinase activity was also decreased in relaxin-treated gilts compared with controls (P < 0.05; Fig. 5CGo, left panel). Collagen IV degradation, measured as a change in fluorescence intensity (Fig. 5Go, A–C, right panel) or a change in mean polarization (data not shown) of the collagen IV substrate, followed a pattern similar to that of the peptide assay. Gelatinase activity was significantly greater (P < 0.05) in uterine secretions from relaxin-treated gilts compared with controls (Fig. 5BGo, right panel). In contrast, gelatinase activity was attenuated in tissue extracts from both the uterus and cervix after relaxin-induced growth in vivo (P < 0.05; Fig. 5Go, A and C, right panel).



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  		Figure 5. Effect of relaxin on gelatinase (MMP-2 and MMP-9) activity in the porcine uterus and cervix. Uterine tissue extracts (A; 20 µg), uterine flushes (B; 10 µl), and cervical tissue extracts (C; 20 µg) from control (C) and relaxin-treated (R) pigs were diluted and incubated with either the peptide substrate (left panel) or collagen IV substrate (right panel) as described in Materials and Methods. Increased fluorescence in the peptide and collagen IV assays indicate enzyme-mediated cleavage of the peptide. *, Values significantly different (P < 0.05) from the control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Relaxin has been shown to have potent uterotropic activities (12, 13). The transient increase in relaxin in the pig endometrium during early pregnancy (30) suggests a role for relaxin in stimulating uterine growth and function during placentation, whereas the growth-promoting effects of relaxin on the uterus may mediate uterine accommodation for rapidly growing fetuses during early pregnancy. On the other hand, at the end of pregnancy, relaxin’s ability to modify the physical properties (12, 31, 32, 33), biochemical composition (34), and histological characteristics (15, 35) of the cervix support a role for relaxin in cervical remodeling and dilation at parturition. In this study we present evidence that relaxin-induced growth of the pig uterus and cervix is associated with secretion of active gelatinases, MMP-2 and MMP-9. In previous studies from our laboratory, a similar relationship was reported between relaxin-induced uterine and cervical growth and urokinase-type plasminogen activator (uPA) activity in the prepubertal pig (19). The data presented here support and extend those studies by showing that the trophic effects of relaxin involve complex and extensive interactions with the enzyme systems responsible for remodeling the extracellular matrix of reproductive connective tissues.

Members of the MMP family are involved in extracellular matrix remodeling of the uterine wall during the cycle, implantation, and pregnancy (3, 4). Similarly, degradation of the extracellular matrix is a critical event in cervical ripening and dilation at parturition (3, 4). Evidence for a role for MMPs in uterine growth in vivo is based on studies in the primate and human uterus demonstrating that an increase in MMP production is associated with cell growth during the proliferative phase of the cycle (36, 37). In addition, MMP expression has been localized in dividing cells of the proliferative endometrium, identified by coexpression of the nuclear antigen Ki67 (37). Several potential roles for the MMPs in promoting reproductive tissue growth have been suggested, including preparing tissue for expansion by altering basement membrane integrity, activation of growth factors, and/or release of matrix-bound growth factors and their receptors to initiate growth stimulatory effects (reviewed in Ref. 3). Given relaxin’s ability to stimulate growth and remodeling in these same tissues, it is likely that a regulatory interaction exists between relaxin and MMP-mediated connective tissue reorganization. Relaxin increases uterine collagenase activity in vitro in rats (16), enhances collagenase production by human amnion-chorion cells in vitro (38), and stimulates collagenolysis in mouse symphysis pubis (39). Conversely, relaxin suppresses collagenase activity in cervical explants from cyclic rats (16), inhibits collagenolysis in the involuting postpartum rat uterus (40), and prevents postpartum collagenase-mediated resorption of the guinea pig symphysis pubis (41). Although these seemingly contradictory effects may be due to experimental design (e.g. in vitro vs. in vivo), it is also possible that these variations are due to tissue-specific temporal and spatial MMP regulation. Furthermore, in an in vivo system, MMP activity can be regulated locally by a number of proteinase inhibitors that may not be present in the in vitro environment.

In this study we found that MMP-2 and MMP-9 protein and activity were significantly enhanced in uterine flushes from relaxin-treated pigs. MMP-2 and MMP-9 degrade various components of the extracellular matrix, including types IV and V collagens, proteoglycan, and elastin (42), and thus are associated with cellular migration, invasion, and tissue remodeling (2, 43). In vivo expression of transcripts for MMP-2 and MMP-9 has been demonstrated in porcine uterine tissue during early pregnancy (44), and human endometrial epithelial and stromal cells produce both MMP-2 and MMP-9 in vitro (45). Furthermore, the increases in MMP-2 and MMP-9 proteins observed in uterine flushes after relaxin administration support and extend studies that show that relaxin increases the secretion of active gelatinases in reproductive tissues. For example, in the rat ovary, relaxin increases granulosa cell secretion of two gelatinases at 63 and 92 kDa (46), which correspond in size to MMP-2 and MMP-9, respectively. In addition, relaxin induces theca-interstitial cell secretion of a 72-kDa gelatinase, corresponding to MMP-2 (46). Likewise, relaxin increases the secretion of active MMP-9 protein in human fetal membrane/decidua explant cultures (47).

Target substrates for MMP-2 and MMP-9, specifically type IV and V collagens, have been localized to the pericellular basement membrane and external lamina surrounding differentiated human decidual cells (48) as well as placental perivascular cells (49). By enhancing the activity of these enzymes, relaxin may facilitate endometrial tissue reorganization during implantation and placentation. In addition, type V collagen is associated with the extracellular matrix of the human uterine stroma (50), and production of both MMPs by fibroblasts, a major component of the stroma, has been demonstrated. Relaxin may increase the expression of these enzymes to promote remodeling of the uterine stroma to accommodate the growing fetuses. Similarly, in sows during the last third of gestation, relaxin promotes reorganization of smooth muscle fiber bundles, a prominent component throughout the length of the pig cervix (15), and increases cervical blood vessel diameter (15). Although the extracellular matrix of the cervix consists mainly of type I and III collagens, hyaluronic acid, proteoglycans, and elastin, type IV collagen, a target substrate for the gelatinases (7, 8), is distributed in a linear fashion between smooth muscle fibers and along the vascular basement membrane of the cervix (51, 52). These observations suggest that relaxin’s ability to soften the cervix during the later stages of pregnancy might involve regulating MMP-2- and MMP-9-mediated remodeling of these structural elements.

The increases in MMP-2 and MMP-9 protein and activity we observed in uterine flushes after relaxin administration are probably due to uterine tissue production and release into the extracellular space and uterine lumen. This is supported by our observation that tissue-associated MMP-2 and MMP-9 activity was lower in both the uterus and cervix after relaxin administration to induce growth. We reported a similar pattern of relaxin-induced increases in uPA activity in uterine luminal fluid with no change in either uterine or cervical tissue-associated uPA (19). We hypothesize that connective tissue remodeling is an early event important for growth. However, to regulate growth once tissue remodeling begins, a means of controlling connective tissue protease activity becomes paramount. This may include, for example, the shifting of connective tissue proteases, such as MMP-2, MMP-9, and uPA, from the tissue into the luminal compartment. On the other hand, relaxin-induced uterine and cervical growth and remodeling may also involve changes in other MMPs or connective tissue proteases not investigated in this study. For example, in the cervix of estrogen-primed rats relaxin increases the activity of degradative enzymes such as collagenase (MMP-1) and proteoglycanase (16). In human fetal membranes, in addition to increasing MMP-9 activity, relaxin induces dose-dependent increases in MMP-1 and MMP-3 gene expression, protein secretion, and enzyme activity into culture medium (47, 53). Another consideration is that the synthesis and activity of MMPs are sensitive to steroid hormones (1, 2, 4, 54, 55, 56, 57). The model used here represents the effects of relaxin on porcine gelatinases in a steroid-deficient environment. It is possible that the expression and/or activity of porcine MMP-2 and MMP-9 in response to relaxin may differ in uterine and cervical tissues of mature sows exposed to systemic steroid hormones and thus may account for the decrease in tissue-associated gelatinase activity observed.

As MMP activity is locally regulated by tissue inhibitors of metalloproteinases (TIMPs), the post-relaxin decline in tissue-associated gelatinase activity may be the result of increased TIMP activity in the uterus and cervix. This is an attractive alternative, given that expression of TIMPs in the pig uterus is selectively enhanced in the endometrial stroma during early pregnancy (44). Furthermore, relaxin may enhance the expression of TIMPs in the cervix to localize and regulate remodeling. For example, the formation of stable gelatinase/TIMP complexes would prevent activation of latent enzyme, and if the peak in TIMP production lags behind that of the enzyme, the increased TIMP/gelatinase ratio could account for the decrease in gelatinase activity. Thus, the impact of relaxin on uterine and cervical TIMPs will be an important area of future research.

In summary, the present study is the first to demonstrate an effect of relaxin on porcine uterine and cervical MMP-2 and MMP-9 production and activity in vivo. We present evidence for the expression of both MMP-2 and MMP-9 in uterine and cervical tissues during relaxin-mediated growth in the prepubertal pig. In addition, we report that the net effect of relaxin administration in vivo was a decrease in tissue-associated gelatinase activity in the uterus and cervix and an increase in uterine secretion of active MMP-2 and MMP-9. These findings support a role for relaxin in promoting MMP-2- and MMP-9-mediated extracellular proteolysis in the pig uterus and cervix to facilitate growth and highlights the complex nature of MMP regulation during this growth and remodeling. The balance between production, activation, and inhibition of these enzymes implies multiple points of regulation. Further studies are needed to determine the nature of relaxin’s contribution to this process.


   Acknowledgments
 
The authors thank Drs. Edward Zambraski, Patricia Schoknecht, and Sylvie Ebner for their assistance with the animal surgery; Ms. Patricia Kraft and Ms. Donna Haynes-Johnson for assistance with the MMP activity assays; and Ms. Katherine McGonigle for technical assistance.


   Footnotes
 
This work was supported by USDA Grant 99-35203-7812 (to C.A.B.) and the New Jersey Agricultural Experiment Station.

1 Present address: Mississippi State University, Mississippi State University, Mississippi 39762. Back

2 Present address: Serono Reproductive Biology Institute, Randolph, Massachusetts 02368. Back

Abbreviations: MMP, Matrix metalloproteinase; PMSF, phenylmethylsufonylfluoride; TBST, Tris-buffered saline; TIMP, tissue inhibitor of metalloproteinases; uPA, urokinase-type plasminogen activator.

Received March 26, 2001.

Accepted for publication May 18, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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