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Endocrinology Vol. 143, No. 1 91-98
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Relaxin Increases Secretion of Tissue Inhibitor of Matrix Metalloproteinase-1 and -2 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, Rutgers University (J.A.L., P.L.R., K.M.O., C.A.B.), 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. E-mail: bagnell{at}aesop.rutgers.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Remodeling of reproductive organs during pregnancy requires degradation and resynthesis of structural barriers to cell invasion. Matrix metalloproteinases (MMPs) are enzymes that break down components of the extracellular matrix (ECM) and are essential for tissue remodeling processes. Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of MMP activity. In the pig, relaxin stimulates growth and remodeling of the uterus and cervix during pregnancy, effects that include the ability to alter elements of the ECM. Therefore, the objective of this study was to determine whether relaxin alters the production and/or activity of TIMP-1 and TIMP-2 in the porcine uterus or cervix. The growth-promoting effects of relaxin were elicited by administering relaxin to prepubertal gilts every 6 h for 54 h. Expression of TIMP-1 and TIMP-2 was characterized by immunoblotting. Total enzyme activity was measured using an MMP-specific fluorescent substrate assay. TIMP-1 and TIMP-2 proteins were present in the uterus and cervix of control and relaxin-treated pigs, and both proteins were increased by relaxin in the uterine flushes and tissues (P < 0.05). Inhibitor activity in uterine tissue extracts and uterine flushes from relaxin-treated animals was greater than that in controls; however, this activity was restricted to inhibition of MMP-2. In the uterine cervix, relaxin enhanced expression of TIMP-1 and TIMP-2 (P < 0.05), whereas expression of both TIMP proteins was similar in the vaginal cervix of control and relaxin-treated animals. Likewise, inhibitor activity against MMP-2 in the uterine cervix was enhanced in response to relaxin (P < 0.05). In contrast, inhibitor activity was attenuated in extracts from the vaginal cervix (P < 0.05). This study highlights the complex nature of MMP/TIMP regulation during reproductive tissue growth and suggests that TIMP-1 and TIMP-2 may be involved in other aspects of the growth process. These data support a role for relaxin in regulating the activity of TIMPs during growth and remodeling of reproductive connective tissue.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UTERINE AND CERVICAL growth requires both cell proliferation and orchestrated remodeling of connective tissue and the extracellular matrix (ECM). Although degradation of the ECM involves a variety of enzymes, the matrix metalloproteinases (MMPs) are primary contributors to this process. MMP activity is locally regulated by tissue inhibitors of metalloproteinases (TIMPs) secreted from the same or closely adjacent cell types as those producing the MMPs. TIMPs form complexes with active and latent MMPs and the concurrent production of proteinases and their inhibitors allows strict regulation and localization of proteolytic activity. The extent of ECM remodeling reflects a balance between the activity of MMPs and that of their inhibitors. TIMP-1, TIMP-2, and TIMP-4 are present in soluble form (1), whereas TIMP-3 is insoluble, bound to the ECM (2). TIMP-1 and TIMP-2 target the gelatinases (MMP-2 and MMP-9), with TIMP-1 preferentially binding to MMP-9, whereas TIMP-2 has a high affinity for MMP-2 (1). TIMP-1 and TIMP-2 are multifunctional proteins with diverse actions in addition to inhibiting enzyme-mediated tissue remodeling. For example, both TIMP-1 and -2 exhibit growth factor-like activity (1, 3) and can inhibit angiogenesis (4, 5). TIMP expression is under the influence of a diverse array of stimuli, including hormones, growth factors, and cytokines (6). As TIMPs are highly expressed in reproductive tissues and hormonally regulated, they are thought to control a variety of connective tissue remodeling events in organs such as the ovary and uterus (7).

Relaxin stimulates growth and remodeling of the pig uterus and cervix (8, 9), and connective tissue is a prime target for relaxin in reproductive tissues. Relaxin’s growth-promoting effects in the uterus and cervix include the ability to modify structural support elements of the ECM, such as the basement membrane, to facilitate remodeling. Degradation of the basement membrane is thought to be crucial for invasion and tissue remodeling (10, 11). Changes in uterine and cervical connective tissue matrix composition (12) and proteolytic enzyme profile (13) are associated with relaxin-induced growth. For example, relaxin-induced uterine and cervical growth in prepubertal gilts is accompanied by increased secretion of serine protease activity and protein (i.e. urokinase-type plasminogen activator) into the uterine lumen (14). Likewise, we recently reported that after relaxin-induced uterine growth in this model, there was a significant increase in the secretion of active gelatinases, MMP-2 and MMP-9 (15), which are the primary MMPs responsible for type IV collagen degradation, the major component of basement membranes. In contrast, tissue-associated gelatinase activity in both the uterus and cervix was lower in relaxin-treated animals compared with controls (15). As TIMPs are important regulators of MMP-mediated remodeling, we were interested in determining whether the postrelaxin decline in tissue-associated MMP-2/MMP-9 activity in this model was the result of increased TIMP activity in uterine and cervical tissues. In addition, although the role of TIMPs during reproductive tissue remodeling has been studied in a number of species (7), TIMP expression and activity in pig reproductive tissues have received less attention. Thus, the objectives of this study were to determine the effect of relaxin on porcine TIMP-1 and TIMP-2 production in vivo during relaxin-induced uterine and cervical growth and remodeling, and to quantify relaxin-mediated changes in TIMP activity in this model using a gelatinase-specific activity assay.


   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 (16). 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 (17), and immunoreactivity was verified by RIA (18). Monoclonal antibovine TIMP-1 (Ab-1) and antihuman TIMP-2 (Ab-1) antibodies and recombinant bovine TIMP-2 and human TIMP-2 proteins were obtained from Oncogene Research Products (Calbiochem, Cambridge, MA). MMP-2 enzyme was acquired from PanVera Corp (Madison, WI). MMP-3 and MMP-9 enzymes were obtained from Biogenesis (Sandown, NH). Goat antimouse IgG-horseradish peroxidase-conjugated antibody was purchased from Transduction Laboratories (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 im with porcine relaxin (0.5 mg) or saline (0.5 ml) every 6 h for 54 h (9). Three hours after the last injection, animals were killed by exsanguination after stunning. Plasma and uterine flushes were collected and processed as described by Ohleth et al. (19). Uterine and cervical tissues were collected and processed as described by Wang-Lee et al. (14). 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 (9, 19) and cervix (14) and the systemic and local concentrations of relaxin (19) achieved after in vivo relaxin administration in this animal model have been reported previously. The prepubertal status of the gilts was confirmed by the absence of 17ß-E2 and progesterone in the plasma and uterine flushes of all animals before and after the treatment regimen (19, 20).

Immunoblot analysis of TIMP-1 and TIMP-2
Uterine and cervical tissues were homogenized in 0.25% (vol/vol) Triton X-100 and 10 mM CaCl2, centrifuged at 9000 x g for 30 min at 4 C to remove insoluble material (21), and stored at -80 C until analysis. Tissue extracts and uterine flushes were desalted before analysis using Micro Bio-Spin chromatography columns (P-6, Bio-Rad Laboratories, Inc., Hercules, CA). Proteins from uterine flushes (10 µl) and uterine and cervical extracts (20 µg) were resolved on 4–12% bis-Tris-HCl-buffered PAGE gels (Novex, San Diego, CA) under reducing conditions. Bovine TIMP-1 (75 ng) and human TIMP-2 (50 ng) served as the positive controls. Proteins were transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). Membranes were blocked in 5.0% (wt/vol) BSA in Tris-buffered saline (TBST), then incubated with anti-TIMP-1 or anti-TIMP-2 antibody (0.5 µg/ml) in TBST/1% (wt/vol) BSA overnight at 4 C. Membranes were washed with TBST and incubated with horseradish peroxidase-conjugated goat antimouse IgG [1:7500 in TBST/5% (wt/vol) 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 MMP-9 to MMP-3 ratio of 40:1 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 ZnSO, and 0.05% (vol/vol) Brij 35] for fluorescent activity analysis.

MMP substrate
The peptide substrate was (Aedens)EAGPRGMAGQFSH(dabcyl)K-amide, a gelatinase-specific, FRET peptide, developed at R. W. Johnson Pharmaceutical Research Institute (Raritan, NJ). The fluorescent peptide was prepared in 100% dimethylsulfoxide at 8 mM, then 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.

Preparation of samples for inhibitor activity analysis
Tissue extracts and uterine flushes were heated to 100 C for 30 min to inactivate endogenous MMPs. Samples were cooled on ice, then incubated with methylamine hydrochloride (0.2 M final) for 30 min at room temperature to destroy {alpha}-macroglobulin inhibitors. Samples were desalted before analysis using Micro Bio-Spin chromatography columns (P-6, Bio-Rad Laboratories, Inc.).

Inhibitor activity assays
Inhibitor activity was measured using a gelatinase-specific activity assay in which the ability of MMP-2 and MMP-9 to degrade the FRET peptide substrate has been demonstrated (22). Tissue extracts (20 µg) and uterine flushes (10 µl) were diluted to a total volume of 20 µl in assay buffer. Equal volumes of uterine flushes were examined to obtain a measure of total inhibitor activity in the uterine lumen of control and relaxin-treated animals. Activated MMP-2 (15 ng) or MMP-9 (15 ng) in assay buffer or assay buffer alone (5 µl) was added, and the final reaction volume was brought up to 100 µl with assay buffer containing the fluorescently labeled peptide substrate (final substrate concentration, 20 µM). The ability of samples to inhibit gelatinase-catalyzed proteolysis of the peptide substrate was quantified by measuring changes in fluorescence intensity (fluorescent intensity units) at 37 C every 30 min for 8 h, using a CytoFluor multiwell plate reader (series 4000, Perseptive Biosystems, Framingham, MA). Linearity was established for enzyme/ inhibitor interaction using serially diluted TIMP-1 and TIMP-2 proteins to inhibit MMP activity. The positive controls were 1,10-phenanthroline (0.1 M), a metalloproteinase inhibitor, and heat- and methylamine hydrochloride-inactivated samples without the addition of exogenous MMP-2 or MMP-9. The negative controls included 1) activated MMP-2 or MMP-9 in assay buffer in the absence of inhibitors and 2) 0.05 mg/ml trypsin diluted in assay buffer with and without the addition of TIMP protein or 1–10 phenanthroline.

Densitometry and statistical analysis
TIMP-1 and TIMP-2 were quantified in immunoblots by scanning densitometry (Sigma Gel, SPSS, Inc., Chicago, IL). Data 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
 
Validation of peptide substrate fluorescent assays
A linear concentration/activity relationship was established for serially diluted MMP-2 (Fig. 1AGo) and MMP-9 (Fig. 1CGo) standards alone and for MMP-2 in the presence of serially diluted TIMP-1 and TIMP-2 (Fig. 1BGo). In contrast, although MMP-9 activity was inhibited by TIMP-1 in a concentration-dependent manner, TIMP-2 inhibition was minimal, and the ability of TIMP-2 to inhibit MMP-9 was not sensitive to the TIMP-2 concentration (Fig. 1DGo).



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  		Figure 1. Inhibition of gelatinase-catalyzed proteolysis of the fluorescent peptide substrate by TIMP-1 and TIMP-2. A, Assay linearity for serially diluted MMP-2. B, Linear concentration/inhibition relationship with MMP-2 (15 ng) in the presence of serially diluted TIMP-1 and TIMP-2. C, Assay linearity for serially diluted MMP-9. D, Linear concentration/inhibition relationship with MMP-9 (15 ng) when incubated with serially diluted TIMP-1. In contrast, inhibition of MMP-9 by TIMP-2 was minimal and was not concentration dependent.

 
Effect of relaxin on uterine TIMP-1 and TIMP-2
Immunoblot analysis. The antibovine TIMP-1 antibody recognized both bovine TIMP-1 (T1; 28 kDa) and human TIMP-2 (T2; 24 kDa) standards (Fig. 2AGo) as well as porcine TIMP-1 (28 kDa) and, to a lesser extent, porcine TIMP-2 (24 kDa; Fig. 2CGo). The antihuman TIMP-2 antibody recognized the human TIMP-2 standard and did not cross-react with the bovine TIMP-1 standard (Fig. 2BGo). In uterine flushes, an immunoreactive band of protein at 28 kDa corresponding to TIMP-1 (Fig. 2AGo) and a 24-kDa band corresponding to TIMP-2 (Fig. 2BGo) were identified in both control and relaxin-treated prepubertal gilts. Relaxin significantly enhanced the secretion of both TIMP-1 (Fig. 2AGo; P < 0.05) and TIMP-2 (Fig. 2BGo; P < 0.05) proteins into the uterine lumen. In uterine tissue, immunoreactive TIMP-1 (Fig. 2CGo) and TIMP-2 (Fig. 2DGo) proteins were also detected in control and relaxin-treated animals, and relaxin significantly increased both TIMP-1 (Fig. 2CGo; P < 0.05) and TIMP-2 (Fig. 2DGo; P < 0.05) expression compared with controls.



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  		Figure 2. Immunoblot analysis of the effect of relaxin on expression of TIMP-1 and TIMP-2 in the porcine uterus. Uterine flush proteins (10 µl), uterine tissue extracts (20 µg), bovine TIMP-1 (T1; 75 ng), and human TIMP-2 (T2; 50 ng) were resolved by SDS-PAGE, and TIMP proteins were detected using monoclonal TIMP-1- and TIMP-2-specific antibodies. Graphs illustrate the quantitative analysis of TIMP protein (mean ± SE) for the corresponding immunoblot. Uterine flush TIMP-1 (A) and TIMP-2 (B) proteins and uterine tissue TIMP-1 (C) and TIMP-2 (D) proteins from control and relaxin-treated gilts are shown. *, Values significantly different (P < 0.05) from control. C, Control; R, relaxin-treated.

 
Uterine inhibitor activity. Inhibitor activity is expressed as the difference between peptide degradation by the enzyme in the absence of inhibitor (0% inhibition) and that in the presence of uterine flush or tissue sample. Uterine flushes from both control and relaxin-treated animals inhibited MMP-2 activity (Fig. 3AGo). However, after relaxin administration, flushes displayed significantly greater inhibitory activity compared with controls (relaxin, 32.9 ± 0.6%; control, 22.4 ± 0.92%; P < 0.02). In contrast, MMP-9 activity was not significantly altered in the presence of uterine secretions from either control or relaxin-treated animals (Fig. 3AGo). Inhibitory activity in uterine tissue protein from control and relaxin-treated pigs was similar to that observed in the flushes. Relaxin administration significantly enhanced uterine tissue-associated MMP-2 inhibition (relaxin, 52.4 ± 0.5%; control, 21.6 ± 0.62%; P < 0.001; Fig. 3BGo), whereas uterine tissue extracts from control and relaxin-treated pigs failed to inhibit MMP-9 activity (Fig. 3BGo).



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  		Figure 3. Effect of relaxin on inhibition of gelatinase activity in the porcine uterus. Endogenous enzymes in uterine flushes (A; 10 µl) and uterine tissue extracts (B; 20 µg) from control and relaxin-treated pigs were inactivated, and the resulting supernatant was assayed for its ability to inhibit peptide substrate degradation in the presence of MMP-2 (15 ng) or MMP-9 (15 ng) as described in Materials and Methods. Data are expressed as the percent inhibition, which is the difference between peptide degradation by the enzyme in the absence of inhibitor (0% inhibition) and in the presence of uterine flush or tissue sample. *, Values significantly different (P < 0.05) from control. C, Control; R, relaxin-treated.

 
Effect of relaxin on cervical TIMP-1 and TIMP-2
Immunoblot analysis. Relaxin administration enhanced the expression of immunoreactive TIMP-1 (Fig. 4AGo; P < 0.05) and TIMP-2 (Fig. 4BGo; P < 0.05) proteins in the uterine cervix compared with controls. In contrast, expression of TIMP-1 and TIMP-2 proteins was similar in the vaginal cervix of control and relaxin-treated animals (Fig. 4Go, C and D).



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  		Figure 4. Immunoblot analysis of the effect of relaxin on TIMP-1 and TIMP-2 expression in the porcine cervix. Cervical tissue extracts (20 µg) from the uterine and vaginal portions of the cervix, bovine TIMP-1 (T1; 75 ng), and human TIMP-2 (T2; 50 ng) were resolved by SDS-PAGE, and TIMP proteins were detected using monoclonal TIMP-1- and TIMP-2-specific antibodies. Graphs illustrate the quantitative analysis of TIMP protein (mean ± SE) for the corresponding immunoblot. Uterine cervix TIMP-1 (A) and TIMP-2 (B) proteins and vaginal cervix TIMP-1 (C) and TIMP-2 (D) proteins from control and relaxin-treated gilts. *, Values significantly different (P < 0.05) from control. C, Control; R, relaxin-treated.

 
Cervical inhibitor activity. MMP-2 activity was inhibited in the presence of tissue protein from both the uterine and vaginal ends of the cervix of control and relaxin-treated gilts. In the uterine cervix (Fig. 5AGo), inhibition of MMP-2 activity was significantly greater (P < 0.05) after relaxin administration (67.3 ± 1.3%) compared with that in controls (34.3 ± 0.7%). The situation was reversed in the vaginal cervix (Fig. 5BGo), where inhibition of MMP-2 activity was significantly less (P < 0.05) in tissues from relaxin-treated gilts (27.0 ± 1.2%) compared with that in control animals (34.0 ± 1.0%). In both uterine and vaginal cervix, MMP-2 activity was much more sensitive to inhibition (ranging from 27.0–67.3%) compared with MMP-9 (<10% inhibition). There were no differences in the inhibition of MMP-9 activity when the uterine and vaginal portions of the cervix were compared or between control and relaxin-treated animals (Fig. 5Go, A and B).



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  		Figure 5. Effect of relaxin on inhibition of gelatinase activity in the porcine cervix. Endogenous enzymes in tissue extracts (20 µg) from the uterine (A) and vaginal (B) cervix of control and relaxin-treated pigs were inactivated, and the resulting supernatant was assayed for its ability to inhibit peptide substrate degradation in the presence of MMP-2 (15 ng) or MMP-9 (15 ng) as described in Materials and Methods. Data are expressed as the percent inhibition, which is the difference between peptide degradation by the enzyme in the absence of inhibitor (0% inhibition) and in the presence of uterine flush or tissue sample. *, Values significantly different (P < 0.05) from control. C, control; R, relaxin-treated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Relaxin has potent uterotropic activities (8, 9, 23) and increases the distensibility of the collagenous framework of the uterus. Likewise, relaxin’s ability to modify the physical properties (9, 24, 25, 26), biochemical composition (27), and histological characteristics (12, 28) of the cervix supports a role for relaxin in cervical dilation at parturition. Although the effects of relaxin on reproductive tissue growth and remodeling are well established, the means by which relaxin exerts its trophic influence has not been fully elucidated. Previous studies from our laboratory have demonstrated that relaxin has tissue-specific effects on reproductive connective tissue enzyme activity in the prepubertal gilt, specifically that of plasminogen activators (14) and gelatinases (15). In this study we present evidence that relaxin regulates TIMP-1 and TIMP-2 production and activity in vivo in the porcine uterus and cervix. The data presented here support and extend those studies by showing that relaxin enhances the expression of TIMPs, which selectively regulates ECM proteolysis by binding to MMPs involved in reproductive tissue remodeling. Concurrent stimulation of proteolytic enzyme and inhibitor activity by relaxin may be a means of locally controlling the growth and degradation important for uterine and cervical expansion.

The process of tissue remodeling requires a balance between the activities of MMPs and TIMPs. TIMPs play an important role in regulating implantation and placentation (29, 30), and TIMP-1, TIMP-2, and TIMP-3 proteins have been identified within various compartments of the human endometrium (31). In addition, TIMPs have been implicated in numerous other reproductive tissue remodeling processes, such as ovulation (32), angiogenesis (4), embryo growth (33) and mammary gland development (34, 35). For example, TIMPs are thought to control the interactions between epithelial cells and the underlying basement membrane during morphogenesis and remodeling of the mammary gland (34, 35), which help to maintain the structural integrity of the basement membrane and protect it from degradation. Although TIMP gene expression has been detected in pig follicular cells (36) and during luteal development (37), this study is the first to demonstrate TIMP-1 and TIMP-2 protein and activity in pig uterine and cervical tissue and secretion of active TIMPs into uterine luminal fluid.

As endometrial TIMP-1 expression varies during the menstrual cycle (31), it may be sensitive to changes in hormone levels. However, hormonal control of TIMPs in the uterus and cervix has not been widely investigated. In vitro, E2 and progesterone have been reported to increase TIMP-1 production in uterine cervical fibroblasts (38). In contrast, although progesterone alone or in combination with E2 down-regulates TIMP-2 production in human breast cancer cells (39), for the most part uterine TIMP-2 expression appears to be independent of steroids. No changes in uterine tissue TIMP-2 have been reported during the menstrual cycle (31). During pregnancy, increased TIMP-2 expression is limited to localized areas of endometrial cells undergoing decidualization (30, 40, 41). Consequently, TIMP-2 may be involved in other processes, such as enhancing growth and/or regulating angiogenesis. Although this is the first report of a role for relaxin in the regulation of uterine and cervical TIMP expression and activity, the effects of relaxin on TIMP expression have been reported in several cell types. In support of the data reported here, relaxin increased TIMP-1 and TIMP-2 activities in medium from equine ovarian stromal cell cultures (42). However, relaxin decreased TIMP-1 in human skin fibroblasts in vitro (43) and had no effect on the production of TIMP-1 mRNA in human fetal membranes (44).

During uterine accommodation, the change in uterine connective tissue from a compact to a more widely separated network of fibers is accomplished through carefully controlled degradation and restructuring of the ECM. Likewise, cervical ripening and dilation at parturition require tightly regulated proteolytic activity. The extensive uterine growth and cervical remodeling elicited by relaxin in this model led us to hypothesize that relaxin would up-regulate the expression and activity of pig MMPs. Although we did observe an increase in gelatinase (MMP-2/MMP-9) activity in uterine flushes, uterine and cervical tissue-associated gelatinase activity was attenuated (17). Therefore, in this study we analyzed the expression of gelatinase-specific inhibitors, specifically TIMP-1 and TIMP-2, to determine whether relaxin enhanced inhibitor expression to regulate enzyme activity. We found that 54 h after the administration of relaxin to induce uterine and cervical growth, the expression and activity of TIMP-1 and TIMP-2 in the uterus and uterine cervix were significantly increased. These results were consistent with the suppression of MMP-2 and MMP-9 activity we observed in these tissues (15). In contrast, in the vaginal cervix relaxin had no effect on the expression of TIMP-1 and TIMP-2 protein and decreased TIMP inhibitor activity. This spatial difference in TIMP expression and inhibitory activity in the pig cervix may be due to temporal differences in the regulation of remodeling between the uterine and vaginal portions of the cervix. For example, the uterine portion of the cervix remains relatively firm throughout most of pregnancy in the pig and is thought to be more important than the vaginal portion of the cervix for retaining the fetuses and protecting the uterus from infection (45). Thus, the balance between MMP/TIMP expression and activity might be shifted in favor of a TIMP-mediated suppression of ECM remodeling in the uterine cervix for a longer period than that of the vaginal cervix.

To our knowledge, this is the first TIMP activity assay based on the ability of endogenous inhibitors to attenuate the degradation of a gelatinase-specific substrate by MMP-2 and MMP-9. Other inhibitor assays used to measure TIMP activity employ less selective MMP substrates, such as Azocoll (46) or reverse gelatin zymography (29). Although these assays are useful for measuring bioactive TIMP, assay sensitivity is an issue (37). The fluorescent assays used in this study allowed us to examine the activities of MMP-2 and MMP-9 on specific matrix substrates in their native conformation rather than on a mix of proteins after denaturation, as is the case in the Azocoll assay. Furthermore, these assays do not readily lend themselves to monitoring TIMP activity over time to ensure that inhibition is not in the plateau phase due to insufficient enzyme or substrate availability. Quantitative analysis of total inhibitor activity requires that the enzyme and substrate both be present in excess. This assay enabled us to quantify TIMP activity at a point on the assay activity curve where neither enzyme nor substrate was limiting. Thus, specific inferences can be made about the level of gelatinase activity in the porcine uterus and cervix in the presence or absence of relaxin. However, the peptide degradation assay used in this study measures total inhibitory activity; therefore, it was not possible to quantify the individual contributions of TIMP-1 and TIMP-2 proteins relative to the total inhibitory activity in each sample. Although this limited our ability to determine the individual effects of each inhibitor in the tissues, we demonstrate that tissue extracts from both the uterus and uterine cervix inhibited MMP-2 activity while having no significant effect on MMP-9. As both TIMP-1 and TIMP-2 standards were able to inhibit MMP-2, but only TIMP-1 significantly attenuated MMP-9 activity, it is likely that the bulk of the inhibitory activity measured in pig uterine and cervical samples was TIMP-2. Further studies will be necessary to confirm these findings.

Although the primary function of the TIMPs is thought to be extracellular matrix homeostasis and maintenance of tissue integrity, additional biological functions of TIMPs have been described. TIMP -1 and TIMP-2 may control activation of latent MMPs, as they form complexes with pro-MMP-9 and pro-MMP-2, respectively (47, 48). TIMPs may also be involved in the potentiation of cell growth (3, 49) and steroidogenesis (50). The transient increase in relaxin in the porcine uterine endometrium during early pregnancy (51) suggests a role for relaxin in stimulating uterine growth and function during placentation. Consequently, the increase in uterine tissue-associated TIMP-1 and TIMP-2 protein and activity in response to relaxin may facilitate uterine accommodation for rapidly growing fetuses by stimulating uterine metabolism and growth. Another reported function of TIMPs is inhibition of MMP-mediated degradation of IGF-binding protein-3 (IGFBP-3) (52). Increased local production of IGFs is pivotal in stimulating uterine growth during pregnancy in the pig (53), and IGFBPs regulate IGF bioavailability. In this study we found that TIMP-1 and TIMP-2 were significantly elevated in uterine flushes from relaxin-treated animals at the same time a significant increase in uterine luminal IGFBP-2 and -3 was observed (19). These findings suggest that the growth-promoting effects of relaxin on the pig uterus may involve interaction of the IGF and MMP/TIMP systems.

In summary, this study is the first to demonstrate an effect of relaxin on porcine uterine and cervical TIMP-1 and TIMP-2 production and activity. We present evidence for the expression of both TIMP-1 and TIMP-2 in uterine and cervical tissues during relaxin-induced growth in the prepubertal pig. These findings further support a role for relaxin in ECM remodeling during uterine growth and cervical ripening. In addition, these results suggest that the roles of TIMP-1 and TIMP-2 in the uterus and cervix may be more complex than simply regulating gelatinase activity. Further studies are needed to more completely assess the involvement of TIMPs in relaxin-induced reproductive tissue growth.


   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, Mississippi 39762. Back

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

Abbreviations: ECM, Extracellular matrix; IGFBP, IGF-binding protein; MMP, matrix metalloproteinase; TBST, Tris-buffered saline; TIMP, tissue inhibitor of metalloproteinases.

Received July 16, 2001.

Accepted for publication September 13, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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