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Biology of Reproduction 65, 1780-1788 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Disruption of the Tissue Inhibitor of Metalloproteinase-1 Gene in Reproductive-Age Female Mice Is Associated with Estrous Cycle Stage-Specific Increases in Stromelysin Messenger RNA Expression and Activity1

Warren B. Nothnick2,a

a Departments of Obstetrics and Gynecology and Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue inhibitors of metalloproteinases (TIMPs) are expressed in the uteri of virtually all species, yet the precise role of these factors in uterine physiology is uncertain. It has been previously demonstrated that disruption of the TIMP-1 gene product in vivo results in altered reproductive cycles and an aberrant uterine phenotype. Because this phenotype may be due to an elevation in uterine matrix metalloproteinase (MMP) activity, the purpose of the following experiments was to identify which uterine MMPs may have their expression altered in response to disruption of the TIMP-1 gene. Mature female TIMP-1 wild-type and null mice were killed during each stage of the estrous cycle, and uterine MMP activity and transcript expression were assessed. Disruption of the TIMP-1 gene product was associated with an increase in total uterine protease activity. Gel zymography further revealed that uterine stromelysin (stromelysin-1, -2, and -3) activity was significantly increased in the TIMP-1 null mice, whereas Northern blot analysis indicated that an up-regulation of stromelysin-1 and -3 mRNA expression may contribute to this increase in activity. It is concluded from this study that TIMP-1 plays a pivotal role in regulating uterine stromelysins both at the level of protease activity and the level of transcript expression.

female reproductive tract, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the course of the reproductive cycle, the uterus continuously undergoes dynamic structural and physiological changes. Structural changes within the uterus involve degradation and remodeling of the extracellular matrix (ECM), which are most prominent during the period of menses. One of the major families of proteases responsible for these changes in tissue turnover is the matrix metalloproteinases (MMPs). Uterine expression of MMPs has been mapped in several species, including the human [1–5], monkey [6, 7], baboon [8], sheep [9], rat [10, 11], and mouse [12]. In menstruating species such as the human, monkey, and baboon, MMPs are implicated to play a role in the active tissue remodeling that occurs during menstruation [1–8]. In nonmenstruating species such as the rat and mouse, and during the proliferative stage of the human menstrual cycle, the exact role of MMPs is uncertain. Matrix metalloproteinases may play a role in the normal tissue remodeling that occurs during the reproductive cycle, which is associated with cell proliferation and/or differentiation [1–8, 13].

Matrix metalloproteinase activity and, hence, tissue remodeling are controlled at three levels: 1) production and secretion of latent enzymes, 2) activation of latent enzymes, and 3) inhibition of enzyme activity by inhibitors of MMPs [14, 15]. The MMP inhibitors can be classified as either serum-borne or tissue-derived. The serum-borne inhibitors include the macroglobulins, such as {alpha}2-macroglobulin, and are found throughout the body. The exact role of this class of inhibitors in regulating MMP activity at the level of the tissue has been questioned based on their large size (>=720 kDa) and ability to traverse endothelial basement membranes. The second class of MMP inhibitors are referred to as the tissue inhibitors of metalloproteinases (TIMPs), which are secreted at the level of the tissue and are postulated to control the site and extent of MMP-induced tissue remodeling/ECM breakdown. Currently, four TIMPs have been identified [16, 17], of which TIMP-1 appears to be the most functionally diverse; TIMP-1 has been implicated to play a role in angiogenesis, steroidogenesis, cell proliferation, and mammary gland development. Most recently, it has been demonstrated that disruption of the TIMP-1 gene product is associated with an alteration in the "normal" uterine lumen structure [18]. In fact, TIMP-1 null female mice exhibit increased uterine luminal epithelial cell density, and the luminal epithelial cells appear to invade the surrounding stromal tissue in an uncontrolled fashion. This aberrant phenotype may be a result of uncontrolled MMP activity, which may lead to breakdown of the stromal cell compartment and/or enhanced ability of the luminal epithelial cells to penetrate the surrounding stromal tissue. The breakdown of the supporting ECM of the stroma could further influence cell proliferation by regulation of specific gene expression and cell-matrix interactions [19, 20].

The purpose of the following set of experiments was to examine the changes in uterine MMP expression and activity during the estrous cycle in TIMP-1 wild-type and null mice to gain insight regarding the mechanisms by which TIMP-1 may regulate murine uterine MMPs and their physiological actions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

The TIMP-1 null and wild-type mice were utilized for all studies. Animals deficient in TIMP-1 (SVTER 129 background) were generated by homologous recombination of a neo-containing, gene-targeting vector in mouse embryonic stem cells. Transmission of the mutant allele and the genotype of mice were determined by polymerase chain reaction analysis of the neo sequences in genomic tail DNA. Deficiency of TIMP-1 was confirmed at the transcript and protein level by Northern blot analysis and protease inhibitor assays, respectively [21].

A breeding colony of both genotypes was generated at the University of Kansas Medical Center. Mice were housed within environmentally controlled conditions under the supervision of a licensed veterinarian. All animal procedures for these experiments were approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee. Mice were maintained on a 14L:10D photoperiod and provided water and mice chow ad libitum. Eight- to twelve-week-old female mice of both genotypes were monitored for reproductive cyclicity through at least two consecutive reproductive cycles, and stage of estrous cycle was determined as previously described [18]. Animals were killed during each of the four stages of the estrous cycle.

Twelve to fifteen animals of each genotype were monitored in each trial, and three separate trials were performed over the course of a 4-mo period. All animals were killed by decapitation, and uteri were removed, trimmed of fat and connexion, and weighed. Uteri were then snap-frozen in liquid nitrogen until utilized for protein or total RNA extraction. Uterine protease activity as well as MMP mRNA expression were determined using a minimum of three separate mice/genotype/stage of cycle (see individual figure legends for the exact number of mice/genotype/stage of cycle used for each experiment).

Extraction of Uterine Proteinases

Uterine proteinases were extracted (1:5, w/v) by homogenizing uteri in homogenization buffer (0.5 M Tris-HCL [pH 7.6], 0.2 M NaCl, 0.01 M CaCl2, and 1.0% [w/v] Triton X-100). Homogenized samples were then placed on ice for 5 min, followed by centrifugation at 12 000 x g for 30 min at 4°C. Supernatants were then removed, and an aliquot was subjected to the DC Protein Assay (Bio-Rad Laboratories, Richmond, CA) to determine protein concentration. The remaining sample was stored at -70°C until analyzed for protease activity.

Protease Assay

Uterine protease activity was assessed using a modification of a previously described MMP-inhibitor assay [21, 22]. Briefly, uterine proteinase activity was determined by adding 50–100 µg of total protein into 0.5 ml of ACA buffer (0.05 M Tris, 0.01 M CaCl2, 0.2 M NaCl, 0.05% Brij 35, and 0.02% NaN3, pH 7.5). Samples were preincubated for 2 h at 37°C in the presence or absence of the MMP-inhibitor TIMP-1 (100 ng of recombinant human TIMP-1, kindly provided by Dr. Hideaki Nagase, University of Kansas Medical Center) or chelating agents known to inhibit MMP activity (5 mM EDTA or 5 mM 1,10-phenanthroline). After the preincubation period, an additional 0.5 ml of ACA buffer containing 2 mg/ml of the colorimetric substrate Azocoll (Calbiochem, Richmond, CA) was added to each sample, and samples were incubated for 18 h at 37°C with continuous shaking. Protease activity (reflected as a breakdown of the Azocoll substrate and liberation of the dye into the buffer) was quantitated using an Ultrospec 2000 spectrophotometer (Pharmacia Biotech, Piscataway, NJ) at a wavelength of 520 nm. Data were expressed as arbitrary units, which reflect the change in OD units per milligram of protein compared to a blank buffer sample.

Collagenase Activity Assay

Uterine extracts (250 µg of total protein) were incubated at 30°C for 48 h with 20 µg of native type I collagen and brought to a final volume of 100 µl with incubation buffer (50 mM Tris-HCl, 5 mM CaCl2, 1% Triton X-100, and 0.02% NaN3). Following incubation, sample volumes were reduced using a speed vacuum, and samples were reconstituted in gel loading buffer to a final volume of 50 µl. Then, SDS-PAGE was used to separate {alpha}1A and {alpha}2A collagen 1 degradation products. Recombinant human interstitial collagenase (1 µg of MMP-1; kindly provided by Dr. Hideaki Nagase) was included as a positive control.

Gelatin and Casein Zymography

To qualitatively identify the types and relative abundances of the gelatinases and stromelysins responsible for the MMP activity, gelatin and casein zymography were performed as previously described but with minor modifications [23]. Briefly, uterine extracts (50–250 µg of total protein) were subjected to substrate gel zymography by electrophoresis in 10% polyacrylamide gels containing 1 mg/ml of gelatin or in precast, 4–16% polyacrylamide gels containing 1 mg/ml of blue casein (Invitrogen, Carlsbad, CA). After electrophoresis, SDS was eluted from the gels in two changes of 2.5% Triton X-100 for 60 min at room temperature (RT). After washing, buffer was replaced with incubation buffer (50 mM Tris [pH 7.5] and 5 mM CaCl2), and gels were incubated for 30 min at RT. Incubation buffer was then replaced with fresh buffer, and gels were allowed to incubate for 18–24 h at 37°C. In some experiments, gels were allowed to incubate for 48 h at 37°C to allow detection of MMP-13 (collagenase-3) activity [24]. Gelatin-impregnated gels were then rinsed with water and stained with Coomassie Blue R250 dye (2.5 mg/ml). Gels were destained, and gelatin-degrading enzymes were qualitatively identified by their ability to digest the gel and compared to molecular weight markers and purified MMPs. Casein-impregnated gels required no further processing (i.e., staining and destaining) to allow detection of bands of casein digestion. The MMPs were identified based on their ability to degrade gelatin (MMP-2 and -9) or casein (MMP-3, -7, and -13) as well as by comparison of their point of migration/molecular weight compared to molecular weight markers and purified human pro-MMP-2, -3, and -9 (kindly provided by Dr. Hideaki Nagase) and semipurified rat MMP-7 (a generous gift from Dr. Thomas E. Curry, Jr., University of Kentucky, Lexington, KY). The level of MMP activity was identified as clear bands of lysis against a blue background. To quantitate the level of activity, zymograms were photographed using Polaroid positive/negative film (Fischer Scientific, Pittsburgh, PA), and the optical density (OD) of each band was quantitated using the GelPro System (Media Cybernetics, Des Moines, IA). The level of activity was then normalized to the amount of protein loaded in each lane by running 50 µg of total protein on a 10% acrylamide gel, staining the gel with Coomassie Blue, and photographing the destained gel with positive/negative film. The negative image for the band of protein that corresponds to albumin (molecular weight, 72 kDa) was used as a reference. The level of expression for this band was shown during preliminary studies not to change across the estrous cycle or to vary between genotypes. Activity for each MMP was then expressed as the ratio of OD units for that MMP to the OD units for albumin.

RNA Isolation and Northern Blot Analysis

Total RNA was isolated from the uterus of each animal (n >= 3 genotypes/cycle stage) by separately homogenizing the tissue in 1 ml of Trizol reagent (Life Technologies/Gibco BRL, Gaithersburg, MD) per 100 mg of tissue wet weight. The RNA was then extracted with chloroform and precipitated with isopropyl alcohol according to the manufacturer's recommendations. Total RNA samples were next electrophoresed through 1.0% agarose gels containing 2.2 M formaldehyde and transferred to nylon membranes (Nytran; Schleicher and Schuell, Keene, NH) as recommended by the manufacturer. The murine MMP-2, -3, and -9 cDNA probes (kindly provided by Dr. Dylan Edwards, University of East Anglia, Norwich, UK) as well as the MMP-7 [25], -10 [12], -11 [12], and -13 [26] probes (kindly provided by Dr. Lynn Matrisian, Vanderbilt University, Nashville, TN) were excised from their respective plasmids using the following restriction endonucleases: MMP-2, EcoRI/HindIII, yielding a 475-base pair (bp) insert; MMP-3, EcoRI/HindIII, yielding a 600-bp insert; MMP-7, HindIII/BamHI, yielding a 700-bp insert; MMP-9, PstI, yielding a 740-bp insert; MMP-10, EcoRI, yielding an approximately 700-bp insert; MMP-11, ApaI, yielding an approximately 1100-bp insert; and MMP-13, KpnI/BamHI, yielding an approximately 800-bp insert. Resulting inserts were labeled using Ready to Go DNA labeling beads (Pharmacia Biotech). Probes were labeled to a specific activity of 5 x 108 to 1 x 109 dpm/µg of DNA using [{alpha}-32P]dCTP (NEN-DuPont, Boston, MA). Membranes were hybridized overnight, washed, and exposed to Kodak-XAR-5 film (Fischer Scientific) for up to 24 h at -75°C. Some of the membranes were stripped of hybridized probe by boiling the blots in a solution of 0.1x SSC (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate) and 0.5% SDS and then reprobed for other MMPs. After all MMP transcripts were analyzed, blots were either stripped of the hybridized MMP probes or the MMP signal was allowed to decay. All membranes were then reprobed for the 18S ribosomal RNA transcript using a rat cDNA probe (kindly provided by Dr. Michael Melner, Vanderbilt University, Nashville, TN), which cross-hybridizes with the mouse transcript [18]. Membranes were then exposed to Kodak XAR-5 film for 2 h at -75°C. In all experiments, MMP data were normalized to the relative expression of the 18S transcript for each of the study groups. Data are expressed as fold changes in OD units.

Statistical Analysis

All data were analyzed across stage of the estrous cycle by one-way ANOVA. When an F test indicated statistical significance, post-hoc analysis was made using the Student-Newman-Keuls procedure. Planned comparisons between genotypes within each stage of the estrous cycle were made using unpaired t-tests. Significance was set at P < 0.05 for all comparisons [27].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TIMP-1 Null Mice Have Elevated Uterine Protease Activity

In wild-type mice, uterine total protease activity was lowest during the periods of diestrus and proestrus and significantly increased during the periods of estrus and metestrus (Fig. 1). Total uterine protease activity in the TIMP-1 null mice displayed a similar pattern of activity, because the lowest levels were detected during diestrus (Fig. 1). Protease activity significantly increased during proestrus, estrus, and metestrus (Fig. 1). When uterine total protease activity was compared within each stage of the estrous cycle between genotypes, TIMP-1 null mice exhibited significantly greater uterine proteases activity during proestrus and estrus (P < 0.05), whereas protease activity in the null mice during metestrus tended to be greater than that of their wild-type counterparts (P = 0.07) (Fig. 1).



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  		FIG. 1. Total uterine protease activity during the estrous cycle in TIMP-1 wild-type and null mice. Uterine proteases were extracted and quantitated as described in Materials and Methods. Data are displayed as the mean ± SEM, with three to four independent observations per cycle stage/genotype. Block letters and italicized letters indicate statistically significant differences (P < 0.05) within wild-type and null mice, respectively, across the estrous cycle by one-way ANOVA. Asterisks indicate statistically significant differences (P < 0.05) within cycle stage between genotypes as determined by planned comparison

To determine the contribution of the MMPs to the total uterine protease activity, biological and chemical inhibitors of MMPs were utilized. Addition of the natural MMP-inhibitor TIMP-1 inhibited protease activity compared to vehicle-treated samples during all stages of the estrous cycle in both wild-type and TIMP-1 null mice (Fig. 2A). When the percentage change in protease activity in response to addition of TIMP-1 was compared within each cycle stage between genotypes, it was noted that the protease activity derived from uteri of TIMP-1 null mice exhibited significantly higher levels of protease activity that could be inhibited by TIMP-1 (Fig. 2A). In fact, TIMP-1 inhibited protease activity in the range of twofold (during proestrus) to threefold more (during diestrus, estrus, and metestrus) in TIMP-1 null mice compared to their respective wild-type counterparts (Fig. 2A). Similar inhibition patterns and proportions of protease activity between genotypes and across the estrous cycle, compared to those obtained with TIMP-1, were obtained with both EDTA (Fig. 2B) and phenanthroline (data not shown), further supporting the postulate that MMP activity is elevated in the uteri of TIMP-1 null mice.



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  		FIG. 2. Uterine MMP activity during the estrous cycle. Uterine MMP activity was assessed as described in Materials and Methods with an aliquot of the same uterine extracts used to assess total protease activity in Figure 1. Data are displayed as the mean ± SEM, with three to four independent observations per cycle stage/genotype. A) TIMP-1-inhibited activity in wild-type and TIMP-1 null mice across the estrous cycle. B) EDTA-inhibited activity in wild-type and TIMP-1 null mice across the estrous cycle. Block letters and italicized letters indicate statistically significant differences (P < 0.05) within wild-type and null mice, respectively, across the estrous cycle by one-way ANOVA. Asterisks indicate statistically significant differences (P < 0.05) within cycle stage between genotypes as determined by planned comparison

TIMP-1 Null Mice Exhibit Estrous Cycle-Specific Increases in Uterine Stromelysin Activity

To ascertain the contribution of each class of MMPs (i.e., collagenases, gelatinases, stromelysins, and matrilysins) to the total protease activity, substrate-specific assays as well as Northern blot analysis were performed. The most abundant uterine MMPs were of the gelatinase family, because activity could be detected with as little as 50 µg of total protein. Gelatin zymography revealed that both MMP-2 (gelatinase A) and MMP-9 (gelatinase-B) were detected across the stages of the estrous cycle in mice of both genotypes (Fig. 3). Both uterine MMP-2 and -9 activity across the estrous cycle was variable and showed no significant differences among the stages of the estrous cycle within genotype or within the cycle stages between genotypes. To assess the level of uterine MMP-2 and -9 mRNA expression, Northern blot analysis was performed. Uterine MMP-2 transcript was detected across the estrous cycle in mice of both genotypes as a single band of approximately 3.1 kilobases (kb) (Fig. 4A). However, the level of expression did not significantly change across the stages of the estrous cycle or between the genotypes within each stage of the cycle, with the exception that, during the period of estrus, uterine MMP-2 transcript expression in TIMP-1 null mice was significantly lower (P < 0.05) compared to wild-type counterparts (Fig. 4A). The MMP-9 transcript was detected as a single band of approximately 2.9 kb, and its expression was highly variable across the estrous cycle. Within genotype across the estrous cycle, wild-type mice exhibited peak MMP-9 expression during estrus, whereas TIMP-1 null mice exhibited peak expression during proestrus (Fig. 4B). When comparisons within cycle stage between genotypes were made, it was determined that TIMP-1 null mice exhibited significantly greater MMP-9 transcript expression during proestrus compared to wild-type counterparts.



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  		FIG. 3. Uterine gelatinase activity during the estrous cycle in TIMP-1 wild-type and null mice. Total uterine protein was extracted and subsequently analyzed for MMP-2 (gelatinase A) and MMP-9 (gelatinase B) activity as described in Materials and Methods using 50 µg of protein per lane. Major bands of proteolytic activity were detected at 96 and 86 kDa, which represent the pro- and active forms of MMP-9, as well as at 72 and 62 kDa, which represent the pro- and active forms of MMP-2. Data are representative of five observations per cycle stage/genotype (n = 5). D, Diestrus; E, estrus; M, metestrus; P, proestrus



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  		FIG. 4. Uterine gelatinase mRNA expression during the estrous cycle in TIMP-1 wild-type and null mice. Total uterine RNA was extracted and subsequently analyzed for MMP-2 (gelatinase A) and MMP-9 (gelatinase B) mRNA expression as described in Materials and Methods using 10 µg (MMP-2) or 20 µg (MMP-9) of total RNA/lane. A) Upper panel is a representative Northern blot for MMP-2; lower panel depicts the expression of the ribosomal 18S RNA. The MMP-2 mRNA was detected as a single transcript of approximately 3.1 kb, whereas the 18S rRNA was detected as a single transcript of approximately 1.9 kb. Bar graph displays normalized data expressed as the mean ratio of MMP-2 transcript to 18S transcript ± SEM and are reported as OD units for four separate observations (n = 4 mice/genotype/stage of cycle). Autoradiographic exposures for MMP-2 membranes were for 4 h at -75°C. B) Upper panel is a representative Northern blot for MMP-9; lower panel depicts the expression of the ribosomal 18S RNA. The MMP-9 mRNA was detected as a single transcript of approximately 2.9 kb. Bar graph displays normalized data expressed as the mean ratio of MMP-9 transcript to 18S transcript ± SEM and are reported as OD units for four separate observations (n = 4 mice/genotype/stage of cycle). Autoradiographic exposures for MMP-9 membranes were for 48 h at -75°C. Different letters indicate statistical significance (P <0.05) across the estrous cycle within genotype as determined by one-way ANOVA (block letters indicate comparisons within wild-type mice, and italicized letters indicate comparisons within null mice). Asterisks indicate statistically significant differences (P < 0.05) within cycle stage between genotypes as determined by planned comparison. D, Diestrus; E, estrus; M, metestrus; P, proestrus

To assess the contribution of stromelysins to the total MMP activity, casein zymography and Northern blot analysis were performed. In contrast to the gelatinases, uterine stromelysin activity was generally low during the periods of diestrus, proestrus, and metestrus, with a peak expression of activity occurring during the period of estrus in wild-type mice (Fig. 5). In contrast, stromelysin activity in the TIMP-1 null mice was significantly higher during both estrus and metestrus (Fig. 5). Specifically, TIMP-1 null mice exhibited an approximately eight- and twofold increase in stromelysin activity during estrus and metestrus, respectively, compared to their wild-type counterparts. Northern blot analysis of the stromelysins revealed that each of the three stromelysin family members displayed distinct patterns of expression during the course of the reproductive cycle (Fig. 6). More specifically, uterine transcript expression of MMP-3 (stromelysin-1) was detected as a single band of approximately 1.9 kb, and its expression was highest during diestrus and proestrus in wild-type mice and declined during estrus and metestrus (Fig. 6A). In contrast, MMP-3 expression in the null mice did not decrease during metestrus, as occurred in the wild-type mice (Fig. 6A). Expression of MMP-3 during metestrus in the null mice was significantly greater compared to that in wild-type mice during metestrus. Uterine MMP-10 (stromelysin-2) was detected as a single band of approximately 1.7 kb in mice of both genotype. In both wild-type and TIMP-1 null mice, uterine MMP-10 transcript levels were highest during proestrus and significantly (P < 0.01) decreased during estrus, metestrus, and diestrus (Fig. 6B). No significant differences in MMP-10 expression were detected within cycle stages between genotypes. Uterine MMP-11 (stromelysin-3) was detected as two transcripts of approximately 2.4 and 3.8 kb, which is consistent with previous reports of multiple MMP-11 transcripts [28, 29]. Expression of MMP-11 was greatest during the period of estrus and showed relatively low levels of expression, which did not change during diestrus, proestrus, or metestrus in wild-type mice (Fig. 6C). In TIMP-1 null mice, MMP-11 mRNA expression was also greatest during the period of estrus, but unlike in the wild-type mice, MMP-11 expression did not decrease during metestrus, remaining significantly elevated (Fig. 6C).



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  		FIG. 5. Uterine stromelysin activity during the estrous cycle in TIMP-1 wild-type and null mice. Total protein was extracted and subsequently analyzed for MMP-3 (stromelysin-1), MMP-10 (stromelysin-2), and MMP-11 (stromelysin-3) activity as described in Materials and Methods using 250 µg of protein per lane. Casein zymography of uterine stromelysin activity revealed major bands at 58, 43, and 28 kDa. Data are representative of four separate observations (n = 4 mice/genotype/stage of cycle). D, Diestrus; E, estrus; M, metestrus; P, proestrus



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  		FIG. 6. Uterine stromelysin mRNA expression during the estrous cycle in TIMP-1 wild-type and null mice. Total mRNA was extracted and subsequently analyzed for MMP-3 (stromelysin-1), MMP-10 (stromelysin-2), and MMP-11 (stromelysin-3) mRNA expression as described in Materials and Methods using 10 µg of total RNA/lane. A) Upper panel is a representative Northern blot for MMP-3; lower panel depicts the expression of the ribosomal 18S RNA. The MMP-3 mRNA was detected as a single transcript of approximately 1.9 kb. Bar graph displays normalized data expressed as the mean ratio of MMP-3 transcript to 18S transcript ± SEM and are reported as OD units for four separate observations (n = 4 mice/genotype/stage of cycle). B) Upper panel is a representative Northern blot for MMP-10; the lower panel depicts the expression of the ribosomal 18S RNA. The MMP-10 mRNA was detected as a single transcript of approximately 1.7 kb. Bar graph displays normalized data expressed as the mean ratio of MMP-10 transcript to 18S transcript + SEM and are reported as OD units for four separate observations (n = 4 mice/genotype/stage of cycle). C) Upper panel is a representative Northern blot for MMP-11; lower panel depicts the expression of the ribosomal 18S RNA. The MMP-11 mRNA was detected as two transcripts of approximately 2.4 and 3.8 kb, and the OD units for both transcripts are expressed together as MMP-11 (no significant differences in expression of either transcript were detected between genotypes across the estrous cycle, so OD data were combined for both transcripts). Bar graph displays normalized data expressed as the mean ratio of MMP-11 transcript to 18S transcript ± SEM and are reported as OD units for four separate observations (n = 4 mice/genotype/stage of cycle). Autoradiographic exposures for MMP-3, -10, and -11 were for 24 h at-75°C. The blot depicted in A (wild-type) for MMP-3 was subsequently stripped after hybridizing for MMP-2 (Fig. 4A, wild-type), and as such, the same 18S rRNA hybridization signal is depicted in both figures. The blot depicted in B for MMP-10 was stripped and rehybridized for MMP-11 (C), and as such, the same 18S rRNA Northern blot is depicted in both B and C. Different letters indicate statistical significance (P < 0.05) across the estrous cycle within genotype as determined by one-way ANOVA (block letters indicate comparisons within wild-type mice, and italicized letters indicate comparisons within null mice). Asterisks indicate statistically significant differences (P < 0.05) within cycle stage between genotypes as determined by planned comparison. D, Diestrus; E, estrus; M, metestrus; P, proestrus

Using gelatin zymography, matrilysin activity could not be detected in uterine extracts of either wild-type or TIMP-1 null mice (data not shown). Semipurified rat matrilysin was included as a positive control and detected as a single 28-kDa band of gelatin-degrading activity, which is consistent with the reported size for this MMP (data not shown). This is a peculiar observation, because a rather robust expression of uterine MMP-7 transcript was detected (a single band of ~1.1 kb) in mice of both genotypes. Transcript levels were low to undetectable during diestrus and proestrus and then significantly increased during estrus and metestrus (Fig. 7). No significant differences in the level of expression of MMP-7 mRNA was detected between genotypes within any stage of the estrous cycle.



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  		FIG. 7. Uterine MMP-7 (matrilysin) mRNA expression during the estrous cycle in TIMP-1 wild-type and null mice. Total mRNA was extracted and subsequently analyzed for MMP-7 (matrilysin) expression as described in Materials and Methods using 10 µg of total RNA/lane. The MMP-7 mRNA was detected as a single transcript of approximately 1.1 kb. The MMP-7 mRNA expression is expressed as the mean ratio of MMP-7 transcript to 18S transcript ± SEM and reported as OD units for four separate observations (n = 4 mice/genotype/stage of cycle). Autoradiographic exposures for MMP-7 were for 24 h at -75°C. Different letters indicate statistical significance (P < 0.05) across the estrous cycle within genotype as determined by one-way ANOVA (block letters indicate comparisons within wild-type mice, and italicized letters indicate comparisons within null mice)

To assess uterine MMP-13 (collagenase-3) activity, collagen type I degradation assays as well as gelatin zymography [24] were utilized. Using either assay for uterine MMP-13 activity, MMP-13 (collagenase-3) activity could not be detected using up to 250 µg of total protein (data not shown). Initial analysis of MMP-13 mRNA using 10 µg of total RNA per lane (the quantity used to assess all other MMPs) produced very faint signals. When 20 µg of total RNA per lane were used to analyze MMP-13 expression, however, a stronger signal was detected, but no significant differences across the estrous cycle or between genotypes within a given stage of the cycle were noted (Fig. 8). The MMP-13 was detected as a single band of approximately 2.8 kb in mice of both genotypes.



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  		FIG. 8. Uterine MMP-13 (collagenase-3) mRNA expression during the estrous cycle in TIMP-1 wild-type and null mice. Total mRNA was extracted and subsequently analyzed for MMP-13 (collagenase-3) mRNA expression as described in Materials and Methods using 20 µg of total RNA/lane. The MMP-13 mRNA was detected as a single transcript of approximately 2.8 kb. The MMP-13 mRNA data are expressed as the mean ratio of MMP-13 transcript to 18S transcript ± SEM and reported as OD units for four separate observations (n = 4 mice/genotype/stage of cycle). Autoradiographic exposures for MMP-13 were for 48 h at -75°C. No statistically significant differences were detected in MMP-13 expression across the estrous cycle within genotypes or within cycle stage between genotypes. The same membrane in Figure 4B that was hybridized for MMP-9 was stripped and reprobed for MMP-13, and as such, the same 18S rRNA Northern blot is depicted in both figures

In summary, the major changes in MMP expression in the TIMP-1 null mice were the increase in stromelysin activity during estrus and metestrus (Fig. 5). This increase in activity during metestrus in the null mice was associated with increased levels of MMPs 3 and 11 mRNA expression (Fig. 6). These data may be interpreted to suggest that the increase in stromelysin activity may not only be at the level of MMP activity (due to the absence of TIMP-1 neutralizing activity) but also at the level of stromelysin (MMPs 3 and 11) mRNA transcript expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A multifunctional protein, TIMP-1 is capable of eliciting a wide variety of biological effects, including cell proliferation, differentiation, and programmed cell death [16, 17, 30–37]. The mechanisms for these diverse functions of TIMP-1 have been shown to be either independent or dependent on TIMP-1's ability to regulate MMP activity. For example, TIMP-1 has been shown to regulate hepatic cell proliferation [30] as well as mammary epithelial cell apoptosis [31] via MMP-dependent mechanisms. In contrast, TIMP-1 has also been reported to regulate cell proliferation and programmed cell death in other cell types via what appears to be MMP-independent pathways [32–36]. Taken together, these studies suggest that TIMP-1 is capable of regulating both cell proliferation and programmed cell death in a wide array of cell types. It appears that, in some cell types, regulation of these processes are dependent on the ability of TIMP-1 to regulate protease activity, whereas in others, TIMP-1 appears to regulate cell proliferation and apoptosis via a protease-independent cellular signaling system.

Within the uterus, TIMP-1 may have more of an antiproliferative effect, because absence of TIMP-1 is associated with aberrant uterine lumen development and luminal epithelial cell proliferation [18]. The mechanisms responsible for this altered pattern of epithelial cell proliferation are uncertain, but the current study indicates an increase in stromelysin activity, suggesting that this mechanism may be MMP-related. This is a particularly exciting finding, because the stromelysins are capable of regulating the bioavailability of membrane- or protein-bound growth factors such as heparin binding-epidermal growth factor (HB-EGF) [37] and insulin-like growth factor-I (IGF-I) [38–40], both of which stimulate (or are proposed to stimulate) uterine epithelial cell proliferation [41–44]. Stromelysin-1 (MMP-3) is capable of cleaving cell surface-expressed HB-EGF [37] as well as degrading both IGF binding protein (BP)-1 [40] and IGFBP-3 [38, 39]. In the mouse, IGFBP-1 and -3 [45] as well as -4 [46] are expressed during the estrous cycle and are postulated to facilitate the bioavailability of IGF-I. The degradation of these IGFBPs by stromelysins within the uterus of the mouse could further dictate the amount of bioavailable IGF-I. However, whether stromelysins degrade IGFBPs and which uterine IGFBPs are degraded or cleaved by the stromelysins remain to be determined. The current data indicate at least an association among absence of TIMP-1, elevated stromelysin activity, and altered uterine phenotype, but more definitive studies are clearly warranted to examine the mechanisms by which TIMP-1 regulates uterine development and growth.

Additionally, TIMP-1 may regulate uterine lumen structure by maintaining the uterine stromal component, which may impose a physical barrier to penetration by proliferating epithelial cells. In the TIMP-1 null mice, the elevated levels of stromelysin activity may lead to a breakdown of the stromal ECM, making the stromal component of the uterus more susceptible to invasion by the proliferating epithelial cells. The elevated levels of stromelysin activity in the TIMP-1 null mice may also contribute to disruption of the basement membrane, which may lead to alterations in the cell-matrix interactions. Cell-matrix interactions are known to influence cell proliferation and/or differentiation as well as to affect the responsiveness of cells to external stimuli [19, 20, 26].

The elevation of uterine stromelysin activity in the TIMP-1 null mice was not surprising, because the alteration in the TIMP:MMP ratio would favor an increased activity of those MMPs that it can normally bind to and neutralize. However, it was surprising that, of all the MMPs that TIMP-1 can bind to and inhibit, only the stromelysins expressed markedly increased activity. Along these lines, it was unexpected that the increase in stromelysin activity may also be due to an increase in stromelysin transcript expression. Whereas TIMP-1 has been reported to increase collagenase (MMP-1) activity [47], the exact mechanism for this increase is uncertain. It is plausible that, because TIMP-1 has been postulated to act via a receptor-ligand signaling system [32–36], TIMP-1 under normal circumstances (i.e., in the wild-type mice) regulates MMP-11 expression at the level of transcription. Alternatively, the elevated levels of stromelysin activity could lead to the liberation of growth factors or other signaling molecules, which could further regulate stromelysin expression at the transcript level, because growth factors have been shown to stimulate uterine stromelysin transcript expression [24].

The present study also provided, to my knowledge, the first characterization of MMP activity across the estrous cycle in the mouse. In the present study, it was demonstrated that the gelatinases appear to be the major active uterine MMPs expressed during the estrous cycle. These findings agree with the mRNA expressions of MMP-2 and -9, which were detected to varying degrees during all stages of the estrous cycle. Whereas the gelatinases appeared to be expressed during the entire estrous cycle, stromelysin activity was most evident during proestrus and estrus in the wild-type mice, with the majority of the activity being detected during estrus. This peak in stromelysin activity agreed with the pattern of stromelysin mRNA expression in the wild-type mice. In TIMP-1 null mice, the pattern of stromelysin activity agreed with that of stromelysin mRNA expression, exhibiting marked expression/activity during estrus and metestrus.

Both collagenase-3 and matrilysin were detected at the mRNA level in mice of both genotypes, but bioactive forms of the MMPs could not be detected using zymography. This inability could be due posttranslational regulation of these MMPs. For example, TIMPs 2 and 3, which are expressed within the uterus during the estrous cycle [18], could possibly bind to these MMPs and neutralize their bioactivity. Similar posttranslational regulation of MMP-9 may also explain the inability to detect elevated levels of MMP-9 activity despite significant increases in MMP-9 transcript expression. It has been well established [14, 15] that concurrent increases in TIMP expression accompany increases in MMP transcript expression and regulate MMP activity. As previously reported [18], uterine TIMPs 1 and 3 increase during proestrus through metestrus and, once translated into active inhibitor, could neutralize MMP activity.

The role of uterine TIMPs during the reproductive cycle is unknown. In both human and nonhuman menstruating species, TIMPs are postulated to control the activity of the MMPs and, hence, to dictate the site and extent of tissue breakdown that occurs during menses. However, TIMPs (and MMPs) are expressed not only during the entire menstrual cycle (i.e., not just during menses) but also within the uterus of "nonmenstruating" species such as the sheep [9], rat [10, 11], and mouse [12, 18]. As such, the precise uterine function of TIMPs in any species is not clear. We have learned from studies using TIMP-1 null mice that TIMP-1 appears to regulate the events that occur during the reproductive cycle at the level of the uterus, because TIMP-1 deficiency is associated with irregular reproductive cycles, increased wet weight gain, decreased TIMP-3 expression, decreased progesterone receptor expression (unpublished results), and abnormal uterine morphology [18]. The current study provides additional insight regarding the mechanisms responsible for this phenotype, and the results suggest that elevated MMP activity, and specifically that of the stromelysins, may be at least in part responsible for these abnormalities. The question now becomes which specific MMPs are responsible, and how do they lead to this altered uterine phenotype? Based on the current findings, coupled with the data in the literature, it is postulated that the stromelysins may be a logical starting point to determine the exact mechanisms by which TIMP-1 controls the events that occur within the uterus during uterine development, growth, and function.


   ACKNOWLEDGMENTS
 
Gratitude is expressed to Dr. Paul Soloway (Roswell Park Cancer Institute) for initial generation of the TIMP-1 deficient mice and his helpful insight. The author would also like to thank Drs. Lynn Matrisian and Michael Melner (Vanderbilt University) for the MMPs 3, 7, 10, 11, and 13 (Dr. Matrisian) and the 18S ribosomal RNA (Dr. Melner) cDNA probes as well as Dr. Dylan Edwards (University of East Anglia) for the MMPs 2 and 9 cDNAs. Additionally, gratitude is expressed to Drs. Hideaki Nagase (University of Kansas Medical Center) and Thomas Curry (University of Kentucky) for the TIMP-1 (Dr. Nagase) and MMP standards.


   FOOTNOTES
 
First decision: 5 February 2001.

1 Supported by a grant award (HD37941) from the Office of Research on Women's Health in conjunction with the NICHD and core support from NICHD center grant HD33994. Back

2 Correspondence: Warren B. Nothnick, University of Kansas Medical Center, Department of Obstetrics and Gynecology, 3901 Rainbow Blvd., Kansas City, KS 66160. FAX: 913 588 6271; wnothnic{at}kumc.edu Back

Accepted: August 2, 2001.

Received: January 11, 2001.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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