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Biology of Reproduction 67, 99-106 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Bovine Membrane-Type 1 Matrix Metalloproteinase: Molecular Cloning and Expression in the Corpus Luteum1

Bo Zhanga, Li Yanb, Marsha A. Mosesb, and Paul C.W. Tsang2,,a

a Department of Animal and Nutritional Sciences, University of New Hampshire, Durham, New Hampshire 03824 b Laboratory for Surgical Research, Department of Surgery, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix metalloproteinase-2 (MMP-2) is produced as a zymogen, which is subsequently activated by membrane-type 1 metalloproteinase (MT1-MMP). The objectives of the present study were to clone bovine MT1-MMP and to investigate its expression in the corpus luteum. Corpora lutea were harvested from nonlactating dairy cows on Days 4, 10, and 16 of the estrous cycle (Day 0 = estrus; n = 3 for each age). The bovine MT1-MMP cDNA contained an open reading frame of 1749 base pairs, which encoded a predicted protein of 582 amino acids. Northern blotting revealed no differences (P > 0.05) in MT1-MMP mRNA levels between any ages of corpora lutea. Western blotting demonstrated that two species of MT1-MMP, the latent form (~63 kDa) and the active form (~60 kDa), were present in corpora lutea throughout the estrous cycle. Active MT1-MMP was lower (P < 0.05) in early stages of the corpus luteum than the mid and late stages, where MMP-2 activity, as revealed by gelatin zymography, was also elevated. Furthermore, immunohistochemistry revealed that MT1-MMP was localized in endothelial, large luteal, and fibroblast cells of the corpus luteum at different stages. Taken together, the differential expression and localization of MT1-MMP in the corpus luteum suggest that it may have multiple functions throughout the course of the estrous cycle, including activation of pro-MMP-2.

corpus luteum, corpus luteum function, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The corpus luteum is a dynamic, transient endocrine gland that develops from the postovulatory follicle [1]. In addition to functional changes, the development, maintenance, and regression of the corpus luteum are associated with structural events such as tissue remodeling and angiogenesis [2, 3]. It is believed that this tissue remodeling process includes breakdown and resynthesis of extracellular matrix (ECM) components [2, 4], which require the participation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) [2, 5, 6]. Although the literature is still emerging, a few of the secreted MMPs and TIMPs have been investigated in corpora lutea and follicles of species with long luteal phases, including the bovine [7–10], ovine [11–13], porcine [14], human [15], and macaque [16].

Matrix metalloproteinases are secreted as latent proenzymes, which are proteolytically cleaved to their active forms [17, 18]. It is proposed that some serine proteinases, such as trypsin, neutrophil elastase, and plasmin, can initiate the extracellular activation process of pro-MMPs by a "cysteine switch" mechanism [19]. Unlike other secreted MMPs, however, pro-MMP-2 is inefficiently activated by this proteolytic activation mechanism, because its sequence is not susceptible to cleavage by serine proteinases [20]. Instead, pro-MMP-2 activation is localized on the cell surface [21], where membrane-type 1 MMP (MT1-MMP) has been identified as its activator in placenta and several different kinds of tumor cells [22, 23].

Currently, 6 different membrane-type MMPs (MT1–MT6) have been identified [24, 25] with MT1-MMP being the most well-studied. Similar to other nonmembrane type MMPs, MT1-MMP is characterized by having a signal peptide at the amino terminus, followed by a propeptide, catalytic domain, hinge region, and hemopexin-like domain at the C terminus [18, 22]. In contrast to secreted MMPs, MT1-MMP contains a distinct transmembrane domain, which has about 25 hydrophobic amino acid residues, enabling it to be expressed on the cell surface [18]. The human MT1-MMP nucleic acid and protein sequence was the first to be deduced [22], and subsequently, rat [26], mouse [27], and rabbit [28] MT1-MMPs were cloned and sequenced.

MT1-MMP possesses a variety of functions. As a matrix-degrading enzyme, MT1-MMP has collagenolytic and gelatinolytic activities during connective tissue remodeling [29, 30]. With the exception of MT4-MMP, the most well known role of MT-MMPs, including MT1-MMP, is their ability to activate pro-MMP-2, a key enzyme associated with a variety of pathological and physiological processes. Thus, it is not surprising that MT1-MMP expression is correlated with wound healing [25] and a variety of disease states, including tumor invasion and metastasis [31], and rheumatoid arthritis [32]. It is interesting that MT1-MMP expression is also associated with several physiological processes, including bone resorption, implantation, and mammary gland development [24]. In rats and mice, MT1-MMP mRNA is expressed during follicular development, ovulation, and formation and regression of the corpus luteum [33–36].

In order to establish the presence and activity of MT1-MMP in a domestic ruminant, the objectives of the present study were to clone and sequence the bovine-specific MT1-MMP, and to determine the pattern of mRNA and protein expression in relation to MMP-2 activity in early, mid, and late stage corpora lutea obtained throughout the estrous cycle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular Cloning and Sequencing of Bovine MT1-MMP cDNA

A bovine UNI-ZAP II cDNA library was constructed using RNA isolated from bovine capillary endothelial cells of adrenal cortex with oligo(dT) primers. The catalytic domain of bovine MT1-MMP cDNA was amplified from this library using polymerase chain reaction (PCR) primers designed according to the conserved regions of MT1-MMP of other species [18]. The remaining cDNA was amplified using primer-walking. PCR products were subsequently cloned into the PCR TA-cloning vector (Invitrogen, Carlsbad, CA) for DNA sequencing. Complementary DNA sequencing was performed with PCR-based automated sequencing methods (Biopolymer Facility and DNA Sequencing Core Facility, Children's Hospital, Boston, MA). Protein database searches were assessed by using the basic local alignment search tool service of the National Center for Biotechnology Information (Bethesda, MD). DNA and protein sequence analyses were conducted using the Mac Vector 6.0 DNA sequence analysis software package (Kodak, Rochester, NY).

Animals and Tissue Collection

Corpora lutea were collected from regularly cycling, nonlactating dairy cows that were housed at the University of New Hampshire Dairy Teaching and Research Center. Luteal tissues were removed on Days 4, 10, and 16 of the estrous cycle (Day 0 = estrus; n = 3 per day). For Day 4 corpora lutea, the ovary was removed by colpotomy after the cow received an epidural anesthetic (2% mepivacaine hydrochloride; 0.01 mL/kg of body weight; Upjohn, Kalamazoo, MI), and the corpus luteum was then dissected from the ovarian stroma. Corpora lutea at Days 10 and 16 were removed from ovaries by enucleation. All animal experimentation protocols were approved by the Institutional Animal Care and Use Committee at the University of New Hampshire.

Tissue Distribution of MT1-MMP

In order to determine the tissue distribution of MT1-MMP, poly(A)+ RNA from bovine liver, brain, kidney, lungs, heart, and spleen was purified using a modified guanidinium thiocyanate method [37] followed by poly(A)+ RNA selection with two rounds of oligo(dT)-cellulose columns. Poly(A)+ RNA (2.5 µg) was fractionated on 1% formaldehyde agarose gels before transfer onto nylon membranes (Schleicher & Schuell, Keene, NH). A 700-base pair (bp) cDNA probe, corresponding to nucleotides 900 to 1600 of the bovine MT1-MMP coding sequence, was labeled with 32P-dCTP using the Rediprime II Random Prime Labeling System (Amersham Pharmacia Biotech, Piscataway, NJ). The blots were then hybridized overnight at 65°C with radiolabeled probes at a concentration of 2 x 107 cpm/10 ml, followed by washing with 1x SSC at room temperature and 0.1x SSC at 65°C.

Northern Blotting of Luteal Tissues

For analysis of luteal tissues, the same cDNA probe described above was labeled with digoxigenin (DIG)-dUTP using the DIG DNA Labeling Kit (Roche Molecular Biochemicals, Indianapolis, IN). Total RNA was isolated from luteal tissues using the guanidine thiocyanate-phenol acid extraction protocol as described by Chomczynski and Sacchi [37]. Following quantification by absorbance at 260 nm, 20 µg of total RNA was fractionated on 1% (v/v) formaldehyde gels and transferred onto Hybond-N+ nylon membranes (Amersham Pharmacia) by capillary blotting using 20x SSC (3 M NaCl, 300 mM sodium citrate pH 7.0). After an overnight transfer, nucleic acids were cross-linked with a UV Crosslinker (Hoefer, San Francisco, CA). Prehybridization was performed at 50°C in standard hybridization buffer containing 50% (v/v) formamide, 5x SSC, 0.2% (w/v) SDS, and 2% (w/v) blocking reagent (Roche) for 2 h. The membranes were then hybridized in the same buffer containing DIG-labeled MT1-MMP cDNA probe overnight at 65°C. The blots were washed twice at room temperature in 2x SSC in 0.1% (w/v) SDS, followed by higher-stringency washes with 0.1x SSC in 0.1% (w/v) SDS at 65°C. The membranes were then equilibrated in washing buffer (0.1 M maleic acid, 0.15 M NaCl, and 0.3% [v/v] Tween-20 pH 7.5) for 1 min, followed by a 30-min incubation in 1% (w/v) blocking solution (1% blocking reagent in washing buffer). The membranes were then incubated in the same blocking solution containing an anti-DIG antibody conjugated to alkaline phosphatase (1:5000 [v/v]; Roche). After two rinses with washing buffer, the blots were incubated with CSPD (Roche), a chemiluminescent substrate for alkaline phosphatase. Signals were visualized after development of Kodak XAR-5 films by a Konica Medical Film Processor (Tokyo, Japan).

Western Blot Analysis

Finely minced luteal tissue was homogenized in an extraction buffer (50 mM Tris-HCl, 150 mM NaCl, 0.02% [w/v] sodium azide, 10 mM EDTA, 1% [v/v] Triton X-100, 10 µg/ml aprotinin, 1 µg/ml pepstatin A, and 1 µg/ml aminoethyl benzenesulphonyl fluoride) described by Lehti et al. [38] with minimal modification (pH 7.5). A ratio of 0.125 g luteal tissue and 1.0 ml extraction buffer was maintained. Following extraction, the samples were centrifuged at 800 x g for 10 min at 4°C to enable collection of the supernatant fraction.

To assess the protein expression pattern of MT1-MMP in different ages of corpus luteum, equivalent amounts of tissue extracts were subjected to SDS-PAGE on 10% (w/v) polyacrylamide gels. Fractionated proteins were then electrophoretically transferred to Protran nitrocellulose membranes (Schleicher & Schuell). Nonspecific binding sites were blocked with 5% (w/v) nonfat powdered dry milk in TBST buffer (0.01 M Tris-HCl, 0.15 M NaCl, and 0.05% [v/v] Triton X-100; pH 8.0) for 2 h. A rabbit anti-human MT1-MMP polyclonal antibody (1:500; Chemicon International Inc, Temecula, CA), which recognizes the conserved hinge region of the bovine molecule, was diluted in 5% (w/v) nonfat dry milk in TBST, applied onto the membranes, and incubated overnight at room temperature. The membranes were then washed 5 times for 15 min each with TBST before a 1-h incubation with anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (1:15 000; Pierce, Rockford, IL) at room temperature. After another 5 washes as described above, the blots were developed with the use of SuperSignal West Pico Chemiluminescent Substrate (Pierce) according to the manufacturer's instructions. The protein bands were finally visualized after exposure to Kodak XAR-5 film. Low range prestained SDS-PAGE Standard (Bio-Rad Laboratories, Hercules, CA) was run in adjacent lanes.

Gelatin Zymography

Gelatinolytic activity in luteal tissue samples was detected using a modified protocol that was previously described [10]. Briefly, equivalent amounts of tissue extracts were electrophoresed on 10% (w/v) polyacrylamide gels impregnated with 0.5 mg/ml gelatin. After electrophoresis, SDS was removed from gels by two 15-min washes with 2.5% (v/v) Triton X-100, and then incubated overnight at 37°C in substrate buffer (0.05 M Tris-HCl, 5 mM CaCl2, and 0.05 M NaCl pH 8.0). The gels were stained with 0.1% (w/v) Coomassie Brilliant Blue G-250 (Bio-Rad) for 30 min, and destained with distilled H2O. Gelatinolytic activity was revealed as clear bands against a blue-stained background. Perfect Protein Markers (Novagen, Madison, WI) and a positive control, the conditioned medium of HT1080 cells, were run in adjacent lanes.

In order to verify that the gelatinolytic activities detected were metalloproteinases, gels were incubated with substrate buffer containing 10 mM 1,10-phenanthroline (Sigma, St. Louis, MO), a specific MMP inhibitor and a zinc ion chelator. By interfering with the zinc-containing active site of MMPs, zones of clearance are prevented from forming. Furthermore, in order to distinguish between the latent and active forms of MMPs, samples and conditioned medium of HT1080 cells were incubated with 2 mM p-aminophenylmercuric acetate (APMA, Sigma) for 2 h at 37°C before zymography. This results in the cleavage of latent MMPs to their "active," lower-molecular-weight forms, which may undergo further processing (i.e., autolytic cleavage).

Immunohistochemistry

Tissue sections (6 µm) were cut onto Superfrost slides (Fisher Scientific, West Chester, PA), and air-dried at room temperature for 30 min. Sections were then fixed in cold acetone for 10 min, and washed with PBS pH 7.4. Endogenous peroxidase activity was quenched by incubating sections with 0.3% (v/v) hydrogen peroxide for 30 min. Nonspecific binding was blocked for 30 min with 5% (w/v) BSA. Sections were subsequently incubated at room temperature for 1 h with a 1:250 dilution of a polyclonal rabbit anti-MT1-MMP (Chemicon). After washing, slides were incubated with biotin-conjugated goat anti-rabbit immunoglobulin G (1:200; Vector Laboratories, Burlingame, CA), before visualization with the Vectastain Elite ABC Kit (Vector Laboratories) according to the manufacturer's instructions. For each tissue sample, an adjacent section placed on the same slide was used as a negative control, with BSA substituting for the primary antibody. After completion of the color reaction, the slides were counterstained with Gill 2 Haematoxylin (Shandon, Pittsburgh, PA).

Densitometry

Northern blots, Western blots, and zymograms were analyzed with UN-SCAN-IT gel automated digitizing system (Silk Scientific Inc, Orem, UT). For Northern blots, densities of MT1-MMP and 28S rRNA were used to calculate the MT1-MMP:28S rRNA density ratio for each sample. Similarly, for zymography, levels of latent and active MMP2, and the HT1080 positive control were used to calculate the MMP2:HT1080 ratio for each sample.

Statistics

Each sample was run in triplicate. Quantitative values were expressed as means ± SEM. All data were analyzed by ANOVA, followed by the Tukey test of pairwise comparisons to determine differences between the three age groups of corpora lutea. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence Analysis of Bovine MT1-MMP

The bovine MT1-MMP cDNA contained an open reading frame of 1749 bp (GenBank accession number AF290429; 27 July 2000), which encoded a protein of 582 amino acids. All three characteristic regions of the MT-MMP subfamily, the 11-amino acid insertion (IS-I) between the propeptide and catalytic domains, the 8-amino acid (IS-II) insertion in the catalytic domain, and the 75-amino acid (IS-III) at the C terminus, were also present at these positions in bovine MT1-MMP (Fig. 1A). Alignment of the predicted protein sequences revealed significant homology between the bovine MT1-MMP protein and other species, with rat being the highest (95.9%), followed by human (95.7%), mouse (95.0%), and rabbit (92.3%) (Fig. 1B).



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  		FIG. 1. Protein structure and homology analysis of bovine MT1-MMP. A) The signal peptide (Signal), propeptide (Pro), catalytic (Cat), hinge (Hinge), hemopexin (Hex), and transmembrane (TM) domains are indicated. The numbers represent positions of amino acids. The location of MT-MMP subfamily inserts (IS-I, IS-II, and IS-III) are indicated by arrows. B) Species names appear on the left. The whole molecule (Overall) and individual domains of MT1-MMPs are compared. The numbers represent percentages. The identity scores are determined by setting the cow MT1-MMP at 100%

Tissue Distribution of MT1-MMP

Northern blotting revealed a 3.5-kilobase (kb) transcript in a variety of bovine tissues, with the strongest signal in the lungs, followed by spleen, kidney, heart, and liver. The signal in the brain was undetectable (Fig. 2).



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  		FIG. 2. Tissue distribution of bovine MT1-MMP mRNA. For Northern blot analysis, mRNA was extracted from bovine brain, heart, kidney, liver, lung, and spleen before probing with bovine MT1-MMP. The positions of 28S and 18S rRNA are indicated on the left. A single 3.5-kb transcript was detected (arrow)

Expression of MT1-MMP in Bovine Corpus Luteum

A single 3.5-kb transcript was detected by Northern blotting in luteal tissues collected at early, mid, and late stages of the estrous cycle (Fig. 3A). However, the levels of MT1-MMP mRNA, expressed as a ratio of the MT1-MMP band density for each corpus luteum sample to its corresponding 28S rRNA band density, were similar (P > 0.05) among the three stages (Fig. 3B).



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  		FIG. 3. Expression of MT1-MMP mRNA in different ages of bovine corpora lutea. A) The upper panel shows that a single 3.5-kb MT1-MMP transcript (arrow) was detected in early (Day 4), mid (Day 10), and late (Day 16) cycle corpora lutea. Corresponding positions of 28S and 18S rRNA are indicated on the left. The lower panel shows ethidium bromide staining of 28S rRNA. B) The levels of MT1-MMP mRNA, expressed as the densitometric ratio of MT1-MMP to 28S rRNA, were not different (P > 0.05) among any age of corpus luteum. Dissimilar letters denote significant difference at P < 0.05

MT1-MMP was studied in bovine corpora lutea by Western blot analysis. In all samples, two bands with a relative molecular mass of ~63 kDa, consistent with the latent form of MT1-MMP, and ~60 kDa, consistent with the active form of MT1-MMP, were observed (Fig. 4A). The level of active MT1-MMP protein in the Day 4, early corpus luteum was significantly lower (P < 0.05) than in mid and late stages (Fig. 4B).



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  		FIG. 4. Expression of MT1-MMP protein in different ages of bovine corpora lutea. A) In early (Day 4), mid (Day 10), and late (Day 16) cycle corpora lutea, the latent species (~63 kDa) and active species (~60 kDa) of MT1-MMP were observed. B) Densitometric analysis (average total pixels x 105) showed that the active form of MT1-MMP in Day 4 corpus luteum was significantly lower (P < 0.05) than in Day 10 and Day 16 corpora lutea. Dissimilar letters denote significant difference at P < 0.05

Expression of MT1-MMP Is Correlated to Pro-MMP-2 Activation

MMP-2 activity in the bovine corpus luteum during the estrous cycle was determined by gelatin zymography (Fig. 5A). Visual observation revealed species of ~68 kDa (pro-MMP-2) and ~62 kDa (active MMP-2) in all luteal samples. Although the intensity of the ~68 kDa species did not vary, the intensity of the ~62 kDa species in the Day 4 early corpus luteum appeared to be less than the other two, older stages. Incubation of samples with 1,10-phenanthroline prevented the appearance of gelatinolytic zones of clearance (data not shown), indicating that these clear zones on the zymograms reflected MMP activities. Lastly, the decrease in band intensity of the ~68 kDa band after treatment of luteal samples and conditioned medium of HT1080 cells with APMA for 2 h at 37°C suggested that this enzyme species was the latent form of MMP-2 (Fig. 5C).



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  		FIG. 5. Gelatin zymographic analysis of MMP-2 activity in bovine corpus luteum. A) Perfect Protein Markers (Novagen, Madison, WI) were loaded in lane 1 (MW). The positive control, phorbol 12-myristate 13-acetate (PMA)-stimulated HT1080 conditioned medium (CM), was loaded in lane 2. Representative Day 4, Day 10, and Day 16 samples were loaded in lanes 3 to 5, respectively. The arrows on the right indicate the pro- and active forms of MMP-2 in the positive control and corpora lutea samples. The relative molecular masses of protein markers are indicated on the left. B) The active form of MMP-2, expressed as the densitometric ratio of corpora lutea samples to HT1080 conditioned medium, was significantly lower (P < 0.05) in early, Day 4 than in Day 10 and Day 16 corpora lutea. Dissimilar letters denote significant differences at P < 0.05. C) Treatment with APMA. Samples of HT1080 conditioned medium (CM) and representative luteal cell extracts (Day 4, Day 10, and Day 16) were incubated at 37°C with (+) or without (-) 2 mM APMA before zymographic analysis. Perfect Protein Markers were loaded in lane 1. The numbers on the left indicate relative molecular mass (kDa). Arrows indicate the pro- and active forms of MMP-2

Densitometric analysis revealed that levels of the ~68 kDa pro-MMP-2 were similar (P > 0.05; data not shown) among the three stages of corpus luteum. However, the level of the ~62 kDa active form of MMP-2 increased during progression of the estrous cycle, being significantly greater (P < 0.05) in Day 10 and Day 16 corpora lutea than in the early stage (Fig. 5B).

Cellular Distribution of MT1-MMP

Immunohistochemistry was used to investigate the cellular localization of MT1-MMP in the bovine corpus luteum. Overall, the cellular distribution of MT1-MMP varied throughout the life span of the corpus luteum. In early (Fig. 6A) and mid cycle (Fig. 6B) corpora lutea, MT1-MMP was localized in endothelial cells, and on the cell membrane and cytoplasm of large luteal cells, which is consistent with cellular processing of the MT1-MMP protein in cytoplasmic organelles before becoming resident on the cell membrane. In late corpora lutea (Fig. 6C), the expression of MT1-MMP was predominantly observed in fibroblasts, whereas endothelial and large luteal cells were weakly stained. No staining was observed when the primary antibody was omitted (Fig. 6D).



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  		FIG. 6. MT1-MMP immunohistochemistry in the bovine corpus luteum. A) In the Day 4 corpus luteum, positive staining is observed in endothelial (black arrows) cells, and the cell membrane and cytoplasm of large luteal (white arrows) cells (x400). B) In the Day 10 corpus luteum, the staining is localized on large luteal cell plasma membranes and cytoplasm (white arrows), and endothelial cells (black arrows; x400). C) In the Day 16 corpus luteum, there was predominant staining in fibroblasts (white arrowheads; x400), and weak staining in endothelial (black arrows) and large luteal (white arrows) cells. D) Negative control of a representative section of Day 10 corpus luteum. No positive staining was observed (x400)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To date, MT1-MMP nucleotide and predicted protein sequences are known for humans [22], mice [27], rats [26], and rabbits [28], whereas there are no reports, to our knowledge, regarding its sequence and expression in agriculturally important species such as domestic ruminants. In the present study, we report the bovine-specific MT1-MMP complementary nucleotide sequence and demonstrate its expression and localization in the bovine corpus luteum. The bovine MT1-MMP cDNA consisted of 1749 bp nucleotides, which encode a 582 amino acid protein. Sequence alignment indicated that the bovine MT1-MMP was highly similar (>92%) to the rat, human, mouse, and rabbit homologues. In addition, bovine MT1-MMP shares characteristics that are typical of other membrane-type metalloproteinases (e.g., the 11-amino acid insertion [IS-I] between the propeptide and the catalytic domains that serve as a putative convertase recognition site, the RXKR motif [IS-II] in the catalytic domain, and the transmembrane segment [IS-III]) [39]. These results strongly suggest that this molecule is highly conserved among the mammals investigated thus far. Furthermore, as in humans [22] and rabbits [28], bovine MT1-MMP had broad tissue distribution, suggesting multiple sites of action in these species.

Because information regarding MT1-MMP and ovarian function are limited, the remainder of the present study focused on the bovine corpus luteum. Previously, MT1-MMP transcripts have been shown to be present in mouse ovaries [35] and in developing and regressing rat corpora lutea [33, 36]. Here, we found that MT1-MMP mRNA was constitutively expressed in all 3 stages of luteal development; however, the level of active MT1-MMP protein was greater in the mid stage and late stage bovine corpora lutea than it was in the early stage. A major function of MT1-MMP is to activate latent MMP-2. In contrast to other MMPs, the activation process of pro-MMP-2 is unusual in that it is not through the extracellular proteolytic cleavage by serine proteinases [40], but rather it is localized on the cell membrane [21]. A ternary molecular complex model is hypothesized in which TIMP-2 binds to MT1-MMP forming a cell surface "coreceptor" for pro-MMP-2 [41, 42]. This cell surface-bound pro-MMP-2 is subsequently activated by the adjacent active MT1-MMP [42, 43]. Indeed, in rats [34], MT1-MMP and MMP-2 mRNA are coordinately expressed and localized during follicular development and ovulation. In the bovine corpus luteum, we demonstrated that active MMP-2 enzyme levels correlated to that of active MT1-MMP. Thus, together with our observation that TIMP-2 was also coordinately expressed [44], we suggest that in the bovine corpus luteum, as in other tissues, MT1-MMP is an endogenous activator of pro-MMP-2.

The cellular localization of MT1-MMP in luteal tissue was also investigated. The formation and development of the corpus luteum are associated with tissue remodeling and angiogenesis [2, 3]. Angiogenesis is the formation of new capillaries from preexisting vessels. This complex, multifactor-regulated process requires angiogenic factors to stimulate production of MMPs, which degrade the basement membrane surrounding endothelial cells [45, 46]. This alteration of the ECM leads to endothelial cell proliferation and migration. As vessels extend, additional MMPs are required to break down the ECM, thereby accommodating the growth of sprouting vessels [45]. MT1-MMP may be mediating these processes because angiogenesis is impaired in MT1-MMP deficient mice [47], and it is highly associated with malignant tumor angiogenesis [48]. In the present study, MT1-MMP was localized in endothelial cells of the young bovine corpus luteum, where it may directly digest ECM proteins [29, 30]. Indeed, coincubation of purified MT1-MMP and collagen types I, II, and III before SDS-PAGE [30] yielded cleavage products, whereas very weak gelatinolytic activity by MT1-MMP mutants lacking a transmembrane domain was observed using zymography [29]. This may explain the lack of a readily detectable band in zymograms of our luteal tissue samples. Besides MT1-MMP, MMP-2 is also required for angiogenesis [46]. In an in vivo tumor system, MMP-2 activity is necessary for the switch to the angiogenic phenotype during tumor development [49]. Consistent with these findings is that injection of ewes with an MMP-2 antibody results in defects of the corpus luteum vasculature [50]. Together with our preliminary finding that MMP-2 is localized in bovine luteal endothelial cells [44], MT1-MMP may mediate the angiogenic process by activating pro-MMP-2, which in turn digests collagen type IV, a major component of the basement membrane [46]. Thus, following ovulation, when the follicle is transformed to become the richly vascularized corpus luteum, MT1-MMP might have a dual role as a proteolytic enzyme and an activator of pro-MMP-2.

As the corpus luteum develops, ongoing angiogenesis is necessary to establish and maintain the mature vascular network [51]. In fact, the density of the vasculature is highest in mid cycle cow [52] and mature human [53] corpora lutea, correlating with the high metabolic rate, and the high rate of blood flow and progesterone production by the corpus luteum [1, 52, 54]. Progesterone either decreases [55, 56] or increases [13, 16, 57] MMP activity and angiogenesis. In part, this may be attributed to cell type or species differences. In the present study, the active form of both MT1-MMP and MMP-2 enzymes increased in mid stage and late stage corpora lutea, consistent with the need for MMP production and activity for vascular maintenance.

In addition to endothelial cells, MT1-MMP was prominently expressed on the membranes of mid cycle large luteal cells. Accompanying the development of the luteal vasculature, there is also a rapid increase in luteal weight and size [58], although this increase is primarily attributed to the proliferation of nonluteal cells such as fibroblasts and endothelial cells [59, 60], and small luteal cells [58]. The sizes of large luteal cells also enlarge from the early to mid cycle stage [59, 61]. To accommodate this growth, MT1-MMP might act directly or indirectly through activation of locally produced pro-MMP-2 to commence the pericellular degradation of the ECM [48]. The activated MMP-2 may then bind to luteal cell membranes through an integrin such as {alpha}vß3 [62]. Thus, in large luteal cells, MT1-MMP and its associated actions might facilitate cell-matrix and cell-cell interactions, which ultimately could regulate steroidogenesis [2].

At the end of the estrous cycle, luteolysis ensues, whereby luteal progesterone production [1], size, and weight [58] decrease dramatically. There is also apoptosis of luteal cells [63]. In the present study, MT1-MMP was localized in fibroblasts of the late stage, Day 16 corpus luteum. At this time, these cells also undergo proliferation [59]. This switch in cellular distribution of MT1-MMP, when compared with the young and mid cycle corpora lutea, might occur in anticipation of the functional and structural changes that are soon to follow. Indeed, fibroblast MT1-MMP may participate in the luteolytic process by degrading connective tissue ECM as part of the structural demise of the corpus luteum.

Although the literature base is relatively small, there are reports of differences or consistent trends in expression patterns of MMPs and TIMPs in species with long luteal phases. Similar to the present study, high MMP-2 activity is observed in the late stage human corpus luteum [15], and in mid and late stages of the porcine corpus luteum [14]. When MT1-MMP is low, MMP-9 [10] and TIMP-1 mRNA [9] are increased early in the cycle, whereas TIMP-2 protein [10, 44] and mRNA [9, 44], along with MT1-MMP (present study), are increased during the mid-luteal phase in the bovine. In the human corpus luteum, MMP-9 peaks in early and late stages, but TIMP-1 and TIMP-2 do not change over the luteal phase [15]. In contrast, TIMP-1 and TIMP-2 mRNA levels decline with age of the porcine corpus luteum, while MMP-1 and MMP-9 are highest in the late phase [14]. However, in the sheep corpus luteum, TIMP-2 mRNA in the early phase is greater than the late phase [12], but TIMP-1 mRNA does not vary over the cycle [11]. Collectively, among the nonlaboratory mammals, a number of MMPs, including MT1-MMP in the bovine, and TIMPs, may work in concert to regulate remodeling of the vasculature and the ECM during the life span of the corpus luteum.

In summary, the present study reports the cDNA and predicted protein sequences of bovine MT1-MMP and the pattern of mRNA and protein expression in corpora lutea harvested over the bovine estrous cycle. Active MT1-MMP and active MMP-2 protein levels were coordinately increased in mid stage and late stage corpora lutea, supporting the role of MT1-MMP as an activator of pro-MMP-2 in vivo. The localization of MT1-MMP in endothelial cells, in large luteal cells, and in late cycle fibroblasts was associated with the angiogenesis and structural remodeling that occur over the life span of the corpus luteum. Collectively, these data indicate that MT1-MMP might act on the ECM to modulate the local environment, which in turn, may influence vascular development and the function of luteal cells (e.g., hormone biosynthesis), by affecting intercellular and cell-matrix communication.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the technical assistance of Ying Yu.


   FOOTNOTES
 
First decision: 3 October 2001.

1 This work was supported by a research grant from American Cancer Society (ACS-RPG-97-013-CB) to M.A.M., the Northeast Regional Project NE-161 to P.C.W.T., and the U.S. Department of Agriculture (98-35208-6654). This work was presented in part at the 33rd Annual Meeting of the Society of the Study for Reproduction at Madison, Wisconsin. This is scientific contribution 2104 from the New Hampshire Agricultural Experiment Station. Back

2 Correspondence. FAX: 603 862 3758; pct{at}cisunix.unh.edu Back

Accepted: January 18, 2002.

Received: August 22, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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