Molecular Human Reproduction, Vol. 9, No. 5, 271-277,
May 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Epithelial expression of matrix metalloproteinase-26 is elevated at mid-cycle in the human endometrium
Submitted on October 17, 2002; accepted on February 8, 2003
1 Departments of Obstetrics & Gynecology and 2 Pathology, University Hospital, S-221 85 Lund, 3 Atherosclerosis Research Unit, King Gustav V Research Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden and 4 Department of Obstetrics & Gynecology, Palacky University, 775 20 Olomouc, Czech Republic
5 To whom correspondence should be addressed. e-mail: bertil.casslen{at}gyn.lu.se
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ABSTRACT |
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The human endometrium is a dynamic tissue, which undergoes extensive tissue remodelling during the menstrual cycle. Due to their involvement in such processes, several well-characterized matrix metalloproteinases (MMP) have previously been studied in the endometrium. MMP-26 is a newly described matrilysin. We studied MMP-26 mRNA in 39 normal endometrial samples obtained across the menstrual cycle. Tissue distribution and cycle variation was examined using in-situ hybridization, Northern blot analyis and real time PCR. The probes for Northern blot analysis and real time PCR recognized non-overlapping sequences. MMP-26 was localized exclusively in epithelial cells of both glands and the luminal surface. Expression increased during the proliferative phase to a maximum at mid-cycle, then decreased to non-detectable levels in the late secretory and menstrual phases. Expression of MMP-26 mRNA in endometrial tissue explants in vitro required stimulation with both estradiol and progesterone. The tissue content of c-jun mRNA was assayed, since c-jun, as part of the enhancer complex AP-1, may be involved in regulation of MMP-26 gene transcription. The pattern of c-jun expression over the menstrual cycle was similar to that of MMP-26. Epithelial expression in the peri- and post-ovulatory stages of the menstrual cycle suggests the involvement of MMP-26 in reproductive processes.
Key words: c-jun/human endometrium/MMP-26/mRNA/proteinase/regulation
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Introduction |
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Matrix metalloproteinases (MMP) are zinc-dependent proteolytic enzymes involved in degradation of extracellular matrix (ECM) components such as collagen, proteoglycans, fibronectin and laminin (Curry and Osteen, 2001). The MMP and their tissue inhibitors (TIMP), together referred to as the MMP system, play important roles in a variety of physiological events, e.g. tissue repair, embryogenesis, extravillous trophoblast invasion and menstruation, as well as pathological processes, e.g. rheumatoid arthritis, atherosclerosis and tumour invasion (Nagase and Woessner, 1999). The MMP family comprises >20 related proteolytic enzymes which are divided in subfamilies based on their domain structure. There are eight distinct structural classes of MMP, five secreted and three membrane-type MMP (MT-MMP) (Egeblad, 2002). With the exception of MT-MMP, which appear to be activated prior to their inclusion in the cell membrane, MMP are secreted as latent pro-enzymes which require subsequent proteolytic activation (Sternlicht and Werb, 2001).
In normal ovulatory cycles, human endometrial tissue undergoes complex changes involving proliferation and subsequent differentiation of both epithelial and stromal components in preparation for embryo implantation. If no pregnancy occurs, the declining plasma levels of progesterone and estradiol initiate a cascade of events ultimately leading to breakdown and shedding of the functional layer of the endometrium as a menstrual bleeding. Disintegration in the late secretory phase of the endometrial basal lamina and cell-to-cell contacts in the stroma (Roberts et al., 1992) suggests that menstruation is preceded by degradation of ECM components (Hampton and Salamonsen, 1994; Rodgers et al., 1994; Marbaix et al., 1995).
MMP that are mainly or exclusively expressed in the peri-menstrual phase of the endometrial cycle include MMP-1 (Marbaix et al., 1995), MMP-2 (Rodgers et al., 1994), MMP-3 (Hampton and Salamonsen, 1994), MMP-7 (Rodgers et al., 1994), MMP-9 (Rodgers et al., 1994), MMP-10 (Rodgers et al., 1994) and MMP-11 (Rodgers et al., 1994). In contrast to MMP-7, which is predominantly found in epithelial cells (Rodgers et al., 1993; Wilson and Matrisian, 1996), MMP-1 (Rawdanowicz et al., 1994), MMP-2 (Rawdanowicz et al., 1994), MMP-3 (Rawdanowicz et al., 1994), MMP-9 (Rawdanowicz et al., 1994), MMP-10 (Rodgers et al., 1994) and MMP-11 (Rodgers et al., 1994) are expressed by stromal cells.
The cyclic pattern of endometrial expression of certain MMPs suggests regulation, direct or indirect, by gonadal steroids. In fact, physiological concentrations of progesterone almost totally suppress MMP-1 release (Lockwood et al., 1998) as well as MMP-2 and MMP-9 activity (Marbaix et al., 1992; Lockwood et al., 1998) in explant cultures of human endometrial tissue. Also, withdrawal of progesterone was followed by increased secretion of MMP-1, MMP-2, MMP-3 and MMP-9 by isolated stromal cells (Marbaix et al., 1992; Salamonsen et al., 1997). In addition, cytokines such as interleukin-1
(IL-1), tumour necrosis factor- (TNF-) or transforming growth factor-ß (TGF-ß) released from adjacent cells influence MMP expression under steroid control (Rawdanowicz et al., 1994; Bruner et al., 1995; Salamonsen and Woolley, 1996; Hulboy et al., 1997; Singer et al., 1997).
Recently, four groups independently identified the human gene encoding a novel metalloproteinase, MMP-26 (endometase; matrilysin-2) (de Coignac et al., 2000; Park et al., 2000; Uria and Lopez-Otin, 2000; Marchenko et al., 2001). This new member shares a number of structural and functional properties with matrilysin-1 (MMP-7) and has 52% amino acid identity with macrophage metalloelastase (MMP-12) (de Coignac et al., 2000). MMP-26 is the smallest MMP identified to date and its structure includes a signal sequence, a prodomain and a catalytic domain with the zinc-binding site. The gene is located on the short arm of chromosome 11 and encodes a 261 amino acid protein product.
Analysis of the proteolytic specificity showed that MMP-26 is able to degrade a wide range of proteins present in the ECM and basement membrane such as fibronectin, fibrinogen, vitronectin, 1-antitrypsin, 2-macroglobulin and denatured collagen (types I–IV). In addition, MMP-26 activates pro-MMP-9 in vitro (Uria, 2000). In contrast, tenascin C, laminin V and native collagen (types I-IV) are resistant to proteolysis by MMP-26 (Marchenko et al., 2001).
Tissue specific regulatory mechanisms have not yet been fully elucidated, but like many other MMP genes, the enhancer region of MMP-26 has a binding site for activator protein-1 (AP-1) (Marchenko et al., 2002). AP-1, which stimulates transcription of a wide variety of genes involved in cell proliferation, apoptosis, survival and differentiation, is not a single protein, but a complex of proteins belonging to the Jun, Fos and ATF subfamilies (Shaulian and Karin, 2002). Binding of AP-1 in the promoter region has been suggested to play an important role in transcriptional regulation of the MMP-26 gene (Marchenko et al., 2002).
In this paper we report the epithelial location and the cyclic pattern of MMP-26 gene expression in human endometrium. By assaying c-jun mRNA we also explored the potential regulation of MMP-26 gene transcription by AP-1. Possible transcriptional effects of estradiol and progesterone were evaluated in endometrial tissue explants.
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Materials and methods |
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Tissue sampling and processing
Endometrial tissue was collected from healthy women, aged 34–50 years, with regular menstrual cycles, between January 1996 and December 2001, at the Department of Gynecology and Obstetrics, Lund University Hospital. Tissue samples were obtained at diagnostic curettage or hysterectomy for benign reasons unrelated to endometrial dysfunction (e.g. leiomyoma, cervical dysplasia, uterine prolapse). Samples with endometrial pathology or samples from patients receiving steroid treatment were excluded from the study. All specimens were classified according to an ideal 28 day reproductive cycle as early (n = 8), mid (n = 6) and late (n = 7) proliferative phase, early (n = 4), mid (n = 4) and late (n = 4) secretory phase, pre-menstrual (n = 3) and menstrual phase (n = 3) (Noyes, 1950; Hendrickson, 1980). Early pregnancy decidua was obtained at legal abortion (n = 5) using vacuum aspiration. All samples were processed for both in-situ hybridization and Northern blot. Sampling was approved by the Review Board for studies in Human Subjects at the Lund University Hospital. All patients gave informed consent. Tissue samples were immediately frozen in dry ice in pieces (max. 4x4x4 mm) and stored at –80°C until analysed. Tissue samples from ovary (n = 2), Fallopian tubes (n = 3), myometrium (n = 1), vagina (n = 1) and skeletal muscle (n = 2) were also obtained for comparison and frozen. Another set of endometrial tissue samples, to be used for explant cultures (n = 7), was transported to the laboratory in sterile phosphate-buffered saline (PBS).
mRNA extraction
One portion of each frozen sample was homogenized with a microdismembrator, weighed and RNA extracted for RNA using Trizol ReagentTM (Life Technologies, Sweden). Frozen samples were homogenized in 1 ml of Trizol per 50 mg of tissue and centrifuged for 15 min at 12 000 g 4°C. After a 10 min incubation at room temperature, 0.2 ml of chloroform per 50 mg of tissue was added to the supernatant. Samples were vortex-mixed for 15 s and centrifuged for 15 min at 12 000 g at 4°C. Supernatant was mixed with isopropanol and salt solution (0.8 mol/l Na-citrate and 1.2 mol/l NaCl), 0.75 ml per 50 mg of tissue, and incubated at –20°C for 60 min. After sedimentation, samples were centrifuged for 30 min at 12 000 g at 4°C. Supernatant was discarded and the pellet was dried with 75% ethanol, 1 ml per 50 mg of tissue. The RNA pellet was air-dried and diluted in RNase-free water. Total RNA was quantified by measuring its absorbance in a spectrophotometer.
The other portion of each endometrial sample was processed for in-situ hybridization.
Preparation and labelling of cRNA probes
Single-stranded RNA probes were prepared from cloned MMP cDNA. For the human MMP-26 mRNA, a probe was used corresponding to 500 nucleotides (nt 225–725), GenBank accession AF248646 (Park et al., 2000).
DNA templates were generated by PCR from the human MMP-26 cDNA using bipartite primers consisting of either a T7 RNA promoter and a downstream gene-specific sequence (anti-sense) or a T3 RNA promoter and an upstream gene-specific primer (sense). PCR reactions using 1 ng human MMP-26 cDNA, 1 µg primers, 200 µmol/l dNTP, 3 mmol/l MgCl2, 10 mmol/l Tris, pH 8.3, 50 mmol/l KCl, 2.5 IU Taq polymerase (Invitrogen) were amplified at 95°C for 1 min, 62°C for 1 min and 72°C for 1 min for 30 cycles with a final extension at 72°C for 10 min. The full-length size of transcripts was verified with electrophoresis. DNA templates were purified from agarose gels using QIAquick Gel Extraction Kit (250) (Qiagen). Complementary RNA (cRNA) probes were transcribed from 5 ng of gel-purified DNA template using [35S]UTP (1300 Ci/mmol; Dupont NEN) and either T3 or T7 RNA polymerase according to the manufacturer’s instructions (Ambion MAXIscript) to generate sense and antisense probes respectively.
mRNA quantification
Northern hybridizations
Electrophoretic separation of RNA and Northern transfers were performed according to standard procedures (Sambrook, 1989). 10 µg of total RNA was separated on a 1% agarose, 0.66 mol/l formaldehyde gel using 1xMOPS running buffer and electrophoresing at 5 V/cm for 3 h. Gels were rinsed five times in distilled water (5 min each rinse) and Northern transfer was set up using 10xsaline sodium citrate (SSC) and Gene Screen Plus nylon membrane (Dupont). After overnight transfer, membranes were baked at 80°C for 2 h. Membranes were prehybridized for 2 h at 42°C in 10 ml ULTRAhyb (Ambion). cDNA probes were labelled using a Megaprime DNA labelling system (Amersham Pharmacia Biotech) and purified using G-50 nick columns (Amersham Pharmacia Biotech). A probe was used corresponding to 500 nt (261–761), GenBank accession AF 248646 (Park et al., 2000) and hybridizations were performed overnight using pre-hybridization buffer containing 5x105 cpm of denatured 32P-labelled probe per ml. Membranes were washed twice in 2xSSPE, then twice in 0.1xSSPE, 0.1% sodium dodecyl sulphate (SDS), each wash at 42°C for 20 min. Washed membranes were exposed to a phosphor image plate (Fujifilm) for between 4 and 20 h, then to X-ray film (Kodak) at –80°C for between 1 and 7 days. Quantification of transcripts following phosphor imaging was performed using ImageGauge software (Fujifilm).
Real time PCR
A total of 20 ng of mRNA from each sample was reverse-transcribed using superscript II according to the manufacturer’s manual (Invitrogen, USA). 2 µl of cDNA were amplified by real time PCR with 1xTaqMan universal PCR mastermix (Applied Biosystems, USA). An MMP-26 Assay on Demand primer and probe kit was used (Hs002(2320 m1; Applied Biosystem). The TaqMan probe included the nucleotide region 122–146. For ß-actin, 200 µmol/l of each primer and 1.25 pmol/l of probe were used and primers were designed using the Primer Express software (Applied Biosystems). ß-actin was used as a housekeeping gene to normalize for RNA loading; the primers for ß-actin were: ß-actin-FW: 5-CTGGCTGCTGACCGAGG-3 and ß-actin-RW: 5-GAAGGTCTCAAACATGATCTGGGT-3 and the probe was: ß-actin-TM: 6FAM5'-CCCTGAACCCCAAGGCCAACCG-3'TAMRA. Each sample was analysed in duplicate using ABI prism 7000 (Applied Biosystems). The PCR amplification was related to a standard curve.
mRNA localization
Cryostat sections 14 µm thick were collected on siliconized glass slides and subsequently stored at –80°C until used for in-situ hybridization. Radiolabelled cRNA probe was transcribed from template using 20 µmol/l 35[S]UTP (800 CI/mmol). The RNA polymerases used were T3 for sense and T7 for antisense. Prior to hybridization, tissue sections were pre-treated as previously described (Young, 1990). Sections were thawed directly in 4% formaldehyde in PBS and fixed for 5 min, rinsed twice in PBS, and acetylated in 0.1 mol/l triethanolamine–HCl, 0.25% acetic anhydride, pH 8 for 10 min. Sections were then rinsed twice in 2xSSC, dehydrated in ethanol 70% for 1 min, 80% for 1 min, 95% for 2 min, 95% for 1 min, and 100% for 1 min, prior to air drying. cRNA hybridization histochemistry was performed as described by Cox et al. (1984) and Whitfield et al. (1990).
[35S]cRNA probes were denatured at 65°C for 5 min and placed on ice for 5 min. Final hybridization buffer, with 1x106 cpm of denatured [35S]cRNA probe per 50 µl, consisted of 20 mmol/l Tris–HCl (pH 7.4), 1 mmol/l EDTA (pH 8.0), 300 mmol/l NaCl, 50% formamide, 10% dextrasulphate, 1xDenhardt’s, 25 g/l yeast tRNA, 100 mg/l salmon sperm DNA, 250 mg/l total yeast RNA (fraction XI), 100 mmol/l dithiothreitol (DTT), 0.1% sodium thiosulphate, and 0.1% SDS.
Hybridization buffer (70 µl/15 cm2) was applied to tissue sections on each slide and covered with untreated glass coverslips. Slides were then incubated at 56°C in chambers humidified with 2xSSC with 50% formamide for 24 h. After hybridization, slides were cooled to room temperature, and coverslips were floated off the slides in 4xSSC. Slides were then rinsed in four rounds of 4xSSC followed by immersion in 20 mg/l RNAse A at 37°C for 30 min. Sections were desalted in graded SSC solutions and washed twice in 0.1xSSC at 65°C for 30 min each. Sections were air-dried after dehydration in graded ethanol solutions.
After hybridization, autoradiography with 1–4 days exposure to Kodak Bio-Max MR was performed. Slides were then coated with undiluted nuclear track emulsion (NTB-3, Kodak) and following exposure for 4–5 weeks at 4°C, slides were developed in D-19 (Kodak) and counterstained with 1% Toluidine Blue.
All slides were evaluated by two independent observers (R.P. and H.D). In order to allow semiquantitative evaluation, the intensity of the signal was graded as negative (–) when the signal was indistinguishable from the background; faint (+) when intensity was distinguishable from the background; moderate (++) when signal intensity was less than half maximal; strong (+++) when the signal was more than half maximal; very strong (++++) when the signal was maximal.
Explant cultures of intact endometrium
Fresh tissue was rinsed with Hanks’ buffered salt solution (HBSS) and cut in 1 mm3 pieces which were equally distributed in two 10 cm2 tissue culture wells. Usually 4–6 pieces were cultured per well. Explant cultures were maintained in M199 (Life Technologies, USA) supplemented with 10% fetal bovine serum, 2 mmol/l glutamine, 100 000 IU/l penicillin, 100 mg/l streptomycin, and 0.25 mg/l fungizone and incubated in humidified air with 5% CO2 at 37°C (Casslen et al., 1995). Phenol red was omitted from HBSS and M199 to avoid interference with the steroid receptors. Stock solutions of the hormones were made 1000 and 10 000 times their final concentration, and the addition of ethanol was thereby kept at 
0.1% in all experiments. One of the wells was treated with estradiol 10–8 mol/l and the other with estradiol 10–8 and progesterone 10–6 mol/l. The medium was replaced every second day with fresh medium together with hormones. After 5 days incubation, tissue samples were frozen at –80°C and subsequently cryocut in 14 µm sections for in-situ hybridization.
Immunohistochemistry
Cryostat sections were fixed in 4% buffered formalin for 15 min, and subsequently treated with 10 mmol/l citrate buffer pH 6.0 in a microwave oven at 550 W for 17 min, for antigen retrieval (Shi et al., 1991). Immunostaining was performed in an automated immunostainer, TechMate 500 (Ventana Biotek, Tuscan, AZ, USA) using the biotin–streptavidin–peroxidase method with diaminobenzidine as the chromogen. Haematoxylin was used for counterstaining (Dako ChemMateTM Detection Kit). The primary antibodies were monoclonals for the estrogen receptor (ER; Clone 1D5, DakoCytomation, Denmark) diluted 1:100 and the progesterone receptor (PR; Clone PR 636, DakoCytomation, USA) diluted 1:500. Immunostaining was evaluated by two independent observers (H.D. and R.P.) as semiquantitative estimates of the total nuclear intensity as 0, +, ++ or +++.
Statistical methods
Results are presented as mean and SEM. Mann–Whitney U-test was used to evaluate the significance of differences between groups. Progressive variation during the menstrual cycle was evaluated with the test for trend.
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Results |
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MMP-26 mRNA was found exclusively in epithelial cells in all positive specimens (Figure 1). The hybridization signal was evenly distributed in all glandular and luminal epithelial cells. No hybridization was detected in the stroma. Sections hybridized with the sense (control) probe had no signal.
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Intensity of the hybridization signal was evaluated in all tissue sections using a semiquantitative scoring system (see Materials and methods). The signal increased gradually from early proliferative to early secretory phase, was lower in the mid-secretory phase and absent in the late secretory, pre-menstrual and menstrual phases (Figure 2). The decidual samples tested had no signal (data not shown).
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MMP-26 mRNA isolated from the same endometrial sample was subjected to both Northern blot analysis and real time PCR. The results were normalized to ß-actin mRNA. Both Northern blot analysis (Figure 3 and Figure 4) and real time PCR (Figure 5) demonstrated a similar cyclic pattern, as seen in the in-situ hybridization experiments (Figure 2). The probes for Northern blot analysis and real time PCR recognized non-overlapping sequences. Peak expression was found in the early secretory phase and the transcript was virtually absent in the late secretory, pre-menstrual and menstrual phases. It appears that the mid secretory phase represents transition between high and low levels in the early versus late secretory phases, since two out of four samples were high whereas the other two were undetectable. RNA prepared from ovarian, tubal and skeletal muscle tissue were negative for MMP-26 mRNA. The amount of MMP-26 mRNA in preparations from vaginal and myometrial tissue was similar to that of early proliferative phase endometrium (data not shown). No MMP-26 mRNA was found in the decidual tissue of early pregnancy.
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Northern blot analysis of c-jun mRNA demonstrated a cyclic pattern with peak levels in the early and mid secretory phases (Figure 3 and Figure 6). RNA preparations from ovarian and tubal tissue were negative for c-jun mRNA, while the amount of c-jun mRNA in skeletal muscle, vaginal and myometrial tissue was similar to that in early proliferative endometrium (data not shown).
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To test a possible steroid regulation of MMP-26 gene expression, endometrial explants were stimulated with estradiol alone or in combination with progesterone. Both ER and PR were detectable in all samples. In explants taken in the proliferative phase we found down-regulation of PR, but not of ER, by progesterone. In contrast, the expression of ER and PR was not affected in secretory phase samples (data not shown). Proliferative phase explants treated with estradiol alone showed no hybridization signal, whereas those treated with the combination of estradiol and progesterone had a weak focal signal in epithelial but not in stromal cells. In contrast, explants obtained in the secretory phase did not express MMP-26 regardless of treatment conditions.
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Discussion |
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MMP-26 has been identified in epithelial cells in a variety of human tissues such as the uterus, placenta, kidney, brain, thymus and skeletal muscle (de Coignac et al., 2000; Park et al., 2000; Uria and Lopez-Otin, 2000) as well as in a variety of tumours of epithelial origin such as endometrial, lung and prostate cancer as well as choriocarcinoma (Uria and Lopez-Otin, 2000; Marchenko et al., 2001; Zhang et al., 2002). In the present study of human endometrium we show that MMP-26 mRNA is exclusively expressed in epithelial cells, glandular as well as luminal, and is not detected in the stroma. Thus, our results support previous observations that MMP-26 is mainly epithelial, and these data together suggest a possible role for MMP-26 in the biology of normal as well as malignant epithelial cells.
Among MMP family members characterized so far, MMP-7 (matrilysin-1) (Rodgers et al., 1993; Wilson and Matrisian, 1996) and MMP-26 (matrilysin-2) are unique in their epithelial expression, which is in contrast to the stromal distribution of most other MMP (Rawdanowicz et al., 1994; Rodgers et al., 1994). Classification of these two MMP as matrilysins is based on similarities in substrate specificity. Moreover MMP-26 shares minimal domain organization with MMP-7, including domains necessary for secretion, latency and catalytic activity. Based on such structural similarities between MMP-7 and MMP-26, the two matrilysins were proposed to constitute a distinct group in a domain-based classification, as an alternative to the traditional substrate specificity based classification (Egeblad, 2002).
Many MMP are reportedly expressed in endometrial tissue, but the cyclic pattern varies. We found the MMP-26 gene to be maximally expressed at mid-cycle, e.g. the late proliferative and early secretory phases. In contrast the expression of MMP-1 (interstitial collagenase), MMP-3 (stromelysin-1), MMP-7, MMP-9 (gelatinase-B), MMP-10 (stromelysin-2) and MMP-11 (stromelysin-3) are associated primarily with the menstrual period, with some of them found also in the proliferative phase (Matrisian et al., 1994; Rodgers et al., 1994; Marbaix et al., 1995). MMP-2 (gelatinase-A) is the only MMP that seems to be constitutively expressed in all phases of the endometrial cycle (Rodgers et al., 1994). Most MMP genes seem to be expressed when progesterone concentrations are low, i.e. during the menstrual and proliferative phases. Thus high progesterone levels in the secretory phase are associated with low expression of most MMP. However, results from some studies (Brenner et al., 1996; Salamonsen and Woolley, 1996) indicate that progesterone may not have a direct effect on MMP reduction during the secretory phase. In fact, MMP concentrations seem to drop before the rise of progesterone at the end of the proliferative phase.
Numerous cytokines are known to work in concert with progesterone to regulate MMP expression (Hulboy et al., 1997). For example, the suppression of stromal-specific MMP-3 and epithelial-specific MMP-7 expression during the secretory phase of the menstrual cycle requires both progesterone and TGF-ß (Osteen et al., 1994; Bruner et al., 1995). In contrast to TGF-ß that works cooperatively with progesterone to limit MMP expression, inflammatory cytokines, e.g. members of the IL-1 family, which are known to regulate MMP expression, may act in opposition to progesterone (Simon et al., 1994; Osteen et al., 1997). For example, IL-1 released by endometrial epithelial cells is capable of stimulating MMP-3 expression in stromal cells. However, following progesterone exposure either in vivo or in vitro, MMP-3 expression in stromal cells becomes insensitive to cytokine stimulation (Osteen et al., 1997; Keller et al., 2000). Furthermore, IL-1 expression appears to be linked to elevated levels of MMP-7 found in epithelial cells at sites of ectopic pregnancy (Osteen et al., 1997). The expression pattern of MMP-26 suggests that IL-1 may contribute as a modulator.
TIMP-4 was recently suggested to be a physiological inhibitor of MMP-26 based on observations that it is a more potent inhibitor of MMP-26 than of MMP-1, -2, -3, -7 and -9 (Liu et al., 1997; Zhang et al., 2002). However, to our knowledge, there are no data concerning endometrial expression of TIMP-4 and no studies of TIMP-4–MMP-26 interactions in endometrial tissue.
The two epithelial matrilysins MMP-7 and MMP-26 seem to be differentially regulated, since MMP-7 peaks in both the menstrual and the proliferative phase. The cyclical pattern of MMP-26 expression suggests steroidal regulation. Endometrial explants taken in the proliferative phase and cultured in the presence of estradiol and progesterone developed weak focal MMP-26 expression in glandular epithelial cells. No such expression was seen in explants treated with estradiol alone. This observation suggests that maximal expression requires progesterone stimulation of estrogen-primed endometrial tissue. The promoter region of MMP-26 lacks traditional progesterone-response elements, suggesting that regulation may involve either additional enhancer elements further upstream, or an indirect effect via induction of as yet unknown intermediary proteins. In fact, cell–cell communication and stromal–epithelial contact appears to be crucial in the regulation of MMP expression following steroid exposure (Curry and Osteen, 2001). Paracrine mediators such as growth factors, cytokines etc have been suggested (Hulboy et al., 1997). In fact, proliferation of endometrial epithelial cells in response to estradiol is not direct but mediated via paracrine signals from the stroma (Cooke et al., 1998).
Increasing levels of MMP-26 mRNA in endometrial tissue during the proliferative phase suggest stimulation by estradiol. However, we found up-regulation of MMP-26 mRNA by estradiol in endometrial tissue explants, and the promoter region does not include sequence homology with estrogen response elements. This makes a direct interaction of activated estrogen receptor with MMP-26 gene transcription less likely.
The promoter region of the MMP-26 gene contains an AP-1 site, and AP-1 function is critical for efficiency of the promoter (Marchenko et al., 2002). In addition, cells transfected with the c-jun gene show up-regulated MMP-26 promoter activity (Marchenko et al., 2002). The AP-1 transcription factor is composed of jun and fos (Shaulian and Karin, 2002). We found c-jun expression to vary over the menstrual cycle with a mid-cycle maximum. This pattern was similar to that of MMP-26 expression. It is thus possible that c-jun expression is under steroid control, and that c-jun subsequently, as part of the AP-1 complex, takes part in the regulation of MMP-26 transcription. In fact, c-jun transcription is regulated by estradiol in the rat uterus (Nephew et al., 1994). These data may suggest that c-jun, via AP-1, is under estrogen control and is involved in the regulation of endometrial MMP-26.
TCF-4 transcription factor, a crucial member of the APC–ß-catenin–TCF pathway, has been described as a potent transcription regulator of MMP-26 in cancer cells of epithelial origin and the MMP-26 promoter has a TCF-4 site (Marchenko et al., 2002). Since endometrial levels of E-cadherin, - and ß-catenin mRNA are lower in the proliferative than in the secretory phase (Fujimoto et al., 1996), it appears less likely that TCF-4 stimulates MMP-26 gene transcription.
MMP-26 has the capability to activate pro-MMP-9 in vitro (Uria and Lopez-Otin, 2000). Furthermore, MMP-9 is immunolocalized within luminal and glandular epithelium during the post-ovulatory period, with progression towards the apical surface of the cells and release of the enzyme into the uterine lumen at the time of implantation (Jeziorska et al., 1996). The temporal and spatial expression suggests a role for MMP-26 and MMP-9 in the process of embryo implantation. During the invasive period of implantation, several barriers must be breached to establish functional connections to the blood supply for the conceptus. These barriers include the basement membrane of uterine epithelium, the interstitial matrix and the endothelium. In concordance with their specificity, several MMP (e.g. MMP-2, MMP-9, MT1-MMP), which are expressed in the secretory phase, were suggested to play a role during implantation (Salamonsen and Nie, 2002). A recent study found MMP-26 in the uterine glands of rhesus monkeys with maximal expression during the first few days after implantation, and suggested a role for this enzyme during the implantation process (Li et al., 2002). Since no data are available from early human implantation sites, all information is obtained from examination of MMP expression in endometrium during the secretory phase of the menstrual cycle and in decidual tissue derived from abortions during the first trimester. We found no expression of MMP-26 in early pregnancy decidua. However, these samples were obtained no earlier than 4 weeks after implantation. This agrees with the finding of Li et al. that MMP-26 was not expressed in the decidua at 26 days after implantation. MMP-26 may play a role during the process of implantation, either directly or as an activator of pro-MMP-9, or as a possible regulator of bioavailability of other molecules such as cytokines, growth factors and vasoactive factors.
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Acknowledgements |
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This project was supported by grants from the Swedish Cancer fund; the Medical faculty at Lund University; the Lund University Hospital fund for cancer research; Nilsson, Persson and Kamprad foundations; the Swedish Medical Research Council (12660; 14358; 14187) and the King Gustaf V and Queen Victoria Foundation.
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REFERENCES |
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Brenner, R.M., Rudolph, L., Matrisian, L. and Slayden, O.D. (1996) Non-human primate models; artificial menstrual cycles, endometrial matrix metalloproteinases and s.c. endometrial grafts. Hum. Reprod., 11 (Suppl. 2), 150–164.
Bruner, K.L., Rodgers, W.H., Gold, L.I., Korc, M., Hargrove, J.T., Matrisian, L.M. and Osteen, K.G. (1995) Transforming growth factor beta mediates the progesterone suppression of an epithelial metalloproteinase by adjacent stroma in the human endometrium. Proc. Natl Acad. Sci. USA, 92, 7362–7366.
Casslen, B., Nordengren, J., Gustavsson, B., Nilbert, M. and Lund, L.R. (1995) Progesterone stimulates degradation of urokinase plasminogen activator (u-PA) in endometrial stromal cells by increasing its inhibitor and surface expression of the u-PA receptor. J. Clin. Endocrinol. Metab., 80, 2776–2784.[Abstract]
Cho, C., Bunch, D.O., Faure, J.E., Goulding, E.H., Eddy, E.M., Primakoff, P. and Myles, D.G. (1998) Fertilization defects in sperm from mice lacking fertilin beta. Science, 281, 1857–1859.
Cooke, P.S., Buchanan, D.L., Lubahn, D.B. and Cunha, G.R. (1998) Mechanism of estrogen action: lessons from the estrogen receptor-alpha knockout mouse. Biol. Reprod., 59, 470–475.
Cox, K.H., DeLeon, D.V., Angerer, L.M. and Angerer, R.C. (1984) Detection of mRNA in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol., 101, 485–502.[CrossRef][ISI][Medline]
Curry, T.E., Jr and Osteen, K.G. (2001) Cyclic changes in the matrix metalloproteinase system in the ovary and uterus. Biol. Reprod., 64, 1285–1296.
de Coignac, A.B., Elson, G., Delneste, Y., Magistrelli, G., Jeannin, P., Aubry, J.P., Berthier, O., Schmitt, D., Bonnefoy, J.Y. and Gauchat, J.F. (2000) Cloning of MMP-26. A novel matrilysin-like proteinase. Eur. J. Biochem., 267, 3323–3329.[ISI][Medline]
Egeblad, M. and Werb, Z. (2002) New functions for the matrix metalloproteinases in cancer progression. Nature Rev. Cancer, 2, 161–174.[ISI][Medline]
Fujimoto, J., Ichigo, S., Hori, M. and Tamaya, T. (1996) Alteration of E-cadherin, alpha- and beta-catenin mRNA expression in human uterine endometrium during the menstrual cycle. Gynecol. Endocrinol., 10, 187–191.[ISI][Medline]
Hagaman, J.R., Moyer, J.S., Bachman, E.S., Sibony, M., Magyar, P.L., Welch, J.E., Smithies, O., Krege, J.H. and O’Brien, D.A. (1998) Angiotensin-converting enzyme and male fertility. Proc. Natl Acad. Sci. USA, 95, 2552–2557.
Hampton, A.L. and Salamonsen, L.A. (1994) Expression of messenger ribonucleic acid encoding matrix metalloproteinases and their tissue inhibitors is related to menstruation. J. Endocrinol., 141, R1–3.[Abstract]
Hendrickson, M.R. and Kempson, R.L. (1980) Surgical Pathology of the Uterine Corpus. Saunders, Philadelphia.
Hulboy, D.L., Rudolph, L.A. and Matrisian, L.M. (1997) Matrix metalloproteinases as mediators of reproductive function. Mol. Hum. Reprod., 3, 27–45.
Jeziorska, M., Nagase, H., Salamonsen, L.A. and Woolley, D.E. (1996) Immunolocalization of the matrix metalloproteinases gelatinase B and stromelysin 1 in human endometrium throughout the menstrual cycle. J. Reprod. Fertil., 107, 43–51.[Abstract]
Keller, N.R., Sierra-Rivera, E., Eisenberg, E. and Osteen, K.G. (2000) Progesterone exposure prevents matrix metalloproteinase-3 (MMP-3) stimulation by interleukin-1alpha in human endometrial stromal cells. J. Clin. Endocrinol. Metab., 85, 1611–1619.
Li, Q., Wang, H., Zhao, Y., Lin, H., Sang, Q.A. and Zhu, C. (2002) Identification and specific expression of matrix metalloproteinase-26 in rhesus monkey endometrium during early pregnancy. Mol. Hum. Reprod., 8, 934–940.
Liu, Y.E., Wang, M., Greene, J., Su, J., Ullrich, S., Li, H., Sheng, S., Alexander, P., Sang, Q.A. and Shi, Y.E. (1997) Preparation and characterization of recombinant tissue inhibitor of metalloproteinase 4 (TIMP-4). J. Biol. Chem., 272, 20479–20483.
Lockwood, C.J., Krikun, G., Hausknecht, V.A., Papp, C. and Schatz, F. (1998) Matrix metalloproteinase and matrix metalloproteinase inhibitor expression in endometrial stromal cells during progestin-initiated decidualization and menstruation-related progestin withdrawal. Endocrinology, 139, 4607–4613.
Marbaix, E., Donnez, J., Courtoy, P.J. and Eeckhout, Y. (1992) Progesterone regulates the activity of collagenase and related gelatinases A and B in human endometrial explants. Proc. Natl Acad. Sci. USA, 89, 11789–11793.
Marbaix, E., Kokorine, I., Henriet, P., Donnez, J., Courtoy, P.J. and Eeckhout, Y. (1995) The expression of interstitial collagenase in human endometrium is controlled by progesterone and by oestradiol and is related to menstruation. Biochem. J., 305, 1027–1030.
Marchenko, G.N., Ratnikov, B.I., Rozanov, D.V., Godzik, A., Deryugina, E.I. and Strongin, A.Y. (2001) Characterization of matrix metalloproteinase-26, a novel metalloproteinase widely expressed in cancer cells of epithelial origin. Biochem. J., 356, 705–718.[CrossRef][ISI][Medline]
Marchenko, G.N., Marchenko, N.D., Leng, J. and Strongin, A.Y. (2002) Promoter characterization of the novel human matrix metalloproteinase-26 gene: regulation by the T-cell factor-4 implies specific expression of the gene in cancer cells of epithelial origin. Biochem. J., 363, 253–262.[CrossRef][ISI][Medline]
Matrisian, L.M., Gaire, M., Rodgers, W.H. and Osteen, K.G. (1994) Metalloproteinase expression and hormonal regulation during tissue remodeling in the cycling human endometrium. Contrib. Nephrol., 107, 94–100.[Medline]
Nagase, H. and Woessner, J.F. Jr (1999) Matrix metalloproteinases. J. Biol. Chem., 274, 21491–21494.
Nephew, K.P., Tang, M. and Khan, S.A. (1994) Estrogen differentially affects c-jun expression in uterine tissue compartments. Endocrinology, 134, 1827–1834.[Abstract]
Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3–25.
Osteen, K.G., Rodgers, W.H., Gaire, M., Hargrove, J.T., Gorstein, F. and Matrisian, L.M. (1994) Stromal–epithelial interaction mediates steroidal regulation of metalloproteinase expression in human endometrium. Proc. Natl Acad. Sci. USA, 91, 10129–10133.
Osteen, K.G., Sierra-Rivera, E., Keller, N.R. and Fox, D.B. (1997) Interleukin-1 alpha opposes progesterone-mediated suppression of MMP-7. A possible role of this cytokine during human implantation. Ann. NY Acad. Sci., 828, 137–145.
Park, H.I., Ni, J., Gerkema, F.E., Liu, D., Belozerov, V.E. and Sang, Q.X. (2000) Identification and characterization of human endometase (Matrix metalloproteinase-26) from endometrial tumor. J. Biol. Chem., 275, 20540–20544.
Rawdanowicz, T.J., Hampton, A.L., Nagase, H., Woolley, D.E. and Salamonsen, L.A. (1994) Matrix metalloproteinase production by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase-A, gelatinase-B and stromelysin-1 and their differential regulation by interleukin-1 alpha and tumor necrosis factor-alpha. J. Clin. Endocrinol. Metab., 79, 530–536.[Abstract]
Roberts, D.K., Parmley, T.H., Walker, N.J. and Horbelt, D.V. (1992) Ultrastructure of the microvasculature in the human endometrium throughout the normal menstrual cycle. Am. J. Obstet. Gynecol., 166, 1393–1406.[ISI][Medline]
Rodgers, W.H., Osteen, K.G., Matrisian, L.M., Navre, M., Giudice, L.C. and Gorstein, F. (1993) Expression and localization of matrilysin, a matrix metalloproteinase, in human endometrium during the reproductive cycle. Am. J. Obstet. Gynecol., 168, 253–260.[ISI][Medline]
Rodgers, W.H., Matrisian, L.M., Giudice, L.C., Dsupin, B., Cannon, P., Svitek, C., Gorstein, F. and Osteen, K.G. (1994) Patterns of matrix metalloproteinase expression in cycling endometrium imply differential functions and regulation by steroid hormones. J. Clin. Invest., 94, 946–953.[ISI][Medline]
Salamonsen, L.A., Butt, A.R., Hammond, F.R., Garcia, S. and Zhang, J. (1997) Production of endometrial matrix metalloproteinases, but not their tissue inhibitors, is modulated by progesterone withdrawal in an in vitro model for menstruation. Clin. Endocrinol. Metab., 82, 1409–1415.
Salamonsen, L.A. and Nie, G. (2002) Proteases at the endometrial–trophoblast interface: their role in implantation. Rev. Endocr. Metab. Disord., 3, 133–143.[CrossRef][Medline]
Salamonsen, L.A. and Woolley, D.E. (1996) Matrix metalloproteinases in normal menstruation. Hum. Reprod., 11 (Suppl. 2), 124–133.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, NY.
Shaulian, E. and Karin, M. (2002) AP-1 as a regulator of cell life and death. Nat. Cell Biol., 4, E131–136.
Shi, S.R., Key, M.E. and Kalra, K.L. (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem., 39, 741–748.[Abstract]
Simon, C., Frances, A., Piquette, G., Hendrickson, M., Milki, A. and Polan, M.L. (1994) Interleukin-1 system in the materno-trophoblast unit in human implantation: immunohistochemical evidence for autocrine/paracrine function. Clin. Endocrinol. Metab., 78, 847–854.
Singer, C.F., Marbaix, E., Kokorine, I., Lemoine, P., Donnez, J., Eeckhout, Y. and Courtoy, P.J. (1997) Paracrine stimulation of interstitial collagenase (MMP-1) in the human endometrium by interleukin 1alpha and its dual block by ovarian steroids. Proc. Natl Acad. Sci. USA, 94, 10341–10345.
Sternlicht, M.D. and Werb, Z. (2001) How matrix metalloproteinases regulate cell behavior. Annu. Rev. Cell. Dev. Biol., 17, 463–516.[CrossRef][ISI][Medline]
Uria, J.A. and Lopez-Otin, C. (2000) Matrilysin-2, a new matrix metalloproteinase expressed in human tumors and showing the minimal domain organization required for secretion, latency and activity. Cancer Res., 60, 4745–4751.
Whitfield, H.J., Brady, L.S., Smith, M.A., Mamalaki, E., Fox, R.J. and Herkenham, M. (1990) Optimization of cRNA probe in situ hybridization methodology for localization of glucocorticoid receptor mRNA in rat brain: a detailed protocol. Cell. Mol. Neurobiol., 10, 145–157.[CrossRef][ISI][Medline]
Wilson, C.L. and Matrisian, L.M. (1996) Matrilysin: an epithelial matrix metalloproteinase with potentially novel functions. Int. J. Biochem. Cell Biol., 28, 123–136.[CrossRef][ISI][Medline]
Young, W.S. (1990) In situ hybridization histochemistry. In Björklund, A., Hökfelt, T., Wouterlood, F.G. and van den Pol, A.N. (eds), Handbook of Chemical Neuroanatomy: Analysis of Neuronal Microcircuits and Synaptic Interactions. Elsevier, Amsterdam.
Zhang, J., Cao, Y.J., Zhao, Y.G., Sang, Q.X. and Duan, E.K. (2002) Expression of matrix metalloproteinase-26 and tissue inhibitor of metalloproteinase-4 in human normal cytotrophoblast cells and a choriocarcinoma cell line, JEG-3. Mol. Hum. Reprod., 8, 659–666.
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