Mol. Hum. Reprod. Advance Access originally published online on June 29, 2006
Molecular Human Reproduction 2006 12(8):497-503; doi:10.1093/molehr/gal055
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Endometrial TIMP-4 mRNA is expressed in the stroma, while TIMP-4 protein accumulates in the epithelium and is released to the uterine fluid
1Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden, 2Department of Obstetrics and Gynecology, Palacky University, Olomouc, Czech Republic and 3Department of Pathology, University Hospital, Lund, Sweden
4 To whom correspondence should be addressed at: BMC C14, SE-221 84 Lund, Sweden. E-mail: bertil.casslen{at}med.lu.se
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Abstract |
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We have previously reported that endometrial mRNA expression of both tissue inhibitors of metalloproteinase-4 (TIMP-4) and matrix metalloproteinase-26 (MMP-26) peaks in the early secretory phase, which implies a role in implantation. The objective of this study was to compare the distribution of TIMP-4 and MMP-26 in endometrial tissue and uterine fluid over the menstrual cycle. Endometrial tissue was analysed with in situ hybridization and immunohistochemistry to localize mRNA and protein for TIMP-4 and MMP-26 in the same set of samples. TIMP-4 mRNA was quantified in separated stromal and epithelial cells using real-time PCR. Uterine fluid was analysed with western blotting. TIMP-4 mRNA was exclusively localized to the stroma, whereas MMP-26 mRNA was expressed by epithelial cells. TIMP-4 protein was only occasionally found in the stroma but was consistently present in granules of the apical part of luminal and glandular epithelial cells. TIMP-4, but not MMP-26, was demonstrated in uterine fluid. Thus, TIMP-4 is produced in the stroma only, secreted by stromal cells, taken up by epithelial cells, accumulated in apical granules and finally secreted to the uterine fluid. Maximal expression of MMP-26, and its strongest inhibitor TIMP-4, in the early and mid-secretory phase suggests a role during implantation. MMP-26 is stored in epithelial cells in its active form, is not released spontaneously and is controlled by TIMP-4 in both stroma and uterine fluid.
Key words: implantation/MMP-26/mRNA/protein/uptake
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Introduction |
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Implantation is a highly coordinated invasive process which is dependent on the ability of the embryo to initiate degradation of extracellular matrix (ECM) proteins including the basement membrane (Graham and Lala, 1992
). Several studies have implicated matrix metalloproteinases (MMPs) in this process (Brenner et al., 1989; Behrendtsen et al., 1992; Shimonovitz et al., 1994). MMPs are zinc-dependent peptidases with broad substrate specificity, allowing them to degrade most components of both interstitial matrix and basement membranes (Woessner and Nagase, 2000). More than 20 known MMPs are divided in eight distinct structural groups, five of which are secreted and three are membrane-bound (Egeblad and Werb, 2002). Endometrial MMPs are mainly expressed during menstruation, and during subsequent regrowth and angiogenesis in the proliferative phase (Hampton and Salamonsen, 1994; Rodgers et al., 1994; Marbaix et al., 1996; Goffin et al., 2003). Human preimplantation embryos express several MMP genes, particularly MMP-1, MMP-2 and MMP-9 (Fisher et al., 1989; Bischof et al., 1991; Librach et al., 1991; Wang et al., 2003).
Tissue inhibitors of metalloproteinases (TIMPs) are endogenous regulators of MMPs (Brew et al., 2000). Four mammalian TIMPs have been identified to date (TIMP-1, TIMP-2, TIMP-3 and TIMP-4) (Welgus and Stricklin, 1983; Stetler-Stevenson et al., 1989; Apte et al., 1995; Greene et al., 1996). Their molecular weight ranges from 20 to 30 kDa and some forms, in particular TIMP-4, can be glycosylated (Douglas et al., 1997). TIMPs inhibit the activity of the MMPs by forming stoichiometric 1 : 1 complexes. Despite the amino acid homology, each TIMP has an individual inhibitory profile of MMPs. For instance, all secreted MMPs are sensitive to TIMP-1 inhibition, whereas membrane-type MMPs are sensitive to TIMP-2 and TIMP-3 (Amour et al., 1998; Stracke et al., 2000; Hernandez-Barrantes et al., 2002). TIMP-1, TIMP-2 and TIMP-3 are present in all cellular compartments of the human endometrium throughout the menstrual cycle. While taking into account, their broad inhibitory specificities are likely to represent a substantial barrier to invasion during implantation (Zhang and Salamonsen, 1997). Moreover, all three are expressed in human preimplantation embryos (Wang et al., 2003).
TIMP-4 has high structural homology with TIMP-2, with which it also shares inhibitory capacity for MMP-19 (Greene et al., 1996; Stracke et al., 2000). In addition, TIMP-4 inhibits the activity of MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9. Kinetic studies have shown that TIMP-4 has higher inhibitory capacity for MMP-26 than for other tested MMPs (Liu et al., 1997; Zhang et al., 2002). TIMP-4 has a limited tissue distribution and has been reported in the heart, the brain, the testis and the ovary (Greene et al., 1996; Young et al., 2002). Recently, we demonstrated that TIMP-4 mRNA is expressed in endometrial stroma with peak levels in the early secretory phase (Pilka et al., 2004a).
MMP-26, which was cloned and characterized by four independent groups (de Coignac et al., 2000; Park et al., 2000; Uria and Lopez-Otin, 2000; Marchenko et al., 2001), is the smallest member of the MMP family. Like MMP-7, it is expressed by epithelial cells and lacks the haemopexin-like domain. MMP-26 is an activator of pro-MMP-9, which when activated is a broad-spectrum protease with affinity for numerous ECM proteins, such as fibronectin, type IV collagen, gelatin, etc. (Uria and Lopez-Otin, 2000; Zhao et al., 2003). We have shown that MMP-26, mRNA as well as protein, is exclusively localized to the endometrial epithelium, and unlike other MMPs, its expression peaks in the periovulatory period (Pilka et al., 2003, 2004b).
Thus, a similar cyclic pattern of endometrial expression was found for MMP-26 and its potent inhibitor TIMP-4. Peak expression at mid-cycle for both MMP-26 and TIMP-4 mRNA indicates regulation by the gonadal steroids and suggests involvement in the implantation process. In this study, we focus on the origin and distribution of TIMP-4 in endometrial tissue and uterine fluid. We also compare the distribution and secretory pattern of TIMP-4 with that of MMP-26.
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Materials and methods |
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Sampling of endometrial tissue and uterine fluid
Samples of endometrial tissue and uterine fluid were obtained from patients undergoing hysterectomy for benign conditions unrelated to endometrial dysfunction (e.g. leiomyoma, cervical dysplasia and uterine prolapse). Patients were 39 (25–45, mean and ranges) years old. Patients using oral contraceptives and intrauterine devices were not included in the study. Samples with endometrial pathology or dysfunctional endometrium were excluded from the study. Endometrial tissue (n = 39) was classified according to an ideal 28-day menstrual cycle as early, mid and late proliferative phase, early, mid and late secretory phase, premenstrual and menstrual phase (Noyes et al., 1950
; Hendrickson and Kempson, 1980). All tissue samples were collected between January 1996 and December 2001 at the Department of Gynecology and Obstetrics, Lund University Hospital. Uterine fluid samples were collected between June 2004 and March 2005 at the Department of Gynecology and Obstetrics, Olomouc University Hospital. Sampling was approved by the Review Board for studies in Human Subjects at the University Hospitals in Lund, Sweden, and Olomouc, Czech Republic. One part of each tissue sample was immediately cut into small pieces (maximum 4 x 4 x 4 mm), frozen on dry ice and stored at –80°C until further processed for in situ hybridization and real-time PCR. The other part was formalin-fixed and paraffin-embedded for histopathological examination and immunohistochemistry. Another set of endometrial tissue samples, which were used for stromal and epithelial cell culture preparation (n = 17), was transported to the laboratory in sterile NaCl.
Uterine fluid (n = 14) was sampled using a baby-feeding tube no. 8 connected to a syringe aspirator as previously described (Casslen et al., 1981). The tube was introduced into the uterine cavity under sterile conditions. A low aspiration pressure was used in order to avoid injury to the endometrium. The free end of the tube was moved slightly within the uterine cavity during the aspiration. With this procedure, it was possible to collect a blood-free sample of uterine fluid. The sample was transferred to a plastic capillary tube and centrifuged for 10 min at 13 000 g. The supernatant was collected by cutting the plastic tube with scissors. Sample volumes ranged from 40 to 135 µl. The tubes with uterine fluid supernatants were stored at –20°C until analysed.
Endometrial cell separation
Endometrial tissue was obtained immediately at operation, transported in saline to the laboratory, rinsed in Hanks’ balanced salt solution (HBSS) to remove blood, cut into 1 mm3 pieces and incubated in 10 ml of dissociation solution (crude collagenase 2.5 g/l, DNAse-I 0.25 g/l and tosyl-L-lysine-chloromethyl-ketone 0.2 µmol/l in HBSS) for 45 min at 37°C on a shaking rotor (ES-20, BioSan, Riga, Latvia) (Casslen et al., 1995). Gentle pipetting up and down assisted dissociation. Following addition of 10 ml of culture medium supplemented with fetal bovine serum (FBS), 10% of cells were fractionated over sterile nylon sieves. The first sieve (350 µm pore size) removed undigested fragments of tissue, and the second sieve (35 µm pore size) retained the glands. Stromal cells were present in the flow-through. Glands were back-washed from the sieve with HBSS, and both cell types were collected by centrifugation at 300 g.
Total RNA extraction
The RNA was extracted from the harvested cells with EZNA® Total RNA Kit (OMEGA Bio-tek, Doraville, GA, USA) according to the manufacturer’s instruction. The concentration of extracted RNA and the purity of each sample were analysed with a spectrophotometer. The quality of the total RNA was verified by 2% agarose gel electrophoresis. All samples included in RT-PCR showed two bands representing the 18S and 28S ribosome subunit.
cDNA synthesis
Intact RNA was converted to cDNA by reverse transcription using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA, Part. No. N808-0234). According to protocols from the manufacturer, one 20-µl reaction contained 0.2 µg of total RNA, final concentration of 1x TaqMan RT buffer, 2.2 mmol/l of MgCl2, 200 µmol/l of dNTP, 1.0 µmol/l of random hexamers, 0.4 U/µl of RNAse inhibitor and 0.5 U/µl of MuLV reverse transcriptase. The reaction was incubated at 25°C for 10 min, at 48°C for 30 min and then 5 min of inactivation at 95°C. The final concentration of cDNA was 10 ng/µl. The samples were then stored at –20°C until further use.
Real-time PCR amplification
For quantification of TIMP-4 mRNA by real-time PCR, ABI PRISM 7000 (Applied Biosystems) was used. The PCR reaction was run in duplicates with the final volume of 25 µl/well containing final concentrations: 1x TaqMan Universal PCR Master Mix (Applied Biosystems, Part. No. 4304437), 1x Assay mix premanufactured probes for TIMP-4 (Applied Biosystems, primer set Hs00162784_m1) or ß-actin (Applied Biosystems, Art. No. Hs99999903_m1 ACTB) and 2.2 µl of 10 ng/µl of sample cDNA. ß-actin was used as a housekeeping gene to normalize the results; 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. The probes were labelled with fluorogenic dye FAM and ROX. The cycling conditions were initiated by UNG activation at 50°C for 2 min, and an initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and annealing at 60°C for 1 min. Two negative controls were included in each amplification. Quantification of the amplification product was performed by comparison with a calibration curve obtained by 10-fold dilution of the template DNA (0.08–80 ng). In order to standardize results, each TIMP-4 mRNA value was divided by the value for the housekeeping gene ß-actin mRNA in the same sample. Thus, the amount of TIMP-4 mRNA is expressed as a relative value.
Preparation and labelling of cRNA probes
For the human TIMP-4 mRNA, a probe corresponding to 440 NT (231–670) was used, Genbank accession nm_003256 (Greene et al., 1996). For the human MMP-26 mRNA, a probe corresponding to 500 NT (225–725) was used, Genbank accession number AF248646
[GenBank]
(Park et al., 2000). DNA templates were generated by PCR amplification from the human TIMP-4 or MMP-26 cDNA using bipartite primers consisting of either a modified T7 RNA promoter and a downstream gene-specific sequence (antisense) or a modified T3 RNA promoter and an upstream gene-specific primer (sense). Single-stranded RNA probes were prepared from cloned MMP complementary deoxyribonucleic acid. PCR reactions using 1 ng of human TIMP-4 or MMP-26 cDNA, 1 µg of primers, 200 µmol/l of dNTPs, 3 mmol/l of MgCl2, 10 mmol/l of Tris, pH 8.3, 50 mmol/l of KCl and 2.5 U Taq polymerase (Invitrogen, Carslbad, CA, USA) 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. Full-length size of transcripts was verified with electrophoresis. DNA templates were purified from agarose gels using QIAquick Gel Extraction Kit (250) (Qiagen, GmbH, Hilden, Germany). Complementary RNA (cRNA) probes were transcribed from 5 ng of gel-purified DNA template using 35S-UTP (Amersham Biosciences, Little Chalfont, UK, 800 Ci/mmol), and either T3 or T7 RNA polymerase, according to instructions by the manufacturer (Ambion MAXIscript, Austin, TX, USA), to generate sense and antisense probes, respectively.
In situ hybridization
One piece of frozen tissue from each sample was processed for in situ hybridization. Cryostat sections, 14 µm thick, were collected on siliconized glass slides and stored at –80°C. The radio-labelled cRNA probe was transcribed from a template using 20 µmol/l of 35S-UTP (800 Ci/mmol). The RNA polymerases used were T3 for sense and T7 for antisense.
Prior to hybridization, tissue sections were pretreated as described below (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 of triethanolamine–HCl, 0.25% acetic anhydride, pH8 for 10 min. Sections were then rinsed twice in 2x saline sodium citrate (SSC), dehydrated in ethanol 70% for 1 min, 80% for 1 min, 95% for 2 min, 100% for 1 min and 95% for 1 min, before air drying. Hybridization with cRNA 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 1 x 106 cpm of denatured 35S-cRNA probe per 50 µl, consisted of 20 mmol/l of Tris–HCl (pH 7.4), 1 mmol/l of EDTA (pH 8.0), 300 mmol/l of NaCl, 50% formamide, 10% dextransulfate, 1x Denhardt’s solution, 25 g/l of yeast tRNA, 100 mg/l of salmon sperm DNA, 250 mg/l of total yeast RNA (fraction XI), 100 mmol/l of dithiothreitol (DTT), 0.1% sodium thiosulfate and 0.1% SDS.
Hybridization buffer (70 µl/slide) 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 2x SSC with 50% formamide for 24 h. After hybridization, slides were cooled to room temperature, and coverslips were floated off the slides in 4x SSC. Slides were then rinsed in four rounds of 4x SSC followed by immersion in 20 mg/l of RNAse A at 37°C for 30 min. Sections were desalted in graded SSC solutions and washed twice in 0.1x SSC at 65°C for 30 min, dehydrated in graded ethanol solutions and air-dried. The slides were then exposed to Kodak Bio-Max MR film for 1–4 days and subsequently coated with undiluted nuclear track emulsion (NTB, Kodak). Following exposure for 4–5 weeks at 4°C, slides were developed in D-19 (Kodak) and counterstained with 1% Giemsa.
All slides were evaluated by two independent observers (R.P. and H.D.). Sections hybridized with the sense probes showed no specific signal.
Immunohistochemistry
Formalin-fixed, paraffin-embedded sections were treated with 10 mmol/l of 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 detection system (ChemMateTM Detection Kit, Dako, Glostrup, Denmark), with diamino-benzidine as the chromogen. Haematoxylin was used for counterstaining. The primary antibodies were rabbit anti-TIMP-4 (Ab-1, Neomarkers, Fremont, CA, USA) and rabbit anti-MMP-26 (RP2, RP3; Triple Point Biologics, Portland, OR, USA) diluted 1 : 50. RP2 recognizes an active form, RP3 both latent and active forms of MMP-26. As a negative control, rabbit non-immune IgG replaced the primary antibodies. Immunostaining was evaluated by two independent observers (H.D. and R.P.).
Western blotting
Uterine fluid samples (2.5 µl) were incubated with LDS sample buffer (2.5 µl) (Invitrogen, cat. NP0007) for 10 min at 70°C before being electrophoresed in a continuous 10% Bis Tris Gel (Invitrogen). Proteins were transferred from the gel to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA) by electroblotting. Non-specific binding sites on the membranes were blocked with blocking buffer [Tris-buffered saline, pH 7.6, with Tween-20, 0.1% (ICN Biomedicals Inc., Aurora, OH, USA) containing non-fat dry milk 50 g/l (Bio-Rad)] for 1 h at room temperature on an orbital shaker. The membranes were subsequently incubated with primary antibodies for TIMP-4 (Ab-1) and MMP-26 (RP2 and RP3) (see Immunohistochemistry). Also, a peptide, which corresponds to the immunogenic epitope on TIMP-4 and blocks specific binding of the TIMP-4 antibody, was preincubated with the antibody as a specificity control (Neomarkers). After incubation with antibodies overnight at 4°C, membranes were washed in TBS–Tween (pH 7.6) and developed for 1 h at room temperature with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (sc-2030, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). After washings in TBS–Tween, blots were incubated with enhanced chemiluminescence substrate solution (ECL, Amersham Biosciences) and exposed to Hyperfilm ECL (Amersham Biosciences) to visualize immunoreactive bands.
Statistical methods
Results are presented as median and percentiles. Significance of differences between groups was evaluated with the Mann–Whitney test. Tests were two-sided and 5% level of significance was used.
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Results |
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Altogether 39 endometrial samples were used for in situ hybridization. They were obtained in the early (n = 8), mid (n = 6) and late (n = 7) proliferative phase, early (n = 4), mid (n = 4) and late (n = 4) secretory phase, and in the premenstrual (n = 3) and menstrual phases (n = 3). The TIMP-4 mRNA signal was evenly distributed within the stroma, and no signal was detected in glandular or luminal epithelial cells (Figure 1). The signal was always weak. For this reason, the exposure time had to be increased, and that caused a slight increase in background. Specific signal was detected in samples obtained from the mid-proliferative to the early secretory phase. The signal in mid and late secretory phase samples was not distinguishable from that with the sense probe. MMP-26 mRNA was present in all endometrial samples from the early proliferative to the mid-secretory phase. The signal was weak or absent in late secretory, premenstrual and menstrual phases. It was exclusively found in epithelial cells, glandular as well as luminal (Figure 2). The hybridization signal was present in the majority of epithelial cells.
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TIMP-4 mRNA was analysed with real-time PCR in stromal and epithelial cells, which were prepared from fresh endometrial tissue samples (n = 17). The amount of TIMP-4 mRNA was higher in endometrial stromal cells than in epithelial cells, which had almost negligible amounts of TIMP-4 mRNA, thus extending the results from in situ hybridization (Figure 3).
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The distribution of TIMP-4 and MMP-26 proteins was assayed with immunohistochemistry in 23 endometrial samples. They were obtained in the early (n = 4), mid (n = 3) and late (n = 3) proliferative phase, early (n = 4), mid (n = 4) and late (n = 5) secretory phase. Occasional stromal cells stained positive for TIMP-4 in five of 23 samples from different parts of the menstrual cycle, but no general stromal staining was observed. TIMP-4 was present in both glandular and luminal epithelial cells (Figure 4). Staining tended to be more intense in glands adjacent to the myometrium. Epithelial immunostaining was not evenly distributed within the samples and some glandular cells and even glands were negative. It was present in samples from the mid-proliferative to the mid-secretory phase but was absent in the early proliferative and late secretory phase. Staining for TIMP-4 was observed both in granule-like structures in the apical part and in the basal part of the epithelial cells in positive samples. Furthermore, positive staining of intraglandular secretion was occasionally seen in the mid-secretory phase.
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MMP-26 staining was present in epithelial cells, glandular as well as luminal (Figure 4). Endothelial cells of small blood vessels, mostly arterioles, were the only cells in the stroma where staining was detected. Staining was strong in samples from the early proliferative to the mid-secretory phase but was weak in the late secretory phase. Staining with the RP3 antibody, which detects both the pro-form and the active form, was weaker than that with the RP2 antibody but showed otherwise the same distribution.
Uterine fluid (n = 14) was obtained in the mid (n = 3) and late (n = 3) proliferative phase, and in the early (n = 4) and mid (n = 4) secretory phase. Western blot membranes probed with the TIMP-4 antibody showed bands at Mr 29 kDa, corresponding to the glycosylated form of TIMP-4, in uterine fluid samples obtained in the late proliferative, early secretory and mid-secretory phase, but not in samples obtained in the mid-proliferative phase. No bands were detected when the TIMP-4 antibody had been preincubated with the blocking peptide. Membranes were also probed with both MMP-26 antibodies, RP2 and RP3, which were used for immunohistochemistry, but no bands were visible corresponding to the position of either the zymogen (30 kDa) or the active form (18 kDa) of MMP-26 (Figure 5).
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Discussion |
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In this study, we show that endometrial TIMP-4 mRNA is exclusively found in stromal cells, whereas TIMP-4 protein locates in the epithelial cells, particularly in apical granules, and is also detected in the uterine fluid. By contrast, for MMP-26 both mRNA and protein are found in epithelial cells and are not released to the uterine fluid. These observations suggest that TIMP-4 is produced in and subsequently released by the stromal cells, taken up by the epithelium and subsequently secreted to the uterine fluid.
Stromal staining for TIMP-4 was restricted to few cells in some of the endometrial samples. The absence of TIMP-4 staining in the stroma is remarkable but may be due to efficient secretion, and once secreted TIMP-4 is no longer bound to membranes or other proteins and could be lost in washings during the process. However, when taken up by the epithelial cells, and especially when incorporated into granules, it is detectable with immunohistochemistry.
Our present finding, taken together with our previous results (Pilka et al., 2004a), of weak expression of TIMP-4 mRNA diffusely in the stroma but not in the epithelial cells, is supported by the present demonstration of TIMP-4 in separated stromal cells, but not in epithelial cells in this study. Stromal and epithelial cells were separated by a standardized technique yielding >95% purity of both cell types (Casslen et al., 1995). Very low expression of TIMP-4 mRNA in epithelial cells is in agreement with <5% contamination with stromal cells. Our localization of TIMP-4 protein in the endometrium is in agreement with the results of Chegini et al. (2003) and Tunuguntla et al. (2003) who found immunostaining for TIMP-4 predominantly in luminal and glandular epithelial cells and to a lesser extent in the endometrial stroma.
This is the first report on the presence of TIMP-4 in uterine fluid. To our knowledge, it is also the first clear demonstration of epithelial secretion to the uterine fluid of a stromal derived protein. Composition of the uterine fluid is complex and poorly understood. For instance, the mechanism behind the very high concentrations of potassium and certain amino acids is still poorly understood (Casslen and Nilsson, 1984; Casslen, 1987). One source of uterine fluid proteins is endometrial secretion, and another source may be migratory cells, which appear in endometrium and uterine fluid (Casslen et al., 1982). Also, a contribution of fluid may reach the uterine cavity from the fallopian tubes or from the peritoneal cavity via the tubes (Casslen, 1986). Some proteins present in the uterine fluid are transudated from serum, e.g. protease inhibitors (Casslen and Ohlsson, 1981), whereas other proteins are locally synthesized and released by the endometrium, e.g. proteases, cytokines and growth factors (Casslen et al., 1981; MacLaughlin and Richardson, 1983; Licht et al., 1998; Inagaki et al., 2003). Whether transudated or secreted, the majority of proteins in the uterine fluid show varying concentrations over the menstrual cycle (Casslen and Astedt, 1981; Casslen et al., 1982; Rotello et al., 1991; von Rango et al., 1998; O’Sullivan et al., 2004). The fate of TIMP-4 with stromal origin, accumulation in epithelial cells and subsequent secretion into the uterine fluid is certainly not unique but has not been detailed for other proteins. However, it seems to be shared by uPA, another stromal derived component of the endometrial secretion (Casslen et al., 1981; Nordengren et al., 2004).
TIMP-4 protein was present in uterine fluid samples obtained in the early and mid-secretory phase. This period coincides with that of staining for MMP-26 protein in the epithelial cells (Pilka et al., 2004b). Because TIMP-4 is the most efficient inhibitor of MMP-26 (Zhang et al., 2002), co-expression of MMP-26 and TIMP-4 suggests a physiological role during implantation.
Zhang et al. (2003) showed that a TIMP-4 antibody increased the percentage of in vitro outgrowth of mouse blastocysts. Also, this TIMP-4 antibody increased indirectly activation of embryonic MMP-2 and MMP-9 via an unknown mechanism. Because MMP-26 is an efficient activator of proMMP-9 (Uria and Lopez-Otin, 2000; Zhao et al., 2003), we hypothesize that epithelial MMP-26 represents such an activator of embryonic proMMP-9 at the time of blastocyst adhesion and penetration. This would provide the preimplantation embryo with active MMP-9. In addition to being a broad-spectrum proteolytic enzyme, MMP-9 can initiate activation of other embryonic MMPs (Salamonsen et al., 2002).
We have reported that MMP-26 is absent in the Fallopian tube (Pilka et al., 2003). This would suggest that activation of embryonic pro-MMP-9 by MMP-26 is restricted to the uterine cavity. That would furthermore imply that activation of embryonic pro-MMP-9 is deficient in cases of tubal pregnancy even though trophoblastic cells in tubal pregnancies show expression of MMP-9 (Bai et al., 2005). Deficient activation of proMMP-9 may be one contributing explanation for shallow implantation in tubal pregnancies.
Membrane type-1 MMP (MT1-MMP) is expressed in the endometrial epithelium from day 20 on in a normal cycle, and it has been suggested to activate embryonic proMMP-2 (Salamonsen, 1999; Zhang et al., 2000). Previous studies have shown that TIMP-4 inhibits MT1-MMP-mediated MMP-2 activation (Bigg et al., 2001). TIMP-4 secreted into uterine fluid may thus block potential activation of embryonic proMMP-2 by epithelial MT1-MMP.
Absence of MMP-26 in the Fallopian tube as well as presence of TIMP-4 in the uterine fluid may constitute protective mechanisms against inappropriate activation of embryonic proteases.
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Acknowledgements |
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This project was supported by grants from the Swedish Cancer fund; the Swedish Medical Research Council; the Medical faculty at Lund University; the Lund University Hospital Funds for cancer research; Nilsson, Craaford and Kamprad foundations; the King Gustaf V and Queen Victoria Foundation.
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References |
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Amour A, Slocombe PM, Webster A, Butler M, Knight CG, Smith BJ, Stephens PE, Shelley C, Hutton M, Knauper V et al. (1998) TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3. FEBS Lett 435,39–44.[CrossRef][Web of Science][Medline]
Apte SS, Olsen BR and Murphy G (1995) The gene structure of tissue inhibitor of metalloproteinases (TIMP)-3 and its inhibitory activities define the distinct TIMP gene family. J Biol Chem 270,14313–14318.
Bai SX, Wang YL, Qin L, Xiao ZJ, Herva R and Piao YS (2005) Dynamic expression of matrix metalloproteinases (MMP-2, -9 and -14) and the tissue inhibitors of MMPs (TIMP-1, -2 and -3) at the implantation site during tubal pregnancy. Reproduction 129,103–113.
Behrendtsen O, Alexander CM and Werb Z (1992) Metalloproteinases mediate extracellular matrix degradation by cells from mouse blastocyst outgrowths. Development 114,447–456.[Abstract]
Bigg HF, Morrison CJ, Butler GS, Bogoyevitch MA, Wang Z, Soloway PD and Overall CM (2001) Tissue inhibitor of metalloproteinases-4 inhibits but does not support the activation of gelatinase A via efficient inhibition of membrane type 1-matrix metalloproteinase. Cancer Res 61,3610–3618.
Bischof P, Friedli E, Martelli M and Campana A (1991) Expression of extracellular matrix-degrading metalloproteinases by cultured human cytotrophoblast cells: effects of cell adhesion and immunopurification. Am J Obstet Gynecol 165,1791–1801.[Web of Science][Medline]
Brenner CA, Adler RR, Rappolee DA, Pedersen RA and Werb Z (1989) Genes for extracellular-matrix-degrading metalloproteinases and their inhibitor, TIMP, are expressed during early mammalian development. Genes Dev 3,848–859.
Brew K, Dinakarpandian D and Nagase H (2000) Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta 1477,267–283.[CrossRef][Medline]
Casslen B (1986) Uterine fluid volume. Cyclic variations and possible extrauterine contributions. J Reprod Med 31,506–510.[Web of Science][Medline]
Casslen BG (1987) Free amino acids in human uterine fluid. Possible role of high taurine concentration. J Reprod Med 32,181–184.[Web of Science][Medline]
Casslen B and Astedt B (1981) Fibrinolytic activity of human uterine fluid. Acta Obstet Gynecol Scand 60,55–58.[Web of Science][Medline]
Casslen B and Ohlsson K (1981) Cyclic variation of proteinase inhibitors in human uterine fluid and influence of an IUD. Contraception 23,425–434.[CrossRef][Web of Science][Medline]
Casslen B and Nilsson B (1984) Human uterine fluid, examined in undiluted samples for osmolarity and the concentrations of inorganic ions, albumin, glucose, and urea. Am J Obstet Gynecol 150,877–881.[Web of Science][Medline]
Casslen B, Thorell J and Astedt B (1981) Effect of IUD on urokinase-like immunoreactivity and plasminogen activators in human uterine fluid. Contraception 23,435–445.[CrossRef][Web of Science][Medline]
Casslen B, Kobayashi TK and Stormby N (1982) Cyclic variation of the cellular components in human uterine fluid. J Reprod Fertil 66,213–218.
Casslen B, Nordengren J, Gustavsson B, Nilbert M and Lund LR (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]
Chegini N, Rhoton-Vlasak A and Williams RS (2003) Expression of matrix metalloproteinase-26 and tissue inhibitor of matrix metalloproteinase-3 and -4 in endometrium throughout the normal menstrual cycle and alteration in users of levonorgestrel implants who experience irregular uterine bleeding. Fertil Steril 80,564–570.[CrossRef][Web of Science][Medline]
de Coignac AB, Elson G, Delneste Y, Magistrelli G, Jeannin P, Aubry JP, Berthier O, Schmitt D, Bonnefoy JY and Gauchat JF (2000) Cloning of MMP-26. A novel matrilysin-like proteinase. Eur J Biochem 267,3323–3329.[Web of Science][Medline]
Cox KH, DeLeon DV, Angerer LM and Angerer RC (1984) Detection of mRNA in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol 101,485–502.[CrossRef][Web of Science][Medline]
Douglas DA, Shi YE and Sang QA (1997) Computational sequence analysis of the tissue inhibitor of metalloproteinase family. J Protein Chem 16,237–255.[CrossRef][Web of Science][Medline]
Egeblad M and Werb Z (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2,161–174.[Web of Science][Medline]
Fisher SJ, Cui TY, Zhang L, Hartman L, Grahl K, Zhang GY, Tarpey J and Damsky CH (1989) Adhesive and degradative properties of human placental cytotrophoblast cells in vitro. J Cell Biol 109,891–902.
Goffin F, Munaut C, Frankenne F, Perrier D’Hauterive S, Beliard A, Fridman V, Nervo P, Colige A and Foidart JM (2003) Expression pattern of metalloproteinases and tissue inhibitors of matrix-metalloproteinases in cycling human endometrium. Biol Reprod 69,976–984.
Graham CH and Lala PK (1992) Mechanisms of placental invasion of the uterus and their control. Biochem Cell Biol 70,867–874.[Web of Science][Medline]
Greene J, Wang M, Liu YE, Raymond LA, Rosen C and Shi YE (1996) Molecular cloning and characterization of human tissue inhibitor of metalloproteinase 4. J Biol Chem 271,30375–30380.
Hampton AL and Salamonsen LA (1994) Expression of messenger ribonucleic acid encoding matrix metalloproteinases and their tissue inhibitors is related to menstruation. J Endocrinol 141,R1–R3.
Hendrickson MR and Kempson RL (1980) Surgical Pathology of the Uterine Corpus. Saunders, Philadelphia.
Hernandez-Barrantes S, Bernardo M, Toth M and Fridman R (2002) Regulation of membrane type-matrix metalloproteinases. Semin Cancer Biol 12,131–138.[CrossRef][Web of Science][Medline]
Inagaki N, Stern C, McBain J, Lopata A, Kornman L and Wilkinson D (2003) Analysis of intra-uterine cytokine concentration and matrix-metalloproteinase activity in women with recurrent failed embryo transfer. Hum Reprod 18,608–615.
Librach CL, Werb Z, Fitzgerald ML, Chiu K, Corwin NM, Esteves RA, Grobelny D, Galardy R, Damsky CH and Fisher SJ (1991) 92-kD type IV collagenase mediates invasion of human cytotrophoblasts. J Cell Biol 113,437–449.
Licht P, Losch A, Dittrich R, Neuwinger J, Siebzehnrubl E and Wildt L (1998) Novel insights into human endometrial paracrinology and embryo-maternal communication by intrauterine microdialysis. Hum Reprod Update 4,532–538.
Liu YE, Wang M, Greene J, Su J, Ullrich S, Li H, Sheng S, Alexander P, Sang QA and Shi YE (1997) Preparation and characterization of recombinant tissue inhibitor of metalloproteinase 4 (TIMP-4). J Biol Chem 272,20479–20483.
MacLaughlin DT and Richardson GS (1983) Analysis of human uterine luminal fluid proteins following radiolabeling by reductive methylation: comparison of proliferative and secretory phase samples. Biol Reprod 29,733–742.[Abstract]
Marbaix E, Kokorine I, Moulin P, Donnez J, Eeckhout Y and Courtoy PJ (1996) Menstrual breakdown of human endometrium can be mimicked in vitro and is selectively and reversibly blocked by inhibitors of matrix metalloproteinases. Proc Natl Acad Sci USA 93,9120–9125.
Marchenko GN, Ratnikov BI, Rozanov DV, Godzik A, Deryugina EI and Strongin AY (2001) Characterization of matrix metalloproteinase-26, a novel metalloproteinase widely expressed in cancer cells of epithelial origin. Biochem J 356,705–718.[CrossRef][Web of Science][Medline]
Nordengren J, Pilka R, Noskova V, Ehinger A, Domanski H, Andersson C, Hoyer-Hansen G, Hansson SR and Casslen B (2004) Differential localization and expression of urokinase plasminogen activator (uPA), its receptor (uPAR), and its inhibitor (PAI-1) mRNA and protein in endometrial tissue during the menstrual cycle. Mol Hum Reprod 10,655–663.
Noyes RW, Hertig AT and Rock J (1950) Dating the endometrial biopsy. Fertil Steril 1,3–25.[Medline]
O’Sullivan CM, Ungarian JL, Singh K, Liu S, Hance J and Rancourt DE (2004) Uterine secretion of ISP1 & 2 tryptases is regulated by progesterone and estrogen during pregnancy and the endometrial cycle. Mol Reprod Dev 69,252–259.[CrossRef][Web of Science][Medline]
Park HI, Ni J, Gerkema FE, Liu D, Belozerov VE and Sang QX (2000) Identification and characterization of human endometase (Matrix metalloproteinase-26) from endometrial tumor. J Biol Chem 275,20540–20544.
Pilka R, Whatling C, Domanski H, Hansson S, Eriksson P and Casslen B (2003) Epithelial expression of matrix metalloproteinase-26 is elevated at mid-cycle in the human endometrium. Mol Hum Reprod 9,271–277.
Pilka R, Domanski H, Hansson S, Eriksson P and Casslen B (2004a) Endometrial TIMP-4 mRNA is high at midcycle and in hyperplasia, but down-regulated in malignant tumours. Coordinated expression with MMP-26. Mol Hum Reprod 10,641–650.
Pilka R, Norata GD, Domanski H, Andersson C, Hansson S, Eriksson P and Casslen B (2004b) Matrix metalloproteinase-26 (matrilysin-2) expression is high in endometrial hyperplasia and decreases with loss of histological differentiation in endometrial cancer. Gynecol Oncol 94,661–670.[CrossRef][Web of Science][Medline]
von Rango U, Classen-Linke I, Krusche CA and Beier HM (1998) The receptive endometrium is characterized by apoptosis in the glands. Hum Reprod 13,3177–3189.
Rodgers WH, Matrisian LM, Giudice LC, Dsupin B, Cannon P, Svitek C, Gorstein F and Osteen KG (1994) Patterns of matrix metalloproteinase expression in cycling endometrium imply differential functions and regulation by steroid hormones. J Clin Invest 94,946–953.[Web of Science][Medline]
Rotello RJ, Lieberman RC, Purchio AF and Gerschenson LE (1991) Coordinated regulation of apoptosis and cell proliferation by transforming growth factor beta 1 in cultured uterine epithelial cells. Proc Natl Acad Sci USA 88,3412–3415.
Salamonsen LA (1999) Role of proteases in implantation. Rev Reprod 4,11–22.[Abstract]
Salamonsen LA, Zhang J and Brasted M (2002) Leukocyte networks and human endometrial remodelling. J Reprod Immunol 57,95–108.[CrossRef][Web of Science][Medline]
Shi SR, Key ME and Kalra KL (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]
Shimonovitz S, Hurwitz A, Dushnik M, Anteby E, Geva-Eldar T and Yagel S (1994) Developmental regulation of the expression of 72 and 92 kd type IV collagenases in human trophoblasts: a possible mechanism for control of trophoblast invasion. Am J Obstet Gynecol 171,832–838.[Web of Science][Medline]
Stetler-Stevenson WG, Krutzsch HC and Liotta LA (1989) Tissue inhibitor of metalloproteinase (TIMP-2). A new member of the metalloproteinase inhibitor family. J Biol Chem 264,17374–17378.
Stracke JO, Hutton M, Stewart M, Pendas AM, Smith B, Lopez-Otin C, Murphy G and Knauper V (2000) Biochemical characterization of the catalytic domain of human matrix metalloproteinase 19. Evidence for a role as a potent basement membrane degrading enzyme. J Biol Chem 275,14809–14816.
Tunuguntla R, Ripley D, Sang QX and Chegini N (2003) Expression of matrix metalloproteinase-26 and tissue inhibitors of metalloproteinases TIMP-3 and -4 in benign endometrium and endometrial cancer. Gynecol Oncol 89,453–459.[CrossRef][Web of Science][Medline]
Uria JA 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.
Wang H, Wen Y, Mooney S, Li H, Behr B and Polan ML (2003) Matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression in human preimplantation embryos. Fertil Steril 80(Suppl 2),736–742.
Welgus HG and Stricklin GP (1983) Human skin fibroblast collagenase inhibitor. Comparative studies in human connective tissues, serum, and amniotic fluid. J Biol Chem 258,12259–12264.
Whitfield HJ, Brady LS, Smith MA, Mamalaki E, Fox RJ 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][Web of Science][Medline]
Woessner JF and Nagase H (2000) Introdution to the matrix metalloproteinases (MMPs). In Woessner JF and Nagase H (eds) Matrix Metalloproteinases and TIMPs. Oxford University Press, New York, pp. 1–10.
Young WS (1990) In situ hybridization histochemistry. In Björklund A, Hökfelt T, Wouterlood FG and van den Pol AN (eds) Handbook of Chemical Neuroanatomy: Analysis of Neuronal Microcircuits and Synaptic Interactions. Elsevier Science Publishers B.V., Amste rdam, pp. 481–512.
Young DA, Phillips BW, Lundy C, Nuttall RK, Hogan A, Schultz GA, Leco KJ, Clark IM and Edwards DR (2002) Identification of an initiator-like element essential for the expression of the tissue inhibitor of metalloproteinases-4 (Timp-4) gene. Biochem J 364,89–99.[Web of Science][Medline]
Zhang J and Salamonsen LA (1997) Tissue inhibitor of metalloproteinases (TIMP)-1, -2 and -3 in human endometrium during the menstrual cycle. Mol Hum Reprod 3,735–741.
Zhang J, Hampton AL, Nie G and Salamonsen LA (2000) Progesterone inhibits activation of latent matrix metalloproteinase (MMP) -2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium. Biol Reprod 62,85–94.
Zhang J, Cao YJ, Zhao YG, Sang QX and Duan EK (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.
Zhang J, Zhao YG, Cao YJ, Sang QX and Duan EK (2003) Expression and implications of tissue inhibitor of metalloproteinases-4 in mouse embryo. Mol Hum Reprod 9,143–149.
Zhao YG, Xiao AZ, Newcomer RG, Park HI, Kang T, Chung LW, Swanson MG, Zhau HE, Kurhanewicz J and Sang QX (2003) Activation of pro-gelatinase B by endometase/matrilysin-2 promotes invasion of human prostate cancer cells. J Biol Chem 278,15056–15064.
Submitted on January 31, 2006; resubmitted on May 16, 2006; accepted on May 23, 2006.
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