Molecular Human Reproduction, Vol. 5, No. 4, 376-381,
April 1999
© 1999 European Society of Human Reproduction and Embryology
Secretion of matrix metalloproteinase-2, matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases into the intrauterine compartments during early pregnancy
1 Department of Obstetrics and Gynaecology, Centre for Reproductive Biology, University of Edinburgh, 37 Chalmers Street, Edinburgh EH3 9EW, and 2 Department of Obstetrics and Gynaecology, St Bartholomew's Hospital, London, UK
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Abstract |
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Matrix metalloproteinases (MMPs) are important enzymes in tissue remodelling, a key event for the development of the fetal membranes and placenta and establishing the feto–maternal interface during early pregnancy. This study has examined the secretion of the gelatinases, MMP-2 (72 kDa) and MMP-9 (92 kDa), and the endogenous tissue inhibitors of metalloproteinases (TIMPs) into extra-embryonic coelomic and amniotic fluids, the two principal intra-uterine compartments of the first trimester, and compared them to amniotic fluid collected later in gestation. In extra-embryonic coelomic fluid, gelatin zymography demonstrated that MMP-2 (72 kDa) was the predominant gelatinase, with some MMP-9 present. A broad range of TIMPs corresponding to TIMP-1 and TIMP-2, glycosylated and unglycosylated TIMP-3 and TIMP-4 was detected in this compartment by reverse zymography and immunoblot analyses. There was little gelatinase or TIMP activity in amniotic fluid in the first trimester. In amniotic fluid from the second trimester after fusion of the membranes obliterating the extra-embryonic coelom, and at term elective caesarean section, MMP-2 is the predominant gelatinase present, with a broad spectrum of TIMPs. These findings demonstrate that predominantly MMP-2 and also MMP-9, regulated by a range of TIMPs, are involved in intra-uterine tissue remodelling during the establishment of pregnancy.
fetus/matrix metalloproteinase/placenta/TIMP/trophoblast
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Introduction |
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Successful pregnancy is dependent upon invasion of trophoblast into the decidua at implantation, and subsequently the further invasion of extravillous trophoblast into the walls of the maternal spiral arterioles (Fisher et al., 1985
; Librach et al., 1991; Cross et al., 1994). These events require the breakdown of extracellular matrix and cellular migration. Little is known about the specific collagenase enzymes involved. The matrix metalloproteinases (MMPs) are an important family of zinc-dependent enzymes with a broad range of substrate specificities capable of the breakdown of extracellular matrix (Hulboy et al., 1997). The activity of secreted MMPs is regulated by specific endogenous inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), of which four have been identified (Hulboy et al., 1997). These TIMPs have a broad specificity for all MMPs and form tight stoichiometric non-covalently bound complexes with the activated MMPs to inhibit their enzymatic activity (Woesnner, 1991).
In the uterus, a role for MMPs and TIMPs in the cyclical remodelling of the endometrium during the menstrual cycle has been established (Hampton and Salamonsen, 1994; Rodgers et al., 1994; Zhang and Salamonsen, 1997). During early pregnancy, MMPs are implicated in the invasion of trophoblast cells at implantation and during the establishment of the placental bed (Bishof et al., 1991; Graham and Lala, 1991; Shimonowitz et al., 1994; Huppertz et al., 1998). However, the intrauterine secretion of MMPs and TIMPs during human pregnancy has been poorly characterized. In the first trimester, within the uterus the extra-embryonic coelom is the space between the amnion and chorion. It becomes obliterated by the start of the second trimester as the amnion and chorion fuse. The amniotic sac and the extra-embryonic coelom are likely to accumulate locally secreted enzymes and inhibitors released by the surrounding amnion and chorion layers of the fetal membranes. Sampling of these fluid spaces offers a convenient window on in-vivo enzyme secretion by the fetal membranes in early pregnancy. We have chosen to examine the secretion of the gelatinases MMP-2 and MMP-9 as these enzymes have substrate specificity for type IV collagen, a major component of basement membranes. At the same time we have also characterized the secretion of TIMPs into the same fluid cavities.
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Materials and methods |
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Collection of samples
Matched samples (n = 7) of extra-embryonic coelomic and amniotic fluids were collected by ultrasound guided needle aspiration from women between 8–11 weeks gestation, prior to the termination of pregnancy by general anaesthesia, as described in detail previously (Wathen et al., 1991
). Fetal viability and gestational age were confirmed by ultrasound. Amniotic fluid samples collected prospectively in the second trimester by amniocentesis were obtained from the regional cytogenetics laboratory. All samples used had normal karyotypes. At term, amniotic fluid samples were collected at the time of elective caesarean section. Samples were stored at –20°C prior to performing zymography. Ethical approval for the collection of all samples used in these studies was granted by the appropriate St Bartholomew's Hospital, and the Lothian Research Ethical Committees.
Detection and measurement of MMP-2 and MMP-9 by gelatinase zymography
MMP-2 and MMP-9 activities were detected by gelatinase zymography, as described previously (Rawdanowicz et al., 1994) with minor modifications. Briefly, amniotic and extra-embryonic coelomic fluid samples (the same volume of 5 µl was consistently used) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE; 7.5% gels; Minigel apparatus; BioRad, Hemel Hempstead, UK) containing gelatin (1 mg/ml) with non-reducing conditions. Caseinolytic activity was also assayed by substituting casein (1mg/ml) for gelatin as substrate. The presence of SDS in the gel activates the latent forms of MMPs, as well as dissociating active forms from inhibitors and so permitting their detection. Gels were washed (twice, 2.5% (v/v) Triton X-100) then incubated in zymography digestion buffer (200 mM NaCl, 50 mM Tris, 5 mM CaCl2, 1 µM ZnCl2, 0.02% (v/v) Brij-35, pH 7.6; all chemicals from Sigma Chemical Co, St Louis MO, USA, except where specified) for 18 h at 37°C. Gels were immersed in staining solution (0.5% Coomassie Blue R250 in 30% methanol/10% glacial acetic acid in H2O) for 3 h at 23°C, then destained (staining solution with Coomassie Blue omitted) to reveal discrete areas where gelatin substrate has been hydrolysed by gelatinase activity.
Detection and measurement of TIMPs by reverse zymography
Detection of TIMPs was performed by reverse zymography using methods described previously, using a commercial kit (University Technologies Inc, Calgary, Canada; Hampton et al., 1995) with some minor modifications. Briefly, samples of extra-embryonic and amniotic fluids (same volume of 7.5 µl used consistently) were separated by PAGE using 12% gels containing 1 mg/ml gelatin and an MMP preparation (from BHK-21 cells that constitutively express proMMP-2; University Technologies Inc). The gels were washed (50 mM Tris, 5 mM CaCl2, 2.5% (v/v) Triton X-100; for 2.5 h at 23°C), then incubated in reverse zymography digestion buffer (50 mM Tris, 5 mM CaCl2) for 17 h at 37°C. The gel was counterstained (as for zymography) with staining buffer then destained to detect the presence of protein, predominantly the incorporated gelatin. The presence of TIMPs was determined by their discrete inhibition of MMP activity, visualized as a dark band on a lighter background. TIMPs were identified and characterized by comparison with molecular weight markers, with control standards of conditioned medium containing mouse TIMP-1, -2 and -3 expressed by transfected BHK cells (University Technologies Inc) and human recombinant TIMP-2 (Calbiochem, Nottingham). This human TIMP-2 standard exhibited different mobility to the mouse form (observed at 21 kDa by reverse zymography under non-reducing conditions and 26 kDa by Western blotting under reducing conditions).
Analysis of MMP-2 and TIMP-4 by Western blotting
Extra-embryonic coelomic and amniotic fluid samples (20 µl) were prepared in reducing buffer, separated by PAGE and transferred to a nitro-cellulose membrane (pore size 0.45 µm; BioRad) by wet blotting (100 V for 1 h). The membrane was blocked with 5% bovine serum albumin (BSA; Sigma) before application of the appropriate antibody. The following primary antibodies were used: MMP-2 (mouse monoclonal; Calbiochem); TIMP-4 (affinity purified rabbit polyclonal; Chemicon, Harrow, UK). These were detected using sequentially a biotinylated horse anti-mouse or goat anti-rabbit second antibody as appropriate and an avidin-peroxidase complex according to the manufacturers instructions (Vector Labs, Burlingame, CA, USA), with 3,3'-diaminobenzidine with Ni2+ enhancement, as chromagen.
Statistical analysis
MMP and TIMP activities as detected by zymography were measured by transmission densitometry (G-700 Densitometer; BioRad) and protein abundance on Western blots measured by reflectance densitometry. Relative intensities were derived from zymogram gels by comparison to parallel background readings of equal area and calculated using dedicated software (Quantity One; BioRad). Densitometric readings were only compared with the other gel examined under precisely the same conditions (i.e. the same electrophoresis run and identical buffers, stains and incubation periods). These densitometry data were analysed using Student's t-test.
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Results |
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MMP-2, MMP-9, and TIMPs in uterine compartments during early pregnancy
In extra-embryonic coelomic fluid, analysis by zymography demonstrated that the predominant gelatinase activity was identified at 72 kDa molecular weight, corresponding to the latent form of MMP-2 (Figure 1
). Lesser amounts of active MMP-2 (66 kDa) were detected. Amounts of the latent form of MMP-2 were significantly higher (P < 0.05; t-test) than the barely detectable activity in the matched amniotic fluid samples (Figure 1). Low levels of gelatinase activity were also detectable in extra-embryonic coelomic fluid at 92 kDa, corresponding to latent MMP-9 with virtually undetectable amounts in the amniotic fluid. The gelatinase activities at 72 and 92 kDa were further characterized as MMP activated proteins by inhibition with EDTA (5 mM) or o-phenanthroline (2.5 mM; data not shown). Another unidentified band of gelatinase activity of ~115 kDa was also present. The presence of MMP-2 in extra-embryonic coelomic fluid and amniotic fluid was confirmed by Western blot analysis (Figure 2a) using a specific antibody which demonstrated a predominant band at 72 kDa, corresponding to the latent form. The abundance of MMP-2 protein by Western blotting was similar to the zymography findings, demonstrating MMP-2 protein in its lowest abundance during the first trimester of pregnancy in amniotic fluid, with higher amounts present in extra-embryonic coelomic fluid and in amniotic fluid from the second and third trimesters. Using zymography with casein as substrate, no caseinolytic activity was detected in extra-embryonic coelomic or amniotic fluids from the first trimester (data not shown).
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A broad spectrum of TIMP activity was detected by reverse zymography predominantly in extra-embryonic coelomic fluid (Figure 3
). Three bands of gelatinase inhibitory activity were present at 27–30 kDa (these correspond to the molecular weight of TIMP-1, glycosylated TIMP-3 and TIMP-4), 24 kDa (corresponding to unglycosylated TIMP-3) and 21 kDa (corresponding to TIMP-2). These bands aligned with standards to TIMP-1, TIMP-2 and glycosylated and unglycosylated TIMP-3. In amniotic fluid samples from the first trimester, very low to undetectable amounts of TIMP activity were present. Reverse zymography is unable to distinguish precisely between the TIMP isoforms of 27–30 kDa molecular weight. The presence of TIMP-4 was, therefore, determined by Western blot analysis (Figure 2b
) which demonstrated only low abundance in extra-embryonic coelomic fluid and in amniotic fluid from the second trimester and at term. Little TIMP-4 was detectable in amniotic fluid collected during the first trimester.
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MMP-2, MMP-9, and TIMPs in amniotic fluid during the second trimester
In amniotic fluid samples collected in the second trimester MMP-2 activated protein corresponding to the latent (72 kDa) form was the predominant gelatinase activity present (Figure 4
). There was a less intense band of activity at 66 kDa, corresponding to the active form of MMP-2. Significantly greater (P < 0.05; t-test) amounts of MMP-2 (latent) compared to MMP-9 (latent) were present in the second trimester amniotic fluid. There was significantly higher (P < 0.05; t-test) secretion of latent MMP-2 into second trimester amniotic fluid compared with samples collected in the first trimester. Active MMP-2 was also present but in lower amounts than the latent isoform. The pattern of TIMPs activity was similar to that present in the extra-embryonic coelom in the first trimester, with three main bands of activity visualized (Figure 5).
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MMP-2, MMP-9, and TIMPs in amniotic fluid in late pregnancy
Amniotic fluid in late pregnancy contained predominantly latent forms of MMP-2 and MMP-9 activated protein, there being significantly more MMP-2 than MMP-9, as was the case for second trimester amniotic fluid (Figure 4
). The pattern of TIMP activity was similar to that in second trimester amniotic fluid (Figure 5a). The secretion of TIMPs into amniotic fluid increased significantly (P < 0.05; analysis of variance) with gestation, as determined by densitometric analysis (Figure 5b). |
Discussion |
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These studies demonstrate the presence of MMP-2 and MMP-9 gelatinases in first trimester extra-embryonic coelomic fluid, as well as in the amniotic fluid at all gestations. The predominant MMP activated protein was the latent form of MMP-2 which was present in increasing concentrations in amniotic fluid from the first to the second trimester. The same fluid compartments contained TIMPs activities corresponding to TIMP-1, TIMP-2, glycosylated and unglycosylated TIMP-3, and TIMP-4 at all gestations. The TIMP activity in the amniotic fluid also increased with gestation.
These studies, of course, cannot precisely identify the cellular origin of the various MMPs and TIMPs. Enzyme activity recovered from the extra-embryonic coelom may arise from mesenchymal cells lining either the chorion or amnion epithelium, from adjacent trophoblast, or even from more distantly placed decidual cells. Similarly, enzyme activity recovered from the amniotic fluid may arise from the amnion epithelium or from the growing fetus. Furthermore, although it seems likely that enzyme present in these fluid compartments is present as overspill from various local tissues which are undergoing remodelling, it is equally possible that the enzymes have been directly secreted into these spaces to mediate specific functions. Gestation related changes in MMP and TIMP activities have been quantified by densitometric analysis of gelatin zymography. The lack of suitable standards, limited number of samples that can be examined at any one time (only gels run directly in parallel are comparable) and inability to compare absolute protein content or activity using this technique, only permits the demonstration of relative changes in amounts of activated gelatinase proteins (Martelli et al., 1993).
There is evidence for the production of MMP-2, MMP-9 and TIMPs in decidua, trophoblast and mesenchymal cells in the fetal membranes as well as from the developing embryo (Hulboy et al., 1997). Several authors have identified MMP-1, -2, -3, and -9 and TIMP-1, -2, and -3 by immunocytochemistry in the placental bed from the first, second and third trimester, but different fixation and tissue preparation techniques influence the observed pattern of distribution (Librach et al., 1991; Ruck et al., 1996; Huppertz et al., 1998; Skinner et al., 1999). In rat and human fetal membranes the amount of MMP-9 is increased with labour (Bryant-Greenwood and Yamamoto, 1995; Draper et al., 1995; Lei et al., 1995). In vitro, decidualized endometrial cells express TIMP-1, -2 and -3 (Higuchi et al., 1995; Zhang and Salamonsen, 1997). Protein and message for TIMP-1, -2 and -3 is also present in human first trimester placenta and decidua and in amnion mesenchymal and epithelial cells (Hurskainen et al., 1996; Rowe et al., 1997) and TIMP-1 increases in maternal serum with gestational age (Clark et al., 1994). Transudation from maternal serum may also be a potential source, as inulin is transferred into the uterine compartments during the first trimester (Jauniaux et al., 1997). There is little information about the expression of MMPs or TIMPs in the developing fetal membranes in first trimester (Hulboy et al., 1997).
Both MMPs and TIMPs are likely to be involved in the tissue remodelling that accompanies the rapid growth and structural changes of the fetal membranes in the first trimester. This may be reflected in the increased amounts of the active form of MMP-2 in amniotic fluid during the second trimester. Proteases may also have an action to cleave inactive growth factor and cytokine precursors to active forms, and to liberate growth factors bound to extracellular matrix (Cross et al., 1994; Damsky et al., 1994). Interestingly, TIMPs stimulate proliferation and may play a direct role in the development of the intra-uterine structures (Hayakawa et al., 1994; Corcoran and Stetler-Stevenson, 1996). A complex of TIMP-1 with procathepsin-L has been shown to upregulate steroidogenesis in the testis (Boujrad et al., 1995) raising the possibility that TIMP-1 may play a role in stimulating the high concentrations of oestradiol 17-ß, progesterone and 17
-progesterone found in the extra-embryonic coelom (Jauniaux et al., 1993, Atkinson et al., 1996).
In conclusion, these studies have demonstrated compartmentalization of MMPs and TIMPs within the uterus in the first trimester of human pregnancy. MMP-2 and -9, and TIMP-1, -2, -3 and -4 are present predominantly in the extra-embryonic coelomic fluid, with barely detectable amounts in amniotic fluid. When the extra-embryonic coelom becomes obliterated the concentrations of MMPs and TIMPs in the amniotic fluid increase. It is not clear if this compartmentalization of enzyme and endogenous inhibitors is related to specific functions within the extra-embryonic coelom, or if proteins have accumulated here as overspill from surrounding rapidly developing tissues.
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Acknowledgments |
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This study was funded by a grant from the Scottish Hospital Endowment's Research Trust (Grant 1389). We wish to thank Dr Janet Brennand and Dr Euan Wallace for supplying amniotic fluid samples.
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3 To whom correspondence should be addressed:
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References |
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Submitted on October 6, 1998; accepted on January 18, 1999.
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M.-A. Ledingham, F. C. Denison, S. C. Riley, and J. E. Norman Matrix metalloproteinases-2 and -9 and their inhibitors are produced by the human uterine cervix but their secretion is not regulated by nitric oxide donors Hum. Reprod., August 1, 1999; 14(8): 2089 - 2096. [Abstract] [Full Text] [PDF]
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