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a State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies have compared cytotrophoblast from the first and third trimesters: for example, in human preimplantation embryos, MMP-2 is the predominant form of type IV collagenases while MMP-9 accounts for a minor amount [12–14]; after the blastocysts implant into the endometrium and before placentation is complete in the first trimester, human trophoblasts produce both MMP-2 and MMP-9 [15–18]. In the third trimester, trophoblast cells primarily secrete MMP-9, while MMP-2 is secreted in minimal amounts [7, 16]. But week-by-week comparisons of MMP-2 and MMP-9 expression in cultured human cytotrophoblast cells during the first trimester have not been made. It has also been demonstrated that the invasiveness exhibited in vitro by human trophoblasts depends on the production of MMP-9 [9]. The studies mentioned made it appropriate to conduct experiments to compare the expression of the two enzymes in trophoblast cells taken from different weeks of gestation in the first trimester to attempt to elucidate whether the expressions of MMPs is developmentally related.
The activity of MMPs is tightly controlled physiologically by tissue inhibitors of metalloproteinases (TIMPs), of which TIMP-1 and TIMP-2 are best understood. TIMP-1 inhibits all the MMPs in their activated form, preferentially binding MMP-9 in both the latent and the active form [19]. TIMP-2 binds either the active or the latent form of MMP-2 with less inhibitory activity to other MMPs [20, 21]. It has been shown that TIMPs are produced by trophoblastic and decidual tissues throughout gestation [22–24].
Immunohistochemistry studies have demonstrated that human placental trophoblast cells can produce matrix proteins such as collagen IV, laminin, and fibronectin in the first trimester [25, 26]. Fibronectin and collagen IV can also be synthesized in vitro by isolated first-trimester human cytotrophoblast cells [26, 27]. These observations suggest that the cytotrophoblast cells themselves secrete substrates, in addition to the degrading enzymes, which may be relevant to the cell invasion.
To elucidate the dynamic expression profiles of MMPs and their inhibitors in detail during the first trimester, we conducted a study to compare the expression and production of MMP-2, MMP-9, MMP-14, TIMP-1, and TIMP-2 in cultured human cytotrophoblast cells at different weeks of gestation. The secretion of several matrix proteins that are substrates for these proteases (collagen I, collagen IV, fibronectin, laminin, and vitronectin) was also investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cells were isolated and maintained as previously described [28, 29]. Briefly, human chorionic villi tissues were obtained from patients who underwent therapeutic termination of pregnancy at 6, 7, 8, 9, and 11 wk of gestation. Informed consent was provided by the patients, and the project was approved by the local ethics committee. The time of gestation was defined according to the first day of the last menstrual period and further examined morphologically by means of stereomicroscope. For one cell culture, trophoblast tissues from 2–3 patients at 6–9 wk and 1–2 patients at 11 wk of gestation were used, and the experiments were repeated three times. Tissues from each week were minced separately and digested with 0.25% trypsin (Sigma Chemical Co., St. Louis, MO) and 15 IU/ml DNase I (Sigma) at 7–9°C for 45–60 min. Trypsinization was stopped by addition of two volumes of FD medium (Ham's F-12/DMEM: 1:1; Gibco BRL, Gaithersburg, MD). After washing, the dispersed cells were filtered through a nylon sieve to remove the gross villous core residues. The filtered cell suspension (1–2 ml) was then added slowly to the top of a BSA gradient (prepared by sequential addition of 3 ml of 3%, 2%, and 1% BSA in FD medium to a 15-ml centrifuge tube). The cells were sedimented for 1 h at unit gravity, and cytotrophoblast cells were collected from the bottom of the tube. The purified cytotrophoblast cells were plated at 1–2 x 105 cells per well in collagen I (Cellmatrix Type I-A; Institute of Biochemistry, Osaka, Japan)-coated 24-well dishes (Corning, Corning, NY) with 1 ml of serum-free FD medium supplemented with 1 ng/ml epidermal growth factor (Collaborative Research, Lexington, MA), 10 µg/ml insulin, 0.1% BSA, 1.75 mM Hepes (Sigma), and 2 mM glutamine (Dongfang Chemical Co., Shanghai, China). Five wells were set up for each group. The cells began to attach within 2 h after plating. They spread and showed a monolayer epithelial cell morphology after 24 h under serum-free culture conditions. Immunocytochemical studies revealed that more than 99% of the cells exhibited positive staining for cytokeratin and GnRH and were vimentin negative, consistent with their identification as cytotrophoblast cells.
Immunocytochemistry
The primary antibodies used were the following: anti-collagen I (MAB 1340, dilution 1:100; Chemicon International, Temecula, CA), anti-collagen IV (MAB 1910, dilution 1:500; Chemicon), anti-fibronectin (F3648, dilution 1:400; Sigma), anti-laminin (L-9393, dilution 1:30; Sigma). The antibody to human vitronectin (dilution 1:500) was a generous gift from Prof. Deane F. Mosher at the Dept. of Medicine, University of Wisconsin (Madison, WI).
Cytotrophoblast cells were cultured in chamber slides (Nalge Nunc International, Naperville, IL). After 48 h of culture, the cells were fixed in 2% formaldehyde in PBS for 20 min at room temperature, washed twice in PBS, and then digested to expose the antigen in 0.01 N HCl containing 0.4% pepsin for 20 min at room temperature. After washing, the cells were blocked in 10% goat serum for 20–30 min at room temperature and then incubated with primary antibodies overnight at 4°C. Subsequently, slides were washed in PBS and placed into fluorescein isothiocyanate-conjugated secondary antibodies (diluted 1:400 in PBS containing 1% BSA; Jackson ImmunoResearch Laboratories, West Grove, PA) at 37°C for 30 min. Then the cells were counterstained with 0.1 mg/ml propidium iodide (PI; Sigma) for 5 min for visualization of the cell nucleus. Slides were then washed and mounted with an aqueous mounting medium. The labeled cells were visualized using a Leica TCS NT confocal system (Leica, Wetzlar, Germany). Immunocytochemical controls consisted of omission of primary antibody.
ELISA
The levels of secreted pro-MMP-2, pro-MMP-9, TIMP-1, and TIMP-2 in the media from cultured 6- to 11-wk gestation cytotrophoblast cells were quantified using sensitive ELISA assay kits purchased from Amersham Life Science (Little Chalfont, Buckinghamshire, England).
Gelatin Zymography
The presence of MMP-2 and MMP-9 in media was demonstrated by zymography [30]. The harvested culture media were standardized according to the protein content of cell lysates, which was measured according to the method of Bradford [31]. Thus, 10–20 µl medium, equivalent to 6 µg protein of cell lysates, was loaded to each lane for zymography. The medium was mixed 3:1 (v:v) with sample buffer (10% [w:v] SDS, 25% [v:v] glycerol, 0.25 M Tris) and then applied to gels for electrophoresis without boiling under nonreducing conditions in a 10% acrylamide gel containing 1 mg/ml gelatin (Difco Laboratories, Detroit, MI). After electrophoresis, the gels were washed at room temperature for 1 h in 2.5% Triton X-100, 50 mM Tris-HCl, at pH 7.5, to remove SDS and incubated overnight in buffer (150 mM NaCl, 5 mM CaCl2, and 50 mM Tris-HCl) at 37°C, pH 7.6. Thereafter gels were stained with 0.1% (w:v) Coomassie Brilliant Blue R-250 in 30% (v:v) isopropyl alcohol, 10% glacial acetic acid for 60 min and destained in 10% (v:v) methanol, 5% (v:v) glacial acetic acid.
Northern Blotting Analysis
Total RNA was extracted from cytotrophoblast cells after 48 h of culture using TRIzol reagent (Gibco BRL) according to the instructions. RNA (20 µg) was mixed with sample buffer (0.5-strength 3-[N-morpholino]propanesulfonic acid [MOPS], 2.2 M formaldehyde, 50% formamide, 5% glycerol, 0.1 mM EDTA, 0.025% bromophenol blue, and 0.025% xylene cyanol FF), heated at 65°C for 15 min, and then separated on formaldehyde-agarose gels (1% agarose, 2.2 M formaldehyde, and single-strength MOPS). After electrophoresis, RNA was transferred to nylon membranes (Amersham Life Science) by vacuum using 20-strength SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) and UV cross-linked. A 420-base pair fragment extending from nucleotide (nt) 210 to nt 630 of the human MMP-14 cDNA sequence described originally by Sato et al. [32] was amplified by polymerase chain reaction from human placenta cDNA. The respective cDNA probe for human MMP-2 and MMP-9 (a generous gift from Dr. Karl Tryggvason, Karolinska Institute, Stockholm, Sweden) was excised from its plasmid with the appropriate endonucleases; the three cDNA probes were labeled with [
-32P]dCTP using a Nick Translation Kit (Gibco BRL) and denatured in boiling water for 10 min. The membranes were prehybridized for 4 h at 65°C in prehybridization buffer (0.2 M sodium phosphate [pH 7.4], 0.1 mM EDTA, 7% [w:v] SDS, 1% [w:v] BSA, and 15% [v:v] formamide) and further hybridized for 24 h at 65°C in fresh prehybridization buffer containing 10% (w:v) dextran sulfate. After hybridization, the membranes were washed in solution A (single-strength SSC, 0.1% [w:v] SDS) at room temperature for 30 min; this was followed by three 30-min washes in solution B (50 mM sodium phosphate, 1 mM EDTA, 1% SDS [w:v]). After washing, the membranes were exposed to x-ray film (Fuji Photo Film, Tokyo, Japan) at -80°C for 24–96 h. The mRNA signal was quantified by densitometric scanning of the autoradiograms. The relative expression was normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, which was detected with rat GAPDH cDNA probe.
Statistics
Results are presented as the average ± SE of at least three separate experiments. Statistical differences were evaluated by analysis with Student's t-test. Values of P < 0.05 were accepted as significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After 48 h in culture, cytotrophoblast cells were fixed and double-stained with anti-matrix antibody and PI as described above. As evidenced by confocal immunofluorescence micrography, cells stained with antibodies against fibronectin, laminin, and vitronectin demonstrated intense staining in their periphery, indicating that the cells had extracellular deposits of fibronectin, laminin, and vitronectin (Fig. 1, A–C). The nuclei were stained red by the PI, and the matrix proteins showed green fluorescence. A yellow color represented the overlap of green and red. The negative control showed PI staining only. We did not observe positive staining when cells were treated with anti-collagen I (Fig. 1D) and anti-collagen IV antibodies (data not shown). No difference was seen in the apparent level of staining of matrix proteins in cytotrophoblast cells from different weeks of gestation during the first trimester.
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Production Patterns of MMP-2 and MMP-9 in Cultured Cytotrophoblast Cells During the First Trimester
Gelatin zymography of the medium of cytotrophoblast cells cultured for 48 h revealed that the production of MMP-9 increased gradually from Week 6 to Week 11, while MMP-2 production decreased during this period (Fig. 2). To further confirm the production patterns of MMP-2 and MMP-9 in cultured cytotrophoblast cells during the first trimester, we used an ELISA method to quantitate the levels of MMP-2 and MMP-9 in the media from 6 to 11 wk of culture. The data showed that the cells at Week 6 secreted no detectable MMP-9, while from Week 7 to Week 11 the MMP-9 secretion increased gradually, producing a substantial amount of MMP-9 (167.7 ± 18.8 ng/ml) at Week 11. This level was a 10-fold increase over the Week 7 production (14.7 ± 3.9 ng/ml). In contrast, MMP-2 secretion declined from Week 6 (205.7 ± 27.2 ng/ml) to Week 11 (32.3 ± 8.1 ng/ml). The MMP-2 production at Week 11 was approximately one sixth that of Week 6 (Fig. 3).
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Production of TIMP-1 and TIMP-2 in Cultured Cytotrophoblast Cells During the First Trimester
Production of TIMP-1 and TIMP-2 in trophoblast cells from Week 6 to Week 11 was measured by ELISA. The results showed that trophoblast cells themselves were able to secrete TIMP-1 and that the amounts of TIMP-1 increased gradually throughout the period studied. Cells secreted 169.1 ± 30.2 ng/ml at Week 11 of pregnancy, which was about 7-fold higher than that of Week 6 cells (23.5 ± 4.8 ng/ml) (Fig. 4). The concentration of TIMP-2 in the medium was nondetectable by ELISA at all times.
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Expression of MMP-2, -9, and -14 mRNAs in Cultured Cytotrophoblast Cells During the First Trimester
To examine whether the different production pattern of MMP-2 and MMP-9 during the first trimester was a result of changes in the mRNA level, total RNA was extracted from cytotrophoblast cells isolated from the villi at 6–11 wk of gestation and cultured for 48 h. As shown in Figure 5, Northern blotting analysis was consistent with the zymography and ELISA assays. No MMP-9 mRNA signal was detected in 20 µg total RNA extracted from cultured Week 6 to Week 8 cells, but there were apparent signals in the Week 9 and Week 11 samples. The MMP-9 mRNA transcripts were especially abundant in Week 11 cells. In contrast, the amounts of MMP-2 mRNA in cytotrophoblast cells started to decline from Week 7 of gestation and decreased significantly at Weeks 9 and 11 (P < 0.05 and P < 0.01, respectively) when compared with that for Week 6. MMP-14 mRNA transcripts in the trophoblast cells were also measured, and the results indicated that MMP-14 was expressed throughout 6–11 wk of gestation with no significant difference among the different times (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been shown that cytotrophoblast cells isolated from preeclamptic placentas express almost no MMP-9 [18]. Genbacev et al. [33, 34] have reported that normal first-trimester human cytotrophoblast cells cultured under hypoxic conditions express an integrin pattern characteristic of cytotrophoblasts in preeclampsia. Additionally, Rodesch et al. [35] reported that prior to 10 wk of gestation the conceptus is in a relatively hypoxic environment. Thus the low level of MMP-9 secreted by the cytotrophoblast cells before 9 wk of gestation that was showed in our results may be related to hypoxic tension.
MMPs secreted by cells are usually in zymogen forms. Subsequently they are activated by proteinases other than the MMPs that we studied. In the zymogram study, as shown in Figure 2, we also observed additional bands that shifted slightly faster than those of pro-MMP-2 and pro-MMP-9. We presume that they are probably the active forms of pro-MMP-2 and pro-MMP-9, 66 kDa and 84 kDa in size, respectively [36].
Many studies have demonstrated, using immunohistochemistry and in situ hybridization, that TIMP-1 and TIMP-2 are expressed by human decidual cells and trophoblast cells [15, 22–24]. Using an ELISA method, we detected much higher levels of TIMP-1 in Week 11 trophoblast cells as compared with cells at Weeks 6 and 7 of pregnancy. This progressive increase in TIMP-1 production parallels that of MMP-9 expression. TIMP-1 inhibits all MMPs in an activated form; however, it preferentially binds to both latent and active MMP-9 [19]. Our work suggested that the coordinate expression for MMPs and TIMPs might be important for the degradation of matrix proteins in a controlled fashion. In our experiments, the amounts of TIMP-2 in the culture medium were below the limit of detection of the ELISA. It seems possible that TIMP-2 detected by others using immunohistochemical staining was produced by other cell types in the placenta [24].
MMP-14 has been reported to be involved in cleaving pro-MMP-2 into the active form, and may function as an important plasma membrane-associated regulator for the activity of MMP-2 [32, 37]. Our Northern blot analysis has indicated that the expression of MMP-14 appears relatively stable throughout Week 6 to Week 11 human cytotrophoblast cells. Hurskainen et al. [38] also found that trophoblast tissue in the early human placenta is the main producer of MMP-14 mRNA and protein. Recently, MMP-14 was reported to be able to digest interstitial collagen and other extracellular matrix proteins [39]. The activation of MMP-2 by membrane-type-MMP is absolutely dependent upon the presence of TIMP-2. Lack of TIMP-2 secretion by cultured cytotrophoblast cells, as shown in this study, would therefore either imply an additional control mechanism to maintain MMP-2 in its inactive form, or indicate that the MT-MMP is acting directly to degrade substrates at the leading edge of invasion.
It has been reported that cultured first-trimester human cytotrophoblast cells could produce fibronectin [26, 27]. Collagen IV, fibronectin, laminin, and vitronectin have been detected around the trophoblast cells in human term placenta by immunohistochemical and immunoelectromicroscopy [40, 41]. In the present studies, we found that cultured first-trimester cytotrophoblast cells could also secrete abundant laminin and vitronectin in addition to fibronectin. The data mentioned above indicate that the human trophoblast cells were surrounded by self-secreted matrix proteins. In another experiment we found that human cytotrophoblast cells exhibited differing adhesion ability to the various matrix proteins and showed variation in morphology and migrating behavior after the cells were cultured on these matrix proteins for 48 h (unpublished results). Thus, whether these matrix proteins can affect the expression of MMPs and TIMPs in trophoblast cells needs to be investigated further.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by the ninety-fifth National Pan-Deng project, the Knowledge Innovation Program from the Chinese Academy of Sciences, the grant 39330130# from National Natural Science Foundation of China, and the grant RF 94025 #30 from the Rockefeller Foundation. 
2 Correspondence: Lin-zhi Zhuang, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 19 Zhongguancun Lu, Beijing, 100080, China. FAX: 86 10 62565689; zhuanglz{at}panda.ioz.ac.cn
Accepted: November 15, 1999.
Received: August 11, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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