Molecular Human Reproduction, Vol. 8, No. 7, 681-687,
July 2002
© 2002 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Prostaglandin F2
, cytokines and cyclic mechanical stretch augment matrix metalloproteinase-1 secretion from cultured human uterine cervical fibroblast cells
1 Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, 2 Research and Development Division, Nippon Organon K.K., 5-90 Tomobuchi-cho 1-chome, Miyakojima-ku, Osaka 534-0016 and 3 Department of Biochemistry, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan
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Human uterine cervical tissue is composed mainly of fibroblast cells and the extracellular matrix in which collagen types I and III predominate. It is hypothesized that these collagens are degraded by matrix metalloproteinases (MMPs) in the initial step of uterine cervical ripening during parturition. Among the MMPs, MMP-1, -8 and -13 have substrate selectivity for collagen types I and III. In the present study, we examined the regulation of MMP-1 secretion from the human uterine cervix. Immunohistochemistry detected strong staining of MMP-1, but not of MMP-8 or -13, in stromal cells of the pregnant uterine cervix. The MMP-1 expression in the pregnant uterine cervix was further confirmed by Western blot analysis and RT–PCR. To clarify the regulation of MMP-1 production, we subsequently investigated the effects of prostaglandins, inflammatory cytokines and cyclic mechanical stretch on the secretion of MMP-1 from cultured human uterine cervical fibroblast cells. Treatment with prostaglandin (PG)F2
(10-7 to 10-5 mol/l) or interleukin (IL)-1 (0.01–1.0 ng/ml) or stimulation with cyclic mechanical stretch increased MMP-1 secretion from cultured human uterine cervical fibroblast cells, with maximal increases of 3.4-, 4.5- and 1.9-fold respectively (24 h of treatment, P < 0.05 for all comparisons). These data suggest that MMP-1 may play a significant role in the degradation of extracellular collagen types I and III in the pregnant uterine cervix during the process of cervical ripening, in response to various stimulations such as PGF2, IL-1 and mechanical stretch.
cervical ripening/interleukin-1/matrix metalloproteinases/mechanical stretch/prostaglandin F2
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During normal pregnancy, the uterine cervical canal remains tightly closed until the onset of labour, although the volume of the intrauterine cavity increases gradually to accommodate the fetal enlargement. After the onset of active labour with vigorous contraction of the uterine corpus, progressive ripening and dilatation of the cervix occur, followed by the expulsion of the conceptus. Thus, the ripening of the uterine cervix is an essential process in normal vaginal delivery.
The human uterine cervix is composed mainly of fibroblast cells and fibrous connective tissues in which collagen and glycosaminoglycans predominate (Uldbjerg et al., 1983a
). Ripening of the uterine cervix is an active biochemical process, consisting of complex reactions (such as degradation and/or remodelling of extracellular matrix proteins and glycoproteins, disruption of tightly aligned collagen fibrils, increase in hydration caused by hyaluronan, etc.) in which degradation of collagens is a critical process (Junqueira et al., 1980; Uldbjerg et al., 1983b; El Maradny et al., 1997). Accordingly, collagenases, recently termed matrix metalloproteinases (MMPs), are key enzymes in cervical ripening.
MMPs are a family of at least 26 different types of zinc-dependent enzymes which are also capable of degrading other extracellular matrix components such as proteoglycans, fibronectins and laminin present in the interstitial matrix and basement membrane [Uldbjerg et al., 1983b; Konttinen et al., 1999; Leppert et al., 2001; Merops protease database (http://www.merops.ac.uk)]. Each MMP has a particular substrate specificity (Hulboy 2et al., 1997). Since the major extracellular matrix proteins in the human uterine cervix are collagen types I and III (Kleissl et al., 1978; Minamoto et al., 1987), it is important to study the expression and regulation of MMPs such as MMP-1, -8 and -13, which have substrate selectivity for collagen types I and III. Although the expression of MMP-1, -2, -3, -9 and -14 has been reported in cultured uterine cervical fibroblast (CxF) cells and/or smooth muscle cells by a number of investigators (Sato et al., 1996; Imada et al., 1997; Ito et al., 1998; Watari et al., 1999; Sugano et al., 2000), there have been few reports on the regulation of expression of MMPs in human uterine cervical tissue (Osmers et al., 1995a,b; Ledingham et al., 1999). The contribution of MMP-8 production from infiltrated neutrophil to the complicated process of cervical ripening has been demonstrated (Osmers et al., 1995a,b; Winkler et al., 1999a,b). However, the tissue distribution of MMP-1, -8 and -13 in the human uterine cervix remains to be elucidated.
Inflammatory cytokines have been reported to activate cervical ripening (Garcia-Velasco and Arici, 1999). Watari et al. reported that inflammatory cytokines increase the expression of MMP-1, -3 and -9 mRNA in human uterine cervical smooth muscle cells (Watari et al., 1999). Since fibroblast cells are the major component of the human uterine cervix (Rorie and Newton, 1967), it is important to investigate the effects of inflammatory cytokines on MMP production in human uterine CxF cells.
Prostaglandin (PG)F2 and PGE2 have also been reported to be involved in cervical ripening in vivo (Platz-Christensen et al., 1997; Mawire et al., 1999), and have recently been used clinically for the purpose of inducing cervical ripening. Both PGF2 and PGE2 modulate MMP expression in T cells (Zeng et al., 1996) and ciliary smooth muscle cells (Lindsey et al., 1996). However, there has been no report on the effects of PGs, including PGF2 and PGE2, on the expression of MMPs in the uterine CxF cells.
Uterine cervical ripening proceeds rapidly during the active phase of labour, when the fetal presenting part descends and the uterine cervix is distended cyclically and vigorously (Cunningham et al., 1997). However, to our knowledge, it remains to be elucidated whether mechanical distension directly affects the production of MMPs in uterine CxF cells.
In the present study, to clarify the biological significance of MMP-1 in the ripening process of the human uterine cervix, we investigated the tissue localization of MMP-1 in the human uterine cervix, and the effects of various species of PGs, inflammatory cytokines and cyclic mechanical stretch on the secretion of MMP-1 from cultured human uterine CxF cells.
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Reagents
All reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA) and were of analytical grade unless otherwise indicated.
Collection of cervical tissues
Uterine cervical tissues were obtained with written informed consent from premenopausal non-pregnant (n = 5) and pregnant women (8–39 weeks gestation, n = 8) at hysterectomy for gynaecological reasons such as uterine cervical cancer (two pregnant women at 37 and 38 weeks gestation), ovarian cancer (one non-pregnant and one pregnant woman at 10 weeks gestation), uterine myoma (three non-pregnant and four pregnant women at 8, 9, 10 and 39 weeks gestation) and/or for obstetrical reasons such as atonic bleeding (one pregnant woman at 39 weeks gestation). All of the tissues from first trimester pregnant women (8, 9, 10 and 10 weeks gestation) were used to establish cultured human uterine CxF cells as well as to carry out immunostaining for MMP-1, -8 and -13. The remaining tissues were snap-frozen by liquid nitrogen and stored at –80°C until assayed.
Western blot analysis of MMP-1 was performed using all of the tissues collected with the exception of one tissue sample from a pregnant woman at 10 weeks gestation. The CxF cells and cervical tissues from a non-pregnant woman and pregnant women at 8 and 39 weeks gestation (before labour) were used to measure MMP-1 gene expression by RT–PCR analysis. The CxF cells and cervical tissues from a pregnant woman at 39 weeks gestation (before labour) were utilized to measure PG receptor gene expression by RT–PCR analysis. The study was approved by the Ethics Committee on Human Research at Kyoto University Graduate School of Medicine (No. 90).
Preparation of cultured human uterine CxF cells
The CxF cells were prepared by the explant method as previously reported (Yoshida et al., 2001). Cervical stromal tissues were obtained from first trimester pregnant women (n = 4), rinsed several times in 10 mmol/l phosphate-buffered saline (PBS) and minced into 
2 mm pieces which were then placed on 6 cm collagen-coated culture dishes and gently covered with a thin microcover glass. The initially out-grown cells were regarded as passage 1 cells. The culture was conducted using minimum essential medium (Gibco Brl, Rockville, MD, USA) containing 10% fetal bovine serum, at 37°C in 95% air and 5% CO2 under humidified conditions. The cells from the tissues were grown to passage 6 and were used at that stage as cultured human uterine CxF cells (Yoshida et al., 2001). An immunofluorescence study showed 99% positive staining for vimentin in CxF cells at passage 6, and <1% positive staining of both cytokeratin and -smooth muscle actin, indicating high purity of the CxF cells (Yoshida et al., 2001). We used these cells in the further experiments in this study.
Immunohistochemical detection of MMP-1, -8 and -13
Specimens were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek Inc., Torrance, CA, USA) and stored at –80°C. Sections of 6 µm were incubated for 1 h at room temperature with anti-MMP-1 (5 µg/ml), anti-MMP-8 (10 µg/ml), anti-MMP-13 (2.5 µg/ml) monoclonal antibodies (Fuji Chemical Industries Ltd, Takaoka, Japan) or normal mouse IgG (10 mg/ml; Dako Co., Carpinteria, CA, USA) as a negative control. Staining was detected using the avidin–biotin–peroxidase method kit for monoclonal antibodies (Elite ABC; Vector Laboratories, Burlingame, CA, USA) with 3,3'-diaminobenzidine as previously described (Itoh et al., 1998).
Western blot analysis of MMP-1 protein expression
The cultured human uterine CxF cells and cervical tissues were homogenized in solubilizing buffer [150 mmol/l NaCl, 50 mmol/l Tris–HCl, 10 mmol/l EDTA (pH = 7.4), 0.1% Tween 20, 0.1% ß-mercaptoethanol, 0.1 mmol/l phenylmethylsulphonylfluoride, 5 µg/ml leupeptine, 5 µg/ml aprotinin] (Itoh et al., 1998). Protein was extracted from culture medium using 10% trichloroacetic acid (final concentration) and applied to polyacrylamide gels (0.48 µg extracted protein/lane). Protein extracts from uterine cervices (20 µg/lane) were fractionated by 12.5% sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (20 µg/lane, 100 V, 2 h) with positive controls (from human placenta) and Rainbow molecular weight markers (BioRad Laboratories, Cambridge, MA, USA), before transfer to an Immobilon P membrane (30 V, 18 h). Immunodetection was achieved by using a monoclonal antibody raised against human MMP-1 (10 µg/ml; 16 h, 4°C; Fuji Chemical Industries) followed by donkey anti-mouse IgG/horseradish peroxidase conjugate (1:3000; 1 h, room temperature; Amersham, Arlington Heights, IL, USA) and enhanced chemiluminescence reagent detection with exposure to hyper-film (Amersham) (Itoh et al., 1998). The antibody recognizes both pro-MMP-1 and active mature form MMP-1.
RT–PCR analysis of MMP-1, PGF2 receptor (FP receptor) and PGE2 receptor (EP3 receptor)
To examine the gene expression of MMP-1, FP receptor, EP3 receptor and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as a control, RT–PCR was carried out. Total RNA was extracted from CxF cells as previously described (Masuzaki et al., 1997). After RT of 2 µg of total RNA from human pregnant and non-pregnant uterine cervices and cultured human uterine CxF cells using oligo(dT) primer (Promega, Madison, WI, USA) and SuperscriptTM II (Gibco), the resulting single-stranded cDNA was subjected to PCR. Forward and reverse primers and programs used for amplifying part of the cDNA of human MMP-1 (Hulboy et al., 1997), human FP receptor (Abramovitz et al., 1994) and human EP3 receptor (Adam et al., 1994) were: MMP-1 forward, 5'-CTGAAGGTGATGAAGCAGCC-3', MMP-1 reverse, 5'-AGTCCAAGAGAATGGCCGAG-3'; FP receptor forward, 5'-AAGACATCAAAGACTGGGAA-3', FP receptor reverse, 5'-GTGCTTGCTGATTTCTCTG-3'; EP3 receptor forward, 5'-TTTTCGGGCTCTCCTCGTTGTT-3', EP3 receptor reverse, 5'-TATTAAGAAGAAGTTGCATT-3'. Forward and reverse primers for human G3PDH coding region were purchased from Clontech (Palo Alto, CA, USA). The programmes used for amplification of MMP-1, FP, EP3 and G3PDH cDNA were: MMP-1, 32 cycles of 94°C for 30 s, 55°C for 60 s and 72°C for 60 s; FP receptor, 30 cycles of 94°C for 30 s, 58°C for 60 s and 72°C for 60 s; EP3 receptor, 35 cycles of 94°C for 30 s, 52°C for 60 s and 72°C for 60 s; and G3PDH, 30 cycles of 94°C for 30 s, 55°C for 60 s and 72°C for 60 s. The final products were extended to full length by incubation at 72°C for 4 min. Primers for EP3 receptor were designed for amplification of cDNA common to at least eight EP3 receptor isoforms (Kotani et al., 1997). The expected final products from human MMP-1, human FP receptor, human EP3 receptor and human G3PDH cDNA were 428, 505, 575 and 452 bp respectively.
Northern blot analysis of MMP-1 mRNA expression
A total of 10 µg of total RNA was separated in each lane on a 1.5% agarose gel containing 2% formaldehyde and transferred to a Hybond-N+ nylon membrane (Amersham, Buckinghamshire, UK). The human proMMP-1 cDNA probes, a kind gift from Professor Hideaki Nagase, Kennedy Institute of Rheumatology, Imperial College, UK, were hybridized sequentially to the membranes using Rapid-Hyb Buffer (Amersham, UK), followed by final washes in 0.1xstandard saline citrate and SDS for 5 min at 65°C. Densitometric analysis was performed using a BAS 2000 II Bioimage Analyzer (Fujix, Tokyo, Japan).
Experimental protocol for cell culture and stretch experiment
When CxF cells at passage 6 reached confluence, the medium was replaced with fresh minimum essential medium without serum, and the cells were then incubated for 24 h with the reagents indicated in the text in 6 cm collagen-coated plates or in 6-well plates with flexible bottoms (Bio Flex collagen-I; Flexcell International Co., McKeesport, PA, USA).
The reagents used were 0.01–1.0 ng/ml interleukin (IL)-1 (a kind donation from Dai-ichi Pharmaceutical Co. Ltd, Tokyo, Japan), 1.0–10.0 ng/ml tumour necrosis factor (TNF)-, 10-8 to 10-5 mol/l PGF2, 10-8 to 10-5 mol/l PGE2, 10-8 to 10-5 mol/l sulprostone (a specific ligand for the PGE2 receptor isoforms EP1 and EP3) (Suda et al., 2000), 10-8 to 10-5 mol/l butaprost (a specific ligand for the PGE2 receptor isoform EP2) (Suda et al., 2000) and 1–5x10-5 mol/l curcumin (a specific inhibitor of the AP-1 site) (Huang et al., 1991). PGF2, PGE2, sulprostone and butaprost were kindly donated by Ono Pharmaceutical Co. Ltd (Osaka, Japan).
The stimulation by cyclic mechanical stretch was applied to the cells seeded on 6-well plates with flexible bottoms by cyclic vacuum extraction from the well bottom (–13 kpa, 15% elongation, repetition of 45 s stretch and 15 s release) using a Flexer Cell 3000 System (Flexcell) with or without 1 h pretreatment with 1, 2 and 5x10-5 mol/l curcumin (Huang et al., 1991; Wung et al., 1997; Park et al., 1999).
After 24 h of incubation, the medium and cells were collected and kept at –20°C and –80°C respectively. The total MMP-1 level in the medium was measured by ELISA (Nippon Organon K.K., Osaka, Japan), which recognizes both pro-MMP-1 and active mature form MMP-1. The PGE2 and PGF2 concentrations in the culture medium were measured by ELISA (Cayman Chem. Co., Ann. Arbor, MI, USA). The expression of MMP-1, FP receptor and EP3 receptor mRNAs in the cells was assessed by RT–PCR or Northern blot analysis.
After 24 h incubation with various stimulating reagents, the number of CxF cells was 2.5 to 2.8x105 cells per 12-well plate, which was similar to that in the vehicle control.
Statistical analysis
Values were expressed as the means ± SEM. The statistical significance was assessed by analysis of variance followed by Fisher's protected least significant difference test. P < 0.05 was regarded as significant.
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Expression of MMP-1, -8 and -13 in non-pregnant and pregnant uterine cervical tissues and cultured CxF cells
Strongly positive MMP-1 staining (brown) was detected in both glandular cells and stromal cells (Figure 1A
), indicating that MMP-1 was expressed widely in cervical tissues. On the other hand, positive staining of MMP-8 (Figure 1B) and MMP-13 (Figure 1C) was observed mainly in glandular cells. A negative control using normal mouse IgG showed greatly reduced staining (Figure 1D).
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Western blot analysis with anti-MMP-1 antibody detected major 52 kDa and minor 41 kDa bands, corresponding to pro-MMP-1 and the mature active form of MMP-1 respectively in the placenta (as a positive control, Figure 2
, lane 1), non-pregnant (Figure 2, lanes 2–5) and pregnant uterine cervices (Figure 2, lanes 6–12). On the other hand, only the 52 kDa band was detected in CxF cells (Figure 2, lane 13). RT–PCR analysis revealed MMP-1 mRNA expression in the placenta (as a positive control, Figure 3A, lane 1), non-pregnant uterine cervix (Figure 3A, lane 2), and first and third trimester pregnant uterine cervices (Figure 3A, lanes 3 and 4 respectively). MMP-1 mRNA expression on CxF cells was more prominent compared with that of non-pregnant and pregnant uterine cervices, suggesting the possibility that MMP-1 is predominantly expressed in fibroblast cells in the cervix. Immunocytochemical analysis also demonstrated protein expression of MMP-1, but not of MMP-8 or MMP-13, in CxF cells (data not shown).
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Effects of PGE2, PGF2
and IL-1 on MMP-1 secretion from cultured CxF cellsPrior to this experiment, we confirmed the expression of FP and EP3 receptor mRNAs in pregnant uterine cervical tissue as well as in CxF cells by RT–PCR (Figure 3B
). Treatment with 10-6 and 10-5 mol/l PGF2 dose-dependently increased MMP-1 secretion from CxF cells up to 9.4 ± 1.3 (mean ± SEM of quadruplicate wells) and 24.2 ± 4.8 ng/ml respectively, levels which were significantly higher than that in the vehicle control, 6.9 ± 0.5 ng/ml (P < 0.05; Figure 4A). In contrast, MMP-1 concentrations in the culture medium of CxF cells after 24 h incubation with the treatment of 10-7, 10-6 and 10-5 mol/l PGE2 were 8.1 ± 0.2, 7.9 ± 0.8 and 8.6 ± 1.0 ng/ml respectively, similar to the levels of the vehicle control, 8.1 ± 0.2 ng/ml. The PGE2 receptor consists of four isoforms, EP1, EP2, EP3 and EP4. Neither sulprostone, a selective agonist for EP1 and EP3, nor butaprost, a selective agonist for EP2, had a significant effect on MMP-1 secretion from CxF cells (data not shown). Treatment for 24 h with 0.1 or 1.0 ng/ml IL-1 dose-dependently increased the MMP-1 level in the culture medium of CxF cells, up to 12.6 ± 1.5 and 45.2 ± 2.4 ng/ml respectively, levels which were significantly higher than that of the vehicle control (8.1 ± 0.2 ng/ml; P < 0.05 for both comparisons; Figure 4B).
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Although it is hypothesized that in human parturition the amnion and decidua mainly secrete PGE2 and PGF2
respectively (Okazaki et al., 1981), CxF cells also secreted considerable amounts of both PGE2 and PGF2 into the culture medium. PGF2 concentrations in the culture medium after 24 h incubation with 1.0 and 10.0 ng/ml IL-1 were 92 ± 9 and 308 ± 37 pg/ml respectively, significantly higher than the levels of the vehicle control, 19 ± 2 pg/ml (P < 0.05 and n = 4 for all). In a similar manner, PGE2 concentrations in the culture medium after 24 h incubation with 1.0 and 10.0 ng/ml IL-1 were 440 ± 122 and 1325 ± 173 pg/ml respectively, significantly higher than the levels of the vehicle control, 139 ± 2 pg/ml (P < 0.05 and n = 4 for all). Treatment with indomethacin, an inhibitor of cyclo-oxygenase, partially but significantly attenuated the IL-1-augmented MMP-1 secretion from CxF cells (Figure 4C). However, indomethacin treatment did not affect the PGF2-augmented MMP-1 secretion from CxF cells (Figure 4C).
The MMP-1 concentration in the culture medium after 24 h of incubation with 1.0, 5.0 and 10.0 ng/ml TNF- was increased dose-dependently to 12.7 ± 0.6, 14.1 ± 0.9 and 18.6 ± 2.3 ng/ml respectively. The MMP-1 level after treatment with 5.0 and 10.0 ng/ml TNF- was significantly higher than the level in the vehicle control (12.0 ± 0.3 ng/ml; P < 0.05 for all comparisons).
Effect of cyclic mechanical stretch on MMP-1 secretion from cultured CxF cells
MMP-1 levels in the medium of CxF cells after 3, 6 and 12 h incubation under stimulation by cyclic mechanical stretch were 3.4 ± 0.1, 4.2 ± 0.3 and 7.0 ± 0.5 ng/ml, which were similar to those without stimulation, 3.5 ± 0.2, 4.3 ± 0.1 and 4.7 ± 0.3 ng/ml respectively (n = 4 for all). On the other hand, MMP-1 level in the medium of CxF cells after 24 h of incubation under stimulation by cyclic mechanical stretch (14.6 ± 1.1 ng/ml, n = 4) was significantly higher than that of the vehicle control, 7.7 ± 0.2 ng/ml (Figure 5, P < 0.05). The MMP-1 mRNA expression in CxF cells with the stimulation of cyclic mechanical stretch for 6 h was 11.5 arbitrary units (AU), more than double compared with that without stimulation, 5.1 AU (Figure 6). However, such stretch-induced augmentation of MMP-1 mRNA expression was not observed after stimulation for 3 h as well as 12 h (Figure 6).
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The MMP-1 levels in the medium of CxF cells with 24 h of stimulation by cyclic mechanical stretch after pretreatment with 10, 20 and 50 µmol/l curcumin, an inhibitor of the AP-1 site (Huang et al., 1991
), were 10.1 ± 0.2, 7.6 ± 0.1 and 7.0 ± 0.2 ng/ml, levels which were significantly and dose-dependently lower than that without the pretreatment, 14.6 ± 1.1 ng/ml (Figure 5, P < 0.05 for all comparisons).
By contrast, PGF2 and PGE2 concentrations in the culture medium after the 24 h stimulation of cyclic stretch were 23 ± 5 and 186 ± 43 pg/ml respectively, similar to the levels of the corresponding vehicle control, 18 ± 4 and 126 ± 30 pg/ml.
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Discussion |
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Contraction of the uterine corpus and dilatation of the cervical canal are essential events in the process of normal vaginal delivery. Degradation of extracellular matrix proteins, such as collagens and glycosaminoglycans, is one of the key processes in the series of complicated biochemical reactions of cervical ripening. The uterine cervical collagens consist mainly of collagen types I and III (Kleissl et al., 1978
; Minamoto et al., 1987), which are specifically hydrolysed by MMP-1, -8 and -13 (Hulboy et al., 1997). Thus, it is plausible that MMP-1, -8 and -13 may play important roles in cervical ripening.
The present study demonstrated that MMP-1, -8 and -13 are all expressed in cervical glandular cells, but that only MMP-1 is widely expressed in cervical stromal cells in specimens from first trimester pregnant women, although the numbers of tissues were limited. Since we did not study MMP-1, -8 and -13 immunostaining in specimens from dilated uterine cervix during labour due to ethical problems in sampling, the present study cannot exclude the possible production of MMP-8 and/or -13 from cervical stromal cells in the process of cervical ripening. Moreover, Winkler et al. and Osmers et al. have demonstrated that MMP-8 production from infiltrated neutrophil contributes to degradation of uterine cervical collagen in parturition (Osmers et al., 1995a,b; Winkler et al., 1999a,b). Nevertheless, in the present study, MMP-1 was widely distributed in uterine cervical stromal cells.
Fibroblast cells are major components of uterine cervical stromal cells, and are initially as well as directly exposed to various kinds of stimulation, such as inflammatory cytokines, prostaglandins and cyclic stretch. Therefore, we examined the production of MMP-1. Western blot analysis revealed the presence of both 52 kDa proMMP-1 and 41 kDa mature active form MMP-1 protein in non-pregnant and pregnant uterine cervical tissues. Moreover, Western blot analysis and ELISA detected MMP-1 protein in the culture medium from CxF cells. The expression of MMP-1 mRNA was also detected in pregnant cervical tissues as well as CxF cells, although the number of the specimens used was rather small. Taken together, these data suggest that MMP-1 may play a key role in the degradation of extracellular collagen in the uterine cervical stroma during the process of cervical ripening at delivery.
The present study demonstrated that PGF2 significantly augmented MMP-1 production by CxF cells. FP receptor mRNA was detected both in pregnant human uterine cervical tissue and in CxF cells. Therefore, PGF2 may contribute to the degradation of collagen in the process of cervical ripening via augmentation of MMP-1 production. To our knowledge, the present study is the first report of prostanoid-mediated MMP-1 secretion from human CxF cells, although there have been several reports of MMP-1 secretion from other tissues (Lindsey et al., 1996; Zeng et al., 1996). Moreover, CxF cells themselves also secrete a considerable amount of PGF2 into the culture medium, especially after IL-1 stimulation, although the major source of PGF2 production during labour is thought to be decidual cells (Okazaki et al., 1981). The PGF2 concentrations in the culture medium after IL-1 treatment were close to the known FP receptor KD value of 1.0 nmol/l (Abramovitz et al., 1994) and could potentially exert a biological effect, although the levels were much less than those needed to directly affect MMP-1 production. Thus, PGF2 produced locally in the uterine cervix, in addition to that produced in decidual tissue, may contribute to the degradation of collagen in the process of cervical ripening via activation of MMP-1 production, in an autocrine/paracrine manner. These results are relevant to the previous clinical reports of the possible effects of PGF2 on cervical ripening (Platz-Christensen et al., 1997; Mawire et al., 1999).
By contrast, PGE2 did not affect MMP-1 production from CxF cells, which is not consistent with the findings of a clinical study on the effect of PGE2 (Platz-Christensen et al., 1997). One possibility might be that CxF cells have lost PGE2 receptors other than EP3 or lost the related signal transduction system during the course of culturing. Another possibility is that PGE2 contributes to cervical ripening in vivo through a mechanism other than the direct augmentation of MMP-1 production from fibroblast cells, such as leukocyte infiltration and MMP-8 secretion from neutrophils. Further in-vitro as well as in-vivo investigations are necessary to clarify the mechanisms involved in the stimulation of cervical ripening by PGE2.
Inflammatory cytokines have been reported to play key roles in the complicated process of cervical ripening (Garcia-Velasco et al., 1999). Watari et al. reported that IL-1 and TNF- augment MMP-1 and MMP-3 mRNA expression in human cervical smooth muscle cells (Watari et al., 1999). However, Rorie et al. reported that smooth muscle cells constituted only 6 and 18% of the lower and middle parts of the human uterine cervix respectively (Rorie and Newton, 1967). In a preliminary immunohistochemical study, we found that a large proportion of uterine cervical stromal cells were fibroblast cells (M.Yoshida, H.Itoh and N.Sagawa, unpublished findings). Thus, it is also important to assess the effect of inflammatory cytokines on MMP-1 production by human CxF cells. The present study revealed that treatment with IL-1 and TNF- increased MMP-1 secretion from cultured human CxF cells, although the effect of IL-1 was more marked than that of TNF-.
Inflammatory cytokines have been reported to initiate cervical ripening in parturition (Garcia-Velasco et al., 1999). Therefore, these data also suggest the physiological importance of MMP-1 in the process of cervical ripening, although the reaction can relate to the mechanical dilation of the non-pregnant and pregnant uterine cervical canal, but not necessarily to that in labour. Moreover, we found that IL-1 treatment also increased PGF2 secretion from CxF cells and that treatment with PGF2 enhanced MMP-1 production from CxF cells. Most PGs, including PGE2 and PGF2, are synthesized from PGH2. Indomethacin inhibits the enzyme activity of both cyclo-oxygenase-1 and -2, which catalyse the conversion of arachidonic acid to PGH2, and thus reduces the production of PGE2 and PGF2 (Masferrer et al., 1999). Treatment with indomethacin partially, but dose-dependently, blocked the IL-1-induced augmentation of MMP-1 secretion from CxF cells. By contrast, indomethacin treatment did not alter PGF2-augmented MMP-1 production from CxF cells. Taken together, these findings indicate that IL-1 may stimulate MMP-1 production from CxF cells at least partly via augmentation of PGF2 secretion.
In parturition, uterine cervical ripening proceeds dramatically in the active phase of labour in accordance with the descent of the fetal presenting part, when the uterine cervix is cyclically distended by myometrial contraction (Cunningham et al., 2001). Therefore, in the present study, we investigated whether cyclic mechanical stretch augments MMP-1 production in CxF cells. Cyclic mechanical stretch significantly augmented MMP-1 secretion as well as MMP-1 mRNA expression in cultured CxF cells. The AP-1 site was reported to mediate stretch-associated signal transduction in several types of cell (Wung et al., 1997; Park et al., 1999). In the present study, pretreatment with curcumin, an inhibitor of the AP-1 site (Huang et al., 1991), suppressed the stretch-induced increase of MMP-1 secretion from CxF cells. Since the AP-1 cascade is related to various types of immediate early genes (Du et al., 1995), it is possible that cyclic mechanical stretch might cause various changes in CxF cells. Indeed, in a pilot study, we found that IL-8 secretion from CxF cells was significantly enhanced by cyclic mechanical stretch (M.Yoshida, H.Itoh, N.Sagawa, unpublished data). Nevertheless, it is interesting to speculate that cyclic distension of the uterine cervix by the fetal presenting part during labour may augment MMP-1 production in CxF cells, thereby contributing to the degradation of collagen in the process of cervical ripening.
On the other hand, it has been reported that nitric oxide accelerates uterine cervical ripening at parturition (Chwalisz and Garfield, 1998; Norman et al., 1998). Previously, we reported that nitric oxide donors augmented MMP-1 production from CxF cells (Yoshida et al., 2001). Thus, all of these data together suggest that MMP-1 plays an important role in the degradation of cervical collagen in the process of cervical ripening.
In summary, the present study demonstrated that MMP-1 is produced by human CxF cells, and that MMP-1 secretion from these cells may be regulated at least partly by PGF2, IL-1 and cyclic mechanical stretch. Based on these findings, we suggest that MMP-1 may play an important role in the degradation of extracellular collagens during the course of human cervical ripening.
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This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports, Japan (No. 12877262, 13470352, 13557139, 13671707), a Grant from the Ministry of Health and Welfare, Japan, and grants from the Smoking Research Foundation and the Kanzawa Medical Research Foundation, Japan. The authors thank Professor Hideaki Nagase, Kennedy Institute of Rheumatology Imperial College, UK, for the generous donation of cDNA probe of proMMP-1. The authors also thank Professor Kazuwa Nakao, Dr Masaki Harada and Dr Yoshihiko Saito, Department of Clinical Science and Medicine, Kyoto University Graduate School of Medicine, for co-operation in conducting the experiment with the Flexer Cell 3000 System. The authors also acknowledge Ms Akiko Kishimoto and Ms Akiko Abe for secretarial and technical assistance with this work.
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4 To whom correspondence should be addressed. E-mail: fetus{at}kuhp.kyoto-u.ac.jp
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Submitted on April 27, 2001; resubmitted on December 27, 2001; accepted on April 18, 2002.
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