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Biology of Reproduction 62, 540-546 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Platelet-Activating Factor Induces an Imbalance Between MatrixMetalloproteinase-1 and Tissue Inhibitor of Metalloproteinases-1 Expressionin Human Uterine Cervical Fibroblasts

Terumasa Sugano1,a, Kaei Nasua, Hisashi Naraharaa, Yasushi Kawanoa, Yoshihiro Nishidaa, and Isao Miyakawaa

a Department of Obstetrics and Gynecology, Oita Medical University, Oita 879-5593, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Platelet-activating factor (PAF) is involved in such reproductive processes as parturition. We investigated the effect of PAF on the expression of matrix metalloproteinase-1 (MMP-1) and that of tissue inhibitor of metalloproteinases-1 (TIMP-1) in human uterine cervical fibroblasts. Uterine cervical tissue was obtained from patients who underwent cesarean section at term. Collagenase-dispersed fibroblasts were cultured and used in the experiments. PAF receptor was identified in the uterine cervical fibroblasts by use of reverse transcription-polymerase chain reaction and Southern blot analysis. Northern blot analysis showed that PAF increased the expression of MMP-1 mRNA in a time-dependent manner, whereas expression of TIMP-1 mRNA was not affected by PAF. Concentration of MMP-1 protein in the PAF-treated culture media significantly exceeded that in control cultures. The PAF-induced production of MMP-1 protein was abolished by treatment with WEB 2170, a specific PAF receptor antagonist. Results suggest that PAF may accelerate collagenolysis in the human uterine cervix by inducing an imbalance in the activity between MMP-1 and TIMP-1, thus contributing to the cervical ripening during parturition.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Uterine cervical ripening during parturition involves changes in the connective tissue of the uterine cervix. Major changes are found in the metabolism of such extracellular matrix (ECM) as collagen, proteoglycans, and glycosaminoglycans [1–4], leading to a reduction in the number of collagen fibers [5]. The concentration of collagenase—interstitial collagen-degrading enzyme—shows a marked increase at term, especially at labor [6]. Matrix metalloproteinases (MMPs) are zinc-dependent enzymes that degrade most, if not all, ECM proteins [7]. This breakdown of ECM is closely correlated with the balance between MMPs and their endogenous inhibitors, which are the tissue inhibitors of metalloproteinases (TIMPs) [8, 9]. In particular, MMP-1 degrades such interstitial collagens as type I collagen, which predominates in the uterine cervix [5]. MMP-1 is produced by leukocytes, fibroblasts, and many other cell types. The cells that secrete proMMPs also produce TIMPs [10]. TIMP-1 is a glycoprotein that binds to and inactivates the MMPs, including MMP-1 [7].

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a potent lipid mediator that is involved in various inflammatory diseases and immune responses [11, 12]. It has recently been shown that PAF is involved in human reproduction, including ovulation, implantation, maturation of the fetal lung, and parturition [13]. PAF concentrations are increased in the amniotic fluid collected from women in labor [14]. PAF stimulates the production of prostaglandin E2 in fetal membranes [15, 16] and causes uterine myometrial contraction [17, 18]. We previously demonstrated the secretion of a PAF-inactivating enzyme, PAF-acetylhydrolase, by human decidual macrophages, and suggested the presence of an autocrine or paracrine regulation of PAF concentration in the decidual tissue [19].

Uterine cervical ripening is considered to resemble inflammation, in which inflammatory cells infiltrate the tissue and produce various bioactive molecules. Such cytokines as interleukin (IL)-1, IL-8, and tumor necrosis factor-{alpha} (TNF-{alpha}) secreted by the inflammatory cells are reportedly involved in the ripening of the cervix of guinea pigs [20], rabbits [21–23], and humans [24–26]. PAF is produced by various cell types including macrophages, polymorphonuclear leukocytes, basophils, platelets, and endothelial cells [13, 27]. It is a potent neutrophil-chemotactic factor [12, 28–31]. However, the role of PAF in uterine cervical ripening is largely unknown.

We investigated whether PAF might modulate the expressions of MMP-1, TIMP-1, and type I collagen and induce MMP-1 production via an interaction with the PAF receptor in human uterine cervical fibroblasts.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Carbamyl-PAF (1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphorylcholine) was purchased from Calbiochem (La Jolla, CA). Recombinant human IL-1ß, TGF-ß1, and TNF-{alpha} were purchased from R&D Systems (Minneapolis, MN). 12-O-tetradecanoyl-phorbol 13-acetate (TPA) was purchased from Sigma Chemical Co. (St. Louis, MO). The following cDNAs were used for Northern hybridization: for MMP-1 mRNA, a 1.5-kilobase (kb) human cDNA; for TIMP-1 mRNA, a 1.5-kb human cDNA; and for pro{alpha}2(I) collagen mRNA, a 2.8-kb human cDNA obtained from the American Type Culture Collection (ATCC; Rockville, MD). The human PAF receptor cDNA for use in reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis was a gift from Dr. Takao Shimizu, The University of Tokyo, Tokyo, Japan. WEB 2170, a PAF receptor antagonist, was kindly provided by Boehringer Ingelheim (Biberach, Germany).

Tissue Preparation and Cell Culture

Human uterine cervical tissue was obtained from 7 patients (aged 25–36 yr) at term, before the onset of labor, when a cesarean section was performed at the Oita Medical University Hospital. Indications for cesarean delivery were cephalopelvic disproportion, history of prior cesarean sections, or fetal distress. The study had the approval of our Ethics Committee. Informed consent for the procedure was obtained from each patient. Tissue samples were immediately washed free of blood in sterile Ca2+- and Mg2+-free PBS supplemented with antibiotics. For the culture of cervical fibroblasts, samples were cut into small pieces and digested with 200 U/ml collagenase (Gibco-BRL, Gaithersburg, MD) in PBS with gentle stirring for 1 h at 37°C. The collagenase-dispersed cells were filtered through a sterile 80-µm-wire sieve and collected by low-speed centrifugation; they were then washed twice with Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical, Tokyo, Japan). Cells were then cultured with DMEM supplemented with 10% (v/v) fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS), 100 U/ml penicillin (Gibco-BRL), and 100 µg/ml streptomycin (Gibco-BRL) at 37°C in a humidified atmosphere of 5% CO2. The culture medium was replaced every 4 days. Cells were passed by standard methods of trypsinization and used in experiments at the 4th to the 6th passage. Cells were identified as fibroblasts by immunostaining. These cells stained positive with a monoclonal antibody against human ß subunit of prolyl 4-hydroxylase, which is specific for human fibroblast (Biomeda, Foster City, CA), and a monoclonal antibody against human vimentin (Dako, Glostrup, Denmark). The cells were negative for staining with a monoclonal antibody against human cytokeratin (Dako), CD68 (Dako), smooth muscle cell actin (Dako), factor VIII (Dako), and leukocyte common antigen (Dako). Therefore, no contamination by epithelial cells, macrophages, smooth muscle cells, or endothelial cells was present in our cultures. Next, the cells were passed by trypsinization and plated in culture dishes of 92-mm diameter (Nalge Nunc International, Naperville, IL) for the extraction of RNA and then plated in 6-well culture plates (Nalge Nunc) for measurement of MMP-1 protein in the medium. Each cell preparation from the various patients was cultured separately, and each experiment was repeated with a minimum of three preparations. There was no difference in the response to PAF among the various cell preparations.

HL-60 cells, a human promyelocytic leukemia cell line, were obtained from ATCC and used as a positive control for RT-PCR, because these cells express PAF receptor after phorbol ester-stimulated differentiation [32]. Cultures were grown in Iscove's modified Dulbecco's medium (Nissui Pharmaceutical) supplemented with 10% (v/v) FBS (JRH Biosciences).

RNA Isolation

Total RNA was isolated from confluent cultures of cervical fibroblasts with Trizol Reagent (Gibco-BRL) using a guanidinium thiocyanate phenol-chloroform method according to the manufacturer's instructions. The quantity of total RNA was determined by measuring absorbance at a wavelength of 260 nm.

RT-PCR and Southern Blot Analysis

Total RNA (3 µg) was reverse transcribed using a cDNA Synthesis Kit (Takara, Tokyo, Japan). In brief, a reaction volume of 20 µl was prepared; it contained 50 U of Rous-associated virus 2 reverse transcriptase; 2 µg of oligo(dT)18 primer; 1 mM each of dATP, dCTP, dGTP, and dTTP; and 20 U of RNase inhibitor in First Strand Synthesis Buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol). The mixture was incubated at 42°C for 60 min. To identify the PAF receptor mRNA, PCR amplification was carried out in a 50-µl reaction that contained 2 µl of each reverse transcription reaction; 5 U of TaKaRa Ex Taq DNA polymerase (Takara); 0.5 µM each of the sense and antisense primers; 0.2 mM each of dATP, dCTP, dGTP, and dTTP (Takara); and Ex Taq Buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, 2 mM MgCl2). The reaction was amplified for 30 cycles as follows: 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min 10 sec. The primer sets used to amplify a 1097-base pair (bp) fragment of the PAF receptor cDNA were designed by Roth et al. [33]. The primer sequences are 5'-GTG GGA TCC ATG GAG CCA CAT GAC TCC-3' (sense primer) and 5'-GTG GAA TCC ATC CCT TCT TCC CCC AGC-3' (antisense primer). The primer sets for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sense primer: 5'-TGA AGG TCG GAG TCA ACG GAT TTG GT-3' and antisense primer: 5'-CAT GTG GGC CAT GAG GTC CAC CAC-3'; Clontech Laboratories, Palo Alto, CA) were also used as internal controls. The PCR reaction mixture containing total RNA (100 ng) before RT was also amplified using primer pairs specific for both PAF receptor and GAPDH (minus RT).

PCR products were validated by Southern blotting. A volume of 10 µl of each PCR product was electrophoretically separated on a 1% agarose gel and transferred to a Hybond-N nylon filter (Amersham Pharmacia Biotech, Buckinghamshire, UK) by capillary action using 0.4 N NaOH and then crosslinked to the membrane by use of ultraviolet light. The membrane was hybridized with 32P-labeled random-primed probe prepared using a human PAF receptor cDNA and a Rediprime II random prime labeling system (Amersham Pharmacia Biotech). Hybridization was carried out for 16 h at 42°C in the hybridization solution Hybrisol I (50% formamide, 100 mM NaCl, 100 mM sodium citrate, 10% dextran sulfate, 1% SDS, sheared DNA, modified Denhardt's solution) (Oncor, Gaithersburg, MD). The membrane was washed twice in double-strength SSC (33.3 mM NaCl, 33.3 mM sodium citrate, pH 7.0)/0.1% (w/v) SDS for 20 min at room temperature, followed by washes of increasing stringency up to 0.2-strength SSC (3.33 mM NaCl, 3.33 mM sodium citrate, pH 7.0)/0.1% (w/v) SDS for 30 min at 65°C. Autoradiography was performed at -80°C using Hyperfilm-MP (Amersham Pharmacia Biotech).

Northern Blot Analysis

Confluent cultures of cervical fibroblasts were placed in DMEM supplemented with 0.1% (w/v) BSA (Sigma) and treated with PAF or such cytokines as IL-1ß, TNF-{alpha}, and TGF-ß1 to examine the expression of mRNAs for MMP-1, TIMP-1, and pro{alpha}2(I) chain. IL-1ß and TNF-{alpha} are known to induce the synthesis of some MMPs [7]. TGF-ß1 stimulates collagen production and inhibits the degradation of collagens [34]. Cultures were washed with PBS, and total RNA was isolated with Trizol Reagent. The denatured total RNA (10 µg in each well) was electrophoretically separated by size on a formaldehyde agarose-denaturing gel, transferred to a Hybond-N by capillary action using 10-strength SSC solution (166.5 mM NaCl, 166.5 mM sodium citrate, pH 7.0), and crosslinked to the membrane by use of ultraviolet light. The membrane was pre-hybridized with Hybrisol I for 2 h at 42°C. Hybridization was conducted for 16 h at 42°C in the same buffer with various 32P-labeled, random-primed cDNA probes that were prepared using Rediprime II (Amersham Pharmacia Biotech) as described above. After the hybridization, the membrane was washed twice in double-strength SSC/0.1% (w/v) SDS for 20 min at room temperature and twice in 0.2-strength SSC/0.1% (w/v) SDS for 30 min at 65°C. Autoradiography was performed at -80°C using Hyperfilm-MP. Autoradiographic bands were quantified by densitometric scanning with a Bio-Imaging Analyzer BAS 2000 (Fuji Film, Tokyo, Japan) and the public domain NIH Image program 1.61 developed at the National Institutes of Health (Bethesda, MD). The expression of mRNA for GAPDH was also examined as an internal control.

Enzyme-Linked Immunosorbent Assay (ELISA) for MMP-1

Approximately 1 x 106 cells per well were seeded on 6-well culture plates (Nalge Nunc) in 2 ml of DMEM with 10% FBS and cultured until fully confluent. After an overnight serum starvation, the supernatants were replaced with 1 ml of DMEM supplemented with 0.1% (w/v) BSA, either alone or containing PAF (10-8 M), and cultured for 24 h and 48 h. Immunoreactive MMP-1 in the culture media of cervical fibroblasts was quantified with an ELISA kit (Amersham Pharmacia Biotech) that detected total MMP-1, including proMMP-1, active MMP-1, and MMP-1/TIMPs complexes. In addition, to demonstrate whether PAF directly increases the MMP-1 production by uterine cervical fibroblasts through interaction with its corresponding receptor, the cells were preincubated for 1 h with 10 µM WEB 2170. The assay was performed in triplicate.

Statistical Analysis

ELISA data are presented as mean ± SD of triplicate samples, and were evaluated by Student's t-test and the Bonferroni/Dunn test with StatView 4.5 (Abacus Concepts, Berkeley, CA). Differences were considered statistically significant at a level of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PAF Receptor mRNA Expression in Human Uterine Cervical Fibroblasts

To identify the PAF receptor in human uterine cervical fibroblasts under basal conditions, we performed RT-PCR of PAF receptor mRNA expression. As described in Materials and Methods, total RNA was isolated from confluent cultures of cervical fibroblasts and was reverse transcribed. Total RNA of HL-60 cells differentiated by treatment with TPA (10 nM) for 24 h was used as a positive control for RT-PCR. EcoRI-cut cDNA for the human PAF receptor was used as a positive control for PCR. As shown in Figure 1A, the expression of PAF receptor mRNA was demonstrated in cervical fibroblasts (lane 3). The size of this amplified RT-PCR fragment was identical to that of TPA-treated HL-60 cells (lane 4) and to that of the human PAF receptor cDNA (lane 5). The PCR products were confirmed by Southern blot analysis (Fig. 1B). PCR using total RNA samples before RT (minus RT) demonstrated no genomic DNA contamination (Fig. 1A).



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  		FIG. 1. RT-PCR and Southern blot analysis of PAF receptor mRNA expression in human uterine cervical fibroblasts. A volume of 10 µl of each PCR product was electrophoretically separated on a 1% agarose gel and stained with ethidium bromide (A). Primer sets used to amplify the PAF receptor cDNA were expected to yield 1097-bp products. Lanes: 1, cervical fibroblasts (minus RT); 2, TPA-treated HL-60 cells (minus RT); 3, cervical fibroblasts; 4, TPA-treated HL-60 cells; 5, positive control for PCR using EcoRI-cut cDNA for the human PAF receptor; 6, 1780-bp EcoRI-cut cDNA for the human PAF receptor; M, molecular weight marker. PCR using total RNA samples before RT (minus RT) demonstrated no genomic DNA contamination. Human GAPDH amplification was equal. The RT-PCR is representative of three experiments. The PCR products loaded to lanes 3 and 4 were confirmed by Southern blot analysis using 32P-labeled cDNA probe for human PAF receptor (B)

Effects of PAF on MMP-1, TIMP-1, and Type I Procollagen mRNA Expression in Human Uterine Cervical Fibroblasts

To assess the effect of PAF on type I collagen turnover by uterine cervical fibroblasts, we examined the expression of mRNAs for MMP-1, TIMP-1, and pro{alpha}2(I). Total RNA was isolated from cervical fibroblasts after incubation for 12 h with PAF or with various cytokines. As shown in Figure 2, MMP-1 mRNA expression was increased by treatment with PAF to an extent similar to that following stimulation with IL-1ß and TNF-{alpha}, substances known to induce MMP-1 production. While TGF-ß1 did not increase the MMP-1 mRNA level, it did increase the pro{alpha}2(I) mRNA level. PAF had only a weak effect on increasing the expression of pro{alpha}2(I) mRNA. TIMP-1 mRNAs were constitutively expressed, despite treatment with these cytokines and PAF.



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  		FIG. 2. Effect of PAF vs. various cytokines on the expression of mRNA for MMP-1, TIMP-1, and type I procollagen in human uterine cervical fibroblasts. Confluent cells were incubated either alone or with IL-1ß (1 ng/ml), TGF-ß1 (1 ng/ml), TNF-{alpha} (10 ng/ml), or PAF (10-8 M) in serum-free DMEM containing 0.1% BSA for 12 h. Total RNA (10 µg/lane) was analyzed by Northern blot hybridization with the cDNA probes encoding human MMP-1, human TIMP-1, human pro{alpha}2(I) chain, or human GAPDH. Lanes: 1, control; 2, IL-1ß; 3, TGF-ß1; 4, TNF-{alpha}; 5, PAF. The Northern blot is representative of three experiments

Figure 3 shows the effect of differing concentrations of PAF on the expression of mRNAs for MMP-1, TIMP-1, and pro{alpha}2(I) in uterine cervical fibroblasts. The MMP-1 mRNA expression was increased markedly in response to 10-10 M of PAF compared with vehicle control, although the expression of TIMP-1 mRNA was not stimulated by PAF. Pro{alpha}2(I) mRNA was not induced except at 10-8 M of PAF.



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  		FIG. 3. Effect of differing concentrations of PAF on the expression of mRNA for MMP-1, TIMP-1, and type I procollagen in human uterine cervical fibroblasts. Confluent cells were incubated either alone or with different concentrations of PAF (10-10–10-8 M) in serum-free DMEM containing 0.1% BSA for 12 h. Total RNA (10 µg/lane) was analyzed by Northern blot hybridization with the cDNA probes shown on the left (A). The densitometric value of each signal for MMP-1 mRNA was normalized to GAPDH mRNA (B). The Northern blot is representative of three experiments

The kinetics of the expression of mRNAs for MMP-1, TIMP-1, and pro{alpha}2(I) are shown in Figure 4. A time-dependent increase in MMP-1 mRNA expression was found by 12 h after the addition of PAF. After incubation with PAF (10-8 M) for 12 h, the level of MMP-1 mRNA increased by 7.5-fold compared with the 0-h control value (Fig. 4B). The level of MMP-1 mRNA after 24 h of treatment resembled that seen with 12 h of treatment. PAF (10-8 M) weakly increased the expression of pro{alpha}2(I) mRNA, but it did not affect TIMP-1 mRNA (Fig. 3A and Fig. 4A).



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  		FIG. 4. Time-dependent effect of PAF on the expression of mRNA for MMP-1, TIMP-1, and type I procollagen in human uterine cervical fibroblasts. Confluent cells were incubated for various periods of time (0–24 h) with 10-8 M PAF in serum-free DMEM containing 0.1% BSA. Total RNA (10 µg/lane) was analyzed by Northern blot hybridization with the cDNA probes shown on the left (A). The densitometric value of each signal for MMP-1 mRNA was normalized to GAPDH mRNA (B). The Northern blot is representative of three experiments

PAF-Induced MMP-1 Protein Production by Human Uterine Cervical Fibroblasts

Cultured fibroblasts were incubated with PAF (10-8 M) and/or WEB 2170 (10 µM). After culture for 24 h and 48 h, we measured the concentration of total MMP-1 proteins including proMMP-1, active MMP-1, and complexes of MMP-1/TIMPs in culture media by ELISA. Control and WEB 2170-treated media were concentrated by centrifugation with Microcon-10 (Millipore, Bedford, MA), in that the amount of MMP-1 produced in the media was below the level of detection (< 6.25 ng/ml). PAF induced the production of MMP-1 protein to a significantly greater extent as compared with vehicle control (PAF treatment vs. control, 24 h: 11.50 ± 2.432 vs. 3.070 ± 1.987 ng/ml, P = 0.0012; 48 h: 48.86 ± 4.992 vs. 7.630 ± 3.462 ng/ml, P < 0.0001; Fig. 5). WEB 2170, a specific PAF receptor antagonist, abolished the MMP-1 protein production induced by PAF (PAF treatment vs. treatment with PAF plus WEB 2170, 24 h: 11.50 ± 2.432 vs. 5.507 ± 1.926 ng/ml, P = 0.0133; 48 h: 48.86 ± 4.992 vs. 7.620 ± 1.061 ng/ml, P < 0.0001; Fig. 5). WEB 2170 alone had no significant effect on MMP-1 production by cervical fibroblasts.



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  		FIG. 5. Production of MMP-1 protein by cervical fibroblasts after treatment with PAF. Confluent cells in 6-well culture plates were incubated with PAF (10-8 M) and/or WEB 2170 (10 µM). Immunoreactive total MMP-1 proteins in culture media were quantified by ELISA. Data are presented as mean ± SD of triplicate samples from four separate representative experiments. * P = 0.0012 vs. control; P = 0.0133 vs. PAF with WEB 2170 treatment. ** P < 0.0001 vs. control; P < 0.0001 vs. PAF with WEB 2170 treatment


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PAF is considered to be involved in parturition [13]. Amnion cells and decidual macrophages synthesize PAF, whereas the decidual macrophages at the maternal-fetal interface produce PAF-acetylhydrolase, which converts PAF into biologically inactive lyso-PAF (1-alkyl-2-lyso-sn-glycero-3-phosphocholine) [13, 19]. PAF is mainly inactivated by this enzyme, which regulates the level of PAF in both the de novo and remodeling pathways of PAF formation [12, 13]. During parturition, the PAF concentration in the amniotic fluid shows a marked increase [14]. This suggests that an imbalance in the metabolism of PAF may occur in the human uterus, leading to uterine contraction via both the PAF-induced stimulation of prostaglandin E2 production by the chorion-decidua [16] and the induction of myosin light-chain phosphorylation following an increase in the intracellular Ca2+ concentration in human myometrial smooth muscle cells [18].

The present study demonstrated that PAF stimulated MMP-1 mRNA expression and MMP-1 protein synthesis in human uterine cervical fibroblasts. The PAF-induced expression of MMP-1 mRNA showed an increase in a time-dependent manner for up to 24 h. Bazan and colleagues [35] reported that PAF induced the expression of mRNA for MMP-1 and the synthesis of protein in corneal epithelial cells. The time course of MMP-1 mRNA expression in the previous reports [35, 36] differed from that in our study; in the earlier studies the expression of MMP-1 mRNA peaked in the corneal cells at 8 h and leveled off at 24 h, whereas in the present study of human uterine cervical fibroblasts, the expression of mRNA increased for up to 24 h. This might be attributable to differences in initiation or degradation of the mRNAs between the corneal cells and cervical fibroblasts. Collagenase activity is reported to be significantly higher in the human uterine cervix during cervical ripening [6]. The major origin of the collagenase is reported to be such inflammatory cells as polymorphonuclear leukocytes and macrophages [37]. Most of the connective tissue cells also produce precursors of MMPs that are activated by some extracellular proteinases [7]. For example, in the human endometrium, MMP-1 mRNA and protein were detected only in non-macrophagic, fibroblast-like stromal cells [38]. Our observation that human uterine cervical fibroblasts produced MMP-1 is consistent with previous reports [39–42], which suggests that these cells could be a cellular source of MMPs.

In contrast to MMP-1, TIMP-1 mRNA was highly expressed in the cervical fibroblasts in the present study. Interestingly, PAF did not increase the expression of TIMP-1 mRNA, although TIMP-1 expression in cultured human uterine cervical fibroblasts is known to be positively regulated by such cytokines and growth factors as IL-1 [43] and epidermal growth factor [44]. PAF may induce an imbalance in the activity of MMP-1 and TIMP-1 in which the activity of MMP-1 overwhelms that of TIMP-1 in cervical tissue, as TIMPs play a key role in the metabolism of ECM by the inhibition of MMPs [7]. The PAF-induced increase in MMP-1 activity could lead to the breakdown of ECM in the human uterine cervix.

We identified PAF receptor mRNA in human uterine cervical fibroblasts by the use of RT-PCR. This suggests that PAF directly binds to the specific receptors and stimulates the production of MMP-1 in these cells. The observation that WEB 2170, a specific PAF receptor antagonist, blocks the stimulatory effect of PAF on MMP-1 production supports this possibility. The biological effects of PAF are mediated through the PAF receptor [45] present in human leukocytes, lung, heart, brain, skin, intestine, spleen, kidney, and placenta [27, 32, 46–48]. The presence of the PAF receptor has also been reported in human uterine myometrium [13, 18] and endometrium [49]. The human PAF receptor gene produces two different species of mRNAs, a tissue-type transcript and a leukocyte-type transcript [48, 50, 51]. Their expression is controlled by distinct promoters, but both transcripts contain the same coding region. Estrogen-responsive elements (ERE) are present in the promoter region of the tissue-type transcript, and estrogen positively regulates the transcriptional activity through ERE [50]. Considering that the uterus is exposed to increased amounts of estrogen during parturition, the PAF receptor could be increased in human uterine cervical fibroblasts, which would augment the PAF-induced production of MMPs.

PAF has been reported to stimulate the production of IL-6 and IL-8 by human lung fibroblasts [33]. We previously reported that PAF stimulates cytokine production, e.g., IL-6, IL-8, macrophage colony-stimulating factor, macrophage inflammatory protein-1{alpha}, and TNF-{alpha}, by human endometrial stromal cells [52]. We have also observed that PAF increases IL-6 and IL-8 production by human uterine cervical fibroblasts (unpublished results). Polymorphonuclear leukocytes, which infiltrate the cervical stroma at the time of parturition, could produce PAF [27] as well as various inflammatory cytokines. The PAF and cytokines produced by the inflammatory cells could stimulate these cells and cervical stromal cells, and accelerate cervical collagenolysis by inducing the production of MMPs. Moreover, PAF and IL-8 might also induce the migration of leukocytes into the cervix [12, 28–31], forming a positive feedback loop among PAF and cytokines. The data suggest that the paracrine and autocrine effects of various inflammatory mediators including PAF enhance collagenolysis and induce uterine cervical ripening.

In summary, we demonstrated the presence of the PAF receptor in human uterine cervical fibroblasts and showed that PAF increased the expression of MMP-1, but not that of TIMP-1, in these cells. PAF may therefore induce an imbalance in the activity between MMP-1 and TIMP-1 to accelerate collagenolysis in the human uterine cervix, contributing to cervical ripening during parturition.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Takao Shimizu and Dr. Satoshi Ishii, The University of Tokyo, Tokyo, Japan, for the gift of the human PAF receptor cDNA. We also thank Ms. Toshie Yoshida for assistance with cell culture and Ms. Yuko Sato for her editorial assistance.


   FOOTNOTES
 
First decision: 30 July 1999.

1 Correspondence: Terumasa Sugano, Department of Obstetrics and Gynecology, Oita Medical University, Hasama-machi, Oita 879-5593, Japan. FAX: 81 97 549 5087; sugano{at}oita-med.ac.jp Back

Accepted: October 7, 1999.

Received: June 29, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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