Molecular Human Reproduction, Vol. 9, No. 2, 103-110,
February 2003
© 2003 European Society of Human Reproduction and Embryology
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Interleukin-1ß stimulates placental leucine aminopeptidase/oxytocinase expression in BeWo choriocarcinoma cells
Submitted on September 20, 2002; accepted on November 25, 2002
Departments of 1 Obstetrics and Gynecology and 2 Public Health, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, 3 Division of Pathology, Clinical laboratory, Nagoya University Hospital, Nagoya, 466-8560 and 4 Laboratory of Cellular Biochemistry, RIKEN (Institute of Physical and Chemical Research), Wako, 351-0148, Japan
5 To whom correspondence should be addressed. e-mail: snomura{at}med.nagoya-u.ac.jp
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In addition to prostaglandins, inflammatory cytokines induce uterine contraction via oxytocin (OT). Placental leucine aminopeptidase (P-LAP), an oxytocinase that is identical to cystine aminopeptidase, destroys OT activity. Patients with spontaneous preterm delivery have higher concentrations of inflammatory cytokines and lower P-LAP activities than those with normal delivery. In addition, the P-LAP promoter region contains putative binding sites for cytokine-induced transcription factors. We therefore postulated that inflammatory cytokines suppress P-LAP expression and examined this notion using BeWo choriocarcinoma cells cultured in the presence of cytokines. However, interleukin-1ß (IL-1ß) increased P-LAP activity in a time- and dose-dependent manner. Furthermore, Western blot analysis showed a dose-dependent increase of P-LAP proteins. We also detected IL-1 type I receptor mRNA in BeWo cells by RT–PCR. Semi-quantitative RT–PCR and Southern blot analysis showed that IL-1ß also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assays did not reveal any regulatory regions that could explain IL-1ß-induced P-LAP mRNA accumulation within 1.1 kb upstream of the P-LAP gene. Immunohistochemical analysis of human placenta with chorioamnionitis demonstrated prominent P-LAP staining at sites of abundant inflammatory cell infiltration.These findings indicated that prolonged exposure to IL-1ß induces P-LAP in the trophoblasts, possibly via other de-novo protein synthesis, which contradicted our initial hypothesis.
Key words: aminopeptidase/cytokines/oxytocin/placenta/promoter
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Introduction |
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During pregnancy, oxytocin (OT) is the most potent uterotonic peptide hormone. Synthesized by both mother and fetus (Chard, 1989), as well as by amnion, chorion, decidua (Chibbar et al., 1993) and placenta (Lefebvre et al., 1992) during pregnancy, OT plays a critical role in the regulation of labour. While less attention has been focused on OT degradation, local concentrations of OT in the feto–placental– maternal unit depend upon a balance between synthesis and degradation. Maternal serum, decidua, chorion and the placenta possess the enzymes responsible for OT degradation (Page et al., 1961; Tsujimoto et al., 1992). At least two types of peptidases metabolize OT; these are post-proline endopeptidase and cystine aminopeptidase. Aminopeptidase completely destroys OT activity, but whether post-proline endopeptidase has similar potential remains unclear (Ferrier et al., 1974; Mizutani et al., 1985; Mitchell and Wong, 1995). Thus, cystine aminopeptidase should be regarded as an oxytocinase. Cystine aminopeptidase is more abundant in the placenta than in the decidua and chorion (Mitchell and Wong, 1995), suggesting that it plays a critical role in regulating endocrine or paracrine OT activity in the placenta. We have previously demonstrated that placental leucine aminopeptidase (P-LAP) is identical to cystine aminopeptidase (Tsujimoto et al., 1992). The findings that P-LAP activities in maternal serum (Mizutani et al., 1976; Yamahara et al., 2000) and placenta (Yamahara et al., 2000) increase with gestational age and decrease in patients with spontaneous preterm delivery (Kozaki et al., 2001) suggest that this enzyme is involved in the maintenance of pregnancy. To obtain insight into the molecular mechanisms underlying the P-LAP gene regulation, we isolated genomic clones containing the 5'-upstream region of the P-LAP gene (Horio et al., 1999). We identified several putative nucleotide consensus sequences such as binding sites for activator protein-2 (AP-2), selective promoter factor 1 (Sp1), nuclear factor-
B (NF-B) and NF-interleukin-6 (NF-IL6). In previous studies we therefore examined the roles of transcription factors in P-LAP expression. Under basal conditions, we found that AP-2 and Ikaros cooperatively enhance P-LAP transcription in trophoblastic cells (Ito et al., 2001, 2002). However, the stimulants involved in P-LAP gene regulation via the action of transcription factors are unknown. Since NF-B and NF-IL6 are cytokine-induced nuclear proteins, the potential binding sequences for those factors in the P-LAP 5'-flanking region suggest that inflammatory cytokines are associated with P-LAP gene expression, but no evidence has yet supported this notion.
Intrauterine infection, typically chorioamnionitis (Hillier et al., 1993; Menon et al., 1995; Arntzen et al., 1998), as well as the normal birth process (Opsjln et al., 1993; Steinborn et al., 1995) are accompanied by the production of cytokines, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumour necrosis factor-
(TNF-). In general, these inflammatory cytokines stimulate prostaglandin (PG) production and release with the induction of cyclooxygenase (Cox) in fetal membranes and myometrium, resulting in uterine contraction (Lundin-Schiller and Mitchell, 1991; Mitchell et al., 1993). However, several reports also suggest that other pathways via OT actions are likely to play roles in cytokine- or PG-associated uterine contraction (Jeng et al., 2000; Pavan et al., 2000). The studies showing that the OT antagonist atosiban delays threatened preterm birth (Romero et al., 2000; Valenzuela et al., 2000) may support this notion. Interestingly, P-LAP activity decreases just before onset of labour (Mizutani and Tomoda, 1996; Yamahara et al., 2000). Therefore, the regulatory mechanisms of P-LAP mediated by cytokines may affect the timing of labour.
We speculated that inflammatory cytokines decrease P-LAP expression in the human placenta, which implies an increase in the relative concentrations of functional OT by protecting OT from degradation. If this speculation is true, the cytokines may stimulate uterine contraction via OT in addition to PG. We also postulated that P-LAP regulation is mediated through the potential binding sites for NF-B and NF-IL6 located in the P-LAP 5'-flanking region. To test these hypotheses, we examined the effects of inflammatory cytokines on P-LAP activity in human choriocarcinoma BeWo cells that have retained several placental properties and are therefore considered to be a suitable model of placental trophoblastic cells (Ringler and Strauss, 1990; Ito et al., 2001). Since IL-1ß exerted significant effects on P-LAP activity, we investigated the relationship between IL-1ß and P-LAP protein and mRNA expression, and functionally analysed the P-LAP promoter region to show how IL-1ß affects the molecular mechanism of P-LAP gene regulation. We also analysed P-LAP expression by immunohistochemical means in the human placenta with chorioamnionitis.
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Materials and methods |
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Chemicals
We purchased IL-1ß from Diaclone Research (Besaçn Cedex, France), IL-6 from Life Technologies Inc. (Gaithersburg, MD, USA), and TNF-
from Chemicon International Inc. (Temecula, CA, USA)
Cell culture
Monolayer BeWo (ATCC CCL-98) human choriocarcinoma cells were maintained in Roswell Park Memorial Institute 1640 medium (Sigma, St Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin (100 U/ml) and streptomycin (100 µg/ml).
P-LAP enzymatic activity
P-LAP activities were measured spectrophotometrically at a wavelength of 405 nm using L-leucine-p-nitroanilide (Sigma) as a substrate as previously described (Mizutani et al., 1976).
Western blot analysis
Cells were manually detached from dishes using a scraper and sonicated in lysis buffer [phosphate-buffered saline (PBS) containing 1% Triton-X, and protease inhibitor cocktail tablets; Complete Mini, EDTA-free, Roche, Mannheim, Germany]. Lysates were clarified by centrifugation at 8000 g for 15 min. The concentration of cellular proteins in the supernatant was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Protein extracts (20 µg) were resolved on 7.5% sodium dodecyl sulphate–polyacrylamide gels and transferred onto nitrocellulose membranes (Millipore Corp., Bedford, MA, USA). Anti-P-LAP immunoreactive proteins were visualized using the ECL plus Western blotting detection kit (Amersham Pharmacia Biotech, NJ, USA) with a rabbit anti-P-LAP polyclonal antibody (1:1000 dilution) raised against recombinant soluble P-LAP (Matsumoto et al., 2000), which was quantified by densitometry.
RNA isolation and semi-quantitative RT–PCR/Southern blot procedure
Total RNA was isolated from the cells using an RNeasy kit (Qiagen K.K., Tokyo, Japan) according to the manufacturer’s instructions. Total RNA (1 µg) was reverse-transcribed with 2.5 µmol/l random hexamers (Applied Biosystems, Foster City, CA, USA) in a total volume of 20 µl as previously described (Nomura et al., 1996). Aliquots (1 µl) of RT reaction samples were amplified by PCR under the following conditions: 94°C for 30 s, 64°C for 30 s and 72°C for 30 s; for P-LAP using the following primers in 50 µl mixtures: P-LAP sense: 5'-GGGCACAGATCAGGCTTCCCACT-3'; P-LAP anti-sense: 5'-GATCTCAGCTTGTTTTTCTTGGCTTG-3'. We performed RT–PCR for ß-actin as the internal control using sense (5'-AACCGCGAGAAG ATGACCCAG-3') and anti-sense (5'-CTCCTGCTTGCTGATCCACAT-3') primers under the same PCR conditions as used for P-LAP. The numbers of PCR cycles were established in preliminary experiments within the exponential phase of the amplification. The PCR products (10 µl) per lane were resolved by electrophoresis on 1.0% agarose gels, then transferred to Hybond-N+ nylon membranes (Amersham Pharmacia Biotech) using a vacuum blotting system and cross-linked to the membrane by UV irradiation. Southern hybridization proceeded using [32P]P-LAP cDNA and [32P]ß-actin cDNA as probes. P-LAP mRNA levels were normalized by ß-actin expression measured with a BAS 2000 Bioimage Analyzer (Fuji Photo Film Co., Kanazawa, Japan) after autoradiography.
Interleukin-1 receptor type I expression was detected by RT–PCR using the following primers: sense: 5'-GATTCAGGACATTACTATTGCG-3'; antisense: 5'-CTGGGATCCCAAGTCTACTTCC-3'.
Construction of luciferase reporter plasmids
We prepared P-LAP promoter-luciferase constructs by subcloning PCR-derived fragments of the P-LAP 5'-flanking region into the pGL3-Basic vector (Promega Corp., Madison, WI, USA) at the KpnI site as previously described (Ito et al., 2001). PCR was performed using the following sense primers: –1170: 5'-GCGGTACCATGTGCTGATGAGGTCCCAGAG-3', –752; 5'-GC GGTACCCAATGCGCATACCGAGAGGATA-3'; –421: 5'-ATGGTACC CCGGGTTTTGTTATCCGCCCG-3'; –242: 5'-ATGGTACCTCTCCCTCG GTCCTTCGAGCAGTGA-3'; and the antisense primers: +49: 5'-AT GGTACCTCCCAGGAGCGGACATTCCAGA-3'. The transcriptional start site was assigned as +1. The underlined KpnI restriction site was added to the 5'-portion of all of the primers. The PCR fragments were digested with KpnI and subcloned into a similarly digested pGL3-Basic vector.
Transfections and luciferase assay
BeWo cells were plated (0.8x106 cells/6-well plate) 24 h before transfection. Firefly luciferase reporter plasmid DNA (1 µg) and 0.1 µg of pRL-TK plasmid DNA as an internal control to standardize transfection efficiency were transiently co-transfected into cultured cells using the LipofectAmine PlusTM Reagent (Life Technologies) for 3 h. Twenty-one hours after transfection, cells were incubated with or without 20 ng/ml of IL-1ß for a further 12 h in serum-free medium and lysed with 500 µl of passive lysis buffer. Firefly and renilla luciferase activities were measured using dual-luciferase reporter assay systems (Promega).
Immunohistochemistry
We obtained written, informed consent from all the patients who participated in this study, which was approved by the Ethics Committee of Nagoya University Graduate School of Medicine. Placentas from patients with chorioamnionitis (n = 5) and from those with placenta previa at similar gestational ages (n = 3) were immediately cut into small pieces, washed in PBS to remove blood, fixed in 10% formalin and embedded in paraffin. Sections of 4 µm thickness were immunostained as previously described (Nagasaka et al., 1997) using rabbit anti-P-LAP polyclonal antibody.
Statistical analysis
All experiments were repeated at least three times in triplicate. Data are expressed as means ± SD. Since the data were not normally distributed, we employed non-parametric statistics. Comparisons among groups were made with a Kruskal–Wallis one-way analysis of variance and between groups with a Mann–Whitney U-test for two independent samples and Bonferroni correction for multiple comparisons. P < 0.05 was considered significant.
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Results |
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Effects of cytokines on P-LAP activity in BeWo cells
We incubated BeWo cells with IL-1ß (20 ng/ml), IL-6 (10 ng/ml) and TNF-
(20 ng/ml) to assess the effects of inflammatory cytokines on P-LAP activity. Our preliminary experiments employing 24 h exposure to cytokines revealed no significant changes in P-LAP activity (data not shown). We therefore incubated the cells with cytokines for 72 h and found that IL-1ß significantly increased P-LAP activity (133.3 ± 26.7%) (P < 0.01), while both IL-6 and TNF- had no effects (Figure 1).
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Effects of IL-1ß on P-LAP protein
Among the inflammatory cytokines investigated, only IL-1ß significantly affected P-LAP activity after 72 h. Therefore, we further investigated the effects of this cytokine. Figure 2 shows the time-course effects of IL-1ß (20 ng/ml) treatment on P-LAP activity. IL-1ß had no effect on P-LAP activity up to 48 h, while it significantly increased P-LAP activity after 72 h. The continuous increase in P-LAP activity both in the presence and absence of IL-1ß for 72 h indicated the reliability of assay and cell viability.
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We then examined the dose response of P-LAP protein levels after a 72 h exposure to IL-1ß. IL-1ß at concentrations of 2 and 20 ng/ml increased P-LAP activity dose dependently and significantly (Figure 3A). We confirmed this by Western blot analysis using anti-P-LAP polyclonal antibody. The signal intensity of a single band at the expected molecular weight of 175 kDa increased in the presence of IL-1ß at concentrations of 2 and 20 ng/ml (Figure 3B). The density of the bands determined by scanning densitometry paralleled the P-LAP activity (Figure 3C). Therefore, IL-1ß dose- and time-dependently increased P-LAP activity. In addition, a dose-dependent increase in P-LAP protein levels was also demonstrated.
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IL-1 receptor type I mRNA expression in BeWo cells
IL-1ß interacts with membrane-bound type I and II receptors. The former is critical to transduction of the intracellular signal, whereas the latter has no signalling properties (Colotta et al., 1994). To confirm involvement of the general pathway with signal transduction, we determined whether the IL-1 type I receptor is present in BeWo cells using RT–PCR for the IL-1 type I receptor (Figure 4). We could detect amplified products of the predicted size (464 bp). For determining the specificity of this band, we digested the products with restriction enzyme DraI, which yielded 287 and 177 bp fragments. These results indicated that the IL-1 receptor type I mRNA is present in BeWo cells.
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Effects of IL-1ß on P-LAP mRNA
We examined whether the induction of P-LAP protein by IL-1ß is associated with an increase of P-LAP mRNA. BeWo cells were incubated with IL-1ß (20 ng/ml) for 24 h, then mRNA was extracted and P-LAP mRNA levels were determined by semiquantitative RT–PCR followed by Southern blot analysis. First, we determined the range of PCR cycles required to provide linearity. We found that 24–27, and 14–17 cycles were required to reach the linear non-saturating increase phase for P-LAP and for ß-actin respectively (Figure 5A). The representative Southern blots of PCR products amplified by 26 cycles for P-LAP and 16 cycles for ß-actin shown in Figure 5B indicate that IL-1ß apparently stimulated P-LAP mRNA expression. The band density for P-LAP increased by 2.6-fold in the presence of IL-1ß (P < 0.01) when normalized to the corresponding band for ß-actin by scanning densitometry (Figure 5C). We also evaluated whether de-novo protein synthesis was required for this P-LAP accumulation. The cells were pre-incubated for 30 min with cycloheximide (10 µg/ml), an inhibitor of translational elongation, then stimulated with IL-1ß. Cycloheximide abolished IL-1ß-enhanced P-LAP expression, suggesting that the induction of P-LAP transcription by IL-1ß requires additional protein synthesis (Figure 5B and C).
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Effects of IL-1ß on luciferase activity
The transcription factors, NF-IL6, NF-
B and AP-1, are activated by IL-1ß. The 1.1 kb 5'-flanking region of P-LAP contains two putative binding sites each for NF-IL6 and NF-B. Table I shows that both NF-IL6 motifs (TTAGGCAAG, TGTTGAAAG) precisely match the consensus sequence (TT/GNNGNAAT/G) and that both NF-B motifs (GGGGCTGCCC, GGGACGCTCC) have a 9/10 match with the consensus (GGGAA/CTNT/CCC) (Faisst and Meyer, 1992). To determine whether IL-1ß enhances P-LAP transcription via these motifs, we investigated the effects of IL-1ß on the luciferase activity of P-LAP deletion constructs in BeWo cells. We incubated transfected BeWo cells with IL-1ß for 12 and 24 h. Data are expressed as fold activation of luciferase activities of individual constructs after a 12 h incubation with IL-1ß (Figure 6). A deletion from –1170 to –752, which removed the two NF-IL6 binding sites, had no significant effect on luciferase activities between cells incubated in the presence or absence of IL-1ß. A further deletion to –421 and to –242 also had no effect on reporter expression after exposure to IL-1ß, suggesting that NF-B also does not participate in IL-1ß-induced P-LAP mRNA expression. The results after 24 h in the presence of IL-1ß were similar (data not shown). The changes in luciferase activity with deletion in the absence of IL-1ß were similar to the previous results (Ito et al., 2001) (data not shown).
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P-LAP immunostaining in human placenta with chorioamnionitis
We investigated whether P-LAP expression changes in the placenta of patients with chorioamnionitis. The gestational age of delivery ranged from 26–34 weeks in such patients and all of them had elevated serum C-reactive protein levels and white blood cell counts. A histological investigation revealed that all patients had stage II or III chorioamnionitis (Blanc, 1981). Figure 7 is a representative result showing that the area adjacent to the focus of chorioamnionitis (A) was more immunoreactive to P-LAP than the distant areas (B). Syncytiotrophoblasts were predominantly stained as demonstrated in the uninfected human placenta (Nagasaka et al., 1997). The P-LAP staining intensity at areas far from infectious focus was similar to that in normal placenta of similar gestational age without chorioamnionitis (data not shown).
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Discussion |
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The uterotonic action of OT depends on metabolism in addition to synthesis and its receptor levels. P-LAP functions as an oxytocinase by opening the ring structure of OT (Matsumoto et al., 2000), the critical motif for OT bioactivity. Our genome analysis suggests the possible involvement of inflammatory cytokines as P-LAP gene regulators. We hypothesized that inflammatory cytokines down-regulate P-LAP, leading to a relative increase of active OT. We used BeWo choriocarcinoma cells because several trophoblastic properties are maintained, indicating that these cells would present a suitable model of the placenta (Ringler and Strauss, 1990) in which to investigate the molecular mechanism of P-LAP gene regulation (Horio et al., 1999; Ito et al., 2001). Contrary to our hypothesis, the present study demonstrated that IL-1ß up-regulates P-LAP expression at both the mRNA and protein levels. Mechanisms other than that predicted, and with de-novo additional proteins synthesis, appeared to mediate P-LAP enhancement.
The concentrations of inflammatory cytokines increase with gestational age as well as in pregnancy with infection. We examined the effects of IL-1ß, IL-6 and TNF- on P-LAP activities, and found that only IL-1ß exerted positive effects. This result seems consistent with the finding that IL-1ß plays a dominant role among inflammatory cytokines including TNF, IL-1ß, IL-6 and IL-8 in the presence of chorioamnionitis (Arntzen et al., 1998). Similar findings concerning cytokines and peptidases have been demonstrated in bronchial epithelial cells, indicating that IL-1ß increases neutral endopeptidase activity and expression more efficiently than TNF- and IL-4 (Ringler and Strauss, 1990; Van der Velden et al., 1998). During normal pregnancy, IL-1ß is produced by infiltrating inflammatory cells, especially monocytes/macrophages, as well as by placental membrane (Romero et al., 1989; Saji et al., 2000). Placentas with chorioamnionitis during labour produce 17-fold more IL-1ß than placentas during labour alone (Taniguchi et al., 1991). Since these data suggested an association of IL-1ß with uterine contractions, the relationship between IL-1ß and OT–OT receptor (OTR) pathway has been extensively investigated. IL-1ß induces OT secretion from the rat neurohypophysis (Christensen et al., 1990) and human uterine smooth muscle cells (Friebe-Hoffmann et al., 2001). On the other hand, IL-1ß down-regulates OT binding capacity, OTR concentrations, and OTR mRNA in the uterus and in uterine smooth muscle cells (Rauk and Friebe-Hoffmann, 2000; Schmid et al., 2001). In addition, OT signalling such as intracellular calcium and inositol triphosphate production in the human myometrium is impaired by IL-1ß (Rauk and Chiao, 2000). Our study showed that prolonged exposure to IL-1ß stimulates P-LAP activity, leading to enhanced OT metabolism. Increased P-LAP immunostaining in the human placenta around sites of abundant inflammatory cell infiltration might support the in-vitro results, although the number of subjects studied is small. Therefore, the previous findings and those of the present study suggest that IL-1ß attenuates the OT–OTR system by several mechanisms such as (i) decreasing the concentration and binding capacity of OTR, (ii) reducing OT-mediated signalling, and (iii) increasing OT metabolism.
Even though the net effects of IL-1ß on OT pathways are obscure, what are the clinical implications of the P-LAP up-regulation by IL-1ß? We detected a significant increase in P-LAP activity in BeWo cells exposed to IL-1ß (2 and 20 ng/ml). These levels of IL-1ß are comparable or slightly higher than those in amniotic fluid from the patients with chorioamnionitis (Hillier et al., 1993). However, in a previous in-vivo experiment employing IL-1ß infusion into the amniotic cavity of pregnant rhesus macaques, IL-1ß infusion at 1–1.5 µg/kg (
100 ng/ml in amniotic fluid) was required to obtain effects similar to experimental intrauterine infection (Bethea et al., 1998). Thus, although speculative, IL-1ß exposure at the concentrations of 2 and 20 ng/ml might mimic the conditions of mild chorioamnionitis. If so, P-LAP seems to suppress labour in the case of mild infection that does not require delivery. As another possibility, up-regulated P-LAP/oxytocinase by IL-1ß may be related to the clinical observations that severe and prolonged chorioamnionitis inhibits labour as a consequence of dystocia and increases the requirement for frequent oxytocin administration (Mark et al., 2000). Of course we could not exclude the possibility that protective mechanisms of P-LAP are unlikely to play a critical role in uterine contraction.
In contrast, the possible role of PG in IL-1ß-induced uterine contraction has been well documented. IL-1ß induces Cox-2 with an increase in PG release, which stimulates uterine contractions (Tetsuka et al., 1994). IL-1ß induces Cox-2 within 1 day, generally within a few hours, through the activation of transcriptional factors including NF-B, NF-IL6 and AP-1 (Allport et al., 2000; Kirtikara et al., 2000) or the stimulation of p38 mitogen-activated protein kinase (Guan et al., 1997). Taking these findings into account, we could also speculate that short- and long-term-IL-1ß exposure has alternative effects on uterine contraction via PG or OT.
Immunohistochemical analysis in this study revealed less intense staining of P-LAP at sites distant from inflammatory cell infiltration, which was compatible with in-vitro results, although the number of placentas examined was small. However, since immunohistochemistry is not sufficiently quantitative, further experiments employing quantitative methods such as Western blot analysis are required to confirm this result.
We have previously isolated a genomic clone containing the promoter region of P-LAP (Horio et al., 1999). The sequence of this region contains several putative binding sites for transcription factors, including AP-2, Sp1, NF-B and NF-IL6. AP-2 is a trophoblast-specific transcription factor located in the promoter region of several genes such as hCG-ß (Johnson et al., 1997; Johnson and Jameson, 1999), hPL (Richardson et al., 2000) and placental aromatase (Yamada et al., 1999). We have demonstrated that AP-2 also plays a critical role in regulating P-LAP basal promoter activity (Ito et al., 2001, 2002). We then shifted our focus of study to searching the P-LAP gene regulators and postulated that inflammatory cytokines would influence P-LAP transcriptional activity through the putative binding sites for NF-B and NF-IL6 located in the P-LAP 5'-flanking region. However, the present study found that none of the luciferase P-LAP constructs investigated was significantly changed after exposure to IL-1ß despite the increase of P-LAP mRNA. Although we did not use any positive controls to confirm the effects of IL-1ß on promoter activity, experiments without IL-1ß treatment showed the results similar to our previous study, indicating, at least, the validity of the luciferase assay and the transfection. Failure to prove our hypothesis suggests that other mechanisms are involved in IL-1ß increasing P-LAP mRNA. Since we detected IL-1ß receptor mRNA, which was further confirmed by DraI digestion in BeWo cells, it is unlikely that the effect of IL-1ß is non-specific. It is noteworthy that cox-2 gene expression induced by IL-1ß in amnion epithelial cells requires activator protein-1 (AP-1) in addition to NF-B (Allport et al., 2000). Therefore, other intermediary transcription factors that bind further upstream regions or introns may be essential for the IL-1ß-dependent increase of P-LAP mRNA. This rationale is consistent with the present findings that P-LAP mRNA increased after prolonged exposure to IL-1ß and that P-LAP mRNA elevation was suppressed by cycloheximide. As another possibility, IL-1ß may induce proteins that stabilize P-LAP mRNA. It has been demonstrated that IL-1ß increases IL-6 transcripts via the synthesis of mRNA stabilizer (Lu-Kuo et al., 1996).
In conclusion, we found that prolonged exposure to IL-1ß stimulated P-LAP BeWo trophoblastic cells to express P-LAP, which contradicted our initial hypothesis. This P-LAP induction required the de-novo synthesis of other proteins. The gene sequences responsible for regulating P-LAP gene expression by IL-1ß were not found within the 1.1 kb upstream region. Although further studies are required to precisely determine the effects of OT administration on uterine contractions under prolonged IL-1ß exposure in vivo, our results suggest that P-LAP functions in a protective mechanism(s) to suppress labour under inflammatory conditions.
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Acknowledgements |
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The work was partly supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and from the Ministry of Public Management, Home Affairs, Posts and Telecommunications of Japan for specific medical research (in collaboration with Nagoya Teishin Hospital).
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