Molecular Human Reproduction, Vol. 5, No. 7, 662-667,
July 1999
© 1999 European Society of Human Reproduction and Embryology
Urinary trypsin inhibitor down-regulates hyaluronic acid fragment-induced prostanoid release in cultured human amnion cells by inhibiting cyclo-oxygenase-2 expression
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka, 431-3192, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We postulated that urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, may inhibit low molecular weight hyaluronic acid (HA) fragment-induced prostanoid release and de-novo expression of the inducible cyclo-oxygenase-2 (COX-2) isoform in human term amnion cells. Purified amnion cultures were obtained from human fetal membranes and were exposed to a HA fragment (molecular weight 35 kDa) in the presence or absence of UTI (0–5.0 µmol/l). Amnion cells treated with the HA fragment (100 nmol/l) released significantly more prostanoids (PGE2 and PGF2
) than controls (PGE2: 2.1 ± 0.13 pg/106 cells/24 h compared with 0.42 ± 0.01, P < 0.05; PGF2: 1.0 ± 0.17 pg/106 cells/24 h compared with 0.13 ± 0.01, P < 0.05). UTI inhibited HA fragment-induced prostanoid release in a dose-dependent manner, with 50% inhibitory concentration values of 0.8 µmol/l for PGE2 and 1.9 µmol/l for PGF2. Western blot analyses demonstrated that protein levels of COX-2 were substantially increased in amnion cells treated with HA fragment. HA fragment-mediated COX-2 production was markedly diminished by pretreatment with UTI (1.0 µmol/l). These results are the first to demonstrate that UTI is a potent inhibitor of HA fragment-induced arachidonic acid metabolism. cyclo-oxygenase/hyaluronic acid/parturition/urinary trypsin inhibitor
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The mechanism by which uterine smooth muscle switches from a quiescent state during pregnancy to contraction at labour has been the subject of investigation for years. The role of innervation, hormones, prostaglandins (Mitchell et al., 1995
), nitric oxide (NO), NO synthase (NOS) (Sladek et al., 1997), corticotrophin-releasing factor (CRF) (Quartero and Fry, 1989), CFR-binding protein (CRF-BP) (Perkins et al., 1993) and CRF receptors (Hillhouse et al., 1993) in mediating this switch have been extensively explored. These substances may keep the physically contracting uterine smooth muscle from delivering the fetus before the optimal term gestation. Parturition occurs through the up-regulation of a group of pro-labour genes, such as oxytocin, oxytocin receptors (Kimura et al., 1996), gap junction proteins (Sakamoto et al., 1983) and CRF (Quartero and Fry, 1989; Perkins et al., 1993; Hillhouse et al., 1993) or through the down-regulation of a group of NO, NOS (Sladek et al., 1997), CRF-BP (Perkins et al., 1993), and amniotic fluid-derived urinary trypsin inhibitor (UTI) (Kanayama et al., 1995a,b, 1996; El Maradny et al., 1996; Kaga et al., 1996a,b) which may decrease uterine contractility and maintain the uterus in a state of quiescence during pregnancy.
Human inter--trypsin inhibitor (II) is comprised of three genetically different polypeptides; two heavy chains and one light chain. The light chain is referred to as the UTI, which appears to regulate protease activity (Schreitmuller et al., 1987; Kobayashi et al., 1996). In addition to protease inhibitory activity, proteins of the II family are considered to have important biological functions. An intriguing function of UTI is to inhibit uterine contractility during pregnancy (El Maradny et al., 1994). The fetal liver, kidney and pancreas (Itoh et al., 1996) as well as amniotic membrane (El Maradny et al., 1994) are considered to be the sources of amniotic fluid-derived UTI.
Hyaluronic acid (HA), a high molecular weight non-sulphated linear glycosaminoglycan, ordinarily synthesized as a molecule of 103 to 104 kDa, is actively synthesized by many cells (Lee and Cowman, 1994). Studies of pregnant animals with parturition show that serum HA concentrations are often strikingly elevated (Rajabi et al., 1992). Lower molecular weight fragment accumulates and induces the production of prostanoids which may be important in the development and maintenance of the parturition. HA fragment significantly stimulates prostanoid production by inducing the expression of cyclo-oxygenase-2 (COX-2) in human term amnion cells (Kobayashi and Terao, 1997; Kobayashi et al. 1998a).
Prostanoids play major roles in uterine cervical dilatation and the labour onset. Amniotic prostanoid concentrations can rise during parturition. High concentrations of prostanoids may affect uterine contractility. The prostanoid cascade is initiated by liberation from membrane phospholipids of the precursor arachidonic acid, which is metabolized by COX to prostaglandins. COX has been identified in two major isoforms; a constitutive form (COX-1), responsible for the release of physiological concentrations of prostanoids, and an inducible form (COX-2), expressed on appropriate stimulation with cytokines and proinflammatory agents (Minghetti et al., 1996). It is interesting that up-regulation of COX-2 mRNA expression was observed in amnion cells during parturition (Fuentes et al., 1996).
In the present study we found for the first time that UTI down-regulates the HA fragment-induced prostanoid production by inhibiting COX-2 expression. We also addressed the problem of the possible interactions between HA fragment and prostanoid release and of the modulatory role exerted by UTI on these systems.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Materials
Purified low molecular weight hyaluronic acid (HA) (molecular weight 35 kDa) was provided by Drs S.Miyauchi and M.Ikeda (Sekagaku Kogyo Co Ltd, Tokyo, Japan). The size of HA fragment was checked as follows: molecular weight distributions of HA fragment were determined by calibrated chromatography on a Sephacryl S-300 column. In brief, 2 ml samples of the HA fragment were chromatographed on a 1.0x100 cm column of Sephacryl S-300 (Pharmacia; Uppsala, Sweden) equilibrated with 10 mmol/l phosphate-buffered saline (PBS), pH 7.4. Fractions of 1 ml were collected and the flow rate was kept at 8 ml/h. Hyaluronic acid concentration was measured by a specific enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Chugai Pharmaceutical Co Ltd, Tokyo, Japan). The Sephacryl S-300 column was calibrated with fractions of HA of known molecular weight, kindly donated by Seikagaku Kogyo and Chugai Pharmaceutical Co Ltd, or available in our laboratory, which contains 2200, 800, 34 and 3.5 kDa HA fractions. Urinary trypsin inhibitor (UTI) purified from human urine was a generous gift of Mochida Pharmaceutical Co Ltd (Tokyo, Japan) (Kobayashi et al., 1996
). It was dissolved in medium to a concentration of 10 mg/ml and kept as a stock solution at –20°C.
Subjects
At weeks 37–40, complete fetal membranes were collected by elective Caesarean section in four cases before the onset of labour. Routine clinical monitoring was performed in all cases to confirm whether there were spontaneous uterine contractions. In the four cases studied, the uterine cervix was firmly closed and there was no contractile activity prior to the Caesarean section. The indication for Caesarean section was a previous Caesarean section or breech presentation in otherwise uncomplicated pregnancies. Placentas and fetal membranes obtained from pregnancies complicated by multiple gestations and fetal anomalies were excluded. Treatment decisions were made by the patients' physicians in all cases and were not affected by participation in this study. All women gave their informed consent to participate in this study which were approved by Hamamatsu University Hospital Ethical Committee.
Amnion cell culture
Amnion cell cultures were established as previously described (Lamont et al., 1990). The amnion cells were suspended in medium 199 containing 1.1 gm/l sodium bicarbonate, antibiotics, 1% L-glutamine, and 10% fetal calf serum (FCS). Cell viability was assessed by 0.5% Trypan Blue exclusion staining (routinely ~80% viability) and the suspension was diluted to 5x105 cells/2 ml. Cells were dispensed in multi-well culture dishes (2 ml/well) and incubated at 37°C in an atmosphere of 5% CO2 in air. Then the culture medium was removed and adherent cells were washed twice with 2 ml of prewarmed PBS before 2 ml of fresh medium containing HA fragment (100 nmol/l) and/or indomethacin (150 nmol/l) was added. The amnion cells from four patients were tested separately. Before and after the cells were exposed to HA fragment, their viability was tested by their exclusion of Trypan Blue.
The amniotic cells with added reagents were incubated at 37°C for 24 h in an atmosphere of 5% CO2 in air, after which the medium was collected, centrifuged to remove debris, and stored at –20°C until tested for prostaglandins. Prostaglandin release was expressed as pg/106 cells/24 h, and the mean ± SD for the four separate experiments was calculated.
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting
Amnion cells (1x105 cells) treated with HA fragment were obtained in the presence of 0.2 mol/l PBS, pH 7.4 containing 45 mmol/l benzamidine HCl, 5 mmol/l N-ethylmaleimide, 1 mmol/l EDTA, 1 mmol/l phenylmethylsulphonyl fluoride, and 5 µg/ml pepstatin and centrifuged at 2000 g at 4°C for 15 min to remove cellular debris. The remaining supernatants were centrifuged at 100 000 g at 4°C for 60 min to purify microsomal proteins. Microsomal proteins were sonicated, and concentrations were determined as described previously (Lowry et al., 1951). Samples were mixed with a SDS sample buffer (10% SDS, 10% glycerol, 0.01% Bromophenol Blue, and 0.625 mol/l Tris–HCl, pH 6.8). The samples were electrophoresed on 1.5 mm thick, 10 cm long, 12% SDS–PAGE slab gels after stacking on a 2 cm SDS (3%) polyacrylamide gel. Electrophoresis was done at constant current (15 mA stacking, 25 mA separating). Following PAGE, the proteins were transferred to polyvinylidine difluoride (PVDF; Bio-Rad) paper using a semi-dry electroblotting apparatus (Marysol, Tokyo, Japan) at 40 mA/gel (90 min, 23°C). The PVDF sheets were incubated in Tris-buffered saline (TBS) containing 2% bovine serum albumin (BSA), and then incubated for 2 h at 23°C in TBS containing 2% BSA and anti-cyclo-oxygenase-2 (anti-COX-2) antibody (1:100 dilution; Cosmo Bio, Tokyo, Japan). Following incubation with anti-COX-2 antibody, the paper was washed and incubated for 1 h at 23°C in the secondary antibody, goat anti-rabbit immunoglobulin (Ig)G conjugated to biotin (1:500 dilution; Dako, Copenhagen, Denmark). This paper was washed again and incubated for 1 h at 23°C in avidin–peroxidase (1:500 dilution; Dako) and immunocomplexes were detected with the chemiluminescent reagent ECL and visualized by autoradiography (Amersham International, Tokyo, Japan). For quantification, computerized scanning and densitometry (Power Macintosh 7600/200-assisted FAS-II and Electronic UV transilluminator; Toyobo Co Ltd, Tokyo, Japan) were used.
ELISA for prostanoids
Prostaglandin E2 (PGE2) and PGF2 were measured by sensitive and specific radioimmunoassays according to the instructions of the manufacturer (Amersham assay kits; PGE2 ELISA system and PGF2 [3H] assay system). Results were expressed as pg PGE2 and PGF2 per 106 cells per 24 h. Cross-reaction with PGE2 = 100%; PGE1 = 7.0%; 6-keto-PGF1 = 5.4%; PGF2 = 4.3%; other prostaglandins (including 15-keto-PGs and 13,14-dehydro-PGs) <2.0%. Cross-reaction with PGF2 = 100%; 6-keto-PGF1 = 1.5 %; other prostaglandins (including 15-keto-PGs and 13,14-dehydro-PGs) <1.0 %. The intra-assay and inter-assay coefficients of variation were <8 and 9% respectively.
Statistical analysis
Statistical analysis was performed using StatView for Macintosh. One-way analysis of variance (ANOVA) was performed with post-hoc analysis using Scheffé's F procedure for statistical interpretation. P < 0.05 was considered to be statistically significant. Prostanoid and cytokine production between groups was compared using the Mann–Whitney U-test. Data are presented as mean ± SD.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Low molecular weight hyaluronic acid fragment stimulates prostanoid release in amnion cells
The low molecular weight (Mw) hyaluronic acid (HA) fragment induced PGE2 release in human amnion cells in a concentration- and time-dependent manner. Data obtained after 24 h of incubation are depicted in Figure 1
. Human amnion cells treated with HA fragment (100 nmol/l) released significantly more PGE2 (mean ± SD, 2.3 ± 0.21 pg/106 cells/24 h) than controls (0.34 ± 0.03 pg/106 cells/24 h). Maximal stimulation of PGE2 release was observed in the presence of 10–100 nmol/l of HA fragment. Similar results were also obtained with respect to PGF2 release. Maximal concentration of PGF2 in the medium was observed at a concentration of over 10 nmol/l of HA fragment. As expected for induced enzymatic activities, HA fragment-induced PGE2 release was prevented in the presence of cycloheximide, a protein synthesis inhibitor, which was active at concentrations as low as 0.1 µg/ml that did not affect cell viability (unpublished data; Kobayashi et al., 1998a).
|
Effect of urinary trypsin inhibitor on low molecular weight hyaluronic acid fragment-induced PGE2 release
To validate the hypothesis that exogenously applied UTI inhibits prostanoid formation, we studied the effect of UTI in amnion cells cultured in conditions in which HA fragment was added. As shown in Figure 1
, 1 µmol/l UTI can inhibit HA fragment-induced PGE2 release. It has been demonstrated that HA fragment-induced PGE2 release was inhibited by UTI in a dose-dependent manner (Figure 2). The inhibition was apparent at a concentration of 1 µmol/l UTI and was more pronounced at higher concentrations of 2 µmol/l UTI. Only partial inhibition of PGE2 release was observed at all concentrations of HA fragment tested, as even high concentrations of UTI (5 µmol/l) did not suppress PGE2 release to basal values. Preincubation with UTI for up to 24 h before addition of HA fragment did not augment the inhibitory effect on PGE2 concentrations compared with the simultaneous addition of UTI and HA fragment (data not shown). UTI alone did not affect the basal PGE2 release. Cell viability was not affected by 5 µmol/l UTI and in agreement with the reported low toxicity of this drug (Kobayashi et al., 1995). Thus, UTI is likely to affect prostanoid release (or probably biosynthesis) at the following steps: release of the precursor arachidonic acid from membrane phospholipids, COX expression or enzymatic activity.
|
= amnion cells were incubated with UTI (5 µmol/l) in the absence of hyaluronic acid. Data are given as mean ± SD (n = 4) from one of three experiments. ––––––- = basal PGE2 value.Time-course experiments showed an early onset of HA fragment-induced release of PGE2 (Figure 3
). Inhibition by UTI was clearly detectable after 4 h incubation, but became more pronounced after longer incubation times. Again, basal PGE2 release was not inhibited by the continuous presence of UTI. A single administration of UTI together with HA fragment was sufficient to sustain inhibition at the level reached after 24 h.
|
Effect of hyaluronic acid fragment and urinary trypsin inhibitor on expression of cyclo-oxygenase proteins
We have previously shown that HA fragment induces prostanoid biosynthesis in amnion cells by increasing COX-2 expression (Kobayashi et al., 1998a
). To further elucidate the molecular mechanism of the induction of prostaglandin release or synthesis, cyclo-oxygenase protein expression was measured by Western blot analysis (Figure 4). Cells were treated for 24 h with HA fragment, in the presence of UTI (0.1– 5.0 µmol/l) with fresh culture medium supplemented with 10 µmol/l arachidonic acid, to provide a large availability of the substrate for PGE2 synthesis. After the culture supernatants were collected for PGE2 content determination, amnion microsomal proteins from each sample were separated by SDS–PAGE and analysed by Western blot using specific antibodies for COX-1 and COX-2. Under basal conditions, a distinct band was obtained with a polyclonal antibody raised against COX-1 protein, while COX-2 protein was barely detectable. The polyclonal antibody raised against COX-1 recognized one protein band of ~70 kDa. The COX-2 antibody recognized two bands of molecular weights of ~70 and 65 kDa. The band with slower electrophoretic mobility (~70 kDa) was more intense. Upon incubation with low Mw HA fragment, the expression of COX-1 protein remained unaltered. To correlate changes in PGE2 release seen in earlier 24 h incubations, we measured COX-2 protein expression after a 24 h incubation following addition of HA fragment. In comparison with untreated cells, HA fragment-stimulated cells showed a higher expression of COX-2 and greater ability to convert arachidonic acid into PGE2, while no change was observed in the expression of COX-1 protein. UTI had no significant effect on the constitutive expression of COX-1 protein in amnion cells, but the induction of COX-2 protein by HA fragment was reduced. Compared with the very low values seen in control amnion cells, COX-2 protein was markedly enhanced by >30-fold by the induction of HA fragment. In the presence of UTI, COX-2 protein expression remained at lower levels. After 24 h, induction was only 5-fold, corresponding to a ~80% inhibition by UTI. Even high concentration of UTI (5 µmol/) did not suppress COX-2 protein induction to basal values.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Prostanoid production by appropriately stimulated amnion cell cultures has been described by several authors (Pendergraft et al., 1994
; Fuentes et al., 1996; Perkins and Kniss, 1997). Amnion cells produce considerable amounts of prostanoids upon stimulation with substances including bacterial lipopolysaccharides (Casey et al., 1989) or cytokines such as interleukin (IL)-1 (Bry and Hallman, 1991). Cytokine had a major role in controlling the level of prostanoids through its stimulatory effect on COX-2 expression (Albert et al., 1994; Spaziani et al., 1996). Thus, amnion cells are target cells for prostanoid production thought to be responsible for the modulation of uterine contraction, cervical ripening and the labour onset. In the present study, low molecular weight hyaluronic acid fragment was chosen to induce prostanoid release (possibly production) from amnion cells, because it is a mediator of cytokine production from uterine cervical fibroblasts and has also been shown to be involved in the inflammatory processes during parturition (Kobayashi and Terao, 1997). Nothing is known about the metabolism of the released prostanoids during the 24 h incubation, and so the amount of prostanoids measured may have been underestimated. Notwithstanding these limitations, HA fragment enhanced the basal prostanoid formation in a concentration- and time-dependent manner.
An increasing number of reports have demonstrated direct and specific effects of UTI on uterine, placental and amnion cells, which suggest that the suppressive activity of uterine contractility may be due to the interference of the UTI with intrinsic regulatory mechanism involved in uterine contraction within the uterus (El Maradny et al., 1994, 1996; Kanayama et al., 1995a,b, 1996, 1997; Imada et al., 1997). Now, we have shown that in primary cultures of amnion cells the HA fragment-induced release of prostanoids is down-regulated by exogenous UTI, through an inhibition of COX-2 expression. It is likely that UTI directly or indirectly inhibits biosynthesis of inducible COX-2. UTI reduced the enhanced prostanoid release of amnion cells by ~70%. In none of the experiments did UTI inhibit HA fragment-induced prostanoid release to control values, even if the UTI was given at higher concentrations up to 5 µmol/l. These observations suggest a molecular mechanism different from the direct COX inhibition by non-steroidal anti-inflammatory drugs (e.g. acetylsalicylic acid) (Donnelly and Hawkey, 1997).
The concentrations used in this study are likely to be within the concentration range which leads to suppression of uterine contractility in vivo. This is in accordance with other studies with uterine myometrial cells or vascular endothelial cells (Imada et al., 1997; Kanayama et al., 1997). Suppression of prostanoid release by UTI was observed at early time points of stimulation by HA fragment, and also during the prolonged release of prostanoid at 12–24 h incubation. This time course suggests a pronounced inhibitory effect on the molecular mechanisms underlying induction of prostanoid release, not just an attenuating effect of UTI on the existing enzymatic machinery.
The mechanism by which UTI interferes with PGE2 release has been discussed at different levels. The formation and release of prostanoids from amnion cells is a multi-step process requiring a series of enzymatic conversions of the arachidonic acid to the final product, PGE2. Arachidonic acid is liberated upon stimulation from plasma membrane phospholipids. It is possible that UTI inhibited the HA fragment-induced phospholipase A2 synthesis and release. We have to investigate an effect of UTI at the level of arachidonic acid liberation.
We have focused on the second step of the arachidonate cascade. Cyclo-oxygenase utilizes arachidonic acid as a substrate to produce a metastable intermediate product, which is then further processed to different prostanoids. Recently, it was shown that HA and its fragment induced synthesis and release of cytokines such as IL-1ß, tumour necrosis factor (TNF)- and IL-8 (Kobayashi and Terao, 1997). Also, IL-1ß and TNF- induced de-novo synthesis of COX molecules (Casey et al., 1989; Spaziani et al., 1996). This was paralleled by an enhancement of PGE2 release into the conditioned media of these cultures and an increased COX-2 protein expression. The effect on COX-2 could benefit from the incorporation of cytokines to this model. Now we have shown that, in amnion cells, HA fragment had no significant effect on COX-1 expression but strongly induced COX-2 protein, which was barely detectable in non-stimulated cells. It has been speculated that UTI would interfere at the mRNA level with the induction of COX-2 without effecting the constitutive expression of COX-1. The missing effect of UTI on the constitutively expressed COX-1 isozyme may explain the minor effects of UTI on basal PGE2 release. The striking concurrence of UTI-mediated inhibition of PGE2 release, COX-2 protein expression strongly suggests that the interference of UTI at this step of the biosynthetic cascade is the most relevant, which, however, does not exclude effects of UTI at other levels. The interference with an induction pathway resulting in inhibition of de-novo PGE2 synthesis may resemble the action of UTI on suppression of uterine contractility.
The effect of UTI was not restricted to the HA fragment-induced COX-2 expression. There are some reports in the literature that UTI, in addition to inhibiting the release of prostanoids, suppressed the intracellular calcium influx, most likely in uterine myometrial cells, vascular endothelial cells, neutrophils and platelets (Kanayama et al., 1995a,b, 1997). One might speculate that the effect of UTI, such as the suppression of intracellular calcium, might be essential in cells without the capacity of de-novo protein biosynthesis, such as platelets. This would explain the another mechanism of UTI function which contributes to the interference of UTI with uterine contractility.
We pursued the hypothesis that UTI inhibits HA fragment-induced prostanoid release, as well as the de-novo expression of the COX-2 isoform in human term amnion cells. The findings of this paper are supported by previous reports in which amniotic fluid UTI concentrations decrease significantly at term (Kobayashi et al. 1998b) and up-regulation of COX-2 mRNA expression was observed in amnion cells during the last trimester of parturition (Fuentes et al., 1996). Taken together, parturition may occur through the down-regulation of UTI which inhibits uterine contractility and maintains the uterus in a state of quiescence during pregnancy. We are currently trying to understand the transduction system mediating the effect of UTI on COX-2.
|
Acknowledgments |
|---|
We are thankful to Doctors H.Morishita and K.Kato for their continuous and generous support of our work with UTI.
|
Notes |
|---|
1 To whom correspondence should be addressed
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Albert, T.J., Su, H.C., Zimmerman, P.D. et al. (1994) Interleukin-1 beta regulates the inducible cyclooxygenase in amnion-derived WISH cells. Prostaglandins, 48, 401–416.[Web of Science][Medline]
Bry, K. and Hallman M. (1991) Synergistic stimulation of amnion cell prostaglandin E2 synthesis by interleukin-1, tumor necrosis factor and products from activated human granulocytes. Prostagl. Leukotr. Ess. Fatty Acids, 44, 241–245.
Casey, M.L., Cox, S.M., Beutler, B. et al. (1989) Cachectin/tumor necrosis factor-alpha formation in human decidua. Potential role of cytokines in infection-induced preterm labor. J. Clin. Invest., 83, 430–436.
Donnelly, M.T. and Hawkey, C.J. (1997) Review article: COX-II inhibitors–a new generation of safer NSAIDs? Aliment. Pharmacol. Therapeut., 11, 227–236.[Web of Science][Medline]
El Maradny, E., Kanayama, N., Halim, A. et al. (1996) Effects of urinary trypsin inhibitor on myometrial contraction in term and preterm deliveries. Gynecol. Obstet. Invest., 41, 96–102.[Web of Science][Medline]
El Maradny, E., Kanayama, N., Halim, A. et al. (1994) Urinary trypsin inhibitor has a protective effect on the amnion. Gynecol. Obstet. Invest., 38, 169–172.[Web of Science][Medline]
Fuentes, A., Spaziani, E.P. and O'Brien, W.F. (1996) The expression of cyclooxygenase-2 (COX-2) in amnion and decidua following spontaneous labor. Prostaglandins, 52, 261–267.[Web of Science][Medline]
Hillhouse, E.W., Grammatopoulos, D., Milton, N.G. and Quartero, H.W. (1993) The identification of a human myometrial corticotropin-releasing hormone receptor that increases in affinity during pregnancy. J. Clin. Endocrinol. Metab., 76, 736–741.[Abstract]
Imada, K., Ito, A., Kanayama, N. et al. (1997) Urinary trypsin inhibitor suppresses the production of interstitial procollagenase/proMMP-1 and prostromelysin-1/ proMMP-3 in human uterine cervical fibroblasts and chorionic cells. FEBS Lett., 417, 337–340.[Web of Science][Medline]
Itoh, H., Tomita, M., Kobayashi, T. et al. (1996) Expression of inter-alpha-trypsin inhibitor light chain (bikunin) in human pancreas. J. Biochem., 120, 271–275.
Kaga, N., Katsuki, Y., Futamura, Y. et al. (1996a) Role of urinary trypsin inhibitor in the maintenance of pregnancy in mice. Obstet. Gynecol., 88, 872–882.[Web of Science][Medline]
Kaga, N., Katsuki, Y., Obata, M. and Shibutani, Y. (1996b) Repeated administration of low-dose lipopolysaccharide induces preterm delivery in mice: a model for human preterm parturition and for assessment of the therapeutic ability of drugs against preterm delivery. Am. J. Obstet. Gynecol., 174, 754–759.[Web of Science][Medline]
Kanayama, N., El Maradny, E., Halim, A. et al. (1995a) Urinary trypsin inhibitor prevents uterine muscle contraction by inhibition of Ca++ influx. Am. J. Obstet. Gynecol., 173, 192–199.[Web of Science][Medline]
Kanayama, N., El Maradny, E., Halim, A. et al. (1995b) Urinary trypsin inhibitor suppresses premature cervical ripening. Eur. J. Obstet. Gynecol. Reprod. Biol., 60, 181–186.[Web of Science][Medline]
Kanayama, N., El Maradny, E., Yamamoto, N. et al. (1996) Urinary trypsin inhibitor: a new drug to treat preterm labor: a comparative study with ritodrine. Eur. J. Obstet. Gynecol. Reprod. Biol., 67, 133–138.[Web of Science][Medline]
Kanayama, N., Maehara, K., Suzuki, M. et al. (1997) The role of chondroitin sulfate chains of urinary trypsin inhibitor in inhibition of LPS-induced increase of cytosolic free Ca2+ in HL60 cells and HUVEC cells. Biochem. Biophys. Res. Commun., 238, 560–564.[Web of Science][Medline]
Kimura, T., Takemura, M., Nomura, S. et al. (1996) Expression of oxytocin receptor in human pregnant myometrium. Endocrinology, 137, 780–785.[Abstract]
Kobayashi, H., Gotoh, J., Hirashima, Y. and Terao, T. (1996) Inter-alpha-trypsin inhibitor bound to tumor cells is cleaved into the heavy chains and the light chain on the cell surface. J. Biol. Chem., 271, 11362–11367.
Kobayashi, H., Sun, G.W., and Terao, T. (1998a) Production of prostanoids via increased cyclo-oxygenase-2 expression in human amnion cells in response to low molecular weight hyaluronic acid fragment. Biochim. Biophys. Acta, 1383, 253–268.[Medline]
Kobayashi, H., Suzuki, K., Sugino, D., and Terao, T. (1998b) Urinary trypsin inhibitor levels in amniotic fluid of normal human pregnancy: Decreased levels observed at parturition. Am. J. Obstet. Gynecol., in press.
Kobayashi, H., Shinohara, H., Gotoh, J. et al. (1995) Anti-metastatic therapy by urinary trypsin inhibitor in combination with an anti-cancer agent. Br. J. Cancer, 72, 1131–1137.[Web of Science][Medline]
Kobayashi, H. and Terao, T. (1997) Hyaluronic acid-specific regulation of cytokines by human uterine fibroblasts. Am. J. Physiol., 273, C1151–C1159.
Lamont, R.F., Anthony, F., Myatt, L. et al. (1990) Production of prostaglandin E2 by human amnion in vitro in response to addition of media conditioned by microorganisms associated with chorioamnionitis and preterm labor. Am. J. Obstet. Gynecol., 162, 819–825.[Web of Science][Medline]
Lee, H.G. and Cowman, M.K. (1994) An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution. Anal. Biochem., 219, 278–287.[Web of Science][Medline]
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265–275.
Minghetti, L., Polazzi, E., Nicolini, A. et al. (1996) Interferon- and nitric oxide down-regulate lipopolysaccharide-induced prostanoid production in cultured rat microglial cells by inhibiting cyclooxygenase-2 expression. J. Neurochem., 66, 1963–1970.[Web of Science][Medline]
Mitchell, M.D., Romero, R.J., Edwin, S.S. and Trautman, M.S. (1995) Prostaglandins and parturition. Reprod. Fertil. Dev., 7, 623–632.[Medline]
Pendergraft, J.S., O'Brien, W.F. and Williams, M.C. (1994) Modulators of prostaglandin E2 synthesis in human amnion. J. Soc. Gynecol. Invest., 1, 131–134.[Web of Science][Medline]
Perkins, A.V., Eben, F., Wolfe, C.D. et al. (1993) Plasma measurements of corticotrophin-releasing hormone-binding protein in normal and abnormal human pregnancy. J. Endocrinol., 138, 149–157.
Perkins, D.J. and Kniss, D.A. (1997) Tumor necrosis factor-alpha promotes sustained cyclooxygenase-2 expression: attenuation by dexamethasone and NSAIDs. Prostaglandins, 54, 727–743.[Web of Science][Medline]
Quartero, H.W. and Fry, C.H. (1989) Placental corticotrophin releasing factor may modulate human parturition. Placenta, 10, 439–443.[Web of Science][Medline]
Rajabi, M.R., Quillen, E.W.Jr., Nuwayhid, B.S. et al. (1992) Circulating hyaluronic acid in nonpregnant, pregnant, and postpartum guinea pigs: elevated levels observed at parturition. Am. J. Obstet. Gynecol., 166, 242–246.[Web of Science][Medline]
Sakamoto, H., Takagi, S., Saito, Y. et al. (1983) Biochemical and morphological study of pregnant rat myometrium: role of steroid receptors and gap junction formation in onset of parturition. Acta Obstet. Gynaecol. Jap., 35, 645–654.
Schreitmuller, T., Hochstrasser, K., Reisinger, P.W. et al. (1987) cDNA cloning of human inter-alpha-trypsin inhibitor discloses three different proteins. Biol. Chem. Hoppe-Seyler, 368, 963–970.[Web of Science][Medline]
Sladek, S.M., Magness, R.R. and Conrad, K.P. (1997) Nitric oxide and pregnancy. Am. J. Physiol., 272, R441–R463.
Spaziani, E.P., Lantz, M.E., Benoit, R.R. and O'Brien, W.F. (1996) The induction of cyclooxygenase-2 (COX-2) in intact human amnion tissue by interleukin-4. Prostaglandins, 51, 215–223.[Web of Science][Medline]
Submitted on July 9, 1998; accepted on April 16, 1999.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||