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a Department of Obstetrics & Gynecology, University of South Florida Health Science Center, Tampa, Florida 33612
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is a large body of evidence supporting the role of prostaglandins in the initiation of term and preterm labor [7]. In combination and correlation with other factors, gradual increases in prostaglandin (PG) E2 are observed in plasma and amniotic fluid during pregnancy with a dramatic rise in PGE2 bioavailability just prior to the onset of labor [8]. The effects of PGE2 are exerted through its binding to specific plasma membrane G protein-linked receptors. There are currently four known subtypes of the PGE2 receptors, designated as EP1–EP4, and each one is associated with a different second messenger system [9]. The EP1 and EP3 receptor subtypes are generally associated with stimulatory functions [10].
The presence of the EP1 and EP3 receptor subtypes in amnion has been demonstrated [11]. In addition, increased expression of EP1 in amnion has been observed in response to cytokine exposure, suggesting that the regulation of PGE2 receptors may be involved in the initiation of preterm labor [11,12]. While the exact mode of function of the EP1 receptor in human amnion has not been clearly defined, results seem to suggest either an autocrine or a paracrine role in events associated with parturition. A potential role for CRH in the initiation and propagation of labor is also not clearly defined. We hypothesized that in amnion, CRH may regulate the expression of the EP1 subtype. The purpose of this study was to further clarify the role of CRH during labor by investigating its effects on the expression of the EP1 receptor subtype and PGE2 production in amnion WISH cell culture.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Amnion WISH cells (AWC; American Type Culture Collection, Rockville, MD) were seeded into sterile vented 25-cm2 polyethylene tissue culture flasks at a concentration of approximately 5 x 106 cells per flask. The culture medium consisted of Dulbecco's modified Eagle's medium F-12 mixture (1:1) containing 15% heat-inactivated fetal calf serum supplemented with 15 mM Hepes, 2 mM L-glutamine, 100 g/ml gentamicin, and 25 µg/ml amphotericin B. Cultures were grown to 80% confluence in a humidified incubator at 37°C and 5% CO2.
After the cells reached 80% confluence, the culture medium was removed and the cells were washed in PBS; cells were incubated for 24 h in increasing concentrations of CRH (5, 10, 25, 50 ng/ml). The experiment was performed on 8 separate flasks per CRH concentration. Cultures incubated in medium alone served as controls. The number of controls was equal to the number of flasks for each concentration. In order to verify that EP1 receptor protein production was not a function of a feedback loop involving PGE2, cultures were also incubated with high CRH (50 ng/ml) with indomethacin (10 µg/ml) in combination.
After incubation with CRH, cells were scraped and homogenized on ice in a glass-polytetrafluoroethylene homogenizer in 50 mM Tris, pH 7.5, containing 2 mM EDTA, 0.25 M sucrose, 10 µg/ml leupeptin, 50 µg/ml pepstatin A, 1 mM PMSF, 1 mM dithiothreitol, and 1% Triton X-100. After 30 min, the crude homogenate was spun at 1000 x g for 15 min. Crude homogenates were assayed for protein content using a micro-bicinchoninic acid method (Sigma Chemical Co., St. Louis, MO) with BSA as standard [13]. An aliquot of culture fluid was collected for PGE2 measurement by enzyme immunoassay (Cayman Chemical Co., Ann Arbor, MI).
Western Blot Analysis
Changes in EP1 receptor protein levels were evaluated by Western blot analysis. Protein (25 µg) from each lysate was added per well of a 7.5% Tris-glycine gel and separated by electrophoresis (SDS-PAGE). Molecular weights were estimated using prestained molecular weight markers (Bio-Rad Labs., Hercules, CA). After SDS-PAGE, proteins were transferred to nitrocellulose membranes for 2 h at 200 mA in 25 mM Tris and 192 mM glycine buffer, pH 8.3, containing 20% methanol.
EP1 receptor bands were detected with EP1 polyclonal antibodies, prepared as previously described [11], and enhanced chemiluminescence (Amersham, Arlington Heights, IL; now Amersham Pharmacia Biotech, Piscataway, NJ). Band intensity was measured after scanning the gels with the Sigmagel analysis program (Jandel Scientific, San Rafael, CA). One Western blot on each flask studied was performed (n = 8 flasks per concentration of CRH).
Northern Blot Analysis
Changes in EP1 mRNA levels were evaluated by Northern blot analysis. After incubation of cultured cells in CRH, total RNA was extracted using the Tri-Reagent procedure (Molecular Research, Cincinnati, OH). Total RNA (15 µg) was separated on a 1% denaturing agarose gel at 90 volts. RNA was transferred to nylon membrane overnight in 20-strength sodium citrate buffer (3 M NaCl, 0.3 M citrate) using a downward transfer system (Schleicher & Schull, Keene, NH) and cross-linked with UV light (0.29 J/cm2). Nucleic acid size was determined using RNA molecular weight markers (Bio-Rad). Equal loading was verified by ethidium bromide staining.
Membranes were hybridized with a 32P-labeled EP1 cDNA probe (Merck Frosst, Quebec, PQ, Canada) in high-efficiency hybridization buffer (Molecular Research) containing 1% SDS and 0.1 M NaCl overnight at 60°C. Blots were washed 3 times in single-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate)/0.1% SDS buffer for 7 min at 55°C and developed by autoradiography. Bands were quantified using image analysis software (Jandel Scientific). One Northern blot on each flask studied was performed (n = 8 flasks per concentration of CRH).
Immunohistochemistry
Six replicates of AWC were seeded into chamber slides (Lab Tech, Naperville, IL) and incubated as described above with or without CRH. Slides incubated in medium alone served as controls.
Once cells reached 80% confluence, they were incubated in medium containing CRH (50 ng/ml) for 18 h. After incubation in CRH, the slides were washed in 20 mM Tris-buffered saline (TBS), pH 7.5, and fixed for 2 min in cold absolute alcohol. Fixed slides were blocked with 1% milk protein, incubated for 15 min in 3% H2O2, and rinsed again in TBS. The slides were then incubated for 30 min at room temperature with a polyclonal rabbit anti-human EP1 antibody, diluted 1:800, as previously described [11]. The specificity of the polyclonal peptide-specific antibody to the EP1 receptor was determined by an ELISA as has been previously reported [11]. Antibody-treated slides were washed twice in TBS and stained by an indirect immunoperoxidase methodology using streptavidin-biotin and diaminobenzidine (DAB) as the color-producing agent (Dako Corp., Carpinteria, CA). All incubations were performed at room temperature. Negative controls were run by the sequential elimination of each immunohistochemical agent and by exposure of cells to both purified rabbit IgG and or whole, nonimmune rabbit serum prior to exposure to the secondary antibody. Positive controls were also performed using human red blood cells.
After immunohistochemical staining, slides were viewed by standard light microscopy either unstained or counterstained with hematoxylin and mounted in glycerine jelly.
Statistical Analysis
Statistical analysis of EP1 receptor protein density, mRNA density, and culture fluid PGE2 levels was performed using a one-way ANOVA. Differences between groups were determined using a Student-Newman-Keuls multiple t-test. A P < 0.05 was considered significant. Correlation values (r) were estimated by performing a linear regression.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Incubation of AWC with CRH also caused a significant increase in EP1 mRNA expression (Fig. 3). A significant but not dose-dependent difference in EP1 mRNA was observed between controls and 25 and 50 ng/ml of CRH (P < 0.01, n = 8 per concentration) (Fig. 4).
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Coincubation of AWC with CRH (50 ng/ml) and indomethacin (10 µg/ml) also resulted in the increased expression of EP1 mRNA relative to control. However, no difference in EP1 mRNA expression was observed between cultures incubated in CRH and indomethacin combined and cultures incubated in 50 ng/ml CRH alone.
Incubation of AWC with CRH resulted in a dose-dependent increase in culture fluid PGE2 levels (r = 0.74) (Table 1). A significant difference in PGE2 concentration (pg/µg protein) was observed between control and concentrations of 10, 25, and 50 ng/ml of CRH (P < 0.01, n = 6 per concentration). A strong correlation was also observed between EP1 protein levels and culture fluid PGE2 concentration (r = 0.85). Coincubation of AWC with CRH (50 ng/ml) and indomethacin (10 µg/ml) reduced PGE2 concentrations to near control levels. A significant difference in PGE2 levels was observed between groups of AWC incubated in CRH alone and in CRH combined with indomethacin (P < 0.01, n = 6 per concentration).
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Immunohistochemical analysis confirmed the expression of the EP1 receptor in both unstimulated and CRH-stimulated AWC (Fig. 5). The receptor appeared to be membrane-associated and cytoplasmic in distribution. In the unstimulated state (Fig. 5A), small nests of immunochemically positive cells were seen adjacent to regions of immunochemically negative amniocytes. Incubation of cells in CRH (50 ng/ml) resulted in increased DAB precipitation per cell as well as an increase in the size of the positive cell "nests" (Fig. 5B). In both stimulated and nonstimulated cultures, the distribution of the EP1 receptors was not homogenous along the cell membrane. Discrete areas void of receptor were visible within the cells and established a polarity that was visible independently of CRH stimulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is a growing body of evidence to suggest that placental CRH may be involved in the initiation of human term and/or preterm labor. CRH is produced in amnion, chorion, placental, and myometrial tissues during pregnancy and especially prior to the onset of labor. [3] The late stages of pregnancy are also associated with a state of "hypercorticalism" in maternal plasma [14] that is believed to be in part due to placental production of CRH [15]. Along these same lines, during the last weeks of pregnancy, plasma CRH-binding protein levels decrease dramatically in association with increased CRH [5]. Production of CRH by human fetal membranes is also regulated by the steroid hormones including dexamethasone and progesterone [16] and is elevated in women with preterm labor [6].
Several roles for CRH during pregnancy and labor have been postulated, but its exact mechanism is still unclear. Incubation of placental tissues with CRH causes the secretion of ß-endorphin and 
-melanocyte-stimulating hormone [17]. CRH also causes the stimulation of PGE2 and PGF2 in fetal membranes and placental tissues [1] and stimulates nitric oxide production and subsequent vasodilation in the endothelium of placental vessels [18].
In this study, CRH increased the expression of the PGE2 EP1 subtype receptor as demonstrated by the increased expression of EP1 protein and mRNA and PGE2 concentrations in amnion cell culture. Immunohistochemical analysis confirmed that an increased number of amnion cells were being "recruited" to express the EP1 receptor protein after exposure to CRH. It is also clear from these results that CRH-induced increased EP1 expression in amnion is independent of PGE2 production and is not a function of a "feedback loop."
Amnion tissue, in addition to its obvious physical role, is the source of several substances believed to be important to the maintenance of pregnancy and the onset of labor. Amnion produces PGE2 [7], endothelin-1 (a vasoconstrictor) [19] parathyroid hormone-related protein (a potent vasodilator) [20], CRH [4], and cytokines [21].
It is not clear at this time what the role of the amnion EP1 receptor is during pregnancy. Stimulation of the EP1 receptor is associated with increased intracellular calcium second messenger systems [9]. In the kidney, EP1 receptor expression is specifically localized to the collecting ducts where PGE2 attenuates the vasopressin-induced osmotic water permeability through calcium mobilization [22]. EP1 receptors also mediate the contraction of smooth muscle in various tissues including the gastrointestinal tract, respiratory tract, vas deferens, myometrium, and iris sphincter [10]. It is possible that stimulation of the amnion EP1 receptors may be responsible for increased production of vasoactive substances in amnion during the initiation of labor or in the regulation of amniotic fluid volume. It is also possible that CRH may be involved in the effect of cytokines during term or preterm labor. Previous work by our laboratory has demonstrated that cytokines increase EP1 and EP3 receptor density in amnion cells [11], and CRH levels are elevated during preterm labor [3]. Studies are currently being conducted in our laboratory to investigate this possibility as well as to examine whether this system is also present in myometrial tissue. Despite these questions, it is now clear that CRH stimulates the dose-dependent expression of the PG EP1 receptor in AWC culture, perhaps by recruitment of cells expressing the receptor. This suggests an additional role for CRH in placental and fetal tissues during pregnancy and also suggests that the CRH-regulated expression of EP1 receptors may be involved in the initiation of labor.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: Eric P. Spaziani, University of South Florida Health Science Center, Department of Obstetrics & Gynecology, Box 18, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612. FAX: 813 974 7026; espazian{at}com1.med.usf.edu 
Accepted: August 19, 1999.
Received: May 14, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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upregulates the prostaglandin E2 EP1 receptor subtype and the cyclooxygenase-2 isoform in cultured amnion WISH cells. J Interferon Cytokine Res 1998; 18:1039–1044.[Medline]This article has been cited by other articles:
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  R. Menon, C. P. Arora, C. J. Hobel, and S. J. Fortunato Corticotrophin-Releasing Hormone in Lipopolysaccharide-Stimulated Term Fetal Membranes and Amniotic Fluid From Term and Preterm Birth in African Americans and Caucasians Reproductive Sciences, May 1, 2008; 15(5): 477 - 483. [Abstract] [PDF]
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