- Email: [email protected]
Modulation of the prostaglandin e receptor: a possible mechanism for infection-induced preterm labor1
Modulation of the Prostaglandin E Receptor: A Possible Mechanism for Infection-Induced Preterm Labor ERIC P. SPAZIANI, PhD, WILLIAM F. O’BRIEN, MD, JOHN C. M. TSIBRIS, PhD, RAYMOND R. BENOIT, AND STANLEY F. GOULD, MD, PhD Objective: To evaluate the modulatory effects of interleukin (IL)-1b and prostaglandin (PG)E2 on the PGE2 receptor subtype EP1 in amnion cell cultures. Methods: Amnion cell cultures were incubated in increasing concentrations of (IL)-1b or PGE2. Cultures were also incubated in high concentrations of IL-1b and PGE2 in combination. Changes in EP1 receptor levels were evaluated by western and northern blot analysis. Culture fluid PGE2 levels were measured by enzyme-linked immunosorbent assay. Results: EP1 receptor protein levels decreased with increasing levels of PGE2 (r 5 2 0.82, P < .05). EP1 receptor protein (r 5 0.95, P < .05), EP1 mRNA (r 5 0.95, P < .01), and culture fluid PGE2 levels (P < .01) were all increased after IL-1b administration. EP1 receptor levels also increased approximately fourfold in response to IL-1b incubation even in the presence of high agonist (PGE2) concentrations (P < .01). Conclusion: The results of this study show that IL-1b might be involved in infection-induced preterm labor by interfering with the normal regulation of EP1 receptor levels and with the promotion of increased PGE2 production in amnion tissue. (Obstet Gynecol 1999;93:84 – 8. © 1999 by The American College of Obstetricians and Gynecologists.)
Although preterm birth is the leading cause of preventable neonatal morbidity and mortality in the United States,1 the underlying mechanisms and causes of the initiation of preterm labor are still poorly understood. Preterm labor is clearly associated with premature rupture of the membranes (PROM), but the cause(s) of PROM also is not well defined.2 Several factors are From the Department of Obstetrics and Gynecology, University of South Florida Health Science Center, Tampa, Florida. Dr. Kathleen Metters at the Merck Centre for Therapeutic Research, Quebec, Canada, provided the EP1 cDNA probe, Dr. Lois Hunt provided assistance in development of the EP1 antibody, and Dr. Allahyar Jazayeri provided assistance with northern blot analysis.
84 0029-7844/99/$20.00 PII S0029-7844(98)00361-5
associated with preterm labor, delivery of low-birthweight infants, and PROM. One factor is a strong correlation with infection.3 Histopathologic or microbiologic evidence of intra-amniotic infection is not uncommon in patients with preterm labor, PROM, or both.4 Increased amniotic fluid concentrations of prostaglandins, including PGE2, are well documented in term and preterm labor.5 The stimulatory effect of PGE2 on myometrial contractility is also well known.6 Although several hypotheses try to explain the relationship between infection and preterm labor-PROM, most biochemical evidence points to infectionassociated or -induced cytokine production, including interleukin (IL)-1b, which leads to increased myometrial activity.7 The relationship between increased cytokine production and myometrial activity is unlikely to be direct, and an intermediary such as PGE2 probably has a sizable influence on the cellular mechanisms that cause regular and sustained premature uterine contractility. The actions of PGE2 are mediated through its binding to specific membrane receptors. When activated, these receptors activate second messenger systems that initiate cellular response. Four subtypes of PGE2 receptor have been identified (EP1, EP2, EP3, and EP4), and these subtypes vary in their response to various agonists and antagonists.8 In general, the EP1 and EP3 receptors are associated with stimulatory functions within cells, and EP1 receptors are associated with changes in intracellular calcium9 and uterine muscle contraction.10 Despite abundant evidence of an association between labor and increased prostaglandins and cytokines in amniotic fluid, the mechanisms for production and regulation of these agents during infection-induced preterm labor are still unknown. The regulation of PGE2 receptors in maternal-fetal tissues has not been ex-
Obstetrics & Gynecology
plored. If the theory that infection-induced cytokine production leads to increased production of prostaglandins is correct, then the most likely initial target for infection-produced cytokines is the fetal amnion, because its cells are anatomically the first to be exposed. Therefore, we investigated the effect of IL-1b on PGE2 production and expression and modulation of the prostaglandin receptor subtype EP1 in amnion cell culture. If PGE2 induces human myometrial contraction and has a major influence on initiation and propagation of premature labor, then stimulation of amnion cells in culture by IL-1b should increase PGE2 production and affect PGE2 receptor levels.
Methods Cell Cultures The amnion cell line WISH (Wistar Institute Susan Hayflick) was used for these experiments. Wistar Institute Susan Hayflick cells (American Type Culture Collection, Washington, DC) were seeded into sterile vented 25-cm2 polyethylene tissue culture flasks at a concentration of approximately 5 3 106 cells per flask. The culture medium consisted of Dulbecco modified Eagle medium F-12 Ham mixture (1:1) containing 15% heat-inactivated fetal calf serum supplemented with 15 mmol HEPES, 2 mmol l-glutamine, 100 g/mL gentamicin, and 25 mg/mL amphotericin B. Cultures were grown to 80% confluence in a humidified incubator at 37C and 5% carbon dioxide. After the cells reached confluence, the culture media were removed, the cells washed in phosphate buffered saline, and incubated for 24 hours in either IL-1b (2, 10, 25, or 50 ng/mL) or PGE2 (100, 200, or 300 nmol). Cultures were also incubated with IL-1b (50 ng/mL) in combination with PGE2 (300 nmol). Cultures incubated in medium alone served as controls. There were six replicates per concentration of IL-1b, PGE2, or combination for western blot analysis, and four replicates per concentration for northern analysis. Western Blot Analysis After incubation in IL-1b and PGE2, cells were scraped and homogenized on ice with a glass-Teflon homogenizer in 50 mmol Tris, pH 7.5, containing 2 mmol ethylenediaminetetra-acetic acid, 0.25 mol sucrose, 10 mg/mL leupeptin, 50 mg/mL pepstatin A, 1 mmol phenylmethylsulfonyl fluoride, 1 mmol dithiothreitol, and 1% Triton-X 100. After 30 minutes, the crude homogenate was subsequently centrifuged at 1000 3 g for 15 minutes. Crude homogenates were assayed for protein content using a microbicinchonicic acid method
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with bovine serum albumin as standard.11 An aliquot of culture fluid was collected for PGE2 measurement by enzyme-linked immunosorbent assay. Changes in EP1 receptor protein levels were analyzed by western blot. Twenty-five micrograms of protein from each lysate were added per well to a 7.5% Trisglycine gel and were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Molecular weights were estimated using prestained molecular weight markers (Bio-Rad Labs, Hercules, CA). After electrophoresis, proteins were transferred to nitrocellulose membranes for 2 hours at 200 mA in 25 mmol Tris and 192 mmol glycine buffer, pH 8.3, containing 20% methanol. EP1 receptor bands were detected with EP1 polyclonal antibodies, prepared as previously described.12,13 As secondary antibody, an antirabbit immunoglobulin G with a horseradish conjugate was used (Promega, Madison, WI). Prestained molecular weight markers were used to identify molecular weights for EP1 receptor proteins. Blots were developed with an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL), and band intensity was measured with the Sigmagel analysis program (Jandel Scientific, San Rafael, CA) after scanning the gels (HewlettPackard Scanjet 3C scanner; Hewlett Packard, Wilmington, DE).
Northern Blot Analysis Changes in EP1 mRNA synthesis were evaluated by northern blot analysis. RNA was extracted from cultured cells by using the Tri-Reagent procedure (Molecular Research, Inc, Cincinnati, OH). Absorbance at 260 nm was used to estimate total RNA. The A260:A280 ratio was used to estimate the purity of the preparation. Total RNA (15 mg) was separated on a 1% denaturing agarose gel at 90 V. RNA was transferred to nylon membranes overnight in 20X saline sodium citrate buffer (3 mol NaCl, 0.3 mol C6H5Na3O7 z 2H2O) using a downward transfer system (Schleicher & Schull, Keene, NH) and cross-linked with ultraviolet light (0.29 J/cm2). Nucleic acid size was determined using RNA molecular weight markers (Bio-Rad Labs). Equal loading was verified by ethidium-bromide staining. Membranes were hybridized with a denatured EP1 32 P-labeled cDNA probe (Merck, Frosst, Quebec) in High Efficiency Hybridization Buffer (Molecular Research, Cincinnati, OH) containing 1% sodium dodecyl sulfate and 0.1 mol NaCl overnight at 60C. Blots were washed 3 times in 1X saline sodium citrate/0.1% sodium dodecyl sulfate buffer for 7 minutes at 55C and developed by autoradiography. Changes in band den-
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Table 1. Effect of Interleukin-1b on Culture Fluid Prostaglandin E2 IL-1b concentration (ng/mL) 10 25
0
2
0.79 6 0.01
1.18 6 0.06
5.34 6 0.06
*
6.32 6 0.32*
50 6.61 6 0.37*
IL 5 interleukin. Values are expressed as picogram/microgram protein per day. Data are expressed as mean 6 standard error, for six replicates per concentration. * Significant difference from control.
sities were estimated using image analysis software (Jandel Scientific, San Rafael, CA).
IL-1b alone (50 ng/mL) and cultures incubated in IL-1b and PGE2 (300 mmol) in combination. Northern blot analysis demonstrated a dosedependent increase in EP1 mRNA (4.6 kb) with increasing concentrations of IL-1b (r 5 0.95). A significant difference in band density was observed for concentrations of IL-1b of 10 ng/mL or greater compared with controls (P , .01) (Figure 2). Incubation of amnion cells in increasing concentrations of PGE2 had no significant effect on EP1 mRNA levels. Co-incubation of amnion cells in high concentrations of both PGE2 and IL-1b resulted in a threefold increase in EP1 mRNA over cells incubated in high PGE2 alone (P , .05) (Figure 3).
Discussion Statistical Analysis Statistical analysis of EP1 receptor protein band density, EP1 mRNA band density, and culture fluid PGE2 levels was performed using a one-way analysis of variance. Differences between groups were determined using Student-Newman-Keuls multiple t test. P , .05 was considered significant. Correlation coefficients (r) were estimated by performing a linear regression between the concentration of IL-1b or PGE2 used and the mean band density calculated for each concentration.
As shown by previous investigators14 and confirmed by us, the exposure of amnion Wistar Institute Susan Hayflick cells to IL-1b produces a dramatic increase in PGE2 production. However, what was not expected and what is physiologically significant, is the observation that, in the presence of IL-1b, the increased production of PGE2 is not accompanied by a concomitant decrease in the PGE2 EP1 receptor level. Stimulation of the IL-1b
Results When Wistar Institute Susan Hayflick cells were incubated in increasing concentrations of IL-1b there was a dramatic increase in culture fluid PGE2 levels (pg/mg protein per day) for concentrations of 10 ng/mL or greater compared with media controls (P , .01) (Table 1). Western blot analysis of cell homogenates showed the presence of the EP1 receptor protein, which appeared as a single band with a molecular weight of approximately 42 kd. Incubation of amnion Wistar Institute Susan Hayflick cells in increasing concentrations of IL-1b resulted in a dose-dependent increase in EP1 receptor protein (r 5 0.95). A significant difference from control in EP1 protein was observed for concentrations of 10 ng/mL and higher (P , .05) (Figure 1A). EP1 receptor protein levels were effectively downregulated by increasing concentration of the PGE2 agonist (r 5 20.82). A significant difference from control in band density was observed for PGE2 concentrations of 200 and 300 mmol (P , .05) (Figure 1B). Co-incubation of amnion cells in high concentrations of both PGE2 (300 nmol) and IL-1b (50 ng/mL) resulted in a fourfold increase in the EP1 receptor protein levels over those of controls (Figure 1B). No significant difference in band density was detected between cultures incubated in
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Figure 1. Western blot analysis for EP1 proteins of WISH cells incubated with prostaglandin (PG) E2 and interleukin (IL)-1b. A) Representative western blot for EP1 proteins incubated with increasing concentrations of IL-1b. B) Representative western blot for EP1 proteins incubated in increasing concentrations of PGE2 alone and in combination with IL-1b. Lane 1, Control. Lanes 2– 4, PGE2 alone in concentrations of 100, 200, and 300 nmol. Lane 5, PGE2 (300 nmol) and IL-1b (50 ng/mL) in combination. Lane 6, IL-1b alone (50 ng/mL).
Obstetrics & Gynecology
Figure 2. Representative northern blot of EP1 messenger RNA for cells incubated in increasing concentrations of interleukin-1b. Top, Graphic representation of band density. Middle, northern blot for EP1 mRNA. The migration of the 28S ribosomal RNA band is indicated in the figure. Bottom, Equal loading was verified by ethidium-bromide staining of the 28s and 18s RNA bands.
receptor results in an increase both in amount and in synthesis of the EP1 receptor as evidenced by increased protein and mRNA levels. Thus, the expected downregulation of the EP1 receptor in response to increasing amounts of PGE2 in the culture medium did not occur when amnion cells were incubated in the presence of IL-1b. When amnion Wistar Institute Susan Hayflick cells were incubated with the agonist PGE2 in the absence of concomitant IL-1b stimulation, the expected reduction of EP1 receptor protein occurred in a dosedependent manner.
Figure 3. Representative northern blot of EP1 mRNA for cells incubated in increasing concentrations of prostaglandin (PG) E2, PGE2 and interleukin (IL)-1b in combination, and IL-1b alone. Top, Graphic representation of band density. Middle, northern blot for EP1 mRNA. The migration of the 28S ribosomal RNA band is indicated in the figure. Bottom, Equal loading was verified by ethidium-bromide staining of the 28s and 18s RNA bands.
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This apparent short-circuiting of the normal pathways of receptor downregulation in the presence of increasing concentrations of agonist provides a possible starting point for investigating amnion-mediated stimulation of the myometrium. It is tempting to hypothesize that cytokine stimulation of amnion cells in vivo would produce the same increase in PGE2 production without a concomitant dose-related downregulation of the EP1 receptor. Continued stimulation by infectioninduced cytokine release would not only produce continued and sustained release of PGE2, but would also cause a decrease in the stimulatory threshold required to affect PGE2 production by the amniocyte. If this conclusion is true, then the production of PGE2 by infection-induced production of endogenous cytokines would fail to respond to normal cellular control mechanisms and would lead to irreversible stimulation of the myometrium. Because there is little evidence of a direct effect of cytokines on myometrial contractility, it is likely that they act through one or more mediators. A likely candidate for this role is PGE2. Unlike oxytocin, which has minimal effect on uterine smooth muscle until close to term, PGE2 stimulates myometrial activity in nonpregnant women as well as throughout gestation. Because the amnion is a well-established source of PGE2 and is in direct contact with both amniotic fluid and the uterine decidua, it is a good candidate for the production of sufficient quantities of PGE2 to induce and sustain continued and coordinated uterine contractions. Stimulation by IL-1b resulted in the increased amount and synthesis of PGE2 EP1 receptors, evidenced by increased protein and mRNA levels. Increased EP1 receptor concentrations in amnion would increase its sensitivity to stimulation by the agonist PGE2. This would require less agonist to elicit the same response and could explain the sensitivity of the myometrium to small quantities of PGE2. The actions of most cytokines are pleiotropic in their biological effects, in that they exert multiple effects on several tissue types.15 The action of IL-1b in amnion Wistar Institute Susan Hayflick cells is consistent in this action by its ability to stimulate both PGE2 production by cyclooxygenase stimulation and increase in PGE2 EP1 receptor levels. Amnion cells co-incubated in high concentrations of PGE2 and IL-1b, a state that might be found in a patient with chorioamnionitis, also resulted in increased EP1 receptor promotion. This highly unusual response indicates that the level of IL-1b stimulation of EP1 receptor production exceeds the rate of internalization of the agonist-bound receptor. It is also possible that IL-1b might slow the rate of agonistinduced receptor internalization. The present study focused specifically on the EP1
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subtype receptor. It is known that four distinct receptor subtypes exist and that the physiologic effect of exposure to PGE2 is determined by which of the four receptor isoforms (and unique second messenger system) is stimulated. The EP1 isoform becomes especially important in the study of infection-associated preterm or term labor because of its known association with the opening of membrane calcium channels and increase in intracellular calcium.10 In view of the fact that calcium channel blockers are presently used in the clinical treatment of preterm labor, any data that further elucidate the role and function of the EP1 receptor is clinically important.
References 1. Cooper RL, Goldberg RL, Creasy RK, DuBard MB, Davis RO, Entman SS, et al. A multicenter study of preterm birth weight and gestational age-specific neonatal mortality. Am J Obstet Gynecol 1993;168:78 – 84. 2. Shapiro S, McCormick MC, Starfield BH, Krischer JP, Bross D. Relevance of correlates of infant deaths for significant morbidity at 1 year of age. Am J Obstet Gynecol 1988;136:363–73. 3. Gibbs RS, Romero R, Hillier SL, Eschenbach DA, Sweet RL. A review of premature birth and subclinical infection. Am J Obstet Gynecol 1992;166:1515–28. 4. Hillier SL, Martius J, Krohn MJ. A case control study of chorioamnionitis infection and histologic chorioamnionitis in prematurity. N Engl J Med 1988;319:972– 8. 5. Romero R, Baumann P, Gomez R. The relationship between spontaneous rupture of membranes, labor, and microbial invasion of the amniotic cavity and amniotic fluid concentrations of prostaglandins, and thromboxane B2 in term pregnancy. Am J Obstet Gynecol 1968;168:1654 – 68. 6. Romero R, Munoz H, Gomez H, Parra H, Polanco M, Valverde V, et al. Increase in prostaglandin bioavailability precedes the onset of human parturition. Prostaglandins Leukot Essential Fatty Acids 1995;54:187–93. 7. Romero R, Brody DT, Oyarzun E, Mazor M, Wu YK, Hobbins JC, et al. Infection and labor III. Interleukin-1: A signal for the onset of parturition. Am J Obstet Gynecol 1989;160:1117–23.
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8. Negishi MY, Sugimotto Y, Ichikawa I. Molecular mechanisms of diverse actions of prostanoid receptors. Biochem Biophys Acta 1995;1259:109 –20. 9. Ichikawa I, Sugimoto Y, Negishi MY. Molecular aspects of the structures and functions of prostaglandin E receptors. Journal of Lipid Mediators and Cell Signalling 1996;14:83–7. 10. Asboth G, Phaneuf S, Europe-Finner N, Toth M, Bernal A. Prostaglandin E2 activates phospholipase C and elevates intracellular calcium in cultured myometrial cells: Involvement of EP1 and EP3 receptor subtypes. Endocrinology 1996;137:2572–9. 11. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provanzano MD, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76 – 85. 12. Spaziani EP, Tsibris JCM, Hunt LT, Benoit RR, O’Brien WF. The effect of Interleukin-1b and interleukin-4 on the expression of prostaglandin receptors EP1 and EP3 in amnion WISH cells. Am J Reprod Immunol 1997;38:279 – 85. 13. Funk CD, Furci L, Fitzgerald GA, Grygorczyk R, Rochett C, Bayne M, et al. Cloning and expression of a cDNA for human prostaglandin E receptor EP1 subtype. J Biol Chem 1993;268:26767–72. 14. Peplow PV. Actions of cytokines in relation to arachidonic acid metabolism and eicosanoid production. Prostaglandins Leukot Essential Fatty Acids 1996;54:303–17. 15. Trotta PP. Cytokines: An overview. Am J Reprod Immunol 1991; 25:137– 41.
Address reprint requests to:
Eric P. Spaziani, PhD University of South Florida Health Science Medical Center Department of Obstetrics and Gynecology Box 18, 12901 Bruce B. Downs Boulevard Tampa, FL 33612 E-mail: [email protected]
Received May 6, 1998. Received in revised form July 14, 1998. Accepted July 30, 1998. Copyright © 1999 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.
Obstetrics & Gynecology
Although preterm birth is the leading cause of preventable neonatal morbidity and mortality in the United States,1 the underlying mechanisms and causes of the initiation of preterm labor are still poorly understood. Preterm labor is clearly associated with premature rupture of the membranes (PROM), but the cause(s) of PROM also is not well defined.2 Several factors are From the Department of Obstetrics and Gynecology, University of South Florida Health Science Center, Tampa, Florida. Dr. Kathleen Metters at the Merck Centre for Therapeutic Research, Quebec, Canada, provided the EP1 cDNA probe, Dr. Lois Hunt provided assistance in development of the EP1 antibody, and Dr. Allahyar Jazayeri provided assistance with northern blot analysis.
84 0029-7844/99/$20.00 PII S0029-7844(98)00361-5
associated with preterm labor, delivery of low-birthweight infants, and PROM. One factor is a strong correlation with infection.3 Histopathologic or microbiologic evidence of intra-amniotic infection is not uncommon in patients with preterm labor, PROM, or both.4 Increased amniotic fluid concentrations of prostaglandins, including PGE2, are well documented in term and preterm labor.5 The stimulatory effect of PGE2 on myometrial contractility is also well known.6 Although several hypotheses try to explain the relationship between infection and preterm labor-PROM, most biochemical evidence points to infectionassociated or -induced cytokine production, including interleukin (IL)-1b, which leads to increased myometrial activity.7 The relationship between increased cytokine production and myometrial activity is unlikely to be direct, and an intermediary such as PGE2 probably has a sizable influence on the cellular mechanisms that cause regular and sustained premature uterine contractility. The actions of PGE2 are mediated through its binding to specific membrane receptors. When activated, these receptors activate second messenger systems that initiate cellular response. Four subtypes of PGE2 receptor have been identified (EP1, EP2, EP3, and EP4), and these subtypes vary in their response to various agonists and antagonists.8 In general, the EP1 and EP3 receptors are associated with stimulatory functions within cells, and EP1 receptors are associated with changes in intracellular calcium9 and uterine muscle contraction.10 Despite abundant evidence of an association between labor and increased prostaglandins and cytokines in amniotic fluid, the mechanisms for production and regulation of these agents during infection-induced preterm labor are still unknown. The regulation of PGE2 receptors in maternal-fetal tissues has not been ex-
Obstetrics & Gynecology
plored. If the theory that infection-induced cytokine production leads to increased production of prostaglandins is correct, then the most likely initial target for infection-produced cytokines is the fetal amnion, because its cells are anatomically the first to be exposed. Therefore, we investigated the effect of IL-1b on PGE2 production and expression and modulation of the prostaglandin receptor subtype EP1 in amnion cell culture. If PGE2 induces human myometrial contraction and has a major influence on initiation and propagation of premature labor, then stimulation of amnion cells in culture by IL-1b should increase PGE2 production and affect PGE2 receptor levels.
Methods Cell Cultures The amnion cell line WISH (Wistar Institute Susan Hayflick) was used for these experiments. Wistar Institute Susan Hayflick cells (American Type Culture Collection, Washington, DC) were seeded into sterile vented 25-cm2 polyethylene tissue culture flasks at a concentration of approximately 5 3 106 cells per flask. The culture medium consisted of Dulbecco modified Eagle medium F-12 Ham mixture (1:1) containing 15% heat-inactivated fetal calf serum supplemented with 15 mmol HEPES, 2 mmol l-glutamine, 100 g/mL gentamicin, and 25 mg/mL amphotericin B. Cultures were grown to 80% confluence in a humidified incubator at 37C and 5% carbon dioxide. After the cells reached confluence, the culture media were removed, the cells washed in phosphate buffered saline, and incubated for 24 hours in either IL-1b (2, 10, 25, or 50 ng/mL) or PGE2 (100, 200, or 300 nmol). Cultures were also incubated with IL-1b (50 ng/mL) in combination with PGE2 (300 nmol). Cultures incubated in medium alone served as controls. There were six replicates per concentration of IL-1b, PGE2, or combination for western blot analysis, and four replicates per concentration for northern analysis. Western Blot Analysis After incubation in IL-1b and PGE2, cells were scraped and homogenized on ice with a glass-Teflon homogenizer in 50 mmol Tris, pH 7.5, containing 2 mmol ethylenediaminetetra-acetic acid, 0.25 mol sucrose, 10 mg/mL leupeptin, 50 mg/mL pepstatin A, 1 mmol phenylmethylsulfonyl fluoride, 1 mmol dithiothreitol, and 1% Triton-X 100. After 30 minutes, the crude homogenate was subsequently centrifuged at 1000 3 g for 15 minutes. Crude homogenates were assayed for protein content using a microbicinchonicic acid method
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with bovine serum albumin as standard.11 An aliquot of culture fluid was collected for PGE2 measurement by enzyme-linked immunosorbent assay. Changes in EP1 receptor protein levels were analyzed by western blot. Twenty-five micrograms of protein from each lysate were added per well to a 7.5% Trisglycine gel and were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Molecular weights were estimated using prestained molecular weight markers (Bio-Rad Labs, Hercules, CA). After electrophoresis, proteins were transferred to nitrocellulose membranes for 2 hours at 200 mA in 25 mmol Tris and 192 mmol glycine buffer, pH 8.3, containing 20% methanol. EP1 receptor bands were detected with EP1 polyclonal antibodies, prepared as previously described.12,13 As secondary antibody, an antirabbit immunoglobulin G with a horseradish conjugate was used (Promega, Madison, WI). Prestained molecular weight markers were used to identify molecular weights for EP1 receptor proteins. Blots were developed with an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL), and band intensity was measured with the Sigmagel analysis program (Jandel Scientific, San Rafael, CA) after scanning the gels (HewlettPackard Scanjet 3C scanner; Hewlett Packard, Wilmington, DE).
Northern Blot Analysis Changes in EP1 mRNA synthesis were evaluated by northern blot analysis. RNA was extracted from cultured cells by using the Tri-Reagent procedure (Molecular Research, Inc, Cincinnati, OH). Absorbance at 260 nm was used to estimate total RNA. The A260:A280 ratio was used to estimate the purity of the preparation. Total RNA (15 mg) was separated on a 1% denaturing agarose gel at 90 V. RNA was transferred to nylon membranes overnight in 20X saline sodium citrate buffer (3 mol NaCl, 0.3 mol C6H5Na3O7 z 2H2O) using a downward transfer system (Schleicher & Schull, Keene, NH) and cross-linked with ultraviolet light (0.29 J/cm2). Nucleic acid size was determined using RNA molecular weight markers (Bio-Rad Labs). Equal loading was verified by ethidium-bromide staining. Membranes were hybridized with a denatured EP1 32 P-labeled cDNA probe (Merck, Frosst, Quebec) in High Efficiency Hybridization Buffer (Molecular Research, Cincinnati, OH) containing 1% sodium dodecyl sulfate and 0.1 mol NaCl overnight at 60C. Blots were washed 3 times in 1X saline sodium citrate/0.1% sodium dodecyl sulfate buffer for 7 minutes at 55C and developed by autoradiography. Changes in band den-
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Table 1. Effect of Interleukin-1b on Culture Fluid Prostaglandin E2 IL-1b concentration (ng/mL) 10 25
0
2
0.79 6 0.01
1.18 6 0.06
5.34 6 0.06
*
6.32 6 0.32*
50 6.61 6 0.37*
IL 5 interleukin. Values are expressed as picogram/microgram protein per day. Data are expressed as mean 6 standard error, for six replicates per concentration. * Significant difference from control.
sities were estimated using image analysis software (Jandel Scientific, San Rafael, CA).
IL-1b alone (50 ng/mL) and cultures incubated in IL-1b and PGE2 (300 mmol) in combination. Northern blot analysis demonstrated a dosedependent increase in EP1 mRNA (4.6 kb) with increasing concentrations of IL-1b (r 5 0.95). A significant difference in band density was observed for concentrations of IL-1b of 10 ng/mL or greater compared with controls (P , .01) (Figure 2). Incubation of amnion cells in increasing concentrations of PGE2 had no significant effect on EP1 mRNA levels. Co-incubation of amnion cells in high concentrations of both PGE2 and IL-1b resulted in a threefold increase in EP1 mRNA over cells incubated in high PGE2 alone (P , .05) (Figure 3).
Discussion Statistical Analysis Statistical analysis of EP1 receptor protein band density, EP1 mRNA band density, and culture fluid PGE2 levels was performed using a one-way analysis of variance. Differences between groups were determined using Student-Newman-Keuls multiple t test. P , .05 was considered significant. Correlation coefficients (r) were estimated by performing a linear regression between the concentration of IL-1b or PGE2 used and the mean band density calculated for each concentration.
As shown by previous investigators14 and confirmed by us, the exposure of amnion Wistar Institute Susan Hayflick cells to IL-1b produces a dramatic increase in PGE2 production. However, what was not expected and what is physiologically significant, is the observation that, in the presence of IL-1b, the increased production of PGE2 is not accompanied by a concomitant decrease in the PGE2 EP1 receptor level. Stimulation of the IL-1b
Results When Wistar Institute Susan Hayflick cells were incubated in increasing concentrations of IL-1b there was a dramatic increase in culture fluid PGE2 levels (pg/mg protein per day) for concentrations of 10 ng/mL or greater compared with media controls (P , .01) (Table 1). Western blot analysis of cell homogenates showed the presence of the EP1 receptor protein, which appeared as a single band with a molecular weight of approximately 42 kd. Incubation of amnion Wistar Institute Susan Hayflick cells in increasing concentrations of IL-1b resulted in a dose-dependent increase in EP1 receptor protein (r 5 0.95). A significant difference from control in EP1 protein was observed for concentrations of 10 ng/mL and higher (P , .05) (Figure 1A). EP1 receptor protein levels were effectively downregulated by increasing concentration of the PGE2 agonist (r 5 20.82). A significant difference from control in band density was observed for PGE2 concentrations of 200 and 300 mmol (P , .05) (Figure 1B). Co-incubation of amnion cells in high concentrations of both PGE2 (300 nmol) and IL-1b (50 ng/mL) resulted in a fourfold increase in the EP1 receptor protein levels over those of controls (Figure 1B). No significant difference in band density was detected between cultures incubated in
86 Spaziani et al
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Figure 1. Western blot analysis for EP1 proteins of WISH cells incubated with prostaglandin (PG) E2 and interleukin (IL)-1b. A) Representative western blot for EP1 proteins incubated with increasing concentrations of IL-1b. B) Representative western blot for EP1 proteins incubated in increasing concentrations of PGE2 alone and in combination with IL-1b. Lane 1, Control. Lanes 2– 4, PGE2 alone in concentrations of 100, 200, and 300 nmol. Lane 5, PGE2 (300 nmol) and IL-1b (50 ng/mL) in combination. Lane 6, IL-1b alone (50 ng/mL).
Obstetrics & Gynecology
Figure 2. Representative northern blot of EP1 messenger RNA for cells incubated in increasing concentrations of interleukin-1b. Top, Graphic representation of band density. Middle, northern blot for EP1 mRNA. The migration of the 28S ribosomal RNA band is indicated in the figure. Bottom, Equal loading was verified by ethidium-bromide staining of the 28s and 18s RNA bands.
receptor results in an increase both in amount and in synthesis of the EP1 receptor as evidenced by increased protein and mRNA levels. Thus, the expected downregulation of the EP1 receptor in response to increasing amounts of PGE2 in the culture medium did not occur when amnion cells were incubated in the presence of IL-1b. When amnion Wistar Institute Susan Hayflick cells were incubated with the agonist PGE2 in the absence of concomitant IL-1b stimulation, the expected reduction of EP1 receptor protein occurred in a dosedependent manner.
Figure 3. Representative northern blot of EP1 mRNA for cells incubated in increasing concentrations of prostaglandin (PG) E2, PGE2 and interleukin (IL)-1b in combination, and IL-1b alone. Top, Graphic representation of band density. Middle, northern blot for EP1 mRNA. The migration of the 28S ribosomal RNA band is indicated in the figure. Bottom, Equal loading was verified by ethidium-bromide staining of the 28s and 18s RNA bands.
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This apparent short-circuiting of the normal pathways of receptor downregulation in the presence of increasing concentrations of agonist provides a possible starting point for investigating amnion-mediated stimulation of the myometrium. It is tempting to hypothesize that cytokine stimulation of amnion cells in vivo would produce the same increase in PGE2 production without a concomitant dose-related downregulation of the EP1 receptor. Continued stimulation by infectioninduced cytokine release would not only produce continued and sustained release of PGE2, but would also cause a decrease in the stimulatory threshold required to affect PGE2 production by the amniocyte. If this conclusion is true, then the production of PGE2 by infection-induced production of endogenous cytokines would fail to respond to normal cellular control mechanisms and would lead to irreversible stimulation of the myometrium. Because there is little evidence of a direct effect of cytokines on myometrial contractility, it is likely that they act through one or more mediators. A likely candidate for this role is PGE2. Unlike oxytocin, which has minimal effect on uterine smooth muscle until close to term, PGE2 stimulates myometrial activity in nonpregnant women as well as throughout gestation. Because the amnion is a well-established source of PGE2 and is in direct contact with both amniotic fluid and the uterine decidua, it is a good candidate for the production of sufficient quantities of PGE2 to induce and sustain continued and coordinated uterine contractions. Stimulation by IL-1b resulted in the increased amount and synthesis of PGE2 EP1 receptors, evidenced by increased protein and mRNA levels. Increased EP1 receptor concentrations in amnion would increase its sensitivity to stimulation by the agonist PGE2. This would require less agonist to elicit the same response and could explain the sensitivity of the myometrium to small quantities of PGE2. The actions of most cytokines are pleiotropic in their biological effects, in that they exert multiple effects on several tissue types.15 The action of IL-1b in amnion Wistar Institute Susan Hayflick cells is consistent in this action by its ability to stimulate both PGE2 production by cyclooxygenase stimulation and increase in PGE2 EP1 receptor levels. Amnion cells co-incubated in high concentrations of PGE2 and IL-1b, a state that might be found in a patient with chorioamnionitis, also resulted in increased EP1 receptor promotion. This highly unusual response indicates that the level of IL-1b stimulation of EP1 receptor production exceeds the rate of internalization of the agonist-bound receptor. It is also possible that IL-1b might slow the rate of agonistinduced receptor internalization. The present study focused specifically on the EP1
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subtype receptor. It is known that four distinct receptor subtypes exist and that the physiologic effect of exposure to PGE2 is determined by which of the four receptor isoforms (and unique second messenger system) is stimulated. The EP1 isoform becomes especially important in the study of infection-associated preterm or term labor because of its known association with the opening of membrane calcium channels and increase in intracellular calcium.10 In view of the fact that calcium channel blockers are presently used in the clinical treatment of preterm labor, any data that further elucidate the role and function of the EP1 receptor is clinically important.
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8. Negishi MY, Sugimotto Y, Ichikawa I. Molecular mechanisms of diverse actions of prostanoid receptors. Biochem Biophys Acta 1995;1259:109 –20. 9. Ichikawa I, Sugimoto Y, Negishi MY. Molecular aspects of the structures and functions of prostaglandin E receptors. Journal of Lipid Mediators and Cell Signalling 1996;14:83–7. 10. Asboth G, Phaneuf S, Europe-Finner N, Toth M, Bernal A. Prostaglandin E2 activates phospholipase C and elevates intracellular calcium in cultured myometrial cells: Involvement of EP1 and EP3 receptor subtypes. Endocrinology 1996;137:2572–9. 11. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provanzano MD, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76 – 85. 12. Spaziani EP, Tsibris JCM, Hunt LT, Benoit RR, O’Brien WF. The effect of Interleukin-1b and interleukin-4 on the expression of prostaglandin receptors EP1 and EP3 in amnion WISH cells. Am J Reprod Immunol 1997;38:279 – 85. 13. Funk CD, Furci L, Fitzgerald GA, Grygorczyk R, Rochett C, Bayne M, et al. Cloning and expression of a cDNA for human prostaglandin E receptor EP1 subtype. J Biol Chem 1993;268:26767–72. 14. Peplow PV. Actions of cytokines in relation to arachidonic acid metabolism and eicosanoid production. Prostaglandins Leukot Essential Fatty Acids 1996;54:303–17. 15. Trotta PP. Cytokines: An overview. Am J Reprod Immunol 1991; 25:137– 41.
Address reprint requests to:
Eric P. Spaziani, PhD University of South Florida Health Science Medical Center Department of Obstetrics and Gynecology Box 18, 12901 Bruce B. Downs Boulevard Tampa, FL 33612 E-mail: [email protected]
Received May 6, 1998. Received in revised form July 14, 1998. Accepted July 30, 1998. Copyright © 1999 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.
Obstetrics & Gynecology