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Regular Article |
a Division of Maternal-Fetal Medicine and Reproductive Biology Unit, Department of Obstetrics and Gynecology and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa Health Research Institute, Ottawa, Ontario, Canada K1H 8L6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B) in IL-1ß signaling, leading to the expression of COX-2 in human amnion cell culture. Fetal amnion was obtained following vaginal delivery and digested with collagenase, and the subepithelial (mesenchymal) cells were isolated. Cultures were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Confluent cells were stimulated with human recombinant IL-1ß, and activation of NFB was assessed by measuring changes in the inhibitory protein IB (total IB and phosphorylated IB) using Western blot analysis as well as by nuclear binding of NFB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1ß, phosphorylated IB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IB and increased nuclear NFB DNA-binding activity. In the IL-1ß-treated group, COX-2 protein began to increase after 6 h; this response was time-dependent, with a significant increase until 24 h after IL-1ß stimulation. When NFB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFB is involved in the IL-1ß-induced COX-2 expression in the mesenchymal cells of human amnion.
cytokines, parturition, placenta, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various substances have been found to regulate COX-2 expression in human fetal membranes, including the cytokine interleukin-1ß (IL-1ß) [9]. IL-1ß is also produced by the fetal membranes and has been shown to increase in concentration in the amniotic fluid of patients with infection during preterm labor [10]. Nuclear factor kappa B (NFB) is a key intermediate in IL-1ß stimulation of various genes and is involved in the stimulation of COX-2 expression in a number of systems [11–13]. NFB is composed of two subunits, p50 and p65, which are localized to the cytoplasm and are bound to a cytoplasmic retention protein, IB. Activation of NFB involves phosphorylation of IB and subsequent translocation of NFB into the nucleus, where it regulates various genes [13, 14].
Previous studies from our laboratory [15] have shown that IL-1ß stimulates COX-2 expression in primary cultures of amnion mesenchymal cells. The regulation of COX-2 expression in these cells is somewhat novel, because glucocorticoids have been shown to stimulate COX-2 expression [16] in contrast to other cell types, including an amnion-derived cell line (WISH cells) in which glucocorticoids inhibit COX-2 expression [17, 18]. Recently, NFB has been shown to be involved in the IL-1ß stimulation of COX-2 expression in WISH cells [18], and in recent studies using transfection, it has been reported that NFB is involved in COX-2 expression in a mixed population of amnion cells [19]. To our knowledge, however, the functional link between IL-1ß-induced NFB activation and COX-2 expression has not yet been described in primary cultures of human amnion mesenchymal cells.
To further characterize IL-1ß stimulation of COX-2 expression in amnion mesenchymal cells, we set out to examine whether the NFB signaling system was involved. The present results provide evidence that NFB activation is required for COX-2 protein expression when these cells are stimulated with IL-1ß.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collagenase A, trypsin inhibitor, and recombinant human IL-1ß were obtained from Boehringer Mannheim Canada (Dorval, PQ, Canada). D-MEM/F-12 medium and fetal bovine serum (FBS) were from Gibco-BRL (Burlington, ON, Canada). Keratin polyclonal rabbit antiserum and vimentin monoclonal mouse antibody were from DAKO (Glostrup, Denmark). The DAKO LSAB kit was obtained from DAKO Corporation (Mississauga, ON, Canada), and the Vectastain Elite Kit was purchased from Vectastain (Burlington, CA). Tris and PMSF were purchased from Sigma Chemical Co. (St. Louis, MO), and the enhanced chemiluminescence (ECL) Western blotting detection kit and [32P]dATP (30 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). Nitrocellulose membrane, acrylamide (electrophoresis grade), N,N'-methylene-bis-acrylamide, ammonium persulfate, dithiothreitol (DTT), glycine, and Bio-Rad protein assay kit were purchased from Bio-Rad Laboratories (Hercules, CA). X-ray film was from Eastman Kodak Company (Rochester, NY). Polyclonal rabbit anti-human COX-2 (PG 27) was purchased from Oxford Biomedical Research, Inc. (Oxford, MI). NFB probe and T4 polynucleotide kinase were from Promega (Madison, WI). Rabbit polyclonal anti-human phosphorylated and total IB-
antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). SN50 and SN50M were from BioMol Research Laboratories, Inc. (Madison, WI).
Isolation of Amnion Cells and Primary Culture
All manipulations were carried out under sterile conditions. Placental tissue was obtained according to the Ethical Guidelines approved by the Ottawa General Hospital. Tissues were isolated and cell cultures carried out as previously described [15]. Briefly, term (38- to 40-wk gestation) placentae with adherent membranes were obtained immediately after spontaneous vaginal delivery from patients with no clinical evidence of infection. Membranes were cut approximately 1 inch above the placental plate. Amnion was peeled from chorion laeve, washed several times in PBS (150 mM NaCl, 10 mM Na2HPO4, 1.5 mM NaH2PO4; pH 7.5) and finely minced with scissors. Tissue was transferred into a sterile tube containing 40 ml of PBS and 1 mg/ml of collagenase A and then incubated at 37°C with gentle shaking for 2 h. After digestion, 3 µg/ml of trypsin inhibitor were added, and the suspension was filtered through nylon screens of 100 and 35 µm. The cells were pelleted by centrifugation at 2000 x g for 10 min, washed once in PBS, counted on a hemocytometer, and suspended in culture medium. Culture medium consisted of a 1:1 (v:v) mixture of Ham F-12, and Dulbecco modified Eagle medium (DMEM/F-12), 10% fetal bovine serum, 1 g/L of BSA, 29 mM NaHCO3, 105 U/L of penicillin, 10 mg/ml of gentamicin, and 8 µg/ml of fungizone. Cells were plated in 35-mm dishes at 1.5 x 106 cells. They were maintained in culture at 37°C with a water-saturated atmosphere and 5% (v/v) CO2 and air. Media were changed after 2 and 24 h, respectively. Forty-eight hours later, cells were treated with IL-1ß (10 ng/ml) for various times. Control cells received media alone.
Quantification of Cell Types
Amnion consists of an epithelial layer of cells and a subepithelial mesenchymal layer of cells. In primary cultures, amnion mesenchymal cells were localized with vimentin monoclonal mouse antibody, and epithelial cells were localized with keratin polyclonal rabbit antiserum. Cultured cells were processed for immunohistochemistry with use of the DAKO LSAB kit. All antibody dilutions and washes for this kit were in PBS (150 mM NaCl, 10 mM Na2HPO4, 1.5 mM NaH2PO4; pH 7.5). Cells were permeabilized in 0.2% Triton X-100 for 1 h before staining. Endogenous peroxidase activity was quenched by 10 min of treatment with 3% H2O2 in PBS. Slides were washed twice for 5 min each time. Blocking serum was applied for 20 min, and the slides were incubated with primary antibody in 0.1% (v/v) BSA overnight at 4°C. The primary antibody dilutions found to be appropriate for this kit were 1:3000 (v/v) antivimentin and 1:4000 (v/v) antikeratin. Slides were washed twice for 5 min each time, incubated with biotinylated secondary antibody for 30 min, washed twice for 5 min each time, incubated with streptavidin kit for 30 min, and washed twice for 5 min each time. Immunoreactive proteins were visualized using the DAKO aminoethyl carbazole substrate system (5–15 min). Slides were counterstained for 1 min in Harris hematoxylin, washed with water, fixed in 10% formalin buffer, and then washed 2 times for 5 min each time with PBS.
At least 300 cells were counted in a field of 1 mm2 at a magnification of 100x. The percentage of mesenchymal cells in each culture was determined from the percentage of cells that were vimentin-immunopositive. Values are expressed as the mean ± SEM of determinations by 3 independent observers.
Immunohistochemical Localization of NFB (p65)
The NFB (p65) was localized in cultured cells using a Vectastain Elite Kit. Methodology was similar to that described above for the immunohistochemistry of vimentin and keratin and has been previously described [15]. Cells were not counterstained to allow clear visualization of NFB in the nucleus.
Protein Extraction and Western Blot Analysis
Cells were sonicated in a lysis buffer (pH 7.4) containing 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40 PBS, and protease inhibitors (1 mM PMSF, 10 µg/ml of aprotinin, and 1 mM sodium orthovanadate). The sonicates were pelleted (15 000 x g for 20 min), and supernatant was retained and stored at -20°C. Protein content of the extracts was determined with the Bio-Rad DC Protein Assay Reagent. Samples were mixed with loading buffer, resolved by 12% SDS-PAGE, and electrotransferred (80 V for 2 h) onto nitrocellulose membranes. After blocking for 1 h with nonfat milk powder (5%) in Tris-buffered saline (TBS; 10 mM Tris and 150 mM NaCl) and Tween-20 (TBS-T; 0.05%), membranes were incubated overnight with primary antibodies in TBS-T containing 5% nonfat milk powder and subsequently with horse radish peroxidase-conjugated goat-anti-mouse secondary antibody (1:2500 [v/v]) in TBS-T with nonfat milk powder (room temperature for 45 min). Immunoreactivity was detected by chemiluminescence autoradiography (ECL kit) in accordance with the manufacturer's instructions.
Electrophoretic Mobility Shift Assay
Nuclear extracts of cultured amnion mesenchymal cells were prepared as described by McKinsey et al. [20] with minor modifications. Briefly, cells were scraped from the culture, and 3 x 106 cells were pelleted (200 x g for 5 min) and resuspended in 30 µl of buffer A (10 mM Hepes [pH 7.9], 10 mM KCl, 1.5 mM MgCl, 0.5 mM PMSF, and 0.67% Nonidet P-40). Cells were allowed to swell and rupture (0°C for 15 min). The suspension was centrifuged (10 000 x g at 4°C for 30 min), and the supernatant was collected and stored at -80°C. The pellet (containing cell nuclei) was resuspended in 30 µl of buffer B (20 mM Hepes, 0.4 M NaCl, 0.2 mM EDTA, 1.5 mM MgCl, 0.5 mM DTT, and 0.5 mM PMSF; pH 7.9) and rocked vigorously at 4°C for 15 min. The nuclear extract was centrifuged (10 000 x g and 30 min) and stored at -80°C. Double-stranded DNA oligonucleotides containing consensus sequences for NFB was 32P-labeled with [
-32P]dATP and T4 polynucleotide kinase. Nuclear proteins (5–8 µg) were incubated with radiolabeled DNA probe (room temperature for 20 min) in the binding buffer. Nuclear acid-protein complexes were resolved on a native 5% polyacrylamide gel in Tris-buffered EDTA (pH 8.0) and detected by autoradiography.
Statistics
Results are expressed as the mean ± SEM of 3 independent experiments. Statistical analyses were carried out by one- or two-way ANOVA. A significant difference between treatment groups was determined by the Tukey test. Statistical significance was inferred at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both epithelial and mesenchymal cells were found in the amnion cell cultures, and the results obtained from 6 separate cultures are shown in Table 1. More than 90% of the cells were mesenchymal. Cultures showing less than 90% purity were excluded from the studies.
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IL-1ß Promotes the Expression of COX-2 Protein
Amnion cells were cultured in serum-free DMEM for 3, 6, 12, and 24 h in the presence of IL-1ß (10 ng/ml), and the COX-2 protein content was determined by Western blot analysis. IL-1ß significantly increased the expression of COX-2 protein in a time-dependent manner (P < 0.01) (Fig. 1), reaching a maximum after 12 h. These findings suggest that COX-2 protein was induced by IL-1ß.
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IL-1ß Activates the IB-NFB Pathway in Amnion Mesenchymal Cells In Vitro
To determine if IL-1ß activates the IB-NFB pathway in amnion mesenchymal cells in vitro, we investigated the temporal changes in phosphorylated IB and total IB content, p65 NFB subunit translocation, and nuclear NFB-binding ability in amnion mesenchymal cells in response to IL-1ß. The results obtained are shown in Figure 2. Addition of IL-1ß (10 ng/ml) to amnion cell cultures resulted in increased levels of phosphorylated IB, which reached a maximum (15 min) and then subsequently dropped to pretreatment levels. Total IB levels remained constant for 5 min following the IL-1ß treatment, after which a marked decrease occurred by 30 min and a gradual increase after 60 min. Nuclear NFB-binding ability, as measured by electrophoretic mobility shift assay, was markedly increased after IL-1ß treatment, reaching a maximum at 30 min, and then decreased gradually after 60 min. The increase in NFB-binding capacity was specific, because it was completely eliminated by the addition of a 50-fold excess of unlabeled NFB probe (Fig. 3). Both the p50 and p65 subunits of NFB were involved in the binding, because supershift occurred following incubations with antibodies to both proteins (Fig. 3). In addition, the nuclear localization of p65 subunit of NFB, as determined by immunohistochemistry, was clearly increased after cells were treated with IL-1ß for 30 min (Fig. 4).
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NFB Activation Is Involved in IL-1ß-Induced COX-2 Expression
To determine if the IL-1ß-induced COX-2 is mediated by NFB activation, the role of NFB nuclear binding on COX-2 expression was examined by adding SN50 (a cell-permeable inhibitory peptide of NFB translocation). Pretreatment of amnion cells with SN50 for 30 min significantly attenuated the NFB activation, as evidenced by suppressed nuclear NFB-binding activity in a concentration-dependent manner. However, the same concentrations (100 and 200 µg/ml) of the inactive peptide SN50M were ineffective (Fig. 5). The IL-1ß-induced COX-2 protein level was also attenuated by SN50, but not by SN50M (P < 0.01) (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B in primary cultures of human amnion mesenchymal cells. When nuclear translocation of NFB was blocked by a specific inhibitory peptide SN50, the nuclear NFB DNA-binding activity as well as the synthesis of COX-2 protein were inhibited in these cells. This supports the contention that IL-1ß-induced activation of NFB is functionally linked to the transcriptional regulation of COX-2 expression in amnion mesenchymal cells.
A ubiquitous transcription factor, NFB is involved in the regulation of many genes, including COX-2 and a number of cytokines (e.g., tumor necrosis factor , IL-1, IL-6) [21, 22]. Its activity is mediated principally by its subunits, p50 and p65, that bind to DNA as hetero- or homodimers. Typically, they are present in the cytoplasm and are in association with IB. Following cell activation, IB is phosphorylated, ubiquinated, and degraded [23], resulting in translocation of the p65/p50 from the cytoplasm to the nucleus, where they bind to the NFB consensus sites in the promoter regions of various genes. The present results clearly support this sequence of events occurring in amnion mesenchymal cells when stimulated with IL-1ß and resulting in increased expression of COX-2. We found that IL-1ß stimulation resulted in a marked increase in phosphorylated IB within 5 min and a 90% decrease in total IB levels shortly thereafter. This was temporarily associated with NFB translocation to the nucleus, increased nuclear NFB-binding activity, and up-regulation of COX-2 protein. SN50 is a cell-permeable peptide containing the nuclear localization sequence of the p50 subunit of NFB that binds to the NFB complex at the nuclear membrane and reduces its translocation into the nucleus [24]. Pretreatment of amnion mesenchymal cells with SN50 attenuated both NFB activation and the increase in COX-2 protein level induced by IL-1ß, suggesting that IL-1ß-induced COX-2 expression in amnion mesenchymal cells is mediated by the IB-NFB signal pathway.
The COX-2 gene possesses two NFB-binding sites in its promoter, and NFB has been shown to be involved in IL-1ß activation of COX-2 in a number of biological systems. IL-1ß produces a 10-fold induction of COX-2 mRNA and an 8-fold increase in COX-2 transcription that was temporally preceded by activation of the transcription factor NFB in type II A549 cells [12]. In rheumatoid synoviocytes, IL-1ß rapidly stimulated translocation of the p65 and p50 subunits of NFB from the cytoplasm to the nucleus and binding to two NFB sites within the COX-2 promoter, suggesting that signaling via the NFB pathway is involved in regulation of COX-2 expression induced by IL-1ß [25]. Recently, in a mixed population of amnion cells, NFB was found to be involved in the regulation of COX-2 using a transfection approach [19]. The present results are consistent with these findings, and using a more direct method, we have clearly shown the involvement of NFB in IL-1ß stimulation of COX-2 expression in the mesenchymal cells. In the amnion epithelial cell line (WISH cells), both NFB-binding sites in the COX-2 promoter region appear to be involved in the regulation of COX-2 gene expression by IL-1ß [18], but NFB does not seem to be involved in the stimulation of COX-2 expression in another amnion cell line, AV-3 cells [26]. Future studies will be required to determine if NFB is involved in the stimulation of COX-2 expression in the epithelial cells of the amnion.
The concentrations of a number of cytokines, including IL-1ß, are increased in amniotic fluid during both preterm labor with infection [27] and term labor [28], and PG production by the fetal membranes may be involved in parturition at term and preterm. Because NFB is involved in the IL-1ß-stimulated increase in COX-2 expression in amnion cultures and is involved in the regulation of cytokine gene expression in other systems, inhibition of NFB action may be a potential site for the pharmacological regulation of PG production in preterm labor by limiting COX-2 and/or cytokine gene expression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by CIHR (grant MOP-37943 to W.G.) and the Chinese Education Commission (scholarship to X.Y.). 
2 Correspondence: William Gibb, Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Ottawa Hospital-General Campus, 501 Smyth Rd., Ottawa, ON, Canada K1H 8L6. FAX: 613 737 8070/8470; wgibb{at}ottawahospital.on.ca or wgibb{at}attcanada.ca
Accepted: December 20, 2001.
Received: August 15, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B and JNK activation. FEBS Lett 1997; 418:135-138[CrossRef][Medline]B in the transcriptional control of COX-2 gene expression by IL-1ß. Biochem Biophys Res Commun 1997; 237:28-32[CrossRef][Medline]B: a specific inhibitor of the NFB transcription factor. Science 1988; 242:540-546B and Rel-participants in a multiform transcriptional regulatory system. Int Rev Cytol 1993; 143:1-62[Medline]B pathway in human amnion derived WISH cells. Prostaglandins Leukot Essent Fatty Acids 1998; 59:63-69[CrossRef][Medline]B activity which mediates cyclo-oxygenase-2 expression and is involved with the "functional progesterone withdrawal". Mol Hum Reprod 2001; 7:581-586B by the tax protein of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of NFB. Mol Cell Biol 1996; 16:2083-2090[Abstract]B-mediated X-linked inhibitor of apoptosis protein expression prevents fat granulosa cells from tumor necrosis factor -induced apoptosis. Endocrinology 2001; 142:557-563B: a mechanism for NFB activation. Mol Cell Biol 1993; 13:3301-3310B in the regulation of COX-2 expression by IL-1 ß in rheumatoid synoviocytes. Arthritis Rheum 1997; 40:226-236[Medline] regulation of prostaglandin H synthase-2 transcription is not through nuclear factor-kappa B in amnion derived AV-3 cells. Placenta 2000; 21:789-798[CrossRef][Medline]This article has been cited by other articles:
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  C.M. Mitchell, R.F. Johnson, W.B. Giles, and T. Zakar Prostaglandin H synthase-2 gene regulation in the amnion at labour: histone acetylation and nuclear factor kappa B binding to the promoter in vivo Mol. Hum. Reprod., January 1, 2008; 14(1): 53 - 59. [Abstract] [Full Text] [PDF]
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 W. E Ackerman IV, T. L.S Summerfield, D. D Vandre, J. M Robinson, and D. A Kniss Nuclear Factor-Kappa B Regulates Inducible Prostaglandin E Synthase Expression in Human Amnion Mesenchymal Cells Biol Reprod, January 1, 2008; 78(1): 68 - 76. [Abstract] [Full Text] [PDF]
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 T. M Lindstrom and P. R Bennett The role of nuclear factor kappa B in human labour Reproduction, November 1, 2005; 130(5): 569 - 581. [Abstract] [Full Text] [PDF]
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W. E. Ackerman IV, X. L. Zhang, B. H. Rovin, and D. A. Kniss Modulation of Cytokine-Induced Cyclooxygenase 2 Expression by PPARG Ligands Through NF{kappa}B Signal Disruption in Human WISH and Amnion Cells Biol Reprod, September 1, 2005; 73(3): 527 - 535. [Abstract] [Full Text] [PDF]
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W. E. Ackerman IV, B. H. Rovin, and D. A. Kniss Epidermal Growth Factor and Interleukin-1{beta} Utilize Divergent Signaling Pathways to Synergistically Upregulate Cyclooxygenase-2 Gene Expression in Human Amnion-Derived WISH Cells Biol Reprod, December 1, 2004; 71(6): 2079 - 2086. [Abstract] [Full Text] [PDF]
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S.-W. Yoon, S.-H. Goh, J.-S. Chun, E.-W. Cho, M.-K. Lee, K.-L. Kim, J.-J. Kim, C.-J. Kim, and H. Poo {alpha}-Melanocyte-stimulating Hormone Inhibits Lipopolysaccharide-induced Tumor Necrosis Factor-{alpha} Production in Leukocytes by Modulating Protein Kinase A, p38 Kinase, and Nuclear Factor {kappa}B Signaling Pathways J. Biol. Chem., August 29, 2003; 278(35): 32914 - 32920. [Abstract] [Full Text] [PDF]
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