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Biology of Reproduction 64, 965-973 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Interferon-Tau Suppresses Prostaglandin F2{alpha} Secretion Independently of the Mitogen-Activated Protein Kinase and Nuclear Factor {kappa} B Pathways1

James K. Prua, Bo R. Ruedab, Kathy J. Austina, William W. Thatcherc, Aydin Guzeloglucand >, and Thomas R. Hansen2,a

a Department of Animal Science, University of Wyoming, Laramie, Wyoming 82071-3684 b Vincent Center for Reproductive Biology, Massachusetts General Hospital, Boston, Massachusetts 02114 c Department of Poultry and Dairy Sciences, University of Florida, Gainesville, Florida 32601

ABSTRACT

Pregnancy is established in ruminants through inhibitory actions of interferon (IFN)-{tau} on the release of prostaglandin F2{alpha} (PGF), which allows the corpus luteum to survive and continue to produce progesterone. Experiments were designed to 1) delineate the signal transduction pathway coordinating the synthesis of PGF, 2) determine how rapidly recombinant bovine (rb) IFN-{tau} attenuated phorbol ester (PDBu)-induced secretion of PGF, and 3) establish the site at which rbIFN-{tau} attenuates the secretion of PGF in cultured bovine endometrial (BEND) cells. BEND cells were untreated (control) or treated for 5, 10, 60, 180, or 300 min with PDBu (100 ng/ml), rbIFN-{tau} (50 or 500 ng/ml), PDBu + rbIFN-{tau}, or PDBu + PD98059 (MEK-1 inhibitor; 50 µM). Secretion of PGF was induced (P < 0.0001) by PDBu within 180 min, but induction was inhibited 74% by the addition of rbIFN-{tau} (P < 0.0001) and was ablated completely by PD98059. Parallel results were obtained for cyclooxygenase (COX)-2 protein expression. PDBu induced (P < 0.05) activation of the Raf-1/MEK-1/ERK-1/2 pathway, which was obligatory for the expression of COX-2 and secretion of PGF but was not altered by cotreatment with rbIFN-{tau}. PDBu induced (P < 0.05) transcription of c-jun and c-fos mRNAs within 30 min; induction was inhibited (P < 0.05) by cotreatment with PD98059 but not by cotreatment with rbIFN-{tau}. Treatment of BEND cells with rbIFN-{tau} also did not attenuate PDBu-induced degradation of I{kappa}B{alpha}, suggesting that the I{kappa}B{alpha}/NF{kappa}B pathway is not a site of IFN-{tau} inhibition of PGF. However, rbIFN-{tau} did block transcription of the COX-2 gene induced by PDBu within 30 min. In conclusion, COX-2 expression and PGF secretion induced by PDBu is mediated through the Raf-1/MEK-1/ERK-1/2 pathway, but this pathway is not disrupted by rbIFN-{tau}. Because rbIFN-{tau} inhibits COX-2 mRNA within 30 min, we hypothesized that transcription factors activated by rbIFN-{tau} rapidly and directly attenuate COX-2 gene expression, thereby suppressing secretion of PGF.

conceptus, mechanisms of hormone action, pregnancy, signal transduction, uterus

INTRODUCTION

Prostaglandins and sex steroids are important regulators of the estrous cycle and early pregnancy in mammals [1]. It is generally accepted that prostaglandin F2{alpha} (PGF) is secreted primarily from the luminal epithelium of the endometrium [2] and is luteolytic in ruminants [3–6]. Secretion of PGF is mediated by oxytocin in vivo [7, 8]. Prior to luteolysis, PGF secretion is maintained by oxytocin of neurohypophyseal origin. At the time of luteolysis, oxytocin of luteal origin initiates an increase in endometrial PGF secretion, which then triggers the release of additional oxytocin from the corpus luteum by a positive feedback loop [9, 10]. Oxytocin induces cyclooxygenase-2 (COX-2) expression, which increases pulse amplitude of PGF production in the endometrium [11]. The ability of oxytocin to stimulate PGF release is higher at the time of luteolysis [12], suggesting that a difference in receptivity to oxytocin exists during the estrous cycle. Prostaglandin F ablates progesterone synthesis by the corpus luteum [13] and initiates a new estrous cycle. The pulsatile secretion of PGF from endometria of estrous cycling cows is stimulated by oxytocin and is mediated through the inositol triphosphate-diacylglycerol second messenger system [14]. Furthermore, Burns et al. [15] suggested that phospholipase C (PLC) activity is high in endometrial caruncular explants treated with oxytocin. PLC activity has since been linked to the dependence of extracellular and intracellular calcium for oxytocin-induced PGF release [16]. Yet, the exact signal transduction pathway by which oxytocin mediates PGF secretion in the endometrium remains undetermined. This is not surprising considering that in other tissues, PGF secretion is elevated in response to ligands that activate protein kinase A, protein kinase C (PKC), nuclear factor kappa B (NF{kappa}B), and the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK)-1/2 [17].

Pregnancy [18], and more specifically interferon (IFN);ch{tau}, attenuates the release of PGF in cows. This antiluteolytic action of IFN-{tau} rescues the corpus luteum and facilitates establishment of early pregnancy. Also, IFN-{tau} has been shown to alter the in vitro PGF:prostaglandin E2 (PGE) ratio in favor of PGE [19]. In contrast to PGF, PGE has luteotrophic action in ruminants [20, 21]. Several in vitro studies using bovine endometrial cells have shown that IFN-{tau} suppresses COX-2 and prostaglandin F synthase [11, 22, 23].

The total amount of PGF is not attenuated during early pregnancy in ewes [24]. IFN-{tau} appears to suppress estrogen receptor gene expression [25], which is obligatory for gene expression of the oxytocin receptor [26]. Therefore, IFN-{tau} exerts its inhibitory actions, at least in part, at the "top" of the signaling pathway that mediates PGF secretion, which might help explain how IFN-{tau} disrupts pulsatility rather than total amount of PGF release in the ewe. This situation may not hold for cattle. Using in situ hybridization, Robinson et al. [27] showed that although there is a slight decrease in oxytocin receptor mRNA in endometrium from Day 16 pregnant cows when compared with estrous cycling cows, estrogen receptor mRNA does not change. Thus, pregnancy can alter oxytocin receptor mRNA expression exclusive of the estrogen receptor in cows. Unlike the promoter for the oxytocin receptor gene of the ewe, the bovine oxytocin receptor gene lacks a classical palindromic estrogen response element [28]. IFN-{tau} also may act independently from the oxytocin receptor. Following in vivo treatment of cows with recombinant bovine (rb) IFN-{tau} via intrauterine catheterization, cultured primary endometrial epithelial cells secreted less PGF, and the inhibition of PGF secretion was not associated with formation of oxytocin receptors based on radioligand binding assay [29]. Primary endometrial epithelial cells treated with phorbol ester and rbIFN-{tau} failed to secrete PGF, whereas cells treated with phorbol ester alone secreted PGF [14]. Furthermore, rbIFN;ch{tau} suppressed gene expression of phospholipase A2 (PLA2) [30], COX-2, PGF synthase [23], and 9-keto-PGE reductase [31] in primary bovine endometrial cell culture. These enzymes are critical components of the PGF synthetic pathway [32].

In this study, a bovine endometrial (BEND) cell line [30, 33] was used to study the effect of rbIFN-{tau} on the signal transduction pathway coordinating the secretion of PGF. To achieve this goal, the phorbol ester phorbol-12,13-dibutyrate (PDBu) was used to stimulate PGF secretion by activation of PKC as described previously [30]. In this way, the estrogen and oxytocin receptors could be circumvented to assess signal transduction events directly downstream from PKC. The first objective was to determine if the MAPK pathway mediated induction of these events by PDBu. The second objective was to establish how rapidly BEND cells expressed COX-2 and secreted PGF in response to PDBu. The third objective was to determine where in this pathway rbIFN-{tau} elicited its inhibitory actions. Because the bovine COX-2 gene contains several putative NF{kappa}B response elements (GenBank accession number AF031699) and because COX-2 has been shown to be regulated by NF{kappa}B [17], the final objective was to determine if PDBu induced degradation of inhibitor of NF kappa light polypeptide gene enhancer in B-cells (I{kappa}B) and if rbIFN-{tau} inhibited this "activation" of NF{kappa}B.

MATERIALS AND METHODS

Materials

All antibodies, enzymes, and kits were used according to each manufacturer's specifications. Restriction endonucleases (New England Biolabs, Beverly, MA), T4 DNA ligase, anti-active MAPK antibody and polynucleotide kinase (Promega, Madison, WI), Random Primers DNA Labeling System and NM522 Escherichia coli (Gibco-BRL, Rockville, MD), nitrocellulose Western blotting membrane and nylon Northern blotting membrane (Micron Separations, Westborough, MA), anti-RAF-1 and anti-I{kappa}B{alpha} antibodies and luminol reagent (Santa Cruz Biotechnology, Santa Cruz, CA), anti-COX-2 antibody (Cayman Chemical, Ann Arbor, MI), PDBu and PD98059 (Calbiochem, La Jolla, CA), and GeneAmp RNA PCR Kit and dRhodamine Terminator Cycle Sequencing Ready Kit (Perkin Elmer, Foster City, CA) were purchased from commercial sources. All other reagents and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Oligonucleotides were synthesized at the DNA core facility at the University of Missouri, Columbia.

BEND Cell Culture Conditions and Experimental Models

BEND cells (passages 6–9; American Type Culture Collection, Manassas, VA) were cultured in T-75 culture flasks or 60-mm culture dishes in Ham Nutrient mixture F-12 (HAMS)/minimal essential medium (MEM) containing 10% fetal calf serum and 10% horse serum as described previously [33]. Once cells reached 90% confluence, they were rinsed with serum-free HAMS/MEM medium and incubated for 24 h in serum-free conditions. Medium was again replaced with serum-free medium 3 h prior to treatment. Where applicable, BEND cells were pretreated with the MAPK (MEK-1) inhibitor PD98059 (50 µM) [34] 30 min prior to the addition of the other treatments. When examining release of PGF and expression and phosphorylation of components of the MAPK and I{kappa}B{alpha}/NF{kappa}B signaling pathways and the PGF synthetic pathway, BEND cells were cultured in 60-mm dishes. BEND cells were untreated or were treated with PDBu (100 ng/ml), PDBu + rbIFN-{tau} (10.9 x 10-7 IU/mg; 500 ng/ml) or PDBu + PD98059 (50 µM) for 5, 10, 30, 60, 180, or 300 min under serum-free conditions. These experiments were completed three times using triplicate cultures. Data from individual experiments were analyzed and graphed as means ± SEM. When examining early COX-2, c-jun, and c-fos mRNA expression using Northern blot analysis, BEND cells were cultured in T-75 flasks and were untreated or were treated with PDBu (100 ng/ml), rbIFN-{tau} (500 ng/ml), PDBu + rbIFN-{tau}, or PDBu + PD98059 (50 µM) for 30 or 60 min under serum-free conditions. This experiment was performed in duplicate. Data were analyzed and graphed as means ± SEM.

Collection of BEND Cell Lysates for Western Blot Analysis

Experiments were immediately stopped at specified times by rinsing cells twice with cold PBS. Cells were lysed with cold nondenaturing lysis buffer (10 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 100 mM NaCL, 1% Triton X-100, 0.5% Nonidet P-40, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin A) on ice for 10 min. Adherent cells were scraped from culture dishes and further lysed by passage through a 22-gauge needle. Cellular debris was cleared via centrifugation (12 000 rpm, 4°C, 15 min). Aliquots (25 µl) were lyophylized and reconstituted in 15 µl of 1x Laemmli buffer and frozen at -80°C until SDS-PAGE and Western blotting.

Proteins obtained from BEND cell lysates were separated via SDS-PAGE and electroblotted onto nitrocellulose (0.2 µm) membranes. Nonspecific antibody interactions were blocked by incubating membranes (2 h, 22°C) in Tris-buffered saline (TBS; 20 mM Tris pH 7.5, 150 mM NaCl) and 5% nonfat milk. Membranes were incubated at room temperature for 2 h with primary antibody (Raf-1, 1:500; I{kappa}B{alpha}, 1:500; phosphoMAPK, 1:20 000; or COX-2, 1:500) and washed four times for 10 min each with TBS. Membranes were incubated (1 h, 22°C) in secondary antibody (1:5000) conjugated to horseradish peroxidase and washed with TBS as before. Blots were incubated in Enhance chemiluminescent substrate (NEN Life Sciences, Boston, MA) for 1 min and exposed to x-ray film for 5–60 sec depending on the abundance of protein. The intensity of protein signals was determined using UnScan-It Automated Digitizing System, Version 5.1 (Silk Scientific Corp., Orem, UT). Raf-1 protein migrated through the gels at different rates according to treatment and time. The difference in migration was considered to be due to phosphorylation that accompanies activation and subsequent negative feedback in the Raf-1/MEK-1/ERK-1/2 pathway [35]. For this reason, Raf-1 blots were scanned for phosphorylated protein. The 5-min control was used as a baseline. Any shift in size above the baseline was scanned, analyzed, and interpreted to represent phosphorylated Raf-1. The 42- and 44-kDa isoforms of ERK were scanned together for analysis. BEND cell lysates were normalized on the basis of equal protein or equal volume. Consistent results were obtained using either PAGE loading method.

Isolation of Total Cellular RNA and Northern Blot Analysis

BEND cells were collected by scraping and centrifugation (1200 rpm, 4°C, 10 min). Supernatant was removed from the cells, and total cellular RNA was isolated according to the manufacturer's specifications (TriReagent, Sigma). Ten micrograms of total cellular RNA was denatured (5 min, 70°C), electrophoresed in a 1.5% agarose:formaldehyde gel, and passively transferred to nylon membranes (0.2 µm) by capillary blotting. Membranes were baked (2 h, 80°C) and prehybridized as described elsewhere [36]. Blots were hybridized (15 h, 42°C) with the appropriate cDNA probes (COX-2, c-jun, or c-fos), which were randomly primed with 50 µCi {alpha}-32P-dCTP. Blots were washed as described previously [36] and exposed to x-ray film for 36 h. Blots were reprobed with radiolabeled murine 18S rRNA (Ambion, Austin, TX) to ensure equal loading. Autoradiographic signals were scanned and quantitated similarly to the method described for Western blotting. Because 18S rRNA did not differ across treatments, there was no need to normalize COX-2, c-jun, and c-fos mRNA data.

Reverse Transcription-Polymerase Chain Reaction and Subcloning of Bovine COX-2

Total cellular RNA was isolated from BEND cells treated for 60 min with PDBu (100 ng/ml). The RNA (1 µg) was reverse transcribed and amplified using the Perkin-Elmer RNA GeneAmp Kit. COX-2 cDNA (1493 base pairs) was amplified using bovine COX-2 primers (5'-ATGCTCGCCCG GGCCCTGCTGCTCTGC, 5'-GGGCTTCTCTACCAG AAGGGCGGG). Primers corresponded to positions +110–136 and 1580–1603 of the bovine COX-2 mRNA (EMBL/GenBank accession number AF031698). The cDNA was subcloned into pBluescript (Stratagene, La Jolla, CA) and amplified in NM522 E. coli. The clone was linearized separately at the 5' and 3' ends and sequenced to confirm identity. The COX-2 cDNA insert was removed (EcoRI/XhoI) from pBluescript and random primed for Northern blotting using 50 µCi {alpha}-32P-dCTP. Efficiency of radionucleotide incorporation was 72% (specific activity = 4.6 x 106 cpm/ng).

RIA for PGF

The secretion of PGF into HAMS/MEM culture medium was measured by a direct RIA developed by Knickerbocker et al. [37]. The antibody used was characterized by Dubois and Bazer [38] and validated by Danet-Desnoyers et al. [39]. Cell culture supernatant was diluted 1:4 in Tris buffer (50 mM Tris-HCl, pH 7.5) to a final assay volume of 100 µl. The assay was validated for serum-free medium by adding 25 pg PGF/100 µl. The average recovery of PGF was 25.1 ± 1.42 pg/100 µl. A standard curve ranging from 1.25 to 1000 pg/tube was used for determining PGF in cell culture supernatants. Inter- and intraassay coefficients of variation were 14% and 13%, respectively, and the minimal detectable concentration was 3.32 pg/tube.

Statistics

Assignment to treatments was made at random. Data were subjected to least squares analysis of variance using general linear models procedures of the Statistical Analyses System [40] followed by t-tests for paired comparisons. The results are expressed as the mean ± SEM.

RESULTS

Secretion of PGF from BEND Cells

Figure 1 shows the secretion of PGF from BEND cells in response to treatment (time x treatment interaction, P < 0.0001). Although the basal secretion of PGF by untreated BEND cells did not change with time, treatment with PDBu induced secretion (P < 0.0001) of PGF starting at 180 min and continuing (P < 0.0001) through 300 min. INF-{tau} attenuated PDBu-induced secretion of PGF by 74% (P < 0.0001) at 180 min and by 58% (P < 0.0001) at 300 min. The PDBu-induced secretion of PGF was completely abolished by the MEK-1 inhibitor PD98059 at all times.



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  		FIG. 1. Secretion of PGF by cultured BEND cells. BEND cells were untreated or treated with PDBu (100 ng/ml), PDBu + rbIFN-{tau} (500 ng/ml), or PDBu + PD98059 (50 µM) for 5, 10, 30, 60, 180, and 300 min. The induction of PGF secretion by PDBu was attenuated (P < 0.0001) by rbIFN-{tau} treatment and was completely blocked by PD98059 (P < 0.0001). Different letters represent differences (P < 0.0001) in PGF secretion within times (time x treatment interaction, P < 0.0001)

Recombinant bIFN-{tau} Suppresses COX-2 mRNA and Protein Expression

A representative Northern blot is shown in Figure 2A and demonstrates that COX-2 mRNA migrated as a single ~4.2-kilobase transcript. Quantitation revealed that COX-2 gene expression is induced in BEND cells (time x treatment interaction, P < 0.0001). PDBu induced COX-2 mRNA by 30 min, and this induction was inhibited (P < 0.0001) by coculture with rbIFN-{tau} or PD98059 (Fig. 2B). Expression of COX-2 mRNA increased eightfold (P < 0.0001) by 60 min following treatment with PDBu. At 60 min, rbIFN-{tau} suppressed PDBu-induced COX-2 expression by 81%, but COX-2 mRNA was still more abundant in treated than in untreated cells (P < 0.0001). The inhibitor PD98059 completely blocked the induction of COX-2 by PDBu at 60 min. Recombinant bIFN-{tau} alone had no effect on COX-2 gene expression. Expression of COX-2 protein closely paralleled the secretion of PGF (time x treatment interaction, P < 0.0001). Figure 3A shows a representative Western blot of COX-2 protein in BEND cells. COX-2 was induced (P < 0.0001) at 180 and 300 min by treatment with PDBu (Fig. 3B). Recombinant bIFN-{tau} suppressed PDBu-induced COX-2 (P < 0.0001) at 180 and 300 min but did not completely block COX-2 (PDBu + rbIFN-{tau} > control, P < 0.0001). In contrast, PD98059 completely blocked COX-2. INF-{tau} alone had no effect on expression of COX-2 protein (not shown).



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  		FIG. 2. Expression of COX-2 mRNA in BEND cells. A) Representative Northern blot showing COX-2 gene expression in BEND cells untreated or treated with PDBu (100 ng/ml), rbIFN-{tau} (500 ng/ml), PDBu + rbIFN-{tau}, or PDBu + PD98059 (50 µM) for 30 or 60 min. In the lower panel, 18S rRNA did not change across time or treatment. B) PDBu induces COX-2 mRNA in BEND cells at 30 and 60 min. This induction is blocked 81% by rbIFN-{tau} at 60 min. Different letters represent differences (P < 0.05) in COX-2 gene expression within times



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  		FIG. 3. Detection of COX-2 protein in BEND cells. A) Western blot of COX-2 protein in BEND cells untreated (1) or treated with PDBu (2; 100 ng/ml), PDBu + rbIFN-{tau} (3; 500 ng/ml), or PDBu + PD98059 (4; 50 µM) for 5, 10, 30, 60, 180, or 300 min. B) Recombinant bIFN-{tau} suppresses the PDBu-induced expression of COX-2 at 180 and 300 min. Different letters represent differences (P < 0.0001) in COX-2 protein expression within times

Raf-1 Phosphorylation

Western blot analysis (Fig. 4A) revealed no change in total Raf-1 protein content in BEND cells for any time or treatment. However, a shift in the electrophoretic pattern of Raf-1 to a higher molecular weight was observed starting at 10 min in BEND cells treated with PDBu alone or in combination with rbIFN-{tau}. Based on previous reports, the increase in molecular weight of Raf-1 resulted from multiple phosphorylation events, which presumably activates Raf-1. Raf-1 becomes increasingly phosphorylated (time x treatment interaction, P < 0.0001; larger arrow in Fig. 4A) in BEND cells treated with PDBu or PDBu + rbIFN-{tau} (Fig. 4B). These results indicate that rbIFN-{tau} does not alter the PDBu-induced phosphorylation of Raf-1. Treatment with PD98059 blocked phosphorylation of Raf-1 induced by PDBu possibly by disrupting hyperphosphorylation of Raf-1 by the ERKs [35].



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  		FIG. 4. Phosphorylation of Raf-1. A) A representative Western blot of Raf-1 protein from BEND cells untreated (1) or treated with PDBU (2), PDBu + rbIFN-{tau} (3), or PDBu + PD98059 (4). Although protein expression was consistent across time and treatments, Raf-1 migrated more slowly from PDBu and PDBu + rbIFN-{tau}-treated BEND cells (large arrow). B) Raf-1 becomes increasingly phosphorylated in BEND cells treated with PDBu alone or in combination with rbIFN-{tau} beginning at 10 min (time x treatment interaction, P < 0.0001). *, A difference in Raf-1 phosphorylation compared with the control within times (P < 0.0001)

Phosphorylation of ERK Isoforms

Figure 5A illustrates detection of phosphorylated ERK phosphoprotein (pp) 42mapk and pp44mapk isoforms using an anti-phosphotyrosine antibody. The antibody detected only the active phosphorylated protein. Both ERK isoforms were combined and analyzed. PDBu increased (P < 0.0001) phosphorylation of ERK isoforms in BEND cells (Fig. 5B). Cotreatment with rbIFN-{tau} had no effect on phosphorylation of the ERKs in response to PDBu. However, as expected, pretreatment with the MEK-1 inhibitor PD98059 blocked (P < 0.0001) phosphorylation of the ERKs in response to PDBu. Culture with rbIFN-{tau} alone had no effect on phosphorylation of the ERKs (not shown). Thus, rbIFN-{tau} does not block the Raf-1/MEK-1/ERK-1/2 pathway or other effectors upstream from ERK (e.g., PLC and PKC).



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  		FIG. 5. Tyrosine phosphorylation of ERK-1/2. A) BEND cell lysates were analyzed for the presence of phosphorylated active ERK-1/2 (pp42mapk/pp44mapk) using Western blot analysis. B) BEND cells treated with PDBu alone or in combination with rbIFN-{tau} generated phosphorylated ERK. With the exception of a 5-min treatment, rbIFN-{tau} did not alter the PDBu-induced phosphorylation of ERK. The phosphorylated isoforms of ERK were not detected in untreated BEND cells or in those treated with PDBu + PD98059

c-fos and c-jun mRNA Expression

Northern blot analysis was used to examine mRNA expression of the early response genes c-fos and c-jun in BEND cells in response to treatments (Fig. 6A). Within each time, c-fos gene expression was induced (P < 0.0001) in BEND cells by PDBu when compared to the controls (Fig. 6B). The PDBu-induced expression of c-fos was unaffected by the addition of rbIFN-{tau}. Recombinant bIFN-{tau}-induced expression of c-fos at 30 and 60 min (P < 0.05). Treatment with PD98059 suppressed the PDBu-induced expression of c-fos by 59% and 44% at 30 and 60 min, respectively. Expression of the c-jun gene also was assessed by Northern blot analysis (Fig. 6C). In untreated BEND cells, mRNA expression of c-jun occurred at low levels. Treatment with PDBu upregulated (P < 0.05) c-jun mRNA at 30 and 60 min relative to controls (Fig. 6D). Treatment with rbIFN-{tau} alone or PDBu + PD98059 for 30 min did not alter c-jun mRNA levels as compared with the controls. However, at 60 min these treatments caused an upregulation (P < 0.05) in the expression of c-jun mRNA.



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  		FIG. 6. Transcription of c-fos mRNA. A) A representative Northern blot showing c-fos gene expression in BEND cells untreated or treated with PDBu (100 ng/ml), rbIFN-{tau} (500 ng/ml), PDBu + rbIFN-{tau}, or PDBu + PD98059 (50 µM) for 30 or 60 min. The 18S rRNA was used to show consistent loading. B) At both times, all treatments of BEND cells caused an increase in c-fos mRNA expression when compared with controls. No difference in c-fos gene expression was observed for these respective treatments with time except for treatment with rbIFN-{tau}, where more c-fos mRNA was detected at 30 min than at 60 min. Different letters represent a difference in c-fos gene expression within time (P < 0.05). C) A representative Northern blot showing c-jun gene expression in BEND cells untreated or treated with PDBu (100 ng/ml), rbIFN-{tau} (500 ng/ml), PDBu + rbIFN-{tau}, or PDBu + PD98059 (50 µM) for 30 or 60 min. The 18S rRNA is shown to illustrate consistent loading. D) At 30 min, c-jun gene expression increased in BEND cells treated with PDBu alone or in combination with rbIFN-{tau} when compared with untreated BEND cells. Recombinant bIFN-{tau} did not alter the PDBu-induced expression of c-jun. By 60 min, treatment of BEND cells with rbIFN-{tau} alone or PDBu + PD98059 caused an upregulation of basal c-jun expression. Treatment with rbIFN-{tau} caused an increase in c-jun expression at 60 min when compared with 30 min. Furthermore, at 60 min rbIFN-{tau} enhanced the PDBu-induced expression of c-jun. *, Differences (P < 0.05) in c-jun gene expression within times as compared with the control

I{kappa}B{alpha} Degradation

Western blot analysis was used to measure the decline in I{kappa}B{alpha} in BEND cells as an indirect assay for NF{kappa}B activation [41]. The I{kappa}B{alpha} protein migrated at approximately 36 kDa (Fig. 7A), showed no change in controls, but decreased over time regardless of treatment (Fig. 7B; time x treatment interaction, P < 0.05). PDBu significantly reduced I{kappa}B{alpha} protein content in BEND cells treated for 30, 60, 180, and 300 min. Addition of rbIFN-{tau} to the PDBu treatment appeared to hasten (P < 0.05) I{kappa}B{alpha} degradation (i.e., 10 min) when compared with treatment with PDBu alone (i.e., 30 min). PD98059 enhanced the PDBu-induced degradation (P < 0.05) of I{kappa}B{alpha} at 10, 30, and 60 min when compared with PDBu treatment alone. I{kappa}B{alpha} degradation was highest in BEND cells treated with PDBu, PDBu + rbIFN-{tau}, and PDBu + PD98059 at 30 min. rbIFN-{tau} alone caused an increase in the degradation of I{kappa}B{alpha} through 1 h (not shown).



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  		FIG. 7. Degradation of I{kappa}B{alpha}. A) A representative Western blot showing the degree of I{kappa}B{alpha} degradation in BEND cells untreated or treated with PDBu (100 ng/ml), rbIFN-{tau} (500 ng/ml), or PDBu + PD98059 (50 µM) for 5, 10, 30, 60, 180, or 300 min. B) Treatment of BEND cells with PDBu alone or in combination with rbIFN-{tau} or PD98059 caused a significant (P < 0.05) loss of I{kappa}B{alpha} protein content. *, An increase in I{kappa}B{alpha} degradation when compared with controls within times (P <0.05)

DISCUSSION

Oxytocin increases the expression of mRNA and protein for COX-2 and mRNA for prostaglandin F synthase, which leads to increased pulsatile release of PGF because these enzymes are rate limiting in the PGF synthetic pathway [22]. However, the exact pathway by which oxytocin mediates PGF secretion is not completely understood, and the mechanism through which rbIFN-{tau} inhibits release of PGF has not been completely delineated. BEND cells are of epithelial origin [42] and were generated from bovine endometrium collected on Day 14 of the estrous cycle [33]. This model differs from that used by Asselin [11] and Xiao [22] in that BEND cells were obtained from a progesterone-dominated uterus rather than an estrogen-dominated uterus (i.e., Days 1–4). IFN-{tau} is antiluteolytic and as such is required during early pregnancy for sustained progesterone release from the corpus luteum. In some instances IFN-{tau} and progesterone act together to coordinate gene expression [43]. The endometrium might respond differently to IFN-{tau} during proliferative and differentiated stages of the estrous cycle.

In the present experiments, BEND cells were stimulated with PDBu, a PKC agonist. Treatment with PDBu was critical for understanding the actions of IFN-{tau} downstream from the oxytocin receptor and PLC activity. PDBu caused an increase in PGF secretion by 3 h that continued to increase through 5 h. These results are in agreement with those of others, who have shown that PGF secretion can be stimulated by oxytocin [22, 29] or phorbol ester [14], and demonstrate that PGF secretion from BEND cells requires activation of PKC. These results also extend and complement those reported by Binelli et al. [30]; earlier times were considered in the present study.

The first objective was to determine the signaling pathway coordinating the synthesis of PGF. Expression of COX-2 and secretion of prostaglandins are regulated by the I{kappa}B{alpha}/NF{kappa}B pathway [44]. The bovine COX-2 promoter contains numerous putative NF{kappa}B response elements (GenBank accession number AF031699). Western blotting revealed that I{kappa}B{alpha} became degraded in BEND cells treated with PDBu. This finding provided a good model to study the actions of rbIFN-{tau} on the I{kappa}B{alpha}/NF{kappa}B pathway. We initially hypothesized that rbIFN-{tau} would block the degradation of I{kappa}B{alpha}, thereby attenuating COX-2 gene expression. Treatment of BEND cells with rbIFN-{tau} (50 or 500 ng/ml) did not suppress the PDBu-mediated degradation of I{kappa}B{alpha}, so this hypothesis was rejected. rbIFN-{tau} alone caused degradation of I{kappa}B{alpha} within 1 h but did not induce expression of COX-2 mRNA or protein. Degradation of I{kappa}B{alpha} mediated by the IFN receptor is likely to involve activation of a cascade of enzymatic events, as has been shown for other receptors. Because rbIFN-{tau} did not inhibit I{kappa}B{alpha} degradation, this probably is not a site of IFN-{tau} action in inhibiting the PGF biosynthetic pathway. The importance of IFN-{tau}-mediated degradation of I{kappa}B{alpha} remains to be determined and is the subject of current investigation in our laboratories.

Treatment of BEND cells with the MEK-1 inhibitor PD98059 completely abolished the PDBu-induced secretion of PGF at all time points, suggesting that MEK-1 is involved in the secretion of PGF. The Raf-1/MEK-1/ERK-1/2 pathway was activated in BEND cells in the present study through PKC-mediated phosphorylation of Raf-1 and subsequent activation of MEK-1 and ERK-1/2. This activation is consistent with PDBU activation of the MAPK pathway in other cell lines [45]. Here, we link the PKC-Raf-1/MEK-1/ERK-1/2 pathway to COX-2 activation and PGF secretion in BEND cells and suggest that this pathway is obligatory.

The second objective was to determine how rapidly rbIFN-{tau} attenuated the PDBu-induced secretion of PGF. Using Northern blotting, it was determined that COX-2 gene expression was induced by PDBu in BEND cells within 30 min. Because PD98059 blocked COX-2 gene expression, it is likely that MEK-1 and its associated pathway coordinate PGF secretion at the level of COX-2 gene expression. Evidence has been provided here to suggest that rbIFN-{tau} acts within minutes to uncouple the PGF synthetic pathway in BEND cells. With some understanding of the signaling cascade controlling PGF secretion downstream from PKC, the third objective was to identify the site within the signaling cascade at which rbIFN-{tau} acted to attenuate PGF secretion and COX-2 expression. Because it is likely that PGF is secreted upon activation of the PKC-Raf-1/MEK-1/ERK-1/2 pathway, it was hypothesized that one site of IFN-{tau} action was cytoplasmic PLA2. PLA2 is a rate-limiting enzyme in the prostaglandin synthetic pathway that liberates arachodonic acid from lipid pools found in plasma membranes [5]. This enzyme probably becomes activated by ERK-1/2 via phosphorylation of serine and threonine residues [46]. Protein expression of PLA2 did not differ by treatment or time in BEND cells (data not shown). This finding is consistent with results reported by Binelli et al. [30] in that it took about 12 h of culture with rbIFN-{tau} to notice inhibitory effects on PLA2. In the present experiments, cultures were terminated within 5 h. Graf et al. [47] showed similar results for PLA2 mRNA expression in the ewe upon treatment with oxytocin. We also investigated the phosphorylation of Raf-1 and ERK-1/2. Recombinant bIFN-{tau} did not perturb the phosphorylation of Raf-1 or phosphorylated ERK isoforms. Thus, the inhibitory actions of rbIFN-{tau} on the release of PGF and activation of COX-2 were determined to be downstream from Raf-1 and ERK-1/2 phosphorylation.

Using chemical inhibitors and dominant negative transfection approaches, several groups have demonstrated that c-jun and c-fos are required for the expression of COX-2 [48, 49]. The PKC-Raf-1/MEK-1/ERK-1/2 pathway is required for COX-2 gene expression and subsequent secretion of PGF in BEND cells. Gene expression of the AP-1 transcription factors c-jun and c-fos were examined using Northern blot analysis. Treatment of BEND cells with PDBu caused induction of both c-jun and c-fos mRNAs. Recombinant bIFN-{tau} failed to block the PDBu-induced transcription of c-jun and c-fos genes. This finding and the early (30 min) suppression of COX-2 gene expression suggest that the COX-2 promoter is directly suppressed in response to rbIFN-{tau} treatment. Because the bovine COX-2 promoter contains multiple putative IFN-stimulated response elements (ISREs), the signal transducers and activators of transcription (STATs) are logical candidates to consider for the regulation of the COX-2 gene expression. The STATs are phosphorylated and translocate to the nucleus within 30 min [42, 50]. Translocated STATs may bind the COX-2 promoter and inhibit gene expression of the COX-2 gene regardless of the presence of activators such as NF{kappa}B or c-jun and c-fos.

In summary (Fig. 8), PDBu was used in BEND cells to circumvent signaling events upstream from PKC, which included PLC, G-proteins, the oxytocin receptor, and the estrogen receptor. Treatment of BEND cells with phorbol ester resulted in phosphorylation of RAF-1, activation of MEK-1, phosphorylation of ERK-1/2, and transcription of c-jun and c-fos genes. Activation of this MAPK pathway led to induction of COX-2 gene transcription within 30 min, presumably through interaction of c-jun and c-fos with one or several putative AP-1 sites on the bovine COX-2 promoter. It was determined that rbIFN-{tau} had no direct inhibitory effect on components of the MAPK pathway described above. Rather, rbIFN-{tau} inhibited the PDBu-induced transcription of the COX-2 gene within 30 min. It is hypothesized that this rapid inhibition of the COX-2 gene by rbIFN-{tau} is through activation of the STATs and interaction with putative ISREs and potentially an interferon regulatory factor element in the bovine COX-2 promoter.



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  		FIG. 8. Proposed mechanism of IFN-{tau}-mediated suppression of PGF secretion in BEND cells. The phorbol ester PDBu was used to initiate PGF secretion through activation of PKC in BEND cells. The primary goal of this study was to determine if rbIFN-{tau} attenuated actions downstream from the oxytocin receptor and PLC activity. BEND cells respond to PDBu through activating PKC, which then initiates the Raf-1/MEK-1/ERK-1/2 phosphorylation cascade. ERK-1/2 presumably activates nuclear transcription factors via phosphorylation that promote gene expression of the early immediate response genes c-jun and c-fos. Consequently, c-jun and c-fos activate COX-2 gene expression. Concurrently, ERK-1/2 activates PLA2 via phosphorylation. Thus, two critical enzymes for synthesis of PGF are activated in response to PDBu through the MAPK pathway, resulting in the secretion of PGF. IFN-{tau} does not inhibit the MAPK pathway. Also, rbIFN-{tau} does not inhibit PDBU-induced degradation of I{kappa}B{alpha}; rbIFN-{tau} alone caused degradation of I{kappa}B{alpha} through some unknown mechanism. However, rbIFN-{tau} rapidly (within 30 min) inhibits PDBu induced transcription of the COX-2 gene. The STATs are the only transcription factors known to be induced/phosphorylated within 30 min of rbIFN-{tau} action in BEND cells [42, 50]. Thus, we hypothesized that IFN-{tau} inhibits the COX-2 gene directly via the STATs within minutes. If one examines the bovine COX-2 gene promoter carefully, an ISRE and potentially an IRFE exist just upstream of the TATA box. Also, several AP-1 and NF{kappa}B sites exist that are upstream of the IFN response elements. The hypothesis that STATs inhibit this gene regardless of binding of c-jun, c-fos, and NF{kappa}B will be tested in future experiments

ACKNOWLEDGMENTS

The authors are grateful to Dr. John S. Davis, Women's Research Institute, Wichita, for providing bovine c-jun and c-fos cDNA clones and to Dr. R. Michael Roberts, Dr. Alan Ealy, and Andrei Alexenko, University of Missouri, Columbia, for providing rbIFN-{tau}.

FOOTNOTES

First decision: 9 June 2000.

1 Supported in part by NIH grant R01-32475-6 awarded to T.R.H. Back

2 Correspondence. FAX: 307 766 2355; thansen{at}uwyo.edu Back

Accepted: October 27, 2000.

Received: May 17, 2000.

REFERENCES

  1. Poyser NL. The control of prostaglandin production by the endometrium in relation to luteolysis and menstruation. Prostaglandins Leukotrienes Essent Fatty Acids 1995; 53:147–195[CrossRef][Medline]
  2. Bazer FW, Ott TL, Spencer TE. Pregnancy recognition in ruminants, pigs and horses: signals from the trophoblast. Theriogenology 1994; 41:79–94
  3. Goding J. The demonstration that PGF2{alpha} is the uterine luteolysin in the ewe. J Reprod Fertil 1972; 38:261–272
  4. McCracken JA, Carlson JC, Glew J, Goding JR, Baird DT, Green K, Samuelsson B. Prostaglandin F2{alpha} identified as a luteolytic hormone in sheep. Nature 1972; 238:129–134
  5. Inskeep EK, Murdoch WJ. Relation of ovarian function to uterine and ovarian secretion of prostaglandins during the estrous cycle and early pregnancy in the ewe and cow. In: Reproductive Physiology II: International Review of Physiology, vol. 22. Baltimore, MD: University Park Press; 1980: 325–356
  6. McCracken JA, Barcikowski B, Carlson JC, Green K, Samuelsson B. The physiological role of prostaglandin F2{alpha} in corpus luteum regression. Adv Biosci 1973; 9:599–624[Medline]
  7. Fairclough RJ, Moore LG, McGowan LT, Peterson AJ, Smith JF, Tervit HR, Watkins WB. Temporal relationship between plasma concentrations of 13, 14-dihydro-15-keto-prostaglandin F and neurophysin I/II around luteolysis in sheep. Prostaglandins 1980; 20:199–208[CrossRef][Medline]
  8. Wathes DC, Denning-Kendall PA. Control of synthesis and secretion of ovarian oxytocin in ruminants. J Reprod Fertil Suppl 1992; 45:39–52[Medline]
  9. Roberts JS, McCracken JA. Does PGF2{alpha} release from the uterus by oxytocin mediate the oxytocin action of oxytocin? Biol Reprod 1976; 15:457–463[Abstract]
  10. Silvia WJ, Lewis GS, McCracken JA, Thatcher WW, Wilson L. Hormonal regulation of uterine secretion of prostaglandin F2{alpha} during luteolysis in ruminants. Biol Reprod 1991; 45:655–663[Abstract]
  11. Asselin E, Drolet P, Fortier MA. Cellular mechanisms involved during oxytocin-induced prostaglandin F2{alpha} production in endometrial epithelial cells in vitro: role of cyclooxygenase-2. Endocrinology 1997; 138:4798–4805[Abstract/Free Full Text]
  12. Lafrance M, Goff AK. Effect of pregnancy on oxytocin-induced release of prostaglandin F2 alpha in heifers. Biol Reprod 1985; 33:1113–1119[Abstract]
  13. Juengel JL, Niswender GD. Molecular regulation of luteal progesterone synthesis in domestic ruminants. J Reprod Fertil Suppl 1999; 54:193–205[Medline]
  14. Tysseling KA, Thatcher WW, Bazer FW, Hansen PJ, Mirando MA. Mechanisms regulating prostaglandin F2{alpha} secretion from the bovine endometrium. J Dairy Sci 1998; 81:382–389[Abstract]
  15. Burns PD, Graf GA, Hayes SH, Silvia WJ. Cellular mechanisms by which oxytocin stimulates uterine PGF2 alpha synthesis in bovine endometrium: roles of phospholipases C and A2. Domest Anim Endocrinol 1997; 14:181–191[CrossRef][Medline]
  16. Burns PD, Hayes SH, Silvia WJ. Cellular mechanisms by which oxytocin mediates uterine prostaglandin F2 alpha synthesis in bovine endometrium: role of calcium. Domest Anim Endocrinol 1998; 15:477–487[CrossRef][Medline]
  17. May MJ, Ghosh S. Signal transduction through NF-kappa B. Immunol Today 1998; 19:80–88[CrossRef][Medline]
  18. Thatcher WW, Bartol FF, Knickerbocker JJ, Curl JS, Wolfenson D, Bazer FW, Roberts RM. Maternal recognition of pregnancy in cattle. J Dairy Sci 1984; 67:2797–2811
  19. Asselin E, Lacroix D, Fortier MA. IFN-{tau} increases PGE2 production and COX-2 gene expression in the bovine endometrium in vitro. Mol Cell Endocrinol 1997; 132:117–126[CrossRef][Medline]
  20. Pratt BR, Butcher RL, Inskeep EK. Antiluteolytic effect of the conceptus and PGE2 in ewes. J Anim Sci 1977; 45:784–791
  21. Magness RR, Huie JM, Hoyer GL, Huecksteadt TP, Reynolds LP, Seperich GJ, Whysong G, Weems CW. Effect of chronic ipsilateral or contralateral intrauterine infusion of prostaglandin E2 (PGE2) on luteal function of unilateral ovariectomized ewes. Prostaglandins Med 1981; 6:389–401[CrossRef][Medline]
  22. Xiao CW, Murphy BD, Sirois J, Goff AK. Down-regulation of oxytocin-induced cyclooxygenase-2 and prostaglandin F synthase expression by interferon-{tau} in bovine endometrial cells. Biol Reprod 1999; 60:656–663[Abstract/Free Full Text]
  23. Xiao CW, Liu JM, Sirois J, Goff AK. Regulation of cyclooxygenase-2 and prostaglandin F synthase gene expression by steroid hormones and interferon-{tau} in bovine endometrial cells. Endocrinology 1998; 139:2293–2299[Abstract/Free Full Text]
  24. Bazer FW, Spencer TE, Ott TL. Interferon tau: a novel pregnancy recognition signal. Am J Reprod Immunol 1997; 37:412–420
  25. Spencer TE, Bazer FW. Ovine interferon tau suppresses transcription of the estrogen receptor and oxytocin receptor genes in the ovine endometrium. Endocrinology 1996; 137:1144–1147[Abstract]
  26. Wathes DC, Lamming GE. The oxytocin receptor, luteolysis and the maintenance of pregnancy. J Reprod Fertil Suppl 1995; 49:53–67[Medline]
  27. Robinson RS, Mann GE, Lamming GE, Wathes DC. The effect of pregnancy on the expression of uterine oxytocin, oestrogen and progesterone receptors during early pregnancy in the cow. J Endocrinol 1999; 160:21–33[Abstract]
  28. Bathgate RAD, Tillmann G, Ivell R. Molecular mechanisms of bovine oxytocin receptor gene regulation. Biol Reprod 1998; 58(suppl 1):121
  29. Meyer MD, Desnoyers GD, Oldick B, Thatcher WW, Drost M, Schalue TK, Roberts RM. Treatment with recombinant bovine interferon-{tau} in utero attenuates secretion of prostaglandin F from cultured endometrial epithelial cells. J Dairy Sci 1996; 79:1375–1384[Abstract]
  30. Binelli M, Guzeloglu A, Badinga L, Arnold DR, Sirois J, Hansen TR, Thatcher WW. Interferon-{tau} modulates phorbol ester-induced production of prostaglandin and expression of cyclooxygenase-2 and phospholipase-A2 from bovine endometrial cells. Biol Reprod 2000; 63:417–424[Abstract/Free Full Text]
  31. Asselin E, Fortier MA. Detection and regulation of the messenger of a putative bovine endometrial 9-keto-prostaglandin E2 reductase: effect of oxytocin and interferon-tau. Biol Reprod 2000; 62:125–131[Abstract/Free Full Text]
  32. Murdoch WJ, Hansen TR, McPherson LA. A review—role of eicosanoids in vertebrate ovulation. Prostaglandins 1993; 46:85–115[CrossRef][Medline]
  33. Staggs KL, Austin KJ, Johnson GA, Teixeira MG, Talbott CT, Dooley VA, Hansen TR. Complex induction of bovine uterine proteins by interferon-tau. Biol Reprod 1998; 59:293–297[Abstract/Free Full Text]
  34. Pang L, Sawada T, Decker SJ, Saltiel AR. Inhibition of MAP kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J Biol Chem 1995; 270:13585–13588[Abstract/Free Full Text]
  35. Wartmann M, Hofer P, Turowski P, Saltiel AR, Hynes NE. Negative modulation of membrane localization of Raf-1 protein kinase by phosphorylation. J Biol Chem 1997; 272:3915–3923[Abstract/Free Full Text]
  36. Johnson GA, Austin KJ, Collins AM, Murdoch WJ, Hansen TR. Endometrial ISG17 mRNA and a related mRNA are induced by interferon-tau and localized to glandular epithelial and stromal cells from pregnant cows. Endocrine 1999; 10:243–252[Medline]
  37. Knickerbocker JJ, Thatcher WW, Bazer FW, Drost M, Barron DH, Fincher KB, Roberts RM. Proteins secreted by day-16 to -18 bovine conceptuses extend corpus luteum function in cows. J Reprod Fertil 1986; 77:381–389[Abstract]
  38. Dubois DH, Bazer FW. Effect of porcine conceptus secretory proteins on in vitro secretion of prostaglandins-F2{alpha} and -E2 from luminal and myometrial surfaces of endometrium from cyclic and pseudopregnant gilts. Prostaglandins 1991; 41:283–289[CrossRef][Medline]
  39. Danet-Desnoyers GD, Wetzels C, Thatcher WW. Natural and recombinant bovine interferon-{tau} regulate basal and oxytocin-induced secretion of prostagladins F2{alpha} and E2 by epithelial cells and stromal cells in endometrium. Reprod Fertil Dev 1994; 6:193–200[CrossRef][Medline]
  40. SAS. Sas User's Guide: Statistics, Version 6. Cary, NC: Statistical Analyses Systems Institute; 1999
  41. Alkalay I, Yaron A, Hatzubai A, Orian A, Ciechanover A, Ben-Heriah Y. Stimulation-dependent I{kappa}B{alpha} phosphorylation marks the NF{kappa}B inhibitor for degradation via the ubiquitin-proteasome pathway. Proc Natl Acad Sci U S A 1995; 92:10599–10603[Abstract/Free Full Text]
  42. Binelli M, Subramaniam P, Diaz T, Johnson GA, Hansen TR, Badinga L, Thatcher WW. Bovine interferon-{tau} stimulates the Jak-STAT pathway in bovine endometrial epithelial cells. Biol Reprod 2001; 64:654–665[Abstract/Free Full Text]
  43. Johnson GA, Spencer TE, Burghardt RC, Joyce MM, Bazer FW. Interferon-tau and progesterone regulate ubiquitin cross-reactive protein expression in the ovine uterus. Biol Reprod 2000; 62:622–627[Abstract/Free Full Text]
  44. von Knethen A, Callsen D, Brune B. Superoxide attenuates macrophage apoptosis by NF-kappa B and AP-1 activation that promotes cyclooxygenase-2 expression. J Immunol 1999; 163:2858–2866[Abstract/Free Full Text]
  45. Magnuson NS, Beck T, Vahidi H, Hahn H, Smola U, Rapp UR. The Raf-1 serine/threonine protein kinase. Semin Cancer Biol 1994; 5:247–253[Medline]
  46. Lin LL, Wartmann M, Lin AY, Knopf JL, Seth A, Davis RJ. cPLA2 is phosphorylated and activated by MAP kinase. Cell 1993; 72:269–278[CrossRef][Medline]
  47. Graf GA, Burns PD, Silvia WJ. Expression of a cytosolic phospholipase A2 by ovine endometrium on days 11–14 of a simulated oestrous cycle. J Reprod Fertil 1999; 115:357–363[Abstract]
  48. Iniguez MA, Martinez-Martinez S, Punzon C, Redondo JM, Fresno M. An essential role of nuclear factor of activated T cells in the regulation of the expression of the cyclooxygenase-2 gene in human T lymphocytes. J Biol Chem 2000; 275:23627–23635[Abstract/Free Full Text]
  49. Subbaramaiah K, Michaluart P, Sporn MB, Dannenberg AJ. Ursolic acid inhibits cyclooxygenase-2 transcription in human mammary epithelial cells. Cancer Res 2000; 60:2399–2404[Abstract/Free Full Text]
  50. Perry DJ, Austin KA, Hansen TR. Cloning of interferon-stimulated gene 17: the promoter and nuclear proteins that regulate transcription. Mol Endocrinol 1999; 13:1197–1206[Abstract/Free Full Text]



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