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Endocrinology Vol. 142, No. 12 5107-5115
Copyright © 2001 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Induction of PG G/H Synthase-2 in Bovine Myometrial Cells by Interferon-{tau} Requires the Activation of the p38 MAPK Pathway

Florence Doualla-Bell and Antonis E. Koromilas

Perinatal Research and Developmental Pharmacology Unit (F.D.-B.) and Terry Fox Molecular Oncology Group (A.E.K.), Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montréal, Québec H3T 1E2, Canada; and Departments of Experimental Medicine (F.D.-B.), Oncology (A.E.K.), Cell Biology and Anatomy (F.D.-B.), Microbiology and Immunology (A.E.K.), McGill University, Montréal, Québec H36 1Y6, Canada

Address all correspondence and requests for reprints to: Dr. Florence Doualla-Bell, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Chemin de la Côte-Ste-Catherine, Montréal, Québec H3T 1E2, Canada. E-mail: fdoualla{at}ldi.jgh.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PGs are regulators of a plethora of uterine functions during reproductive processes, including uterine contractility. In bovine uterus, the rate-limiting step in PG synthesis is catalyzed by the PG endoperoxide G/H synthase (PGHS) enzymes. It has previously been established that PGHS-2 isoform expression is affected by the ruminant-specific interferon (IFN)-{tau} in bovine endometrial cells. Here, we show that PGHS-2 mRNA and protein levels are induced by IFN-{tau} in primary cell cultures from bovine myometrium. Treatment with recombinant bovine IFN-{tau} induces the activation of the JAK-STAT and p38 MAPK pathways in bovine myometrial cells. Inhibition of the p38 pathway by the specific inhibitor SB203580 strongly decreases PGHS-2 mRNA and protein expression without affecting the phosphorylation and DNA-binding of transcription factors STAT-1 and STAT-2. The p38 pathway regulates PGHS-2 expression at the posttranscriptional level, because the presence of SB203580 results in the destabilization of IFN-{tau}-induced PGHS-2 mRNA. Taken together, these data demonstrate the ability of IFN-{tau} to induce the activation of the JAK-STAT pathway in a manner similar to other types of IFN (i.e. {alpha}, ß, and {gamma}) and to regulate PGHS-2 mRNA stability through the activation of the p38 pathway. These findings provide new insights into the physiological function of IFN-{tau}, in regard to regulation of specific genes associated with myometrial contractility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MANY REPRODUCTIVE processes, including uterine contractility (1, 2), PGs are of central physiologic importance. The rate-limiting step in PG synthesis is orchestrated by PG endoperoxide G/H synthases (PGHS). These enzymes exist in two isoforms, PGHS-1 and PGHS-2 (3). PGHS-2 is inducible by several stimuli, including growth factors, cytokines, phorbol ester, PG, or gonadotrophin (4, 5, 6, 7).

Interferon (IFN)-{tau} has first been described as responsible for maternal recognition of pregnancy in ruminants. This cytokine is released at high levels by the conceptus trophectoderm (8, 9), and its main action occurs in endometrium, where it prevents luteolysis by suppressing endometrial PGF2{alpha} secretion (10, 11). In this tissue, different and contradictory effects of IFN-{tau} on PGHS-2 gene expression and activity have been described, depending on the origin of cells, the stage of estrous cycle chosen, or the doses of IFN-{tau} used (12, 13, 14). Although other biological properties of IFN-{tau}, such as its potent antiviral or antiproliferative activities, have been described (15), no consideration has been given to its possible role in the portion of the uterus responsible for its contractile status: the myometrium. We previously reported, in bovine midcycle myometrium, that phorbol myristate acetate (PMA) preferentially mediated production of prostacyclin (PGI2), the myorelaxant PG, via both cytosolic PLA2 (cPLA2) and PGHS-2 enzyme pathways (4). Given the potential role of IFN-{tau} in the maintenance of a quiescent status in the uterus, we were interested in examining a possible function of IFN-{tau} in the biosynthesis of myometrial PGs in cyclic cow myometria and examining the signaling pathway(s) mediating this process.

IFN-{tau} shares 50% amino acid sequence homology with IFN-{alpha} (16) and can bind to IFN-{alpha} receptor (17). Interestingly, it has been shown that in bovine endometrium, IFN-{tau} uses the same signaling pathway as IFN-{alpha} (18). IFNs are divided into two subtypes, which induce signaling pathways leading to activation of transcription via different cell surface receptors. Binding of both type I (IFN-{alpha}, ß, and {omega}) and type II IFN (IFN-{gamma}) to their cognate receptors results in the differential activation of the Janus family of tyrosine kinases (JAKs) that phosphorylate the latent cytoplasmic signal transducer and activator of transcription factors (STATs) (19, 20). Phosphorylated STATs translocate to the nucleus, whereby they bind to specific DNA elements and induce transcription. Type I IFN induces the tyrosine phosphorylation of STAT-1 and STAT-2 proteins by the receptor-associated tyrosine kinases JAK1 and TYK2. After phosphorylation, STAT-1 and STAT-2 heterodimers form the transcriptionally active IFN-stimulated gene (ISG) factor 3 (ISGF3) by association with a 48-kDa protein (i.e. p48) known as IFN regulatory factor-9 (21). The specificity of the transcriptional activation by ISGF3 is mediated by specific elements termed IFN-stimulatory response elements (ISRE) located in the promoter region of IFN- inducible genes (22). Gene induction by type II IFN involves the tyrosine phosphorylation of STAT-1 by JAK1 and JAK2 kinases, STAT-1 homodimerization, and binding to the IFN-{gamma}-activated site (GAS) to induce transcription (23).

Although JAKs are primary components of the phosphorylation-related-IFN signaling pathway, other phosphorylation mechanisms have been shown to be essential for IFN biological effects (20, 24). For example, IFN-inducible phosphorylation of STAT-1{alpha} on serine 727 has been demonstrated to play an important role in gene transactivation. Indeed, Goh et al. (25) have recently shown that, in HeLa cells, STAT-1 serine-727 phosphorylation by p38 mitogen-activated protein (MAP) is a prerequisite for the subsequent transcriptional gene activation induced by IFN-{gamma}. The p38 MAPK is a serine-threonine protein kinase strongly activated in response to various cellular stresses and cytokine stimulation (26, 27). Interestingly, it has been shown that cPLA2 activation, necessary for the release of PGHS substrate (arachidonic acid), is not only involved in ISGF3-dependent gene activation (28, 29) but is also directly activated by p38 MAPK (30, 31).

In this paper, we demonstrate that IFN-{tau} induces PGHS-2 RNA and protein expression in primary bovine myometrial cells. We also show that, in analogy to type I and II IFNs, treatment of myometrial cells with IFN-{tau} induces the activation of STAT proteins through mechanisms that are mediated by both types of IFN. Furthermore, we demonstrate that IFN-{tau} activates the p38 MAPK signaling pathway, which mediates the induction of PGHS-2 expression through a mechanism that requires the stabilization of PGHS-2 mRNA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Tissue culture reagents and FCS were purchased from ICN Biochemicals, Inc. (Costa Mesa, CA). Bovine PGHS-2 cDNA was donated by Dr. J. Sirois (Québec, Canada). The recombinant bovine IFN-{tau} was purchased from Research Diagnostic Inc. (Flanders, NJ). Monoclonal antibody to PGHS-2 was obtained from Cayman Chemical Co. (Ann Arbor, MI). Polyclonal antibody to cPLA2 was a gift of Merck Frosst Canada Inc. (Pointe-Claire, Québec, Canada). Monoclonal antibodies against STAT-1{alpha} and STAT-2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-pS727-STAT-1 was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Alpha [32P] deoxy-CTP and {gamma} [32P] deoxy-GTP (>3,000 Ci/mmol) were purchased from Perkin-Elmer Canada (Mississauga, Ontario, Canada). Poly (dI-dC) was purchased from Pharmacia Biotech (Montréal, Canada). 4-(4-Fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridinyl) imidazole (SB203580) was from Calbiochem (La Jolla, CA). Polyclonal antibodies to phosphorylated and unphosphorylated p38 MAPK were purchased from Pharmacia Biotech. All other chemicals were supplied by Sigma (St. Louis, MO).

Cell culture
Bovine uteri were collected at local slaughterhouses, and the stage of the estrous cycle at which the uteri were collected (the end of midluteal phase) was estimated by examining the ovarian morphology (32). Myocytes from the longitudinal layer of bovine myometrium were cultured in RPMI 1640 supplemented with 10% FBS as previously described (4, 33, 34). After cell cultures reached confluency (d 7–10 after plating), the culture medium was replaced with fresh medium, without serum, for 24 h, and cells were stimulated in the absence or presence of 100 ng/ml recombinant bovine IFN-{tau}, 1 µM SB203580,or 0.5 µg/ml actinomicyn D for various periods of time, as indicated in each experiment.

Cell extracts
EMSAs were performed with whole-cell extracts (WCE), as previously described (35). Briefly, cells were washed with ice-cold PBS, then incubated for 10 min in a lysis buffer containing 0.5% NP-40, 50 mM Tris (pH 8.0), 10% glycerol, 0.1 mM EDTA, 150 mM NaCl, 0.1 mM Na3VO4, 50 mM NaF, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonylfluoride, 3 µg/ml apoprotein, 1 µg/ml pepstatin, and 1 µg/ml leupeptin. The lysate was centrifuged at 14,000 x g for 10 sec, and the supernatant was stored at -80 C. For EMSAs, 10 µg WCE were added to [{alpha}-32P]deoxy-GTP-labeled double-stranded DNA (dsDNA) oligonucleotide (1 ng), containing approximately 9 x 105 cpm. Binding reactions were performed in a buffer containing 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.9; 40 mM KCl; 1 mM MgCl2; 0.1 mM EGTA; 0.5 mM dithiothreitol; 10% glycerol; 240 ng/ml poly(dI-dC). The protein-DNA complexes formed during 30 min incubation at room temperature were subsequently electrophoresed on a 6% nondenaturing polyacrylamide gel in 0.2x TBE (20 mM boric acid; 0.4 mM EDTA, pH 8.0) at 400 V and 4 C. The specificity of the reaction was confirmed using 200-fold excess of unlabeled dsDNA oligonucleotide in cold competition reactions. DNA-protein complexes were visualized by autoradiography on Kodak film (Interscience, Markham, Ontario, Canada). For supershift experiments, 4 µg antibodies against STAT-1, STAT-2, phosphotyrosine (PY), or mouse IgG1 (negative control) were incubated with WCE for 30 min before the addition of DNA.

Two double-stranded oligonucleotides were used: the ISRE of the IFN-{alpha}/ß-inducible ISG-15 gene (5'-GATCGGGAAAGGGAAACCGAAACTGAAGCC-3'; the underlined sequence shows the ISGF3 binding site; Ref. 36) and the GAS of the IFN-{gamma}-inducible IFP-53 gene (5'-GATCCAGATTCTCAGAA-3'; the underlined sequence shows the binding site of STAT-1 homodimer; Ref. 37). All the oligonucleotides tested were purchased from Invitrogen (Burlington, Ontario, Canada).

Immunoprecipitation
WCE were incubated with 1 µg mouse IgG1 and 50 µl 50% suspension of protein G-Sepharose (Pharmacia Biotech Inc.) for 1 h. After centrifugation, the supernatant was incubated with 1 µg anti-STAT-1 antibody and 50 µl 50% suspension of protein G-Sepharose for 2 h. The Sepharose beads were then washed three times with lysis buffer and centrifuged at 14,000 x g for 15 min. The supernatant was loaded on an SDS-7% polyacrylamide gel and transferred onto polyvinylidine difluoride membrane for 1 h at 125 V. Immunoblotting analyses with anti-STAT-1{alpha}, anti-STAT-2, anti-PY, or anti-pS727-STAT-1 antibodies (1:1000) were performed on the same stripped membrane. Blots were incubated with sheep antimouse IgG peroxidase-linked secondary antibody, and bands were visualized by enhanced chemiluminescence according to the manufacturer’s specification (NEN Life Science Products, Inc., Boston, MA).

PG assays
Accumulation of 6-keto-PGF1{alpha} (the stable metabolite of PGI2) in the incubation medium was determined by ELISA, as described previously (4, 34).

Northern blot analysis
Total RNA was prepared by the method of Chomczynski and Sacchi (38). Total RNA (10 µg) was loaded on a 1% agarose-2.2-M formaldehyde gel, transferred to nylon membranes (Magna, Micron Separations Inc., Westboro, MA), then cross-linked by UV irradiation. After 4 h of prehybridization in 50% formamide at 42 C, membranes were sequentially hybridized overnight at 42 C with {alpha} [32P]deoxy-CTP-labeled cDNA probes corresponding to bovine PGHS-2 mRNA, and {alpha} [32P]deoxy-CTP-labeled 18S ribosomal DNA. Membranes were washed and exposed to x-ray films with an intensifying screen, at -80 C, for 4–72 h. For rehybridization with a different probe, membranes were boiled in 0.1% SDS solution.

Western blot analysis
Cells were rinsed with PBS, and proteins were extracted with lysis buffer containing Tris 62.5 mM (pH 6.8), 2% SDS, 10% glycerol, and 5% ß-mercaptoethanol. For immunoblot analysis, 50 µg protein (39) from cell lysates were separated by SDS-PAGE. Proteins were transferred onto polyvinylidine difluoride membranes (Micron Separations Inc.). Membranes were blocked in 5% nonfat milk-Tris-buffered saline with 0.1% Tween 20 before incubating with either antibody against human PGHS-2, cPLA2, p38, or phosphorylated (Thr180/Tyr182)-p38 MAPK (dilutions: 1/1000). Blots were incubated with sheep antimouse IgG or antirabbit Ig peroxidase-linked secondary antibody, in Tris-buffered saline with 0.1% Tween 20. Chemiluminescent detection was performed using reagents from NEN Life Science Products, and bands were visualized on Kodak film (Interscience).

Statistical analysis
All experimental data are presented as means ± SEM. For statistical comparison among groups, ANOVA or unpaired t test was applied. Differences between groups were determined by a Bonferroni’s multiple-comparison test. A P value of less than or equal to 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{tau} up-regulates myometrial PG biosynthesis
To investigate the potential role of IFN-{tau} in the regulation of myometrial PG pathway, we examined the effect of the cytokine on PGHS-2 gene expression and PG production (Fig. 1Go). First, we determined the concentrations of IFN-{tau} that can induce myometrial PGHS-2 gene expression in vitro. Cells were left untreated or treated with 1, 10, and 100 ng/ml IFN-{tau}, and PGHS-2 protein levels were detected by immunoblotting analysis (Fig. 1AGo, top). We observed that PGHS-2 protein was induced by both 10 and 100 ng/ml IFN-{tau} (lanes 3 and 4), with the latter concentration exhibiting a higher effect. Next, we examined how IFN-{tau}-induces PGHS-2 expression in myometrial cells. Cells cultured in serum-free medium were treated for the indicated periods of time, in the presence of 100 ng/ml IFN-{tau}, then subjected to Northern blot analysis (Fig. 1BGo) or immunoblotting (Fig. 1CGo) for PGHS-2 mRNA and protein expression, respectively. We observed that PGHS-2 mRNA levels were increased in a time-dependent manner after IFN-{tau} treatment (Fig. 1BGo, top), reaching a maximum (approximately 3-fold increase over control value in lane 1) within 6 h (lane 3; P < 0.01, n = 3). Longer periods of IFN-{tau} treatment did not further increase PGHS-2 mRNA levels (see 24-h stimulation in lane 4; P < 0.01, n = 3). At the protein level (Fig. 1CGo), we observed an increase similar to that of mRNA (~3-fold), which was obvious at 24 h after stimulation (lane 4, P < 0.01, n = 3). PGHS-2 protein expression correlated well with prostacyclin accumulation measured by ELISA in the culture supernatants (Fig. 1DGo). The maximal induction of PGI2 accumulation by IFN-{tau} was approximately 50-fold over the control value; that is, PGI2 accumulation in the absence of IFN-{tau} treatment, and was obtained 24 h after treatment (P < 0.001, n = 4). These data clearly demonstrate that IFN-{tau} induces PGHS-2 gene expression and activity of bovine myometrial cells.



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  		Figure 1. Induction of PGHS-2 expression and activity by IFN-{tau}. A, Dose-dependent effects of IFN-{tau} on PGHS-2 protein expression. Primary bovine myometrial cells were left untreated (lane 1) or treated with 1 (lane 2), 10 (lane 3), or 100 ng/ml of IFN-{tau} (lane 4) for 24 h. Protein extracts (50 µg) were subjected to immunoblot analysis with a monoclonal antibody against PGHS-2 (top panel) or {alpha}-actin (bottom panel). The bands were quantified by densitometry using the NIH Scion Image Software, and the relative optical densities (ROD) of the bands are indicated. B and C, Analysis of PGHS-2 expression by IFN-{tau}. Primary bovine myometrial cells were treated with 100 ng/ml IFN-{tau} for 3, 6, and 24 h; and PGHS-2 expression levels were determined either by Northern blot analysis (Fig. 1BGo, top) or immunoblot analysis using a monoclonal antibody against PGHS-2 (Fig. 1CGo, top). PGHS-2 RNA and protein levels from three different experiments were normalized to the levels of 18S rRNA (B, bottom) and {alpha}-actin protein (C, bottom), respectively. Blot signals were quantified from three individual experiments derived from different myometrial preparations. *, P < 0.01 significantly different from time 0 values. Induction of PGHS-2 activity by IFN-{tau} was assessed by measuring PGI2 accumulation in the supernatant myometrial cell culture medium by ELISA, and results are shown as means ± SEM (n = 4); *, P < 0.01; **, P < 0.001 (Fig. 1DGo).

 
IFN-{tau} induces both I SGF3 and STAT-1/STAT-1 complex formation in bovine myometrium
To better understand the molecular mechanism(s) underlying IFN-{tau} action in bovine myometrium, we examined whether IFN-{tau} is capable of activating the JAK-STAT pathway in bovine myometrium. To do so, we first tested the ability of IFN-{tau} to induce STAT binding to DNA oligonucleotides encompassing the ISG-15-ISRE and IFP-53-GAS motifs used by IFN-{alpha}/ß and IFN-{gamma}, respectively (Fig. 2Go). DNA-binding was analyzed by EMSAs. When the ISG-15-ISRE oligonucleotide was used, we found that IFN-{tau} induced the formation of a protein/DNA complex with the migration and specificity of ISGF3 (Fig. 2AGo, lane 2). The specificity of this complex was confirmed by competition with an excess of unlabeled oligonucleotide (lane 3), whereas its composition was analyzed by supershift with an antibody against STAT-1{alpha} (lane 4) or disappearance with antibodies against STAT-2 (lane 5) or PY (lane 7). Note that incubation with mouse IgG1 did not affect the DNA binding of the complex (lane 6). Similarly, when the IFP-53-GAS oligonucleotide was used, we observed the formation of a specific DNA/protein complex (Fig. 2AGo, lane 9), which was competed by the presence of an excess of unlabeled oligonucleotide (lane 10). This complex consisted of STAT-1 homodimers judged by its competition by anti-STAT-1 (lane 11) and anti-PY antibodies (lane 14) but not by STAT-2 (lane 12) or mouse IgG1 (lane 13).



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  		Figure 2. Induction of STAT DNA binding and phosphorylation by IFN-{tau}. A, WCE (10 µg) from bovine myometrium cells, uninduced (lanes 1 and 8) or induced with 100 ng/ml IFN-{tau} for 15 min (lanes 2–7 and 9–14), were used for EMSA binding either to ISG-15 ISRE (lanes 1–7) or to IFP-53 GAS (lanes 8–14) 32P-labeled probe. A 200-fold excess of the corresponding unlabeled dsDNA oligonucleotide was added in cold competition reactions (lanes 3 and 10). For supershift assays, protein extracts were preincubated with 4 µg anti-STAT-1{alpha} (lanes 4 and 11), anti-STAT-2 (lanes 5 and 12), anti-PY (lanes 7 and 14), or mouse IgG1 antibody (lanes 6 and 13). B, WCE (10 µg) from primary myometrial cells treated with 100 ng/ml IFN-{tau}, for 15 min, were tested for EMSA binding either to 32P-labeled IFP-53 GAS (lanes 1–3) or to 32P- labeled ISG-15 ISRE (lanes 4–6) in the absence (lanes 1 and 4) or presence of 200-fold excess of unlabeled IFP-53 (lanes 2 and 5) or ISG-15 oligonucleotide (lanes 3 and 6). The bands were quantified by densitometry using the NIH Scion Image Software, and the ROD of the bands are indicated. Results represent the means ± SEM of three separate experiments (*, P < 0.01, n = 3). C, WCE (150 µg) from myometrial cells, untreated (lanes 1, 3, and 5) or treated with 100 ng/ml IFN-{tau} for 15 min, were immunoprecipitated with anti-STAT-1{alpha} antibody, followed by Western blotting with anti-PY antibodies (lanes 1 and 2), anti-STAT-1{alpha} antibody (lanes 3 and 4), or anti-STAT-2 antibody (lanes 5 and 6). mIgG1, Mouse IgG1.

 
The specificity of the DNA/protein complexes induced by IFN-{tau} for each oligonucleotide probe was further ascertained by cold competition assays (Fig. 2BGo). Specifically, binding to 32P-labeled IFP-53 GAS DNA oligonucleotide (lane 1) was completely abolished by the presence of unlabeled IFP-53 GAS (lane 2) and partially by the presence of unlabeled ISG-15 (lane 3). On the other hand, binding to 32P-labeled ISG-15-ISRE DNA was not affected by the presence of an excess of unlabeled IFP-53 GAS (lane 5) but was completely abolished by the presence of ISG-15 ISRE (lane 6). The partial, but significant (P < 0.01, n = 3), competition of binding to IFP-53 GAS by ISG-15 ISRE (lane 3 vs. lane 1) may be explained by the different affinities by which STAT proteins bind to ISRE and GAS motifs (21).

The activation of the JAK-STAT pathway by IFN-{tau} was further demonstrated by the tyrosine phosphorylation of activated STATs (Fig. 2CGo). Protein extracts from bovine myometrial cells cultured in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 100 ng/ml IFN-{tau} for 15 min were subjected to immunoprecipitation with an anti-STAT-1{alpha} antibody followed by immunoblotting with anti-PY (lanes 1 and 2), anti-STAT-1{alpha} (lanes 3 and 4), and anti-STAT-2 antibodies (lanes 5 and 6). We observed that IFN-{tau} induced the association of two tyrosine phosphorylated proteins after immunoprecipitation with anti-STAT-1{alpha} antibody (lane 2). These proteins were STAT-1 and STAT-2, judged by the immunoblotting of the same blot with anti-STAT-1{alpha} (lane 4) and anti-STAT-2 (lane 6) antibodies. These data demonstrate that activation of the JAK-STAT pathway by IFN-{tau} proceeds through the same mechanisms used by type I and type II IFNs.

IFN-{tau} induces p38 MAPK activation
The p38 MAPK pathway has been recently implicated in the activation of the JAK-STAT pathway and transcriptional activation by IFNs (25, 40). Therefore, we examined whether p38 MAPK is induced by IFN-{tau}, and its possible role in PGHS-2 expression. Primary bovine myometrium cells were left untreated (Fig. 3AGo, lane 1) or treated 24 h with 100 ng/ml IFN-{tau} (lane 2), and activation of p38 kinase was tested first by immunoblotting with an antirabbit phospho(Thr180/Tyr182)-p38 MAPK antibody followed by immunoblotting with an antirabbit p38 MAPK antibody. As control to p38 activation, we used protein extracts from the Jurkat leukemia T cells, untreated (Fig. 3AGo, lane 3) or PMA-treated (lane 4). Quantitative analysis of these data (Fig. 3BGo) indicated that, in bovine myometrium, IFN-{tau} is indeed an inducer of p38 phosphorylation.



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  		Figure 3. Induction of p38 MAPK kinase by IFN-{tau}. A, Fifty micrograms of protein extracts from primary myometrial cells, untreated (lane 1) or treated with 100 ng/ml IFN-{tau} for 24 h (lane 2), and protein extracts from untreated (lane 3) or PMA-treated Jurkat T leukemia cells (lane 4) were subjected to immunoblotting with an antibody against the phosphorylated (Thr180/Tyr182) form of p38 MAP kinase (upper panel), followed by immunoblotting with an antibody against p38 MAPK (lower panel). B, The quantification of p38 MAPK activation, before and after IFN-{tau} stimulation for 24 h, from multiple experiments, was done by densitometry using the NIH Scion Image Software; and the ROD of the bands are shown. The graph shows the means ± SEM from three separate experiments (*, P < 0.05, n = 3).

 
Next, we examined the effect of p38 MAPK in activation of STAT induced by IFN-{tau} (Fig. 4Go). To this end, we tested STAT DNA-binding by EMSA in the absence or presence of the specific p38 kinase inhibitor SB203580. As shown in Fig. 4AGo, IFN-{tau} induced the binding of ISGF3 factor to ISG-15 ISRE DNA (lane 1). However, ISGF3 DNA binding was not affected by the presence of the SB203580 inhibitor (lane 3). The identity of ISGF3 was verified by supershift analysis with anti-STAT-1{alpha} antibodies (lanes 2 and 4). Like ISGF3, DNA-binding of STAT-1 homodimers to IFP-53-GAS DNA was readily induced by IFN-{tau} (lane 5) and was not affected by the presence of SB203580 (lane 7). The identity of the complex was also verified by performing competition with anti-STAT-1 antibody (lanes 6 and 8).



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  		Figure 4. Activation of p38 MAPK by IFN-{tau} does not modulate STAT DNA-binding. A, Primary bovine myometrial cells were treated for 15 min with 1 µM SB203580, followed by a stimulation with 100 ng/ml IFN-{tau} for 15 min. WCE (10 µg of protein) was incubated with 32P-labeled ISG-15-ISRE (lanes 1–4) or IFP-53-GAS (lanes 5–8) probe. The effect of SB203580 on STAT-1 DNA binding was determined by preincubating WCE for 30 min, in the presence of STAT-1 antibody (lanes 4 or 8, compared with lanes 2 and 6, respectively). B, WCE were prepared from myometrial cells, untreated (lane 1) or treated (lanes 2 and 3) for 15 min with 100 ng/ml IFN-{tau}. Myometrial cells were treated with 1 µM SB203580 for 15 min before the addition of IFN-{tau} (lane 3). Protein extracts (150 µg) were immunoprecipitated with anti-STAT-1 antibody; immunoprecipitates were resolved by SDS-7%PAGE and analyzed by immunoblotting for STAT-1 protein (top panel) or phosphorylation with anti-PY (middle panel) or antiphosphoserine 727 antibody (bottom panel).

 
It has been previously shown that phosphorylation of STAT-1 on serine 727 by IFN-{gamma} in human HeLa cells requires p38 MAPK activation and is inhibited by the presence of the SB203580 (25). Based on this, we wished to examine the possibility that activation of p38 kinase by IFN-{tau} in primary myometrial cells induces STAT-1 phosphorylation (Fig. 4BGo). To do so, STAT-1 was immunoprecipitated from protein extracts from myometrial cells, unstimulated (lane 1) or stimulated with IFN-{tau} in the absence (lane 2) or presence (lane 3) of SB203580. Immunoprecipitated STAT-1 was then subjected to immunoblot analysis with either anti-STAT-1 antibody (top panel), anti-PY antibody (middle panel), or an antibody to phosphoserine 727 of STAT-1 (bottom panel). In these experiments, we observed that phosphorylation of STAT1 on tyrosine 701 was induced by IFN-{tau} (lane 2) but was not affected by the presence of the p38 MAPK inhibitor (lane 3). We also observed that STAT1 is constitutively phosphorylated on serine 727 (lane 1) in the absence of IFN-{tau}. Stimulation with IFN-{tau} resulted in a modest 25% induction (n = 2) of serine 727 phosphorylation (lane 2), whose levels were not affected by the presence of the SB203580 inhibitor (lane 3). These data show that the p38 MAPK pathway plays no role in STAT-1 serine 727 phosphorylation, as has been demonstrated for many different cell lines and tissues (24), with the exemption of HeLa cells (25).

Induction of PGHS-2 synthesis by IFN-{tau} requires p38 MAPK activation
The activation of p38 MAPK by IFN-{tau} prompted us to examine the role of this pathway in the induction of PGHS-2 synthesis (Fig. 5Go). Primary myometrial cells were stimulated with IFN-{tau}, for 24 h, in the absence (Fig. 5AGo, lane 1) or presence of SB203580 for various periods of time (Fig. 5AGo, lanes 2–4). The production of PGHS-2 was monitored by immunoblot analysis with an anti-PGHS-2 monoclonal antibody (top panel), whereas the activation of p38 MAPK was tested by immunoblotting the membrane with an antirabbit phospho(Thr180/Tyr182)-p38 MAPK antibody (middle panel), followed by immunoblotting with an antirabbit p38 MAPK antibody (bottom panel), respectively. We found that PGHS-2 protein expression was gradually inhibited by the presence of SB203580, reaching a 30% inhibition after a 24 h treatment (top panel, lane 4; P < 0.05, n = 3). On the other hand, the activation (middle panel) and protein levels of p38 (bottom panel) remained constant throughout the period of treatment. This is in accord with previous findings that SB203580 acts by inhibiting the intrinsic ATPase activity of p38 kinase rather than diminishing its phosphorylation levels (41). The inhibition of PGHS-2 synthesis correlated well with a significant decrease (P < 0.005) of IFN-{tau}-induced PGI2 accumulation after 24 h of SB203580 treatment (Fig. 5BGo).



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  		Figure 5. Induction of PGHS-2 expression by IFN-{tau} requires the activation of p38MAPK. A, Primary myometrial cells, grown to confluency, were serum-starved for 24 h in medium containing 100 ng/ml IFN-{tau}, followed by incubation in the absence (lane 1) or presence of 1 µM SB203580 for 3 h (lane 2), 6 h (lane 3), or 24 h (lane 4). WCE (50 µg protein) was subjected to immunoblot analysis with antibodies against PGHS-2 (top panel), phosphorylated p38 (middle panel), or p38 (bottom panel). Results are shown as means ± SEM [*, P < 0.01 significantly different from time 0 value (without SB203580 treatment)]. B, Primary myometrial cells were treated with 100 ng/ml IFN-{tau}, in the absence or presence of 1 µM SB203580, and the levels of PGI2 in the culture supernatant were measured by ELISA. The results are expressed as means ± SEM (*, P < 0.005, n = 3). C, Primary myometrial cells were left untreated (lane 1) or treated with 100 ng/ml IFN-{tau} for 6 h (lanes 2–8), in the absence (lane 2) or presence of 0.5 µg/ml actinomycin D (Act. D; lanes 2–5) or in the presence of 0.5 µg/ml actinomycin D and 1 µM SB203580 (lanes 6–8) for the indicated periods of time. Total RNA (10 µg) was subjected to Northern blot analysis using either [32P]-labeled PGHS-2 cDNA (top panel) or [32P]-labeled 18S DNA as a probe (bottom panel). D, Radioactive bands from C were quantified by densitometric analysis of autoradiograms, at the linear range of exposure, using NIH Image 1.57 software; and data represent the mean values ± SEM of three independent experiments (*, P < 0.001, n = 3).

 
Because IFN-{tau} treatment is associated with an induction of PGHS-2 mRNA levels (Fig. 1AGo), next we assessed the effect of SB203580 on PGHS-2 mRNA expression (Fig. 5CGo). Because the induction of PGHS-2 mRNA expression could be a result of enhanced transcription and/or mRNA stabilization, we measured PGHS-2 mRNA levels in myometrial cells treated with the inhibitor of transcription actinomycin D in the absence or presence of SB203580 (Fig. 5CGo). Treatment with actinomycin D blocked the de novo transcription of PGHS-2 mRNA within the first 15 min (top panel, lane 3) reaching a 40% inhibitory effect after 1-h treatment (lane 5). The presence of SB203580 further enhanced the inhibition of PGHS-2 mRNA levels by actinomycin D (top panel, lanes 6–8), reaching a 95% inhibition after 1 h of the combined treatment (lane 8). Quantification of PGHS-2 mRNA levels after normalization to 18S rRNA levels (Fig. 5CGo, bottom) clearly indicated a higher rate of inhibition of PGHS-2 mRNA expression in the presence of actinomycin D and SB203580, compared with actinomycin D alone (Fig. 5DGo). These data clearly indicate that induction of p38 MAPK activity results in stabilization of PGHS-2 mRNA; and this may account, at least in part, for the induction of PGHS-2 mRNA expression by IFN-{tau} in bovine myometrial cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The role of PGs in the control of uterine contractility has been fully demonstrated (1, 2). Though their action as positive and/or negative modulators of calcium channel activity and Ca2+ sensitivity of proteins associated with contractile apparatus is well documented (42), little is known about species-specific local modulators affecting myometrial PG biosynthesis. Given the potential role of IFN-{tau} in the maintenance of a quiescence state in the uterus, we attempted to examine the effect of IFN-{tau} on the myometrial PG pathway. In this study, we clearly demonstrate that IFN-{tau} significantly induces PGHS-2 gene expression in primary culture of bovine myometrial cells, resulting in increased prostacyclin production. In regard to physiological relevance of our findings, increased PGHS-2 expression by IFN-{tau} may play a role in the quiescence of myometrium in vivo, which is essential for the establishment of pregnancy. Such a function would be consistent with previous observations concerning the action of IFN-{tau} in bovine endometrium. That is, IFN-{tau} decreases PGF2{alpha} secretion and PGHS-2 expression in endometrial cells (13, 43), and this is considered essential for the prevention of luteolysis during pregnancy in cows. Thus, our results may indicate a new role for IFN-{tau} in modulation of myometrial contractility.

The sequence and functional similarities between IFN-{tau} and IFN-{alpha} (16, 17) have implied similar signaling properties between the two molecules IFN-{tau} and IFN-{alpha}. This prediction has been supported by a recent report showing the activation of the JAK-STAT pathway by IFN-{tau} in bovine endometrial cells (18). However, many distinct functional properties made IFN-{tau} stand apart from the type I IFN family (44, 45, 46, 47). In this report, we demonstrate that IFN-{tau} induces two distinct DNA-binding factors associated with both type I and type II IFN signaling pathways. Indeed, depending on the DNA recognition elements, IFN-{tau} treatment of primary myometrial cells induced the formation of ISGF-3 complex encompassing IFN regulatory factor-9 with STAT-1/STAT-2 heterodimers and STAT-1 homodimer associated with GAS element. The IFN-{tau}-activated state of these complexes was confirmed by EMSAs and immunoprecipitation studies revealing the presence and interaction of two tyrosine-phosphorylated STAT proteins, STAT-1 and STAT-2. These results confirm those obtained on the human Burkitt lymphoma cell line, describing that induction of STAT-1 and STAT-2 phosphorylation by IFN-{tau} (48).

It has been recently demonstrated that, in addition to the classical and mandatory tyrosine phosphorylation of STAT-1 protein necessary for its nuclear translocation and subsequent gene regulatory effect, other distinct phosphorylation mechanisms were required for the full transcriptional activity of STAT (24). Indeed, Goh et al. (25) showed that STAT-1{alpha}-Ser727 phosphorylation by p38 MAPK was a prerequisite only for type II IFN-associated transcriptional gene activation. Interestingly, it has been described that p38 MAPK was able to phosphorylate cPLA2, a prerequisite mechanism for this enzyme activation (31, 49). In this study, we show that SB203580, a potent inhibitor of p38 MAPK, strongly inhibits IFN-stimulated myometrial PGHS-2 gene expression and subsequent PGI2 production, clearly indicating that p38 MAPK activation modulates the myometrial PG biosynthesis pathway. However, whereas SB203580 strongly decreases the steady-state levels of PGHS-2 transcript and protein, it has no effect on ISGF3 or STAT-1 homodimer formation, or on STAT-1 Ser727 phosphorylation, suggesting that p38 MAPK activation of IFN-{tau}-induced PGHS-2 gene expression is independent of JAK/STAT signaling pathway in this system. It has recently been established that bovine PGHS-2 mRNA carries AUUUA motifs in its 3'-untranslated region (50). These motifs have been shown to play an important role in the regulation of mRNA stability (51, 52). On the other hand, p38 MAPK pathway has recently been associated with cytokine-induced mRNA stabilization (53). Similar results were recently found concerning the stability of a chimeric ß-globin-PGHS-2 transcript (54). In this study, the authors clearly demonstrated that the stable ß-globin mRNA, which was rendered unstable by the insertion of AUUUA motifs from the PGHS-2 3' untranslated region, was stabilized by an activator of p38 MAPK. Indeed, our findings indicate that, whereas a link exists between IFN-{tau}-induced PGHS-2 gene expression and p38 MAPK activation, IFN-{tau}-mediated STAT activation is not dependent on MAPK pathway, suggesting that: 1) IFN-{tau} modulation of PGHS-2 gene expression requires a specific signaling pathway independent on the JAK-STAT pathway; and 2) p38 MAPK activation acts downstream of the JAK-STAT pathway, affecting directly PGHS-2 RNA stability. Although there is no evidence that IFN-{tau} is present in the general circulation or in lymphatic fluid draining uterus, stabilization of IFN-{tau}-induced PGHS-2 mRNA by the stress-associated MAPK remains a possible mechanism for myometrial cells to maintain the needed levels of PGI2 necessary for myometrial quiescence at this stage of the estrous cycle.


   Acknowledgments
 
We thank Dr. J. M. Moutquin for his generous contribution of PGI2 antisera, Dr. J. Sirois for the gift of the bovine PGHS-2 cDNA probe, and Mr. M. Hamadeh for his excellent assistance in the statistical analysis of these results. We thank L. Marchand for his technical assistance and A. H.-T. Wong for his suggestions. We gratefully acknowledge the generosity of Merck Frosst Canada Inc. for cPLA2 antibody.


   Footnotes
 
This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (to F.D.-B.) and the Canadian Institutes of Health Research, and the Human Frontier Science Program Organization (to A.E.K.). A.E.K. is a recipient of a Canadian Institutes of Health Research Scientist Award.

Abbreviations: cPLA2, Cytosolic PLA2; dsDNA, double-stranded DNA; IFN, interferon; ISG, IFN-stimulated gene; ISGF, ISG factor; ISRE, IFN-stimulatory response element; JAK, Janus family of tyrosine kinases; PGHS, PG endoperoxide G/H synthase; PMA, phorbol myristate acetate; PY, phosphotyrosine; ROD, relative optical densities; STAT, signal transducer and activator of transcription factor; WCE, whole-cell extracts.

Received April 5, 2001.

Accepted for publication August 10, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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