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Endocrinology, doi:10.1210/en.2008-0744
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Endocrinology Vol. 150, No. 1 485-491
Copyright © 2009 by The Endocrine Society

Development and Characterization of a Simian Virus 40 Immortalized Bovine Endometrial Stromal Cell Line

Narayanan Krishnaswamy, Pierre Chapdelaine, Jacques P. Tremblay and Michel A. Fortier

Centre Hospitalier Universitaire de Québec, Centre Hospitalier de l’Université Laval, and Centre de Recherche en Biologie de la Reproduction et Département d’Obstétrique et Gynécologie (N.K., P.C., M.A.F.), Département de Physiologie (J.P.T.), Université Laval, Québec, Canada G1K 7P4

Address all correspondence and requests for reprints to: Dr. Michel A. Fortier, Ontogénie et Reproduction, Room T-1-49, Centre Hospitalier Universitaire de Québec (CHUL), 2705 Boulevard Laurier, Québec, G1V 4G2 Québec, Canada. E-mail: MAFortier{at}crchul.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In ruminants, interferon-{tau} (IFN{tau}) is the maternal recognition signal inhibiting prostaglandin (PG) F2{alpha} production by endometrial epithelial cells and stimulating interferon-stimulated genes in the stroma. Stromal cells mediate the action of progesterone on epithelial cells during pregnancy. Our working hypothesis is that IFN{tau} acts as a molecular switch that turns on PGE2 production in endometrial stromal cells while suppressing PGF2{alpha} production from epithelial cells. In this report we document immortalization and functional characterization of a bovine stromal cell line from the caruncular region of the endometrium [caruncular stromal cell (CSC)]. Primary stromal cells were immortalized by nucleofection with simian virus 40 large T antigen and integrase. The resulting cell line, CSC, expresses stromal cell-specific vimentin, estrogen, and progesterone receptors, and is amenable for transient transfection. Basal and stimulated production of PGE2 is higher than PGF2{alpha} and associated with cyclooxygenase (COX) 2 expression. Phorbol myristate acetate (PMA) and IFN{tau} up-regulate COX2 and PG production in a dose-dependent manner. When added together, low concentrations of IFN{tau} inhibit PMA-induced COX2 expression; whereas this inhibition is lost at high concentrations. Expression of signal transducer and activator of transcription 1 is induced by IFN{tau} at all concentrations studied but is not modulated by PMA. Because expression of signal transducer and activator of transcription 1 does not exhibit the biphasic response to IFN{tau}, we investigated the p38 MAPK pathway using the selective inhibitor SB203580. Inhibition of the p38 MAPK pathway abolishes IFN{tau} action on PG production. In summary, CSC appears as a good stromal cell model for investigating the molecular mechanisms related to IFN{tau} action and PG production in the bovine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Epithelial and stromal cells of the endometrium are the site of recognition of pregnancy. In ruminants, epithelial cells are the target of oxytocin (OT) to generate luteolytic pulses of prostaglandin (PG) F2{alpha} (1). However, spatiotemporal expression of oxytocin receptor is coregulated with that of estrogen and progesterone receptors [ERs (ER{alpha}) and PRs, respectively], and may involve paracrine interactions between epithelial and stromal cells (2, 3, 4). In addition, PGF2{alpha} of stromal origin, secreted in response to TNF{alpha}, may contribute to the initiation of luteolysis (5).

Interferon-{tau} (IFN{tau}) is the maternal recognition signal in ruminants. Apart from inhibiting PGF2{alpha} pulses of epithelial origin, IFN{tau} stimulates a set of genes (interferon-stimulated genes) in the endometrial stroma (6). Using bovine primary stromal cell cultures, we have shown that IFN{tau} stimulates the production of PGE2 (7). Generation of stable in vitro endometrial culture systems appears as the logical next step for investigating the complex signaling pathways and transcriptional mechanisms regulated by IFN{tau} in the bovine. At present, a spontaneously derived bovine endometrial epithelial cell line, bovine endometrial cell (BEND), is used as a model to investigate the mechanisms regulating PG production, but it expresses both epithelial and stromal cell markers, suggesting a mixed phenotype (8). Immortalized cell lines of luminal and glandular epithelial cells and stromal cells have been generated and characterized in sheep (9), but no bovine stromal cells are available. In this report we document the generation of a stromal cell line and show its utility in studying the regulation of PG biosynthesis in response to the embryonic signal IFN{tau}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immortalization and clone selection
Primary stromal cell cultures were prepared as described previously with minor modifications (10). Stromal cells were transfected by nucleofection with a plasmid expressing simian virus 40 large T antigen (SV40 TAG) and a gene resistant to aminoglycoside G418 (neomycin) and another plasmid coding for bacteriophage phiC31 integrase (11). The cytomegalovirus promoter of the SV40 TAG transgene was flanked by an attB sequence to improve plasmid integration into the genome. Subconfluent cultures of caruncular and intercaruncular stromal cells (ICSCs) were trypsinized and resuspended in serum free media, and 5 µg plasmid DNA coding for integrase and 5 µg vector DNA containing SV40 TAG were added to 1 x 106 cells and nucleofected using the T16 program. Nucleofection efficiency was 60% as assessed by green fluorescence protein. After 3 d, the cells were trypsinized and cultured in 150 x 20-mm petri plates for 7 d in presence G418 (200 µg/ml) to select resistant colonies. A total of 33 clones (seven caruncular and 26 intercaruncular) was picked using O-ring and clonally propagated in separate T-25 flasks up to 10 passages (P10). We then selected one caruncular (CSC) (clone no. CAR7) and one intercaruncular (ICSC) (clone no. ICAR6) stromal cell clone according to basal and TNF{alpha} (6 nM) induced PGE2 and PGF2{alpha} production, growth rate, and stromal morphology, and passed the cell lines until P50.

Immunofluorescence analysis
CSCs and ICSCs were cultured on Lab Tek 4-chamber slides (Nalge Nunc Intl., Rochester, NY) and analyzed for expression of cytokeratin (Sigma-Aldrich Corp., St. Louis, MO), vimentin (in house antibody), and SV40 TAG (Oncogene Research Products, San Diego, CA) by immunofluorescence as described previously (12). Lipofectamine-mediated transfection of green fluorescent protein was done in CSCs as per the manufacturer’s instructions (Invitrogen Life Technologies Inc., Burlington, Ontario, Canada).

Experimental protocols
The CSC line was selected for the subsequent studies because it exhibited optimal growth rate and a PG production profile representative of all clones tested, including those from intercaruncular areas (ICSC). Typically, CSC cultures were initiated from a frozen aliquot and grown to confluency in a T75 flask for 60–72 h at 37 C and 5% CO2. The monolayer was trypsinized, extended with RPMI 1640 supplemented with 10% fetal bovine serum, divided into two equal volumes, and centrifuged at 1500 rpm for 10 min. One fraction was frozen and stored at –150 C for future experiments. The other fraction was diluted at 4 x 104 cells per ml and seeded in 24-well plates. Confluent cultures were exposed to steroid free medium overnight before treatment. At the end of the experiment, culture medium was harvested and stored at –20 C until analysis for PG. Protein extraction and estimation were done as described previously (13). All experiments were replicated three times, and each treatment was run in quadruplicate unless indicated otherwise. For functional characterization, cells were treated with IFN{tau} (10 µg/ml), lipopolysaccharide (LPS) (10 ng/ml), phorbol myristate acetate (PMA) (10 nM), OT (500 nM), TNF{alpha} (6 nM), and cyclooxygenase (COX) 2 inhibitor NS-398 (1 µM). The concentrations used were based on previously published conditions (7). PGE2 and PGF2{alpha} production by CSCs was then associated with expression levels of key enzymes of the biosynthetic cascade. Experiment 2 aimed at comparing PG biosynthesis after treatment with increasing concentrations of IFN{tau} and PMA from 0.02–20 µg/ml and 1 pM to 100 nM, respectively, and their interactions on COX2 and signal transducer and activator of transcription (STAT) 1 genes in CSCs. In experiment 3 the involvement of p38 MAPK was tested by treating CSCs with high concentration of IFN{tau} (10 µg/ml) in the presence or not of the selective inhibitor of p38 MAPK, SB203580 (1 µM).

Enzyme immunoassays of PGE2 and PGF2{alpha}
PGs were assayed by competitive enzyme immunoassay using acetylcholinesterase-linked PG tracers (Cayman Chemical Co., Ann Arbor, MI) as described previously using rabbit anti-PGE2 (kindly provided by Dr. T. G. Kennedy, University of Western Ontario, London, Ontario, Canada) and sheep anti- PGF2{alpha} (Bio-Quant, Ann Arbor, MI) (10).

Western blot analysis
An aliquot of 10 µg protein was loaded in each lane, resolved on 12.5% SDS-PAGE, and electrotransferred onto 0.45 µm nitrocellulose membrane (Bio-Rad Laboratories, Inc., Mississauga, Ontario, Canada). However, for detection of cytosolic phospholipase A2 (cPLA2), 7% gel was used, and for microsomal prostaglandin E synthase (mPGES)-1, 0.2 µm nitrocellulose membrane was used. The membranes were blocked in 5% (wt/vol) nonfat dried milk in PBS containing 0.05% Tween 20 for 1 h at room temperature and incubated overnight at 4 C with respective primary antibodies. The primary antibody dilutions were as follows: 1:500 for anti-cPLA2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); 1:3000 for anti-COX1 and anti-COX2 (kindly provided by Dr. S. Kargman, Merck Frosst Canada Ltd., Kirkland, Quebec, Canada); 1:250 for anti-mPGES-1 (Cedarlane, Burlington, Ontario, Canada); 1:500 for mPGES-2 and cytosolic prostaglandin E synthase (cPGES), and 1:2000 for anti-aldoketoreductase 1 B5 (AKR1B5), a polyclonal serum raised in our laboratory using recombinant protein; 1:1000 for STAT1 (BD Biosciences, Mississauga, Ontario, Canada) and anti-pS727 STAT1 (Upstate Biotechnology Inc., Lake Placid, NY); 1:1000 for phosphorylated and unphosphorylated p38 antibodies (Upstate Biotechnology); and 1:5000 for β-actin (Sigma-Aldrich). After three washes of 10 min each in PBS, the membranes were incubated for 1 h at room temperature with appropriate secondary antibody. The membranes were washed three times in PBS containing 0.05% Tween 20, treated for 1 min with enhanced chemiluminescent substrate (PerkinElmer Life and Analytical Sciences, Inc., Waltham, MA), and exposed to Bio-Max film (PerkinElmer Life and Analytical Sciences). Relative OD (ROD) of three different immunoblots from each experiment was quantitated by densitometry (Alpha imager; Fisher Scientific Co., Ottawa, Ontario).

RT-PCR
Total RNA was extracted using TRIZOL (Invitrogen Life Technologies), reverse transcribed with Superscript II RT (Invitrogen Life Technologies). To demonstrate ER{alpha} and PR in the cell line, the following specific sets of primers were used. For ER{alpha}, the sense and antisense primers were 5'-ATGACCCTACCAGACCTTTCAGT-3' and 5'-ATTTGAGGCACACAAACTCTTC-3', respectively. Similarly, for PR, the forward and reverse primers were 5'-ATTGTTGATAAAATCCGCAGAAA-3' and 5'-GAGGTATCAGGTTTGCTGTTGTC-3', respectively. ER{alpha} primers were deduced from accession no. NM_001001443, whereas PR specific primers were designed based on the accession no. AY656812.

Statistical analysis
Randomized block design was used in all the experiments with treatment as the main factor and plate as random effect. The resulting data on PG production were transformed into fold stimulation (except See Fig. 2Go) by dividing each observation by the mean of control. The StatView program (SAS Institute Inc., Cary, NC) was used for analyzing the transformed data. The group mean of different treatments was tested by two-way ANOVA with Fisher’s projected least significant difference as the post hoc test to find the critical difference between pairs of treatment means. The confidence level was set at 95% (P < 0.05) to determine statistical significance. Data are presented as the mean ± SEM.


Figure 2
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  		FIG. 2. PGE2 and PGF2{alpha} production in immortalized endometrial stromal cells. A, Immortalized cell lines from all caruncular (CAR) (n = 7) and intercaruncular (ICAR) (n = 26) clones were grown to confluency and stimulated or not with TNF{alpha} 6 nM for 24 h. Results are the mean ± SEM of PG levels from all clones. B, Effect of NS398, a COX2 inhibitor, on TNF{alpha}-induced PG production in CSCs. Bars with different superscripts differ significantly (P < 0.05).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of bovine endometrial stromal cell lines and phenotype characterization
The selected stromal cell line could be propagated until at least P50 while maintaining all phenotypical characteristics of primary cultures and expression of ER{alpha} and PR mRNA (Fig. 1CGo). CSCs and ICSCs expressed SV40 TAG, mesenchyme-specific vimentin but were negative for the epithelial-specific cytokeratin (Fig. 1Go, A and B). The cell line is also amenable for transient transfection. Average basal and TNF{alpha}-stimulated PGE2 and PGF2{alpha} production of all bovine endometrial stromal clones from either caruncles (n = 7) or intercaruncles (n = 26) is shown in Fig. 2AGo. PGE2 production was higher (P < 0.05) than PGF2{alpha} under basal and TNF{alpha}-stimulated conditions (Fig. 2AGo), which was abolished in the presence of the COX-2 inhibitor NS-398 (Fig. 2BGo). PGE2 production was also significantly higher in the caruncular clones (P < 0.05).


Figure 1
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  		FIG. 1. A, Characterization of cytoskeletal proteins expressed in the selected CSC line. Subconfluent endometrial cells were stained with cytokeratin and vimentin antibodies, and detected with a fluorescent secondary antibody. Left panels represent phase-contrast illumination, whereas center and right panels show cytokeratin and vimentin fluorescence, respectively (magnification, x100). B, Integration of SV40 TAG within the genome of bovine caruncular and intercaruncular stromal clones. 1, Demonstration by immunofluorescence (magnification, x100) 2, Detection of SV40 TAG by immunoblot. Lanes 1–3 represent primary stromal cells, CSC and ICSC. C, Demonstration of ERs and PRs in CSCs by RT-PCR. Lanes 1–3 represent primary stromal cells, CSC and ICSC.

 
Regulation of PG biosynthesis and expression of corresponding genes
In the caruncular cell line CSC, PGE2, and PGF2{alpha} production increased significantly after stimulation with IFN{tau}, TNF{alpha}, LPS, and PMA (P < 0.05), but not with OT (Fig. 3AGo). Note that in Fig. 3Go and the following figures, the scale used for PGE2 is higher than that of PGF2{alpha}. Analysis of the enzymes in the PGE2 and PGF2{alpha} biosynthetic pathway indicated that increased PG production was mostly coupled with increased COX2 and cPLA2 expression. Terminal synthases, although all expressed at a significant level, did not exhibit any modulation (Fig. 3BGo). Increased expression of COX2 together with inhibition of TNF{alpha} stimulation by NS398 (Fig. 2BGo) confirms COX2 as a rate-limiting step.


Figure 3
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  		FIG. 3. Effect of IFN{tau}, TNF{alpha}, LPS, PMA, and OT on PG production in CSCs. A, PGE2 and PGF2{alpha} production. Values represent the mean ± SEM of three different experiments run in quadruplicate. Bars with different superscripts differ significantly (P < 0.05). B, Representative immunoblots of cPLA2, COX 1 and 2, AKR1B5, mPGES 1 and 2, cPGES, and β-actin. Lanes 1–6 refer to control, IFN{tau} 10 µg/ml, TNF{alpha} 6 nM, LPS 0.01 µg/ml, PMA10 nM, and OT 0.5 µM, respectively.

 
CSC as a model for functional studies
As was reported for primary stromal and BEND cells, PMA induced COX2 expression and PGE2 and PGF2{alpha} production in a dose-dependent manner in CSCs (Fig. 4Go). Similarly, IFN{tau} increased (P < 0.05) PGE2 and PGF2{alpha} production, as well as COX2 expression (Fig. 5Go, A and B) in a dose-dependent manner. Expression of COX2 closely parallels PG production (Fig. 5CGo). Treatment with IFN{tau} stimulated STAT1 phosphorylation, and total STAT1 expression (Fig. 5Go, B and C). IFN{tau} was able to inhibit PMA-stimulated PG production at low concentrations, but this response disappeared at higher concentrations (Fig. 6AGo). The same effect was observed at the level of COX2 expression (Fig. 6Go, B and C). However, PMA had no effect on STAT1 expression or on its stimulation by IFN{tau}. When opposite conditions were tested, the effect of increasing concentrations of PMA stimulated further PG production and COX2 expression (Fig. 7Go) but did not impact on IFN{tau}-induced STAT1 expression (Fig. 7CGo). Because a dose-dependent biphasic effect of IFN{tau} on COX2 was not observed at the level of STAT1 in CSC, we hypothesized that MAPK could be involved as demonstrated for COX2 expression in the myometrium (14). Accordingly, IFN{tau} stimulated phosphorylation of p38 and inhibition of the p38 MAPK pathway with SB203580 reduced COX2 expression and PG production (Fig. 8Go). These results support a contribution of the p38 MAPK pathway in endometrial IFN{tau} signaling.


Figure 4
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  		FIG. 4. Effect of PMA on PG production and COX2 expression in CSCs. CSCs were treated with increasing concentrations of PMA (0–100 nM) for 24 h, and PGs were measured in the culture medium. Values represent the mean ± SEM of three different experiments run in quadruplicate. Bars with different superscripts differ significantly (P < 0.05). A, PGE2 and PGF2{alpha} production. B, Representative immunoblots of COX2 and β-actin. Numbers 1–6 indicate different concentrations of PMA: 0, 0.01, 0.1, 1.0, 10, and 100 nM, respectively. C, ROD values are the ratio between COX2 and β-actin.

 

Figure 5
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  		FIG. 5. Effect of IFN{tau} on PG production and COX2 expression in CSCs. CSCs were treated with increasing concentrations of IFN{tau} (0–20 µg/ml) for 24 h, and PGs were measured in the culture medium. Values represent the mean ± SEM of three different experiments run in quadruplicate. Bars with different superscripts differ significantly (P < 0.05). Lanes 1–6 on B and the x-axis of C indicate different concentrations of IFN{tau}: 0, 0.02, 0.2, 2.0, 10, and 20 µg/ml, respectively. A, PGE2 and PGF2{alpha} production. B, Representative immunoblots of COX2, phosphorylated and unphosphorylated STAT1 and β-actin. C, ROD values are the ratio between COX2/β-actin and phosphorylated to total STAT1.

 

Figure 6
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  		FIG. 6. Interaction between PMA and IFN{tau} in CSCs. CSCs were treated with PMA 10 nM and various concentrations of IFN{tau} (0–20 µg/ml) for 24 h, and PGs were measured in the culture medium. Values represent the mean ± SEM of three different experiments run in quadruplicate. Bars with different superscripts differ significantly (P < 0.05). Numbers on B and the x-axis of C indicate: control (1 ); PMA 10 nM (2 ); PMA 10 nM plus IFN{tau} 0.02 µg/ml (3 ); PMA 10 nM plus IFN{tau} 0.2 µg/ml (4 ); PMA 10 nM plus IFN{tau} 2.0 µg/ml (5 ); PMA 10 nM plus IFN{tau} 10 µg/ml (6 ); and PMA 10 nM plus IFN{tau} 20 µg/ml (7 ). A, PGE2 and PGF2{alpha} production. B, Representative immunoblots of COX2, STAT1, and β-actin. C, ROD values are the ratio between COX2, STAT1, and β-actin.

 

Figure 7
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  		FIG. 7. Interaction between IFN{tau} and PMA in CSCs. CSCs were treated with IFN{tau} 10 µg/ml and various concentrations of PMA (0.01–100 nM) for 24 h, and PGs were measured in the culture medium. Values represent the mean ± SEM of three different experiments run in quadruplicate. Bars with different superscripts differ significantly (P < 0.05). Numbers on B and the x-axis of C indicate: control (1 ); IFN{tau} 10 µg/ml (2 ); IFN{tau} 10 µg/ml plus PMA 0.01 nM (3 ); IFN{tau} 10 µg/ml plus PMA 0.1 nM (4 ); IFN{tau} 10 µg/ml plus PMA 1.0 nM (5 ); IFN{tau} 10 µg/ml plus PMA 10 nM (6 ); and IFN{tau} 10 µg/ml plus PMA 20 nM (7 ). A, PGE2 and PGF2{alpha} production. B, Representative immunoblots of COX2, STAT1, and β-actin. C, ROD values are the ratio between COX2 or STAT1 and β-actin.

 

Figure 8
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  		FIG. 8. Effect of the p38 MAPK inhibitor, SB203580, (1 µM) on IFN{tau} (10 µg/ml) induced PG production in CSCs. CSCs were treated with IFN{tau} 10 µg/ml in the presence or absence of SB203580 (1 µM) for 24 h. A, PGE2 and PGF2{alpha} production. Values represent the mean ± SEM of three different experiments run in quadruplicate. Bars with different superscripts differ significantly (P < 0.05). B, Representative immunoblots of COX2, β-actin, phosphorylated and unphosphorylated p38 MAPK and STAT1. Lanes 1–4 indicate control, IFN{tau} 10 µg/ml, IFN{tau} 10 µg/ml plus SB203580 1 µM, and SB203580 1 µM, respectively. C, ROD values are the ratio between COX2/β-actin and phosphorylated to total p38MAPK and STAT1.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The presence of SV40 TAG in CSCs and ICSCs and repeated passage without apparent senescence confirmed the permanent status of the selected cell lines. Vimentin-positive and cytokeratin-negative features support the stromal phenotype and mesenchymal origin of the cell lines. TNF{alpha} was used to stimulate PG production during initial screening of the stromal clones because this cytokine has been ascribed a role in the initiation of luteolysis (15). Both caruncular and intercaruncular clones produced PGE2 levels 10- to 30-fold higher than PGF2{alpha}, a feature distinguishing endometrial stromal from epithelial cells (10). Basal and TNF{alpha}-stimulated PGE2 production was significantly higher in the caruncular than the intercaruncular stromal clones, as we reported previously (16). Increased PG production in response to IFN{tau}, TNF{alpha}, LPS, and PMA, and nonresponsiveness to OT observed in this study is in agreement with our previous observations on primary stromal cell cultures (7, 10). Among biosynthetic enzymes, only COX2 and cPLA2 are regulated in parallel with PG production. This was confirmed using NS-398, a COX2-specific inhibitor that blocked TNF{alpha}-stimulated PG production. These observations are also in agreement with what was observed by us in primary stromal cells for COX2 (7) and by others in BEND cells for cPLA2 (17). Although increased prostaglandin E synthase expression was seen at the transcriptional level previously (7), we report here that PGE and PGF terminal synthases are expressed but not modulated at the protein level. A possible explanation is that prostaglandin E synthase expression level is higher in CSCs than it was in primary stromal cells. Because mPGES1 is known to be associated with COX2 in situations of increased PGE2 production, constant expression of the former with induced expression of the latter appears to be sufficient to explain our observations in CSCs. Together, these results show that the PG enzymatic machinery is intact and functional in CSCs where COX2 appears as the rate-limiting enzyme. Because the CSC is also amenable for transfection, this cell line can be used in combination with gene-silencing experiments with small interfering RNA to confirm the contribution of the different members of the PG biosynthetic cascade. Stromal cell-specific phenotype, the presence of ER{alpha} and PR, preferential production of PGE2 over PGF2{alpha}, and responsiveness to known PG production inducers validate CSC as a bona fide stromal cell model to study the molecular aspects of in vitro regulation of PG production in bovine.

Using this validated model, we studied the regulation of COX2 and PG production in response to PMA and IFN{tau} and their interactions, and attempted to correlate it with the expression of STAT1 involved in IFN{tau}-mediated signaling (8, 9). Phorbol ester was chosen because it is used as a surrogate for OT response in BEND cells. It is clear that both PMA and IFN{tau} induce COX2 expression and PGE2 and PGF2{alpha} production in a dose-dependent manner. However, IFN{tau} alone is able to phosphorylate and up-regulate STAT1. Phosphorylation of STAT1, which is still detectable after 24 h in CSCs, is comparable with the persistent tyrosine phosphorylation of STAT1 observed in the ovine endometrial epithelial cell line and may be involved in sustained effects of IFN{tau} (18). Interaction studies showed that PMA- induced COX2 expression can be inhibited by low but not high concentrations of IFN{tau}. Second, PMA does not affect IFN{tau}-induced up-regulation of STAT1. Because the apparent biphasic effect of IFN{tau} on COX2 could not be correlated at the level of STAT1, we chose to probe the p38 MAPK pathway known to be involved in other systems. Interestingly, the p38 MAPK inhibitor SB203580 blocked the effect of IFN{tau} on COX2 expression and PG production. This result is supported by the observation that IFN{tau} confers transcriptional stability to COX2 in bovine myometrial cells through p38 MAPK (14).

The biphasic effect or dose-dependent dichotomy of IFN{tau} on COX2 expression may throw some light on the up-regulation of COX2 observed during the maternal recognition window in ruminants (19, 20, 21) as well as after intrauterine infusions of IFN{tau} (22). Given that copious production of IFN{tau} occurs during recognition of pregnancy (23) and because interferon-stimulated genes are mainly present in the endometrial stroma (6), it is possible that the up-regulation of COX2 by high concentrations of IFN{tau} reflects the in vivo conditions (24). COX2 mediates inflammation and tumorigenesis (25), and is traditionally viewed as pathological, but it is also necessary for normal female reproductive function (26, 27). In humans and rodents, implantation is associated with elevated levels of PGE2 by the decidualizing stromal cells (28). Although the implantation is superficial and its onset is relatively late in ruminants compared with human, it is associated with up-regulation of COX2 (19, 20, 21, 22). Our preliminary results with CSCs suggest that IFN{tau} may influence PGE2 and COX2 through the p38 MAPK pathway to mediate its pro-gestation effects in the endometrial stroma. In this respect, the CSC may serve as an ideal model for investigating the paradigm of counteraction of the luteolytic PGF2{alpha} and the immunomodulatory PGE2, at the time of maternal recognition of pregnancy.


   Acknowledgments
 
We thank the Central Sheep and Wool Research Institute, Indian Council of Agricultural Research, and Department of Agriculture Research and Education, India, for granting study leave to N.K.


   Footnotes
 
This work was supported by Grant 44276 from Natural Sciences and Engineering Research Council, Canada.

Disclosure Statement: The authors have nothing to declare.

First Published Online September 4, 2008

Abbreviations: AKR1B5, Aldoketoreductase 1 B5; BEND, bovine endometrial cell; COX, cyclooxygenase; cPGES, cytosolic prostaglandin E synthase; cPLA2, cytosolic phospholipase A2; CSC, caruncular stromal cell; ER, estrogen receptor; ICSC, intercaruncular stromal cell; IFN{tau}, interferon-{tau}; LPS, lipopolysaccharide; mPGES, microsomal prostaglandin E synthase; OT, oxytocin; PG, prostaglandin; PMA, phorbol myristate acetate; PR, progesterone receptor; ROD, relative OD; STAT, signal transducer and activator of transcription; SV40 TAG, simian virus 40 large T antigen.

Received May 19, 2008.

Accepted for publication August 22, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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