HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Binelli, M. Articles by Thatcher, W. W. Search for Related Content PubMed PubMed Citation Articles by Binelli, M. Articles by Thatcher, W. W. Agricola Articles by Binelli, M. Articles by Thatcher, W. W.

Interferon- Modulates Phorbol Ester-Induced Production of Prostaglandin and Expression of Cyclooxygenase-2 and Phospholipase-A₂ from Bovine Endometrial Cells¹

^a Department of Dairy and Poultry Sciences, University of Florida, Gainesville, Florida 32611 ^b Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, Quebec, Canada J2S 7C6 ^c Department of Animal Science, University of Wyoming, Laramie, Wyoming 82071

ABSTRACT

Antiluteolytic actions of bovine interferon-tau (bIFN-) require suppression of prostaglandin F₂ (PGF₂) production. Our objective was to test whether bIFN- could block PGF₂ production and synthesis of phospholipase A₂ (PLA₂) and cyclooxygenase-2 (COX-2) enzymes induced by a protein kinase C (PKC) stimulator (phorbol 12,13 dibutyrate; PDBu). Bovine endometrial epithelial (BEND) cells were treated with PDBu in the presence or absence of bIFN-. Medium samples were analyzed for concentrations of PGF₂, whole-cell extracts were analyzed for abundance of PLA₂ and COX-2 by immunoblotting, and RNA extracts were examined for steady-state levels of COX-2 mRNA by Northern blotting. The PDBu stimulated production of PGF₂ between 3 and 12 h, levels of COX-2 mRNA by 3 h and protein expression of COX-2 and PLA₂ by 6 and 12 h, respectively. Added concomitantly with PDBu, bIFN- suppressed PGF₂ production, steady-state levels of COX-2 mRNA, and expression of COX-2 and PLA₂ proteins. Added after a 3-h stimulation with PDBu alone, bIFN- suppressed PGF₂ production after 1 h. Bovine IFN- inhibited intracellular mechanisms responsible for PGF₂ production in BEND cells, and this could be through both cytosolic and nuclear actions.

conceptus, hormone action, kinases, pregnancy, uterus

INTRODUCTION

Both in vivo [1] and in vitro [2–4] experiments demonstrated that bovine interferon-tau (bIFN-) is able to attenuate endometrial production of prostaglandin F₂ (PGF₂). A variety of experimental models have been used to demonstrate this effect of bIFN-, including explants [2] and primary cultures of endometrial epithelial cells collected on Days 1 to 4 [4] or 15 of the estrous cycle [3]. The aforementioned approaches involve synchronization and slaughter of animals at specific stages of the estrous cycle or collecting reproductive tracts from commercial slaughter facilities in order to obtain cells to conduct experiments. These approaches are both time and resource consuming. Moreover, production of PGF₂ is highly variable among animals, which makes interpretation of results challenging. An alternative experimental model for studying effects of bIFN- in the endometrium is use of bovine endometrial (BEND) cells. BEND cells are a line of spontaneously replicating epithelial cells originating from Day 14 cyclic cows [5]. The overall goal of this study was to characterize the effect of bIFN- on production of PGF₂ from BEND cells, and the working hypothesis was that bIFN- has an inhibitory effect on stimulated synthesis of PGF₂.

Oxytocin stimulates production of PGF₂ from primary endometrial epithelial cells in culture [3, 4]. Oxytocin binds to a seven-transmembrane domain and to G protein-coupled receptors and activates phospholipase C (PLC). The PLC cleaves membrane phosphatidylinositol bisphosphate, yielding inositol triphosphate (IP₃) and diacylglycerol (DAG). The IP₃ binds to specific receptors in the endoplasmic reticulum, resulting in release of calcium from internal stores into the cytosolic compartment. The DAG activates protein kinase C (PKC), leading to serine phosphorylation of cytosolic, calcium-dependent phospholipase A₂ (PLA₂) through a MAP kinase-dependent pathway [6]. The IP₃-induced increase in cytosolic calcium acts to further stimulate PLA₂ activity [7]. Stimulated PLA₂ translocates to the membrane, where phospholipids are cleaved to yield arachidonic acid (AA; [7]). Free AA is converted to prostaglandin H₂ (PGH₂) by the enzyme cyclooxygenase-2 (COX-2). Prostaglandin F₂ synthase converts PGH₂ into PGF₂, which is then released into the uterine vein [8].

In a preliminary experiment (unpublished results) it was determined that 1-h treatment with oxytocin failed to stimulate production of PGF₂ from BEND cells. Furthermore, Arnold and coauthors [9] demonstrated that treatment of Day 15 endometrial explants with phorbol 12,13 dibutyrate (PDBu; an stimulator of PKC activity), but not oxytocin, induced production of PGF₂. Using endometrial cells collected on Days 1 to 4 of the estrous cycle, Xiao and coworkers [4] determined that phorbol 12-myristate 13-acetate (PMA) increased both production of PGF₂ and expression of COX-2 protein. In addition, these authors reported that bIFN- reduced both of these PMA-induced effects. Objectives of the present study were to 1) test whether PDBu could stimulate PGF₂ production from BEND cells and whether bIFN- could block such an effect, 2) study the time-response dynamics of PDBu-induced PGF₂ production in the presence or absence of bIFN-, and 3) examine the effects of PDBu on steady-state levels of COX-2 mRNA and on PLA₂ and COX-2 protein expression in the presence or absence of bIFN-.

MATERIALS AND METHODS

Materials

Recombinant bIFN- (1.08 x 10⁷ units of antiviral activity per milligram) was a generous gift from Dr. R. Michael Roberts (University of Missouri, Columbia, MO). Polystyrene tissue culture Costar six-well plates and culture dishes (100 x 20) were purchased from Corning (Corning Glass Works, Corning, NY). Polystyrene cell-culture flasks (175 cm²) were from Sarstedt, Inc. (Newton, NC). Acrylamide, N,N'-methylenebisacrylamide, sodium dodecyl sulfate, and Nonidet-P40 were from BDH Laboratory Supplies (Poole, U.K.). Coomassie brilliant blue, bromophenol blue, ß-mercaptoethanol, NaOH, Tris, Tris-HCl, TEMED, ammonium persulfate, agarose, formaldehyde, acetic acid, Tween 20, isopropyl alcohol, chloroform, diallyltartardiamide (DTT), NaCl, EDTA, NaF, glycerol, glycine, methanol, and gelatin, were purchased from Fisher Scientific (Pittsburgh, PA). The PDBu, rabbit IgG, monoclonal anti-vimentin clone V9 antibody, monoclonal anti-pan cytokeratin antibody, Ham F-12, Eagle minimum essential medium, antibiotic-antimycotic solution (AbAm), insulin, D-valine, horse serum, aprotinin, leupeptin, pepstatin, Na₄P₂O₇, EGTA, Na₃VO₄, PMSF, and bovine serum albumin were from Sigma Chemical Co. (St. Louis, MO). Isotopically labeled [5, 6, 8, 11, 12, 14, 15-³H]-PGF₂ (212 Ci/mol) and nitrocellulose membranes (Hybond-ECL) were from Amersham Corp. (Arlington Heights, IL). BioTrans nylon membrane was from ICN (Irvine, CA), and TRIzol reagent was from Life Technologies (Grand Island, NY). X-ray film was from Eastman Kodak Co. (X-Omat Blue XB-1; Rochester, NY). Nonfat dried milk was from Mid-America Farms (Springfield, MO). Fetal bovine serum was acquired from Atlanta Biologicals (Norcross, GA). Enhanced chemiluminescence (ECL) kit (Renaissance Western Blot Chemiluminescence Reagent Plus) was from NEN Life Science Products (Boston, MA). Anti-COX-2 polyclonal antibody was from Cayman Chemical (Ann Arbor, MI). Cytosolic PLA₂ polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cell Culture and Experimental Designs

Isolation of BEND cells from a primary cell culture is described by Staggs et al. [5]. The cell line is deposited and characterized by the American Type Culture Collection (ATCC number CRL-2398; ATCC, 10801 University Boulevard, Manassas, VA 20110-2209). The ATCC describes methodology for subculturing, propagation, and freezing and for products produced by the cells. BEND cells were plated on 100-mm tissue culture dishes (8.5 x 10⁵ cells per plate) in culture medium (40% Ham F-12, 40% MEM, 10 ml AbAm/L, 200 U insulin/L, 0.0343g D-valine/L, 10% fetal bovine serum, and 10% horse serum) for experiments 1, 2, 3, and 4 or on 35-mm wells on six-well plates (2 x 10⁵ cells per well) for experiment 5. Cells were grown to confluency at 37°C under a humidified atmosphere containing 95% O₂ and 5% CO₂, washed in serum-free medium, and cultured for an additional 24 h in serum-free medium. After this time (designated as 0 h), a sample of medium was collected and stored at -20°C (experiments 1, 2, and 5). Then cells were washed again, and serum-free medium was mixed with appropriate treatments and added to cells in the same volumes listed above. Samples of medium (500 µl in experiments 1, 2, and 3; 250 µl in experiment 5) were collected at specified times and stored at -20°C. After sampling, the same volume of medium removed was replaced with medium containing the appropriate treatment so that a constant volume was maintained throughout each experiment. Concentrations of PGF₂ were measured in medium as described below.

Experiment 1 Cultures (20 ml of culture medium, n = 8) were assigned randomly to receive bIFN- (0 or 50 ng/ml) or PDBu (0 or 100 ng/ml) in duplicate, in a 2 x 2 factorial design, for 24 h. Medium was sampled after 12 and 24 h of treatment. After the 24-h sample, remaining medium was discarded and cells harvested for extraction of proteins.

Experiment 2 Cultures (10 ml of culture medium, n = 10) were assigned randomly to receive medium alone (control, three plates), PDBu (100 ng/ml, four plates) or PDBu in combination with bIFN- (50 ng/ml, four plates) for 12 h. Medium was sampled after 3, 6, 9, and 12 h of treatment. After collection of 12-h samples, remaining medium was discarded and cells were harvested for extraction of proteins.

Experiment 3 Cultures (10 ml of culture medium, n = 27) were assigned randomly to receive medium alone (control, nine plates), PDBu (100 ng/ml, nine plates), or PDBu in combination with bIFN- (50 ng/ml, nine plates). Medium was sampled after 1, 2, 3, 4, 5, and 6 h of treatment. After collecting the 6-h sample, three plates per treatment were used for protein extracts. Cells on additional plates were harvested for RNA extraction after 3 h (three plates per treatment) or 6 h (three plates per treatment).

Experiment 4 Cultures (10 ml of culture medium, n = 16) were assigned randomly to receive medium alone (control, four plates), PDBu (100 ng/ml, six plates), or PDBu in combination with bIFN- (50 ng/ml, six plates). Whole-cell extracts (WCE) were prepared from control cultures (0 h; four plates) and from treated plates after 6, 12, and 24 h of exposure to treatments (two plates per treatment per time) and used for immunoblots.

Experiment 5 Cultures (3 ml of culture medium, n = 12) were assigned in triplicate to receive medium alone (control), PDBu (100 ng/ml), PDBu in combination with bIFN- (50 ng/ml), or PDBu for 3 h and PDBu in combination with bIFN- for an additional 3 h. Medium was sampled after 1, 2, 3, 4, 5, and 6 h of treatment. This experiment was replicated once.

Preparation of Cell Extracts and Immunoblotting Analysis

At the end of culture periods, plates were transported to a cold room (4°C), culture medium was discarded, and cells were rinsed twice in ice-cold PBS containing 1 mM Na₃VO₄ and 5 mM NaF. Cells were washed briefly in 1 ml of WCE buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 20 mM NaF, 1 mM Na₃VO₄, 1 mM Na₄P₂O₇, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM PMSF, 10% v/v glycerol, 0.5% v/v NP-40, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin) and allowed to drip dry. Cells were scraped from plates in the presence of 1 ml of WCE buffer, incubated on ice for 10 min, and disrupted by aspiration through a 25-gauge needle. Lysates were then processed by centrifuge (13 000 x g for 2 min), and protein concentrations were determined in supernatants by the Bradford method [10]. Protein (20 µg) from each plate was loaded onto duplicate 7.5% acrylamide gels, submitted to SDS-PAGE, and electrophoretically transferred to nitrocellulose membranes. Membranes were blocked for 2 h in 5% (w/v) nonfat dried milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST), washed for 15 min in TBST and separately probed with antibodies against either COX-2 (1:1000) or PLA₂ (1:250) diluted in 5% nonfat dried milk in TBS. Secondary antibody was anti-rabbit IgG (1:5000 dilution in 5% nonfat dried milk in TBST). Proteins were detected by ECL and analyzed by densitometry (AlphaImager 2000; Alpha Innotech Corporation, San Leandro, CA).

RNA Isolation and Northern Blot Analysis

To obtain RNA (experiment 3), plates were transported to a cold room (4°C) and washed twice with ice-cold PBS; total RNA was isolated from confluent BEND cells with Trizol according to the manufacturer's specifications. Total cellular RNA (30 µg per lane) was fractionated in a 1.5% agarose-formaldehyde gel, stained with ethidium bromide, blotted to BioTrans nylon membrane, and hybridized with a homologous bovine COX-2 cDNA probe. The bovine COX-2 cDNA was cloned from an ovarian follicular cDNA library (unpublished data; GenBank accession number AF031698), and its sequence was shown to be identical to that of the corresponding exonic sequences of the bovine COX-2 gene [11]. The probe was labeled by nick translation as described elsewhere [12]. The COX-2 mRNA transcript was identified by autoradiography and hybridization signals analyzed by densitometry. To normalize loading of samples, 28s ribosomal RNA was also quantified, and densitometric values were used as a covariate in the analysis of variance.

Radioimmunoassay

Concentrations of PGF₂ were measured in medium undiluted (control-, bIFN-- and PDBu + bIFN--treated cells) or diluted 1:2 in medium (PDBu-treated cells) as described by Danet-Desnoyers and coworkers [3]. Assay was validated for serum-free medium by adding 25 pg/0.1 ml PGF₂ to medium. Average recovered PGF₂ was 25.1 ± 1.42 pg/0.1 ml (100.25%). Anti-PGF₂ antiserum utilized was characterized by Dubois and Bazer [13] and diluted 1:5000 (Tris buffer). Minimum detectable concentration of PGF₂ was 3.32 pg per tube. Inter- and intraassay coefficients of variation were 13.99 and 12.61%, respectively. Because multiple samples were removed and medium was added back to plates, concentrations of PGF₂ were adjusted to account for PGF₂ removed from previous samples. Adjustment consisted of adding the amount of PGF₂ removed in previous samples to the amount measured in the current sample. Total cell protein was determined, and total PGF₂ values per well were expressed as micrograms of protein.

Statistical Analysis

Data were analyzed by least-squares analysis of variance, using the GLM procedure from SAS [14]. Data for each experiment were analyzed separately. The PGF₂ data were analyzed as a split-plot design. For experiments 1, 2, and 3, independent variables in the mathematical models were as follows: treatment, dish (or well) within treatment, time, treatment x time, and residual error. The term dish-within-treatment was the error term for effects of treatment. For experiment 5, independent variables in the mathematical models were as follows: experiment, treatment, experiment x treatment, well within experiment x treatment, time, experiment x time, treatment x time, and residual error. The term well within experiment by treatment was the error term for the effects of treatment and experiment. Heterogeneity of variance among treatments was tested by the maximum F-ratio test (F_max; [15]) and found significant in all experiments. Therefore, data were transformed to log₁₀ and retested for heterogeneity of variance. Transformations effectively eliminated heterogeneity of variance for experiments 2, 3, and 5 but not for experiment 1. In experiment 1, the Box-Cox procedure for data transformation was applied [16], and a value of 0.4 was found to eliminate heterogeneity of variance. Data were analyzed after appropriate transformations. However, for the sake of clarity, data are presented as untransformed values. Means were compared as a series of preplanned orthogonal contrasts of treatment, time, and treatment by time.

Prostaglandin-F₂ production data were further analyzed by homogeneity of regression to determine regression equations for each treatment within experiments and to perform orthogonal comparisons of PGF₂ production among treatments.

For abundance of COX-2 and PLA₂, the mathematical model included the effects of treatment, time, and treatment by time (Northern blots in experiment 3 and immunoblots in experiment 4). Abundance of mRNA for Northern blots of COX-2 in experiment 3 was adjusted for 28s RNA as a covariate. Orthogonal contrasts were used for mean comparisons.

RESULTS

Effects of PDBu and bIFN- on PGF₂ Production

Production of PGF₂ at time 0 was low (<0.3 pg per microgram protein) for all treatments (Fig. 1, a and b; Fig. 2). Accumulation of PGF₂ for the control group was negligible in all experiments (Figs. 1 and 2; P < 0.01). Similarly, incubations with bIFN- alone had no effect on PGF₂ production (Fig. 1, panel a). Treatment of BEND cells with PDBu induced production of PGF₂ compared with the case of controls (Figs. 1 and 2; P < 0.01). Initial stimulation by PDBu was detected after 2 h of exposure (Fig. 1c), and increased at a rate of 47.4 pg per microgram of protein per 6 h between 0 and 6 h (Fig. 1b). A further increase in PGF₂ production was detected but occurred at a lower rate between 6 and 12 h (14.7 pg per microgram of protein per 6 h; Fig. 1b), but no more accumulation occurred after 12 h of exposure to PDBu (Fig. 1a). Incubations with bIFN- attenuated the PDBu-induced production of PGF₂ at all time points studied (Figs. 1 and 2; treatment x time interaction, P < 0.01). Furthermore, analysis of homogeneity of regression confirmed changes in production rates of PGF₂ during the experiments. Equations for each experiment were calculated (data not shown), and fit of regression curves were R² = 0.968 (P < 0.01), R² = 0.982 (P < 0.01), and R² = 0.954 (P < 0.01) for experiments 2, 3, and 5, respectively. Orthogonal comparisons of curves confirmed that PDBu stimulated PGF₂ production and that bIFN- reduced that effect (P < 0.01). The fact that inhibitory effects of bIFN- on PDBu-induced PGF₂ production were noted as early as 2 h (time when first PDBu-induced rise in PGF₂ is noted; experiment 3; Fig. 1c) prompted us to examine how quickly bIFN- would be able to suppress synthesis of PGF₂ in cells exposed previously to PDBu. We chose to add bIFN- after 3-h exposure to PDBu because 1) PDBu-induced PGF₂ production was increasing at a fast rate by this time (Fig. 1, b and c), which indicates that the PGF₂-synthesizing machinery was present and functional and 2) maximum production rate was noticed in the first 6 h of PDBu stimulation (experiment 2; Fig. 1b). In experiment 5, addition of bIFN- after a 3-h exposure to PDBu caused a decrease in PDBu-induced PGF₂ production, which was noticeable at the 5-h sample and became even more pronounced at the 6-h sample (Fig. 2). Moreover, rate of accumulation of PGF₂ in the medium between 4 and 6 h was greater in absence of bIFN- (1201 pg per milliliter per 2 h for PDBu vs. 442 pg per milliliter per 2 h for PDBu + bIFN--3h). Furthermore, when PDBu and PDBu + bIFN--3h treatments were analyzed without other treatments, there was a time x treatment interaction (P < 0.01), indicating that bIFN- effectively suppressed PDBu-stimulated PGF₂ production, even when added after PDBu treatment had been initiated. Orthogonal comparison of curves representing production of PGF₂ induced by PDBu and by PDBu + bIFN--3h indicated a treatment effect (P < 0.01).

View larger version (29K): [in this window] [in a new window]   FIG. 1. Total PGF2 in medium from BEND cells treated with medium alone (Con), bIFN- (50 ng/ml; IFN), phorbol 12,13 dibutyrate (100 ng/ml; PDBu) or bIFN- and PDBu. a) Experiment 1. Samples were removed before treatments were added (0 h), 12 and 24 h after. b) Experiment 2. Samples were removed before treatments were added (0 h), 3, 6, 9, and 12 h after. c) Experiment 3. Samples were removed before treatments were added (0 h), 1, 2, 3, 4, 5 and 6 h after. Details of statistical differences are described in Results

View larger version (24K): [in this window] [in a new window]   FIG. 2. Experiment 5. Least-squares means and SEM of concentrations of PGF2 in medium conditioned by BEND cells treated with medium alone (Con), phorbol 12,13 dibutyrate (100 ng/ml; PDBu), PDBu and bIFN- (50 ng/ml; IFN) added concomitantly, and PDBu for 3 h followed by PDBu and bIFN- (see text). Samples were removed before treatments were added (0 h), 1, 2, 3, 4, 5, and 6 h after. Details of statistical differences are described in Results

Effects of PDBu and bIFN- on COX-2 and PLA₂ Protein Expression: COX-2 and PLA₂ Immunoblotting

There was a low abundance of COX-2 and PLA₂ in cells incubated with medium alone or with bIFN- in experiments 1 to 3 (data not shown) and 4 (Fig. 3). However, treatment with PDBu increased synthesis of both COX-2 (P < 0.01) and PLA₂ (P < 0.1; experiment 4). The PDBu induced a sharp increase in the abundance of COX-2 to reach maximum levels by 6 h of treatment, compared to controls (Fig. 3a). The abundance of COX-2 remained elevated at 12 h but decreased by 24 h. PBDu stimulated an increase in the abundance of PLA₂ to reach a maximum at 12 h but decreased by 24 h (effect of time; P < 0.02; Fig. 3b). In experiment 4, presence of bIFN- reduced the PDBu-induced increase in COX-2 (experiment 4; Fig. 3a; treatment effect; P < 0.01). However, the ability of bIFN- to suppress the PDBu-stimulated COX-2 tended to decrease over time (treatment x time interaction; P < 0.09). Presence of bIFN- tended to attenuate the PDBu stimulation of PLA₂ (effect of treatment; P < 0.1). Most of the difference in abundance of PLA₂ between treatments was noted between 12 and 24 h.

View larger version (26K): [in this window] [in a new window]   FIG. 3. Experiment 4. Immunoblotting analysis of COX-2 and PLA2 in whole-cell extracts from BEND cells treated with phorbol 12,13 dibutyrate (100 ng/ml; PDBu) or bIFN- (50 ng/ml; IFN) and PDBu for 0, 6, 12, or 24 h. a) Representative-enhanced ECL exposure of abundance of total COX-2 and least-squares means (±SEM) of abundance of total COX-2 arbitrary densitometric units (ADU). b) Representative ECL exposure of abundance of total PLA2 and least-squares means (± SEM) of abundance of total PLA2 ADU. Details of statistical differences are described in Results. ** Differences between means within time significant at P < 0.01

Effects of PDBu and bIFN- on Steady-State Levels of COX-2 mRNA: COX-2 Northern Blotting

There was an effect of treatment on the abundance of COX-2 mRNA (P < 0.01, Fig. 4a). In the absence of bIFN-, PDBu increased steady-state levels of the COX-2 mRNA compared with controls. However, this effect was attenuated when cells were incubated with a combination of bIFN- and PDBu. Similar responses were observed both after 3 or 6 h of exposure to treatments (treatment by time interaction, P > 0.1). A similar abundance of 28S ribosomal RNA indicated that a similar amount of RNA was loaded per lane (Fig. 4b).

View larger version (40K): [in this window] [in a new window]   FIG. 4. Experiment 3. Northern blotting analysis of COX-2 mRNA in total RNA extracts from BEND cells treated with medium alone (Con), phorbol 12,13 dibutyrate (100 ng/ml; PDBu), or bIFN- (50 ng/ml; IFN) and PDBu for 3 or 6 h. a) Autoradiography exposure of abundance of COX-2 mRNA. b) Ethidium bromide staining of 28S ribosomal RNA. c) Least-squares means and SEM of abundance of COX-2 mRNA arbitrary densitometric units. Details of statistical differences described in Results. PDBu stimulated (** P < 0.01) abundance of COX-2 mRNA, which was blocked by IFN at both 3 and 6 h

DISCUSSION

BEND cells provide a model for studying regulation of PGF₂ in the endometrium. Previous studies utilized endometrial epithelial cells in primary culture harvested from uteri collected from Day 15 [3] or from Days 1–4 cyclic cows [4, 17, 18], but collection procedures are time and resource consuming. BEND cells can be used repeatedly for at least 25 passages and still maintain phenotype (unpublished observations). Moreover, BEND cells originated from Day 14 cyclic cows, which provided endometrial cells that were primed, in vivo, with progesterone. This stage of the estrous cycle is appropriate to study mechanisms of bIFN- action in suppressing release of PGF₂. Using BEND cells, it was determined that bIFN- suppresses PGF₂ probably through inhibiting steady-state levels of COX-2 mRNA, protein expression and enzymatic activity of COX-2 and PLA₂. Moreover, bIFN- suppression of PDBu-induced PGF₂ production was achieved within 2 h.

Phorbol esters such as PMA stimulate PGF₂ production in vitro [4]. In the present study, PDBu increased PGF₂ production for 12 h but failed to maintain induction from 12 to 24 h of culture. The loss of action of PDBu after 12 h may have been because of depletion of AA (precursor for PGF₂ synthesis) or down-regulation of the PKC pathway in response to long-term stimulation by PDBu. The PDBu induced maximum production of PGF₂ during the first 6 h, followed by a decrease between 6 and 12 h. The induction of PGF₂ required a 2-h period. This suggests that PDBu activity is, at least initially, dependent on de novo protein synthesis and not on stimulation of activity of pre-existing enzymes involved in PGF₂ production. Nam and others [19], reported that the protein synthesis inhibitors actinomycin D and cycloheximide blocked the ability of PDBu to stimulate PGF₂ production in astroglial cells.

When added alone to BEND cells, bIFN- had no effect on PGF₂ production. Thus, treatment with bIFN- alone was excluded from subsequent experiments. In contrast, bIFN- effectively suppressed the PDBu-induced production of PGF₂ in all experiments. However, bIFN- was never able to completely suppress PDBu-stimulated PGF₂. Perhaps other products of the conceptus are required to abolish PGF₂ completely in vivo. These findings confirm and expand those of Xiao and coworkers [4]. However, attenuation of PGF₂ by bIFN- in the present study was much more dramatic.

Treatment with PDBu strongly induced synthesis of both COX-2 and PLA₂ protein compared with the case of controls. This was detected as early as 6 h and lasted for at least 12 h. PDBu-stimulated COX-2 expression was also related to an increased expression of the COX-2 gene, which was elevated as early as 3 h and remained high for at least 6 h (experiment 3). Increased abundances of COX-2 and PLA₂ probably were related to their increased cellular activity, as evaluated through increased PGF₂ accumulation in medium. Presence of bIFN- reduced PDBu-stimulated PLA₂ and COX-2 protein expression, but such effects were time dependent. Responses of PLA₂ were somewhat variable but were consistently lower than PDBu treatment alone. When related to time trends of PGF₂ production, results from experiment 4 suggest that maximum protein levels of COX-2 (6 to 12 h) were related to maximum production of PGF₂. Moreover, the decrease in COX-2 protein noted at 24 h corresponded to a cessation in the production of PGF₂, probably because of down-regulation of the PKC system [20, 21]. In contrast, bIFN- delayed both the increased expression of COX-2 and the accumulation of PGF₂ induced by PDBu. Presence of bIFN- may have inhibited, but did not abolish, the PDBu-stimulated PKC pathway. As a consequence, there was an increasing accumulation of PGF₂ over time, and this may be due to an inhibition of the PDBu down-regulation of the PKC system by bIFN-. Thereby, similar levels of COX-2 noted at 24 h represented different levels of PGF₂ secretory activity (i.e., cessation in PDBu-treated cells but increase in PDBu+bIFN--treated cells). Altogether, these observations are consistent with those of Xiao and others [4], who reported that PMA increased COX-2 protein expression (about fivefold higher than that of controls) and that bIFN- attenuated that response (to about 2.5-fold higher than that of controls). Presence of bIFN- reduced all responses measured at all time points. In contrast, Asselin et al. and others [17] reported no changes in PLA₂ gene expression after treatment of endometrial and stromal cells with bIFN-. Moreover, COX-2 expression was up-regulated by bIFN-. Large doses of bIFN- (1 to 20 µg/ml [17] vs. 50 ng/ml in the current study) could explain this discrepancy of results. Collectively, our results suggest that bIFN- exerts a complex regulation of both PLA₂ and COX-2 gene expression and activity to modulate PGF₂ production in BEND cells. The upstream regulator of both PLA₂ and COX-2 is PKC. Phorbol esters mimic the action of diacylglycerol, a product of PLC activity, which has the effect of activating PKC activity. The PKC may stimulate PGF₂ production via stimulation of synthesis and/or activity of both PLA₂ [22, 23] and COX-2 [24, 25]. Therefore, effects of bIFN- could be at the level of PKC to inhibit the ability of this enzyme to stimulate PGF₂ synthesis through modulation of its mediators, PLA₂ and COX-2. Because not all PDBu-induced PKC effects were suppressed (i.e., there was still PGF₂ production, even in presence of bIFN-), it is unlikely that bIFN- effects were exerted upstream from PKC (e.g., a block on PDBu ability to bind PKC). It is likely that bIFN- blocked some, but not all, PDBu effects downstream from PKC.

Decreased rate of production of PGF₂ at 2 h after addition of bIFN- (PDBu + bIFN--3 h treatment, experiment 5) indicated that bIFN- was able to quickly suppress PDBu-induced PGF₂. The exact mechanism whereby bIFN- exerted its effects is unknown; however, it is tempting to speculate that such a quick action is independent of protein synthesis and can occur through novel, previously undescribed cytosolic (i.e., nuclear independent) actions of bIFN-.

The observation that attenuation of PGF₂ synthesis occurred within 2 h is consistent with activation of the janus kinase (JAK) signal transducers and activators of transcription (STAT) pathway [26–28] but also suggests that bIFN- may act through additional pathways (Fig. 5). The JAK-STAT pathway involves phosphorylation and nuclear translocation of cytoplasmic STAT proteins, as a result of bIFN- binding to its receptor [26-28]. In the nucleus, STAT proteins act as transcription factors to stimulate expression of interferon-stimulated genes. Interferon-induced proteins may act in a variety of ways to produce the interferon-induced phenotype. Presence and function of this classical mode of action in the endometrium has been the prevalent dogma of laboratories studying effects of bIFN- on production of PGF₂ during maternal recognition of pregnancy in cattle [5–30] and sheep [31]. For example, interferon-stimulated gene 17 (ISG17; a ubiquitin cross-reactive protein) is induced by bIFN- in endometrial explant culture [5, 32]. This protein forms complexes with other cytosolic proteins and could modulate their activity and turnover rate [32]. In BEND cells, complex formation requires 3 h of exposure to bIFN- (unpublished observations), which makes ISG17 a possible mediator of early bIFN- suppression of PGF₂. Other possible mediators of bIFN- actions include the interferon-induced transcription factors IRF-1 and IRF-2 [31, 33]. IRFs are rapidly synthesized in response to bIFN- [28] and could induce synthesis of specific proteins to suppress PGF₂ synthesis. However, it is possible that a protein synthesis-independent mode of action for bIFN- operates in the endometrium to suppress PGF₂ production. Bovine IFN- could activate intracellular second messengers other than STAT proteins, which may have modulatory effects on the PGF₂ synthesizing machinery. Critical studies to dissect such alternative pathways induced by bIFN- warrant investigation.

View larger version (51K): [in this window] [in a new window]   FIG. 5. Hypothetical model of actions of bovine interferon-tau (bIFN-) to prevent protein kinase C (PKC)-induced production of PGF2 in endometrial epithelial cells. Activation of PKC stimulates synthesis (white squares) and enzymatic activity (gray diamonds) of specific endometrial proteins, such as phospholipase A2 (PLA2) and cyclooxygenase-2 (COX-2) to ultimately stimulate synthesis of PGF2. PKC may stimulate synthesis or activity of other cellular mediators, such as kinases, lipases, and transcription factors to further enhance synthesis and activity of enzymes directly involved in the PGF2 production. Binding of bIFN- to the type I receptor, here represented with a hypothetical, bIFN--specific chain (R), may have protein synthesis-dependent (through activation of the JAK-STAT pathway; right side of diagram) or -independent (left side of diagram) actions to decrease production of PGF2. STAT dimers may translocate to the nucleus to stimulate transcription of interferon-inducible genes and to repress transcription of genes related to synthesis of PGF2. The bIFN--induced proteins (crossed circles) may act as transcription factors to regulate transcription of genes, or may have cytosolic actions to regulate activity of enzymes involved in production of PGF2. Alternatively, bIFN- may regulate activity of preexisting cellular mediators (black circles) to negatively affect the PGF2 synthesizing machinery

ACKNOWLEDGMENTS

The authors would like to thank Dr. R. Michael Roberts for providing the recombinant bIFN- and Marie-Joelle Thatcher for assistance with the RIA for PGF₂.

FOOTNOTES

First decision: 27 January 2000.

¹ This work was supported partially by grant 98–35203-6367, NRI Competitive Grants Program/USDA, Florida Agricultural Experimental Station Journal Series R-07419, by NIH grant NIH HD 32475–06, and by MRC of Canada, grant MT-13190.

² Correspondence: William W. Thatcher, Dept. of Dairy and Poultry Sciences, University of Florida, Shealy Dr. and Ritchie Rd., Gainesville, FL 32611. FAX: 352 392 5595; thatcher{at}dps.ufl.edu

³ Current address: Dept. of Animal and Range Sciences, North Dakota State University, Fargo, ND 58105.

Accepted: March 7, 2000.

Received: December 31, 1999.

REFERENCES

1. Meyer MD, Hansen PJ, Thatcher WW, Drost M, Badinga L, Roberts RM, Li J, Ott TL, Bazer FW. Extension of corpus luteum lifespan and reduction of uterine secretion of prostaglandin F₂ of cows in response to recombinant interferon-. J Dairy Sci 1995; 78:1921–1931.[Abstract]
2. Helmer SD, Gross TS, Newton GR, Hansen PJ, Thatcher WW. Bovine trophoblast protein-1 complex alters endometrial protein and prostaglandin secretion and induces an intracellular inhibitor of prostaglandin synthesis in vitro. J Reprod Fertil 1989; 87:421–430.[Abstract]
3. Danet-Desnoyers G, Wetzels C, Thatcher WW. Natural and recombinant bovine interferon regulate basal and oxytocin-induced secretion of prostaglandins F₂ and E₂ by epithelial cells and stromal cells in the endometrium. Reprod Fertil Dev 1994; 6:193–202.[CrossRef][Medline]
4. Xiao CW, Murphy BD, Sirois J, Goff AK. Down-regulation of oxytocin-induced cyclooxygenase-2 and prostaglandin F synthase expression by interferon- in bovine endometrial cells. Biol Reprod 1999; 60:656–663.[Abstract/Free Full Text]
5. Staggs KL, Austin KJ, Johnson GA, Teixeira MG, Talbott CT, Doosley VA, Hansen TR. Complex induction of bovine uterine proteins by interferon-tau. Biol Reprod 1998; 59:293–297.[Abstract/Free Full Text]
6. Lin L-L, 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]
7. Clark JD, Lin L-L, Kriz RW, Ramesha CS, Sultzman LA, Lin AY, Milona N, Knopf JL. A novel arachidonic acid-selective cytosolic PLA₂ contains a Ca²⁺-dependent translocation domain with homology to PKC and GAP. Cell 1991; 65:1043–1051.[CrossRef][Medline]
8. Smith WL, Marnett LJ, DeWitt DL. Prostaglandin and thromboxane biosynthesis. Pharmacol Ther 1991; 49:153–179.[CrossRef][Medline]
9. Arnold DA, Binelli M, Vonk J, Alexenco AP, Drost M, Thatcher WW. Intracellular regulation of endometrial PGF_2a production in dairy cows during early pregnancy and following treatment with recombinant interferon-tau. Domest Anim Endocrinol 2000; 18:199-216.[CrossRef][Medline]
10. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248–254.[CrossRef][Medline]
11. Liu J, Antaya M, Boerboom D, Lussier JG, Silversides DW, Sirois J. The delayed activation of the prostaglandin G/H synthase-2 promoter in bovine granulosa cells is associated with down-regulation of truncated upstream stimulatory factor-2. J Biol Chem 1999; 274:35037–35045.[Abstract/Free Full Text]
12. Badinga L. Michel FJ, Fields MJ, Sharp DC, Simmen RCM. Pregnancy-associated endometrial expression of antileukoproteinase gene is correlated with epitheliochorial placentation. Mol Reprod Dev 1994; 38:357–363.[CrossRef][Medline]
13. Dubois DH, Bazer FW. Effect of porcine conceptus secretory proteins on in vitro secretion of prostaglandin-F₂ and -E₂ from luminal and myometrial surfaces of endometrium from cyclic and pseudopregnant gilts. Prostaglandins 1991; 41:283–301.[CrossRef][Medline]
14. SAS. SAS/STAT User's Guide (Release 6.03). Cary, NC: Statistical Analysis System Institute Inc.; 1988.
15. Hartley HO. The maximum F-ratio as a short-cut test for heterogeneity of variance. Biometrika 1950; 37:308–312.[Free Full Text]
16. Peltier MR, Wilcox CJ, Sharp III DC. Technical note: application of the Box-Cox data transformation to animal science experiments. J Anim Sci 1998; 76:847–849.[Abstract/Free Full Text]
17. Asselin E, Lacroix D, Fortier MA. IFN- increases PGE₂ production and COX-2 gene expression in the bovine endometrium in vitro. Mol Cell Endocrinol 1997; 56:402–408.
18. Asselin E, Drolet P, Fortier MA. In vitro response to oxytocin and interferon-tau in bovine endometrial cells from caruncular and inter-caruncular areas. Biol Reprod 1998; 59:241–247.[Abstract/Free Full Text]
19. Nam MJ, Thore C, Busija D. Effects of protein kinase C activation on prostaglandin production and cyclooxygenase mRNA levels in ovine astroglia. Prostaglandins 1996; 51:203–213.[CrossRef][Medline]
20. Akiguchi I, Izumi M, Nagataki S. Effects of phorbol ester on protein kinase C activity and effects of depletion of its activity on thyrotrophin, forskolin and 8'-bromoadenosine 3',5'-cyclic monophosphate-induced [3H] thymidine incorporation in rat FRTL-5 cells. J Endocrinol 1993; 138:379–389.[Abstract]
21. Turner NA, Walker JH, Ball SG, Vaughan PF. Down-regulation or long term inhibition of protein kinase C (PKC) reduces noradrenaline release evoked via either PKC-dependent or PKC-independent pathways in human SH-SY5Y neuroblastoma cells. Neurosci Lett 1996; 220:37–40.[CrossRef][Medline]
22. Mayer RJ, Marshall LA. New insights on mammalian phospholipase A2(s); comparison of arachidonyl-selective and -nonselective enzymes. FASEB J 1993; 7:339–348.[Abstract]
23. Karimi K, Lennartz MR. Protein kinase C activation precedes arachidonic acid release during IgG-mediated phagocytosis. J Immunol 1995; 15:5786–5794.
24. DeWitt DL. Prostaglandin endoperoxide synthase: regulation of enzyme expression. Biochem Biophys Acta 1991; 1083:121–134.[Medline]
25. Vezza R, Habib A, Li H, Lawson JA, FitzGerald GA. Regulation of cyclooxygenase by protein kinase C. J Biol Chem 1996; 271:30028–30033.[Abstract/Free Full Text]
26. Darnell JE Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994; 264:1415–1421.[Abstract/Free Full Text]
27. Binelli M, Diaz T, Hansen TR, Thatcher WW. Bovine interferon-tau induces phosphorylation and nuclear translocation of STAT-1, -2 and -3 in endometrial epithelial cells [abstract]. Biol Reprod 1998; 58(suppl 1):214.
28. Perry DJ, Austin KJ, 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]
29. Binelli M, Subramaniam PS, Thatcher WW. Bovine interferon-tau promotes transient phosphorylation of STAT-1 protein in endometrium [abstract]. Biol Reprod 1996; 54(suppl 1):169.
30. Hansen TR, Austin KJ, Perry DJ, Pru JK, Teixeira MG, Johnson GA. Mechanism of interferon-tau action in the uterus during early pregnancy. J Reprod Fertil 1999; 54(suppl):329–339.
31. Spencer TE, Ott TL, Bazer FW. Expression of interferon regulatory factors one and two in the ovine endometrium: effects of pregnancy and interferon-tau. Biol Reprod 1998; 58:1154–1162.[Abstract/Free Full Text]
32. Johnson GA, Austin KJ, Van Kirk EA, Hansen TR. Pregnancy and interferon tau induce conjugation of bovine ubiquitin cross-reactive protein to cytosolic uterine proteins. Biol Reprod 1998a; 58:898–904.
33. Harada H, Fujita T, Miyamoto M, Kimura Y, Maruyama M, Furia A, Miyata T, Taniguchi T. Structurally similar but functionally distinct factors, IRF-1 and IRF-2, bind to the same regulatory elements of IFN and IFN-inducible genes. Cell 1989; 58:729–739.[CrossRef][Medline]

This article has been cited by other articles:

				Y. Chen, E. Antoniou, Z. Liu, L. B Hearne, and R M. Roberts A microarray analysis for genes regulated by interferon-{tau} in ovine luminal epithelial cells Reproduction, July 1, 2007; 134(1): 123 - 135. [Abstract] [Full Text] [PDF]

				C. Rodriguez-Sallaberry, C. Caldari-Torres, E. S. Greene, and L. Badinga Conjugated Linoleic Acid Reduces Phorbol Ester-Induced Prostaglandin F2{alpha} Production by Bovine Endometrial Cells. J Dairy Sci, October 1, 2006; 89(10): 3826 - 3832. [Abstract] [Full Text] [PDF]

				C. Caldari-Torres, C. Rodriguez-Sallaberry, E. S. Greene, and L. Badinga Differential effects of n-3 and n-6 fatty acids on prostaglandin F2alpha production by bovine endometrial cells. J Dairy Sci, March 1, 2006; 89(3): 971 - 977. [Abstract] [Full Text] [PDF]

				S. A. Balaguer, R. A. Pershing, C. Rodriguez-Sallaberry, W. W. Thatcher, and L. Badinga Effects of Bovine Somatotropin on Uterine Genes Related to the Prostaglandin Cascade in Lactating Dairy Cows J Dairy Sci, February 1, 2005; 88(2): 543 - 552. [Abstract] [Full Text] [PDF]

				S.-Z. Wang and R. M. Roberts Interaction of Stress-Activated Protein Kinase-Interacting Protein-1 with the Interferon Receptor Subunit IFNAR2 in Uterine Endometrium Endocrinology, December 1, 2004; 145(12): 5820 - 5831. [Abstract] [Full Text] [PDF]

				A. Guzeloglu, F. Michel, and W. W. Thatcher Differential Effects of Interferon-{tau} on the Prostaglandin Synthetic Pathway in Bovine Endometrial Cells Treated with Phorbol Ester J Dairy Sci, July 1, 2004; 87(7): 2032 - 2041. [Abstract] [Full Text] [PDF]

				A. Guzeloglu, P. Subramaniam, F. Michel, and W. W. Thatcher Interferon-{tau} Induces Degradation of Prostaglandin H Synthase-2 Messenger RNA in Bovine Endometrial Cells Through a Transcription-Dependent Mechanism Biol Reprod, July 1, 2004; 71(1): 170 - 176. [Abstract] [Full Text] [PDF]

				V. Emond, L. A. MacLaren, S. Kimmins, J. A. Arosh, M. A. Fortier, and R. D. Lambert Expression of Cyclooxygenase-2 and Granulocyte-Macrophage Colony-Stimulating Factor in the Endometrial Epithelium of the Cow Is Up-Regulated During Early Pregnancy and in Response to Intrauterine Infusions of Interferon-{tau} Biol Reprod, January 1, 2004; 70(1): 54 - 64. [Abstract] [Full Text] [PDF]

				K. Okuda, Y. Kasahara, S. Murakami, H. Takahashi, I. Woclawek-Potocka, and D. J. Skarzynski Interferon-{tau} Blocks the Stimulatory Effect of Tumor Necrosis Factor-{alpha} on Prostaglandin F2{alpha} Synthesis by Bovine Endometrial Stromal Cells Biol Reprod, January 1, 2004; 70(1): 191 - 197. [Abstract] [Full Text] [PDF]

				J. Parent, C. Villeneuve, A. P. Alexenko, A. D. Ealy, and M. A. Fortier Influence of Different Isoforms of Recombinant Trophoblastic Interferons on Prostaglandin Production in Cultured Bovine Endometrial Cells Biol Reprod, March 1, 2003; 68(3): 1035 - 1043. [Abstract] [Full Text] [PDF]

				J. K. Pru, K. J. Austin, A. L. Haas, and T. R. Hansen Pregnancy and Interferon-{tau} Upregulate Gene Expression of Members of the 1-8 Family in the Bovine Uterus Biol Reprod, November 1, 2001; 65(5): 1471 - 1480. [Abstract] [Full Text] [PDF]

				A. D. Ealy, S. F. Larson, L. Liu, A. P. Alexenko, G. L. Winkelman, H. M. Kubisch, J. A. Bixby, and R. M. Roberts Polymorphic Forms of Expressed Bovine Interferon-{{tau}} Genes: Relative Transcript Abundance during Early Placental Development, Promoter Sequences of Genes and Biological Activity of Protein Products Endocrinology, July 1, 2001; 142(7): 2906 - 2915. [Abstract] [Full Text] [PDF]

				J. K. Pru, B. R. Rueda, K. J. Austin, W. W. Thatcher, A. Guzeloglu, and T. R. Hansen Interferon-Tau Suppresses Prostaglandin F2{{alpha}} Secretion Independently of the Mitogen-Activated Protein Kinase and Nuclear Factor {{kappa}} B Pathways Biol Reprod, March 1, 2001; 64(3): 965 - 973. [Abstract] [Full Text]

				M. Binelli, P. Subramaniam, T. Diaz, G. A. Johnson, T. R. Hansen, L. Badinga, and W. W. Thatcher Bovine Interferon-{{tau}} Stimulates the Janus Kinase-Signal Transducer and Activator of Transcription Pathway in Bovine Endometrial Epithelial Cells Biol Reprod, February 1, 2001; 64(2): 654 - 665. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Binelli, M.