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BOR - Papers in Press, published online ahead of print November 13, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.008961
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BIOLOGY OF REPRODUCTION 68, 1015–1026 (2003)
DOI: 10.1095/biolreprod.102.008961
© 2003 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Transcriptional Regulation of the Bovine Oxytocin Receptor Gene1

Ralph Telgmann4,a, Ross A.D. Bathgate3,4,a, Stefanie Jaegera, Gina Tillmanna, and Richard Ivell2,a

a Institute for Hormone and Fertility Research University of Hamburg, 22529 Hamburg, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The oxytocin receptor (OTR) is expressed in the cow uterus at high levels at estrus and at term of pregnancy. This expression appears to be controlled mostly at the transcriptional level and correlates with increasing estrogen concentration and progesterone withdrawal. Approximately 3200 base pairs of the upstream region of the bovine OTR gene were cloned and analyzed using a combination of bioinformatic, electrophoretic mobility shift (EMSA), and transfection analyses. Using nuclear proteins from high- and low-expressing tissues, EMSA indicated no significant quantitative or qualitative changes in specific DNA-protein binding, suggesting that transcription is probably controlled by signalling systems targeting constitutive factors. Using various cell types, including primary and immortalized ruminant endometrial epithelial cells, as hosts for transfection of promoter-reporter constructs showed that endogenous activity resided only in the longest, i.e., 3.2-kb, construct but not in those shorter than 1.0 kb. While estrogen appears to be important in vivo, no effect of estradiol was found on any construct directly; only when the longest 3.2-kb construct was used in combination with some cotransfected steroid receptor cofactors, e.g., SRC1e, was an estradiol-dependent effect observed. A putative interferon-responsive element (IRE) was found at approximately -2,400 from the transcription start site. This element was shown to bind mouse IRF1 and IRF2 as well as similar proteins from bovine endometrial and myometrial nuclear extracts. This element also responded to these factors when cotransfected into various cell types. The bovine equivalents to IRF1 and IRF2 were molecularly cloned from endometrial tissue and shown to be expressed in a temporal fashion, supporting the role of interferon-tau in maternal recognition of pregnancy. Of many factors tested or analyzed, these components of the IFN system are the only ones found to significantly influence the transcription of the bovine OTR gene.

estradiol receptor, female reproductive tract, gene regulation, oxytocin, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The receptor for the small peptide hormone oxytocin is a crucial element in the cascade of events leading to parturition and expulsion of the fetus. In most mammals, its expression is acutely upregulated in the uterus and cervix immediately before birth, thus enabling a powerful uterotonic signal transduction pathway to be activated in response to the progressively increasing amplitude and frequency of oxytocin pulses, presumably of neurohypophyseal origin [1–3]. In earlier studies, we and others have shown that part of the oxytocin receptor (OTR) upregulation is due to a dramatic increase in specific gene transcription [4, 5]. In some species, this occurs predominantly in the myometrial cells of the uterus, and in others, such as ruminants, it occurs both in the myometrium and in the endometrium. Similarly in the ruminant cervix, both mucosal and myoid components express high concentrations of OTR gene transcripts at term. In turn, the endometrial or mucosal receptors induce the local synthesis of PGF2{alpha} or PGE2, thus reinforcing local contractility or relaxation in the uterus and cervix, respectively.

In rodents and ruminants, OTR gene upregulation correlates in vivo with high circulating estrogen levels, particularly in association with a decline in progesterone, giving rise to the notion that sex steroids might directly regulate the OTR gene [6]. A lack of such correlation in the human uterus, and for OTR expression in other tissues such as the kidney, makes a direct effect of estrogen or progesterone on the OTR gene highly unlikely [7]. This is supported by transfection studies using different promoter-reporter constructs of the human and bovine OTR genes and appropriate steroid receptor expression systems, where no significant effect of steroids can be demonstrated [8].

In order to learn what molecular mechanisms may indeed be involved in the direct regulation of the OTR gene, we have developed a model making use of the bovine OTR gene promoter. The bovine system offers several advantages. Not only are tissues easily available, but in the cow, the OTR gene is also massively upregulated in the epithelial cells of the endometrium from the uterus of the estrous cycle to levels equivalent to those pertaining at parturition, but only within a narrow time window around the period of estrus [5]. This allows the use of tissues taken from nongravid animals. Interestingly, epithelial cells prepared from uteri of metestrous cows spontaneously upregulate the OTR gene to express high concentrations of receptors (ca. 30 000 receptors per cell) in vitro, showing first that, in vivo, this gene is under general inhibition during the cycle and second that cells can be prepared for experimental use from uteri of any stage of the cycle [9].

The reason for the high expression of OTR in the endometrial epithelium of the ruminant estrous cycle is the key role played by luteal oxytocin in the maternal recognition of pregnancy [10]. In a nonconceptive cycle, the late corpus luteum secretes oxytocin, which interacts with OTR on the endometrial epithelium to cause activation of prostaglandin synthase and the production of luteolytic PGF2{alpha}. This in turn causes more oxytocin to be secreted from the late corpus luteum, but at the same time inducing luteolysis and the withdrawal of steroidal support for the uterus. In the conceptive cycle, the young blastocyst from about Day 16 onward produces large amounts of a type 1 interferon (IFN-{tau}), which acts on the endometrial epithelium to suppress the expression of OTR, thus interrupting the normal luteolytic cascade [10–12]. To date, IFN-{tau} is the only effector shown to influence the expression of the endogenous OTR gene in isolated endometrial epithelial cells [9] as well as in the intact ruminant endometrium [13].

In the present extensive study, we have systematically analyzed the bovine OTR gene in order to identify cis- and trans-acting factors specifically involved in gene regulation in vivo and in vitro. In preliminary studies, uterine and other bovine tissues wherein OTR is either never expressed or is differentially expressed were used for electrophoretic mobility shift assay (EMSA) analysis of nuclear protein binding to different regions of the gene. Subsequently, a transfection approach was employed, making use of primary bovine epithelial cell cultures, wherein the OTR gene is endogenously expressed in high amounts, as well as various cell lines mostly of reproductive epithelioid origin where, however, only low, if any, endogenous expression of the OTR gene can be detected. In these studies, particular emphasis has been placed on the postulated mechanism of action of IFN-{tau} as a factor known to have a direct effect on OTR gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissues and Cells

Bovine endometrial epithelial (bEE) cells were prepared and cultured exactly as previously described [9], using uteri of Holstein cows collected from the local slaughterhouse. All cycle stages were represented, though in the earlier study, we showed that this had no effect on the final number of expressed oxytocin receptors. Otherwise, frozen tissues were used that had been collected in the context of other research programs from Angus half-bred cows held at the University of Florida, Gainesville, FL [14]. Other cell lines used were the immortalized ovine glandular epithelial (OGE) and ovine stromal (OS) cells [15] (which were a generous gift of Dr. Thomas Spencer, Center for Animal Biotechnology and Genomics, Texas A&M University, College Station, TX), the breast carcinoma cell lines MCF7 and MDA-MB231 [16], and the uterine carcinoid cell line Skut-1b [17].

Genomic Subclones and DNA Constructs

The bacteriophage clone #20 from a bovine genomic library, which includes a large region of the OTR locus upstream of the transcription start site [18], was used as the source for the construction of subclones and double-stranded DNA fragments. Restriction enzymes HindIII, EcoRI, and BamHI were used to create subclones, from which approximately 3200 base pairs (bp) upstream of the transcription start site were sequenced on both strands by using the dideoxy termination method. Sequence was confirmed, particularly in the regions of restriction enzyme cleavage, by using gene-specific oligonucleotides to prime sequence reactions within the 3200-bp region. The resulting promoter sequence was subjected to transcription factor binding site analysis using the AliBaba2 algorithm [19] (wwwiti.cs.uni-magdeburg.de/~grabe/alibaba2/). As indicated in Figure 1, double-stranded DNA fragments were prepared for electrophoretic mobility shift analysis (EMSA) either by polymerase chain reaction (PCR; fragments F1–F6), using gene-specific oligonucleotide primers (Table 1), or by restriction digestion (fragments PF1–PF5) using the enzymes indicated in Figure 1. The PCR products were cloned into the plasmid pGEM-Teasy (Promega, Mannheim, Germany), whereas the restriction fragments were cloned into appropriate sites in the vector pBS.KS (Stratagene, Palo Alto, CA). For the more detailed EMSA evaluation of fragment F4, subfragments were prepared by PCR using various upstream oligonucleotides F4-1 to F4-5 (Table1; see later, Fig. 3A), together with a common downstream oligonucleotide (F4-down; Table 1). In addition, double-stranded oligonucleotides (BS1/2, DSO1, DSO2, and DSO3) were prepared by chemically synthesizing the respective oligonucleotides (Table 1) of the positive and negative strands and allowing these to anneal in vitro.



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  		FIG. 1. Schematic organization of genomic DNA upstream of the transcription start site of the bovine oxytocin receptor gene. Geometric symbols indicate putative transcription factor binding motifs as indicated. The transcription start site at nucleotide +1 is indicated by a filled arrow above the line. Primers used to produce the promoter deletion reporter constructs by PCR are shown as open arrowheads below the line. The DNA fragments used as probes for EMSA experiments are shown above the line (PF1–PF5; F1–F6). The region of high primary sequence homology between the bovine gene and the human and rodent genes is shown as a hatched bar


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  		TABLE 1. List of oligonucleotide primers used.*



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  		FIG. 3. A) Diagram to show the positions of double- and single-stranded oligonucleotides used either as EMSA competitors (above; BS1/2, DSO1–3) or PCR primers (below), respectively, in relation to the fragment F4 DNA-protein complex. For details of oligonucleotides, see Table 1. B) EMSA experiment using fragment F4 or subfragments F4-1 and F4-2 as radiolabeled probes. Double-stranded oligonucleotides (DSO1–3) or DNA fragments, as indicated, were used in the given amounts (ng) as competitors. Nuclear proteins were from term endometrium in this experiment

For the construction of nested deletion promoter-reporter vectors, upstream primers (LV-3.2 to LV-0.1; Table 1) were chosen as indicated in Figure 1 (open arrowheads), together with a common downstream primer (LV-inv; Table 1) 51 bp downstream of the start site of transcription (TSS) in exon 1, and used for PCR reactions. The blunt-ended PCR products were then cloned into the SmaI site of the plasmid pGL3-basic (Promega), which uses luciferase as reporter enzyme. These and all other PCR-generated constructs were fully sequenced to confirm orientation and that no mutations had occurred consequent to the PCR reaction.

Expression constructs for mouse IRF1 and IRF2 in the pCDM8 vector [20] were a generous gift of Professor Tadatsugu Taniguchi (Dept. Immunology, University of Tokyo, Tokyo, Japan) and were recloned into the vector pRc/CMV prior to use. An expression construct for human CREB (cyclic AMP response element binding protein)-binding protein (CBP) in vector pRc/CMV was kindly provided by Dr. Jan Brosens (Inst. Reproductive and Developmental Biology, Imperial College, University of London, London, UK), and for human SRC1a and SRC1e in vector pSG5 by Dr. Benita Katzenellenbogen (Dept. Molecular and Integrative Physiology, University of Illinois, Urbana, IL).

Electrophoretic Mobility Shift Assays

Electrophoretic mobility shift assays (EMSA) were carried out exactly as previously described [16, 21]. Nuclear proteins were prepared from frozen tissues by the method of Deryckere and Gannon [22] and from cells by the method of Andrews and Faller [23]. As probes, the double-stranded longer (100–250-bp) DNA fragments were excised from their pGEM-Teasy plasmids using EcoRI and were end-labeled by the Klenow reaction in the presence of [{alpha}32P]dATP (Amersham Biosciences, Freiburg, Germany). When chemically synthesized double-stranded oligonucleotides were used as probes, these were labeled by T4 polynucleotide kinase in the presence of [{gamma}32P]ATP (Amersham Biosciences). The sequence of the double-stranded 24-mer oligonucleotide corresponding to the IRE from the bovine OTR gene promoter is indicated in Table 1 (bIRE), as also is that derived from the human ß-interferon gene (hIFNß) [24]. Radioactive probes were gel purified on 6.0% nondenaturing polyacrylamide gels run in 0.25x Tris-borate-EDTA (TBE) and eluted into Tris-EDTA (TE) buffer. Typical binding reactions comprised, in a 25-µl volume, 10 mM Hepes (pH 8.0), 0.5 mM EDTA, 100 mM NaCl, 4% (w/v) Ficoll, 0.5 mM DTT, 50 µM Na4P2O7, and 1 µg poly(dAdT) (Sigma, Deisenhofen, Germany) as nonspecific competitor, together with 1.5 µg nuclear proteins and approximately 10 000 cpm of radiolabeled probe (equivalent to 1–10 fmol DNA fragment). Where used, 1–3 µl of in vitro-translated proteins (see below) were added. Unless otherwise indicated in the figure legends, 100-fold molar excess of unlabeled competing oligonucleotides were added to the reaction mix prior to the addition of the radiolabeled probe. Similarly, where antibodies were used to interfere with DNA-protein complex formation, these were added at 1 µl serum per reaction volume prior to addition of the radiolabeled probe. Anti-mouse IRF1 (sc-640) and anti-mouse IRF2 (sc-498) polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Preincubation of components was for 15 min at room temperature, followed (after addition of the radiolabeled probe) by a further incubation at room temperature for 30 min before loading reactions onto 6% polyacrylamide nondenaturing gels and electrophoresing in 0.25x Tris-borate-EDTA at room temperature. Gels were dried and exposed to autoradiography film at -80°C overnight.

RNA Analysis

RNA was extracted from tissues using a modification of the guanidinium thiocyanate extraction procedure followed by CsCl ultracentrifugation exactly as described previously [14]. For cells, RNA was extracted using the RNA-Clean reagent (Peqlab, Erlangen, Germany) in a modification of the one-step procedure of Chomszinzky and Sacchi [25]. Northern hybridization analysis employed the formaldehyde/MOPS (morpholino-propane sulfonic acid)/agarose procedure as described in Sambrook et al. [26]. As probes, PCR fragments specific for the bovine IRF1 and IRF2 gene transcripts were prepared by reverse transcription PCR using primers derived from the mouse IRF1 and IRF2 sequences (Table 1), chosen because of high conservation between mouse, human, and duck cDNA sequences. The resulting PCR products were subcloned into pGEM-Teasy and sequenced (accession numbers AJ490935 and AJ490936) to verify their identities as the bovine homologues of IRF1 and IRF2. The DNA inserts were excised from plasmids, gel purified, and radiolabeled by the random priming method of Feinberg and Vogelstein [27] in the presence of [{alpha}32P]dCTP (Amersham Bioscience). In order to test whether these probes were able to cross-hybridize with each other, dot blot hybridizations were carried out using each radiolabeled probe with increasing amounts of the unlabeled plasmid. This experiment showed that, under standard hybridization conditions, neither probe could cross-react with the paralogous IRF cDNA, even with the target in large molar excess (not shown). For Northern hybridizations, 10 µg of total RNA from endometrial tissues of various time points, as indicated, were loaded. As hybridization control for loading, blots were cohybridized with a similarly radiolabeled probe for the constitutively expressed transcript of the small ribosomal protein S15 [28]. After hybridization and preliminary examination by autoradiography, the resulting blots were quantified by PhosphorImager (Storm 860; Molecular Dynamics, Sunnyvale, CA). The bands corresponding to IRF1 and IRF2 were normalized by reference to the intensity of the S15 signal. Preliminary experiments (not shown) had established that the signal-to-target ratio for all probes remained linear over the range of values encountered in the Northern experiments.

Immunological Analysis of IRF Expression in Bovine Cells and Endometrium

As controls to validate the anti-IRF1 and anti-IRF2 antibodies and for use in the EMSA analysis (see above), the cDNA clones encoding mouse IRF1 and IRF2, which were cloned into the vector pRc/CMV, were used for in vitro protein production using the combined translation/transcription system (TnT reticulocyte lysate kit; Promega). Following exactly the manufacturer's protocol, per 50-µl reaction, 1 µg cDNA was used to program protein synthesis in the presence of [35S]methionine (Amersham Bioscience) and drive the transcription from the T7 RNA polymerase promoter. For Western blot analysis, either 5 µl of the in vitro translated proteins or 15 µg protein extracts from the bEE, OGE, or OS cells prepared in RIPA buffer (1x phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) and quantified using the Bradford assay (BioRad Laboratories, München, Germany) were electrophoresed on 10% denaturing polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene fluoride membrane (Roche Applied Science, Mannheim, Germany) by semidry blotting. Immunodetection followed a standard protocol, with the primary rabbit polyclonal antibodies (100 µg/ml) being applied at dilutions of 1:500 (anti-IRF1) or 1:1000 (anti-IRF2). As second antibody, peroxidase-conjugated anti-rabbit Fab IgG from goat (Jackson ImmunoResearch Laboratories, West Grove, PA) was used at a dilution of 1:5000. Specific immunocomplexes were visualized using the enhanced chemiluminescent reagent (Amersham Bioscience).

Immunohistochemistry was performed on 10-µm sections of bovine endometrium, which had been immersion fixed in Bouin solution and embedded in paraffin. Sections were dewaxed and processed exactly as described previously using the combination PAP (peroxidase-anti-peroxidase)-ABC (avidin-biotin complex) technique [29] and with the primary polyclonal anti-IRF1 and anti-IRF2 antibodies at dilutions of 1:50.

Transient Transfection Analysis

After preliminary testing and optimization of various transfection procedures (including electroporation, gene-gun, adenovirus, calcium phosphate, and various proprietary lipofection reagents), the primary bEE cells were found to transfect best using the LipofectAmin LF2000 reagent (Gibco-Invitrogen, Karlsruhe, Germany). For all other cell types, we used a modified calcium phosphate precipitation procedure. The LipofectAmin procedure essentially followed the recommendations of the manufacturer. One hour prior to transfection, cells were washed with Dulbecco PBS and transferred to new culture medium without antibiotics. The LF2000 reagent (6 µl per reaction) was emulsified with OPTIMEM medium (100 µl per reaction; Gibco-Invitrogen) together with the DNA to be transfected (as indicated, also in 100 µl OPTIMEM) and incubated at room temperature for 20 min before adding to the cells at 70% confluence in 12-well plates (Nunc, Roskilde, Denmark). Cultures were incubated for a further 48 h until harvesting. The calcium phosphate coprecipitation procedure used the ProFection Mammalian Transfection System (Promega), as described in Stedronsky et al. [16], with the modification that cells were harvested 48 h after transfection.

All transfections were performed in triplicate using molar equivalents of DNA such that, for the deletion-promoter reporter constructs, 1.0 µg of the 3.2-kb construct was used but only 0.55 µg of the much shorter 0.115-kb construct, etc. Unless otherwise indicated, cotransfected IRF1 and IRF2 expression plasmids were applied all at 1 µg per reaction. For the parallel reactions of basal activity where these expression plasmids were not transfected, an equivalent amount of the promoterless reporter gene construct p0GH (Nichols Institute Diagnostics, San Clemente, CA) was used. An internal normalization control construct was not used because the generally employed viral promoters of such constructs respond specifically to activators of the interferon signaling system. Preliminary experiments showed that the transfections had no effect on growth and survival rates of the cells in culture, so no correction for this was necessary. Where different cell types were compared, values are given relative to the expression of the same constitutively positive control construct pGL3-C (Promega), which is driven by a strong viral promoter, thus correcting for cell-specific variations in transfection efficiency. Other assays within a cell type are expressed relative to the unstimulated basal activity with the empty pGL3-B basal vector alone. Where indicated, results were subjected to ANOVA and mean values compared by a post hoc Newman-Keuls test. All experiments were repeated at least twice, with identical results. Data shown are means ± SEM from triplicate wells of a single representative experiment.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Differential EMSA Analysis Using Different Bovine Tissues

The preliminary sequence of the bovine OTR gene [18] comprised, besides the coding exons, only about 1200 bp of genomic sequence upstream of the transcription start site. By further sequencing of the XhoI-BamHI and HindIII-BamHI restriction fragments of the original genomic clone #20 in bacteriophage lambda [18], this has been extended here to approximately 3200 bp (EMBL accession number AJ490937), and the complete gene has been subjected to computer analysis for putative transcription factor binding sites using the program AliBaba2 [19] (Fig. 1). A previous comparison of this upstream region with the equivalent regions from the rat, mouse, and human OTR genes highlighted several regions of high sequence conservation, which were, however, restricted to only about 800 nucleotides 5' to the transcription start site [30]. None of the potential motifs conserved across all species corresponded to any of the sites identified in the AliBaba2 computer analysis except for ubiquitous sequences for general transcription factors such as Sp1 (not shown). In Figure 1, only those motifs are indicated that are unambiguous and could be relevant in the context of specific uterine physiology.

Bovine tissues were selected corresponding to situations where OTR are either never expressed (liver, adrenal gland), expressed at low levels only (Day 7 endometrium, Day 20 pregnant endometrium), or expressed at high levels (Day 0 endometrium, Day 20 nonpregnant endometrium, term myometrium). Other tissues with different expression levels were also used (not shown). EMSA analysis was carried out using approximately 200-bp overlapping fragments (Fig. 1) from both the high-homology region (-800 bp) of the bovine OTR gene, where gene-specific binding is expected, as well as regions further upstream, together with nuclear protein extracts from the respective tissues. All regions indicated moderate tissue-specific nuclear protein binding with both endometrial (Fig. 2) and myometrial (not shown) samples, which was generally absent using liver samples. However, this differential display EMSA analysis failed to indicate any significant quantitative differences in binding between low- and high-expressing tissues or between myometrium and endometrium, implying that all binding probably represented general constitutive factors. Hence, in Figure 2, only the results for Day 0 endometrium are shown because these are qualitatively and quantitatively similar to the other uterine samples; other tissue samples from other time points gave very similar results (not shown). Fragment PF3 failed to show any DNA-protein complex formation (omitted). Fragment F4, from approximately -500 bp, consistently gave rise to a prominent protein-DNA complex that was absent from liver samples (not shown) but also present in both myometrium and endometrium from all stages (Figs. 2 and 3B, and data not shown) as well as from Hela cells (Fig. 2). Using various subfragments of DNA from this region (Fig. 3A) combined with competition by specific oligonucleotides showed that there were probably several repeats of the responsible cis element within the 5' half of fragment F4, whereby only the competitor DSO1 (-TTTTCTTTCTGCCATTTTTT-) indicated significant affinity (Fig. 3B). Subfragments F4-3, F4-4 and F4-5 failed to generate DNA-protein complexes (not shown). Although it was not possible to define a transcription factor whose binding properties correspond to this element, it seems most likely that a factor related to the constitutive SRY family would fulfill the requirements.



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  		FIG. 2. EMSA for fragments PF1–PF5 and F1–F6 using nuclear proteins from a negative control tissue (liver) and a highly expressing tissue (Day 0 endometrium; d0 endo). For probe F4, complexes formed with Hela cell nuclear extract are illustrated rather than those with liver. Probe PF3 showed no binding and was omitted

Within the -3200 region, there were also three ERE (estrogen response element) half sites identified as well as a corrupted palindrome at -2600 (-gTGATTctcGGTCActcTGTCGg-; Fig. 1) and an intact ERE palindrome downstream of the transcription start site within the coding region. Although special attention was given to these sites, none were conserved across species and none showed any evidence for binding of specific estrogen receptor (ER)-related nuclear proteins (not shown). The downstream palindromic element had previously been tested also for transactivation activity in a transfection assay and found to be inactive [31].

A Motif at -2400 Is an Interferon-Responsive Element

The computer analysis indicated a site at -2441 with features typical of known interferon-responsive elements (IRE; cf., bovine OTR element: -GAAAAATGAAAGC- and consensus IRE -RAAAGTGAAAGCY-). First, when using this motif from the bovine OTR gene as probe for EMSA analysis, both as a larger ~100-bp sequence (PF2; Fig. 1) and as a 24-mer oligonucleotide, specific binding was observed using endometrial nuclear extracts (Figs. 2 and 4). In this assay, we also observed no significant differences between nuclear extracts from different time points (not shown), even though Northern analysis (see below) suggested that there might be differences in transcript concentration for interference response element binding factors (IRF). Consistently, two specific binding complexes were observed, which could be competed by an excess of the unlabeled IRE-containing bovine oligonucleotide as well as by a classic IRE from the human ß-interferon (IFN-ß) gene (Fig. 4, left panel). A similar pattern of specific binding was seen when the IRE from the human IFN-ß gene promoter was used as radiolabeled probe with the same nuclear extracts (Fig. 4, right panel). This sequence was also able to compete effectively for binding of the endometrial nuclear proteins to the radiolabeled bovine IRE sequence and vice versa (Fig. 4). Application of specific antibodies, raised against murine IRF1 and IRF2, showed that only the latter were effective in producing a supershift (Fig. 5), though this was most noticeable when using the control human IFN-ß element as probe (Fig. 5C). For the bovine IRE probe, either no supershift was observed or only a very weak shift was seen when using the IRF2 antibodies (Fig. 5B, star). In these EMSA analyses, control proteins were also used (Fig. 5, B and C, lanes 7 and 8) derived by in vitro transcription/translation of mouse IRF1 and IRF2 expression plasmids (see below). Because the tissue samples from which the nuclear extracts were derived comprised many different cell types, nuclear extracts were also used derived from primary cultures of endometrial epithelial cells (Fig. 5A). These showed the same two DNA-protein complexes formed with the putative bovine IRE element of the OTR gene, which could be specifically competed by an excess of the unlabeled IRE oligonucleotide (Fig. 5A, lane 4) but could be influenced by either the mouse IRF1- (Fig. 5A, lane 3) or IRF2-specific (not shown) antibodies.



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  		FIG. 4. EMSA experiment using either the putative IRE of the bovine OTR gene (24-mer; left panel) or the classic double IRE of the human ß-interferon gene (right panel) as radiolabeled probes. For details of the oligonucleotides used, see Table 1. These probes were competed with increasing amounts of either the same bovine putative IRE (bIRE) or the double element of the human ß-interferon gene (hIFNß), as indicated. Nuclear proteins were from Day 0 endometrium in this experiment



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  		FIG. 5. EMSA experiment using either the putative IRE of the bovine OTR gene (24-mer; A, B) or the classic double IRE of the human ß-interferon gene (C) as radiolabeled probes. As source of transcription factors, either nuclear extracts from freshly prepared bEE cells (A,), from Day 0 endometrium (B, C), or as pure in vitro translated mouse (m) IRF1 and IRF2 (T/T; B, C) were used. The protein-DNA complexes formed were competed either with 100 ng of the double-stranded oligonucleotides hIFNß or bIRE or were reacted with 1 µl of the polyclonal antisera against mouse IRF1 and IRF2 or a nonspecific double-stranded oligonucleotide (ns oligo). The star (lane B6) indicates a weak shifted complex induced by the anti-IRF2 antibody when using the bIRE as probe. The equivalent shifted complex is much stronger when using the hIFNß oligonucleotide as probe (lane C6). The open arrowhead indicates a nonspecific complex formed with endogenous products of the in vitro translation system

As a further control to validate the identification of the -2441 element as a bona fide IRE, in vitro-translated mouse IRF1 and IRF2 were produced and tested in EMSA using both the bovine IRE as well as the human IFN-ß element as probes. Both murine IRF1 and IRF2 gave rise to specific DNA-protein complexes (Fig. 6), which could be competed by the unlabeled IRE motifs but not by similarly sized nonspecific oligonucleotides and could be induced to supershift by applying the specific IRF1 and IRF2 antibodies. It should be noted that the IRF proteins bind both as monomers and dimers to the IFN-ß element (Fig. 6).



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  		FIG. 6. Control EMSA experiment using either the putative IRE of the bovine OTR gene (24-mer; A) or the classic double IRE of the human ß-interferon gene (B) as radiolabeled probes. In vitro translated mouse IRF1 (lanes A1–6, B1–4) and IRF2 (lanes A7–12, B5–8) are the source of transcription factors for complex formation. The reaction mixtures were preincubated with either the bIRE, hIFNß, or ns oligo competing double-stranded oligonucleotides or with antibodies (Ab) against mouse IRF1 or IRF2, as indicated

Northern Blot, Reverse Transcription-PCR, and Western Blot Analyses for Transcripts Encoding Interferon Responsive Factors (IRF) 1 and 2 in Bovine Tissues and Cells

Oligonucleotide sequences conserved between mouse and human IRF1 and IRF2 were used to clone by reverse transcription-PCR (RT-PCR) the equivalent transcript regions encoding bovine IRF1 and IRF2 (accession numbers AJ490935, AJ490936). The resulting fragments were then used as probes to detect specific mRNA in different bovine tissues by Northern blot analysis. Specific transcripts were detected for IRF1 and IRF2 at approximately 2500 bp (Fig. 7A). Quantification of endometrial samples suggested a higher expression of IRF1 and IRF2 at Day 20 of pregnancy, than at estrus or Day 20 of the nonconceptive cycle (Fig. 7, B and C). Although Northern analysis was not sufficiently sensitive to detect either transcript in RNA from primary cultures of bovine endometrial epithelial cells, RT-PCR analysis showed that both transcripts were expressed in these cells over 9 days of culture (data not shown).



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  		FIG. 7. Northern hybridization analysis of total RNA from bovine endometrial tissues of defined times. A) Representative Northern blots using the specific bovine IRF1 (lane 1) and IRF2 (lane 2) cDNAs as probes. Blots were cohybridized with a probe for the constitutively expressed transcript of the bovine small ribosomal protein S16. B) Quantitative analysis of the specific hybridization signals from different Northern blots estimated using a PhosphorImager and corrected (normalized) using the signals from the cohybridized S16 transcripts (n = 4 for each sample category; mean ± SEM). The asterisks (*) indicate a significant difference (P < 0.05) from the Day 16–18 pregnancy sample (Student t-test)

As further supportive evidence for the expression of IRF1 and IRF2 in bovine endometrial epithelial cells, Western blots were carried out using the anti-mouse IRF antibodies, discussed above. Both IRF1- and IRF2-like immunoreactive proteins can be detected in primary cultures of bovine endometrial epithelial cells (Fig. 8A). While these are approximately of similar size as the in vitro-translated proteins, their identity as the bovine homologues of mouse IRF1 and IRF2 must remain speculative. The same antibodies applied in immunohistochemisty of bovine uterine tissue sections indicated strong specific signals, particularly in the glandular epithelium of the endometrium (Fig. 8B), where both OTR and the type I IFN receptors are known to be expressed at high levels [18, 32].



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  		FIG. 8. Immunochemical analysis of IRF-related epitopes in bovine cells and tissues. A) Western blots of in vitro translated (T/T) mIRF1 and mIRF2 proteins as well as total protein extracts from primary cultures of bEE cells cultured for 1 and 2 days, as indicated. Blots were reacted with either anti-mIRF1 (lanes 1–6) or anti-mIRF2 (lanes 7–12). Whereas the mouse IRF2 antibodies appear to react with similarly sized proteins in the bEE cells (lanes 11, 12) to the pure mouse IRF2 (lane 9), the IRF1 antibodies appear to cross-react with a protein in bEE cells (lanes 5, 6) marginally larger than the in vitro-translated mIRF1 (lane 2). B) Immunohistochemistry of term bovine endometrium using the anti-IRF1 antibodies. C) Immunohistochemistry of term bovine endometrium using the anti-IRF2 antibodies. D) Negative control where the primary antibodies have been replaced by an equivalent amount of rabbit preimmune serum

Transcriptional Activity of the Upstream 3200 bp of the Bovine OTR Gene

A series of promoter-deletion reporter constructs were prepared by PCR in the vector pGL3-basic. For the bovine endometrial epithelial cells, a variety of transfection procedures had been tested. Electroporation, gene-gun, adenovirus, and calcium phosphate procedures did not give reliable transfection in our hands. Figure 9 shows results using the LipofectAmin reagent, which proved most effective for this cell type. In addition to the primary bovine endometrial epithelial cells, also the immortalized sheep cell lines OS and OGE were used, together with the endometrial adenoma cell line Skut1b and the human breast cancer cell lines MCF7 and MDA-MB231, all being transfected by the calcium phosphate method.



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  		FIG. 9. Transient transfection of different cell types, as indicated, using the nested promoter deletion reporter constructs (upper scheme) for the bovine OTR upstream region. Values are expressed relative to the luciferase activity generated by the positive-control construct (pGL3-C), which is driven by a strong constitutive viral promoter. The horizontal broken lines mark the level of activity attained by the negative control ‘empty’ reporter vectors

Figure 9 indicates that the whole promoter region is relatively inactive by comparison with the positive control construct used, which is driven by a strong viral promoter. Assuming that the primary bEE cells, in which there is high endogenous OTR expression [9], are closest to the in vivo situation, then most specific activation potential resides in the full-length -3200 construct, with relatively little activity in the shorter proximal promoter constructs, even though this latter region is highly conserved across species. Of the cell lines tested, the OGE, MCF7, and Skut-1b cells appear to be the most useful, whereas the OS cells were fully inert with respect to the bovine OTR constructs and the MDA-MB231 cells were somewhat aberrant. None of the cell lines expresses endogenous OTR transcripts to a level above that detectable by a sensitive RT-PCR assay (not shown). Because it has been shown that the OTR gene can be upregulated in some but not all cell cultures by application of cAMP-elevating agents [33, 34], presumably acting by phosphorylation of constitutive transcription factors, both 8Br-cAMP (0.5 mM) and/or phorbol myristacetate (100 ng/ml) were applied to both OGE- and OS-transfected cell systems without, however, having any effect on reporter gene activity (not shown).

Because the MCF7 cell line is furnished with a relatively high concentration of functionally active estrogen receptor {alpha} (ER{alpha}), this cell line was also used to test for any effects of estradiol on these bovine promoter constructs. At two different concentrations of steroid (10-8 M and 10-9 M), no effect could be detected, although a control construct driven by the ERE of the vitellogenin A2 gene showed at least a 2-fold stimulation (not shown). Similar results were obtained also using additionally cotransfected expression vectors encoding ER{alpha} (not shown). We had previously shown that estradiol had no effect on the endogenous OTR expression in primary bEE cells [9], although the cells do express ER{alpha} transcripts [9] and transfected ERE-driven reporter constructs do respond to estradiol (unpublished results). Here we have also tested for an effect of estrogen on the full-length transfected bOTR-3.2 promoter-reporter construct in these cells. Confirming the previous results, there was no effect of 10-8 M estradiol.

A lack of estrogenic effect could be due to an imbalance in levels of steroid receptor cofactors. To test this, different relative amounts of the three cofactors CBP, SRC1a, and SRC1e were cotransfected into the OGE cell system, together with the bOTR-3.2 full-length promoter-reporter construct (Fig. 10). Whereas each factor alone was without effect, certain combinations of the cofactors, particularly SRC1e and/or CBP, caused a significant induction of reporter activity by estradiol (Fig. 10).



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  		FIG. 10. Transient transfection of the OGE cell line using the full-length 3.2-kb promoter-reporter construct of the bovine OTR gene, cotransfected with different combinations of expression vectors for the steroid receptor cofactors CBP, SRC1a, and SRC1e, as indicated. Figures indicate DNA in µg added; expression vector DNA was supplemented by the promoterless reporter plasmid pGH0 to provide always a constant amount of DNA transfected. Where indicated, cultures were treated or not with 10-8 M estradiol

The bovine endometrial epithelial primary cells and the OGE cells were used for further studies to investigate the functionality of the putative IRE in the distal promoter region.

The factors IRF1 and IRF2 have been shown to act as repressor or enhancer of interferon-regulated genes, respectively, where these include a classic IRE motif [20]. In preliminary experiments (not shown), using the mouse IRF1 and IRF2 expression constructs in cotransfection experiments with Hela cells first verified this function for the control IRE from the human ß-interferon gene. When instead a reporter construct was used wherein the equivalent 24-bp element from the bovine OTR gene is used, there is a clear, though smaller, effect of both IRF1 and IRF2, but in both cases, this is stimulatory and additive (not shown). This initial experiment made use of the IRE taken out of context from the bovine OTR gene and demonstrates only the potential interferon responsiveness of this cis element. In order to test whether this element functions in this manner in a more natural context, the same deletion promoter-reporter constructs as used before were applied in bEE, OGE, and OS cells with cotransfection of the IRF1 and IRF2 expression vectors (Fig. 11). There is no or only minimal effect of the cotransfection on any of the constructs containing 1000 bp or less of the upstream promoter region of the bovine OTR gene. However, with the 3.2-kb construct, which contains the putative IRE motif, there is a clear effect of the additional IRFs. Here IRF1 acts as a significant suppressor in both bEE and OGE cell types (Fig. 11), while IRF2 acts as an enhancer. For the OS cells, which are already inert, the IRF1 construct can have no further suppressive effect, whereas the IRF2 construct also significantly induces reporter activity (Fig. 11). In other experiments (not shown), the steroids estradiol and progesterone were also applied to test for any influence on the effects of the IRFs on the 3.2-kb OTR construct. In no case were any significant effects of the steroids detected.



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  		FIG. 11. Transient transfections of bEE, OGE, and OS cells with the nested deletion promoter-reporter constructs of the bovine OTR gene (see Fig. 9). Cells were cotransfected with expression vectors for mouse IRF1 or mouse IRF2, as indicated. The untreated controls (0) were cotransfected with the promoterless plasmid pGH0 to provide equivalent amounts of DNA. C) Basal reporter construct pGL3-B. Asterisks indicate values significantly different (P < 0.05) from the respective basal control


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Studies on the functionality of gene promoters are fraught with artefactual effects created by an imbalance of transcription factors and cofactors within cells (e.g., squelching) as well as by problems resulting from the dissection of promoter regions out of their natural chromatin context. For this reason, we have chosen here to work with systems as close as possible to the in vivo situation. Initially, we have applied a differential EMSA approach in an attempt to identify cis elements in the high homology region up to 800 bp upstream of the transcription start site, shared with the OTR genes from other species, and where we would anticipate specific binding of nuclear proteins related to the dramatic up- and down-regulation of the OTR gene in vivo. While specific binding was evident throughout this region, there was no evidence of any differential effects (time or tissue specific) correlating with the transcriptional activity of the OTR gene. This is possibly not surprising being that complex tissues were used, wherein only a small proportion of the cells (e.g., endometrial epithelial cells) would be expressing the OTR gene. Nevertheless, this experiment also shows that the OTR gene is probably not regulated by highly cell-specific factors but involves factors (e.g., constitutive ones) shared in common in many cell types.

The issue of whether or not the OTR gene is directly regulated by steroid hormones is equally elusive. While obvious direct effects of estradiol can be eliminated in the present study, as in previous ones [7], more subtle effects are plausible. For example, cotransfection of the cofactors CBP, SRC1a, and SRC1e leads to a small but significant estradiol-dependent effect in the OGE cell system. There are no functional palindromic EREs in the OTR promoter region, but three ERE half sites have been identified as well as a corrupted palindrome. It is now known that such half sites can interact with estrogen receptors through the agency of other cooperatively acting transcription factors and/or cofactors such that a potential for estrogen responsiveness is given. Although the bEE cells express the ER{alpha} gene, the endogenous OTR appears to be nonresponsive to estradiol [9]. However, an effect of estradiol on OT-induced prostaglandin production is reported [35]. Thus, the estrogen-dependent upregulation observed in the present study may not be biologically relevant and should be regarded with caution. Most evidence to date suggests that steroid sensitivity of the OTR gene is a function of the intact tissue, probably requiring additional factors from the stromal compartment [36, 37].

From their phenotype, the immortalized OGE cells are quite similar to the bEE primary cells and show a similar level of unstimulated induction of the full-length (3.2-kb) OTR promoter region, although only the latter cells show substantial endogenous OTR expression. Shorter constructs (<1 kb) are less active in these and all other cells tested. Both cell types are also responsive to type 1 interferons, such as IFN-{tau}. The region from -1.0 to -3.2 kb includes a consensus motif at -2441 for binding members of the IRF family of transcription factors. Fleming and coworkers have elaborated the putative pathway by which IFN-{tau} is able to suppress the transcription of the ER{alpha} gene in ruminant endometrial cells [38]. As in other interferon-responsive cells, activation of a type 1 interferon receptor causes the induction of the gene for IRF1, which in turn can stimulate or inhibit other genes with an IRE motif in their promoter regions. IRF2 is supposedly secondarily induced by IRF1. In the meantime, there are now known to be at least nine different IRF genes in mammals [39], such that the mouse IRF1 and IRF2 expression constructs used in the present study must be seen as surrogates also for other members of this family.

In the present study, we have shown that the bovine endometrium and in particular the endometrial epithelial cells express IRF1- and IRF2-like molecules, detectable at both the mRNA and protein level. In vivo, the concentration of the two transcripts reflects what we should expect for an IFN-{tau}-regulated physiology, with an induction in the endometrium of both factors during maternal recognition of pregnancy. However, these experiments used complex tissues, and preliminary studies using purified bEE cells (not shown) suggest that such an IFN-dependent induction might not occur in these cells. Because the immunohistochemical evidence suggests that IRF-like entities are present mostly in the epithelial cells, the Northern results might also reflect the increased proportion of glandular and epithelial components in the secretory-phase endometrium. Second, we have shown that the putative IRE at -2400 of the bovine OTR gene promoter is indeed able to bind mouse IRF1 and IRF2 specifically and is functional because it responds to these factors in a transfection assay, both as an isolated fragment and in the context of the 3.2-kb promoter construct. Finally, EMSA analysis shows that nuclear extracts from endometrial tissue as well as from bEE cells also contain IRF-like proteins, which are able to bind specifically to the -2400 element. Both OGE and OS cell nuclear extracts also formed DNA-protein complexes with the bovine IRE, which were indistinguishable from those formed using the bEE cells (data not shown), confirming recent findings from the intact sheep endometrium [40]. The identity of these proteins is not clear, though that in the faster moving complex appears to react weakly with mouse IRF2 antibodies. Neither native complex can be influenced by mouse IRF1 antibodies. This lack of effect by the antibodies most likely reflects a difference between species, with the antibodies raised against the mouse proteins not, or only poorly, reacting with their bovine counterparts. The observation of an IRF1 immunoreactive entity in the endometrial epithelium by immunohistochemistry and by Western blotting (Fig. 8) may indeed reflect a different IRF-like molecule that is not able to bind the bovine IRE. It should be noted that, recently, IRF9 has been identified and characterized from the ovine endometrium [40] such that one of the endogenous proteins detected by EMSA could be IRF9. A further point of interest is that, whereas IRF1 appears to be stimulatory and IRF2 inhibitory on various IRE-containing promoters, such as that for human ß-interferon [20] or for sheep ER{alpha} [38], the IRE of the bovine OTR gene responds in the converse direction, with IRF1 being inhibitory and IRF2 stimulatory. However, on the promoter for the cytochrome b558 heavy chain of the NADPH-dependent oxidase, IRF1 and IRF2 are both stimulatory [41]. It is important to emphasize that, from our experiments, we can only prove that the bovine OTR gene indeed possesses a functional IRE motif that is able to respond to IRF proteins. Which proteins are interacting here in vivo and under which conditions (stimulation or not by IFN-{tau}) remain a subject for future studies.

In summary, the evidence presented here suggests, first, that the proximal promoter of the bovine OTR gene, which shares high homology with the same regions of the OTR gene in other species, does not appear to bind any highly cell- or gene-specific transcription factors but rather constitutive factors commonly found in many cell types and which might be regulated as the end points of kinase pathways. This would agree well with studies on the human OTR promoter, where the latest study suggests that chromatin methylation of CpG islands may play a significant role in the tissue-specific expression of the OTR gene [42]. While in general there is no evidence for direct action of estradiol via its specific receptors (ER{alpha} or ERß) on a region 3.2 kb upstream of the transcription start site, cotransfection experiments with steroid receptor cofactors, such as SRC1e, imply a possibility for estrogenic action by an unconventional mechanism, which may make use of ERE half sites. While this aspect requires more detailed study, the failure to find any effect of estrogen on the endogenous OTR expression or on the transfected reporter constructs in the OGE cells pleads for extreme caution in overinterpreting this result. The only specific effect that we have been able to elaborate involves the direct action of the IFN-{tau} responsive factors, for which mouse IRF1 and IRF2 are surrogates, on an element at -2400 in the bovine OTR gene promoter. Comparable factors are expressed in the OTR-producing cells and tissues of the bovine uterus, and the mouse factors can mimic some of the effects expected during maternal recognition of pregnancy.

The induction of the OTR gene remains a major factor in the acute mechanisms leading to parturition and for ruminants is an essential component in blastocyst recognition. Research on OTR regulation is massively hampered by a lack of suitable cell systems in which to study its gene expression; the best cells we have found, the bEE cells from the nonpregnant uterus, which have a high endogenous OTR expression, unfortunately prove to be very difficult to transfect and manipulate, such that further research must await new methodological developments in this area.


   ACKNOWLEDGMENTS
 
We should like to acknowledge the excellent technical assistance of Ms. Marga Balvers as well as contributions in preliminary experiments by Dr. David Stewart (College Station, TX), Dr. Katrin Stedronsky (Hamburg, Germany), and Mr. Simon Draper (Oxford, UK). We are very grateful to Dr. B.S. Katzenellenbogen (Urbana, IL), Professor Tadatsugu Taniguchi (Tokyo, Japan), and Dr. Jan Brosens (London, UK) for the kind gifts of expression constructs. We should also like to thank Professors Michael Fields (Gainesville, FL) and Anna-Riitta Fuchs (New York) for provision of dated bovine tissues and Professor Freimut Leidenberger (Hamburg) for his continued support of this project.


   FOOTNOTES
 
1 This research was supported by a grant from the Deutsche Forschungsgemeinschaft (Iv7/8-3) to R.I. R.A.D.B. was a fellow of the Alexander-von-Humboldt Foundation, Bonn, Germany. Back

2 Correspondence: Richard Ivell, Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany. FAX: 49 40 56190864; e-mail: ivell{at}rrz.uni-hamburg.de Back

3 Current address: Howard Florey Institute, University of Melbourne, Australia Back

4 R.T. and R.A.D.B. are equal-ranking first authors Back

Received: 1 July 2002.

First decision: 31 July 2002.

Accepted: 3 October 2002.


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