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Endocrinology Vol. 145, No. 10 4452-4460
Copyright © 2004 by The Endocrine Society

Regulation of Interferon-{tau} (IFN-{tau}) Gene Promoters by Growth Factors that Target the Ets-2 Composite Enhancer: A Possible Model for Maternal Control of IFN-{tau} Production by the Conceptus during Early Pregnancy

Toshihiko Ezashi and R. Michael Roberts

Department of Animal Sciences, University of Missouri, Columbia, Missouri 65211

Address all correspondence and requests for reprints to: R. Michael Roberts, 158 Animal Sciences Research Center, University of Missouri-Columbia, 920 East Campus Drive, Columbia, Missouri 65211-5300. E-mail: robertsrm{at}missouri.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of interferon-{tau} gene (IFNT) by conceptuses of cattle and sheep must be in phase with the physiological state of the mother if the pregnancy is to be successful. IFNT has a close-to-consensus AP1 site (–71 to –64), overlapping a binding site for Ets-2 (–79 to –70), the key transcription factor controlling IFNT expression. When a bovine IFNT gene control region-luciferase (luc) construct was transfected into mouse 3T3 fibroblasts in the presence of Ets-2 and oncogenic Ras, luc expression was activated (50- to 100-fold). Mutations in either the activator protein 1 (AP1) site or the Ets-2 site of this construct abolished this effect. Similarly, a mutation of Thr72 of the Ets-2 or the addition of a specific inhibitor for the MAPK signal transduction pathway also markedly reduced expression. IFNT promoter activity was up-regulated in response to colony-stimulating factor-1 in 3T3 cells expressing the colony-stimulating factor-1 receptor c-fms. This response did not occur when the AP1 site or the Ets-2 binding sites were mutated. Nor was the response observed in 3T3 cells expressing an inactive form of c-fms. The experiments indicate that IFNT can be activated by growth factors operating through the Ras/MAPK pathway. The Ets-2 and AP1 binding sites are essential for such effects. The AP1 site, however, is noncanonical and unable to bind either cJun or cFos. These data emphasize the importance of a complex Ets-2 enhancer for expression of IFNT and suggest a means whereby the mother can exert control over conceptus IFN-{tau} production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTERFERON-{tau} (IFN-{tau}) PLAYS a key role in maternal recognition of pregnancy in mammalian species, such as cattle, sheep, and deer, within the Ruminantia suborder (1, 2, 3, 4). Conceptuses of these species produce IFN-{tau} to prevent the regression of the maternal corpus luteum (CL), an event that would normally occur at the end of the estrous cycle if the animal were not pregnant. In this sense, IFN-{tau} acts in an analogous manner to human chorionic gonadotropin in the human, but rather than being targeted directly to the CL and having a luteotrophic effect, it acts locally on the uterine endometrium and prevents the pulsatile release of the luteolytic hormone, prostaglandin F2{alpha}.

The IFN-{tau} are related structurally to other type 1 IFNs, including IFN-{alpha} and -ß, and especially IFN-{omega}, from which they diverged approximately 36 million years ago (5, 6). The hallmark of the entire grouping is their potent antiviral activity, which is usually evident in the picomolar range of concentrations (7, 8). However, all are pleiotropic in action, typically possessing antiproliferative and complex immunomodulatory as well as antiviral properties. Where the IFN-{tau} differ most markedly from the IFN-{alpha}, -ß, and -{omega} is not in their biological activity but in the lack of inducibility of their genes in response to virus, their localized expression in one epithelial cell layer (trophectoderm), and their very high rate of production and persistence of expression over a few days at a crucial time during early pregnancy when the CL is wavering on the point of regression (9). It is likely that what sets the IFN-{tau} apart from other IFN and has provided them with the status of hormones of pregnancy is their production by the conceptus in unusually large amounts at the correct time to prevent CL regression.

The IFN-{tau} genes (IFNT) are only expressed weakly in trophectoderm of cattle at the blastocyst stage of development (10, 11, 12, 13). Expression per cell increases markedly as the blastocyst enlarges and begins to elongate (1, 10, 11, 13), and an association has been observed among the growth of the conceptuses in vivo, their production of IFN-{tau}, and the levels of progesterone in the serum of the mother (14). Moreover, the production of IFN-{tau} by in vitro-produced blastocysts can be markedly increased if the culture medium is supplemented with uterine flush material from ewes in the secretory phase of their estrous cycles (6, 15). Finally, absence of endometrial gland secretions, as occurs in ewes in which endometrial gland development has been compromised, leads to a failure of both conceptus elongation and a marked reduction in IFN-{tau} production (16). Together, these data suggest that factors present in uterine secretions might be capable of up-regulating IFNT gene expression, thereby coordinating the growth and activity of the conceptus with the hormonal state of the mother. A reasonable inference is that these factors bind to receptors on the surface of trophectoderm and activate signal transduction pathways that up-regulate the already constitutively active IFNT.

Control of expression of IFNT-reporter constructs transfected into the human choriocarcinoma cell line, JAr, is mediated primarily by a complex of regulatory sequences, containing an Ets-2 binding site, placed –79 to –70 bp upstream of the transcription start site (17, 18). Ets-2 enhancers are not just characteristic of the IFNT but are a feature of many genes, including the one for urokinase-type plasminogen activator (uPA; Fig. 1AGo), that are expressed in trophectoderm (19, 20, 21, 22, 23). Several such Ets-2-responsive genes have an AP1-binding site placed close to the Ets site (17, 19, 21, 22). Mutation of this AP1 site typically abrogates responsiveness to Ras/MAPK signaling (19). We have noted that all IFNT control regions have a close-to-consensus AP1 site partially overlapping the Ets-2 binding site (Ref. 17 and Fig. 1AGo), but we were unable to demonstrate that it had any functional significance in controlling promoter transactivation by Ets-2 in JAr choriocarcinoma cells (Ref. 17 and unpublished data).



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  		FIG. 1. Coexpression of Ets-2 and Ras cause superactivation of murine uPA and bovine IFNT promoters. A, A comparison of the sequence of the Ets/AP1 elements of the boIFNT1 and the murine uPA (19 ) control regions. The former is located at –77/–64 of the bovine gene, the latter at –2446/–2356 of murine uPA. The Ets core binding sequences are boxed. Sequences within the putative AP1 binding site that are also found in a consensus AP1 binding site (37 ) (shown below) are underlined. Bases introduced into mutated AP1 and IFNT reporter constructs and DNA probes (µAP1 and µEts) are shown with lowercase letters. B, Cartoon illustration of the uPA-luc reporter (upper panel) and the stimulatory effects of Ets-2 and Ras expression on uPA promoter activity (lower panel). A short DNA fragment containing the distal Ets/AP1 element of uPA (–2446 to –2356) was placed immediately upstream of the proximal uPA control region at –114 (19 ) and linked to a firefly luc-reporter gene (pGL2 basic; Promega). The uPA reporter was cotransfected with expression plasmids for Ets-2 and activated Ras (alone or in combination) into 3T3 cells. C, Cartoon illustration of the bovine IFNT –126 luc reporter, containing the entire –126/+50 gene control region (upper panel), and the stimulatory effects of Ets-2 and Ras expression on –126 luc reporter activity (lower panel). The –126 luc reporter was cotransfected with expression plasmids for Ets-2 and activated Ras (alone or in combination) into 3T3 cells. In both B and C, luc activity was normalized relative to ß-galactosidase activity from the reference reporter pRSVLTR-ßgal. The data are expressed as fold activation (means ± SEM; n = 3) relative to controls.

 
Although Ets-2 and related transcription factors can bind specifically to Ets binding elements on DNA, the proteins generally seem to require activation through phosphorylation before they participate in gene regulatory events (24, 25, 26, 27, 28, 29). Activation of Ets-2, for example, requires the MAPK-mediated phosphorylation of Thr72 in its so-called pointed domain (24, 25, 26, 30). It is for this reason that Ets-2 is frequently cotransfected with an activated Ras construct before measurable effects on gene transcription can be observed (30) and why mutation of Thr72 negates Ets-2 effects (24, 25, 26, 30). Curiously, in the case of Ets-2 transactivation of IFNT promoter constructs, we failed to observe any significant effects of Ras cotransfection in JAr choriocarcinoma cells (17). Again, however, those experiments were carried out in JAr cells, a line of tumor-derived cells that might already possess an activated Ras oncogene component (31).

Three hypotheses have been tested in the present paper. The first is that the IFNT control region does, in fact, possess a Ras-responsive enhancer (RRE), despite the fact that Ras responses cannot be demonstrated in choriocarcinoma cells. The second is that the AP1-like sequence adjacent to the Ets-2 binding site on the IFNT control region is functionally important for promoter activity. The third is that IFNT promoters are responsive to growth factors that depend on the Ras/MAPK signal transduction pathway and hence are potentially activated by components present in maternal uterine secretions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reporter gene constructs and expression plasmids
Bovine IFNT1 (boIFNT1)-reporter constructs, –126 luc, and –457 luc (containing the gene control regions –126 to +50 and –457 to +66 bp, respectively) have been described previously (17, 18). A –1675 boIFNT1 control region (–1675 to +66 bp) (32) was subcloned into the XhoI site of the luciferase (luc) reporter plasmid, pGL2-Basic (–1675 luc; Promega, Madison, WI). Site-directed mutagenesis of the AP1 binding site at –71 on the reporters was achieved with primers mAP1-1 (5'-cgcTCTagagaaattttcgg; the uppercase letters show the mutation sites) and mAP1–2 (5'-gctctAGAacttcctgtttgtgt) and standard PCR procedures (33). Mutagenesis of the Ets binding site at –79 has been described previously (18). The mutated sequences are shown in Fig. 1Go. Fidelity of all constructs was verified by DNA sequencing.

The Ets-2 expression plasmids (pCGNEts-2 T72 and pCGNEts-2 A72) and the activated ras expression vector (pHO6T1) have been described previously (17). The reporter plasmid for uPA was a gift from Dr. Michael Ostrowski (The Ohio State University, Columbus, OH) (30).

Either the promoterless Renilla luc reporter plasmid (pRL-null; Promega) or ß-galactosidase gene driven by the Rous sarcoma virus long terminal repeat (pRSVLTR-ßgal) was used as an internal control in transfection experiments (17, 18).

Cell culture and transfections
Mouse NIH3T3 cells were maintained in DMEM (11965-092; Invitrogen, Carlsbad, CA) supplemented 10% fetal bovine serum (FBS). Transfections were performed by using either the calcium phosphate method (17, 18) or the FuGene 6 product as recommended by the manufacturer (Roche Molecular Biochemicals, Indianapolis, IN). When using the calcium phosphate method, cells were plated in either 60-mm cell culture dishes (2 x 105 cells/dish; Figs. 1Go and 2Go) or six-well plates (1 x 105 cells/well; Fig. 3Go). With the FuGene 6 reagent, 24-well plates were used (2 x 104 cells/well; Fig. 4Go). The amounts of DNA (micrograms) used in each transfection are listed in Table 1Go. Total amounts of transfected DNA were kept constant within experiments by adding corresponding empty vectors.



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  		FIG. 2. Replacement of Thr72 of Ets-2 with alanine and mutation of the putative AP1 and Ets-2 binding sites on the IFNT control region prevent superactivation by Ets-2 and Ras. A, Comparative representation of the structures of the mutated –126 luc reporters (µAP1 and µEts) and the wt reporter. B, Reporter constructs were transfected into 3T3 cells with expression plasmids Ets-2-T72, Ets-2-A72, and activated Ras alone or in combination. Normalized activities are presented as fold activation (means ± SEM, n = 3) relative to control values.

 


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  		FIG. 3. The putative AP1 site at –71 plays a key role in the superactivation of the boIFNT promoter relative to other AP1 sites present further upstream. A, Diagrammatic representation of the –1675 luc reporter and the locations of its putative AP1 sites (left). A cartoon of the mutated AP1 site (µAP1) is shown (right). B, The reporters were transfected either alone or in combination with expression plasmids for Ets-2-T72 (black bars), Ets-2-A72 (gray bars), and activated Ras (underlined). The MEK1/2 inhibitor PD98059 (40 µM) was added to some cultures after transfection (hatched bars). Normalized luc activities are presented as fold activation (means ± SEM, n = 3) relative to control values.

 


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  		FIG. 4. CSF-1 stimulates IFNT promoter activity in 3T3 cells expressing the CSF-1 receptor c-fms. A, Seven different reporter constructs, –1675 luc (wt and µAP1), –457 luc (wt, µAP1, and µEts), and –126 luc (wt and µAP1), were transfected into 3T3 cells stably transfected with c-fms. After transfection, the cells were cultured either in the presence of 0.1 µg/ml CSF-1 for 48 h (black bars) or in its absence (white bars). CSF-1 significantly (**, P < 0.01) increased luc reporter activity in cells transfected with all three wt constructs. B, Three different reporters [–1675 luc, –457 luc, and –126 luc (wt only)] were transfected into 3T3 cells expressing a form of c-fms with a Phe substituted for Tyr-809 (Y809F). After transfection, cells were treated as in Fig. 4AGo. No differences were observed between any treatments. C, Activation of the wt CSF-1 (c-fms) receptor leads to phosphorylation of MEK1/2. Relative concentrations of phosphorylated and total MEK1/2 (left panels) and phosphorylated Ets-2 (T72) (right panel) were analyzed by Western blotting of cell extracts (8 µg protein) from 3T3 cells expressing either wt or Y809F c-fms, cultured in presence (+) or absence (–) of 0.1 µg CSF-1 for 1.5 h. Mobilities of molecular weight markers (Mrx 10–3) are shown alongside each gel. The relative amounts of phosphorylated Ets-2 in the c-fms-positive and -negative cells in response to CSF-1 treatment are compared.

 

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  		TABLE 1. The amounts of DNA (micrograms) used in each transfection are listed

 
Cells were cultured for approximately 20 h in presence of transfection reagents and washed with PBS, and complete culture media containing 10% FBS were added. After 24 h, the culture media were replaced with media containing 0.5% FBS and incubated for an additional 24 h. In the inhibitor studies, 40 µM PD98059 (Cell Signaling Technology, Beverly, MA) was added 4 h after transfection. Reporter activities present in cell extracts were measured 48 h after transfection (18).

NIH3T3 cells expressing the wild-type (wt) human colony-stimulating factor-1 (CSF-1) receptor, c-fms, (34), and/or a mutant form with a point mutation at tyrosine-809 (Y809F c-fms) (35) were gifts from Dr. Martine Roussel and Dr. Charles Sherr of St. Jude’s Children’s Hospital (Memphis, TN). Purified recombinant human CSF-1 was purchased from PeproTech (Rocky Hill, NJ). Cells were provided with 0.1 µg CSF-1 per milliliter immediately following transfection. Following the change of media at 24 h, the cells were restimulated with 0.04 µg/ml CSF-1 for 6 h before cell harvest (36). Individual transfections were performed in triplicate and repeated two or three times.

Western blot analyses
Cells were extracted in Passive Lysis Buffer (Promega). After centrifugation to remove particulate material, protein in the extracts [8 µg for phospho-MAPK kinase (MEK)1/2 and phospho-Ets-2; 1.6 µg for total-MEK1/2] was analyzed by SDS-PAGE. Protein in the gels was transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). Phosphorylated MEK1/2 proteins at Ser217/221 were identified by using phospho-MEK1/2 antibody (diluted 1:1,000; Cell Signaling Technology, catalog no. 9121). Total MEK1/2 proteins were assessed on an identical blot by means of MEK1/2 antibody (diluted 1:1000; Cell Signaling Technology, catalog no. 9122). Ets-2 protein, specifically phosphorylated at Thr72, was detected by using an affinity-purified anti-phospho-Ets-2 antibody (24, 31) at a dilution of 1:1000. Immune complexes were detected with alkaline phosphatase-conjugated antirabbit IgG, diluted 1:10,000, which was used in conjunction with the Western-Star chemiluminescence kit from Applied Biosystems (Bedford, MA). The band intensities of Thr72-phosphorylated Ets-2 were measured using the Kodak Electrophoresis Documentation and Analysis System 290 (Eastman Kodak, Rochester, NY) as the sum of the background subtracted pixel values in the band rectangle.

EMSA
NIH3T3 cell nuclear extracts were prepared with Nuclear Extract Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions. Annealed oligonucleotides –76/–58 IFNT (Fig. 5AGo), –76/–58 µAP1 (Fig. 5BGo), and the consensus AP1 oligonucleotide (see Fig. 5Go for sequences) were labeled with [{gamma}-32P] ATP as described in the Promega kit (Gel Shift Assay System). Reaction mixtures (10 µl) included 5 µg nuclear extract, plus 10 fmol DNA probe (22,000~30,000 cpm). Precise procedures for EMSA have been described previously (17). For competition-binding assays, the indicated competitor DNA (175-fold molar excess, 1.75 pmol) was added before incubation with labeled probe. For supershift assays, the indicated antibody reagents from Santa Cruz Biotechnology (Santa Cruz, CA) (0.2 µg anti-c-Jun, sc-1694; 0.2 µg anti-AP2{gamma}, sc-8977; 0.2 µg anti-c-Fos, sc-52; 1 µg anti-pan-Jun, sc-44X; 1 µg pan-Fos, sc-413X) were added to the mixture before the incubation with labeled probe.



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  		FIG. 5. EMSA analysis of the sequence encompassing the putative AP1 site at –71/–58 of the boIFNT gene. Top, Sequences of the probes used in EMSA: A, wt 19-bp probe; B, mutated probe. Panel I, radiolabeled duplex probes (a, 22,224 cpm; b, 30,710 cpm) were incubated with 5 µg 3T3 cell nuclear extracts. Binding products were subject to electrophoresis on a 4% polyacrylamide gel. Panel II, formation of the wt DNA-protein complex was examined by competition analysis with a 175-fold molar excess of unlabeled duplex probes a and b, and by supershift analysis following addition of anti-c-Jun, anti-AP2{gamma}, and anti-c-Fos antibodies (0.2 µg) to the reaction mixture. Panel III, formation of wt DNA-protein complexes was examined by supershift analysis following addition of anti-pan-Fos and anti-pan-Jun antibodies (1 µg), and by competition analysis with a 175-fold molar excess of consensus AP1 binding site duplex DNA. Panel IV, A 21-bp AP1 consensus radiolabeled duplex probe (10 fmol, 18,430 cpm) was incubated with 5 µg 3T3 cell nuclear extract. The effect of addition of 0.2 µg anti-c-Jun antibody to the reaction mixture caused supershift (shown by arrow).

 
Data analysis
Data are presented as the mean ± SEM. Data were analyzed by one-way ANOVA followed by a Dunnett’s multiple comparison test to compare selected pairs of experimental groups (Fig. 4AGo) or t test for the paired groups (Fig. 4BGo) with the PRISM statistical analysis software (version 4) (GraphPad Software Inc., San Diego, CA). Differences of P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Potential role of the AP1 binding site (–71 to –64) adjacent to the Ets binding site at –79 to –70 in the superactivation of IFNT promoters
Many cellular genes, including that for uPA, contain an RRE comprised of binding sites for both Ets and AP1 (19). Figure 1AGo provides a comparison of the nucleotide sequences of the RRE in murine uPA with the established regulatory region of IFNT (–77 to –64). The IFNT control region sequence contains an eight-base motif immediately adjacent to the core Ets binding motif GGAA, which corresponds at six of its eight bases to a consensus AP1 binding sequence (37). On the other hand, the AP1 site of uPA has a one-base insertion when compared with the consensus and closely resembles a cAMP-response element (TGACGTCA).

To examine whether the IFNT control region exhibits the typical behavior of an RRE, a transfection experiments were conducted in mouse 3T3 fibroblasts, which were selected for use because the basal Ras-MAPK signal transduction pathway is not fully activated in this cell line (31). Accordingly, the effects of a transfected activated Ras construct on reporter gene activity can be assessed more easily than in a cell line in which Ras is constitutively active, such as choriocarcinoma cells. As anticipated from the experiments of others (30), a combination of Ets-2 and activated Ras transfected into 3T3 cells up-regulated the uPA promoter approximately 16-fold, whereas either protein alone had only modest effects (~1.5-fold with Ets-2 and ~3-fold with activated Ras; Fig. 1BGo). A comparable outcome was noted with the IFNT promoter (Fig. 1CGo). Although Ets-2 and activated Ras individually increased reporter gene activity only 3- to 4-fold, in combination they increased expression from the IFNT promoter over 50-fold. These data show that up-regulation of transcriptional activity from the IFNT promoter in 3T3 cells is dependent upon the combined action of Ets-2 and activated Ras. By analogy with the uPA control region, we conclude that the putative AP1 adjacent to the Ets-2 binding site plays a role in controlling expression from the IFNT promoter and that –126 IFNT contains a robust RRE.

Essential nature of the putative AP1 site at –71 for superactivation of the –126 IFNT promoter by Ras and Ets-2
To examine whether the putative AP1 site (TGAGAGA) adjacent to the Ets-2 binding sequence within the –126IFNT control region was implicated in the superactivation of the wt –126IFNT by Ras and Ets-2, this sequence was mutated at three bases (TTCTAGA) (Fig. 1AGo), and the mutated construct (µAP1; illustrated in Fig. 2AGo) cotransfected with the Ets-2 and Ras expression constructs into 3T3 cells. As shown in Fig. 2BGo, basal expression of luc expression from µAP1 was slightly reduced (28%) compared with that observed with the –126 wt. The mutated construct, like the wt, responded modestly to Ets-2 or Ras overexpression, whereas the effect of a combination of Ras and Ets-2 was reduced approximately 80% relative to the control. When the Ets-2 binding site was mutated (–126 µEts; see the sequence shown in Fig. 1AGo and illustrated in Fig. 2AGo), the reporter construct became almost totally unresponsive to a combination of Ets-2 and Ras. This experiment emphasizes the preeminent role of Ets-2 in controlling the transcriptional activity of the IFNT promoter. In addition, it is evident that the putative AP1 sequence adjacent to the Ets-binding site is required for full superactivation of –126 wt. We conclude that the sequence encompassing the Ets binding site and the putative AP1 binding site, i.e. bases –79 to –65, contains an RRE, as inferred from Fig. 1AGo.

Phosphorylation of Thr72 on Ets-2 contributes to the superactivation of the IFNT promoter through its RRE
Ras generally regulates the activity of Ets-2 by targeting a MAPK phosphorylation site at Thr72 on the transcription factor (24, 25, 30). This threonine as well as the surrounding sequences are well conserved in ovine Ets-2 (17) (Fig. 6BGo). Mutation of this site abolishes responsiveness to activated Ras cotransfection (25, 30). To test whether the Ras/Ets-2 superactivation of the –126IFNT control region involves phosphorylation of this threonine residue, 3T3 cells were transfected with the –126 luc reporter and either wt Ets-2 or a form in which Thr72 had been mutated to alanine (Ets-2-A72) in the presence or absence of activated Ras expression (Fig. 2BGo). Ets-2A72 alone provided a slight (2.4-fold) increase in reporter activity. A combination of activated Ras and the mutated Ets-2 (Ets-2A72) expression in the cells increased reporter expression 16-fold relative to the control, a value only about one fifth of that observed with the Ets-2-T72 and Ras. These data suggest that mutation of T72 reduces but does not abolish the superactivation of the –126IFNT reporter by the combination of Ets-2 and activated Ras expression.



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  		FIG. 6. A model for IFNT gene regulation through the Ras/MAPK signaling pathway. A, Many activated receptor tyrosine kinases (RTKs) stimulate Ras/MAPK signaling, which can lead to the phosphorylation of downstream targets, including Thr72 of Ets-2. The wt IFNT promoter contains a putative bipartite Ets/AP1 enhancer sequence that responds either to activation of the RTK after it binds ligand or to overexpression of Ets-2 and activated Ras (I). When a mutant form of Ets-2 (Ets-2-A72) is overexpressed, the full superactivation effects are not observed (II). If the AP1-like binding site adjacent to the Ets binding site is mutated, the superactivation effects are also reduced (III). Similarly, if the Ets binding site is mutated, basal activity is reduced and the promoter fails to respond to Ras/MAPK signaling (IV). B, Comparison of the amino acid sequences surrounding the putative kinase substrate region of human and ovine Ets-2. The conserved Thr72, a MAPK site, is boxed for the sequences for both species.

 
The AP1 site at –71 plays an exclusive role in the superactivation of the long –1675 IFNT promoter
This laboratory previously reported the sequence of –1675 bp of 5' flanking region from the bovine IFNT1 gene (referred as BTP-1.8) (32). An analysis of this region with the MatInspector V2.2 (http://transfac.gbf.de) software program revealed the presence of several AP1-like sites in addition to the one beginning at –71 bp. They are TGACTCA at –867 of the top strand, TGACCCC at –695 of the bottom strand, TGACTGT at –603 (top), TGACTCT at –403 (top), TGACATA at –332 (top), and TGACAAA at –188 (top) (Fig. 3AGo).

To assess whether these additional AP1 sites contribute to the Ras responsiveness of the IFNT promoter, we compared reporter activities from a –1675 wt and a –1675 µAP1 mutant. The latter was mutated only at the –71 AP1 site (as illustrated in Fig. 1AGo), whereas the other potential AP1 sites were left intact (Fig. 3AGo). Ets-2T72 up-regulated the –1675 wt reporter 8.5-fold, whereas Ras and Ets-2 together provided a 108-fold activation relative to basal activity. When the AP1 site at –71 was mutated (–1675 µAP1), basal activity was reduced (~25%) relative to the wt reporter. When coexpressed with Ras and Ets-2-T72, luc expression from the mutated gene was increased, but only to about 9% of the activity observed with the –1675 wt reporter in the presence of Ets-2-T72 plus Ras. The experiment demonstrates the importance of the putative AP1 site at –71 relative to the other potential AP1 sites present in the –1675 gene control region and the dominant nature of the Ras/Ets-2 downstream enhancer. Importantly, treatment with selective MEK1/2 inhibitor, PD98059, reduced the Ras/Ets-2 superactivation of the –1675 wt reporter by over 80% (Fig. 3BGo), illustrating the likely involvement of the MAPK pathway in the activation of the IFNT promoter.

CSF-1 stimulates IFNT reporter activity in a 3T3 cell line expressing the CSF-1 receptor, c-fms
To explore the effects of a growth factor that stimulates the MAPK pathway, we used a 3T3 cell line that had been stably transfected with the CSF-1 cognate receptor, c-fms. When CSF-1 was provided in the culture medium, reporter activities from the transfected wt IFNT reporters, –1675, –475, and –126, were significantly stimulated (2.1-, 2.9-, and 2.2-fold, respectively; P < 0.01) in the wt c-fms cells (Fig. 4AGo). By contrast, CSF-1 failed to exert a significant effect on any of the three reporters in a comparable cell line (Y809F c-fms) (Fig. 4BGo), in which an autophosphorylation site on c-fms at tyrosine 809 residue involved in ligand-dependent mitogenic signal transduction had been mutated to a phenylalanine (35).

When the AP1 site at –71 was mutated in the –1675, –475, and –126 gene control regions (Fig. 4AGo) in the wt c-fms expressing cells, basal activities were reduced, and all three promoters failed to respond to CSF-1 treatment (Fig. 4AGo). Mutation of the Ets site (shown here for only the –457 reporter) led to even lower basal activity and negated the stimulatory response of CSF-1.

The up-regulation of the wt IFNT reporters by CSF-1, although significant, was relatively modest (only 2.2-fold for the –126 wt reporter). This response to CSF-1 could be doubled to 4.2-fold if Ets-2 was overexpressed by transfection (0.1 µg Ets-2 expression plasmid per 60-mm dish) before addition of CSF-1 to the culture medium (data not shown). In other words, the concentration of Ets-2 in the cells limited the ability of CSF-1 to up-regulate IFNT transactivation. The enhanced CSF-1 effects by the Ets-2 expression plasmid on reporter gene expression were not observed with either the –126 µAP1 or the –126 µEts mutated reporter constructs (data not shown). We conclude that the –79/–71 Ets/AP1 site in the IFNT control region is targeted after CSF-1 engages its cognate receptor.

To characterize the intracellular signaling pathway used by CSF-1 and its receptor, the concentration of phosphorylated MEK1/2 was analyzed by Western blot analysis. Cell extracts prepared from 3T3 cells that expressed wt c-fms increased their concentration of phosphorylated MEK1/2 in response to CSF-1 (Fig. 4CGo). In contrast, phosphorylated MEK1/2 concentration did not change in cells expressing the mutated receptorY809F c-fms.

Thr72 of Ets-2 is generally considered to be the downstream target for the MAPK pathway (24, 25, 30). Western blotting with an immunopurified antibody preparation raised against the phosphorylated Thr72 epitope of Ets-2 (24, 31) demonstrated only a slight (28% in pixel values) increase in Ets-2 phosphorylation at Thr72 in the c-fms-expressing cells after they had been exposed to CSF-1 (Fig. 4CGo, bottom panel). As expected, no increase was observed in the 3T3 cells that expressed Y809F c-fms.

Neither Jun nor Fos bind to the AP1 site at –71 of the IFNT control region
The AP1 transcription factors are comprised of members of the Fos and Jun protein families and, like Ets-family members, are activated by Ras-mediated signaling (38). Nuclear extracts from 3T3 cells were, therefore, subjected to EMSA to characterize the nature of the proteins that bound to the AP1 site at –71 on the IFNT control region. A DNA fragment from –76 to –58 of boIFNT1 containing the AP1 site (Fig. 5AGo) and a mutated AP1 oligonucleotide (Fig. 5BGo) were used as probes in the assay. The wt probe (a, 10 fmol, 22,224 cpm) formed at least two complexes with 3T3 cell nuclear extracts, whereas the mutated probe b, which had a higher specific radioactivity (10 fmol, 30,710 cpm) than probe a, failed to form any complexes (gel I; Fig. 5Go). The intensity of the wt DNA-protein complex was reduced in the presence of a 175-fold molar excess of unlabeled probe a, but not by the addition of mutated competitor b (gel II; Fig. 5Go). This experiment showed that the putative AP1-binding sequence formed specific associations with proteins present in nuclear extracts of 3T3 cells.

However, a polyclonal antibody (0.2 µg; sc-1694; Santa Cruz Biotechnology) raised against the N-terminal region of the c-Jun protein failed either to supershift or to prevent association of the nuclear protein complexes formed with probe a (gel II; Fig. 5Go), despite the ability of an equal volume of this antiserum to supershift a complex formed with a consensus AP1 DNA probe (core sequence: TGAGTCA) (gel IV; Fig. 5Go). An equivalent volume of antiserum against a nonrelevant transcription factor (anti-AP2{gamma}) had no effect on the complex formed between probe a and proteins in 3T3 cell nuclear extracts. Equally as surprising as the negative result with the c-Jun antiserum discussed above was the failure of an anti-c-Fos antiserum (sc-52; Santa Cruz Biotechnology) either to supershift or to prevent association of the complex formed with probe a (gel II; Fig. 5Go). These results showed that the protein factor that bound the putative AP1 binding site in 3T3 cells was most probably not the expected c-Jun/c-Fos heterodimer.

To determine whether other Jun or Fos family members might be present in the complex formed with probe a, attempts were made to alter the mobility of the complex by the addition of anti-pan-Jun (sc-44X; Santa Cruz Biotechnology), which is reactive against c-Jun, Jun B, and Jun D; and anti-pan-Fos (sc-413X; Santa Cruz Biotechnology), which is reactive against c-Fos, Fos B, Fra-1, and Fra-2 (gel III; Fig. 5Go). Neither of these broadly specific reagents affected the DNA-protein complexes. Furthermore, a 175-fold molar excess of unlabeled oligonucleotide representing a consensus AP1 binding site failed to dissociate the wt DNA-protein complex (gel III; Fig. 5Go).

Together, these experiments show that the AP1-like site in the IFNT promoter, although Ras-responsive and having a near to consensus binding sequence, does not bind a typical AP1 protein.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The experiments described here are entirely consistent with the model shown in Fig. 6AGo, which suggests that a factor or factors present in maternal uterine secretions bind to cognate receptors on the surface of trophectoderm, activates an effector pathway that involves a Ras-GTP, MEK1/2, and MAPK, and ultimately targets the IFNT genes through activation of the transcription factor Ets-2. A classic inhibitor of MEK1/2, PD98059, largely reverses the ability of activated Ras and Ets-2 to up-regulate the IFNT promoter (Fig. 3Go), thereby further implicating this particular enzyme cascade. The pathway shown in Fig. 6AGo, where transcription factors of the Ets family are MAPK substrates, regulates cell proliferation in some cells and cellular differentiation in others (27, 28, 29). In the case of Ets-2, the substrate for the pathway is usually considered to be Thr72, whose phosphorylation leads to enhanced transcriptional activation of Ets-2-regulated genes. This threonine residue and the peptide sequence surrounding it are highly conserved across mammalian species (Fig. 6BGo). We have previously demonstrated increased phosphorylation of Thr72 on Ets-2 in response to activated Ras expression in 3T3 cells when studying the regulation of the human chorionic gonadotropin ß subunit gene (31). When Thr72 is mutated to Ala, the ability of Ets-2 to respond to MAPK activation is diminished, and transcriptional activity of the IFNT promoter is curtailed (Fig. 6AGo, I and II). In addition to effects on Ets-2-regulated genes, Ras-mediated signal transduction cascades frequently modulate the activities of genes controlled by the Fos/Jun (AP1) family of transcription factors. Not unexpectedly, enhancers that contain both Ets and AP1 binding sites, such as the ones for uPA and matrix metalloproteinases (19, 21, 30), appear to be particularly responsive to Ras-mediated up-regulation (Fig. 1Go). As predicted, mutation of the AP1 site on the IFNT control region (Fig. 6AGo, III) largely abolished the superactivation by Ras and Ets-2 (Figs. 2Go and 3Go) and caused a failure to respond to CSF-1 (Fig. 4Go), thereby indicating that the putative AP1 binding site also plays a role as a downstream target site for the Ras/MAPK signaling pathway. Mutation of the Ets binding site markedly reduced basal promoter activity and led to a failure of the promoter to respond to Ras/MAPK signaling and to CSF-1 (Figs. 2Go and 4Go). This binding site is therefore essential for MAPK up-regulation as well as basal expression of the IFNT gene (Fig. 6AGo, IV). Together, our data show that IFNT expression is under the control of the Ras/MAPK signal transduction cascade and hence likely to be responsive to factors present in maternal uterine secretions that trigger this cascade. Such a process of control operating via extracellular factors likely allows the production of IFN-{tau} by the conceptus to remain coordinated with the physiological state of the mother, whose production of uterine secretions is under the control of steroid hormones.

The situation is undoubtedly more complex that that diagrammed in Fig. 6AGo. First, mutation of Thr72 fails to ablate Ras-mediated activation of the IFNT promoter as effectively as the inhibitor PD98059 (Fig. 3Go). Conceivably, there are one or more additional phosphorylation sites on the protein that contribute to its ability to up-regulate the IFNT promoter. Indeed, our data show that phosphorylation of Thr72 is only barely increased when the cells respond to CSF-1 (Fig. 4CGo). A second puzzle relates to the nature of the transcription factor present in 3T3 cells that binds at the AP1-like site (Fig. 5Go). These proteins do not appear to be among the better-characterized Fos or Jun family members and could be completely unrelated transcription factors. A future goal will be to define the proteins in nuclear extracts of conceptuses that associate with this apparent AP1 site, because these may not be the same as those that bind the site in 3T3 cells. A final puzzle is to reconcile our data with those of others (39), who have implicated an upstream AP1 site (–654 to –555) in the transcriptional control of an ovine IFNT promoter. This distal element is in a part of IFNT genes that is poorly conserved across species and is not represented in the bovine gene control region used in our work. Indeed, our experiments, performed here and earlier (17, 18, 32), have consistently indicated that these far-upstream regions of the gene are not necessary for IFNT expression. Moreover, only the most proximal AP1-like site appears to contribute to the responsiveness of the bovine IFNT promoter to Ras (Fig. 3Go) and CSF-1 (Fig. 4Go).

Our choice of CSF-1 as a possible regulatory factor for IFNT expression was largely empirical and primarily based on our access to 3T3 cell lines that had been engineered to express either the wt human CSF-1 receptor c-fms, or a mutant form of c-fms, whose signal transduction capacity had been crippled through mutation of a critical tyrosine residue (Fig. 4Go). Because CSF-1 is known to activate the Ras/MAPK pathway (40), we hypothesized that the factor would up-regulate the IFNT promoter and increase the amount of phosphorylated MEK1/2 and Ets-2 only in those cells that expressed functional receptors. These predictions were entirely borne out (Fig. 4Go) and provided additional evidence that the IFNT promoter can be regulated through external growth factors. Whether CSF-1 is the only or even the main factor that causes IFN-{tau} expression to increase at the time of trophoblast elongation in vivo is unknown. CSF-1 is expressed in endometrium of cattle, with expression increasing markedly between d 14 and 17 after previous estrus (41), at the time that IFN-{tau} production by the conceptus is massively up-regulated (11). In addition, its cognate receptor, c-fms, is present on bovine trophoblast at about the same developmental stage of pregnancy (42). On the other hand, cow reproductive tract secretions likely contain other factors, such as granulocyte-macrophage CSF (43) and IGFs (44, 45), both of which have been implicated in increasing the production of IFN-{tau} by ovine conceptuses (46, 47), although the results with granulocyte-macrophage CSF have been disputed (48). In addition, we have preliminary evidence that the IFNT promoter might also respond to growth factors and hormones that operate through the protein kinase A signal transduction pathway (49). Whatever protein factors and signaling pathway are eventually implicated, these maternal products clearly provide a means for the mother to exert some control over conceptus IFN-{tau} expression and hence the outcome of her pregnancy.


   Acknowledgments
 
The authors thank Dr. Michael Ostrowski for uPA reporter construct and Drs. Martine Roussel and Charles Sherr for 3T3-fms cells.


   Footnotes
 
This work was supported by National Institutes of Health Grant HD21896.

Abbreviations: AP1, Activator protein 1; CL, corpus luteum; CSF, colony-stimulating factor; FBS, fetal bovine serum; IFN, interferon; IFNT, IFN-{tau} genes; luc, luciferase; MEK1/2, MAPK kinase; RRE, Ras-responsive enhancer; uPA, urokinase-type plasminogen activator; wt, wild-type.

Received May 12, 2004.

Accepted for publication June 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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