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Molecular Endocrinology, doi:10.1210/me.2007-0300
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Molecular Endocrinology 22 (2): 331-343
Copyright © 2008 by The Endocrine Society

Combinatorial Roles of Protein Kinase A, Ets2, and 3',5'-Cyclic-Adenosine Monophosphate Response Element-Binding Protein-Binding Protein/p300 in the Transcriptional Control of Interferon-{tau} Expression in a Trophoblast Cell Line

Padmalaya Das, Toshihiko Ezashi, Rangan Gupta and R. Michael Roberts

Division of Animal Sciences (P.D., T.E., R.M.R.) and Veterinary Pathobiology (R.G., R.M.R.) and Biochemistry (R.M.R.), University of Missouri-Columbia, Columbia, Missouri 65211

Address all correspondence and requests for reprints to: R. Michael Roberts, University of Missouri-Columbia, 240b Christopher S. Bond Life Sciences Center, 1201 East Rollins Street, Columbia, Missouri 65211-7310. E-mail: robertsrm{at}missouri.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In ruminants, conceptus interferon-{tau} (IFNT) production is necessary for maintenance of pregnancy. We examined the role of protein kinase A (PKA) in regulating IFNT expression through the activation of Ets2 in JAr choriocarcinoma cells. Although overexpression of the catalytic subunit of PKA or the addition of 8-bromo-cAMP had little ability to up-regulate boIFNT1 reporter constructs on their own, coexpression with Ets2 led to a large increase in gene expression. Progressive truncation of reporter constructs indicated that the site of PKA/Ets2 responsiveness lay in a region of the promoter between –126 and –67, which lacks a cAMP response element but contains the functional Ets2-binding site and an activator protein 1 (AP1) site. Specific mutation of the former reduced the PKA/Ets2 effects by more than 98%, whereas mutation of an AP1-binding site adjacent to the Ets2 site or pharmacological inhibition of MAPK kinase 2 led to a doubling of the combined Ets2/PKA effects, suggesting there is antagonism between the Ras/MAPK pathway and the PKA signal transduction pathway. Although Ets2 is not a substrate for PKA, lowering the effective concentrations of the coactivators, cAMP response element-binding protein-binding protein (CBP)/p300, known PKA targets, reduced the ability of PKA to synergize with Ets2, suggesting that PKA effects on IFNT regulation might be mediated through CBP/p300 coactivation, particularly as CBP and Ets2 occupy the proximal promoter region of IFNT in bovine trophoblast CT-1 cells. The up-regulation of IFNT in the elongating bovine conceptus is likely due to the combinatorial effects of PKA, Ets2, and CBP/p300 and triggered via growth factors released from maternal endometrium.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INTERFERON-{tau} (IFNT) IS regarded as a crucial signal from the conceptus that triggers early maternal responses to pregnancy in ruminants (1). One role of IFNT is to prolong the lifespan of the corpus luteum (CL) by suppressing the pulsatile release of the luteolysin, prostaglandin F2{alpha} from the uterine endometrium, thereby preventing a return to ovarian cyclicity (2, 3, 4, 5). The production of large amounts of IFNT by the still unattached, early elongating, conceptus is believed to be critical to the establishment of pregnancy by modulating the uterine output of the luteolytic factor, prostaglandin F2{alpha} and adjusting gene expression locally in the endometrium to accommodate the growing conceptus (6, 7).

The genes encoding the IFNT proteins (IFNT) differ most markedly from other type I IFN in their lack of inducibility in response to virus, their localized transcription in trophectoderm before firm attachment of the conceptus to the uterine wall, and the high rate and persistence of their expression over a critical period of pregnancy when progesterone production by the CL must be maintained if the conceptus is to survive (1, 2, 3, 4, 5). IFNT is the major secretory product of conceptus between d 13 and d 21 of pregnancy in sheep (8, 9) and between d 14 and d 24 in cattle (10, 11), but a low rate of production can be measured as the blastocyst forms (12, 13). However, there is a marked increase in IFNT synthesis coinciding with the morphological transition of the blastocyst from a spherical to a filamentous form, rather than a strict correlation with day of pregnancy (14, 15).

Hormones and growth factors secreted by the maternal endometrium may be required for the optimal production of IFNT, because supplementation of culture medium with uterine flushing from ewes in the late luteal phase of estrous cycles increases the production of IFNT by cultured, in vitro-produced blastocysts (13, 16, 17). Moreover, when endometrial glands fail to form in ewes, conceptuses release lower amounts of IFNT (18), suggesting that maternal secretions produced by the glands might be responsible for the up-regulation of production. When uterine secretions in bred ewes are stimulated after mating by progesterone administration, conceptus IFNT production is increased more than 50-fold at d 12 of pregnancy compared with the controls (19). Similarly, early progesterone supplementation promotes IFNT production by conceptuses in the uterine tract of the cow (20). We (16) and others (21, 22, 23) have suggested that factors present in maternal glandular secretions trigger these events by binding their cognate receptors on trophectoderm and activating key intracellular signaling pathways For example, the growth factor, colony-stimulating factor-1, which operates through the Ras/MAPK pathway, is able to up-regulate IFNT promoter activity (16). On the other hand, uterine secretions probably contain a complex mixture of growth factors and hormones, and the increase in IFNT production during the period in which the trophoblast elongates is massive and sustained, raising the possibility that more than a single hormone and intracellular signaling pathway are involved.

Considerable evidence has accumulated to suggest that the transcription factor, Ets2, through its interaction with the proximal promoter region of IFNT, is the key transcription factor governing IFNT expression in trophectoderm (24, 25). It is, for example, a target for the Ras/MAPK pathway discussed above and, as such, probably mediates the action of hormones, such as colony-stimulating factor-1, on IFNT gene transcription (16). In the case of ovine trophoblast Kunitz domain protein-1, which has an almost identical expression pattern to IFNT during ovine conceptus development, Ets2 interacts with a second transcription factor, CCAAT enhancer binding protein-β to transactivate the trophoblast Kunitz domain protein-1 gene (26). Ets2 is essential for full placental development in the mouse, with deletion of the ets2 gene leading to embryonic mortality before d 8.5 of embryonic development (27). Ets factors, including Ets2, can up-regulate the promoters of several genes known to be expressed in trophoblast of species other than cattle and sheep, including CGA (hCG{alpha}) (28) and CGB5 (hCGβ) (29, 30), CYP11A1 (31), MMP1 (32), MMP3 (33, 34, 35), urokinase-type plasminogen activator (PLAU) (36, 37), and MMP9 (38), rat placental lactogen II (39), prolactin related protein (40), and porcine pregnancy associated glycoprotein 2 (PAG2) (41), suggesting that Ets2, and probably other key transcription factors, play dual roles. One is in the regulation of genes encoding specific trophoblast products; the other is ensuring proper functional differentiation of the trophoblast (42, 43).

The cAMP signaling system is another key regulator of trophoblast differentiation and function (44). Mononuclear cytotrophoblast cells aggregate and fuse to form multinucleated synctiotrophoblasts in the human placenta, and this behavior may be controlled by cAMP (45). Single cytotrophoblast-like cells of the human choriocarcinoma (BeWo) cell line fuse and undergo extensive morphological differentiation to yield syncytia in the presence of the cAMP analog, forskolin, (45, 46). Finally, although only about 5% of JAr cells normally produce measurable human chorionic gonadotropin (hCG), the addition of 8-bromo-cAMP increases the number of hCG-producing cells several fold, as well as stimulating hCG synthesis (47, 48). These data suggest that the expression of the IFNT, like many other genes associated with trophoblast function, might be regulated by the cAMP/protein kinase A (PKA) signal transduction pathway and hormones and growth factors that activate that pathway.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Synergistic Effects of Ets2 and PKA on IFNT Promoter Expression
First we investigated the effect of the constitutively active, {alpha} catalytic subunit of PKA on the expression of the bovine IFNT1 promoter in JAr choriocarcinoma cells. The bovine –126 IFNT1 promoter, which includes the proximal enhancer sequence (–91 to –61), is sufficient to drive basal expression (49), as well as Ets2-mediated up-regulation of luciferase (luc) reporter activity (25). This promoter-reporter construct was modestly up-regulated (usually between 2- to 4-fold) by ectopic expression of PKA (Fig. 1AGo). Under the same transfection conditions, overexpression of Ets2 increased luc reporter activity from the –126 promoter approximately 58-fold, although this value can vary according to the plasmid preparation and cell culture parameters (data not shown). Cotransfection of the Ets2 expression construct with the activated PKA construct up-regulated the –126 IFNT reporter by about 500-fold (Fig. 1AGo). A qualitatively similar, but lower up-regulation of reporter activity was observed when JAr cells were exposed to 0.5 mM 8-bromo-cAMP, rather than to ectopically expressed PKA (Fig. 1BGo). When coexpressed in a second choriocarcinoma cell line, JEG3, Ets2 and PKA provided a dramatic, synergistic increase on reporter activity of even larger magnitude (~2000-fold) than that noted in JAr cells (Fig. 1CGo). Curiously, PKA alone had a much larger effect in JEG3 than in the JAr cells (~200-fold vs. 2-fold). The reason for this difference is not clear but may relate to the relative basal activities of the PKA signal transduction pathway in the cells.


Figure 1
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  		Fig. 1. Coexpression of Ets2 and PKA Synergistically Up-Regulates the IFNT Promoter in Choriocarcinoma Cells

A, The –126luc promoter was cotransfected with expression plasmids for Ets2 and constitutively active catalytic subunit of PKA alone (PKA) or in combination into JAr cells. B, The –126luc promoter was cotransfected with the Ets2 expression plasmid into JAr cells. After 24 h, cells were treated with 0.5 mM 8-bromo-cAMP for 36 h before collection of cell lysates. C, The –126luc promoter was cotransfected with expression plasmids for Ets2 and PKA alone or in combination into JEG3 cells. In all experiments, the luc activity was normalized relative to β-galactosidase activity from the reference reporter, pRSVLTR-βgal, and the normalized luc activities are presented relative to the activity of –126luc from nontreated cells (means ± SEM; n = 3), with fold activation shown above each bar. If letters above bars are different, there was a significant effect of treatment (P < 0.05).

 
Deletion and Mutational Analysis of the IFNT Promoter
To define which region of the IFNT promoter was responsible for the ability of PKA and cAMP to act synergistically with Ets2 and provide the large up-regulation of luc reporter activity noted in Fig. 1Go, we progressively truncated the –126 IFNT promoter and cotransfected these shorter constructs either with the PKA expression plasmid (Fig. 2AGo) or with the combination of PKA/Ets2 (Fig. 2BGo) expression plasmids into JAr cells. In addition, we examined a series of longer promoter constructs to determine whether regions upstream of –126 were influenced by Ets2/PKA overexpression (Fig. 2Go, A and B). Basal luc activity from the boIFNT1 –457luc and –1675luc promoter constructs was up-regulated approximately 2-fold when coexpressed with the constitutively active catalytic subunit of PKA (Fig. 2AGo). Reporter activity from the three shorter constructs was quite low and varied between experiments. Although there was a tendency for ectopic PKA expression to increase reporter expression with all three promoter constructs, the differences observed were not significant (P > 0.05). It would appear that the entire IFNT upstream region lacks a strong PKA target sequence. The slight positive effects are possibly the results of nonspecific effects on cell metabolism rather than direct effects on the promoter. Cotransfection of Ets2 and PKA transactivated the –1675luc, –457luc, and –126luc promoters to similar extents, namely 445- and 450- and 501-fold, respectively (Fig. 2BGo). By contrast, the –67luc and –49luc promoters, which lack the Ets binding site, were much less responsive (a 93% decrease compared with –126luc) when Ets2 and PKA were coexpressed. This experiment suggests that the proximal enhancer region between –126 and –67 bp of the IFNT gene is involved in mediating PKA responsiveness, but only when Ets2 is overexpressed.


Figure 2
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  		Fig. 2. A Truncation Analysis of the IFNT Promoter to Define the Gene Control Region Responsible for the Activation of the IFNT Promoter by PKA

A, JAr cells were cotransfected with the –49, –67, –126, –457, and –1675luc promoters and either empty vector or the expression plasmid for the constitutively active catalytic subunit of PKA, and relative luc activities were measured (left scale and values above each bar). Values with asterisks indicate where the treatment (ectopic expression of PKA) had a significant effect on reporter expression from a particular promoter construct. A significant effect was only noted with the –457 and –1675 promoter constructs. B, JAr cells were transfected with –126, –457, –1675, –67, and –49luc promoters with and without the expression plasmids for Ets2 and PKA. In Fig. 2AGo, luc activities are compared with average luc activity of –49luc from cells nontreated with PKA. In Fig. 2BGo, luc activities are compared with averaged luc activity of –126luc from cells nontreated with Ets2 and PKA (means ± SEM; n = 3). Fold activation over the activity of –126luc from nontreated cells is shown above each bar. If letters above bars are different, there was a significant effect of treatment (P < 0.05). bIFNT, bovine IFNT.

 
Dependency of Ets2/PKA Synergism on the Kinase Activity of PKA
We next examined whether the IFNT promoter was up-regulated when Ets2 was coexpressed with an inactive form of PKA, which has a methionine substitution for a lysine in the ATP-binding region. As also observed in Figs. 1Go and 2Go, the overexpression of Ets2 and PKA synergistically up-regulated the IFNT promoter (Fig. 3AGo). Luc reporter activity was reduced approximately 93% when the mutant form of PKA was substituted for the wild-type enzyme. We also examined the effects of coexpressing an inhibitor of PKA, PKI. In its presence, the synergistic effect of Ets2 and PKA was markedly reduced (Fig. 3AGo). Together, these experiments suggest that the PKA-mediated enhancement of Ets2 effects require a catalytically active kinase.


Figure 3
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  		Fig. 3. Cooperative Transactivation of the IFNT Promoter by Ets2 and Activated PKA in JAr Cells is Dependent on the Kinase Activity of PKA but Not on the ERK1/ERK2 MAPK Pathway and the Presence of the Phosphorylatable Thr 72 (T72) of Ets2

A, The –126luc promoter was cotransfected with combinations of expression plasmids for Ets2, PKA, mutated PKA (mPKA), and a specific inhibitor of PKA (PKI). B, The –126luc promoter was cotransfected with expression plasmids for Ets2 and PKA in the presence and absence of the PKA inhibitor H89 and the MEK1/MEK2 inhibitor PD 98059 (PD). C, The –126luc promoter was cotransfected with expression plasmids for Ets2T72, Ets2A72, and PKA alone or in combination. D, The –126luc promoter was cotransfected with expression plasmids for Ets2T72, Ets2A72, and PKA in the presence and absence of the MEK1/2 inhibitor PD 98059 (PD). In all three experiments, the normalized luc activities are presented relative to control values (means ± SEM; n = 3), with fold activation shown above each bar. If letters above bars are different, there was a significant effect of treatment (P < 0.05).

 
As a further test of whether the PKA signal transduction pathway was involved in the transactivation of the IFNT promoter by Ets2, we tested a pharmacological inhibitor (H-89) of the pathway. PD98059, an inhibitor of the Ras/MAPK signal transduction pathway, which is also known to target Ets2 at Thr72 (16, 25, 50), was used as a control. Ets2 and PKA caused a major (469-fold) up-regulation of reporter activity (Fig. 3BGo). This stimulatory effect was inhibited approximately 93% by H-89, which binds to the ATP-binding site of the catalytic subunit of PKA. Surprisingly, the addition of MAPK kinase (MEK) inhibitor, PD98059, rather than acting as an inhibitor, almost doubled reporter gene activity (from 469-fold to more than 900-fold) (Fig. 3BGo). Again, it is clear that there is a requirement for PKA catalytic activity in the synergistic interaction of PKA and Ets2. In addition, it would appear that the Ras/MAPK pathway antagonizes the PKA-induced stimulatory effects.

The Effect of Mutating Thr 72 of Ets2 on the Synergistic Interaction of Ets2 and PKA
Thr72 represents a well-defined site for phosphorylation on Ets2 and is a downstream target for the Ras/MAPK signal transduction pathway (16, 25, 50). Here, we tested whether a form of Ets2 with Thr72 replaced with Ala (Ets2A), was as effective as wild-type Ets2 (Ets2T) in transactivating the –126luc promoter in the presence of PKA. The up-regulation of the promoter in the presence of the ectopically expressed mutant form of Ets2A was not significantly different from that observed with overexpressed control protein (510-fold vs. 600-fold) (Fig. 3CGo). Furthermore, the MEK inhibitor PD98059 increased transcription from the mutated Ets2A promoter as effectively as it did from the wild-type Ets2T promoter (Fig. 3DGo). This experiment demonstrates that Thr72 is not essential for the enhancement of Ets2 effects by PKA and, second, that this amino acid residue is probably not a target for PKA-catalyzed phosphorylation.

Role of the Activator Protein 1 (AP1) Site Adjacent to the Ets2-Binding Site in Ets2/PKA Synergistic Regulation of the IFNT Promoter
Transcription factors of the AP1 family have been implicated as downstream mediators for several signal transduction pathways, including the Ras/MAPK (51, 52) and the PKA pathways (53, 54). Accordingly, we examined the effects of mutating the AP1-binding site that lies adjacent to the Ets2-binding site in the –126luc promoter (Fig. 4AGo). As expected, the combination of Ets2 and PKA provided the usual large up-regulation (490-fold) of promoter activity compared with either factor on its own (43-fold and 5-fold, respectively) (Fig. 4BGo). Mutation of the Ets-binding site again caused a major drop in reporter activity (>98% compared with the wild-type construct). Unexpectedly, a mutation designed to disrupt the AP1-binding site doubled (to nearly 900 fold) reporter activity resulting from Ets2/PKA overexpression compared with the wild-type promoter (Fig. 4BGo). This effect was almost identical to that observed with PD98059 (Fig. 3Go, B and D). These data suggest that the presence of a factor, presumably an AP1 family member, normally occupying this site and targeted by the Ras/MAPK pathway, may have an inhibitory effect on the PKA-signaling pathway.


Figure 4
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  		Fig. 4. The Effect of Mutating Transcription Factor Binding Sites within the IFNT Proximal Promoter on Ets2/PKA Cooperative Effects on Reporter Gene Expression in JAr Cells

A, A diagrammatic illustration of the –126luc reporter containing the –126/+50 regulatory region. The positions of the binding sites for Ets2 and AP1 are marked. B, The –126luc promoter construct (–126WT) and its two mutated forms (–126mEts2 and –126mAP1) were cotransfected with expression plasmids for Ets2 and PKA alone or in combination. In both experiments, the normalized luc activities are presented relative to control values (means ± SEM; n = 3), with fold activation shown above each bar. If letters above bars are different, there was a significant effect of treatment (P < 0.05). bIFNT, Bovine IFNT; mAP1, mutant AP1; WT, wild type.

 
Ets2 Is Not a Direct Target of PKA Phosphorylation
The consensus target sites for PKA are RXS and RXXS (55). The Ets2 amino acid sequence provides two such potential PKA phosphorylation sites (RLS245 and RVPS310). We therefore tested whether truncated Ets2 constructs, some carrying these sites, others lacking them, were substrates for PKA in an in vitro assay. As a positive control, we employed ovine IFNT11, which had been engineered to carry a PKA target site at its COOH terminus (56). Products of the reactions were separated by SDS-PAGE and detected by autoradiography (supplemental Fig. 1, A and B, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).

In the initial experiment, we compared similar amounts of full-length Ets2 and IFNT11 as substrates for PKA in the in vitro reaction and employed a 15-min exposure to film to detect the radioactive bands. Whereas IFNT11 (Mr~18,000) was clearly a substrate for PKA, the incorporation of 32P into full-length Ets2 was very low (supplemental Fig. 1A), with a faint doublet (Mr ~58,000) visible on the gel. As expected, there was no incorporation of 32P in the absence of PKA. These data suggest that if Ets2 is a substrate for PKA, it is a very poor one.

To determine whether Ser245, Ser310, or both are targets for PKA, a series of truncations were employed (supplemental Fig. 1B). In the analysis of the reaction products, the gels were exposed to film for 1 h rather than 15 min to provide a more complete identification of reaction products. Several bands of radioactivity of similar molecular weight were detected in each lane, even though the substrate proteins, Ets2 truncations, encompassed a range of sizes. The bands in some lanes, e.g. for the amino acids 225–469 substrate, were much more prominent than in others. The most likely explanation for these data is that contaminating bacterial proteins present in trace quantities are the substrates for PKA rather than Ets2 fusion proteins. This likelihood is reinforced by the observation that one truncated form (Ets2 322–469), which lacks both the putative PKA phosphorylation sites, provided the same radioactive products as the other truncations. The variation between lanes probably reflects the relative amount of contaminating bacterial protein present. These observations strongly suggest that Ets2 is not a direct target for PKA phosphorylation.

CBP/p300 Acts in Association with Ets2 to Mediate the Effect of PKA
Here we examined the role of the coactivators cAMP response element (CRE)-binding protein (CREB)-binding protein (CBP)/p300 and the transcription factor CREB in the Ets2-mediated activation of IFNT gene by PKA. Contrary to a recent report (57), which indicated that CBP acted as a direct coactivator in the regulation of IFNT gene transcription, in our hands CBP suppressed both basal and Ets2-mediated up-regulation of the IFNT promoter (Fig. 5AGo). Overexpression of CREB had a similar suppressive action. Conceivably, CBP (and its homolog p300), as well as CREB, are not rate-limiting in JAr cells and, when overexpressed, have a slight squelching effect on transcription. Therefore, instead of overexpressing these potential activators of Ets2-mediated transcription, we attempted to reduce their effective concentrations in the cells.


Figure 5
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  		Fig. 5. The Coactivator CBP/p300 Influences the Ets2/PKA-Mediated Transactivation of the IFNT Promoter in Choricarcinoma Cells

A, The –126luc promoter was transfected into JAr cells with combinations of expression plasmids for Ets2, PKA, CBP, and CREB. Note that overexpression of CBP and CREB significantly suppressed the cooperative transactivation of the –126luc promoter by Ets2 and PKA. B, The –126luc promoter was transfected into JAr cells with combinations of the expression plasmids for Ets2, PKA, CBP, E1A 12S, and E1A 12S ({Delta}2–36). C, The –126luc promoter was transfected into JEG3 cells with combinations of expression plasmids for Ets2, PKA, CBP, E1A 12S, and E1A 12S ({Delta}2–36). In all three experiments, the normalized luc activities are presented relative to the activity of –126luc from nontreated cells (means ± SEM; n = 3), with fold activation shown above each bar. If letters above bars are different, there was a significant effect of treatment (P < 0.05).

 
To overcome the problem of excess endogenous CBP, we first made use of the adenoviral E1A 12S protein, which inhibits CBP/p300 interaction by binding to the same region of CBP/p300 as transcription factor IIB (58). When E1A 12S was coexpressed with the Ets2 and PKA expression vectors, luc activity from the –126 boIFNT1 promoter was reduced by approximately 50% (Fig. 5BGo). This reduction was not observed when a mutated form of E1A 12S possessing a deletion ({Delta}2–36), which removed the CBP/p300-binding region, was substituted for the wild-type protein. Similar results to those obtained in JAr cells were observed in JEG3 cells (Fig. 5CGo). Again CBP/p300 reduced luc reporter expression slightly, whereas E1A 12S had a significant inhibitory effect, presumably by reducing the effective basal concentration of CBP/p300 in the cells.

A second tactic to examine whether suppression of basal CBP/p300 would influence Ets2/PKA effects on the promoter was to use an RNA interference approach with short interfering RNA (siRNA) duplexes directed against CBP and p300 (59) designed to knock down endogenous concentration of endogenous CBP and p300 transcripts and hence protein (Fig. 6AGo). Western blot analyses indicated that the lowest concentration of siRNA (25 nM) directed against CBP was highly effective in reducing the amount of CBP in the cells, whereas the control siRNA (200 nM) had no effect. In the case of p300, the optimal concentration of the specific siRNA was rather higher (100 nM), although an effect was still observed with 25 nM (Fig. 6AGo). When they were transfected with the expression constructs for Ets2 and PKA, the siRNAs directed against CBP and p300 reduced reporter gene expression from the –126luc by about 50% (Fig. 6BGo). When both siRNAs were included in the transfection mixture, the ability of Ets2 and PKA to transactivate the promoter was reduced by more than 75%, suggesting that both coactivators were capable of interacting either directly or indirectly with Ets2. In contrast, no such effect was observed when the control siRNA duplex was transfected.


Figure 6
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  		Fig. 6. Effects of CBP and p300 Expression on the Ability of Ets2 to Transactivate the IFNT Promoter in JAr Cells

A, Cells were either mock transfected (lane 1) or transfected with a control siRNA (si-ve) and increasing concentrations of siRNA directed against the CBP (upper panel) and p300 (lower panel) mRNA. Whole-cell lysates were analyzed by SDS-PAGE, and relative levels of CBP and p300 protein were determined by SDS-PAGE and Western blotting. Asterisks indicate the concentration of siRNA used to knock down expression of CBP and p300 (25 nM and 100 nM, respectively) in panel B. B, The –126luc promoter was cotransfected with combinations of expression plasmids for Ets2 and PKA, siRNAs directed against mRNAs for CBP and p300 (25 nM and 100 nM, respectively), and a negative siRNA control (si-ve, 200 nM) that is not known to target any known human gene. In panel B, the normalized luc activities are presented relative to the activity of –126luc from nontreated cells (means ± SEM; n = 3), with fold activation shown above each bar. If letters above each bar are different, there was a significant effect of treatment (P < 0.05). siCBP, Short interfering CBP; sip300, short interfering p300.

 
The siRNA targeted against CREB mRNA had a modest but specific ability to reduce the concentration of CREB protein in JAr cells (supplemental Fig. 2A), but had no effect on the ability of Ets2 and PKA to transactivate the –126 IFNT promoter (supplemental Fig. 2B). These data are consistent with the possibility that CREB, which has no consensus binding site on the IFNT promoter, does not play a direct role in PKA/Ets2 up-regulation of IFNT.

Association of CBP/p300 and Ets2 with the IFNT Proximal Promoter Region in CT-1 Cells
We then sought to determine whether CBP/p300 was associated with Ets2 on the proximal (–188 to +3 region) of actively transcribed IFNT by classical chromatin immunoprecipitation (ChIP) assays. Sheared chromatin was prepared from bovine CT-1 cells, which actively secrete IFNT into the culture medium. DNA collected in immunocomplexes after addition of affinity-purified rabbit anti-p300 and anti-Ets2 immunoglobulin, respectively, was subjected to PCR analysis with specific primers (Fig. 7Go). Both antibodies provided DNA that contained the IFNT proximal regulatory region, whereas a nonspecific immunoglobulin was unable to do so. These data strongly suggest that CBP/p300 is associated with transcription factor complexes, including Ets2, bound to the IFNT promoter.


Figure 7
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  		Fig. 7. Association of CBP/p300 and Ets2 with the Regulatory Region Actively Transcribed IFNT from Bovine CT-1 Cells

Sheared chromatin prepared from formaldehyde-fixed CT-1 cells was exposed to no antibody (lane 1), anti-p300 (lane 2), anti-Ets2 (lane 3), and purified rabbit IgG (lane 4). The DNA recovered from each immunocomplex (lanes 1–4), 10% of the total in-put chromatin (lane 5), and 0.6 ng of plasmid DNA containing the –457IFNT promoter (lane 6) were analyzed by PCR with primers specific for a region (–188 to +3) containing the Ets2/AP1 enhancer element within the IFNT promoter.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
IFNT is known only to be expressed in a single tissue, trophectoderm, and for a limited period during early pregnancy when its production, whether assessed either on a per cell basis or as total amount produced by the conceptus (1, 11, 12, 60), rises dramatically in the period before the conceptus makes firm attachment to the uterine wall to form the definitive placenta. Unlike other type I IFN, the IFNT genes are not responsive to virus and do not contain a conserved viral response element, even though some sequence similarities with the virally inducible IFNW and IFNA remain evident (61). Conversely, the virally responsive IFNW and IFNA are not up-regulated during trophoblast differentiation in cattle (62). Therefore, the up-regulation of the IFNT reflects unique features of the IFNT-regulatory region, a permissive combination of transcription factors in cells of trophectoderm, and input from the external environment that prompts superinduction of expression. One of the key transcription factors is Ets2, which appears to play a central command role in regulating the IFNT (25, 63) and other trophoblast-associated genes in ruminants (26).

The IFNT is only expressed weakly in trophectoderm of cattle at the blastocyst stage of development (11, 13, 60, 64). The increase that follows is correlated with the initial rapid elongation of the conceptus and the rise in progesterone accompanying the full maturation of the CL of the mother (14, 15). As argued in the introduction to this paper, the rise in IFNT production is probably dependent upon maternal factors released into the immediate environment of the conceptus, which provide a means of coordinating the growth and activity of the conceptus with the hormonal state of the mother. The data presented here suggest that the PKA signal transduction pathway, presumably activated by receptors in response to binding with exocrine factors of maternal origin, participates in the up-regulation of the IFNT in the preimplantation conceptus. Clearly, a robust luteal phase in the mother will be required if conceptus IFNT is produced in time and in sufficient quantities to rescue the CL of pregnancy. What has also become clear is that the timing of the increase in progesterone in the early luteal phase (d 4 to d 5) may be a key factor in setting the stage for these subsequent events because it is necessary to program the secretory activity of the reproductive tract (86). Management strategies, including nutrition, that optimize this early rise in progesterone in the mother are likely, therefore, to be crucial for ensuring an adequate IFNT response by the conceptus.

Ets2 can be activated directly by the Ras/MAPK signal transduction pathway in many different kinds of cells (37, 50, 65, 66). Ets2 is not, however, a well established target of the cAMP/PKA signal transduction pathway, although PKA and Ets2 have been implicated in the regulation of the CGB5 subunit gene (30), which, like the IFNT, lacks a classical CRE (67). Our laboratory has shown that 8-bromo-cAMP and overexpressed PKA up-regulated CGB5 promoter activity primarily through a previously unrecognized proximal Ets2 enhancer on the promoter (28). The Ets2-binding sites on the promoter for the CGB5 partner gene, CGA, are also crucial for mediating cAMP/PKA effects on CGA gene expression (29, 30). Mutation of the Ets2-binding sites, for example, virtually abolishes cAMP responsiveness of the CGA subunit gene. Similarly, inactivation of the CREs by either truncation or point mutation, abrogates any ability of Ets2 to up-regulate the promoter (29). These were the first reports that implicated Ets2 as a target for the cAMP/PKA signal transduction pathway, a connection that has been further strengthened by the results presented here for the regulation of the IFNT genes.

Evidence in favor of Ets2 being the downstream target for PKA includes the following observations: 1) Coexpression of Ets2 and PKA greatly up-regulated the IFNT promoter in a synergistic manner (Figs. 1Go and 2Go); 2) Either a deletion of the Ets2-binding sequence (Fig. 2BGo) or a point mutation within its core (Fig. 4BGo) largely eliminated the PKA effects; 3) Inhibitors of the PKA signal transduction pathway prevented the synergistic up-regulation of the promoter by the combination Ets2 and PKA (Fig. 3Go, A and B). Finally PKA catalytic activity is required to observe any effect (Fig. 3AGo). Ets2 is not, however, a substrate for PKA, at least in vitro (supplemental Fig. 1), suggesting that some other transcription factor closely linked to the action of Ets2 is the direct target.

Our data argue against a role for CREB itself in promoting the synergistic action of PKA and Ets2. The IFNT promoter lacks an obvious binding site for CREB, and overexpression of CREB depresses the Ets2/PKA combinatorial effect, possibly because it provides an alternative substrate for PKA and may divert PKA activity from the pathway that transactivates the IFNT promoter. In addition, silencing of CREB by using an RNA interference approach had little effect (supplemental Fig. 2).

Our experiments are consistent with the possibility that CBP or its close relative p300 mediate the action of PKA on the transactivation of IFNT. CBP (CREB-binding protein) and p300 are two homologous, conserved, nuclear phosphoproteins that function as transcriptional coactivators by bridging a very large number of DNA-bound transcription factors with the basal transcription complex to activate transcription of genes (68, 69). CBP can serve as a link between the basal transcription machinery and many DNA-binding factors, including Ets1 and Ets2 (35). Although p300 and CBP had a negative effect on the –126 IFNT promoter when they were ectopically expressed in JAr cells, this unexpected phenomenon could be due to the fact that both proteins were already present in optimal concentrations and that overexpression, rather than promoting increased transcription, had an, as yet, ill-defined squelching effect. Although others have described positive effects of CBP/p300 on IFNT promoters in choriocarcinoma cells (57), such tumor-derived cell lines may not maintain a constant phenotype. In our experiments, the evidence for a positive involvement of p300/CBP arose from three observations. The first was that ectopic expression of the adenoviral E1A 12S protein, which inhibits CBP/p300 interaction with transcription factor IIB and components of the transcriptional machinery (70), also blocks reporter expression. The second was that the siRNA approach was effective in inhibiting the ability of PKA to promote Ets2-based transactivation of the IFNT promoter. The third was that CBP, along with Ets2, occupies the crucial enhancer region of actively transcribed IFNT in CT-1 cells, a bovine trophoblast cell line that produces IFNT. Finally, a search of EST databases reveals the presence of transcripts of PKA subunits, CBP/p300 and CREB in bovine trophoblast (data not shown). Although not proving a role for CBP/p300 in regulating IFNT expression, these data are quite compelling.

Ets2 is believed to interact with AP1 family members to mediate some of the downstream effects of the MAPK pathway on the IFNT and other Ets2-responsive promoters (71), so that a positive effect of mutating the AP1 site adjacent to the Ets2-binding site was unexpected (Fig. 4Go). Because the MEK-specific inhibitor, PD98059, also doubled Ets2/PKA stimulation of luc reporter activity (Fig. 3Go, B and D), it would appear that an operational Ras/MAPK pathway is antagonistic to activation of IFNT genes by the PKA signal transduction pathway. Cross talk is known to exist between these two signaling pathways (72, 73). Sometimes, the PKA pathway stimulates ERK signaling, whereas on other occasions it inhibits. There is at least one report where the inhibitor, PD98059, has been reported to up-regulate CRE-dependent gene activity (74). In addition, activation of PKA can lower MAPK activity (75) whereas its inhibition can increase it (76). One explanation for this phenomenon may be the ability of the cAMP pathway to target, and presumably inactivate, c-Raf, a component of Ras/MAPK signaling (73). Alternatively, the two pathways may converge on the transcription factors that bind at the Ets2/ AP1-like site and interfere with each other’s ability to drive promoter expression. What adaptive significance, if any, such antagonism might have is unclear, because factors that activate the Ras/MAPK pathway, e.g. CSF1 (77), and the cAMP/PKA pathway, e.g. FGF2 (23), are likely present simultaneously in maternal uterine secretions (19, 78, 79, 80), with the possibility that one can partially counteract the other in terms of their control of IFNT expression. This antagonistic relationship between PKA and MAPK and the precise role played by CBP/p300 in the context of IFNT regulation is of interest, but clearly complex. Although our experiments, for example, suggest that the effects of PKA are independent of Ets2 phosphorylation at Thr72 (Fig. 3Go, C and D), there is at least one reported example in which MAPK-mediated Ets2 phosphorylation at Thr72 augments the interaction of Ets2 with CBP/p300 (81). Further experiments are needed to understand the phenomenon. Nevertheless, we conclude that the most likely explanation for the steep increase in IFNT production at the time of conceptus elongation is an increase in intracellular cAMP, activation of PKA, and subsequent downstream effects on transcription factors already driving transcription of the IFNT at a low rate.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reporter Gene Constructs
Bovine IFNT1 promoters-luc gene reporter constructs –49luc, –126luc, –457luc, and –1675luc, containing the gene control regions –49 to +66 bp, –126 to +50 bp, –457 to +66 bp, and –1675 to +66 bp, respectively, and mutated sequences have been described previously (24, 25). Control region –67 to +66 bp of the boIFNT1 gene was generated by XbaI digestion and after self-ligation of the mutated AP1 binding site at –71 on the reporter (–126µAP1) (16). Fidelity of all constructs was verified by DNA sequencing.

Expression Vectors
The expression plasmids for Ets2 and its mutant form (pCGNEts2T72 and pCGNEts2A72), PKA expression plasmids, constitutively active catalytic subunit [Rous sarcoma virus (RSV)-PKA], and an expression vector of the specific inhibitor of PKA (RSV-PKI), have been described previously (25, 29, 82). A mutant form of PKA expression plasmid with a lysine replaced by methionine in the ATP-binding region, which results in an inactive catalytic subunit was gift from Dr. Richard Maurer (Oregon Health and Science University, Portland, OR) (82). The expression vectors for CBP and p300 were provided by Dr. Tony Kouzarides (University of Cambridge, Cambridge, UK) (83). The expression plasmid for human CREB (84) was obtained from Dr. T. F. Osborne (University of California-Irvine), and the CREB coding sequence was cloned into BamHI and NotI sites of pCDNA3.0 vector (Invitrogen, Carlsbad, CA). Either the β-galactosidase gene driven by the Rous sarcoma virus long-terminal repeat (pRSVLTR-βgal) or the Renilla luc gene driven by the cytomegalovirus (CMV) promoter (pRL-CMV; Promega, Madison, WI) was used as an internal control in all transfection experiments.

Cell Cultures and Transfections
JAr and JEG3 cells (HTB-144 and HTB-36; American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum and MEM supplemented with 2 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 1 mM sodium pyruvate, 10% fetal bovine serum, respectively. The cells were transfected either by the calcium phosphate method as described previously (25) or using Lipofectamine Plus (Invitrogen) as per manufacturer’s instructions. JAr or JEG3 cells were plated in six-well plates (1 x 105 cells per well) overnight and transfected with 0.5 µg of reporter gene constructs and 1.5 µg of expression vector DNA per well in the presence of 25 ng of pRSVLTR-βgal or 5 ng of pRL-CMV of internal control plasmid. Total amount of transfected DNA was kept constant by including the insert-free parental vectors.

After 36 h exposure to the transfection agent, cells were washed twice with PBS, (Invitrogen) and lysed with Passive Lysis Buffer (Promega). Luciferase activities were measured by injecting luciferase assay reagent (Promega) into cell extracts and recording chemiluminescence (a 10-sec light output) in a 20/20n Luminometer (Turner Biosystems, Mountain View, CA). β-Galactosidase activities were measured by using Tropix Galacto-Light substrate (Applied Biosystems, Foster City, CA) added to extracts after they had been heated at 48 C for 50 min to inactivate endogenous eukaryotic β-galactosidase. The Renilla luciferase activity as internal control was assayed with the Dual Luciferase Reporter Assay System (Promega). The transcriptional activity of each promoter-luc reporter construct was normalized with either the β-galactosidase or Renilla luciferase activity (25).

siRNA Transfections
siRNA duplexes against human CBP and p300 (59), human CREB (85), and human Ets2 (23) were purchased from Dharmacon (Chicago, IL). JAr cells were plated on six-well plates at a density of 1 x 105 per well and transfected in triplicate in OptiMEM by using Lipofectamine2000 (Invitrogen). Cells were transfected with 2 µg of plasmid DNA either alone or with duplex siRNA together with 25 ng of β-galactosidase as an internal control. A siCONTROL RISC-Free siRNA (Dharmacon) was used as negative control. Luciferase and β-galactosidase activities were measured as mentioned in the previous section.

For biochemical analyses, 1 x 105 JAr cells per well were transfected with 0, 25, 50, 100, and 200 nM CBP-siRNA and p300-siRNA and CREB-siRNA, respectively, or 100 nM siCONTROL RISC-Free siRNA in Opti-MEM. Cells were lysed 36 h later in Passive Lysis Buffer (Promega), and 30 µg of each lysate was separated on either 8% or 10% SDS-PAGE gels (for CBP or CREB, respectively). Western blot procedures have been described previously (28). Detection was performed with either rabbit polyclonal CBP antibody (sc-583) or CREB antibody (Cell Signaling Technology, Beverly, MA) in a blocking solution (25 mM Tris-HCl, pH 7.3; 150 mM NaCl; 0.1% Tween 20; and 5% nonfat dry milk). After overnight incubation with primary antibody at 4 C, the blot was incubated with antirabbit IgG coupled to horseradish peroxidase (Cell Signaling Technology) at 1:1000 dilution in blocking solution. Membranes were developed with the Phototype-horseradish peroxidase Western Blot Detection System (Cell Signaling Technology), and images were acquired with the Fuji LAS 3000 Imaging System (Fujifilm Medical Systems, Stamford, CT).

In Vitro PKA Phosphorylation Assay
Full-length human Ets2 (469 amino acids) and truncations, Ets2 (1–261 amino acids), Ets2 (225–469), Ets2 (295–469), and Ets2 (322–469), were expressed as GST fusion proteins in DH5{alpha} Escherichia coli and purified after thrombin cleavage to remove a putative PKA phosphorylation site at the junction of the Ets2 and GST polypeptides. Purified proteins (0.5 µg) were tested for their ability to serve as PKA substrates by incubating in the presence of the catalytic subunit of PKA from bovine heart (Sigma Chemical Co., St. Louis, MO) (15 U) and 10 µCi/µl [{gamma}-32P]ATP in a buffer (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; 12 mM MgCl2; and 1 mM dithiothreitol) for 1 h at 30 C. Ovine IFNT11, with an introduced PKA phosphorylation site at its carboxyl terminus (56), served as a positive control. At the end of the incubation, the reaction was terminated by the addition of equal volume of 2x SDS-PAGE loading buffer to the sample. The proteins were resolved in a 10% SDS-PAGE gel and dried, and 32P-labeled proteins were detected by autoradiography on BioMax MS (maximum sensitivity) x-ray film (Eastman Kodak Co., Rochester, NY).

ChIP Analysis
ChIP analysis on bovine CT-1 cells was conducted essentially as described by Ghosh et al. (29). In brief, sheared chromatin prepared from approximately 107 CT-1 cells, was precleared with a Protein G-Agarose bead slurry (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Twenty percent of the preparation was saved as "total input" control. The remaining chromatin was either left untreated ("no antibody" control), or treated with 2 µg of p300 antibody (sc-585, Santa Cruz Biotechnology), rabbit anti-Ets2 antibody (raised in our laboratory), and purified nonspecific IgG (Active Motif, Carlsbad, CA), respectively. The immune complexes were collected on Protein G-Agarose beads, eluted, and prepared for PCR analysis (29). A volume of 5 µl of the ChIP DNA was used as template for each PCR. The primers used were: forward, 5'-tga caa acc caa att tta ttg gga aa; reverse, 5'-tct gat gat gat cgt tct aag caa gg, and were designed to amplify a region of the IFNT proximal promoter (–188 to +3) containing the Ets2/AP1 enhancer. PCR conditions were as follows: 95 C for 2 min for one cycle, 33 cycles of 95 C for 30 sec, 52 C for 30 sec, 72 C for 2 min, followed by 72 C for 10 min. PCR products were visualized by ethidium bromide staining after electrophoresis in 2% agarose.

Statistical Analyses
Each transfection was carried out in triplicate, and the experiment was repeated either three or four times. Values from individual experiments were log transformed. Statistical analyses (for at least three replicated experiments) were performed by one-way ANOVA, with multiple data set comparisons analyzed by Tukey postcomparison test on Prism analytical software (GraphPad Prism version 4.0; GraphPad Software, Inc., San Diego, CA).


   ACKNOWLEDGMENTS
 
We thank Drs. Debjani Ghosh and Shrikesh (Rick) Sachdev (University of Missouri, Columbia, MO) and Anindita Chakrabarty (Vanderbilt University, Nashville, TN) for their input during the course of the experiments; Drs. Michael F. Smith, David Setzer, and Jonathan A Green for their critical review of the data; Drs. R. A. Maurer (University of Iowa, Iowa City, IA), A. Kouzarides (University of Cambridge, Cambridge UK), and T. Osborne (University of California-Irvine) for providing us with constructs for transfection experiments; and Norma McCormack for her assistance in formatting the manuscript and figures for submission.


   FOOTNOTES
 
This research was supported by National Institutes of Health Grants R01 HD21896 and R01 HD42201 (to R.M.R.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 1, 2007

Abbreviations: AP1, Activator protein 1; CBP, cAMP response element-binding protein (CREB)-binding protein; ChIP, chromatin immunoprecipitation; CL, corpus luteum; CMV, cytomegalovirus; CRE, cAMP response element; CREB, CRE-binding protein; IFNT, interferon-{tau}; hCG, human chorionic gonadotropin; MEK, MAPK kinase; PKA, protein kinase A; PKI, inhibitor of PKA; RSV, Rous sarcoma virus; siRNA, short interfering RNA.

Received for publication June 15, 2007. Accepted for publication October 23, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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