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Molecular Endocrinology, doi:10.1210/me.2004-0320
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Molecular Endocrinology 19 (4): 1049-1066
Copyright © 2005 by The Endocrine Society

Coordinate Regulation of Basal and Cyclic 5'-Adenosine Monophosphate (cAMP)-Activated Expression of Human Chorionic Gonadotropin-{alpha} by Ets-2 and cAMP-Responsive Element Binding Protein

Debjani Ghosh, Shrikesh Sachdev, Mark Hannink and R. Michael Roberts

Departments of Animal Sciences (D.G., R.M.R.) and Biochemistry (S.S., M.H., R.M.R.), University of Missouri, Columbia, Missouri 65211

Address all correspondence and requests for reprints to: R. Michael Roberts, 105 Life Sciences Center, University of Missouri, Columbia, 1201 Rollins Road, Columbia, Missouri 65211-7310. E-mail: RobertsRM{at}missouri.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ets-2 controls the activities of many genes characteristically up-regulated in trophoblast. One apparent exception has been the gene for the human chorionic gonadotropin subunit {alpha} (hCG{alpha}). Here, we show that the hCG{alpha} gene contains two overlapping Ets binding sites adjacent to an activator protein-1-like site in its proximal promoter. Transactivation by Ets-2 is susceptible to truncation and mutation of these sites, which bind Ets-2 during in vitro mobility shift assays, as well as in vivo as determined by chromatin immunoprecipitation in choriocarcinoma cells. Knockdown of Ets-2 with short interfering RNA decreases both promoter activity and synthesis of hCG{alpha}. Ets-2 acts in combination with the protein kinase A (PKA) signal transduction pathway to activate the hCG{alpha} promoter expression. Mutation of the Ets-2 binding sites dramatically reduces up-regulation by PKA, whereas mutations within the two cAMP-responsive elements abolish responsiveness of the promoter to Ets-2. cAMP-responsive element binding protein (CREB) and Ets-2 form a complex that can be coimmunoprecipitated from choriocarcinoma cells, and association of CREB and Ets-2 is increased by activation of PKA. Regulation of hCG{alpha} subunit gene activity by cAMP involves the binding of CREB and Ets-2 to discrete elements in the promoter as well as a physical interaction between the two proteins. We propose that regulation of hCG{alpha} by Ets-2 and CREB enables coordinated expression of hCG{alpha} with its partner hCGß subunit.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CHORIONIC GONADOTROPIN (CG) is the primary signal for maternal recognition of pregnancy in primates where it acts as a luteotrophic hormone and maintains the output of progesterone from the corpus luteum (CL), which, without luteotrophic stimulation, would otherwise regress (1). The early onset of human CG (hCG) expression and its subsequent up-regulation are events crucial in early pregnancy when the timely production of the hormone is essential if the embryo is to survive. CG directly stimulates progesterone output from luteal cells (2, 3), and exogenous CG can extend the functional lifespan of the CL of women and female monkeys who are not pregnant (4, 5, 6, 7). Moreover, immunoneutralization of CG during pregnancy leads to abortion (8, 9, 10, 11). Such data support the view that CG is essential for maintaining pregnancy in higher primates.

CG is a heterodimeric molecule. It possesses the common {alpha}-subunit of the glycoprotein hormone family and a unique ß-gene product (12). During dimer formation, the two subunits assemble roughly parallel to each other, with both contributing to the receptor-binding site (13, 14). Thus, coexpression of the {alpha}- and ß-chains is rate limiting for active hormone production. Because hCG synthesis is regulated primarily by the rate at which the genes are transcribed (15), common control mechanisms might be anticipated. Both genes are up-regulated by cAMP, albeit with different kinetics (16), yet the promoter sequences of both subunit genes are unique and contain few elements that provide any clue as to how their transcription might be coordinated.

CG is produced by the trophectoderm of the developing embryo. Several genes that are first expressed in early trophoblast cells from a number of different species are under the control of the Ets-2 protein (17, 18, 19, 20, 21, 22, 23), suggesting that this transcription factor might have a directive role in regulating differentiation of trophectoderm (24). Ets-2 is also essential for the development of trophoblast in the mouse (25). The characteristic feature of Ets-2 and its relatives is the ETS domain, a highly conserved helix-turn-helix sequence of about 85 amino acids responsible for DNA binding (26, 27). Ets family members recognize a central GGA core sequence, usually located within a (C/A)(C/A)GGA(A/T)(A/G) motif (27). We recently demonstrated by mutational analysis that the hCGß gene possesses two closely spaced, functional Ets binding sites (EBS) and an adjacent activator protein-1 (AP-1)-like site, which are located just upstream of its initiator element (23). These Ets-2 binding sites are crucial for regulating hCGß promoter activity in the choriocarcinoma cell lines JAr and JEG-3, where they govern the responsiveness of the hCGß promoter to both cAMP and MAPK regulation.

The promoter of the hCG{alpha} subunit gene has been intensively analyzed for its ability to control transcriptional activity of reporter genes in cells of trophoblast origin, but no functional Ets-2 binding sites have been identified (22), making hCG{alpha} something of an anomaly among genes up-regulated as trophectoderm differentiates. The proximal 180 bp of the promoter (Fig. 1Go) is sufficient for both basal and cAMP-stimulated transcription (28, 29, 30, 31, 32) in choriocarcinoma cells. The DNA sequences responsive to cAMP have been localized to two adjoining repeats (29, 33, 34, 35), known as cAMP-responsive elements (CREs), which act synergistically (36, 37). The core sequence of the CRE is found in a variety of other gene regulatory elements that are activated by cAMP. The major transcription factor that binds to these CREs is the CRE-binding protein (CREB), which is a substrate for phosphorylation by cAMP-dependent protein kinase A (PKA) (38, 39). An extended upstream regulatory element (URE), which binds several additional transcription factors, may also contribute to control of hCG{alpha} expression (29, 30, 31, 36, 40, 41, 42). The URE can be divided into a –172/–151 URE1 and an overlapping –177/–156 URE2 region. Mutations that separately abolish protein binding at either URE1 or URE2 do not reduce promoter activity, although mutations that destroy both binding sites significantly reduce basal promoter activity (31). A second control element (–162/–141) located distal to the CRE, which partially overlaps the URE, is known as the {alpha}-ACT region (15, 41). It binds a GATA factor (43) and AP-2{gamma} (44, 45).



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  		Fig. 1. The Nucleotide Sequence of the Proximal hCG{alpha} Gene Promoter from the Initiator Element to –235

The transcription initiation site is indicated with the arrow. Transcription factor binding sites, including two CRE, four EBS, an AP-1 site, and the TATA box, are underlined and bold.

 
A recent report has suggested that Ets-2 does not contribute to regulation of hCG{alpha} expression in JEG-3 cells (22). However, these cells express Ets-2 and possess an activated MAPK signaling pathway, resulting in high constitutive levels of Ets-2, dependent transcription that may mask any contribution of ectopically expressed Ets-2 protein toward transcription of the hCG{alpha} promoter (19, 22, 23). Accordingly, we have reinvestigated the effects of Ets-2 on this promoter. Our results indicate that the hCG{alpha} promoter contains two functional binding sites for Ets-2 that mediate responsiveness to Ets-2. Furthermore, we have identified physical and functional interactions between Ets-2 and CREB that control hCG{alpha} expression. Our results suggest that cooperative interactions between Ets-2 and CREB provide a mechanism whereby expression of the two hCG subunit genes is coordinated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Synergistic Effects of Ets-2 and Ras on hCG{alpha} Promoter Expression
A search of the hCG{alpha} proximal promoter sequence with the computer software analysis program Mat Inspector V2.2 (genomatix.de/cgi-bin/matinspector.pl) revealed four potential EBS, each containing a core EBS motif of either GGAA or AGGA (Fig. 1Go). Two adjacent EBS (EBS1, –81 to –78; EBS2, –75 to –72) were of particular interest because these sites are located on opposite strands immediately upstream of a candidate binding site for AP-1, and functional interaction between adjacent Ets and AP-1 binding sites is frequently observed (18, 20).

To determine the role of Ets-2 in activation of hCG{alpha} expression, we first used transient transfection reporter gene assays in NIH 3T3 cells that have low endogenous MAPK pathway activity (23) and express low levels of Ets family proteins. Two hCG{alpha} promoter constructs, containing either 254 or 1443 bp of upstream promoter-derived sequences, were used. Basal promoter activity of either hCG{alpha} promoter construct was extremely low in NIH 3T3 cells, approximately 1% of basal promoter activity observed in both JAr and JEG-3 choriocarcinoma cells (Fig. 2AGo). Ectopic expression of Ets-2 in NIH 3T3 cells resulted in 8- to 11-fold elevation of reporter gene activity from either promoter (Fig. 2AGo). Although basal levels of hCG{alpha} promoter activity were higher in both JAr and JEG-3 choriocarcinoma cells, a low but consistent elevation (3- to 11-fold, respectively) of hCG{alpha} promoter activity was obtaining after ectopic Ets-2 expression (Fig. 2Go, B and C).



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  		Fig. 2. Transactivation of the hCG{alpha} Promoter by Ets-2 and Ras

3T3 (A), JAr (B), and JEG-3 (C) cells were transfected with the indicated hCG{alpha} promoter-Luc plasmid (–254 or –1443), or cotransfected with the promoter-Luc plasmid and expression vectors for Ets-2 and activated Ras. The data are provided as relative Luc activities to illustrate the differences in basal and stimulated activities for the two promoters in the three cell lines. Numbers above each bar are fold-stimulation of reporter activity resulting from expression of Ets-2 and Ras alone or in combination relative to basal rate for the particular promoter for a particular cell line. Results from five separate experiments (each performed in triplicate on different days) have been calculated as means ± SEM. If letters above bars are different, there was a significant effect of treatment (P < 0.05).

 
Because the ability of Ets family proteins to activate transcription can be regulated by Ras-dependent MAPK signal transduction pathways (18, 27), levels of hCG{alpha} promoter activity were measured after ectopic expression of Ras, either alone or in combination with Ets-2. Expression of Ras alone resulted in 3- to 5-fold elevation of hCG{alpha} promoter activity in NIH 3T3 cells. However, when expressed together, Ets-2 and Ras dramatically increased reporter gene activity, resulting in a 250-fold increase in reporter gene activity for the 254-bp promoter construct and a 140-fold increase in reporter gene activity for the 1443-bp promoter (Fig. 2AGo).

Two approaches were used to determine whether synergistic activation of the hCG{alpha} promoter by Ras and Ets-2 in NIH 3T3 cells was dependent on MAPK signaling. First, because Thr 72 located within the pointed domain of Ets-2 is a known target of the activated MAPK pathway (46), the ability of a mutant Ets-2 protein containing an Ala substitution for Thr 72 to activate hCG{alpha} promoter expression was determined. The Ets-2-(T72A) protein was markedly impaired in its ability to activate hCG{alpha} promoter expression in NIH 3T3 cells, when expressed either alone or in combination with Ras (Fig. 3AGo). Second, the MAPK pathway inhibitors PD9805 and U0126 (47, 48, 49) were potent inhibitors of synergistic activation of the hCG{alpha} promoter by Ras and Ets-2 (Fig. 3AGo). An inhibitor of the stress-activated protein kinase pathway, SB202190 (50), resulted in only modest inhibition of activation of hCG{alpha} promoter by Ras and Ets-2. Thus, synergistic cooperation between Ras and Ets-2 in activation of hCG{alpha} promoter expression in NIH 3T3 cells is mediated, at least in part, by the MAPK signal transduction pathways (51, 52).



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  		Fig. 3. The MAPK Pathway Contributes to Activation of the hCG{alpha} Promoter by Ets-2 and Ras

A, 3T3 cells were transfected with the –254 promoter-Luc plasmid or cotransfected with the promoter-Luc plasmid and expression vectors for Ets-2 and activated Ras. Expression vectors for the wild-type Ets-2 protein and for the mutant Ets-2(T72A) protein were used in parallel transfections. In some cases, the transfected cells were treated with the MEK1/2 inhibitors PD98059 (25 µM) and U0126 (1 µM) or the p38 inhibitor SB202190 (5 µM) before collection of cell lysates. B and C, JAr or JEG-3 cells, as indicated, were transfected with the –254 promoter-Luc plasmid or cotransfected with the –254 promoter-Luc plasmid and expression vectors for Ets-2 and activated Ras. In some cases, the transfected cells were treated with the MEK1/2 inhibitors PD98059 (25 µM) and U0126 (1 µM) or the p38 inhibitor SB202190 (5 µM) before collection of cell lysates. The data are provided as relative Luc activities (mean ± SEM). The numbers above each bar represent fold-stimulation of reporter activity resulting from expression of Ets-2 and Ras alone or in combination. If letters above bars are different, there was a significant effect of treatment (P < 0.05).

 
Parallel experiments were performed to determine whether Ras and Ets-2 cooperate to activate hCG{alpha} promoter expression in JAr and JEG-3 choriocarcinoma cells. Ectopic expression of Ras had no effect, either alone or in combination with Ets-2 (Fig. 2Go, B and C). However, although ectopic expression of Ras did not further increase Ets-2-mediated transactivation of the hCG{alpha} promoter, the MAPK inhibitor U0126 completely blocked activation by Ras and Ets-2 of hCG{alpha} promoter expression (Fig. 3Go, B and C). Thus, a functioning MAPK pathway is required for Ets-2-mediated transactivation of the hCG{alpha} promoter. Furthermore, basal expression of the hCG{alpha} promoter in JAr cells was reduced approximately 50% by U0126 (data not shown), consistent with the notion that these choriocarcinoma cell lines contain a fully activated MAPK pathway. Taken together, these experiments indicate that Ets-2, acting in concert with a Ras-activated MAPK signal transduction pathway, can transactivate expression of the hCG{alpha} promoter.

Deletion and Mutational Analysis of the hCG{alpha} Promoter
The results shown in Fig. 2Go demonstrate that the proximal 254 bp of the hCG{alpha} promoter contains one or more DNA elements that mediate transactivation by Ets-2. To further define the region(s) of the hCG{alpha} promoter that mediate responsiveness to Ets-2, several additional hCG{alpha} promoter constructs were characterized in both NIH 3T3 cells and the two choriocarcinoma cells (Fig. 4Go). For each of the hCG{alpha} promoter constructs, basal reporter gene expression was compared with reporter gene expression in the presence of ectopically expressed Ets-2 and Ras.



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  		Fig. 4. Deletion Analysis of the Proximal hCG{alpha} Promoter

A set of DNA fragments from the hCG{alpha} promoter (–1443/+48, –677/+48, –254/+48, –107/+48, –47/+48) were cloned into the pGL2 Basic-Luc vector and tested for Luc expression in 3T3 (A), JAr (B) and JEG-3 (C) cells. Each reporter construct was either transfected alone or cotransfected with expression vectors for Ets-2 and Ras. The data are provided as relative Luc activities (mean ± SEM). The number above each bar represents fold-stimulation of reporter activity for each hCG{alpha} promoter construct resulting from coexpression of Ets-2 and Ras. If letters above bars are different, there was a significant effect of treatment (P < 0.05).

 
Deletion of sequences located between –1443 and –254 in the hCG{alpha} promoter resulted in a modest reduction in basal expression of the hCG{alpha} promoter. However, Ets-dependent transactivation of the hCG{alpha} promoter was increased 3-fold in NIH 3T3 cells and 2-fold in the JAr and JEG-3 cells (Fig. 4Go). Thus, the potential EBS located in the distal regions of the hCG{alpha} promoter are not the major determinants of Ets-2-dependent hCG{alpha} promoter expression. Deletion of sequences located between –254 and –107, including two adjacent CREs, reduced the ability of Ets-2 to activate expression from the hCG{alpha} promoter by 50–75%. An additional deletion to –47, resulting in the removal of two Ets sites and a nearby AP-1 site, resulted in a further reduction of Ets-2-dependent activation of hCG{alpha} promoter-dependent reporter gene activity to approximately 10% of that observed with the –254 promoter construct (Fig. 4Go). These results suggest two important points. First, that the two adjacent EBS located between –107 and –47 likely contribute to Ets-2-dependent transactivation of the hCG{alpha} promoter. Second, that the promoter region located between –254 and –107, which contains the two adjacent CREs, also contributes to the Ets-2-dependent transactivation effect. Thus Ets-2 may mediate its stimulatory effects not just through one or more of the EBS located between –107 and –47, but also through the CREs, which are generally regarded as the major enhancers that drive hCG{alpha} expression in trophoblast (36, 37).

To determine more precisely the Ets-2-responsive elements in the hCG{alpha} promoter, a systematic site-directed mutational analysis was carried out (Fig. 5Go, A–C). All experiments were performed with the –254 promoter in the presence of ectopically expressed Ets-2 and Ras proteins. For each of the hCG{alpha} promoter constructs, reporter gene expression in the presence of ectopically expressed Ets-2 and Ras was compared with wild-type promoter in absence of Ets-2 and Ras. In each of the three cell lines, mutations that abolished the adjacent EBS located between –107 and –47 resulted in marked reduction of Ets-2-dependent activation of hCG{alpha} promoter-dependent reporter gene activity. The effect of these mutations was most dramatic in the JAr cell line, where the functional elimination of either one or both EBS reduced Ets-dependent transactivation of the promoter to less than 5% of that observed with the wild-type –254 promoter. Mutation of the putative AP-1 binding site located immediately downstream of the two EBS also lessened Ets-2-dependent activation of the hCG{alpha} promoter, suggesting that one or more members of the Fos/Jun family may contribute to expression of hCG{alpha}. Finally, and consistent with the results obtained with the deletion constructs, mutations that abolished one or both of the CRE motifs located between –254 and –107 reduced Ets-dependent transactivation of the hCG{alpha} promoter to as low as 5% of the level obtained with the wild-type promoter. Although some differences were observed between the three cell lines in the degree to which individual mutations reduced hCG{alpha} promoter-dependent reporter gene activity, the overall pattern was very similar.



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  		Fig. 5. Mutational Analysis of the Proximal hCG{alpha} Promoter

A series of mutations that altered individual transcription factor binding sites within the proximal hCG{alpha} promoter were constructed. The mutant reporter constructs were cotransfected with expression vectors for Ets-2 and Ras into 3T3 (A), JAr (B) and JEG-3 (C) cells. In A-C, the wild-type reporter construct was either singly transfected or cotransfected with expression vectors for Ets-2 and Ras. Numbers above the bars represent fold-stimulation of reporter activity above the values obtained with the wild-type reporter construct in the absence of Ets-2 and Ras. The data are provided as relative Luc activities from five different experiments, each performed in triplicate. In panel D, the wild-type or mutant reporter plasmids or the pGL2 Basic-Luc plasmid were singly transfected into JAr cells. The data are provided as relative Luc activities (mean ± SEM). Values were calculated from five different transfections, each performed in triplicate on different days. The numbers above the bars represent the fold increase of reporter gene activity over that obtained with empty pGL2 Basic-Luc plasmid. If letters above bars are different, there was a significant effect of treatment (P < 0.05).

 
To determine whether the same mutations had similar effects on basal promoter activities, the experiment was repeated in the absence of ectopic expression of Ets-2 and Ras proteins. Here, promoter-dependent reporter gene activities were compared with those obtained with a promoterless construct. The basal reporter gene expression from the mutant hCG{alpha} promoters could be reliably measured in both JAr and JEG-3 cells but was below the level of detection in 3T3 cells. Because the overall pattern of expression from the mutant hCG{alpha} promoters was similar in JAr (Fig. 5DGo) and JEG-3 cells (data not shown), only data with the former are shown. Mutations within either one or both Ets sites markedly reduced basal promoter activity to approximately 10% of that observed with the wild-type promoter, confirming that these EBS are of functional relevance for activation of the hCG{alpha} promoter. Mutation of the AP-1 site also reduced basal promoter activity. Finally, consistent with previously published results, mutations within either one or both of the CREs reduced basal promoter activity to less than 1% as compared with the wild-type promoter.

Together, these data demonstrate that the EBS located between –107 and –47, along with the adjacent AP-1 site, contribute to both basal and Ets-dependent expression of the hCG{alpha} promoter. Most intriguingly, these results suggest that Ets-2 requires a functional CRE motif to exert its full effects on the promoter.

Analysis of Ets-2 Binding to the Naked hCG{alpha} Proximal Promoter
To demonstrate that the EBS located between –107 and –47 are capable of binding Ets-2, a series of EMSA experiments were performed. Two oligonucleotides that contained the core sequences of each individual EBS were used, while the third oligonucleotide was designed to include both binding sites (Table 1Go). The ability of these oligonucleotides to bind Ets-2 was first tested by using a soluble glutathione-S-transferase (GST) fusion protein containing the full-length Ets-2 open reading frame purified from bacteria. The oligonucleotides formed specific protein-DNA complexes with the GST-Ets-2 fusion protein but not with GST alone, as evidenced by the ability of unlabeled oligonucleotides to displace the radioactive oligonucleotide from the protein-DNA complexes. The binding specificity of purified Ets-2 protein was verified by Ets-2 antibody and Ets site mutated oligonucleotides (supplemental Fig. 1Go, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). The oligonucleotides containing the individual EBS bound to the GST-Ets-2 protein with comparable affinity (Kd 0.25 and 0.20 µM, respectively; Fig. 6Go, A and B). The oligonucleotide containing both binding sites bound the GST-Ets-2 protein with slightly higher affinity (Kd 0.13 µM; Fig. 6CGo).


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  		Table 1. Synthetic Oligonucleotides Used for PCR and EMSA and the Nucleotide Sequences for siRNA Experiments

 


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  		Fig. 6. Sequence-Specific Binding of Ets-2 to the hCG{alpha} Promoter

Protein-DNA binding was assessed by EMSA using GST-Ets-2 and double-stranded 32P-labeled oligonucleotides encompassing Ets site 1 (nucleotides –91 to –74; A), Ets site 2 (nucleotides –79 to –60; B) and both Ets sites (nucleotides –85 to –67; C) from the hCG{alpha} promoter. The 32P-labeled oligonucleotides were incubated with 2 µg of purified GST-Ets-2 in the absence or presence of increasing amounts of unlabeled competitor oligonucleotide (5-, 10-, 50-, 100-, 250-, and 500-fold molar-excess). Saturation binding curves with an inset Scatchard plot and calculated Kd are shown. To assess binding of nuclear proteins, 32P-labeled oligonucleotides containing Ets site 1 (D), Ets site 2 (E) or the combined Ets sites (F) were incubated with 10 µg of JAr cell nuclear extracts in the absence or presence of unlabeled competitor nucleotide (50-, 100-, 250-, and 500-fold molar excess). To confirm that the protein-DNA complex formed with JAr nuclear extracts contained the Ets-2 protein, 32P-labeled oligonucleotides containing Ets site 1 (G), Ets site 2 (H) or the combined Ets sites (I) were incubated with 10 µg of JAr cell nuclear extracts in the absence (lane 1) or presence of normal rabbit serum (lane 2) or in the presence of affinity-purified anti-Ets-2 antibodies (lane 3). To assess sequence-specific binding of nuclear Ets-2 proteins, JAr cell nuclear extracts were incubated with 32P-labeled oligonucleotides containing Ets site 1 (J), Ets site 2 (K) or the combined Ets sites (L) in the absence (lane 1) or presence of either the unlabeled wild-type (lane 3) or unlabeled mutant oligonucleotides (lane 4) (500-fold molar excess). JAr cell nuclear extracts were also incubated with mutant 32P-labeled oligonucleotides (lane 2).

 
The ability of these oligonucleotides to bind proteins present in nuclear extracts from JAr cells was determined. Specific protein-DNA complexes were observed with the oligonucleotides containing either one or both of the EBS (Fig. 6Go, D–F). The presence of Ets-2 protein in these protein-DNA complexes was confirmed by the ability of affinity-purified antibodies against Ets-2 to reduce the mobility of the protein-DNA complex in native acrylamide gels (Fig. 6Go, G–I). Normal rabbit serum had no effect on the formation or mobility of the protein-DNA complexes. Sequence-specific binding of Ets-2 to the hCG{alpha} promoter-derived oligonucleotides was confirmed by the ability of unlabeled wild-type, but not mutated, oligonucleotides to compete for binding (Fig. 6Go, J–L; compare lanes 1, 3, and 4). Furthermore, radiolabeled mutant oligonucleotides were unable to form a protein-DNA complex when added to nuclear extracts from JAr cells (Fig. 6Go, J–L; compare lanes 1 and 2). Identical results were obtained with nuclear extracts prepared from JEG-3 cells (see supplemental Fig. 2Go, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).

Endogenous Ets-2 Binds to the hCG{alpha} Promoter and Regulates Both Basal Promoter Expression and cAMP-Stimulated Production of hCG{alpha} Protein
Chromatin immunoprecipitation assays were performed to determine whether these EBS within the hCG{alpha} promoter are occupied by the endogenous Ets-2 protein. Sheared chromatin isolated from JAr cells that had been fixed with formaldehyde before cell lysis was immunoprecipitated with an affinity-purified antibody directed against Ets-2. The presence of the DNA derived from the proximal hCG{alpha} promoter element in the immunoprecipitate was determined by PCR amplification. As shown in Fig. 7AGo, DNA derived from the proximal hCG{alpha} promoter was specifically immunoprecipitated with anti-Ets-2 antibody but not with a nonspecific antibody (antimouse hemagglutinin antibody). Unexpectedly, the immunoprecipitates recovered with an anti-CREB antibody did not contain DNA sequences from the proximal hCG{alpha} promoter although the primers used in the PCR were designed to amplify the region of the promoter containing the two CREs, as well as the EBS. By contrast, DNA sequences from the proximal hCGß promoter were immunoprecipitated with either anti-Ets-2 antibody or anti-CREB antibody. These results demonstrate that the endogenous Ets-2 protein binds to sequences located within both the hCG{alpha} and hCGß promoters, consistent with the notion that Ets-2 contributes to coordinated expression of both subunits of hCG.



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  		Fig. 7. The Endogenous Ets-2 Protein Binds to the Proximal hCG{alpha} Promoter and Regulates hCG{alpha} Expression

A, Sheared chromatin prepared from formaldehyde-fixed JAr cells was immunoprecipitated with the indicated antibodies. The DNA was recovered from immunoprecipitates and analyzed by PCR using primers specific for the hCG{alpha} promoter (top panel, lane 4) and the hCGß promoter (bottom panel, lanes 4 and 5). B, JAr cells were transfected with empty pGL2 Basic vector or the –254 promoter-Luc plasmid in the absence or presence of increasing amounts of an siRNA against the Ets-2 mRNA. A siRNA molecule against the human Keap1 mRNA was included in one sample as a control. The data are provided as relative Luc activities (mean ± SEM). If letters above bars are different, there was a significant effect of treatment (P < 0.05). C, JAr cells were either mock-transfected or transfected with increasing amounts of an siRNA against the Ets-2 mRNA. One sample contained a siRNA molecule against the human Keap1 mRNA as a control. Whole cell lysates were collected, and steady-state levels of endogenous hCG{alpha} (top), hCGß (middle), and Ets-2 (bottom) were determined by immunoblot analysis.

 
To confirm a functional role for the endogenous Ets-2 protein in regulation of hCG{alpha} gene expression, short interfering RNA (siRNA) duplexes targeted against human Ets-2 transcripts were used to knock down levels of the endogenous Ets-2 protein. In one set of experiments, siRNA molecules directed against Ets-2 reduced reporter gene expression from the –254 hCG{alpha} promoter in a dose-dependent manner (Fig. 7BGo). In contrast, a siRNA molecule directed against the human Keap1 mRNA had no effect on reporter gene expression driven from the –254 hCG{alpha} promoter.

In a second set of experiments, the ability of Ets-2 siRNA molecules to inhibit hCG{alpha} protein expression was determined. These experiments were performed with cAMP-stimulated cells, because amounts of endogenous hCG{alpha} in the absence of cAMP stimulation were too low for detection in the assay. Transfection of the Ets-2 siRNA into JAr cells, followed by stimulation with 8-bromoadenosine-cAMP (8-bromo-cAMP), resulted in a dose-dependent reduction in the steady-state concentrations of the endogenous hCG{alpha} protein, as determined by immunoblot analysis of whole cell extracts (Fig. 7CGo, top panel). Steady-state concentrations of hCGß protein were also decreased in a dose-dependent manner by the Ets-2 siRNA molecule, (Fig. 7CGo, middle panel). As expected, amounts of the endogenous Ets-2 protein were also decreased in a dose-dependent manner by transfection of the Ets-2 siRNA molecule into JAr cells (Fig. 7CGo, lower panel). A nonspecific siRNA molecule was ineffective in reduction of steady-state levels of hCG{alpha}, hCGß, or Ets-2 (Fig. 7CGo, lane 6).

Interaction of Ets-2 and the PKA Signal Transduction Pathway in the Activation of the hCG{alpha} Promoter
Previous studies on the cAMP regulation of the hCG{alpha} promoter have implicated the two CREs as the dominant enhancer for the entire promoter element and as the major target for the PKA pathway (35, 36, 37). Therefore, we examined the possibility that Ets-2 and CREB might participate in physical and functional interactions that regulate hCG{alpha} expression. In one set of experiments, the ability of ectopic Ets-2 expression in combination with 8-bromo-cAMP to activate expression from the proximal hCG{alpha} promoter was determined. Activation of PKA signaling with 8-bromo-cAMP resulted in a 20-fold increase in reporter gene activity in 3T3 cells and a 15-fold increase in JAr or JEG-3 cells (data not shown), consistent with published reports that hCG{alpha} promoter expression is increased by activation of PKA signaling (33, 34). Ectopic expression of Ets-2 in combination with 8-bromo-cAMP treatment increased hCG{alpha} promoter-dependent reporter gene expression more than 52-fold in JAr cells, 46-fold in JEG-3 cells, and approximately 114-fold in 3T3 cells, suggesting that the two treatments provided optimal up-regulation of the promoter (Fig. 8Go, A–C). Mutation of either of the Ets-2 sites or one of the CREs reduced activation of hCG{alpha} promoter-dependent reporter gene expression to less than 20% relative to the wild-type promoter, whereas mutation of both CRE and the first Ets-2 site virtually eliminated responsiveness to the combined effects of Ets-2 and 8-bromo-cAMP in all three cell lines. Loss of the putative AP-1 element also partially reversed the combined stimulatory effects of Ets-2 and 8-bromo-cAMP. These experiments confirm the interdependence of the CREs and the sequence encompassing the Ets-2 and AP-1-like element.



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  		Fig. 8. Multiple Sites within the hCG{alpha} Promoter Mediate Responsiveness to Ets-2 and 8-Bromo-cAMP

The wild-type and mutant reporter constructs were cotransfected with expression vectors for Ets-2 into 3T3 (A), JAr (B), and JEG-3 (C) cells. The transfected cells were treated with 500 µM 8-bromo-cAMP for 36 h before collection of cell lysates. JAr cells singly transfected with the wild-type reporter construct and not treated with 8-bromo-cAMP were included in all transfections for comparison. The data are provided as relative Luc activities (mean ± SEM). Values were calculated from five different transfections, each performed in triplicate on different days. The numbers above the bars represent fold-stimulation of reporter activity above the values obtained with the wild-type reporter construct in the absence of Ets-2 expression and treatment with 8-bromo-cAMP. If letters above bars are different, there was a significant effect of treatment (P < 0.05).

 
Two PKA inhibitors, H-89 and 14–22 amide (53), largely reversed the stimulatory effects of 8-bromo-cAMP and Ets-2 expression, confirming that the effect of cAMP was mediated via activation of PKA. In contrast, the MAPK inhibitor PD98059 had no effect of activation of hCG{alpha} promoter by Ets-2 and cAMP in the choriocarcinoma cells, although an approximately 30% inhibition was observed in 3T3 cells (Fig. 9Go, A–C). In addition, ectopic expression of the constitutively active catalytic subunit of PKA along with Ets-2 resulted in more than 100-fold induction of hCG{alpha} promoter-dependent reporter activity in all three cell lines (Fig. 9Go, D–F). Activation of hCG{alpha} promoter by expression of the catalytic subunit of PKA was completely abolished by coexpression of a synthetic gene encoding the heat-stable inhibitor of catalytic subunit of PKA (PKI), suggesting that active catalytic subunit is important for PKA-mediated activation of promoter (54, 55) (Fig. 9Go, D–F).



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  		Fig. 9. Activation of the hCG{alpha} Promoter by Ets-2 and 8-Bromo-cAMP Requires the Activity of PKA

The –254 hCG{alpha} promoter construct was either singly transfected or cotransfected with an expression vector for Ets-2 into 3T3 (A), JAr (B), and JEG-3 (C) cells. The transfected cells were treated with 500 µM 8-bromo-cAMP for 36 h before collection of cell lysates. In some cases, the transfected cells were exposed to the indicated inhibitors (10 µM H-89, 2 µM 14–22 amide, or 25 µM PD98059) before collection of cell lysates. The –254 hCG{alpha} promoter construct was also singly transfected or cotransfected with expression vectors for both Ets-2 and PKA into 3T3 (D), JAr (E), or JEG-3 (F) cells. Some samples, as indicated, also contained an expression vector for PKI. The data are provided as relative Luc activities (mean ± SEM). The numbers above the bars represent fold-stimulation of reporter activity resulting from expression of Ets-2 and PKA. If letters above bars are different, there was a significant effect of treatment (P < 0.05).

 
Phosphorylation Status of CREB and in Vivo Association of CREB with Ets-2
To determine whether activation of the hCG{alpha} promoter by the PKA pathway involved phosphorylation of CREB, we first assessed whether the amount of CREB phosphorylated at Ser-133 changed in response to various treatments (Fig. 10Go, A and B). As expected, levels of phosphorylated CREB were markedly increased in 3T3 (Fig. 10AGo, upper panel) cells exposed to 8-bromo-cAMP (~7.5-fold increase) or transfected with PKA (~30-fold increase; supplemental Fig. 3CGo, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Ectopic expression of Ets-2 had no effect on CREB phosphorylation in the absence of 8-bromo-cAMP treatment or PKA expression, but, in the presence of an activated PKA pathway, there was a further elevation in CREB phosphorylation (~18-fold and ~40-fold increases, respectively; supplemental Fig. 3CGo). As expected, the PKA inhibitor H-89 reversed the effects of Ets-2 and 8-bromo-cAMP. Steady-state levels of CREB were not altered by either activation of PKA or overexpression of Ets-2 (Fig. 10AGo, bottom panel).



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  		Fig. 10. Association between Ets-2 and CREB Is Increased by Phosphorylation of CREB

To assess the phosphorylation status of CREB, whole cell lysates from 3T3 (A) or JAr (B) cells were subjected to immunoblot analysis using anti-phosphoCREB (top panels) or an antibody against CREB that recognized both nonphosphorylated and phosphorylated CREB (bottom panels). To determine whether CREB and Ets-2 associate with each other, proteins from JAr cells that immunoprecipitated with anti-Ets-2 antibodies were subjected to immunoblot analysis using anti-CREB antibodies (C). Proteins from JAr cells that immunoprecipitated with anti-CREB antibodies were subjected to immunoblot analysis with anti-Ets-2 antibodies (D). The coimmunoprecipitation experiments were performed with both nontransfected JAr cells (C and D, lane 2) and with JAr cells transfected with expression vectors for both Ets-2 and CREB (C and D, lanes 4 and 5). Some samples were treated with 500 µM 8-bromo-cAMP before cell lysis as indicated (C and D, lanes 3 and 5). Samples were eluted without using any reducing materials and subjected to electrophoresis in the presence of reducing agents. Lane 6 indicates immunoprecipitation by using a nonspecific IgG.

 
In JAr cells, levels of phosphorylated CREB were elevated approximately 3-fold after exposure to 8-bromo-cAMP or ectopic expression of PKA (Fig. 10BGo, upper panel, and supplemental Fig. 3DGo). However, expression of Ets-2 did not further increase CREB phosphorylation (Fig. 10BGo, upper panel, and supplemental Fig. 3DGo). Steady-state levels of total CREB protein were unchanged (Fig. 10BGo, bottom panel).

To examine whether Ets-2 physically interacts with CREB, the presence of CREB in anti-Ets-2 immunoprecipitates prepared from JAr cells was determined. CREB was detected both in total cell lysates and in anti-Ets-2 immunoprecipitates from untreated JAr cells (Fig. 10CGo, lanes 1 and 2). Activation of the PKA signaling pathway with 8-bromo-cAMP increased levels of CREB present in anti-Ets-2 immunoprecipitates (Fig. 10CGo, lane 3). Increased levels of CREB in anti-Ets-2 immunoprecipitates were also observed in cell lysates from JAr cells transfected with expression vectors for both proteins and treated with 8-bromo-cAMP (Fig. 10CGo, lanes 4 and 5, and supplemental Fig. 4Go, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). To confirm that activation of PKA signaling results in increased association of CREB and Ets-2, the presence of Ets-2 in anti-CREB immunoprecipitates was also determined. Treatment of JAr cells with 8-bromo-cAMP markedly increased the levels of Ets-2 that were present in anti-CREB immunoprecipitates, both in nontransfected cells and in cells transfected with expression vectors for both plasmids (Fig. 10DGo and supplemental Fig. 4Go).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Regulation of gene expression requires the integration of inputs from multiple cellular signaling pathways (56, 57). Accordingly, many genes have multiple cis-acting regulatory elements in their promoter regions, so that it is the assembly of unique combinations of activated transcription factors that regulate expression of any particular gene in a cell-type specific and temporally regulated manner. Efficient transcription of the hCG{alpha} gene requires several different cis-acting elements, but, most importantly, an 18-base nucleotide sequence containing two CREs. The adjacent CRE motifs, which are required for cAMP responsiveness of hCG{alpha} expression, bind CREB and are the downstream targets for hormones and growth factors that operate through the PKA signal transduction pathway. Because the CREs are essential for efficient transcription of the hCG{alpha} gene, they have generally been regarded as the dominant enhancer elements controlling promoter activity, with other regions such as the URE and {alpha}-ACT regions having subordinate roles (29, 33, 34, 35, 36, 37). The data presented in this paper demonstrate that Ets-2, a transcription factor not previously implicated in hCG{alpha} expression, has an important functional role in both basal and cAMP-stimulated expression of hCG{alpha}. We have identified two adjacent binding sites for Ets-2 within the proximal hCG{alpha} promoter and demonstrated their involvement in both basal and cAMP-stimulated expression from the hCG{alpha} promoter. Ectopic expression of Ets-2 is sufficient to increase expression from the proximal hCG{alpha} promoter and, in the presence of an activated PKA pathway, enables maximal stimulation of hCG{alpha} expression. Finally, we demonstrate that the endogenous Ets-2 protein is recruited to the proximal hCG{alpha} promoter, and that elimination of endogenous Ets-2 protein reduces both basal expression from the hCG{alpha} promoter and cAMP-stimulated expression of hCG{alpha} protein.

Examination of the 1.4-kb hCG{alpha} promoter revealed the presence of at least seven candidate binding sites for members of the Ets transcription factor family. Three EBS located in the distal hCG{alpha} promoter between nucleotides –1443 and –254, are not likely to contribute to activation of hCG{alpha} expression by Ets-2, because the proximal promoter region contained within the first 254 bp was sufficient for full responsiveness to both Ets-2 and cAMP. Therefore, our mutational analysis was focused on the four EBS present in the proximal promoter. Deletion analysis of the proximal promoter revealed that two adjacent EBS immediately upstream of a consensus AP-1 binding site are required for Ets-2-mediated transactivation (Fig. 4Go). Importantly, both of these EBS are capable of binding Ets-2 in a sequence-specific manner. It is not clear from our Scatchard analysis whether one or both sites are occupied simultaneously by two Ets-2 protein molecules. However, our mutational analysis supports the notion that both binding sites are functional, because mutation of either individual motif reduced expression from the hCG{alpha} promoter whereas mutation of both binding sites resulted in a further reduction of promoter activity.

The two functional Ets-2 binding sites in the hCG{alpha} promoter are located approximately three helical turns downstream from the two adjacent CREs previously identified in the hCG{alpha} promoter. The CREs are bound by CREB, which requires phosphorylation by PKA at Ser133 for full transactivation potential (38, 39, 58, 59). As expected, we found that activation of PKA signaling increased CREB phosphorylation in JAr cells. Surprisingly, we found that ectopic expression of Ets-2 also increased steady-state levels of CREB phosphorylation, suggesting crosstalk between PKA signaling and Ets-2-dependent transcription. Because Ets-2 and CREB can be coimmunoprecipitated from JAr cell extracts, our results are consistent with the notion that CREB and Ets-2 proteins coexist in a complex in vivo. Because the amount of the Ets-2-CREB complex increases after activation of PKA signaling, it is possible that phosphorylation of one or both transcription factors stabilizes the complex. The formation of a complex between Ets-2 and CREB in a phosphorylation-dependent manner may facilitate binding of the respective proteins to their cognate sites in the hCG{alpha} promoter. Surprisingly, we failed to note the presence of CREB on the hCG{alpha} promoter by chromatin immunoprecipitation in unstimulated JAr cells. In contrast, association of CREB to the hCGß5 promoter was readily detected, as was binding of Ets-2 to both the hCG{alpha} and hCGß5 promoters. It is not known whether Ets-2 and CREB contact each other directly or whether other proteins, such as CREB binding protein may bring together both Ets-2 and CREB into a multimeric transcriptional activation complex (39, 58, 59). It is likely that Ets-2 and CREB act together to activate a number of additional genes in addition to hCG{alpha} and hCGß, because functional Ets and CRE motifs have been implicated in the up-regulation of at least two other genes (60, 61).

Ets-2 has a central coordinating role as a regulator of several genes that are characteristically up-regulated in trophoblast as it approaches functional maturity (20, 62, 63), including the one for hCGß5 which is the main contributor to ß-subunit synthesis in the human placenta (23). The hCGß5 gene is a particularly interesting example, because, if active hormone is to be produced in quantity, hCGß must be expressed coordinately with hCG{alpha}. This expectation is confounded by the fact that the promoter regions thought to be responsible for controlling the expression of the two genes in trophoblast show no regions of sequence identity indicative of common cis-regulatory codes. For example, the hCGß5 subunit gene lacks a CRE and a well-defined cAMP responsive region. Nevertheless, careful functional analysis of the two promoters has indicated that each possesses an Ets-2 binding element placed just upstream of a possible AP-1 site. Both promoters contain two EBS. In the hCGß5 gene, the core GGAA/T sequences are on the sense strand of the promoter and located one helical turn apart. In contrast, in the hCG{alpha} promoter the two sites are immediately adjacent to each other and on opposite strands (Fig. 1Go). The putative AP-1 sites on the two promoters are also dissimilar, and neither represents a consensus sequence. Importantly, and in view of the need for coordinate expression of hCG{alpha} and hCGß, the Ets-2/AP-1 enhancer in the hCGß5 promoter, like that in the hCG{alpha} promoter, is crucial for full responsiveness of the genes to cAMP and also enables an input from the MAPK signal transduction pathway (64, 65).

Another trophoblast-expressed gene matrix metalloproteinase-3 uses a bipartite Ets-2 binding site (66) somewhat similarly organized to that for hCGß, whereas others, such as those for interferon-{tau} (19, 62), urokinase plasminogen activator (63), placental lactogen II (20), and human collagenase (67), have just a single EBS with a single GGAA motif. Each of these genes contain an AP-1 site located adjacent to the Ets site, and the compound element confers responsiveness to both Ets and Ras. It is not known whether these complex Ets-2/AP-1 enhancers mediate sensitivity of these genes to cAMP, although our results imply that this may be the case. Ets-2 may function as a master switch for gene activation in trophoblast, required both for basal transcription and, in at least some instances, for a robust response to elevated cAMP levels.

Ets-2 expression is necessary for development of the placenta in mice (25), with null embryos dying at around d 7.5 as a result of trophoblast failure. Nevertheless, blastocysts do form and initiate implantation, suggesting that Ets-2 is not necessary for specifying trophectoderm before its emergence at the morula-blastocyst transition. More likely, Ets-2 coordinates early expression of genes that provide functional characteristics to cytotrophoblast, including the production of key enzymes and hormones, after trophectoderm commitment but before full cytodifferentiation begins. In human trophoblasts, cAMP-dependent processes appear to play an important role in late differentiation events (68, 69), and promote the formation of the syncytial trophoblast where hCG production becomes maximal (70). It is likely that Ets-2 acts before this late stage of the differentiation pathway that is dependent upon cAMP signaling. As part of this early coordinating role, Ets-2 would appear to regulate the expression of both hCG{alpha} and -ß, but full production of the hormone in end-stage cells is probably driven by cAMP/CREB-mediated events.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Constructs
A 1500-bp hCG{alpha} promoter fragment was subcloned into the HindIII and XhoI restriction sites of the Luciferase (Luc) reporter plasmid, pGL2 Basic (Promega Corp., Madison, WI). A series of deletion mutants with HindIII and XhoI linkers was generated from the 1500-bp fragment by PCR with promoter-specific primers –1443 XhoI sense (se), –677 XhoI se, –254 XhoI se, –107 XhoI se, –47 XhoI se, and hCG{alpha} HindIII antisense (as) (Table 1Go) (71). hCG{alpha} antisense oligonucleotide in combination with all sense oligonucleotides will generate –1443/+48, –677/+48, –254/+48, –107/+48, –47/+48 hCG{alpha} promoter fragments, respectively. Each was subcloned into the same pGL2 Basic vector as above. PCR was used to introduce StuI restriction sites to mutate the EBS at –81 to –78 (EBS1) and –75 to –72 (EBS2) (Fig. 1Go). StuI restriction sites were introduced similarly into each of the two cAMP-responsive elements (CRE 1, –146 to –129; CRE 2, –128 to –111) on the –254 Luc hCG{alpha} promoter construct (Fig. 1Go). A double Ets site mutant construct was prepared by introducing a ClaI site into the first site (EBS1) and a StuI site into the second (EBS2). A double CRE site mutant construct was prepared by introducing a ClaI site into CRE 1 and a StuI site into CRE 2. A ClaI site was also used to disrupt the AP-1 like sequence (Fig. 1Go). Combinations of CRE and Ets site mutations were accomplished similarly, e.g. introduction of a ClaI site into CRE 2 and a StuI site into EBS 2; a StuI site into both CRE 2 and EBS 2; and a ClaI site into CRE 1 (23, 72). The mutated PCR fragments were amplified in the pBluescriptSK vector (Stratagene, La Jolla, CA), released by HindIII and XhoI digestion, and ligated into pGL2 Basic vector. Fidelity of all constructs was verified by DNA sequencing with Luc vector-specific primers (GL se and GL as). Primers (MEts site1 se and as, MEts site2 se and as, MAP1 se and as, MCRE1 se and as, MCRE2 se and as, Mdouble Ets se and as, Mdouble CRE se and as) used for generating the mutants by PCR are listed in Table 1Go. The wild-type Ets-2 (Ets-2T72), a mutant construct encoding an alanine in Ets-2 (Ets-2A72), the activated Ras construct (pHO6T1), and its parental vector (Homar6) have been described previously (46, 73, 74). The ß-galactosidase gene driven by Rous Sarcoma virus long terminal repeat (pRSVLTR-ßgal) was used as an internal control in experiments performed on choriocarcinoma cell lines. A Renilla luciferase control vector (pRL-Simian virus 40; Promega Corp.) was used for the 3T3 cell experiments. The expression vector for wild-type PKA catalytic subunit (RSV-PKA), a synthetic gene encoding the heat-stable inhibitor of catalytic subunit of cAMP-dependent PKA (RSV-PKI), and an empty vector (RSV-globin) were a gift from Mark S. Roberson (Cornell University, Ithaca, NY). The RSV-PKA and RSV-PKI constructs preparation has been described previously (75, 76).

Cell Culture
JAr cells [HTB 144, American Type Culture Collection (ATCC) Manassas, VA] were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. JEG-3 (HTB 36; ATCC) cells were cultured in MEM supplemented with 2 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 1 mM sodium pyruvate, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. NIH 3T3 cells (CRL-1658; ATCC) were cultured in DMEM supplemented with 2 mM L-glutamine, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Conditions for Transfection
Transfections of JEG-3 and JAr cells, except in the case of siRNA experiments, were performed by the standard calcium phosphate method (23). Briefly, cells were plated at a cell density of 1 x 105 per 60-mm dish, cultured overnight, and incubated with DNA/Ca3 (PO4) precipitates in DMEM supplemented with 10% FBS for 7–8 h. Concentrations of plasmids were: hCG{alpha} promoter (2.5 µg/dish), Ets-2 (0.5 µg/dish), Ets-2A (0.5 µg/dish), PKA (0.5 µg/dish), PKI (0.5 µg/dish), Ras (1.2 µg/dish), and ß-galactosidase (50 ng/dish). DNA concentrations were normalized with either the PCGN expression vector or, in the case of PKA constructs, with RSV-globin. Cells were washed with PBS and maintained for an additional 36 h on complete medium before extraction with "Galacto-light plus" lysis solution (Tropix, Bedford MA). Luciferase activities were determined by adding Luc assay reagent (Promega Corp.) and recording light emission every 15 sec in a Turner luminometer (Turner Biosystems, Sunnyvale, CA).

Transfection of 3T3 cells was performed based on the procedures described previously (77). Concentrations of plasmid were: hCG{alpha} promoter (3.5 µg/dish), Ets-2 (0.5 µg/dish), Ets-2A (0.5 µg/dish), PKA (0.5 µg/dish), PKI (0.5 µg/dish), Ras (1.2 µg/dish), and Renilla Luc (50 ng/dish). PD98059 and U0126 (Calbiochem, San Diego, CA) were used as MAPK kinase (MEK) 1/2 inhibitors and SB 202190 as a p38 stress kinase inhibitor; H-89 (dihydrochloride) and 14–22 amide (cell permeable, myristoylated) (Calbiochem, San Diego, CA) were used as PKA inhibitors. After transfection (8 h for JAr and JEG-3 cells and 20 h for NIH 3T3 cells) and removal of DNA/Ca3 (PO4) precipitates, cells were washed with PBS, incubated for 2–3 h on complete culture medium, and then treated with individual inhibitors (25 µM PD98059; 1 µM U0126; 5 µM SB202190; 10 µM H-89; and 2 µM 14–22 amide, each dissolved in dimethylsulfoxide). Dimethylsulfoxide alone was used as the vehicle control. In a series of separate experiments, cells were exposed to 8-bromo-cAMP (Sigma, St. Louis, MO) by diluting a stock solution (dissolved in PBS) in medium to a final concentration of 500 µM for 40 h, beginning 8 h after transfection for JAr and JEG-3 cells and 20 h for 3T3 cells.

EMSA
Double-stranded synthetic oligonucleotides were annealed and labeled using [{gamma}32P]ATP and T4 polynucleotide kinase (78). Two separate probes were designed to encompass each individual EBS: EBS1, nucleotides –91 to –74; EBS2, nucleotides –79 to –60. A third probe, EBS1/2, was designed to encompass both EBS1 and EBS2 and contained sequences from –85 to –67. Oligonucleotide sequences are listed in Table 1Go. GST-Ets-2 fusion protein was synthesized as described previously (79). Bacterial growth, isopropyl-ß-D-thiogalactopyranoside treatment and preparation of extracts followed the Amersham Biosciences (Piscataway, NJ) protocol. Protein concentration was measured by the procedure of Bradford (80). Either GST-Ets-2 (2 µg) or GST alone was incubated with labeled, double-stranded oligonucleotides as probes (10,000 to 20,000 cpm; 25 fmol) in the presence of 1 µg of nonspecific carrier DNA (polydeoxyinosine:deoxycytidine) (23). Annealed double-stranded unlabeled oligonucleotides were used as a specific competitor for probe binding at 5, 10, 50, 100, 250, and 500 molar excess. Competition assays for each of the three probes were performed separately. After drying, gels were exposed to BioMax MS (maximum sensitivity) film (Eastman Kodak Company, Rochester, NY) in the presence of Kodak TranScreen HE (high energy) intensifying screens. After developing the films, densitometry was performed by using Kodak ID image analysis software (Kodak Scientific Imaging System, New Haven, CT). Saturation binding curves (as band intensities), Scatchard plots, and dissociation constants (Kd) were calculated on GraphPad Software, GraphPad Prism version 4.0 (GraphPad Software, Inc., San Diego, CA). In some experiments, affinity-purified antibodies against Ets-2 (Ets-2 antibodies raised against the amino terminal fragment of human Ets-2 having amino acids 1–326; Ezashi, T., and R. M. Roberts, unpublished observations) were used to supershift the protein-DNA complex. Annealed double-stranded oligonucleotides containing mutations within the core EBS were used to confirm specificity of binding. EMSAs were also performed with 10 µg of JAr and JEG-3 nuclear extracts (81) under the conditions described above.

Chromatin Immunoprecipitation (ChIP) Assays
ChIP analysis on human JAr choriocarcinoma cells was conducted essentially as described previously (82). Briefly, 2 x 107 JAr cells were treated with 1% formaldehyde for 10 min at room temperature, and the chromatin was prepared. The diluted chromatin was incubated with rabbit preimmune serum and with a salmon testes DNA/protein A agarose/BSA slurry (Sigma) for 3 h at 4 C with rotation. Twenty percent of the precleared chromatin was saved as "total input" control. The remaining chromatin was either left untreated ("no antibody" control), treated with 2 µg of antimouse hemagglutinin antibody (Covance Research Products, Berkeley, CA; "nonspecific antibody" control), treated with 2 µg of antirabbit affinity-purified Ets-2 antibody (described above), or treated with 2 µg of antirabbit CREB antibody (Cell Signaling Technology, Beverly, MA). The immune complexes were collected with a salmon testes DNA/protein A agarose/BSA slurry, and consecutively washed with low salt wash buffer [0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl], high salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl], LiCl wash buffer [250 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.0)] and three times with 1 x TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA]. Complexes were eluted with freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3). The formaldehyde crosslinks were reversed in the presence of 10 µg RNAse and 0.3 M NaCl at 65 C for 6 h. The DNA and debris were precipitated with 100% ethanol at –20 C overnight; pelleted by centrifugation; resuspended in 100 µl of 40 mM Tris-HCl (pH 6.5), 10 mM EDTA, 20 µg proteinase K; and incubated at 45 C for 2 h. The DNA was purified using QIAQuick spin columns (Qiagen, Germantown, MD) and eluted in 50 µl of 10 mM Tris-HCl (pH 8.0). A total of 2.5 µl of ChIP DNA was used in each PCR. The primers used in the ChIP assay are listed in Table 1Go. The PCR conditions were 95 C for 2 min and 95 C for 30 sec, 52 C for 30 sec, 72 C for 2 min for 35 cycles followed by prolonged extension at 72 C for 10 min. The PCR products were visualized by ethidium bromide staining after electrophoresis.

siRNA Transfections
siRNA duplexes (0.05 µmol) against human Ets2 (J04102) and against a nonspecific control, human Keap1 (BC002930), were designed using software available from Dharmacon (Chicago, IL). The sequences designed for this study are mentioned in Table 1Go. JAr cells were plated on 24-well plates at a density of 2 x 104 per well and transfected in triplicate in OptiMEM medium using Lipofectamine2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Cells were transfected with 200 ng of empty vector or 200 ng of –254 hCG{alpha} reporter construct either alone or with increasing concentrations (5, 15, 50, and 100 nM) of duplex siRNA together with 50 ng of Renilla luciferase as an internal control. A 100 nM concentration of siRNA against Keap1 was used as a nonspecific control. Cell lysates were analyzed by the Dual-Light Luciferase reporter assay (Promega Corp.) according to the manufacturer’s protocol.

For biochemical analyses, 8 x 104 JAr cells per well were transfected with 0, 15, 50, 100, and 200 nM siEts2-siRNA or 200 nM siKeap1-siRNA described above. At 6 h post transfection, cells were re-fed with complete RPMI medium and treated with 500 µM 8-bromo-cAMP to elevate hCG{alpha} and ß protein production for 36 h. Cells were washed in PBS and harvested in 100 µl of 1x sample buffer at 40 h post transfection. Equal amounts of protein were resolved by electrophoresis through 17.5% SDS-PAGE gels; transferred to nitrocellulose; blocked in 1x PBS, 0.1% Tween 20, and 5% nonfat dry milk at 4 C overnight; and detected with antimouse hCG{alpha} (Abcam, Cambridge, MA) or antimouse hCGß antibodies (Spring Bioscience, Fremont, CA) at 1:1000 dilution. The same blots were stripped and reprobed with anti-Ets-2 antibody at 1:10,000 dilution.

Western Blotting of Phosphorylated CREB and Total CREB Protein
The relative concentrations of phosphorylated CREB protein in whole cell lysates were assessed by comparing the relative band intensities of phosphorylated and nonphosphorylated CREB on Western blots from 50 µg of total cellular extracts from 3T3 and JAr cells. Affinity purified antibody that specifically recognizes the phosphorylated CREB epitope (Ser133) was obtained from Cell Signaling Technology and diluted 1:1000 before use. Antibody to CREB (Cell Signaling Technology) was diluted 1:2000. Cell extracts were centrifuged, and the supernatant was analyzed by SDS-PAGE on 10% gels (23). Proteins were transferred to polyvinylidene fluoride membranes, and detection of bound IgG was performed as described previously (23). The relative amount of phosphorylated CREB was measured by densitometry with Kodak ID image analysis software (Kodak Scientific Imaging System).

Coimmunoprecipitation Assays
JAr cells (on 100-mm dishes) were transfected with 1 µg cytomegalovirus Ets-2 and 1 µg cytomegalovirus CREB after standard calcium phosphate method (23). Some groups of transfected cells and controls were treated with 500 µM cAMP as described above. In each experiment, which was repeated three times, extracts were prepared from three dishes in a lysis buffer containing detergent plus protease and phosphatase inhibitors (23). Cell lysates were cleared by centrifugation at 14,000 rpm for 30 min at 4 C. About 500 µg of freshly isolated protein (in 1 ml of buffer) was incubated overnight with 5 µg of affinity purified antibody (anti-Ets-2 from Santa Cruz Biotechnology, anti-CREB from Cell Signaling Technology, or purified nonspecific IgG from Sigma). The binding buffer was identical to the lysis buffer except that it contained 500 mM NaCl. After centrifugation, supernatants were mixed with 15 µl of swollen, washed Immunopure immobilized Protein G-Agarose beads (Pierce, Rockford, IL) for 6 h. The beads were then washed five to six times in cold binding buffer. Bound immune complexes were eluted in sample buffer [2% SDS in 0.063 M Tris-HCl (pH 6.75), 80 C for 20 min] in the absence of reducing agent, and analyzed by SDS-PAGE under both reducing and nonreducing conditions. Analysis was performed on 8–16% gradient gels (Pierce) and by Western blotting as described above and elsewhere (23). The immune complexes formed in the presence of Ets-2 antibody were detected on the Western blots with CREB antibody. Those formed by addition of anti-CREB were detected by using Ets-2 antibody preparations. Control immunoprecipitations were carried out with a nonspecific IgG and analyzed in parallel. These controls also indicated the absence of IgG contamination in samples eluted from Protein-G Agarose beads, an important consideration in the detection of Ets-2, which has an apparent Mr similar to that of heavy chain IgG under reducing conditions. A sample of 20 µg protein extracted directly from the cells was analyzed in parallel on each SDS-PAGE gel to determine the relative amount of Ets-2 and CREB present. Band intensity was quantitated by scanning densitometry (Kodak Scientific Imaging System).

Statistical Procedures
Each transfection experiment was run in triplicate, and values were averaged. Experiments were replicated on at least three to five occasions. Values are presented as means ± SEM for the different replicates and expressed both as relative Luc activity over basal expression and fold activation. Statistical analyses were performed by ANOVA followed by a least square means (LSM) t test (SAS version V-6, TS 020; SAS Institute Inc., Cary, NC). Significance was accepted at P < 0.05.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Mark S. Roberson (Cornell University, Ithaca, NY) for his gift of RSV-PKA and RSV-PKI expression vector constructs; and Drs. Michael Ostrowski (Ohio State University, Columbus, OH), Toshihiko Ezashi (University of Missouri, Columbia, MO), and Richard Maurer (Oregon Health and Science University, Portland, OR) for providing some of the other constructs used in these experiments.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grants HD 42201 and HD 21896.

First Published Online January 6, 2005

Abbreviations: AP-1, Activator protein-1; as, antisense; 8-bromo-cAMP, 8-bromoadenosine cAMP; CG, chorionic gonadotropin; ChIP, chromatin immunoprecipitation; CL, corpus luteum; CRE, cAMP responsive-element; CREB, CRE-binding protein; EBS, Ets binding site(s); FBS, fetal bovine serum; GST, glutathione-S-transferase; hCG, human CG; Luc, luciferase; MEK, MAPK kinase; PKA, protein kinase A; PKI, heat-stable inhibitor of PKA; se, sense; siRNA, short interfering RNA; SDS, sodium dodecyl sulfate; URE, upstream regulatory element.

Received for publication August 9, 2004. Accepted for publication December 22, 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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