A Central Role for Ets-2 in the Transcriptional Regulation and Cyclic Adenosine 5'-Monophosphate Responsiveness of the Human Chorionic Gonadotropin-ß Subunit Gene

Debjani Ghosh, Toshihiko Ezashi, Michael C. Ostrowski and R. Michael Roberts

Department of Animal Sciences (D.G., T.E., R.M.R.), University of Missouri, Columbia, Missouri 65211; and Department of Molecular Genetics (M.C.O.), Ohio State University, Columbus, Ohio 43210

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ets-2 has an important role in controlling the differentiation of the placenta. Here we show by truncation and mutational analysis that two closely spaced Ets-2 binding sites in the proximal promoter of the human chorionic gonadotropin ß5 (hCGß5) gene constitute a major enhancer for hCGß gene expression in JAr and JEG-3 human choriocarcinoma cells and in mouse NIH3T3 cells. Contrary to a previous report, we also demonstrate that the ability of Ets-2 to enhance transcription is subject to control by the Ras/MAPK pathway, although this relationship is less easily demonstrable in JAr and JEG-3 choriocarcinoma cells than in the 3T3 cells because the former already possess a fully activated MAPK pathway and contain Ets-2 phosphorylated at threonine residue at T72. Coexpression of Ets-2 and activated Ras in 3T3 cells led to activation of MAPK/ERK kinase 1/2, phosphorylation of Ets-2 at T72, and an approximately 120-fold up-regulation of reporter gene expression from a short (-175) hCGß promoter. Fold activation in JAr and JEG-3 cells was rather less (20- to 30-fold), but basal activity was much higher. These effects on promoter activity were largely reversed in presence of the MAPK inhibitor PD98059, which prevents ERK1/2 activation, and partially reversed by mutating T72 on Ets-2. We finally show that the ability of 8-bromoadenosine-cAMP to stimulate hCGß promoter activity in JAr and JEG-3 cells occurs with a short promoter lacking the upstream elements previously considered to be essential for cAMP activation of the gene and, through mutational analysis, confirm that the major cAMP effects on the hCGß promoter are mediated through the proximal Ets-2 enhancer. The data are consistent with the hypothesis that Ets-2 has a general and possibly essential role in controlling the activity of genes associated with trophectoderm differentiation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CHORIONIC GONADOTROPIN (CG) is a product of the trophoblast of higher primates (1, 2). In these species, human CG (hCG) is necessary to maintain the viability of the corpus luteum, which, in an animal that is not pregnant, would normally regress before the next ovulatory cycle. Luteal rescue ensures the continued production of progesterone and the maintenance of a uterine environment appropriate for the continued development of the conceptus. CG synthesis is regulated primarily by the rate at which its two subunit genes are transcribed (3). In the human, there is a single {alpha}-subunit gene on human chromosome 9, and six closely similar ß-subunit genes (hCGß-7, -8, -5, -1, -2, and -3) linked in tandem on chromosome 19 (4). Of the latter, hCGß5 represents the protein isoform expressed predominantly in placenta and choriocarcinoma cells and has been studied most extensively (5, 6). Curiously, however, the promoter regions of the single hCG{alpha} subunit and the hCGß-5 gene, which must be coordinately up-regulated in trophoblast, have little or no sequence identity and appear to carry few common enhancer elements that might explain how their expression might be synchronized, particularly in early pregnancy when production of the intact hormone is vital. Both genes are silenced by the embryonic transcription factor, Oct-4, which might explain why neither subunit gene is expressed until the trophoblast first forms (7, 8), but, again, the silencing mechanisms operating are very different. Similarly, the expression of both hCG subunit genes is responsive to cAMP, but the ß-gene lacks recognizable cAMP control elements [cAMP response element (CRE); TGACGTCA], which are present in the proximal promoter of the {alpha}-subunit gene.

The hCGß5 gene, the main focus of the present paper, unlike the {alpha}-subunit gene, lacks a canonical TATA box (Fig. 1Go), and a relatively short -78-bp upstream region of the initiator element is sufficient for basal expression (9, 10). Transcription in choriocarcinoma cells is enhanced when a -3.5 kb to -1.7 kb region is present on promoter constructs, but this distal segment appears to play no role in either trophoblast-specific expression or in cAMP responsiveness (11, 12). Instead, cAMP effects are believed to be targeted through a more proximal (-311 to -202) region of the promoter (10, 11). A trophoblast-specific element has been defined in the same region (-305 to -279) (10), as well as two Sp1/activating protein (AP)-2 binding sites that have been implicated in both basal activity and cAMP responsiveness (13).



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Figure 1. The Nucleotide Sequence of the hCGß Gene Promoter from the Initiator Element to Position -325

This promoter has no TATA element but contains a possible initiator element (C) at the transcription start site. The underlined region (-311 to -202) has been implicated in cAMP regulation of the promoter. Two potential Ets factor-binding sites are boxed and designated Site-1 and Site-2. Other possible Ets factor-binding sites possessing a core GGA sequence on either the positive or negative DNA strand are circled. A possible AP-1-like sequence immediately proximal to the Site-1 Ets site and overlapping the initiator element is also marked.

 
One transcription factor that can up-regulate the promoters of several genes that are expressed in differentiating trophectoderm (14, 15, 16, 17, 18, 19, 20) and that is also essential for placental development in the mouse (21) is the transcription factor Ets-2, a member of an extensive family of proteins that resemble the v-ets oncogene in the E26 retrovirus (22). The characteristic feature of the family is the ETS domain, a highly conserved helix-turn-helix sequence of about 85 amino acids responsible for DNA binding (23, 24). Ets family members recognize a central GGA core sequence, usually within a (C/A)(C/A)GGA(A/T)(A/G) motif (24), which is found in the promoters for large numbers of genes, including that for hCGß (Fig. 1Go). However, Ets-2 and its relatives need to be activated before they are functional in transactivation (25, 26, 27, 28). The best understood, but not necessarily the only mechanism, for Ets-2 activation involves MAPK-mediated phosphorylation of a threonine (T72) within the Ets-2 transactivation domain (29). Consequently, growth factors and other signaling pathways that operate through the Ras, Raf, and ERK1/2 signal transduction pathway, generally appear to control Ets-2 transactivation of its target genes (30, 31, 32).

Johnson and Jameson (33) recently drew attention to one potential Ets-binding site (Site-2, Fig. 1Go) within the proximal promoter region of the hCGß gene, plus a second in the 5'-untranslated region, and showed that Ets-2 transfected into human JEG-3 cells caused a modest 3- to 6-fold increase in hCGß promoter activity. Unexpectedly an activated Ras construct, when coexpressed with Ets-2, did not increase Ets-mediated transactivation of the promoter. Moreover, Ets-2 appeared not to bind to either of the putative Ets-2 binding sites within the hCGß promoter, although deletion of the region containing Site-2 of Fig. 1Go reduced Ets-2 transactivation by about one-half. Deletion of a second site in the 5'-untranslated region of the gene had no effect. The atypical Site-1 (Fig. 1Go) was not studied. Johnson and Jameson (33) concluded that Ets-2 probably did not bind directly to DNA within the proximal promoter but, instead, either interacted with other transcription factors to increase transcription or operated through a more distal binding site, which was not defined. Importantly, however, they demonstrated that coexpression of Ets-2 and the protein kinase A (PKA) catalytic subunit synergistically up-regulated the hCGß promoter. These data suggested that a pathway involving PKA, rather than Ras, was responsible for modulating the ability of Ets-2 to control hCGß subunit gene expression in choriocarcinoma cells.

This laboratory has previously demonstrated that Ets-2 has a major responsibility in controlling the expression of interferon-{tau} (IFN-{tau}) genes in trophectoderm of cattle and sheep (15). This IFN plays an analogous role in large ruminant species to hCG in primates, in that its primary function is in preventing the regression of the corpus luteum during early pregnancy. Consequently, IFN-{tau} genes, like those for the hCG{alpha} and ß subunits, must be rapidly and efficiently up-regulated in trophoblast if the developing embryo is to survive, but just as Jameson and Johnson failed to find any synergism between Ets-2 and Ras in the regulation of the hCGß promoter in JEG-3 cells, we noted that Ras failed to increase Ets-2 transactivation effects on the IFN-{tau} gene promoter in human JAr cells (15), which, like JEG-3 cells, are derived from a chorionic tumor. As an explanation, we suggested that the MAPK signal transduction pathways might be already up-regulated in such transformed cells. In the present paper, we have attempted to resolve some of these contradictions. We show that there are two closely associated Ets-2 binding sites functioning in the hCGß promoter and that Ras and Ets-2 act synergistically to stimulate reporter gene activity. We also validate the prediction that choriocarcinoma cells have a fully active MAPK signal transduction pathway that probably accounts for much of their basal hCGß promoter expression. Finally, we show that cAMP acts primarily, through the proximal Ets-2 enhancer to up-regulate the hCGß promoter.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Effects of Ets-2 and Ras Overexpression on the Activity of Transfected hCGß Promoters in JAr, JEG-3, and 3T3 Cells
Johnson and Jameson (33) demonstrated that, in JEG-3 human choriocarcinoma cells, Ets-2 over-expression provided a modest (3- to 5-fold) stimulatory effect on transcription from a -345 hCGß promoter (Fig. 1Go). These workers were unable to obtain any further up-regulation of this promoter by cotransfecting Ets-2 with an activated Ras construct. Here, we repeated the experiments with JEG-3 cells (Fig. 2BGo), but included additional transfections into a second choriocarcinoma cell line, JAr (Fig. 2AGo), as well as into mouse 3T3 cells (Fig. 2CGo). We employed three hCGß promoters (-325, -295, and -175), but because results with the two longer promoters were essentially identical, data from the -295 construct have been excluded. In JEG-3 and JAr cells basal Luc activity from the short (-175) promoter was only slightly less than from the longer construct. Ets-2 overexpression stimulated transcription from each promoter approximately 20- to 30-fold, significantly higher values than the ones reported earlier in JEG-3 cells (33). Ras alone had a small but significant effect (approximately 2- to 4-fold) on basal Luc activities, but had only a minor effect when coexpressed with Ets-2. These data are consistent with the conclusion (33) that Ras fails to synergize strongly with Ets-2 in choricarcinoma cells.



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Figure 2. Luciferase Expression from hCGß Gene Promoter Elements Transiently Transfected into JAr (A), JEG-3 (B), and NIH3T3 (C)

Cells were transfected with either promoter-Luc constructs (-175 and -325) alone, or in combination with expression constructs for Ets-2, activated Ras, or Ets-2 and Ras in combination. 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. Values above each bar are fold stimulation of reporter activity relative to the basal rate for the particular promoter in a particular cell line. Results from five separate experiments have been calculated as mean ± SE. If letters above bars are different, there was a significant effect of treatment (P < 0.05). In D, Western blotting indicates the relative concentrations of Ets-2 in the three cell lines (3T3, JEG-3, and JAr) before (lanes 1, 3, and 5) and 36 h after (lanes 2, 4, and 6) transfection with the Ets-2 expression construct. Lane 7 is a control and indicates the presence of murine Ets-2 in F9 cells.

 
The results with 3T3 cells were somewhat different (Fig. 2CGo). Basal activities of the promoters were very low, approximately 10% of those observed in the choriocarcinoma cells, and were not significantly influenced by promoter length. Ets-2 overexpression had a modest (6- to 12-fold) stimulatory effect, whereas Ras alone barely increased Luc reporter activity from either promoter construct. When Ets-2 and Ras were transfected together, however, activities from the -175 and -325 constructs were increased 117- and 72-fold, respectively, relative to basal values. This result strongly suggested that the Ras pathway contributes to the ability of Ets-2 to function as a transcriptional activator in 3T3 cells.

Ets-2 Is Expressed in Choriocarcinoma Cells but Poorly in 3T3 Cells
Western blotting experiments performed on 50 µg of cell extracts with anti-Ets-2 antiserum showed that both JEG-3 and JAr cells express Ets-2 when cultured under standard conditions (Fig. 2DGo, lanes 3 and 5), whereas the protein was barely detectable in an equivalent amount of cell extract from 3T3 cells (Fig. 2DGo, lane 1). This low amount of Ets-2 in 3T3 cells does not appear to be an artifact arising from the fact that the antiserum had been prepared against the human protein because Ets-2 was readily detectable, as expected (34) in mouse F9 cells (Fig. 2DGo, lane 7). Transfection with the pCGN-Ets-2 expression construct raised Ets-2 concentrations in 3T3, JEG-3, and JAr cells (Fig. 2DGo, lanes 2, 4, and 6) relative to the control cultures.

Identification of Potential Ets-2 Binding Sites in the hCGß Promoter
A search of the hCGß proximal promoter sequence with the computer software analysis program Mat-Inspector V2.2 (www.gene-regulation.de) revealed several potential Ets-factor binding sites, each containing one of the two hallmark core sequences, GGAA or GGAG (circled or boxed in Fig. 1Go). Six of these (three on the sense strand, three on the antisense strand) were present in the -325 promoter. Two of them (Site-2, with a core GGAA sequence at -37 to -34, and Site-1, with a GGAG sequence at -20 to -17; Fig. 1Go) are the main focus of this paper. Site-2 (CAGGAAA) conforms closely to the Ets-2 consensus binding sequence, whereas Site-1 (GAGGAGG) is atypical. Johnson and Jameson (33), in their experiments, recognized the likely importance of Site-2, but overlooked Site-1, possibly because it is unusual.

Ets-2 Transactivation of Mutated hCGß Promoter Constructs
To make a preliminary test of which of the putative Ets-2 sites in the -325 hCGß promoter were functional, several truncated constructs were prepared and cotransfected with Ets-2 and activated Ras expression plasmids into 3T3 cells (Fig. 3Go). The mouse cells were chosen for these initial experiments because they demonstrated little endogenous Ets-2 protein in cell extracts and were more responsive to Ets-2 and Ras overexpression than either choriocarcinoma cell line (Fig. 2Go). Truncation of the promoter from -325 to -175 increased rather than decreased Luc reporter activity in 3T3 cells, thereby ruling out a functional role for the three most distal sites (Fig. 3AGo). Deletion to position -34 reduced reporter activity by about 80%, whereas further deletion to -17 essentially abolished activity. These data indicated that a major Ets-2/Ras responsive site lay between -175 and -34 and that a second was probably present between -34 and -17. This result was consistent with the hypothesis that Sites-1 and-2 (Fig. 1Go) might be functional.



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Figure 3. Deletion (A) and Mutational (B–D) Analysis of the Proximal hCGß Gene Promoter

A, A series of deletion fragments from the hCGß proximal promoter (-325/+65, -295/+65, -175/+65 -34/+65, -17/+65) were cloned into pGL2 Basic-Luc vector and tested for expression in 3T3 cells, either alone or in association with cotransfected Ets-2, Ras, or Ets-2/Ras expression constructs. The values shown here were obtained in association with Ets-2 and Ras coexpression. B, Ets-2 binding Sites-1 and -2 (Fig. 1Go) were mutated in the -175/+65 promoter as described in Materials and Methods and tested for expression by transient transfection in 3T3 cells after Ets-2/Ras cotransfection. In C and D, the experiment described in B with 3T3 cells was repeated in JAr (C) and JEG-3 (D) cells, respectively. In A and B, values were calculated from four different transfections (each performed in triplicate on different days) and are shown as means ± SE. In C and D, experiments were performed in triplicate on two different days. Data are displayed as fold activation relative to basal expression obtained in absence of Ets-2 and Ras cotransfection in that particular cell line. Where letters differ above individual bars, differences are significant (P < 0.05).

 
To determine which, if any, of the two potential Ets-2 binding sites in the proximal promoter region were responsible for Ets-2/Ras transactivation, each was mutated separately by introducing a StuI restriction site. Mutations introduced into either Sites-1 or -2 (Fig. 1Go) reduced Ets-2/Ras transactivation of the -175 hCGß promoter by about one half in 3T3 cell. Mutations in both sites, however, led to loss of more than 90% of Luc reporter activity (Fig. 3BGo).

The experiments with the promoters mutated at the two putative Ets-2 binding sites were repeated with JAr and JEG3 choriocarcinoma cells (Fig. 3Go, C and D). Again, mutation of either Site-1 or Site-2 reduced Ets-2 transactivation by about half, whereas mutation of both sites had a more pronounced effect. Why the consequences of the double mutation were more severe in JAr cells than in JEG-3 cells is unclear.

The Site-1 and Site-2 mutations also reduced basal promoter activity in JAr and JEG-3 cells in absence of Ets-2 transfection (Fig. 4Go). The effects of the mutations were of a similar magnitude to those noted after Ets-2 overexpression (Fig. 3Go, C and D) and were not unexpected because both cell lines express Ets-2.



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Figure 4. The Effect of Mutations in the Site-1 and Site-2 Ets-2 Binding Sites on Basal Activity of the -175 hCGß Promoter Transfected into JAr and JEG-3 Choriocarcinoma Cell Lines

These experiments, in contrast to those in Fig. 3Go, were carried out without Ets-2/Ras cotransfection, and values are compared with luciferase activity of the plasmid lacking the -175 hCGß promoter. A–E, Experiments in JAr cells; b–e, JEG-3 cells. A, Empty, vector; B, b, wild-type, nonmutated -175 hCGß promoter; C, c, Site-1 mutated promoter; D, d, Site-2 mutated promoter; E, e, Site-1 and -2 mutated promoter. Where letters differ above individual bars, values are significantly different (P < 0.05).

 
Together, these results confirm that Sites-1 and -2 have an important role in controlling hCGß promoter responses to Ets-2 and Ras and that the two sites may be inter-dependent in their action.

Ets-2 Binding to Sites-1 and -2 of the hCGß Proximal Promoter
To provide further confirmation that Sites-1 and -2 bound Ets-2, EMSA experiments were performed with recombinant Ets-2-glutathione-S-transferase (GST) fusion protein and double-stranded oligonucleotides (Table 1Go) encompassing the core sequences of each site (Fig. 5Go, A–D). GST failed to bind a probe representing either site. Ets-2-GST gave a strong band with both labeled probes, and, in each case, the complexes could be dissociated by increasing the concentration of unlabeled oligonucleotide in the reaction mixture (Fig. 5Go, A and C). A 500-fold excess of nonspecific competitor oligonucleotide (double-stranded, annealed oligonucleotide, mutated in the Ets site) could not dissociate the complex (Fig. 5Go, B and D). Double-stranded synthetic oligonucleotides, with disrupted Ets binding sites, did not form complexes (Fig. 5Go, B and D). A purified antibody against Ets-2 failed to supershift the complexes, but did cause their dissociation (Fig. 5Go, B and D). These experiments indicate that Ets-2 can form specific associations with oligonucleotides representing Sites-1 and -2.


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Table 1. Synthetic Oligonucleotides Used for PCR and EMSA

 


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Figure 5. EMSA Analysis to Assess the Ability of Ets-2 Protein (A–D) and Nuclear Extracts from JAr Cells (E–H) to Bind Specifically to Double-Stranded Oligonucleotides Representing Putative Ets-2 Sites-1 and -2

Two oligonucleotide probes encompassing sequences from the hCGß promoter from -27 to -6 and -45 to -25, respectively, were designed to encompass Site-1 and Site-2 (Fig. 1Go). A, The annealed, double-stranded, and 32P-labeled oligonucleotide for Site-1 (AAG TAG AGG AGG GTT GAG GCT T) was incubated with 1–2 µg of either affinity purified GST-Ets-2 or GST alone, in absence or presence of an excess of unlabeled competitor (5, 10, 50, 100, 250 molar excess). B, Binding was carried out in absence (lane 1) or presence (lane 2) of antibody directed against Ets-2. A nonspecific antiserum (lane 4) and an excess of nonspecific competitor DNA (annealed double-stranded Ets site mutated oligonucleotide) (lane 5) failed to dissociate the Ets-2-oligonucleotide complex. There was only weak binding of Ets-2 to an oligonucleotide mutated in its core sequence (lane 3). C and D, Experiments in A and B were repeated with the annealed, double-stranded, and 32P-labeled oligonucleotide for Site-2 (GTG GTG CAG GAA AGC CTC AAG). E and G, EMSA analysis of JAr cell nuclear extracts (15 µg protein) in absence or in presence of increasing concentration of unlabeled competitor nucleotide (50, 100, 250, 500 molar excess) representing Site-1 (E) and Site-2 (G), respectively. F and H show the effects of added antiserum on the complexes formed between Site-1 and Site-2 oligonucleotides with JAr cell nuclear extracts. Lane 1, No additions; lane 2, after addition of antiserum to Ets-2; lane 3, after addition of nonimmune antiserum. The positions of the supershifted bands are shown with the arrows.

 
Nuclear extracts from untransfected JAr cells were also able to form specific complexes with the oligonucleotides representing Site-1 (Fig. 5Go, E and F) and Site-2 (Fig. 5Go, G and H). Essentially identical results were obtained with JEG3 cell nuclear extracts (data not shown). The complexes were dissociated by addition of unlabeled oligonucleotide competitors, and a supershift was induced by addition of an antiserum against Ets-2 but not by addition of nonimmune serum. Why the complexes from nuclear extracts displayed a supershift with anti-Ets-2 (Fig. 5Go, E–H), whereas those complexes formed with recombinant Ets-2 dissociated in presence of antibody is unclear. The supershift could be due to the presence of additional binding proteins in nuclear extracts. Alternatively, Ets-2 from nuclear extracts is most likely phosphorylated (Fig. 7Go), probably binds more tightly to the labeled oligonucleotide, and consequently is less easily dissociated from the DNA by competing antibodies.



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Figure 7. Western Blot Analysis of Phosphorylated Ets-2 and MEK1/2 Level

A, Relative concentrations of phosphorylated Ets-2 (T72-phosphate) in nuclear extracts prepared from control and Ets-2/Ras-transfected 3T3 and JAr cells. Nuclear extracts (50 µg) from control (lane 1) or Ets-2/Ras-transfected (lane 2) 3T3 cells were analyzed by SDS-PAGE and Western blotting with an antiserum directed against a peptide representing the sequence encompassing T72-phosphate of human Ets-2. Lanes 3 and 4 represent nuclear extracts from control and Ets-2/Ras-transfected JAr cell. B, Relative concentrations of phosphorylated and total MEK1/2 in 3T3 cells as determined by Western blotting. Panel I shows the relative amounts of phosphorylated MEK1/2 in 50 µg of total cell extracts from control 3T3 cells (lane 1), from Ets-2/Ras transfected cells (lane 2), and from cells treated with PD98059 and U0126 (lanes 3 and 4). Panel II is identical to panel I, except the antibody used was directed against total MEK1/2 rather than the phosphorylated peptide.

 
Interaction between Ets-2 and the MAPK Pathway in Regulation of hCGß Promoter Activity
Ras regulates Ets-2 and related transcription factors as a result of its ability to up-regulate the MAPK-mediated phosphorylation of a specific threonine residue (T72 in the case of Ets-2) within the pointed domain of the protein (29, 30, 35). To determine whether this residue plays a role in the regulation of the hCGß promoter by Ets-2, 3T3 cells were transiently transfected with a construct designed to overexpress a form of Ets-2 mutated at T72 (Ets-2A72). This mutated form of Ets-2 led to a 50% reduction, but not a complete ablation, of the usual approximately 100-fold Ets2/Ras activation of -175 hCGß promoter (Fig. 6AGo). Control 3T3 cells appeared to contain little phosphorylated Ets-2 (Fig. 7AGo, lane 1), and the concentration of phosphorylated Ets-2 protein was increased in 3T3 cells after Ras and Ets-2 coexpression (Fig. 7AGo, lane 2) compared with the controls. In contrast, untransfected JAr cells showed a distinct band of phosphorylated Ets-2 protein, and the intensity of this band was only slightly increased in the Ets-2/Ras-transfected cells (Fig. 7AGo, lanes 3 and 4). The relatively high concentration of phosphorylated Ets-2 in control JAr cells supports the hypothesis that choriocarcinoma cells have an activated MAPK pathway.



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Figure 6. The Effect of T72 Mutation and MAPK Inhibitors on Ras- and Ets-2-Induced hCGß Promoter Activity

A, Luciferase expression from the -175/+65 hCGß gene promoter element transiently transfected into 3T3 cells in association with overexpression of wild-type (T72) and mutated (A72) Ets-2A. 3T3 cells were cotransfected with hCGß-Luc and expression vectors for Ras and either Ets-2 or Ets-2A72 under standard conditions. Data have been calculated as means ± SE and expressed as fold activation. Where letters differ above bars, differences are significant. B–D, Effect of MAPK inhibitors to influence the ability of a combination of Ras and Ets-2 to up-regulate activity of the -175/+65 hCGß gene promoter element. 3T3 (B), JAr (C), and JEG-3 (D) cells were cotransfected with -175/+65 hCGß-Luc and Ets-2 and Ras expression constructs under standard conditions. After exposure to DNA/Ca3(PO4) precipitates (8 h for JAr and JEG-3 cells, 20 h for 3T3 cells), cells were washed with Tris-buffered saline, placed for 2–3 h on complete culture medium, and then treated with either the MEK1/2 inhibitors PD98059 (25 and 50 µM for 3T3 cells; 50 µM only for JAr and JEG-3 cells) and U0126 (1 µM), or the p38 inhibitor SB202190 (5 µM). Data are expressed as fold activation (means ± SE) relative to values obtained with -175/+65 hCGß-Luc in absence of Ets-2/Ras cotransfection. Where letters above bars differ within figures, values are significantly different (P < 0.05).

 
Three different MAPK inhibitors were then used to determine which MAPK pathway was involved in the Ras-mediated activation of Ets-2 in 3T3 cells. PD98059, a relatively specific inhibitor of MAPK/ERK kinase 1/2 (MEK1/2) (36, 37), reduced hCGß promoter transactivation by about 45% at 25 µM, and by more than 80% at 50 µM. A second MEK 1/2 inhibitor, U0126 (38), when used at 1 µM, inhibited promoter activation by Ets-2/Ras by about 70% (Fig. 6BGo). By contrast, SB202190, a potent and relatively selective inhibitor of the P38 stress-activated pathway (39), failed to inhibit the Ets-2/Ras effects on the hCGß promoter (Fig. 6BGo). The concentrations of inhibitor selected for the above experiments were based on preliminary experiments designed to test for cell toxicity and were comparable to ones used previously by others (40, 41). At the concentrations employed, the compounds caused no visible changes in phenotype relative to control cells treated with dimethyl sulfoxide, the vehicle for the drugs.

Western blot analysis of extracts from Ets-2/Ras cotransfected 3T3 cells revealed an elevated expression of phosphorylated MEK1/2 relative to nontransfected control cells (Fig. 7BGo, panel I). As, anticipated, the concentrations of the phosphorylated form(s) decreased when the transfected cells were treated with the MEK1/2 inhibitors, PD98059 and U0126 (Fig. 7BGo, panel I). The total amount of MEK1/2 protein in 3T3 cells was unaffected by transfection of Ets-2 and Ras, and by the treatment with the two inhibitors (Fig. 7BGo, panel II).

Inhibitors PD98059 and U0126 reduced the ability of Ets-2 and Ras to transactivate the hCGß promoter in both JAr and JEG-3 cells (Fig. 6Go, C and D). With JEG-3 cells, the magnitude of the effects were comparable to those observed in 3T3 cells. In JAr cells, the inhibition by PD98059 and U0126 was not as pronounced as in JEG-3 and 3T3 cells, and the stress kinase inhibitor SB202190 was also able to reduce Ets-2/Ras transactivation of the promoter. It seems likely that Ets-2 can be activated via the MEK1/2 MAPK pathways in all three cell lines, but that the situation in JAr cells is less straightforward, with at least one other activation pathway participating.

Interaction between Ets-2 and cAMP in the Regulation of hCGß Promoter Activity
Despite not containing a classical CRE and not binding the transcription factor CRE binding protein, the hCGß promoter is up-regulated by cAMP in JEG-3 cells (11, 13, 33, 42). The regions of the promoter responsible for this effect have been mapped to sequences between -311 and -202 (11). Unexpectedly, in our experiments, addition of 8-bromoadenosine-cAMP (8-bromo-cAMP) to JAr cells increased Luc reporter activity from the short -175 hCGß promoter as effectively as it did from the longer -325 promoter (~7-fold in each case) (Fig. 8AGo). Quite similar results were noted with JEG-3 cells (Fig. 8BGo), except the up-regulation of both promoters was rather less (~4-fold). Addition of 8-bromo-cAMP to either JAr or JEG-3 cells that were also overexpressing Ets-2 had synergistic and roughly proportional effects on both promoters (Fig. 8Go, A and B). In the case of JAr cells, the Ets-2 effect was increased a further 8- to 10-fold by cAMP. For JEG-3 cells, values were quite similar. Identical results were obtained when dibutyryl cAMP was substituted for 8-bromo-cAMP (data not shown). As in the earlier experiments (Fig. 2Go), Ras had little measurable effect on luciferase-reporter expression from the hCGß promoters when it was expressed either alone or in association with Ets-2 in either the presence or absence of 8-bromo-cAMP (Fig. 8Go, A and B). Neither the -325 nor -175 promoter responded to 8-bromo-cAMP in 3T3 cells (Fig. 8CGo).



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Figure 8. The Effect of 8-Bromo-cAMP, Ets-2, Ras, and Ets-2 and Ras in Combination on Luc Reporter Expression from hCGß Gene Promoter Elements Transiently Transfected into JAr (A), JEG-3 (B), and NIH3T3 (C) Cells

Cells were transfected with either promoter-Luc constructs (-175 and -325), alone, or in combination with expression constructs for Ets-2, activated Ras, or Ets-2 and Ras together. After transfection, cells were either left untreated or exposed to 8-bromo-cAMP (500 µM) for 36 h. The data are provided as relative Luc activities. Values above each bar are fold stimulation of reporter activity relative to the basal rate for the particular promoter in a particular cell line. Results have been calculated as mean ± SE. If letters above each bar are different, there was a significant effect of treatment (P < 0.05).

 
The dependence of cAMP on the presence of the Ets-2 binding sites in the promoter was demonstrated by comparing cAMP effects on choriocarcinoma cells after transfection with the wild-type and mutated promoters. Data are shown for JAr cells only (Fig. 9Go) because comparable results were obtained with both cell lines. Again, a combination of Ets-2/Ras over expression and 8-bromo-cAMP addition stimulated promoter activity over 200-fold (Fig. 9AGo). Mutation of either Ets-2 Sites-1 or -2 (Fig. 1Go) reduced luciferase activity by about 45% (Fig. 9BGo) and 75% (Fig. 9CGo), respectively. Mutation of both sites led to a loss of 95% of reporter activity (Fig. 9DGo). The same double mutation, virtually abolished cAMP effects on basal promoter activity when Ets-2/Ras were not over expressed (compare Fig. 9Go, A and D).



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Figure 9. The Effect of Ets-2 Binding Site Mutations on the Ability of 8-Bromo-cAMP, Ets-2, Ras, and Ets-2 and Ras in Combination to Stimulate Luc Reporter Expression from -175 hCGß Gene Promoter Elements Transiently Transfected into JAr Cells

The different treatment combinations are shown below the bar graphs. All values are expressed relative to the basal luciferase activity of cells in which Ets-2 and Ras were not cotransfected with the -175 hCGß promoter and without cAMP treatment. Experiments are shown for the wild-type -175 hCGß promoter (A); the Site-1 mutated promoter (B); the Site-2 mutated promoter (C) and the doubly mutated Site-1/-2 promoter (D). Values above the bars are fold increases in reporter expression relative to luciferase values obtained when Ets-2 and Ras were not cotransfected with the hCGß-Luc promoter and without cAMP treatment. Where lowercase letters differ above values, the differences are significant (P < 0.05). Essentially identical results were obtained with JEG-3 cells and are, therefore, not shown.

 
Together, these data indicate that cAMP effects on the hCGß promoter are primarily dependent on the Ets-2 enhancer present in the proximal region of the promoter.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hypothesis motivating these studies was that the transcription factor Ets-2 regulates the early activation of genes specified in mammalian trophectoderm. Our specific objective in the present experiments has been to evaluate Ets-2 engagement in the transcriptional control of hCGß5, a gene that is expressed early in the development of the human embryo and that has been recruited for a role in trophoblast-maternal signaling only within the primate order. This gene and its associated trophoblast-specific, transcriptional-control sequences have, therefore, evolved quite recently (3, 43, 44).

The report by Johnson and Jameson (33), which appeared after our work had begun, confirmed that Ets-2 might indeed play a role in regulation of hCGß expression in human placenta because this transcription factor modestly up-regulated the hCGß promoter in the human choriocarcinoma cell line JEG-3 and had a major effect when the PKA catalytic subunit was coexpressed. However, a direct interaction of the Ets-2 protein with the proximal promoter region of the hCGß gene was not observed, and the Ras/MAPK signal transduction pathway was inferred not to be involved in Ets-2 activation. By contrast, our experiments have shown that there are two functional Ets-2 binding sites placed 10 bp apart just upstream of the initiator element (Figs. 1–5GoGoGoGoGo). Moreover, overexpression of Ets-2 in JAr and JEG-3 cells resulted in approximate 20-fold up-regulation (rather than the 3- to 4-fold value noted by Johnson and Jameson) of a cotransfected hCGß promoter (Fig. 2Go, A–C). These differences in expression could have arisen for technical reasons. For example, we assayed reporter activity 36 h after transfection, whereas Johnson and Jameson chose shorter elapsed periods before assaying Luc activity. Another explanation is that the two JEG-3 cell populations had diverged genetically.

At least one other Ets-2-responsive gene expressed strongly in trophoblast, the matrix degrading proteinase matrix metalloproteinase-3 utilizes a pair of Ets-2 binding sites, which together confer both Ets and Ras responsiveness (29, 45, 46) (Fig. 10Go). Other Ets-2 responsive promoters, including those for the IFN-{tau} genes (15), urokinase plasminogen activator (20), placental lactogen II (14) and human collagenase (47) have an AP1 site placed either close to or adjacent to the Ets binding site (Fig. 10Go). Examination of the hCGß gene reveals a sequence TGAGGCT immediately proximal to Ets Site-1, which could constitute a site for AP1 binding (Fig. 1Go). This sequence motif, which is quite similar to the AP-1 site on the urokinase plasminogen activator gene (Fig. 10Go), partially overlaps the 5' end of the transcription initiation element of the TATA-less promoter of the hCGß gene. Further experiments will be necessary to determine whether this site has any functional significance and, if it does, whether its activity is linked to that of the downstream Ets-2 enhancer, to transcription initiation, or to both.



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Figure 10. A Comparison of the Ets Binding Sequences of the hCGß Promoter with Those of Several Genes in which There Is a Combined Ets/AP1 Enhancer Element within the Proximal Promoter Region

The Ets-2 binding sites, usually (C/A)(C/A)GGA(A/T)(G/A), and the AP1 sites, usually TGA(G/C)T(C/A)AG, are marked for each sequence. Data were generated from Refs.14 15 20 and 47 .

 
Ras coexpression with Ets-2 had only a minor ability to increase hCGß promoter activity in choriocarcinoma cells, especially in the JEG-3 cell line (Fig. 2Go). These tumor cells, not only express Ets-2, which might account for much of their basal hCGß promoter activity and the modest effects of transfected Ets-2, but also possess an active MAPK pathway, as evident from the phosphorylation state of MEK1/2 (data not shown) and from reports of others (48, 49, 50). Such cells may also express an active Ras oncogene (51). It was for these reasons that we undertook studies in 3T3 cells, which have low amounts of Ets-2 and activated MEK1/2. Using these cells, we were able to demonstrate a dramatic synergistic effect of Ras on Ets-2 transactivation of the hCGß promoter (Fig. 2CGo), and, that associated with this up-regulation, there was increased phosphorylation of T72 in the Ets-2 pointed domain (Fig. 7AGo, lanes 1 and 2) and of MEK1/2 (Fig. 7BGo, panel I). Finally, the PD98059 inhibitor of MEK1/2 reduced the effects of Ets-2 and Ras coexpression by more than 80% (Fig. 6BGo). Similar data were obtained in JEG-3 cells (Fig. 6DGo), also emphasizing the likely cooperation of Ets-2 and the MAPK pathway in control of hCGß expression in trophoblast. One apparent inconsistency was the ability of the stress kinase inhibitor SB202190 to reduce Ets-2 effects in JAr compared with JEG-3 and 3T3 cells. That observation, coupled with the less pronounced inhibitory effects of PD98059 and U0126 in JAr cells, suggests that more than one activation pathway converges on Ets-2 as a transcriptional target molecule (Fig. 6CGo). Such an interpretation is consistent with the failure of the Ala/Thr substitution at residue 72 to reduce the Ets-2/Ras stimulation by more than 50% (Fig. 6AGo). This result suggests that T72 is perhaps not the only activation site on Ets-2 for the MAPK signal transduction pathway. Together, these data explain why a large number of growth factors able to act through MAPK signaling up-regulate hCG production in human trophoblast-derived cells (49, 52, 53, 54).

As initially suggested (33), cAMP effects on the hCGß promoter in choriocarcinoma cells may be mediated through Ets-2 (Fig. 8Go, A and B). What made these data especially puzzling were the results from preceding work (3, 11, 13, 55) showing the apparent target for cAMP to be a region well upstream (-311 to -202) of the proximal Ets-2 enhancer identified here. However, it is clear from our experiments in JAr cells that the region of the promoter beyond -175 had little role in mediating the cAMP effects. Even in JEG3 cells, the -325 promoter had only about twice the basal and cAMP-stimulated activity as the shorter -175 promoter, and the stimulatory effect of 8-bromo-cAMP addition was identical (~200-fold). Again the basis for the differences between this and earlier work (33) may relate to the time of treatment exposure, in this case to cAMP. Previous experiments have clearly shown that cAMP effects on the hCGß promoter are considerably delayed and likely indirect compared with the immediate and pronounced effects on hCG{alpha}, which level off within a few hours (11). By contrast, the stimulatory effects of cAMP on the ß-promoter continue to increase beyond 16 h after treatment. In addition, untreated JAr cells express only low amounts of hCG, with only about 6% staining positively with an antiserum that is targeted primarily against hCGß (56). Initiation of further expression may require additional cells in the population to progress to a more differentiated state, a process thought to be accelerated by cAMP (44, 56). With such a progression of cell phenotype in cAMP-treated choriocarcinoma cells, the hCGß promoter could become more responsive to hormones in the culture medium whose effects on transcriptional activity depend upon the MAPK pathway and Ets-2.

The fact that the cAMP effects on the hCGß promoter are largely abolished when both Ets-2 binding sites are mutated (Fig. 9Go) are also entirely consistent with the hypothesis that the Ets-2 enhancer is central to the control of hCGß expression and plays an analogous role to that of the dual CREs in the hCG{alpha} promoter. How cAMP is acting is unclear. One possibility is that certain additional transcription factors are up-regulated during cAMP exposure and exert Ets-2 cooperative effects on the proximal enhancer. An alternative explanation is that the effect of cAMP is more immediate than the differentiation model discussed above, and that there is direct activation of either Ets-2 itself or some other transcription factor that cooperates with Ets-2 via the action of PKA. Consistent with the later hypothesis, Kievit et al. (57) implicated the MAPK pathway and possibly also Ets factor binding sites within the proximal promoter region of the PRL gene in mediating cAMP stimulation of PRL synthesis in pituitary GH3 cells. Conceivably, such an activation pathway extends to genes other than that for PRL.

In summary, the experiments described herein suggest that a pair of Ets-2 sites in the proximal promoter region of the hCGß gene acts as a major enhancer for hCGß expression. This site is subject to control through activation of Ras and by factors that act through the Ras/MAPK signal transduction pathway, as well as by cAMP. The data are consistent with the hypothesis that Ets-2 has a general role in controlling the transcriptional activity of genes that are expressed in trophectoderm at the time of or soon after lineage specification has occurred. Remarkably, two genes, those for IFN-{tau} and hCGß5, whose primary responsibilities are in maintaining maternal progesterone production in early pregnancy of ruminants and primates, respectively, are both under the control of Ets-2, although their promoters otherwise bear no resemblance to each other.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Preparation of Expression Vectors and Reporter Genes
An hCGß promoter fragment (-325/+65) (7) was subcloned into 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 -325/+65 fragment by PCR with promoter specific primers -295 XhoI sense (se), -175 XhoI se, -34 XhoI se, -17 XhoI se, and hCGß HindIII antisense (as) (Table 1Go). Each was subcloned into the same pGL2 basic vectors as above. PCR was used to introduce StuI restriction sites and mutate the Ets binding sites at -23 to -17 (Site-1) and -39 to -33 (Site-2) on -175 Luc promoter (Fig. 1Go). A double Ets site mutant construct was prepared by introducing a ClaI site into the first site and a StuI site into the second (58, 59). The mutated PCR fragments were amplified in the pBluescript SK vector (Stratagene, La Jolla, CA), released by HindIII and XhoI digestion and ligated into pGL2 Basic vector. Fidelity of constructs was verified by DNA sequencing with Luc vector specific primers (GL-se and GL-as). Primers (Site-1 StuI se and as, Site-2 StuI se and as, Site-1 cla1 se and as) used for generating mutants by PCR are listed in Table 1Go. The wild-type Ets-2 (Ets-2T72), the mutant alanine at 72 (Ets-2A72,), the activated Ras (pH06TI) and its parental vector (Homer6) have been described previously (29, 60, 61). The ß-galactosidase gene driven by Rous sarcoma virus long terminal repeat (pRSVLTR-ß-gal) (15) was used as an internal control in experiments performed on choriocarcinoma cell lines and Renilla luciferase control vector, pRL-Simian virus 40 (Promega Corp.) for 3T3 cell experiments.

Cell Culture
NIH3T3 cells (ATCC, Manassas, VA; CRL-1658) were cultured in DMEM supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. JAr cells (ATCC, HTB-144) were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. JEG-3 (ATCC, HTB-36) cells were cultured in Eagle’s MEM supplemented with 2 mM L-glutamine, 1.5 g/liter sodium bicarbonate 1 mM sodium pyruvate, 10% FBS, and 100 U/ml penicillin and 100 µg/ml streptomycin.

Transfection
The transfections of JEG-3 and JAr cells were performed by the standard calcium phosphate method (15). Briefly, cells were plated at a cell density of 2 x 105/60-mm dish, cultured overnight, and incubated with DNA/Ca3(PO4) precipitates in DMEM supplemented with 10% FBS for 7–8 h. Concentrations of plasmid were: hCGß promoter constructs (3.5 µg/dish), Ets-2 (0.5 µg/dish), and Ras (1.2 µg/dish), ß-galactosidase (50 ng/dish). DNA concentrations were normalized with pCGN expression vector. Cells were washed with PBS and maintained for a further 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.

Transfection of 3T3 cells was performed based on the procedures described previously (62). Concentrations of plasmid were: hCGß promoter constructs (3.5 µg/dish), Ets-2 (0.5 µg/dish), and Ras (1.2 µg/dish), Renilla Luc (50 ng/dish). Briefly, cells (same density as above) were incubated with DNA/Ca3(PO4) precipitates for 16–20 h, washed with saline buffered with 50 mM Tris-HCl, pH 7.5 and harvested 48–50 h later. The medium, which contained 10% FBS, was replaced with one containing only 0.5% FBS 16 h before harvest.

PD98059 and U0126 (Calbiochem, San Diego, CA) were used as MEK1/2 inhibitors, and SB202190 as a p38 stress kinase inhibitor. After transfection (8 h for JAr or JEG-3 and 20 h for NIH3T3 cells) and removal of DNA/Ca3(PO4) precipitates, cells were washed with Tris-buffered saline, incubated for 2–3 h on complete culture medium, and then treated with individual inhibitors (50 µM and 25 µM PD98059, 1 µM U0126 and 5 µM SB202190 dissolved in dimethyl sulfoxide). Dimethyl sulfoxide alone was used as 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.

EMSA
Double-stranded synthetic oligonucleotides were annealed and labeled with [{gamma}32P]ATP by using T4 polynucleotide kinase (63). Probes were deigned to encompass two potential Ets-2 binding sites: Site-1, -27 to -6, and Site-2, -45 to -25. Primers (Site-1 se and as, Site-2 se and as) are listed in Table 1Go. GST-Ets-2 fusion protein was synthesized as described previously (15). Bacterial growth, isopropyl-ß-D-thiogalactopyranoside treatment, and preparation of extracts followed the Amersham Biosciences (Piscataway, NJ) protocol. Either GST-Ets-2 (1 to 2 µg) or GST protein was incubated with labeled, double-stranded oligonucleotide as probes (10,000 to 20,000 cpm; 25 fmol) in presence of 1 µg of nonspecific carrier DNA (poly deoxyinosine:deoxycytidine). Protein concentrations were measured by the procedure of Bradford (64). Buffer composition was 20% (vol/vol) glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM dithiothreitol, 250 mM NaCl, 50 mM Tris-HCl of pH 8.0 containing 2% (vol/vol) CHAPS detergent and 10 mg/ml BSA (both from Sigma). Annealed, double-stranded, unlabeled oligonucleotides were used as specific competitors for probe binding at 5, 10, 50, 100, and 250 molar excess.

In a separate experiment, Ets-2 sites on the complementary strands of the competitor/probe were mutated (M Site-1, se and as; M Site-2, se and as; Table 1Go) to verify the protein binding specificity of the wild-type sequence. A 500-molar excess of these mutated oligonucleotides were used as nonspecific competitors. Experiments were also conducted to attempt to supershift the Ets-2-oligonucleotide complexes with antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; catalog no. sc-351) to Ets-2 protein as described by Ezashi et al. (15). EMSA was repeated with the Site-1 and Site-2 probe by using JAr and JEG-3 choriocarcinoma cell nuclear extracts under the similar conditions as described above. The nuclear extracts for EMSA were prepared after the procedure described by Dignam et al. (65). About 15 µg of JAr or JEG-3 cell nuclear extract protein was used in the binding reaction. Annealed, double-stranded, unlabeled oligonucleotides were used as specific competitors for probe binding at 50, 100, 250, and 500 molar excess. In a separate reaction, Ets-2 antiserum (described later) was used to verify the binding specificity.

Western Blot Analysis
The relative amounts of activated MAPKs (MEK1/2) in whole cell lysate was assessed by comparing the relative staining intensities of phosphorylated and nonphosphorylated MEK1/2 on Western blots. Affinity-purified antibodies to phosphorylated MEK1/2 (Ser217/221) and unmodified MEK1/2 were obtained from Cell Signaling Technology (Beverly, MA) and diluted 1:5000 in blocking buffer (see below). The former antibody does not cross-react with related family members, including phosphorylated MKK4, MKK3, and MKK6. After either cell transfection or after treatment with inhibitors, cells were lysed in 20 mM imidazol buffer (pH 6.8), containing 100 mM KCl, 1 mM MgCl2, 5 mM EDTA, 0.2% (vol/vol) Triton X-100, 10 mM NaF, 1 mM sodium vanadate, 0.25% (vol/vol) sodium deoxycholate, 10 mM sodium pyrophosphate, 10% (vol/vol) glycerol, 0.1 mM dithiothreitol, and proteinase inhibitors (1 mg/ml phenylmethylsulphonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml aprotinin). After one freeze-thaw cycle, cell extracts were centrifuged, and 50 µg of cell supernatant analyzed by SDS-PAGE on 12.5% gels. Proteins were transferred to polyvinylidene fluoride membranes. Open sites were blocked with 5% (wt/vol) BSA (fraction V) dissolved in 0.1% (vol/vol) Tween 20 and 1% (wt/vol) polyvinyl pyrrolidone in Tris-buffered saline, and the blot probed with respective antibodies. The blots were exposed to goat antirabbit IgG (diluted 1:2000) conjugated to horseradish peroxidase and bound antibodies detected on x-ray film by light emission from Enhanced Luminescence Substrate (Cell Signaling Technology, Beverly, MA).

The content of Ets-2 protein in transfected and nontransfected cells was assayed by Western blotting of cell extracts as above by using a rabbit antiserum to Ets-2 raised against the amino-terminal fragment (amino acids between 1 and 326) of human Ets-2 fused to GST (Ezashi, T., and R. M. Roberts, unpublished results). This sequence does not contain the Ets domain, which is highly conserved in members of Ets family of transcription factors. The phosphorylation status of Ets-2 was assessed in nuclear extracts by Western blotting with an affinity purified antibody against the phosphopeptide LPLL(phospho-T)CSKA, as described previously (30). The rabbit antiserum was first incubated with Ets-2 "pointed" domain (recombinant Ets-2 peptide corresponding to amino acids 60–167, which included the pointed domain and the MAPK phosphorylation site) that had been immobilized on nitrocellulose. Antibodies that failed to bind to this unmodified polypeptide were subsequently incubated with immobilized Ets-2 pointed domain that had been phosphorylated in vitro at T-72 with recombinant MAPK p44 (New England Biolabs, Inc., Beverly, MA). The antibodies that bound to the phosphorylated peptide were eluted with 0.2 M glycine, pH 2.0, the eluate neutralized with Tris base, and the protein solution stored at -70 C before use. This affinity-purified antiphosphoEts-2 antibody was used at a dilution of 1:1000 for western blotting. Nuclear extracts for the detection of phosphorylated Ets-2 protein by Western blot were prepared from cell monolayers, which had been exposed to lysis buffer as described previously (35), and protein concentration measured (64). Western blotting was performed as described above.

Statistical Analyses
Each transfection was carried out in triplicate and values averaged. Transfection experiments were replicated either four or five times on different days, with the exception of the experiments summarized in Fig. 3Go, C and D, and Fig. 9Go where only two separate replicates (each in triplicate) were performed. Values are presented as means ± SE for the different replicates and expressed as both relative Luc activity and fold-induction over the basal expression of the promoter. Statistical analyses were performed by analysis of variance (ANOVA) followed by LSM t test, using an SAS computer program (SAS system, version V-6.12, TS 020). Significance was accepted at P < 0.05.


    ACKNOWLEDGMENTS
 
The authors are thankful to Dr. Jonathan A. Green for his suggestions. The authors are also thankful to Mr. James A. Bixby for his kind technical help.


    FOOTNOTES
 
This work was supported by NIH Grant HD-21896.

Abbreviations: AP, Activating protein; as, antisense; 8-Br-cAMP, 8-bromoadenosine cAMP; CG, chorionic gonadotropin; CRE, cAMP-responsive element; FBS, fetal bovine serum; GST, glutathione-S-transferase; hCG, human CG; IFN-{tau}, interferon-{tau}; Luc, luciferase; MEK1/2, MAPK/ERK kinase 1/2; PKA, protein kinase A; se, sense.

Received for publication July 1, 2002. Accepted for publication October 17, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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