Mechanistic Differences in the Activation of Estrogen Receptor-alpha (ERalpha )- and ERbeta -dependent Gene Expression by cAMP Signaling Pathway(s)*

Kevin M. ColemanDagger , Martin DutertreDagger , Abeer El-Gharbawy§, Brian G. Rowan§, Nancy L. WeigelDagger , and Carolyn L. SmithDagger

From the Dagger  Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030-3498 and the § Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614-5804

Received for publication, December 4, 2002, and in revised form, January 30, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although increases in intracellular cAMP can stimulate estrogen receptor-alpha (ERalpha ) activity in the absence of exogenous hormone, no studies have addressed whether ERbeta can be similarly regulated. In transient transfections, forskolin plus 3-isobutyl-1-methylxanthine (IBMX), which increases intracellular cAMP, stimulated the transcriptional activities of both ERalpha and ERbeta . This effect was blocked by the protein kinase A inhibitor H89 (N-(2-(p-bromocinnamylamino)-ethyl)-5-isoquinolinesulfonamide) and was dependent on an estrogen response element. A 12-O-tetradecanoylphorbol-13-acetate response element (TRE) located 5' to the estrogen response element was necessary for cAMP-dependent activation of gene expression by ERbeta but not ERalpha , indicating that the former subtype requires a functional interaction with TRE-interacting factor(s) to stimulate transcription. Both p160 and CREB-binding protein coactivators stimulated cAMP-induced ERalpha and ERbeta transcriptional activity. However, mutation of the two cAMP-inducible SRC-1 phosphorylation sites important for cAMP activation of chicken progesterone receptor or all seven known SRC-1 phosphorylation sites did not specifically impair cAMP activation of ERalpha . The E/F domains of ERalpha are sufficient for activation by forskolin/IBMX, and this is accompanied by an increase in receptor phosphorylation. In contrast, cAMP signaling reduces the phosphorylation of the corresponding region of ERbeta , and this correlates with the lack of forskolin/IBMX stimulated transcriptional activity. Our data suggest that cAMP activation of ERalpha transcriptional activity is associated with receptor instead of SRC-1 phosphorylation. Moreover, differences in the cofactor requirements, domains of ERalpha and ERbeta sufficient for forskolin/IBMX activation, and the effect of cAMP on receptor phosphorylation indicate that this signaling pathway utilizes distinct mechanisms to stimulate ERalpha and ERbeta transcriptional activity.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The biological effects of estrogens are mediated by two estrogen receptors (ERs),1 ERalpha and ERbeta , which belong to a large superfamily of nuclear hormone receptors. These ligand-regulatable transcription factors possess six structural domains labeled A through F (1). The A/B domain encompasses activation function-1 (AF-1); the C and D domains correspond to the DNA binding domain and the hinge region, respectively; the E region encompasses a second activation function (AF-2) and an overlapping ligand binding domain; and the F domain, located at the extreme carboxyl terminus, is thought to play a modulatory role in ER activity. Both ERs possess similar binding affinities for estradiol and their cognate DNA binding site (estrogen response element; ERE), which is likely caused by the high degree of sequence homology they share in their ligand and DNA binding domains (2-6). The AF-2 domain of each receptor is regulated by ligand-induced changes in receptor conformation, but the activities of the poorly conserved AF-1 domains are ligand-independent and can be modulated by phosphorylation (7-10). Notable for ERalpha , in most cases the AF-1 and the AF-2 domains interact functionally to enhance transcription in a cooperative manner (7, 11).

In the best studied mechanism of ERalpha and ERbeta activation, hormone diffuses into the cell, binds to the receptor, and induces a conformational change in the receptor ligand binding domain (1). Receptors, bound to their EREs either as ERalpha or ERbeta homodimers or ERalpha :ERbeta heterodimers, can then recruit coactivators to the promoter region of estrogen target genes via their interaction with the receptor activation domains (2, 3, 12, 13). There, these molecules can stimulate transcription by bridging ERs to the general transcriptional machinery and promoting the formation of a stable preinitiation complex (12, 13). Notably, various coactivators also possess ubiquitin ligase, arginine methyltransferase, or histone acetyltransferase enzymatic activities that may facilitate chromatin remodeling and gene activation (14-17).

In addition to this relatively well characterized mode of activation, ERalpha can be activated via the cAMP signaling pathway in the apparent absence of estrogens (for review, see Ref. 18). In MCF-7 cells, endogenous ERalpha target genes, including the progesterone receptor (PR; Ref. 19), pS2 (20), Liv-1 (20), and cathepsin-D (21), can be stimulated either with the cAMP analog 8-bromo-cAMP or a combination of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and cholera toxin, which increases cAMP production via a G protein-mediated signal transduction pathway. Importantly, in these experiments as well as in later studies the cAMP-induced responses are inhibited by treatment with the pure antiestrogen ICI 164,384, signifying their receptor dependence (19-21). Demonstrating the need to transfect ERalpha into cells lacking endogenous receptor further supported this receptor dependence (22).

In addition to these ERalpha studies a number of reports indicate that the transcriptional activities of several other nuclear receptors can be modulated by the cAMP signaling pathway. For example, the chicken PR (23), androgen receptor (24), retinoic acid receptor (25), retinoid X receptor (26), and peroxisome proliferator-activated receptor-delta (27) can be activated by cAMP signaling, demonstrating that this mode of ligand-independent activation is not exclusive to ERalpha . However, human PR (28) cannot be activated ligand-independently via this mechanism, nor can the unliganded glucocorticoid receptor, although cAMP stimulation increases the hormone-dependent responses of these receptors (29, 30). Collectively, these reports demonstrate the specific nature by which the cAMP signaling pathway can cross-talk with different nuclear receptors.

Some progress has been made in the effort to understand the mechanisms involved in cAMP activation of nuclear receptor-dependent transcription. Although an increase in receptor phosphorylation accompanies the cAMP-mediated activation of ERalpha (31, 32), there is no increase in chicken PR phosphorylation associated with its activation in cells treated with 8-bromo-cAMP (33). Rather, cAMP/protein kinase A (PKA) signaling enhances chicken PR-dependent transcription, in part by increasing phosphorylation of a receptor-interacting coactivator, steroid receptor coactivator-1 (SRC-1), and thereby promotes a more stable interaction between this coactivator and p300/CREB-binding protein (CBP)-associated factor and facilitates functional synergism between SRC-1 and CBP (34). Although it is still unknown how the unliganded ERalpha is activated by cAMP/PKA, there is evidence that the transcription factor, CREB, can interact functionally with ERalpha and thereby mediate synergism between the 17beta -estradiol (E2)-dependent and cAMP-dependent signaling pathways (35).

The identification of ERbeta has increased our awareness of the diversity of potential mechanisms by which ER-dependent and estrogenic responses may be achieved (6). Notably, the relative magnitude of ERalpha - and ERbeta -mediated estrogen activation of ERE-containing reporters typically varies depending on the cell type and promoter context (36). In the absence of ligand, both ERalpha - and ERbeta -dependent transcription can be modulated by a mitogen-activated protein kinase (MAP kinase) signaling pathway (8-10). It is unknown, however, whether ERbeta can be activated by the cAMP/PKA signaling pathway, and we therefore examined this using transient transfection assays and synthetic agents that increase intracellular cAMP levels. In so doing, we have defined mechanistic differences between cAMP activation of ERalpha and ERbeta , particularly with respect to phosphorylation and the influence of cross-talk with AP-1 transcription factors. Moreover, we report that all of the p160 coactivators, as well as CBP, can coactivate ERalpha and ERbeta transcriptional activity stimulated by cAMP pathways, and we demonstrate that the importance of SRC-1 phosphorylation to cAMP activation of gene expression is receptor-dependent.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- E2, IBMX, and N-(2-(p-bromocinnamylamino)-ethyl)-5-isoquinolinesulfonamide (H89) were obtained from Sigma. Forskolin was obtained from Calbiochem (San Diego).

Plasmid DNAs-- The mammalian expression vectors for human ERalpha , pCMV5-hERalpha (31) and pCR3.1-hERalpha (15), and human ERbeta , pCXN2-hERbeta (37) have been described previously. The synthetic target genes pERE-E1b-CAT (38), pE1b-CAT (38), pERE-E1b-CAT(mTRE) (39), 17mer-E1b-CAT (40), and pS2-CAT (41) were used in previous studies, as were pATC0, pATC1, and pATC2 (42). The pCR3.1-hSRC-1a (43) and pCR3.1-hSRC-1a-Ala1179/1185 (34) expression plasmids have been published, as were the pCR3.1-TIF2, pCR3.1-RAC3, and pCR3.1-CBP vectors (44). The pBind expression plasmid encoding the Gal4 DNA binding domain (amino acids 1-147) was obtained from Promega (Madison, WI).

The SRC-1a phosphorylation mutant pCR3.1-hSRC-1a-7Ala, which has alanine substitutions at positions 372, 395, 517, 569, 1033, 1179, and 1185, was generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the appropriate mutagenic primers. The plasmids were sequenced to ensure that errors did not occur during mutagenesis. The constructs for hERalpha -179C (pCR3.1-hERalpha -179C) and hERbeta -143C (pCR3.1-hERbeta -143C) were made by PCR using the primers 5'-ACCATGGCCAAGGAGACTCGCTACTGT-3' and 5'-CTCTCAGACTGTGGCAGGGAAACC-3' to amplify the segment of pCMV5-hERalpha encoding amino acids 179-595 and the primers 5'-ACCATGAAGAGGGATGCTCACTTCTGC-3' and 5'-GCGTCACTGAGACTGTGGGTTCTG-3' to PCR amplify the segment of pCXN2-hERbeta encoding residues 143-530, respectively. Each of the resulting PCR fragments was subcloned into the pCR3.1 expression plasmid using the TA cloning kit (Invitrogen). The expression plasmids encoding Gal-ERalpha EF (pBind-ERalpha EF) and Gal-ERbeta EF (pBind-ERbeta EF) were generated by PCR using the primers 5'-GGGATCCGTAAGAAGAACAGCCTGGCCTTGTTCC-3' and 5'-TCTAGAGACTGTGGCAGGGAAACCCTCTGCC-3' to amplify the segment of pCMV5-hERalpha corresponding to amino acids 302-595 and the primers 5'-CGGGATCCGAGTGCGGGAGCTGCTGCTGG-3' and 5'-ATAGTTTAGCGGCCGCTCACTGAGACTGTGGGTTCTG-3' to amplify the portion of pCXN2-hERbeta encoding amino acids 254-530. Each of the resulting fragments was subcloned into the pCR3.1 expression plasmid via the TA cloning kit and subsequently transferred to the pBind expression vector via a BamHI-XbaI restriction fragment for pBind-ERalpha EF and BamHI-NotI fragment for pBind-ERbeta EF. The pCR3.1-FLAG-hERalpha construct was generated by PCR using a 5'-primer (5'-GATATTGCTAGCATGGACTACAAGGACGACGATGACAAGACCCTCCACACCAAAGCATCT-3'), which incorporated the FLAG epitope (underlined), and 5'-CGCCGCAGCCTCAGACCCGGGGCC-3' to amplify the 5'-region of a hERalpha cDNA within pCR3.1-hERalpha , and the resulting PCR fragment was substituted back into the pCR3.1-hERalpha expression vector via NheI and XmaI restriction sites. The pCR3.1-FLAG-hERbeta expression vector was constructed as follows. First, the coding region for hERbeta was removed from pCXN2-hERbeta via a partial digest with EcoRI and transferred to pCR3.1, which yielded pCR3.1-hERbeta . The 5'-primer (5'-CGTGACCGTGCTAGCATGGACTACAAGGACGACGATGACAAGGATATAAAAAACTCACCATCTAGC-3'), which encompasses a coding sequence for the FLAG peptide (underlined), and 3'-primer (5'-CACAAGGCGGTACCCACATCTCTC-3') were used to PCR amplify a portion of pCR3.1-hERbeta corresponding to the 5'-end of the hERbeta cDNA. The resulting PCR product was substituted into pCR3.1-hERbeta via NheI and KpnI restriction sites. All of the expression vectors that were made using PCR amplification were sequenced to ensure that no errors occurred during their synthesis. The 17mer-E1b-CAT(Delta TRE) reporter plasmid was generated by an Eco0109-HindIII digest of the 17mer-E1b-CAT, which was religated after blunting the restriction ends.

Cell Culture and Transfections-- HeLa (human cervical carcinoma) cells were routinely maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). 24 h prior to transfections for transactivation assays, cells were plated in six-well culture dishes at a density of 3 × 105 cells/well in phenol red-free DMEM with 5% charcoal-stripped fetal bovine serum (sFBS). For transfections, medium was replaced with serum-free medium, and DNA was introduced into cells in the indicated amounts using LipofectAMINE (Invitrogen) according to the manufacturer's guidelines. 5 h later, serum-free medium was replaced with phenol red-free DMEM supplemented with 5% sFBS. 12 h thereafter, cells were treated with the indicated amounts of various hormones. After 24 h of hormone treatment (12 h for inhibitor experiments), cells were harvested, and extracts were assayed for CAT activity, as described previously (45, 46) using butyryl-coenzyme A (Amersham Biosciences) and [3H]chloramphenicol (PerkinElmer Life Sciences). The quantity of resulting radiolabeled product was determined by scintillation counting using biodegradable counting scintillant (Amersham Biosciences) and a Beckman LS 6500 scintillation counter, and then normalized to total cellular protein measured by Bio-Rad protein assay. Experiments were done in duplicate, and values represent the average ± S.E. of at least three individual experiments.

Western Blot Analysis-- To determine ER expression levels, cells were transfected as above and harvested. Cell pellets were resuspended in a 50 mM Tris (pH 8.0) buffer containing 5 mM EDTA, 1% Nonidet P-40, 0.2% Sarkosyl, 0.4 M NaCl, 100 µM sodium vanadate, 10 mM sodium molybdate, and 20 mM NaF and incubated on ice for 1 h. The lysates were subsequently centrifuged at 21,000 × g for 10 min at 4 °C. The resulting supernatants were mixed with SDS-PAGE loading buffer, resolved on a 7.5% SDS-polyacrylamide gel, and subsequently transferred to nitrocellulose membrane. The membranes were blocked using 1% nonfat dried milk in 50 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween 20, and sequentially incubated with an anti-FLAG M2 antibody (Sigma) and a horseradish peroxidase-conjugated, anti-mouse antibody. Blots were visualized using enhanced chemiluminescence (ECL) reagents as recommended by the manufacturer (Amersham Biosciences).

Gel Mobility Shift Assays-- HeLa cells were harvested in phosphate-buffered saline using a cell scraper and subsequently resuspended in ice-cold lysis buffer (10 mM Hepes (pH 7.9), 1 mM EDTA, 60 mM KCl, 1 mM dithiothreitol, Complete Mini-Tablet protease inhibitor (Roche Diagnostics), and 0.5% Nonidet P-40), and incubated on ice for 5-15 min to lyse the cell membranes. The resulting lysates were centrifuged for 5 min at ~ 1,400 × g to pellet the nuclei. Nuclei were washed in 2.5 ml of lysis buffer lacking Nonidet P-40 and repelleted as before. Thereafter, the nuclei were resuspended in a minimum volume of 250 mM Tris (pH 7.8) buffer containing 60 mM KCl and protease inhibitors and subjected to three cycles of rapid freeze/thaw. The nuclear lysates were centrifuged for 15 min at 21,000 × g and 4 °C, and the protein concentrations of the resulting supernatants were determined by Bradford assay.

Complementary oligonucleotides corresponding to the region of the ERE-E1b-CAT plasmids containing the 12-O-tetradecanoylphorbol-13-acetate response element (TRE) (5'-TGAAAACCTCTGACACATGCAGCTCCC-3' and 5'-GGGAGCTGCATGTGTCAGAGGTTTTCA-3') or mTRE (5'-TGAAAACCTCGGACTCATGCAGCTCCC-3' and 5'-GGGAGCTGCATGAGTCCGAGGTTTTCA-3') were synthesized by Invitrogen and annealed in 10 mM Tris (pH 7.5) buffer containing 1 mM EDTA and 50 mM NaCl to create double-stranded oligonucleotides. These DNA fragments were labeled with [gamma -32P]ATP (7,000 Ci/mmol; ICN Biomedicals Inc., Irvine, CA) and T4 polynucleotide kinase. Unincorporated [gamma -32P]ATP was removed with a MicroSpin G25 column (Amersham Biosciences) according to the manufacturer's recommendation.

The radiolabeled oligonucleotides were combined with nuclear extract in binding reaction buffer (50 mM Tris (pH 7.5) containing 2.5 mM EDTA, 5 mM MgCl2, 2.5 mM dithiothreitol, 250 mM NaCl, 20% glycerol, and 0.25 mg/ml poly(dI-dC)) and incubated for 15 min at room temperature. For oligonucleotide binding competition and antibody supershift experiments, unlabeled oligonucleotides or antibody (anti-c-Jun (Upstate Biotechnology, Lake Placid, NY) or anti-c-Fos (Santa Cruz Biotechnology, Santa Cruz, CA)), respectively, were incubated with the nuclear extract for 30 min on ice before the addition of 32P-labeled probe. Free and complexed DNAs were resolved on a nondenaturing acrylamide gel. To avoid any possible influence of the loading dye on complex mobility, the gel-loading buffer containing 250 mM Tris (pH 7.5), 0.2% bromphenol blue, and 40% glycerol was added to the minus-extract control sample only. After electrophoresis at 350 V, the gel was transferred to 3MM Whatman paper, vacuum dried, and exposed to X-Omat film for autoradiography.

Assessment of Forskolin/IBMX-induced Phosphorylation-- For metabolic labeling studies, 2 × 106 HeLa cells were plated onto 150-mm Petri dishes, and 24 h thereafter they were transfected with pBind-ERalpha EF or pBind-ERbeta EF using a nonrecombinant adenovirus DNA transfer procedure (47). Briefly, plasmid DNA (200 ng/plate) was mixed with polylysine-coupled adenovirus and incubated for 30 min at room temperature. Before the addition of the adenovirus-plasmid particles to the cells, the medium was aspirated and replaced with DMEM. The adenovirus-plasmid particles were added to the cells at a 400:1 adenovirus:cell ratio. After incubation with the cells for 2 h at 37 °C, an equal volume of DMEM with 5% sFBS was added to each dish resulting in a final concentration of 2.5% sFBS. 24 h after transfection, the medium was aspirated from the dishes and replaced with phosphate-free DMEM, and the cells were incubated for 1 h at 37 °C. The medium was removed and replaced with phosphate-free DMEM containing 1% dialyzed FBS before the addition of 0.14 mCi/ml [32P]H3PO4. After a 14-16-h incubation, the plates were incubated for 90 min with either 0.1% dimethyl sulfoxide (vehicle) or 10 µM forskolin + 100 µM IBMX.

After treatments, cells were harvested and incubated with lysis buffer (10 mM Tris (pH 8.0), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM beta -mercaptoethanol, 50 mM potassium phosphate, 50 mM sodium fluoride, 0.1% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor mixture of 1 µg/ml each leupeptin, antipain, aprotinin, benzamidine HCl, chymostatin, and pepstatin), vortexed, and centrifuged at 20,000 × g for 10 min at 4 °C. Protein A-Sepharose beads (Amersham Biosciences) were incubated with 2 µg of antibody to the Gal4 DNA binding domain (sc-577; Santa Cruz Biotechnology) for 1 h at room temperature and then incubated with the cell lysate supernatant for 2 h while rotating at 4 °C. The beads were washed with 100 volumes of phosphate-buffered saline. After the addition of Laemmli sample buffer and boiling for 5 min, the samples were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane for autoradiography. The same membrane was subsequently used for Western blotting to detect pBind-ERalpha EF and pBind-ERbeta EF using a horseradish peroxidase-conjugated antibody to the Gal4 DNA binding domain (sc-510HRP; Santa Cruz Biotechnology) and ECL. Autoradiography signals were quantitated by densitometric scanning of films or quantitation of the ECL signal using a Kodak Image Station 440CF.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of ERalpha and ERbeta by a cAMP Signaling Pathway-- Several studies have demonstrated that agents that stimulate increases in cAMP can promote ERalpha -dependent gene expression in the apparent absence of hormone (19-22, 32, 48). To determine whether ERbeta could be activated in a similar manner, HeLa cells were transiently transfected with expression vectors for either human ERalpha or ERbeta along with an ERE-E1b-CAT synthetic target construct that possesses an ERE from the Xenopus vitellogenin A2 promoter linked to the adenoviral E1b TATA box and CAT reporter gene. Cells were subsequently stimulated with either E2 or a combination of forskolin and IBMX (forskolin/IBMX), which increases cAMP production by activating adenylate cyclase and inhibiting phosphodiesterases, respectively, and CAT activity was measured. As expected, E2 stimulated both ERalpha - and ERbeta -dependent transcription of this reporter (Fig. 1). Consistent with previous reports (22, 32), stimulation with forskolin/IBMX also resulted in a robust stimulation of ERalpha transcriptional activity, although a longer (24 h) hormone treatment greatly enhanced the E2-stimulated relative to forskolin/IBMX-stimulated response (compare Figs. 1 and 2). Importantly, under the same conditions ERbeta was also activated upon stimulation of cells with forskolin/IBMX (Fig. 1). Pretreatment with a PKA-selective inhibitor, H89 (49), blocked forskolin/IBMX-induced gene expression by both receptor subtypes, thus demonstrating that forskolin/IBMX induction of ERalpha and ERbeta activity is mediated by a cAMP/PKA signaling pathway in these cells. Moreover, this inhibition was specific for forskolin/IBMX induction because H89 treatment did not significantly alter basal or E2-stimulated responses for either ERalpha or ERbeta .


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Fig. 1.   Forskolin and IBMX-induced ERalpha and ERbeta transcriptional activity are dependent upon PKA signaling. HeLa cells were transiently transfected with 10 ng of expression plasmid for ERalpha (pCMV5-ERalpha ) or ERbeta (pCXN2-ERbeta ) and 1 µg of ERE-E1b-CAT reporter plasmid. Cells were subsequently treated for 12 h with vehicle (0.1% ethanol), 1 nM E2, or 10 µM forskolin + 100 µM IBMX (F/I) after a 1-h pretreatment with either 10 µM H89 (+) or dimethyl sulfoxide (-). Values are normalized to the activity of ERalpha in the absence of hormone and represent the average ± S.E. of three independent experiments.


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Fig. 2.   cAMP/PKA stimulation of ER-dependent transcription requires ER binding to its cognate hormone response element. HeLa cells were transiently transfected with either 10 ng of expression plasmid for ERalpha (pCMV5-ERalpha ) or ERbeta (pCXN2-ERbeta ) along with 1 µg of ERE-E1b-CAT or E1b-CAT. Cells were subsequently treated with vehicle, 1 nM E2, or 10 µM forskolin + 100 µM IBMX (F/I) for 24 h. Values are normalized to ERE-E1b-CAT reporter activity for ERalpha in the absence of hormone and represent the average ± S.E. of three experiments.

An ERE Is Required for ERalpha and ERbeta Activation of ERE-E1b-CAT Expression by the cAMP/PKA Signaling Pathway-- To test whether the cAMP signaling response was mediated through an ER genomic mechanism in which the ER binds directly to the promoter, the effects of forskolin/IBMX on ERalpha and ERbeta was assessed on the E1b-CAT reporter, which lacks an ERE. The expected ERalpha - and ERbeta -dependent responses were observed for the ERE-E1b-CAT reporter. As anticipated, basal transcription of the E1b-CAT promoter was minimal, and E2 did not further increase CAT gene expression in cells transfected with either ERalpha or ERbeta (Fig. 2). Moreover, forskolin/IBMX did not stimulate E1b-CAT reporter gene activity in the presence of transfected ERalpha , and only a very weak forskolin/IBMX-dependent E1b-CAT expression was observed in the presence of ERbeta . These data indicate that ERalpha - and ERbeta -dependent transcription in response to forskolin/IBMX requires the presence of an ERE.

CBP and p160/SRC Coactivators Enhance cAMP-stimulated ERalpha and ERbeta Responses-- ERalpha - and ERbeta -dependent transcriptional differences are partly attributable to the selectivity these receptors possess for the different coactivators in the presence of various ligands (50, 51). To extend this concept, we overexpressed the p160 coactivators (SRC-1, TIF2, and RAC3) as well as the general coactivator/cointegrator, CBP, in cells to determine whether any of them might distinguish between ERalpha and ERbeta in their ability to potentiate activation by cAMP-dependent signaling. Each of these coactivators strongly enhanced ERalpha - and ERbeta -dependent transcription compared with reporter activity in the absence of exogenous coactivator (Fig. 3). However, none of the coactivators selectively enhanced the activity of estrogen-activated receptor over cAMP-activated receptor, nor of one receptor subtype over the other. These results suggest that each of the p160 coactivators and CBP can contribute significantly to the forskolin/IBMX-induced activation of both ERalpha and ERbeta .


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Fig. 3.   The p160 and CBP coactivators enhance cAMP/PKA-mediated ERalpha - and ERbeta -dependent transcription. HeLa cells were transiently transfected with 10 ng of expression plasmid for ERalpha (pCMV5-ERalpha ) or ERbeta (pCXN2-ERbeta ) along with 250 ng of expression plasmid for SRC-1e, TIF2, RAC3, or the empty vector (pCR3.1) and 1 µg of ERE-E1b-CAT reporter. Cells were subsequently treated with vehicle, 1 nM E2, or 10 µM forskolin + 100 µM IBMX (F/I). CAT measurements were standardized to total protein, and the results are the average ± S.E. of three experiments.

Activation of ERalpha and ERbeta by cAMP Depends on Promoter Context-- In general, ER-mediated transcription depends on the promoter context in which an ERE is found (11, 52, 53). We therefore tested the extent to which forskolin/IBMX could stimulate ER-dependent gene expression in the context of various synthetic and natural ERE-containing promoters. The pS2 construct contains the -1100 to +10 region of the E2-responsive human pS2 promoter subcloned into a CAT reporter plasmid (41). The pATC0, pATC1, and pATC2 are synthetic reporters that possess 0, 1, or 2 EREs, as indicated in their nomenclature. The ERE-E1b-CAT reporter construct was included in these experiments as a control. There was no E2-induced response for pATC0 because it had no ERE, a weak response for pATC1, and a synergistic E2-dependent response for pATC2, for both ER subtypes (Fig. 4, A and B). Notably, there was no forskolin/IBMX-induced response on either of these promoters, demonstrating that the number of EREs, in and of itself, had no effect on the forskolin/IBMX-induced activation. Moreover, although the expected E2-dependent increases were present for both ERalpha and ERbeta on the pS2 promoter, there was no significant increase in reporter activity in response to forskolin/IBMX. Thus, the cAMP/PKA signaling pathway mediates ERalpha - and ERbeta -dependent gene expression in a promoter-dependent fashion.


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Fig. 4.   cAMP activation of ERalpha and ERbeta depends on promoter context. HeLa cells were transiently transfected with 10 ng of expression plasmid for ERalpha (pCMV5-ERalpha ) (A) or ERbeta (pCXN2-ERbeta ) (B) along with 1 µg of the indicated CAT reporter plasmids. Cells were subsequently treated with vehicle, 1 nM E2, or 10 µM forskolin + 100 µM IBMX (F/I). Values for ERE-E1b-CAT and pS2-CAT are normalized to their respective vehicle-treated samples, which were arbitrarily set as 100. Values for pATC0, pATC1, and pATC2 are normalized to vehicle treatment for pATC2, which was set as 100. The results are the averages ± S.E. of three experiments.

The Upstream TRE Enhancer in the ERE-E1b-CAT Reporter Is Required but Not Sufficient for Activation of ERbeta -dependent Gene Activation by Forskolin/IBMX-- Promoter differences in forskolin/IBMX-induced responses suggested that cis-acting factor(s) in addition to EREs contributed to cAMP-stimulated ERalpha and ERbeta transcriptional activities. It has been reported that ERalpha and ERbeta can mediate ligand-dependent responses at TREs, independent of EREs, when they are tethered to the promoter via AP-1 transcription factor complexes (54, 55). Previous sequence analysis (56) revealed that a putative TRE (TGACACA) that differs from the consensus TRE sequence (TGAGTCA) by two nucleotides resides in the backbone of many plasmid vectors. In ERE-E1b-CAT such a putative TRE is located ~255 bp upstream from the ERE. To determine whether this TRE played any role in the ability of forskolin/IBMX to stimulate the activity of either ER, we examined the ability of forskolin/IBMX to increase CAT gene expression from a reporter plasmid (ERE-E1b-CAT(mTRE)) in which the TRE was mutated to GGACTCA, a mutation previously demonstrated to abolish AP-1 binding (39, 57). As shown in Fig. 5A, the TRE mutation decreased overall reporter gene activity in the presence of both ERalpha and ERbeta whether cells were treated with vehicle, E2, or forskolin/IBMX. However, E2 was still able to increase ERE-E1b-CAT(mTRE) activity above basal for both ERs, and forskolin/IBMX stimulated this reporter activity in the presence of ERalpha . In contrast, forskolin/IBMX was unable to stimulate ERbeta -dependent transcription of ERE-E1b-CAT(mTRE), suggesting that this putative TRE was necessary for cAMP-induced ERbeta -mediated increases in ERE-E1b-CAT reporter gene expression. Similar results were obtained when this TRE was removed by an NdeI to Eco0109I deletion of a 195-bp region surrounding the site (39) as opposed to mutating it (data not shown). Western blot analysis indicated that cAMP activation of ERalpha on the ERE-E1b-CAT reporter (with or without the TRE) was not caused by an increase ERalpha protein levels (Fig. 5B). In contrast to ERalpha , ERbeta protein expression is elevated modestly by forskolin/IBMX. However, this does not permit cAMP-stimulated ERE-E1b-CAT(mTRE) activity by ERbeta . Thus, cAMP activation of ERbeta is dependent on a putative TRE site, but loss of this cis element only partially attenuates ERalpha activity and indicates that activation of ERalpha and ERbeta by cAMP signaling is distinct. Taken together with the above data that demonstrated a requirement for an ERE, these data suggest that forskolin/IBMX promotes synergism between either ERalpha or ERbeta and a factor(s) that is bound to a neighboring DNA response element (i.e. the putative TRE).


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Fig. 5.   A putative AP-1 response element in the target gene promoter is essential for cAMP/PKA-mediated transcription by ERbeta . A, HeLa cells were transiently transfected with 10 ng of expression plasmid for ERalpha (pCMV5-ERalpha ) or ERbeta (pCXN2-ERbeta ) along with 1 µg ERE-E1b-CAT or ERE-E1b-CAT(mTRE). Cells were subsequently treated with vehicle, 1 nM E2, or 10 µM forskolin + 100 µM IBMX (F/I). Values are normalized to ERE-E1b-CAT reporter activity for ERalpha in the absence of hormone and represent the average ± S.E. of three experiments. B, HeLa cells were transfected with 1 µg of either FLAG-ERalpha or 3xFLAG-ERbeta expression plasmid, and receptor expression was detected with anti-FLAG (M2) antibody. The blot shown is representative of three experiments.

To determine whether c-Jun and/or c-Fos expressed in HeLa cells could bind to the putative TRE site, an electrophoretic mobility shift assay was performed. As shown in Fig. 6, a 27-bp oligonucleotide corresponding to the TRE-containing region of the target gene bound to factors present in a HeLa cell nuclear extract, and this binding could be competed with an excess of cold oligonucleotide. The addition of antibodies against c-Jun or c-Fos to the binding reaction resulted in the supershift of the oligonucleotide-protein complex, indicating that both of these proteins associate with this DNA fragment. Notably, equivalent levels of oligonucleotide-bound material were observed regardless of whether the nuclear extract was prepared from vehicle- or forskolin/IBMX-treated cells, indicating that the forskolin/IBMX-induced activation of ER-dependent gene expression was not a result of increased Jun/Fos occupancy of the promoter region. This is consistent with the comparable levels of c-Jun and the small increase in c-Fos expression detected by Western blot analyses of extracts prepared from vehicle- and forskolin/IBMX-treated cells (data not shown).


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Fig. 6.   c-Jun and c-Fos binding to the putative TRE is similar in vehicle- and forskolin/IBMX-treated HeLa cells. Nuclear extracts prepared from vehicle (-) or 10 µM forskolin/100 µM IBMX-treated cells (+) were subjected to electrophoretic mobility shift assays using a 32P-labeled TRE as probe. Competitions with a 100-fold excess of unlabeled probe (TRE) or a DNA fragment containing a mutation in the TRE site (mTRE) are shown in lanes 3 and 6, and 4 and 7, respectively. Supershift assays are shown with c-Jun (lanes 9 and 11) or c-Fos (lanes 8 and 10) antibodies. Lane 1 contains no nuclear extract. A representative experiment is shown.

The Carboxyl Terminus of ERalpha and ERbeta Mediates Forskolin/IBMX-induced Transcription, Which Can Be Enhanced Further by the Amino Terminus of ERalpha but Not ERbeta -- Two previous reports have characterized the physical interactions between ERalpha and the AP-1 transcription factor family member, c-Jun (55, 58). Although one of these studies demonstrated that ERalpha interaction with c-Jun is predominantly mediated by the centrally located hinge region (domain D) of the receptor (58), both indicated that an interaction with the amino terminus of ERalpha is also possible. Therefore, to assess the potential contribution of the amino-terminal A/B domain in mediating forskolin/IBMX-induced responses, ERalpha and ERbeta deletion mutants lacking their A/B domains (ERalpha -179C and ERbeta -143C) were constructed, and the ability of the resulting receptors to stimulate ERE-E1b-CAT reporter activity in response to forskolin/IBMX stimulation was examined. In the absence of transfected ER (Fig. 7A, Empty), there is very weak CAT gene expression in response to forskolin/IBMX treatment. This minimal promoter activity is substantially less compared with activity in the presence of transfected ERs. As shown by comparison of ERalpha -179C (ERalpha amino acids 179-595) with wild type ERalpha , deletion of the amino terminus of ERalpha reduced forskolin/IBMX as well as basal and E2-stimulated activities, which is consistent with the constitutively active amino-terminal AF-1 domain of this receptor contributing to E2-dependent ERalpha responses (7, 11). In contrast, removing the A/B region of ERbeta to generate ERbeta -143C (ERbeta amino acids 143-530) did not reduce forskolin/IBMX induction of ERbeta -dependent target gene expression. Notably, the E2-stimulated activity of ERbeta -143C is much higher than that of wild type ERbeta , which is in agreement with a previously reported inhibitory function for the amino terminus of ERbeta (59).


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Fig. 7.   Different ERalpha and ERbeta domains mediate promoter-specific gene expression in response to the cAMP/PKA signaling pathway. HeLa cells were transiently transfected with 10 ng of expression plasmid for ERalpha (pCR3.1-ERalpha ), ERalpha -179C (pCR3.1-ERalpha -179C), ERbeta (pCR3.1-ERbeta ), ERbeta -143C (pCR3.1-ERbeta -143C), or empty vector (pCR3.1) along with 1 µg of either ERE-E1b-CAT (A) or ERE-E1b-CAT(mTRE) (B). Cells were subsequently treated with vehicle, 1 nM E2, or 10 µM forskolin + 100 µM IBMX (F/I). Values are the average ± S.E. of three experiments.

As shown in Fig. 7B, ERalpha but not ERbeta retained the ability to stimulate CAT expression from the TRE-minus reporter (ERE-E1b-CAT(mTRE)) in response to forskolin/IBMX (see also Fig. 5). Interestingly, much of the E2-induced and all of the remaining forskolin/IBMX-induced ERalpha activity are lost when the A/B domain is removed, as shown for ERalpha -179C, thus supporting the above data that this domain can mediate cAMP-dependent activation of ERalpha . Taken together, these results demonstrate that domains C through F of ERalpha and ERbeta are sufficient for cAMP/PKA signaling pathway activation of either receptor provided that an AP-1 DNA binding site is present on the promoter; these results also indicate that this functional interaction is enhanced by the A/B domain of ERalpha but not ERbeta .

Several studies have focused on the ability of the MAP kinase signaling pathway to stimulate the AF-1 activity of ERalpha (8, 10). Because it is known that the cAMP/PKA signaling pathway can cross-talk with the MAP kinase signaling pathway (34, 60), we examined whether the MAP kinase-directed phosphorylation site in the amino terminus of ERalpha (Ser118) might be important for the forskolin/IBMX-induced activity of the ERalpha AF-1 domain. However, mutating this serine to an alanine (ERalpha -S118A) did not alter the ability of ERalpha to stimulate transcription of the ERE-E1b-CAT(mTRE) reporter in response to forskolin/IBMX (data not shown). Similarly, alanine mutation of the other known amino-terminal phosphorylated residues in ERalpha (Ser104/106/118 or Ser167) did not inhibit forskolin/IBMX induction of reporter gene expression (data not shown), indicating that these phosphoserine residues did not account for the forskolin/IBMX-dependent activity of the ERalpha AF-1 domain. Thus, ERalpha AF-1 activity in response to forskolin/IBMX is likely to be mostly the result of cAMP/PKA signaling to a factor(s) that can interact with the A/B domain rather than altering the phosphorylation of the ERalpha amino terminus itself.

Functional Interactions with the TRE-bound Factor(s) Can Be Mediated by the EF Region of ERalpha but Not ERbeta -- It has been demonstrated that amino acids 259-302 of ERalpha constitute a major interaction site with c-Jun (58), and because the ability of ERalpha -179C to mediate forskolin-induced activation of CAT expression was dependent on a TRE site within the reporter gene that has been shown to support c-Jun physical and functional interactions (39, 56), we investigated the possibility that cAMP activation of ERalpha -179C was caused by a direct interaction between the hinge region of the receptor and c-Jun. To test this hypothesis, the EF domain of ERalpha (amino acids 302-595) and the corresponding ERbeta fragment (amino acids 254-530) were fused to the Gal4 DNA binding domain (Gal-ERalpha EF and Gal-ERbeta EF) and examined for their abilities to stimulate expression of 17mer-E1b-CAT, which contains four Gal4 binding sites upstream from the TATA box and CAT gene. This reporter also possesses the TRE site upstream of the Gal4 binding sites. As expected, both Gal-ERalpha EF and Gal-ERbeta EF were stimulated by E2 (Fig. 8). Importantly, forskolin/IBMX stimulated Gal-ERalpha EF-dependent expression of the 17mer-E1b-CAT but not the TRE-minus reporter, 17mer-E1b-CAT(Delta TRE). This demonstrates that an ERalpha construct lacking all of the c-Jun binding sites can still be activated; this indicates that the interaction between the TRE-binding factor and ERalpha could be indirect and may be mediated via a trans-acting factor that can bind to both the EF domain of ERalpha and a TRE-binding factor such as c-Jun. In contrast, the EF region of ERbeta is insufficient to activate reporter gene expression regardless of the presence or absence of a TRE site, indicating that regions within the DNA binding domain and/or hinge of ERbeta are important for activation of target gene expression in response to cAMP signaling. Taken together, these results indicate that the EF domains of ERalpha and ERbeta differ in their abilities to mediate ER cooperation with a TRE-bound factor in response to cAMP.


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Fig. 8.   Mapped c-Jun interaction sites in the ERalpha A/B domain and hinge are not required for forskolin/IBMX stimulation. HeLa cells were transiently transfected with 100 ng of expression plasmid for Gal-ERalpha EF (pBind-ERalpha EF), Gal-ERbeta EF (pBind-ERbeta EF), or Gal4 DNA binding domain (pBind) along with 1 µg of either 17mer-E1b-CAT or 17mer-E1b-CAT(Delta TRE). Cells were subsequently treated with vehicle, 1 nM E2, or 10 µM forskolin + 100 µM IBMX (F/I). Values are normalized to Gal-ERalpha EF activity for 17mer-E1b-CAT in the presence of vehicle and represent the average ± S.E. of three experiments.

To assess the effect of forskolin/IBMX treatment on Gal-ERalpha EF and Gal-ERbeta EF phosphorylation, expression vectors for Gal-ERalpha EF or Gal-ERbeta EF were transfected into HeLa cells using an adenovirus-mediated DNA transfer technique to increase protein expression levels (47) followed by in vivo labeling with [32P]H3PO4 and incubation with forskolin/IBMX or vehicle for 90 min. The results of a representative experiment are shown in Fig. 9, A and B, and demonstrate that when 32P signals were normalized to protein levels that there was an increase in the overall level of Gal-ERalpha EF phosphorylation after incubation with forskolin/IBMX, whereas the same treatment induced a decrease in Gal-ERbeta EF phosphorylation. This experiment was repeated three times, and the averaged results reveal that forskolin/IBMX increased Gal-ERalpha EF phosphorylation by 44 ± 6% but decreased Gal-ERbeta EF phosphorylation by 45 ± 17% (Fig. 9C).


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Fig. 9.   Effect of forskolin/IBMX treatment on Gal-ERalpha EF and Gal-ERbeta EF phosphorylation. HeLa cells were transfected with expression vectors for Gal-ERalpha EF (A) or Gal-ERbeta EF (B), incubated with 0.14 mCi/ml [32P]H3PO4 followed by incubation for 90 min with either vehicle (veh) or 10 µM forskolin + 100 µM IBMX (F/I). Gal-ERalpha EF and Gal-ERbeta EF were immunopurified and then electrophoresed on a 10% SDS-polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane, and the membrane was exposed to X-AR film for autoradiography (left panels). After autoradiography, the membrane was subjected to Western blotting with anti-Gal-horseradish peroxidase antibody and detection using enhanced chemiluminescence (right panels). C, autoradiography signals were quantitated by densitometric scanning of films, and Western blot signals were quantitated by either densitometric scanning of films or quantitation of the ECL signal using a Kodak Image Station 440CF. Normalized phosphorylation values for Gal-ERalpha EF and Gal-ERbeta EF were calculated by dividing the value for the 32P signal by the value for the protein signal. Data are plotted as change in phosphorylation relative to vehicle treatment (set at a value of 1). The experiment was repeated three times with the mean ± S.E. reported.

Forskolin/IBMX-stimulated ERalpha Activity Is Not Caused by cAMP/PKA-dependent SRC-1 Phosphorylation-- It had been demonstrated previously that the chicken PR is not phosphorylated in response to cell treatment with 8-bromo-cAMP, but rather cAMP activation of transcription seems to be mediated by an increase in SRC-1 phosphorylation (34). Moreover, SRC-1 binds to both the EF domain of ERalpha as well as c-Jun (61, 62) and is therefore a good candidate for mediating indirect interactions between these two transcription factors, as described above. Therefore, we examined whether the ability of cAMP/PKA to modulate SRC-1 phosphorylation might also contribute to forskolin/IBMX-dependent activation of ERalpha . SRC-1 expression vectors containing substitutions for the two cAMP-induced phosphorylatable residues (T1179/S1185A) or substitutions for all of the seven previously mapped phosphorylation sites (at positions 372, 395, 517, 569, 1033, 1179, and 1185; Ref. 63) were introduced into cells, and the abilities of these mutant coactivators to enhance ERalpha -dependent reporter activity was assessed. Compared with wild type SRC-1 there is an ~20 and ~35% decrease in the ability of the SRC-1T1179/S1185A and the seven-alanine mutant (SRC-17Ala), respectively, to potentiate forskolin/IBMX-induced ERalpha activity (Fig. 10, A and B). Nonetheless, decreases in ERalpha coactivation by both the SRC-1T1179/S1185A and the SRC-17Ala mutants were equal for basal as well as for E2-stimulated and forskolin/IBMX-stimulated responses, suggesting that SRC-1 phosphorylation does not specifically modulate cAMP-dependent activation of ERalpha .


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Fig. 10.   Alanine mutation of SRC-1 phosphorylation sites decreases its coactivation of ERalpha but is not specific to cAMP/PKA-dependent signaling. A, a representative experiment in which HeLa cells were transiently transfected with 10 ng of expression plasmid for ERalpha (pCMV5-ERalpha ) along with 1 µg of expression plasmid for either wild type or mutant SRC-1a (SRC1T1179/S1185A or SRC-17Ala) or the empty vector (pCR3.1) and 1 µg ERE-E1b-CAT reporter. Cells were subsequently treated with vehicle, 1 nM E2, or 10 µM forskolin + 100 µM IBMX (F/I). B, combined results from seven experiments. Relative coactivation was determined by dividing reporter activity in the presence of wild type SRC-1 by values obtained in the absence of coactivator (pCR3.1) for each treatment group (vehicle, E2, and F/I) and defining this value as 100. Values for mutant SRC-1 coactivation are given relative to wild type SRC-1 coactivation. Results are the averages ± S.E. of seven experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report, we demonstrated that forskolin/IBMX, through increased intracellular cAMP and activation of PKA, can stimulate ERbeta -dependent transcription, as was shown previously to be the case for ERalpha . However, there are significant differences in the ability of ERalpha and ERbeta to be ligand-independently activated by this mechanism. In particular, a TRE upstream from the ERE was necessary for ERbeta -dependent transcription in response to stimulation with forskolin/IBMX, whereas this sequence contributed to, but was not required for, activation of full-length ERalpha . Furthermore, functional interactions with the TRE-bound factor(s) could be mediated by the EF region of ERalpha but not ERbeta , and forskolin/IBMX treatment increased the phosphorylation of Gal-ERalpha EF but decreased the phosphorylation of the corresponding ERbeta construct. Overexpression of the p160 and CBP coactivators enhanced forskolin/IBMX-induced ERalpha and ERbeta activity, indicating that these coactivators can form functional complexes with the unliganded receptor. However, in contrast to the previously reported importance of SRC-1 phosphorylation for cAMP-mediated activation of chicken PR (34), the contribution of these phosphorylation sites to SRC-1 coactivation of cAMP-induced ERalpha was minor and not specific to ER activation by forskolin/IBMX. Taken together, these data demonstrate that the cAMP signaling pathway can stimulate ER-dependent transcription by promoting functional interactions among TRE-bound transcription factor(s), coactivators, and either ERalpha or ERbeta bound to an ERE.

There has been considerable interest in understanding the relative contributions of the ERalpha and ERbeta AF-1 domains in mediating ER-dependent transcription. The A/B domain of ERalpha , which encompasses the AF-1, is generally more active than that of ERbeta (59). Interestingly, although a MAP kinase signaling pathway can induce ERalpha and ERbeta AF-1 activity in the absence of ligand (8-10), our results demonstrate that cAMP signaling can stimulate the AF-1 activity of ERalpha , but not ERbeta . Although it has been reported that the cAMP signaling pathway can stimulate MAP kinase activity, mutation of the previously identified amino-terminal MAP kinase phosphorylation site in ERalpha (8, 10) does not inhibit its activation by forskolin/IBMX. Consistent with this result, our work and that of LeGoff et al. (31) demonstrates cAMP-induced phosphorylation of the ERalpha carboxyl-terminal domain; however, the location of these phosphorylation sites in vivo remains to be identified. It is therefore likely that forskolin/IBMX activation of ERalpha via the AF-1 domain is caused by cAMP/PKA signaling effects on the recruitment and/or activity of a coactivator(s) that interacts selectively with the AF-1 domain of ERalpha . Interestingly, there are several coactivators, including the p72/p68 RNA-binding DEAD box proteins (64, 65) and the RNA coactivator, SRA,2 which have been found to interact selectively with the AF-1 domain of ERalpha but not that of ERbeta . Moreover, in addition to p68 being a phosphorylated protein (66), all three of these coactivators are found in complexes with other coactivator molecules, such as CBP, SRC-1, TIF2, and AIB1 (64, 65), which are known to be phosphoproteins (34, 67-69).

The carboxyl-terminal ligand binding domains of ERalpha and ERbeta possess considerably higher sequence homology than do the A/B domains. Interestingly, however, there are also differences in the abilities of the ERalpha and ERbeta EF domains to be activated by cAMP signaling, as indicated by the ability of Gal-ERalpha EF but not Gal-ERbeta EF to stimulate 17mer-E1b-CAT activity in response to forskolin/IBMX. Interestingly, forskolin/IBMX induced opposite changes in overall phosphorylation for these two constructs. Consistent with a previous demonstration of cholera toxin and IBMX-induced phosphorylation of an ERalpha mutant lacking its A/B domains (31), forskolin/IBMX increased the phosphorylation of the Gal-ERalpha EF chimera. There are four protein kinase A consensus sites (XRRXSX or SKKIXSIX) in the carboxyl terminus of ERalpha : Ser236, Ser305, Ser338, and Ser518. However, mutation of the Ser236, Ser305, and Ser518 sites to alanines does not block cAMP-induced ERalpha transcriptional activity (35), and the Ser236 site is not present in the Gal-ERalpha EF construct. Although a S338A mutation does inhibit cholera toxin/IBMX-induced ERalpha activity, substitution of a glutamic acid at this position does not mimic the ligand-independent response, suggesting that mutation of this residue may affect cAMP signaling to the receptor by inducing structural perturbations (35). The only other phosphorylation sites identified in the ERalpha carboxyl terminus, Thr311 (70) and Tyr537 (71), are also unlikely to be the targets of the forskolin/IBMX-induced phosphorylation site because cAMP signaling has been reported to induce only serine phosphorylation of ERalpha (31). The location of the phosphorylation site(s) induced by forskolin/IBMX in vivo, as well as their importance for cAMP activation of ERalpha transcriptional activity therefore remain to be characterized; this is however, beyond the scope of this report. It should be noted that the forskolin/IBMX-induced site need not lie within a protein kinase A consensus sequence because the cAMP signaling has been shown to cross-talk with other kinase pathways (34, 60). Apart from an in vitro demonstration of p38-induced phosphorylation of the AF2 domain of ERbeta (72), little is known about phosphorylation of the ERbeta EF domain. Our results therefore provide the first demonstration that the ERbeta carboxyl-terminal region is phosphorylated in vivo, as well as novel information indicating that the basal level of ERbeta EF domain phosphorylation is reduced by forskolin/IBMX treatment in HeLa cells. Although this may seem paradoxical, cAMP has been shown to inhibit MAP kinase pathways through cross-talk with the G protein Rap1 and Raf-1 (60).

The inability of the AF-1-deletion mutants (ERalpha -179C and ERbeta -143C) and the Gal-ERalpha EF chimera to stimulate the activities of reporters lacking TREs (ERE-E1b-CAT(mTRE) and 17mer-E1b-CAT(Delta TRE), respectively) in response to forskolin/IBMX also suggests that activation of domains EF of ERalpha and C through F of ERbeta require a functional interaction between these receptor domains and a TRE-bound transcription factor. Based on sequence information, this TRE-binding factor most likely belongs to the AP-1 transcription factor family. This includes Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra1, Fra2), and ATF (ATFa, ATF2, ATF3) proteins, which can form homo/heterodimers among themselves and promote transcription via binding to palindromic sequences (TGA(C/G)TCA) that are found in a number of promoters (for review, see Ref. 73). Although the sequence (TGACACA) present in our ERE-E1b-CAT reporter diverges from the consensus TRE sequence, it has been shown to bind to in vitro translated Jun and Fos (56), and our gel mobility shift assays reveal that the levels of both c-Jun and c-Fos expressed in HeLa cells are sufficient to bind to this DNA. Moreover, our laboratory demonstrated previously through overexpression studies that c-Jun can enhance ERalpha -mediated transcription of ERE-E1b-CAT but not of the corresponding TRE mutant reporter, ERE-E1b-CAT(mTRE) (39). Because we demonstrate a requirement for the ERE and TRE DNA sites, our data suggest that cooperation between ER and AP-1 transcription factors bound to their respective target promoter sequences results in robust forskolin/IBMX activation of target gene expression. Based on the ability of just the EF domains of ERalpha to be activated by cAMP even though this portion of ERalpha does not bind c-Jun or c-Fos (58), it is likely that the interactions between ER and AP-1 transcription factors need not be direct, although we cannot rule out the possibility of other TRE-binding factors directly contacting ERalpha and/or ERbeta .

One potential mechanism through which TRE and ERE binding factors could interact indirectly with one another is through coactivators. We have demonstrated that CBP as well as all three p160 coactivators can enhance forskolin/IBMX-induced transcription of ERE-E1b-CAT by ERalpha and ERbeta , suggesting that these coactivators can form functional complexes with ERs in the absence of hormone. Moreover, overexpression of these coactivators does not compensate for the inability of forskolin/IBMX to stimulate ERbeta -dependent transcription of the ERE-E1b-CAT(mTRE) target construct (data not shown), indicating that their ability to stimulate cAMP-induced ER function is derived from ER and TRE-binding factor interactions. There are a number of potential candidates that possess the ability to bind to c-Jun as well as ERs. These include the coactivators SRC-1, JAB1, and CAPER as well as the integrator protein, CBP (61, 74-77). In all cases but CAPER, the coactivator protein utilizes distinct sites to bind to c-Jun and nuclear receptors, suggesting that these coactivators are well suited to act as physical bridges between these two classes of transcription factors. Although all of the p160 coactivators and CBP contributed to forskolin/IBMX-induced activation of ER target gene expression, we were unable to observe a similar activity by the c-Jun activation-binding protein, JAB1 (data not shown).

Cyclic AMP signaling leads to phosphorylation of SRC-1 at Thr1179 and Ser1185 residues contributing to stabilizing interactions between CBP-P and p300/CBP-associated factor and functional synergy between CBP and SRC-1 (34). Moreover, mutation of these two amino acids to alanines reduced both progesterone-stimulated and, in an even more marked fashion, cAMP-stimulated chicken PR activity in COS cells. However, these mutations did not completely block the ability of SRC-1 to enhance cAMP-dependent activation of chicken PR, suggesting that phosphorylation of another cofactor(s) may contribute to activation of this receptor by cAMP. The same mutations only slightly impaired the ability of SRC-1 to enhance ERalpha activity stimulated by E2 and cAMP. Moreover, mutation of all seven SRC-1 phosphorylation sites identified by Rowan et al. (63) also reduced the overall efficacy of this coactivator but, again, regardless of receptor stimulus. These data suggest that SRC-1 is not specifically involved in the activation of ERalpha by cAMP and that this ligand-independent activity can be mediated by another ERalpha -interacting cofactor(s). It should be noted that growth factor and protein kinase C signal transduction pathways have been shown to alter the phosphorylation and/or coactivation potential of GRIP1/TIF2, AIB1/RAC3, and p300/CBP coactivators (67-69), and it is possible that cAMP cross-talk with one or more of these factors is critical for activation of ER transcriptional activity. An examination of this possibility awaits identification of cAMP-induced phosphorylation sites in these coactivators. Taken together, our data indicate that cAMP activation of cPR and ERalpha differ in the extent to which SRC-1 phosphorylation is required for this process as well as whether the respective receptors are themselves phosphorylated. In addition, ERalpha and ERbeta also differ in their phosphorylation, their dependence on promoter TRE sites, and the minimal region of receptor required to respond to cAMP signaling. Overall, this argues that multiple mechanisms contribute to cAMP activation of nuclear receptor transcriptional activity.

The promoters of endogenous genes typically consist of binding sites for many distinct transcription factors. Importantly, the human pS2 promoter contains binding sites for ERs as well as AP-1 transcription factors (78), indicating that expression of this gene, which had previously been demonstrated to be activated by cAMP in an ICI 164,384-inhibited manner, might involve cross-talk between ER and AP-1. Unexpectedly, pS2-CAT was not activated by ERalpha or ERbeta in response to forskolin/IBMX treatment. This could be the result of cell type differences and/or loss of a promoter region critical for cAMP activation of ER during construction of the pS2-CAT reporter. DNA sequence analyses have enabled us to identify several other target gene promoters containing both TRE and ERE sites. Thus, the ability of the cAMP signaling pathway to stimulate ER-dependent transcription via ER-AP-1 interactions might be applicable to many other ER target genes. Based on our studies, it is clear that ER-dependent responses cannot be predicted on the presence of an ERE alone, but consideration must be given to the complexity of such promoters and how these receptors interact with various nonreceptor transcription factors, either directly or through coactivator/cointegrator molecules. Undoubtedly, the ability of coactivators to integrate responses through various classes of transcription factors adds another level of control and specificity to regulation of gene expression.

    ACKNOWLEDGEMENTS

We are grateful to Francesca Gordon and Drs. Pierre Chambon, Basem Jaber, Benita Katzenellenbogen, David Lonard, Masami Muramatsu, and David Shapiro for providing the plasmids.

    FOOTNOTES

* This work was supported by a UNCF/Merck fellowship and National Institutes of Health Training Fellowship HD07165 (to K. M. C.), Department of Defense Breast Cancer Research Program Fellowship DAMD17-00-1-0136 (to M. D.), Department of Defense Breast Cancer Research Program Awards DAMD17-02-1-0531 and DAMD17-02-1-0530 (to B. G. R.), and Department of Defense Grant DAMD17-98-1-8282 and National Institutes of Health Grant DK53002 (to C. L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. Molecular and Cellular Biology, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-6235; Fax: 713-790-1275; E-mail: carolyns@bcm.tmc.edu.

Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M212312200

2 K. M. Coleman and C. L. Smith, unpublished data.

    ABBREVIATIONS

The abbreviations used are: ER(s), estrogen receptor(s); AF, activation function; AIB1, amplified in breast cancer-1; AP-1, activated protein-1; CAT, chloramphenicol acetyltransferase; CBP, CREB-binding protein; CMV, cytomegalovirus; CREB, cAMP response element-binding protein; DMEM, Dulbecco's modified Eagle's medium; E2, 17beta -estradiol; ERE, estrogen response element; FBS, fetal bovine serum; GRIP1, glucocorticoid receptor-interacting protein-1; H89, N-(2-(p-bromocinnamylamino)-ethyl)-5-isoquinolinesulfonamide; IBMX, 3-isobutyl-1-methylxanthine; MAP kinase, mitogen-activated protein kinase; PKA, protein kinase A; PR, progesterone receptor; RAC3, receptor-associated coactivator-3; sFBS, charcoal-stripped fetal bovine serum; SRC-1, steroid receptor coactivator-1; TIF2, transcription intermediary factor-2; TRE, tetradecanoylphorbol-13-acetate response element.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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