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Ligand-Independent Activation of Pituitary ER: Dependence on PKA-Stimulated Pathways

Department of Internal Medicine, Division of Endocrinology and Metabolism (D.A.S., M.A.S.), and Department of Pharmacology (E.M.R., V.Y.L.), University of Virginia, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Margaret A. Shupnik, Ph.D., Box 800578, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail: mas3x{at}virginia.edu

	Abstract

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In pituitary and other target tissues, estrogen acts through ERs, which are ligand-activated nuclear transcription factors. ERs can also be activated by intracellular signaling pathways in a ligand-independent manner in some cells. Because the pituitary is the target of several cAMP-activating factors, we examined the ability of cAMP to activate ERs in the T3 gonadotrope cell line. Forskolin, 8-bromo-cAMP, and pituitary adenylate cyclase-activating polypeptide all enhanced ER-dependent promoter activity, which was inhibited by antiestrogen or a pituitary-specific inhibitory ER variant. Activation was PKA dependent and was blocked by the PKA inhibitor H89 or cotransfection of the inhibitor PKI. Although cAMP activated MAPK in T3 cells, inhibition of MAPK with the MEK inhibitor PD98059 did not prevent forskolin-induced ER activation. Similarly, epidermal growth factor did not stimulate ER activity, although it increased MAPK activation. Forskolin-induced activation of ER was enhanced by cotransfection of steroid receptor coactivator-1 and was inhibited by the repressor of ER action, suggesting that cAMP does not alter the normal interactions between ER and cofactors. In contrast to results with estrogen, cAMP treatment did not decrease ER protein levels. These results demonstrate that in the pituitary, cAMP activates ER in a ligand-independent manner exclusively through PKA.

	Introduction

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ESTROGEN INFLUENCES pituitary hormone secretion directly and through actions on hypothalamic releasing factors. In particular, estrogen regulation of gonadotropin and PRL synthesis and secretion from gonadotropes and lactotropes has been studied extensively (1, 2, 3, 4). In pituitary cells and other estrogen target tissues, estrogen acts primarily through ERs, which are ligand-activated nuclear transcription factors (5). As in other target tissues two full-length ER isoforms, ER and ERß, are expressed in the pituitary (6, 7, 8). ERs share a common domain structure with other steroid receptors with a centrally located DNA-binding domain (DBD), a C-terminal ligand-binding/ligand-dependent trans-activation domain (AF-2), and an N-terminal ligand independent trans-activation domain (AF-1). Upon ligand binding, these receptors undergo conformational changes that enhance dimerization, DNA binding to estrogen response elements (EREs) in target gene promoters, recruitment of cofactors, and trans-activation (9). In addition, the rodent pituitary expresses a truncated form of ER, truncated ER product (TERP) that has biphasic actions on ER-dependent transcription (10, 11, 12).

The pituitary is the target of hypothalamic factors that stimulate several second messenger pathways, including cAMP. Peptides such as pituitary adenylate cyclase-activating polypeptide (PACAP), VIP, GHRF, and CRF all increase intracellular cAMP (13, 14, 15). Other hypothalamic factors, such as dopamine and melatonin, can inhibit pituitary cAMP production (16, 17). In uterus and breast, increases in intracellular cAMP lead to phosphorylation of ER protein and activation of ER transcription (18). The effects of cAMP on ER activity may also result from the interaction of cAMP with other signaling pathways. For example, VIP- and PACAP-induced increases in cAMP and cAMP analogs stimulate MAPK kinase activity in pituitary cells (15, 19), and MAPK is a well known mediator of ligand-independent ER and ERß activation through AF-1 phosphorylation (20, 21, 22). In other cell types, such as astrocytes and cerebellar neurons, cAMP also stimulates MAPK activity (23, 24).

Cross-talk between intracellular signaling pathways, including cAMP, to influence ER transcriptional activity is tissue and cell specific. Ligand-independent activation of ER by cAMP could result from direct PKA or MAPK phosphorylation of ER isoforms (20, 25, 26) or other proteins that bind to and modulate ER transcription, including coactivators, corepressors, and integrator proteins such as cAMP response element-binding protein-binding protein (27, 28, 29, 30). Any such modifications in pituitary cells could result in enhanced functional ER interactions with coactivators or reduced interactions with corepressors, thus increasing overall ER-mediated transcription. Because ERs are important regulators of pituitary function and may be influenced by several intracellular signaling pathways activated by hypothalamic peptides and neurotransmitters, we sought to determine whether cAMP could activate ER-dependent transcription in the T3 pituitary gonadotrope cell line (31). The T3 cells express functional GnRH and PACAP receptors, and biological responses to these peptides, including activation of intracellular signaling cascades and stimulated gene expression, are essentially the same as those in normal gonadotropes (32, 33, 34, 35, 36, 37). These cells also have functional ER; express transcripts for ER, ERß, and the pituitary-specific isoform TERP-1; and are transcriptionally responsive to estrogen (8, 12, 31, 36). We found that increased levels of intracellular cAMP stimulated ER-mediated transcription in T3 cells, and that PKA, but not MAPK, pathways are involved.

	Materials and Methods

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Plasmids
A reporter plasmid (ERETkluc) containing two consensus EREs upstream of a minimal promoter and the firefly luciferase gene was used. ERETkluc and the parental vector Tkluc contain a minimal thymidine kinase promoter. Human steroid receptor coactivator-1 (SRC-1) and the parental pBK-cytomegalovirus expression plasmids were gifts from Dr. Bert O’Malley (Baylor College of Medicine, Houston, TX). The human repressor of ER activity (REA) expression vector was obtained through high fidelity RT-PCR (Roche, Indianapolis, IN) cloning from MCF-7 cell total RNA using primers (Operon Technologies, Alameda, CA) to the published full-length coding sequence (30). The full-length REA PCR product was ligated into the PCR2.1 cloning vector (Invitrogen, San Diego, CA), sequenced, and subcloned into the pcDNA3.1AMP expression vector (Invitrogen) under the control of a cytomegalovirus promoter. The expression vector for TERP-1 in pcDNA3.1AMP has been described previously (12). An additional TERP-1 expression vector containing a mutation that inhibits dimerization to full-length ERs (L509R) has also been described previously (38). Expression vectors for the PKA inhibitory protein PKI and an inactive mutant PKI were donated by Dr. Richard N. Day (University of Virginia, Charlottesville, VA) (39).

Cells lines, transient transfection, and treatments
Mouse T3 pregonadotrope cells were originally obtained from Dr. Pamela Mellon (University of California, San Diego, CA). They express functional GnRH and PACAP receptors and the pituitary gonadotropin -subunit, but not intact gonadotropins. Cells were maintained in DMEM/10% FBS (Life Technologies, Inc., Grand Island, NY) containing 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Inc.). These cells contain functional ER, and significant levels of ER and TERP-1 protein comparable to or greater than those in other clonal pituitary cell lines or a mixed population of intact female rat pituitary cells (8, 31). Cells (5 x 10⁵) were plated in 35-mm six-well tissue culture plates in phenol red-free DMEM/5% charcoal-stripped normal calf serum (NCS). On the following day the medium was changed 3 h before transfection. Cells were transiently transfected by calcium phosphate for 16–18 h. Cells were then washed with PBS, and media was replaced with DMEM/1% charcoal-stripped NCS treatment medium for 7 h before collection. Cells were washed with PBS and collected in 1 x cell lysis buffer (Promega Corp., Madison, WI) for assessment of luciferase activity and total lysate protein. Luciferase activity was assessed using a Turner 20e luminometer (Sunnyvale, CA), and light units were normalized for total lysate protein using protein dye (Bio-Rad Laboratories, Inc., Richmond, CA). Treatments are detailed in Results. 17ß-Estradiol, hydroxytamoxifen, pituitary adenylate cyclase 38, forskolin, and epidermal growth factor (EGF) were obtained from Sigma (St Louis, MO). ICI 182,780 was obtained from Tocris, and H89, Kn92, and Kn93 were obtained from Calbiochem (La Jolla, CA).

Immunoblot analysis
ER and ERß protein expression was determined by immunoblot analysis using rabbit polyclonal antibodies. The ER antibody (C1355) was generated against amino acids 586–600 of the rat ER and has been characterized previously (40). The ERß antibody (Z8P) was obtained from Zymed Laboratories, Inc. (South San Francisco, CA) and is directed against the C-terminus of mouse ERß. The monoclonal ß-actin antibody was obtained from Sigma. The antibody and conditions for Western analysis were determined using in vitro translated proteins. For analysis of cellular expression, cells were collected in 2 x gel loading buffer containing 100 mM Tris (pH 6.8), 2% SDS, 20% glycerol, and a cocktail of protease inhibitors (antipain, aprotinin, chymostatin, leupeptin, and pepstatin A). Total lysate protein was determined with a bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL). Twenty-five micrograms of total protein were separated on 12% polyacrylamide-SDS gels, transferred to nitrocellulose, and subjected to immunoblotting. After overnight blocking in Tris-buffered saline with 0.1% Tween and 5% milk at 4 C, blots were incubated with primary antibodies for 1 h at room temperature. C1355 was used at 1:5,000, and Z8P was used at 1 µg/ml. After rinsing, blots were incubated for 1 h in a horseradish peroxidase (HRP)-conjugated donkey antirabbit IgG secondary antibody (1:5,000; Amersham Pharmacia Biotech, Arlington Heights, IL), followed by detection on x-ray film (Kodak X-OMAT, Eastman Kodak Co., Rochester, NY) with chemiluminescence (Pico-West, Pierce Chemical Co.). Blots were then blocked overnight in 5% milk at 4 C and reprobed with the ß-actin antibody at 1:5,000. After rinsing, blots were incubated for 1 h in an HRP-conjugated goat antimouse IgG secondary antibody (1:20,000; Amersham Pharmacia Biotech) followed by detection on x-ray film with enhanced chemiluminescence (Amersham Pharmacia Biotech). Densitometry was performed with a Personal Densitometer SI and analyzed with ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).

Activated p42/44 MAPK (ERK1/ERKk2 MAPK) was detected with a phospho-specific MAPK polyclonal antibody (Cell Signaling Technology, Beverly, MA) and normalized for total MAPK expression with a polyclonal antibody directed against a different epitope of p42/44 MAPK (Cell Signaling Technology). Cells (2.5–3 x 10⁶) were plated in 60-mm tissue culture dishes in phenol red-free DMEM/5% charcoal-stripped NCS. The following day medium was changed to serum-free DMEM for 16–18 h. Cells were treated with various agents (estrogen, forskolin, and EGF) for 2 min to 6 h. Cells were rapidly lysed and collected in boiling 2 x gel loading buffer without protease inhibitors. Lysates were boiled for an additional 5 min and then frozen at -70 C. Total lysate proteins were determined using a bicinchoninic acid assay kit (Pierce Chemical Co.), and 15 µg of each lysate were separated on 10% polyacrylamide gels and transferred to nitrocellulose. Immunoblotting was performed for 1 h with the total MAPK (1:1000) as described above using the HRP-conjugated donkey antirabbit IgG secondary antibody at 1:1000. Proteins were detected with enhanced chemiluminescence (Amersham Pharmacia Biotech). Blots were subsequently stripped at 50 C in 62.5 mM Tris buffer (pH 6.7) containing 100 mM ß-mercaptoethanol and 2% SDS. Blots were reprobed with the phospho-specific MAPK antibody at 1:1000 and the HRP-conjugated donkey antirabbit IgG secondary antibody at 1:1000. Proteins were detected by enhanced chemiluminescence. Phospho-specific and total MAPK films were analyzed by densitometry, and phospho-MAPK was normalized to total MAPK for analysis.

Data presentation and statistical analysis
Luciferase values in duplicate or triplicate samples were normalized for total cell lysate protein. Control or vehicle treatments were normalized to 1, and treatment values are expressed as a function of the normalized controls, except where noted. Data from at least four individual experiments were analyzed by one-way ANOVA, followed by a priori pairwise comparisons between treatment groups and controls using t tests. In some instances data were log transformed before statistical analysis due to unequal variance.

	Results

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cAMP activates ER-dependent transcription in T3 cells
In transiently transfected T3 cells, increased intracellular cAMP activated transcription from an ERE-containing reporter gene (Fig. 1). The adenylate cyclase activator forskolin (FSK; 1 µM) and the cell-permeable cAMP analog 8-bromo-cAMP (8-Br-cAMP; 2 mM) significantly activated the ERETkluc reporter up to 8-fold (Fig. 1). Activation by 100 nM FSK (not shown) was equivalent to that observed with 10 nM estrogen (E), whereas 1 µM FSK and 2 mM 8-Br-cAMP were more effective than E. PACAP (200 nM, a maximally effective dose for T3 and pituitary cells) (33) increased transcription 2.6-fold, demonstrating that a cAMP signal generated by a physiologically relevant peptide also stimulates ER-dependent transcription in these cells. In contrast, 100 ng/ml EGF did not enhance transcription of the reporter gene in T3 cells, suggesting that AF-1 activation by MAPK does not play a major role in ER action in the pituitary. We used two methods to demonstrate that cAMP-induced transcriptional activation was ER dependent. The ER antagonist ICI 182,780 (1 µM) inhibited FSK and 8-Br-cAMP actions on the ERETkluc reporter (Fig. 2A). To further demonstrate a dependence on ER, T3 cells were cotransfected with the reporter and a dominant negative form of ER called TERP or a TERP mutant that cannot dimerize with full-length ERs (DimMut). TERP, a pituitary-specific ER variant, inhibits ER-dependent transcription at ratios greater than 1:1 by forming inactive heterodimers with both ER isoforms (38). TERP inhibited E- and FSK-induced activation of ERETkluc (Fig. 2B), but the TERP dimerization mutant failed to inhibit E- or FSK-induced transcription, demonstrating that FSK actions in our model system require active full-length ERs.

View larger version (17K): [in this window] [in a new window]   Figure 1. Activation of an estrogen-responsive model promoter in T3 cells by cAMP. Cells were transfected with 2 µg ERETkluc reporter plasmid as described and treated with vehicle (Con), 10 nM 17ß-estradiol (E2), 1 µM FSK, 2 mM 8-Br-cAMP (cAMP), 200 nM PACAP, or 100 ng/ml EGF for 24 h. Data shown represent the normalized mean ± SEM from five to seven independent experiments performed in duplicate. Significant differences from control are denoted with asterisks (*, P < 0.05; **, P < 0.01).

View larger version (27K): [in this window] [in a new window]   Figure 2. Activation of an estrogen-responsive model promoter in T3 cells by cAMP is ER dependent. A, Cells were transfected with 2 µg ERETkluc reporter plasmid as described and treated with vehicle, 0.1 or 1 µM FSK, or 2 mM 8-Br-cAMP (cAMP) with (; ICI) or without (; Control) the ER antagonist ICI 182,780 (ICI; 1 µM) for 7 h. Data shown represent the normalized mean ± SEM from 5–10 independent experiments performed in duplicate. Significant differences from vehicle are denoted with asterisks (*, P < 0.05; **, P < 0.01), and significant suppression from paired controls are denoted with crosses (, P < 0.05). B, Cells were cotransfected with 2 µg ERETkluc reporter plasmid and 4 µg pcDNA3.1AMP (), a TERP-1 expression vector (TERP; ), or a TERP-1 dimerization mutant (TERP DimMut; ) and treated for 7 h with vehicle, 10 nM 17ß-estradiol (E2), or 1 µM FSK. Data shown represent the mean ± SEM from five or six independent experiments performed in duplicate. Significant differences from vehicle are denoted with asterisks (*, P < 0.05), and significant suppression from paired controls is denoted with crosses (, P < 0.05).

View larger version (25K): [in this window] [in a new window]   Figure 3. Activation of an estrogen-responsive model promoter in T3 cells by cAMP is PKA dependent. A, Cells were transfected with 2 µg ERETkluc reporter plasmid as described and treated with vehicle (), 0.1 µM FSK (7M; ), or 1 µM FSK (FSK) (6M; ) in the absence or presence of two doses of the PKA antagonist H89 for 7 h. Data shown represent normalized means from four independent experiments performed in triplicate. Significant differences from vehicle controls are denoted with asterisks (*, P < 0.05). B, Cells were cotransfected with 2 µg ERETkluc reporter plasmid and 5 µg plasmid encoding the PKA inhibitor PKI, an inactive mutant of PKI (PKImut), or the PKI parental vector Puc19. Cells were treated for 7 h with vehicle () or 1 µM FSK (). Data shown represent the normalized mean ± SEM from four independent experiments performed in triplicate. Significant differences from vehicle controls are denoted with asterisks (**, P < 0.01).

View larger version (21K): [in this window] [in a new window]   Figure 4. Activation of an estrogen-responsive model promoter in T3 cells by cAMP is not CAMK II dependent. Cells were transfected with 2 µg ERETkluc reporter plasmid as described and treated with vehicle () or 1 µM forskolin () in the absence or presence of three doses of Kn92 () or Kn93 () for 7 h. Data shown represent the normalized mean ± SEM from six independent experiments performed in duplicate and are expressed as the fold FSK response in the presence of vehicle. FSK significantly activated the reporter gene (**, P < 0.01), and Kn92 and Kn93 did not alter this stimulation.

View larger version (34K): [in this window] [in a new window]   Figure 5. Activation of an estrogen-responsive model promoter in T3 cells by cAMP is not MAPK dependent. Activated MAPK (ERK1/ERK2-P) and total MAPK (ERK1/ERK2-T) were detected by immunoblotting. A, Cells were serum starved for 16 h and treated with 1 µM FSK in the presence or absence of a 30-min pretreatment with the MAPK kinase inhibitor PD98059 (PD; 50 µM). Similar results were obtained in three experiments. B, Cells were serum starved for 16 h and treated with 100 ng/ml EGF from 5 min to 6 h before collection and MAPK immunoblotting. Similar results were obtained in three experiments. C, Cells were transfected with 2 µg ERETkluc reporter plasmid as described and treated with vehicle () or 50 µM PD98059 () in the absence or presence of two doses of FSK for 7 h. Data shown represent the normalized mean ± SEM from six independent experiments performed in duplicate and are expressed as the fold FSK response. FSK significantly activated the reporter gene (*, P < 0.05; **, P < 0.01), and PD98059 did not alter this stimulation.

View larger version (23K): [in this window] [in a new window]   Figure 6. Interaction of coregulators with cAMP-induced ER activation in T3 cells. A, Cells were transfected with 2 µg ERETkluc reporter plasmid and 0, 100, or 1000 ng SRC-1 expression vector. Total DNA was brought to 3 µg with the SRC-1 parental vector pBK-cytomegalovirus. Cells were treated for 7 h with vehicle, 10 nM 17ß-estradiol (E2), or 1 µM FSK. Data shown are the mean ± SEM from three independent experiments performed in duplicate. SRC-1 significantly enhanced FSK-induced ER activation at 1000 ng (*, P < 0.05). B, Cells were transfected with 2 µg ERETkluc reporter plasmid and 0, 0.5, 1, or 2 µg REA expression vector. Total DNA was brought to 4 µg with pcDNA3.1-AMP. Cells were treated for 7 h with vehicle, 10 nM E2, or 1 µM FSK. Data shown are the mean ± SD from an example experiment performed in triplicate. Significant differences from vehicle are denoted with asterisks (*, P < 0.05; **, P < 0.01), and significant suppression from E2- or FSK-stimulated expression are denoted with crosses (, P < 0.01; , P < 0.05). Similar results were obtained in four independent experiments.

FSK and E differentially regulate ER expression

View larger version (42K): [in this window] [in a new window]   Figure 7. FSK and E differentially regulate ER expression. T3 cells were plated in phenol red-free DMEM/5% charcoal-stripped NCS for 18 h and treated with vehicle (Con), 10 nM 17ß-estradiol (E2), or 1 µM FSK for 1, 3, or 6 h. Cell lysates (25 µg) were separated on 12% polyacrylamide gels and immunoblotted for ER with the C1355 antibody followed by ß-actin immunoblotting. ER expression was normalized for actin levels, and the percent expression relative to the untreated control is noted below the blots. Data are the mean ER (average of four independent experiments).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study we demonstrate that T3 gonadotrope cells are responsive to cAMP-dependent ER activation, but not to stimulation by the growth factor EGF. Similar stimulatory effects of FSK, but not of growth factors such as EGF, were observed in other pituitary cell lines, such as sommatolactotrope GH₃ cells and gonadotrope LßT2 cells (not shown), but these responses were not extensively characterized. The cAMP stimulation of ER in T3 cells occurred via PKA-dependent pathways and not through other signaling mechanisms, such as MAPK or CAMK. Although FSK and cAMP can activate MAPK in the pituitary and pituitary cell lines (15, 19) and did so in T3 cells in the present study, this activation alone does not stimulate ligand-independent activation of ER in T3 pituitary cells. EGF stimulated MAPK with no effect on ER-dependent transcription, and blockade of MAPK phosphorylation by the MEK inhibitor PD98059 did not alter FSK-induced stimulation of ER. Because the pituitary is the target of several physiological factors that stimulate cAMP levels, cAMP could provide a physiological mechanism to modulate ER activity in the pituitary.

Several investigators have begun to show differential cell type responsiveness to ligand-independent activation of ER, and the relative roles of the receptor AF-1 and AF-2 domains in this response (43, 44). Growth factors such as EGF phosphorylate specific serine residues in the AF-1 domain of both ER and ERß, and this phosphorylation is crucial to the transcriptional response (20, 21, 22, 27). The absence of a stimulatory EGF effect supports a predominant role for the ER AF-2 domain in pituitary cells. In agreement with this concept, increases in intracellular cAMP lead to phosphorylation of rat and human ER residues outside the AF-1 domain in ER, and deletion mutants lacking the AF-1 domain can still be activated by cAMP (18, 20, 25, 26). Thus, either the DBD or the C-terminus of the receptor, including the AF-2 function, are likely sites for cAMP regulation.

In T3 pituitary cells, activation of PKA is essential for cAMP-dependent activation of ER. The PKA effects could occur by phosphorylation events on the ER itself or any of the regulatory proteins that interact with the AF-2 region. At the level of ER, at least two potential PKA-dependent phosphorylation sites are present C-terminal to AF-1, including one in the DBD and another proposed in the hinge region (25, 26). The first site, serine 236, is in the DBD of human ER, and the homologue to serine 236 is present in mouse ER and ERß (22). Phosphorylation of this site in vitro inhibits ER dimerization and DNA binding, but the effects have not been measured in any cellular context, and the potential contribution of this phosphorylation to pituitary activity is unknown (26). PKA phosphorylation of another nuclear receptor, the PPAR in 293 cells, within the DBD and AF-2 domains enhances both ligand-dependent and -independent activities (45).

Alternatively, modification of coregulatory proteins, including coactivator, corepressor, and integrator proteins interacting with the AF-2 region, may contribute to cAMP-dependent ER activation. Nothing is known of the structure of unliganded, but transcriptionally active, ER, although E-induced structural changes in the AF-2 domain are known to be critical for transcriptional activity (9). Similarly, little is known of ER interactions with coactivators or corepressors in ligand-independent pathways. The coactivator SRC-1 cannot be phosphorylated directly by PKA, and activation of the progesterone receptor by cAMP has been proposed to occur via enhanced interaction between the receptor and SRC-1 through MAPK actions (28). The MAPK pathway, however, does not appear important in the cAMP response in pituitary cells. Potential PKA phosphorylation sites have been predicted, but not demonstrated, on the ER-specific corepressor REA (30). The present study clearly demonstrates that both SRC-1 and REA modulate the activity of cAMP-activated pituitary ER. FSK treatment does not simply substitute for SRC-1 in ER activation, nor does it prevent REA-suppressive effects. Thus, cAMP-mediated ER activation appears to use the same general regulatory pathways as estrogen-mediated activation. It is possible, however, that pituitary-specific coactivator and corepressor proteins exist and that cAMP-mediated modifications of such proteins are critical for pituitary ER activation. Finally, cointegrator proteins such as cAMP response element-binding protein-binding protein and p300 are also downstream targets of cAMP signaling cascades and are critical in ER transcriptional activation (9, 46, 47), and FSK may act via this pathway. The combination of PKA-dependent phosphorylation of numerous proteins may be required to mediate the pituitary ER response to cAMP.

Recent evidence from this laboratory (8) and others (48, 49) demonstrates that several steroid hormones, including estrogen, rapidly down-regulate their own receptors through proteolytic degradation. This mechanism probably acts as a brake on transcription and may allow ER cycling. Unlike estrogen, cAMP activation did not lead to a rapid degradation of ER protein. The effects of cAMP activation on ER protein levels have not, to our knowledge, been quantified in any other cell type. However, the overall pituitary transcriptional response to FSK may be greater than the response to estrogen, in part by maintaining ER levels after activation. Phosphorylation of ERs or regulatory proteins may also inhibit degradation and provide one mechanism to explain additive or synergistic effects of cAMP and estrogenic ligands. These data suggest that any conformational changes in ER induced by FSK allow functional interactions with cofactors, but do not target the ER for degradation. The observation that ER-dependent transcription in pituitary cells, which are physiological targets of cAMP activators, can be stimulated by cAMP activation and not MAPK-dependent pathways, further demonstrates the unique cell- and tissue-specific actions that govern steroid actions.

	Acknowledgments

 
The authors thank the Molecular Core Laboratory of the Center for the Study of Reproduction at the University of Virginia.

	Footnotes

 
This work was supported by a grant from the Lalor Foundation (to D.A.S.) and NIH Grant RO1-DK57082 (to M.A.S.). The Molecular Core Laboratory of the Center for the Study of Reproduction at the University of Virginia was supported by NICHHD/NIH Cooperative Agreement U54-HD-28934 as part of the Specialized Cooperative Centers Program in Reproductive Research. Portions of this work were presented at the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000.

Abbreviations: AF-1, N-Terminal ligand independent trans-activation domain; AF-2, C-terminal ligand-binding/ligand-dependent trans-activation domain; 8-br-cAMP, 8-bromo-cAMP; CAMK, calmodulin kinase; DBD, DNA-binding domain; E, estrogen; EGF, epidermal growth factor; ERE, estrogen response element; FSK, forskolin; HRP, horseradish peroxidase; NCS, normal calf serum; PACAP, pituitary adenylate cyclase-activating polypeptide; REA, repressor of ER activity; SRC-1, steroid receptor coactivator-1; TERP, truncated ER product.

Received January 17, 2001.

Accepted for publication April 12, 2001.

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