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Transcription Does Not Require Proteasome Activity and Protects the Receptor from Ligand-Mediated Degradation
Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia (H.-W.T., M.A.S.), Charlottesville, Virginia 22908; and Departments of Chemistry (J.A.K.) and Molecular and Integrative Physiology (B.S.K.), University of Illinois, Urbana, Illinois 61801
Address all correspondence and requests for reprints to: Dr. Margaret A. Shupnik, Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia, Box 800578, Charlottesville, Virginia 22908. E-mail: mas3x{at}virginia.edu.
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) transcription is accompanied by protein degradation via the 26S-proteasome pathway. Inhibition of proteasome activity stabilizes ER protein and abolishes E2-activated transcription, suggesting functional linkages between transcription and degradation. It is not known whether ligand-independent ER activation is coupled to proteolysis. In pituitary cells, forskolin (FSK) stimulates ER transcription through the protein kinase A (PKA) pathway. This study examined interactions between E2-dependent and PKA-stimulated pathways in GH3 cells by measuring transcription of a transfected reporter gene and endogenous ER levels. E2 stimulated estrogen response element-mediated transcription 2- to 3-fold and decreased ER protein levels to 40%. In contrast, FSK stimulated ER transcription without decreasing ER protein. Treatment with FSK plus E2 resulted in synergistic ER transactivation, and FSK specifically prevented E2-induced ER degradation. PKA is required for protection and was prevented by H89 (a PKA inhibitor), but not PD98059 (a MAPK kinase inhibitor). Propyl-pyrazole-triol and R,R-diethyl-tetrahydrochrysene, selective ER agonists, reduced ER protein by 50% while stimulating ER transcriptional activity 4- to 8-fold. The antagonist ICI 182,780 similarly decreased ER levels, but prevented ER activation. FSK prevented all ligand-induced ER degradation. Lactacystin, a proteasome inhibitor, abolished E2-stimulated, but not FSK-stimulated, ER transcription. Thus, stimulation of ER transcription by the PKA-dependent pathway is dissociated from receptor degradation and proteasome activity. These data suggest a mechanism of ER transcriptional activation by PKA that is distinct from E2 activation and that may contribute to the synergistic transcriptional activation of ER by ligand-dependent and PKA-dependent pathways. |
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and ERß (1, 2, 6), and ER is predominantly expressed in the pituitary (7). Like the other members of the steroid/nuclear receptor superfamily, ER protein contains an amino-terminal, ligand-independent, transactivation domain (AF-1), a central DNA-binding domain (DBD), a hinge region, and a carboxyl-terminal ligand-binding (LBD) and ligand-dependent transactivation (AF-2) region (1, 6). In the presence of E2, ER monomers undergo a conformational change, dimerize, and bind to estrogen response elements (EREs) on the promoters of target genes (6, 8, 9). The dynamic recruitment of coactivators and other regulatory proteins is altered on ligand-bound ER, thus activating the receptor and modifying the activity of the transcriptional machinery (6, 10).
Concomitant with increased ER transcriptional activity, E2 rapidly decreases ER protein levels in a variety of cells (7, 11, 12, 13, 14). This ligand-induced ER degradation is primarily mediated by the 26S-proteasome pathway, and the proteasome inhibitors, MG132 and lactacystin, prevent ER down-regulation by E2 (11, 12, 13, 15, 16). Before degradation by the 26Sproteasome, protein is covalently conjugated with ubiquitin by a series of enzymes, including E1 ubiquitin-activating enzymes (Uba), E2 ubiquitin-conjugating enzymes (Ubc), and E3 ubiquitin ligases (17, 18). E2-bound ER is demonstrated to be covalently conjugated with ubiquitin in vitro (16) and in vivo (12, 16, 19), thus targeting the protein for destruction. Mutation of the ubiquitin-activating enzyme Uba abolishes ligand-induced ER degradation (13). Thus, E2-induced ER down-regulation is tightly controlled by the ubiquitin-proteasome system.
Ligand-induced ER degradation also occurs upon treatment with the inactive isomer 17-E2 as well as antiestrogens such as ICI 182,780, GW7604, and RU58668, even though these compounds are unable to activate ER (12, 15, 20, 21, 22). One exception is the partial antagonist/agonist, 4-hydroxyltamoxifen (4-OHT), which does not induce ER degradation (12). Nevertheless, these observations suggest that binding of ER to ligands, including E2 and antiestrogens, induces specific conformational changes in or recruits specific proteins to the ER LBD, and this is required for protein degradation (12). In support of this hypothesis, ER constructs with deletions in the LBD show no protein degradation in response to E2 (13). A variety of mutations on the residues of the LBD that disrupt the ability of ER to bind to ligand (G521R) (20) or coactivators (L539A/L540A, I358D, V376D, D538A/E542A/D545A, K362D, and L539A) also prevent E2-induced degradation (12, 13, 23).
Increasing evidence indicates a connection between transcription and the ubiquitin-proteasome system (17). Prevention of ER degradation by the proteasome inhibitors, MG132 and latacystin, disrupts E2-induced ER transactivation in HeLa cervical cancer and MCF7 breast cancer cells, suggesting a functional linkage between protein degradation and transcriptional activity of ER (13, 15). Chromatin immunoprecipitation studies of endogenous E2-sensitive genes demonstrate that ER is continuously turned over on these promoters, and suggest that this is required for continued responsiveness to E2 (15, 24). Cyclic occupancy of ER on an ERE is disrupted upon blocking the proteasome activity (15). Among various ER-interacting cofactors, E6-AP and TRIP1/SUG1 are an E3 ubiquitin-ligase and a subunit of the 26S proteasome complex, respectively (10, 18). Thus, ligand binding and transactivation of ER are tightly linked to its degradation (12).
Although the link between ligand binding, ER-mediated transcription, and receptor degradation is currently under intensive investigation, much less is known about ligand-independent activation of the receptor and degradation pathways. We previously found in pituitary cells that forskolin (FSK), acting though PKA, stimulated ER activity without an apparent decrease in ER protein levels (14). In the present study we investigated whether FSK activation of ER required proteasome activity, and how ligand-dependent and -independent pathways converged on transcriptional activation and receptor degradation in this system. We found that stimulation of ER transcription by the PKA ligand-independent pathway is dissociated from receptor degradation and proteasome activity. This suggests a mechanism of transcriptional activation distinct from that induced by ligands and may contribute to the synergistic activation of ER by ligand-dependent and -independent pathways. In addition, FSK can protect ER from degradation by ligand binding for both agonists and antagonists, suggesting that PKA-dependent pathways may act to stabilize ER and prevent degradation.
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were purchased from Calbiochem (San Diego, CA). ICI 182,780 (a complete ER antagonist) and diarylpropionitrile (DPN; an ERß agonist) were purchased from Tocris (Ellisville, MO). Latacystin, a proteasome inhibitor, was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). For all experiments, lactacystin was freshly dissolved in water before use. E2, FSK, 4-OHT, and -amanitin were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Propyl-pyrazole-triol (PPT) and R,R-diethyl-tetrahydrochrysene (R,R-THC) were synthesized as previously described (25, 26, 27, 28, 29).
Cell culture, transfection, and luciferase assays
Rat-derived somatolactotroph pituitary cells, GH3 cells, were maintained in Cellgro DMEM (Mediatech/Fisher, Herndon, VA) containing 10% fetal bovine serum (Life Technologies, Inc./Invitrogen, Grand Island, NY) as well as 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Inc./Invitrogen). As previously reported, these cells express significant levels of ER protein, but not ERß (7). For each experiment, GH3 cells were first plated in phenol red-free DMEM with 5% charcoal-stripped newborn calf serum at a density of 4 x 105 cells/ml in 12-well plates (d 0). To monitor ER transcriptional activity, cells were transiently transfected with the pGL3–2ERE reporter plasmid (500 ng/well) on the following day for 18–22 h using FuGene 6 (Roche, Indianapolis, IN). The pGL3–2ERE reporter contains two consensus estrogen ERE upstream of a prolactin TATA box and the firefly luciferase gene (14). Cells were then treated with vehicle, E2, FSK, or other receptor ligands for the times indicated in Results. Later, cells were washed with PBS (pH 7.4) and then collected in 200 µl 1x cell culture lysis reagent (Promega Corp., Madison, WI). Cell lysates were stored at –20 C until assayed for luciferase activity. Using a Turner TD-20e luminometer (Sunnyvale, CA), luciferase activity from each sample (100 µl lysate) was measured, and the light units were normalized for total lysate protein using a protein assay (Bio-Rad Laboratories, Richmond, CA). The luciferase activity of the vehicle-treated cells was expressed as 100% activity, and other treatments were expressed as the fold increase. Each treatment was performed in duplicate or triplicate, and each experiment was repeated at least three times. In some experiments, cells were also cotransfected with cytomegalovirus-ß-galactosidase (30), as described below.
ß-Galactosidase assay
To access the toxicity of lactacystin in GH3 cells, cells were plated as described above and transiently transfected with the cytomegalovirus-ß-galactosidase expression vector (500 ng/well). Cells were pretreated with lactacystin (10 µM) for 2 h, followed by the addition of vehicle, 10 nM E2, 1 µM FSK, or both. Eighteen to 20 h later, cell lysates were collected and stored at –20 C until measurement of ß-galactosidase activity. Using a ß-galactosidase assay kit (Invitrogen, Carlsbad, CA), the ß-galactosidase activity from each sample (10 µl lysate) was measured in a ELx800 Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT), expressed as OD units at 420 nm, and normalized for total lysate protein.
Immunoblotting
GH3 cells were plated in 12-well plates as described above. Two days later cells were treated with vehicle, E2, FSK, or other receptor ligands for the indicated times up to 24 h. In experiments to test the effects of FSK or E2 treatment in the absence of transcription, cells were treated with 10 µM -amanitin for 2 h before the addition of vehicle, E2, or FSK for an additional 6 h (15). In studies to test specificity of FSK protection, cells were pretreated with FSK for 15 min, then with TNF (100 ng/ml) for an additional 15 min (20). For all experiments, cells were collected in 2x gel loading buffer containing 100 mM Tris (pH 6.8), 2% sodium dodecyl sulfate, 20% glycerol, and a mixture of protease inhibitors (antipain, aprotinin, chymostatin, leupeptin, and pepstatin A) as previously described (14). Total lysate protein was determined with the bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL). Total cell lysate proteins (
30 µg/lane) were separated on 12% polyacrylamide-sodium dodecyl sulfate gels and transferred to nitrocellulose membranes. Membranes were first incubated overnight at 4 C in Tris-buffered saline with 0.1% Tween containing 10% milk. Blots were then incubated with primary ER antibody (1:7,500), for 1 h at room temperature. The ER antibody (C1355) was generated against amino acids 586–600 of rat ER and has been characterized previously (31). After rinsing, blots were incubated for 1 h with horseradish peroxidase-conjugated donkey antirabbit IgG secondary antibody (1:10,000; Amersham Pharmacia Biotech, Arlington Heights, IL), followed by detection on x-ray film (X-OMAT, Eastman Kodak Co., Rochester, NY) with SuperSignal West Pico Chemiluminescence (Pierce Chemical Co.). Later, the same blots were reprobed with the ß-actin antibody at 1:100,000 (Sigma-Aldrich Corp.). After rinsing, blots were incubated for 1 h in an HRP-conjugated goat antimouse IgG secondary antibody (1:50,000; Jackson ImmunoResearch Laboratories, West Grove, PA), followed by detection on x-ray film with chemiluminescence. The intensities of ER and ß-actin on individual films were measured by densitometry and analyzed with ImageQuant (Molecular Dynamics, Inc., Sunnyvale, CA). ER protein levels were normalized to the ß-actin level in each sample. ER protein levels in samples were normalized to the vehicle-treated controls and expressed as a percentage of the control. Each treatment was performed in duplicate wells, and each experiment was repeated at least three times.
Semiquantitative RT-PCR
GH3 cells were plated in 5% newborn calf serum-DMEM at ae density of 8 x 106 cells/100-mm cell culture dish (Corning Glass, Corning, NY). Cells were treated with vehicle, E2, FSK, or both E2 and FSK. At 6 or 24 h of treatment, total RNA was extracted from these cells using the RNeasy Mini Kit (Qiagen, Valencia, CA). PCR primer sequences, the expected product sizes, and the conditions for RT-PCR were previously determined and described (7, 32). RNA (1 µg) was reverse transcribed in a 20-µl mixture consisting of 5 mM MgCl2 (PerkinElmer, Palo Alto, CA), PCR buffer II, 1 mM deoxyribonucleotides, 1 U ribonuclease inhibitor, 2.5 µM random hexamers, and 2.5 U murine leukemia virus reverse transcriptase. RT reactions were incubated for 10 min at room temperature, for 15 min at 42 C, and for 5 min at 99 C and cooled to 4 C for 5 min in an Eppendorf Mastercylcer Gradient (Westbury, NY). For each PCR, MgCl2 was adjusted to 2.5 mM, and buffer, water, 0.3 µM primer oligonucleotides (Operon Technologies, Alameda, CA), and 2.5 U/100 µl Taq DNA polymerase (Invitrogen) were added to a final volume of 16 µl. PCR conditions consisted of a single cycle of 3 min at 95 C, 3 min at 55 C, and 3 min at 68 C. An additional 29 cycles were performed with 45-sec steps and a final 10-min extension step at 68 C. After PCR, 8 µl of each reaction were separated on 1% agarose gels containing ethidium bromide (0.7 µg/ml). Gels were photographed and analyzed with ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
Statistical analyses
Both luciferase activities and ER protein levels were compared by one-way or two-way ANOVA to assess the effects of treatments. A confidence level of P < 0.05 was considered significant, and if there was a significant interaction, the Tukey test was used for post hoc comparisons.
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degradation protein levels in GH3 cells in response to E2 or FSK over time. Compared with vehicle-treated controls, treatment with E2 (10 nM) resulted in a 2.79 ± 0.22-fold increase in luciferase activity by 6 h (P < 0.05), which remained elevated after 24 h of treatment with E2 (2.52 ± 0.17-fold; P < 0.05; Fig. 1A
, left panel). FSK (1 µM) stimulated ER-mediated transcription significantly after 24 h (2.91 ± 0.13-fold; P < 0.05), but not at 6 h (1.72 ± 0.29-fold) of treatment (Fig. 1A, right panel). This suggests a distinct molecular mechanism for the ligandindependent activation of ER by FSK compared with E2.
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protein levels with time, as shown in Fig. 1B
(E lanes). Compared with vehicle-treated controls (100%), ER expression in E2-treated groups was reduced to 79%, 66%, 38%, and 34% at 1, 3, 6, and 24 h, respectively (Fig. 1C, left panel). In contrast, ER protein remained relatively constant over time in FSK-treated groups (85%, 87%, 110%, and 123% at 1, 3, 6, and 24 h, respectively; Fig. 1, B and C). Thus, FSK-stimulated ER transactivation is distinguished from E2 stimulation in two ways: FSK stimulation requires a longer time course for full activity and does not result in ER degradation.
FSK synergistically enhances E2-dependent ER activation and specifically prevents E2-induced ER protein degradation
In the presence of E2 and FSK, ER transcriptional activity in GH3 cells was stimulated synergistically (8.59 ± 0.27-fold), compared with that after treatment with E2 (2.51 ± 0.05-fold) or FSK (2.09 ± 0.32-fold) alone (Fig. 2A). As shown in Fig. 1, FSK alone did not cause a significant decrease in ER protein levels over time, but E2 did (Fig. 2B). Interestingly, treatment of GH3 cells with E2 plus FSK prevented the E2-induced ER protein degradation, indicating that FSK may protect ER from the degradation pathway (Fig. 2B). Importantly, this also occurred with the continuous presence of FSK and E2 at 24 h (Fig. 2C), when transcriptional synergy was most pronounced. The protective effect of FSK is specific, as FSK treatment did not protect the transcription factor I
from degradation induced by TNF (Fig. 2D), which also occurs via a proteasome-mediated pathway (20). FSK protection of ER protein occurred even in the presence of the transcriptional inhibitor -amanitin (Fig. 3A), and there was no stimulation of ER mRNA levels by FSK after either 6 or 24 h of treatment (Fig. 3B). These data suggest that the ability of FSK to protect ER from E2-dependent degradation is a posttranscriptional process and does not occur via increased levels of ER mRNA.
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transcriptional activity and protection from E2-induced degradation are mediated by PKA from degradation. To determine which signaling pathway is responsible, we treated GH3 cells with either H89 (a PKA inhibitor) or PD98059 (a MEK inhibitor) along with E2, FSK, or both compounds. The effectiveness of FSK in stimulating the PKA and MAPK pathways and the ability of H89 and PD98059 to inhibit these pathways were assessed by phosphorylation of cAMP response element binding protein and ERK1/2, respectively (data not shown). In agreement with our previous findings (14), H89, but not PD98059, inhibited FSK-induced ER transcription (Fig. 4A). H89 alone did not alter E2-induced ER down-regulation (Fig. 4B). However, inhibition of PKA reduced the ability of FSK to protect ER from E2-induced degradation (Fig. 4B). In contrast, in GH3 cells treated with PD98059, FSK was fully effective in protecting ER protein levels from E2-induced degradation (Fig. 4C, lanes 5 and 7). These data indicate that the PKA, but not the MAPK, pathway is involved in the protective effect of FSK on E2-stimulated ER degradation.
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protein degradation and ERß, a variety of ER subtype-selective ligands have been developed to target ER transcriptional activity, including R,R-THC (ER agonist/ERß antagonist), PPT (ER agonist), and DPN (ERß agonist). We found that both R,R-THC and PPT increased ER transcriptional activity in GH3 cells, with R,R-THC appearing less potent than PPT (Fig. 5A, top and middle panels). As with E2, both ligands decreased ER protein levels (Fig. 5B, top and middle panels). Although high levels of DPN did not induce robust ER activation (Fig. 5A, bottom panel), consistent with its low affinity and only weak activity via ER (26, 27, 28, 29), treatment with the high concentration of DPN did trigger ligand-dependent ER degradation (Fig. 5B, bottom panel). These observations suggest that stimulation of transcriptional activity by other estrogenic ligands binding to the LBD may be coupled to ER degradation.
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protein from ER subtype-selective, ligand-induced degradation from degradation induced by E2, we investigated whether FSK had similar protective activity with ER subtype-selective ligands. As with E2, FSK increased ER transcriptional activity induced by R,R-THC, PPT, and DPN (Fig. 6A). For each ligand, FSK protected ER protein levels from the ligand-induced degradation that accompanied receptor transcriptional activation (Fig. 6B). Thus, the ability of FSK to protect ER from degradation can be extended to other activating ligands.
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protein from degradation by the antiestrogen ICI 182,780 protein from degradation by ligands that generally antagonize ER transcription. Both ICI 182,780 and 4-OHT are antagonists to E2 action in pituitary cells. However, ICI 182,780 reduces ER protein in target cells by a mechanism that is proposed to be distinct from that of E2-mediated degradation (12, 15). As we previously reported (14), FSK-induced ER activation (4.0 ± 0.2-fold) was blocked by both ICI 182,780 (Fig. 7A, left panel) and 4-OHT (Fig. 7A, right panel), and ICI 182,780 also reduced basal ER activation (Fig. 7A, 
, left panel). Although FSK-induced ER activation was inhibited by ICI 182,780, FSK effectively protected ER from degradation (Fig. 7B, right panel). Although 4-OHT treatment did not reduce ER protein levels, treatment with FSK plus 4-OHT resulted in slightly increased receptor levels compared with 4-OHT treatment alone (Fig. 7B, right panel). Thus, these data extend the actions of FSK to protect ER from degradation by both activating ligands and antiestrogens.
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protein, whereas E2- or subtype-specific ligand activation is invariably linked to ER degradation. Several investigators have shown that inhibition of 26S proteasome activity protects ER protein and disrupts E2-mediated ER transactivation (13, 15). We examined whether ligand-independent ER activation by FSK similarly required the 26S-proteasome pathway. GH3 cells were treated with the proteasome inhibitor lactacystin, and transfected reporter gene activity and ER protein levels were measured after E2 and FSK treatment. Treatment with 10 µM inhibitor prevented E2-induced degradation of ER protein (Fig. 8B), but was not toxic to these cells, as shown by consistent levels of transfected ß-galactosidase activity in all treatment groups (Fig. 8A). Concentrations of lactacystin that prevented E2-induced ER degradation suppressed ER transcriptional activity in response to E2, but not FSK (Fig. 8A). In agreement with previous data, treatment of cells with E2 plus FSK prevented the E2-mediated degradation of ER protein. In the presence of lactacystin, which prevents the ligand-induced ER degradation, FSK had little additional effect on protein levels. Lactacystin partially suppressed the transcriptional activation of ER in response to FSK plus E2 to a level similar to that observed with FSK alone (Fig. 8A). These data demonstrate that FSK stimulation of ER transcription does not require the proteasome pathway and suggest that this mechanism of transcriptional activation is distinct from that of ligand-stimulated transcription.
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activation is also coupled to increased receptor turnover, whereas FSK-induced ER activation is not linked with ER degradation. Further, inhibition of proteasome activity suppresses E2-induced ER transcriptional activity, but ER remains fully responsive to FSK stimulation. Thus, these observations clearly suggest that the FSK-induced and E2-stimulated ER activation may be controlled by distinct mechanisms, and that FSK-stimulated transcription does not require receptor proteolysis.
Several previous studies demonstrated that a variety of ER subtype-selective ligands, such as PPT, R,R-THC, and DPN, selectively modulate the transcriptional activity of ER and ERß (25, 27). As E2-stimulated ER activation is coupled to receptor degradation, we examined the possibility that ligand-stimulated ER activation is also linked with ER proteolysis. Our findings show that among these ligands, PPT and R,R-THC significantly increase ER transcription activity, but all three ligands lower ER protein levels. Thus, the linkage between ER transactivation and ER degradation by ligand may be applied to other active estrogenic compounds in addition to E2. However, this association does not hold true for all ligands, because some ER antagonists, such as ICI 182,780, RU58668, and GW7604 (12, 15, 20, 21, 22), also decrease ER protein levels without transcriptional activation, and DPN can decrease receptor levels even without significantly stimulating transcription. These data have formed the basis for the hypothesis that changes in the conformation of ligand-bound ER are critical for ER degradation rather than its transcriptional activity (12).
Linkage between ligand-induced activation and proteasome activity-dependent receptor turnover is not consistent among all nuclear receptors. Inhibition of proteasome activity stimulates ligand-activated glucocorticoid receptor transcription (33), but suppresses ligand-mediated transcription of the progesterone receptor (34), thyroid hormone receptor, and androgen receptor (35). For the human progesterone receptor, ligand and MAPK activator treatment results in receptor transcriptional hyperactivity, and ligand- and proteasome-dependent turnover is enhanced by direct MAPK phosphorylation of the receptor (34). Thus, cross-talk between ligand-dependent and ligand-independent pathways can occur at the level of both transcriptional activation and receptor turnover.
Our studies clearly show that ER transcriptional activation through the PKA pathway does not require proteasome activity or linkage with receptor turnover. In addition, in the presence of E2, FSK not only synergistically enhances ER transcriptional activity, but also protects ER protein from E2-induced degradation. FSK does not increase ER mRNA levels in these cells, and the protective effect of FSK occurs even in the presence of -amanitin, demonstrating that FSK protection of ER levels in the presence of E2 occurs via a posttranscriptional mechanism. The ability of FSK to protect ER occurs with all of the activating ligands tested and ICI 182,780, but does not eliminate the suppressive effect of the antagonist on transcription. The protective effect of FSK on ligand-bound ER protein is mediated exclusively by the PKA pathway and not via MEK. This unique feature of PKA activation is supported by data demonstrating that although ER-mediated transcription can be stimulated by PMA, a protein kinase C activator, PMA decreases ER levels (36) and does not protect the receptor from ligand-mediated degradation (our unpublished data). In addition, other investigators showed that MAPK activation facilitated ER degradation in breast cancer cells (21, 37). A recent report showed that thyroid hormone (T3) also protects ER from E2-induced degradation in PR1 pituitary cells; however, protection by T3 appears to be limited to E2-bound ER, because T3 cannot protect ER from degradation induced by ICI 182,780 (38). Furthermore, T3 does not synergize with E2 to stimulate transcription, but has either a suppressive effect (on an ERE) or no effect (on the prolactin promoter) in this system.
At present, it is not known whether PKA-stimulated ER transcription and the protection of ligand-bound ER from degradation by PKA occur by the same, related, or distinct mechanisms. The two actions of PKA could occur by completely different pathways, such as synthesis or modification of receptor coactivators to stimulate transcription and modification of the receptor or specific enzymes that result in decreased receptor degradation. The fact that the two processes may occur with different time courses, with longer times required for transcriptional stimulation, supports this idea. It is also not known whether the transcriptional synergy between E2 and FSK requires PKA-mediated protection of the receptor from ligand-dependent degradation. However, increased ER levels alone are insufficient for transcriptional synergy, as lactacystin treatment prevents ER degradation, but does not allow transcriptional synergy by E2 and FSK.
These data reinforce the idea that there may be distinct mechanisms by which E2 and FSK/PKA can stimulate ER transcription. PKA stimulation of ER activity occurs through the C-terminal AF-2 region, as does E2 stimulation (15, 39, 40, 41), and AF-2 activity is predominant in pituitary cells (14). PKA can phosphorylate several serine residues in the DBD and AF-2 region, and analogous residues exist in both rat and human ER (42, 43). Mutation of these residues does not abolish cAMP stimulation of ligand-activated ER activity in SK-BR-3 breast cancer cells (44). Other investigators have found that PKA can phosphorylate coactivators such as steroid receptor coactivator-1, and that this may contribute to ER activation under these conditions (45), but have also proposed that ER phosphorylation may be important (46). PKA has been proposed to increase the association between ER and cyclin D1 and to stimulate receptor transcription by this mechanism (47). The exact mechanism for transcriptional activity may depend on the cell type and features of the specific promoter used for the reporter assay. For example, in breast cancer and ovarian cells, PKA and protein kinase C have little effect alone, but synergistically activate ER in the presence of E2 (36). This was postulated to occur by stabilization of ER interactions with components of the transcriptional machinery. In SK-BR-3 breast cancer cells, synergistic activation of ER by cAMP and E2 required CREB and CBP, and the addition of a CRE element near an ERE in the promoter of the luciferase reporter. In contrast, our investigations in pituitary cells show significant PKA-dependent stimulation of an ERE-fused to a TATA box, without cAMP response element or AP-1 sites. Further, this stimulation does not require proteasome activity.
Several studies have demonstrated that ligand-induced ER degradation requires only the carboxyl-terminal region of the receptor (12, 13). In addition, specific mutations within helix 3, 5, or 12 of ER confers resistance to degradation by E2 or the antagonists ICI 182,780 and GW7604 (12, 15, 20, 21, 22). Ligand-mediated degradation of ER has been shown to occur through the proteasome pathway, and ligand binding increases ubiquitination of ER (12, 15). 4-OHT, which does not induce degradation, similarly does not increase ubiquitination (12). The sites of ubiquitination on the ER have not been defined. Similarly, it is not known whether potential ER protein modifications could alter this ubiquitination. In our studies, addition of the proteasome inhibitor lactacystin prevents E2-mediated ER degradation and E2-stimulated transcription. In addition to ER itself, coactivators binding to the receptor have been shown to undergo degradation along with ligand-bound ER (48); this implies that some ER-interacting proteins might be involved in the ligand-induced degradation. One interesting possibility is that PKA stimulation of transcription might occur with different coactivator preference or other different ER-protein associations than with E2, and that this might contribute to the lack of receptor degradation with FSK treatment. As ER ubiquitination seems to be the signal to direct ER into the degradation pathway, we speculate that PKA might act via modification of ER itself, coactivators binding to the receptor, or the specific enzymes that mediate ubiquitination, such as E1, E2, or E3-ligases, or by other mechanisms.
A recent study suggested that PKA stabilizes ligand-free human ER in breast cancer cells (21). In our studies, PKA stimulation also appears to stabilize the transcriptionally active ER, and H89 inhibition of PKA results in slightly lower protein levels. More importantly, PKA nearly completely prevents ligand-induced ER degradation by either ER agonists or antagonists. Thus, our studies demonstrate that a unique feature of ligand-independent activation of ER through the PKA pathway is the relative stabilization of the ER protein and its independence of proteasome activity for transcriptional activity. This suggests at least two pathways for ER-mediated transcription, both of which can be activated during cross-talk between the E2 and PKA pathways for synergistic activation of ER transcriptional activity.
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Parts of this work were presented at the 85th Annual Meeting of The Endocrine Society, Philadelphia, PA, 2003.
Abbreviations: AF-1, Amino-terminal ligand independent activation domain; AF-2, carboxyl-terminal ligand-binding/ligand-dependent activation domain; DBD, DNA-binding domain; DPN, diarylpropionitrile; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; FSK, forskolin; LBD, ligand-binding domain; MEK, MAPK kinase; 4-OHT, 4-hydroxytamoxifen; PKA, protein kinase A; PPT, propyl-pyrazole-triol; R,R-THC, R,R-diethyl-tetrahydrochrysene; SERM, selective estrogen receptor modulator.
Received October 31, 2003.
Accepted for publication March 8, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the anterior pituitary gland. Mol Endocrinol 11:674–681 protein and estrogen responsiveness. Endocrinology 141:2174–2184 is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators. J Biol Chem 276:35684–35692 and coactivator turnover and for efficient estrogen receptor- transactivation. Mol Cell 5:939–948[CrossRef][Medline]
on responsive promoters is an integral feature of estrogen signaling. Mol Cell 11:695–707[CrossRef][Medline]
: regulation by synthesis, modification and degradation. Cell Mol Life Sci 59:821–831[CrossRef][Medline]
. Am J Physiol 282:E891–E898
(ER), differentially affect ER extractability, proteasome-mediated stability and transcriptional activity in human breast cancer cells. Mol Endocrinol 17:2013–2027. function and stability by tamoxifen and a critical amino acid (Asp-538) in helix 12. J Biol Chem 278:7630–7638 or estrogen receptor-ß. Endocrinology 140:800–804-selective agonists. J Med Chem 43:4934–4947[CrossRef][Medline]
- and ß-selective ligands at diverse estrogen responsive gene sites mediating transactivation or transrepression. Mol Cell Endocrinol 206:13–22[CrossRef][Medline]
(ER) but not estrogen receptor-ß modulates estrogen stimulation of the ER truncated variant, TERP-1. Endocrinology 143:4196–4202 expression in breast cancer cells. Mol Endocrinol 15:1344–1359 protein: estrogen-dependent proteolysis is not essential for receptor transactivation function in the pituitary. Endocrinology 144:3469–3476 by protein kinase A regulates dimerization. Mol Cell Biol 19:1002–1015 (ER)- and ERß-dependent gene expression by cAMP signaling pathway(s). J Biol Chem 278:12834–12845This article has been cited by other articles:
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  I. S. Fenne, T. Hoang, M. Hauglid, J. V. Sagen, E. A. Lien, and G. Mellgren Recruitment of Coactivator Glucocorticoid Receptor Interacting Protein 1 to an Estrogen Receptor Transcription Complex Is Regulated by the 3',5'-Cyclic Adenosine 5'-Monophosphate-Dependent Protein Kinase Endocrinology, September 1, 2008; 149(9): 4336 - 4345. [Abstract] [Full Text] [PDF]
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 N. Vasudevan and D. W. Pfaff Membrane-Initiated Actions of Estrogens in Neuroendocrinology: Emerging Principles Endocr. Rev., February 1, 2007; 28(1): 1 - 19. [Abstract] [Full Text] [PDF]
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 M. H. Al-Dhaheri and B. G. Rowan Protein Kinase A Exhibits Selective Modulation of Estradiol-Dependent Transcription in Breast Cancer Cells that Is Associated with Decreased Ligand Binding, Altered Estrogen Receptor {alpha} Promoter Interaction, and Changes in Receptor Phosphorylation Mol. Endocrinol., February 1, 2007; 21(2): 439 - 456. [Abstract] [Full Text] [PDF]
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E. T. Alarid Lives and Times of Nuclear Receptors Mol. Endocrinol., September 1, 2006; 20(9): 1972 - 1981. [Abstract] [Full Text] [PDF]
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 G. B Silberstein, K. Van Horn, E. Hrabeta-Robinson, and J. Compton Estrogen-triggered delays in mammary gland gene expression during the estrous cycle: evidence for a novel timing system. J. Endocrinol., August 1, 2006; 190(2): 225 - 239. [Abstract] [Full Text] [PDF]
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M. Fan, A. Park, and K. P. Nephew CHIP (Carboxyl Terminus of Hsc70-Interacting Protein) Promotes Basal and Geldanamycin-Induced Degradation of Estrogen Receptor-{alpha} Mol. Endocrinol., December 1, 2005; 19(12): 2901 - 2914. [Abstract] [Full Text] [PDF]
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 S K Nair, T J Thomas, N J Greenfield, A Chen, H He, and T Thomas Conformational dynamics of estrogen receptors {alpha} and {beta} as revealed by intrinsic tryptophan fluorescence and circular dichroism J. Mol. Endocrinol., October 1, 2005; 35(2): 211 - 223. [Abstract] [Full Text] [PDF]
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K. M. Olesen, H. M. Jessen, C. J. Auger, and A. P. Auger Dopaminergic Activation of Estrogen Receptors in Neonatal Brain Alters Progestin Receptor Expression and Juvenile Social Play Behavior Endocrinology, September 1, 2005; 146(9): 3705 - 3712. [Abstract] [Full Text] [PDF]
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) for 6 and 24 h, respectively. For each treatment, luciferase activity (mean ± SEM) was normalized and expressed as the fold stimulation over that in vehicle-treated controls (1.0) for triplicate samples in each group from three independent experiments. *, P < 0.05 vs. controls. B, Representative immunoblots of ER
) in each group; #, P < 0.05 vs. FSK alone-treated cells. B, ER
