Various Phosphorylation Pathways, Depending on Agonist and Antagonist Binding to Endogenous Estrogen Receptor
(ER
), Differentially Affect ER
Extractability, Proteasome-Mediated Stability, and Transcriptional Activity in Human Breast Cancer Cells
Véronique Marsaud,
Angélique Gougelet,
Sébastien Maillard and
Jack-Michel Renoir
Pharmacologie Cellulaire et Moléculaire des Anticancéreux, Unité Mixte de Recherche 8612, Centre National de la Recherche Scientifique, 92296 Châtenay-Malabry, France
Address all correspondence and requests for reprints to: Jack-Michel Renoir, Pharmacologie Cellulaire et Moléculaire des Anticancéreux, Unité Mixte de Recherche 8612, Centre National de la Recherche Scientifique, 92296 Châtenay-Malabry, France. E-mail: Michel.Renoir{at}cep.u-psud.fr.
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ABSTRACT
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Estrogen receptor-
(ER) is down-regulated in the presence of its cognate ligand, estradiol (E2), as well as in the presence of antiestrogens, through the ubiquitin proteasome pathway. Here, we show that, at pharmacological concentrations, the degradation rate of pure antagonist/endogenous ER complexes from human breast cancer MCF-7 cells is 10 times faster than that of ER-E2 complexes, while 4-hydroxy-tamoxifen (4-OH-T)-ER complexes are stable. Whereas pure antagonist-ER complexes are firmly bound to a nuclear compartment from which they are not extractable, the 4-OH-T-ER accumulates in a soluble cell compartment. No difference was observed in the fate of ER whether bound to pure antiestrogens ICI 182,780 or RU 58668. Cycloheximide experiments showed that, while the proteasome-mediated destruction of E2-ER (unlike that of RU 58668- and ICI 182,780-ER) complexes could implicate (or not) a protein synthesis-dependent process, both MAPKs (p38 and ERKs p44 and p42) are activated. By using a panel of kinase inhibitors/activators to study the impact of phosphorylation pathways on ER degradation, we found that protein kinase C is an enhancer of proteasome-mediated degradation of both ligand-free and ER bound to either E2, 4-OH-T, and pure antagonists. On the contrary, protein kinase A, MAPKs, and phosphatidyl-inositol-3 kinase all impede proteasome-mediated destruction of ligand free and E2-bound ER while only MAPKs inhibit the degradation of pure antiestrogens/ER species. In addition, no correlation was found between the capacity of kinase inhibitors to affect ER stability and the basal or E2-induced transcription. These results suggest that, in MCF-7 breast cancer cells, ER turnover, localization, and activity are maintained by an equilibrium between various phosphorylation pathways, which are differently modulated by ER ligands and protein kinases.
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INTRODUCTION
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IN ADDITION TO IMPACTING ON the reproductive system, estradiol (E2) exerts multiple biological effects, such as the regulation of bone structure, of cardiovascular function, and of the central nervous system. At present, it is generally accepted that estradiol acts predominantly by binding to its cognate intracellular receptors. The estrogen receptors (ERs) (1) are members of the nuclear receptor superfamily of small lipophilic ligand-activated transcription factors (1). Like several other members of this class of DNA binding proteins, ER is thought to be matured after sequential and dynamic interactions with different subsets of molecular chaperone complexes organized around the heat shock protein of 90 kDa molecular mass [heatshock protein 90 (hsp90)] and the cochaperones p23 and immunophilins (see Refs. 2 and 3 for reviews). Traditionally, it is believed that after E2 binding, ER
undergoes conformational changes leading to its release from a molecular chaperone. As a consequence, the E2-ER complex binds to DNA and promotes gene transcription via the concerted action of ER activation functions (4) and the recruitment of coactivator proteins (5). Subsequently, the complex stimulates the expression of estrogen-responsive genes via histone acetyltransferase activity of coactivators such as cAMP response element-binding protein (CREB)-binding protein/p300 and other accessory factors, which is thought to remodel chromatin and to allow access to the transcriptional machinery (6).
Crystal structure of the ER ligand binding domain revealed that the conformation of the agonist-ER complex is distinct from that formed in the presence of the selective ER modulators, tamoxifen and raloxifen (7, 8). The use of a mammalian two-hybrid assay demonstrated that different ER ligands induce a different ER domain exposure at the surface of the receptor (9, 10), rendering ER capable of interacting with different coactivators. Thus, different ligands induce a unique ER conformation, and target cells selectively recognize these particular complexes. Such a ligand-induced ER conformational change initiates, in addition to the transcription process, a cascade of events among which phosphorylation through the activation of different kinases is involved (see Ref. 11 for a review).
Another important feature triggered by the binding of a ligand to the receptor is the decrease of the cellular ER content. ER half-life is shorter in the presence than in the absence of E2 (12, 13). Although the molecular understanding of this process remained unclear for years, rat uterine ER has been shown to be ubiquitinated (14). In various systems including breast cancer cells and ER transiently transfected cells, ER-ligand complexes are ubiquitinated and degraded by the 26S-proteasome (15, 16, 17, 18) in a ligand binding-dependent manner (19). In addition, the proteasome-mediated destruction of E2-ER complexes occurs after DNA binding of the receptor (19), contrary to that after ICI 182,780 (ICI) (20) binding. Indeed, ICI still induces the degradation of the ER variant that lacks the DNA binding domain (19); because ER-ICI complexes do not recruit coactivators, this indicates that other factors, in addition to the ability to activate transcription, are able to direct the receptor to proteolysis. As noted by many groups, tamoxifen increases the ER half-life, whereas ICI effectively reduces the cellular ER content (18, 19, 21, 22). Although being poorly ubiquitinated in MCF-7 cells, ligand-free ER is also likely to be degraded through the proteasome pathway (19).
In addition to hormone-dependent ER activation, hormone-independent activation also occurs in human breast cancer cells. As an example, MAPKs activated by epidermal growth factor (EGF) directly phosphorylate ER in the absence of ligand (23, 24) and induce ER transactivation. Similarly, phosphatidylinositol 3-kinase (PI3K) and AKT/PKB (human homolog of v-akt oncogene, isolated from an AKR mouse T cell lymphoma, also called protein kinase B) kinase activate ER in the absence of hormone (25). Other phosphorylations induced by various extracellular signals such as those involved in the dopamine (26)-, cAMP (27)-, and HER-2 Neu (28)-mediated pathways also phosphorylate ER and activate its transcriptional activity. More recently, ER from MCF-7 and progesterone receptor (PR) from T47-D cells were shown to be degraded through the proteasome pathway after hyperactivation of MAPKs (29, 30). Thus, different stimuli, in addition to the receptor ligands and emanating or not from cell surface receptors, are able to alter the receptor turnover.
In the present work, we address the question whether the two pure antiestrogens ICI and RU 58 668 (RU) (31) induce differences in endogenous ER degradation and receptor localization, and how kinase modulators influence ER expression and gene transcription in MELN (MCF-7 cells stably transfected with an estrogen response element-ß globin-LUC construct) breast cancer cells. Initially, we compared the impact of RU and ICI on ER stability. Both ICI and RU are E2-derived compounds that differ essentially by the position of the substitution chain: 11ß for RU vs. 7
for ICI, i.e. at opposing points of the E2 molecule, and with opposite spatial orientation with regard to the plane of the molecule (Fig. 1
). Like ICI, RU can inhibit the growth of a subpopulation of tamoxifen-resistant tumors (32, 33, 34). Surprisingly, in spite of similar affinities for human ER (31), they possess different antitumor activities in MCF-7 tumors implanted in mice, RU having been demonstrated to induce a total regression in 30% of MCF-7 cell xenografts (33, 34). We have performed a series of experiments to decipher differences between the behavior of ER-RU and ER-ICI complexes. High concentrations of pure antagonists were used intentionally to mimic the pharmacological effects of the drugs. The use of different ER ligands, including a partial agonist 4-hydroxy-tamoxifen (4-OH-T) and the two pure antiestrogens RU and ICI, alone or in association with modulators of kinases in cell cultures, led us to conclude that the endogenous ER turnover is stabilized by a basal level of phosphorylation affecting either the receptor itself and/or associated proteins, or both. Several kinases participate to maintain this balance, and modification of their activities through inhibition (or activation) has a powerful effect on the ligand-free as well as ligand-bound receptor turnover.
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RESULTS
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Extractability of Different ER-Ligand Complexes
MCF-7 cells were incubated for 18 h with either low (10 nM) or high (1 µM) concentrations of the four ER ligands, the structures of which are shown in Fig. 1
. Three types of extracts were then prepared: cell lysates in low-salt conditions (LSEs); high-salt extracts (HSEs), and total cell extracts (TCEs) (see Materials and Methods for details). In LSEs from cells exposed to E2, RU, or ICI (Fig. 2A
), no ER protein was detected by Western blotting. In contrast, an increase of the receptor content in LSE was observed after 4-hydroxy-tamoxifen (4-OH-T) treatment. HSEs of cells treated with either E2 or 4-OH-T were rich in ER, whereas only a weak signal was detected in RU- or ICI-treated cells (Fig. 2B
). Analysis of TCEs revealed a small, but nevertheless much lower, ER amount in RU- and ICI-treated cells as compared with cells incubated with E2 or 4-OH-T, both at 1 µM (Fig. 2C
) and 10 nM (Fig. 2D
). As expected (35, 36), the ER level was highest in 4-OH-T-treated cells. Similar data were obtained in T47-D cells, another ER-rich human breast cancer cell line (not shown).

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Fig. 2. Differences in the Extractability of ER According to the Ligand
MCF-7 (60% confluence) cells were exposed for 18 h or not (C) to 1 µM or 10 nM (the same results, data not shown) of E2, RU, 4-OH-T, or ICI. LSE or HSE was prepared. In a parallel experiment, steroid-exposed cells were pelleted, rinsed, and directly boiled in Laemmli sample buffer for obtaining the TCE. Proteins (30 µg per lane) were separated on 10% acrylamide SDS-PAGE and Western blotted using the D12 monoclonal antibody. Arrows on the right indicate ER whose migration is identified by a standard of receptor sample (1 µg) translated in rabbit reticulocyte lysate (*). A nonspecific protein (NS) migrating slower than ER is visualized and serves as an internal loading control. Similar data were obtained with the H222 or with C314 antibodies (not shown). In the experiment shown in panel D, cells were incubated overnight (+) or not (-) with 10 nM E2 in the presence of either 1 µM RU or 4-OH-T or ICI and extracted proteins contained in TCE were loaded on 8% SDS-PAGE. The protein profile shown was obtained with H222. Identical data were obtained with D12 (not shown).
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Thus, according to the ligand, ER is differentially extractable. In addition, pure antiestrogens RU and ICI induce a stronger receptor degradation than E2, whereas the partial agonist 4-OH-T protects ER
against degradation.
To mimic the conditions in which antagonists can be clinically used, cells were incubated with 10 nM E2 in the presence or not of 1 µM antagonist (Fig. 2D
). Analysis of ER content in TCEs indicated that pure antiestrogens maintain the strong degradation of the receptor whether E2 was present or not, contrary to what was observed with 4-OH-T.
Similarities in Pure Antagonist-Induced Proteasome-Mediated ER Degradation
Incubation of MCF-7 cells with drugs inhibiting the proteasome by different mechanisms [MG132, lactacystin, and epoxomycin (37)] induced a decrease in the content of ER in LSEs, whereas ER in TCEs remained constant (Fig. 3
, A and B). This indicates that proteasome inhibitors relocalize the ER, an observation made also by others (15, 18, 19, 22) but also suggests that functional ER could be permanently ubiquitinated and degraded. ER was undetectable in LSEs of cells treated with either RU or ICI, both in the presence and absence of the proteasome inhibitors. However, in TCEs from similarly treated cells, MG132, as well as epoxomycin (data not shown), blocked the ligand-induced receptor degradation, including that induced by pure antagonists (Fig. 3B
). This suggests that proteasome-mediated ER destruction takes place in the nonextractable cell compartment. Similar results were obtained in both MCF-7 and T47-D cell lines (data not shown) at 10 nM as well as 1 µM steroid concentrations. These results are in agreement with previous reports that identified the nuclear matrix, a dynamic structure involved in DNA replication, transcription, repair, and RNA processing, as the cell compartment from which the ER complexes with ICI (22) or with E2 are nonextractable even by high ionic strength (38, 39, 40).

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Fig. 3. Proteasome-Mediated ER Degradation and Difference in the Extractability of ER Depending on the Ligand
MCF-7 cells were exposed or not (C) to the different drugs at either 10 nM (data not shown, identical results) or 1 µM in the presence or absence of the three proteasome inhibitors, MG132 (5 µM), lactacystin (lact, 10 µM), or epoxomycin (epoxo, 10 µM). Both LSEs (panel A) and TCEs (panel B) were prepared, and the detection of blotted ER was performed with the D12 antibody. The upper band detected in the upper gel is the NS band as described in Fig. 2 .
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RU and ICI Induce ER Elimination with Similar Dose Dependence and Kinetics
The effects of the two pure antagonists on ER down-regulation, in comparison with those of E2 and 4-OH-T, were studied in MCF-7 cells. After treatment with 1 µM E2, more than 50% of ER was eliminated after a 3-h incubation (Fig. 4A
). An even stronger decrease of ER level was obtained with RU or ICI, whereas 4-OH-T induced an increase of ER content. In experiments using cells exposed to these steroids at high concentrations during different periods of time (Fig. 4B
), RU induced a 50% decrease of ER content after 12 min of treatment, a degradation rate similar to that observed with ICI, and faster than with E2 (t1/2 = 100 min) [Fig. 4
, B and C (inset)]. In cells incubated with E2, ER level decreased until 6 h and increased thereafter, to reach approximately 60% of the initial value by 48 h. In contrast, 4-OH-T induced an increase of ER to almost twice the initial level at 15 h, and remained stable at 48 h.

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Fig. 4. Kinetics and Dose Response of Ligand-Induced ER Degradation
MCF-7 cells were exposed for 3 h to variable concentrations (01 µM) of E2 or RU or 4-OH-T or ICI. The TCEs were prepared, and protein samples (30 µg) were loaded onto SDS-PAGE. ER was identified after blotting as in Fig. 3 . In panel B, 1 µM E2, RU, 4-OH-T, or ICI was added to 50% confluent MCF-7 cells for various periods of time. Actin was detected as a control of constant protein loading. In panel C, densitometric analysis of Western blots of ER protein from panel B were normalized to actin and plotted as percentage of the control (time 0). Points are the average of a duplicate analysis. The inset is a magnification of times 03 h, and the results are means of two different experiments.
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Contrary to a previous work suggesting that RU was a more effective inducer of ER down-regulation than ICI (41), we did not observe any significant difference between these two pure antagonists in promoting ER destruction. Our data (Figs. 24

) confirm that E2 is a much weaker promoter of ER degradation than pure antagonists that relocalize ER to a nonextractable compartment with similar efficiency. In contrast, 4-OH-T protected the ER, even at 1 µM, without inducing relocalization as revealed by the easy extractability of 4-OH-T-ER.
Reversibility of ER Elimination
Northern blot analysis indicated no variation of the ER mRNAs in cells treated with 1 µM RU and ICI (Fig. 5
, A and B). In contrast, in MCF-7 cells exposed to E2, ER mRNA decreased until 4 h and returned consistently to its initial level at 24 h with the replenishment of the protein level observed in Fig. 5C
. Such results are similar to previous data obtained at physiological concentrations of E2 (13, 35, 36). This indicates that the regulation of ER protein content after ligand binding depends on the type of the ligand, agonists acting through a dual transcriptional/posttranscriptional mechanism, and pure antagonists and selective ER modulators (such as 4-OH-T), acting predominantly or exclusively via a posttranscriptional process. However, the effects of RU and ICI on ER mRNA were indistinguishable.

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Fig. 5. Replenishment of ER in Cells Exposed to Ligands
MCF-7 cells were grown in phenol red-free DMEM supplemented with 10% dextran-charcoal-stripped fetal calf serum for 5 d. In panel A are shown the variations of both ER and 36B4 mRNAs after time-dependent exposure of MCF-7 cells to 1 µM E2, 4-OH-T, RU, or ICI. Densitometric analyses of the autoradiographs from Northern blots were normalized to the internal standard 36B4 and plotted (panel B) as percentage of the normalized control (time 0). Points are average of duplicate experiments. In panel C, cells were grown as in panel A, after which the medium was replaced by a fresh steroid-free medium (NT) or the same medium containing 1 µM E2, RU, or ICI for overnight exposure; the medium was further replaced by steroid-free medium for various times (0, 24, 48, 72 h). In panel D, cells grown as in panel A were incubated overnight with 1 µM RU or ICI, after which the medium was replaced by medium containing 10 nM E2 for various periods of time before TCE preparation. Western blots were revealed with the D12 antibody.
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The replenishment of ER was studied in cells preincubated with different pharmacological concentrations of ligands and subsequently placed in estrogen-free culture medium (Fig. 5C
) or in medium containing physiological concentrations of E2 for different periods of time (Fig. 5D
). The depletion of the receptor after RU and ICI binding persisted for at least 72 h after the withdrawal of the medium containing the steroids; similarly, when cells treated with 1 µM pure antagonists were subsequently exposed to 10 nM E2, they did not resynthesize the receptor to the level of untreated cells, even after 48 h. Thus, the destruction of the receptor after its binding to pure antagonists is a long lasting phenomenon.
Comparison of ER Turnover after Binding to Various Ligands
In cells exposed to cycloheximide (CHX), the ER level was affected neither by E2 nor by 4-OH-T exposure (17). We confirmed this observation in the present work: Fig. 6
is representative of three identical experiments that gave similar results. However, the pure antagonist-induced ER degradation persisted despite the inhibition of protein synthesis and despite an increased half-life as compared with that measured in the absence of CHX shown in Fig. 4C
(t1/2 = 1 h for RU and 90 min for ICI as compared with 12 min for both ligands in the absence of CHX). This indicates that ligand binding-induced ER protein loss is not a process totally independent of protein synthesis and strengthens the notion that factors that are different from those required for E2-mediated receptor degradation are involved in the elimination of RU- and ICI-ER complexes. Because CHX and many other inhibitors of protein synthesis have been described as activators of MAPK (42, 43), we examined its influence (as well as that of puromycin, 50 µg/ml; same results; data not shown) on both p38 and ERKs. Indeed, CHX activates these kinases in MCF-7 cells as revealed by the increase of phospho-p38 and phospho-p44 and -p42 (Fig. 6A
) in non-hormone-treated cells. Densitometry analyses (Fig. 6
, B and C) revealed that, after CHX exposure, an increased activation of MAPK occurring maximally at 12 h and decreasing thereafter was occurring in nontreated and E2-treated cells. This process was weak and did not appear within minutes (results not shown) but much more later (at least after 30 min), suggesting that E2 is not an activator of MAPK as indicated previously (44, 45) and contrary to what has been claimed by others (46, 47). Interestingly, whereas both pure antiestrogens do not influence significantly the CHX-modified profile of both p38 and ERK1,2 activation, 4-OH-T strongly induces and maintains it to a relatively high level.

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Fig. 6. CHX Experiments Analysis of ER Turnover
MCF-7 cells grown in phenol red-free DMEM containing stripped serum were preincubated or not with CHX (50 µg/ml) for 30 min before the addition of 1 µM E2, RU, ICI, or 4-OH-T for various periods of time. TCEs were prepared and equal amounts of proteins were electrophoretically separated and analyzed with D12 antibody to detect ER and with antibodies specific for total and activated ERK1, ERK2, and p38. The total amount of ERK1 and ERK2 as well as p38 (panel A) was used as a control of constant protein loading and serve as a reference to estimate the relative index of phosphorylation of MAPK after densitometry analysis (panels B and C).
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Influence of Protein Kinase Inhibitors on Ligand-Induced ER Degradation
To decipher the phosphorylation activities that could be involved in the modification of ER turnover induced by ligand binding, we investigated the influence of several kinase inhibitors on the ligand-free, agonist-, 4-OH-T-, and pure antagonist-bound ER protein levels.
Modulation of PKC Activity.
Because 4-OH-T at very high concentration has been shown to inhibit PKC in vitro (48, 49), we investigated the role of this kinase in the stability and extractability of ER complexed or not with a ligand. Either the PKC inhibitor iso-H7 or the PKC activator mezerein (50) was added to the culture medium of MCF-7 cells, exposed or not to ER ligand. As shown in Fig. 7A
, mezerein induced a strong MG132-inhibited destruction of nonliganded ER. The stability of the 4-OH-T-ER, as well as that of E2-ER complexes, was also decreased by mezerein (Fig. 7
, E and C, compare lanes 2 and 8), an effect blocked by MG132 (Fig. 7
, E and C, lanes 9).

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Fig. 7. Modulation of ER Content by Inhibitors of PKA and PKC
MCF-7 were preincubated (+) or not (-) with MG132 (5 µM) for 30 min before the addition of IsoH7 (10 µM) or H89 (10 µM) or mezerein (5 µM) for 1 h (data not shown, the same results) or 16 h. TCEs and CLs were prepared for receptor analysis by blotting with the D12 antibody. In panels CF, 1 µM steroid was added to the culture medium simultaneously with the kinase modulators and incubated overnight. Constant protein amounts (25 µg, panels A and B, 30 µg, panels CF) were loaded on 8% SDS-PAGE.
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Similar results were obtained after either 1-h (data not shown) or overnight cell exposure to the drugs and at 1 µM or 10 nM ER ligands (data not shown, same results). These data suggest that the ER degradation is accelerated by the activation of PKC activity in breast cancer cells. Whether this process requires a PKC-mediated phosphorylation affecting directly ER remains to be established.
In our experimental conditions, we could not ascertain whether or not the RU-induced proteasome-mediated ER degradation was further accelerated by mezerein (Fig. 7D
, lanes 8 and 9); however, the ICI-induced degradation of the receptor appeared to be faster in the presence of the PKC activator (compare lanes 8 and 2 in Fig. 7F
).
The PKC inhibitor IsoH7 (Fig. 7 C
, lane 8) enhanced the cell content of the receptor complexed with E2 (Fig. 7 C
, compare lanes 2 and 4) but did not alter that of pure antiestrogen-ER complexes or of 4-OH-T-ER complexes (Fig. 7E
, compare lanes 4 to 5). Actin signal used as control confirmed that equal amounts of protein were loaded throughout the experiment shown in panels AF of Fig. 7
(data not shown).
Inhibition of Protein Kinase A (PKA) Activity.
Inhibition of PKA by H89 decreased the expression of ligand-free receptor when analyses were performed in the TCEs (Fig. 7A
); surprisingly, this effect was not observed in the LSE, suggesting that a PKA-mediated phosphorylation process stabilizes the ligand-free ER protein in a cell compartment in which it is tightly bound to insoluble structures. The inhibition of PKA did not affect the ER content of cells exposed to any receptor ligand, agonist, or antagonist (Fig. 7
, CF, compare lanes 2 and 6).
Inhibition of MAPKs.
In T47-D-YB cells (T47-D cells engineered to stably express the PR-B isoform), the ERK1/ERK2 inhibitor PD98059 (PD) inhibits promegestone-induced progesterone receptor degradation by the proteasome-mediated pathway (30). Similarly, hyperactivation of MAPK resulted in down-regulation of ER that was reversible by PD in MCF-7 cells overexpressing ER (29). In our work with MCF-7 cells, U0126, a more potent ERK inhibitor than PD, induced the MG132-sensitive degradation of ligand-free endogenous ER as well as of ER occupied by 4-OH-T. The same results were obtained using PD (data not shown). In contrast, U0126 had no effect on the RU-, ICI-and E2-ER complexes (Fig. 8
, BD). These results suggest that the Raf/Ras/ERK pathway participates at the basal phosphorylation level of the ligand-free apo-ER and of 4-OH-T-ER complexes or that it affects a factor(s) interacting with these complexes. The p38 MAPK inhibitors SB203580 (SB580) and SB202190 (SB190) caused the MG132-inhibited degradation of all ER-ligand complexes in MCF-7 cells, as well as that of ligand-free ER (Fig. 8
, A and E). Similar effects were observed at 1 µM and 10 nM (not shown) ligand concentration. However, there were differences in the extent of the destruction of the receptor according to the ligand bound. Although both SB inhibitors potentiated pure antagonist- as well as 4-OH-T-bound ER degradation (Fig. 8E
), their effects were not identical. In contrast to SB190, SB580 enhanced E2-induced degradation (Fig. 8E
, compare lanes 3 and 4). Because SB580 is a p38-specific inhibitor by opposition to SB190, which also inhibits p44-p42 (30), this may indicate that E2-induced degradation is inhibited by p38. In contrast to the E2-ER complexes, the proteasome-mediated turnover of pure antagonist-ER complexes is modulated by both Ras/Raf/ERK and p38 MAPK pathways (Fig. 8E
, compare lanes 5 with 6 and 9 with 10, respectively). Thus, the inhibition of both pathways accelerated the antiestrogen-induced degradation of the receptor, whereas that of the p38 pathway accelerated only the E2-induced receptor degradation. All the effects of MAPK inhibitor-induced ER degradation were inhibited by MG132 (Fig. 8
, AE), suggesting that a basal MAPK-dependent phosphorylation of the receptor itself (or of an associated factor) is necessary to maintain ER expression level.

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Fig. 8. Effects of MAPK Inhibitors on ER Content
MCF-7 cells were preincubated (30 min) (+) or not (-) with MG132 (5 µM) (panels AD) and then SB202190 (40 µM), SB 203580 (20 µM), or UO126 (10 µM) was added for 1 h (panels AD) or 16 h (data not shown, the same results). Steroids (1 µM) were added at the same time as MAPK inhibitors as follows; E2 (B), RU or 4-OH-T (C), and ICI (D). In panel E, either SB203580 or SB202190 was incubated in cell culture medium (+) or not (-) for 1 h (or 16 h, similar patterns) in the presence (+) or the absence (-) of 1 µM steroid. Western blots of TCEs (25 µg proteins) were probed with the D12 antibody. C in panel E corresponds to the level of ER in control (nonsteroid)-treated cells.
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Inhibition of PI3K.
PI3K has been reported to phosphorylate in vivo ER at Ser(167) (25). In the experiments shown in Fig. 9
, LY 294002 (LY) decreased the level of ligand-free receptor as well as that of the receptor bound to E2 and to 4-OH-T; this degradation process was blocked by MG132 and epoxomycin (data not shown). These data also suggest that the pure antagonist-induced proteasome-mediated ER degradation is enhanced by inhibition of the PI3K; this is deduced from the weaker inhibition of ER degradation by MG132 in cells incubated with both LY and pure antagonists as compared with pure antagonists alone.

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Fig. 9. Effects of PI3K Inhibitors on ER Content
MCF-7 cells were treated with the PI3K inhibitor LY 294002 (20 µM) for 2 h (+) or not (-) with (+) or without (-)MG132 (5 µM) in the presence (+) or the absence (-) of 1 µM steroids as indicated. The incubation was carried out overnight before preparation of TCEs and analysis by the D12 antibody. The nonspecific upper band serves as an internal control of constant protein loading.
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Influence of ER Ligands and Kinase Modulators on ER-Mediated Transcription in MELN Cells
Both ICI and RU efficiently inhibited E2-induced transcription in MELN cells (Fig. 10
). In dose response experiments not detailed here we found that 0.1 nM pure antagonists reduced by 50% the luciferase activity induced by 0.1 nM E2, agonist concentration sufficient for maximal transactivation. Inhibition by MG132 of the proteasome slightly, but significantly, inhibited the induction of the reporter gene activity. Similarly, activation of PKC by mezerein, which provoked receptor degradation (Fig. 7
), decreased the E2-induced transcription. Because in different ER-containing cells, the E2-induced transcription was found to be enhanced through activation of the MAPKs (Refs. 23 and 24 and review in Ref. 11), it was surprising that inhibition of MAPK increased both the basal and the E2-induced LUC activity. It is possible that in these cells the inhibition of MAPK activity affects participants in the transcription other than the receptor itself. Similarly to the MAPK inhibitor U0126, which induced a degradation of apo-ER (Fig. 8
, A and B), the PKA inhibitor H89 also accelerated the degradation of the receptor, but both inhibitors significantly induced both the basal and the E2-induced transcription in MELN cells (Fig. 10
). These apparently contradictory effects could be the consequence of the E2-induced activation of the PKC and PKA described to occur in many cells (see Ref. 51 for a review and references therein). Of note was the strong decrease of the basal transactivation activity induced by LY, supporting the finding that phosphorylation by the PI3K-AKT pathway, in the absence of hormone, affects factors involved in constitutive transcription; the inhibition by LY of both the basal and E2-induced transcription correlates with the strong receptor degradation shown in Fig. 9
.

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Fig. 10. Effects of Various Modulators of ER Stability on Basal and E2-Induced Transcription in MELN Cells
MELN cells grown as described in Materials and Methods were treated or not (control) with RU or ICI (1 µM each), MG132 (5 µM), UO126 (10 µM), LY (20 µM), Mezerein (MEZ; 20 nM), IsoH7 (100 µM), and H89 (10 µM) for 1 h before (open bars) or not (gray bars) an overnight incubation with 1 nM E2. Antiestrogens were added to a final concentration of 1 µM with E2. Luciferase activity is expressed as fold induction relative to the value in untreated cells. The means of triplicates ± SEM are shown.
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DISCUSSION
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The changes of nuclear localization of ER have been largely documented. For example, it has been proposed that the ER level decrease after binding to ICI 164,384 results from an inhibition of its nuclear uptake (21); however, this mislocalization of ER is likely the consequence of its overexpression after transient transfection, resulting in misfolded ER incapable of nuclear translocation (40). In fact, in most experiments using transiently expressed receptor, high levels of proteins are recovered in the cytoplasm and in perinuclear clusters (52). Thus, in this work, we have chosen to study the behavior of only endogenous ER, avoiding transient transfection experiments that do not reflect physiological conditions. In addition, we used intentionally pharmacological concentrations of antiestrogens to mimic clinical situations.
Initially, our aim was to find an explanation for the difference between the in vivo antitumor activities of the two antiestrogens RU and ICI. We found that proteasome-mediated proteolysis of both ER complexes with pure antagonists occurs at a similar rate in a compartment from which receptor-ligand complexes are not extractable by conventional lysis buffers (Figs. 2
and 4
). Nevertheless, subtle differences in the structure of the two ER-antagonist complexes, although undetectable by classical biochemical techniques, may lead to some differences in ER pharmacology. Comparisons between the crystal modelization structures of the receptor ligand-binding domain/ICI with that of the receptor ligand-binding domain/RU could help to solve this point.
An important question relates to the duration of pure antiestrogen-induced ER loss. We did not observe any ER replenishment after 72 h withdrawal of either RU or ICI except if E2 was added (Fig. 5
). The rate at which ER recovered after E2 supplementation was slower for RU than for ICI pretreatment. Whether this contributes to the different in vivo effects observed with these two pure antiestrogens in MCF-7 cell xenografts remains to be established.
The cellular compartment in which ER destruction occurs has been suggested by others to be the nuclear matrix (22, 39, 40). With the use of the recovery of fluorescent ER after photobleaching, it has been shown that ICI acts more efficiently than E2 to immobilize the receptor in the nuclear matrix, where its proteasome-mediated degradation takes places (40). From our experiments, the inhibition by proteasome inhibitors of the ER degradation induced by pure antagonists was detectable only in TCEs (Fig. 3
). This indicates that when bound to any ligand as well as when cells are incubated with proteasome inhibitors alone, the receptor is directed to a compound(s) that immobilize(s) it with nuclear matrix structures.
Because high concentrations of tamoxifen were shown to inhibit PKC in vitro (48, 49), we hypothesized that the resistance of 4-OH-T-ER complexes to degradation may be due in part to the capacity of 4-OH-T to inhibit PKC. To verify this hypothesis, we tested the effects of PKC modulators on ER stability and extended our work to the effects of other kinase inhibitors. Indeed, after PKC activation, degradation of 4-OH-T-ER complexes occurred and took place in the nonextractable cell compartment (see Fig. 7A
), a compartment to which the PKA inhibitor H89 also directed this complex. This suggests that the redistribution of ER into the nuclear matrix is not sufficient for the receptor to undergo the degradation process and that posttranslational modifications such as phosphorylations could be involved. Altogether, these data support the notion that, depending on the ligand, ER is attached to structures associated with the nuclear matrix, pure antagonists being the strongest inducers of such binding. Among the candidates are ubiquitin ligases such as the coactivators E6-AP (53) and TRIP1/SUG1 (thyroid hormone receptor-interacting protein/suppressor for galactose) (54), a component of the proteasome (55), the glucocorticoid receptor-interacting protein 120 (56) or carboxyl terminus of Hsc70-interacting protein sharing a C-terminal ubiquitin-like/proteasome-binding site and a N-terminal tetratricopeptide repeat domain (57, 58). Because N-terminal tetratricopeptide repeats allow interactions of proteins with hsps and because hsp90 is the key molecular chaperone with which ER associates before binding to any ligand, it is tempting to speculate that some of the coactivators or molecular chaperones like carboxyl terminus of Hsc70-interacting protein could act as mediators of the proteasome-mediated degradation of ER as suggested for the glucocorticoid receptor (59). Another candidate for maintaining ER associated with the nuclear matrix is the nuclear matrix protein/scaffold attachment factor HET/SAF-B (60), which has been reported to bind ER strongly in the presence of tamoxifen and to be important for its antagonist effect. Moreover, it is rather more likely that ER, like many other proteins shuttling from the nucleus to the cytoplasm, can be the target of the kinase activity of COP-9 signalosome (61), a well conserved proteasome-related 450-kDa structure, known to participate in both the CRM1-mediated nuclear export of nuclear factors and in their proteasome-mediated degradation (Ref. 62 and references therein). This is supported by preliminary data indicating that leptomycin B, an inhibitor of CRM-1-mediated nuclear export, affects the E2-ER complex extractability but not that of pure antiestrogen-ER complexes (data not shown). Taken in concert with the recent observation that the ubiquitin-like neural precursor cell-expressed developmentally down-regulated pathway 8, essential for protein processing and cell cycle progression, is required for proteasome-mediated degradation of ER and essential for the antiproliferative activity of ICI (63), it could be possible that at least two systems are involved in the degradation of ER: one using the signalosome when ER is bound to agonists, and the other one using the neural precursor cell-expressed developmentally down-regulated pathway 8 when ER is linked to pure antiestrogens.
As a summary of studies with kinase modulators, Fig. 11
shows that the apo-ER turnover is accelerated by PKC and reduced by PKA, by ERK and p38 MAPK pathways, and by PI3K. This decrease of ER protein content by activation of PKC occurs whether the receptor is liganded or not. The alterations of the receptor level induced by kinase activity modifications were all blocked in the presence of a proteasome inhibitor, strongly suggesting that in MCF-7 cells, a basal phosphorylation of ER or of factors involved in its activity, maintains the integrity of the receptor and that any change in this level targets receptor for proteolysis. However, modification of the kinase status produces different effects on the receptor expression according to the ligand bound. In the presence of E2, PKC may have a dual effect: first, an increase of phosphorylation leading to an increased ER association with coactivators and an enhancement of ER-mediated transcription (see Ref. 11 for review), and second, an enhancement of the rate of ER destruction by the proteasome. This is in agreement with the E2-induced transcription process described to occur before E2-induced degradation of ER through the ubiquitin/proteasome pathway (19) and to different chromatin remodeling of the pS2 gene occurring after 4-OH-T and ICI binding to ER (64). Data from Fig. 6
indicate that the inhibition of protein synthesis, although not affecting either ligand-free or pure antagonist-induced ER destruction, does not impede pure antiestrogen-induced ER loss, suggesting that factors that are affected neither by CHX (nor by puromycin or any other protein synthesis inhibitors) act on the ER protein expression level. Interestingly, these inhibitors induce transient activation of MAPKs, which is maintained by further cell exposure to 4-OH-T, in agreement with the maintenance of ER level due to inhibition of PKC by 4-OH-T. In fact, PKC has been well described for activating MAPKs and the JNK signal pathway (Refs. 65 and 66 and references therein), and the accumulation of ER after 4-OH-T binding is consistent with the schedule described in Fig. 11
.

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Fig. 11. Schematic Summary of the Involvement of Various Kinase Activities in the Proteasome- and Ligand-Dependent Degradation of ER
In its ligand-free form ER is associated in a complexed structure with molecular chaperones (Ch) that dissociate upon ligand binding. The ER is degraded through the ubiquitin proteasome pathway. This process is enhanced by PKC ( ) and impeded (I) by PKA, MAPKs, and PI3K. Each ligand induces a different conformation to the ER. Differently phosphorylated ER species are differently extractable (*, extractable; **, unextractable), and associated with different cofactors in the nuclear matrix, E2-ER complexes being targeted to specific DNA sequences to enhance transcription before being degraded at the proteasome level. Whereas MAPK and PI3K synergize with E2 to enhance transcription, p38 and PI3K with PKA but not with PKC, inhibit the degradation of E2-ER complexes. 4-OH-T-ER complexes are directed to the proteasome only when PKC is activated. Pure antiestrogen-ER complexes are rapidly targeted to the nuclear matrix, remain insoluble (**), and are rapidly eliminated. Their degradation is accelerated by PKC and inhibited by p38 and ERKs activities.
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The role of PKA in E2/ER degradation is not similar to that of PKC; PKA is involved in the activation of ER-mediated transcription because it enhances the association of ER with cyclin D1, a putative enhancer of ER-mediated transcription via the cAMP-dependent pathway (67). In that case, PKA at a low level of activation does not enhance either ER-mediated transcription or the proteasome-regulated ER destruction but helps to maintain ER expression (Fig. 11
). MG132 alone slightly inhibited the E2-induced estrogen response element-dependent indicator gene expression, whereas it has been described as a strong inhibitor of both ER (18)-, progesterone receptor (30)-, and glucocorticoid receptor-mediated transactivation (68); nevertheless, the MG132 treatment interferes with luciferase reporter assays as shown by the enhancement of glucocorticoid-induced activation of the mouse mammary tumor virus promoter linked to a chloramphenicol acetyltransferase reporter gene, while in the same cells, treatment with MG132 inhibited glucocorticoid-induced activation of mouse mammary tumor virus linked to a firefly luciferase or to a ß-galactosidase reporter gene (68).
Inhibition of MAPKs induces opposite effects on different ligand-bound receptor complexes: whereas blockage of the ERK pathway does not alter the receptor level when bound to E2, it inhibits the stabilization induced by 4-OH-T; on the contrary, inhibition of the p38 pathway accelerates the degradation of the receptor with any type of ligand bound although to different degrees. This may appear to be in contrast to the down-regulation of ER reported to occur after MAPK activation by EGF (29). This was observed in a ER-negative cells with overexpressed EGF and c-erbB2 receptors, contrary to the MCF-7 cells used in this work. Our data suggest that in ER-positive breast cancer cells, the level of the receptor is maintained by a low level of MAPK activation. Indeed, the basal phosphorylation of the p42 and p44 MAPKs in MCF-7 cells is low (Fig. 6
and Ref. 45).
Inhibition of the PI3K activity has no effect on E2- and a weak effect on 4-OH-T-bound receptor and no detectable effect on pure antagonist-receptor complexes. However, it could modify the activity of ubiquitin ligases, altering in turn the extent of ubiquitination of the receptor. In fact, the degradation of proteins (like the c-Mos, c-Fos, and c-Jun protooncogenes) can be initiated or inhibited from phosphorylation of consensus sites (see Ref. 69 for a review). Thus, different phosphorylated receptor sites and different degrees of phosphorylation triggered by ligand binding or by external signals, in addition to agonist-induced transcription or antagonist-repressed transcription, may initiate a slow or rapid proteasome-mediated ER destruction.
However, due to the pharmacological concentrations of ER antagonists used in this study, the fast rapid ER destruction induced by pure antagonists could implicate nongenomic effects through requirement of G protein-coupled receptors. Thus, breast cancer treatments using pharmacological concentrations of antiestrogens triggers a number of events and cross-talks mediated through different pathways that vary according to the ER ligand.
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MATERIALS AND METHODS
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Cell Culture and Drug Treatment
MCF-7, MELN (70, 71), and T47-D cells were grown in DMEM in the presence of 10% fetal calf serum. At least 4 d before cell exposure to steroids or other drugs, the culture medium was replaced by phenol red-free DMEM containing 10% stripped serum (charcoal Norit A 1%, Dextran 0.1%, 30 min at room temperature). The ERK p4244 inhibitors PD 98059 and UO126 were from Calbiochem (La Jolla, CA) and Promega Corp. (Madison, WI), respectively. p38 MAPK inhibitors SB 203190 and SB 203580 were from Calbiochem, and the PI3 kinase inhibitor LY 294002 was obtained from Sigma (St. Louis, MO). RU 58668 was a gift from P. Van de Velde (Roussel-Uclaf, Romainville, France) and ICI 182780 was provided by Zeneca (Maclesfield, UK). 4-OH-T was obtained from Besins Iscovesco (Paris, France). The proteasome inhibitors lactacystin, MG132, and epoxomycin, as well as the PKC activator mezerein, the PKC inhibitor iso-H7, and the PKA inhibitor H89, were from Calbiochem. All other products were reagent grade.
Preparation of Cellular Extracts
Cells (35.106) were harvested and pelleted by centrifugation in cold PBS. To obtain low-salt cellular extracts (LSE), 120 µl of cell lysate buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton, 1.5 mM MgCl2, 1 mM EGTA, 20 mM NaP, 10 mM Na pyrophosphate, 200 µM Na3VO4], plus protease inhibitors (Complete reagent, Roche Diagnostics, Indianapolis, IN) were added. Cells were allowed to stand for 1 h on ice before being centrifuged 10 min at 13,000 rpm at 2 C. The supernatant was boiled for 5 min in Laemmli sample buffer to obtain 1% sodium dodecyl sulfate (SDS) and 1% ß-mercaptoethanol.
HSEs were prepared by resuspending the pellet remaining after LSE in 100 µl high salt-containing buffer [50 mM HEPES, (pH 7.5), 10% glycerol, 1 mM dithiothreitol, 400 mM NaCl, 20 mM Na2WO4, containing protease inhibitors] and allowed to stand for 30 min on ice. After centrifugation at 30,000 rpm for 15 min at 2 C, the supernatant was removed, and boiled for 5 min in Laemmli sample buffer as above.
TCEs were obtained from pelleted cells by resuspension in 100 µl lysis buffer for 30 min at 2 C, and boiling for 5 min in Laemmli sample buffer.
Protein concentration was determined by the Bio-Rad Assay (Bio-Rad Laboratories, Hercules, CA). For TCEs, BSA standards were complemented with SDS to the same concentration as that contained in the samples.
SDS-PAGE and Western Blot
SDS-PAGE was performed in 8% polyacrylamide gels unless otherwise specified. After migration, the proteins were electrotransferred onto a polyvinylidene difluoride (Immobilon-P, Millipore Corp., Bedford, MA) membrane. To prevent nonspecific interactions, the membranes were blocked for at least 2 h at 37 C in 10% dried nonfat milk in PBS containing 0.1% Tween 20. Three different mouse monoclonal anti-ER antibodies were used at 1 µg/ml in PBS-Tween 200.4% milk overnight at 4 C: H222 (ER epitope, amino acids 463526) gift from Dr. G. Greene, C314 (ER epitope, amino acids 120170) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and D12 (ER epitope, amino acids 2185) (Santa Cruz Biotechnology, Inc.). Phospho-p44, -p42 (Thr 202Tyr 204), phospho-p38 (Thr 180Tyr 182), and total ERK1, ERK2, and p38 MAPKs were detected with rabbit antibodies (Cell Signaling, Beverly, MA) all diluted at 1 µg/ml. For ER detection, the antigen-antibody complexes were amplified with the binding of a second biotinylated antimouse antibody followed by revelation with the avidin-peroxidase system of the Vectastain ABC Kit (Vector laboratories, Inc., Burlingame, CA) and enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL). For the detection of total and phospho-MAPK proteins, a second horseradish-labeled antirabbit antibody was used. The signals from the immunobands corresponding to ER as well as to activated MAPK were analyzed utilizing the Bio-Profil V99 BIO 1D software from Wilbert Lourmat Fisher BioScientific, Marne-la-vallée, France) and quantified with reference to actin and total MAPK, respectively.
Northern Blot Experiments
Total mRNA was extracted from 5 x 106 cells using the Trizol (Life Technologies, Gaithersburg, MD) reagent according to the manufacturers instructions. RNA (25 µg) was electrophoresed on a 1% agarose-2.2 M formaldehyde gel and then transferred onto a nylon membrane (Hybond N, Amersham Pharmacia Biotech). RNAs were cross-linked using UV irradiation and hybridized for at least 18 h with a 32P-labeled probe from ER and the ubiquitary human acidic ribosomal phosphoprotein mRNA 36B4. Blots were washed for 30 min at 65 C, with 2x standard saline citrate (150 mM NaCl, and 15 mM Na3C6H3, pH 7.0) containing 1% SDS and then with 0.1x standard saline citrate-0.1% SDS before autoradiography at -80 C with x-ray film (X-OMAT, Eastman Kodak, Rochester, NY). Quantification of RNA levels was performed as detailed above, with 36B4 mRNA serving as internal standard.
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ACKNOWLEDGMENTS
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We thank J. Mester for criticism and reading of the manuscript; S. Maillard for art work; G. Greene for the Gift of H222; P. Van de Velde and J. Brémaud (ROUSSEL-UCLAF, Romainville, France) for the gift of RU 58668; M. Pons for the gift of MELN cells; Astra-ZENECA for the gift of ICI 182,780; and H. Richard-Foy for the gift of 36B4.
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FOOTNOTES
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This work was supported by the Association pour la Recherche sur le Cancer (Grants 9863 and 5970 to J.-M.R.) and the Ligue Nationale contre le Cancer (Comités du Cher et des Yvelines) grants to J.-M.R. V.M. was a postdoctoral fellow from the Ernst Schering Fundation and the Ligue Nationale contre le Cancer (Comité de lIndre).
Abbreviations: CHX, Cycloheximide; E2, estradiol; EGF, epidermal growth factor; ER, estrogen receptor; 4-OH-T: 4-hydroxy-tamoxifen; hsp, heat shock protein; HSE, high-salt extract; ICI, ICI 182,780 or 7
-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)-nonyl]estra-1,3,5(10)-triene-3,17ß-diol; LSE, low-salt extract; MELN, MCF-7 cells stably transfected with an estrogen response element-ß globin-LUC construct; PD, PD98059; PI3K, phosphatidylinositol 3-kinase; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; RU, RU 58668 or 11ß-[4-[5-[(4,4,5,5,5-pentafluoropentyl)sulfonyl]pentyloxy]phenyl]-estra-1,3,5,(10)triene-3,17ß-diol; SB190, SB202190; SB580, SB203580; SDS, sodium dodecyl sulfate; TCE, total cell extract.
Received for publication August 2, 2002.
Accepted for publication June 27, 2003.
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