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Molecular Endocrinology, doi:10.1210/me.2005-0111
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Molecular Endocrinology 19 (12): 2901-2914
Copyright © 2005 by The Endocrine Society

CHIP (Carboxyl Terminus of Hsc70-Interacting Protein) Promotes Basal and Geldanamycin-Induced Degradation of Estrogen Receptor-{alpha}

Meiyun Fan, Annie Park and Kenneth P. Nephew

Medical Sciences (M.F., A.P., K.P.N.), Indiana University School of Medicine, Bloomington, Indiana 47405; and Indiana University Cancer Center (K.P.N.) and Department of Cellular and Integrative Physiology (K.P.N.), Indiana University School of Medicine, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Kenneth P. Nephew, Ph.D., Medical Sciences, Indiana University School of Medicine, 302 Jordan Hall, 1001 East 3rd Street, Bloomington, Indiana 47405-4401. E-mail: knephew{at}indiana.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In estrogen target cells, estrogen receptor-{alpha} (ER{alpha}) protein levels are strictly regulated. Although receptor turnover is a continuous process, dynamic fluctuations in receptor levels, mediated primarily by the ubiquitin-proteasome pathway, occur in response to changing cellular conditions. In the absence of ligand, ER{alpha} is sequestered within a stable chaperone protein complex consisting of heat shock protein 90 (Hsp90) and cochaperones. However, the molecular mechanism(s) regulating ER{alpha} stability and turnover remain undefined. One potential mechanism involves CHIP, the carboxyl terminus of Hsc70-interacting protein, previously shown to target Hsp90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, a role for CHIP in ER{alpha} protein degradation was investigated. In ER-negative HeLa cells transfected with ER{alpha} and CHIP, ER{alpha} proteasomal degradation increased, whereas ER{alpha}-mediated gene transcription decreased. In contrast, CHIP depletion by small interference RNA resulted in increased ER{alpha} accumulation and reporter gene transactivation. Transfection of mutant CHIP constructs demonstrated that both the U-box (containing ubiquitin ligase activity) and the tetratricopeptide repeat (TPR, essential for chaperone binding) domains within CHIP are required for CHIP-mediated ER{alpha} down-regulation. In addition, coimmunoprecipitation assays demonstrated that ER{alpha} and CHIP associate through the CHIP TPR domain. In ER{alpha}-positive breast cancer MCF7 cells, CHIP overexpression resulted in decreased levels of endogenous ER{alpha} protein and attenuation of ER{alpha}-mediated gene expression. Furthermore, the ER{alpha}-CHIP interaction was stimulated by the Hsp90 inhibitor geldanamycin (GA), resulting in enhanced ER{alpha} degradation; this GA effect was further augmented by CHIP overexpression but was abolished by CHIP depletion. Finally, ER{alpha} dissociation from CHIP by various ER{alpha} ligands, including 17ß-estradiol, 4-hydroxytamoxifen, and ICI 182,780, interrupted CHIP-mediated ER{alpha} degradation. These results demonstrate a role for CHIP in both basal and GA-induced ER{alpha} degradation. Furthermore, based on our observations that CHIP promotes ER{alpha} degradation and attenuates receptor-mediated gene transcription, we suggest that CHIP, by modulating ER{alpha} stability, contributes to the regulation of functional receptor levels, and thus hormone responsiveness, in estrogen target cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PRIMARY MEDIATORS of 17ß-estradiol (E2) action, the major female sex steroid hormone, are the estrogen receptors ER{alpha} and ERß. These receptors function as ligand-activated transcription factors, regulating expression of genes coordinating most physiological and many pathophysiological processes in estrogen target tissues (1). Tissue sensitivity, and the overall magnitude of response to E2 and other estrogens, is strongly influenced by a combination of factors, including cellular levels of ER{alpha} and its various coactivators and corepressors (2, 3).

To strictly control cellular responses, the cellular synthesis and turnover of the ER{alpha} protein dynamically fluctuates with changing cellular environments (4). For example, in the absence of ligand, ER{alpha} is a short-lived protein (half-life of 4–5 h) and undergoes constant degradation (5). In the presence of ligand, by contrast, the turnover rate of ER{alpha} can be increased or decreased, depending upon the ligand, thus modulating receptor protein levels. Turnover-inducing factors and conditions include the cognate ligand E2, pure antiestrogens [ICI 164,384, ICI 182,780 (ICI), RU 58,668], heat shock protein (Hsp) 90 inhibitors [geldanamycin (GA) and radicicol], ATP depletion (oligomycin and hypoxia) and aryl hydrocarbon agonists; these all induce degradation and rapid down-regulation of ER{alpha} levels (6, 7, 8, 9, 10, 11, 12). In contrast, the partial agonist/antagonist 4-hydoxytamoxifen (OHT), thyroid hormone, and protein kinase K activators (forskolin, 8-bromo-cAMP) all block receptor degradation, subsequently increasing ER{alpha} protein levels (13, 14, 15).

Although both basal and ligand-induced ER{alpha} degradation are mediated by the ubiquitin-proteasome pathway (12, 13, 16, 17, 18, 19, 20, 21), regulation of this pathway, at the molecular level, remains unclear. Emerging evidence suggests that multiple ER{alpha} degradation pathways exist, and the engagement of one pathway over another depends on the nature of the stimulus (19, 21, 22, 23). For example, E2-induced receptor degradation is coupled with transcription and requires new protein synthesis (17, 19, 22, 24); conversely, neither ER{alpha} transcriptional activity nor new protein synthesis are needed for ICI-induced ER{alpha} degradation (19, 20, 22). In addition, various stimuli induce distinct changes in the conformation and cellular compartmentalization of ER{alpha} (22, 25, 26, 27), and these may be associated with receptor ubiquitination.

Like other members of the steroid receptor superfamily, unliganded ER{alpha}, by associating with various Hsp90-based chaperone complexes, is maintained in a ligand-binding competent conformation (28). Although these associations do not influence ER{alpha} ligand-binding affinity, Hsp90 chaperone complexes appear to regulate ER{alpha} stability because Hsp90 disruption induces rapid ER{alpha} degradation through the ubiquitin proteasome pathway (9, 28, 29). For regulation of such complexes, recent studies have identified the carboxyl terminus of Hsc70-interacting protein (CHIP) as a ubiquitin ligase that directs chaperone substrates for ubiquitination and proteasomal degradation (30, 31). CHIP interacts with Hsp/Hsc70 and Hsp90 through an amino-terminal TPR domain and catalyzes ubiquitin conjugation through a carboxyl-terminal U-box domain (30). As recent observations demonstrate that CHIP targets a number of Hsp70/90-associated proteins for ubiquitination and degradation, including the glucocorticoid receptor, androgen receptor, Smad1/4, and ErbB2 (30, 31, 32, 33), we investigated a regulatory role for CHIP in ER{alpha} stability. Our results demonstrate that CHIP, likely through a chaperone intermediate, associates with ER{alpha} and consequently facilitates both basal and GA-induced receptor degradation in human cancer cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CHIP Overexpression Decreases and CHIP Knockdown Increases ER{alpha} Protein Levels
To investigate the effect of CHIP overexpression on steady-state levels of ER{alpha}, ER-negative HeLa cells were cotransfected with constructs expressing CHIP (pcDNA-His6-CHIP) and ER{alpha} (pSG5-ER{alpha}). ER{alpha} protein levels were subsequently determined by immunoblot analysis. Overexpression of CHIP decreased ER{alpha} protein levels in a dose-dependent manner (Fig. 1AGo). To control for transfection efficiency, the green fluorescent protein (GFP) was also included in transfection. No effect of CHIP on GFP expression level was observed (Fig. 1AGo), demonstrating that CHIP-induced down-regulation of ER{alpha} was specific. Next, we examined whether CHIP-induced ER{alpha} down-regulation could be inhibited by CHIP-specific small interference RNA (siRNA). Compared with cells transfected with CHIP only, cotransfection of pBS/U6/CHIPi, a CHIP-siRNA expression construct (33), dramatically decreased the level of exogenous CHIP (Fig. 1BGo, upper panel). However, pBS/U6/CHIPi had no effect on GFP level, confirming that the CHIP-siRNA specifically blocks CHIP expression (Fig. 1BGo). The effect of CHIP-siRNA on CHIP-induced ER{alpha} down-regulation was then examined. As shown in Fig. 1BGo (lower panel), cotransfection of CHIP-siRNA, in a dose-dependent fashion, attenuated ER{alpha} down-regulation induced by exogenous CHIP. Collectively, these results demonstrate that CHIP overexpression can down-regulate ER{alpha} protein level in HeLa cells.



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  		Fig. 1. CHIP Overexpression Decreases and CHIP Knockdown Increases ER{alpha} Protein Levels

A, Overexpression of CHIP down-regulates ER{alpha} protein levels. HeLa cells were transfected with 250 ng pSG5-ER{alpha}, 100 ng CMV-GFP, and various amounts (0, 50, 100, and 250 ng) of pcDNA-his6-CHIP. B, Expression of CHIP-siRNA attenuates CHIP-induced ER{alpha} down-regulation. In the upper panel, HeLa cells were transfected with 250 ng pcDNA-his6-CHIP, with or without 250 ng pBS/U6/CHIPi, as indicated. In the lower panel, HeLa cells were transfected with 250 ng pSG5-ER{alpha}, 250 ng pcDNA-his6-CHIP, and various doses (150, 300, 500, and 1000 ng) of pBS/U6/CHIPi. C, Knockdown of endogenous CHIP increases ER{alpha} level. HeLa cells were transfected with 250 ng pSG5-ER{alpha} and either 250 ng pcDNA-his6-CHIP or 250 ng pBS/U6/CHIPi, as indicated. For all experiments, 3 x 105 HeLa cells were plated in 60-mm dishes, cultured in hormone-free medium for 3 d, and then transfected with LipofectAMINE Plus Reagent. Cell lysates were prepared 24 h after transfection. Protein levels were determined by immunoblotting with specific antibodies. Exogenous His6-CHIP and endogenous CHIP were detected by anti-His6 and anti-CHIP, respectively. GFP and GAPDH were used as transfection control and SDS-PAGE loading controls, respectively. Representative results of two independent experiments, each performed in duplicate, are shown.

 
To examine a role for endogenous CHIP in regulation of ER{alpha} protein levels, HeLa cells, which are known to express CHIP (30), were cotransfected with pBS/U6/CHIPi and ER{alpha}. Expression of CHIP-siRNA decreased the level of endogenous CHIP by 60%, and correspondingly increased ER{alpha} protein level by 1.6-fold (Fig. 1CGo), indicating that endogenous CHIP plays a role in controlling ER{alpha} level in HeLa cells.

CHIP Down-Regulates ER{alpha} Levels through the Ubiquitin Proteasome Pathway
To determine whether proteasome activity is required for CHIP-induced ER{alpha} down-regulation, HeLa cells were cotransfected with pcDNA-His6-CHIP and pSG5-ER{alpha}, treated with the protease inhibitor MG132, and subjected to immunoblotting. As shown in Fig. 2AGo, a 6-h treatment with MG132 completely blocked CHIP-induced down-regulation of ER{alpha}. To examine whether polyubiquitination is required for CHIP-induced ER{alpha} degradation, a mutant ubiquitin, UbK0, with all lysines replaced by arginines (34), was used. Previously, we showed that the UbK0 protein could efficiently block E2-induced ER{alpha} degradation (35). Expression of UbK0, but not wild-type ubiquitin, restored ER{alpha} protein levels (Fig. 2BGo), demonstrating that CHIP stimulates ER{alpha} degradation through the ubiquitin and proteasome pathway.



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  		Fig. 2. The Proteasome Inhibitor MG132, Partial ER{alpha}-Antagonist OHT, and Ubiquitin Mutant UbK0, All Block CHIP-Induced ER{alpha} Degradation

A, The proteasome inhibitor MG132 and the partial ER{alpha} antagonist OHT block CHIP-induced ER{alpha} down-regulation. HeLa cells were transfected with 250 ng pSG5-ER{alpha} and 100 ng CMV-GFP, along with 250 ng pcDNA (vector control) or pcDNA-His6-CHIP, then treated with DMSO (vehicle), 10 µM MG312 or 1 µM OHT for 6 h before immunoblot analysis. Protein levels of ER{alpha}, CHIP and GFP were determined by immunoblotting with anti-ER{alpha}, anti-His6, and anti-GFP, respectively. GFP was used as a control for transfection efficiency and SDS-PAGE loading. B, Expression of the ubiquitin mutant UbK0 blocks CHIP-induced ER{alpha} down-regulation. HeLa cells were transfected with 250 ng pSG5-ER{alpha}, with or without 250 ng pcDNA-His6-CHIP, pcDNA-Ub, or pCS2-UbK0, as indicated. ER{alpha} protein levels were determined by immunoblotting with anti-ER{alpha}. GAPDH was used as a loading control for SDS-PAGE. For all experiments, 3 x 105 HeLa cells were plated in 60-mm dishes, cultured in hormone-free medium for 3 d, and then transfected with LipofectAMINE Plus Reagent. Cell lysates were prepared 24 h after transfection. The band density of exposed films was evaluated with ImageJ software. Relative ER{alpha} levels were presented as the mean ± SE of three independent experiments, each performed in duplicate.

 
CHIP Targets Mature ER{alpha} for Degradation
It has been proposed that CHIP functions as a general ubiquitin ligase, responsible for ubiquitinating unfolded or misfolded proteins in a chaperone-dependent process (31). To examine whether ER{alpha} down-regulation by CHIP was due to the selective ubiquitination of unfolded or misfolded receptor protein, we examined the effect of OHT, a selective ER modulator, on CHIP-mediated ER{alpha} degradation. It has been shown that OHT can dissociate ER{alpha} from its chaperone complex and protect the receptor from both basal turnover and degradation induced by Hsp90-binding agents (8, 13, 21). We reasoned that if CHIP selectively targets immature or misfolded ER{alpha} (with no functional OHT-binding pocket), then, in the presence of CHIP, OHT treatment should not restore ER{alpha} levels. On the other hand, if CHIP targets mature ER{alpha}, OHT treatment should rescue the receptor protein from CHIP-induced degradation. HeLa cells were thus cotransfected with pcDNA-His6-CHIP and pSG5-ER{alpha} and treated with OHT for 6 h before lysate preparation. OHT treatment completely abolished CHIP-induced ER{alpha} down-regulation (Fig. 2AGo) but had no effect on protein levels of CHIP and GFP excluding the possibility that OHT treatment affects protein degradation in general. These results demonstrate that CHIP induces degradation of correctly folded, ligand-binding competent ER{alpha}.

Both the TPR and U-Box Domains Are Essential for CHIP-Induced ER{alpha} Down-Regulation
To examine whether the ubiquitin ligase activity and chaperone interaction domain are required for CHIP-induced ER{alpha} degradation, two mutant CHIP constructs were used: 1) CHIP(K30A), a TPR domain mutant unable to interact with Hsp/Hsc70 or Hsp90; and 2) CHIP(H260Q), a U-box domain mutant unable to catalyze protein ubiquitin conjugation (36). In contrast to wild-type CHIP, neither CHIP(K30A) nor CHIP(H260Q) overexpression decreased ER{alpha} protein levels (Fig. 3AGo). These results establish that both the chaperone interaction and ubiquitin ligase activity of CHIP are required for CHIP-targeted degradation of ER{alpha} protein.



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  		Fig. 3. Both the TPR and U-Box Domains Are Required for CHIP-Induced ER{alpha} Down-Regulation

A, Both the TRP and U-box domains are required for CHIP to down-regulate ER{alpha}. HeLa cells were transfected with 250 ng pSG5-ER{alpha}, 100 ng CMV-GFP, along with 250 pcDNA (control) or various CHIP constructs, as indicated. ER{alpha} and GFP protein levels were determined by immunoblotting with anti-ER{alpha} and anti-GFP, respectively. GFP was used as control for transfection efficiency and SDS-PAGE loading. B, The TRP domain is required for CHIP-ER{alpha} interaction. HeLa cells were transfected with 250 ng pSG5-ER{alpha}, along with 250 ng pcDNA-His6-CHIP or pcDNA-His6-CHIP(K30A). ER{alpha} protein in cell lysates was precipitated with anti-ER{alpha}. The presence of CHIP in the precipitated ER{alpha} complex was determined by immunoblotting with anti-His6. The same blot was reprobed with anti-ER{alpha} to assess the amount of ER{alpha} in the precipitated immunocomplex. The expression levels of CHIP or CHIP(K30A) in whole cell lysates was determined by immunoblotting with anti-His6 (lower panel). For all experiments, HeLa cells were plated in 60-mm dishes at a density of 3 x 105 cells/dish, cultured in hormone-free medium for 3 d, and then transfected with LipofectAMINE Plus Reagent. Cell lysates were prepared 24 h after transfection. Representative results of two independent experiments, each performed in duplicate, are shown. IP, Immunoprecipitation.

 
The TPR Domain of CHIP Is Required for the CHIP-ER{alpha} Interaction
As CHIP appears to be linked to ER{alpha} degradation, we investigated whether CHIP associates with the receptor. HeLa cells were cotransfected with ER{alpha} and CHIP, and coimmunoprecipitation analysis performed using an ER{alpha}-specific antibody. The results revealed a complex containing both CHIP and ER{alpha} (Fig. 3BGo). Because CHIP(K30A) exhibited no effect on ER{alpha} turnover (Fig. 3AGo), we examined whether the TPR domain is required for the CHIP-ER{alpha} interaction. In HeLa cells cotransfected with ER{alpha} and CHIP(K30A), the CHIP mutant was not detected in the precipitated ER{alpha} complex (Fig. 3BGo), demonstrating a requirement for the TPR domain in the CHIP-ER{alpha} interaction. Because it is known that CHIP interacts with Hsp90 or Hsc/Hsp70 through the TPR domain (30), our results suggest that a chaperone intermediate is involved in CHIP-induced ER{alpha} degradation.

CHIP Interacts with Endogenous ER{alpha}, in Breast Cancer Cells, to Induce Receptor Ubiquitination and Degradation
Having demonstrated a role for CHIP (possibly in association with chaperones) in degradation of exogenous ER{alpha} in HeLa cells, it was of interest to examine the effect of CHIP on stability and function of endogenous ER{alpha} in breast cancer cells. In human breast cancer MCF7 cells, overexpression of CHIP resulted in a dose-dependent ER{alpha} down-regulation (Fig. 4AGo). Coimmunoprecipitation analysis of MCF7 cells transfected with pcDNA-His6-CHIP revealed both CHIP and ER{alpha} in the immunocomplexes precipitated by either an ER{alpha}-specific or anti-His6 antibody (Fig. 4BGo), suggesting that CHIP associates with endogenous ER{alpha}. In addition, both Hsc70 and Hsp90 were detected in the precipitated ER{alpha} complex (Fig. 4BGo). These results indicate that CHIP can associate with endogenous ER{alpha}-Hsp90/Hsc70 complexes to down-regulate ER{alpha} level in breast cancer cells.



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  		Fig. 4. CHIP Interacts with Endogenous ER{alpha} and Induces ER{alpha} Ubiquitination and Degradation in Breast Cancer MCF7 Cells

A, Overexpression of CHIP down-regulates endogenous ER{alpha} levels in MCF7 cells. MCF7 cells were plated in 100-mm dishes at a density of 1 x 106 cells/dish, cultured in hormone-free medium for 3 d, and transfected with various amounts (0, 5, or 10 µg) of pcDNA-His6-CHIP using FuGENE. Twenty-four hours after transfection, whole cell lysates were prepared, and protein levels of ER{alpha} and CHIP determined by immunoblotting with anti-ER{alpha} and anti-His6, respectively. GAPDH was used as an SDS-PAGE loading control. B, CHIP associates with ER{alpha}-Hsp complex in MCF7 cells. MCF7 cells were transfected as in panel A and subjected to coimmunoprecipitation analysis. ER{alpha} and CHIP were precipitated with anti-ER{alpha} and anti-His6, respectively. The presence of CHIP, Hsc70, Hsp90, or ER{alpha} in the precipitated complexes was determined by immunoblotting with anti-His6, anti-Hsc70, anti-Hsp90, or anti-ER{alpha}, respectively. C, Expression of CHIP enhances endogenous ER{alpha} polyubiquitination in MCF7 cells. MCF7 cells were plated in 60-mm dishes at a density of 5 x 105 cells/dish cultured in hormone-free medium for 3 d, and transfected with 250 ng pcDNA-HA-Ub and 250 ng pcDNA or pcDNA-His6-CHIP. Twenty-four hours after transfection, whole cell lysates were prepared and ER{alpha} protein was precipitated with anti-ER{alpha}. The presence of ubiquitin-conjugated ER{alpha} in the immunocomplex was detected by immunoblotting with anti-HA (upper panel). The same membrane was reprobed with anti-ER{alpha} to assess the amount of precipitated ER{alpha} (middle panel). Whole cell lysates were separated by SDS-PAGE and probed with HA antibody to determine the amount of total ubiquitinated proteins (lower panel). D, CHIP increases ER{alpha} ubiquitination in MCF7 cells expressing UbK0. MCF7 cells were plated as in panel C and transfected with 500 ng pcDNA-HA-Ub or 500 ng pCS2-UbK0, along with 250 ng pcDNA or pcDNA-His6-CHIP, as indicated. Twenty-four hours after transfection, whole cell lysates were prepared and ER{alpha} protein was detected by immunoblotting with anti-ER{alpha}. E, Knockdown of endogenous CHIP increases ER{alpha} level. MCF7 cells were plated as in panel C and transfected with 2 µg vector or pBS/U6/CHIPi using FuGENE. Forty-eight hours after transfection, whole cell lysates were prepared, and protein levels of CHIP and ER{alpha} were determined by immunoblotting with anti-CHIP and anti-ER{alpha}, respectively. GAPDH was used as an SDS-PAGE loading control. For all experiments, representative results of two independent experiments, each performed in duplicate, are shown. IP, Immunoprecipitation.

 
To determine whether CHIP promotes polyubiquitination of endogenous ER{alpha}, we examined the ubiquitination status of ER{alpha} in MCF7 cells transfected with hemagglutinin-tagged ubiquitin (HA-Ub), plus a vector control (pcDNA) or a CHIP-expressing construct. To block proteasomal degradation of polyubiquitinated proteins, transfected cells were treated with MG132 for 6 h before lysate preparation. An ER{alpha}-specific antibody was then used for immunoprecipitation, and the presence of ubiquitinated ER{alpha} in the immunocomplex was detected by immunoblotting with an HA antibody. To assess overall levels of protein ubiquitination, whole cell lysates were immunoblotted using an HA antibody. The polyubiquitinated ER{alpha} exhibited a typical high-molecular-weight smear on the blot membrane, and overexpression of CHIP markedly increased smear intensity, suggesting elevated receptor polyubiquitination (Fig. 4CGo, upper panel). In contrast, CHIP had no effect on overall protein ubiquitination (Fig. 4CGo, lower panel).

A possible limitation of in vivo ubiquitination assays is that the immunocomplex may contain multiple polyubquitinated species, not just the target protein of interest. To corroborate the observation that CHIP promotes ER{alpha} ubiquitination, we examined the effect of CHIP on ER{alpha}-ubiquitination in MCF7 cells transfected with UbK0. This mutant ubiquitin competes with endogenous ubiquitin and terminates ubiquitin chains, resulting in the accumulation of oligoubiquitin-ER{alpha} conjugates, which upon immunoblotting with ER{alpha} antibody can be detected as mobility-shifted bands. In MCF7 cells transfected with wild-type ubiquitin, overexpression of CHIP had no effect on the intensity of ER{alpha}-ubiquitination (Fig. 4DGo, left panel), presumably due to the rapid degradation of polyubiquitinated ER{alpha}. However, in cells transfected with UbK0, overexpression of CHIP remarkably increased the amount of oligoubiquitinated ER{alpha} (Fig. 4DGo, right panel), confirming that overexpression of CHIP promotes ER{alpha} ubiquitination. Together, these results suggest that CHIP, by facilitating receptor ubiquitination, targets endogenous ER{alpha} for proteasome-mediated degradation.

Knockdown of Endogenous CHIP by siRNA Increases ER{alpha} Level in MCF7 Cells
The above experiments showed that overexpression of CHIP promotes ER{alpha} polyubiquitination and degradation in breast cancer cells. Conversely, we wanted to examine whether knockdown of endogenous CHIP protein by CHIP-siRNA could increase endogenous ER{alpha} level. Transfection of MCF7 cells with pBS/U6/CHIPi decreased the level of endogenous CHIP by 60% (Fig. 4EGo, upper panel) and increased the level of ER{alpha} level by 1.5-fold (Fig. 4EGo, lower panel), indicating that endogenous CHIP plays a role in basal turnover of ER{alpha} in breast cancer cells.

CHIP Down-Regulates ER{alpha}-Mediated Gene Expression
Having established a role for CHIP in ER{alpha} ubiquitination and receptor turnover, we next examined the effect of CHIP on ER{alpha}-mediated gene transactivation. HeLa cells were transiently transfected with ER{alpha} and an estrogen-responsive reporter (ERE-pS2-Luc), plus various CHIP (CHIP, H260Q, K30A, CHIP-siRNA) or control (pcDNA) constructs. Twenty-four hours after transfection, cells were treated for 6 h with vehicle [dimethylsulfoxide (DMSO)] or E2 (10 nM) and luciferase activity then measured. In a parallel experiment, a constitutive reporter [simian virus 40 promoter-firefly luciferase (SV40-Luc)] was used to monitor transcription efficiency, as well as any general effects of the various CHIP constructs might have on luciferase expression. The ERE-pS2-Luc activities were then normalized to the corresponding SV40-Luc activities. Expression of wild-type CHIP decreased (P < 0.05) E2-induced ERE-pS2-Luc expression, whereas the CHIP mutants had no effect on ER{alpha}-mediated gene transactivation (Fig. 5AGo). Conversely, depletion of endogenous CHIP by siRNA increased both basal and E2-induced ERE-pS2-Luc expression (P < 0.05, Fig. 5BGo). Similarly, in MCF7 cells, overexpression of CHIP, but not U-box or TPR mutant, attenuated ER{alpha}-mediated reporter gene expression (Fig. 6AGo), whereas knockdown of endogenous CHIP by siRNA augmented ER{alpha}-mediated reporter gene expression (Fig. 6BGo). To examine the effect of knocking down CHIP on the expression of an endogenous ER{alpha} target gene, MCF7 cells were transfected with CHIP-siRNA, and pS2 mRNA levels were examined. As shown in Fig. 6CGo, both basal and E2-induced expression of pS2 mRNA were significantly increased. Together, these results demonstrate that CHIP coordinately regulates ER{alpha} protein levels and ER{alpha}-mediated gene transactivation.



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  		Fig. 5. CHIP Down-Regulates ER{alpha}-Mediated Reporter Gene Expression in HeLa Cells

HeLa cells were plated in 12-well dishes at a density of 1 x 105/well, grown in hormone-free medium for 3 d, and transfected with 10 ng pSG5-ER{alpha}, 250 ng ERE-pS2-Luc, 250 ng various CHIP constructs (A) or pBS/U6/CHIPi (B). Twenty-four hours after transfection, cells were treated for 6 h with DMSO or 10 nM E2 and then assayed for luciferase activity. The ERE-pS2-Luc activity was normalized to SV40-Luc activity, which was determined in a parallel experiment where ERE-pS2-Luc was replaced with SV40-Luc. The results are expressed as means ± SE from three independent experiments, with each performed in quadruplicate. *, P < 0.05 (Student’s t test, vs. pcDNA treated with E2).

 


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  		Fig. 6. CHIP Down-Regulates ER{alpha}-Mediated Gene Expression in MCF7 Cells

A, Overexpression of CHIP Inhibits ER{alpha}-Mediated Reporter Gene Expression. MCF7 cells were plated in 12-well dishes at a density of 1 x 105/well, grown in hormone-free medium for 3 d, and transfected with 250 ng ERE-pS2-Luc, along with 250 ng various CHIP constructs. Twenty-four hours after transfection, cells were treated with DMSO or 10 nM E2 for 6 h and then assayed for luciferase. The ERE-pS2-Luc activity was normalized to SV40-Luc activity (determined in a parallel experiment where ERE-pS2-Luc was replaced with SV40-Luc). Results are expressed as the mean ± SE from three independent experiments, each performed in quadruplicate. *, P < 0.05 (Student’s t test, vs. pcDNA treated with E2). B, Knockdown of CHIP by siRNA increases ER{alpha}-mediated reporter gene expression. MCF7 cells were plated as in panel A and transfected with 250 ng ERE-pS2-Luc and various amounts (0, 250, and 500 ng) of pBS/U6/CHIPi. Twenty-four hours after transfection, cells were treated and subjected to luciferase analysis, as in panel A. C, Knockdown of CHIP by siRNA increases expression of pS2 mRNA. MCF7 cells were plated in 100 mm dishes at a density of 1 x 106/dish, grown in hormone-free medium for 3 d, and transfected with 5 µg vector or pBS/U6/CHIPi using FuGENE. Forty-eight hours after transfection, cells were treated for 6 h with DMSO or 10 nM E2. The mRNA level of pS2 was determined by real-time quantitative PCR. The relative pS2 mRNA levels were normalized with ß-actin mRNA and expressed as mean ± SE from three independent experiments, each performed in duplicate. *, P < 0.05 (Student’s t test, CHIP-siRNA vs. pcDNA).

 
GA Induces ER{alpha} Degradation through a CHIP-Dependent Mechanism
The Hsp90 inhibitor, GA, binds to the amino-terminal ATP/ADP-binding domain of Hsp90, locking this chaperone protein in its ADP-bound conformation (9, 29, 37). CHIP has been reported to play a role in GA-induced degradation of ErbB2, a Hsp90 client protein (36, 38), and recent studies have shown that GA stimulates ER{alpha} degradation through the ubiquitin-proteasome pathway (9, 29, 37). Whether CHIP plays a role in GA-induced ER{alpha} degradation has not been previously investigated. Thus, we examined the effects of CHIP overexpression and depletion on GA-induced ER{alpha} degradation. In HeLa cells transfected with ER{alpha}, GA treatment resulted in a time-dependent ER{alpha} down-regulation (Fig. 7AGo); this effect was enhanced by CHIP overexpression (Fig. 7AGo). Conversely, CHIP depletion by siRNA completely abolished GA-induced ER{alpha} down-regulation (Fig. 7AGo).



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  		Fig. 7. Disruption of Hsp90 Function Induces ER{alpha} Degradation through a CHIP-Dependent Mechanism in HeLa Cells

A, CHIP overexpression augments and CHIP depletion by siRNA blocks GA-induced ER{alpha} degradation. HeLa cells were plated in 60-mm dishes at a density of 3 x 105 cells/dish, cultured in hormone-free medium for 3 d, and transfected with 250 ng pSG5-ER{alpha}, along with 250 ng pcDNA, pcDNA-His6-CHIP or pBS/U6/CHIPi by using LipofectAMINE Plus Reagent. Twenty-four hours after transfection, the cells were treated with 1 µM GA for 0, 0.5, 1, and 3 h. Cell lysates were immunoblotted with anti-ER{alpha}. GAPDH was used as an SDS-PAGE loading control. The band density of exposed films was evaluated with ImageJ software. Relative ER{alpha} levels were presented as mean ± SE from three independent experiments. B, GA enhances CHIP-ER{alpha} interaction. HeLa cells were plated as in panel A and transfected with 250 ng pSG5-ER{alpha} and 250 ng pcDNA-His6-CHIP. Twenty-four hours after transfection, cells were untreated or treated with 1 µM GA or 10 mM MG132 for 1 h before lysate preparation. ER{alpha} protein was precipitated by anti-ER{alpha} and the presence of CHIP determined by immunoblotting with anti-His6. The same membrane was then reprobed with anti-ER{alpha} to assess the amount of precipitated ER{alpha} in the same complex. Representative results of three independent experiments, each performed in duplicate, are shown. IP, Immunoprecipitation.

 
To investigate the effect of GA on the CHIP-ER{alpha} interaction, HeLa cells were transfected with ER{alpha} and CHIP, and coimmunoprecipitation was performed with an ER{alpha}-specific antibody. The amount of CHIP in the precipitated ER{alpha} complex increased after a 1-h GA treatment (Fig. 7BGo), suggesting that GA promotes ER{alpha} degradation by recruiting CHIP to the chaperone-ER{alpha} complex. Because CHIP can associate with ubiquitinated proteins through its U-box domain (31), ER{alpha} ubiquitination may play a role in the GA-induced ER{alpha}-CHIP interaction. We thus examined the interaction between ER{alpha} and CHIP in the presence of the proteasome inhibitor MG132. We reasoned that if CHIP preferentially interacts with ubiquitinated ER{alpha}, then MG132, by enhancing the accumulation of polyubiquitinated ER{alpha}, would increase the ER{alpha}-CHIP interaction. However, MG132 treatment did not increase the amount of CHIP precipitated with the ER{alpha} complex (Fig. 7BGo), suggesting that the GA-induced ER{alpha}-CHIP interaction occurs before ER{alpha} polyubiquitination.

To establish a role for CHIP in GA-induced ER{alpha} degradation under physiologically relevant conditions, the consequence of knocking down endogenous CHIP by siRNA on ER{alpha} degradation was examined in MCF7 cells. GA induced rapid ER{alpha} down-regulation in MCF7 cells transfected with a pcDNA control plasmid (Fig. 8AGo), consistent with previous reports (9, 29). However, expression of CHIP-siRNA significantly impaired GA-induced ER{alpha} down-regulation (Fig. 8AGo). In addition, we performed a coimmunoprecipitation analysis to examine the effect of GA treatment on the association between endogenous CHIP and ER{alpha}. As shown in Fig. 8BGo, GA treatment increased the amount of CHIP that coimmunoprecipitated with ER{alpha}. Based on these results, we suggest that GA induces ER{alpha} degradation by enhancing the recruitment of CHIP to ER{alpha}-chaperone complexes.



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  		Fig. 8. CHIP Is Required for GA-Induced ER{alpha} Degradation in Breast Cancer MCF7 Cells

A, CHIP depletion by CHIP-siRNA eliminates GA-induced ER{alpha} degradation. MCF7 cells were plated in 60-mm dishes at a density of 3 x 105 cells/dish, cultured in hormone-free medium for 3 d, and transfected with 500 ng pcDNA (control) or pBS/U6/CHIPi. Twenty-four hours after transfection, cells were treated with 1 µM GA for 0, 1, 2.5, and 4 h, and subjected to immunoblotting with anti-ER{alpha}. ß-Tubulin was used as SDS-PAGE loading control. The band density of exposed films was evaluated with ImageJ software. Relative ER{alpha} levels are presented as mean ± SE from three independent experiments (lower panel). B, GA stimulates CHIP-ER{alpha} interaction. MCF7 cells were plated at 1 x 106 cells in 100-mm dishes, cultured in hormone-free medium for 3 d, and treated with 1 µM GA for 0, 1, and 3 h before lysate preparation. ER{alpha} protein was precipitated by anti-ER{alpha} and the presence of CHIP examined by immunoblotting with anti-CHIP. The same membrane was then reprobed with ER{alpha} antibody to assess the amount of precipitated ER{alpha} in the same complex. Representative results of two independent experiments, each performed in duplicate, are shown. IP, Immunoprecipitation.

 
Effects of Ligand Binding on GA-Induced ER{alpha} Degradation
Ligand binding results in disassembly of the ER{alpha}-Hsp90 chaperone complex, due to competition for overlapping binding sites and conformational changes within the ER{alpha} protein (28). Because GA stimulated the CHIP-ER{alpha} interaction (Figs. 7BGo and 8BGo), we investigated whether ligand binding, by interrupting the CHIP-ER{alpha} interaction, could interfere with GA-induced ER{alpha} degradation. Toward this, ER{alpha} protein levels were examined in MCF7 cells: 1) exposed to E2, ICI or GA alone; 2) pretreated with vehicle, E2, OHT, or ICI for 30 min, followed by a 6-h treatment with GA; and 3) pretreated with vehicle or GA for 30 min, followed by a 5.5-h treatment with E2, OHT, or ICI. As expected, E2, ICI and GA treatment, but not OHT, dramatically down-regulated ER{alpha} levels in MCF7 cells (Fig. 9AGo, upper panel). Exposure to E2 or OHT, either before (Fig. 9AGo, middle panel) or shortly after (Fig. 9AGo, lower panel) GA treatment, completely abolished GA-induced ER{alpha} degradation. In contrast to what was observed with E2 and OHT, ICI exposure, neither before (Fig. 9AGo, middle panel) nor shortly after (Fig. 9AGo, lower panel) GA treatment, failed to protect ER{alpha} against degradation.



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  		Fig. 9. Effect of Ligand Binding on Geldanamycin-Induced ER{alpha} Degradation

A, ER{alpha} protein levels in MCF7 cells treated with GA before or after ligand exposure. MCF7 cells were plated in 60-mm dishes at a density of 3 x 105 cells/dish and cultured in hormone-free medium for 3 d. Upper panel, Cells were treated with vehicle, 10 nM E2, 1 µM OHT, 100 nM ICI or 1 µM GA for 6 h; middle panel, cells were exposed to indicated ligand for 30 min before a 6-h GA treatment; lower panel, 30 min after GA treatment, cells were exposed to indicated ligand for 5.5 h. For all experiments, ER{alpha} levels were determined by immunoblotting with anti-ER{alpha}. ß-Tubulin was used as SDS-PAGE loading control. B, Effect of ligands on GA-induced CHIP-ER{alpha} interaction. MCF7 cells were plated in 100-mm dishes at a density of 1 x 106 cells/dish, cultured in hormone-free medium for 3 d, and then transfected with 5 µg pcDNA-His6-CHIP by using FuGENE. Twenty-four hours after transfection, the cells were treated with 1 µM GA for 30 min, followed by a 30-min treatment with indicated ligands (100 nM E2, 1 µM OHT, and 100 nM ICI). ER{alpha} protein from the cell lysates was precipitated using anti-ER{alpha}. CHIP presence in the precipitated ER{alpha} complex was determined by immunoblotting with anti-His6. The same membrane was reprobed with ER{alpha} antibody to assess the amount of precipitated ER{alpha}. Representative results of two independent experiments, each performed in duplicate, are shown. IP, Immunoprecipitation.

 
To examine the effect of these ligands on the CHIP-ER{alpha} interaction, coimmunoprecipitation analysis was performed on MCF7 cells transfected with CHIP. Cells were pretreated with GA for 30 min, followed by a 30-min treatment with E2, OHT, or ICI. GA treatment alone increased the amount of CHIP detected in the precipitated ER{alpha} complex; however, this amount was substantially reduced by treatment with E2, OHT, or ICI (Fig. 9BGo). These results demonstrate that all three ligands can interfere with the interaction between CHIP and ER{alpha}. Because these ligands have dramatically different effects on ER{alpha} stability, our results indicate that after dissociation from the Hsp90 chaperone complex, distinct downstream pathways exist for ER{alpha} degradation. Because E2 alone can induce ER{alpha} degradation through a transcription coupled mechanism (17, 19, 22, 24), it was somewhat unexpected to observe that ER{alpha} was stable during the combined treatment of GA and E2 (Fig. 9AGo). One explanation is that Hsp90 activity (inhibited by GA) is required for transcription-coupled ER{alpha} degradation. The OHT-ER{alpha} complex lacks transcriptional activity in MCF7 cells and thus is not a substrate for the transcription-coupled degradation pathway. Consequently, the ability of OHT to block GA-induced ER{alpha} degradation is likely due to disruption of the CHIP-ER{alpha} interaction (Fig. 9BGo). ICI also interrupts the GA-induced CHIP-ER{alpha} interaction (Fig. 9BGo) but fails to stabilize ER{alpha} (Fig. 9AGo), suggesting that the ER{alpha}-ICI complex is targeted for degradation through a CHIP-independent, GA-insensitive pathway.

Effect of CHIP and GA on ER {alpha} Cellular Localization
CHIP and Hsp90 are located primarily in the cytoplasm (30), whereas ER{alpha} is primarily a nuclear-localized protein (39). To determine whether CHIP overexpression, or GA treatment, could affect the cellular distribution of ER{alpha}, HeLa cells were transfected with a GFP-ER{alpha} fusion protein (40) and the cellular distribution of green fluorescence was examined. In control cells, fluorescence was restricted to the nuclei (Fig. 10AGo, top left panel). CHIP coexpression or GA treatment did not affect the nuclear localization of GFP-ER{alpha} (Fig. 10AGo). In contrast, ICI treatment, either alone or in the presence of transfected CHIP, resulted in the appearance of green fluorescence in the cytoplasm (Fig. 10AGo, bottom two panels). This observation is consistent with a previous study by Dauvois et al. (7) showing that ICI induces cytoplasmic retention of ER{alpha}. In addition, in HeLa cells transfected with GFP-ER{alpha} only, treatment with GA resulted in the appearance of GFP foci in the nuclei of approximately 20% of transfected cells (Fig. 10AGo, left middle panel). These GFP foci were not observed in GA-treated cells cotransfected with CHIP (Fig. 10AGo, right middle panel). Although the identity of the GFP foci is unknown, one possibility is that these represent aggregated GFP-ER{alpha}, resulting from the combined effect of Hsp90 inhibition and high expression levels of GFP-ER{alpha}. CHIP overexpression may promote both basal and GA-induced ER{alpha} degradation, preventing GFP-ER{alpha} aggregate formation. Consistent with this interpretation, we found that expression of CHIP decreased the number of GFP-ER{alpha}-expressing cells (Fig. 10BGo). Based on our results, and a recent finding that a small fraction of nuclear-localized CHIP can promote nuclear protein degradation (41), we suggest that CHIP-mediated ER{alpha} degradation occurs within the nucleus.



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  		Fig. 10. Effect of CHIP and GA on ER{alpha} Cellular Localization

HeLa cells were plated in six-well dishes at a density of 1 x 105 cells/dish, cultured in hormone-free medium for 3 d, and transfected with 250 ng GFP-ER{alpha} and 250 ng pcDNA or pcDNA-His6-CHIP by using LipofectAMINE Plus Reagent. Twenty-four hours after transfection, the transfected cells were treated with 1 µM GA or 100 nM ICI for 6 h. The fluorescence of GFP-ER{alpha} was then examined using an inverted microscope (Axiovert 40 CFL) (A). The number of cells expressing GFP-ER{alpha} from 10 microscope fields is shown in the histogram (B). Representative results of two independent experiments, each performed in triplicate, are shown. Open bar, Vehicle-treated controls; gray bar, GA treatment (1 µM, 6 h).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The cellular level of ER{alpha} determines both estrogen sensitivity and responsiveness (2, 35, 42). Steady-state levels of ER{alpha} protein are tightly regulated through a rapid balance between receptor synthesis and turnover, according to changing cellular conditions (4). Although it has been well documented that ER{alpha} degradation is primarily mediated by the ubiquitin proteasome pathway, the molecular mechanism(s) by which cells regulate ER{alpha} stability are largely unknown. Here we report that the Hsc70/Hsp90-interacting protein CHIP plays a key role in both basal and Hsp90 inhibitor-induced ER{alpha} turnover. Furthermore, CHIP-induced receptor degradation occurs through the ubiquitin proteasome pathway. Overexpression of CHIP promotes ER{alpha} degradation, accompanied by a decrease in ER{alpha}-mediated gene transactivation. Conversely, inhibition of CHIP by siRNA increases ER{alpha} levels and up-regulates ER{alpha}-mediated gene transactivation. Thus, this is the first report that CHIP, by modulating the cellular concentration of ER{alpha}, plays a role in regulating estrogen action.

During the preparation of this report, Tateishi and colleagues (43) reported a similar finding, that CHIP plays a role in basal ER{alpha} turnover. Our findings agree with several conclusions from that study, including: 1) CHIP, through its TPR domain, associates with ER{alpha}-chaperone complexes; 2) CHIP promotes, through its TPR and U-box domains, both polyubiquitination and proteasomal degradation of unliganded ER{alpha}; 3) CHIP-mediated ER{alpha} degradation occurs in the nucleus; and 4) ligand binding blocks CHIP-mediated ER{alpha} degradation by disrupting CHIP-ER{alpha} interaction. Here, we further extend the study of Tateishi et al. (43) in two significant aspects: 1) CHIP is required for Hsp90 inhibitor-induced ER{alpha} degradation; and 2) CHIP targets functional ER{alpha} (correctly folded, ligand-binding competent receptor protein) for degradation.

Several lines of evidence from our study support the conclusion that CHIP targets functional ER{alpha} for degradation. First, OHT treatment completely blocked CHIP-induced ER{alpha} degradation, suggesting that ER{alpha} reaches a correctly folded conformation, competent for ligand binding, before CHIP-directed degradation. Secondly, CHIP overexpression down-regulated ER{alpha} levels and decreased ER{alpha}-mediated gene expression, whereas CHIP depletion by siRNA up-regulated ER{alpha} levels and increased ER{alpha}-mediated gene transcription. This coordinate regulation of ER{alpha} levels and activity suggests that CHIP targets functional ER{alpha} for degradation. Thirdly, CHIP plays a role in GA-induced ER{alpha} degradation by primarily targeting Hsp90-associated, transcriptionally competent ER{alpha} (29). Although originally believed to function as a general ubiquitin ligase, responsible for ubiquitinating unfolded or misfolded proteins in a chaperone-dependent process (31), more recent studies have demonstrated that CHIP also targets mature Hsp90 client proteins for degradation (33, 36).

Tateishi et al. (43) observed that CHIP overexpression increased ER{alpha} transcriptional activity. Although this was not observed in our study, the use of different estrogen response element (ERE) and control reporter constructs for the functional analyses of ER{alpha} could account for this discrepancy. In the present study, an estrogen-responsive reporter construct (ERE-pS2-Luc), possessing two ERE copies within the pS2 promoter (44), was used. Our previous study demonstrated a close correlation between ERE-pS2-Luc expression and cellular concentration of ER{alpha} (35). In the present study, we also used a constitutively active construct, SV40-Luc, to monitor and normalize the effects of both CHIP and CHIP-siRNA on transfection efficiency and luciferase expression. In the study by Tateishi et al. (43), pRSVßGal was used as an internal control. When we used a similar construct, CMVßGal, we found that overexpression of either wild-type CHIP or TPR mutant (K30A), but not U-box mutant (H260Q), dramatically decreased CMVßGal expression in a dose-dependent manner (data not shown). Based on these observations, we suggest that ßGal is not a suitable control reporter for studying the effect of CHIP on gene transcription.

Our results, with data from Tateishi et al. (43), suggest a role for the Hsp90 chaperone complex in the regulation of cellular ER{alpha} levels. A summary of distinct ER{alpha} degradation pathways is depicted in Fig. 11Go. Nascent ER{alpha} is translocated into nucleus, and by associating with Hsp90, receptor protein is maintained in a ligand-binding competent conformation, ready for subsequent activation (28). In the absence of ligand or other activation signals, CHIP constantly targets chaperone-associated ER{alpha} for degradation, thereby limiting cellular concentrations of receptor protein. Ligand binding disassembles the ER{alpha}-Hsp90 complex and thus protects ER{alpha} from CHIP-mediated degradation. However, depending on the ligand, ER{alpha} stability can vary considerably, suggesting that different downstream destructive pathways exist. Furthermore, the ER{alpha}-ligand interaction could play a definitive role in pathway use. For example, when activated by E2, ER{alpha} is degraded through a transcription-coupled mechanism (17, 19, 22, 24). Pretreatment with GA, however, abolished E2-induced ER{alpha} degradation (Fig. 9AGo), suggesting that Hsp90 activity is required for transcription-coupled ER{alpha} degradation. In support of this possibility, the Hsp90-p23 complex has been shown to play a role in disassembling the nuclear receptor transcriptional complex from chromatin, a process believed to be a prerequisite for degradation of activated transcription factors (45, 46, 47). Conversely, through an unknown mechanism, the nuclear ER{alpha}-ICI complex is immobilized to the nuclear matrix and undergoes rapid degradation, in association with cytoplasmic retention of aggregated nascent ER{alpha} (7, 8, 22, 27, 40, 48). Although it is not clear how intracellular localization influences receptor degradation, the unique distribution pattern of ER{alpha} after treatment with ICI-182,780, together with the fact that ICI-induced receptor degradation is independent of ER{alpha} transcription activity, support the possibility that the pure antiestrogen and E2 use distinct degradation pathways for ER{alpha}. Taken together with our previous observation that an intact NEDD8 conjugation pathway is essential for ICI-induced ER{alpha} degradation in breast cancer cells (49), we suggest that destruction of the ICI-liganded receptor requires a cullin-based ubiquitin ligase.



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  		Fig. 11. Schematic Summary of Distinct ER{alpha} Degradation Pathways

Nascent ER{alpha} is translocated into nucleus. There, by associating with Hsp90, the receptor is maintained in a ligand-binding competent conformation, ready for subsequent activation. In the absence of ligand or other activation signals, CHIP constantly targets Hsp90-associated ER{alpha} for degradation. Ligand binding disassembles the ER{alpha}-Hsp90 complex and thus protects ER{alpha} from CHIP-mediated degradation. However, depending upon the ligand, distinct downstream destructive pathways are engaged in the degradation of liganded ER{alpha}. When activated by E2, ER{alpha} is degraded through a transcription-coupled mechanism. In response to ICI, nuclear ER{alpha}-ICI complex is immobilized to the nuclear matrix and undergoes rapid proteasomal degradation. In addition, ICI induces cytoplasmic retention and aggregation of nascent ER{alpha} OHT-ER{alpha} complexes are stable, likely due to the lack of transcriptional activity.

 
Abnormal expression of ER{alpha} has long been associated with both the initiation and progression of breast cancer (50). An increase in the number of ER{alpha}-positive cells, as well as increased individual cell ER{alpha} content, have frequently been detected in malignant breast tumors (42). Furthermore, increased ER{alpha} content has been shown to augment the magnitude of estrogen-stimulated gene expression, providing a growth advantage to breast cancer cells (2, 35, 49, 51). A recent study demonstrated a correlation between the loss of ERK7, a regulator of estrogen-induced ER{alpha} degradation, and breast cancer progression (52). Collectively, these observations indicate that alterations in ER{alpha} degradation pathways may contribute to deregulation of ER{alpha}, perhaps leading to enhanced estrogen action in breast tumors. Based on our results, the chaperone/CHIP pathway, by regulating ER{alpha} levels, likely contributes to the development/progression of that disease; and such a possible role merits further examination.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
The following antibodies and reagents were used in this study: anti-ER{alpha} (HC20) and anti-ß-tubulin (SC9104) (Santa Cruz Biotechnology, Santa Cruz, CA); anti-HA tag (3F10; Roche Molecular Biochemicals, Indianapolis, IN); anti-ER{alpha} (Ab-10) and anti-GFP (GFP01) (NeoMarkers, Inc., Fremont, CA); anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Chemicon International, Inc., Temecula, CA); anti-CHIP (PA1-015, Affinity Bioreagents, Golden, CO); anti-Hsp90 (SPA-830) and anti-Hsc70 (SPA-816) (Stressgene, Victoria, British Columbia, Canada); anti-His6 (8906-1, BD Biosciences, Palo, Alto, CA); protein G-agarose beads (Oncogene Research Products, San Diego, CA); horseradish peroxidase-conjugated second antibodies and SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL); protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA); protease inhibitor cocktail set III (Calbiochem-Novabiochem Corp., San Diego, CA); LipofectAMINE Plus Reagent (Life Technologies, Inc., Logan, UT); FuGENE (Roche Molecular Biochemicals, Indianapolis, IN); 17ß-estradiol, OHT, GA and MG132 (Sigma Chemical Co., St. Louis, MO); ICI (Tocris Cookson Ltd., Ellisville, MO); passive lysis buffer and luciferase assay system (Promega Corp., Madison, WI); fetal bovine serum (FBS) and dextran-coated charcoal-stripped FBS (Hyclone Laboratories, Inc., Logan, UT); cell culture supplementary reagents (Life Technologies, Inc., Rockville, MD).

Plasmid Construction
The construction of pSG5-ER{alpha}(HEGO), ERE2-pS2-Luc, SV40-Luc, pcDNA-HA-Ub, pCS2-UbK0 and cytomegalovirus promoter (CMV)-GFP have all been described previously (35). The pcDNA-His6-CHIP, pcDNA-His6-CHIP(K30A), and pcDNA-CHIP(H260Q) constructs were kindly provided by Drs. Neckers and Patterson (36), the pBS/U6/CHIPi construct by Dr. Chang (33), and the GFP-ER{alpha} construct by Dr. Stenoien (40).

Cell Lines and Transient Transfection
The human cervical carcinoma cell line HeLa and the breast cancer cell line MCF-7 were purchased from ATCC (Manassas, VA). HeLa cells were maintained in MEM with 2 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 0.1 mM non- essential amino acids, 1.0 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% FBS. MCF7 cells were maintained in the same medium, with the addition of 6 ng/ml insulin. Before experiments, cells were cultured in hormone-free medium (phenol red-free MEM with 3% dextran-coated charcoal-stripped FBS) for 3 d. For transfection, cells (80% confluence) were transfected with an equal amount of total plasmid DNA (adjusted with the corresponding empty vectors) by using LipofectAMINE Plus Reagent or FuGENE according to the manufacturer’s guidelines.

Immunoblotting, Immunoprecipitation, and Luciferase Assay
For immunoblot analysis, whole cell extracts were prepared by suspending cells (~2 x 106) in 0.1 ml SDS lysis buffer [62 mM Tris (pH 6.8), 2% SDS, 10% glycerol, and protease inhibitor cocktail III]. After 15 min incubation on ice, extracts were sonicated (3 x 20 sec), insoluble material removed by centrifugation (15 min at 12,000 x g), and supernatant protein concentration determined using a Bio-Rad protein assay kit. Five percent ß-mercaptoethanol was added to the protein extracts before heating at 90 C for 5 min. Protein extracts (50 µg per lane) were fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies. Primary antibody was detected by horseradish peroxidase-conjugated second antibody and visualized using an enhanced SuperSignal West Pico Chemiluminescent Substrate. The band density of exposed films was evaluated with National Institutes of Health ImageJ software (http://rsb.info.nih.gov/ij/). Immunoprecipitation was performed as described previously (49). For luciferase assays, cell lysates were prepared with passive lysis buffer and luciferase activity determined using the Luciferase Assay System.

CHIP siRNA Construct
The pBS/U6/CHIPi construct was kindly provided by Dr. Zhijie Chang (33). The siRNA expressed by the pBS/U6/CHIPi construct starts with GGG (position 233–251 bp relative to the ATG start site in the CHIP cDNA).

Quantitative Real-Time PCR
Total RNA was prepared by a RNAeasy Mini Kit (QIAGEN, Valencia, CA), according to the manufacturer’s protocol. RNA (2 µg) was reverse-transcribed in a total volume of 40 µl containing 400 U Moloney murine leukemia virus reverse transcriptase (New England Biolabs, Beverly, MA), 400 ng random hexamers (Promega), 80 U ribonuclease inhibitor and 1 mM deoxynucleotide triphosphates. The resulting cDNA was used in subsequent quantitative real-time PCRs, performed in 1x iQ SYBR Green Supermix (Bio-Rad) with 5 pmol forward and reverse primers as previously described (35).


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Drs. Len Neckers (National Cancer Institute, Rockville, MD) and Cam Patterson (University of North Carolina, Chapel Hill, NC) for providing pcDNA-His6-CHIP, pcDNA-His6-CHIP(K30A) and pcDNA-CHIP(H260Q); Dr. Zhijie Chang (Tsinghua University, Beijing, China) for pBS/U6/CHIPi; Dr. Michele Pagano (New York University Cancer Institute, New York, NY) for pCS2-UbK0 and Dr. Michael A. Mancini (Baylor College of Medicine, Houston, TX) for GFP-ER{alpha}. We thank Dr. Curt Balch (Indiana University School of Medicine) for his critical review of this manuscript.


   FOOTNOTES
 
This work was supported by the U.S. Army Medical Research Acquisition Activity, award numbers DAMD 17-02-1-0418 and DAMD17-02-1-0419; American Cancer Society Research and Alaska Run for Woman Grant TBE-104125; and the Walther Cancer Institute.

First Published Online July 21, 2005

Abbreviations: CHIP, Carboxyl terminus of Hsc70-interacting protein; CHIPi, CHIP-siRNA expression construct; CMV, cytomegalovirus promoter; DMSO, dimethylsulfoxide; E2, 17ß-estradiol; ER{alpha}, estrogen receptor-{alpha}; ERE, estrogen response element; FBS, fetal bovine serum; GA, geldanamycin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HA, hemagglutinin; Hsp, heat shock protein; ICI, ICI 182,780; Luc, firefly luciferase; OHT, 4-hydroxytamoxifen; siRNA, small interference RNA; SV40, simian virus 40 promoter; TPR, tetratricopeptide repeat.

Received for publication March 4, 2005. Accepted for publication July 14, 2005.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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