Alternative Effects of the Ubiquitin-Proteasome Pathway on Glucocorticoid Receptor Down-Regulation and Transactivation Are Mediated by CHIP, an E3 Ligase
Xinjia Wang and
Donald B. DeFranco
Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Address all correspondence and requests for reprints to: Donald B. DeFranco, Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261. E-mail: dod1{at}pitt.edu.
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ABSTRACT
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The ubiquitin/proteasome-dependent protein degradation pathway (UPP) is responsible for the accelerated down-regulation of glucocorticoid receptor (GR) levels in cells subjected to chronic glucocorticoid exposure. Whereas hormone-dependent down-regulation of GR operates in most cells, the receptor is not down-regulated after long-term glucocorticoid treatment of either cultured embryonic hippocampal neurons or the HT22 hippocampal cell line. In this report, we show that stable overexpression of the carboxy terminus of heat shock protein 70-interacting protein (CHIP) E3 ligase can restore hormone-dependent down-regulation of GR in HT22 cells. Proteasome inhibitor studies establish that ubiquitylated GR can be efficiently engaged with the proteasome upon CHIP overexpression, unlike the case in parental HT22 cells. In addition to its impact on GR down-regulation, CHIP overexpression alters the coupling between the UPP and GR transactivation. Unlike other steroid receptors whose transactivation properties are typically reduced upon proteasome inhibition, GR transactivation in HT22 cells and other cell lines is enhanced upon proteasome inhibition. However, in HT22 cells overexpressing CHIP, proteasome inhibition leads to a reduction in GR transactivation activity. Thus, the divergent response of a single transactivator (i.e. GR) to the UPP can be dictated by CHIP, an E3 ligase that also functions as a proteasome-targeting factor.
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INTRODUCTION
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GLUCOCORTICOID HORMONES serve important homeostatic roles in a variety of tissues and cell types impacting glucose, fat, and mineral metabolism as well as physiological responses to a variety of physical and emotional stresses (1). The primary mode of glucocorticoid action requires hormone binding to the glucocorticoid receptor (GR), which elicits cellular responses predominantly through the regulation of unique sets of target genes (2). GR is a member of a large superfamily of nuclear receptors that regulates gene transcription both positively and negatively either through direct DNA binding or interactions with select DNA-bound transcription factors (3). When associated with its target sites, GR, like all nuclear receptors, facilitates the recruitment of various coregulator proteins and chromatin remodeling factors that influence the efficiency of RNA polymerase II-directed transcription from linked promoters (4, 5, 6).
Glucocorticoid responsiveness in cultured cells and intact tissues is influenced by the steady state levels of GR (7, 8, 9, 10). For example, modest overexpression of GR in transgenic mice leads to defects in neuroendocrine, inflammatory, immune, and stress response systems, which were shown in some cases to be associated with specific alterations in glucocorticoid regulated gene transcription (10). Whereas GR expression is regulated by a variety of factors in physiological and pathophysiological conditions (11), the mechanisms responsible for such regulation has been best characterized in cells chronically exposed to hormone. In most cultured cells and intact tissue, prolonged hormone treatment leads to reductions in GR levels and subsequent diminution of glucocorticoid responsiveness (7, 8). Hormone-activated GR limits its own expression through effects on both GR gene transcription and GR protein stability (11, 12, 13). Hormone-dependent down-regulation of GR may be one factor that contributes to acquired glucocorticoid resistance in patients subjected to chronic glucocorticoid exposure (11).
GR, like many other nuclear receptors, is a substrate for the ubiquitin/proteasome-dependent protein degradation pathway (UPP) (14, 15, 16). Proteins targeted to the proteasome for degradation are covalently linked at specific lysine residues to a chain of ubiquitin moieties, which serve as a recognition motif for specific ubiquitin-binding subunits of the proteasome such as Rpn10/S5a or Rpt5/S6' (17). The attachment of the 76-amino acid ubiquitin peptide to proteins ultimately destined for proteasome degradation requires the concerted action of at least three sets of enzymes. Single ubiquitin chains are first linked to E1 ubiquitin-activating enzymes (i.e. UBAs) before their transfer to E2 ubiquitin-conjugating enzymes (UBCs) (17, 18, 19). The specificity of the final transfer of ubiquitin from UBCs is brought about a diverse family of E3 ubiquitin-protein ligases (UBLs), which provide specific substrate recognition (19). Two different classes of UBLs exist that use distinct mechanisms to bring about substrate-specific transfer of ubiquitin. UBLs of the RING finger family facilitate the transfer of ubiquitin from an UBC to the substrate, whereas HECT domain E3 ligases transfer to the substrate a covalently linked ubiquitin that was acquired from an associated UBC (19).
The association of nuclear receptors and other transcription factors with various components of the UPP can exert direct or indirect effects on their transcriptional regulatory activity (20, 21). For example, whereas the coactivator function of the E6-AP E3 ligase is independent of its ubiquitin ligase activity (22), coactivation by UbcH7 requires its enzymatic activity (23). The recruitment of specific proteasome subunits to an active promoter, first demonstrated in yeast (24), has been further elaborated for steroid hormone-regulated genes and revealed the cyclic association of various proteasome components and E3 ligases with estrogen receptor (ER)- (25) and androgen receptor (AR)- (26) regulated genes. In fact, inhibition of the proteolytic activity of the proteasome disrupts the dynamic association of ER, AR, and associated factors with the chromatin of target genes (25, 26). Thus, proteasome-dependent degradation is critical to maintain the appropriate dynamics of steroid receptor and cofactor interactions with chromatin that is essential for efficient hormone-induced transcription (27, 28, 29, 30, 31).
Whereas the proteasome has emerged as an important participant in the process of transcription, its impact on transcription factor function can be quite diverse (20). Furthermore, it seems clear that effects of the UPP on transcription may not be linked exclusively to protein degradation. For example, proteasome inhibitors disrupt dynamic chromatin cycling of ER on target sites (25) and thereby reduces ER transactivation, despite bringing about elevations in overall receptor levels (32). Conversely, proteasome inhibition that elevates GR levels enhances its transactivation activity both on transiently transfected (14, 16, 33) and native chromatin templates (16). These divergent roles for the proteasome in transcriptional regulation has been observed in other systems and indicates that the proteasome, like other transcriptional regulators, may use a variety of mechanisms to bring about gene-specific changes in transcription (20).
In a previous report, we found that GR was not down-regulated in response to chronic glucocorticoid treatment in primary embryonic hippocampal neurons and the HT22 hippocampal cell line (33). Because GR had the capacity to be ubiquitylated in HT22 cells when hormone-dependent down-regulation was restricted (33), we hypothesized that limitations in specific targeting factors may be responsible for the failure of ubiquitylated GR to efficiently engage with the proteasome. In this report, we show that overexpression of CHIP [the E3 ligase carboxy terminus of heat shock protein (Hsp) 70-interacting protein (34)] is able to restore hormone-dependent down-regulation of GR in HT22 cells. In addition, in the presence of overexpressed CHIP, GR transactivation is reduced upon treatment with proteasome inhibitors, rather than enhanced as observed in parental HT22 cells. Thus, the coupling between the transactivation properties of GR and its UPP-dependent degradation can be dictated by the relative abundance of a single E3 ligase and proteasome-targeting factor, CHIP.
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RESULTS
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CHIP Overexpression in HT22 Cells Restores Hormone-Dependent Degradation of GR
CHIP functions as an E3 ligase and selectively enhances the degradation of numerous targets including GR (15). In addition, CHIP has been postulated to facilitate the delivery of ubiquitylated substrates to the proteasome via its C-terminal U-Box domain (15). Because endogenous CHIP protein was undetectable in HT22 cells (Fig. 1A
), CHIP effects on GR degradation were assessed in stable HT22 cell transfectants (e.g. the Chip-6B line) expressing Myc-tagged CHIP (Fig. 1A
). Overexpression of CHIP did not affect the growth of HT22 cells (data not shown), although we have not examined whether the response of HT22 cells to various toxic insults was altered by CHIP overexpression, as observed in other cell types (35). The Myc-tagged CHIP in Chip-6B cells was associated with endogenous GR (Fig. 1B
) analogous to results obtained in transiently transfected COS-7 cells (15). The binding of GR to Myc-tagged CHIP in the Chip-6B cells was not significantly affected by dexamethasone (Fig. 1B
). Myc-tagged CHIP/GR complexes form in COS-7 cells not exposed to hormone, although the impact of GR hormone binding on the stability of this complex was not assessed (15).

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Fig. 1. CHIP Overexpression and Interaction with GR in HT22 Cells
A, Chip-6B cells were recovered after the stable transfection of HT22 cells with a Myc-tagged CHIP expression plasmid. Total protein in whole cell lysates prepared from HT22 cells or Chip-6B cells was separated by SDS-PAGE and subjected to Western blot analysis to detect CHIP (upper left panel), myc-tagged CHIP (upper right panel) or actin (bottom panel). A nonspecific band is sometimes detected in the Myc-antibody Western blots of HT22 cells that comigrates with the Myc-CHIP protein. Blot shown is representative of two separate experiments. B, HT22 and Chip-6B whole cell extracts (4% of input) were either directly subjected to SDS-PAGE and Western blot analysis to detect Myc-tagged CHIP or subjected to an immunoprecipitation (IP) with the BuGR2 GR antibody before SDS-PAGE and Western blot analysis (IB) to detect Myc-tagged CHIP associated with GR. In Western blots of immunoprecipitated material, the IgG heavy chain (HC) subunit is detected. Blot shown is representative of two separate experiments. In B, cells were either untreated () or treated with 1 µM dexamethasone (Dex) for 2 h before harvesting.
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The effects of CHIP overexpression on steady-state levels of GR were examined using Western blot analysis. As shown in Fig. 2A
, base line levels of GR in cells not exposed to hormone were similar in the parental HT22 cells and the Chip-6B cell line. In contrast to our findings, GR levels in transiently transfected COS-7 cells were reduced when CHIP was cotransfected (15), whereas in transiently transfected HeLa cells, CHIP overexpression exerted minimal effects on transfected GR levels (36). Our experiment differs from these previous reports in that endogenous GR levels were examined in cells that stably overexpress CHIP.

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Fig. 2. CHIP Overexpression in HT22 Cells Restores Hormone-Dependent Down-Regulation of GR
Total protein in whole cell lysates prepared from HT22 cells or Chip-6B cells was separated by SDS-PAGE and subjected to Western blot analysis to detect GR, actin or hsp70. GR levels in untreated HT22 and Chip-6B cells are visualized in A, whereas GR is visualized in extracts prepared from HT22 (B), HT22-hygro (C), or Chip-6B (D) cells after various lengths of Dex exposure (i.e. between 0 and 54 h). Blots were stripped and reprobed to detect actin (AD) or hsp70 (D). Blots shown are representative of three separate experiments.
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As reported previously (33), chronic hormone treatment did not trigger GR down-regulation in the parental HT22 cells (Fig. 2B
). Furthermore, GR levels were not affected by dexamethasone in a hygromycin-resistant HT22 stable transformant that does not express Myc-tagged CHIP (Fig. 2C
). However, overexpression of CHIP in HT22 stable transformants led to hormone-dependent GR down-regulation (Fig. 2D
). This recovery of hormone-dependent GR down-regulation was also observed in an independently derived HT22 stable transformant overexpressing CHIP (data not shown). Thus, stable CHIP overexpression in HT22 cells exerts selective effects to stimulate GR down-regulation in cells exposed to chronic hormone treatment and has minimal effects on steady-state levels of GR in untreated cells.
GR Engages with the Proteasome in CHIP-Overexpressing HT22 Cells
The failure of chronic hormone treatment to trigger GR down-regulation in HT22 cells is not due to the inability of endogenous E3 ligases to promote GR ubiquitylation (33). Based upon this result, we hypothesized that ubiquitylated GR in HT22 cells may not efficiently engage with the proteasome. The recovery of GR down-regulation in Chip-6B cells suggests that CHIP can function as a proteasome-targeting factor for GR. One approach to assess the recruitment of GR to the proteasome is to examine the effects of proteasome inhibitors (e.g. MG132) on GR levels. MG132 treatment of HT22 cells does not affect GR levels (33) consistent with the inefficient association of GR with the proteasome. However, as shown in Fig. 3
, MG132 treatment reduces the hormone-dependent down-regulation of GR that occurs in the Chip-6B cell line. MG132 treatment enhanced hsp70 expression in the CHIP 6B cells (Fig. 3
), consistent with its effects in parental HT22 cells (33). Thus, the overexpression of CHIP allows GR that is ubiquitylated in hormone-treated cells to effectively target to the proteasome for subsequent degradation.

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Fig. 3. Proteasome Inhibition Blocks Hormone-Dependent Down-Regulation of GR
Chip-6B cells were treated with 2.5 µM MG132 for 16 h followed by an 8-h treatment with 1 µM Dex. Total protein in whole cell lysates was separated by SDS-PAGE and subjected to Western blot analysis to detect GR (top panel) Myc-tagged CHIP (middle panel), or hsp70 (bottom panel). The identity of the low molecular weight band that appears in Myc-CHIP Western blots of MG132-treated Chip-6B cells is unknown. Blot shown is representative of two separate experiments.
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CHIP Overexpression Enhances GR Ubiquitylation in HT22 Cells
GR ubiquitylation in vitro or in transiently transfected COS-7 cells is enhanced by CHIP addition or overexpression, respectively (15). To assess the extent of GR ubiquitylation in HT22 cells overexpressing CHIP, cells were transiently transfected with a hemagglutinin (HA)-tagged ubiquitin expression plasmid. The detection of ubiquitylated GR in the HT22 cells (33) as well as in COS-7 cells transiently transfected with CHIP (15) is facilitated by treatments with the proteasome inhibitor MG132. As shown in Fig. 4
, the ubiquitylation of immunoprecipitated GR was increased upon stable overexpression of CHIP in HT22 cells. Thus, overexpressed CHIP in Chip-6B cells can exhibit E3 ligase activity toward a specific substrate (i.e. the GR) in addition to its functioning as a putative proteasome-targeting factor.

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Fig. 4. CHIP Overexpression Enhances GR Ubiquitylation in HT22 Cells
HT22 and Chip-6B cells were transiently transfected with an HA-tagged ubiquitin expression plasmid or empty vector and then incubated with 10 µM MG-132 or vehicle for 9 h. Total protein in whole cell lysates was immunoprecipitated (IP) with the anti-GR BuGR2 antibody and then subjected to Western blot analysis (IB) after SDS-PAGE to detect GR (upper panel) or HA-tagged ubiquitin (lower panel). Nonspecific bands indicated (*) are detected by the anti-HA antibody in nontransfected HT22 cells. HA-ubiquitylated GR species are detected as a high molecular weight smear in MG132-treated cells. Blot shown is representative of two separate experiments.
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CHIP Effects on GR Transactivation in HT22 Cells
GR appears to differ from other nuclear receptors in its transcriptional response to proteasome inhibition (14, 16, 32, 33). Specifically, glucocorticoid-induced transcription is typically enhanced upon proteasome inhibition rather than reduced as observed for PR and ER (32). Because CHIP overexpression affects GR interactions with the UPP in HT22 cells, we examined whether such changes in GR processing had any impact on GR transactivation as assessed from a transiently transfected luciferase reporter plasmid driven by the glucocorticoid-responsive promoter of the mouse mammary tumor virus (MMTV). As shown in Fig. 5A
, GR transactivation activity was significantly enhanced in HT22 cells that stably overexpresses CHIP. Enhancement of GR transactivation occurred even though stable CHIP overexpression led to hormone-dependent degradation of GR in HT22 cells (see Fig. 2
). This is not unexpected because hormone-dependent GR degradation occurs in most cells that exhibit efficient GR transactivation. The reduction in GR transactivation activity in transiently transfected COS-7 cells that overexpress CHIP may be due to diminished GR expression that occurs even in the absence of hormone treatment (15). GR transactivation from the MMTV promoter is also enhanced in HT22 cells transiently transfected with another GR E3 ligase, Mdm2 (37) (Fig. 5B
). Thus, two distinct GR E3 ligases have the capacity when overexpressed to enhance GR transactivation in HT22 cells.

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Fig. 5. Alterations in GR Transactivation upon CHIP and Mdm2 Overexpression in HT22 Cells
HT22 and/or Chip-6B cells were transiently transfected with a glucocorticoid-responsive luciferase reporter plasmid (MMTV-Luc). Cells were treated with 1 µM Dex or vehicle for 10 h where indicated. Whole cell extracts were assayed for firefly luciferase activity, which was normalized to total protein concentration and the activity of a nonhormone-regulated Renilla luciferase reporter plasmid. Panel A shows GR transactivation activity in HT22 and Chip-6B cells, whereas B shows GR transactivation in HT22 cells transiently transfected with an Mdm2 expression plasmid or control empty plasmid vector (i.e. pCMV5) along with MMTV-Luc. Shown are the mean values ± SD of six determinations. Double asterisk, P < 0.001, single asterisk, P < 0.05 as assessed by a Students t test. The data shown are representative of three independent experiments.
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As an additional assessment of the impact of CHIP overexpression, GR transactivation was examined in the presence of proteasome inhibition. GR transactivation in increased upon proteasome inhibition in a variety of cell types (14, 16, 33) irrespective of the receptor down-regulation response that is triggered by hormone. Increased GR transactivation after MG132 treatment of HT22 cells is illustrated in Fig. 6A
and confirms previous results in HT22 cells (33) and other cell types (14, 16). Interestingly, in Chip-6B cells, MG132 treatment leads to a reduction in GR transactivation activity (Fig. 6B
). Thus, the response of GR transactivation activity to proteasome inhibition in HT22 cells is switched upon CHIP overexpression to resemble that observed for PR and ER (i.e. reduced transactivation; Ref. 32).

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Fig. 6. Reversal of MG132 Effects on GR Transactivation upon CHIP Overexpression
HT22 (A) or Chip-6B (B) cells were transiently transfected with a glucocorticoid-responsive luciferase reporter plasmid (MMTV-Luc). Cells were treated with 1 µM Dex or vehicle in the presence or absence of 5 µM MG-132 for 10 h where indicated. Whole cell extracts were assayed for firefly luciferase activity, which was normalized to total protein concentration and the activity of a nonhormone-regulated Renilla luciferase reporter plasmid. Shown are the mean values ± SD of six determinations. Double asterisk, P < 0.001; single asterisk, P < 0.05 as assessed by a Students t test. The data shown are representative of three independent experiments.
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DISCUSSION
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Down-regulation of GR protein is a characteristic response of most cells to chronic glucocorticoid exposure serving to limit the duration of glucocorticoid action and contributing in part to acquired glucocorticoid resistance (11). Whereas long-term glucocorticoid treatment does not lead to GR down-regulation in all cell types, the mechanisms that account for this diversity in receptor processing are not known. In this report, we identified a single component of the UPP machinery (i.e. the E3 ligase CHIP) that alters GR processing and generates a down-regulation response in cells (i.e. HT22 cells) where steady-state levels of GR were unaffected by prolonged hormone treatment. The overexpression of CHIP in HT22 cells also alters the coupling between GR transactivation and the UPP pathway, as evidenced by both enhanced transcription of a GR-responsive promoter (i.e. the MMTV promoter) in the presence of hormone and the reduction in this response upon proteasome inhibition. GR-mediated transactivation from the MMTV promoter in various cell lines, including parental HT22 cells, is stimulated upon proteasome inhibition. Thus, the impact of the UPP on the processing and transactivation activity of even a single transcription factor (e.g. GR) in a single cell type (e.g. HT22 cells) can be influenced by the relative expression of select components (e.g. CHIP) of the UPP machinery.
Differences in expression of another GR-targeted E3 ligase (i.e. Mdm2) have previously been shown to impact GR degradation and transactivation. Specifically, in breast cancer cells, estrogen treatment leads to an induction of Mdm2 expression and a resultant enhancement of GR degradation through the UPP (38). CHIP expression is not affected by estrogen in breast cancer cells (38) and therefore it is unlikely that endogenous CHIP impacts the response of GR degradation to estrogen in breast cancer cells. The decrease in GR levels in estrogen-treated breast cancer cells does not require the binding or activation of GR by glucocorticoid hormone but is associated with reduced GR transactivation. These effects (i.e. reduction in both steady-state GR levels in unstimulated cells and in hormone-dependent transactivation) resemble those observed in COS-7 cells transiently transfected with GR and CHIP (15) but differ from results of stable CHIP overexpression in HT22 cells where enhanced GR degradation occurs only in the presence of hormone. Mdm2 overexpression in transiently transfected HT22 cells enhances GR transactivation from the MMTV promoter, but the capacity for Mdm2 to be regulated by steroids (38) or cellular stress (37) and impact GR degradation in HT22 cells awaits future investigation.
Because GR has the capacity to be ubiquitylated in the parental HT22 cells (33), it is not restricted from accessing the ubiquitylation machinery. Thus, either residual CHIP or some other E3 ligase (e.g. Mdm2) can act in HT22 cells to promote GR ubiquitylation. However, in the absence of sufficient levels of some proteasome-targeting factor such as CHIP, ubiquitylated GR does not effectively engage the proteasome. In coimmunoprecipitation assays with Chip-6B cells, CHIP/GR complexes were detected in both hormone-treated and untreated cells. The lack of hormone dependence has been observed for a CHIP/GR association in transfected COS-7 cells and for the interaction between CHIP and AR in vitro (39). The CHIP-binding domain of GR has not been identified, but it could be related to a conserved region within the amino-terminal domain of AR that is recognized by CHIP (39). Importantly, the interaction between CHIP and GR in untreated Chip-6B cells is not sufficient to promote GR degradation as steady-state levels of GR are not significantly altered in the Chip-6B cells. Thus, CHIP is mobilized to deliver GR to the proteasome only upon hormone activation of the GR. Given the involvement of CHIP in coupling the UPP to GR transactivation, it is tempting to speculate that CHIP interactions with GRs engaged in transcription are required to uncover its proteasome-targeting activity.
Our results support the notion that GR down-regulation by CHIP overexpression results from its acting as a proteasome-targeting factor (15) rather than strictly as an E3 ligase. A number of proteins have been found to possess polyubiquitin-binding domains (UBA domains) and therefore proposed to function as proteasome-targeting factors (40). Whereas some UBA-domain proteins are stoichiometric components of the proteasome and therefore likely to function as polyubiquitin chain receptors (41), UBA domains are also found within some soluble E3 ligases (40). Thus, soluble polyubiquitin chain binding proteins could also function in targeting ubiquitin-modified substrates to the proteasome. CHIP does not contain a UBA domain but has been found to bind directly to the S5a proteasome subunit in vitro and associate with the proteasome in vivo (15). However, it is unclear whether CHIP plays a direct role in targeting ubiquitylated substrates to the proteasome or an indirect role through its association with other proteasome-targeting factors such as Bag-1 (42).
After the initial demonstration of UPS involvement in transcriptional activation (24), numerous models have been proposed in attempts to reconcile the divergent effects that proteasome and UPS components exert on RNA polymerase II-directed transcription (20). It has been particularly difficult to assign a mechanism to could for the apparent opposing effects of the UPS on highly related steroid receptors (14, 16, 21, 32, 33). However, in this report we demonstrate that the divergent response of a single transactivator (i.e. GR) to the UPP can be dictated by CHIP, an hsp70-interacting E3 ligase. Specifically in HT22 cells overexpressing CHIP, the hormone-dependent transactivation activity of GR, which has been found in numerous cell types to be enhanced upon proteasome inhibition, is diminished when the proteasome is inhibited. GR is therefore not unique among steroid receptors in its response to proteasome inhibition, but acquires the phenotype of other steroid receptors (i.e. reduced transactivation upon proteasome inhibition) when expressed in cells with elevated CHIP levels. Thus, it may not be appropriate to group individual transcription factors into unique categories depending upon the nature of the coupling of the UPP to their transactivation activity and degradation (20). Rather, many transcription factors may have the capacity to respond in different ways to the UPP, depending upon the relative expression of particular components of the UPP.
The genetic manipulation of steady-state GR levels has a profound impact on neuronal cell responses to stress (43, 44). Whereas stress responses in other tissues are also altered when GR expression is increased or decreased, the brain may be particularly sensitive to GR levels because many regions of the brain express mineralocorticoid receptor that will respond to physiological levels of natural glucocorticoids (45). The UPP is likely to play an important role in GR action in the brain, as in other tissues, given its influence on GR protein levels and transcriptional responses. Because overexpression of a single component of the UPP (i.e. CHIP) can alter the UPPs action both on GR degradation and GR transactivation in a neuronal cell line, modulation of the expression of select UPP components (46) could provide an important mechanism to regulate glucocorticoid responsiveness.
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MATERIALS AND METHODS
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Cell Lines and Plasmids
The Chip-6B cell line was derived from the mouse HT22 hippocampal cell line (provided by David Schubert, Salk Institute, La Jolla, CA) after stable transfection with a full-length human CHIP cDNA (provided by Cam Patterson, University of North Carolina). Specifically, HT22 cells were cotransfected with a Myc-tagged human CHIP expression plasmid and a hygromycin resistance plasmid (pTK-hygro; BD Biosciences Clontech, Palo Alto, CA), and stable transformants selected that survived growth in 250 µg/ml hygromycin B (Invitrogen Life Technologies, Carlsbad, CA). The HT22-hygro cell line was derived by an analogous stable transfection into HT22 cells with the pTK-hygro plasmid alone. HT22 cells were grown at 37 C with 5% CO2 in DMEM (Invitrogen Life Technologies), supplement with 10% fetal bovine serum. Chip-6B and HT22-hygro cells were grown under the same conditions but maintained in the presence of 250 µg/ml hygromycin B. The Mdm2 expression plasmid (pMdm2) and the pCMV5 control plasmid vector were a gift of David Livingston (Dana-Farber Cancer Institute, Boston, MA).
Luciferase Assays
Luciferase activities in extracts from transiently transfected cells were determined essentially as described previously (33). For transient transfection, HT22 and Chip-6B cells were plated onto 100-mm diameter plates for 24 h and then exposed for 8 h to a lipofectamine (Life Technologies Inc., Rockville, MD)/DNA mixture that included 1 µg of the MMTV-Luc reporter plasmid, 0.05 µg of pCMV-Renilla and where indicated 6 µg of an Mdm2 expression plasmid or empty pCMV5 plasmid vector (33). Cells were allowed to recover for another 6 h and then replated onto six 35-mm diameter plates for 8 h before initiating 10-h treatments with or without 1 µM dexamethasone (Sigma Corp., St. Louis, MO) and/or 5 µM MG132 (Biosciences Inc., Darmstadt, Germany). Whole-cell extracts were prepared using the Luciferase Assay Buffer, as recommended by the supplier, and firefly and Renilla luciferase activities measured using the Dual-Luciferase Reporter Assay System (Promega Corp, Madison, WI). All assays were carried out three independent times using six samples per group.
Western Blot Analysis
Whole cell extracts for Western blot analysis were prepared as described previously (33). Fifty to 80 µg of total lysate protein was separated by sodium dodecyl sulfate (SDS)-7.5 or 12% SDS-PAGE, transferred to a Immobilon-P membranes (Millipore Corp., Bedford, MA), and immunoblotted with the following antibodies: anti-GR (BuGR2 monoclonal antibody (47), antiactin (C-2, sc-8432; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-hsp 70 (catalog no. SPA-810, Stressgen Biotechnologies Corp., San Diego, CA), anti-CHIP (catalog no. PC711, Oncogene Research Products, San Diego, CA), anti-c-myc (9E 10, sc-40; Santa Cruz Biotechnology, Inc.), and anti-HA (F-7, sc-7392; Santa Cruz Biotechnology, Inc.).
Immunoprecipitation
Immunoprecipitation reactions were performed using equal amounts of protein extracts as described previously (33). For analysis of endogenous GR interactions with Myc-tagged CHIP, Chip-6B cells (and HT22 cells as a negative control) were collected and then disrupted in 50 µl of lysis buffer (33). After lysis, the extract was diluted with 950 µl of dilution buffer (33) and DNA sheared by passing the lysate several times through a 22-gauge needle. The extracts were incubated with 20 µl of protein A/G-agarose (sc-2003; Santa Cruz Biotechnology, Inc.) for 1 h at 4 C, and then pelleted again. The resultant supernatants were added to 5µl of the anti-GR BuGR2 antibody prebound to 15 µl of protein A/G-agarose overnight at 4 C. Pelleted immune complexes were washed three times with washing buffer (33) and resuspended in 1x SDS-sample buffer (33). Samples were separated on 6% polyacrylamide gels by SDS-PAGE. For analysis of GR ubiquitylation, HT22 and Chip-6B cells grown in 100 mm-diameter tissue culture plates were transfected with 8 µg of either an HA-tagged ubiquitin expression plasmid (33) or an empty plasmid vector using lipofectamine (Life Technologies, Inc.). After 24 h, cells were either untreated or treated for 9 h with 10 µM MG132 where indicated. Cells were collected, lysed, and subjected to immunoprecipitation with the BuGR2 antibody as described above.
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ACKNOWLEDGMENTS
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We thank Drs. D. Livingston, C. Patterson, and D. Schubert for supplying DNA or cells. We also thank Dr. C. Patterson for many thoughtful discussions.
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FOOTNOTES
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This work was supported by National Institutes of Health Grant CA43037 (to D.B.D.).
First Published Online March 10, 2005
Abbreviations: AR, Androgen receptor; ER, estrogen receptor; GR, glucocorticoid receptor; HA, hemagglutinin; hsp, heat shock protein; MMTV, mouse mammary tumor virus; SDS, sodium dodecyl sulfate; UBAs, E1 ubiquitin-activating enzymes; UBCs, E2 ubiquitin-conjugating enzymes; UBLs, E3 ubiquitin-protein ligases; UPP, ubiquitin/proteasome-dependent protein degradation pathway.
Received for publication September 27, 2004.
Accepted for publication March 3, 2005.
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REFERENCES
|
---|
- Munck A, Guyre PM, Holbrook NJ 1984 Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocr Rev 5:2544[Abstract]
- Yamamoto KR 1985 Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet 19:209252[CrossRef][Medline]
- Tsai M-J, OMalley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451486[CrossRef][Medline]
- Glass CK, Rosenfeld MG 2000 The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 4:121141
- McKenna NJ, OMalley BW 2002 Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108:465474[CrossRef][Medline]
- Hsiao PW, Deroo BJ, Archer T 2002 Chromatin remodeling and tissue-selective responses of nuclear hormone receptors. Biochem Cell Biol 80:343351[CrossRef][Medline]
- Oakley RH, Cidlowski JA 1993 Homologous down regulation of the glucocorticoid receptor: the molecular machinery. Crit Rev Eukaryot Gene Expr 3:6388[Medline]
- Bellingham DL, Sar M, Cidlowski JA 1992 Ligand-dependent down-regulation of stably transfected human glucocorticoid receptors is associated with the loss of functional glucocorticoid responsiveness. Mol Endocrinol 6:20902102[Abstract]
- Vanderbilt JN, Miesfeld R, Maler BA, Yamamoto KR 1987 Intracellular receptor concentration limits glucocorticoid-dependent enhancer activity. Mol Endocrinol 1:6874[Abstract]
- Reichardt HM, Umland T, Bauer A, Kretz O, Schutz G 2000 Mice with an increased glucocorticoid receptor gene dosage show enhanced resistance to stress and endotoxic shock. Mol Cell Biol 20:90099017[Abstract/Free Full Text]
- Schaaf MJM, Cidlowski JA 2003 Molecular mechanisms of glucocorticoid action and resistance. J Steroid Biochem Mol Biol 83:3748[CrossRef]
- Burnstein KL, Jewell CM, Sar M, Cidlowski JA 1994 Intragenic sequences of the human glucocorticoid receptor complementary DNA mediate hormone-inducible receptor messenger RNA down-regulation through multiple mechanisms. Mol Endocrinol 8:17641773[Abstract]
- Rosewicz S, McDonald AR, Maddux BA, Goldfine ID, Miesfeld RL, Logsdon CD 1988 Mechanism of glucocorticoid receptor down-regulation by glucocorticoids. J Biol Chem 263:25812584[Abstract/Free Full Text]
- Wallace AD, Cidlowski JA 2001 Proteasome-mediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids. J Biol Chem 276:4271442721[Abstract/Free Full Text]
- Connell P, Ballinger CA, Jiang J, Wu Y, Thompson LJ, Hohfeld J, Patterson C 2001 The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nat Cell Biol 3:9396[CrossRef][Medline]
- Deroo BJ, Rentsch C, Sampath S, Young J, DeFranco DB, Archer TK 2002 Proteasomal inhibition enhances glucocorticoid receptor transactivation and alters its subnuclear trafficking. Mol Cell Biol 22:41134123[Abstract/Free Full Text]
- Hershko A, Ciechanover A 1998 The ubiquitin system. Annu Rev Biochem 67:425479[CrossRef][Medline]
- Thrower JS, Hoffman L, Rechsteiner M, Pickart CM 2000 Recognition of the polyubiquitin proteolytic signal. EMBO J 19:94102[Abstract/Free Full Text]
- Pickart CM 2001 Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503533[CrossRef][Medline]
- Lipford JR, Deshaies RJ 2003 Diverse roles for ubiquitin-dependent proteolysis in transcriptional activation. Nat Cell Biol 5:845850[CrossRef][Medline]
- Nawaz Z, OMalley BW 2004 Urban renewal in the nucleus: is protein turnover by proteasomes absolutely required for nuclear receptor-regulated transcription? Mol Endocrinol 18:493499[Abstract/Free Full Text]
- Nawaz Z, Lonard DM, Smith CL, Lev-Lehman E, Tsai SY, Tsai MJ, OMalley BW 1999 The Angelman syndrome-associated protein, E6-AP, is a coactivator for the nuclear hormone receptor superfamily. Mol Cell Biol 19:11821189[Abstract/Free Full Text]
- Verma S, Ismail A, Gao X, Fu G, Li X, OMalley BW, Nawaz Z 2004 The ubiquitin-conjugating enzyme UBCH7 acts as a coactivator for steroid hormone receptors. Mol Cell Biol 24:87168726[Abstract/Free Full Text]
- Gonzalez F, Delahodde A, Kodadek T, Johnston SA 2002 Recruitment of a 19S proteasome subcomplex to an activated promoter. Science 296:548550[Abstract/Free Full Text]
- Reid G, Hubner MR, Metivier R, Brand H, Denger S, Manu D, Beaudouin J, Ellenberg J, Gannon F 2003 Cyclic, proteasome-mediated turnover of unliganded and liganded ER
on responsive promoters is an integral feature of estrogen signaling. Mol Cell 11:695707[CrossRef][Medline]
- Kang Z, Pirskanen A, Janne OA, Palvimo JJ 2002 Involvement of proteasome in the dynamic assembly of the androgen receptor transcription complex. J Biol Chem 277:4836648371[Abstract/Free Full Text]
- McNally JG, Muller WG, Walker D, Wolford R, Hager GL 2000 The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science 287:12621265[Abstract/Free Full Text]
- Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M 2000 Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 103:843852[CrossRef][Medline]
- Stenoien DL, Patel K, Mancini MG, Dutertre M, Smith CL, OMalley BW, Mancini MA 2001 FRAP reveals that mobility of oestrogen receptor-
is ligand- and proteasome-dependent. Nat Cell Biol 3:1523[CrossRef][Medline]
- Metivier R, Penot G, Hubner MR, Reid G, Brand H, Kos M, Gannon F 2003 Estrogen receptor-
directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115:751756[CrossRef][Medline]
- Schaaf MJ, Cidlowski JA 2003 Molecular determinants of glucocorticoid receptor mobility in living cells: the importance of ligand affinity. Mol Cell Biol 23:19221934[Abstract/Free Full Text]
- Lonard DM, Nawaz Z, Smith CL, OMalley BW 2000 The 26S proteasome is required for estrogen receptor-
and coactivator turnover and for efficient estrogen receptor-
transactivation. Mol Cell 5:939948[CrossRef][Medline]
- Wang X, DeFranco DB 2002 Glucocorticoid receptors in hippocampal neurons that do not engage proteasomes escape from hormone-dependent down-regulation but maintain transactivation activity. Mol Endocrinol 16:19871998[Abstract/Free Full Text]
- Ballinger CA, Connell P, Wu Y, Hu Z, Thompson LJ, Yin LY, Patterson C 1999 Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions. Mol Cell Biol 19:45354545[Abstract/Free Full Text]
- Dai Q, Zhang C, Wu Y, McDonough H, Whaley RA, Godfrey V, Li H-H, Madamanchi N, Xu W, Neckers L, Cyr D, Patterson C 2003 CHIP activates HSF1 and confers protection against apoptosis and cellular stress. EMBO J 22:54465458[Abstract/Free Full Text]
- Cardozo CP, Michaud C, Ost MC, Fliss AE, Yang E, Patterson C, Hall SJ, Caplan AJ 2003 C-terminal Hsp-interacting protein slows androgen receptor synthesis and reduces its rate of degradation. Arch Biochem Biophys 410:134140[CrossRef][Medline]
- Sengupta S, Wasylyk B 2001 Ligand-dependent interaction of the glucocorticoid receptor with p53 enhances their degradation by Hdm2. Gen Dev 15:23672380[Abstract/Free Full Text]
- Kinyamu HK, Archer TK 2003 Estrogen receptor-dependent proteasomal degradation of the glucocorticoid receptor is coupled to an increase in mdm2 protein expression. Mol Cell Biol 23:58675881[Abstract/Free Full Text]
- He B, Bai S, Hnat AT, Kalman RI, Minges JT, Patterson C, Wilson EM 2004 An androgen receptor NH2-terminal conserved motif interacts with the COOH terminus of the Hsp70-interacting protein (CHIP). J Biol Chem 279:3064330653[Abstract/Free Full Text]
- Verma R, Oania R, Graumann J, Deshaies RJ 2004 Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system. Cell 118:99110[CrossRef][Medline]
- Deveraux Q, Ustrell V, Pickart C, Rechsteiner M 1994 A 26 S protease subunit that binds ubiquitin conjugates. J Biol Chem 269:70597061[Abstract/Free Full Text]
- Demand J, Alberti S, Patterson C, Hohfeld J 2001 Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteasome coupling. Curr Biol 11:15691577[CrossRef][Medline]
- Wei Q, Lu XY, Liu L, Schafer G, Shieh K-R, Burke S, Robinson TE, Watson SJ, Seasholtz AF, Akil H 2004 Glucocorticoid receptor overexpression in forebrain: a mouse model of increased emotional lability. Proc Natl Acad Sci USA 101:1185111856[Abstract/Free Full Text]
- Kaufer D, Ogle WO, Pincus ZS, Clark KL, Nicholas AC, Dinkel KM, Dumas TC, Ferguson D, Lee AL, Winters MA, Sapolsky RM 2004 Restructuring the neuronal stress response with anti-glucocorticoid gene delivery. Nat Neurosci 7:947953[CrossRef][Medline]
- De Kloet ER, Vreugdenhil E, Oitzl MS, Joels M 1998 Brain corticosteroid receptor balance in health and disease. Endocr Rev 19:269301[Abstract/Free Full Text]
- El-Khodor BF, Kholodilov NG, Yarygina O, Burke RE 2001 The expression of mRNAs for the proteasome complex is developmentally regulated in the rat mesencephalon. Brain Res Dev Brain Res 129:4756[CrossRef][Medline]
- Gametchu B, Harrison RW 1984 Characterization of a monoclonal antibody to the rat liver glucocorticoid receptor. Endocrinology 114:274279[Abstract]