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Endocrine Sciences Research Group and Centre for Molecular Medicine, Faculty of Medicine, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK
1 Molecular Medicine Unit, CSB, St. James’s University Hospital, University of Leeds, Leeds LS9 7TF, UK
(Requests for offprints should be addressed to D Ray; Email: david.w.ray{at}man.ac.uk)
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Abstract |
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Introduction |
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Upon ligand binding, the GR translocates from the cytoplasm to the nucleus where it binds to specific DNA sequences termed glucocorticoid-response elements (GREs) to regulate target gene transcription. A number of studies have shown that cellular sensitivity to glucocorticoids is influenced by glucocorticoid receptor density (Cidlowski & Cidlowski 1981, Vanderbilt et al. 1987, Hoeck et al. 1989, Silva et al. 1994) with ligand-binding initiating a process of glucocorticoid receptor downregulation. This ligand-dependent receptor downregulation is mediated both at the level of gene transcription as well as protein stability (Svec & Rudis 1981, McIntyre & Samuels 1985, Dong et al. 1988, Vedeckis et al. 1989, Oakley & Cidlowski 1993).
The GR is subject to a number of post-translational modifications, which include phosphorylation and ubiquity-lation. Activation of the receptor by ligand binding initiates N-terminal phosphorylation (Weigel 1996, Bodwell et al. 1998). The phosphorylation state of proteins can regulate their stability (Webster et al. 1997), by targeting the protein for polyubiquitylation and subsequent degradation via the 26S proteasome (Fuchs et al. 1998, Kornitzer & Ciechanover 2000, Wallace & Cidlowski 2001, Deroo et al. 2002). The recognition of a phosphorylated substrate relies on its interaction with a specific ubiquitin-protein ligase (E3). Protein ubiquitylation is an energy-dependent process, which requires sequential transfer of ubiquitin from an ubiquitin-activating enzyme (E1), to an ubiquitin-conjugating enzyme (E2) to target protein generally facilitated by an E3. Ubiquitylation of the GR is regulated by at least three different E3s: CHIP (Connell et al. 2001, Wang & DeFranco 2005), E6-AP (Nawaz & O’Malley 2004), and hmdm2 (human homolog of mdm2) via the formation of a trimeric complex with p53 (Sengupta & Wasylyk 2001).
Interestingly, inhibition of proteasomal activity decreases the ligand-induced transcriptional activities of the estrogen and thyroid hormone receptors (Dace et al. 2000, Lonard et al. 2004). This observation suggests that protein ubiquitylation of these receptors, and/or regulatory co-factors, is required for continued transcriptional activity. By contrast, chemical inhibition of the proteasome increased the ligand-induced transactivation potential of the GR, while blocking the ligand-dependent downregulation of GR levels (Wallace & Cidlowski 2001, Wang & DeFranco 2005). These data point to a GR-specific mode of regulation by the ubiquitin–proteasome pathway. Importantly, the E3 ligase, CHIP, may determine the GR response to ubiquitylation by regulating the coupling to the ubiquitin/proteasome-dependent protein degradation pathway (Wang & DeFranco 2005).
Recently, Perissi et al.(2004) demonstrated that the E2, UbcH7, was recruited to DNA by activated nuclear receptors. This interaction enhanced their transcriptional activities. Moreover, UbcH7 modulated nuclear receptor transactivation, including GR function, by interaction with the co-activator SRC-1 (Verma et al. 2004). Independently, we identified UbcH7 as the physical interacting partner of the GR in a yeast two-hybrid screen. Here, we demonstrate that UbcH7 interacts directly with the GR in vitro and inhibits its transactivation function via targeting the protein for proteasome degradation. Therefore, UbcH7 interacts with multiple members of the nuclear receptor superfamily, and with their co-modulator proteins, but results in differential effects on their transcriptional activity depending on the model used. This may indicate the underlying differences in the mechanisms of transcriptional regulation used by these closely related proteins.
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Methods |
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Full-length UbcH7 was cloned into pcDNA3-GFP to create UbcH7.pcDNA3 (Ardley et al. 2001). The UbcH7 C89S mutant was generated from UbcH7.pcDNA3 to create C89SUbcH7.pcDNA3 using the QuikChange site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands) according to the manufacturer’s protocol (Ardley et al. 2003). Mouse mammary tumor virus long terminal repeat luciferase reporter gene (MMTV-luc) and pcDNA3-GR have been previously described (Stevens et al. 2003a, Waters et al. 2004). Cytomegalovirus (CMV)-renilla vector was obtained from Promega, and used to control the transfection efficiency as previously described (Stevens et al. 2003a,b).
Yeast two-hybrid screen
Total RNA was extracted from 1 x 109 untreated CORL103 cells using TRIzol reagent (GIBCO BRL). An mRNA was isolated from 3 mg total RNA using GenElute mRNA isolation (SIGMA), according to the manufacturer’s guidelines. A cDNA library was constructed in B42 AD vector of the MATCHMAKER LexA hybrid screen system (Clontech) using the restriction endonucleases EcoRI and XhoI to ensure the correct orientation of the insert cDNA using the cDNA synthesis kit as per the manufacturer’s instruction (Stratagene). This library formed the ‘prey’ for the yeast two-hybrid screen. Construction of the GR ‘bait’ construct (LexA-GR525–777) was previously described (Stevens et al. 2003a).
Saccharomyces cerevisiae (EGY48) were sequentially transformed with reporter, bait, and then prey DNA constructs. Transformations were performed using a standard lithium acetate procedure (Stevens et al. 2003a). The transfected yeast cells were replica-plated onto synthetic dropout/Gal/ Raf plates with X-gal, containing the appropriate treatment (50 µM RU486 (mifepristone) or dimethylsulfoxide vehicle as the solvent control). The cDNA library plasmid DNA was isolated from positive colonies and re-introduced into fresh S. cerevisiae to confirm a positive interaction between the C-terminus of the GR (GR525–777) and prey. Positive prey DNA were isolated, sequenced, and identified using BLAST.
Immunoprecipitation
Cells were lysed in radioimmunoprecipitation assay buffer (RIPA) buffer (50 mM Tris–HCl buffer (pH 7.4) containing 1% (v/v) NP-40, 0.25% (w/v) sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, and complete protease-inhibitor cocktail). Protein concentration was determined using the Bio-Rad Bradford assay reagent. Total cellular lysate protein, 500 µg, was incubated with 2.0 µg antibodies and 50 µl resuspended protein A agarose beads (Santa Cruz Biotechnology, Santa Cruz, USA) for 16 h at 4 °C on a tube rotator. The beads were captured by centrifugation, and washed thrice in RIPA buffer. The bound proteins were released by boiling in SDS-PAGE loading buffer for 5 min.
Immunoblotting
GR (M20; Santa Cruz; Waters et al. 2004), UbcH7 (Ardley et al. 2003), and ß-actin (Sigma; anti-actin) antibodies were employed. Samples were resolved on SDS-PAGE gels, and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were blocked for 6 h with 5% (w/v) non-fat dry milk in Tris-buffered saline with 0.05% (v/v) Tween 20 and incubated for 16 h with either M20 (1:2000), UbcH7 (1:3000) or ß-actin (1:2000) primary antibodies diluted in blocking buffer. The membranes were washed thrice, and blotted with secondary antibody conjugated with horse-radish peroxidase. After three more washes, the membrane was developed with Pierce Supersignal West Pico reagent. Membranes were subsequently stripped and blotted for ß-actin to confirm equal loading of samples and transfer of protein.
Transfection
COS7 cells, and HeLa cells were cultured in Dulbecco’s modification of Eagle’s medium (DMEM) with Glutamax-1 (Invitrogen Life Technologies), and 10% (v/v) fetal calf serum before being seeded at 5 x 105 cells/10 cm tissue culture dish. All transfections were performed using Fugene 6 according to the manufacturers’ instructions (Roche). For glucocorticoid reporter gene studies, cells were transfected with 2.0 µg MMTV-luc, 0.5 µg pcDNA3-GR, 0.3 or 1.0 µg pcDNA3-UbcH7-GFP or empty vector control, and 0.2 µg CMV-Renilla. Post-transfection cells were transferred to DMEM with Glutamax-1 and 10% (v/v) charcoal/dextran-stripped calf serum, divided into 24-well plates and treated in triplicate with steroid for 18 h before harvest. For chemical inhibition of the proteasome, cells were transfected with 2.0 µg MMTV-luc, 0.5 µg pcDNA3-GR, 1.0 µg pcDNA3-UbcH7-GFP, the C89S.UbcH7 mutant or the empty vector control, and 0.2 µg CMV-Renilla. Cells were then transferred into 10% (v/v) charcoal/dextran-stripped fetal calf serum containing media and split into 12-well plates. They were then pre-treated for 1 h with 1.0 µM MG-132 (Calbiochem) or vehicle then with 100 nM dexamethasone as appropriate. Cell lysates were subjected to dual luciferase assay as per the manufacturer’s instructions (Promega). Firefly luciferase results were normalized using renilla luciferase as control for differences in transfection efficiency. All transfections were performed on at least three occasions with similar results.
UbcH7 localization
COS7 cells were sedimented, resuspended in medium supplemented with 5% (v/v) charcoal/dextran-stripped serum (Hyclone, UK) and seeded onto 22 mm glass coverslips at a density of 3 x 105 cells/slide. After 24 h, cells were transfected with 0.5 µg pcDNA3-GR and either 0.5 µg UbcH7-green fluorescent protein (GFP) or 0.5 µg C89S-GFP using Fugene 6 (Roche). Eighteen hours post-transfection, cells were treated with vehicle (dimethylsulphoxide; DMSO) or dexamethasone (100 nM) for 1 h before fixing with 4% (w/v) paraformaldehyde. To visualize the GR, cells were then washed thrice with tris buffered saline (TD) buffer (10 mM Tris–HCl buffer (pH 8.0) containing 150 mM NaCl) and treated with blocking buffer (TD plus 1% (w/v) BSA, 0.2% (v/v) Triton X-100) for 1 h at 20 °C. A 1:200 dilution of primary antibody, P20 sc-1002 (Santa Cruz) in washing buffer (TD plus 1% (w/v) BSA, 0.05% (v/v) Triton X-100), was added for 2 h at 20 °C. Cells were then washed thrice with wash buffer. A 1:200 dilution of the appropriate secondary antibody (Alexa Fluor 568 goat anti-rabbit IgG; Molecular Probes, Eugene, OR, USA) in washing buffer was added for 1 h at 20 °C. The cells were then washed thrice and mounted on slides using Citifluor (glycerol/PBS; Citifluor Ltd, UK). The coverslips were sealed and stored at 4 °C. Images were taken using a Leica TCS-4D confocal microscope (Leica Microsystems, Heidelberg, Germany) using a 63 x water immersion objective. To visualize Alexa 568, cells were viewed using an excitation filter of 568 nm and the emission was collected using a 590 nm long pass filter. To visualise GFP, cells were excited with an argon laser at 488 nm and emission was collected using a band-pass filter of 525 ± 25 nm.
Statistical analysis
Comparison of group data was done by ANOVA followed by Bonferroni t-test. Significance was taken as < 0.05, and all significant differences found are marked on the graphs. The package SPSS for windows 11.5 was used for analysis.
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Results |
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We used the C-terminal domain of the GR (amino acid residues 525–777) to screen a yeast two-hybrid library generated from the human small cell lung cancer cell line CORL103. We were interested in finding proteins that interacted in the presence of the GR antagonist RU486. Fifteen clones were identified as interacting with the GR bait in the presence of RU486. Interactions were confirmed in re-transformed yeast cells. Clones were sequenced, and revealed that one, XP15, had 100% identity with residues 41–154 of the coding sequence of UbcH7. The interaction between XP15 and the GR was strongest in the presence of RU486. These results identify UbcH7 as interacting with the C-terminus of the GR.
In vivo interaction between the GR and UbcH7
We studied the interaction between UbcH7 and the GR in COS7 cells in order to ensure that binding also occurred under physiological conditions in mammalian cells. These cells were chosen for two reasons. First, we demonstrated that transfected GR levels were reduced when the cells were cultured in the presence of glucocorticoid (Fig. 1a
), and second that they expressed endogenous UbcH7 (Fig. 1b).
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). Therefore, the mechanism of action of glucocorticoid is not through a modulation of UbcH7 levels.
Immunoprecipitation with GR antibodies of COS7 cell lysates previously transfected with either UbcH7-GFP or C89S.UbcH7-GFP was followed by immunoblotting for UbcH7. Initial studies performed in the absence of MG-132, the 26S proteosome inhibitor, or dominant negative UbcH7, did not show co-immunoprecipitated UbcH7 with the GR. UbcH7 was only detected in lysates prepared from those cells transfected with the C89S.UbcH7-GFP construct, and co-incubated with MG-132. These levels were enhanced in the presence of dexamethasone (Fig. 1c). Cells were co-incubated with MG-132 to inhibit proteasomal degradation of ubiquitinated proteins.
Analysis of the effect of glucocorticoid on the localization of UbcH7 and GR in COS7 cells
We employed a combined immunofluorescence, transfected fluorophore procedure to examine if UbcH7 and GR shared an overlapping intracellular distribution. This would not only provide further evidence of interaction, but may also evidence for an interaction that was restricted to specific subcellular compartments. It would be a prerequisite for functional interaction in vivo.
An intense nuclear and relatively weaker cytoplasmic UbcH7 expression was noted in COS7 cells transfected with the UbcH7-GFP (Fig. 2a). The localization of (C89S).UbcH7 was indistinguishable from that of UbcH7 (Fig. 2b). By immunofluorescence analysis, the GR is expressed principally in the cytoplasm in the absence of ligand (Fig. 2a and b; 0), but almost exclusively in the nucleus in the presence of it (Fig. 2a and b; Dex). The pattern of intranuclear distribution is similar to that we and other researchers, have reported before (Garside et al. 2004). It is clear from the overlapping staining patterns that the site of functional interaction could be either in the cytoplasm or in the nucleus, or both (Fig. 2a and b).
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The data presented earlier indicate that UbcH7 and the GR physically interact within living cells. Consequently, a transient transfection system employing a glucocorticoid sensitive luciferase reporter gene construct was used to examine the functional effects of UbcH7 on glucocorticoid action. We observed that UbcH7 inhibited glucocorticoid-induced transactivation of the reporter in a concentration-dependent manner (Fig. 3). It caused both a significant reduction in the maximum activation and a significant increase in the EC50 (Fig. 3).
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We next determined whether the repressive effect of UbcH7 on dexamethasone-induced transactivation was dependent on the ubiquitin-26S proteasome pathway. As noted earlier (Fig. 3), transactivation was significantly lower when UbcH7 was overexpressed in COS7 cells (Fig. 4a, compare column 1 with column 2). The UbcH7-mediated inhibition of this transactivation was abolished when cells were cultured in the presence of the 26S proteasomal inhibitor, MG132 (Fig. 4a, compare columns 1 and 2 with columns 3 and 4 respectively). Indeed, MG132 enhanced dexamethasone-dependent trans-activation in the absence of exogenous UbcH7 (Fig. 4a, compare column 1 with column 3). These data indicated that protein degradation via the 26S proteasome was necessary for repression of GR signaling by UbcH7.
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, compare column 1 with column 2). There was no additive effect of the MG132 and the dominant negative UbcH7 on glucocorticoid transactivation (Fig. 4b, compare column 2 with column 4).
Transfection of HeLa cells, which express endogenous GR, also showed that UbcH7 impaired and the dominant negative UbcH7 enhanced dexamethasone-dependent transactivation (Fig. 5).
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Discussion |
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Inhibition of either UbcH7, with dominant negative expression, or of the proteosome, with MG132, potentiated glucocorticoid action. Interestingly, other nuclear receptors, notably estrogen receptor, show the opposite effect, with proteasomal inhibition reducing transactivation (Lonard et al. 2004). Hence, we examined directly the effect of UbcH7 on GR protein expression levels. These showed that over-expression of dominant negative UbcH7 resulted in higher GR levels. This further strengthens the conclusion that UbcH7 primarily modifies glucocorticoid sensitivity by regulating glucocorticoid receptor stability in the presence of its ligand. Disruption of this regulatory step, as for example, by overexpression of a dominant negative UbcH7 or inhibition of 26S proteasomal activity by MG132 leads to enhancement of glucocorticoid sensitivity. This mechanism is likely to be an important component in the autoregulation of GR expression as UbcH7 expression is ubiquitous and may, therefore, modulate glucocorticoid sensitivity throughout the body.
Whether UbcH7 acts independent of other factors such as E3s in mediating the downregulation remains to be determined. There may be redundancy in the targeting process as E3s, CHIP, E6-AP, and hmdm2 also interact with the GR (Connell et al. 2001, Sengupta & Wasylyk 2001, Wallace & Cidlowski 2001, Nawaz & O’Malley 2004). The choice of E3 may be dependent on cell type, and indeed, there is evidence that CHIP may contribute not only to the ligand-dependent degradation of GR via the proteasome, but also to transactivation, with overexpression of CHIP reversing the potentiation of transactivation seen with MG132 (Wang & DeFranco 2005). As the GR dissociates from the heat-shock protein complex, it exposes new surfaces. Its conformation, particularly in the C-terminal ligand-binding domain, changes markedly to allow effective recruitment of co-modulator proteins (Stevens et al. 2003a). In addition, the N-terminal region becomes hyperphosphorylated. One or more of these changes could provide a signal for ubiquitylation.
Regulation of GR expression is a key determinant for cell and tissue glucocorticoid sensitivity. There is clearly a complex relationship between GR and its targeting by the ubiquitin system, as evidenced by our studies presented here, and those recently published (Verma et al. 2004, Wang & DeFranco 2005). As the dominant negative UbcH7 appears to enhance GR protein concentration, this suggests that UbcH7 acts to suppress such expression in vivo. Whether this modulation of GR protein expression is sufficient to completely explain the actions of UbcH7 is called into question by the findings of Verma et al.(2004). However, we have demonstrated direct interaction between GR and UbcH7, and showed that proteosomal activity is important for the UbcH7 effect, whereas earlier work showed interaction between UbcH7 and SRC-1 that resulted in UbcH7 potentiating GR transactivation, essentially the opposite result to those presented here (Verma et al. 2004). Therefore, it is possible that UbcH7 is acting both directly on the GR to mediate degradation, and also on SRC-1 to enhance transactivation. If so, the relative expression of GR, co-activator, or E3 (for example, CHIP; Wang & DeFranco 2005) may be critical for determining whether UbcH7 enhances or diminishes GR transactivation. It is also possible that the degree of ubiquitylation is important. For example, monoubiquitylation may be required for transactivation, but once transcription is triggered then polyubiquitylation and proteasomal degradation of the GR may follow. Indeed, there is evidence that the GR contains multiple ubiquitylation sites, but quantification of levels of ubiquitylation has proved difficult (Wallace & Cidlowski 2001). Extending our studies to HeLa cells, those used in the Verma study, showed similar effects of the UbcH7 overexpression to those found in COS cells. Taken together, our results and those of Verma et al.(2004) show that UbcH7 influences GR transactivation but other factors, as has previously been shown for the E3, CHIP, may determine the magnitude and direction of such change. UbcH7 acts on multiple proteins in the assembly of the GR-catalyzed transcription regulatory complex, and interactions between GR and UbcH7 are likely to be of low affinity, and transitory, as evidenced by co-immunoprecipitation studies. Therefore, the net result of altered UbcH7 on transactivation is likely to be dependent on the expression levels of GR, and multiple co-modulator proteins, including SRC-1 (Kaul et al. 2002, Verma et al. 2004).
Modulation of GR expression levels, particularly in response to agonist ligand, plays an important role in determining glucocorticoid sensitivity. Aberrant regulation of these key enzymatic steps may explain pathological alterations in glucocorticoid sensitivity such as those found within sites of inflammation. In addition, the divergent effects of UbcH7 on different members of the nuclear receptor super family with considerable structural homology has implications for understanding underlying differences in the mode of action of these related ligand-activated transcription factors.
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Acknowledgements |
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References |
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Received 8 February 2006
Received in final form 19 May 2006
Accepted 20 June 2006
Made available online as an Accepted Preprint 14 July 2006
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