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J. Biol. Chem., Vol. 279, Issue 47, 49307-49314, November 19, 2004
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From the Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
Received for publication, June 29, 2004 , and in revised form, August 26, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-ol-20-one-16
-carbonitrile (PCN), followed by an increase in Cyp3a11 mRNA. Expression of wild type PXR and various mutants as green fluorescent fusion proteins in mouse livers showed that PXR was retained in the cytoplasm from where PCN treatment translocated PXR into the nucleus. Furthermore, the xenochemical response signal, the nuclear translocation signal, and the activation function 2 domain were all required for the nuclear translocation to occur. Immunoprecipitation experiments using the hsp90 antibody demonstrated the presence of PXR in a complex with the endogenous cytoplasmic constitutive active/androstane receptor retention protein (CCRP) in HepG2 cells. Fluorescence resonance energy transfer analysis of mouse liver sections after co-expression of cyan fluorescent protein-CCRP and yellow fluorescent protein-PXR also indicated that CCRP and PXR were closely associated in vivo. Overexpression of exogenous CCRP increased the cytoplasmic level of the PXR·CCRP·hsp90 complex, whereas a decrease in endogenous CCRP by treatment with small interfering RNA for CCRP repressed the PXR-mediated reporter activity in HepG2 cells. We conclude that the CCRP mediates the retention of PXR in the cytosol and modulates the activation of PXR in response to PCN treatment. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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helices that mediate protein-protein interactions and the assembly of multiprotein complexes (10).
The movement of proteins between the nucleus and the cytoplasm is an energy-dependent process determined by both nuclear localization sequence (NLS) and nuclear export sequence on the protein (11). The typical NLS consists of a single or repeat cluster of basic amino acids that associate with factors such as importins, which carry the proteins into the nucleus through a nuclear pore complex. The typical nuclear export sequence is a leucine-rich region and has been identified on a number of proteins, including the aryl hydrocarbon receptor (12, 13). After binding to ligand, the nuclear receptor GR moves along cytoskeletal tracks toward the nucleus by connecting with dynein motors (reviewed in Ref. 13). The C-terminal AF2 domain is required for ligand-dependent transcriptional activation by steroid hormone and many other nuclear receptors (14), and it is displaced upon the binding of ligand, allowing it to interact with co-activators. The removal of the AF2 domain prevents the nuclear translocation of the VDR and GR (15, 16). However, the AF2 domain is not required for the nuclear translocation of CAR following PB exposure (17). The nuclear translocation of CAR following drug exposure is dependent on the xenochemical response sequence (XRS), a leucine-rich sequence near the C terminus conserved as (L/M)XXLXXL (17) in mouse and human CAR and PXR. However, mutations in the key leucine residues in the XRS did not affect the formation of heterodimers between CAR and RXR or the co-activation of CAR by SRC-1. This suggests that the XRS regulates the translocation but not the activation of CAR.
In contrast to CAR, PXR is generally thought to be primarily retained in the nucleus where it activates the expression of genes such as Cyp3a11 after binding ligands such as PCN. Thus, the possibility that co-chaperones could regulate the cellular localization of PXR has not been investigated. However, recent results of the immunostaining of mouse liver sections using a commercially available antibody suggest that mPXR may be located in the cytosol of untreated liver cells (18); however, additional supporting experiments, such as Western blot analysis, were not presented. Moreover, in transformed cells, human hPXR (SXR) is located primarily in the nucleus, and mutation of specific amino acids in the so-called NLS region of the DNA binding domain of hPXR resulted in cytoplasmic localization of the receptor in transformed cells (18). However, the role of the NLS as well as other specific motifs (AF2 and XRS) in the nuclear translocation of mPXR in the liver following drug treatment remains unexplored.
Our goal was to use more definitive methods to determine whether mPXR was located in the cytoplasm of untreated liver and translocated to the nucleus only after exposure to xenochemicals. To achieve this, we produced specific antibodies against the hinge region of mPXR and used these antibodies in the Western blotting of liver nuclear extracts prepared from control mice and those treated with PCN. We also investigated the interaction of mPXR with CCRP in the cytosol to determine whether CCRP played a role in maintaining mPXR in the cytosol. We then looked at the NLS, XRS, and the AF2 domain, which may be involved in the nuclear translocation of mPXR in response to drug treatment, to determine whether these components also affected the association of mPXR with CCRP. Finally, we investigated the role of endogenous CCRP in the activation of mPXR in response to drug treatment.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Plasmids—cDNAs encoding full-length mPXR, mPXR
AF2 (residues 1–416), hPXR, mCAR, and CCRP were PCR-amplified using the appropriate primers and cloned into pcDNA3.1/V5-His-TOPO (Invitrogen) to produce pcDNA3.1/mPXR-V5-His, pcDNA3.1/mPXRAF2-V5-His, pcDNA3.1/hPXR-V5-His, pcDNA3.1/mCAR-V5-His, and pcDNA3.1/CCRP-V5-His. mPXR and CCRP were also amplified using 5' primers containing an in-frame XhoI site and 3' primers containing an EcoRI site and cloned into pEYFP-C1 or pECFP-C1 expression vectors (BD Biosciences) to produce an N-terminal fusion with green fluorescent protein. The XREM-3A4-Luc (p3A4–362(7836/7208ins)) reporter plasmid was kindly provided by Dr. Bryan Goodwin (GlaxoSmithKlein) (20). The following mutants were constructed using the QuikChange site-directed mutagenesis kit (Stratagene, Cedar Creek, TX) and the appropriate primers: pcDNA3.1/mPXR-V5-His or pEYFP-mPXR containing either the mutations M391A, L394A, or L397A in the XRS region and R63/R64A/R85/K86A or R63/R64A/R88A/R89A in the NLS region. All plasmids were verified by nucleotide sequencing and prepared using a Qiagen plasmid maxi kit (Qiagen, Valencia, CA).
Expression of Fluorescent Protein-tagged PXR and CCRP in Mouse Liver in Vivo—Expression of pEYFP-mPXR, its various mutants, and pECFP-CCRP in mouse liver in vivo and their detection by microscopy was performed as described previously (7, 21). Plasmids were injected via the tail veins of male CD-1 mice using the TransIT in vivo gene delivery system (Mirus, Maddison, WI) according to the manufacturer's instructions. Two h later, the mice were injected intraperitoneally with either 15 mg/kg PCN, 100 mg/kg PB, or Me2SO vehicle and killed 6 h later. Liver sections were analyzed by confocal laser scanning microscopy using a Zeiss LSM510 META microscopy system. For FRET analysis, 458-nm excited signals were captured twice before and twice after YFP photobleaching using twenty-five pulses of a 514-nm laser. Wavelength scanning images between 450 and 540 nm were collected to extract simultaneously each of the YFP and CFP emissions using software provided by Zeiss. Dequenching of CFP was quantified for multiple liver cells, and FRET efficiency E was calculated with the equation, E = 1 - (Ii/Ii0), where Ii is fluorescence intensity before the bleaching and Ii0 is that of after the bleaching. The distance between the CFP donor and YFP acceptor was calculated using an R0 value (the distance at which energy transfer efficiency is 50% in ideal conditions) of 4.9 nm (22).
Cells and Transfection Assay—HepG2 cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum and antibiotics (100 units/ml of penicillin and 100 µg/ml streptomycin). At 
50% confluence, the cells were transfected with plasmids using LipofectAMINE 2000 (Invitrogen) according to manufacturer's instructions. Twenty-four h later, the cells were given fresh medium, and a further 24 h later, the cells were harvested, washed twice with phosphate-buffered saline, and homogenized with a Dounce homogenizer in buffer A (10 mM HEPES buffer, pH 7.6, containing 10 mM KCl, 1.5 mM MgCl2, 20 mM Na2MoO4, 0.3% Nonidet P-40 (Calbiochem, La Jolla, CA), 1 mM dithiothreitol, and Complete protease inhibitor (Roche Diagnostics) as described in Refs. 7 and 23). The homogenate was centrifuged at 4,000 x g for 10 min to obtain a nuclear pellet, and the supernatant was centrifuged at 17,800 x g for 10 min to obtain a clear cytosolic fraction for use in immunoprecipitation. The pellet was washed once in buffer A, once in buffer A without Nonidet P-40, and then suspended in lysis buffer (10 mM HEPES buffer, pH 7.6, containing 10% glycerol, 100 mM KCl, 3 mM MgCl2, 1 mM Na3VO4, 1 mM dithiothreitol, and Complete protease inhibitor). The resulting suspension was mixed at 4 °C for 12 h in the presence of 0.4 M NaCl and centrifuged for 10 min at 17,800 x g to obtain a clear nuclear extract.
Preparation of Mouse Liver Nuclear Extracts—Nuclear extracts were prepared based on published methods (24, 25). Liver was homogenized in 20 volumes of 10 mM HEPES buffer, pH 7.6, containing 2 M sucrose, 10% glycerol, 25 mM KCl, 0.15 mM spermidine, 0.5 mM spermine, and 1 mM EDTA using a motor-driven glass Teflon homogenizer kept on ice. The homogenate was then layered over a cushion of the same buffer and centrifuged at 85,000 x g for 60 min at 4 °C. The pellet was suspended in 1 ml of lysis buffer (as described above), mixed at 4 °C for 30 min in the presence of 0.4 M NaCl, and centrifuged at 100,000 x g for 30 min. The supernatant was dialyzed overnight against 20 mM HEPES buffer, pH 7.6, containing 20% glycerol, 0.2 mM EDTA, 1 mM Na2MoO4, 1 mM dithiothreitol, and Complete protease inhibitor.
Immunoprecipitation—Anti-hsp90 (5 µl) or IgM (1 µg), as a control, was added to 2 mg of cytosolic protein from HepG2 cells and incubated overnight at 4 °C. Twenty µl of a 50% slurry of protein L-Sepharose (ImmunoPure immobilized protein L, Pierce) previously washed in buffer A was added, and the mixture was incubated for 1 h at 4 °C. The resin was recovered by centrifugation and washed five times with 1 ml of 50 mM Tris buffer, pH 7.5, containing 0.15 M NaCl, 20 mM Na2MoO4, and 0.2% Nonidet P-40. The immunoprecipitated proteins were extracted from the resin with NuPAGE LDS buffer (Invitrogen) and subjected to Western blotting.
Western Blotting—Proteins were separated either on a NuPAGE 4–10% BisTris gel in NuPAGE MOPS SDS running buffer (Invitrogen) or a 10% SDS gel in Tris-glycine buffer and transferred to nitrocellulose membrane using the SemiPhor semi-dry transfer unit (Hoefer Scientific Instruments, San Francisco, CA). The membrane was then incubated for 1 h in a mixture of TBS and 0.1% Tween 20 containing 5% Blotto milk powder followed by1hof incubation with primary antibody (rabbit anti-mPXR, rabbit anti-mCAR) in the same medium and 1 h with secondary antibody (donkey anti-rabbit IgG horseradish peroxidase conjugate, Santa Cruz Biotechnology). For the detection of V5-tagged proteins, the nitrocellulose membranes were incubated with anti-V5-horseradish peroxidase conjugate in a mixture of TBS and Tween 20 with 5% Blotto. The protein bands were visualized on the membranes using Lumigen PS-3 ECL detection reagent (Amersham Biosciences). In some cases, the immunoblots were stripped with Restore Western blot stripping buffer (Pierce) and restained with antibodies.
GST Pull-down Assay—The GST-CCRP fusion protein was expressed and purified using glutathione-Sepharose 4B (Amersham Biosciences) (7). 35S-labeled mPXR and mCAR were produced from pcDNA3.1/mPXR-V5-His and pcDNA3.1/mCAR-V5-His using the TNT T7 quick coupled transcription/translation system (Promega) along with 35S-labeled methionine. GST-CCRP or GST coupled to the glutathione-Sepharose 4B was incubated with the 35S-labeled mPXR or mCAR in 50 mM HEPES buffer, pH 7.5, containing 0.1 M NaCl and 0.1% Triton X-100 for 20 min at room temperature. The resin was then recovered by centrifugation and washed three times in the same buffer. Proteins were extracted from the resin by heating for 10 min at 70 °C in NuPAGE LDS sample buffer (Invitrogen) and separated on a NuPAGE 4–10% BisTris gel in NuPAGE MOPS SDS running buffer for1hat150 volts. The gel was then stained with Coomassie Blue, destained and dried under vacuum, and the proteins detected by autoradiography.
siRNA Treatment and Reporter Assay—HepG2 cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum and antibiotics (100 units/ml of penicillin and 100 µg/ml streptomycin) to about 50% confluence. The cells were transfected with pcDNA3.1/mPXR-V5-His or pcDNA3.1/hPXR-V5-His, the XREM-3A4-Luc reporter, and phRL-tk as a transfection control (Promega) using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. For RNA interfering experiments, HepG2 cells were transfected with siRNA against CCRP (SMART pool siRNA M-019566–00 from Dharmacon, Inc., Boulder, CO) or an unrelated scramble (5'-ACUCUAUCGCCAGCGUGACUUdTdT-3') using LipofectAMINE 2000 reagent according to the manufacturer's directions. After 48 h, half of the wells were treated with PCN or rifampicin, and after a further 24 h, the luciferase activity of the XREM-3A4-Luc reporter and the control phRL-tk was measured using the Dual Luciferase reporter assay system (Promega). The ratio of XREM-3A4-Luc reporter activity to Renilla luciferase control was calculated for triplicate transfections.
Real-time PCR—To measure CYP3A11 mRNA, liver samples were homogenized in TRIzol reagent (Invitrogen), and total RNA was prepared according to the manufacturer's instructions. Five µg of RNA was used as a template for cDNA synthesis using the SuperScript first strand synthesis system (Invitrogen) with random hexamers as primers. One-twentieth of the cDNA was used for real-time PCR with an ABI Prism 7700 sequence detector using TaqMan Universal PCR reaction mix and primers for mouse CYP3A11 (Applied Biosystems, Foster City, CA) or glyceraldehyde-3-phosphate dehydrogenase as a control.
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RESULTS |
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Structural Components of mPXR That Affect Nuclear Translocation—We investigated the cellular localization of mPXR by in vivo transfection of YFP-tagged mPXR in mouse liver. In untreated liver, YFP-mPXR was localized throughout the cytoplasm of the liver cell (Fig. 2A). However, in liver sections from mice that had been treated with PCN, YFP-mPXR was present predominantly in the nucleus. This cytoplasmic retention and nuclear translocation of YFP-mPXR are reminiscent of what occurs with the endogenous receptor. To identify the structural features of mPXR that affect nuclear translocation in vivo, we expressed mPXR and its various mutants as YFP fusion proteins in mouse livers. These mPXR mutants contained substitutions in the XRS region or the NLS region in the DNA binding domain or had the C-terminal AF2 region deleted (Fig. 2B). We then measured the percentage of cells with mPXR expressed predominantly in the cytoplasm, the percentage with equal nuclear and cytoplasmic localization, and the percentage with primarily nuclear localization of mPXR (Fig. 2A). Wild type YFP-mPXR was located primarily in the cytoplasm in untreated mouse liver and translocated to the nucleus after treatment with PCN. However, mutations in key amino acid residues in the XRS or NLS region or deletion of the AF2 domain prevented nuclear translocation in vivo in response to treatment with PCN. The distribution of the NLS and XRS mutants remained almost entirely cytoplasmic, whereas the AF2 deletion mutant had slightly more nuclear distribution, which was not affected by PCN treatment. These results suggest that the XRS, NLS, and AF2 regions are all required for the nuclear translocation of mPXR after PCN treatment in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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PXR transfected into transformed cells normally accumulates spontaneously in the nucleus without drug treatment. Using this model, Kawana and his associates (18) identified a NLS region in hPXR (SXR) that, when mutated, resulted in accumulation of the receptor in the cytosol of transformed cells. They concluded that the NLS is essential in the ligand-independent translocation of human PXR in HeLa cells. They also found that hPXR with the AF2 domain deleted was accumulated in the nucleus, suggesting that the AF2 domain was not necessary for nuclear translocation. The results of our experiments, in which various mutants of mPXR were expressed in mouse liver in vivo, demonstrate that the PCN-dependent nuclear translocation requires not only the NLS but also the AF2 domain. In addition to the NLS and the AF2 domain, mPXR contains the XRS motif that is known to regulate the nuclear translocation of both mouse and human CAR in mouse liver in vivo (17). Our experiments indicate that the XRS motif is also required for the nuclear translocation of mPXR to occur in response to PCN treatment. The differences between the findings of Kawana and associates (18) and the present work may be due to differences between hPXR and mPXR, cell types used, and/or differences between the model of spontaneous accumulation of PXR in the nucleus and nuclear translocation of PXR in response to drug treatment. Because all of the NLS, XRS, and AF2 domains are required for nuclear translocation of mPXR in vivo, they may all function in the mechanism of nuclear translocation of mPXR.
In contrast to mPXR, hCAR does not need either the NLS or the AF2 domain for nuclear translocation, requiring only the ligand binding domain containing the XRS region. CAR lacking its AF2 domain or everything except the C-terminal half of the LB domain (residues 181–348) translocated to the nucleus in mouse liver in vivo following treatment with CAR activators (17, 21). The NLS region of CAR may not be effective for nuclear translocation because of the substitution of a highly conserved lysine by serine in human, rat, and mouse CAR compared with PXR and VDR (18). In fact, a chimeric hPXR containing the NLS of hCAR underwent nuclear translocation as observed with hCAR.2 The AF2 domain regulates receptor function and is activated by the direct binding of ligand. The direct binding of agonistic ligand initiates GR and VDR to translocate into the nucleus (16). In contrast, the mechanism of nuclear translocation of CAR may be distinct, because the CAR activator PB does not bind to CAR, and the AF2 domain is not required for translocation of CAR. Because PCN binds directly to mPXR and the translocation requires the AF2 domain, the regulatory mechanism for nuclear translocation may be similar to that of GR and VDR.
CCRP (a mouse ortholog of human TPR2 (26)), a co-chaperone of the class III TPR family, which includes proteins that are involved in antiviral interferon response, the stress response, and protein import, was identified as a CAR binding protein by yeast two-hybrid screening of a mouse liver cDNA library using CAR as bait. CCRP, acting as a co-chaperone, mediates the formation of the CAR·CCRP·hsp90 complex in the cytosol of HepG2 cells (7). We have shown here that mPXR can also bind to the co-chaperone CCRP to form a complex within the cytosol along with hsp90, increasing the retention of mPXR in the cytosol of HepG2 cells. In addition, our FRET analysis indicates that CCRP and mPXR are closely associated in mouse liver in vivo. CCRP has also been reported to bind to the GR and is required at a narrowly defined expression limit for maximal activation of the GR (27). Similar to the GR activity, we have found that the mPXR-mediated trans-activation of genes is modulated by CCRP in HepG2 cells. These observations suggest that CCRP may play a role not only in the cytoplasmic retention of the receptors, but also in their activation in the nucleus. The role of hsp90 and TPR proteins in the formation of complexes with signaling proteins, such as steroid receptors, and their movement to the nucleus has recently been reviewed (14). The GR forms a complex with hsp90 and immunophilins, which are TPR proteins that link the complex to dynein motor proteins for retrograde movement along microtubules to the nucleus. However, the mechanism by which CCRP regulates the retention and activation of these receptors remains an interesting target for further investigations.
In summary, we have shown that mPXR is located in the cytoplasm of untreated liver cells and is concentrated in the nucleus following drug treatment. mPXR forms a complex with CCRP and hsp90 that maintains the receptor in the cytosol. The formation of the mPXR·CCRP·hsp90 complex is not dependent on the XRS, NLS, or AF2 domains in mPXR. However, these regions are all required for the nuclear translocation of mPXR. Thus, although CCRP is involved in maintaining mPXR in the cytosol, the binding of mPXR to CCRP does not regulate the nuclear translocation of mPXR in response to PCN treatment. However, CCRP does modulate the activation of mPXR in response to drug treatment. This demonstration of the cytoplasmic localization of mPXR in untreated liver and the role of CCRP should stimulate future research on the mechanism of nuclear localization and activation of PXR following drug treatment.
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FOOTNOTES |
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Present address: Dept. of Animal and Poultry Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
To whom all correspondence should be addressed. Tel.: 919-541-2404; Fax 919-541-0696; E-mail: negishi{at}niehs.nih.gov.
1 The abbreviations used are: PXR, pregnane X receptor; hPXR, human PXR; mPXR, mouse PXR; AF2, activation function 2; CAR, constitutive active/androstane receptor; mCAR, mouse CAR; CCRP, cytoplasmic CAR retention protein; FRET, fluorescence resonance energy transfer; GR, glucocorticoid receptor; hsp90, heat shock protein 90; NLS, nuclear localization sequence; PB, phenobarbital; PCN, 5-pregnen-3-ol-20-one-16-carbonitrile; TPR, tetratricopeptide repeat; VDR, vitamin D receptor; XRS, xenochemical response sequence; Me2SO, dimethyl sulfoxide; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MOPS, 4-morpholinepropanesulfonic acid; GST, glutathione S-transferase; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; siRNA, small interfering RNA; ROI, region of interest.
2 T. Sueyoshi and M. Negishi, unpublished observation.
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