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Molecular and Cellular Biology, November 2004, p. 9705-9725, Vol. 24, No. 22
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.22.9705-9725.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Retinoid X Receptor Regulates Nur77/Thyroid Hormone Receptor 3-Dependent Apoptosis by Modulating Its Nuclear Export and Mitochondrial Targeting

Cancer Center, The Burnham Institute, La Jolla, California

Received 11 March 2004/ Returned for modification 27 May 2004/ Accepted 10 August 2004

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
Retinoid X receptor (RXR) plays a central role in the regulation of intracellular receptor signaling pathways by acting as a ubiquitous heterodimerization partner of many nuclear receptors, including the orphan receptor Nur77 (also known as thyroid hormone receptor 3 or NGFI-B), which translocates from the nucleus to mitochondria, where it interacts with Bcl-2 to induce apoptosis. Here, we report that RXR is required for nuclear export and mitochondrial targeting of Nur77 through their unique heterodimerization that is mediated by dimerization interfaces located in their DNA-binding domain. The effects of RXR are attributed to a putative nuclear export sequence (NES) present in its carboxyl-terminal region. RXR ligands suppress NES activity by inducing RXR homodimerization or altering RXR/Nur77 heterodimerization. The RXR NES is also silenced by RXR heterodimerization with retinoic acid receptor or vitamin D receptor. Consistently, we were able to show that the mitochondrial targeting of the RXR/Nur77 heterodimer and its induction of apoptosis are potently inhibited by RXR ligands. Together, our results reveal a novel nongenotropic function of RXR and its involvement in the regulation of the Nur77-dependent apoptotic pathway.

	INTRODUCTION

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Retinoid X receptors (RXRs) belong to the nuclear receptor superfamily, consisting of a large number of ligand-regulated transcription factors that mediate the diverse physiological functions of their ligands, such as steroid hormones, retinoids, thyroid hormone, and vitamin D₃, in embryonic development, growth, differentiation, apoptosis, and homeostasis (29, 46). The superfamily also includes many orphan receptors whose ligands remain to be identified. All nuclear receptors consist of three major domains: the variable length N-terminal domain, the well-conserved DNA-binding domain (DBD), and the ligand-binding domain (LBD) (29, 46). The C-terminal LBD is multifunctional and, in addition to harboring a ligand-binding site, contains regions for receptor dimerization and the ligand-dependent transactivation function (AF-2). The DBD also contains a dimerization interface that determines target gene specificity (38, 55, 60, 85). RXRs mediate retinoid signaling through the RXR/retinoic acid receptor (RAR) heterodimer and the RXR/RXR homodimer (29, 46, 89). In addition, RXRs form heterodimers with many members of the subfamily 1 nuclear receptors, including vitamin D receptor (VDR), peroxisome proliferator-activated receptor (PPAR), and thyroid hormone receptor (TR), as well as several orphan receptors, such as liver X receptor, pregnane X receptor, constitutively activated receptor, and Nur77 (TR3 or NGFI-B) (29, 46). RXRs, therefore, play an essential role in the regulation of multiple nuclear hormone-signaling pathways through their unique and potent dimerization capacity. The vitamin A metabolite, 9-cis-retinoic acid (9-cis-RA), is a high-affinity ligand for RXRs (22, 39). It induces transactivation of the RXR homodimer (26, 29, 46, 88, 90) and certain RXR heterodimers, such as the RXR/Nur77 heterodimer (16, 54, 76).

Heterodimerization of RXR with its partners dramatically enhances their DNA binding and subsequently transcriptional regulation (29, 46, 89). On binding DNA, some nuclear receptors repress transcription of target genes through their interaction with transcriptional corepressors in the absence of ligands. Ligand binding by a transcriptional agonist causes a conformational change in the receptors, allowing dissociation of transcriptional corepressors and association of transcriptional coactivators (80). In addition to DNA binding and interaction with receptor cofactors, recent studies suggest that subcellular distribution of RXR and its dimerization partners represents another mechanism that regulates their biological activity. Despite being localized in the nucleus in many cell types, RXR (12, 27) and its partners, including RARs (12). TRs (2, 6, 92), VDR (57, 58), and Nur77 (30, 40), were found in the cytoplasm in certain cell types and stages during development or under different cellular environments. Interestingly, RXR heterodimerization promotes nuclear localization of TR (2) and VDR (57).

Orphan receptor Nur77 (7, 20, 51) is an immediate-early response gene whose expression is rapidly induced by a variety of extracellular stimuli, including growth factors, the phorbol ester 12-O-tetradecanoyl-13-phorbol acetate (TPA) and cyclic-AMP-dependent pathways. Nur77 and its closely related family members, Not-1 (also called Nurr1 and RNR-1) (36, 45) and NOR-1 (also called MINOR and TEC) (21, 47, 52), constitute a distinct subfamily within the nuclear receptor superfamily (29, 46, 48). Nur77 was originally recognized for its role in cell proliferation and differentiation. Paradoxically, Nur77 was later found to be a potent proapoptotic molecule (48, 73, 84, 86). The expression of Nur77 was rapidly induced during the apoptosis of immature thymocytes and T-cell hybridomas, as well as various types of cancer cells (24, 25, 28, 40, 41, 44, 62, 66, 74, 78). Overexpression of a dominant-negative Nur77 protein or inhibition of Nur77 expression by antisense Nur77 inhibited apoptosis, whereas constitutive expression of Nur77 resulted in massive apoptosis (40, 41, 44, 66, 69, 74, 75).

Nur77 can function in the nucleus as a transcription factor to regulate the gene expression necessary to alter the cellular phenotype in response to various stimuli. Consistently, Nur77 response elements (NBRE or NurRE) have been identified (56, 72). In addition to its heterodimerization with RXR (16, 54), Nur77 can interact with the orphan receptor COUP-TF (77) that binds to the RARß promoter and is required for efficient RARß expression (42). Through its interaction with RXR and COUP-TF, Nur77 can modulate RARß expression and the growth response of cells to retinoids (8, 77). Interestingly, the EWS/TEC (Nor-1) fusion protein generated by the t(9;22) chromosomal translocation in extraskeletal myxoid chondrosarcoma is 270-fold more active than the native receptor in activating a reporter containing the NBRE (34, 35). This result suggests that the cancer-associated TEC (Nor-1) fusion receptor exerts its oncogenic activity by inducing the expression of target genes involved in cell proliferation. Thus, Nur77 may confer its growth-promoting activities through its action in the nucleus. This is supported by our recent observation that DNA binding and transactivation of Nur77 are required for its induction of cell proliferation (31).

Recent studies have demonstrated that Nur77 can also act outside the nucleus to mediate several important biological functions, including apoptosis and differentiation. Nur77, in response to apoptotic stimuli, translocates from the nucleus to the cytoplasm where it targets mitochondria to induce cytochrome c release and apoptosis (40). Our investigation of this phenomenon demonstrated that the proapoptotic effect of Nur77 does not require its transcriptional activity and DNA binding. Furthermore, Nur77 targets mitochondria through its interaction with Bcl-2, resulting in conversion of Bcl-2 from an antiapoptotic to a proapoptotic molecule (43). Nur77 targets mitochondria in prostate cancer (40, 71), lung cancer (10, 31), colon cancer (71), ovarian cancer (24), and gastric cancer (28, 79) cells, and its mitochondrial localization is involved in Sindbis virus-induced apoptosis (37). The cytoplasmic action of Nur77 was also demonstrated by the translocation of NGFI-B from the nucleus to the cytoplasm in response to nerve growth factor (NGF) treatment in PC12 pheochromocytoma cells, suggesting that the cytoplasmic action of NGFI-B is involved in NGF-induced PC12 cell differentiation (30). Thus, the diverse biological activities of Nur77 depend on its subcellular localization. The importance of TR3 pathways in regulating cancer cell growth is supported by the positive correlation between Nor-1 expression and survival in diffuse large B-cell lymphoma patients on chemotherapy (63) and a recent observation that TR3 is 1 of the 17 signature genes associated with metastasis of primary solid tumors (59).

Because Nur77 heterodimerizes with RXR, we investigated the role of RXR and its ligands in the regulation of the Nur77-dependent apoptotic pathway. Here, we report that RXR migrates from the nucleus to mitochondria as an RXR/Nur77 heterodimer in response to apoptotic stimuli. The migration is mediated by a putative nuclear export sequence (NES) present in the LBD of RXR. The RXR NES is active in the RXR/Nur77 heterodimer formed through dimerization interfaces in their DBDs. Interestingly, in the presence of RXR ligands, the RXR/Nur77 heterodimer is formed through dimerization interfaces in their LBDs. Such an RXR ligand-induced switch of RXR/Nur77 heterodimerization interfaces silences the RXR NES. Consistently, RXR ligands effectively inhibit the mitochondrial targeting of the RXR/Nur77 heterodimer and apoptosis. Together, our results demonstrate that RXR and its ligands plays a critical role in the regulation of the Nur77-dependent apoptotic pathway through their heterodimerization.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmid constructs. The construction of green fluorescent protein (GFP)-RXR and Nur77 fusion, as well as GFP-Nur77/DBD and GFP-Nur77/1, has been described (40). GFP-RXR and GFP-Nur77 mutants were generated by cloning RXR or Nur77 mutants into pGFP-N2 or pGFP-C2 vector (Clontech). RXR and Nur77 mutants were generated by standard restriction enzyme digestion or amplified by PCR. The following oligonucleotides were used as primers: GAA GAT CTT CGA CAC CAA ACA TTT CCT GCC GCT C (upstream) and CGG AAT TCC GGC AGG CCT AAG TCA TTT GGT GCG G (downstream) for RXR, CGG AAT TCC GTC GAG AGC CCC TTG GAG TCA GGG (downstream) for RXR/385, CGG AAT TCC GCA AAG ATG GCG CCC ACC CCT GCG C (downstream) for RXR/347, CCG GAA TTC CGG GAT GTG CTT GGT GAA GGA AGC (downstream) for RXR/135, GAA GAT CTT CAC CAG CAG CGC CAA CGA GGA CAT G (upstream) and CGG AAT TCC GGC AGG CCT AAG TCA TTT GGT GCG G (downstream) for RXR/C1, 5'-CGG GAT CCC GGT GCG CCA TCT GCG GGG ACC GC-3' (upstream) and 5'-CGG AAT TCG ACC CGC TTG TCA ATC AGG CAG TC-3' (downstream) for RXR/182, 5'-CGG AAT TCG ACC ATG CCC ATG GCC ACG CAC TTC-3' (downstream) for RXR/200, 5'-CGG AAT TCG ACG CCA CGC TGC CGC TCC TCC TG-3' (downstram) for RXR/212, 5'-GAA GAT CTT CAT GCC CTG TAT CCA AGC CCA ATA TG-3' (upstream) and 5'-CGG AAT TCC GGC ACC AAG TCC TCC AGC TTG AGG TAG-3' (downstream) for Nur77, 5'-CGG AAT TCC GGT AGC ACC AGG CCT GAG CAG AAG ATG-3' (downstream) for Nur77/467, 5'-CGG AAT TCC GCG GAG AGC AGG TCG TAG AAC TGC TG (downstream) for Nur77/410, 5'-CGG AAT TCG CCC ACC GCC AGG CAC TTC TGG-3' (downstream) for Nur77/332, 5'-CGG AAT TCC GCT CCA CTG TGC GCT TGA AGA AGC CCT G-3' (downstream) for Nur77/296, 5'-CGG AAT TCC GGC AGG CCT TCG TAA GTC TGG CTG GGC (downstream) for Nur77/170, 5'-GAA GAT CTT CAT GGC CCT GTC CTC CAG TGG CTC TGA C-3' (upstream) for Nur77/2, 5'-GAA GAT CTT CTC CGG TTC TCT GGA GGT CAT CCG C-3' (upstream) and 5'-CGG AAT TCC AGC ACC AGG CCT GAG CAG AAG ATG-3' (downstream) for Nur77/C2, and 5'-GAA GAT CTT CAT GGG CCT GGT GCT ACA CCG GCT G-3' (upstream) and 5'-CGG AAT TCC GGC ACC AAG TCC TCC AGC TTG AGG TAG-3' (downstream) for Nur77/C3. The BamHI-EcoRI fragment (324 to 462), BamHI and StuI fragment (324 to 402), and BglII and BamHI fragment (1 to 235) of RXR were cloned into pGFP-C2 to generate GFP-RXR/C2, GFP-RXR/C3, and GFP-RXR/235, respectively. Nur77 or RXR mutants were also cloned into pcDNA3, pECE-Flag, or pCMV-myc vector. For NES-RXR, 5'-GAT CTT CAG GGT GCT GAC GGA GCT TGT GTC CAA GAT GCG GGA CAT GCA GAT GGA CAA GAC GGA GCT GGG CG-3' and AAT TCG CCC AGC TCC GTC TTG TCC ATC TGC ATG TCC CGC ATC TTG GAC ACA AGC TCC GTC AGC ACC CTG AA-3' were annealed and ligated into pGFP-C2. NESm-RXR was generated with QuikChange site-directed mutagenesis kit (Stratagene) with primers 5'-AAG GCA CGG GAC GCA CAG ATG GAC AAG-3' and CTG TGC GTC CCG TGC CTT GGA CAC AAG-3'. RXR/LLL was described previously (90). Bcl-2 expression vector and antibody were kindly provided by John C. Reed at the Burnham Institute Cancer Center.

RXR siRNA. Small interfering RNAs (siRNAs) used in the experiments were obtained from Dharmacon Research, Inc. The following siRNA sequences were used: RXR siRNA, 5'-AAG CAC UAU GGA GUG UAC AGC-3', 5'-GCU GUA CAC UCC AUA GUG CUU-3'. A 2.5-µl aliquot of 20 µmol of siRNA/liter/per well was transfected into cells grown in 12-well plates by using oligofectamine reagent (Invitrogen) according to the manufacturer's recommendations. Two days after transfection, the cells were harvested for Western blotting or prepared for immunostaining.

Cell culture. LNCaP prostate cancer cells and H460 lung cancer cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). CV-1 cells and HEK293T embryonic kidney cells were grown in Dulbecco modified Eagle’s medium supplemented with 10% FBS.

Protein-protein interaction assays. Glutathione S-transferase (GST)-pull-down and gel shift assays have been described previously (77, 87). For GST pull-down assays, GST and GST fusion proteins were expressed in Escherichia coli BL21 and purified by using glutathione-agarose affinity chromatography. The GST fusion proteins were analyzed by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-10% PAGE) for integrity. [³⁵S]methionine-labeled RXR or Nur77 was produced by TNT-coupled transcription-translation system (Promega) and visualized by SDS-PAGE. In vitro binding assays were performed with glutathione-agarose beads (20 µl) coated with 10 µg of GST fusion protein and 2 to 5 µl of [³⁵S]methionine-labeled protein in 200 µl of binding buffer (20 mM HEPES [pH 7.9], 100 mM KCI, 1 mM MgC1₂, 10 µM ZnC1₂ 2 mM dithiothreitol, 10% glycerol, 0.05% Triton X-100, 40 µg of leupeptin/ml). The reaction was allowed to proceed overnight at 4°C with rocking. Beads were then collected by centrifugation and washed five times with 1 ml of NETN buffer (100 mM NaC1, 1 mM EDTA, 20 mM Tris-HCl [pH 8.0], 0.5% NP-40, 40 µg of leupeptin/ml). The beads were resuspended in 20 µl of SDS-PAGE sample buffer and boiled for 5 min. The eluted proteins were loaded on an SDS-PAGE gel, dried, and autoradiographed at –70°C. For coimmunoprecipitation assays, HEK293T cells grown in 10-cm dishes were transfected with 7.5 µg of Flag-tagged Nur77 and 7.5 µg of GFP-tagged RXR/385. Transfected HEK293T cells were lysed in 300 µl of TAE buffer (10 mM Tris-HCI [pH 8.0], 10 mM NaC1, 10 mM EDTA, and 1% NP-40 containing protease inhibitors [Sigma]). Lysate (300 µl) was incubated with 2 µg of anti-Flag monoclonal antibody (M2; Sigma) at 4°C for 2 h. Immunocomplexes were then precipitated with 40 µl of protein A/G-Sepharose (Santa Cruz Biotechnology, Inc.). After an extensive washing with TAE buffer, the beads were boiled in 40 µl of loading buffer and analyzed by Western blotting. Mouse immunoglobulin G (IgG; Santa Cruz Biotechnology) was used as a negative control.

Nondenaturing gel electrophoresis. Nuclear and cytoplasmic fractions of HEK293T cells expressing GFP-RXR/C1 treated with or without 9-cis-RA were prepared and separated on a 8% nondenaturing Tris-glycine gel (Invitrogen), followed by immunoblotting.

Confocal microscopy. Cells were seeded on the chamber slides overnight and treated with apoptotic agents in medium containing 0.5% FBS. After treatment, the cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min and washed twice with PBS. Cells were then permeabilized with 1% triton X-100 in PBS for 5 min. Fixed cells were preincubated for 30 min in PBS containing 5% bovine serum albumin at room temperature. Cells were stained with polyclonal anti-RXR antibody (D20 [Santa Cruz Biotechnology] at 1:500) or monoclonal anti-Nur77 antibody (1:500; Abgent) before detection of fluorescein isothiocyanate (FITC)-labeled anti-rabbit IgG (1:500; Sigma). For mitochondrial staining, cells were incubated with anti-Hsp60 goat IgG (1:500; Santa Cruz Biotechnology), followed by anti-goat IgG conjugated with Cy3 (1:1,000; Sigma). For cytochrome c staining, cells were incubated with monoclonal anti-cytochrome c IgG (1:300; Pharmingen), followed by anti-mouse IgG conjugated with Cy5 (1:500; Amersham). For staining transfected protein, 1 µg of Myc-tagged TR3, Flag-tagged RXR, or Bcl-2 was transfected into HEK293T cells grown in chamber slides in six-well plates overnight and treated with apoptotic agents. After treatment, cells were fixed and incubated with monoclonal anti-Myc IgG antibody (1:300; 9E10; Santa Cruz Biotechnology), followed by FITC-conjugated anti-mouse IgG (1:500; Sigma) or Cy3-conjugated anti-mouse IgG (1:1,000; Sigma). For Bcl-2 staining, cells were incubated with polyclonal anti-Bcl-2 antibody (67), followed by Cy5-conjugated goat anti-rabbit antibody (1:500; Sigma).

Cell fractionation. Subcellular fractionation was performed as described with minor modifications (40). Briefly, cells (10⁷ cells) suspended in 0.5 ml of hypotonic buffer (250 mM sucrose, 20 mM HEPES-KOH [pH 7.4], 10 mM KCl, 10 mM MgCl₂, 0.5 mM EGTA, 1.5 mM EDTA [pH 8.0], 1 mM dithiothreitol) with proteinase inhibitors were homogenized, and cell extracts were centrifuged at 800 x g for 10 min. The pellet containing nuclei was resuspended in 200 µl of 1.6 M sucrose in hypotonic buffer plus protease inhibitors and laid over 1 ml of 2.0 M sucrose in the same buffer and then centrifuged at 150,000 x g for 90 min at 4°C to obtain the nuclear fraction. The supernatant was centrifuged at 10,000 x g for 30 min at 4°C to obtain the heavy membrane (HM) and cytosolic fractions. Nuclear and HM fractions were resuspended in 100 µl of lysis buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 5 mM EDTA [pH 8.0]) with a cocktail of proteinase inhibitors for Western blotting analysis as described previously (40).

Transient-transfection assays. Cells (10⁵ cells/well) seeded in 24-well plates were transiently transfected by using a modified calcium phosphate precipitation procedure as described previously (42). For GFP studies, 1 µg of GFP or GFP constructs was transfected into cells seeded in six-well plates by calcium phosphate precipitation.

Apoptosis assays. For terminal deoxynucleotidyltransferase (TdT) assays, cells were treated with or without indicated agents overnight after pretreatment with RXR ligand 9-cis-RA or SR11237 for 12 h, treated with trypsin, washed with PBS, fixed in 1% formaldehyde, and then resuspended in 70% ice-cold ethyl alcohol. Cells were then labeled with biotin-16-deocyuridine 5' triphosphate by TdT and stained with avidin-fluorescein isothiocyanate (Boehringer Mannheim). For nuclear morphological change analysis, LNCaP cells were treated with trypsin, washed with PBS, fixed with 3.7% paraformaldehyde, and stained with DAPI (4',6'-diamidino-2-phenylindole; 50 µg/ml) to visualize nuclei by fluorescence microscopy.

Western blotting. Cell lysates were boiled in SDS sample buffer, resolved by SDS-12.5% PAGE, and transferred to nitrocellulose. After transfer, the membranes were blocked in 5% milk in TBST (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween 20) containing antibody. The membranes were then washed three times with TBST and then incubated for 1 h at room temperature in TBST containing horseradish peroxide-linked anti-immunoglobulin. After three washes in TBST, immunoreactive products were detected by using enhanced chemiluminescence (Amersham). Anti-GFP antibody was purchased from Santa Cruz Biotechnology.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
RXR targets mitochondria in response to apoptotic stimuli. Nur77 migrates from the nucleus to mitochondria to induce apoptosis in response to certain apoptotic stimuli (10, 24, 31, 37, 40, 43, 71, 78). Since RXR heterodimerizes with Nur77 (16, 54), we studied whether RXR also targeted mitochondria. Subcellular localization of RXR in LNCaP prostate cancer cells in the absence or presence of TPA, which potently induces LNCaP cell apoptosis (40), was examined by confocal microscopy analysis. Immunostaining showed that RXR predominantly localized in the nucleus in the absence of TPA treatment (Fig. 1A). However, when cells were treated with TPA, RXR was found in the cytoplasm. To study whether RXR was associated with mitochondria, cells were stained for heat shock protein 60 (Hsp60), a mitochondrion-specific protein (Fig. 1A). The extensive overlap in the distribution patterns of Hsp60 and RXR suggested the association of RXR with mitochondria. Similarly, treatment with TPA also resulted in mitochondrial localization of Nur77 (Fig. 1B), as previously reported (40). The enhanced staining of Nur77 in TPA-treated cells was due to induction of endogenous Nur77 expression by TPA. Thus, RXR and Nur77 associate with mitochondria in LNCaP cells undergoing apoptosis.

View larger version (53K): [in this window] [in a new window]   FIG. 1. Nur77 and RXR comigrate from the nucleus to the cytoplasm. (A/B) RXR (A) or Nur77 (B) targets mitochondria in response to apoptotic stimulus. LNCaP prostate cancer cells were treated with TPA (100 ng/ml) for 1 h and then immunostained with either anti-RXR (Santa Cruz Biotechnology) (A) or anti-Nur77 (Active Motif) (B) antibody, followed by Cy3-conjugated secondary antibody (Sigma) to detect RXR or Nur77 or with anti-Hsp60 (Santa Cruz Biotechnology), followed by FITC-conjugated secondary antibody (Sigma) to detect mitochondria. RXR, Nur77, and mitochondria (Hsp60) were visualized by using confocal microscopy, and the images of RXR or Nur77 with those of mitochondria were overlaid (overlay). About 80% of cells displayed mitochondrial targeting of RXR and Nur77 when cells were treated with TPA. One of three similar experiments is shown. (C) Nur77 and RXR comigrate from the nucleus to the cytoplasm. LNCaP cells were treated with or without TPA for 1 h and then immunostained with anti-RXR antibody, followed by FITC-conjugated secondary antibody, or with anti-Nur77 antibody (Abgent, San Diego, Calif.), followed by Cy3-conjugated secondary antibody. RXR and Nur77 were visualized by using confocal microscopy and the images were overlaid (overlay). Approximately 80% of TPA-treated cells showed RXR colocalization with Nur77. One of three similar experiments is shown. (D) 3-Cl-AHPC induces mitochondrial localization of transfected RXR and Nur77. Expression vectors for myc-Nur77 and RXR weretransfected into LNCaP cells. Cells were then treated with 3-Cl-AHPC for 3 h and then immunostained with anti-myc antibody (9E10; Santa Cruz Biotechnology), followed by FITC-conjugated secondary antibody, anti-RXR antibody, followed by Cy3-conjugated secondary antibody or anti-Hsp60 antibody, followed by Cy5-conjugated secondary antibody (Jackson Immunoresearch). Myc-Nur77, RXR, and Hsp60 were visualized and the images were overlaid. Overlay 1 is the merger of myc-Nur77 and RXR images, and overlay 2 is the merger of myc-Nur77, RXR, and Hsp60 images. About 30% of transfected cells exhibited the colocalization presented. One of three similar experiments is shown. (E) Mitochondrial localization of RXR is Nur77 dependent. LNCaP cells or LNCaP cells stably expressing Nur77 antisense RNA (Nur77/antisense) (40) were treated with or without TPA for 1 h and then immunostained with anti-RXR antibody, followed by Cy3-conjugated secondary antibody. Approximately 70% of cells displayed the effect presented. One of two similar experiments is shown. (F) Effect of RXR siRNA on RXR levels and Nur77 localization. LNCaP cells were transfected with or without RXR siRNA or control siRNA for 72 h. Cell extracts were prepared and analyzed for RXR expression by Western blotting. Cells were also analyzed for subcellular localization of RXR and Nur77 by confocal microscopy. One of two similar experiments is shown. (G) Nur77/1 prevents RXR mitochondrial targeting. LNCaP cells were transfected with the GFP-Nur77/1 expression vector and then treated with TPA for 1 h and immunostained with anti-RXR antibody. RXR and GFP-Nur77/1 distributions were analyzed by confocal microscopy. About 80% of nontransfected cells showed cytoplasmic localization of RXR after treatment with TPA, whereas >70% of cells transfected with GFP-Nur77/1 showed RXR nuclear localization with the same treatment. One of three similar experiments is shown. (H) 3-Cl-AHPC induces mitochondrial localization of RXR and Nur77 in H460 lung cancer cells. H460 cells were treated with 3-Cl-AHPC (10–6 M) for 3 h and then immunostained with anti-RXR antibody, followed by FITC-conjugated secondary antibody, anti-Nur77 followed by Cy3-conjugated secondary antibody, or anti-Hsp60 antibody, followed by Cy5-conjugated secondary antibody (Jackson Immunoresearch). RXR, Nur77, and Hsp60 were visualized by confocal microscopy, and the images were overlaid. Overlay 1 represents the merger of RXR and Nur77 images. Overlay 2 indicates merged RXR, Nur77, and Hsp60 images. Approximately 80% of 3-Cl-AHPC-treated cells showed the patterns presented. One of three similar experiments is shown.

RXR and Nur77 mitochondrial targeting are mutually dependent.

To further characterize the mitochondrial targeting of RXR and Nur77, we conducted a time course analysis of their targeting in LNCaP cells treated with TPA (Fig. 2A). Immunoblotting of mitochondrion-enriched HM fractions showed that both RXR and Nur77 began to be associated with mitochondria as early as 1 h after cells were treated with the apoptotic stimulus. This result is consistent with our previous observation showing that Nur77 targeted mitochondria after LNCaP cells were treated with TPA for 1 h (40). The simultaneous targeting of RXR and Nur77 to mitochondria again suggested that they targeted mitochondria as a heterodimer. We previously reported that mitochondrial targeting of Nur77 initiated the release of cytochrome c from mitochondria (40). Consistently, we observed significant amounts of cytochrome c in the cytosolic fractions after LNCaP cells were treated with TPA for 1.5 h. These results indicate that mitochondrial targeting of RXR and Nur77 precedes the release of cytochrome c, further demonstrating the role of Nur77/RXR mitochondrial targeting in triggering cytochrome c release. The requirement of RXR in Nur77-dependent apoptosis is also illustrated by our observation that inhibition of RXR expression by the expression of RXR siRNA diminished the apoptotic effect of TPA in LNCaP cells (Fig. 2B). Similar results were also obtained in H460 lung cancer cells treated with 3-Cl-AHPC (Fig. 2C).

View larger version (29K): [in this window] [in a new window]   FIG. 2. Time course analysis of mitochondrial targeting of RXR and Nur77 and the release of cytochrome c from mitochondria. (A) Time course analysis of LNCaP cells in response to TPA. Cells were treated with TPA (100 ng/ml) for the indicated times. Mitochondrion-enriched HM and cytosolic fractions were prepared and analyzed for the presence of RXR, Nur77, and cytochrome c as indicated. As a control, the whole-cell extract was also analyzed. Expression of mitochondrion-specific Hsp60 protein and nucleus-specific PARP protein was determined to control the purity of HM fractions. One of two similar experiments is shown. (B) Inhibition of RXR expression suppresses the apoptotic effect of TPA. LNCaP cells were transfected with RXR siRNA, followed by treatment with TPA (100 ng/ml) for 3 h. Cells were stained with DAPI and analyzed for nuclear morphological changes. Apoptotic cells were scored by examining 300 cells for apoptotic morphology from three different experiments. (C) Time course analysis of H460 cells in response to 3-Cl-AHPC. Cells were treated with 3-Cl-AHPC (10–6 M) and analyzed as described for panel A. One of two similar experiments is shown.

Subcellular localization of RXR and Nur77 mutants.

View larger version (54K): [in this window] [in a new window]   FIG. 3. Identification of domains in RXR and Nur77 required for their nuclear export. (A) Cytoplasmic localization of RXR/Nur77 is mediated by CRM1-dependent nuclear export. LNCaP cells were treated with TPA (100 ng/ml) in the absence (control) or presence of LMB (2.5 ng/ml; Sigma) and analyzed by confocal microscopy as described for Fig. 1C. About 80% cells showed the cytoplasmic localization of RXR and Nur77 after treatment with TPA, whereas >50% of cells showed nuclear localization of RXR and Nur77 when pretreated with LMB. One of two similar experiments is shown. (B) Schematic representations of RXR and Nur77 mutants. The DBD, LBD, and A to F domains are indicated. (C) Analysis of subcellular localization of Nur77 and RXR mutants. The indicated plasmids were transfected into HEK293T cells and analyzed by confocal microscopy as described in Fig. 1. More than 90% of transfected cells showed diffused distribution of GFP and GFP-RXR/135. Nuclear localization of GFP-RXR, RXR/235, and RXR/347 was found in 80, 85, and 60% of transfected cells, respectively. More than 90% of transfected cells showed exclusive cytoplasmic localization of RXR/385, RXR/C2, and RXR/C3, whereas cytoplasmic localization of RXR/C1 was found in 70% of transfected cells. Nuclear localization of GFP-Nur77 and its mutants, GFP-Nur77/2, GFP-Nur77/1, GFP-Nur77/467, and GFP-Nur77/410, was found in >90% of transfected cells, whereas cytoplasmic localization of GFP-Nur77/DBD, cytoplasmic localization of GFP-Nur77/DBD, GFP-Nur77/C2, and GFP-Nur77/C3 was observed in 80, 60 and 70% of transfected cells, respectively. One of four similar experiments is shown.

Identification of a putative NES in RXR.

View larger version (43K): [in this window] [in a new window]   FIG. 4. Identification of a nuclear export sequence in RXR. (A) Effect of LMB on subcellular localization of RXR mutants. The indicated expression vector for GFP fusion proteins was transfected into HEK293T cells and analyzed by confocal microscopy. Approximately 70% of transfected cells displayed diffused distribution of RXR/C2 and RXR/C3 after treatment with LMB. One of four similar experiments is shown. (B) RXR/C3 exclusively resides in the cytoplasm. GFP-RXR/C3 was transfected into HEK293T cells, and its localization was analyzed by cellular fractionation, followed by Western blotting with anti-GFP antibody (Santa Cruz Biotechnology). One of three similar experiments is shown. (C) The effect of LMB on subcellular localization of RXR/C3. RXR/C3 was transfected into HEK293T cells, which were then treated with or without LMB (2.5 ng/ml) for 6 h. Localization of RXR/C3 was analyzed as described in panel B. One of three similar experiments is shown. (D) Schematic representation of the RXR NES. The identified RXR NES is compared to known NESs identified in the indicated genes. The boldface letters indicate conserved amino acid residues. (E) The RXR NES is capable of directing GFP to the cytoplasm. The putative RXRNES (RVLTELVSKMRDMQMDKTELG) or its mutant (RVLTELVSKARDAQMDKTELG) (NESm) was fused to GFP, and the expression vectors were transfected into HEK293T cells. Cells were treated with or without LMB for 6 h and then stained for Hsp60 and analyzed by confocal microscopy. Approximately 90% of GFP-NES-transfected cells exhibited cytoplasmic localization, whereas <20% of GFP-NESm-transfected cells displayed the same cytoplasmic localization. One of three similar experiments is shown. (F) Mutation of the RXR NES impairs the 3-Cl-AHPC-induced mitochondrial localization of RXR/Nur77 heterodimer. Myc-Nur77 was cotransfected with RXR/NESm into LNCaP cells, which were then treated with 3-Cl-AHPC (10–6 M) for 3 h. Cells were stained for Nur77 by anti-myc antibody and for RXR/NESm by anti-RXR antibody and analyzed by the confocal microscopy. About 90% of transfected cells showed nuclear localization of Myc-Nur77 and RXR/NESm, even after treatment with 3-Cl-AHPC. (G) RXR NES is required for cytoplasmic localization of Nur77. The indicated expression vectors were cotransfected into HEK293T cells. Their subcellular localization was analyzed by confocal microscopy as described in Fig. 1A. Less than 10% of transfected cells showed cytoplasmic localization of GFP-Nur77, Nur77/467, and Nur77/1, which was increased to 30, 40, and 40% upon cotransfection with Flag-RXR/385, respectively. About 80% of transfected cells showed nuclear localization of Flag-RXR, with or without Nur77/C3 cotransfection. One of three similar experiments is shown.

Our observation that RXR/385 was exclusively confined to the cytoplasm even in the absence of an apoptotic stimulus (Fig. 3C) was intriguing since this mutant contains both the NLS and NES. This result suggests that removal of the C-terminal sequences (amino acid residues 386 to 462) strongly activates the RXR NES and/or silences its NLS. To determine whether the superactivation of RXR NES in RXR/385 could confer cytoplasmic localization to Nur77, RXR/385 was cotransfected into HEK293T cells with Nur77 or its mutant Nur77/1 or Nur77/467, all of which alone resided in the nucleus (Fig. 3C). Our data showed that coexpression of RXR/385 with Nur77 or either mutant resulted in their colocalization in the cytoplasm (Fig. 4G). The extensive colocalization of RXR/385 with Nur77, Nur77/467, or Nur77/1 suggests that they comigrated to the cytoplasm as a heterodimer. Thus, RXR/385 was able to shuttle Nur77 to the cytoplasm. In contrast, Nur77/C3 could not confer its cytoplasmic localization to RXR when they were coexpressed (Fig. 4G). Thus, the RXR NES plays a critical role in the nuclear export of the RXR/Nur77 heterodimer.

Role of Bcl-2 in RXR mitochondrial targeting. We recently reported that TR3 mitochondrial targeting requires its interaction with Bcl-2 (43). Therefore, we examined whether Bcl-2 expression modulated RXR mitochondrial targeting. GFP-RXR/385 was cotransfected with Bcl-2, and their distribution patterns were visualized by confocal microscopy. Figure 5A shows that expression of Bcl-2 did not alter the diffuse distribution of GFP-RXR/385. However, when Nur77 was cotransfected, GFP-RXR/385 colocalized with both Nur77 and Bcl-2, as evidenced by punctate staining. This result indicated that Bcl-2 interacted with Nur77 but not RXR. The requirement of Nur77 and Bcl-2 for RXR mitochondrial targeting was also illustrated by our cellular fractionation assay (Fig. 5B). GFP-RXR/385 expressed in HEK293T cells did not show any accumulation in the mitochondrion-enriched HM fraction in the absence or presence of Bcl-2 coexpression. However, when Nur77/2, a Nur77 mutant lacking the N-terminal 122 amino acid residues, was coexpressed, a significant amount of GFP-RXR/385 was found in the HM fraction, whereas its cytosolic level decreased (Fig. 5B). Together, these results demonstrate that mitochondrial targeting of RXR requires both Nur77 and Bcl-2 and that the RXR/Nur77 heterodimer interacts with Bcl-2.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Role of Bcl-2 in mitochondrial targeting of RXR and Nur77 mutants. (A) Mitochondrial targeting of RXR/385 in HEK293T cells requires Nur77 and Bcl-2. Expression vectors of GFP-RXR/385 and Bcl-2 were transfected, together with or without myc-Nur77 vector into HEK293T cells. Cells were then immunostained with anti-myc antibody (9E10; Santa Cruz Biotechnology), followed by Cy3-conjugated secondary antibody, or with anti-Bcl-2 antibody, followed by Cy5-conjugated secondary antibody. GFP-RXR/385, myc-Nur77, and Bcl-2 were visualized by confocal microscopy. Overlay 1 represents the merger of the GFP-RXR/385 and myc-Nur77 images, and overlay 2 is a merge of the GFP-RXR/385, myc-Nur77, and Bcl-2 images. Approximately 70% of cells transfected with GFP-RXR/385 and myc-Nur77 exhibited colocalization with Bcl-2, whereas <5% of cells transfected with GFP-RXR/385 alone showed colocalization with Bcl-2. (B) Accumulation of RXR/385 at mitochondria in HEK293T cells requires both Nur77 and Bcl-2. Flag-RXR/385, GFP-Nur77/2, and Bcl-2 were transfected into HEK293T cells as indicated. HM and cytosolic fractions were prepared and levels of Flag-RXR/385, GFP-Nur77/2, and Bcl-2 were determined by immunoblotting with anti-Flag (Sigma), anti-GFP, and anti-Bcl-2 antibody, respectively. For control, levels of Hsp60 and PARP were also determined. One of two similar experiments is shown.

Mitochondrial targeting and apoptotic effect of RXR and Nur77 mutants.

View larger version (41K): [in this window] [in a new window]   FIG. 6. Mitochondrial targeting and apoptosis effects of cytoplasmic RXR and Nur77 mutants. (A) Cytoplasmic Nur77 mutants but not RXR mutants induce apoptosis. The indicated GFP-RXR or GFP-Nur77 mutant was transfected into LNCaP cells. After 48 h, cells were stained by DAPI and analyzed for nuclear morphological change by microscopy. Arrows indicate cells with extensive nuclear condensation or fragmentation. Percentages of apoptotic cells were determined by examining 200 GFP-positive cells for nuclear fragmentation and/or chromatin condensation. Bars represent averages ± the means from three experiments. (B) Cytoplasmic Nur77 mutant but not RXR mutant induces cytochrome c (Cyt) release. GFP-Nur77/DBD or GFP-RXR/C1 expression vector was transfected into LNCaP cells. Cells were then stained for mitochondria (Hsp60) and cytochrome c and analyzed by confocal microscopy. Cytochrome c release was observed in 80% of cells showing Nur77/DBD mitochondrial targeting, whereas it was not found in cells transfected with RXR/C1. One of two similar experiments is shown.

Regulation of the RXR NES activity by ligand binding and RXR homodimerization.

View larger version (39K): [in this window] [in a new window]   FIG. 7. Regulation of RXR nuclear export by its ligands and homodimerization. (A and B) Analysis of subcellular localization of RXR/C1 in the absence or presence of retinoids. (A) GFP-RXR/C1 was transfected into HEK293T cells, which were then treated with the indicated retinoid, stained with Hsp60, and analyzed by confocal microscopy. The inhibitory effect of RXR ligands on the cytoplasmic localization of RXR/C1 was observed in 80% of transfected cells, whereas >90% of transfected cells failed to respond to RAR ligands. (B) Nuclear and cytoplasmic extracts were also prepared and analyzed for expression of GFP-RXR/C1 by Western blotting with anti-GFP antibody. One of three similar experiments is shown. (C) RXR/C1 dimerization status determines its subcellular localization. GFP-RXR/C1 was transfected into HEK293T cells, which were not treated or treated with 9-cis-RA (10–7 M). Nuclear and cytoplasmic extracts were prepared and analyzed by using nondenaturing PAGE and anti-GFP antibody. The same extracts were analyzed by denaturing PAGE for the expression of PARP and Hsp60 to ensure fraction purity. One of two similar experiments is shown. (D) Confocal microscopy analysis of RXR homodimerization-defective mutants. GFP-RXR/385, GFP-RXR/LLL, or GFP-RXR/C1/C432R was transfected into HEK293T cells. Cells were treated with or without 9-cis-RA (10–7 M) and analyzed by confocal microscopy. Approximately 80% of transfected cells showed nuclear localization of GFP-RXR, which was slightly increased to 85% after treatment with 9-cis-RA. Cytoplasmic localization of GFP-RXR/385 (90%), GFP-RXR/LLL (65%), and GFP-RXR/C1/C432R (85%) was not affected by 9-cis-RA treatment. One of three similar experiments is shown.

Regulation of RXR NES activity by heterodimerization. Heterodimerization of RXR with other receptors, such as RAR and VDR, is required for their efficient DNA binding and transactivation (29, 46, 89). Therefore, we analyzed the subcellular localization of RXR/VDR and RXR/RAR heterodimers. GFP-RXR was transfected alone or with either VDR (Fig. 8A) or RAR (Fig. 8B) into HEK293T cells. Without cotransfection, GFP-RXR was distributed in both the cytoplasm and the nucleus. However, on cotransfection of VDR or RAR, the cytoplasmic localization of GFP-RXR was completely abolished and the levels of nuclear GFP-RXR increased. Treatment of cells with the VDR ligand 1,25-dihydroxyvitamin D₃ or the RAR ligand trans-RA did not further modulate the cellular distribution of cotransfected GFP-RXR. The inhibitory effect of VDR was also examined by studying the subcellular localization of RXR/C1 in the absence or presence of VDR coexpression. In the absence of VDR, RXR/C1 exclusively localized in the cytoplasm of HEK293T cells. However, when VDR was cotransfected, RXR/C1 was confined to the nucleus and its distribution pattern overlaid with that of VDR. Again, addition of 1,25-dihydroxyvitamin D₃ had no effect on localization of both VDR and RXR/C1 (Fig. 8C). Together, these results demonstrate that heterodimerization of RXR with RAR and VDR suppresses RXR NES activity in a ligand-independent manner.

View larger version (19K): [in this window] [in a new window]   FIG. 8. Regulation of RXR nuclear export by heterodimerization. (A and B) Regulation of RXR nuclear export by VDR (A) and RAR (B). Expression vector for VDR or RAR was transfected into HEK293T cells, together with GFP-RXR expression vector. Cells were then treated with the indicated ligand, Vit D3 (10–7 M) or Am80 (10–6 M). Nuclear and cytoplasmic extracts were prepared and analyzed for expression of transfected GFP-RXR by anti-GFP antibody. Expression of transfected VDR or RAR was also determined. One of two similar experiments is shown. (C) Confocal analysis of the effect of VDR expression on RXR localization. The GFP-RXR/C1 expression vector was transfected into HEK293T cells together with or without the VDR expression vector. Cells were then treated with VD3 (10–7 M),stained with anti-VDR antibody (Santa Cruz Biotechnology), followed by Cy3-conjugated secondary antibody (Sigma) and analyzed by confocal microscopy. About 70% of transfected cells showed cytoplasmic localization of RXR/C1, whereas >80% of cotransfected cells showed nuclear localization of RXR/C1 and VDR, which was not enhanced by VD3 treatment. One of three similar experiments is shown.

Unique RXR heterodimerization with Nur77.

View larger version (36K): [in this window] [in a new window]   FIG. 9. C termini of RXR and Nur77 are not required for RXR/Nur77 interaction in solution. (A and B) GST pull-down assays for determination of RXR/Nur77 heterodimerization. GST-Nur77, GST-RXR, or GST control protein immobilized on glutathione-Sepharose (20 µl) was incubated with in vitro synthesized 35S-labeled Nur77, RXR or their mutants (5 µl) as indicated. Bound proteins were analyzed by SDS-PAGE and autoradiography. One of three similar experiments is shown. (C) Coimmunoprecipitation assay for Nur77 and RXR/385 interaction. Expression vectors for Flag epitope-tagged-Nur77 (Flag-Nur77) and GFP-RXR/385 were cotransfected into HEK293T cells. The expressed Flag-Nur77 and GFP-RXR/385 were then immunoprecipitated by using either anti-Flag antibody or control IgG, and immunoprecipitates were examined by Western blotting with anti-GFP antibody. The same membranes were also blotted with anti-Flag antibody to determine precipitation specificity and efficiency. Input represents 5% of total cell extract used in the precipitation assays. One of two similar experiments is shown. (D) Reportergene assay. (NurRE)2-tk-chloramphenicol acetyltransferase (CAT) (100 ng), ß-galactosidase (ß-Gal; 100 ng), and Nur77 (25 ng) expression vectors were transiently transfected into HEK293T cells together with or without RXR or RXR/385. Cells were treated with or without 9-cis-RA (10–7 M) as indicated. CAT activity was determined and normalized relative to ß-Gal activity. Bars represent averages ± deviations from three different experiments. (E) Schematic representations of RXR and Nur77 mutants. The DBD, LBD, and A to F domains are indicated. (F) GST pull-down assays for determination of RXR/Nur77 heterodimerization. The indicated GST-RXR, its mutants, or GST control protein was immobilized on glutathione-Sepharose (20 µl) and incubated with in vitro-synthesized 35S-labeled Nur77 or its mutants (5 µl) as indicated. Bound proteins were analyzed by SDS-PAGE and autoradiography. One of three similar experiments is shown.

Modulation of RXR/Nur77 heterodimerization by RXR ligands.

View larger version (31K): [in this window] [in a new window]   FIG. 10. 9-cis-RA modulates RXR/Nur77 heterodimerization and promotes DNA binding. (A) The C-terminal domains of Nur77 and RXR are required for the formation of RXR/Nur77 heterodimer on DNA. The indicated Nur77, RXR, or their mutants were synthesized in vitro and analyzed for binding to the ßRARE by gel shift assays. One of three similar experiments is shown. (B) Differential requirement of the Nur77 C terminus for transactivation of the Nur77 response element NurRE and the ßRARE. (NurRE)2-tk-CAT (100 ng) or ßRARE-tk-CAT (100 ng), ß-Gal expression vector (100 ng), and the expression vector fo