β-Apo-13-carotenone Regulates Retinoid X Receptor Transcriptional Activity through Tetramerization of the Receptor*

Background: β-Apo-13-carotenone, a naturally occurring apocarotenoid, functions as an antagonist of the retinoid X receptor (RXR). Results: β-Apo-13-carotenone inhibits transactivation of RXRα but does not interfere with coactivator binding to the receptor like the known antagonist UVI3003. Conclusion: β-Apo-13-carotenone induces the formation of a transcriptionally silent RXR tetramer. Significance: β-Apo-13-carotenone is a naturally occurring rexinoid with a novel mechanism of antagonism. Retinoid X receptor (RXRα) is activated by 9-cis-retinoic acid (9cRA) and regulates transcription as a homodimer or as a heterodimer with other nuclear receptors. We have previously demonstrated that β-apo-13-carotenone, an eccentric cleavage product of β-carotene, antagonizes the activation of RXRα by 9cRA in mammalian cells overexpressing this receptor. However, the molecular mechanism of β-apo-13-carotenone's modulation on the transcriptional activity of RXRα is not understood and is the subject of this report. We performed transactivation assays using full-length RXRα and reporter gene constructs (RXRE-Luc) transfected into COS-7 cells, and luciferase activity was examined. β-Apo-13-carotenone was compared with the RXRα antagonist UVI3003. The results showed that both β-apo-13-carotenone and UVI3003 shifted the dose-dependent RXRα activation by 9cRA. In contrast, the results of assays using a hybrid Gal4-DBD:RXRαLBD receptor reporter cell assay that detects 9cRA-induced coactivator binding to the ligand binding domain demonstrated that UVI3003 significantly inhibited 9cRA-induced coactivator binding to RXRαLBD, but β-apo-13-carotenone did not. However, both β-apo-13-carotenone and UVI3003 inhibited 9-cRA induction of caspase 9 gene expression in the mammary carcinoma cell line MCF-7. To resolve this apparent contradiction, we investigated the effect of β-apo-13-carotenone on the oligomeric state of purified recombinant RXRαLBD. β-Apo-13-carotenone induces tetramerization of the RXRαLBD, although UVI3003 had no effect on the oligomeric state. These observations suggest that β-apo-13-carotenone regulates RXRα transcriptional activity by inducing the formation of the “transcriptionally silent” RXRα tetramer.

Retinoid X receptor (RXR␣) is activated by 9-cis-retinoic acid (9cRA) and regulates transcription as a homodimer or as a heterodimer with other nuclear receptors. We have previously demonstrated that ␤-apo-13-carotenone, an eccentric cleavage product of ␤-carotene, antagonizes the activation of RXR␣ by 9cRA in mammalian cells overexpressing this receptor. However, the molecular mechanism of ␤-apo-13-carotenone's modulation on the transcriptional activity of RXR␣ is not understood and is the subject of this report. We performed transactivation assays using full-length RXR␣ and reporter gene constructs (RXRE-Luc) transfected into COS-7 cells, and luciferase activity was examined. ␤-Apo-13-carotenone was compared with the RXR␣ antagonist UVI3003. The results showed that both ␤-apo-13-carotenone and UVI3003 shifted the dose-dependent RXR␣ activation by 9cRA. In contrast, the results of assays using a hybrid Gal4-DBD:RXR␣LBD receptor reporter cell assay that detects 9cRA-induced coactivator binding to the ligand binding domain demonstrated that UVI3003 significantly inhibited 9cRA-induced coactivator binding to RXR␣LBD, but ␤-apo-13-carotenone did not. However, both ␤-apo-13-carotenone and UVI3003 inhibited 9-cRA induction of caspase 9 gene expression in the mammary carcinoma cell line MCF-7. To resolve this apparent contradiction, we investigated the effect of ␤-apo-13-carotenone on the oligomeric state of purified recombinant RXR␣LBD. ␤-Apo-13-carotenone induces tetramerization of the RXR␣LBD, although UVI3003 had no effect on the oligomeric state. These observations suggest that ␤-apo-13-carotenone regulates RXR␣ transcriptional activity by inducing the formation of the "transcriptionally silent" RXR␣ tetramer.
Retinoid X receptors (RXR␣, 2 RXR␤, and RXR␥) are members of the nuclear receptor family and play a central role in nuclear receptor-regulated signaling pathways. RXRs are involved in biological processes, including cell growth and differentiation, metabolism, morphogenesis, and embryogenic development (1)(2)(3)(4)(5)(6)(7) The active form of RXR is a dimer or heterodimer (8,9). Besides the RXR homodimer, RXR also forms heterodimers with other nuclear receptor family members, including retinoic acid receptors, the vitamin D receptor, peroxisome proliferator-activated receptors, the farnesoid X receptor, and the liver X receptors (10,11). RXR naturally forms into tetramers that are transcriptionally inactive (12).
RXRs are primarily made up of two modular domains as follows: a central DNA binding domain (DBD) and a carboxylterminal ligand binding domain (LBD). In addition to its role in binding of ligands, the LBD contains dimerization motifs and an activation function 2 (AF-2) domain (13,14). Ligand-free RXR represses transcription of target genes through interaction with corepressor proteins. Ligand binding induces a conformational change of the AF-2 helix that releases corepressor protein and allows recruiting of coactivator complexes. Numerous compounds synthesized as antagonists, such as UVI3003, target the AF-2 helix (13).
Ligand-free RXR tends to associate into homotetramers both in solution and when bound to DNA. However, RXR tetramers rapidly dissociate into active dimers upon binding of an agonist such as 9-cis-retinoic acid (9cRA). RXR heterodimers bind in regulatory regions of their target genes by associating with response elements. RXR homodimers bind to a retinoid DNAresponse element (RXRE). Activation of DNA-bound dimers by ligands promotes the recruitment of transcriptional coactivators to the promoters of target genes and enhances transcription rate. In vitro studies have indicated that full-length RXR self-associates into tetramers, and the LBD alone is sufficient to mediate tetramer formation with 3-5 nM affinity between the dimers (12,15). Studies have substantiated the existence in vivo of an RXR tetramer (12,15). It also has been shown that the RXR tetramer is transcriptionally silent based on the correla-tion between the transcriptional activity of RXR mutants and their ability to form tetramers (16).
The vitamin A metabolite 9cRA is a ligand of RXR (17). Binding of 9cRA as an agonist induces the dissociation of the tetramer into dimer, which is the first step for RXR activation (18,19). Carotenoids are polyisoprenoids that are biosynthesized in plants, fungi, and bacteria. Approximately 50 -60 carotenoids that contain at least one unsubstituted ␤-ionone ring and the correct number and position of methyl groups in the polyene chain exhibit provitamin A activity (20,21). Dietary provitamin A, ␤-carotene, can be metabolized in mammals through two pathways (22). ␤-Carotene oxygenase 1 (BCO1) catalyzes the cleavage of the 15,15Ј double bond resulting in two retinaldehyde molecules, and the eccentric cleavage takes place at double bonds other than the central 15,15Ј double bond to produce ␤-apocarotenoids with different chain lengths. ␤-Apocarotenoids have been detected in foods (23) and the blood of both humans (24) and animals (25). Recently, ␤-apo-8Ј-carotenal was detected in plasma after ingestion of ␤-carotene by a healthy human subject (24). Our previous studies demonstrated that ␤-apo-13-carotenone functioned as an antagonist in transactivation assays using full-length RXR␣ (26) and the retinoic acid receptors ␣, ␤, and ␥ (27). We have reported that ␤-apo-13-carotenone competes for 9cRA binding to RXR␣ with an affinity (7-8 nM) identical to 9cRA itself (27). However, the molecular mechanism of ␤-apo-13-carotenone's modulation of transcriptional activity is not understood yet. This study focused on the mechanism by which ␤-apo-13-carotenone antagonizes 9cRA-induced activation of RXR␣. Our results show that ␤-apo-13-carotenone induces formation of the RXR␣ transcriptionally silent tetramer but does not inhibit coactivator recruitment to the isolated LBD.

EXPERIMENTAL PROCEDURES
Materials-COS-7 African green monkey kidney cells and MCF-7 ((Michigan Cancer Foundation-7 (a human mammary cancer cell line)) mammary carcinoma cells from ATCC (Rockville, MD) were cultured in DMEM supplemented with 10% FBS. Cells were maintained at 37°C with 10% CO 2 . 9-cis-Retinoic acid and UVI3003 were purchased from Santa Cruz Biotechnology. ␤-Apo-13-carotenone was synthesized as described previously (27). All other chemicals were from Sigma.
Nuclear Receptor Reporter Cell Assay with Full-length hRXR␣-COS-7 cells were cultured in 96-well plates overnight. cDNA of full-length human RXR␣ in pSV sport vector (Addgene) was cotransfected with Renilla (pRL-tk) and Firefly luciferase (RXRE-Luc) reporter constructs into COS-7 cells in serum-free DMEM with X-tremeGENE 9 DNA (Roche Applied Science). Twenty four hours after transfection, COS-7 cells in DMEM with 10% charcoal-stripped FBS were then treated with 9cRA in the presence or absence of ␤-apo-13-carotenone or UVI3003 for an additional 24 h. Cell lysates were used in the dual-luciferase assay (Promega) to determine the activation of hRXR␣ by 9cRA and the inhibition by ␤-apo-13-carotenone and UVI3003. For each experiment, the firefly luciferase (experimental reporter) activity was normalized to Renilla luciferase (control reporter).
Quantitative Real Time PCR-MCF-7 breast cancer cells were cultured in 6-well plates and starved for 24 h in serum-free DMEM. MCF-7 cells were then treated with ligands in serumfree medium for 4 h. Total RNA was isolated using Nucleo-Spin RNA II (Macherey-Nagel). Two micrograms of RNA was reverse-transcribed into cDNA using the high capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative real time PCR analysis was performed in quadruplicate with TaqMan chemistry and probed for caspase 9 (Hs00154260-m1) (Applied Biosystems). Eukaryotic 18S rRNA (43337607) was used as a housekeeping gene. The comparative C t method (⌬⌬C t ) was used to analyze results.
mRXR␣LBD Expression in E. coli and Protein Purification-N-His-tagged mouse RXR␣LBD (pET15b) was transformed into BL21(DE3). The E. coli culture was grown at 37°C to A 600 of 0.6. After induction with 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside, cells was incubated for another 2-4 h at 25°C. Cells were harvested and lysed in lysis buffer (20 mM Tris, 500 mM NaCl, 5 mM imidazole, and 3 mM DTT, pH 8.0). The supernatant was loaded onto a HisPur Ni-NTA affinity column followed by extensive washing with 20 mM imidazole in lysis buffer. His-mRXR␣LBD was eluted with 500 mM imidazole in lysis buffer. The concentrated protein peak fraction was then applied to HiLoad Superdex 200 gel filtration column for isolation of mRXR␣LBD dimer and tetramer. The gel filtration column was calibrated with protein standards of 13.7, 25, 43, 67, 158, 232, and 440 kDa and blue dextran 2000 to confirm the molecular weights of the mRXR␣LBD dimer and tetramer. Protein concentration was determined with the Bradford reagent (Bio-Rad) with bovine serum albumin as a standard. The purity of protein was assessed by SDS-PAGE and Coomassie Blue staining.
Gel Filtration Chromatography for Detection of mRXR␣LBD Dimer and Tetramer-Purified mRXR␣LBD dimer (50 M in monomer concentration) was incubated with ␤-apo-13carotenone or UVI3003 in various concentrations on ice for 3 h or overnight. Tween 40 was added to increase ligand solubility in the aqueous buffer. In other experiments, mRXR␣LBD tetramer (50 M in monomer concentration) was first saturated with 100 M ␤-apo-13-carotenone and then was incubated with 9cRA. After ligand treatment, mRXR␣LBD was subjected to gel filtration chromatography on a Superdex 200 HR column controlled by an AKTA FPLC system (GE Healthcare). The running buffer contained 20 mM Tris, 150 mM NaCl, pH 7.5, at 4°C. Protein chromatograms were monitored at 280 nm. As above, this gel filtration column was also calibrated with proteins of known molecular weight to confirm the retention volumes of the mRXR␣LBD dimers and tetramers.

RESULTS
␤-Apo-13-carotenone and UVI3003 Antagonize 9cRA-induced Transactivation of Full-length RXR␣-To investigate the effect of ␤-apo-13-carotenone and UVI3003 on the functional role of RXR␣, an RXRE-luciferase receptor/reporter transactivation assay was performed. Full-length hRXR␣ was transiently cotransfected in COS-7 cells with two reporter plasmids, a firefly luciferase reporter containing RXRE from CRBP-II and Renilla luciferase as an internal control. In transfected cells, 9cRA induced luciferase activity in a dose-dependent manner over a concentration range of 5 ϫ 10 Ϫ5 M (50 M) to 3.2 ϫ 10 Ϫ10 M (0.32 nM), as shown in Fig. 1. To determine the antagonist function of ␤-apo-13-carotenone and UVI3003, cells were treated with 9cRA in the presence of ␤-apo-13-carotenone or UVI3003 at a constant concentration of 200 nM. We observed a shift in the 9cRA dose-response curve induced by both ␤-apo-13-carotenone and the known antagonist UVI3003. ␤-Apo-13carotenone alone did not induce the activation of RXR␣ (data not shown). This suggests that ␤-apo-13-carotenone antagonizes 9cRA activation of full-length hRXR␣ with a similar efficiency as the known antagonist UVI3003.
UVI3003 Inhibits 9cRA-induced Coactivator Binding to the RXR␣ Ligand Binding Domain but ␤-Apo-13-carotenone Does Not-To further characterize the mechanisms of ␤-apo-13carotenone and UVI3003 as antagonists for RXR␣, we used cells that stably express a fusion protein containing the Gal4-DBD linked to the ligand binding domain (LBD) of RXR␣. The luciferase reporter gene utilized in these assays contains the Gal4 upstream activation sequence linked to the luciferase reporter gene. 9cRA activated the transcription; however, ␤-apo-13-carotenone alone did not activate the RXR␣ reporter assay (Fig. 3A). Although we tested cotreatment with ␤-apo-13carotenone at several different concentrations (5, 10, 100, 200, 500, and 1000 nM) with 9cRA, no marked shift of the 9cRA dose-response curve was observed (Fig. 3B). In contrast, 200 or 500 nM UVI3003 prominently shifted the 9cRA dose-response curve, as shown in Fig. 3C. In this assay the activation of RXR␣ does not require the formation of RXR␣LBD dimer. 9cRA binding to the ligand binding domain of RXR␣ provokes a conformational change of the AF-2 motif that produces a suitable binding surface for recruitment of coactivators. Previous structural studies have shown that the binding of antagonist UVI3003 to LBD of RXR␣ disturbs the conformation of helix 12 (H12) and leads to inhibition of coactivator recruitment (31). Strikingly, in the experiments reported here using the hybrid receptor, ␤-apo-13-carotenone had no effect on coactivator binding to the RXR␣LBD.
␤-Apo-13-carotenone Regulates RXR␣ through Tetramerization of the Receptor-To further elucidate the mechanism underlying regulation of the RXR␣ transcriptional activity by

␤-Apo-13-carotenone as an RXR Antagonist
␤-apo-13-carotenone, we investigated the dimer-tetramer equilibrium of RXR␣LBD after exposure to ligand. Mouse RXR␣LBD was expressed in Escherichia coli and purified to homogeneity as shown in Fig. 4. Gel filtration chromatography on calibrated columns of Superdex 200 was used to isolate the mRXR␣LBD dimer and tetramer used in the following experiments. Recombinant mouse RXR␣LBD dimer 50 M (calculated as monomer concentration) was incubated with increasing concentrations (100, 250, and 500 M) of ␤-apo-13-carotenone on ice for 3 h or overnight. Gel filtration chromatography demonstrated ␤-apo-13-carotenone-induced formation of RXR␣LBD tetramer (Fig. 5). Treatment with 500 M ␤-apo-13-carotenone for 3 h, in the molar ratio to monomer receptor of 5:1, led to 33% tetramer formation of the RXR␣LBD, although if treatment was extended overnight, 50% tetramer RXR␣LBD formed. In contrast, the antagonist UVI3003 did not induce tetramer formation at any of the tested concentrations (Fig. 6) even if incubations were extended overnight (data not shown). Finally, ␤-apo-13-carotenone-saturated RXR␣LBD tetramer, in molar ratio 2:1 (␤-apo-13carotenone/monomer RXR␣LBD), was incubated with the agonist 9cRA. RXR␣LBD tetramer dissociated to dimer with the  Histidine-tagged mRXR␣LBD (theoretical molecular mass for monomer ϭ 28,821 Da) was expressed in E. coli strain BL21(DE3) and purified first with an Ni-NTA affinity column. Elution fraction 1 of the affinity column was then applied onto a gel filtration column of HiLoad Superdex 200 16 ϫ 60 column to separate the mRXR␣LBD tetramer and dimer. A, SDS-PAGE of the fractions from Ni-NTA affinity column. Lane 9 was the mRXR␣LBD elution fraction collected for second step purification with gel filtration. B, separation of mRXR␣LBD tetramer and dimer with gel filtration column. mRXR␣LBD tetramer was collected between the retention volume of 61.4 ml to 68.4 ml. mRXR␣LBD dimer was collected between retention volume 72.4 and 84.4 ml. The gel filtration column was calibrated with standard proteins of known molecular weight as described in the text.
␤-Apo-13-carotenone as an RXR Antagonist NOVEMBER 28, 2014 • VOLUME 289 • NUMBER 48 addition of agonist 9cRA. At 50 M, in an equal molar concentration to monomer RXR␣LBD, 9cRA induced ϳ55% of dimer, whereas higher concentrations of 9cRA almost completely converted tetramer to dimer (Fig. 7). The gel filtration chromatography results showed that ␤-apo-13-carotenone induced the tetramerization of RXR␣LBD, which was reversed with addition of 9cRA. In contrast, the antagonist UVI3003 did not influence the tetramer-dimer equilibrium of RXR␣LBD.

DISCUSSION
In this study, we characterize the activity of ␤-apo-13-carotenone as an antagonist to RXR␣ and reveal the mechanism of RXR␣ antagonism by ␤-apo-13-carotenone-induced tetramerization of the receptor. The comparison of experimental data of ␤-apo-13-carotenone and UVI3003 indicated that these RXR␣ antagonists use two distinct mechanisms. ␤-Apo-13-carotenone, a naturally occurring ␤-apocarotenoid that can be obtained from either the diet directly or from eccentric cleavage of ␤-carotene functioned as an antagonist of RXR␣. UVI3003 is a selective antagonist of RXR␣ whose inhibitory effect results from an interference of its long side chains with Leu-451 of helix-12 (31). Both ␤-apo-13-carotenone and UVI3003 inhibited 9cRA-induced transactivation of full-length RXR␣ in a dual-luciferase assay, whereas higher concentrations of 9cRA overcame the inhibition by the two antagonists. Both antagonists inhibited 9cRA induction of the expression of the caspase 9 gene in MCF-7 cells.
The tetramerization of RXR␣ induced by ␤-apo-13-carotenone is supported by reporter cell-based assays. The "Gal4-DBD:RXR␣LBD" receptor expressed in the reporter cells will bind ligand, translocate to the nucleus, bind to the Gal4 upstream activation sequence on the reporter gene, recruit coactivator proteins, and lead to the transcription of luciferase. It is important to note that the whole process of transcription in these reporter cells does not require dimerization of RXR␣. The conformational change of RXR␣LBD due to ligand binding is sufficient to activate coactivator recruitment and subsequent luciferase transcription. ␤-Apo-13-carotenone is inactive in this assay. In contrast, full-length RXR␣ expressed in COS-7 cells undergoes nuclear translocation, dimer formation upon agonist binding, binding to the RXRE, coactivator recruitment,   ␤-Apo-13-carotenone as an RXR Antagonist and luciferase transcription. The distinctive difference in the mechanism between the reporter cell assay with Gal4-DBD: RXR␣LBD and the transactivation assay with full-length RXR␣ is that dimer formation is obligatory for the latter; and ␤-apocarotenone is only effective as an antagonist in this assay. These observations suggest that ␤-apo-13-carotenone inhibits 9cRAinduced RXR␣ transcription through the formation of the RXR␣ tetramer.
In an x-ray crystal study, atRA has been shown to bind to the transcriptionally silent tetrameric RXR␣ in a unique conformation (32). Previously, we showed using molecular modeling that when this bound atRA is computationally removed from the tetrameric RXR protein and redocked, it assumes the identical position as in the crystal structure (26). In addition, when ␤-apo-13-carotenone is built in a similar conformation to this atRA and is docked into this RXR␣ tetramer, it occupies the same position and has the same conformation as the bound atRA. Alternatively, when we built ␤-apo-13-carotenone in a conformation similar to RXR␣-bound 9cRA (33) and attempted to dock this molecule into the dimeric RXR␣, it assumes a very different position than the agonist ligand. Thus, we suggested that ␤-apo-13carotenone should be capable of acting as an antagonist of RXR␣ by stabilizing the transcriptionally silent tetramer, but we had no direct biochemical evidence for that suggestion at that time.
The crystal structure study of the ligand binding domain of the RXR␣ suggested that a cavity corresponded to the RXR ligand-binding site and that 9cRA binding triggered a conformational modification of helix 11, which led to ligand-dependent transactivation by AF-2 (33). It has also been reported that the tetramerization domain is located in helix 11 at the RXR␣LBD and tetramerization does not interfere with the function of helix12 (34). Thus, ␤-apo-13-carotenone could cause tetramerization of RXR␣ by interacting with helix 11 and not affecting helix 12 (or coactivator binding). In contrast, inhibition of 9cRA-induced RXR␣ transcription by UVI3003 is due to the blockage of helix 12 as pointed out above (31). RXR␣ tetramer formation induced by ␤-apo-13-carotenone was confirmed biochemically in this study by the observations of gel filtration chromatography with purified recombinant mouse RXR␣LBD. Comparison between the ␤-apo-13-carotenone and UVI3003-treated dimeric RXR␣LBD indicates that ␤-apo-13-carotenone regulates RXR␣ transcription through tetramerization, whereas inhibition by the antagonist UVI3003 is due to interference with helix 12. The complete dissociation of the tetramer RXR␣LBD saturated with ␤-apo-13-carotenone to dimer by 9cRA shows that tetramerization is reversible when the agonist is in sufficient concentration. Thus, the equilibrium of RXR␣ dimer and tetramer could be controlled by the availability of ligands. A model of these various effects of ligands on RXR␣ is shown in Fig. 8.
In summary, this study revealed the mechanism of ligand-dependent regulation of RXR␣ transcriptional activity by the antagonist ␤-apo-13-carotenone. The findings imply that tetramerization of RXR and factors that modulate the oligomer state may contribute to regulation of cellular signaling. ␤-Apo-13-carotenone-induced tetramerization could conserve RXR␣ as an inactive nuclear receptor pool that can rapidly supply dimeric or monomeric RXR␣ upon 9cRA generation. This may also suggest a ligand-dependent modulation controlling the availability of RXR␣ for the heterodimerization with other nuclear receptor partners engaged in multiple signaling pathways.