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• W1999446842 abstract "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 (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 receptors (RXRα, 2The abbreviations used are: RXRretinoid X receptor9cRA9-cis-retinoic acidLBDligand binding domainDBDDNA binding domainRXREretinoid X-response elementhRXRαhuman RXRαAF-2activation function 2Ni-NTAnickel-nitrilotriacetic acidatRAall-trans-retinoic acid. RXRβ, and RXRγ) 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 (1Sakashita A. Kizaki M. Pakkala S. Schiller G. Tsuruoka N Tomosaki R. Cameron J.F. Dawson M.I. Koeffler H.P. 9-cis-Retinoic acid: effects on normal and leukemic hematopoiesis in vitro.Blood. 1993; 15: 1009-1016Crossref Google Scholar, 2Robertson K.A. Emami B. Mueller L. Collins S.J. Multiple members of the retinoic acid receptor family are capable of mediating the granulocytic differentiation of HL-60 cells.Mol. Cell. Biol. 1992; 12: 3743-3749Crossref PubMed Scopus (98) Google Scholar, 3Sanz M.J. Albertos F. Otero E. Juez M. Morcillo E.J. Piqueras L. Retinoid X receptor agonists impair arterial mononuclear cell recruitment through peroxisome proliferator-activated receptor-γ activation.J. Immunol. 2012; 189: 411-424Crossref PubMed Scopus (31) Google Scholar, 4Qian L. Zolfaghari R. Ross A.C. Liver-specific cytochrome P450 CYP2C22 is a direct target of retinoic acid and a retinoic acid-metabolizing enzyme in rat liver.J. Lipid Res. 2010; 51: 1787-1792Abstract Full Text Full Text PDF Scopus (23) Google Scholar, 5Thaller C. Hofmann C. Eichele G. 9-cis-Retinoic acid, a potent inducer of digit pattern duplications in the chick wing bud.Development. 1993; 118: 957-965Crossref PubMed Google Scholar, 6Pijnappel W.W. Hendriks H.F. Folkers G.E. van den Brink C.E. Dekker E.J. Edelenbosch C. van der Saag P.T. Durston A.J. The retinoid ligand 4-oxo-retinoic acid is a highly active modulator of positional specification.Nature. 1993; 366: 340-344Crossref PubMed Scopus (246) Google Scholar, 7Carter C.J. Farrar N. Carlone R.L. Spencer G.E. Developmental expression of a molluscan RXR and evidence for its novel, nongenomic role in growth cone guidance.Dev. Biol. 2010; 343: 124-137Crossref PubMed Scopus (51) Google Scholar) The active form of RXR is a dimer or heterodimer (8Zhang X.K. Lehmann J. Hoffmann B. Dawson M.I. Cameron J. Graupner G. Hermann T. Tran P. Pfahl M. Homodimer formation of retinoid X receptor induced by 9-cis-retinoic acid.Nature. 1992; 358: 587-591Crossref PubMed Scopus (521) Google Scholar, 9Zhang X.K. Hoffmann B. Tran P.B. Graupner G. Pfahl M. Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors.Nature. 1992; 355: 441-446Crossref PubMed Scopus (793) Google Scholar). 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 (10Lefebvre P. Benomar Y. Staels B. Retinoid X receptors: common hetero-dimerization partners with distinct functions.Trends Endocrinol. Metab. 2010; 21: 676-683Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 11Evans R.M. Mangelsdorf D.J. Nuclear Receptors, RXR, and the Big Bang.Cell. 2014; 157: 255-266Abstract Full Text Full Text PDF PubMed Scopus (753) Google Scholar). RXR naturally forms into tetramers that are transcriptionally inactive (12Kersten S. Kelleher D. Chambon P. Gronemeyer H. Noy N. Retinoid X receptor α forms tetramers in solution.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 8645-8649Crossref PubMed Scopus (92) Google Scholar). retinoid X receptor 9-cis-retinoic acid ligand binding domain DNA binding domain retinoid X-response element human RXRα activation function 2 nickel-nitrilotriacetic acid all-trans-retinoic acid. RXRs are primarily made up of two modular domains as follows: a central DNA binding domain (DBD) and a carboxyl-terminal 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 (13Dawson M.I. Xia Z. The retinoid X receptors and their ligands.Biochim. Biophys. Acta. 2012; 1821: 21-56Crossref PubMed Scopus (256) Google Scholar, 14de Lera A.R. Bourguet W. Altucci L. Gronemeyer H. Design of selective nuclear receptor modulators: RAR and RXR as a case study.Nat. Rev. Drug Discov. 2007; 6: 811-820Crossref PubMed Scopus (220) Google Scholar). 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 (13Dawson M.I. Xia Z. The retinoid X receptors and their ligands.Biochim. Biophys. Acta. 2012; 1821: 21-56Crossref PubMed Scopus (256) Google Scholar). 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 DNA-response 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 (12Kersten S. Kelleher D. Chambon P. Gronemeyer H. Noy N. Retinoid X receptor α forms tetramers in solution.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 8645-8649Crossref PubMed Scopus (92) Google Scholar, 15Kersten S. Pan L. Chambon P. Gronemeyer H. Noy N. Role of ligand in retinoid signaling. 9-cis-Retinoic acid modulates the oligomeric state of the retinoid X receptor.Biochemistry. 1995; 34: 13717-13721Crossref PubMed Scopus (49) Google Scholar). Studies have substantiated the existence in vivo of an RXR tetramer (12Kersten S. Kelleher D. Chambon P. Gronemeyer H. Noy N. Retinoid X receptor α forms tetramers in solution.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 8645-8649Crossref PubMed Scopus (92) Google Scholar, 15Kersten S. Pan L. Chambon P. Gronemeyer H. Noy N. Role of ligand in retinoid signaling. 9-cis-Retinoic acid modulates the oligomeric state of the retinoid X receptor.Biochemistry. 1995; 34: 13717-13721Crossref PubMed Scopus (49) Google Scholar). It also has been shown that the RXR tetramer is transcriptionally silent based on the correlation between the transcriptional activity of RXR mutants and their ability to form tetramers (16Kersten S. Dong D. Lee Wy Reczek P.R. Noy N. Auto-silencing by the retinoid X receptor.J. Mol. Biol. 1998; 284: 21-32Crossref PubMed Scopus (36) Google Scholar). The vitamin A metabolite 9cRA is a ligand of RXR (17Mangelsdorf D.J. Borgmeyer U. Heyman R.A. Zhou J.Y. Ong E.S. Oro A.E. Kakizuka A. Evans R.M. Characterization of three RXR genes that mediate the action of 9-cis-retinoic acid.Genes Dev. 1992; 6: 329-344Crossref PubMed Scopus (1066) Google Scholar). Binding of 9cRA as an agonist induces the dissociation of the tetramer into dimer, which is the first step for RXR activation (18Heyman R.A. Mangelsdorf D.J. Dyck J.A. Stein R.B. Eichele G. Evans R.M. Thaller C. 9-cis-Retinoic acid is a high affinity ligand for the retinoid X receptor.Cell. 1992; 68: 397-406Abstract Full Text PDF PubMed Scopus (1567) Google Scholar, 19Levin A.A. Sturzenbecker L.J. Kazmer S. Bosakowski T. Huselton C. Allenby G. Speck J. Kratzeisen C. Rosenberger M. Lovey A. 9-cis-Retinoic acid stereoisomer binds and activates the nuclear receptor RXRα.Nature. 1992; 355: 359-361Crossref PubMed Scopus (1100) Google Scholar). Carotenoids are polyisoprenoids that are biosynthesized in plants, fungi, and bacteria. Approximately 50–60 carotenoids that contain at least one unsubstituted β-ionone ring and the correct number and position of methyl groups in the polyene chain exhibit provitamin A activity (20Olson J.A. Krinsky N.I. Introduction: the colorful, fascinating world of the carotenoids: important physiologic modulators.FASEB J. 1995; 9: 1547-1550Crossref PubMed Scopus (188) Google Scholar, 21Parker R.S. Absorption, metabolism, and transport of carotenoids.FASEB J. 1996; 10: 542-551Crossref PubMed Scopus (585) Google Scholar). Dietary provitamin A, β-carotene, can be metabolized in mammals through two pathways (22Harrison E.H. Mechanisms of digestion and absorption of dietary vitamin A.Annu. Rev. Nutr. 2005; 25: 87-103Crossref PubMed Scopus (195) Google Scholar). β-Carotene oxygenase 1 (BCO1) catalyzes the cleavage of the 15,15′ double bond resulting in two retinaldehyde molecules, and the eccentric cleavage takes place at double bonds other than the central 15,15′ double bond to produce β-apocarotenoids with different chain lengths. β-Apocarotenoids have been detected in foods (23Fleshman M.K. Lester G.E. Riedl K.M. Kopec R.E. Narayanasamy S. Curley Jr., R.W. Schwartz S.J. Harrison E.H. Carotene and novel apocarotenoid concentrations in orange-fleshed Cucumis melo melons: determinations of β-carotene bioaccessibility and bioavailability.J. Agric. Food Chem. 2011; 59: 4448-4454Crossref PubMed Scopus (80) Google Scholar) and the blood of both humans (24Ho C.C. de Moura F.F. Kim S.H. Clifford A.J. Excentral cleavage of β-carotene in vivo in a healthy man.Am. J. Clin. Nutr. 2007; 85: 770-777Crossref PubMed Scopus (58) Google Scholar) and animals (25Shmarakov I. Fleshman M.K. D'Ambrosio D.N. Piantedosi R. Riedl K.M. Schwartz S.J. Curley Jr., R.W. von Lintig J. Rubin L.P. Harrison E.H. Blaner W.S. Hepatic stellate cells are an important cellular site for β-carotene conversion to retinoid.Arch. Biochem. Biophys. 2010; 504: 3-10Crossref PubMed Scopus (54) Google Scholar). Recently, β-apo-8′-carotenal was detected in plasma after ingestion of β-carotene by a healthy human subject (24Ho C.C. de Moura F.F. Kim S.H. Clifford A.J. Excentral cleavage of β-carotene in vivo in a healthy man.Am. J. Clin. Nutr. 2007; 85: 770-777Crossref PubMed Scopus (58) Google Scholar). Our previous studies demonstrated that β-apo-13-carotenone functioned as an antagonist in transactivation assays using full-length RXRα (26Eroglu A. Hruszkewycz D.P. Curley Jr., R.W. Harrison E.H. The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα.Arch. Biochem. Biophys. 2010; 504: 11-16Crossref PubMed Scopus (63) Google Scholar) and the retinoic acid receptors α, β, and γ (27Eroglu A. Hruszkewycz D.P. dela Sena C. Narayanasamy S. Riedl K.M. Kopec R.E. Schwartz S.J. Curley Jr., R.W. Harrison E.H. Naturally occurring eccentric cleavage products of provitamin A β-carotene function as antagonists of retinoic acid receptors.J. Biol. Chem. 2012; 287: 15886-15895Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). We have reported that β-apo-13-carotenone competes for 9cRA binding to RXRα with an affinity (7–8 nm) identical to 9cRA itself (27Eroglu A. Hruszkewycz D.P. dela Sena C. Narayanasamy S. Riedl K.M. Kopec R.E. Schwartz S.J. Curley Jr., R.W. Harrison E.H. Naturally occurring eccentric cleavage products of provitamin A β-carotene function as antagonists of retinoic acid receptors.J. Biol. Chem. 2012; 287: 15886-15895Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). However, the molecular mechanism of β-apo-13-carotenone's modulation of transcriptional activity is not understood yet. This study focused on the mechanism by which β-apo-13-carotenone antagonizes 9cRA-induced activation of RXRα. Our results show that β-apo-13-carotenone induces formation of the RXRα transcriptionally silent tetramer but does not inhibit coactivator recruitment to the isolated LBD. 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% CO2. 9-cis-Retinoic acid and UVI3003 were purchased from Santa Cruz Biotechnology. β-Apo-13-carotenone was synthesized as described previously (27Eroglu A. Hruszkewycz D.P. dela Sena C. Narayanasamy S. Riedl K.M. Kopec R.E. Schwartz S.J. Curley Jr., R.W. Harrison E.H. Naturally occurring eccentric cleavage products of provitamin A β-carotene function as antagonists of retinoic acid receptors.J. Biol. Chem. 2012; 287: 15886-15895Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). All other chemicals were from Sigma. COS-7 cells were cultured in 96-well plates overnight. cDNA of full-length human RXRα 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 β-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 hRXRα by 9cRA and the inhibition by β-apo-13-carotenone and UVI3003. For each experiment, the firefly luciferase (experimental reporter) activity was normalized to Renilla luciferase (control reporter). Reporter cells expressing human RXRαLBD fused to the GAL-4 DBD (Indigo Biosciences, State College PA) were treated according to the manufacturer's protocol. Reporter cells were incubated with 0, 0.32, 1.6, 8, 40, 200, 1000, and 5000 nm 9cRA for 24 h at 37 °C in the presence or absence of fixed concentrations of either β-apo-13-carotenone or UVI3003. Luminescence was detected with Glomax96 luminometer (Promega). 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 serum-free medium for 4 h. Total RNA was isolated using NucleoSpin 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 Ct method (ΔΔCt) was used to analyze results. N-His-tagged mouse RXRαLBD (pET15b) was transformed into BL21(DE3). The E. coli culture was grown at 37 °C to A600 of 0.6. After induction with 0.5 mm isopropyl 1-thio-β-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-mRXRαLBD 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 mRXRαLBD 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 mRXRαLBD 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. Purified mRXRαLBD dimer (50 μm in monomer concentration) was incubated with β-apo-13-carotenone 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, mRXRαLBD tetramer (50 μm in monomer concentration) was first saturated with 100 μm β-apo-13-carotenone and then was incubated with 9cRA. After ligand treatment, mRXRαLBD 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 mRXRαLBD dimers and tetramers. To investigate the effect of β-apo-13-carotenone and UVI3003 on the functional role of RXRα, an RXRE-luciferase receptor/reporter transactivation assay was performed. Full-length hRXRα 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 × 10−5 m (50 μm) to 3.2 × 10−10 m (0.32 nm), as shown in Fig. 1. To determine the antagonist function of β-apo-13-carotenone and UVI3003, cells were treated with 9cRA in the presence of β-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 β-apo-13-carotenone and the known antagonist UVI3003. β-Apo-13-carotenone alone did not induce the activation of RXRα (data not shown). This suggests that β-apo-13-carotenone antagonizes 9cRA activation of full-length hRXRα with a similar efficiency as the known antagonist UVI3003. It was previously reported that caspase 9 is one of the direct target genes for RAR-RXR heterodimers (28Donato L.J. Noy N. Suppression of mammary carcinoma growth by retinoic acid: proapoptotic genes are targets for retinoic acid receptor and cellular retinoic acid-binding protein II signaling.Cancer Res. 2005; 65: 8193-8199Crossref PubMed Scopus (95) Google Scholar, 29Donato L.J. Suh J.H. Noy N. Suppression of mammary carcinoma cell growth by retinoic acid: the cell cycle control gene Btg2 is a direct target for retinoic acid receptor signaling.Cancer Res. 2007; 67: 609-615Crossref PubMed Scopus (84) Google Scholar, 30Yasmin R. Kannan-Thulasiraman P. Kagechika H. Dawson M.I. Noy N. Inhibition of mammary carcinoma cell growth by RXR is mediated by the receptor's oligomeric switch.J. Mol. Biol. 2010; 397: 1121-1131Crossref PubMed Scopus (18) Google Scholar). Thus, we asked whether β-apo-13-carotenone and UVI3003 would inhibit the 9cRA-induced transcription of the endogenous gene caspase 9. For these experiments, MCF-7 cells were serum-starved for 24 h and followed by incubation with 9cRA, β-apo-13-carotenone, or UVI3003 at concentrations of 200 nm for 4 h. As shown in Fig. 2, 9cRA up-regulated the expression of mRNA for caspase 9 3–4-fold. Both β-apo-13-carotenone or UVI3003 inhibited 9cRA-induced gene expression. These data support the results shown above that β-apo-13-carotenone and UVI3003 antagonize 9cRA-induced transactivation of RXRα in transactivation assays. To further characterize the mechanisms of β-apo-13-carotenone and UVI3003 as antagonists for RXRα, we used cells that stably express a fusion protein containing the Gal4-DBD linked to the ligand binding domain (LBD) of RXRα. 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, β-apo-13-carotenone alone did not activate the RXRα reporter assay (Fig. 3A). Although we tested cotreatment with β-apo-13-carotenone 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 RXRα does not require the formation of RXRαLBD dimer. 9cRA binding to the ligand binding domain of RXRα 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 RXRα disturbs the conformation of helix 12 (H12) and leads to inhibition of coactivator recruitment (31Nahoum V. Pérez E. Germain P. Rodríguez-Barrios F. Manzo F. Kammerer S. Lemaire G. Hirsch O. Royer C.A. Gronemeyer H. de Lera A.R. Bourguet W. Modulators of the structural dynamics of the retinoid X receptor to reveal receptor function.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 17323-17328Crossref PubMed Scopus (130) Google Scholar). Strikingly, in the experiments reported here using the hybrid receptor, β-apo-13-carotenone had no effect on coactivator binding to the RXRαLBD. To further elucidate the mechanism underlying regulation of the RXRα transcriptional activity by β-apo-13-carotenone, we investigated the dimer-tetramer equilibrium of RXRαLBD after exposure to ligand. Mouse RXRαLBD 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 mRXRαLBD dimer and tetramer used in the following experiments. Recombinant mouse RXRαLBD dimer 50 μm (calculated as monomer concentration) was incubated with increasing concentrations (100, 250, and 500 μm) of β-apo-13-carotenone on ice for 3 h or overnight. Gel filtration chromatography demonstrated β-apo-13-carotenone-induced formation of RXRαLBD tetramer (Fig. 5). Treatment with 500 μm β-apo-13-carotenone for 3 h, in the molar ratio to monomer receptor of 5:1, led to 33% tetramer formation of the RXRαLBD, although if treatment was extended overnight, 50% tetramer RXRαLBD 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, β-apo-13-carotenone-saturated RXRαLBD tetramer, in molar ratio 2:1 (β-apo-13-carotenone/monomer RXRαLBD), was incubated with the agonist 9cRA. RXRαLBD tetramer dissociated to dimer with the addition of agonist 9cRA. At 50 μm, in an equal molar concentration to monomer RXRαLBD, 9cRA induced ∼55% of dimer, whereas higher concentrations of 9cRA almost completely converted tetramer to dimer (Fig. 7). The gel filtration chromatography results showed that β-apo-13-carotenone induced the tetramerization of RXRαLBD, which was reversed with addition of 9cRA. In contrast, the antagonist UVI3003 did not influence the tetramer-dimer equilibrium of RXRαLBD.FIGURE 5Gel filtration analysis for tetramerization of RXRαLBD induced by β-apo-13-carotenone. Purified mRXRαLBD dimer (50 μm in monomer concentration) was incubated with β-apo-13-carotenone in concentrations of 0, 100, 250, and 500 μm on ice for 3 h or overnight. Tween 40 of 0.75% (w/v) was added to increase ligand solubility in the aqueous buffer. Superdex 200 HR column and elution buffer were well equilibrated at 4 °C with elution buffer consisting of 20 mm Tris, 150 mm NaCl, and 3 mm DTT, pH 7.4. Protein chromatograms were monitored at 280 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6UVI3003 does not induce tetramerization of RXRαLBD. Purified mRXRαLBD dimer (50 μm in monomer concentration) was incubated with UVI3003 in concentrations of 0, 250, and 500 μm on ice for 3 h. Superdex 200 HR column and elution buffer were well equilibrated at 4 °C with elution buffer consisting of 20 mm Tris, 150 mm NaCl, and 3 mm DTT, pH 7.4. Protein chromatograms were monitored at 280 nm. Extending the incubation time with 500 μm UVI3003 overnight did not lead to tetramer formation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 79cRA dissociates the tetramer of RXRαLBD induced by β-apo-13-carotenone. mRXRαLBD tetramer (50 μm in monomer concentration) was first saturated with 100 μm β-apo-13-carotenone and then was incubated with 9cRA in concentrations of 0, 50, 100, and 250 μm on ice for 3 h. Protein chromatograms were monitored at 280 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this study, we characterize the activity of β-apo-13-carotenone as an antagonist to RXRα and reveal the mechanism of RXRα antagonism by β-apo-13-carotenone-induced tetramerization of the receptor. The comparison of experimental data of β-apo-13-carotenone and UVI3003 indicated that these RXRα antagonists use two distinct mechanisms. β-Apo-13-carotenone, a naturally occurring β-apocarotenoid that can be obtained from either the diet directly or from eccentric cleavage of β-carotene functioned as an antagonist of RXRα. UVI3003 is a selective antagonist of RXRα whose inhibitory effect results from an interference of its long side chains with Leu-451 of helix-12 (31Nahoum V. Pérez E. Germain P. Rodríguez-Barrios F. Manzo F. Kammerer S. Lemaire G. Hirsch O. Royer C.A. Gronemeyer H. de Lera A.R. Bourguet W. Modulators of the structural dynamics of the retinoid X receptor to reveal receptor function.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 17323-17328Crossref PubMed Scopus (130) Google Scholar). Both β-apo-13-carotenone and UVI3003 inhibited 9cRA-induced transactivation of full-length RXRα 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 RXRα induced by β-apo-13-carotenone is supported by reporter cell-based assays. The “Gal4-DBD:RXRαLBD” 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 co-activator 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 RXRα. The conformational change of RXRαLBD due to ligand binding is sufficient to activate coactivator recruitment and subsequent luciferase transcription. β-Apo-13-carotenone is inactive in this assay. In contrast, full-length RXRα expressed in COS-7 cells undergoes nuclear translocation, dimer formation upon agonist binding, binding to the RXRE, coactivator recruitment, and luciferase transcription. The distinctive difference in the mechanism between the reporter cell assay with Gal4-DBD:RXRαLBD and the transactivation assay with full-length RXRα is that dimer formation is obligatory for the latter; and β-apocarotenone is only effective as an antagonist in this assay. These observations suggest that β-apo-13-carotenone inhibits 9cRA-induced RXRα transcription through the formation of the RXRα tetramer. In an x-ray crystal study, atRA has been shown to bind to the transcriptionally silent tetrameric RXRα in a unique conformation (32Gampe Jr., R.T. Montana V.G. Lambert M.H. Wisely G.B. Milburn M.V. Xu H.E. Structural basis for autorepression of retinoid X receptor by tetramer formation and the AF-2 helix.Genes Dev. 2000; 14: 2229-2241Crossref PubMed Scopus (125) Google Scholar). 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 (26Eroglu A. Hruszkewycz D.P. Curley Jr., R.W. Harrison E.H. The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα.Arch. Biochem. Biophys. 2010; 504: 11-16Crossref PubMed Scopus (63) Google Scholar). In addition, when β-apo-13-carotenone is built in a similar conformation to this atRA and is docked into this RXRα tetramer, it occupies the same position and has the same conformation as the bound atRA. Alternatively, when we built β-apo-13-carotenone in a conformation similar to RXRα-bound 9cRA (33Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-α.Nature. 1995; 375: 377-382Crossref PubMed Scopus (1065) Google Scholar) and attempted to dock this molecule into the dimeric RXRα, it assumes a very different position than the agonist ligand. Thus, we suggested that β-apo-13-carotenone should be capable of acting as an antagonist of RXRα 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 RXRα 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 (33Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-α.Nature. 1995; 375: 377-382Crossref PubMed Scopus (1065) Google Scholar). It has also been reported that the tetramerization domain is located in helix 11 at the RXRαLBD and tetramerization does not interfere with the function of helix12 (34Kersten S. Reczek P.R. Noy N. The tetramerization region of the retinoid X receptor is important for transcriptional activation by the receptor.J. Biol. Chem. 1997; 272: 29759-29768Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Thus, β-apo-13-carotenone could cause tetramerization of RXRα by interacting with helix 11 and not affecting helix 12 (or coactivator binding). In contrast, inhibition of 9cRA-induced RXRα transcription by UVI3003 is due to the blockage of helix 12 as pointed out above (31Nahoum V. Pérez E. Germain P. Rodríguez-Barrios F. Manzo F. Kammerer S. Lemaire G. Hirsch O. Royer C.A. Gronemeyer H. de Lera A.R. Bourguet W. Modulators of the structural dynamics of the retinoid X receptor to reveal receptor function.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 17323-17328Crossref PubMed Scopus (130) Google Scholar). RXRα tetramer formation induced by β-apo-13-carotenone was confirmed biochemically in this study by the observations of gel filtration chromatography with purified recombinant mouse RXRαLBD. Comparison between the β-apo-13-carotenone and UVI3003-treated dimeric RXRαLBD indicates that β-apo-13-carotenone regulates RXRα transcription through tetramerization, whereas inhibition by the antagonist UVI3003 is due to interference with helix 12. The complete dissociation of the tetramer RXRαLBD saturated with β-apo-13-carotenone to dimer by 9cRA shows that tetramerization is reversible when the agonist is in sufficient concentration. Thus, the equilibrium of RXRα dimer and tetramer could be controlled by the availability of ligands. A model of these various effects of ligands on RXRα is shown in Fig. 8. In summary, this study revealed the mechanism of ligand-dependent regulation of RXRα transcriptional activity by the antagonist β-apo-13-carotenone. The findings imply that tetramerization of RXR and factors that modulate the oligomer state may contribute to regulation of cellular signaling. β-Apo-13-carotenone-induced tetramerization could conserve RXRα as an inactive nuclear receptor pool that can rapidly supply dimeric or monomeric RXRα upon 9cRA generation. This may also suggest a ligand-dependent modulation controlling the availability of RXRα for the heterodimerization with other nuclear receptor partners engaged in multiple signaling pathways. We thank Dr. Noa Noy, Case Western Reserve University, for the mRXRαLBD-pET-28a plasmid." @default.
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• W1999446842 title "β-Apo-13-carotenone Regulates Retinoid X Receptor Transcriptional Activity through Tetramerization of the Receptor" @default.
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