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.
Highlights
Retinoid X receptors (RXR␣,2 retinoid X receptor (RXR), and RXR␥) are members of the nuclear receptor family and play a central role in
The gel filtration chromatography results showed that -apo-13-carotenone induced the tetramerization of RXR␣ligand binding domain (LBD), which was reversed with addition of FIGURE 7. 9-cis-retinoic acid (9cRA) dissociates the tetramer of RXR␣LBD induced by -apo13-carotenone. mRXR␣LBD tetramer (50 M in monomer concentration) was first saturated with 100 M -apo-13-carotenone and was incubated with 9cRA in concentrations of 0, 50, 100, and 250 M on ice for 3 h
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
Summary
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. Twenty four hours after transfection, COS-7 cells in DMEM with 10% charcoal-stripped FBS were treated with 9cRA in the presence or absence of -apo-13-carotenone or UVI3003 for an additional 24 h. 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 -apo13-carotenone or UVI3003. The concentrated protein peak fraction was 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. This gel filtration column was calibrated with proteins of known molecular weight to confirm the retention volumes of the mRXR␣LBD dimers and tetramers
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
