β‐apo‐13‐carotenone regulates retinoid X receptor transcriptional activity through tetramerization of the receptor

Retinoid X receptor (RXRα) is activated by 9‐cis‐retinoic acid (9cRA) and regulates transcription as a homodimer or as a heterodimer with other receptors. It has been shown that RXRα self‐associates to a transcriptionally silent homotetramer in absence of 9cRA. 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 of transcriptional activity is not understood. Here, we investigated β‐apo‐13‐carotenone induced change in the oligomeric state of purified RXRα ligand binding domain (RXRαLBD). We also compared β‐apo‐13‐ carotenone with the RXRα antagonist UVI3003 using a Cis‐trans nuclear reporter cell assay (INDIGO) that detects 9cRA‐induced coactivator binding to the RXRαLBD. Reporter gene constructs(RXRE‐Luc) and wild type RXRα and mutants that impair the formation of RXRα tetramers (F318A) or dimers (R321A), were transfected into COS‐7 cells and luciferase activity was examined. Our data indicate that β‐apo‐13‐carotenone induces tetramerization of the RXRαLBD whereas UVI3003 had no effect on oligomeric state. INDIGO assay results demonstrated that UVI3003 significantly inhibited 9cRA‐induced coactivator binding to RXRαLBD, but β‐apo‐13‐carotenone did not. In contrast, transactivation assays using full length RXRα showed that β‐apo‐ 13‐carotenone shifted the dose‐dependent RXRα activation by 9cRA. These observations suggest that β‐apo‐13‐carotenone regulates RXRα transcriptional activity through modulating the RXRα tetramer‐dimer equilibrium.