RETINOID-X-RECEPTOR-ALPHA-INDEPENDENT BINDING OF VITAMIN-D-RECEPTOR TO ITS RESPONSE ELEMENT FROM HUMAN OSTEOCALCIN GENE

We have studied the role of retinoid X receptor alpha (RXR alpha) in v itamin D receptor (VDR) responsive interactions using nuclear extracts from human osteoblast-like MG-63 osteosarcoma cells and its specific response element from human osteocalcin gene (OC-VDRE). An RXR alpha-s pecific antibody was not capable of recognizing the two VDR responsive complexes formed with the OC-VDRE. Addition of in-vitro-produced RXR alpha to the binding reaction resulted in decreased binding of the two VDR-responsive interactions and, simultaneously, formation of a new c omplex, which was identified with RXR alpha- and VDR-specific antibodi es. A similarly migrating RXR alpha- and VDR-responsive complex was al so formed when baculovirus-expressed VDR was used with the in-vitro-pr oduced RXR alpha in the absence of a nuclear extract or when VDRE from mouse osteopontin gene (OP-VDRE) was used as a binding site. Characte rization of DNA binding properties for this VDR-RXR alpha complex reve aled that both half sites of OC-VDRE are required for DNA binding. The se results indicate that RXR alpha is probably not the physiological a ccessory factor in the MG-63 osteosarcoma cells needed for the VDR-VDR E interactions within the human osteocalcin gene promoter, although it is capable of dimerizing with recombinant VDR and the native VDR from these cells and although these dimers are capable of binding in vitro to the OC-VDRE.