INHIBITION OF VITAMIN-D RECEPTOR RETINOID-X-RECEPTOR VITAMIN-D RESPONSE ELEMENT COMPLEX-FORMATION BY NUCLEAR EXTRACTS OF VITAMIN-D-RESISTANT NEW-WORLD PRIMATE CELLS

Most New World primate (NWP) genera evolved to require high circulatin g levels of steroid hormones and vitamin D. We hypothesized that an in tracellular vitamin D binding protein (IDBP), present in both nuclear and cytoplasmic fractions of NWP cells, or another protein(s) may caus e or contribute to the steroid hormone-resistant state in NWP by disru ption of the receptor dimerization process and/or by interference of r eceptor complex binding to the consensus response elements present in the enhancer regions of steroid-responsive genes. We employed electrom obility shift assay (EMSA) to screen for the presence of proteins capa ble of binding to the vitamin D response element (VDRE). Nuclear and p ost-nuclear extracts were prepared from two B-lymphoblastoid cell line s known to be representative of the vitamin D-resistant and wild type phenotypes, respectively. The extracts were compared for their ability to retard the migration of radiolabeled double stranded oligomers rep resentative of the VDREs of the human osteocalcin and the mouse osteop ontin gene promoters. A specific, retarded band containing VDR-RXR was identified when wild type cell but not when vitamin D-resistant cell nuclear extract was used in the binding reaction with either probe. In addition, vitamin D-resistant cell nuclear extract contained a protei n(s) which was bound specifically to the VDRE and was capable of compl etely inhibiting WR-RXR-VDRE complex formation; these effects were not demonstrated with nuclear extract from the wild type cell line or wit h the post-nuclear extract of the vitamin D-resistant cell line. We co nclude that a VDRE-binding protein(s), distinct from IDBP and present in nuclear extract of cells from a prototypical vitamin D-resistant NW P, is capable of inhibiting normal VDR-RXR heterodimer binding to the VDRE.