CONTROL OF 1,25-DIHYDROXYVITAMIN D-3 RECEPTOR-MEDIATED ENHANCEMENT OFOSTEOCALCIN GENE-TRANSCRIPTION - EFFECTS OF PERTURBING PHOSPHORYLATION PATHWAYS BY OKADAIC ACID AND STAUROSPORINE

The 1,25-dihydroxyvitamin D-3 (vitamin D) receptor (VDR) is a key tran s-activating protein that mediates calcium regulation as well as cellu lar proliferation and differentiation. Phosphorylation of the VDR cont ributes significantly to its functional activity, but the specific mec hanisms that mediate this regulation are not well understood. Phosphor ylation may influence DNA binding, ligand binding, and protein-protein interactions, including heterodimerization and/or transactivation fun ctions. We used a protein kinase C inhibitor, staurosporine (ST), and an inhibitor of serine-threonine phosphatases, okadaic acid (OA), to e lucidate the contribution of VDR phosphorylation to vitamin D-mediated transcription of the osteocalcin (OC) gene. Vitamin D-induced transcr iption was assayed in transfected ROS 17/2.8 osteosarcoma cells using chloraminphenicol acetyltransferase constructs containing the vitamin D-responsive element (VDRE) at its native locus in the rat OC promoter as well as fused to a heterologous promoter. Both ST and OA inhibit V DRE-mediated and vitamin D-dependent enhancement of OC gene transcript ion as well as OC biosynthesis, as assessed by RIAs. Results from gel mobility shift and Western blot analyses using nuclear proteins from R OS 17/2.8 cells show that binding of the VDR-retinoid-X receptor heter odimer complex to the OC VDRE is not inhibited in the presence of ST. In contrast, OA does inhibit the formation of complexes inter acting w ith both the OC and osteopontin VDREs; immunoprecipitation studies usi ng P-32-labeled ROS 17/2.8 cells reveal that OA treatment result in li gand-independent hyperphosphorylation of the VDR. Our results suggest that two distinct phosphorylation events modulate rat VDR function. On e event is related to transactivation, and the other is also critical to the VDRE-binding activity of VDR-retinoid X receptor-DNA complexes with consequential effects on transactivation.