Development of a biologically active fluorescent-labeled calcitriol and its use to study hormone binding to the vitamin D receptor.

To gain better insight into the mechanism of steroid receptor activation and calcitriol action, we have developed the first pharmacologically relevant fluorescent-labeled ligand for the vitamin D receptor (VDR). Purity and structure of three BODIPY-labeled calcitriol derivatives were characterized by TLC, HPLC, and 1H-NMR spectroscopy. 3 beta-BODIPY-calcitriol was the most potent derivative to induce 25-hydroxyvitamin D3 24-hydroxylase activity and to inhibit cell proliferation. It was taken up rapidly and specifically and was not cleaved by endogenous esterases. 3 beta-BODIPY-calcitriol also retained high-affinity binding to the VDR. Hormone binding to the receptor was measured by spectrofluorometry in high-salt extracts from cultured cells with wild-type VDR, from cells virally over-expressing the human VDR, and in intact cells with and without VDR. Results from fluorescent binding studies agreed with results from radioligand assays. The most useful feature of this reagent is that its fluorescence emission increases severalfold upon binding to VDR. This allows direct monitoring by microscopy of ligand receptor interactions in living cells. Fluorescent-labeled calcitriol can be a valuable diagnostic tool for cancer research and is essential for exploring the subcellular localization of VDRs.