Transcriptional Activity of a Fluorinated Vitamin D Analog on VDR-RXR-Mediated Gene Expression

• Published in: Biochemistry (Easton) Vol. 34; no. 1; pp. 370 - 377
• Main Authors: Sasaki, Haruna, Harada, Hideyuki, Handa, Yuki, Morino, Hikaru, Suzawa, Miyuki, Shimpo, Emiko, Katsumata, Takashi, Masuhiro, Yoshikazu, Matsuda, Kouichiro
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10.01.1995
• Subjects: Animals; Base Sequence; Calcitriol - analogs & derivatives; Calcitriol - metabolism; Calcitriol - pharmacology; Chloramphenicol O-Acetyltransferase - genetics; DNA-Binding Proteins - physiology; Gene Expression Regulation - drug effects; HeLa Cells; Humans; Molecular Sequence Data; Rats; Receptors, Calcitriol - metabolism; Receptors, Calcitriol - physiology; Receptors, Retinoic Acid - physiology; Regulatory Sequences, Nucleic Acid - physiology; Retinoid X Receptors; Transcription Factors - physiology; Transcription, Genetic - drug effects; Transfection
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00001a045

The transcriptional activity of the hexafluorinated derivative of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 [F6-1,25-(OH)2D3], was examined in cultured cells by a transient expression assay (CAT assay) using expression vectors for the rat nuclear vitamin D3 receptor (VDR) and the rat 9-cis-retinoic acid receptor (RXR beta), and a reporter plasmid containing a consensus vitamin D3 response element (VDRE) consisting of two directly repeated AGGTCA motifs spaced by 3 bp (DR3). At physiological concentrations, the transcriptional activity of F6-1,25-(OH)2D3 was 2-4 times more potent than that of 1,25-(OH)2D3 in both nontarget (HeLa) and target (UMR106) cells for 1,25-(OH)2D3. The transcriptional activity of F6-1,25-(OH)2D3 was also higher when the endogenous target gene (osteopontin), which has a VDRE related to the DR3 in its promoter, was induced. A gel-shift assay using DR3 as a probe and in vitro synthesized receptors showed that the ligand-induced DNA binding of VDR required RXR to form a heterodimer. Moreover, in this assay we found that F6-1,25-(OH)2D3 induced the receptor-DNA complex at a 10-fold lower concentration than 1,25-(OH)2D3 without influencing the dissociation kinetics. However, the binding affinity of F6-1,25-(OH)2D3 for VDR was slightly lower than that of 1,25-(OH)2D3. The increased DNA binding of ligand-bound VDR by introducing hexafluorines into 1,25-(OH)2D3 may potentiate the transcriptional activity. Thus, the higher biological activity of F6-1,25-(OH)2D3 may be exerted at least in part by enhanced transcriptional activity.