Highly potent transcriptional activation by 16-ene derivatives of 1,25-dihydroxyvitamin D3. Lack of modulation by 9-cis-retinoic acid of response to 1,25-dihydroxyvitamin D3 or its derivatives

• Published in: The Journal of biological chemistry 1994-01, Vol.269 (4), p.2971-2981
• Main Authors: Ferrara, J, McCuaig, K, Hendy, G N, Uskokovic, M, White, J H
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Calcitriol - analogs & derivatives; Calcitriol - pharmacology; Cell Line; Chloramphenicol O-Acetyltransferase - biosynthesis; Chlorocebus aethiops; Gene Expression - drug effects; Genetic Vectors; Kidney; Kinetics; Mice; Molecular Sequence Data; Oligodeoxyribonucleotides; Osteoblasts - metabolism; Osteopontin; Osteosarcoma; Phosphoproteins - genetics; Promoter Regions, Genetic - drug effects; Rats; Restriction Mapping; Sialoglycoproteins - genetics; Structure-Activity Relationship; Thymidine Kinase - biosynthesis; Transcription, Genetic - drug effects; Transfection; Tretinoin - pharmacology; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)42035-7

Although several studies have been performed on the biological activities of analogs of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3) at the whole animal and cellular levels, little work has been done to analyze their transcriptional activation properties. A highly inducible 1,25-(OH)2 D3-responsive promoter composed of three copies of the mouse osteopontin vitamin D3 response element (VDRE3) inserted upstream of a herpes simplex virus thymidine kinase promoter has been constructed, and its transcriptional properties have been analyzed by transient transfection into the monkey kidney cell line COS-7 and the rat osteoblast-like osteosarcoma line ROS 17/2.8. We have studied systematically transcriptional activation by a number of 1,25-(OH)2 D3 analogs, particularly those substituted at positions 16, 23, 26, and 27, sites that are targets for metabolism. Strikingly, except for derivatives that bind the 1,25-(OH)2 D3 receptor (VDR) very weakly, we find no parallel between the potency of action of a derivative as a transcriptional inducer and its affinity for the VDR. Derivatives substituted by multiple bonds at positions 16 and/or 23, although having varying affinities for the VDR, all stimulate transcription more potently than D3, in some cases at 100-fold lower concentrations. The peak transcriptional activity observed varies by only approximately 20% among different active analogs, indicating little difference in the activity of the VDR once bound to ligand. Gel retardation assays with ROS 17/2.8 nuclear extracts suggest that the VDR binds to the mouse osteopontin VDRE predominantly as a heterodimer with retinoid X receptor(s) (RXR(s)). We find that 9-cis-retinoic acid, the cognate ligand for RXRs, does not have a significant effect on the response of the VDRE3 promoter to 1,25-(OH)2 D3 or a number of its derivatives in ROS 17/2.8 or in COS-7 cells, under conditions in which promoters containing retinoid X response elements are activated. This suggests that 9-cis-retinoic acid may not act on the response to 1,25-(OH)2 D3 or its derivatives by directly influencing the transcriptional activity of VDR/RXR heterodimers. This promoter/reporter system should be useful for analyzing the tissue-specific transcriptional activity of 1,25-(OH)2 D3 and its derivatives in any cell type amenable to transient transfection.