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.
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.
Ferrara
J
J
Department of Physiology, McGill University, Montreal, Québec, Canada.
McCuaig
K
K
Hendy
G N
GN
Uskokovic
M
M
White
J H
JH
D000818:Animals; D001483:Base Sequence; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D002522:Chlorocebus aethiops; D015870:Gene Expression / Q000187:drug effects*; D005822:Genetic Vectors; D007668:Kidney; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010006:Osteoblasts / Q000378:metabolism; D053495:Osteopontin; D012516:Osteosarcoma; D010750:Phosphoproteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D051381:Rats; D015183:Restriction Mapping; D012795:Sialoglycoproteins / Q000235:genetics*; D013329:Structure-Activity Relationship; D013937:Thymidine Kinase / Q000096:biosynthesis; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; C506897:Spp1 protein, rat; D053495:Osteopontin; D014212:Tretinoin; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase; D002117:Calcitriol