Vitamin D(3) augments osteoclastogenesis via vitamin D-responsive element of mouse RANKL gene promoter.
Receptor activator of NF-kappaB ligand (RANKL) is a membrane-bound signal transducer necessary for the induction and maintenance of osteoclasts. To clarify the molecular mechanism by which 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) augments osteoclasts, we characterized the promoter region of the mouse RANKL gene. Mirroring in vitro osteoclastogenesis demonstrated by a coculture of bone marrow macrophages with ST2 stromal cells, Northern blot, and nuclear run-on analyses showed that 1,25-(OH)(2)D(3) upregulate RANKL gene expression at the transcriptional level. Using a series of deletion mutants of mouse RANKL promoter-luciferase reporter gene constructs, transient transfection studies revealed that the inductive effect of 1,25-(OH)(2)D(3) was abolished when the region up to -723 was deleted. An electrophoretic motility shift assay demonstrated that the VDR-RXRbeta heterodimer bound to AGGTCAGCCTGGTTCA (-937/-922), and VDRE/nuclear protein supershift complexes that bound to anti-VDR and -RXRbeta antibodies were detected in the nuclear extract of 1,25-(OH)(2)D(3)-treated ST2 cells. Furthermore, induction of mutation to the putative VDRE also diminished the inductive effect of 1,25-(OH)(2)D(3). We therefore concluded that mouse RANKL gene is one of the target genes of 1,25-(OH)(2)D(3) containing a functional VDRE in the promoter region.
Kitazawa
Riko
R
Division of Molecular Pathology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
Kitazawa
Sohei
S
D000818:Animals; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002762:Cholecalciferol / Q000494:pharmacology*; D018920:Coculture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D017930:Genes, Reporter; D008264:Macrophages / Q000166:cytology; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects; D030181:Vitamin D Response Element / Q000187:drug effects / Q000502:physiology*
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D002762:Cholecalciferol