Vitamin D3 augments osteoclastogenesis via vitamin D-responsive element of mouse RANKL gene promoter

Receptor activator of NF-B-kappa 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. Am electrophoretic motility shift assay demonstrated that the VDR-RXRbeta heterodimer bound to (AGGTCA) under bar GCC (TGGTTCA) under bar (-937/-922), and VDRE/nuclear protein super-shift 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. (C) 2002 Elsevier Science.