ANALYSIS OF THE GENE PROMOTERS OF MOUSE OSTEOCLAST DIFFERENTIATION FCTOR (RANKL)

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Receptor activator of NF-kB ligand (RANKL) is a membrane-bound signal transducer necessary for the induction of osteoclasts. We characterized the promoter region of the mouse RANKL gene, identifying inverted TATA- and CAAT-boxes, putative Runx2 binding sites (-190, -205 and -365), and a putative vitamin D response element (VDRE, -935). Northern blot and nuclear run-on analyses showed that vitamin D_3 upregulates RANKL gene expression at the transcriptional level, and transient transfectikon studies revealed that the inductive effect of vitamin D_3 was abolished by mutation of the putative VDRE (-937/-922) and deletion of the region up to -723. An elecrophoretic motility shift assay demonstrated that the VDR-RXR_heterodimer binds to AGGTCAGCCTGGTTCA (-937/-922) ; VDRE/nuclear protein supershift complexes bound to anti-VDR and RXRβ_antibodies were detected in vitamin D_3-treated ST2 cells. Furthermore, when cocultured with mouse bone marrow macrophages, later-passage ST2 cells showed a decrease in osteoclastogenesis because of decreased RANKL mRNA expression. However, earlier- and later-passage ST2 cells transfected with a RANKL promoter construct showed the same level of luciferase activity and vitamin D_3 induction, suggesting that a cis-regulatory mechanism might be involved in the dependence of RANKL gene expression on passage number. Since CpG loci (-66/+246) were predominantly methylated in later-passage ST2 cells, CpG methylation around the transcription and translation start sites probably regulates, in part, RANKL gene expression.We have presented our data at various international as well as domestic meeting, and published scientific papers for academic journals.