Modulation of Mouse RANKL Gene Expression by Runx2 and Vitamin D3

The expression of receptor activator of nuclear factor-kappa B ligand (RANKL) is regulated by bone-seeking hormones such as PTH and 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3). Runx2, a master gene for osteoblastic differentiation, also modulates osteoclastogenesis by regulating the RANKL gene. To elucidate the mechanism whereby runx2 and 1,25(OH)(2)D-3 regulate RANKL expression, we studied the function of runx2 on the chromatin structure and on the proximal binding sites using osteoblastic cell lines derived from normal (ST2) and runx2-deficient mice (RD-C6). Although the expression of RANKL in the steady-state was higher in RD-C6 than in ST2, 1,25(OH)(2)D-3-treatment of the cells increased it 20-fold in ST2 but only 1.8-fold in RD-C6. Transient transfection studies with proximal RANKL 2kb promoter, runx2 knock-down in ST2, and forced expression of runx2 in RD-C6 all confirmed that runx2 set the steady-state expression of the RANKL gene at a low level, but exerted a positive effect on enhanced transcriptional activity in response to 1,25(OH)(2)D-3. Also, assessment of the acetylation status of the area spanning 40 kb upstream of the basic promoter in ST2 and RD-C6 by ChIP assay revealed that whereas H3 and H4 historic acetylation was detected even in the steady-state in RD-C6, it was detected only with 1,25(OH)(2)D-3 in ST2. In the steady-state, runx2 may suppress RANKL gene by condensing the chromatin structure; however, it exerts a positive effect on 1,25(OH)(2)D-3-induced RANKL transcription when the proximal runx2 sites are accessible. Thus, RANKL expression in stromal/osteoblastic cells is keenly regulated by 1,25(OH)(2)D-3 which transactivates the gene at two different levels. J. Cell. Biochem. 105: 1289-1297, 2008. (c) 2008 Wiley-Liss, Inc.