Fig. 5. (A) Schematic representation of the human RANKL promoter illustrating putative TCF/LEF transcription factor binding sites. MattInspector software was used to examine the human RANKL promoter (GenBank accession number AF333234) for the presence of the consensus TCF/LEF binding sequence ctttgww. Relative positions and nucleotide sequences of putative binding sites are shown (a-d: a, ctttgaa; b, ttcaaag; c, ctttgat; d, ctttgta). Positions indicated are relative to the translational start codon of the RANKL mRNA. Functional vitamin D₃ response elements (VDRE) are shown for comparison (Kitazawa et al., 2003). (B) Mimicking Wnt signalling in osteoblasts inhibited expression of receptor activator of NFB ligand (RANKL). Inhibition of GSK3ß with LiCl (20 mM) potently inhibited RANKL mRNA expression by MC3T3-E1 cells and protein expression by MG-63 osteoblastic cells compared with the NaCl control. (C) RT-PCR analysis illustrating the inhibitory effects of recombinant Wnt on RANKL mRNA expression by MC3T3-E1 cells. Exposure to Wnt3a (100 ng/ml) inhibited expression at levels comparable with LiCl (20 mM). Expression was unaffected by treatment with recombinant Wnt5a (500 ng/ml). (D) Overexpression of full-length ß-catenin inhibited basal activity of the RANKL promoter in ST2 stromal cells. Inhibitory effects were dependent on the C-terminal transactivation domain of ß-catenin deleted in ß-catenin C695-781. Values are mean ± s.e.m., n=8; ^***P0.001; n.s., not significant.