Figure 4.

1,25(OH)₂D and 25(OH)D stimulate VDR and RXR recruitment to the VDRE site on the promoters of osteoblastic proteins. Chromatin was extracted from intact MC3T3 or UMR106 osteoblastic cells that had been treated with vehicle or 10^–7 M 1,25(OH)₂D or 10 ^–6 M 25(OH)D for 2 hours, in the presence of 25 mM ketoconazole to inhibit 1-hydroxylation of 25(OH)D. Extracts were then crosslinked and subjected to immunoprecipitation with VDR or RXR antibody. Nonimmunoprecipitated (Input) and Immunoprecipitated DNA from MC3T3-E3 cells were subjected to PCR using specific primers designed according to the VDRE site located in the promoter region of the target genes osteopontin, osteocalcin, and Cyp24a1 (A). PCR products were analyzed using 2% agarose gels, and representative agarose gels of 2–3 independent experiments are shown. Control was DNA immunoprecipitated with IgG. For assessment of VDR and RXR recruitment to the promoter of the FGF23 gene chromatin extracted from UMR106 cells and DNA immunoprecipitated by ChIP assay as above were analyzed by qPCR using a SsoFast-EvaGreen real-time PCR kit. Expression was normalized to the expression of input. Values represent results of three independent experiments (B).