Functional up-regulation of VDR induced by β2-AR stimulation. (A) Luciferase reporter gene assay. MC3T3-E1 cells transfected with promoter-reporter plasmid containing VDRE (firefly luciferase) and Renilla luciferase plasmid DNAs were treated with or without clenbuterol for 2 hours and then washed and cultured in the presence of 1,25(OH)₂D₃ for indicated periods. Each bar shows mean value with error bar of duplicate data. A representative result from 3 independent experiments is shown. (B) Induction of Rankl and Vdr expression. MC3T3-E1 cells were pretreated with clenbuterol for 2 hours and further incubated in the presence or absence of 1,25(OH)₂D₃ for indicated periods. The expression levels were determined by real-time RT-PCR. Data were normalized to β-actin expression. Each bar shows mean value with error bar of duplicate data. A representative result from 2 independent experiments is shown. (C) Induction and stabilization of VDR by 1,25(OH)₂D₃. MC3T3-E1 cells were pretreated with clenbuterol for 2 hours and then stimulated by 1,25(OH)₂D₃ for indicated periods. VDR was detected by immunoblot analysis. A representative result from 3 independent experiments is shown.