FIG. 8.

The mRL-VDRE confers significant VDR-dependent, 1,25(OH)₂D₃ response to the heterologous TK promoter. (A) Transcriptional activity of the mRL-VDRE or its two-component VDREs in ST2 cells. ST2 cells were transfected with pCH110-βgal (50 ng), pcDNA-hVDR (50 ng), and either the pTK control vector (250 ng), pTK-mRL-VDRE, pTK-mRL-VDRE1, or pTK-mRL-VDRE2. Cells were treated with either vehicle or increasing concentrations of 1,25(OH)₂D₃ (10⁻¹⁰ to 10⁻⁷ M) and evaluated after 24 h for both luciferase and β-galactosidase activities. (B) Effect of half-site mutations within the mRL-VDRE on response to 1,25(OH)₂D₃. Duplex mRL-VDRE oligonucleotides containing either the wild-type sequence or individual triplet mutations as depicted in Fig. 7A were cloned into the TK vector and transfected into ST2 cells, and their activity was evaluated in response to 1,25(OH)₂D₃ (10⁻⁷ M) after 24 h. Due to baseline differences, induction (fold) is reported. (C) Ability of mVDR siRNA to block activation of mRL-VDRE-mediated transcription by 1,25(OH)₂D₃ is rescued through the addition of a functional VDR expression vector. ST2 cells were cotransfected with 10 ng pCH110-βgal, pTK-mRL-VDRE, pcDNA-hVDR(wtp) (wild type), or pcDNA hVDR(m) (mutant) and 50 nM either nontargeted siRNA or mVDR siRNA as described in the legend to Fig. 5. Transfected cells were cultured for 48 h and then treated for an additional 24 h with either vehicle or 1,25(OH)₂D₃ (10⁻⁷ M) and processed as in panel B. Each point represents the normalized relative light unit average ± standard error of the mean for a triplicate set of transfections.