Figure 1. Basal and 1α,25(OH)₂D₃-induced PPARδ mRNA expression in different cancer cell lines. (a) Real-time quantitative PCR was used to measure the basal levels of PPARδ, VDR, cyclin C and CYP24 mRNA in representative breast cancer (MCF-7 and MDA-MB453) and prostate cancer (PC-3 and LNCaP) cell lines in relation to the control gene ARP0. A logarithmic scale is employed on the y-axis to better present the data. (b) In the same four cell lines the induction of PPARδ mRNA levels after three hours of treatment with 100 nM 1α,25(OH)₂D₃ was measured. (a) Data points and (b) columns indicate the means of at least three independent cell treatments and the tips of the bars represent standard deviation. Student's t-test was performed to determine the significance of the inductions in reference to solvent-treated controls (*p<0.05, **p<0.01, ***p<0.001).

Figure 2. The proximal promoter of the human PPARδ gene contains a functional VDRE. In silico analysis of the 20,840 base-pairs upstream of the human PPARδ gene revealed the location of a DR3-type VDRE at positions −347 to −363 with respect to the TSS. (a) The sequence of the RE was aligned to the known rat ANF DR3-type VDRE; the hexameric core sequence is shown in bold. Reporter gene assays were performed with extracts from MCF-7 cells that were transiently transfected in the absence or presence of an expression vector for human VDR with luciferase reporter constructs containing two copies of either (b) the rat ANF or the human PPARδ VDRE or (d) different fragments of the human PPARδ promoter. Cells were treated for 16 hours with either 100 nM 1α,25(OH)₂D₃ or solvent. Relative luciferase activity is shown and fold inductions are indicated above the columns. Columns represent means of at least three experiments and the tips of the bars indicate standard deviations. (c) Gel-shift experiments were performed with one copy of the ³²P-labeled rat ANF and human PPARδ VDRE and unprogrammed reticulocyte lysate (–) or in vitro translated VDR-RXR heterodimers. Protein–DNA complexes were resolved from free probe through non-denaturing 8% polyacrylamide gels. Representative gels are shown. The relative amount of VDR-RXR heterodimer complex formation was quantified on an FLA-3000 reader. NS indicates non-specific complexes.

Figure 3. Protein components of the 1α,25(OH)₂D₃ signaling machinery associate with the VDRE-containing region of the proximal PPARδ promoter. Chromatin was extracted from MCF-7 cells that had been treated for 0, 30, 60, 120, 180 and 240 minutes with 10 nM 1α,25(OH)₂D₃. ChIP assays using specific antibodies directed against VDR (quantifications shown in (d) and (i)), (e) RXRα, (f) pol II, (g) CBP and (h) AcH4. PCR was performed using primers that spanned the 1α,25(OH)₂D₃-responsive regions of the human PPARδ promoter (a) and the proximal human CYP24 promoter (positive control, region 1 in (c)) and an adjacent non-responsive region of the CYP24 promoter (negative control, region 2 in (c)). The location of the investigated promoter regions are schematically depicted on top of (a) and (c). (b) Re-ChIP experiments were performed with a first immunoprecipitation with anti-VDR antibody and a second precipitation with either anti-RXR or anti-phosphorylated pol II antibody for the proximal PPAR promoter. Representative agarose gels are shown in (a)–(c). The amounts of PCR products were quantified on a FLA-3000 reader in relation to chromatin input ((d)–(i)). Columns indicate the means of at least three independent cell treatments and the tips of the bars represent standard deviation. Student's t-test was performed to determine the significance of the association of the proteins with the proximal PPAR promoter in reference to time-point 0 minute (*p<0.05, **p<0.01, ***p<0.001).

Figure 4. PPARδ gene activation modulates VDR and CYP24 mRNA levels. Quantitative real-time PCR using primers for the VDR ((a)–(c) and (g)–(i)) and the CYP24 ((d)–(f) and (j)–(l)) gene was performed on cDNA derived from MCF-7 breast cancer cells ((a)–(f)) and PC-3 prostate cancer cells ((g)–(l)). The cells had been treated with 100 nM 1α,25(OH)₂D₃ or 1 μM L783483 alone or in combination for two hours ((a), (d), (g) and (j)), six hours ((b), (e), (h) and (k)) and 24 hours ((c), (f), (i) and (l)). Columns indicate the means of at least three independent cell treatments and the bars represent standard deviation. A two-tailed, Student's t-test was used to probe the significance of the results (*p<0.05, **p<0.01, ***p<0.001). NS indicates non-significance.