Induction of proline oxidase by PPARγ
and its pharmacologic ligand, troglitazone. (a) Activation of the
POX promoter using a luciferase reporter assay. HEK 293
colorectal cancer cells were transfected with equivalent amounts
of cDNA of PPARγ or vector plasmid as control. The cells
were also transfected with POX-Luc and
pRL-null. Troglitazone (25 μM) or Me2SO in
control was added after 10 hours as indicated. At 24–36
hours after transfection, the cell lysates were harvested, and the
POX promoter luciferase activity was determined
using the Dual Luciferase Assay kit. (b) Troglitazone increases
the binding of PPARγ to the PPRE in the POX
promoter. HCT 116 colorectal cancer cells were treated with or
without 25 μM troglitazone for 36 hours and nuclear extracts
were prepared. The binding of PPARγ to the PPRE was
evaluated by an electrophoretic mobility shift analysis assay
using the double-stranded POX-PPRE oligonucleotide probe. Unlabeled POX-PPRE probe (100x) was used as a competitor (lane 4).
(c) Chromatin immunoprecipitation assay of the
POX promoter in troglitazone-treated HCT 116
cells.
HCT 116 cells were incubated with 1% formaldehyde to fix protein-DNA
complexes. DNA was sheared by sonication. Soluble chromatin-DNA
complexes were immunoprecipitated using PPARγ antibody and
immunoprecipitates were analyzed by PCR with specific primers for
the POX promoter region containing the
POX-PPRE.
