Figure 2.

Pioglitazone-mediated downregulation of Egr-1 and Snail induces 15-PGDH. A, cells were treated with indicated concentrations of pioglitazone for 12 hours. Total RNA was prepared and 15-PGDH mRNA was quantified by real-time PCR. Values were normalized to the levels of β-actin. In the inset, Western blotting was conducted and the blot was probed with antibodies to 15-PGDH and β-actin. B and C, cells were transfected with 2 μg of control siRNA (GFP) or 15-PGDH siRNA as indicated. 24 hours after transfection, cells were treated with vehicle or 10 μmol/L pioglitazone for an additional 24 hours. B, levels of PGE₂ in the cell culture medium were quantified by enzyme immunoassay. In the inset, Western blotting was conducted and the blot was probed with antibodies to 15-PGDH and β-actin. C, aromatase activity was measured. Enzyme activity is expressed as femtomoles/μg protein/min. D and E, cells were treated with indicated concentrations of pioglitazone for 8 and 10 hours, respectively. Cells were then lysed and 100 μg of cell lysate protein was subjected to Western blotting. The blots were probed with antibodies to Egr-1, Snail, and β-actin as indicated. F–H, cells were transfected with 2 μg of control (GFP) or Egr-1 siRNA. Western blotting was performed and blots were probed as indicated with antibodies to Egr-1, Snail, 15-PGDH and β-actin. I, ChIP assays were conducted. Cells were treated with vehicle (control) or 10 μmol/L pioglitazone for 10 hours. Chromatin fragments were immunoprecipitated with antibody against Snail and the 15-PGDH promoter was amplified by real-time PCR. DNA sequencing was carried out, and the PCR product was confirmed to be the 15-PGDH promoter. The 15-PGDH promoter was not detected when normal IgG was used or antibody was omitted from the immunoprecipitation step (data not shown). Mean ± SD are shown, n = 3.^*, P < 0.01 compared with vehicle-treated cells. J, transient transfections were conducted. Cells were transfected with 2 μg of empty vector or Snail expression vector. Subsequently, cells were treated with vehicle or 10 μmol/L pioglitazone for 24 hours. Levels of PGE₂ in the cell culture medium were quantified by enzyme immunoassay. Western blotting (top) was conducted and the blot was probed with antibodies to 15-PGDH and β-actin. K, cells were transfected with 0.9 μg of CYP19 1.3/II promoter and 0.2 μg of pSVβgal. Cells also received 0.9 μg of empty vector or Snail expression vector as indicated. Following transfection, cells were treated with vehicle or 10 μmol/L pioglitazone as indicated for an additional 24 hours. Luciferase activity was measured. Aromatase promoter activity represents data that have been normalized to β-galactosidase activity. In A, B, C, I, J and K, mean ± SD are shown, n = 6. ^*, P < 0.01.