Pioglitazone, a PPARγ agonist, suppresses CYP19 transcription: evidence for involvement of 15-hydroxyprostaglandin dehydrogenase and BRCA1

Abstract

Estrogen synthesis is catalyzed by cytochrome P450 aromatase, which is encoded by the CYP19 gene. In obese postmenopausal women, increased aromatase activity in white adipose tissue is believed to contribute to hormone-dependent breast cancer. Prostaglandin E(2) (PGE(2)) stimulates the cAMP→protein kinase A (PKA) pathway leading to increased CYP19 transcription and elevated aromatase activity in inflamed white adipose tissue. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) plays a major role in the catabolism of PGE(2). Here, we investigated the mechanism by which pioglitazone, a ligand of the nuclear receptor PPARγ suppressed aromatase expression. Treatment of human preadipocytes with pioglitazone suppressed Snail, a repressive transcription factor, resulting in elevated levels of 15-PGDH and reduced levels of PGE(2) in the culture medium. Pioglitazone also inhibited cAMP→PKA signaling leading to reduced interaction between phosphorylated cAMP responsive element-binding protein, p300, and CYP19 I.3/II promoter. BRCA1, a repressor of CYP19 transcription, was induced by pioglitazone. Consistent with these in vitro findings, treatment of mice with pioglitazone activated PPARγ, induced 15-PGDH and BRCA1 while suppressing aromatase levels in the mammary gland. Collectively, these results indicate that the activation of PPARγ induces BRCA1 and suppresses the PGE(2)→cAMP→PKA axis leading to reduced levels of aromatase. PPARγ agonists may have a role in reducing the risk of hormone-dependent breast cancer in obese postmenopausal women.

Figure 1

Pioglitazone inhibits aromatase expression in human preadipocytes. A, cells were transfected with 1.8 μg PPRE-luciferase and 0.2 μg pSVβgal. Following transfection, cells were treated with the indicated concentrations of pioglitazone for 24 hours. In A, D, and E, cells were lysed and luciferase activity was measured in cell lysates. Firefly luciferase activity was normalized to β-galactosidase activity. B, cells were treated with the indicated concentration of pioglitazone for 24 hours and then aromatase activity was assayed in cell lysates. Enzyme activity is expressed as femtomoles/μg protein/min. C, total RNA was prepared from cells that had been treated with pioglitazone for 24 hours. Levels of aromatase mRNA were quantified by real-time PCR. Values were normalized to levels of β-actin. D, cells were transfected with 1.8 μg CYP19 I.3/II promoter and 0.2 μg pSVβgal. Cells were then treated with indicated concentrations of pioglitazone for 24 hours. E, cells were transfected with 0.9 μg CYP19 I.3/II promoter and 0.2 μg pSVβgal. Cells also received 0.9 μg siRNA to GFP (control siRNA) or PPARγ. 24 hours after transfection, cells were treated with vehicle or 10 μmol/L pioglitazone. Aromatase promoter activity was measured 24 hours after treatment. In the inset, Western blotting was conducted on cell lysates that were treated with siRNA to GFP (control siRNA) or PPARγ. The blot was probed with antibodies to PPARγ and β-actin. F, cells were treated with indicated concentrations of PGE₂. 24 hours later, aromatase activity was determined. G, cells were treated with indicated concentrations of pioglitazone for 16 hours. Subsequently, the concentration of PGE₂ in the cell culture medium was measured using enzyme immunoassay. In H and I, cells were treated with pioglitazone for 18 hours. Subsequently, cellular levels of cAMP (H) and PKA activity (I) were determined. A–I, mean ± SD are shown, n = 6.^*, P < 0.01 compared with vehicle-treated cells.

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.

Figure 3

Pioglitazone induces BRCA1 in preadipocytes. A, cells were treated with indicated concentrations of pioglitazone for 24 hours. Subsequently, total RNA was isolated and BRCA1 mRNA was quantified by real-time PCR. Values were normalized to levels of β-actin. B, cells were transfected with 1.8 μg BRCA1 promoter-luciferase. C, ChIP assays were conducted. Chromatin fragments were immunoprecipitated with antibody against PPARγ and the BRCA1 promoter was amplified by PCR (top) or real-time PCR (bottom). DNA sequencing was carried out, and the PCR product was confirmed to be the BRCA1 promoter. The BRCA1 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. D, cells were transfected with either 1.8 μg wild-type (Wt) BRCA1 promoter-luciferase or a BRCA1 promoter-luciferase construct in which the PPRE was mutated. In B and D, cells also received 0.2 μg pSVβgal. Subsequently, cells were treated with vehicle or 10 μmol/L pioglitazone for 24 hours. Luciferase activity was measured. BRCA1 promoter activity represents data that have been normalized to β-galactosidase activity. In A, B and D, mean ± SD are shown, n = 6.^*, P < 0.01 compared with vehicle-treated cells.

Figure 4

Pioglitazone modulates binding of pCREB, BRCA1, and p300 to CYP19 I.3/II promoter and thereby suppresses aromatase expression in preadipocytes. A, cells were treated with vehicle (control) or 10 μmol/L pioglitazone for 18 hours. Chromatin fragments were then immunoprecipitated with antibodies against pCREB, BRCA1, or p300 and the CYP19 I.3/II promoter was amplified by PCR (top) or real-time PCR (bottom). DNA sequencing was carried out, and the PCR product was confirmed to be the CYP19 1.3/II promoter. The CYP19 I.3/II promoter was not detected when normal IgG was used or antibody was omitted from immunoprecipitation step (data not shown). Mean ± SD are shown, n = 3. ^*, P < 0.01 versus vehicle-treated cells. B, cells were transfected with 0.9 μg CYP19 1.3/II promoter and 0.2 μg of pSVβgal. Cells also received 0.45 μg active CREB or 0.45 μg p300 expression vectors as indicated. The total amount of DNA received by cells in all treatment groups was maintained at 2 μg by using vector DNA. Subsequently, cells were treated with vehicle or 10 μmol/L pioglitazone for 24 hours. C, cells were transfected with 0.9 μg CYP19 1.3/II promoter and 0.2 μg of pSVβgal. Cells also received 0.9 μg of either control siRNA (GFP) or BRCA1 siRNA. Cells were then treated with vehicle or 10 μmol/L pioglitazone for 24 hours. Top, Northern blotting was conducted and the blot was probed for BRCA1 and 18S rRNA. B and C, luciferase activity was measured. Aromatase promoter activity represents data that have been normalized to β-galactosidase activity. B and C, mean ± SD are shown, n = 6. ^*, P < 0.01. D, cells were treated with vehicle (control) or 10 μmol/L pioglitazone for 24 hours. Cell lysates (500 μg) were subjected to immunoprecipitation with p300 antiserum and Western blotting was conducted for p300, pCREB, PPARγ, and BRCA1 as indicated. These proteins were not immunoprecipitated with control IgG. Input is p300. E, cells were treated with vehicle (control) or 10 μmol/L pioglitazone for 24 hours. Western blotting on cell lysate protein (100 μg/lane) was conducted and the immunoblot was probed as indicated.

Figure 5

Pioglitazone induces 15-PGDH and BRCA1, and suppresses aromatase levels in mouse mammary gland. Mice were fed control diet or control diet supplemented with 0.05% or 0.1% (w/w) pioglitazone for 2 weeks before being sacrificed. Levels of aromatase activity (A), aromatase mRNA (B), 15-PGDH mRNA (C), PGE₂ (D) 13,14-dihydro-15-keto PGE₂ (E), and BRCA1 mRNA (F) were measured in mammary glands. A, aromatase activity is expressed as femtomoles/μg protein/h. Total RNA was prepared and aromatase (B), 15-PGDH (C), and BRCA1 (F) mRNA levels were quantified by real-time PCR. D and E, PGE₂ and 13,14-dihydro-15-keto-PGE₂ levels were measured in mammary glands. The biomarker levels under each experimental condition (n = 10) were summarized in terms of mean ± SD ■, P < 0.1; ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001 versus mice that received control diet.

Figure 6

Pioglitazone activates PPARγ and induces aP2 in murine mammary gland. Mice were fed control diet or control diet supplemented with 0.05% or 0.1% (w/w) pioglitazone for 2 weeks prior to being sacrificed. A, Northern blotting was conducted on total RNA (10 μg/lane) and the blots were probed for aP2 and 18S rRNA. B, electrophoretic mobility shift assays were conducted using nuclear protein isolated from the mammary glands. 5 μg of nuclear protein from individual mammary glands were incubated with a ³²P-labeled oligonucleotide containing a PPRE canonical site. Left, (control diet), C represents purified PPARγ protein and served as a positive control. Right, (0.1% w/w pioglitazone), lane 6 represents nuclear protein incubated with a ³²P-labeled PPRE containing oligonucleotide and a 100-fold excess of unlabeled oligonucleotide; lanes 7 and 8, nuclear protein incubated with a ³²P-labeled oligonucleotide and IgG (lane 7) or antibody to PPARγ (lane 8). The protein–DNA complex that formed was separated on a 4% PAGE. The arrow shows a supershift for PPARγ.