p53 modulates Hsp90 ATPase activity and regulates aryl hydrocarbon receptor signaling

Abstract

The aryl hydrocarbon receptor (AhR), a client protein of heat shock protein 90 (Hsp90), is a ligand-activated transcription factor that plays a role in polycyclic aromatic hydrocarbon (PAH)-induced carcinogenesis. Tobacco smoke activates AhR signaling leading to increased transcription of CYP1A1 and CYP1B1, which encode proteins that convert PAHs to mutagens. Recently, p53 was found to regulate Hsp90 ATPase activity via effects on activator of Hsp90 ATPase (Aha1). It is possible, therefore, that AhR-dependent expression of CYP1A1 and CYP1B1 might be affected by p53 status. The main objective of this study was to determine whether p53 modulated AhR-dependent gene expression and PAH metabolism. Here, we show that silencing p53 led to elevated Aha1 levels, increased Hsp90 ATPase activity, and enhanced CYP1A1 and CYP1B1 expression. Overexpression of wild-type p53 suppressed levels of CYP1A1 and CYP1B1. The significance of Aha1 in mediating these p53-dependent effects was determined. Silencing of Aha1 led to reduced Hsp90 ATPase activity and downregulation of CYP1A1 and CYP1B1. In contrast, overexpressing Aha1 was associated with increased Hsp90 ATPase activity and elevated levels of CYP1A1 and CYP1B1. Using p53 heterozygous mutant epithelial cells from patients with Li-Fraumeni syndrome, we show that monoallelic mutation of p53 was associated with elevated levels of CYP1A1 and CYP1B1 under both basal conditions and following treatment with benzo[a]pyrene. Treatment with CP-31398, a p53 rescue compound, suppressed benzo[a]pyrene-mediated induction of CYP1A1 and CYP1B1 and the formation of DNA adducts. Collectively, our results suggest that p53 affects AhR-dependent gene expression, PAH metabolism, and possibly carcinogenesis.

Figure 1. p53 regulates AhR signaling

A and B, EB-1 cells were transfected with 1.8 μg of p53 luciferase construct (A) or 1.8 μg of XRE-luciferase construct (B) and 0.2 μg of pSVβgal. 24 hours later, cells were treated with the indicated concentrations of ZnCl₂ for 12 hours and then cells were harvested. In A, cell lysates were also subjected to Western blotting and the blot was probed as indicated. C and D, cells were treated with the indicated concentrations of ZnCl₂ for 12 hours. C, RNA was isolated from cells and relative expression of CYP1A1 and CYP1B1 was quantified by real-time PCR. E, EB-1 cells were treated with vehicle or 75 μmol/L ZnCl₂ for 12 hours. Subsequently, cells were treated with DMSO (Control) or 1 μmol/L B[a]P for 12 hours. D and E, cell lysates were subjected to Western blotting and the blots were probed as indicated. F, MSK-Leuk1 cells were transfected with 2 μg of p53 siRNA or control siRNA. Cells were then treated with vehicle or 1 μmol/L B[a]P for 12 hours. Cell lysates were prepared and subjected to Western blotting and probed as indicated. G and H, cells were transfected with 1.8 μg of p53 luciferase construct (G) or 1.8 μg of XRE-luciferase (H) construct and 0.2 μg of pSVβgal for 24 hours. I, RNA was isolated from cells and relative expression of CYP1A1 and CYP1B1 mRNA was quantified by real-time PCR. J, cells were treated with vehicle or 1 μmol/L B[a]P for 12 hours. Cell lysates were subjected to Western blotting and probed as indicated. In A, B, G and H, luciferase and β-galactosidase activities were measured. Luciferase activity was normalized to β-galactosidase activity. In A–C, G–I, mean ± SD are shown, n = 6. *, P < 0.01 compared with vehicle treated cells (A–C) or BEAS-2B cells (G–I).

Figure 2. CP-31398, a p53 rescue compound, inhibits B[a]P-mediated induction of CYP1A1 and CYP1B1

A and C, cells were transfected with 1.8 μg of p53 luciferase construct and 0.2 μg of pSVβgal. Subsequently, the cells were treated with the indicated concentrations of CP-31398 for 24 hours and then luciferase and β-galactosidase activities were measured. Luciferase activity was normalized to β-galactosidase activity. A and C, mean ± SD are shown, n = 6. *, P < 0.01 compared with vehicle-treated cells. B and D, cells were treated with vehicle or the indicated concentrations of CP-31398 for 2 hours. Subsequently, cells were treated with vehicle or 1 μmol/L B[a]P. Twelve hours later cells were harvested for Western blot analysis. The blots were probed as indicated.

Figure 3. Aha1 and Hsp90 are important for p53-mediated regulation of AhR signaling in MSK-Leuk1 cells

A–C, cells were transfected with 2 μg of control siRNA or siRNA to p53 for 48 hours. A, cell lysates were subjected to Western blotting and the blots probed as indicated. B, cell lysate protein (500 μg) was subjected to immunoprecipitation with an antibody to Hsp90. Immunoprecipitates were then subjected to Western blotting and the blots probed for Aha1 and Hsp90 as indicated. C, cell lysates were used to measure Hsp90 ATPase activity. D and E, cells were transfected with 2 μg of Aha1 siRNA or control siRNA. D, Hsp90 ATPase activity was measured in cell lysates. Inset, cell lysates were subjected to Western blotting for Aha1 and β-actin. E, cell lysate protein (2 mg) was subjected to immunoprecipitation separately with antibodies to CYP1A1, CYP1B1 and β-actin, respectively. The immunoprecipitates were then subjected to Western blotting and the blots probed as indicated. F and G, cells were stably transfected with control vector or Aha1 expression vector. F, cell lysates were used to measure Hsp90 ATPase activity. Inset, cell lysates were subjected to Western blotting and the blots probed for Aha1 and β-actin as indicated. G, cells were treated with vehicle or 1 μmol/L B[a]P. Twelve hours later, cells were harvested for Western blot analysis and the blots probed as indicated. H, cells were treated with 1 μmol/L 17-AAG or 0.5 μmol/L PUH-H71 for 24 hours. Cell lysate protein (2 mg) was subjected to immunoprecipitation separately with antibodies to CYP1A1, CYP1B1 and β-actin, respectively. The immunoprecipitates were then subjected to Western blotting and the blots probed as indicated. In C, D and F, mean ± SD are shown, n = 6. *, P < 0.01.

Figure 4. p53 regulates the localization of AhR in MSK-Leuk1 cells

In A and B, cells were transfected with 2 μg of control siRNA or siRNA to p53 for 48 hours. A, cells were lysed, cytosolic (Cyt) and nuclear (Nuc) fractions were isolated and subjected to Western blotting and the blots probed as indicated. B, ChIP assays were performed. Chromatin fragments were immunoprecipitated with antibodies against AhR, and the CYP1A1 and CYP1B1 promoters were amplified by PCR. C, cells were treated vehicle or 20 μmol/L CP-31398 for 2 hours. Subsequently, the cells were treated with vehicle (control) or 1 μmol/L B[a]P for 2 hours. Cytosolic (Cyt) and nuclear (Nuc) fractions were isolated and subjected to Western blotting and the blots were probed as indicated. D, cells were treated with vehicle or 20 μmol/L CP-31398 for 2 hours. Subsequently, cells received vehicle (control) or 1 μmol/L B[a]P for an additional 4 hours. ChIP assays were then performed. Chromatin fragments were immunoprecipitated with antibodies against AhR, and the CYP1A1 and CYP1B1 promoters were amplified by PCR. In B and D, DNA sequencing was carried out, and the PCR products were confirmed to be the correct promoters. In B and D, mean ± SD are shown, n = 6. *, P < 0.01 compared with control siRNA (B) or B[a]P-treated cells (D).

Figure 5. AhR signaling is activated in cells derived from Li-Fraumeni Syndrome patients

LFS epithelial cells (HME50, IUSM/LFS/HME) and epithelial cells wild type for p53 (HME32) were compared. A, Cell lysate protein (1 mg) was subjected to immunoprecipitation separately with antibodies to CYP1A1, CYP1B1 and β-actin, respectively. The immunoprecipitates were then subjected to Western blotting and the blots probed as indicated. In B–E, cells were treated with DMSO (control) or 1 μmol/L B[a]P for 12 hours. Cell lysates were then subjected to immunoblotting and the blots probed as indicated. F, cytosolic and nuclear fractions were isolated and subjected to Western blotting. The blots were probed as indicated.

Figure 6. CP-31398 inhibits B[a]P-induced DNA adduct formation

MSK-Leuk1 cells were treated with vehicle, 20 μmol/L CP-31398 or 2 μmol/L αNF for 2 hours. Subsequently, the cells received vehicle or 1 μmol/L B[a]P for 10 hours. DNA was isolated for quantification of DNA adducts (A); B[a]P-tetrol formation was measured in the media (B). In A and B, mean ± SD are shown, n = 4. *, P < 0.01 compared with B[a]P treated cells.