Zearalenone-Induced Interaction between PXR and Sp1 Increases Binding of Sp1 to a Promoter Site of the eNOS, Decreasing Its Transcription and NO Production in BAECs

Abstract

Zearalenone (ZEN) is a non-steroidal mycotoxin that has various toxicological impacts on mammalian health. Here, we found that ZEN significantly affected the production of nitric oxide (NO) and the expression of endothelial NO synthase (eNOS) of bovine aortic endothelial cells (BAECs). A promoter analysis using 5'-serially deleted human eNOS promoter revealed that the proximal region (-135 to +22) was responsible for ZEN-mediated reduction of the human eNOS promoter activity. This effect was reversed by mutation of two specificity protein 1 (Sp1) binding elements in the human eNOS promoter. A chromatin immunoprecipitation assay revealed that ZEN increased Sp1 binding to the bovine eNOS promoter region (-113 to -12), which is homologous to -135 to +22 of the human eNOS promoter region. We also found that ZEN promoted the binding of the pregnane X receptor (PXR) to Sp1 of the bovine eNOS, consequently decreasing eNOS expression. This reduction of eNOS could have contributed to the decreased acetylcholine-induced vessel relaxation upon ZEN treatment in our ex vivo study using mouse aortas. In conclusion, our data demonstrate that ZEN decreases eNOS expression by enhancing the binding of PXR-Sp1 to the eNOS promoter, thereby decreasing NO production and potentially causing vessel dysfunction.

Keywords: NO; PXR; Sp1; eNOS; zearalenone.

Figure 1

ZEN decreases NO production in a dose- or time-dependent manner, which is accompanied with the decreased protein and mRNA expressions of eNOS. BAECs were exposed to various concentrations of ZEN (0, 10, 20, 40 µM) for 24 h or 20 µM of ZEN for various time points (0, 4, 8, 16, 24 h). (a) NO production from the BAECs was measured by using the Griess method. (b) The protein expressions of eNOS relative to tubulin in the BAECs were quantified using Western blot analyses. (c) The mRNA expressions of eNOS relative to GAPDH in the BAECs were quantified using RT-PCR analyses. The plots depict the mean fold alterations below the controls (± S.D.) from at least four independent trials. Statistical significances are denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 2

Estrogen receptor is not responsible for decreasing eNOS protein expression by ZEN. After pretreatment with various concentrations of (a) a genomic ER antagonist ICI 182,780 (0, 5, 10 µM), (b) a nongenomic ER antagonist G-15 (1 µM) or G-36 (1 µM) for 1 h, BAECs were exposed to 20 µM of ZEN for 24 h. The eNOS protein expression relative to tubulin was quantified using Western blot analyses. The plots depict the mean fold alterations below the controls (± S.D.) from at least four independent trials. Statistical significances are denoted as * p < 0.05, *** p < 0.001, and N.S. (not statistically significant).

Figure 3

The proximal eNOS promoter (–135 to +22) is involved in ZEN-mediated decrease in eNOS transcriptional activation. (a) Luciferase activities of eNOS(−1600) vector and CMV promoter (renilla) in the BAECs exposed to 20 µM of ZEN for 4, 8, 16, 24 h. (b) Luciferase activities of the serially 5′-deleted promoters of human eNOS and CMV promoter (renilla) in the BAECs exposed to 20 µM of ZEN for 16 h. The plots depict the mean fold alterations below the controls (± S.D.). Statistical significances are denoted as * p < 0.05, ** p < 0.01, *** p < 0.001, and N.S. (not statistically significant).

Figure 4

The Sp1 binding regions of the human eNOS promoter are related to the decreased eNOS promoter activity by ZEN. (a) The positions of the amino acid mutated, Sp1(mut1) and Sp1(mut2), are indicated with a black arrowhead, and the mutated amino acid is shown as a bolded uppercase with an asterisk (*). The method for site mutagenesis was described in Materials and Methods. (b) Luciferase activities of eNOS(−135) wild type or the mutants and CMV promoter (renilla) in the BAECs exposed to 20 µM of ZEN for 8 h. (c) The Sp1 protein expression relative to tubulin was quantified using Western blot analyses. The plots depict the mean fold alterations below the controls (± S.D.) from at least four independent trials. Statistical significances are denoted as *** p < 0.001 and N.S. (not statistically significant).

Figure 5

ZEN increases the binding of Sp1 to the bovine eNOS promoter region (−113 to −12), thereby decreasing the promoter activity and protein expression of eNOS. (a) Human and bovine eNOS promoters were aligned using the Multalin online tool (http://multalin.toulouse.inra.fr/multalin/). The unmatched amino acid residues within the homologous regions between human (▼) and bovine () are indicated in bold. (b) After pretreatment with vehicle or 20 nM of the Sp1 inhibitor mithramycin A for 1 h, BAECs were further co-treated with or without 20 μM of ZEN for 8 h. Binding of Sp1 to the bovine eNOS promoter gene relative to the total chromatin extract (Input) was quantified using a ChIP assay, as described in Materials and Methods. (c) Luciferase activities of the bovine eNOS(−135) vector and CMV promoter (Renilla) in BAECs treated with vehicle or 20 nM mithramycin A for 1 h, followed by further co-treatment with 20 µM of ZEN for 8 h. (d) Protein expression of eNOS relative to tubulin was quantified using Western blot analyses, and (e) NO production was quantified using the Griess method in BAECs treated with vehicle or 20 nM mithramycin A followed by further co-treatment with 20 µM ZEN for 4, 8, 16, and 24 h. (f) Protein expression of eNOS relative to tubulin was quantified using Western blot analyses of BAECs transfected with siRNA targeting Sp1 for 24 h and then exposed to 20 µM of ZEN for 8 h. N.C., negative control. Plots depict mean fold alterations from the control (± S.D.) from at least four independent trials. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and N.S. (not statistically significant).

Figure 6

HDACs, NCoR1, and SMRT do not affect the ZEN-mediated decrease in mRNA and protein expression of eNOS. (a) The protein expression of eNOS relative to tubulin was quantified using Western blot analyses in the BAECs exposed to 20 µM of ZEN for 24 h after pretreatment with vehicle or HDAC inhibitor TSA for 1 h. The plots are representative of four independent experimental trials. (b) The mRNA expression of eNOS relative to GAPDH or (c) the protein expression of eNOS relative to tubulin was quantified using RT-PCR and Western blot analyses in the BAECs transfected with siRNA of NCoR1 or SMRT for 24 h, respectively, 20 µM of ZEN exposure for 24 h. N.C., negative control. The plots depict the mean fold alterations below the controls (± S.D.) from four independent trials. Statistical significances are denoted as * p < 0.05, ** p < 0.01, and N.S. (not statistically significant).

Figure 7

PXR is associated with the ZEN-mediated decrease in mRNA and protein expression of eNOS. (a) mRNA expression of eNOS relative to GAPDH and (b) protein expression of eNOS relative to tubulin were quantified using RT-PCR and Western blot analyses, respectively, in BAECs transfected with siRNA targeting PXR with or without 20 µM of ZEN exposure for 8 h. N.C., negative control. Plots depict mean fold alterations from the control (± S.D.) from at least four independent trials. Statistical significance is denoted as * p < 0.05 and ** p < 0.01.

Figure 8

ZEN induces the binding of PXR to Sp1, increasing Sp1 binding to the bovine eNOS promoter region. (a) BAECs were exposed to 20 μM ZEN for 8 h, and nuclear fractions were extracted. Nuclear fractions were subjected to co-IP assays using antibodies against PXR, Sp1, or non-immune IgG. Binding of proteins was measured by Western blot analyses, and the blots shown are representative of four independent experimental trials. The arrowhead indicates PXR protein, while the filled arrow and the blank arrow indicate Sp1 protein and heavy chain, respectively. (b) After the transfection of BAECs with siRNA targeting PXR, cells were incubated with or without 20 μM ZEN for 8 h. Binding of Sp1 to the bovine eNOS promoter relative to the total chromatin extract (Input) was quantified using a ChIP assay as described in Materials and Methods. N.C., negative control. Plots depict mean fold alterations from the control (± S.D.) from at least four independent trials. Statistical significance is denoted as follows: * p < 0.05 and ** p < 0.01.

Figure 9

ZEN reduces endothelium-dependent vessel relaxation and eNOS protein expression in mouse aortas. Endothelium-intact aortas were dissected from male C57BL/6 mice at 8 weeks old. Mouse thoracic aortas were prepared, and a vessel relaxation assay was performed as described in Materials and Methods. (a) Vessel relaxation indexes of endothelium-intact aorta rings exposed to 20 μM of ZEN or vehicle for 16 h. Aorta rings were contracted with 0.1 μM phenylephrine (PE), followed by cumulative treatment with acetylcholine (ACh, 0.001–1 μM). The line plots represent the mean ± S.E. at each point (n = 6). (b) eNOS protein expression relative to tubulin was quantified by Western blot analyses of total protein from each aorta ring exposed to 20 µM of ZEN or vehicle for 16 h. Plots depict mean fold alterations from the control (± S.D.) from three independent trials. Statistical significance is denoted as follows: * p < 0.05 and ** p < 0.01.

Figure 10

A schematic illustration of the molecular mechanism underlying the ZEN-mediated decrease in eNOS gene expression and NO production in BAECs and mouse aortas. ZEN increases the interaction between PXR and Sp1, which decreases eNOS gene transcription through increasing binding of the PXR-Sp1 complex to a G/C-rich region (−113 to −12) in the bovine eNOS promoter. This effect decreases eNOS protein expression and NO production in BAECs, and vessel relaxation in mouse aortas.