Abstract
Transcriptional activation of the human CYP1A1 gene by halogenated and polycyclic aromatic hydrocarbons is mediated by the aryl hydrocarbon receptor (AhR) complex, a ligand-dependent transcription factor. A competent AhR comprises at least two components following nuclear translocation and DNA binding, the AhR and the AhR nuclear translocator (Arnt) protein, whose combined action on human CYP1A1 gene transcription is shown to be dependent upon functional protein kinase C (PKC). In the present study, we examined the effects of phorbol 12-myristate 13-acetate, a potent PKC activator, on the ligand-induced transcriptional activation of the CYP1A1 gene and cellular function of the AhR in human HepG2 101L cells. The 101L cells carry a stable transgene consisting of 1800 bases of 5'-flanking DNA and the promoter of the human CYP1A1 gene linked to the firefly luciferase structural gene (Postlind, H., Vu, T. P., Tukey, R. H. & Quattrochi, L. C. (1993) Toxicol. Appl. Pharmacol. 118, 255-262). Pretreatment of cells with 12-myristate 13-acetate enhanced ligand-induced CYP1A1 gene expression 2-3-fold. Inhibition of PKC activity blocked directly the transcriptional activation and the transactivation of the CYP1A1 gene, indicating a role for PKC in the AhR-mediated transcriptional activation process. However, the DNA binding activities of the in vitro activated and the induced nuclear AhR as measured by electrophoretic mobility shift analysis were not affected when CYP1A1 transcription was inhibited, indicating the actions of PKC to be a nuclear event that works in concert with or precedes AhR binding to the gene. These results illustrate that PKC is absolutely essential for the cellular and molecular events that control induction of CYP1A1 gene transcription.
Highlights
Exposure to environmental contaminants such as polycyclic aromatic hydrocarbons, which are found in places such as cigarette smoke and smog, as well as halogenated derivatives like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)1 and some polychlorinated biphenyls (PCBs), leads to the induction in animals of cytochromes P450 1A1 and 1A2 [1, 2]
Effects of Various aryl hydrocarbon receptor (AhR) Agonists on CYP1A1 Gene Induction in 101L Cells—It has previously been demonstrated that the treatment of 101L cells with AhR ligands such as TCDD, 3MC, and omeprazole leads to a rapid and dose-dependent induction of the CYP1A1-luciferase activity [16, 23]
Exposure of 101L cells to PMA, a potent protein kinase C (PKC) activator, dramatically enhances transcriptional activation of the CYP1A1 gene induced by various AhR ligands (Figs. 1 and 2)
Summary
Exposure to environmental contaminants such as polycyclic aromatic hydrocarbons, which are found in places such as cigarette smoke and smog, as well as halogenated derivatives like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and some polychlorinated biphenyls (PCBs), leads to the induction in animals of cytochromes P450 1A1 and 1A2 [1, 2]. The ligand-activated AhR migrates rapidly to the nucleus, where it associates with the bHLH AhR nuclear translocator (Arnt) protein and binds to specific enhancer sequences flanking the CYP1A1 gene, dioxin responsive elements (DREs). Recent observations demonstrate that Arnt is a nuclear protein and most likely participates only in the nucleus to facilitate ligand-dependent AhR binding to DNA [7]. While it has been suggested that ligand binding does not directly influence the phosphorylation state of the AhR [12, 15], PKC activity may play a central role in additional cellular processes that coordinate the AhR1⁄7Arnt complex in facilitating gene regulation. 13-acetate (PMA), a model phorbol ester and PKC activator, on the biological function of the cytosolic and nuclear AhR complex and its contribution to the induction of the CYP1A1 gene
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