Cross-coupling of signal transduction pathways: the dioxin receptor mediates induction of cytochrome P-450IA1 expression via a protein kinase C-dependent mechanism.
Signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) is mediated by the intracellular dioxin receptor which, in its dioxin-activated state, regulates transcription of target genes encoding drug-metabolizing enzymes, such as cytochrome P-450IA1 and glutathione S-transferase Ya. Exposure of the dioxin receptor to dioxin leads to an apparent translocation of the receptor to the nucleus in vivo and to a rapid conversion of the receptor from a latent, non-DNA-binding form to a species that binds to dioxin-responsive positive control elements in vitro. This DNA-binding form of receptor appears to be a heterodimeric complex with the helix-loop-helix factor Arnt. In this study, we show that activation of the cytochrome P-450IA1 gene and minimal dioxin-responsive reporter constructs by the dioxin receptor was inhibited following prolonged treatment of human keratinocytes with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Inhibition of the receptor-mediated activation response was also achieved by treatment of the cells with a number of protein kinase inhibitors, one of which, calphostin C, shows selectivity for protein kinase C. Taken together, these data suggest that protein kinase C-dependent phosphorylation may play an essential role in the dioxin signaling pathway. This hypothesis is supported by the observation that pretreatment of the cells with 12-O-tetradecanoylphorbol-13-acetate inhibited the DNA-binding activity of the dioxin receptor in vivo. In vivo, the dioxin receptor was found to be a phosphoprotein. In vitro, dephosphorylation of the ligand-activated, heteromeric dioxin receptor form or dephosphorylation of the individual ligand-binding and Arnt receptor subunits inhibited the xenobiotic response element-binding activity. Moreover, dephosphorylation experiments with the individual receptor subunits prior to assembly of the xenobiotic response element-binding receptor form indicated that phosphorylation seemed to be important for the DNA-binding activity per se of the receptor, whereas Arnt appeared to require phosphorylation to interact with the receptor. Finally, a protein kinase C inhibitor-sensitive cytosolic catalytic activity that could restore the DNA-binding activity of the dephosphorylated dioxin receptor form was identified.
Center for Biotechnology, Karolinska Institute, Huddinge, Sweden.
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A
Berghard
K
K
Gradin
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I
Pongratz
M
M
Whitelaw
L
L
Poellinger
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004790:Enzyme Induction; D006801:Humans; D015603:Keratinocytes / Q000502:physiology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D011506:Proteins / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000502:physiology*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors
C497396:ARNT protein, human; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D003577:Cytochrome P-450 Enzyme System; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate