Superinduction of CYP1A1 gene expression. Regulation of 2,3,7, 8-tetrachlorodibenzo-p-dioxin-induced degradation of Ah receptor by cycloheximide.
Cycloheximide superinduces the transcription of CYP1A1 in the presence of an agonist for the Ah receptor (AhR). To investigate the molecular target for "superinduction," we analyzed the agonist-induced degradation of AhR. Whereas 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), a potent agonist of AhR, induces a rapid reduction of the AhR protein, cycloheximide blocks the down-regulation of steady state AhR. Analyses of the turnover of AhR reveal that cycloheximide blocks the shortening of the half-life of AhR by TCDD. Blocking of the TCDD-induced AhR degradation requires inhibition of protein synthesis, because (a) cycloheximide inhibits protein synthesis at the concentration at which it causes superinduction and inhibition of AhR degradation; and (b) puromycin, an inhibitor of protein synthesis by mimicking aminoacyl-tRNA, also blocks the TCDD-induced AhR degradation. The blocking of the TCDD-induced AhR degradation correlates with the superinduction of CYP1A1 gene expression in a time- and dose-dependent manner. Furthermore, cycloheximide is shown to increase the accumulation of the TCDD-activated AhR and the functional AhR x Arnt complex in nucleus. Collectively, our results reveal a mechanism of superinduction by cycloheximide by enhancing the stability of agonist-activated AhR. The finding that inhibition of protein synthesis blocks the TCDD-induced AhR turnover implicates a cycloheximide-sensitive, labile factor (designated as AhR degradation promoting factor, or ADPF) in controlling the removal of agonist-activated AhR in nucleus.
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Molecular Toxicology Laboratory, Toxicology and Molecular Biology Branch, Health Effects Laboratory Division, NIOSH, National Institutes of Health, Centers for Disease Control and Prevention, Morgantown, West Virginia 26505, USA.
Renzelli
A J
AJ
Baldwin
K T
KT
Antonini
J M
JM
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology*; D019363:Cytochrome P-450 CYP1A1 / Q000187:drug effects / Q000235:genetics*; D015536:Down-Regulation; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015151:Immunoblotting; D051379:Mice; D018613:Microscopy, Confocal; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D011233:Precipitin Tests; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors
D000072317:Polychlorinated Dibenzodioxins; D011500:Protein Synthesis Inhibitors; D018336:Receptors, Aryl Hydrocarbon; D011691:Puromycin; D012313:RNA; D003513:Cycloheximide; D019363:Cytochrome P-450 CYP1A1