Specific blockage of ligand-induced degradation of the Ah receptor by proteasome but not calpain inhibitors in cell culture lines from different species.
To firmly establish the pathway involved in ligand-induced degradation of the AHR, cell lines derived from mouse rat or human tissues were exposed to inhibitors specific to the proteasome or calpain proteases and exposed to TCDD. The level of endogenous AHR and CYP1A1 protein was then evaluated by quantitative Western blotting. Treatment of cells with the calpain inhibitors: calpeptin, calpain inhibitor III, or PD150606 either individually or in combinations up to 75 microM did not reduce TCDD-induced degradation of the AHR, the induction of endogenous CYP1A1 or the nuclear accumulation of the AHR. The activity of the inhibitors was verified with an in vivo calpain assay. In contrast, exposure of cells to the specific proteasome inhibitors: epoxomicin (1-5 microM), proteasome inhibitor I (5-10 microM) or lactacystin (5-15 microM) completely inhibited TCDD-induced degradation of the AHR. Inhibition of AHR degradation with these compounds did not reduce the induction of endogenous CYP1A1. In addition, exposure of the Hepa-1 line to the various proteasome inhibitors caused an accumulation of the AHR in the nucleus in the absence of TCDD exposure. Finally, Western blot analysis of the DNA bound AHR showed that its molecular mass was unchanged in comparison to the unliganded (cytoplasmic) AHR. Thus, these studies conclusively implicate the proteasome and not calpain proteases in the ligand-induced degradation of the mouse, rat and human AHR and suggest that the pharmacological use of proteasome inhibitors may impact the time course and magnitude of gene regulatory events mediated through the AHR.
Pollenz
Richard S
RS
Division of Cell Biology, Microbiology and Molecular Biology, Department of Biology, University of South Florida, SCA 110, 4202 E Fowler Avenue, Tampa, FL 33620, United States. pollenz@cas.usf.edu <pollenz@cas.usf.edu>
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000179:Acrylates / Q000494:pharmacology; D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects*; D004336:Drug Antagonism; D004338:Drug Combinations; D004785:Environmental Pollutants / Q000633:toxicity*; D006801:Humans; D051379:Mice; D009842:Oligopeptides / Q000494:pharmacology; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity*; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000378:metabolism*
D000179:Acrylates; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D004338:Drug Combinations; D004785:Environmental Pollutants; D009842:Oligopeptides; C100442:PD 150606; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; C090535:benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal; C067713:lactacystin; C071482:calpeptin; D019363:Cytochrome P-450 CYP1A1; C058076:calpain inhibitor III; D000111:Acetylcysteine; C078846:epoxomicin