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• W2027153670 abstract "Cycloheximide superinduces the transcription ofCYP1A1 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·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 degradationpromoting factor, or ADPF) in controlling the removal of agonist-activated AhR in nucleus. Cycloheximide superinduces the transcription ofCYP1A1 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·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 degradationpromoting factor, or ADPF) in controlling the removal of agonist-activated AhR in nucleus. 2,3,7,8-tetrachlorodibenzo-p-dioxin aryl hydrocarbon receptor AhR nuclear translocator dimethyl sulfoxide lipopolysaccharide cycloheximide transcription activation basic helix loop helix AhR degradation promoting factor electrophoretic mobility shift assay digitonin polyacrylamide gel electrophoresis 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD,1 dioxin) represents the prototype for a class of structurally related halogenated aromatic hydrocarbons, including polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls (1.$$$$$$ ref data missingGoogle Scholar, 2.Whitlock Jr., J.P. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 103-125Crossref PubMed Scopus (1001) Google Scholar). These man-made compounds are mostly by-products of industrial processes involving chlorine chemistry and combustion of fuels. Many such chemicals are also widespread and persistent environmental contaminants. TCDD is the most potent among the chemicals; animals exposed to TCDD exhibit a wide range of toxic and adaptive responses, including a wasting syndrome, tumor promotion in skin and liver, cleft palate, chloracne, immune and endocrine dysfunctions, and induction of drug metabolizing enzymes (2.Whitlock Jr., J.P. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 103-125Crossref PubMed Scopus (1001) Google Scholar, 3.Hankinson O. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 307-340Crossref PubMed Scopus (1432) Google Scholar, 4.Poland A. Knutson J.C. Annu. Rev. Pharmacol. Toxicol. 1982; 22: 517-554Crossref PubMed Scopus (2337) Google Scholar, 5.Luster M. Faith R. Clark G. Ann. N. Y. Acad. Sci. 1979; 31: 473-486Crossref Scopus (30) Google Scholar, 6.Safe S.H. Annu. Rev. Pharmacol. Toxicol. 1986; 26: 371-399Crossref PubMed Google Scholar). The health effect of TCDD on human beings remains a matter of debate. Humans exposed to TCDD exhibit certain skin lesions such as chloracne; the possibility that TCDD exposure causes certain neuro- and psychopathological alterations (7.Oliver R. Br. J. Indust. Med. 1975; 32: 49-53PubMed Google Scholar, 8.Klawans H.L. Acta Neurol. Scand. 1987; 2: 255-261Google Scholar), some forms of cancers and diabetic conditions (9.Fingerhut M.A. Halerpin W.E. Marlow D.A. Piacitelli L.A. Honchar P.A. Sweeney M.H. Greife A.L. Dill P.A. Steenland K. Suruda A.J. N. Engl. J. Med. 1991; 324: 212-218Crossref PubMed Scopus (502) Google Scholar, 10.Calvert G.M. Sweeney M.H. Deddens J. Wall D.K. Occup. Environ. Med. 1999; 56: 270-276Crossref PubMed Scopus (115) Google Scholar), and reproductive lesions is a particular concern of public health. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with a basic helix-loop-helix PAS (bHLH/PAS) modular structure (2.Whitlock Jr., J.P. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 103-125Crossref PubMed Scopus (1001) Google Scholar, 3.Hankinson O. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 307-340Crossref PubMed Scopus (1432) Google Scholar, 11.Burbach K.M. Poland A. Bradfield C.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8185-8189Crossref PubMed Scopus (723) Google Scholar, 12.Ema M. Sogawa K. Wantabe N. Chujoh Y. Matsushita N. Gotoh O. Funae Y. Fujii-Kuriyama Y. Biochem. Biophys. Res. Commun. 1992; 184: 246-253Crossref PubMed Scopus (357) Google Scholar). Mouse genetic studies implicate AhR in most of the biological responses to TCDD, presumably by affecting the expression of target genes (13.Poland A. Glover E. Mol. Pharmacol. 1980; 17: 86-94PubMed Google Scholar, 14.Fernandez-Salguero P.M. Hilbert D.M. Rudikoff S. Ward J.M. Gonzalez F. Toxicol. Appl. Pharmacol. 1996; 140: 173-179Crossref PubMed Scopus (707) Google Scholar, 15.Mimura J. Yamashita K. Nakamura K. Morita M. Takagi T.N. Nakao K. Ema M. Sogawa K. Yasuda M. Katsuki M. Fujii-Kuriyama Y. Genes Cells. 1997; 2: 645-654Crossref PubMed Scopus (545) Google Scholar). Recent observations also imply that AhR plays certain roles in embryonic development and liver and immune functions in mice (16.Fernandez-Salguero P.M. Pineau T. Hilbert D.M. McPhail T. Lee S.S.T. Kimura S. Nebert D.W. Rudikoff S. Ward J.M. Gonzalez F.J. Science. 1995; 268: 722-726Crossref PubMed Scopus (942) Google Scholar, 17.Schmidt J.V. Su G.H. Reddy J.K. Simon M.C. Bradfield C.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6731-6736Crossref PubMed Scopus (748) Google Scholar), and modulate the growth, differentiation, and apoptotic processes in certain cell lines and mouse liver (18.Kolluri S. Weiss C. Koff A. Gottlicher M. Genes Dev. 1999; 13: 1742-1753Crossref PubMed Scopus (308) Google Scholar, 19.Weis C. Kolluri S.K. Kiefer F. Gottlicher M. Exp. Cell Res. 1996; 226: 154-163Crossref PubMed Scopus (151) Google Scholar, 20.Ma Q. Whitlock Jr., J.P. Mol. Cell. Biol. 1996; 16: 2144-2150Crossref PubMed Scopus (243) Google Scholar, 21.Reiners Jr., J.J. Cliff R.E. J. Biol. Chem. 1999; 274: 2502-2510Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 22.Zaher H. Fernandez-Salguero P.M. Letterio J. Sheikh M.S. Fornace Jr., A.J. Roberts A.B. Bonzalez F.J. Mol. Pharmacol. 1998; 54: 313-321Crossref PubMed Scopus (129) Google Scholar); these activities or functions of AhR were observed in the absence of known exogenous agonists, implicating a mechanism(s) of activating AhR under physiological conditions in vivo. Because of the broad range and the complexity of the biological responses that AhR contributes to, it is conceivable that the signal transduction of AhR involves a complex process during which AhR is regulated through different cellular mechanisms in a tissue-, species-, and developmental stage-dependent manner. The TCDD-inducible CYP1A1 gene encodes cytochrome P4501A1, a major inducible form of microsomal P450 in mammalian species; P4501A1 oxygenates polycyclic aromatic hydrocarbons, such as the carcinogen benzo(a)pyrine (23.Conney A.H. Cancer Res. 1982; 42: 4875-4917PubMed Google Scholar), as the initial step in the metabolism of the chemicals to water soluble metabolites for excretion from body. Studies on the induction of CYP1A1 gene expression by TCDD provided major mechanistic understanding of the mechanism of action and regulation of AhR (2.Whitlock Jr., J.P. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 103-125Crossref PubMed Scopus (1001) Google Scholar, 3.Hankinson O. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 307-340Crossref PubMed Scopus (1432) Google Scholar). In uninduced cells, AhR is localized in the cytoplasm, complexed with hsp90 (24.Perdew G.H. J. Biol. Chem. 1988; 263: 13802-13805Abstract Full Text PDF PubMed Google Scholar) and AIP, an immunophillin-type chaperon protein (25.Ma Q. Whitlock Jr., J.P. J. Biol. Chem. 1997; 272: 8878-8884Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 26.Carver L., A. Bradfield C.A. J. Biol. Chem. 1997; 272: 11452-11456Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 27.Meyer B.K. Pray-Grant M.G. Vanden Heuvel J.P. Perdew G.H. Mol. Cell. Biol. 1998; 18: 978-988Crossref PubMed Scopus (307) Google Scholar). Binding with an agonist triggers the dissociation of AhR from the associated proteins and translocation into nucleus, where AhR dimerizes with Arnt, another bHLH/PAS transcription factor (28.Hoffman E.C. Reyes H. Chu F. Sander F. Conley L.H. Brooks B.A. Hankinson O. Science. 1991; 252: 954-958Crossref PubMed Scopus (843) Google Scholar). The AhR/Arnt dimer binds to a specific nucleotide sequence termed DRE (dioxin responsive element) in the enhancer region of theCYP1A1 gene (29.Denison M.S. Fisher J.M. Whitlock Jr., J.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2528-2532Crossref PubMed Scopus (238) Google Scholar); the transcription activation domains of AhR are essential for the subsequent transcriptional events, including alterations in chromatin structure, binding of general transcription factors to the promoter, and induction of transcription of the gene (30.Ma Q. Dong L. Whitlock Jr., J.P. J. Biol. Chem. 1995; 270: 12697-12703Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 31.Ko H.P. Okino S.T. Ma Q. Whitlock Jr., J.P. Mol. Cell. Biol. 1996; 16: 430-436Crossref PubMed Scopus (144) Google Scholar, 32.Ko H.P. Okino S.T. Ma Q. Whitlock Jr., J.P. Mol. Cell. Biol. 1997; 17: 3497-3507Crossref PubMed Scopus (60) Google Scholar). Several cellular mechanisms have been recognized for the regulation of the AhR activity during the induction of CYP1A1. For example, cycloheximide enhances the induction of CYP1A1 gene expression by TCDD, a phenomenon termed “superinduction.” Early studies established that the superinduction involves an increase in the rate of transcription of the gene, requires functional DREs, but does not change several measurable properties of the TCDD-receptor complex such as the sedimentation velocity of the complex (33.Israel D.I. Estolano M.G. Galeazzi D.R. Whitlock Jr., J.P. J. Biol. Chem. 1985; 260: 5648-5653Abstract Full Text PDF PubMed Google Scholar, 34.Lusska A. Wu L. Whitlock Jr., J.P. J. Biol. Chem. 1992; 267: 15146-15151Abstract Full Text PDF PubMed Google Scholar). Since cycloheximide is known to inhibit protein synthesis, it is assumed that a labile, inhibitory protein factor regulates the AhR activity. However, the nature and the mechanism of action of the putative “labile” factor remain unknown. In another scenario, treatment with TCDD shortens the half-life of the AhR protein from 28 to 3 h (35.Ma Q. Baldwin K.T. J. Biol. Chem. 2000; 275: 8432-8438Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). The TCDD-induced turnover of AhR is mediated through the 26 S proteasome, involves ubiquitination of AhR, and requires the transcription activation domain of AhR (35.Ma Q. Baldwin K.T. J. Biol. Chem. 2000; 275: 8432-8438Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Moreover, inhibition of the 26 S proteasome by proteasome inhibitors increases the induction ofCYP1A1 by TCDD; these findings implicate the agonist-induced AhR degradation in the regulation of AhR function. To identify the molecular target of cycloheximide, we analyzed the TCDD-induced AhR turnover in the superinduction. We show here that cycloheximide blocks TCDD-induced degradation of AhR. Inhibition of the TCDD-induced AhR degradation requires inhibition of protein synthesis and correlates with the superinduction in a time- and dose-dependent manner. Furthermore, cycloheximide is shown to increase the accumulation of the AhR and the functional AhR·Arnt complex in nucleus. In addition, we show that inhibition of the 26 S proteasome superinduces CYP1A1 expression in a similar fashion to cycloheximide. To our knowledge, this report is the first study demonstrating that cycloheximide blocks the agonist-induced degradation of the AhR protein. Our findings provide a novel mechanism of superinduction of CYP1A1 in which a cycloheximide-sensitive, labile protein factor (designated asAhR degradation promotingfactor, or ADPF) negatively regulates the stability of agonist-activated, nuclear AhR. AmpliTaq polymerase was from Perkin-Elmer (Foster City, CA). Restriction endonucleases and other DNA-modifying enzymes were from New England Biolabs (Beverly, MA). Radioactive compounds were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Cell culture materials were from Life Technologies, Inc. (Grand Island, NY). Cycloheximide (CHX), puromycin, dimethyl sulfoxide (Me2SO), lipopolysaccharide (LPS), aprotinin, leupeptin, and phenylmethylsulfonyl fluoride were from Sigma. Lactacycstin and MG132 were from Calbiochem-Novabiochem Corp. (San Diego, CA). TCDD was purchased from AccuStandard (New Haven, CT). Reagents for immunoblotting and Northern blotting are as described below. The mouse hepa1c1c7 cells were gifts from Dr. J. P. Whitlock, Jr. (Stanford University). The cells were grown as monolayer in α-minimal essential medium, containing 10% fetal bovine serum and 5% CO2 at 37 °C, as described previously (36.Miller A.G. Israel D.I. Whitlock Jr., J.P. J. Biol. Chem. 1983; 258: 3523-3527Abstract Full Text PDF PubMed Google Scholar). The cells were treated with TCDD or other agents as described in figure legends; Me2SO was used as the solvent control for TCDD. Nuclear extracts were prepared according to published procedures (29.Denison M.S. Fisher J.M. Whitlock Jr., J.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2528-2532Crossref PubMed Scopus (238) Google Scholar). Briefly, wild type hepa1c1c7 cells, after treatment, were washed in a hypotonic buffer and homogenized in a Dounce homogenizer; the nuclei were obtained by differential centrifugation; nuclear extracts were prepared by incubation of the nuclei with a high salt buffer, followed by centrifugation at 100,000 × gfor 1 h. For preparation of total cell lysate, cells were grown to near confluency in a 60-mm dish, washed twice with phosphate-buffered saline, and scraped into 300 μl of a reporter lysis buffer (Promega, Madison, WI). The cells were disrupted by brief sonication; total cell lysate was obtained by centrifugation at 13,000 × gfor 10 min in a refrigerated microcentrifuge. EMSA was carried out using nuclear extract from hepa1c1c7 cells, as described previously (29.Denison M.S. Fisher J.M. Whitlock Jr., J.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2528-2532Crossref PubMed Scopus (238) Google Scholar), except that 6% polyacrylamide gels were used. The DNA probe contains the DNA recognition sequence for the AhR/Arnt heteromer designated as DRE D (37.Lusska A. Shen E. Whitlock Jr., J.P. J. Biol. Chem. 1993; 268: 6575-6580Abstract Full Text PDF PubMed Google Scholar). The probe was labeled with [γ-32P]ATP using T4 polynucleotide kinase (New England Biolabs). The nuclear extracts were incubated with poly(dI-dC) for 15 min at room temperature. The 32P-labeled probe was then added and incubated for another 15 min at room temperature, followed by nondenaturing gel electrophoresis; the AhR·Arnt·DRE complexes were visualized by autoradiography. For immunoblotting, total cell lysate or nuclear extract of 5 μg was fractionated on SDS-polyacrylamide gels, and transferred to nitrocellulose membranes according to established procedures (38.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The blots were blocked with 5% dry milk, 0.1% Tween 20 in phosphate-buffered saline for 1 h with shaking. Blots were then incubated with a polyclonal antibody against AhR (25.Ma Q. Whitlock Jr., J.P. J. Biol. Chem. 1997; 272: 8878-8884Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar) for 1 h, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for an additional 1 h. Signals were visualized by chemiluminescence using an ECL kit (Amersham Pharmacia Biotech). To ensure equal loading of the samples, the same blots were reprobed with a monoclonal anti-mouse actin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by incubation with alkaline phosphatase-conjugated secondary antibodies (Promega) and color visualization with the nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate system (Promega). For quantitation of the blotting results, the visualized results were scanned and analyzed by using the ImageQuaNT (version 4.2) program (Molecular Dynamics, San Jose, CA). All data were corrected for loading variations by comparing the amount of actin of each sample analyzed. For Northern blotting of CYP1A1, a cDNA fragment (∼700 base pairs) encoding the 5′-untranslated region of the mouse CYP1A1 messenger RNA was used to generate a riboprobe for CYP1A1. To prepare an actin probe, a cDNA fragment of mouse actin was generated by reverse transcriptase-polymerase chain reaction with primers specific for mouse actin (Stratagene, La Jolla, CA), subcloned into pCRII (Invitrogen, Carlsbad, CA), and used as a template for riboprobe synthesis. Riboprobes were synthesized in the presence of DIG-UTP using a DIG RNA-labeling kit (Roche Molecular Biochemicals, Indianapolis, IL). Total RNA was isolated from cells using a Qiagen total RNA isolation kit (Qiagen, Valencia, CA). RNA samples of 5 μg each were electrophoresed in a 1% agarose-formaldehyde gel and transferred to a Nytran membrane. After cross-linking, the membranes were hybridized with the DIG-labeled riboprobes at 68 °C overnight; signals were visualized by chemiluminescence using a DIG RNA detection kit with CDP star as a substrate (Roche Molecular Biochemicals). For all samples analyzed, parallel blots were assayed at the same time for bothCYP1A1 and actin mRNAs. Quantitation of the blotting results were performed by using the ImageQuaNT program as described above. All data were corrected for loading variations by comparing the amount of actin of each sample analyzed. Cells grown to near confluence were incubated in methionine-free medium with 10% dialyzed fetal bovine serum (Life Technologies, Inc.) for 1 h and incubated for another hour in fresh methionine-free medium with 10% dialyzed fetal bovine serum plus [35S]methionine (100 μCi/ml, Amersham Pharmacia Biotech). The cells were then incubated in α-minimal essential medium with 10% fetal bovine serum and treated with Me2SO, cycloheximide (10 μg/ml), TCDD (1 nm), or TCDD plus cycloheximide for various time periods. The cells were scraped into RIPA buffer (1% Ipegal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 100 μm phenylmethylsulfonyl fluoride, and 10 μg/ml aprotinin in phosphate-buffered saline). The35S-labled AhR was precipitated with the anti-AhR antibodies, fractionated by SDS-PAGE (10%), and visualized by fluorography. AhR was precipitated with anti-AhR antibodies according to a standard method (39.Ausubel F. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1998Google Scholar). Briefly, cells grown in 6-well plates were scraped into RIPA buffer. Cell extracts were prepared by centrifugation at 13,000 × g for 10 min, followed by preclearing by incubation with normal rabbit IgG (Santa Cruz Biotechnology, Inc.) and protein A-agarose (Life Technologies, Inc.) for 30 min at 4 °C. The extracts were then incubated with the anti-AhR antibodies (20.Ma Q. Whitlock Jr., J.P. Mol. Cell. Biol. 1996; 16: 2144-2150Crossref PubMed Scopus (243) Google Scholar, 40.Dong L. Ma Q. Whitlock Jr., J.P. J. Biol. Chem. 1996; 271: 7942-7948Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) for 1 h and with protein A-agarose for an additional hour. The precipitated agarose beads were washed 3 times with the RIPA buffer and resuspended in a loading buffer for analysis by SDS-PAGE. Immunofluorescent staining of cells with anti-AhR IgG was performed according to standard procedures (39.Ausubel F. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1998Google Scholar). Briefly, cells grown on coverslips were washed with 1 × phosphate-buffered saline, fixed in 3.7% formaldehyde for 10 min, and permeabilized with methanol at −20 °C for 6 min. The cells were then blocked in 1% bovine serum albumin for 30 min with shaking, and blotted with an affinity-purified polyclonal anti-mouse AhR IgG (Biomol, Plymouth Meetings, PA) in 1% bovine serum albumin for 1 h, followed by incubation with a fluorescein-conjugated anti-rabbit IgG (Chemicon International Inc., Temecula, CA) for an additional 1 h in the dark. The glass coverslips were mounted onto slides with Prolong (Molecular Probes, Eugene, OR), an anti-fade mounting medium. Fluorescence was visualized using a Sarastro 2000 laser scanning confocal microscope fitted with an argon-ion laser (Molecular Dynamics, Inc., Sunnyvale, CA) and an Optiphot-2 microscope (Nikon, Inc., Melville, PA). Confocal images were recorded through a ×60 lens objective using a 488-nm laser line. Cycloheximide superinduces the transcriptional gene expression of CYP1A1 in the presence of an agonist of AhR. As shown in Fig. 1 A, cycloheximide alone does not affect CYP1A1 gene expression (lane 3), whereas co-treatment of hepa1c1c7 cells with TCDD (1 nm) and cycloheximide (10 μg/ml) for 5 h increases the induction of CYP1A1 by TCDD by 6-fold (comparelanes 2 and 4). These results indicate that the superinduction requires activation of AhR by an agonist, suggesting that AhR or a component of the AhR signaling pathway serves as a primary target of cycloheximide. In a recent study on the turnover of the AhR protein, we showed that TCDD shortens the half-life of AhR from 28 to 3 h through ubiquitin-proteasome mediated proteolysis (35.Ma Q. Baldwin K.T. J. Biol. Chem. 2000; 275: 8432-8438Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Furthermore, inhibition of the 26 S proteasome by using proteasome inhibitors enhances the induction of CYP1A1 by TCDD. These findings raised the question of whether cycloheximide modulates the agonist-induced degradation of AhR as a mechanism of superinduction. Therefore, we analyzed the effect of cycloheximide on the protein level of AhR during the superinduction by immunoblotting. As shown in Fig. 1,B and C, treatment of the cells with TCDD (1 nm, 4 h) down-regulates the steady state AhR to less than 20% of the control. Cycloheximide alone (10 μg/ml) does not affect the protein level of AhR (lane 2). However, the level of the AhR protein in cells treated with TCDD plus cycloheximide (TCDD, 1 nm; CHX, 10 μg/ml; 4 h) is nearly the same as the controls (compare lane 4 with 1 and2). Thus, cycloheximide completely blocks the reduction of the steady state AhR protein by TCDD. Since TCDD down-regulates the AhR protein by increasing its turnover, we next tested if cycloheximide inhibits the TCDD-induced degradation of AhR by measuring the half-life (t 12) of AhR. Pulse-chase labeling experiments reveal that AhR in Me2SO-treated cells is relatively stable with at 12 of 28 h (Fig.2, A and B, Ref.35.Ma Q. Baldwin K.T. J. Biol. Chem. 2000; 275: 8432-8438Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar); cycloheximide alone does not affect the t 12 of AhR. TCDD shortens the t 12 of AhR to ∼3 h. However, the t 12 value of AhR in cells treated with TCDD plus cycloheximide is comparable with that in Me2SO-treated cells (i.e. ∼28 h). Therefore, cycloheximide fully inhibits the TCDD-induced turnover of the AhR protein. Cycloheximide inhibits protein synthesis by blocking the peptidyl synthetase activity of eukaryotic ribosomes. To explore the mechanism of inhibition of TCDD-induced AhR degradation by cycloheximide, we analyzed the role of protein synthesis in AhR degradation. IκBα, a regulatory subunit of the NFκB transcription factor, is known to undergo a rapid, signal-induced degradation during the activation of NFκB, followed by recovery of the protein through protein synthesis (41.Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1170) Google Scholar). Therefore, we first used the signal-induced degradation and synthesis of IkBα as a control to test if cycloheximide inhibits protein synthesis under the experimental condition for superinduction. As shown in Fig.3, treatment with LPS (5 μg/ml), an activator of NFκB, results in a rapid reduction of IκBα (lanes 1–5); the reduction is followed by recovery of the protein level through protein synthesis (lane 6). However, co-treatment with LPS and CHX (10 μg/ml) blocks the recovery of IκBα (compare lane 12 with lane 6). These results reveal that cycloheximide inhibits the synthesis of labile proteins, such as IκBα, at a concentration at which it superinducesCYP1A1 and inhibits the TCDD induced AhR degradation. Others have reported inhibition of total protein synthesis by cycloheximide at a similar concentration (34.Lusska A. Wu L. Whitlock Jr., J.P. J. Biol. Chem. 1992; 267: 15146-15151Abstract Full Text PDF PubMed Google Scholar). These findings implicate inhibition of protein synthesis in the action of cycloheximide on AhR degradation. These results also imply that inhibition of protein degradation by cycloheximide exhibits certain specificity toward target proteins, because cycloheximide does not inhibit the LPS-induced degradation of IκBα. We next examined if inhibition of protein synthesis is sufficient for blocking of AhR degradation. Puromycin, an analog of aminoacyl-tRNA, inhibits protein synthesis and superinduces CYP1A1 with a similar potency to cycloheximide (33.Israel D.I. Estolano M.G. Galeazzi D.R. Whitlock Jr., J.P. J. Biol. Chem. 1985; 260: 5648-5653Abstract Full Text PDF PubMed Google Scholar). Therefore, we tested if puromycin blocks AhR degradation by TCDD. As shown in Fig.4, while puromycin alone does not affect the AhR protein level, co-treatment of cells with TCDD and puromycin fully inhibits the degradation of AhR by TCDD. Since puromycin inhibits protein synthesis through a different mechanism from that of cycloheximide, these results indicate that inhibition of protein synthesis is sufficient for inhibition of TCDD-induced AhR turnover. Together, these data support the mechanism of inhibition of AhR degradation through inhibition of protein synthesis. These results implicate a cycloheximide-sensitive, labile, or inducible factor in promoting the TCDD-induced degradation of AhR; hence, we designated the factor as ADPF. Cloning of ADPF will provide new insights into the interplay between inhibition of protein synthesis and inhibition of protein degradation. The finding that cycloheximide blocks TCDD-induced AhR degradation at the concentration of superinduction suggests a mechanism of superinduction by which cycloheximide enhances the stability of agonist-activated AhR through inhibition of AhR degradation. To test this notion, we examined the time and dose curves of the inhibition of AhR degradation and the superinduction of CYP1A1 by cycloheximide. Treatment of cells with TCDD causes a time-dependent reduction of the level of the AhR protein (Fig. 5,A and B, lanes 6–10), which approaches the maximum reduction at 5 h. Cycloheximide blocks the TCDD-induced reduction (lanes 11–15) and increases the AhR protein to levels comparable to those treated with cycloheximide alone (lanes 1–5). Analyses of the induction of CYP1A1under similar conditions reveal that TCDD induces CYP1A1time dependently with the maximum induction at 5 h. Cycloheximide enhances the maximal induction (Fig. 5, C and D, compare lanes 9 and 10 with lanes 13–15). Furthermore, cycloheximide treatment shortens the time to reach the maximal induction (i.e. 2.5 h), indicating that inhibition of AhR degradation at early time points markedly increases the rate of the induction. The block of the AhR degradation by cycloheximide" @default.
• W2027153670 created "2016-06-24" @default.
• W2027153670 creator A5010478851 @default.
• W2027153670 creator A5012794872 @default.
• W2027153670 creator A5018053837 @default.
• W2027153670 creator A5031329587 @default.
• W2027153670 date "2000-04-01" @default.
• W2027153670 modified "2023-10-15" @default.
• W2027153670 title "Superinduction of CYP1A1 Gene Expression" @default.
• W2027153670 cites W1485553398 @default.
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