FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2015472059> ?p ?o ?g. }

• W2015472059 endingPage "24397" @default.
• W2015472059 startingPage "24388" @default.
• W2015472059 abstract "The aryl hydrocarbon receptor (AHR) is the ligand-activated transcription factor responsible for mediating the toxicological effects of dioxin and xenobiotic metabolism. However, recent evidence has implicated the AHR in additional, nonmetabolic physiological processes, including immune regulation. Certain tumor cells are largely nonresponsive to cytokine-mediated induction of the pro-survival cytokine interleukin (IL) 6. We have demonstrated that multiple nonresponsive tumor lines are able to undergo synergistic induction of IL6 following combinatorial treatment with IL1β and the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin. Such data implicate the AHR in tumor expansion, although the mechanistic basis for the AHR-dependent synergistic induction of IL6 has not been determined. Here, we demonstrate that ligand-activated AHR is involved in priming the IL6 promoter through binding to nonconsensus dioxin response elements located upstream of the IL6 start site. Such binding appears to render the promoter more permissive to IL1β-induced binding of NF-κB components. The nature of the AHR-dependent increases in IL6 promoter transcriptional potential has been shown to involve a reorganization of repressive complexes as exemplified by the presence of HDAC1 and HDAC3. Dismissal of these HDACs correlates with post-translational modifications of promoter-bound NF-κB components in a time-dependent manner. Thus the AHR plays a role in derepressing the IL6 promoter, leading to synergistic IL6 expression in the presence of inflammatory signals. These observations may explain the association between enhanced expression of AHR and tumor aggressiveness. It is likely that AHR-mediated priming is not restricted to the IL6 promoter and may contribute to the expression of a variety of genes, which do not have consensus dioxin response elements. The aryl hydrocarbon receptor (AHR) is the ligand-activated transcription factor responsible for mediating the toxicological effects of dioxin and xenobiotic metabolism. However, recent evidence has implicated the AHR in additional, nonmetabolic physiological processes, including immune regulation. Certain tumor cells are largely nonresponsive to cytokine-mediated induction of the pro-survival cytokine interleukin (IL) 6. We have demonstrated that multiple nonresponsive tumor lines are able to undergo synergistic induction of IL6 following combinatorial treatment with IL1β and the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin. Such data implicate the AHR in tumor expansion, although the mechanistic basis for the AHR-dependent synergistic induction of IL6 has not been determined. Here, we demonstrate that ligand-activated AHR is involved in priming the IL6 promoter through binding to nonconsensus dioxin response elements located upstream of the IL6 start site. Such binding appears to render the promoter more permissive to IL1β-induced binding of NF-κB components. The nature of the AHR-dependent increases in IL6 promoter transcriptional potential has been shown to involve a reorganization of repressive complexes as exemplified by the presence of HDAC1 and HDAC3. Dismissal of these HDACs correlates with post-translational modifications of promoter-bound NF-κB components in a time-dependent manner. Thus the AHR plays a role in derepressing the IL6 promoter, leading to synergistic IL6 expression in the presence of inflammatory signals. These observations may explain the association between enhanced expression of AHR and tumor aggressiveness. It is likely that AHR-mediated priming is not restricted to the IL6 promoter and may contribute to the expression of a variety of genes, which do not have consensus dioxin response elements. The aryl hydrocarbon receptor (AHR) 2The abbreviations used are: AHRaryl hydrocarbon receptorARNTaryl hydrocarbon receptor nuclear translocatorDREdioxin response elementHDAChistone deacetylaseTCDD2,3,7,8-tetrachlorodibenzo-p-dioxinB[a]Pbenzo[a]pyreneCYPcytochrome P450PBSphosphate-buffered salineTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineChIPchromatin immunoprecipitationsiRNAsmall interfering RNA. is a ligand-activated transcription factor of the basic helix-loop-helix, Per-Arnt-Sim class of proteins, historically studied as a mediator of xenobiotic response and metabolism. The AHR-mediated signaling pathway has been documented extensively, as exemplified in the review by Beischlag et al. (1Beischlag T.V. Luis Morales J. Hollingshead B.D. Perdew G.H. Crit. Rev. Eukaryot. Gene Expr. 2008; 18: 207-250Crossref PubMed Scopus (554) Google Scholar). Residing in the cytoplasm prior to activation, the AHR is complexed with a dimer of hsp90 and XAP2. The AHR binds an agonist, which induces translocation to the nucleus, followed by release of its chaperones and subsequent heterodimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT). This heterodimer exhibits an ability to bind dioxin response elements (DREs) at the promoters of target genes and plays a role in transcription. The most common ligand studied that mediates AHR activation is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), although it binds a variety of xenobiotics including polycyclic aromatic hydrocarbons such as benzo[a]pyrene (B[a]P). Polycyclic aromatic hydrocarbons are common environmental pollutants that result from car exhaust, manufacturing, iron foundries, cigarette smoke, etc. As a xenobiotic receptor, activated AHR binds to DREs in the promoters of cytochrome P4501A genes, which express enzymes that act in phase I drug metabolism. However, the AHR has recently been shown to have numerous physiological roles aside from drug metabolism. Such endogenous activities include differentiation of Th17 immune cells, regulation of acute phase response genes, antiestrogenic activities, and modulation of NF-κB protein activity (2Kimura A. Naka T. Nohara K. Fujii-Kuriyama Y. Kishimoto T. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 9721-9726Crossref PubMed Scopus (416) Google Scholar, 3Patel R.D. Murray I.A. Flaveny C.A. Kusnadi A. Perdew G.H. Lab. Invest. 2009; 89: 695-707Crossref PubMed Scopus (86) Google Scholar, 4Kharat I. Saatcioglu F. J. Biol. Chem. 1996; 271: 10533-10537Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 5Furness S.G. Whelan F. Pharmacol. Ther. 2009; 124: 336-353Crossref PubMed Scopus (52) Google Scholar). These activities occur through DRE binding, as well as through protein-protein interactions. aryl hydrocarbon receptor aryl hydrocarbon receptor nuclear translocator dioxin response element histone deacetylase 2,3,7,8-tetrachlorodibenzo-p-dioxin benzo[a]pyrene cytochrome P450 phosphate-buffered saline N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine chromatin immunoprecipitation small interfering RNA. We have previously shown that, in both the human MCF-7 breast cancer cell line and the ECC1 endocervical cancer cell line, IL6 production is synergistically increased following concomitant exposure to an AHR ligand and pro-inflammatory IL1β (6Hollingshead B.D. Beischlag T.V. Dinatale B.C. Ramadoss P. Perdew G.H. Cancer Res. 2008; 68: 3609-3617Crossref PubMed Scopus (95) Google Scholar). The low level of AHR activation needed to mediate this response, combined with the fact that increased IL6 protein secretion continued for 72 h after the initial dose of ligand, points to a potentially low threshold that cancer cells must pass before producing and releasing extensive amounts of a known pro-growth signal. Furthermore, release of IL6 from tumor cells can lead to an autocrine loop that enhances other inflammatory and anti-apoptotic signaling pathways. IL6 is an NF-κB-regulated gene; thus our initial findings verified that the p65 (RELA) subunit was involved in the synergistic increase. However, a more detailed transcriptional mechanism was not fully explored. Elucidating the means by which IL6 is synergistically activated by the AHR in MCF-7 cells has the potential to provide insight useful in other tissue and tumor situations. MCF-7 cells express low basal and low IL1β-induced levels of IL6 expression, but this is not the case for all cell lines (7Sasser A.K. Sullivan N.J. Studebaker A.W. Hendey L.F. Axel A.E. Hall B.M. FASEB J. 2007; 21: 3763-3770Crossref PubMed Scopus (178) Google Scholar). It has been postulated that the lack of IL6 inducibility in MCF-7 cells is due to the presence of co-repressors and a closed chromatin structure at the promoter (8Armenante F. Merola M. Furia A. Tovey M. Palmieri M. Nucleic Acids Res. 1999; 27: 4483-4490Crossref PubMed Scopus (25) Google Scholar). An understanding of how the AHR can derepress this gene is important because of the pleiotropic nature of IL6 in the tumor microenvironment. For example, numerous carcinomas have shown pro-growth, anti-apoptotic, and pro-invasive abilities upon exposure to increased IL6 levels (9Cavarretta I.T. Neuwirt H. Untergasser G. Moser P.L. Zaki M.H. Steiner H. Rumpold H. Fuchs D. Hobisch A. Nemeth J.A. Culig Z. Oncogene. 2007; 26: 2822-2832Crossref PubMed Scopus (92) Google Scholar, 10Conze D. Weiss L. Regen P.S. Bhushan A. Weaver D. Johnson P. Rincón M. Cancer Res. 2001; 61: 8851-8858PubMed Google Scholar, 11Asgeirsson K.S. Olafsdóttir K. Jónasson J.G. Ogmundsdóttir H.M. Cytokine. 1998; 10: 720-728Crossref PubMed Scopus (84) Google Scholar, 12Badache A. Hynes N.E. Cancer Res. 2001; 61: 383-391PubMed Google Scholar, 13Sasser A.K. Mundy B.L. Smith K.M. Studebaker A.W. Axel A.E. Haidet A.M. Fernandez S.A. Hall B.M. Cancer Lett. 2007; 254: 255-264Crossref PubMed Scopus (105) Google Scholar, 14Sansone P. Storci G. Tavolari S. Guarnieri T. Giovannini C. Taffurelli M. Ceccarelli C. Santini D. Paterini P. Marcu K.B. Chieco P. Bonafè M. J. Clin. Invest. 2007; 117: 3988-4002Crossref PubMed Scopus (656) Google Scholar). The observation that NF-κB signaling is involved in both oncogenic and immune pathways has led to numerous reviews on the functional role of prominent family members (e.g. p65, RELB) and, to a lesser extent, other related family members such as IκBζ (15Yamamoto M. Takeda K. J. Infect. Chemother. 2008; 14: 265-269Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 16Perkins N.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 49-62Crossref PubMed Scopus (1963) Google Scholar). There are a number of pathways by which the NF-κB family of proteins are regulated that can lead to transcriptional activation, the most prevalent being the canonical pathway. In this cascade of events, the p50 and p65 family members are sequestered in the cytoplasm by IκBα. Activating signals, including IL1β receptor signaling, lead to IKKα and IKKβ phosphorylating IκBα and dismissing it from the complex. This allows for nuclear localization of the p50-p65 heterodimer, which then binds to κB response elements in target gene promoters. Several tangential events also take place to maximize NF-κB activity, including acetylation of various p65 residues that enhance DNA binding and increase transcriptional activity (reviewed in Ref. 17Quivy V. Van Lint C. Biochem. Pharmacol. 2004; 68: 1221-1229Crossref PubMed Scopus (202) Google Scholar). Nuclear IKKα can phosphorylate histones as well as neighboring transcription factors, and members of the IκB family have been shown to act as both repressors and activators when recruited to this DNA-bound complex (18Chen L.F. Greene W.C. J. Mol. Med. 2003; 81: 549-557Crossref PubMed Scopus (247) Google Scholar, 19Kuwata H. Matsumoto M. Atarashi K. Morishita H. Hirotani T. Koga R. Takeda K. Immunity. 2006; 24: 41-51Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The fact that NF-κB is intricately involved in cytokine regulation pointed to the REL family of proteins as likely targets for further study to uncover their role in AHR-mediated synergistic induction of IL6. Upon discovering that AHR activation leads to greatly increased IL6 production in MCF-7 breast cancer cells, we then set out to investigate the mechanism by which this event occurs. The results revealed that activated AHR can bind imperfect DREs upstream of the IL6 promoter, leading to a regulatory region primed for NF-κB-mediated induction. The relative lack of IL6 induction in these cells following IL1β signaling alone appears to be due to the presence of co-repressors at the promoter, which is alleviated by the binding of AHR to its cognate response elements. Furthermore, AHR recruitment to the IL6 promoter results in a loss of HDAC1 occupancy, which coincides with an increase in acetylated p65 levels, a hallmark of optimal NF-κB-mediated transcriptional activity. MCF-7 breast tumor cells were maintained at 37 °C in 5% CO2 in a high glucose Dulbecco’s modified Eagle’s medium (Sigma), supplemented with 7% fetal bovine serum (Hyclone Laboratories), 1,000 units/ml penicillin, and 0.1 mg/ml streptomycin (Sigma). CV-1 cells were maintained in α-minimum essential medium supplemented with 8% fetal bovine serum and penicillin/streptomycin under identical incubation conditions. pGL3-promoter vector was subjected to digest with the restriction enzymes SacI and XhoI and subsequently ligated with sequences containing appropriate restriction sites. First, the pGL3–3.0kb vector was made by amplifying a 255-bp sequence spanning the region from −2897 to −3152 of the IL6 promoter with the primers 5′-TCACGCCTGTAAACCCAGCACTTT-3′ and 5′-GCGGTTGAAGTGAGCCAAGATCAT-3′. Second, the pGL3–3.0kb.synth vector was made by designing forward and reverse complimentary oligonucleotides containing three copies of a 15-base pair stretch of the IL6 promoter centered on the nonconsensus DRE found at −3050 bp. These sequences were 5′-GAGGCGCGTGGATCAGAGGCGCGTGGATCAGAGGCGCGTGGATCA-3′ and 5′-TGATCCACGCGCCTCTGATCCACGCGCCTCTGATCCACGCGCCTC-3′ (produced by Integrated DNA Technologies). Both inserts contained the appropriate restriction enzyme digest sites and were ligated into the pGL3-promoter vector. Vectors with insert were sequenced to verify PCR amplification fidelity or synthesis. A synthetic, codon-optimized cDNA sequence encoding the wild-type human AHR (produced by GenScript) was inserted into pcDNA3 vector to create pcDNA3-AHR. This same sequence was modified to create the human AHR-GS DNA-binding mutant. Construction and characterization of the AHR-GS DNA-binding mutant is outlined in the supplemental text. This modified sequence was inserted into the vector to create pcDNA3-AHR-GS. MCF-7 cells were serum-starved 18 h before treatment. Treatment of cells was performed by diluting compounds to the desired working concentration in serum-free medium supplemented with 5 mg/ml bovine serum albumin. Total RNA was extracted from the cells using TRI reagent (Sigma) as specified by the manufacturer. The ABI high capacity cDNA archive kit (Applied Biosystems) was used to prepare cDNA from isolated RNA. mRNA expression for all samples was measured by quantitative real time PCR using the Quanta SYBR Green kit on an iCycler DNA engine equipped with the MyiQ single color real time PCR detection system (Bio-Rad). The expressed quantities of mRNA were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels and plotted using GraphPad Prism 4.0 (GraphPad Software). Histograms are plotted as the mean values of biological replicates, and the error bars represent the standard deviation of replicates. Real time primers used are listed in the supplemental text. Whole cell extracts were prepared by lysing cells in 1× radioimmunoprecipitation assay buffer (10 mm Tris-HCl, pH 8.0, 1 mm EDTA, 0.5 mm EGTA, 140 mm NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) supplemented with 1% Nonidet P-40, 300 mm NaCl, and protease inhibitor mixture (Sigma). Homogenates were centrifuged at 21,000 × g for 30 min at 4 °C, and the soluble fraction was collected as whole cell extract. Protein concentrations were determined using the detergent-compatible DC protein assay kit (Bio-Rad). Protein samples were resolved by Tricine-SDS-PAGE and transferred to polyvinylidene difluoride membrane. Primary antibodies used to detect specific proteins are shown in the supplemental text and were visualized using biotin-conjugated secondary antibodies (Jackson Immunoresearch) in conjunction with [125I]streptavidin (Amersham Biosciences). MCF-7 cells were grown to ∼90% confluency in 150-cm2 dishes and serum-starved 18 h before treatment. The cells were treated in serum-free medium supplemented with 5 mg/ml bovine serum albumin by diluting compounds to the desired working concentration for specified time. Following treatment, the cells were washed once with warm PBS, and chromatin complexes were chemically cross-linked using a 1% formaldehyde/PBS solution (final concentration) for 10 min at room temperature. Cross-linking was stopped by the addition of glycine solution to a final concentration of 0.125 m; the cells were then washed twice with ice-cold PBS and collected in 2 ml of harvest buffer (100 mm Tris, pH 8.3, 10 mm dithiothreitol). The cells were centrifuged, washed in ice-cold PBS, and resuspended in 600 μl of lysis buffer (1% SDS, 50 mm Tris-HCl, pH 8.1, 10 mm EDTA). Chromatin was sheared with the Bioruptor water bath sonicator (Diagenode, Sparta, NJ) to an average size of 500 bp to 1 kb. The complexes were precleared with protein A-agarose (Pierce) and incubated overnight with specific antibodies, which are listed in the supplemental text. Immunoadsorbed complexes were captured on protein A-agarose (exception: RNA polymerase II mouse monoclonal antibody was bound to streptavidin-agarose (Thermo) previously incubated with 5 μg/immunoprecipitation of goat anti-mouse IgG) and washed once with TE8 (10 mm Tris-HCl, pH 8.0, and 0.5 m EDTA). Agarose-bound complexes were then resuspended in TE8, layered on top of a sucrose solution (1 m sucrose, 200 mm NaCl, 1% Nonidet P-40), and centrifuged for 3 min. Agarose-bound complexes were then washed once with 0.5× radioimmunoprecipitation assay buffer, followed by four washes with TE8. The samples were eluted off the agarose using 200 μl of elution buffer (100 mm NaHCO3, 1%SDS), and cross-links were reversed at 65 °C overnight. Eluted DNA was isolated, washed, and concentrated using the ChIP DNA Clean & Concentrator kit (ZYMO Research). Immunoadsorbed DNA was analyzed by PCR and/or quantitative real time PCR. Specific protein levels were decreased using the Dharmacon small interfering RNA (siRNA) (control oligonucleotide D001810-0X, AHR oligonucleotide J004990-07, ARNT oligonucleotide D007207-01, RELB oligonucleotide J004767-06, HDAC1 oligonucleotide J003493-10, and HDAC3 oligonucleotide J003496-09). Electroporation/nucleofection was performed using the Amaxa nucleofection system essentially as described in the manufacturer protocols. Briefly, the cells were washed and suspended at a concentration of 2.0 × 106/100 μl of nucleofection solution. Control or targeted siRNA was added to the sample for a final concentration of 1.5 μmol/liter. The samples were electroporated using the manufacturer’s MCF-7 high efficiency program and plated into six-well dishes in complete medium. The experiments were carried out as described by DiNatale and Perdew (20DiNatale B.C. Perdew G.H. Cytotechnology. 2010; (in press)PubMed Google Scholar). Briefly, MCF-7 cells were electroporated with a control or AHR-targeted siRNA, along with control pcDNA3, pcDNA3-AHR, or pcDNA3-AHR-GS. CV-1 cells were grown in penicillin/streptomycin-free medium and transfected using the Mirus TransIT-TKO transfection system. Transfection was performed with control pGL3-promoter, pGL3–3.0kb, or pGL3–3.0kb.synth vectors. Beginning 24 h after transfection, the cells were treated for an additional 24 h, rinsed with PBS, and lysed in cell culture lysis buffer (2 mmtrans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 2 mm dithiothreitol, 10% glycerol, and 1% Triton X-100). Cytosol was assayed for luciferase activity using the a luciferase assay system (Promega, Madison, WI) as specified by the manufacturer. Light production was measured using a TD-20e luminometer (Turner Designs, Sunnyvale, CA). Having previously shown that IL6 is synergistically induced in MCF-7 cells via AHR activation in conjunction with IL1β treatment, the signaling mechanism by which this event occurs was explored. MCF-7 cells pretreated with 250 ng/ml of IL1β receptor antagonist (R & D Systems; 280-RA/CF) for 1 h were subsequently treated with vehicle, 10 ng/ml IL1β, 1 nm TCDD, or co-treated with IL1β and TCDD. Pretreatment with IL1β receptor antagonist prevented IL1β signaling and inhibited any significant increase in IL6 mRNA production (Fig. 1A). These data indicate that IL1β is working through its membrane receptor to activate IL6 expression. Our prior research has shown that siRNA-mediated ablation of AHR protein prevents synergistic IL6 induction (6Hollingshead B.D. Beischlag T.V. Dinatale B.C. Ramadoss P. Perdew G.H. Cancer Res. 2008; 68: 3609-3617Crossref PubMed Scopus (95) Google Scholar). To clarify the manner in which the AHR participates in IL6 induction, the AHR signaling pathway was dissected, beginning with its heterodimerization partner, ARNT. siRNA-mediated ablation of ARNT protein levels was carried out, and MCF-7 cells were treated for 2 h with vehicle, IL1β, TCDD, or with a combination of these substances. Electroporation of MCF-7 cells with ARNT-targeting siRNA leads to nearly complete ablation of protein levels (Fig. 1B). As shown in Fig. 1C, the loss of ARNT prevents TCDD-induced CYP1A1 expression, as expected. Similarly, ARNT is shown to be necessary for synergistic IL6 induction (Fig. 1D). This finding suggests that AHR/ARNT heterodimerization is required for AHR-mediated induction of IL6 expression. The AHR has been shown to play a role in regulatory pathways via AHR/ARNT-mediated binding to DRE sequences, as well as through protein-protein interactions. To determine whether the induction of IL6 required the AHR/ARNT heterodimer to bind to the gene promoter region, we replaced AHR protein expression with that of a DNA-binding mutant in MCF-7 cells. A characterized DNA-binding variant of the murine AHR that contains GS amino acid sequence inserts between residues has been shown not to have altered ligand binding, interaction with chaperones, or heterodimerization but is not capable of binding to DREs (21Bunger M.K. Glover E. Moran S.M. Walisser J.A. Lahvis G.P. Hsu E.L. Bradfield C.A. Toxicol. Sci. 2008; 106: 83-92Crossref PubMed Scopus (110) Google Scholar). This mutation was created in human AHR (AHR-GS) and similarly characterized (supplemental text and Fig. S1). Having optimized electroporation conditions to attain nearly complete siRNA-mediated ablation of AHR protein in MCF-7 cells, receptor levels were reduced, and cells were co-transfected with a vector containing a synthetic, codon-optimized AHR cDNA construct that was not targeted by AHR siRNA. The AHR constructs expressing AHR or AHR-GS were utilized. This method of transient protein replacement has been previously characterized (20DiNatale B.C. Perdew G.H. Cytotechnology. 2010; (in press)PubMed Google Scholar). Co-transfection with a control vector resulted in minimal change in AHR protein ablation. AHR expression and AHR-GS expression were equivalent although higher than basal AHR protein expression (Fig. 2A). The prototypical AHR target gene examined following ligand activation is CYP1A1. Although AHR knockdown resulted in the loss of CYP1A1 induction following TCDD treatment, the replacement with ectopic AHR expression rescued induction at a higher level because of the higher receptor expression. Treatment with TCDD following replacement of endogenous AHR protein with the AHR-GS mutant failed to induce CYP1A1 because of the loss of DRE binding in the CYP1A1 enhancer (Fig. 2B). Similarly, a loss of AHR expression prevents the synergistic induction of IL6 following combined IL1β and TCDD treatment, whereas replacement with a fully functional AHR protein allows for synergy to occur. Replacement of endogenous AHR with AHR-GS fails to rescue the induction of IL6 (Fig. 2C). Thus DRE binding appears to be required for the AHR to play a role in synergistic IL6 induction, as opposed to simply being the result of AHR-protein interactions. DRE sequences have been characterized for their AHR binding ability, and the optimal nucleotide sequence has been determined. Having established that AHR/ARNT binding to a DRE is required for AHR ligand-mediated induction of IL6, we wanted to determine which imperfect DRE(s) in the IL6 promoter are functional. Integral to receptor binding is the core (G/T)CGTG sequence, with flanking nucleotides being less important but increasing the affinity of the AHR for the DNA. Functional analyses have shown that having a G as the 5′ base of the core enhances binding and function of receptor (22Denison M.S. Elferink C.F. Phelan D. Denison M.S. Helferich W.G. Toxicant-Receptor Interactions in the Modulation of Signal Transduction and Gene Expression. Taylor and Francis, London1998: 3-33Google Scholar). However, studies have shown a lack of correlation between AHR binding to imperfect DREs in gel shift analyses and AHR-mediated induction of imperfect DRE-driven luciferase assays (23Swanson H.I. Chan W.K. Bradfield C.A. J. Biol. Chem. 1995; 270: 26292-26302Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 24Gillesby B.E. Stanostefano M. Porter W. Safe S. Wu Z.F. Zacharewski T.R. Biochemistry. 1997; 36: 6080-6089Crossref PubMed Scopus (128) Google Scholar). This led to the conclusion that, with a modification of the 5′ core nucleotide, there is still potential for some receptor binding in the genomic context, thus creating the need to assess sequences containing only the four central bases of CGTG. Sequence analysis of the IL6 promoter reveals seven imperfect DREs in the span from the transcription start site to 5 kb upstream. All seven DREs contain the core CGTG sequence, but many have less than optimal flanking sequences (Fig. 3A). Initial attempts at luciferase assays using large stretches of the IL6 promoter show an inability to mimic the regulation observed in cells. This finding is not surprising, because previous research has encountered the same problem (25Faggioli L. Costanzo C. Merola M. Bianchini E. Furia A. Carsana A. Palmieri M. Eur. J. Biochem. 1996; 239: 624-631Crossref PubMed Scopus (25) Google Scholar), such that the length of the promoter used in the assay is inversely proportional to the level of induction (supplemental Fig. S2). Because these limitations prevented a full promoter analysis utilizing reporter vectors, a different approach was adopted. MCF-7 cells treated with either vehicle or combinatorial TCDD and IL1β for 2 h were subjected to ChIP analysis of the IL6 promoter. Quantitative real time PCR was carried out using primers that scanned the 5 kb upstream from the transcription start site in 500-bp fragments. Maximal occupancy increases following treatment were observed in the region of the multiple DREs between −3.kb and −3.5 kb and, to a lesser extent, in the −4.5-kb region (Fig. 3B). Further analysis of the −3.0-kb region was carried out, because it included imperfect DREs at −2962 and −3050 bp (Fig. 3A). Both of these DREs contain a 5′ G next to the core CGTG, and their close proximity to each other could prompt a greater AHR-mediated effect. pGL3–3.0kb vector, which contained both the −2962-bp and the −3050-bp DREs, was transfected into the CV-1 cell line. The combination of a low level of basal activated AHR in CV-1 cells (26Chiaro C.R. Patel R.D. Marcus C.B. Perdew G.H. Mol. Pharmacol. 2007; 72: 1369-1379Crossref PubMed Scopus (78) Google Scholar) and the promoter contained within the pGL3 vector led to luciferase activity in the absence of transfected AHR or exogenous ligand (Fig. 4A, first column). Co-transfection with the WT AHR-expressing construct led to a significant increase with the −3.0-kb vector (Fig. 4A, second column), whereas co-transfection of the luciferase vector and the AHR-GS construct led to a significant decrease in luciferase activity compared with basal levels (Fig. 4A, third column) and, therefore, a significant difference between co-transfection with WT AHR compared with mutant AHR-GS. This is believed to be due to the mutant AHR binding a portion of the putative endogenous ligand and heterodimerizing with ARNT, sequestering part of the ARNT pool, and thus decreasing basal luciferase activity. Co-transfection of the reporter vector and WT AHR-expressing vector, followed by 24 h of treatment with 5 μm of the AHR ligand B[a]P, resulted in a significant 1.7-fold increase in luciferase activity, which is not observed upon expression of the DNA-binding mutant AHR-GS (Fig. 4A, fourth and fifth columns). Increases in AHR protein levels following co-transfection with both the AHR and AHR-GS were detected by Western blot (Fig. 4B). To further validate the functionality of the −3050 bp DRE, pGL3–3.0kb.synth vector, which contained three copies of the DRE in tandem, was transfected into CV-1 cells. As seen with the endogenous promoter construct, transfection of the vector alone resulted in basal luciferase activity caused by the low level of constitutively active AHR in CV-1 cells (Fig. 4C, second column). Addition of the WT AHR construct resulted in greater basal luciferase activity (Fig. 4C, third column), which was increased nearly 3-fold following B[a]P treatment (Fig. 4C, fourth column). The lower, albeit significant level of induction mediated by the promoter construct in Fig. 4A is not altogether surprising, because studies utilizing luciferase vectors containing a single DRE have shown low levels of AHR-mediated inducibility (27Gouédard C. Barouki R. Morel Y. Mol. Cell Biol. 2004; 24: 5209-5222Crossref PubMed Scopus (202) Google Scholar). Nevertheless, these studies clearly establish that the imperfect DREs at" @default.
• W2015472059 created "2016-06-24" @default.
• W2015472059 creator A5039936196 @default.
• W2015472059 creator A5051351244 @default.
• W2015472059 creator A5068840599 @default.
• W2015472059 creator A5069439344 @default.
• W2015472059 creator A5080630073 @default.
• W2015472059 date "2010-08-01" @default.
• W2015472059 modified "2023-10-16" @default.
• W2015472059 title "Mechanistic Insights into the Events That Lead to Synergistic Induction of Interleukin 6 Transcription upon Activation of the Aryl Hydrocarbon Receptor and Inflammatory Signaling" @default.
• W2015472059 cites W1501881225 @default.
• W2015472059 cites W1534753181 @default.
• W2015472059 cites W1539659593 @default.
• W2015472059 cites W1575868719 @default.
• W2015472059 cites W1577722824 @default.
• W2015472059 cites W1794369351 @default.
• W2015472059 cites W1860436011 @default.
• W2015472059 cites W1929257083 @default.
• W2015472059 cites W1971494855 @default.
• W2015472059 cites W1973576115 @default.
• W2015472059 cites W1979976356 @default.
• W2015472059 cites W1980870391 @default.
• W2015472059 cites W1987531976 @default.
• W2015472059 cites W1991900884 @default.
• W2015472059 cites W1997215979 @default.
• W2015472059 cites W2004335766 @default.
• W2015472059 cites W2005283959 @default.
• W2015472059 cites W2006334693 @default.
• W2015472059 cites W2008984410 @default.
• W2015472059 cites W2013488214 @default.
• W2015472059 cites W2027959125 @default.
• W2015472059 cites W2028893303 @default.
• W2015472059 cites W2039242242 @default.
• W2015472059 cites W2050068580 @default.
• W2015472059 cites W2050784092 @default.
• W2015472059 cites W2052908553 @default.
• W2015472059 cites W2058266230 @default.
• W2015472059 cites W2059124237 @default.
• W2015472059 cites W2079003739 @default.
• W2015472059 cites W2079934671 @default.
• W2015472059 cites W2084800620 @default.
• W2015472059 cites W2098184580 @default.
• W2015472059 cites W2106461424 @default.
• W2015472059 cites W2114817620 @default.
• W2015472059 cites W2116367493 @default.
• W2015472059 cites W2117475605 @default.
• W2015472059 cites W2123738327 @default.
• W2015472059 cites W2132613286 @default.
• W2015472059 cites W2140199421 @default.
• W2015472059 cites W2142259273 @default.
• W2015472059 cites W2144929276 @default.
• W2015472059 cites W2148529156 @default.
• W2015472059 cites W2151998204 @default.
• W2015472059 cites W2156203870 @default.
• W2015472059 cites W2159722075 @default.
• W2015472059 cites W2316580498 @default.
• W2015472059 cites W2614988191 @default.
• W2015472059 doi "https://doi.org/10.1074/jbc.m110.118570" @default.
• W2015472059 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2915674" @default.
• W2015472059 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20511231" @default.
• W2015472059 hasPublicationYear "2010" @default.
• W2015472059 type Work @default.
• W2015472059 sameAs 2015472059 @default.
• W2015472059 citedByCount "95" @default.
• W2015472059 countsByYear W20154720592012 @default.
• W2015472059 countsByYear W20154720592013 @default.
• W2015472059 countsByYear W20154720592014 @default.
• W2015472059 countsByYear W20154720592015 @default.
• W2015472059 countsByYear W20154720592016 @default.
• W2015472059 countsByYear W20154720592017 @default.
• W2015472059 countsByYear W20154720592018 @default.
• W2015472059 countsByYear W20154720592019 @default.
• W2015472059 countsByYear W20154720592020 @default.
• W2015472059 countsByYear W20154720592021 @default.
• W2015472059 countsByYear W20154720592022 @default.
• W2015472059 countsByYear W20154720592023 @default.
• W2015472059 crossrefType "journal-article" @default.
• W2015472059 hasAuthorship W2015472059A5039936196 @default.
• W2015472059 hasAuthorship W2015472059A5051351244 @default.
• W2015472059 hasAuthorship W2015472059A5068840599 @default.
• W2015472059 hasAuthorship W2015472059A5069439344 @default.
• W2015472059 hasAuthorship W2015472059A5080630073 @default.
• W2015472059 hasBestOaLocation W20154720591 @default.
• W2015472059 hasConcept C104317684 @default.
• W2015472059 hasConcept C138885662 @default.
• W2015472059 hasConcept C151730666 @default.
• W2015472059 hasConcept C170493617 @default.
• W2015472059 hasConcept C179926584 @default.
• W2015472059 hasConcept C185592680 @default.
• W2015472059 hasConcept C203014093 @default.
• W2015472059 hasConcept C2777093003 @default.
• W2015472059 hasConcept C2778690821 @default.
• W2015472059 hasConcept C33594762 @default.
• W2015472059 hasConcept C41895202 @default.
• W2015472059 hasConcept C45815257 @default.
• W2015472059 hasConcept C55493867 @default.
• W2015472059 hasConcept C62478195 @default.
• W2015472059 hasConcept C74172505 @default.

• next