ARNT Complex Research Articles

Molecular Dynamics Reveals Altered Interactions between Belzutifan and HIF-2 with Natural Variant G323E or Proximal Phosphorylation at T324.

In patients with von-Hippel Lindau (VHL) disease, hypoxia-independent accumulation of HIF-2α leads to increased transcriptional activity of HIF-2α:ARNT that drives cancers such as renal cell carcinoma. Belzutifan, a recently FDA-approved drug, is designed to prevent the transcriptional activity of HIF-2α:ARNT, thereby overcoming the consequences of its unnatural accumulation in VHL-dependent cancers. Emerging evidence suggests that the naturally occurring variant G323E located in the HIF-2α drug binding pocket prevents inhibitory activity of belzutifan analogs, though the mechanism of inhibition remains unclear. Interestingly, proximal phosphorylation at neighboring T324, previously shown to regulate HIF-2 protein interactions, has also been proposed to affect HIF-2 drug binding. Here, we used molecular dynamics (MD) simulations to understand and compare the molecular-level effects of G323E and phospho-T324 (pT324) on the belzutifan bound-HIF-2α:ARNT complex. We find that both G323E and pT324 increase structural flexibility within the drug binding site and reduce the apparent binding affinity for belzutifan. Whereas the effects of G323E are concentrated in the binding pocket Fα helix within the HIF-2α PAS-B domain, pT324 decreased the belzutifan binding affinity and stabilized the HIF-2 heterodimer through an alternate mechanism involving polar interactions between the HIF-2α PAS-B and PAS-A domains. Further analysis via ensemble machine learning uncovered important and distinct interchain residue interactions modified by G323E and pT324. These findings reveal a molecular mechanism of G323E-induced drug resistance and suggest that pT324 may also affect the efficacy of HIF-2 drug binding interactions via allosteric effects.

Jasmone Is a Ligand-Selective Allosteric Antagonist of Aryl Hydrocarbon Receptor (AhR).

Herbal extracts represent a wide spectrum of biologically active ingredients with potential medical applications. By screening minor constituents of jasmine essential oil towards aryl hydrocarbon receptor (AhR) activity using a gene reporter assay (GRA), we found the antagonist effects of jasmone (3-methyl-2-[(2Z)-pent-2-en-1-yl]cyclopent-2-en-1-one). It inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-, benzo[a]pyrene (BaP)-, and 6-formylindolo[3,2-b]carbazole (FICZ)-triggered AhR-dependent luciferase activity in a concentration-dependent manner. However, the inhibition differed markedly between TCDD, BaP, and FICZ, with the latter being significantly less inhibited. The dose-response analysis confirmed an allosteric type of AhR antagonism. Furthermore, jasmone efficiently inhibited AhR activation by AhR agonists and microbial catabolites of tryptophan (MICTs). TCDD- and FICZ-inducible CYP1A1 expression in primary human hepatocytes was inhibited by jasmone, whereas in the human HepG2 and LS180 cells, jasmone antagonized only TCDD-activated AhR. Jasmone only partially displaced radiolabeled TCDD from its binding to mouse Ahr, suggesting it is not a typical orthosteric ligand of AhR. TCDD-elicited AhR nuclear translocation was not affected by jasmone, whereas downstream signaling events, including the formation of the AhR:ARNT complex and enrichment of the CYP1A1 promoter, were inhibited by jasmone. In conclusion, we show that jasmone is a potent allosteric antagonist of AhR. Such discovery may help to find and/or clarify the use of jasmone in pharmaco- and phytotherapy for conditions where AhR plays a key role.

TCDD-Induced Allosteric Perturbation of the AhR:ARNT Binding to DNA.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of structurally diverse chemicals, including halogenated aromatic hydrocarbons. In this work, we investigate the effects of the binding of the AhR prototypical ligand, TCDD, on the stability of the AhR:ARNT complex, as well as the mechanisms by which ligand-induced perturbations propagate to the DNA recognition site responsible for gene transcription. To this aim, a reliable structural model of the overall quaternary structure of the AhR:ARNT:DRE complex is proposed, based on homology modelling. The model shows very good agreement with a previous one and is supported by experimental evidence. Moreover, molecular dynamics simulations are performed to compare the dynamic behaviour of the AhR:ARNT heterodimer in the presence or absence of the TCDD. Analysis of the simulations, performed by an unsupervised machine learning method, shows that TCDD binding to the AhR PASB domain influences the stability of several inter-domain interactions, in particular at the PASA-PASB interface. The inter-domain communication network suggests a mechanism by which TCDD binding allosterically stabilizes the interactions at the DNA recognition site. These findings may have implications for the comprehension of the different toxic outcomes of AhR ligands and drug design.

Activation of aryl hydrocarbon receptor signaling by gallic acid suppresses progression of human breast cancer in vitro and in vivo

BackgroundDespite the significant advances in diagnosis and treatment, breast cancer remains the most common malignancy and the second cause of death in women. Increasingly, preclinical evidence has suggested aryl hydrocarbon receptor (Ahr), a ligand activated transcription factor, a promising therapeutic target in breast cancer. PurposeThis study aims at screening a number of phenolic compounds to identify an Ahr ligand with suppressive effects on human breast cancer. MethodsPotential interactions between Ahr and phenolic compounds were predicted in silico, and physical interaction was examined by ligand competitive binding in vitro. The MDA-MB-231 and T47D breast cancer cell lines were used to examine the expression of Ahr downstream genes and progression of breast cancer cells in vitro. Binding of Ahr/Ahr nuclear transporter (Arnt) complex to the xenobiotic-responsive element (XRE)-box was examined by DNA-protein interaction (DPI)-ELISA, promoter activity was assessed using luciferase reporter system, and RNA interreference was carried out using electroporation. The real-time PCR and/or immunoblotting were used to quantify gene expressions. Tumor growth in vivo was assessed using a murine orthotopic model. ResultsA combined computational modeling and in vitro approaches identified gallic acid (GA) as an Ahr ligand with agonistic properties. It induced binding of Ahr/Arnt to the XRE-box, enhanced the promoter activity and expression of Ahr downstream genes including cytochrome P450 1A1 (CYP1A1), and SRY-related HMG-box4 (SOX4)-targeting miR-212/132 cluster and miR-335 in both MDA-MB-231 and T47D cells. GA increased apoptosis while decreased proliferation, migration and invasion capacities of breast cancer cells in an Ahr-dependent fashion. Furthermore, it reduced the levels of B-cell lymphoma 2 (BCL-2), cyclooxygenase-2 (COX-2) and SOX4, while selectively increased that of tumor protein 53 (P53), in an Ahr-dependent and -independent fashions. In an in vivo orthotopic model, GA activated Ahr signaling and reduced the growth of breast cancer cells. ConclusionWe identified GA as an Ahr phenolic ligand, and provided evidence on the role of Ahr in mediating its anti-breast cancer effects, indicating that GA, and possibly other phenolic compounds, have important therapeutic implications in human breast cancer through activation of Ahr signaling.

Transcriptional Regulation of gga-miR-451 by AhR:Arnt in Mycoplasma gallisepticum (HS Strain) Infection

MicroRNAs (miRNAs) have been determined to be important regulators for pathogenic microorganism infection. However, it is largely unclear how miRNAs are triggered during pathogen infection. We previously reported that the up-regulation of gga-miR-451 negatively regulates the Mycoplasma gallisepticum (MG)-induced production of inflammatory cytokines via targeting tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein zeta (YWHAZ). The aim of this study was to investigate the mechanism regulating gga-miR-451 in MG infection in chickens. Analysis of gga-miR-451 precursor, pri-miR-451, and pre-miR-451 indicated that the regulation occurred transcriptionally. We also identified the transcriptional regulatory region of gga-miR-451 that contained consensus-binding motif for aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (Arnt) complex, which is known as the transcription factor that regulates gene expression. Luciferase reporter assays combined with chromatin immunoprecipitation (ChIP) demonstrated that AhR:Arnt bound directly to the promoter elements of gga-miR-451, which were responsible for gga-miR-451 transcription in the context of MG infection. Furthermore, upregulation of AhR:Arnt significantly induced gga-miR-451 and inhibited YWHAZ expression, suggesting that AhR:Arnt may play an anti-inflammatory role in MG infection. This discovery suggests that induced gga-miR-451 expression is modulated by AhR:Arnt in response to MG infection.

Activation of aryl hydrocarbon receptor signaling by a novel agonist ameliorates autoimmune encephalomyelitis.

BackgroundMultiple sclerosis (MS) is a widespread neurological autoimmune disease that includes episodes of demyelination in the central nervous system (CNS). The accumulated evidence has suggested that aryl hydrocarbon receptor (Ahr), a ligand-activated transcription factor, is a promising treatment target for MS. Thus, the current study aimed to identify a novel Ahr ligand with anti-inflammatory potential in experimental autoimmune encephalomyelitis (EAE).MethodsAn in silico analysis was carried out to predict interactions between Ahr and potential natural ligands. The effects of a predicted interaction were examined in vitro using CD4+ T cells under T helper17 (Th17) cell-polarizing conditions and lipopolysaccharide (LPS)-stimulated macrophages. Silencing Ahr and microRNA (miR)-132 was achieved by electroporation. Myelin oligodendrocyte glycoprotein (MOG)35-55 and the adoptive transfer of encephalitogenic CD4+ T cells were used to induce EAE.ResultsMolecular docking analysis and in vitro data identified gallic acid (GA) as a novel Ahr ligand with potent activation potential. GA induced the expression of Ahr downstream genes, including cytochrome P450 family 1 subfamily A member 1 (Cyp1a1) and the miR-212/132 cluster, and promoted the formation of the Ahr/Ahr nuclear translocator (Arnt) complex. In vivo, GA-treated mice were resistant to EAE and exhibited reduced levels of proinflammatory cytokines and increased levels of transforming growth factor-β (TGF-β). Furthermore, GA reduced infiltration of CD4+CD45+ T cells and monocytes into the CNS. The anti-inflammatory effects of GA were concomitant with miR-132-potentiated cholinergic anti-inflammation and the regulation of the pathogenic potential of astrocytes and microglia. Inducing EAE by adoptive transfer revealed that CD4+ T cells were not entirely responsible for the ameliorative effects of GA.ConclusionOur findings identify GA as a novel Ahr ligand and provide molecular mechanisms elucidating the ameliorative effects of GA on EAE, suggesting that GA is a potential therapeutic agent to control inflammation in autoimmune diseases such as MS.

Structural modeling of the AhR:ARNT complex in the bHLH-PASA-PASB region elucidates the key determinants of dimerization.

Elucidation of the dimerization process of the aryl hydrocarbon receptor (AhR) with the AhR nuclear translocator (ARNT) is crucial for understanding the mechanisms underlying the functional activity of AhR, including mediation of the toxicity of environmental contaminants. In this work, for the first time a structural model of the AhR:ARNT dimer encompassing the entire bHLH-PASA-PASB domain region is proposed. It is developed by using a template-based modeling approach, relying on the recently available crystallographic structures of two dimers of homologous systems in the bHLH-PAS family of proteins: the CLOCK:BMAL1 and the HIF2α:ARNT heterodimers. The structural and energetic characteristics of the modeled AhR:ARNT protein-protein interface are determined by evaluating the variations in solvent accessible surface area, the total binding free energy and the per-residue free energy contributions obtained by the MM-GBSA method and the Energy Decomposition Analysis. The analyses of the intricate network of inter-domain interactions at the dimerization interfaces provide insights into the key determinants of dimerization. These are confirmed by comparison of the computational findings with the available experimental mutagenesis and functional analysis data. The results presented here on the AhR:ARNT dimer structure and interactions provide a framework to start analyzing the mechanism of AhR transformation into its functional DNA binding form.

CK1δ restrains lipin-1 induction, lipid droplet formation and cell proliferation under hypoxia by reducing HIF-1α/ARNT complex formation

Proliferation of cells under hypoxia is facilitated by metabolic adaptation, mediated by the transcriptional activator Hypoxia Inducible Factor-1 (HIF-1). HIF-1α, the inducible subunit of HIF-1 is regulated by oxygen as well as by oxygen-independent mechanisms involving phosphorylation. We have previously shown that CK1δ phosphorylates HIF-1α in its N-terminus and reduces its affinity for its heterodimerization partner ARNT. To investigate the importance of this mechanism for cell proliferation under hypoxia, we visually monitored HIF-1α interactions within the cell nucleus using the in situ proximity ligation assay (PLA) and fluorescence recovery after photobleaching (FRAP). Both methods show that CK1δ-dependent modification of HIF-1α impairs the formation of a chromatin binding HIF-1 complex. This is confirmed by analyzing expression of lipin-1, a direct target of HIF-1 that mediates hypoxic neutral lipid accumulation. Inhibition of CK1δ increases lipid droplet formation and proliferation of both cancer and normal cells specifically under hypoxia and in an HIF-1α- and lipin-1-dependent manner. These data reveal a novel role for CK1δ in regulating lipid metabolism and, through it, cell adaptation to low oxygen conditions.

Antiangiogenic effect of combined treatment with curcumin and genistein on human prostate cancer cell line

High cost and toxicity, low success rate and patient incompliance associated with existing anticancer drugs necessitated for finding new anticancer drugs which can overcome the aforementioned drawbacks. Toward this direction effort has been made to understand the anticancer activity of curcumin (cur) and genistein (gen) combination in human prostate cancer cell line (PC3) cells with respect to their antiangiogenic effect. Cur and gen has shown dose and time dependent decrease in cell viability, increase in apoptosis and cell cycle arrest at G0 phase. These effects were more noticeable when cur (20 µM) and gen (100 μM) were used in combination. To understand antiangiogenic effect of this combination, expression of ARNT and HIF-1α was studied. Significant decline in expression of ARNT and HIF-1α protein level was seen in comparison to control group and their respective monotherapy treated groups. Cur and gen are shown to be effective in abrogating the VEGF production by evading ARNT and HIF-1α complex formation as proved by immunoprecipitation assay. Thus this combination seems to be promising toward cancer treatment.

Nucleotide specificity of DNA binding of the aryl hydrocarbon receptor:ARNT complex is unaffected by ligand structure.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and a wide variety of structurally diverse ligands through its ability to translocate into the nucleus and bind to a specific DNA recognition site (the dioxin-responsive element [DRE]) adjacent to responsive genes. Although the sequence of the DRE is well defined, several reports suggested that the nucleotide specificity of AhR DNA binding may vary depending on the structure of its bound ligand. Given the potential toxicological significance of this hypothesis, an unbiased DNA-selection-and-PCR-amplification approach was utilized to directly determine whether binding and activation of the AhR by structurally diverse agonists alter its nucleotide specificity of DNA binding. Guinea pig hepatic cytosolic AhR activated in vitro by equipotent concentrations of TCDD, 3-methylcholanthrene, β-naphthoflavone, indirubin, L-kynurenine, or YH439 was incubated with a pool of DNA oligonucleotides containing a 15-base pair variable region consisting of all possible nucleotides. The AhR-bound oligonucleotides isolated by immunoprecipitation were PCR amplified and used in subsequent rounds of selection. Sequence analysis of a total of 196 isolated oligonucleotides revealed that each ligand-activated AhR:ARNT complex only bound to DRE-containing DNA oligonucleotides; no non-DRE-containing DNA oligonucleotides were identified. These results demonstrate that the binding and activation of the AhR by structurally diverse agonists do not appear to alter its nucleotide specificity of DNA binding and suggest that stimulation of gene expression mediated by direct DNA binding of ligand-activated AhR:ARNT complexes is DRE dependent.

Erythropoietin Lacking Hypoxia Responsive Element in 3′ Untranslated Region Stimulates Hepatic Erythropoiesis in Anemic Xenopus.

Recent studies on EPO-EPOR systems in non-mammalian vertebrate including frog and teleost fishes demonstrate extramedullary adult erythropoiesis instead of bone marrow. In adult Xenopus laevis (African clawed frog), an animal model of hepatic erythropoiesis should give an opportunity for understanding the microenvironment of vascular niche. Therefore, we investigate on erythrocyte development, gene expressions and gene regulations of EPO and EPOR molecules in adult Xenopus liver. In situ hybridization and immunostaining revealed that Xenopus EPOR (xlEPOR) expressing erythrocyte progenitors, which have low hemoglobin content, were localized among liver sinusoids. The maximum number of xlEPOR expressing cells was observed when peripheral RBC count reached a nadir after hemolytic anemia. Flowcytometric analysis of peripheral blood cells and dispersed liver cells using anti-xlEPOR antibodies also indicate that the xlEPOR+ cells in liver were increased as decreasing count of peripheral RBC. After a nadir of RBC count, xlEPOR+ immature nucleated erythrocytes were emerged in the circulation. The count of xlEPOR+ immature erythrocytes was gradually decreased as increasing count of xlEPOR− mature erythrocytes. Since mature erythrocytes are still nucleated in Xenopus, we used xlEPOR molecules as erythroid differentiation marker. Meanwhile real time RT-PCR analysis of Xenopus EPO (xlEPO) mRNA revealed that xlEpo gene expression was significantly induced in anemic liver compared to normal liver. These data suggest that xlEPO-xlEPOR signaling between erythrocyte progenitors and liver cells progress the proliferation and differentiation of erythrocyte progenitors, and the mobilization of immature erythrocytes into the circulation. Our previous study showed that the anemic serum of phenylhydrazine administrated Xenopus contains erythroid colony forming activity; however, there is no information about the relationship between anemia and hypoxia enough to stimulate erythropoiesis in adult Xenopus. In mammalian species, Epo gene expression is upregulated by binding of hypoxia inducible factor-1a (HIF-1a) and ARNT complex to hypoxia response element (HRE) located in 3′ enhancer region of Epo gene. In frog and fishes, EPO mRNAs are expressed even in normoxia condition. In fish Epo genes, consensus HRE sequence (ACGTG) were not found in 3′UTR, as well as the reporter assay failed to show Epo upregulation respond to hypoxia. Since any consensus HRE sequence was not found in 3′ UTR of xlEpo gene, we examined whether HIF-1a mediates xlEpo gene regulation. By western blot analysis of HIF-1a, we assessed whether HIF-1a is stabilized in anemia; meanwhile binding capacity of HIF-1a to 5′, 3′ UTRs and intron regions of xlEpo gene was analyzed by gel shift mobility assay. The findings in non-mammalian animal model demonstrate the basis of erythropoietic gene regulations, as well as molecular mechanism underlying adult extramedullary erythropoiesis.

The mechanism of AH receptor protein down-regulation (degradation) and its impact on AH receptor-mediated gene regulation

The proteolytic degradation of transcription factors is an established mechanism of regulating signal transduction pathways. Recent reports have suggested that the aryl hydrocarbon receptor (AHR) protein is rapidly downregulated (degraded) following ligand binding. The downregulation of AHR has been observed in nine distinct cells culture lines derived from human and rodent tissues and has also been observed in rodent models following exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). The downregulation of AHR appears to be ubiquitin mediated and occurs via the 26S proteasome pathway following nuclear export of AHR. The consequence of blocking AHR degradation in cell culture appears to be an increase in both the magnitude and duration of gene regulation by the AHR·ARNT complex. Thus, the physiological role of AHR degradation may be to modulate AHR-mediated gene regulation. This review provides analysis of the studies that have focused on the degradation of AHR in vivo and in vitro and the hypothesis that the downregulation of AHR is critical in the attenuation of AHR-mediated gene regulation.

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.

Regulation of constitutive gene expression through interactions of Sp1 protein with the nuclear aryl hydrocarbon receptor complex.

The region of residues -145 to -119 (CD/L) of the cathepsin D gene promoter contains a GC-rich motif that binds Sp1 protein and an adjacent pentanucleotide (CACGC) that corresponds to the core sequence of a dioxin responsive element (DRE) and binds the aryl hydrocarbon receptor (AhR)-AhR nuclear translocator (Arnt) complex. This Sp1(N)(4)DRE(core) motif has been identified in promoters of several genes in which Sp1 plays an important role in basal gene expression. In transient transfection assays with MCF-7 human breast cancer cells using wild-type pCD/L and constructs mutated in the core DRE (pCD/L(m1)) and Sp1 (pCD/L(m2)) sites, it was shown that both motifs were required for maximal basal activity. The requirements for AhR-Arnt interactions with Sp1 protein for maximal activity of pCD/L were confirmed in wild-type MCF-7 and Hepa 1c1c7 cells and Arnt-deficient Hepa 1c1c7 cells using antisense Arnt and Arnt expression plasmids. The functional interactions of Sp1 with AhR-Arnt were paralleled by physical interactions showing that AhR-Arnt and Sp1 proteins were co-immunoprecipitated and AhR-Arnt enhanced Sp1-[(32)P]CD/L binding in electrophoretic mobility shift assays. The physical and functional interactions of Sp1 with AhR-Arnt proteins bound to the Sp1(N)(4)DRE(core) motif were also dependent on the proximity of these sites, and both the activity and the extent of Sp1-DNA binding decreased as the number of intervening nucleotides increased from 4 to 20. These studies show that regulation of basal expression of some genes by Sp1 may also require interactions with AhR-Arnt.

Role of Estradiol Receptor-α in Differential Expression of 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Inducible Genes in the RL95-2 and KLE Human Endometrial Cancer Cell Lines

The present study was conducted to investigate the mechanism of the response of human uterine endometrial carcinoma cells, RL95-2 and KLE, to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). RL95-2 cells were highly responsive to TCDD in terms of cytochrome P4501A1 (CYP1A1), cytochrome P4501B1 (CYP1B1), and plasminogen activator inhibitor-2 (PAI-2), whereas KLE cells showed little stimulatory effects only at high doses. Neither showed any growth inhibition upon exposure to TCDD. KLE cells expressed higher levels of aryl hydrocarbon receptor (AhR) than RL95-2 and gel mobility shift assay also identified more liganded AhR–ARNT complex bound to xenobiotic response elements (XRE). TCDD had no downregulatory effects on the expression of either AhR or the estradiol receptor (ER). Though both cell types expressed ER-α almost equally, immunofluorescence demonstrated a defect in its nuclear translocation in KLE cells where ER-α was mainly cytoplasmic and estradiol-17β (E2) was unable to translocate it to the nucleus. However, both cells were nonresponsive to E2 in terms of transcriptional activation and transient expression of normal ER-α restored the E2 responsiveness. Transient expression of ER-α in KLE cells also restored its responsiveness to TCDD on transcriptional activation. Collectively, these results indicate that ER-α acts as a positive modulator in regulation of the TCDD-inducible genes.

Cross-talk between the Aryl Hydrocarbon Receptor and Hypoxia Inducible Factor Signaling Pathways: DEMONSTRATION OF COMPETITION AND COMPENSATION

The aryl hydrocarbon receptor (AHR) and the alpha-class hypoxia inducible factors (HIF1alpha, HIF2alpha, and HIF3alpha) are basic helix-loop-helix PAS (bHLH-PAS) proteins that heterodimerize with ARNT. In response to 2,3,7,8-tetrachlorodibenzo-p-dioxin, the AHR. ARNT complex binds to "dioxin responsive enhancers" (DREs) and activates genes involved in the metabolism of xenobiotics, e.g. cytochrome P4501A1 (Cyp1a1). The HIF1alpha.ARNT complex binds to "hypoxia responsive enhancers" and activates the transcription of genes that regulate adaptation to low oxygen, e.g. erythropoietin (Epo). We postulated that activation of one pathway would inhibit the other due to competition for ARNT or other limiting cellular factors. Using pathway specific reporters in transient transfection assays, we observed that DRE driven transcription was markedly inhibited by hypoxia and that hypoxia responsive enhancer driven transcription was inhibited by AHR agonists. When we attempted to support this cross-talk model using endogenous loci, we observed that activation of the hypoxia pathway inhibited Cyp1a1 up-regulation, but that activation of the AHR actually enhanced the induction of Epo by hypoxia. To explain this unexpected additivity, we examined the Epo gene and found that its promoter harbors DREs immediately upstream of its transcriptional start site. These experiments outline conditions where inhibitory and additive cross-talk occur between the hypoxia and dioxin signal transduction pathways and identify Epo as an AHR-regulated gene.

Inhibition of the Transcription of CYP1A1 Gene by the Upstream Stimulatory Factor 1 in Rabbits: COMPETITIVE BINDING OF USF1 WITH AhR·Arnt COMPLEX

A xenobiotic-responsive element (XRE)-binding factor(s) other than the AhR.Arnt complex was found to inhibit the transcription of CYP1A1 gene in the liver from adult rabbits, known to be nonresponsive to CYP1A1 inducers. The constitutive factor(s) in liver nuclear extracts bound to the core sequence of XRE. The binding was eliminated by the presence of an excess amount of the AhR.Arnt complex synthesized in vitro. To identify the constitutive factor(s), a sequence similar to rabbit XRE was sought. It was found that the sequence of rabbit XRE overlapped with that of the upstream stimulatory factor 1 (USF1)-binding site in the mouse metallothionein I promoter. In fact, a super shift assay using a specific antibody against human USF1 indicated that USF1 was capable of binding to rabbit XRE. Additionally, the AhR.Arnt-mediated activation of XRE-TK/Luc reporter gene in RK13 cells was blocked by the transfection with a USF1 expression vector with the amounts of the expression vector transfected. These results indicate that the XRE of the rabbit CYP1A1 gene is recognized by the basic helix-loop-helix proteins to regulate the expression of CYP1A1 in both an agonistic (AhR.Arnt) and an antagonistic (USF1) manner.

Upstream Stimulatory Factor 1 (USF1) Suppresses Induction of CYP1A1 mRNA by 3-Methylcholanthrene (MC) in HepG2 Cells

In this study, an endogenous factor(s) involved in the suppression of the induction of CYP1A1 was studied. Analyzing the sequences, we found that the sequence of xenobiotic responsive element (XRE) in the upstream region of the human CYP1A1gene was overlapped with that of the upstream stimulatory factor 1 (USF1)-binding site in mouse metallothionein I promoter. In fact, a gel shift assay using a specific competitor or mutant probes showed that the core sequence of human XRE was specifically recognized by USF1. The amount of USF1 in the nuclear extracts from HepG2 cells was smaller than that from rat and rabbit livers as assayed by the binding to XRE. To determine whether or not USF1 could inhibit the interaction of aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (Arnt) complex with XRE, we transfected USF1-SRα expression vector into HepG2 cells. The results showed that no interaction of AhR/Arnt complex with XRE occurred even when the cells were treated with 2,3,7,8-tetrachlorodibenzofuran (TCDF). Furthermore, the S1 nuclease protection assay showed that the induction of CYP1A1 mRNA by 3-methylcholanthrene (MC) was depressed by the transfection of USF1-SRα into HepG2 cells. Thus, it is highly possible that USF1 negatively regulates the induction of CYP1A1 in humans.

Expression of aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (Arnt) in adult rabbits known to be non-responsive to cytochrome P-450 1A1 (CYP1A1) inducers.

Induction of aryl hydrocarbon hydroxylase by aryl hydrocarbons occurs only in neonatal rabbits and not in adult rabbits [Kahl, G. F., Friederich, D. E., Bigelow, S. W., Okey, A. B. & Nebert, D. W. (1980) Dev. Pharmacol. Ther. 1,137-162]. In the present study, we isolated cDNA clones encoding aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (Arnt) from adult rabbits. The deduced amino acid sequences of rabbit AhR and Arnt showed 80% and 94% identities with those of human AhR and Arnt, respectively. Rabbit AhR mRNA was predominantly expressed in the lung and liver. In contrast, rabbit Arnt mRNA was expressed at almost the same level in all tissues except for the heart, liver, and small intestine. Gel shift analysis showed that the AhR. Arnt complex could bind to the consensus xenobiotic-responsive element, which indicates that AhR expressed in adult rabbit liyers possessed binding activity to the consensus xenobiotic-responsive element in vitro, although aryl hydrocarbons did not induce the activity of AHH in adult rabbits. We propose that the incapability of adult rabbits to induce cytochrome P-450 1A1 (CYP1A1) is caused by factors other than AhR and Arnt.