Multiple roles of ligand in transforming the dioxin receptor to an active basic helix-loop-helix/PAS transcription factor complex with the nuclear protein Arnt.

The dioxin receptor is a ligand-activated transcription factor belonging to an emerging class of basic helix-loop-helix/PAS proteins which show interaction with the molecular chaperone hsp90 in their latent states and require heterodimerization with a general cofactor, Arnt, to form active DNA binding complexes. Upon binding of polycyclic aromatic hydrocarbons typified by dioxin, the dioxin receptor translocates from the cytoplasm to the nucleus to allow interaction with Arnt. Here we have bypassed the nuclear translocation step by creating a cell line which expresses a constitutively nuclear dioxin receptor, which we find remains in a latent form, demonstrating that ligand has functional roles beyond initiating nuclear import of the receptor. Treatment of the nuclear receptor with dioxin induces dimerization with Arnt to form an active transcription factor complex, while in stark contrast, treatment with the hsp90 ligand geldanamycin results in rapid degradation of the receptor. Inhibition of degradation by a proteasome inhibitor allowed geldanamycin to transform the nuclear dioxin receptor to a heterodimer with Arnt (DR-Arnt). Our results indicate that unchaperoned dioxin receptor is extremely labile and is consistent with a concerted nuclear mechanism for receptor activation whereby hsp90 is released from the ligand-bound dioxin receptor concomitant with Arnt dimerization. Strikingly, artificial transformation of the receptor by geldanamycin provided a DR-Arnt complex capable of binding DNA but incapable of stimulating transcription. Limited proteolysis of DR-Arnt heterodimers indicated different conformations for dioxin versus geldanamycin-transformed receptors. Our studies of intracellular dioxin receptor transformation indicate that ligand plays multiple mechanistic roles during receptor activation, being important for nuclear translocation, transformation to an Arnt heterodimer, and maintenance of a structural integrity key for transcriptional activation.

Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation.

Gene regulation by dioxins is mediated via the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/PAS transcription factor. The latent dioxin receptor responds to dioxin signalling by forming an activated heterodimeric complex with a specific bHLH partner, Arnt, an essential process for target DNA recognition. We have analyzed the transactivating potential within this heterodimeric complex by dissecting it into individual subunits, replacing the dimerization and DNA-binding bHLH motifs with heterologous zinc finger DNA-binding domains. The uncoupled Arnt chimera, maintaining 84% of Arnt residues, forms a potent and constitutive transcription factor. Chimeric proteins show that the dioxin receptor also harbors a strong transactivation domain in the C terminus, although this activity was silenced by inclusion of 82 amino acids from the central ligand-binding portion of the dioxin receptor. This central repression region conferred binding of the molecular chaperone hsp90 upon otherwise constitutive chimeras in vitro, indicating that hsp90 has the ability to mediate a cis-repressive function on distant transactivation domains. Importantly, when the ligand-binding domain of the dioxin receptor remained intact, the ability of this hsp90-binding activity to confer repression became conditional rather than irreversible. Our data are consistent with a model in which crucial activities of the dioxin receptor, such as dimerization with Arnt and transactivation, are conditionally repressed by the central ligand- and-hsp90-binding region of the receptor. In contrast, the Arnt protein appears to be free from any repressive activity. Moreover, within the context of the dioxin response element (xenobiotic response element), the C terminus of Arnt conferred a potent, dominating transactivation function onto the native bHLH heterodimeric complex. Finally, the relative transactivation potencies of the individual dioxin receptor and Arnt chimeras varied with cell type and promoter architecture, indicating that the mechanisms for transcriptional activation may differ between these two subunits and that in the native complex the transactivation pathway may be dependent upon cell-specific and promoter contexts.

Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor.

The dioxin receptor is a ligand-regulated transcription factor that mediates signal transduction by dioxin and related environmental pollutants. The receptor belongs to the basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family of factors, which, in addition to the bHLH motif, contain a PAS region of homology. Upon activation, the dioxin receptor dimerizes with the bHLH-PAS factor Arnt, enabling the receptor to recognize xenobiotic response elements in the vicinity of target genes. We have studied the role of the PAS domain in dimerization and DNA binding specificity of the dioxin receptor and Arnt by monitoring the abilities of the individual bHLH domains and different bHLH-PAS fragments to dimerize and bind DNA in vitro and recognize target genes in vivo. The minimal bHLH domain of the dioxin receptor formed homodimeric complexes, heterodimerized with full-length Arnt, and together with Arnt was sufficient for recognition of target DNA in vitro and in vivo. In a similar fashion, only the bHLH domain of Arnt was necessary for DNA binding specificity in the presence of the dioxin receptor bHLH domain. Moreover, the bHLH domain of the dioxin receptor displayed a broad dimerization potential, as manifested by complex formation with, e.g. , the unrelated bHLH-Zip transcription factor USF. In contrast, a construct spanning the dioxin receptor bHLH domain and an N-terminal portion of the PAS domain failed to form homodimers and was capable of dimerizing only with Arnt. Thus, the PAS domain is essential to confer dimerization specificity of the dioxin receptor.

Distinct roles of the molecular chaperone hsp90 in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains.

The intracellular dioxin receptor mediates signal transduction by dioxin and functions as a ligand-activated transcription factor. It contains a basic helix-loop-helix (bHLH) motif contiguous with a Per-Arnt-Sim (PAS) homology region. In extracts from nonstimulated cells the receptor is recovered in an inducible cytoplasmic form associated with the 90-kDa heat shock protein (hsp90), a molecular chaperone. We have reconstituted ligand-dependent activation of the receptor to a DNA-binding form by using the dioxin receptor and its bHLH-PAS partner factor Arnt expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with Arnt. In contrast, this receptor mutant showed low levels of xenobiotic response element-binding activity, indicating that the PAS domain may be important for DNA-binding affinity and/or specificity of the receptor. It was not possible to reconstitute dioxin receptor function with proteins expressed in wheat germ lysate. In line with these observations, reticulocyte lysate but not wheat germ lysate promoted the association of de novo synthesized dioxin receptor with hsp90. At least two distinct domains of the receptor mediated interaction with hsp90: the ligand-binding domain located within the PAS region and, surprisingly, the bHLH domain. Whereas ligand-binding activity correlated with association with hsp90, bHLH-hsp90 interaction appeared to be important for DNA-binding activity but not for dimerization of the receptor. Several distinct roles for hsp90 in modulating dioxin receptor function are therefore likely: correct folding of the ligand-binding domain, interference with Arnt heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system provides a complex and interesting model of the regulation of transcription factors by hsp90.

A cellular factor stimulates ligand-dependent release of hsp90 from the basic helix-loop-helix dioxin receptor.

In response to dioxin, the nuclear basic helix-loop-helix (bHLH) dioxin receptor forms a complex with the bHLH partner factor Arnt that regulates target gene transcription by binding to dioxin-responsive sequence motifs. Previously, we have demonstrated that the latent form of dioxin receptor present in extracts from untreated cells is stably associated with molecular chaperone protein hsp90, and Arnt is not a component of this complex. Here, we used a coimmunoprecipitation assay to demonstrate that the in vitro-translated dioxin receptor, but not Arnt, is stably associated with hsp90. Although it showed ligand-binding activity, the in vitro-translated dioxin receptor failed to dissociate from hsp90 upon exposure to ligand. Addition of a specific fraction from wild-type hepatoma cells, however, to the in vitro-expressed receptor promoted dioxin-dependent release of hsp90. This stimulatory effect was mediated via the bHLH dimerization and DNA-binding motif of the receptor. Moreover, ligand-dependent release of hsp90 from the receptor was not promoted by fractionated cytosolic extracts from mutant hepatoma cells which are deficient in the function of bHLH dioxin receptor partner factor Arnt. Thus, our results provide a novel model for regulation of bHLH factor activity and suggest that derepression of the dioxin receptor by ligand-induced release of hsp90 may require bHLH-mediated concomitant recruitment of an additional cellular factor, possibly the structurally related bHLH dimerization partner factor Arnt. In support of this model, addition of in vitro-expressed wild-type Arnt, but not a mutated form of Arnt lacking the bHLH motif, promoted release of hsp90 from the dioxin receptor in the presence of dioxin.

Effect of ARA9 on dioxin receptor mediated transcription.

The dioxin (Aryl hydrocarbon) receptor (DR) is a unique bHLH transcription factor which is activated by binding of planar aromatic hydrocarbons typified by dioxin (TCDD). The active receptor is key to metabolism of aryl hydrocarbon xenobiotics by being a potent inducer of CYP1A1 gene activity. Chlorinated dioxins are inert to metabolism and initiate multifarious toxicities, including potent tumour promotion. These ill-effects are mediated by the activated DR and we are studying the mechanisms by which the ligand binding domain of the DR controls activity of the protein. The DR ligand binding domain resides within a PAS (Per/Arnt/Sim homology) region which is contiguous with the bHLH. The latent bHLH/PAS dioxin receptor (DR) is found in the cytoplasm of most mammalian cell types in a complex with heat shock protein 90, a novel immunophilin like protein termed ARA9/XAP2/AIP, and the co-chaperone p23. Here we use antisense ARA9 constructs to reveal that in the absence of ARA9, the DR is unable to form a transcriptionally active complex. Co-expression of antisense ARA9 with a form of the DR which is constitutively targeted to the nucleus leads to dramatically decreased levels of the nuclear DR protein, implying that ARA9 may function beyond its currently proposed role in cytoplasmic retention of the latent DR.

The HIF1alpha-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element.

The short isoform of single-minded 2 (SIM2s), a basic helix-loop-helix/PAS (bHLH/PAS) transcription factor, is upregulated in pancreatic and prostate tumours; however, a mechanistic role for SIM2s in these cancers is unknown. Microarray studies in prostate DU145 cells identified the pro-cell death gene, BNIP3 (Bcl-2/adenovirus E1B 19 kDa interacting protein 3), as a novel putative target of SIM2s repression. Further validation showed BNIP3 repression in several prostate and pancreatic carcinoma-derived cell lines with ectopic expression of human SIM2s. BNIP3 levels are enhanced in prostate carcinoma cells upon short interfering (si)RNA-mediated knockdown of endogenous SIM2s. Chromatin immunoprecipitation and promoter studies show that SIM2s represses BNIP3 through its activities at the proximal promoter hypoxia response element (HRE), the site through which the bHLH/PAS family member, hypoxia-inducible factor 1alpha (HIF1alpha), induces BNIP3. SIM2s attenuates BNIP3 hypoxic induction via the HRE, and increased hypoxic induction of BNIP3 occurs with siRNA knockdown of endogenous SIM2s in prostate PC3AR+ cells. BNIP3 is implicated in hypoxia-induced cell death processes. Prolonged treatment of PC3AR+ cells with hypoxia mimetics, DP and DMOG, confers hypoxia-induced autophagy, measured by enhanced LC3-II levels and SQSTM1/p62 turnover. We show that PC3AR+ cells expressing ectopic SIM2s have enhanced survival in these conditions. Induction of LC3-II and turnover of SQSTM1/p62 are attenuated in PC3AR+/SIM2s DMOG and hypoxia-treated cells, suggesting that SIM2s may attenuate autophagic cell death processes, perhaps through BNIP3 repression. These data show, for the first time, SIM2s cross-talk on an endogenous HRE. SIM2s' functional interference with HIF1alpha activities on BNIP3 may indicate a novel role for SIM2s in promoting tumourigenesis.

Evidence that the 90-kDa heat shock protein (HSP90) exists in cytosol in heteromeric complexes containing HSP70 and three other proteins with Mr of 63,000, 56,000, and 50,000.

Monoclonal antibody (mAb) 8D3 and 3G3 are unique antibodies capable of precipitating both free 90-kDa heat shock protein (HSP90) and HSP90-protein complexes. Immunoprecipitation of [35S]methionine-labeled Hepa 1c1c7 cytosolic extracts were performed using mAb 8D3 or 3G3. The resulting immunoprecipitates can be dissociated from the mAb with a 500 mM NaCl wash. These washes were subjected to both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Five major protein spots were detected in addition to HSP90 with the following relative molecular weights: 68,000, 63,000, 56,000, 50,000, and 188,000. On Western blots mAb 3G3 was capable of specifically binding to HSP90. Each of these proteins was localized on two-dimensional gels. Using one-dimensional gel electrophoresis and immunochemical localization on Western blots, the p68 spot was identified as HSP70, and the p56 spot was found to cross-react with polyclonal antibody JP-1 raised against a 59-kDa protein. This 59-kDa protein has been found previously to be associated with several steroid hormone receptors in rabbit uterine cytosol. Immunoprecipitation of [32P]orthophosphate-labeled Hepa 1c1c7 cytosol with mAb 8D3 or 3G3 revealed two major phosphorylated proteins with relative molecular weights of 90,000 and 50,000. The identities of p63 and p188 are currently unknown. This is the first report examining the major proteins that are complexed with HSP90 in mammalian cells.

Degradation of the basic helix-loop-helix/Per-ARNT-Sim homology domain dioxin receptor via the ubiquitin/proteasome pathway.

The basic helix-loop-helix/Per-ARNT-Sim homology domain dioxin receptor (DR) translocates to the nucleus upon binding of aromatic hydrocarbon ligands typified by dioxin, whereupon it partners the Ah receptor nuclear translocator and initiates transcription. Concurrently, ligand binding down-regulates receptor levels via an unknown mechanism. In this study we show that receptor levels are dependent upon cellular compartmentalization, with entry into the nucleus leading to the rapid destruction of the DR. Ligand-induced DR translocation was bypassed by adding a heterologous nuclear localization signal to the DR, creating a constitutively nuclear form of the dioxin receptor (DRNLS). The DRNLS protein was shown to be unstable with a half-life of

Protein-protein interaction via PAS domains: role of the PAS domain in positive and negative regulation of the bHLH/PAS dioxin receptor-Arnt transcription factor complex.

Gene regulation by dioxins is mediated by the dioxin receptor-Arnt heterodimer, a ligand generated complex of two basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) transcription factors. By using dioxin receptor chimeras where the dimerization and DNA binding bHLH motif has been replaced by a heterologous DNA binding domain, we have detected an ability of Arnt to interact with the dioxin receptor via the PAS domain in a mammalian 'hybrid interaction' system. By coimmunoprecipitation assays, we have confirmed the ability of PAS domains of the dioxin receptor and Arnt to mediate independent heterodimerization in vitro. Selectivity for PAS dimerization was noted in our hybrid interaction system, as dioxin receptor or Arnt PAS-mediated homodimers were not detected. Surprisingly, however, the PAS domain of Per could dimerize with both the dioxin receptor and Arnt subunits in vitro, and disrupt the ability of these subunits to form a DNA binding heterodimer. Moreover, ectopic expression of Per blocked dioxin signalling in mammalian cells. The PAS domains of the dioxin receptor and Arnt are therefore novel dimerizing regions critical in formation of a functional dioxin receptor-Arnt complex, while the PerPAS domain is a potential negative regulator of bHLH/PAS factor function.

The hypoxia-inducible factors: key transcriptional regulators of hypoxic responses.

Oxygen depravation in mammals leads to the transcriptional induction of a host of target genes to metabolically adapt to this deficiency, including erythropoietin and vascular endothelial growth factor. This response is primarily mediated by the hypoxia-inducible factors (HIFs) which are members of the basic-helix-loop-helix/Per-ARNT-Sim (bHLH/PAS) transcription factor family. The HIFs are primarily regulated via a two-step mechanism of HIF post-translational modification, increasing both protein stability and transactivation capacity. This review aims to summarise our current understanding of these processes, and discuss the important role of the HIFs in the pathophysiology of many human diseases.

A redox mechanism controls differential DNA binding activities of hypoxia-inducible factor (HIF) 1alpha and the HIF-like factor.

Hypoxia-inducible factor 1alpha (HIF-1alpha) and the HIF-like factor (HLF) are two highly related basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) homology transcription factors that undergo dramatically increased function at low oxygen levels. Despite strong similarities in their activation mechanisms (e.g. they both undergo rapid hypoxia-induced protein stabilization, bind identical target DNA sequences, and induce synthetic reporter genes to similar degrees), they are both essential for embryo survival via distinct functions during vascularization (HIF-1alpha) or catecholamine production (HLF). It is currently unknown how such specificity of action is achieved. We report here that DNA binding by HLF, but not by HIF-1alpha, is dependent upon reducing redox conditions. In vitro DNA binding and mammalian two-hybrid assays showed that a unique cysteine in the DNA-binding basic region of HLF is a target for the reducing activity of redox factor Ref-1. Although the N-terminal DNA-binding domain of HIF-1alpha can function in the absence of Ref-1, we found that the C-terminal region containing the transactivation domain requires Ref-1 for full activity. Our data reveal that the hypoxia-inducible factors are subject to complex redox control mechanisms that can target discrete regions of the proteins and are the first to establish a discriminating control mechanism for differential regulation of HIF-1alpha and HLF activity.

Transcriptional activation of cytochrome P450 genes by different classes of chemical inducers.

1. We review here the molecular mechanisms underlying the xenobiotic induction of genes encoding cytochrome P450 (CYP) enzymes in the liver and other tissues. We will focus on four major families of CYP genes. 2. Members of the CYP1 gene family are induced by polycyclic aromatic hydrocarbons and this process is mediated by the basic helix-loop-helix proteins: the Ah receptor and its heterodimeric partner Arnt. Considerable progress has been made in elucidating the molecular details of this induction process. 3. CYP4 genes are activated by peroxisomal proliferators, a group of structurally diverse chemicals that also induce peroxisome proliferation. The transcriptional response is dependent on the peroxisome proliferator-activated receptor and its partner RXR, both members of the nuclear receptor superfamily; their role in the induction process has been well characterized at the molecular level. 4. In contrast, the mechanism of gene induction of CYP2 genes by phenobarbital and other structurally diverse inducers is not well understood and a specific phenobarbital-responsive receptor has not been identified. 5. Induction of the CYP3 gene family by the glucocorticoid dexamethasone appears to involve the glucocorticoid receptor, but this receptor is not apparently required for induction by metapyrone and a complete molecular understanding of the induction processes is lacking at present.

A 50-kDa cytosolic protein complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus.

We have previously shown that a 50-kDa protein is one component of a heteromeric complex immunoprecipitated by the 90-kDa heat shock protein (hsp90) monoclonal antibodies 8D3 and 3G3 (Perdew, G. H., and Whitelaw, M. L. (1991) J. Biol. Chem. 266, 6708-6713). In this report, we compare the 50-kDa protein with that found in pp60v-src-hsp90-p50 complexes immunoprecipitated from Rous sarcoma virus-transformed cells with antibodies to pp60v-src. 35S- and 32P-labeled p50 proteins from each system were identical in their mobilities by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The profile of N-chlorosuccinimide cleavage products derived from each 32P-labeled p50 protein were also identical when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have developed a mouse monoclonal antibody, 3M/1B5p50, capable of detecting p50 on Western blots. This antibody detected the 50-kDa protein which co-purified with the pa104 pp60v-src mutant of the avian sarcoma virus oncoprotein in 44A rat fibroblasts. We did not detect p50 in association with native glucocorticoid receptor in L cells or with the overexpressed glucocorticoid receptor in Chinese hamster ovary cells. Two experiments utilizing immunochemical staining implied that essentially all cytosolic p50 is associated with hsp90. Firstly, immunoprecipitating hsp90 from Hepa 1 cytosol with monoclonal antibody 3G3 left the cytosol depleted of p50. Secondly, cytosol fractionated by sucrose gradient revealed that p50 cosedimented with hsp90, confirming the existence of p50 only in association with hsp90.

The basic helix-loop-helix/PAS factor Sim is associated with hsp90. Implications for regulation by interaction with partner factors.

Sim is a Drosophila developmental basic helix-loop-helix (bHLH) transcription factor containing a Per-Arnt-Sim (PAS) region of homology. Here we demonstrate that Sim, in analogy to the structurally related bHLH/PAS dioxin receptor, was stably associated with the molecular chaperone hsp90. In the case of the dioxin receptor, release of hsp90 and derepression of receptor function appear to be regulated by ligand binding and dimerization with Arnt, a non-hsp90-associated bHLH/PAS factor. Dimerization with Arnt very efficiently disrupted Sim-hsp90 interaction, a process that required both the bHLH and PAS dimerization motifs of Arnt. Moreover, hsp90 was also released upon dimerization of Sim with the Drosophila PAS factor Per, whereas the hsp90-associated dioxin receptor failed to interact with Sim. These results indicate that hsp90 may play a role in conditional regulation of Sim function, and that Per and possibly bHLH/PAS partner factors may activate Sim by inducing release of hsp90 during the dimerization process.

Heat shock protein hsp90 regulates dioxin receptor function in vivo.

The dioxin (aryl hydrocarbon) receptor is a ligand-dependent basic helix-loop-helix (bHLH) factor that binds to xenobiotic response elements of target promoters upon heterodimerization with the bHLH partner factor Arnt. Here we have replaced the bHLH motif of the dioxin receptor with a heterologous DNA-binding domain to create fusion proteins that mediate ligand-dependent transcriptional enhancement in yeast (Saccharomyces cerevisiae). Previously, our experiments indicated that the ligand-free dioxin receptor is stably associated with the 90-kDa heat shock protein, hsp90. To investigate the role of hsp90 in dioxin signaling we have studied receptor function in a yeast strain where hsp90 expression can be down-regulated to about 5% relative to wild-type levels. At low levels of hsp90, ligand-dependent activation of the chimeric dioxin receptor construct was almost completely inhibited, whereas the activity of a similar chimeric construct containing the structurally related Arnt factor was not affected. Moreover, a chimeric dioxin receptor construct lacking the central ligand- and hsp90-binding region of the receptor showed constitutive transcriptional activity in yeast that was not impaired upon down-regulation of hsp90 expression levels. Thus, these data suggest that hsp90 is a critical determinant of conditional regulation of dioxin receptor function in vivo via the ligand-binding domain.

Constitutive function of the basic helix-loop-helix/PAS factor Arnt. Regulation of target promoters via the E box motif.

Arnt is a nuclear basic helix-loop-helix (bHLH) transcription factor that, contiguous with the bHLH motif, contains a region of homology (PAS) with the Drosophila factors Per and Sim. Arnt dimerizes in a ligand-dependent manner with the bHLH dioxin receptor, a process that enables the dioxin-(2,3,7,8-tetrachlorodibenzo-p-dioxin)-activated Arnt-dioxin receptor complex to recognize dioxin response elements of target promoters. In the absence of dioxin, Arnt does not bind to this target sequence motif. The constitutive function of Arnt is presently not understood. Here we demonstrate that Arnt constitutively bound the E box motif CACGTG that is also recognized by a number of distinct bHLH factors, including USF and Max. Importantly, amino acids that have been identified to be critical for E box recognition by Max and USF are conserved in Arnt. Consistent with these observations, full-length Arnt, but not an Arnt deletion mutant lacking its potent C-terminal transactivation domain, constitutively activated CACGTG E box-driven reporter genes in vivo. These results indicate a role of Arnt in regulation of a network of target genes that is distinct from that regulated by the Arnt-dioxin receptor complex in dioxin-stimulated cells.

Agonistic and antagonistic effects of alpha-naphthoflavone on dioxin receptor function. Role of the basic region helix-loop-helix dioxin receptor partner factor Arnt.

The dioxin receptor is a ligand-dependent transcription factor that binds to target DNA sequences (xenobiotic responsive elements, XREs) following ligand-dependent dimerization with its partner factor, Arnt (aryl hydrocarbon receptor nuclear translocator). Both factors contain an N-terminal basic region helix-loop-helix motif mediating dimerization and subsequent DNA binding. In this study we investigate the possible role of Arnt in agonistic and antagonistic effects of the dioxin receptor ligand alpha-naphthoflavone (ANF). Using specific antisera for the ligand binding dioxin receptor and Arnt, respectively, we show that exposure of the dioxin receptor to ANF in vitro induced recruitment of Arnt, thus stimulating binding of the heteromeric complex to XRE. In transient transfection assays, ANF at high concentrations stimulated expression of an XRE-driven reporter gene. This agonistic effect of ANF is, therefore, most likely attributable to ANF stimulation of dioxin receptor-Arnt heterodimerization and subsequent binding of the complex to XRE. Using a minimal XRE-driven reporter gene construct, we could further confirm earlier studies showing that ANF antagonizes the effect of a dioxin receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Next we employed chimeric receptor constructs containing amino acids 1-500 of the human glucocorticoid receptor fused to dioxin receptor fragments lacking the very N-terminal basic region helix-loop-helix dimerization and DNA binding motif. These chimeric receptor constructs show dioxin responsiveness upon transient transfection into mutant Arnt-deficient hepatoma cells and are, thus, functionally uncoupled from Arnt. Importantly, dioxin-dependent activation of the chimeric receptors was inhibited in the presence of ANF, demonstrating that dimerization of dioxin receptor with Arnt was not necessary for manifestation of the antagonistic effect of ANF. Rather, dioxin receptor sequences, which confer dioxin regulation upon a heterologous DNA binding and transactivating domain, also mediated the antagonistic effects of ANF.

A tyrosine kinase-dependent pathway regulates ligand-dependent activation of the dioxin receptor in human keratinocytes.

Signal transduction by dioxin is mediated by the intracellular basic helix-loop-helix dioxin receptor which, in its ligand-activated state, binds to target DNA as a heteromeric complex with the partner factor Arnt. In contrast, the repressed form of the receptor is a complex with hsp90 which appears to maintain the receptor in an inducible conformation. In human keratinocytes dioxin receptor activation has previously been shown to depend on phosphorylation processes. To further dissect mechanisms regulating dioxin receptor function the importance of tyrosine phosphorylation was investigated by the use of specific tyrosine kinase inhibitors. Here we report that the inhibitor genistein inhibited dioxin-dependent induction of expression of the target gene cytochrome P-450IA1. This effect was rapid and reversible and did not lead to altered levels of dioxin receptor protein. Analyses of dioxin receptor or Arnt fusion proteins that function independently of one another showed that the target for genistein action was the dioxin receptor, and, more specifically, a region of the receptor harboring its ligand-binding domain. In addition, function of an unrelated transactivator, the glucocorticoid receptor, was inhibited by genistein while a truncated form lacking the ligand-binding domain was not. A common denominator between the ligand-binding domains of both receptors is their ability to interact with hsp90. Importantly, co-immunoprecipitation experiments showed that genistein inhibited ligand-induced release of hsp90 from the glucocorticoid receptor. Thus, the interaction of these transactivators with hsp90 may be regulated by a tyrosine kinase-dependent pathway.

Definition of a novel ligand binding domain of a nuclear bHLH receptor: co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor.

The dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) and binds to DNA target sequences as a heterodimer of the approximately 100 kDa ligand binding receptor and the approximately 85 kDa auxiliary factor, Arnt. Both of these factors encompass an N-terminal basic helix-loop-helix (bHLH) motif required for DNA binding and dimerization. In this study we describe the construction of glucocorticoid/dioxin receptor fusion proteins which allow the regulation of glucocorticoid receptor activity by dioxin in transient transfections of CHO and hepatoma cells. Thus, in the absence of dioxin, chimeric receptor constructs which contain large 500-720 amino acid C-terminal dioxin receptor fragments, but lack the N-terminal bHLH motif, confer repression upon the transcriptional activity of a glucocorticoid receptor derivative, tau DBD, containing its N-terminal strong transactivating signal (tau) and its DNA binding domain (DBD). In the presence of dioxin, this repression is reversed. Importantly, these chimeric receptors did not require the bHLH Arnt co-factor for function. A considerably smaller region of the dioxin receptor, located between amino acids 230 and 421, showed specific dioxin binding activity in vitro. Moreover, dioxin binding in vitro correlated with the ability of receptor fragments to form stable complexes in vitro with the molecular chaperone hsp90. These findings support the notion that hsp90 may be important for folding of a dioxin binding configuration of the receptor. Finally, tau DBD activity was constitutively repressed in a dioxin non-responsive manner by dioxin receptor fragments which failed to bind ligand but also failed to bind hsp90 in vitro, indicating that alternative mechanisms in addition to hsp90 binding may contribute to the inactivation function. In summary, the dioxin receptor system provides a novel and complex model of regulation of bHLH factors that may also give important insights into the mechanism of action of ligand-activated nuclear receptors.