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.

Purification of the DNA binding form of dioxin receptor. Role of the Arnt cofactor in regulation of dioxin receptor function.

The basic region/helix-loop-helix dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin). Upon ligand binding the dioxin receptor is converted from a latent, non-DNA binding form to a form that directly interacts with target genes by binding to dioxin-responsive transcriptional control elements. We have purified by conventional and DNA affinity chromatographic procedures the ligand-activated, DNA binding form of dioxin receptor to examine its architecture and functional properties. We observed that the DNA binding activity of the receptor was labile. Most notably, this activity was lost following DNA affinity purification. In complementation experiments we have identified an auxiliary factor(s) that exhibited very poor, if any, intrinsic affinity for the DNA target sequence in vitro but strongly increased the DNA binding activity of the purified receptor-containing material identified by immunoblot analysis. In a similar fashion the in vitro expressed basic region/helix-loop-helix factor Arnt (that has been postulated to modulate the nuclear translocation function of the receptor) reconstituted the DNA binding function of the purified receptor, and the purified auxiliary factor reconstituted receptor activity upon addition to an extract from mutant, Arnt-deficient hepatoma cells. Conversely, purified dioxin receptor reconstituted DNA binding activity in extracts from receptor-deficient hepatoma cells which express bona fide levels of Arnt. Interestingly, UV cross-linking studies using a BrdU-substituted DNA target sequence indicated that primarily the receptor protein was bound to DNA. Moreover, we demonstrate that purified receptor or Arnt exhibited virtually no detectable affinity for the target sequence individually but, in the presence of one another, showed a strong synergy in DNA binding activity in vitro. Importantly, simultaneous expression of the receptor and Arnt resulted in synergistic induction of gene expression in vivo. These data demonstrate that Arnt plays a central role in control of dioxin receptor function by cooperatively modulating the DNA binding activity of the receptor in vitro and dioxin-dependent transactivation in vivo.

Dioxin receptor and C/EBP regulate the function of the glutathione S-transferase Ya gene xenobiotic response element.

The rat glutathione S-transferase Ya gene xenobiotic response element (XRE) has both constitutive and xenobiotic-inducible activity. We present evidence that the XRE is regulated by both the constitutive C/EBP transcription factor and the xenobiotic-activated dioxin receptor. A ligand-activated XRE-binding protein was shown to be dioxin receptor by specific antibody immunodepletion and binding of highly purified receptor. Identification of C/EBP alpha as the constitutive binding protein was demonstrated by competition with a C/EBP binding site, protein-DNA cross-linking to determine the molecular weight of the constitutive protein(s), specific antibody immunodepletion, and binding of purified bacterially expressed C/EBP alpha. Mutational analysis of the XRE revealed that the constitutive factor (C/EBP alpha) shares a nearly identical overlapping binding site with the dioxin receptor. In functional testing of the putative C/EBP-XRE interaction, cotransfected C/EBP alpha activated an XRE test promoter in the non-xenobiotic-responsive HeLa cell line. Unexpectedly, cotransfected C/EBP alpha had no effect on basal activity but significantly increased the xenobiotic response of the XRE test promoter in the xenobiotic-responsive, C/EBP-positive HepG2 cell line. Furthermore, inhibition of C/EBP-binding protein(s) in HepG2 cells by transfection of C/EBP oligonucleotides suppressed the xenobiotic response. These results suggest that C/EBP alpha and dioxin receptor recognize the same DNA sequence element and that transcriptional regulation can occur by cooperative interactions between these two transcription factors.

Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor.

The intracellular basic region/helix-loop-helix (bHLH) dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) and functions as a ligand-activated DNA binding protein directly interacting with target genes by binding to dioxin response elements. Here we show that the partially purified, ligand-bound receptor alone could not bind target DNA. In contrast, DNA binding by the receptor could be induced by addition of a cytosolic auxiliary activity which functionally and biochemically corresponded to the bHLH factor Arnt. While Arnt exhibited no detectable affinity for the dioxin response element in the absence of the dioxin receptor, it strongly promoted the DNA binding function of the ligand-activated but not the ligand-free receptor forms. Arnt also functionally reconstituted in vitro the DNA binding activity of a mutant, nuclear translocation-deficient dioxin receptor phenotype in cytosolic extracts from a dioxin-resistant hepatoma cell line. Importantly, coimmunoprecipitation experiments showed that Arnt physically interacted in solution with the ligand-activated dioxin receptor but failed to heterodimerize with the ligand-free, hsp90-associated receptor form. Mutational analysis suggested that the functional interaction between these two factors occurred via the bHLH motif of Arnt. These data suggest that dioxin receptor activity is governed by a complex pattern of combinatorial regulation involving repression by hsp90 and then by ligand-dependent recruitment of the positive coregulator Arnt. The dioxin receptor system also provides the first example of signal-controlled dimerization of bHLH factors.

Nonresponsiveness of normal human fibroblasts to dioxin correlates with the presence of a constitutive xenobiotic response element-binding factor.

Polychlorinated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzofuran (TCDF) have been shown to induce transcription of the cytochrome P-450IA1 gene by activating an intracellular receptor protein (the Ah- or dioxin receptor) to bind to specific DNA sequences, termed xenobiotic response elements (XREs). However, the expression and inducibility of the cytochrome P-450IA1 activity exhibit tissue-specific differences. With regard to the TCDF induction response, we have examined three human cell types of endodermal (the hepatoma cell line HepG2), ectodermal (normal keratinocytes), and mesodermal origin (normal fibroblasts). DNase I hypersensitivity analysis of the 5' flank and first intron of the P-450IA1 gene showed that in the nonresponsive fibroblasts the chromatin structure lacked open regions while in the two responsive cell types (keratinocytes and HepG2) several constitutive hypersensitive sites as well as TCDF-induced alterations in the chromatin structure could be detected. This observation might correlate with the fact that the XRE, in either the context of the P-450IA1 gene sequences or in front of a heterologous promoter, was inefficient in directing a TCDF induction response in fibroblasts. In in vitro DNA binding studies, the dioxin receptor was activated to a DNA-binding nuclear form in all three cell types. However, in fibroblast nuclear extracts two novel constitutive protein-XRE complexes were detected. The fibroblast factor(s) were immunochemically distinct from the receptor but exhibited indistinguishable DNA binding specificity. These data are compatible with a model where the P-450IA1 is noninducible in fibroblasts due to the presence of a putative repressor(s) which may compete effectively with the receptor for binding to the response element as indicated by in vitro DNA-binding off-rate experiments.

Liver cells contain constitutive DNase I-hypersensitive sites at the xenobiotic response elements 1 and 2 (XRE1 and -2) of the rat cytochrome P-450IA1 gene and a constitutive, nuclear XRE-binding factor that is distinct from the dioxin receptor.

Dioxin stimulates transcription from the cytochrome P-450IA1 promoter by interaction with the intracellular dioxin receptor. Upon binding of ligand, the receptor is converted to a form which specifically interacts in vitro with two dioxin-responsive positive control elements located in close proximity to each other about 1 kb upstream of the rat cytochrome P-450IA1 gene transcription start point. In rat liver, the cytochrome P-450IA1 gene is marked at the chromatin level by two DNase I-hypersensitive sites that map to the location of the response elements and exist prior to induction of transcription by the dioxin receptor ligand beta-naphthoflavone. In addition, a DNase I-hypersensitive site is detected near the transcription initiation site and is altered in nuclease sensitivity by induction. The presence of the constitutive DNase I-hypersensitive sites at the dioxin response elements correlates with the presence of a constitutive, labile factor which specifically recognizes these elements in vitro. This factor appears to be distinct from the dioxin receptor, which is observed only in nuclear extract from treated cells. In conclusion, these data suggest that a certain protein-DNA architecture may be maintained at the response elements at different stages of gene expression.

Role of the ligand in intracellular receptor function: receptor affinity determines activation in vitro of the latent dioxin receptor to a DNA-binding form.

To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits signal- (dioxin-)induced activation of the latent cytosolic dioxin receptor to an active DNA-binding species. The DNA-binding properties of the in vitro-activated form were qualitatively indistinguishable from those of in vivo-activated nuclear receptor extracted from dioxin-treated cells. In vitro activation of the receptor by dioxin was dose dependent and was mimicked by other dioxin receptor ligands in a manner that followed the rank order of their relative affinities for the receptor in vitro and their relative potencies to induce target gene transcription in vivo. Thus, in addition to triggering the initial release of inhibition of DNA binding and presumably allowing nuclear translocation, the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex.

Activation of the dioxin and glucocorticoid receptors to a DNA binding state under cell-free conditions.

The activation in vitro of dioxin and glucocorticoid receptors from a non-DNA binding to a DNA binding state was characterized. Ligand-free dioxin and glucocorticoid receptors were partially co-purified from rat liver cytosol, and both receptors sedimented at 9 S following labeling with the respective ligand. The 9 S forms of the dioxin and glucocorticoid receptors have previously been shown to represent heteromeric complexes containing the Mr approximately equal to 90,000 heat shock protein. The 9 S ligand-free or ligand-bound glucocorticoid receptor was converted to the monomeric 4-5 S form upon exposure to 0.4 M NaCl even in the presence of the stabilizing agent molybdate. Under identical conditions, the 9 S ligand-free and ligand-bound dioxin receptor forms remained essentially intact. However, in the absence of molybdate, the dioxin receptor could be converted to a 4-5 S form upon exposure to high concentrations of salt. These results indicate that the glucocorticoid receptor readily dissociates from the 9 S to the 4-5 S form even in the absence of hormone, whereas both the ligand-free and ligand-occupied 9 S dioxin receptor forms represent more stable species. Gel mobility shift experiments revealed that the 4-5 S glucocorticoid receptor interacted with a glucocorticoid response element both in the absence and presence of ligand. On the other hand, occupation of the dioxin receptor by ligand greatly enhanced the ability of the receptor to be activated to a form that binds to its target enhancer element. Once dissociated, the monomeric form of the dioxin receptor was also able to interact with its DNA target sequences even in the absence of ligand. Thus, ligand binding efficiently facilitates subunit dissociation of the dioxin receptor but is not a prerequisite for DNA binding per se. Given the apparent stability of its non-DNA binding 9 S form, the dioxin receptor system might be a useful model for the investigation of the mechanism of activation of soluble receptor proteins.

The specific DNA binding activity of the dioxin receptor is modulated by the 90 kd heat shock protein.

The dioxin receptor is a gene regulatory protein which exhibits many structural and functional similarities to steroid hormone receptors. In this study we compare the subunit composition of two forms of the dioxin receptor, sedimenting at approximately 9S and approximately 6S respectively, which are present in nuclear extract from wild-type Hepa 1c1c7 mouse hepatoma cells following treatment in vivo with dioxin. The nuclear approximately 9S receptor form contained the 90 kd heat shock protein, hsp90. As assessed by a gel mobility shift assay, this receptor form did not bind to the xenobiotic response element (XRE) of the target gene cytochrome P-450 IA1. In contrast, the smaller approximately 6S receptor form did not contain any immunochemically detectable hsp90. Moreover, this receptor form specifically bound to the XRE recognition sequence. Thus, the specific DNA binding activity of the dioxin receptor was inhibited by association with hsp90, and the approximately 9S dioxin receptor species could be regarded as a nonactive receptor form. Neither the approximately 9S nor the approximately 6S receptor forms were detected in nuclear extract from a dioxin treated mutant clone of Hepa 1 that expresses a nuclear translocation deficient receptor phenotype. We conclude that activation of the dioxin receptor is, at least, a two step process involving binding of the ligand and dissociation of hsp90 from the ligand-binding receptor protein. Inhibition of the DNA binding activity of transcription factors by protein--protein interaction has also been described for several steroid hormone receptors and for the NF kappa B factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural differences between the glucocorticoid, dioxin and oxysterol receptors from rat liver cytosol.

The rat hepatic glucocorticoid, dioxin and oxysterol receptors were subjected to high performance liquid chromatography on size-exclusion and anion-exchange columns. Both the glucocorticoid receptor and the dioxin receptor had a Stokes radius Rs approximately 7.5 nm, expected value for heteromeric complexes containing a dimer of the Mr approximately 90,000 heat shock protein, hsp90 (Rs approximately 7.0 nm). The oxysterol receptor represented a much smaller entity (Rs approximately 6.0 nm). When analyzed on a Mono Q anion-exchange column, the molybdate-stabilized glucocorticoid receptor and dioxin receptor eluted as single peaks at approximately 0.30 M and 0.26-0.28 M NaCl, respectively, whereas the oxysterol receptor represented a less negatively charged species (0.11-0.14 M NaCl). Following washing of the Mono Q column with molybdate-free buffer, the activated monomeric glucocorticoid receptor was detected (0.10-0.12 M NaCl). In contrast, no modification in the elution pattern of the dioxin receptor and the oxysterol receptor was observed. These data demonstrate differences in the physico-chemical properties of the glucocorticoid, dioxin and oxysterol receptors, respectively, which might reflect structural differences.

Ligand-dependent interaction of the dioxin receptor with target DNA.

Wild type and nuclear transfer deficient mouse hepatoma cell lines were used to study the specific DNA binding of a dioxin inducible factor. This factor interacts with XRE only after dioxin treatment and is absent in receptor mutant containing cells even after treatment. Thus, evidence is provided to substantiate the claim that the dioxin receptor is involved in the specific DNA interaction with dioxin response enhancer elements. It is also shown that the molybdate stabilised dioxin-receptor interacts with hsp90 suggesting that, in similarity to the glucocorticoid receptor, the dioxin receptor is kept in a non-transformed state in the absence of ligand.

The dioxin receptor: a comparison with the glucocorticoid receptor.

The physico-chemical properties of the dioxin and glucocorticoid receptors from rat liver and wild-type and mutant cell lines were investigated and compared. In rat liver, the receptors are virtually indistinguishable. Both are highly asymmetrical proteins with axial ratios of 12-15, have Stokes radii of 6 nm and sedimentation coefficients of approximately 4 S. This results in a calculated apparent mol. wt of approximately 100,000. The dioxin receptor from the mouse hepatoma cell line Hepa 1c1c7 represents an atypical form of the dioxin receptor with a pronounced tendency to aggregate to form Mr approximately equal to 300,000 complexes in high ionic strength and in the absence of sodium molybdate. In the presence of sulphydryl reducing agents, however, the Hepa 1c1c7 dioxin receptor dissociates to an Mr approximately 100,000 species. In analogy to the nt- mutant glucocorticoid receptor in mouse lymphoma cells, there is no gross change in the structure of the nt- dioxin mutant in mouse hepatoma cells compared with the wild-type receptor. The nt- dioxin receptor does, however, have a reduced affinity for DNA.

The dioxin receptor: characterization of its DNA-binding properties.

The binding of the rat hepatic dioxin and glucocorticoid receptors to the polyanionic matrices heparin-Sepharose and DNA-cellulose in vitro and to cell nuclei in vivo was studied under various conditions. In a non-liganded and non-activated state both receptors eluted from heparin-Sepharose at a low ionic strength and were not retained on DNA-cellulose. Following ligandation and activation in vitro both receptors showed an increased affinity for heparin-Sepharose and were retained on DNA-cellulose. In analogy to these in vitro data, it was found that a high salt concentration (0.4 M KCl) was required to extract in vivo liganded dioxin receptor from purified nuclear preparations in contrast to that previously reported for non-liganded nuclear receptors. Limited proteolysis of both dioxin and glucocorticoid receptors resulted in molecular species of similar binding properties with regard to DNA-cellulose and heparin-Sepharose. We conclude that, in addition to the dioxin and glucocorticoid receptors showing considerable similarities in their physicochemical properties, they may also share a similar structural organization with regard to functional domains.

Polyanionic-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. A comparison with the glucocorticoid receptor.

The interaction of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) with immobilized heparin (heparin-Sepharose) or DNA (DNA-cellulose) has been compared to the polyanionic-binding properties of the rat hepatic glucocorticoid receptor. Both the nonoccupied and in vitro occupied forms of the receptors interacted with heparin-Sepharose but with varying strength, as determined by ligand binding assays or an enzyme-linked immunosorbent assay based on a monoclonal antibody against the steroid- and DNA-binding Mr approximately 94,000 glucocorticoid receptor protein. In the absence of ligand, both the dioxin and glucocorticoid receptors eluted from heparin-Sepharose at 0.1-0.2 M KCl, in contrast to the in vitro occupied receptor forms which eluted at 0.3-0.4 M KCl. Following elution of the in vitro occupied dioxin receptor from heparin-Sepharose, it was efficiently retained on DNA-cellulose and eluted at an ionic strength of approximately 0.2 M KCl. In the presence of 20 mM sodium molybdate which is known to inhibit the activation of steroid hormone receptors to a DNA-binding form, both the dioxin and glucocorticoid receptors eluted at 0.1-0.2 M KCl from heparin-Sepharose. In analogy to what has previously been shown for the glucocorticoid receptor, sodium molybdate stabilized a large dioxin-receptor complex with a sedimentation coefficient, S20,w, of 9-10 S, a Stokes radius of approximately 7.5 nm, and a calculated Mr of 290,000-310,000. Limited proteolysis of both the dioxin and glucocorticoid receptors with trypsin which is known to eliminate the DNA-binding property of both receptor forms also resulted in a decreased strength in the interaction of both in vitro occupied receptors with heparin-Sepharose (elution at 0.1-0.2 M KCl). In line with these data, calf thymus DNA in solution competed for receptor binding to heparin-Sepharose. In conclusion, the chromatographic properties of the dioxin receptor on heparin-Sepharose are indistinguishable from those of the glucocorticoid receptor, and both receptors appear to be structurally and functionally closely related proteins.