Regulation of mouse CYP1A1 gene expression by dioxin: requirement of two cis-acting elements during induction.

The mouse cytochrome P1450 (CYP1A1) gene is responsible for the metabolism of numerous carcinogens and toxic chemicals. Induction by the environmental contaminant tetrachlorodibenzo-p-dioxin (TCDD) requires a functional aromatic hydrocarbon (Ah) receptor. We examined the 5'-flanking region of the CYP1A1 gene in mouse hepatoma Hepa-1 wild-type cells and a mutant line having a defect in chromatin binding of the TCDD-receptor complex. We identified two cis-acting elements (distal, -1071 to -901 region; proximal, -245 to -50 region) required for constitutive and TCDD-inducible CYP1A1 gene expression. Three classes of DNA-protein complexes binding to the distal element were identified: class I, found only in the presence of TCDD and a functional Ah receptor, that was heat labile and not competed against by simian virus 40 (SV40) early promoter DNA; class II, consisting of at least three constitutive complexes that were heat stable and bound to SV40 DNA; and class III, composed of at least three constitutive complexes that were thermolabile and were not competed against by SV40 DNA. Essential contacts for these proteins were centered at -993 to -990 for the class I complex, -987, -986, or both for the class II complexes, and -938 to -927 for the class III complexes. The proximal element was absolutely essential for both constitutive and TCDD-inducible CYP1A1 gene expression, and at least two constitutive complexes bound to this region. These data are consistent with the proximal element that binds proteins being necessary but not sufficient for inducible gene expression; interaction of these proteins with those at the distal element was found to be required for full CYP1A1 induction by TCDD.     

Effects of SH-modifying reagents on the rat hepatic Ah receptor: inhibition of ligand binding and transformation, and disruption of the ligand-receptor complex

The importance of sulfhydryl (SH) groups in maintenance of physicochemical properties of the rat hepatic Ah receptor was demonstrated using a variety of sulfhydryl (SH)-modifying reagents. Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding was approximately equivalent by 5,5'-dithiobis(2-nitrobenzoate), mersalyl, N-ethylmaleimide, and p-chloromercuriphenylsulfonate, whose inhibition curves were steep in the concentration range close to that of nonprotein SH groups in cytosol (ED50 values 50-200 microM or 13-48 nmol/mg cytosolic protein). Inhibition by p-hydroxymercuribenzoate (PHMB), although exhibiting a lower ED50, was more gradual over this range; iodoacetamide was an order of magnitude less potent. The ability of dithiothreitol to reverse binding inhibition induced by 150 microM (approximately 60 nmol/mg protein) mersalyl diminished with time; it decreased more rapidly in the simultaneous presence of TCDD and mersalyl than when mersalyl was present alone, consistent with increased accessibility of key SH group(s) due to conformational changes attending TCDD-receptor complex formation. Brief exposure of unoccupied receptor to mersalyl prior to TCDD binding caused slower sedimentation of the complex in 0-KCl sucrose gradients and alterations in its elution profiles on DEAE- and DNA-Sepharose suggestive of some impairment of the transformation process. When reagents were added to the transformed TCDD-receptor complex, loss of binding was observed only at concentrations which were an order of magnitude higher than those inhibiting TCDD binding. Loss of binding by each reagent was biphasic, and except for that caused by mersalyl, was not complete even after 6-8 h. Dithiothreitol was able to reverse the effects of mersalyl or PHMB only partially and only if added during the early phase (10-30 min) of binding loss. Mersalyl was much more potent in disrupting the untransformed than the transformed TCDD receptor complex. Physical alteration of the mersalyl-treated TCDD-receptor complex was evident from gel filtration, sucrose gradients, and DNA- and DEAE-Sepharose chromatography. Our results are in striking contrast to the effects of these reagents on steroid receptors, whose bound steroid hormone ligand is rapidly and reversibly displaced by lower concentrations of reagent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the DNA-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin

The DNA-binding properties of the receptor for 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated using chromatography on DNA-cellulose columns. A maximal binding of about 40% of the total receptor complex to DNA-cellulose was observed. In order to interact with DNA, the receptor must first bind TCDD. A heat-activation step followed by gel permeation chromatography using Sephadex G-25 increased the binding of the cytosolic receptor to DNA. The DNA-binding ability of the receptor was almost lost following mild proteolysis using trypsin or alpha-chymotrypsin, although these treatments did not reduce its ligand binding capacity and had no apparent effect on its size. Furthermore, pre-treatment of the DNA-cellulose column with an intercalating drug, ethidium bromide, resulted in inhibition of the binding of the TCDD-receptor complex to DNA, indicating that not only electrostatic interactions but also the configuration of DNA are of importance in receptor-DNA interactions.     

Interaction of the hepatic receptor protein for 2,3,7,8-tetrachlorodibenzo-p-dioxin with DNA

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds to a specific, high-affinity, low-capacity protein in rat liver cytosol. The TCDD-receptor complex is a large molecule with a Stokes radius of 6.6 nm as determined by gel filtration on calibrated columns. The receptor complex sediments at 5.0 S on glycerol gradients. The calculated molecular weight from the physical parameters was 136 000 and the frictional ratio 1.79.The TCDD-receptor complex binds to DNA-cellulose without preceding heat activation or incubation at high ionic strength. The receptor must first bind TCDD before it can interact with DNA. The DNA-binding ability can be removed from the TCDD receptor by limited proteolysis with trypsin. This treatment does not affect the TCDD-binding site of the receptor. The proteolytic fragment of the TCDD-receptor complex containing the TCDD-binding site but not the ability to bind to DNA appears to be approximately the same size as the native receptor, as judged from chromatography of Sepharose CL-6B and glycerol gradient centrifugation.     

Author index

Binding of dexamethasone · receptors with isolated nuclei, DNA-cellulose and cellulose has been compared with respect to dependence on salt concentration and resistance to KCl extraction and DNAase I digestion. A solution of cytoplasmic dexamethasone-receptor complexes was prepared by the incubation of rat thymus cells with steroid at 3°C and breaking the cells by hypotonic lysis. Activation of the complexes was accomplished by warming the solution at 25°C for 15 min. Activation significantly increased the ability of dexamethasone · receptors to bind to nuclei and DNA-cellulose but not to cellulose. Dexamethasone-receptor complexes bound to nuclei at 3°C are completely resistant to extraction with 0.1 M KCl, 76% resistant to 0.2 M KCl and 20% resistant to 0.4 M KCl. Dexamethasone · receptors bound to DNA-cellulose are 45% resistant to extraction with 0.1 M and 0.2 M KCl and 29% resistant to 0.4 M KCl extraction. Cellulose-bound dexamethasone · receptors are not resistant to any of these extractions. DNAase I treatment releases 60% of the dexamethasone · receptors bound to DNA-cellulose but only 13% of those bound to nuclei, though at least 60% of the nuclear DNA is solubilized. The presence of 0.15 M KCl decreases binding of activated dexamethasone · receptors to nuclei by 73% but to DNA-cellulose by only 17%. Pretreatment of nuclei with 0.1–0.4 M KCl reduces their capacity to bind activated dexamethasone · receptors by 90% whereas similar treatment reduces the capacity of DNA-cellulose to bind dexamethasone · receptors by only 29%. Nuclei extracted with 0.1 M KCl appear to have a limited capacity to accept dexamethasone · receptors. These studies demonstrate that binding of dexamethasone · receptors to nuclei and DNA-cellulose differs by (a) the higher resistance of nuclear complexes to KCl and DNAase I treatment; (b) the much greater sensitivity of nuclei to KCl treatment.     

Ligand-independent homomeric and heteromeric complexes between interleukin-2 or -9 receptor subunits and the gamma chain

Signaling via interleukin-2 (IL-2) and interleukin-9 receptors (IL-2R and IL-9R) involves heteromeric interactions between specific interleukin receptor subunits, which bind Janus kinase 1 (JAK1) and the JAK3 binding common gamma chain (gamma c). The potential existence and roles of homomeric and heteromeric complexes before ligand binding and their modulation by ligand and JAK3 are unclear. Using computerized antibody-mediated immunofluorescence co-patching of epitope-tagged receptors at the surface of live cells, we demonstrate that IL-2Rbeta, IL-9Ralpha, and gamma c each display a significant fraction of ligand-independent homomeric complexes (24-28% co-patching), whereas control co-patching levels with unrelated receptors are very low (7%). Heteromeric complex formation of IL2-Rbeta or IL-9Ralpha with gamma c is also observed in the absence of ligand (15-30%). Ligand binding increases this hetero-oligomerization 2-fold but does not affect homo-oligomerization. Co-expression of IL-2Ralpha does not affect the hetero-oligomerization of IL-2Rbeta and gamma c. Recruitment of gamma c into heterocomplexes is partly at the expense of its homo-oligomerization, suggesting that a functional role of the latter may be to keep the receptors inactive in the absence of ligand. At the same time, the preformed complexes between gamma c and IL-2Rbeta or IL-9Ralpha promote signaling by the JAK3 A572V mutant without ligand, supporting a pathophysiological role for the constitutive oligomerization in triggering ligand-independent activation of JAK3 (and perhaps other JAK mutants) mutants identified in several human cancers.     

Mediation of elicitin activity on tobacco is assumed by elicitin-sterol complexes

Elicitins secreted by phytopathogenic Phytophthora spp. are proteinaceous elicitors of plant defense mechanisms and were demonstrated to load, carry, and transfer sterols between membranes. The link between elicitor and sterol-loading properties was assessed with the use of site-directed mutagenesis of the 47 and 87 cryptogein tyrosine residues, postulated to be involved in sterol binding. Mutated cryptogeins were tested for their ability to load sterols, bind to plasma membrane putative receptors, and trigger biological responses. For each mutated elicitin, the chemical characterization of the corresponding complexes with stigmasterol (1:1 stoichiometry) demonstrated their full functionality. However, these proteins were strongly altered in their sterol-loading efficiency, specific binding to high-affinity sites, and activities on tobacco cells. Ligand replacement experiments strongly suggest that the formation of a sterol-elicitin complex is a requisite step before elicitins fasten to specific binding sites. This was confirmed with the use of two sterol-preloaded elicitins. Both more rapidly displaced labeled cryptogein from its specific binding sites than the unloaded proteins. Moreover, the binding kinetics of elicitins are related to their biological effects, which constitutes the first evidence that binding sites could be the biological receptors. The first event involved in elicitin-mediated cell responses is proposed to be the protein loading with a sterol molecule.     

Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2

The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 +/- 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [3H]TCDD and 3-[3H]methylcholanthrene. 3-[3H]Methylcholanthrene did not bind to any component besides that at approximately 9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Hep G2 Ah receptor was 9 nM by Woolf plot analysis, about an order of magnitude weaker than the affinity of [3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [3H]TCDD.Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [3H]TCDD at 37 degrees C in culture, sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC50 for AHH induction was 5.3 microM for benz(a)anthracene and 1.3 microM for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 mM sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drug-metabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.     

Dioxin alters the human low-density and very low-density lipoprotein structure with evidence for specific quenching of Trp-48 in apolipoprotein C-II

The intercellular transport of cholesterol and triglycerides via lipoproteins interacting with their receptors is a critical component in human lipid metabolism. The delivery of cholesterol to cells is accomplished primarily through low-density lipoproteins (LDLs), while the transport of fatty acids to adipose and muscle tissue is accomplished primarily through the actions of very low-density lipoproteins (VLDLs). Disruption of lipoprotein structure leading to impaired binding between these lipoproteins and their obligate receptors is a known risk factor for cardiovascular disease. Because of recent investigations linking 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in humans with coronary artery disease, investigations have been carried out by fluorescence and circular dichroism to evaluate conformational changes in LDL and VLDL structure upon binding of TCDD. These studies demonstrate that, at a molar ratio of three TCDD molecules to one lipoprotein molecule, TCDD binds and disrupts the secondary and tertiary lipoprotein structure. Circular dichroism studies show that residues within the inner core of apoC-II, which compose a four-alpha-helix bundle when this apolipoprotein is associated with VLDL, are directly affected upon binding TCDD. Fluorescence also indicates the specific interaction of Trp-48 within apoC-II upon TCDD binding. We found that the TCDD/apoC-II complex suffers a 5-fold reduction in its ability to bind lipoprotein lipase compared to untreated apoC-II. The interaction of TCDD with LDL markedly altered the secondary structure of apoB reducing its alpha-helical content. These cumulative responses in lipoprotein structure may impair the LDL and VLDL cellular uptake leading to a buildup of serum lipoproteins and fats thus hastening the development of coronary artery disease.     

Intestinal 1,25-dihydroxyvitamin D3 receptors: hormone-dependent uptake and saturability of nuclear components in vitro

An $\text{in vitro}$ system has been designed to study the interaction between intestinal cytoplasmic 1, 25-dihydroxyvitamin D receptors and highly purified homologous nuclei. Cytosolic receptors, labeled with 1, 25-dihydroxyvitamin D, specifically associate with intestinal nuclei as ternary chromatin complexes, an event which proceeds in the absence of a demonstrable change in sedimentation coefficient. This interaction is a saturable process with a dissociation constant of 2.2 × 10^?10 M at 0° C, and is specific for the 3.3S cytoplasmic receptor since neither the 4.1S nor the 6S vitamin D binding proteins associate in a similar fashion. In addition, the interaction is hormone-dependent, since free receptors also do not bind. The development and characterization of an $\text{in vitro}$ system such as this is the beginning in an attempt to assess the molecular action of these receptors on intestinal nuclei.