Two-step synergism between the progesterone receptor and the DNA-binding domain of nuclear factor 1 on MMTV minichromosomes.

In contrast to its behavior as naked DNA, the MMTV promoter assembled in minichromosomes can be activated synergistically by the progesterone receptor and NF1 in a process involving ATP-dependent chromatin remodeling. The DNA-binding domain of NF1 is required and sufficient for stable occupancy of all receptor-binding sites and for functional synergism. Activation of purified minichromosomes is observed in the absence of SWI/SNF and can be enhanced by recombinant ISWI. Receptor binding to minichromosomes recruits ISWI and NURF38, but not brahma. We propose a two-step synergism in which the receptor triggers a chromatin remodeling event that facilitates access of NF1, which in turn stabilizes an open nucleosomal conformation required for efficient binding of further receptor molecules and full transactivation.

The DNA and steroid binding domains of the glucocorticoid receptor are not altered in mononuclear cells of treated CLL patients.

The aim of this study was to investigate whether mutations in the glucocorticoid receptor could account for the increasing unresponsiveness of patients with chronic lymphatic leukemia (CLL) to combination chemotherapy. The receptor was tested immunocytochemically, in steroid binding assays, and by a mutation screening (denaturing gradient gel electrophoresis) of the receptor-cDNA. The receptor concentration, as measured by staining and steroid binding test, varied considerably but showed no clear correlation to clinical response. Using a highly sensitive mutation screening assay of the DNA- and the steroid-binding region, none of the treated patients revealed any mutation, suggesting that the glucocorticoid receptor in the CLL patients tested is not altered in these domains. In one individual who had not been treated before analysis a silent mutation was found in one receptor allele. The results suggest that mechanisms other than altered ligand or DNA binding of the receptor may be responsible for the lack of response to chemotherapy. This conclusion is discussed in relation to the mechanism of corticoid resistance in mouse and human lymphoma cells in culture.

Developmental changes in the expression and compartmentalization of the glucocorticoid receptor in embryonic retina.

Inducibility by glucocorticoids of the glutamine synthetase gene in chicken embryo retina and the transcriptional activity of the glucocorticoid receptor (GR) greatly increase between embryonic days 6 and 10 (E6, E10), although the level of GR does not markedly change during that time. This apparent discrepancy was investigated by examining the pattern of GR expression in undifferentiated E6 retina and in E10 retina, which consists mostly of differentiated cells. Two GR isoforms, 90 and 95 kDa, were found to be expressed at both of these ages at a similar total level but in different proportions: in E6 retina the level of the 90-kDa isoform was higher, whereas in E10 retina the 95-kDa receptor was higher. However, following treatment of the retinas with cortisol, the 95-kDa isoform became the predominant receptor at both ages. Immunohistochemical analysis revealed that the cellular localization of GR markedly changed in the course of development: in the undifferentiated E6 retina GR was expressed in virtually all cells, whereas in the more differentiated E10 and E12 retina, GR was detected only in Müller glia cells. The latter represent approximately 20% of the cells in this tissue and are the only cells in which glucocorticoid hormone induces the glutamine synthetase gene. We suggest that the compartmentalization of GR in Müller glia is a major aspect of the mechanism that modulates receptor activity during retina development and results in the temporal increase in the inducibility of glutamine synthetase and its specific localization in Müller glia cells.

Expression and functional analysis of steroid receptor fragments secreted from Staphylococcus aureus.

Fragments of the DNA-binding domain of the rat glucocorticoid receptor (rGR) and the human estrogen receptor (hER) were expressed in Staphylococcus aureus as a chimeric fusion to protein A by using a modified expression vector with an artificial factor X-cleavage site. The secreted product was isolated by hydrophobic chromatography on Phenyl-Sepharose and purified on DNA-cellulose or by anion-exchange chromatography. After cleavage of the protein A moiety, the purified rGR DNA-binding domain from amino acids 406 to 523 (rGR406-523), binds specifically to a glucocorticoid responsive element as a homodimer but cannot form heterodimers with the DNA-binding domain of the hER. Amino acids 510 to 523 following the zinc finger region, as well as free sulfhydryl-groups are necessary for DNA-binding, which is more efficient when the tripeptide Gly-Gly-Cys is added to the carboxy terminal end. Despite its specific interaction with DNA, rGR406-523 does not activate transcription from the MMTV promoter in a cell-free system that efficiently responds to addition of native GR, suggesting that regions essential for transcriptional activation in vitro are located outside of the DNA-binding domain.

Monoclonal antipeptide antibodies to the glucocorticoid receptor.

The generation of monoclonal antibodies to synthetic peptides of the glucocorticoid receptor is described. Two antibodies to sequences from the DNA binding region are IgMs. Two other antibodies to sequences in the steroid binding region and the C-terminus belong to the IgG class. The specificity of the IgG binding to the receptor in an ELISA assay is demonstrated by competition with the relevant peptides. Both IgGs are able to recognize the receptor in Western blots, but do not form stable complexes in sucrose gradients. Steroid binding to the receptor is not influenced by preincubation with antibodies. This indicates that denaturation or distortion of the receptor is necessary for the accessibility of these antibodies to their epitopes. Both antibodies can be used to stain the glucocorticoid receptor in neoplastic cells of patients suffering from chronic lymphatic leukemia.

Immunolocalization of gluco- and mineralocorticoid receptors in rabbit kidney.

The localization of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) was determined in the rabbit kidney by immunohistochemistry with the use of a monoclonal, anti-GR antibody and a monoclonal, anti-idiotypic, anti-MR antibody. Immunostaining was performed on serial histological sections from normal and adrenalectomized rabbits. The specificity of immunostaining was assessed for MR by in situ competition studies with steroids and for GR by presaturation of the antibody with GR preparation. Immunostaining by both the anti-MR and the anti-GR antibodies was present in all parts of the distal nephron (beyond proximal tubule) and absent in the glomerulus and proximal tubule. The absence of staining by the anti-GR antibody in the proximal tubule suggests that the effects of glucocorticoids in this structure involve either a GR different from that of distal structures or a non-receptor mediated mechanism of action. MR immunostaining predominates in the distal and all along the collecting tubule in its cortical, medullary, and papillary portions. GR immunostaining was most abundant in the medullary ascending limb and distal tubule. Immunostaining by both antibodies was present in papillary interstitial cells and cells of the epithelium lining the papilla. Fifteen to twenty percent of the cells of the cortical collecting tubule, presumably intercalated cells, were devoid of MR and GR immunostaining. Immunostaining was present in both nuclear and cytoplasmic cell compartments. No clear difference was observed between normal and adrenalectomized rabbits. This study is the first report on renal immunolocalization of GR compared with MR. In addition, we show evidence for new targets for corticosteroid hormones such as papillary interstitial cells and papillary epithelium.

Distribution of glucocorticoid receptor-like immunoreactivity in the brain, and its relation to CRF and ACTH immunoreactivity in the hypothalamus of the japanese quail, Coturnix coturnix japonica.

Monoclonal antibody (MAb49) against the rat liver glucocorticoid receptor was used to evaluate glucocorticoid receptor (GR) immunoreactive structures in the brain of the japanese quail, Coturnix coturnix japonica. Using the avidin-biotin technique, the immunoreaction was present in the nerve cell nuclei in intact male birds. High density of glucocorticoid receptor-like immunoreactivity was found in the tubero-infundibular area, nucleus paraventricularis, posteromedialis and lateralis hypothalami, lateral septum and in some brainstem nuclei. Cerebellar cortex was also immunopositive. In contrast to mammals, no immunoreactive cell nuclei were found in the hippocampal region. The glucocorticoid receptors were colocalized with adrenocorticotropin (ACTH) immunoreactivity in the tubero-infundibular region, while corticotropin releasing factor (CRF) positive cells in the paraventricular nucleus did not contain glucocorticoid receptor-like immunoreactivity in their cell nuclei.

Protein-DNA interactions at steroid hormone regulated genes.

This work summarizes binding data that were obtained with partially purified glucocorticoid and progesterone receptors, as well as with a crude nuclear protein extract, to DNA sequences in and around hormonally regulated genes. The sequence recognition by the glucocorticoid receptor at the different defined glucocorticoid regulatory elements (GRE) is discussed and a consensus sequence formulated. A three dimensional representation gives an impression of the mode of interaction between the protein and the double helix of DNA. In the promoters of mouse mammary tomour virus (MTV) and chicken lysozyme overlapping binding sites for both, glucocorticoid and progestine-receptors are found that are responsible for the hormonal inducibility of the genes. In crude extract from rat liver nuclei, a nonhistone protein is found that specifically binds to sequences on the MTV-LTR region overlapping the GRE. The possible implication of this protein in hormonal regulation of transcription is discussed.

Glucocorticoid receptor in magnocellular neurosecretory cells.

Quantitative in vitro autoradiography, cytosol receptor assay in punched brain tissue, and immunocytochemistry have revealed that the glucocorticoid receptor is present in the rat supraoptic nucleus (SON). Based on its binding characteristics the receptor appears to be the type II glucocorticoid receptor. With the use of a monoclonal antibody against purified liver glucocorticoid receptor, immunostaining was found in magnocellular neurosecretory neurons in the SON, but not in magnocellular neurons in the paraventricular nucleus. Immunoreactive cells seem to be concentrated in ventral parts of the SON where vasopressin cells were previously shown to be located. One to 2 weeks after bilateral adrenalectomy, there was a substantial decrease in glucocorticoid receptor immunostaining in magnocellular as well as other types of neurons in various brain regions. Administration of synthetic glucocorticoids (RU 28362 or dexamethasone) induced a robust increase in the intensity of immunostaining in cell nuclei of neurosecretory cells. The presence of glucocorticoid receptors in the SON suggests that glucocorticoids may affect vasopressin synthesis or/and secretion through a direct action on magnocellular neurons.

Immunocytochemical study on the intracellular localization of the type 2 glucocorticoid receptor in the rat brain.

The localization of the glucocorticoid receptor (GR) (type 2) in the rat brain was studied with immunocytochemistry using a monoclonal antibody against the rat liver GR. Strong GR immunoreactivity (GR-ir) was observed in neurons of limbic and brainstem structures known to be associated with the stress-activated circuitry, which suggest that these sites are responsive to glucocorticoid feedback. The intracellular localization of GR-ir was examined in CA1 and CA2 pyramidal neurons of the hippocampus. In intact rats GR-ir is predominantly present in the cell nucleus. Adrenalectomy (ADX) caused a slow depletion of the GR-ir signal from the cell nucleus until near detection limits at two weeks postsurgery. At that time, 1 h after administration to longterm ADX rats the synthetic glucocorticoid (type 2) agonist RU 28362 as well as a moderate and high dose of corticosterone (CORT) markedly enhanced the cell nuclear GR-ir. The type 2 antagonist RU 38486 also caused an increase of GR immunostaining in cell nuclei upon acute administration to ADX rats. The mineralocorticoid aldosterone (ALDO) and a low dose of CORT, which bind almost exclusively to type 1 corticosteroid receptors, were ineffective. In conclusion, our data suggest that in the hippocampal CA1-2 neurons type 1 and type 2 corticosteroid receptors may coexist. The steroid-induced changes in cell nuclear immunoreactive GR staining intensity suggest possible cytoplasmic-cell nuclear translocation of GR and/or exposure of immunogenic GR domains.

Molecular model of the interaction between the glucocorticoid receptor and the regulatory elements of inducible genes.

Binding sites for the glucocorticoid receptor in an ecdysone-inducible gene from Drosophila melanogaster and in the chicken vitellogenin gene are described. A comparison with other binding sites for the glucocorticoid receptor, which have been analyzed by methylation protection experiments, shows they can be classified into three groups. The first group exhibits two blocks of contact points in two subsequent turns of the DNA helix, and includes only functional regulatory elements. The second group shows an identical contact with the hexanucleotide 5'-TGTYCT-3', but only half the contact points in the other turn of the helix, whereas the third group of sites exhibits only the contact points within the conserved hexanucleotide. An analysis of the hydrogen-bonding potential of the DNA base pairs along the major groove of 10 binding sites shows a very well-conserved pattern and a twofold rotational symmetry, suggesting that the array of hydrogen bonds may be a relevant aspect of sequence recognition by hormone receptors. A representation of the binding sites and contact points by computer graphics suggests the interaction of a receptor dimer, in a head-to-head arrangement, with two subsequent turns of the B-DNA helix within the glucocorticoid regulatory elements.

Mechanism of gene regulation by steroid hormones.

A first understanding of the molecular events on the DNA level, underlying transcriptional regulation by steroid hormones, has been approached in the last 3 years by means of protein/DNA interaction studies, using purified receptors. This work summarizes our knowledge of how purified glucocorticoid and progestine receptors interact with their cognate regulatory elements associated with polymerase II dependent genes like mouse mammary tumour virus, the genes encoding human metallothionein IIA, chicken lysozyme, human growth hormone and rabbit uteroglobin. The resulting data agree with those of functional test systems, that have been gene-transfer experiments using stable transformants or transient expression. A consensus sequence for the regulatory element of the glucocorticoid receptor could be deduced that, in its three-dimensional representation, gives an impression of the steric mode of interaction. The regulatory elements of the progestine receptor overlap in two analysed cases with those of the glucocorticoid receptor, but are not identical. Furthermore, also a polymerase I transcribed gene encoding ribosomal RNA in the mouse could be shown to contain a glucocorticoid regulatory element that is functional in in vitro transcription experiments. Finally, the latest strategies are the cloning of the glucocorticoid receptor gene and the analysis of receptor-mediated topological effects.

The molybdate-stabilized L-cell glucocorticoid receptor isolated by affinity chromatography or with a monoclonal antibody is associated with a 90-92-kDa nonsteroid-binding phosphoprotein.

We have previously reported that molybdate-stabilized cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90-92- and a 98-100-kDa protein) that elute from an affinity resin of deoxycorticosterone-derivatized agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor (Housley, P. R., and Pratt, W. B. (1983) J. Biol. Chem. 258, 4630-4635). In the present work we report that both the 90-92- and 98-100-kDa 32P-labeled proteins are also extracted from molybdate-stabilized cytosol by incubation with a monoclonal antibody and protein A-Sepharose. Only the 98-100-kDa protein is specifically labeled when either L-cell cytosol or L-cell cytosol proteins bound to the affinity resin are labeled with the glucocorticoid binding site-specific affinity ligand [3H]dexamethasone 21-mesylate. The 98-100-kDa protein labeled with [3H]dexamethasone mesylate is adsorbed to protein A-Sepharose in an immune-specific manner after reaction with the monoclonal antibody. Sodium dodecyl sulfate-polyacrylamide gel analysis of the protein A-Sepharose-bound material resulting from incubating the monoclonal antibody with a mixture of 32P-labeled cytosol and [3H]dexamethasone mesylate-labeled cytosol demonstrates identity of the 98-100-kDa [3H]dexamethasone mesylate-labeled band with the 98-100-kDa 32P-labeled band and clear separation from the nonsteroid-binding 90-92-kDa phosphoprotein. The results of immunoblot experiments demonstrate that the 90-92-kDa protein is structurally distinct from the 98-100-kDa steroid-binding protein. As the 90-92-kDa nonsteroid-binding phosphoprotein co-purified with the 98-100-kDa uncleaved form of the glucocorticoid receptor by two independent methods, one of which is based on recognizing a steroid-binding site and the other of which is based on recognizing an antibody binding site, we propose that the 90-92-kDa phosphoprotein is a component of the molybdate-stabilized, untransformed glucocorticoid-receptor complex in L-cell cytosol.

The nucleotide sequences recognized by the glucocorticoid receptor in the rabbit uteroglobin gene region are located far upstream from the initiation of transcription.

Although the expression of the uteroglobin gene in the lung is regulated by glucocorticoids, no binding sites for the glucocorticoid receptor are found in the promoter region nor can they be observed in the coding sequences. Instead a fragment situated 2.6 kb upstream from the start of transcription of the uteroglobin gene shows a high affinity for the receptor. Deoxyribonuclease I and methylation protection studies show three contiguous binding sites located within this fragment. All three sites show homology to the glucocorticoid receptor binding sites described for other genes. Two of them encompass the hexanucleotide 5'-TGTTCT-3', and the other binding site contains the homologue hexanucleotide, 5'-AGTCCT-3', but the contacts between the receptor and the hexanucleotides are equivalent to those found in other functional regulatory elements for glucocorticoids. These elements may therefore be responsible for the glucocorticoid regulation of uteroglobin gene expression by acting over a relatively long stretch of nucleotide sequences.

Immunochemical characterization of wild-type and variant glucocorticoid receptors by monoclonal antibodies.

Monoclonal antibodies raised against the rat liver glucocorticoid receptor were used to investigate receptors of wild-type and glucocorticoid-resistant variants of mouse lymphoma cells. Two of the variant types contained receptors of 'nuclear transfer deficient' (nt-) and 'increased nuclear transfer' (nti) phenotypes, respectively, while the third was of the 'receptorless' (r-) phenotype with negligible hormone binding activity. Three monoclonal antibodies of the IgM class and one of the IgG class reacted with both wild-type and nt- receptors but not with the steroid binding form of nti receptors. Some of the antibodies bound the wild-type and nt- receptors more efficiently after activation at 20 degrees C. By use of an immuno-competition assay we were able to detect cross-reacting material in considerable amounts in extracts of nti and r- cell variants. This material was further characterized by gel filtration and immunoblotting. The immunoreactive material of wild-type, nti and r- cells gave a major band of mol. wt. 94 000 upon SDS-gel electrophoresis while the steroid-binding polypeptides of wild-type and nti receptors have mol. wts. of 94 000 and 40 000, respectively. The data show that in S49.1 mouse lymphoma cells the products of two receptor alleles can be distinguished.

Characterization of DNA sequences through which cadmium and glucocorticoid hormones induce human metallothionein-IIA gene.

Deletion experiments have defined two stretches of DNA (genetic elements), lying close to the promoter for a human gene for metallothionein, that separately mediate the induction of the gene by heavy metal ions, particularly cadmium, and by glucocorticoid hormones. The element responsible for induction by cadmium is duplicated, yet a single copy is fully functional; the element responsible for induction by glucocorticoid hormones is coincident with the DNA-binding site for the glucocorticoid hormone receptor.

The glucocorticoid receptor binds to defined nucleotide sequences near the promoter of mouse mammary tumour virus.

Glucocorticoids are known to induce the transcription of integrated proviral mouse mammary tumour virus (MMTV) genes in a variety of cell lines derived from mouse mammary tumours. Chimaeric genes in which selectable markers are linked to the long terminal repeat (LTR) region of MMTV can be induced by the synthetic glucocorticoid dexamethasone after introduction into mouse fibroblasts. This suggests that the regulatory elements required for hormonal induction are located within the cloned LTR fragments. The idea is supported by the observation that glucocorticoid receptors bind to certain cloned fragments of MMTV DNA in vitro. Using filter binding studies and monoclonal antibodies to the glucocorticoid receptor we have now delimited the receptor binding region to a DNA segment of 152 base pairs (bp) that has been shown to be relevant for hormonal induction. In nuclease protection experiments we have identified partially homologous receptor binding sequences located in this region, all of which share the hexanucleotide 5'-TGTTCT-3'.

Monoclonal antibodies to the rat liver glucocorticoid receptor.

Monoclonal antibodies against the 90 000 mol. wt. form of the activated rat liver glucocorticoid receptor were generated from mice immunized with a partially purified receptor preparation. The screening assay was based on the precipitation of liver cytosol, labelled with [3H]triamcinolone acetonide, with monoclonal antibodies bound to immobilized rabbit anti-mouse IgG. Out of 102 hybridomas obtained, 76 produced immunoglobulin and eight of them were found to react with the receptor molecule. Only one of the positive clones secreted IgG whereas the other seven produced IgM. The complexes of receptor and antibodies were identified by sucrose density gradient centrifugation. All seven monoclonal antibodies tested reacted with the 90 000 mol. wt. form of the receptor but not with the 40 000 mol. wt. form that contains the steroid and DNA binding domains. None of the monoclonal antibodies interfered with the binding of the receptor to DNA cellulose, thus suggesting that the antigenic determinants are located in a region of the receptor that is not directly implicated in either steroid binding or DNA binding. These antigenic determinants were common to glucocorticoid receptors from several tissues of the rat, whereas glucocorticoid receptors from other species react only with some of the antibodies.

Glucocorticoid receptors recognize DNA sequences in and around murine mammary tumour virus DNA.

In several rodent cell lines, glucocorticoids increase the transcription of murine mammary tumour virus (MMTV) proviral DNA in a process mediated by the glucocorticoid receptor. To investigate whether a direct interaction between the receptor and specific sequences on the induced genes can be implicated in the hormonal regulation of transcription, filter binding studies were performed with partially purified glucocorticoid receptor of rat liver and eight cloned MMTV proviral probes. Both the 40 000 and the 90 00 mol. wt. forms of the receptor do bind preferentially to restriction fragments containing the right 400-500 nucleotides of the MMTV long terminal repeat units (LTR). Using LTR deletion mutants, we confirm that the right end of the LTR contains at least one binding site for the glucocorticoid receptor. In addition, the receptor binds preferentially to the mouse genomic sequences flanking at least three endogenous proviral copies, and to sequences within the env genes in some of the endogenous and exogenous proviruses. These findings are compatible with the hypothesis that steroid hormones regulate specific gene expression through a direct interaction of the hormone-receptor complex with DNA sequences in and around the induced genes.

Photoaffinity labeling of steroid binding proteins with unmodified ligands.

Photoactivation of the alpha,beta-unsaturated ketones of natural and synthetic steroid molecules by light of lambda greater than or equal to 330 nm allows their covalent attachment to steroid-binding proteins. The general validity of this method is demonstrated with two steroid hormone receptors and the steroid-binding protein uteroglobin. Progesterone can be covalently attached to the partially purified progesterone receptor and to uteroglobin, and comigrates with the binding proteins upon electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Similarly the synthetic glucocorticoid triamcinolone acetonide can be covalently bound to the partially purified glucocorticoid of rat liver. This method allows the identification of steroid hormone receptors after electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Labeling with radioactive steroids is specific since it can be prevented by the addition of an excess of non-radioactive ligand. Digestion of the labeled binding proteins with trypsin or chymotrypsin yields a defined pattern of radioactive peptides, demonstrating that covalent attachment takes place at specific binding sites.