Endogenous Glucocorticoid Receptor Research Articles

Glucocorticoids Stimulate Growth of Human Papillomavirus Type 16 (HPV16)-Immortalized Human Keratinocytes and Support HPV16-Mediated Immortalization without Affecting the Levels of HPV16 E6/E7 mRNA

We investigated the effects of the glucocorticoids hydrocortisone and dexamethasone on human papillomavirus type 16 (HPV16)-mediated human cell carcinogenesis using normal human keratinocytes (HKc) and HKc immortalized by transfection with HPV16 DNA (HKc/HPV16). Normal HKc did not require glucocorticoids for proliferation. In contrast, growth of early passage HKc/HPV16 strictly required these hormones, although glucocorticoid dependence became less stringent duringin vitroprogression. Glucocorticoid dependence was acquired by HKc early after immortalization with HPV16 DNA, and glucocorticoids were required for efficient HKc immortalization. However, treatment of HKc/HPV16 with hydrocortisone or dexamethasone did not increase the steady-state levels of HPV16 E6/E7 mRNA or protein. Firefly luciferase activity expressed under the control of the HPV16 upstream regulatory region and P97 promoter increased by about fourfold following dexamethasone treatment of HeLa, but only twofold in HKc/HPV16, and less than twofold in SiHa. However, all of these cell lines expressed sufficient endogenous glucocorticoid receptors to allow for a dexamethasone response of the mouse mammary tumor virus promoter. These results indicate that mechanisms other than a direct influence by glucocorticoids on HPV16 early gene expression may contribute to the striking biological effects of these steroids on HPV16-mediated human cell carcinogenesis.

Point mutation in the ligand-binding domain of the progesterone receptor generates a transdominant negative phenotype.

A short conserved alpha-helix in the carboxyl-terminal activation function of the ligand-binding domain of steroid hormone receptors, called AF2, is important for ligand-dependent transactivation of inducible genes. We have generated two AF2 mutants of the B isoform of human progesterone receptor (PRB): a point mutant, PRBE911A, and a short deletion, PRB delta907-913R. The two mutants are expressed at levels comparable to the wild type receptor in transfected cells. The PRBE911A mutant showed similar hormone- and DNA- binding affinities as the wild type receptor, whereas the PRB delta907-913R mutant was defective in hormone and DNA binding. Both mutants were inactive when transiently transfected in CV-1 cells, which do not express endogenous PR. However, the point mutant, but not the deletion mutant, inhibited transactivation by cotransfected wild type PRB in a hormone-dependent fashion. The activity of endogenous PR in T47D cells or of endogenous glucocorticoid receptor in HeLa cells was also inhibited by the PRBE911A, but not by the deletion mutant. The point mutant was less active when introduced into an N-terminal truncated form of PR, where it gave rise to proteins that formed homodimers with poor affinity for DNA, but were able to form heterodimers with PRB. The negative dominant phenotype of the PRBE911A mutant likely originates from competition with wild type receptors for binding to DNA and will be useful for mechanistic studies of receptor function.

Inhibition of mineralocorticoid and glucocorticoid receptor function by the heat shock protein 90-binding agent geldanamycin

The effects of mineralocorticoids and glucocorticoids are mediated by the intracellular mineralocorticoid glucocorticoid receptor (MR) and glucocorticoid receptor (GR), respectively. Several studies suggest that hormone binding and, thus, receptor activation depend on the association of both MR and GR with the 90-kDa heat shock protein (hsp 90). However, there are few reports analyzing the functional relevance of this association in vivo. The present study was designed to determine how the new hsp 90-binding agent geldanamycin, which was previously shown to disrupt the formation of steroid receptor/hsp complexes, interferes with MR- and GR-mediated transactivation in intact cells. We show that geldanamycin inhibits aldosterone-dependent transactivation of a mineralocorticoid-responsive reporter genes in a concentration-dependent manner. Similar effects were observed for the dexamethasone-activated GR. However, geldanamycin did not affect transcription from a retinoic acid-dependent reporter gene. Inhibition of GR-mediated transactivation was observed both in HeLa cells expressing endogenous GR and in COS-7 cells transfected with a GRa expression vector. Binding studies indicate that geldanamycin disrupts receptor function by reducing hormone binding affinity without lowering intracellular receptor protein levels. Our data support the current model of hsp 90-dependent steroid receptor activation. Furthermore, we show for the first time that MR function also depends on the interaction with hsp 90 in intact cells. Finally, we demonstrate that the function of endogenous is thought to keep the receptor protein in an inactive, yet ligand-activable state (9–17). Ligand binding induces a conformational change in the receptor molecule, which causes it to dissociate from the hsp complex, to translocate to the cell nucleus, and, finally, to interact with specific hormone response elements in the promoter regions of hormone-responsive genes (6–8). Both MR and GR bind as homodimers to identical palindromic sequences on the target DNA, termed glucocorticoid response elements (GREs) (18). The formation of GR/MR heterodimers has also been described (19,20) and may have profound functional consequences (21). The current model of MR and GR function holds that these receptors are unable to bind their respective hormones as long as they are not associated with the hsp complex (9–17). However, experimental support for this model is mainly based on in vitro work. There are few reports analyzing the functional relevance of GR/hsp interactions in mammalian cells. In the most recent study, Whitesell et al. showed that the hspÊ90-binding agent geldanamycin can specifically disrupt GR/hsp association, thus inhibiting glucocorticoid-mediated transcriptional activation (22). MR is even less well studied in this respect. To our knowledge, there have not been any data supporting a functional role for proper MR/hsp interaction in intact cells. In this study, we show for the first time that MR function depends on the interaction with hsp 90 in intact human cells. Furthermore, we demonstrate that geldanamycin inhibits GR-mediated transcriptional activation in two human cells lines, confirming the results by Whitesell et al. and extending them to transfected as opposed to endogenous GR.

The glucocorticoid receptor is a key regulator of the decision between self-renewal and differentiation in erythroid progenitors.

During development and in regenerating tissues such as the bone marrow, progenitor cells constantly need to make decisions between proliferation and differentiation. We have used a model system, normal erythroid progenitors of the chicken, to determine the molecular players involved in making this decision. The molecules identified comprised receptor tyrosine kinases (c-Kit and c-ErbB) and members of the nuclear hormone receptor superfamily (thyroid hormone receptor and estrogen receptor). Here we identify the glucocorticoid receptor (GR) as a key regulator of erythroid progenitor self-renewal (i.e. continuous proliferation in the absence of differentiation). In media lacking a GR ligand or containing a GR antagonist, erythroid progenitors failed to self-renew, even if c-Kit, c-ErbB and the estrogen receptor were activated simultaneously. To induce self-renewal, the GR required the continuous presence of an activated receptor tyrosine kinase and had to cooperate with the estrogen receptor for full activity. Mutant analysis showed that DNA binding and a functional AF-2 transactivation domain are required for proliferation stimulation and differentiation arrest. c-myb was identified as a potential target gene of the GR in erythroblasts. It could be demonstrated that delta c-Myb, an activated c-Myb protein, can functionally replace the GR.

Glucocorticoids enhance oxidative stress-induced cell death in hippocampal neurons in vitro.

In patients with Alzheimer's disease, hippocampal cells are among the first neuronal cells of the brain to degenerate. Both rat primary hippocampal neurons and cells of the clonal mouse hippocampal cell line HT22 express endogenous functional glucocorticoid receptors (GRs), as shown by transient transfection of cells with a luciferase reporter plasmid containing GR-responsive elements. The influence of activated GRs on oxidative stress-induced neuronal cell death in vitro was investigated employing these hippocampal model systems. Two oxidative stressors were investigated, the free radical-inducing Alzheimer's disease-associated amyloid beta-protein, which is toxic to hippocampal neurons, and the excitatory amino acid glutamate, which induces oxidative cell death in HT22 cells via an increase in intracellular peroxides. Cellular viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test and trypan exclusion staining, followed by microscopical cell counting. Glucocorticoids strongly increased the vulnerability of the hippocampal cells to amyloid beta-protein and glutamate. This increase could be blocked by the specific GR antagonist RU486. Our data suggest that changes in hippocampal GR homeostasis and regulation may render hippocampal neurons more vulnerable to oxidative stress-induced neuronal degeneration.

Glucocorticoid receptor lacking the tau 1 transactivation domain is a gene-specific regulator of the wild-type glucocorticoid-receptor activity.

The glucocorticoid receptor (GR) contains a major transactivation function (tau 1), located in the N-terminal domain. tau 1 contributes to about 80% of the ligand-inducible transcriptional activity of GR. In this study, we show that GR devoid of tau 1 (symbol: see text] GR) can inhibit activation of gene expression by wild-type GR but this does not occur for all target genes. Activation of the mouse mammary tumor virus promoter by wild-type GR in transiently transfected chinese hamster ovary (CHO) cells lacking endogenous GR was repressed by cotransfecting [symbol: see text] GR. This effect was proportional to the amount of transfected [symbol: see text] GR and was not due to squelching. A moderate expression level of stably transfected [symbol: see text] GR mutant was also shown to repress the transcriptional activity of endogenous GR present in rat skeletal myoblast L8 cells. Glucocorticoid mediated down regulation of endogenous GR gene expression can be blocked by the [symbol: see text] GR mutant in stably transfected L8 cells. In contrast, no inhibition was observed on glucocorticoid induction of the endogenous glutamine synthetase gene in L8 cells. However, glucocorticoid induction of a reporter gene driven by the chicken glutamine synthetase promoter was inhibited by [symbol: see text] GR in L8 cells. Stable expression of wild-type GR in CHO cells rendered the cells glucocorticoid responsive with regard to glutamine synthetase induction but coexpression of [symbol: see text] GR did not repress induction of the endogenous glutamine synthetase gene expression by wild-type GR. Expression of [symbol: see text] GR alone in CHO cells did not render the glutamine synthetase gene glucocorticoid responsive, indicating that [symbol: see text] GR has no transcriptional activity on the glutamine synthetase gene. We conclude from these results that the structure of glucocorticoid-response elements within target genes may be very critical for the ability of the mutant receptor to exhibit a dominant negative effect.

Disruption of hypothalamic-pituitary-adrenocortical system in transgenic mice expressing type II glucocorticoid receptor antisense ribonucleic acid permanently impairs T cell function: effects on T cell trafficking and T cell responsiveness during postnatal development.

We used transgenic mice with impaired corticosteroid receptor function, caused by expression of type II glucocorticoid receptor (GR) antisense RNA, to study the role of glucocorticoid feedback during the developmental maturation of hypothalamus-pituitary-adrenal-immune functions. These mice have increased plasma concentrations of ACTH and corticosterone as well as reduced GR binding capacity. In control mice, a strong sex dimorphism in the development of GR gene expression is apparent, and in males between postnatal days 9-36, the GR gene transcript concentration is approximately twice that in female mice. Endogenous GR messenger RNA levels were markedly reduced in transgenic mice, and the sex dimorphism was abolished. An abnormal developmental pattern of adrenal secretory activity accompanied the postnatal maturation of the hypothalamic-pituitary-adrenocortical system of the transgenic mice, and high plasma corticosterone levels were measured at early postnatal ages through adulthood. Inefficient glucocorticoid inhibitory action on the immune axis was supported by both the inability of high circulating levels of corticosterone to reduce thymus weight and the failure of dexamethasone to influence in vitro thymocyte and splenocyte cell proliferation. Alterations in thymocyte trafficking/migration in transgenic mice was supported by flow cytometric analysis of the distribution of phenotypically distinct lymphocyte subsets accompanying the postnatal maturation of the thymus. A marked increase in CD4+CD8+ double positive cells and a 2-fold increase in the CD4/CD8 (helper/suppressor) ratio caused by a 40-60% increase in the CD4+CD8- (T helper) subset and a decrease in the CD4-CD8+ (T suppressor) subset, was seen. Moreover, in transgenic mice, an absence of sexual dimorphism and a significantly increased immune reactivity were observed. The present study shows that disruption of the hypothalamic-pituitary-adrenocortical system has both developmental and permanent effects on T cell function characterized by a shifting of the T cell balance toward the CD4+CD8- helper-inducer phenotype coupled with hyperresponsiveness of the T (helper) cell compartment. These findings point to the GR as a major factor in the counterregulatory feedback loop controlling autoaggressive immune responses and underline the potential modulatory role of sex steroids in this feedback regulation and in the pathogenesis of autoimmune diseases.

Disruption of hypothalamic-pituitary-adrenocortical system in transgenic mice expressing type II glucocorticoid receptor antisense ribonucleic acid permanently impairs T cell function: effects on T cell trafficking and T cell responsiveness during postnatal development

We used transgenic mice with impaired corticosteroid receptor function, caused by expression of type II glucocorticoid receptor (GR) antisense RNA, to study the role of glucocorticoid feedback during the developmental maturation of hypothalamus-pituitary-adrenal-immune functions. These mice have increased plasma concentrations of ACTH and corticosterone as well as reduced GR binding capacity. In control mice, a strong sex dimorphism in the development of GR gene expression is apparent, and in males between postnatal days 9-36, the GR gene transcript concentration is approximately twice that in female mice. Endogenous GR messenger RNA levels were markedly reduced in transgenic mice, and the sex dimorphism was abolished. An abnormal developmental pattern of adrenal secretory activity accompanied the postnatal maturation of the hypothalamic-pituitary-adrenocortical system of the transgenic mice, and high plasma corticosterone levels were measured at early postnatal ages through adulthood. Inefficient glucocorticoid inhibitory action on the immune axis was supported by both the inability of high circulating levels of corticosterone to reduce thymus weight and the failure of dexamethasone to influence in vitro thymocyte and splenocyte cell proliferation. Alterations in thymocyte trafficking/migration in transgenic mice was supported by flow cytometric analysis of the distribution of phenotypically distinct lymphocyte subsets accompanying the postnatal maturation of the thymus. A marked increase in CD4+CD8+ double positive cells and a 2-fold increase in the CD4/CD8 (helper/suppressor) ratio caused by a 40-60% increase in the CD4+CD8- (T helper) subset and a decrease in the CD4-CD8+ (T suppressor) subset, was seen. Moreover, in transgenic mice, an absence of sexual dimorphism and a significantly increased immune reactivity were observed. The present study shows that disruption of the hypothalamic-pituitary-adrenocortical system has both developmental and permanent effects on T cell function characterized by a shifting of the T cell balance toward the CD4+CD8- helper-inducer phenotype coupled with hyperresponsiveness of the T (helper) cell compartment. These findings point to the GR as a major factor in the counterregulatory feedback loop controlling autoaggressive immune responses and underline the potential modulatory role of sex steroids in this feedback regulation and in the pathogenesis of autoimmune diseases.

Characterization of Mechanisms Involved in Transrepression of NF-κB by Activated Glucocorticoid Receptors

Glucocorticoids are potent immunosuppressants which work in part by inhibiting cytokine gene transcription. We show here that NF-kappa B, an important regulator of numerous cytokine genes, is functionally inhibited by the synthetic glucocorticoid dexamethasone (DEX). In transfection experiments, DEX treatment in the presence of cotransfected glucocorticoid receptor (GR) inhibits NF-kappa B p65-mediated gene expression and p65 inhibits GR activation of a glucocorticoid response element. Evidence is presented for a direct interaction between GR and the NF-kappa B subunits p65 and p50. In addition, we demonstrate that the ability of p65, p50, and c-rel subunits to bind DNA is inhibited by DEX and GR. In HeLa cells, DEX activation of endogenous GR is sufficient to block tumor necrosis factor alpha or interleukin 1 activation of NF-kappa B at the levels of both DNA binding and transcriptional activation. DEX treatment of HeLa cells also results in a significant loss of nuclear p65 and a slight increase in cytoplasmic p65. These data reveal a second mechanism by which NF-kappa B activity may be regulated by DEX. We also report that RU486 treatment of wild-type GR and DEX treatment of a transactivation mutant of GR each can significantly inhibit p65 activity. In addition, we found that the zinc finger domain of GR is necessary for the inhibition of p65. This domain is also required for GR repression of AP-1. Surprisingly, while both AP-1 and NF-kappa B can be inhibited by activated GR, synergistic NF-kappa B/AP-1 activity is largely unaffected. These data suggest that NF-kappa B, AP-1, and GR interact in a complex regulatory network to modulate gene expression and that cross-coupling of NF-kappa B and GR plays an important role in glucocorticoid-mediated repression of cytokine transcription.

Cell to cell contacts control the transcription activity of the glucocorticoid receptor.

Contact interactions between glia and neurons are required for hormonal induction of glutamine synthetase in Müller glial cells. Glucocorticoids induce a pronounced increase in glutamine synthetase gene transcription in the intact retinal tissue but not in separated retinal cells. However, if the separated cells are reaggregated and glial cells reestablish contacts with neurons, glutamine synthetase inducibility is restored. This study examines the possible involvement of the glucocorticoid receptor (GR) in cell contact control of glutamine synthetase induction. Using the glucocorticoid-inducible reporter construct, p delta G46TCO, and control constructs that are not inducible by glucocorticoids, we demonstrated that the trans-activating capability of GR markedly declines upon cell separation. Analysis of GR protein revealed that cell separation results in a pronounced decrease in GR expression. This decrease temporally correlated with the decline in glutamine synthetase gene transcription. Cell separation also results in a marked increase in c-Jun expression. This increase might be related to the decline in GR activity since elevation of c-Jun expression in the intact tissue inhibits the transcription activity of GR. Over-expression of GR by transfection of a GR expression vector or activation of endogenous GR molecules by 8-bromo-cAMP enhanced the responsiveness of separated retinal cells to glucocorticoids. These results demonstrate that transcription activity of the receptor protein depends on contact interactions between retinal cells and suggest that GR is involved in cell contact control of glutamine synthetase induction.

The role of corticosteroids in the acquisition of sensitization to locomotor stimulant effects of MK-801

In the present study, we investigated the role of corticosterone and glucocorticoid receptors in the acquisition of sensitization to locomotor stimulant effects of MK-801 in rats. MK-801 (two doses, 0.4 mg/kg i.p. each, given twice, 48 h apart) evoked sensitization, observed as enhancement of the locomotor activity to a challenging dose of MK-801 (0.4 mg/kg) but not of a stereotypy-like activity. Pharmacological manipulations which deplete endogenous corticosterone, i.e., administration of the corticosterone synthesis inhibitor metyrapone (two injections, 150 and 50 mg/kg, given at 24 and 2 h before the first injection of MK-801, 4 mg/kg) or blockade of glucocorticoid receptors by administration of the antiglucocorticoid RU 38486 (20 mg/kg, 45 min before MK-801, 0.4 mg/kg) abolished the acquisition of sensitization. The obtained results indicate that the endogenous corticosterone and glucocorticoid receptors (type II) are involved in the acquisition of sensitization to locomotor stimulant effects of MK-801. Final experiments showed that MK-801 in doses used in the present study (0.4 mg/kg) enhanced the plasma concentration of corticosterone and that single injection of exogenous corticosterone (10 mg/kg s.c.) enhanced the locomotor stimulant effects of MK-801 (0.2 mg/kg). The obtained data indicate that the acquisition of sensitization to locomotor stimulant effects of MK-801 involves secretion of corticosteroids which probably act through glucocorticoid receptors, as was found previously for amphetamine and its congeners.

Mouse Thioredoxin Gene Maps on Chromosome 4, whereas Its Pseudogene Maps on Chromosome 1

Thioredoxins are involved in various biochemical systems and mediate both redox and nonredox functions. In mammalian cells, thioredoxin functions as an endogenous glucocorticoid receptor activating factor and as an adult T-cell leukemia-derived factor (ADF) that stimulates expression of interleukin-2 receptor in human T-cell leukemia virus (HTLV)-I transformed T cells. We have mapped the thioredoxin gene ( Txn) and its processed pseudogene ( Txn-ps1) in the mouse using a panel of interspecific backcross mice. Txn maps to Chr 4, whereas Txn-ps1 maps to the proximal region of Chr 1.

Glucocorticoid receptor conversion to high affinity nuclear binding and transcription enhancement activity in chinese hamster ovary cells subjected to heat and chemical stress

The untransformed glucocorticoid receptor (GR) is a heteromeric complex containing two molecules of the 90-kDa heat shock protein (hsp90) and one molecule of the 56-kDa heat shock protein (hsp56). In the absence of hormone, this complex is found in the cytosolic fraction of cells, and upon hormone-binding the complex dissociates and the GR is recovered in the nuclear pellet fraction. Given the association of heat shock proteins with the cytosolic form of the GR, we have examined the effects of heat shock on GR subcellular localization and transcription enhancement activity in a series of Chinese hamster ovary (CHO) cells which express either low levels of endogenous GR (CHOd cells), high levels of the mouse GR (WCL2 cells), or high levels of mutated mouse GR unable to bind DNA (NB cells). It was found that heat shock treatment of WCL2 cells results in wild-type mouse GR that is recovered almost entirely within the nuclear pellet fraction, a response similar to that seen in hormone-treated cells. In contrast, heat shock treatment of NB cells results in complete loss of GR from the cytosolic fraction, but almost no shift of GR to the nuclear pellet. These results indicate that heat shock-mediated conversion to high-affinity nuclear binding by the wild-type GR requires a functional DNA-binding domain, and that heat shock will result in loss of GR to proteolysis in the absence of nuclear sequestration. Analysis of MMTV-CAT reporter gene expression in these cells revealed that heat or chemical shock, in comparison to hormone-treatment, results in a small induction of MMTV-CAT expression in the WCL2 cells, but not in the CHOd or NB cells. These results indicate that cellular stress can cause at least a partial induction of hormone-independent GR-mediated gene expression.

Purification of the endogenous glucocorticoid receptor stabilizing factor

A ubiquitous, low molecular weight, heat-stable component of cytosol stabilizes the glucocorticoid receptor in its untransformed state in association with hsp90. This heat-stable factor mimics molybdate in its effects on receptor function, and it has the heat stability, charge, and chelation properties of a metal oxyanion [Meshinchi, S., Grippo, J.F., Sanchez, E.R., Bresnick, E.H., & Pratt, W.B. (1988) J. Biol. Chem. 263, 16809-16817]. In this paper, we describe the further purification of the endogenous factor from rat liver cytosol by anion-exchange HPLC (Ion-110) after prepurification by molecular sieving, cation absorption, and charcoal absorption. Elution of the factor with an isocratic gradient of ammonium bicarbonate results in recovery of all of the bioactivity in a single peak which coelutes with inorganic phosphate and contains all of the endogenous molybdenum. The bioactivity can be separated from inorganic phosphate by chromatography of the partially purified endogenous factor on a metal-chelating column of Chelex-100. The chelating procedure results in complete loss of bioactivity with recovery of 98% of the inorganic phosphate in both the column drop-through and a subsequent 1 M NaCl wash. The factor preparation purified through the Ion-110 HPLC step inhibits temperature-mediated dissociation of the immunopurified glucocorticoid receptor-hsp90 complex, but it is considerably more effective at stabilizing the unpurified receptor-hsp90 complex in a Chelex-treated cytosol system that has been depleted of metal components. These observations support the proposal that an endogenous metal can stabilize the binding of hsp90 to the receptor but it is likely that other cytosolic components that are not present in the immunopurified complex must contribute to the stability of the soluble protein-protein complex in cytosol.

The mechanism for glucocorticoid-resistance in a rat hepatoma cell variant that contains functional glucocorticoid receptor

The mechanism of glucocorticoid resistance has been studied in a rat hepatoma cell variant (6.10.2) ,which contains low levels of glucocorticoid receptor (GR). These cells seem to have lost all the glucocorticoid-induced transcriptional responses as measured by the lack of induction of expression of stably integrated mouse mammary tumor virus (MMTV) and the endogenous gene tyrosine aminotransferase (TAT), as well as the transcriptional suppression of GR gene expression. Physico-chemical characterization of the GR in the glucocorticoid resistant 6.10.2 cells revealed that the receptor is indistinguishable from the wild-type receptor with regard to size hormone- and DNA-binding. The levels of the receptor mRNA and the total immunoreactive protein found in 6.10.2 cells were about 20% of those found in wild-type cells. Further analysis of 6.10.2 cells demonstrated that the receptor was indeed biologically functional. Treatment of 6.10.2 cells with 8-bromo-cAMP, which induced the endogenous GR level two-fold, restored responsiveness to glucocorticoids. Secondly, pretreatment of the cells with cycloheximide also led to reacquisition of cellular responsiveness to glucocorticoids. We propose that there exists a “threshold” level of GR, which is required for responsiveness and that under normal culture conditions, the level of GR in 6.10.2 cells is below this threshold. Glucocorticoid responsiveness can be restored by raising the GR level above the threshold with 8-bromo-cAMP or, alternatively, by removing the threshold barrier (repressor protein) with cycloheximide. Finally, the existence of such a repressor protein for MMTV induction was shown by in vivo titration with an isolated negative cis-element from the MMTV promoter.

A glucocorticoid-resistant rat hepatoma cell variant contains functional glucocorticoid receptor.

The mechanism of glucocorticoid resistance was studied in a rat hepatoma cell variant (6.10.2) which contains low levels of glucocorticoid receptor. These cells seem to have lost glucocorticoid-induced transcriptional responses as measured by the induction of expression of stably integrated mouse mammary tumor virus gene and the endogenous tyrosine aminotransferase gene, as well as the transcriptional suppression of glucocorticoid receptor gene expression. However, characterization of the glucocorticoid resistance in 6.10.2 cells revealed that the receptor is indistinguishable from the wild-type receptor with respect to hormone binding and affinity for both nonspecific and specific DNA sequences. The levels of the receptor mRNA and the total immunoreactive protein found in 6.10.2 cells were about 20% of those found in wild-type cells. Further analysis of 6.10.2 cells demonstrated that the receptor was indeed biologically functional. First, treatment of 6.10.2 cells with 8-bromo-cAMP elevated the endogenous glucocorticoid receptor levels 2-fold and restored responsiveness to glucocorticoids. Second, pretreatment of the cells with cycloheximide also led to acquisition of cellular responsiveness to glucocorticoids. We propose that there exists a "threshold" level of glucocorticoid receptor which is required for responsiveness and that under normal culture conditions, the level of glucocorticoid receptor in 6.10.2 cells is below this threshold. However, glucocorticoid responsiveness can be restored by raising the glucocorticoid receptor level above the threshold with 8-bromo-cAMP or, alternatively, by removing the threshold barrier with cycloheximide.

Hormonal induction of transfected genes depends on DNA topology.

Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors.

Hormonal Induction of Transfected Genes Depends on DNA Topology

Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors.

Stable overproduction of intact glucocorticoid receptors in mammalian cells using a selectable glucocorticoid responsive dihydrofolate reductase gene.

We have generated several mammalian cell lines that stably express high levels of intact glucocorticoid receptor. These cells were created by cotransfecting a glucocorticoid-dependent dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary (CHO) cells together with a plasmid directing the expression of human glucocorticoid receptor. Using this approach, transfection frequencies indicate that the inclusion of glucocorticoid receptor cDNA increased the efficiency of DHFR transformation greater than 10-fold over nonreceptor control DNA. When a stably cotransfected line (designated MG/hGR) was subjected to short term growth in cytotoxic concentrations of the antifolate methotrexate, these cells strongly resisted growth inhibition when dexamethasone was present in the medium. This effect was steroid specific and was inhibited by the glucocorticoid antagonist RU38486. In an effort to exploit the methotrexate-induced coamplification properties of the DHFR gene as a means of creating cell lines having increased levels of glucocorticoid receptor, MG/hGR cells were chronically exposed to a relatively low concentration of methotrexate (50 nM). After this treatment a resistant line was isolated (MG/hGR/MTX50) that displayed complete dependence on exogenous glucocorticoid for growth. To investigate the molecular basis for the enhanced ability of MG/hGR/MTX50 cells to resist the cytotoxic effects of methotrexate in the presence of dexamethasone, glucocorticoid receptor protein in these cells was characterized and compared to parental CHO cells and methotrexate sensitive MG/hGR cells. Affinity labeling with [3H]dexamethasone mesylate and Western blot analysis with antiglucocorticoid receptor antiserum revealed that nontransfected CHO cells have virtually undetectable levels of glucocorticoid receptor protein whereas cotransfected MG/hGR cells contain at least 3 times more intact monomeric receptor protein of Mr 94,000. Correspondingly, analysis of receptor protein in MG/hGR/MTX50 cells indicated that these cells contain 8 to 10 times more glucocorticoid receptor than nontransfected CHO cells. Scatchard analysis of steroid binding curves revealed that these increases correspond to 6,600, 22,000 and 63,000 dexamethasone binding sites per cell for nontransfected CHO cells, cotransfected MG/hGR cells, and MG/hGR/MTX50 cells, respectively. Sedimentation profiles of native receptor in transfected and methotrexate-resistant cells further support the progressive increase in receptor content and demonstrate that glucocorticoid receptor exists in cotransfected cels as an oligomeric complex under hypotonic conditions (9S complex in the presence of 20 mM sodium molybdate, 7S in the absence of molybdate), which dissociates to a monomeric 4S species in the presence of 0.4 M KCl. These physicochemical properties are indistinguishable from those observed for the endogenous hamster glucocorticoid receptor and suggest that stably transfected human glucocort

Evidence that the endogenous heat-stable glucocorticoid receptor stabilizing factor is a metal component of the untransformed receptor complex.

Boiled cytosols prepared from a wide variety of sources contain a low Mr factor that inhibits glucocorticoid receptor transformation to the DNA-binding state (Leach, K.L., Grippo, J.F., Housley, P.R., Dahmer, M.K., Salive, M.E., and Pratt, W.B. (1982) J. Biol. Chem. 257, 381-388). In this work, we show that this endogenous factor, which is partially purified from rat liver, produces all of the effects of the group VI-A transition metal oxyanions molybdate and vanadate on the structure and function of glucocorticoid receptors in cytosol preparations. Like molybdate, the endogenous factor behaves as a strong anion with an apparent Mr of 340 on Bio-Gel P-2, and it binds to both hydroxylapatite and Chelex 100 resins. The receptor stabilizing activity of the factor is completely stable to heating at 320 degrees C for 1 h. The small size, profound heat stability, and absorption by a metal chelating resin strongly suggest that the factor is an endogenous metal anion. As reduction of the concentration of the factor in cytosol promotes generation of the DNA-binding form of the receptor, we suggest that this endogenous metal anion interacts with the receptor to stabilize the 9 S complex and maintain the receptor in its untransformed, non-DNA-binding state. We propose that molybdate and vanadate may exert their effects on the untransformed receptor by interacting with the binding site for the endogenous metal anion.