Identification of human glucocorticoid receptor complementary DNA clones by epitope selection.

Steroid hormones regulate cellular differentiation and physiologic functions predominantly through gene transcription. Regulation is achieved by the interaction of specific steroid receptor proteins and target genes. Expression cloning techniques were used to select human glucocorticoid receptor complementary DNA clones in order to define the mechanism by which the receptor exerts its transcriptional control. Immobilized fusion proteins from individual clones were used to select epitope-specific antibody which was subsequently eluted and identified by binding to protein blots of cellular extracts. Three cross-hybridizing clones containing inserts expressing antigenic determinants of the human glucocorticoid receptor were isolated.

Isolation and characterization of dexamethasone-resistant mutants from human lymphoid cell line CEM-C7.

Fifty-four independent dexamethasone-resistant clones were isolated from the clonal, glucocorticoid-sensitive human leukemic T-cell line CEM-C7. Resistance to 1 microM dexamethasone was acquired spontaneously at a rate of 2.6 X 10(-5) per cell per generation as determined by fluctuation analysis. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the phenotypic expression time for dexamethasone resistance was determined to be 3 days. Spontaneous acquisition of resistance to 0.1 mM 6-thioguanine appeared to occur at a much slower rate, 1.6 X 10(-6) per cell per generation. However, the expression time after MNNG mutagenesis for this resistant phenotype was greater than 11 days, suggesting that the different rates of acquisition for the two phenotypes measured by fluctuation analysis were the results of the disparate expression times. The mutagens ICR 191 and MNNG were effective in increasing the dexamethasone-resistant fraction of cells in mutagenized cultures; ICR 191 produced a 35.6-fold increase, and MNNG produced an 8.5-fold increase. All the spontaneous dexamethasone-resistant clones contained glucocorticoid receptors, usually less than half of the amount found in the parental clone. They are therefore strikingly different from dexamethasone-resistant clones derived from the mouse cell lines S49 and W7. Dexamethasone-resistant clones isolated after mutagenesis of CEM-C7 contained, on the average, lower concentrations of receptor than did those isolated spontaneously, and one clone contained no detectable receptor. These results are consistent with a mutational origin for dexamethasone resistance in these human cells at a haploid or functionally hemizygous locus. They also suggest that this is a useful system for mutation assay.

Hormone-independent repression of AP-1-inducible collagenase promoter activity by glucocorticoid receptors.

The role of the ligand in glucocorticoid receptor-mediated transactivation and transrepression of gene expression was investigated. Half-maximal transactivation of a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene in transfected cells expressing the human glucocorticoid receptor mutant GRL753F, from which the rate of ligand dissociation is four to five times higher than the rate of dissociation from normal receptors, required a 200- to 300-fold-higher concentration of dexamethasone than was required in cells expressing the normal receptor. Immunocytochemical analysis demonstrated that this difference was not the result of a failure of the mutant receptor to accumulate in the nucleus after steroid treatment. In contrast, in cells cotransfected with a reporter gene containing the AP-1-inducible collagenase gene promoter, the concentration of dexamethasone required for 50% transrepression was the same for mutant and normal receptors. Efficient receptor-mediated transrepression was also observed with the double mutant GRL753F/C421Y, in which the first cysteine residue of the proximal zinc finger has been replaced by tyrosine, indicating that neither retention of the ligand nor direct binding of the receptor to DNA is required. RU38486 behaved as a full agonist with respect to transrepression. In addition, receptor-dependent transrepression, but not transactivation, was observed in transfected cells after heat shock in the absence of the ligand. Taken together, these results suggest that unlike transactivation, transrepression of AP-1 activity by the nuclear glucocorticoid receptor is ligand independent.

Biochemical evidence that glucocorticoid-sensitive cell lines derived from the human leukemic cell line CCRF-CEM express a normal and a mutant glucocorticoid receptor gene.

To characterize the immunoreactive glucocorticoid receptor (GR) protein present in "receptorless" (r-) mutants isolated from the glucocorticoid-sensitive (dexs) human leukemic cell line CEM-C7, binding of [3H]dexamethasone was determined in extracts prepared from the sensitive cell line 6TG1.1 and the r- mutant ICR27TK.3 after gentle freeze-thaw lysis and low-speed centrifugation. Under these conditions there was significant high-affinity binding activity in r- extracts assayed at 4 degrees C but not at 23 degrees C. Loss of binding at 23 degrees C was not a function of GR proteolysis or denaturation of the steroid-binding site and could be prevented by the addition of sodium molybdate. Dissociation of ligand from either activated or unactivated receptors in r- extracts was significantly more rapid than from receptors in extracts prepared from normal cells, suggesting that the defect in receptors in r- cells is the result of mutation in the ligand-binding site. While the rate of dissociation from unactivated receptors in r- extracts was linear, dissociation from receptors in extracts of 6TG1.1 cells was biphasic. Analysis of these dissociation curves, as well as dissociation from receptors in the B-cell line IM-9, indicated that the mutant gene present in r- cells is also present in the dexs parental cell line. This conclusion is consistent with our previous hypothesis (J.M. Harmon et al., Mol. Endocrinol., 3:734-743, 1989) that glucocorticoid-sensitive CCRF-CEM cells express both a normal (GR+) and a mutant (GR*) allele.

Use of high-resolution two-dimensional gel electrophoresis and affinity labeling to probe glucocorticoid receptor structure and function.

The possible role of posttranslational modification in glucocorticoid receptor regulation was investigated. Glucocorticoid receptor (GR), prepared from the human B-cell line IM-9 and affinity labeled with [3H]-dexamethasone 21-mesylate, was examined by a combination of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-resolution two-dimensional gel electrophoresis. Two-dimensional electrophoresis of immunopurified [3H]dexamethasone 21-mesylate-labeled GR revealed the presence of two isoelectric species (apparent pI approximately to 5.7, and 6.0-6.5). Both forms were present in preparations of unactivated receptor. After GR activation, the pI of neither isoform was altered, indicating that activation does not involve covalent charge modification of the steroid-binding protein. However, only the pI 6.0-6.5 isoform bound to DNA, suggesting that covalent charge modification of the GR can alter its ability to bind to DNA. Two-dimensional electrophoresis of tryptic and chymotryptic fragments showed that the charge heterogeneity responsible for the two GR isoforms is located in a Mr 26,500 tryptic fragment derived from the steroid-binding domain of the protein. In addition, analysis of [3H]dexamethasone 21-mesylate-labeled tryptic fragments suggests that the Mr 26,500 fragment corresponds to residues 499-743 of the human GR. These results demonstrate that posttranslational modification of the steroid-binding domain may regulate the ability of the protein to bind to DNA.

Non-glucocorticoid receptor-mediated effects of the potent glucocorticoid deacylcortivazol.

Deacylcortivazol (DAC), a potent glucocorticoid, contains a phenyl-pyrazole moiety fused to the 2--3 position of the traditional steroid nucleus. When incubated with glucocorticoid-resistant mutants derived from the glucocorticoid-sensitive human leukemic cell line CEM-C7, DAC caused significant growth inhibition. However, this effect required 1 microM DAC, a concentration 50 times higher than that necessary for glucocorticoid receptor saturation. Cytotoxicity was observed in both mutants containing high-affinity glucocorticoid receptors defective in nuclear translocation and a mutant completely devoid of receptors. Further, in dexamethasone-resistant clones, DAC elicited only marginal increases in the activity of the glucocorticoid-inducible enzyme glutamine synthetase. Clones resistant to high concentrations of DAC could not be directly isolated from CEM-C7. However, stable DAC-resistant clones could be isolated from dexamethasone-resistant subclones of CEM-C7 with a frequency of 1 to 8 x 10(-4). These data are consistent with resistance to DAC being acquired in a two-step process. Our results suggest that the cytotoxicity of DAC at concentrations higher than necessary for glucocorticoid receptor saturation is not mediated by glucocorticoid receptors. Thus, DAC may be a bifunctional compound having both steroid receptor-mediated and receptor-independent cytotoxicity.

Analysis of glucocorticoid-resistant human leukemic cells by somatic cell hybridization.

Glucocorticoid-resistant mutants isolated form the glucocorticoid-sensitive human leukemic cell line CEM-C7 can be divided into three phenotypes: those with almost no glucocorticoid-binding activity (r-); those whose steroid-receptor complexes are unstable during attempted activation but are stabilized by the presence of sodium molybdate (actl:molybdate-sensitive); and those whose steroid-receptor complexes are unstable during attempted activation but are insensitive to the presence of molybdate (actl:molybdate-resistant). To determine if these phenotypes represent different mutations within the glucocorticoid receptor locus itself or reflect alterations in other components modifying receptor function, somatic hybrids were constructed between wild-type cells and all three classes of resistant mutants, as well as between various classes of resistant mutants. Hybrids were analyzed for chromosome content, steroid-induced growth inhibition, induction of the enzyme glutamine synthetase, and glucocorticoid receptor content. Hybrids constructed between wild-type cells and any of the three classes of resistant cells were growth-inhibited in the presence of dexamethasone, displayed normal levels of glutamine synthetase induction, and contained a quantity of glucocorticoid receptor approximately equal to the sum of the glucocorticoid receptor concentrations of the parental cell lines. Hybrids constructed between various classes of resistant cells were not growth-inhibited by dexamethasone, displayed no glutamine synthetase induction, and also contained the sum of the glucocorticoid receptor concentration of the individual parents. Thus, each phenotype is recessive, and there is no complementation between phenotypes. We conclude that the three phenotypes are the result of different mutations within the glucocorticoid receptor locus itself and do not represent the presence of dominant receptor-inactivating factors or the absence of positive regulatory components.

Glucocorticoid-resistant human acute lymphoblastic leukemic cell line with functional receptor.

A receptor-containing, steroid-resistant clone of CEM cells, CEM-C1, was isolated without selective pressure from the wild-type population. The biological and physicochemical properties of glucocorticoid receptors in CEM-C1 cells were compared to those from a clone (CEM-C7) sensitive to glucocorticoid-mediated lysis. In a whole-cell binding assay, CEM-C1 cells exhibited high affinity for [3H]dexamethasone (Kd, 22 nM), nuclear translocation of steroid:receptor complex (nt, 43%) and were found to contain, on the average, 12,000 receptor sites/cell (R0). These steroid-binding parameters were similar to those displayed by wild-type CEM-C7 cells: Kd, 19 nM; nt, 47%; and R0, approximately 14,000 sites/cell. The ion-exchange and gel permeation profiles were indistinguishable from those of identically treated CEM-C7 cytosols. Thus, diethylaminoethyl cellulose chromatography of CEM-C1 cytosol showed that [3H]triamcinolone acetonide:receptor complex was eluted at 50 mM phosphate and 220 mM phosphate under "activating" and "nonactivating" conditions, respectively. Receptor complex of activated CEM-C1 cytosol bound to DNA-cellulose and was eluted at 100 mM salt. Filtration of unactivated CEM-C1 cytosol over Sephacryl S-300 generated a single peak of radioactivity for receptor complex with a calculated Stokes' radius of 55 to 59 A. Dexamethasone induced glutamine synthetase in CEM-C1. The dose dependence (50% effective dose, approximately 20 nM) and maximal fold increase (1.9, 1 microM dexamethasone) were comparable to those observed in CEM-C7. Since CEM-C1 cells contain apparently normal, functional cytosolic receptor, the results suggest that resistance to glucocorticoid in these cells involves a defect(s) at another locus.

Human glucocorticoid receptor gene deletion following exposure to cancer chemotherapeutic drugs and chemical mutagens.

The sensitivity of the human glucocorticoid receptor (hGR) gene to mutagenesis by the cancer chemotherapeutic drugs adriamycin, bleomycin, and chlorambucil was evaluated using glucocorticoid-sensitive (dexs) subclones of the human leukemic cell line CEM-C7. Treatment of cells with either bleomycin or chlorambucil increased the frequency of glucocorticoid-resistant (dexr) clones 3.3- and 10-fold, respectively. Measurement of steroid-binding activity in intact dexr cells demonstrated that the predominant phenotype of drug-induced dexr clones was receptorless (r-). dexs CEM cells express only one functional hGR allele and, in addition, are heterozygous for a BclI restriction fragment length polymorphism in the hGR gene (L. A. Palmer and J. M. Harmon, Cancer Res., 51:5224-5231, 1991). To determine the basis of the r- phenotype, BclI digests of genomic DNA isolated from r+ and r- cell lines were examined for the presence of the polymorphic 2.4- and 4.4-kilobase digestion products. A deletion of all or part of the hGR gene was demonstrated by the absence of the 4.4-kilobase fragment in one of two bleomycin-induced dexr clones, as well as the ICR191-induced dexr cell line ICR27TK.3. Cytogenetic analysis of ICR27TK.3 showed that the distal portion of the long arm of one chromosome 5 had been replaced with a portion of chromosome 15. Thus, in at least two dexr cell lines, deletions and/or chromosome breaks in the hGR locus appear to account for the r- phenotype.

Glucocorticoid-induced cell death requires autoinduction of glucocorticoid receptor expression in human leukemic T cells.

In contrast to the negative autoregulation of glucocorticoid receptor (GR) expression seen in most cells and tissues, GR expression is positively autoregulated in human leukemic T cells and in other cells sensitive to glucocorticoid-induced cell death. To determine whether positive autoregulation is a necessary component of glucocorticoid-induced cell death, a wild-type GR gene under the control of a tetracycline-regulated promoter was stably transfected into glucocorticoid-resistant cells lacking endogenous functional receptor. Transfectants grown in the presence of tetracycline contained about 15,000 receptors/cell, a value approximately equal to basal level GR expression in glucocorticoid-sensitive 6TG1.1 cells before steroid treatment. Under these conditions, dexamethasone had a minimal effect on cell growth, elicited little internucleosomal DNA fragmentation, and induced no cell cycle perturbation. In the absence of tetracycline, GR mRNA and protein expression increased 2-3-fold, and cells expressed 48,000 receptors, a level nearly equivalent to that present in 6TG1.1 cells after 18 h of autoinduction. Under these conditions, dexamethasone markedly inhibited cell growth, caused G1 arrest, and induced significant internucleosomal DNA fragmentation. These studies therefore suggest that basal level GR expression is inadequate to mediate glucocorticoid-induced apoptosis in glucocorticoid-sensitive T cells and that positive autoregulation is a necessary component of this process.

Cloning and expression of mutant glucocorticoid receptors from glucocorticoid-sensitive and -resistant human leukemic cells.

The molecular basis for the receptorless (r-) and activation-labile (act1) phenotypes of glucocorticoid-resistant mutants isolated from glucocorticoid-sensitive human leukemic CEM-C7 cells was determined. Clones isolated from a complementary DNA library prepared from r- ICR27TK.3 cells, in which one glucocorticoid receptor (GR) gene has been deleted, contained a single adenosine to thymidine transversion in the third position of codon 753, resulting in the substitution of phenylalanine for leucine. This mutant gene (GR753F) had only 13% of the trans-activating activity of the normal gene and produced a M(r) 92,000 receptor protein with the same r- phenotype seen in ICR27TK.3 cells. Analysis of complementary DNA clones isolated from a library prepared from parental glucocorticoid-sensitive 6TG1.1 cells showed that these cells express both a normal GR gene (GR+) and the GR753F gene. Thus, their genotype is GR+/GR753F. Analysis of clones isolated from a complementary DNA library prepared from glucocorticoid-resistant activation-labile 3R7. 6TG.4 cells revealed the presence of the GR753F gene and a second mutant gene (GR421Y) containing a guanosine to adenosine transition in the second position of codon 421, resulting in the replacement of the first cysteine of the proximal zinc finger of the DNA-binding domain by tyrosine. This mutant had no trans-activating activity but normal ligand-binding characteristics. Thus, the genotype of act1 3R7.6TG.4 cells is GR421Y/GR753F. Consequently, the sequence-specific DNA-binding activity of receptors in act1 cells is attributable to the GR753F gene, while the ligand-binding activity seen in intact cells is attributable to the GR421Y gene. These results provide a direct explanation for the r- and act1 phenotypes of glucocorticoid-resistant cells and demonstrate that glucocorticoid-sensitive cells derived from CEM-C7 cells contain a heterogeneous population of normal and mutant receptors.

Glucocorticoid receptor gene mutations in leukemic cells acquired in vitro and in vivo.

Glucocorticoid resistance was investigated in human leukemic CCRF-CEM cells. A mutation (L753F), which renders the human glucocorticoid receptor (hGR) gene functionally hemizygous, was identified in all CEM-derived cell lines analyzed. Allele-specific PCR identified the same mutation in lymph node biopsy material from patient CEM cells. Given the correlation between hGR concentration and glucocorticoid sensitivity, this suggests that loss of functional heterozygosity may result in resistance to glucocorticoid-based chemotherapy. The L753F mutation was probably not responsible for the ontogeny of the disease because it did not appear to be present in all leukemic cells. Thus, it is unlikely that hGR mutations would be detected in leukemic patients at presentation, but they may occur, and be selected for, during treatment. Deletions and point mutations in the hGR gene of cells selected for steroid resistance in vitro were investigated by PCR-single strand conformation polymorphism analysis. Loss of hGR mRNA expression resulted from 5'-deletion of the hGR gene and nonsense mutations in exon 6. These results provide the first evidence for somatic mutation in the hGR gene of a patient with acute lymphoblastic leukemia, offer a potential in vivo mechanism for acquisition of steroid resistance in leukemia, and suggest that screening for additional in vivo mutations will require analysis of genomic DNA.

Identification of human leukemic glucocorticoid receptors using affinity labeling and anti-human glucocorticoid receptor antibodies.

Antisera raised against human lymphoid glucocorticoid receptors were used in combination with the glucocorticoid receptor affinity label [3H]dexamethasone 21-mesylate [( 3H]DM) to identify the glucocorticoid receptors of the human B-lymphoblastoid cell line IM-9 and the human T-cell leukemic cell line CEM-C7. Antisera were obtained following immunization of New Zealand White rabbits with [3H]triamcinolone acetonide [( 3H]TA)-glucocorticoid receptor complexes partially purified by two-stage DNA-cellulose chromatography. The presence of anti-human glucocorticoid receptor antibodies was verified by: (a) adsorption of [3H]TA-receptor-antibody complexes to Protein A; (b) a shift to higher apparent molecular weight in the elution position from Sephacryl S300 of [3H]TA-receptor complexes incubated with immune serum; and (c) the ability of immune serum to displace [3H]TA-receptor complexes on sucrose gradients. These antibodies also recognized rat liver and murine S49 cell glucocorticoid receptors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]DM-labeled IM-9 cytosol identified a major competable band with a molecular weight of approximately 90,000, three minor competable components with molecular weights of approximately 78,000, approximately 51,000, and approximately 38,500, and at least 21 other noncompetable components. Following immunoprecipitation of [3H]DM-labeled cytosol with immune serum, only the Mr 90,000 and 78,000 components were seen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]DM-labeled CEM-C7 cytosol revealed a larger number of [3H]DM-labeled components. However, after immunoprecipitation of [3H]DM-labeled CEM-C7 cytosol, a predominant competable component with a molecular weight of 90,000 was easily identified. This component was markedly diminished when cytosols from the glucocorticoid receptor-deficient cell line ICR-27 were used. Thus, the combination of affinity labeling and anti-human glucocorticoid receptor antibodies is capable of providing direct physical identification of human lymphoid glucocorticoid receptors.

Region-specific antiglucocorticoid receptor antibodies selectively recognize the activated form of the ligand-occupied receptor and inhibit the binding of activated complexes to deoxyribonucleic acid.

A synthetic 18-amino acid peptide (Cys500-Lys517) was used to raise polyclonal antibodies in rabbits to the glucocorticoid receptor (GR). The sequence of this peptide is identical to that of residues 500-517 of the rat and 481-498 of the human GR. This sequence overlaps the carboxy-terminal end of the core DNA-binding domain and the amino-terminus of the hinge region of the receptor. Antiserum (AP64) was obtained which recognized both human and rat GR, as determined by immunoblots of receptors immunopurified with authentic anti-GR antibodies, immunoadsorption of both specific [3H]dexamethasone-bound GR and 98K receptors that were specifically covalently labeled by [3H]dexamethasone mesylate, and AP64-induced shifts in the elution position of monomeric [3H]dexamethasone-bound GR from Sephacryl S-300. The specificity of AP64 was demonstrated by the ability of the immunizing peptide, but not a peptide of similar length, to inhibit both the antibody-induced change in elution position from Sephacryl S-300 and the antibody-mediated immobilization of [3H]dexamethasone-bound complexes by protein-A. Further studies indicated that AP64 did not react with native steroid-free GR or with steroidbound (or affinity-labeled) unactivated GR, but did selectively associated with monomeric activated, steroid-bound (or affinity labeled) complexes. AP64 also inhibited the DNA binding of activated complexes in a manner that was specifically blocked by the immunizing peptide. Collectively, these data allow the direct localization of a structural region of the GR that is occluded in the unactivated complex but exposed as a result of activation.

Glucocorticoid receptor expression in receptorless mutants isolated from the human leukemic cell line CEM-C7.

The molecular basis for the loss of steroid binding activity in receptorless (r-) glucocorticoid-resistant (dexr) mutants isolated from the glucocorticoid-sensitive (dexs) cell line CEM-C7 was investigated. Although there was little binding of the reversibly associating ligand [3H]dexamethasone in r- mutants, labeling with the covalent affinity ligand [3H] dexamethasone 21-mesylate revealed significant amounts of a 92 kilodalton human glucocorticoid receptor (hGR) protein. Immunoblots of hGR protein in r- and normal cells showed that r- mutants expressed approximately half the amount of immunoreactive hGR protein seen in dexs cells. Comparison of the genomic organization of the hGR genes in normal and mutant cells revealed no discernable differences in the structure, or dosage, indicating that the r- phenotype was not the result of gross deletion or rearrangement of the hGR genes. In addition, r- cells expressed the same 7 kilobase mRNA as normal cells. More importantly, the amount of hGR mRNA expressed in r- cells was never significantly less, and in some cases was greater than, that seen in normal cells, indicating that the decrease in immunoreactive hGR protein seen in r- cells is not the result of loss of hGR mRNA expression. Taken together with the known mutation rate of the hGR gene(s) in these cells, these results suggest that the hGR genes in dexs CEM-C7 cells are allelic and that dexs cells express both a normal hGR protein and one with an altered steroid binding site. Furthermore, they suggest that the r- phenotype is acquired as the result of mutation within the coding region of the originally functional allele, leading to loss of ligand binding and expression of immunoreactive product.

Transfection with aFGF cDNA improves wound healing.

Somatic gene therapy is a potentially useful strategy for the delivery of growth factors or cytokines to enhance wound healing. Experimental excisional and incisional wounds in impaired-healing diabetic mice (db/db) were treated with aFGF and with a plasmid coding for aFGF. A eukaryotic expression plasmid composed of the Hst signal peptide sequence in-frame with the human aFGF sequence was used. Transfection of tissues was accomplished either by direct plasmid uptake or by uptake facilitated with cationic liposomes. The results show that the closure of excisional wounds was significantly accelerated (p < 0.05) by topical application of human recombinant aFGF or by transfection with the aFGF plasmid but not by vehicle or control plasmid not containing the aFGF sequence. In incisional wounds, aFGF or transfection with the plasmid significantly increased the wound-breaking strength compared to their corresponding controls (p < 0.05). Quantitative histology of the plasmid-treated incisional wound sections revealed improved wound quality. The transcription of mRNA from human aFGF cDNA in the incisional wound tissue extracts was confirmed by RT-PCR, and the expressed aFGF was detected by immune dot blot and immunohistochemistry assays. The transfection was a transient process with a peak at 9 d in db/+ (littermates of the diabetic mice) incisional wounds, at 36 d in db/db incisional wounds, and at 27 d in db/db excisional wounds. Cells transfected with human aFGF occupied up to 6.4% of the transectional area in the wound sites. Thus, aFGF gene delivery resulted in both gene expression and a functional improvement in healing.

Activation of the rat kidney mineralocorticoid receptor.

Activation of the rat kidney mineralocorticoid receptor was investigated using diethylaminoethyl (DEAE)-cellulose, DNA-cellulose, and gel permeation chromatography. Specific labeling of the mineralocorticoid receptor was achieved by labeling with [3H]aldosterone in the presence of the pure glucocorticoid RU28362. The specificity of labeling was confirmed by the lack of immunoreactivity of [3H]aldosterone-labeled material with the monoclonal antiglucocorticoid receptor antibody BURG-1. The unactivated aldosterone-mineralocorticoid receptor complex did not bind to DNA-cellulose, was eluted from DEAE-cellulose at relatively high salt (195 mM KCl) concentration, and had an apparent Stokes radius when chromatographed on Sephacryl S300 of 6.3 nm. After activation, the aldosterone-mineralocorticoid receptor complex had increased affinity for DNA-cellulose and decreased affinity for DEAE-cellulose and appeared as a smaller complex when chromatographed on Sephacryl S300. These changes were blocked by sodium molybdate. Our results indicate that activation of the rat kidney mineralocorticoid receptor is analogous to activation of the glucocorticoid receptor and suggest that activation of the mineralocorticoid receptor involves dissociation of a multimeric receptor form.

Glucocorticoid-induced apoptosis and regulation of NF-kappaB activity in human leukemic T cells.

Glucocorticoid-induced apoptosis was investigated in glucocorticoid-sensitive 6TG1.1 and resistant ICR27TK.3 human leukemic T cells. Following glucocorticoid treatment of 6TG1.1 cells, chromatin fragmentation was observed after a delay of 24 h. Fragmentation was not observed in ICR27TK.3 cells containing mutant glucocorticoid receptors (L753F) that are activation-deficient but retain the ability to repress AP-1 activity. Nor was fragmentation observed after treatment with RU38486, indicating that repression of AP-1 activity is not involved. As described in other systems, fragmentation required ongoing protein synthesis. However, inhibition of protein synthesis with cycloheximide anytime during the first 18 h of steroid treatment was as effective in blocking chromatin fragmentation as inhibition for the entire period, suggesting that synthesis of a component with a rapid turnover rate is required. Dexamethasone treatment completely blocked 12-O-tetradecanoylphorbol 13-acetate induction of nuclear factor-kappaB (NF-kappaB) activity and elicited an increase in the amount of immunoreactive IkappaB alpha in sensitive 6TG1.1 cells but not in resistant ICR27TK.3 cells. In addition, mild detergent treatment of cell extracts indicated that a substantial amount of cytoplasmic NF-kappaB is complexed with IkappaB alpha or some other inhibitory factor. These results suggest that induction of a labile inhibitory factor such as IkappaB alpha may contribute to glucocorticoid-induced apoptosis.

Differential autoregulation of glucocorticoid receptor expression in human T- and B-cell lines.

Regulation of glucocorticoid receptor (GR) expression by its cognate ligand was examined in the glucocorticoid-sensitive human leukemic T-cell line 6TG1.1 and in the human B-cell line IM-9. In contrast to the decrease in GR mRNA seen in IM-9 cells after treatment with 1 microM dexamethasone for 16-18 h, treatment of 6TG1.1 cells resulted in an 8-fold increase in GR mRNA, as determined by Northern blot and RNase protection analysis, with a corresponding 3- to 4-fold increase in GR protein. Half-maximal induction of GR mRNA and protein in 6TG1.1 cells was observed between 10-100 nM dexamethasone, and inclusion of 1 microM RU 38486 completely blocked the effects of 100 nM dexamethasone, demonstrating that positive autoregulation of GR expression in 6TG1.1 cells is a receptor-mediated response. Positive autoregulation of GR expression was also observed in glucocorticoid-resistant CEM-C1 cells, which contain functional GR, but whose growth is unaffected by glucocorticoids. Thus, positive autoregulation is neither a consequence nor the sole cause of growth arrest. The degree of negative autoregulation in IM-9 cells and positive autoregulation in 6TG1.1 cells was unaffected by inhibition of protein synthesis with cycloheximide. Measurement of GR mRNA turnover in 6TG1.1 cells treated with actinomycin-D revealed a half-life of 2.5 h, which was unaffected by dexamethasone treatment. A similar half-life was determined in IM-9 cells and was also unaffected by steroid treatment. These results are consistent with the interpretation that glucocorticoid-mediated autoregulation of GR expression is a tissue-specific primary transcriptional response.

Role of serine phosphorylation of Stat5a in prolactin-stimulated beta-casein gene expression.

Milk production remains suppressed in mammals during late pregnancy despite high levels of lactogenic polypeptide hormones. At parturition, associated with a precipitous fall in circulating progesterone, rising glucocorticoid levels synergize with prolactin to initiate copious milk production. This synergy is mediated at least in part through the coordinated activation of glucocorticoid receptors and transcription factor Stat5, particularly Stat5a. Here we show that two proline-juxtaposed serine residues within the transactivation domain of Stat5a are phosphorylated in the mammary gland during late gestation and lactation, and that these phosphorylation sites inhibit the transcriptional activity of Stat5a in the absence of glucocorticoid receptor costimulation. Specifically, transfection assays revealed that phosphorylation of residues S725 and S779 of Stat5a cooperatively suppressed prolactin-stimulated transcription from the beta-casein promoter in both COS-7 kidney and MCF-7 mammary cells. This suppression was associated with shortened duration and reduced amplitude of nuclear DNA binding activity of wild type Stat5a relative to that of the serine phosphorylation-defective Stat5 mutant. However, costimulation of glucocorticoid receptors completely reversed the suppressive effect of Stat5a serine phosphorylation on beta-casein gene transcription. We propose that serine phosphorylation within the transactivation domain may limit the activity of Stat5a in the absence of proper coactivation by glucocorticoid receptors.