Complex human glucocorticoid receptor dim mutations define glucocorticoid induced apoptotic resistance in bone cells

A mutation in the D-loop of the second zinc finger of the DNA-binding domain of the human glucocorticoid receptor (hGR), A458T (GR(dim)), has been suggested to be essential for dimerization and DNA binding of the GR, and genetically altered GR(dim) mice survive, whereas murine GR knockout mice die. Interestingly, thymocytes isolated from the GR(dim) mice were reported to be resistant to glucocorticoid-induced apoptosis. To further evaluate the dim mutations in glucocorticoid-induced apoptosis, we stably expressed either the hGR(dim) (A458T) or the hGR(dim4) (A458T, R460D, D462C, and N454D) mutant receptors in human osteosarcoma (U-2 OS) cells that are devoid of hGR and unresponsive to glucocorticoids. We analyzed these cell lines by comparison with a stable expression hGRα U-2 OS cell line, which undergoes apoptosis after glucocorticoid treatment. Transient reporter gene assays with glucocorticoid response element-driven vectors revealed that the hGR(dim) mutation had diminished steroid responsiveness and cells carrying the hGR(dim4) mutation were unresponsive to steroid, whereas glucocorticoid-induced nuclear factor κB repression was unaffected by either mutation. Interestingly, both the hGR(dim) and hGR(dim4) receptors readily formed dimers as measured by immunoprecipitation. Examination of GR-mediated apoptosis showed that hGR(dim) cells were only partially resistant to apoptosis, whereas hGR(dim4) cells were completely resistant to glucocorticoid-induced cell death despite remaining sensitive to other apoptotic stimuli. Global gene expression analysis revealed that hGR(dim4) cells widely regulated gene expression but differentially regulated apoptotic mRNA when compared with cells expressing wild-type hGRα. These studies challenge conclusions drawn from previous studies of GR dim mutants.     

A novel human glucocorticoid receptor SNP results in increased transactivation potential

Glucocorticoids are one of the most widely used therapeutics in the treatment of a variety of inflammatory disorders. However, it is known that there are variable patient responses to glucocorticoid treatment; there are responders and non-responders, or those that need higher dosages. Polymorphisms in the glucocorticoid receptor (GR) have been implicated in this variability. In this study, ninety-seven volunteers were surveyed for polymorphisms in the human GR-alpha (hGRα), the accepted biologically active reference isoform. One isoform identified in our survey, named hGR DL-2, had four single nucleotide polymorphisms (SNPs), one synonymous and three non-synonymous, and a four base pair deletion resulting in a frame shift and early termination to produce a 743 amino acid putative protein. hGR DL-2 had a decrease in transactivation potential of more than 90%. Upon further analysis of the individual SNPs and deletion, one SNP, A829G, which results in a lysine to glutamic acid amino acid change at position 277, was found to increase the transactivation potential of hGR more than eight times the full-length reference. Furthermore, the hGRα-A829G isoform had a differential hyperactive response to various exogenous steroids. Increasing our knowledge as to how various SNPs affect hGR activity may help in understanding the unpredictable patient response to steroid treatment, and is a step towards personalizing patient care.     

Disruption of microtubules leads to glucocorticoid receptor degradation in HeLa cell line

The role of microtubules (MTs) in steroid hormone-dependent human glucocorticoid receptor (hGR) activation/translocation is controversial. It was demonstrated recently that colchicine (COL) down-regulates hGR-driven genes in primary human hepatocytes by a mechanism involving inhibition of hGR translocation to the nucleus. To investigate whether inhibition of hGR translocation is the sole reason for its inactivation, we used human cervical carcinoma cells (HeLa) as a model. Herein we present evidence that perturbation of microtubules in HeLa cells leads to rapid time- and dose-dependent degradation of hGR protein. Degradation is proteasome mediated as revealed by its reversibility by proteasome inhibitor MG132. Moreover, degradation was observed for neither wt-hGR nor hGR mutants S226A and K419A in transiently transfected COS-1 cells. On the other hand, c-jun N-terminal kinase (JNK) seems not to be involved in the process because JNK inhibitor 1,9-Pyrazoloanthrone (SP600125) does not reverse hGR degradation. Similarly, another hGR functional antagonist, nuclear factor kappa beta (NFkappaB), did not play any role in the degradation process.     

Glucocorticoid receptor represses the Dex-mediated induction of human androgen response element-linked Luc activity

A human androgen response element (hARE), identified within intron 8 of the human sterol regulatory element-binding protein cleavage-activating protein, interacts with both glucocorticoid receptor (GR) and androgen receptors (AR). The aim of this study was to test the hypothesis that human GR (hGR) might modulate the expression of a hARE-linked reporter gene by dexamethasone (Dex). The hypothesis was tested by: a) co-transfecting HepG2 cells with a hGR and a luciferase (Luc)-reporter gene for performing in vitro investigations and b) by their co-injection into the tail vein of mice for in vivo investigation. In vitro co-transfected cells and the in vivo co-injected mice were then treated with Dex. Our results have led us to concluded that both transfection and injection of the hGR leads to a repression in the Dex-mediated induction of hARE-linked Luc activity both in vitro and in vivo settings. These findings suggest that this assay system allows screening of drug candidates affecting to a signal transduction pathway of the GR and AR and may help in the future discovery and analysis of novel and selection of GR and AR agonists.     

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.     

Random silent mutagenesis in the initial triplets of the coding region: a technique for adapting human glutathione reductase-encoding cDNA to expression in Escherichia coli

The introduction of random silent mutations into the 5'-coding region of a human cDNA as the basis for successful expression in Escherichia coli is demonstrated in four steps. (1) Plasmid pUB200 containing the pRpL promoters of phage lambda was found not to serve as an expression vector for a unchanged human glutathione reductase (hGR)-encoding cDNA. (2) When this cDNA was expressed in a two-cistron context using high-copy-number plasmids, recombinant protein was detected in low yield (0.03% of the total cell protein). (3) Silent mutations were introduced into the triplets coding for the N-terminal amino acids. When screening E. coli colonies transformed with expression plasmids containing cDNA mutants, we identified adapted clones that produced hGR in up to 70-fold higher yield than the clone containing the unchanged cDNA. Sequence analyses of adapted cDNA species revealed lower G + C contents in the modified regions, suggesting altered mRNA structures. (4) When the adapted cDNA sequences were recloned in the vector which had failed to express unchanged hGR cDNA in step 1, synthesis of recombinant protein was as high as in step 3. This means that the yield of expression for adapted cDNA was at least 1000-fold higher than for unchanged cDNA. In conclusion, random silent mutations introduced into the translation initiation region of cDNA might be a useful technique for designing sequence features which favour gene expression.     

A new, lineage specific, autoup-regulation mechanism for human glucocorticoid receptor gene expression in 697 pre-B-acute lymphoblastic leukemia cells

Glucocorticoid (GC) steroid hormones induce apoptosis in acute lymphoblastic leukemia (ALL). Autoup-regulation of human GC receptor (hGR) levels is associated with sensitivity to GC-mediated apoptosis. Among the major hGR promoters expressed in 697 pre-B-ALL cells (1A, 1B, 1C, and 1D), only promoters 1C and 1D are selectively activated by the hormone. Promoter 1B is unresponsive, and promoter 1A is down-regulated by dexamethasone (Dex) in 697 cells, whereas they are both up-regulated in CEM-C7 T-ALL cells. Autoup-regulation of promoter 1C and 1D in 697 cells requires sequences containing GC response units (GRUs) (1C GRU, -2915/-2956; 1D GRU, -4525/-4559) that were identified previously in CEM-C7 cells. These GRUs potentially bind GR, c-myeloblastosis (c-Myb), and E-twenty six (Ets) proteins; 697 cells express high levels of c-Myb protein, as well as the E-twenty six family protein members, PU.1 and Spi-B. Dex treatment in 697 cells elevates the expression of c-Myb and decreases levels of both Spi-B and PU.1. Chromatin immunoprecipitation assays revealed the specific recruitment of GR, c-Myb, and cAMP response element-binding protein binding protein to the 1C and 1D GRUs upon Dex treatment, correlating to observed autoup-regulated activity in these two promoters. These data suggest a hormone activated, lineage-specific mechanism to control the autoup-regulation of hGR gene expression in 697 pre-B-ALL cells via steroid-mediated changes in GR coregulator expression. These findings may be helpful in understanding the mechanism that determines the sensitivity of B-ALL leukemia cells to hormone-induced apoptosis.     

Molecular modelling of the human glucocorticoid receptor (hGR) ligand-binding domain (LBD) by homology with the human estrogen receptor alpha (hERalpha) LBD: quantitative structure-activity relationships within a series of CYP3A4 inducers where induction is mediated via hGR involvement

The results of homology modelling of the human glucorticoid receptor (hGR) ligand-binding domain (LBD) based on the ligand-bound domain of the human estrogen receptor alpha (hERalpha) are reported. It is shown that known hGR ligands which induce the human cytochrome P450 enzyme CYP3A4 are able to fit the putative ligand-binding site of the nuclear hormone receptor and form hydrogen bonds with key amino acid residues within the binding pocket. Quantitative structure-activity relationships (QSARs) have been derived for hGR-mediated CYP3A4 induction which involve certain molecular structural and physicochemical properties of the ligand themselves, yielding good correlations (R=0.96-0.98) with fold induction of CYP3A4 known to be mediated via hGR involvement.     

Evaluation of the role of ligand and thermal activation of specific DNA binding by in vitro synthesized human glucocorticoid receptor

We have used a DNA-binding/immunoprecipitation assay to analyze the capacity of human glucocorticoid receptor (hGR), generated in rabbit reticulocyte lysates, to bind DNA. In vitro translated hGR was indistinguishable from native hGR, as determined by migration on sodium dodecyl sulfate-polyacrylamide gels, sedimentation on sucrose density gradients, and reactivity with antipeptide antibodies generated against hGR. In addition, cell-free synthesized hGR was capable of specific binding to glucocorticoid response element (GRE)-containing DNA fragments. Using this assay system, we have evaluated the contributions of ligand binding and heat activation to DNA binding by these glucocorticoid receptors. In vitro translated hGR was capable of selective DNA binding even in the absence of glucocorticoid. Treatment with dexamethasone or the antiglucocorticoid RU486 had no additional effect on the DNA-binding capacity when receptor preparations were maintained at 0 C (no activation). In contrast, addition of either ligand or antagonist in combination with a heat activation step promoted DNA binding by approximately 3-fold over that of heat-activated unliganded receptors. Agonist (dexamethasone) was slightly more effective in supporting specific DNA binding than antagonist (RU486). DNA binding by in vitro synthesized GR was blocked by the addition of sodium molybdate to the receptor preparations before steroid addition and thermal activation. Addition of KCl resulted in less DNA binding either due to blockage of DNA-receptor complex formation or disruption of the complexes. The specificity of DNA binding by cell-free synthesized hGR was analyzed further by examining the abilities of various DNAs to compete for binding to a naturally occurring GRE found in the mouse mammary tumor virus-long terminal repeat. Oligonucleotides containing the consensus GRE were the most efficient competitors, and fragments containing regulatory sequences from glucocorticoid-repressible genes were somewhat competitive, whereas single stranded oligonucleotides were unable to compete for mouse mammary tumor virus-long terminal repeat DNA binding, except when competitor was present at extremely high concentrations. Together these studies indicate that hGR synthesized in rabbit reticulocyte lysates displays many of the same properties, including GRE-specific DNA binding, observed for glucocorticoid receptor present in cytosolic extracts of mammalian cells and tissues. Similarities between the effects of dexamethasone and RU486 suggest that the antiglucocorticoid properties of RU486 do not occur at the level of specific DNA binding.     

Overexpression of full-length human glucocorticoid receptor in Spodoptera frugiperda cells using the baculovirus expression vector system

We have expressed a full-length human glucocorticoid receptor (hGR) in Spodoptera frugiperda (Sf9) cells using the baculovirus expression vector system (BEVS). The level of expression is approximately 100-fold greater than in CEM-C7 cells. Between 0.5-1.0 mg hGR can be generated per liter of Sf9 cell culture. The expressed hGR is capable of binding glucocorticoids with specificity and high affinity. Covalent labeling with 3H-dexamethasone mesylate and Western blot analysis using a polyclonal antibody indicate that the molecular weight of the expressed protein is approximately 94 k. The nonactivated receptor sediments as a 8-9S complex in sucrose gradients and can be heat activated to a 4S form. The activated receptor is capable of retarding the migration of a 23 base-pair DNA fragment containing the glucocorticoid response element from the tyrosine aminotransferase gene. These data indicate that the expressed GR displays characteristics identical to those of GR from mammalian cells. By scaling up this culture we can, for the first time, obtain enough purified full-length receptor for crystallographic and functional studies which could provide new insight into exactly how hGR works.