Christine M. Jewell

Characterization of mechanisms involved in transrepression of NF-kappa B 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.

The Dominant Negative Activity of the Human Glucocorticoid Receptor β Isoform SPECIFICITY AND MECHANISMS OF ACTION

Alternative splicing of the human glucocorticoid receptor gene generates a nonhormone binding splice variant (hGRβ) that differs from the wild-type receptor (hGRα) only at the carboxyl terminus. Previously we have shown that hGRβ inhibits the transcriptional activity of hGRα, which is consistent with reports of elevated hGRβ expression in patients with generalized and tissue-specific glucocorticoid resistance. The potential role of hGRβ in the regulation of target cell sensitivity to glucocorticoids prompted us to further evaluate its dominant negative activity in other model systems and to investigate its mode of action. We demonstrate in multiple cell types that hGRβ inhibits hGRα-mediated activation of the mouse mammary tumor virus promoter. In contrast, the ability of the progesterone and androgen receptors to activate this promoter is only weakly affected by hGRβ. hGRβ also inhibits hGRα-mediated repression of an NF-κB-responsive promoter but does not interfere with homologous down-regulation of hGRα. We show that hGRβ can associate with the heat shock protein hsp90 although with lower affinity than hGRα. In addition, hGRβ binds GRE-containing DNA with a greater capacity than hGRα in the absence of glucocorticoids. Glucocorticoid treatment enhances hGRα, but not hGRβ, binding to DNA. Moreover, we demonstrate that hGRα and hGRβ can physically associate with each other in a heterodimer. Finally, we show that the dominant negative activity of hGRβ resides within its unique carboxyl-terminal 15 amino acids. Taken together, our results suggest that formation of transcriptionally impaired hGRα-hGRβ heterodimers is an important component of the mechanism responsible for the dominant negative activity of hGRβ.

Proinflammatory cytokines regulate human glucocorticoid receptor gene expression and lead to the accumulation of the dominant negative beta isoform: a mechanism for the generation of glucocorticoid resistance.

Inflammatory responses in many cell types are coordinately regulated by the opposing actions of NF-κB and the glucocorticoid receptor (GR). The human glucocorticoid receptor (hGR) gene encodes two protein isoforms: a cytoplasmic alpha form (GRα), which binds hormone, translocates to the nucleus, and regulates gene transcription, and a nuclear localized beta isoform (GRβ), which does not bind known ligands and attenuates GRα action. We report here the identification of a tumor necrosis factor (TNF)-responsive NF-κB DNA binding site 5′ to the hGR promoter that leads to a 1.5-fold increase in GRα mRNA and a 2.0-fold increase in GRβ mRNA in HeLaS3 cells, which endogenously express both GR isoforms. However, TNF-α treatment disproportionately increased the steady-state levels of the GRβ protein isoform over GRα, making GRβ the predominant endogenous receptor isoform. Similar results were observed following treatment of human CEMC7 lymphoid cells with TNF-α or IL-1. The increase in GRβ protein expression correlated with the development of glucocorticoid resistance.

Molecular Origins for the Dominant Negative Function of Human Glucocorticoid Receptor Beta

ABSTRACT This study molecularly elucidates the basis for the dominant negative mechanism of the glucocorticoid receptor (GR) isoform hGRβ, whose overexpression is associated with human glucocorticoid resistance. Using a series of truncated hGRα mutants and sequential mutagenesis to generate a series of hGRα/β hybrids, we find that the absence of helix 12 is neither necessary nor sufficient for the GR dominant negative phenotype. Moreover, we have localized the dominant negative activity of hGRβ to two residues and found that nuclear localization, in addition to heterodimerization, is a critical feature of the dominant negative activity. Molecular modeling of wild-type and mutant hGRα and hGRβ provides structural insight and a potential physical explanation for the lack of hormone binding and the dominant negative actions of hGRβ.

Human Glucocorticoid Receptor β Binds RU-486 and Is Transcriptionally Active

ABSTRACT Human glucocorticoid receptor (hGR) is expressed as two alternately spliced C-terminal isoforms, α and β. In contrast to the canonical hGRα, hGRβ is a nucleus-localized orphan receptor thought not to bind ligand and not to affect gene transcription other than by acting as a dominant negative to hGRα. Here we used confocal microscopy to examine the cellular localization of transiently expressed fluorescent protein-tagged hGRβ in COS-1 and U-2 OS cells. Surprisingly, yellow fluorescent protein (YFP)-hGRβ was predominantly located in the cytoplasm and translocated to the nucleus following application of the glucocorticoid antagonist RU-486. This effect of RU-486 was confirmed with transiently expressed wild-type hGRβ. Confocal microscopy of coexpressed YFP-hGRβ and cyan fluorescent protein-hGRα in COS-1 cells indicated that the receptors move into the nucleus independently. Using a ligand binding assay, we confirmed that hGRβ bound RU-486 but not the hGRα ligand dexamethasone. Examination of the cellular localization of YFP-hGRβ in response to a series of 57 related compounds indicated that RU-486 is thus far the only identified ligand that interacts with hGRβ. The selective interaction of RU-486 with hGRβ was also supported by molecular modeling and computational docking studies. Interestingly, microarray analysis indicates that hGRβ, expressed in the absence of hGRα, can regulate gene expression and furthermore that occupation of hGRβ with the antagonist RU-486 diminishes that capacity despite the lack of helix 12 in the ligand binding domain.

Autoregulation of glucocorticoid receptor gene expression

Glucocorticoid receptors are members of a highly conserved family of steroid receptor proteins, which are ligand-dependent transcription factors. Previous studies have shown that the presence of functional glucocorticoid receptors is a prerequisite for manifestation of cellular responses to hormone. Glucocorticoid receptors undergo down-regulation following treatment with glucocorticoids. To define the molecular mechanisms that are involved in this process we have analyzed the down-regulation of glucocorticoid receptors both in HeLa cells, which contain endogenous receptors, and in cells containing receptors that have been introduced by DNA transfection. Our results show that cells that contain glucocorticoid receptors--either endogenous or transfected--undergo down-regulation of steroid-binding capabilities, as well as reductions in receptor protein and mRNA levels, in a remarkably similar fashion. DNA sequences in the coding region of the human glucocorticoid receptor cDNA appear to be sufficient to account for down-regulation of receptor. This novel finding suggests that unique mechanisms are involved in controlling glucocorticoid receptor homeostasis.

Regulation of the human glucocorticoid receptor by long-term and chronic treatment with glucocorticoid.

HeLa S3 cells that contain endogenous glucocorticoid receptors (GR) were treated with dexamethasone (DEX) for periods of time ranging from 24 h to 2 weeks or chronically over a 2-year period. Regulation of GR protein and mRNA were examined by affinity labeling, Western blotting, and Northern blotting. Relatively short-term treatment of cells with DEX for 24 or 48 h revealed more profound down-regulation of GR protein than of GR mRNA. However, by 2 weeks of DEX treatment, the levels of both receptor protein and mRNA were both maximally down-regulated. Cells that had been chronically DEX treated (for up to 2 years) had no measurable GR protein or mRNA. The down-regulation of receptor protein and RNA that occurred after 2 weeks of DEX treatment is completely reversible upon DEX removal, whereas reversibility did not occur with cells that had been chronically treated with DEX. Furthermore, transfection of a glucocorticoid responsive reporter plasmid into these chronically DEX-treated cells demonstrated that these cells were no longer responsive to steroid treatment. However, cotransfection of a plasmid encoding the human GR into these chronically DEX-treated cells resulted in restored production of GR and responsiveness to hormone, indicating that the defect in these cells occurs only at the receptor level.

Generating diversity in human glucocorticoid signaling through a racially diverse polymorphism in the beta isoform of the glucocorticoid receptor

Alternative splicing of the human glucocorticoid receptor gene generates two isoforms, hGRα and hGRβ. hGRβ functions as a dominant-negative regulator of hGRα activity and but also has inherent transcriptional activity, collectively altering glucocorticoid sensitivity. Single-nucleotide polymorphisms in the 3′ UTR of hGRβ have been associated with altered receptor protein expression, glucocorticoid sensitivity, and disease risk. Characterization of the hGRβ G3134T polymorphism has been limited to a relatively small, homogenous population. The objective of this study was to determine the prevalence of hGRβ G3134T in a diverse population and assess the association of hGRβ G3134T in this population with physiological outcomes. In a prospective cohort study, 3730 genetically diverse participants were genotyped for hGRβ G3134T and four common GR polymorphisms. A subset of these participants was evaluated for clinical and biochemical measurements. Immortalized human osteosarcoma cells (U-2 OS), stably transfected with wild-type or G3134T hGRβ, were evaluated for receptor expression, stability, and genome-wide gene expression. Glucocorticoid-mediated gene expression profiles were investigated in primary macrophages isolated from participants. In a racially diverse population, the minor allele frequency was 74% (50.7% heterozygous carriers and 23.3% homozygous minor allele), with a higher prevalence in Caucasian non-Hispanic participants. After adjusting for confounding variable, carriers of hGRβ G3134T were more likely to self-report allergies, have higher serum cortisol levels, and reduced cortisol suppression in response to low-dose dexamethasone. The presence of hGRβ G3134T in U-2 OS cells increased hGR mRNA stability and protein expression. Microarray analysis revealed that the presence of the hGRβ G3134T polymorphism uniquely altered gene expression profiles in U-2 OS cells and primary macrophages. hGRβ G3134T is significantly present in the study population and associated with race, self-reported disease, and serum levels of glucocorticoids. Underlying these health differences may be changes in gene expression driven by altered receptor stability.

Regulation of Glucocorticoid Receptor Protein and Gene Expression by Glucocorticoids

Glucocorticoids, members of the highly conserved steroid hormone family in evolution, exert numerous physiological effects on the developmental and adaptational processes of eukaryotic organisms. In general, free circulating glucocorticoids released from the adrenal cortex in response to adrenocorticotropic hormone (ACTH) from the pituitary enter target cells by a passive diffusion process conferred by the high lipid solubility common to all steroids. Although there have been some reports on membrane effects of steroid hormones, actions of steroid hormones appear to be largely mediated by their intracellular receptors. These receptors act much like ligand-dependent transcription factors that regulate gene expression. The magnitude of steroid hormone responses, therefore, is determined not only by the hormone concentration but also by cellular levels of functional receptor proteins. Proposed models for the structure of the glucocorticoid receptor (GR) have the common feature that the receptor exists as an oligomeric protein complex that consists of one or more subunits complexed with non-steroid-binding proteins such as the heat shock protein 90 (hsp90). Dissociation of the oligomeric complex takes place following binding of the hormone and permits translocation of the steroid-receptor complex into the nucleus where it associates with enhancer-like glucocorticoid response elements (GREs) in the genome. This could either induce the expression of normally silent genes or change the transcription of constitutively expressed housekeeping genes (such as the GR gene itself) by up- or down-regulation. Thus to fully understand the mechanism underlying glucocorticoid action, the study of GR autoregulation becomes an imperative issue.