Inhibition of the production of mediators of inflammation by corticosteroids is a glucocorticoid receptor-mediated process

In order to find an explanation for corticosteroid resistance we assessed whether inhibition by dexamethasone (DEX) of the stimulated production of TNF- proportional, variant, IL-6, PGE(2) and LTB(4) by peripheral blood mononuclear cells (MNC) depends on binding to the glucocorticoid receptor (GR), and whether it is determined by the number or the affinity of the GR of these cells. GR number and affinity of MNC were determined by means of a whole cell DEX binding assay. MNC were incubated with DEX and LPS or A23187 in the absence or presence of RU486, a potent steroid antagonist. DEX caused a concentration dependent inhibition of TNF- proportional, variant, IL-6 and PGE(2) production but had no effect on LTB(4) production. RU486 significantly blocked the effect of DEX, but no correlations were found between the inhibition of mediator release and the K(d) or receptor number.     

Glucocorticoid receptor expression in human bronchial epithelial cells: effects of smoking and COPD.

Previously, we found that inflammatory mediators modulated the number and binding affinity of glucocorticoid receptors (GR) in human bronchial epithelial cell lines. In this study we investigated whether smoking and chronic obstructive pulmonary disease (COPD), both characterized by airway inflammation with increased levels of inflammatory mediators, affect GR characteristics in cultured human bronchial epithelial cells (HBEC). A statistically significant difference was found between the dissociation constant (Kd) values in HBEC from smoking (Kd = 0.98+/-0.08 nM; n = 6) and nonsmoking controls (Kd = 0.76+/-0.10 nM, P = 0.03; n = 5), but no significant difference was found between the mean number of binding sites. Our results are the first indication that cultured HBEC from smokers possess GR with a lower binding affinity. This may result from the inflammation found in the airways from smokers. Furthermore, these results provide further evidence that the bronchial epithelium may be an actual target for inhaled glucocorticoid therapy.     

The responses of rat liver glucocorticoid receptors and genes for tyrosine aminotransferase, alpha-2-macroglobulin and gamma-fibrinogen to adrenalectomy-, dexamethasone- and inflammation-induced changes in the levels of glucocorticoids and proinflammatory cytokines

The responses of liver glucocorticoid receptor (GR) and genes coding for a glucocorticoid-inducible tyrosine aminotransferase (TAT) and two acute-phase proteins (APP) [alpha2-macroglobulin (alpha2-M) and gamma-fibrinogen (Fb)] to changes in glucocorticoid (GC) and proinflammatory (AP) cytokine contents have been examined in rats after single or combined treatments with turpentine oil, dexamethasone (Dex) and adrenalectomy. Activation of two APP genes in turpentine-induced inflammation was accompanied by an increase in the level of GR mRNA and a preferential translocation of GR-GC complexes to the nucleoplasm, while the expression of TAT remained unaltered. Dex alone caused a decrease in the levels of GR and Fb mRNAs, activation of TAT and alpha2-M genes, a decrease in the affinity of hormone binding sites and redistribution of translocated GR-Dex complexes within the nuclei. Inflammation potentiated the effect which Dex alone exerted on the GR content and the number of GR binding sites but counteracted its influence on the affinity of GR binding sites and nuclear distribution of GR-Dex complexes. Adrenalectomy promoted a fall in TAT mRNA, no changes in the GR and Fb mRNA, a decrease in the affinity of GR hormone binding sites and redistribution of GR-hormone complexes within the nuclei. The AP cytokines released in response to inflammation exerted a counteracting effect on the adrenalectomy-induced changes in the affinity of hormone binding sites and nuclear distribution of GR-hormone complexes. They potentiated a fall of TAT mRNA but promoted full expression of the Fb gene. These results argue strongly for the influence of AP cytokines on the functional state of the GR and GC signaling pathways.     

Discovery of nonsteroidal glucocorticoid receptor ligands based on 6-indole-1,2,3,4-tetrahydroquinolines

A series of nonsteroidal glucocorticoid receptor (GR) ligands based on a 6-indole-1,2,3,4-tetrahydroquinoline scaffold are reported. Structure-activity relationship (SAR) of the pendent indole group identified compound 20 exhibiting good GR binding affinity (K(i)=1.5nM) and 100- to 1000-fold selectivity over MR, PR, and AR while showing activity in an E-selectin repression assay.     

Somatostatin increases glucocorticoid binding and signaling in macrophages by blocking the calpain-specific cleavage of Hsp 90

Somatostatin has direct anti-inflammatory actions and participates in the anti-inflammatory actions of glucocorticoids, but the mechanisms underlying this regulation remain poorly understood. The objective of this study was to evaluate whether somatostatin increases glucocorticoid responsiveness by up-regulating glucocorticoid receptor (GR) expression and signaling. Somatostatin promoted a time- and dose-dependent increase in [(3)H]dexamethasone binding to RAW 264.7 macrophages. Cell exposure to 10 nM somatostatin for 18 h promoted a 2-fold increase in the number of GR sites per cell without significant modification of the affinity. Analysis of GR heterocomplex components demonstrated that somatostatin increased the level of heat shock protein (Hsp) 90, whereas the level of GR remained almost unchanged. The increase in Hsp 90 was associated with a decrease in the cleavage of its carboxyl-terminal domain. Evidence for the involvement of calpain inhibition in this process was obtained by the demonstration that 1) somatostatin induced a dose-dependent decrease in calpain activity and 2) calpain inhibitors, calpain inhibitor I and calpeptin, both abolished the cleavage of Hsp 90 and induced a dose-dependent increase in [(3)H]dexamethasone binding. Increases in glucocorticoid binding after somatostatin treatment were associated with similar increases in the ability of GR to transactivate a minimal promoter containing two glucocorticoid response elements (GRE) and to interfere with the activation of nuclear factor-kappaB (NF-kappaB). Thus, the present findings indicate that somatostatin increases glucocorticoid binding and signaling by limiting the calpain-specific cleavage of GR-associated Hsp 90. This mechanism may represent a novel target for intervention to increase glucocorticoid responsiveness.     

Accessory factors facilitate the binding of glucocorticoid receptor to the phosphoenolpyruvate carboxykinase gene promoter

Glucocorticoid induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene requires a glucocorticoid response unit (GRU) comprised of two non-consensus glucocorticoid receptor (GR) binding sites, GR1 and GR2, and at least three accessory factor elements (gAF1-3). DNA-binding accessory proteins are commonly required for the regulation of genes whose products play an important role in metabolism, development, and a variety of defense responses, but little is known about why they are necessary. Quantitative, real time homogenous assays of cooperative protein-DNA interactions in complex media (e.g. nuclear extracts) have not previously been reported. Here we perform quantitative, real time equilibrium and stopped-flow fluorescence anisotropy measurements of protein-DNA interactions in nuclear extracts to demonstrate that GR binds to the GR1-GR2 elements poorly as compared with a palindromic or consensus glucocorticoid response element (GRE). Inclusion of either the gAF1 or gAF2 element with GR1-GR2, however, creates a high affinity binding environment for GR. GR can undergo multiple rounds of binding and dissociation to the palindromic GRE in less than 100 ms at nanomolar concentrations. The dissociation rate of GR is differentially slowed by the gAF1 or gAF2 elements that bind two functionally distinct accessory factors, COUP-TF/HNF4 and HNF3, respectively.     

Efficient binding of glucocorticoid receptor to its responsive element requires a dimer and DNA flanking sequences

A combination of the gel retardation assay and interference by hydroxyl radical modification (missing nucleoside technique) was used to analyze the interaction of the glucocorticoid receptor (GR) with various glucocorticoid responsive elements (GRE). Short oligonucleotides containing the 15-bp GRE and 1 to 3 flanking base pairs on each side, are bound with very low affinity. The same GREs, when positioned in the center of a large DNA fragment (40-50 bp), show high affinity for the receptor. However, when the GRE is positioned at the border of a 54-bp fragment, the affinity of the GR for the GRE decreases markedly. The DNA binding affinity increases linearly with each added flanking base pair and optimal binding is observed with 8-10 flanking bp. Thus, the nonconserved DNA sequences flanking the GRE contribute significantly to the free energy of receptor binding to DNA. Using larger DNA fragments (greater than 100 bp) and a smaller form of the receptor (40 kD), two retarded complexes are found that correspond to monomeric and homodimeric receptor DNA complexes. The DNA-binding domain of the GR (20 kD), expressed in bacteria, binds to the GRE as a monomer as well as a dimer and can form heterodimers with the native 94-kD GR. Insertion or deletion of one single base pair between the two halves of the GRE reduces the affinity for the homodimeric form of the native GR, and inhibits the function of the GRE in gene transfer experiments, suggesting that a dimer of the GR is the functional entity that binds to the GRE.     

Quantitative analysis of the glucocorticoid receptor-DNA interaction at the mouse mammary tumor virus glucocorticoid response element

Purified glucocorticoid receptor (GR) from rat liver was used for a quantitative analysis of the protein-DNA interaction at specific GR-binding segments within the 5'-long terminal repeat of the mouse mammary tumor virus. A truncated receptor was generated and used to demonstrate formation of heterodimeric GR, which furthermore was shown to be in rapid equilibrium with receptor-monomer. The relative affinity for GR binding to specific GR sites versus random calf thymus DNA was approximately 2 x 10(3). At equilibrium a free GR concentration of 3 x 10(-10) M was required for half-maximal saturation of the two functionally important DNA sites within the mouse mammary tumor virus 5'-long terminal repeat. Although these two DNA segments act synergistically in mediating hormonal response, we did not detect cooperative GR binding to these regions in vitro. However, GR bound cooperatively within the downstream binding region. Similarly, GR was unable to facilitate factor binding to a neighboring nuclear factor 1 site, another essential element in the promoter. In contrast, nuclear factor 1 binding was inhibited slightly by GR.     

Assembly of a glucocorticoid receptor complex prior to DNA binding enhances its specific interaction with a glucocorticoid response element

Gel retardation analysis with full- and half-palindromic sequences using partially purified glucocorticoid receptor (GR) resulted in GR-glucocorticoid response element (GRE) species of identical mobilities, suggesting that formation of the dimeric GR protein complex is not catalyzed by DNA binding. These results are in contrast to the behavior of the isolated DNA binding domain of the glucocorticoid receptor where dimerization occurred on the GRE. Density gradient centrifugation of cytosolic GR resulted in two forms, a 4 S peak characteristic of the monomeric GR and a fraction which sediments at 6 S which is consistent with the observed size of the dimeric GR. These two forms were found to differ in their ability to bind to specific DNA sequences with the 6 S species having a higher affinity for a GRE. Taken together our results are consistent with a two-step model for hormone-induced transformation of GR: dissociation of the multimeric untransformed complex and dimerization of the GR to yield a high affinity DNA binding species.     

Circadian and seasonal variations of glucocorticoid receptors in normal human lymphocytes

Circulating human lymphocytes are known to contain specific glucocorticoid receptors. Using a competitive binding whole cell assay, we have examined the binding of [3H] dexamethasone to peripheral lymphocytes of normal male subjects. Lymphocytes were found to contain 2000-10000 glucocorticoid receptor sites/cell and the Kd value was in the range of 0.5-9 X 10(-9) M. The number and affinity of glucocorticoid receptors did not change throughout a 1-year observation time. In contrast, there was a significant diurnal variation in receptor content (38% higher at 11 p.m. than at 8 a.m.), while receptor affinity did not change.     

Interindividual glucocorticoid sensitivity in young healthy subjects: the role of glucocorticoid receptor alpha and beta isoforms ratio.

Only few studies have addressed the interindividual variation and tissue specificity of glucocorticoid (GC) sensitivity in healthy individuals, a phenomenon observed in pathological conditions. Alternative splicing of the glucocorticoid receptor (GR) produces alpha and beta isoforms. GRbeta has dominant-negative effects on hormone-induced GRalpha effects, and an increased expression of the GRbeta has been associated with glucocorticoid resistance. We determined, using a simple, rapid, and accurate Real-Time PCR assay, the individual mRNAs expression of GRalpha and GRbeta in 26 normal subjects (mean+/-SE, age 30+/-6 years; 12 males and 14 females), in order to evaluate the role of these isoforms in glucocorticoid sensitivity in health. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as a housekeeper gene. GRalpha/GAPDH, GRbeta/GAPDH and GRalpha/GRbeta ratios showed a normal distribution. We observed a higher expression of GRalpha compared to GRbeta and an interindividual variability in the GRalpha, GRbeta, and GAPDH gene expressions in the young healthy population. In addition, no correlation was observed between GRalpha/GRbeta ratio and the dexamethasone (DEX) doses needed to suppress plasma cortisol, GRalpha/GRbeta ratio and the concentration of DEX that caused inhibition of Con-A stimulated cell proliferation, and GRalpha/GRbeta ratio and the affinity of GR (Kd) of each subject. Therefore, the variability of GC sensitivity observed in normal subjects can not be ascribed to the variation in the GRalpha and GRbeta expression.     

Size and steroid-binding characterization of membrane-associated glucocorticoid receptor in S-49 lymphoma cells.

The precise mechanism for glucocorticoid-mediated lymphocytolysis is not understood, although it is presumed to be receptor mediated. We have recently presented evidence that this response is mediated by a specialized form of the glucocorticoid receptor (GR) that resides in the plasma membrane (mGR). Confirmation of the previous receptor identification studies in a population of S-49 cells enriched for mGR is now made using another antibody specific for the rodent GR, BUGR-2. The membrane resident receptor could be labeled competitively with the affinity ligand dexamethasone 21-mesylate, and Scatchard analysis of whole cell binding revealed that receptor number, but not the affinity for hormone, varied between the mGR-enriched and -deficient cell populations. Steroid specificity displacement analyses showed an order of affinities as follows: triamcinolone acetonide greater than progesterone greater than dexamethasone greater than testosterone = estrogen. Studies of mGR by one- and two-dimensional gel electrophoresis, immunoblot, autoradiography, and density gradients revealed a species with an equivalent size to cytosolic receptor as well as multiple higher molecular weight species, confirming earlier studies. To offer a possible explanation for the nucleic acid origins of the mGR, RNA from the mGR-enriched cells was probed with rat GR cDNA; mGR-enriched cells contained higher levels of GR mRNA. Possible molecular etiologies of larger receptor species in membrane are discussed.     

Discovery and SAR study of novel dihydroquinoline containing glucocorticoid receptor ligands

We report the discovery of a novel class of glucocorticoid receptor (GR) ligands based on 1,2-dihydroquinoline molecular scaffold. The compounds exhibit good GR binding affinity and selectivity profile against other nuclear hormone receptors.     

2-Benzenesulfonyl-8a-benzyl-hexahydro-2H-isoquinolin-6-ones as selective glucocorticoid receptor antagonists

The 2-azadecalin ring system was evaluated as a scaffold for the preparation of glucocorticoid receptor (GR) antagonists. High affinity, selective GR antagonists were discovered based on a hypothetical binding mode related to the steroidal GR antagonist RU-43044. 2-Benzenesulfonyl substituted 8a-benzyl-hexahydro-2H-isoquinolin-6-ones exemplified by (R)-37 had low nanomolar affinity for GR with moderate functional activity (200 nM) in a reporter gene assay. These compounds were devoid of affinity for other steroidal receptors (ER, AR, MR, and PR). Analogues based on an alternative putative binding mode (CP-like) were found to be inactive.