Mercury inhibits rat liver and kidney glucocorticoid receptor hormone binding activity

The present study was focused on the influence of mercury on the rat liver and kidney glucocorticoid receptor (GR) binding properties. The time-course and dose-dependence of mercury effects, as well as possible involvement of thiol groups were examined after in vivo and in vitro administration of the metal in the form of HgCl2. Mercury led to reduction of the liver and kidney GR hormone binding capacity. In both examined tissues maximal reduction was noticed 4 h after administration of the metal at 2 and 3 mg Hg/kg bw, but the effect was more prominent in kidney as compared to liver. On the other hand, binding affinity in the two tissues was similar. The complete reversal of mercury effects on GR binding capacity by 10 mmol/L DTT was achieved in liver and partially in kidney. The reversal by DTT suggested that mercury caused the decrease of GR binding activity by interacting with thiol groups. The difference in the response of the two tissues reflected the fact that kidney contained a higher mercury concentration and a lower thiol content in comparison to liver. The implicated thiols probably belong to GR, since when applied in vitro at 0 degrees C, mercury produced reduction of the receptor binding activity similar to that observed in vivo. GR protein level examined by quantitative Western blot was either unchanged, when determined by polyclonal antibody, or reduced, when determined by BuGR2 antibody, suggesting that Hg might affect BuGR epitope availability.     

Steroid derivatives for electrophilic affinity labelling of glucocorticoid binding sites: interaction with the glucocorticoid receptor and biological activity

To investigate the possible use of electrophilic affinity labelling for the characterization of glucocorticoid receptors, different chemically reactive derivatives of deoxycorticosterone (deoxycorticosterone 21-mesylate and deoxycorticosterone 21-(1-imidazole) carboxylate), dexamethasone (dexamethasone 21-mesylate, dexamethasone 21-iodoacetate and dexamethasone 21-bromoacetate) and progesterone (21-chloro progesterone) were tested for their ability to bind irreversibly to the glucocorticoid receptor from goat lactating mammary gland. Using partially purified receptor, only one of the steroids tested, dexamethasone 21-mesylate (DXM-M) was found more effective than dexamethasone (DXM) in preventing exchange of radioactive dexamethasone in the receptor binding site. The affinity of DXM-M for the glucocorticoid receptor, measured by competitive binding assay, was 1/15 that of DXM. Polyacrylamide gel electrophoresis in sodium dodecyl sulphate of the [3H]-DXM-M labeled glucocorticoid receptor revealed a specific covalently radiolabeled fraction corresponding to an apparent molecular weight of 75,000 to 80,000. The biological activity of DXM-M was studied in RPMI 3460-clone 6 Syrian hamster melanoma cells, a cell line which is sensitive to growth inhibition by glucocorticoids. Like DXM, DXM-M inhibits the growth of RPMI 3460-clone 6 cells and it acts as a slowly reversible glucocorticoid agonist at concentrations which correlate with the affinity of DXM-M for the glucocorticoid receptor in vitro.     

Discovery of novel phosphorus-containing steroids as selective glucocorticoid receptor antagonist

Mifepristone is a non-selective antagonist of 3-oxosteroid receptors with both abortifacient and anti-diabetic activities. For glucocorticoid receptor (GR) program, we sought an unexplored, synthetically accessible phosphorus-containing steroidal mimetic of mifepristone, suitable for parallel synthesis of analogues. One compound 4a, with high oral bioavailability (59%) in rat, exhibited functional antagonism of GR in oral glucose tolerance test (OGTT). Thus this series of compounds might be potentially useful for the treatment of type II diabetes.     

The involvement of receptro sulphydryl groups in the binding of steroids to the cytoplasmic glucocorticoid receptor from rat thymus.

The glucocorticoid receptor protein present in the high-speed supernant fraction of rat thymus tissue is extremely unstable, having a half-life of about 2 h at 4 degrees C. It was found that the decline in steroid-binding capacity could be slowed, though not arrested completely, by the addition of sulphydryl-protecting agents such as 2-mercaptoethanol or dithiothreitol, and by EDTA. The inactivation was also partly reversed by these agents. 0.5 mM N-ethylmaleimideor p-chloromercuriphenylsulphonic acid inactivated the recptor at 4 degrees C, but the presence of bound steroid protected the receptor against this inactivation. Bound steroid did not protect the receptor against the action of higher concentrations of these reagents. Treatment of intact thymus cells with 2,4-dinitrophenol resulted in a reduction in the steroid-binding capacity of the supernatant fraction derived from these cells. This effect of 2,4-dinitrophenol could not be reversed by the presence of dithiothreitol in the extraction buffer. It is concluded that the inactivation of the receptor in vitro is at least partly due to the oxidation of one or more sulphydryl groups necessary for steroid binding; the process of oxidation does not account for the reduction in steroid binding observed in intact thymus cells under conditions of energy deprivation.     

Redox manipulation of DNA binding activity and BuGR epitope reactivity of the glucocorticoid receptor.

Treatment of the transformed glucocorticoid receptor with hydrogen peroxide promotes the formation of disulfide bonds and inhibits the ability of the receptor to bind to DNA (Tienrungroj, W., Meshinchi, S., Sanchez, E. R., Pratt, S. E., Grippo, J. F., Holmgren, A., and Pratt, W. B. (1987) J. Biol. Chem. 262, 6992-7000). It has not been determined whether the inhibition of DNA binding activity is due to disulfide bonds formed within the DNA binding domain or between the DNA binding domain and another region of the receptor. In this paper, we examined the ability of hydrogen peroxide to inactivate the DNA binding activity of the mouse glucocorticoid receptor. We show that inhibition of DNA binding activity caused by hydrogen peroxide can be accounted for entirely by the formation of disulfide bonds between cysteine residues lying within the 15-kDa tryptic fragment containing the DNA binding domain of the receptor. Reversal of the peroxide-induced inactivation of DNA binding activity requires both zinc and a thiol-disulfide exchange reagent, such as dithiothreitol. Peroxide also eliminates recognition of the intact receptor and the 15-kDa tryptic fragment by the BuGR monoclonal antibody, and the reactivity of the BuGR epitope is restored by reduction without a requirement for zinc. Pretreatment of the receptor with methyl methanethiosulfonate inhibits much of the peroxide-mediated inactivation of the BuGR epitope but pretreatment with N-ethylmaleimide does not. Similarly, DNA binding activity of the receptor is inhibited by methyl methanethiosulfonate but not by N-ethylmaleimide. These results are consistent with the proposal that peroxide promotes the formation of disulfide bonds between thiols that lie spatially close to one another in the 15-kDa tryptic fragment, resulting in rapid elimination of zinc. Restoration of the zinc finger structure restores DNA-binding activity but restoration of the BuGR epitope requires only reduction without restoration of the zinc fingers.     

Mapping the HSP90 binding region of the glucocorticoid receptor

In animal cells, unliganded steroid receptors are complexed with a 90-kDa heat shock protein, HSP90; hormone binding by the receptor leads to the release of HSP90. We found that the 795-amino acid rat glucocorticoid receptor protein formed oligomeric complexes in vitro upon synthesis in rabbit reticulocyte lysates; these oligomers also dissociated in the presence of hormone. Similar complexes formed when X795, a receptor derivative containing only the C-terminal half (amino acids 407-795) of the protein, was translated in vitro. Moreover, X795 was co-immunoadsorbed from the reticulocyte lysates together with HSP90 by three different anti-HSP90 monoclonal antibodies, indicating that the in vitro translated receptor binds HSP90 and that the interaction occurs within the C-terminal half of the receptor. To localize the HSP90 binding region in greater detail, various deletion mutants of X795 were translated in vitro and assayed for oligomer formation and for co-immunoadsorption with HSP90. The results indicated that HSP90 interacted with the receptor within a subregion of the hormone binding domain, between amino acids 568 and 616. These findings are consistent with the proposal that HSP90 may participate in the mechanism of signal transduction by steroid receptors.     

Insights into functional modulation of catalytic RNA activity

RNA molecules play critical roles in cell biology, and novel findings continuously broaden their functional repertoires. Apart from their well-documented participation in protein synthesis, it is now apparent that several noncoding RNAs (i.e., micro-RNAs and riboswitches) also participate in the regulation of gene expression. The discovery of catalytic RNAs had profound implications on our views concerning the evolution of life on our planet at a molecular level. A characteristic attribute of RNA, probably traced back to its ancestral origin, is the ability to interact with and be modulated by several ions and molecules of different sizes. The inhibition of ribosome activity by antibiotics has been extensively used as a therapeutical approach, while activation and substrate-specificity alteration have the potential to enhance the versatility of ribozyme-based tools in translational research. In this review, we will describe some representative examples of such modulators to illustrate the potential of catalytic RNAs as tools and targets in research and clinical approaches.     

Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids.     

Thermodynamics of steroid binding to the human glucocorticoid receptor.

The thermodynamics of the interaction of glucocorticoids with their receptor were studied in cytosol from human lymphoblastoid cells. The rate and affinity constants of dexamethasone and cortisol between 0 degree and 25 degrees C were calculated by curve-fitting from time-course and equilibrium kinetics. The data were consistent with a simple reversible bimolecular interaction. Arrhenius and Van't Hoff plots were curvilinear for both steroids. At equilibrium, the solution for the equation delta G = delta H - T X delta S (eqn. 1) was (in kJ X mol-1) -47 = 36 - 83 (dexamethasone) and -42 = -9 - 33 (cortisol) at 0 degree C. Enthalpy and entropy changes decreased quasi-linearly with temperature such that, at 25 degrees C, the respective values were -50 = -75 + 25 and -43 = -48 + 5. Thus, for both steroids, the interaction was entropy-driven at low temperature and became entirely enthalpy-driven at 20 degrees C. Thermodynamic values for the transition state were calculated from the rate constants. For the forward reaction, eqn. (1) gave 45 = 84 - 39 (dexamethasone) and 46 = 60 - 14 (cortisol) at 0 degree C, and 44 = 24 + 20 (dexamethasone) and 46 = 28 + 18 (cortisol) at 25 degrees C. These data fit quite well with a two-step model [Ross & Subramanian (1981) Biochemistry 20, 3096-3102] proposed for ligand-protein interactions, which involves a partial immobilization of the reacting species governed by hydrophobic forces, followed by stabilization of the complex by short-range interactions. On the basis of this model, an analysis of the transition-state thermodynamics led to the conclusion that no more than half of the steroid molecular area is engaged in the binding process.     

In vitro modulation of rat liver glucocorticoid receptor by urea

We have examined the influence of urea on the properties of the rat liver glucocorticoid receptor (GR). A 1-h incubation of hepatic cytosol with 1-3 M urea at 0 or at 23 degrees C caused a progressive decrease in the steroid binding efficiency of GR. Urea treatment of cytosol incubated with 20 nM [3H]triamcinolone acetonide caused transformation of glucocorticoid-receptor complexes (GRc) and resulted in an increase in the binding of GRc to DNA-cellulose and ATP-Sepharose. The transforming effect was maximal with 2.5 M urea at 0 degrees C for 1 h, and it caused a shift in the rate of sedimentation of the 9 S untransformed GRc to a 4 S form, similar to that observed upon incubation of the cytosol GRc at 23 degrees C. This 9 to 4 S transformation could also be observed in the presence of Na2MoO4. The Stokes radii of the GRc eluted from a Bio-Gel-A-0.5m agarose column were determined to be 5.9 and 4.9 nm in the absence and presence of 2.5 M urea. The aqueous two-phase partitioning analysis revealed a significant change in surface properties of GR following urea treatment; the observed partition coefficient values (cpm upper phase/bottom phase) were 0.022, 0.208, and 0.60 for GRc, GRc + 23 degrees C, and GRc + 2.5 M urea, respectively. Furthermore, the urea treatment rendered the GRc less negatively charged, forcing their appearance in the flow-through fractions of a DEAE-Sephacel column. These results suggest that urea is a potent in vitro modulator of the physicochemical behavior of GR, influencing both the steroid binding and the process of receptor transformation.     

Submicromolar free calcium modulates dexamethasone binding to the glucocorticoid receptor

The relative distribution of bound cis- and trans-(NH₃)₂PtCl₂ at specific sites in SV40 DNA is evaluated by monitoring the extent to which five restriction endonucleases, each of which cleave at a single, unique site, are inhibited as a result of the DNA modification. The order of cleavage inhibition is Bgl 1 ? Bam HI > Hpa II, Kpn I > Eco RI. Both isomers produce a comparable effect for any particular endonuclease. Inhibition correlates with the % (G+C) content within and about the recognition sequences. That modified sequences immediately adjacent to the recognition sequence influence cleavage is further supported by differential cleavage observed with the multicut Hind III endonuclease. The binding of cis-(NH₃)₂PtCl₂ at the hyper-reactive Bgl 1 site may well be directly responsible for inhibiting SV40 replication.     

DNA binding specificity of mutant glucocorticoid receptor DNA-binding domains

Mutation of a small number of amino acids in the DNA-binding domain of the estrogen receptor to the corresponding sequence of the glucocorticoid receptor switches the specificity of the receptor in transactivation assays (Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989) Nature 338, 271-274). We have made the corresponding reciprocal mutations in the context of the glucocorticoid receptor DNA-binding domain and studied the binding of wild type and mutant purified proteins to palindromic glucocorticoid and estrogen response elements as well as to elements of intermediate sequence, using gel mobility shift assays. We show here that a protein with two altered amino acids binds glucocorticoid and estrogen response elements with a low but equal affinity, whereas a protein with an additional changed residue has a high affinity for estrogen response elements but still retains a considerable affinity for glucocorticoid response elements. Using binding sites of intermediate sequence we have further characterized the interaction with DNA. The in vitro DNA binding results are confirmed by in vivo transactivation assays in yeast. Finally we suggest a testable model for amino acid/base pair interactions involved in recognition by the glucocorticoid receptor DNA-binding domain of its target sequence.     

DNA binding analysis of glucocorticoid receptor specificity mutants.

The glucocorticoid receptor (GR) DNA binding domain consists of several conserved amino acids and folds into two zinc finger-like structures. Previous transactivation experiments indicated that three amino acids residing in this region, Gly, Ser and Val, appear to be critical for target-site discrimination. Based on the solved crystal structure, these residues are at the beginning of an amphipathic alpha-helix that interacts with the DNA's major groove; of these, only valine, however, contacts DNA. In order to examine their functional role directly, we have substituted these residues for the corresponding amino acids from the estrogen receptor (ER), overexpressed and purified the mutant proteins, and assayed their binding specificity and affinity by gel mobility shifts using glucocorticoid or estrogen response elements (GRE or ERE, respectively) as DNA probes. We find that all three residues are indeed required to fully switch GR's specificity to an ERE. The contacting valine in GR is of primary importance. The corresponding residue in ER, alanine, is less important for specificity, while glutamic acid, four amino acids towards the N-terminus, is most critical for ER discrimination. Finally, we show that the GR DNA binding domain carrying all three ER-specific mutations has a significantly higher affinity for an ERE than the ER DNA binding domain itself. We interpret these results in the context of both the data presented here and the crystal structure of the GR DNA binding domain complexed to a GRE.     

Localization of pyrodoxal phosphate binding site on the mero-receptor domain of the glucocorticoid receptor

Previous studies have demonstrated that the vitamin pyridoxal phosphate can alter the physicochemical properties of glucocorticoid receptors. We now report the localization of a pyridoxal phosphate binding site within the mero-receptor domain of this glucocorticoid receptor. Mero-glucocorticoid receptors that are generated by trypsin (10 μg/ml) or chymotrypsin (100 μg/ml) digestion of intact receptors sediment as 2.6 S species on 5–20% sucrose gradients in the presence or absence of pyridoxal phosphate. Mero-glucocoritcoid receptors prepared by exogenous proteinases are hydrophobic and show no affinity for DEAE Bio-Gel A. Treating either trypsin-generated or chymotrypsin-generated mero-receptors with pyridoxal phosphate rapidly converts the proteins (60 and 35%, respectively) into forms that bind to DEAE Bio-Gel A. Induction of DEAE binding is specific to pyridoxal phosphate, for treating mero-receptors with pyridoxal, pyridoxamine or pyridoxine phosphate is ineffective. Furthermore, DEAE binding cannot be induced by adding other pyridoxal phosphate-treated cytosols to untreated mero-receptors. High-resolution polyacrylamide gel isoelectric focussing studies indicated that treating mero-receptor generated by either proteinase with pyridoxal phosphate shifted the isoelectric points of lower pH values. The conversion of the mero-receptor to a more acidic form also occurred when the intact glucocorticoid receptor was treated with the vitamin prior to proteolysis. These studies localize at least one pyridoxal phosphate binding site on the mero-receptor domain of the rat thymocyte glucocorticoid receptor.