Cross-reactivity of a monoclonal antiglucocorticoid receptor antibody BuGR1 with glucocorticoid and mineralocorticoid receptors of various species

The reactivity of a monoclonal antibody BuGR1, raised against glucocorticoid receptors of rat liver, with glucocorticoid and mineralocorticoid receptors of mammalian (rabbit) and amphibian (A6 cells) origin was examined. The glucocorticoid receptors of rabbit kidney and liver and of A6 cells were labeled with tritiated dexamethasone. The mineralocorticoid receptors were labeled with tritiated aldosterone in the presence or absence of RU26988, depending on whether aldosterone was bound to glucocorticoid receptors (A6 cells) or not (rabbit kidney), in addition to its binding to mineralocorticoid receptors. BuGR1 did not recognize mineralocorticoid receptors of A6 cells and rabbit kidney. BuGR1 cross-reacted with glucocorticoid receptors of rabbit liver and kidney but not of A6 cells, suggesting that the domain of glucocorticoid receptors recognized by BuRG1 could be present only in the mammalian species. The findings indicate that BuGR1 shows species differences as well as receptor class specificity.     

Affinity of corticosteroids for mineralocorticoid and glucocorticoid receptors of the rabbit kidney: effect of steroid substitution

Corticosteroid derivatives coupled in the C3, C7 or C17 position with a long aliphatic chain were synthesized in order to select a suitable ligand for the preparation of a biospecific affinity adsorbent for mineralocorticoid receptor purification. The affinity of these derivatives for mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) was explored in rabbit kidney cytosol. In this model, aldosterone bound to a single class of receptors with high affinity (Kd 1 nM) and mineralocorticoid specificity. RU26988, a highly specific ligand for GR, did not compete for these sites. The C7 and C17 positions were found to be of crucial importance in the steroid's interaction with the mineralocorticoid receptors, since the linkage of a long side chain in these positions induced complete loss of affinity. Hence, deoxycorticosterone no longer bound to MR after 17 beta substitution with a 9-carbon aliphatic chain. This loss of affinity was not observed for glucocorticoids. The 17 beta nonylamide derivative of dexamethasone still competed for GR. Increasing the length of the C7 side of the spirolactone SC26304 suppressed its affinity for MR. Finally, C3 was an appropriate position for steroid substitution. The 3-nonylamide of carboxymethyloxime deoxycorticosterone bound to MR but not to GR, and therefore constitutes a suitable ligand for the preparation of a mineralocorticoid adsorbent.     

Modulation of aldosterone receptors in rat kidney: effects of steroid treatment and potassium diet

The numbers of type I and type II aldosterone receptors in the kidney cytosol of adrenalectomized rats were estimated after animals were treated with various steroids, or fed with high or low potassium diets. Oestradiol and 5 beta-pregnane-3,20 dione, which exhibited no affinity for aldosterone receptors, did not modify the levels of type I or type II receptors. Cortisol, corticosterone, progesterone and spirolactones, which all competed with aldosterone for both types of receptor, reduced the number of type I sites, as does aldosterone itself. Steroid treatment has no appreciable effect on type II receptors. We conclude that type I receptors are modulated by steroids able to bind to aldosterone receptors and that steroid-receptor interaction is an essential step in the receptor modulation process. The effects of potassium on aldosterone receptor modulation were tested in adrenalectomized rats on hypo- or hyperkalaemic diets. No change in receptor levels was observed in the rats on a low potassium diet, but the number of type I receptors increased in animals on a high potassium diet. However, the effects of potassium on receptor modulation were of lesser magnitude than those of aldosterone agonists and antagonists.     

Corticosteroid hormones: mechanisms involved in the recognition of aldosterone by mineralocorticoid receptors

Aldosterone and cortisol, the major mineralocorticoid and glucocorticoid hormones in humans, are structurally very closed. Both hormones bind to the mineralocorticoid receptor (MR) with the same affinity. Nevertheless MR is preferentially activated by aldosterone, suggesting that the binding of these two hormones to MR involved some distinct contacts. We constructed a tridimensional model of the ligand-binding domain of the human MR, by taking as a template the structural data of the retinoid receptor associated with its ligand. The MR model allowed the identification of several residues involved in the interaction with aldosterone and cortisol. The residues Gln 776 and Arg 817 make hydrogen bonds with the 3-keto function and the residue Asn 770 with the C21-hydroxyl group. Analyses of the wild type and mutant MRs activities in response to corticosteroids bearing hydroxyl groups at various steroid skeleton position led to the following conclusions: 1) the interaction between the residue Asn 770 and the C21-hydroxyl group of corticosteroids is determinant for stabilizing the active MR conformation and 2) the stability of this conformation is enhanced by the 11-18 hemiketal group of aldosterone whereas it is decreased by the 11 beta- and 17 alpha-hydroxyl groups of cortisol. These results are discussed in the light of a model for the MR activation process.     

Conformational change in the human glucocorticoid receptor induced by ligand binding is altered by mutation of isoleucine 747 by a threonine

Limited proteolysis experiments were performed to study conformation changes induced by ligand binding on in vitro produced wild-type and I747T mutant glucocorticoid receptors. Dexamethasone-induced conformational changes were characterized by two resistant proteolysis fragments of 30 and 27 kDa. Although dexamethasone binding affinity was only slightly altered by the I747T substitution (Roux, S., Térouanne, B., Balaguer, P., Loffreda-Jausons, N., Pons, M., Chambon, P., Gronemeyer, H., and Nicolas, J.-C. (1996) Mol. Endocrinol. 10, 1214-1226), higher dexamethasone concentrations were required to obtain the same proteolysis pattern. This difference was less marked when proteolysis experiments were conducted at 0 degrees C, indicating that a step of the conformational change after ligand binding was affected by the mutation. In contrast, RU486 binding to the wild-type receptor induced a different conformational change that was not affected by the mutation. Analysis of proteolysis fragments obtained in the presence of dexamethasone or RU486 indicated that the RU486-induced conformational change affected the C-terminal part of the ligand binding domain differently. These data suggest that the ligand-induced conformational change occurs via a multistep process. In the first step, characterized by compaction of the ligand binding domain, the mutation has no effect. The second step, which stabilizes the activated conformation and does not occur at 4 degrees C, seems to be a key element in the activation process that can be altered by the mutation. This step could involve modification of the helix H12 position, explaining why the conformation induced by RU486 is not affected by the mutation.     

UV-C response of the ribonucleotide reductase large subunit involves both E2F-mediated gene transcriptional regulation and protein subcellular relocalization in tobacco cells

E2F factors are implicated in various cellular processes including specific gene induction at the G1/S transition of the cell cycle. We present in this study a novel regulatory aspect for the tobacco large subunit of ribonucleotide reductase (R1a) and its encoding gene (RNR1a) in the UV-C response. By structural analyses, two E2F sites were identified on the promoter of this gene. Functional analysis showed that, in addition to their role in the specific G1/S induction of the RNR1a gene, both E2F sites were important for regulating specific RNR1a gene expression in response to UV-C irradiation in non-synchronized and synchronized cells. Concomitantly, western blot and cellular analyses showed an increase of a 60 kDa E2F factor and a transient translocation of a GFP-R1a protein fusion from cytoplasm to nucleus in response to UV irradiation.     

Finerenone Impedes Aldosterone-dependent Nuclear Import of the Mineralocorticoid Receptor and Prevents Genomic Recruitment of Steroid Receptor Coactivator-1

Aldosterone regulates sodium homeostasis by activating the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. Hyperaldosteronism leads todeleterious effects on the kidney, blood vessels, and heart. Although steroidal antagonists such as spironolactone and eplerenone are clinically useful for the treatment of cardiovascular diseases, they are associated with several side effects. Finerenone, a novel nonsteroidal MR antagonist, is presently being evaluated in two clinical phase IIb trials. Here, we characterized the molecular mechanisms of action of finerenone and spironolactone at several key steps of the MR signaling pathway. Molecular modeling and mutagenesis approaches allowed identification of Ser-810 and Ala-773 as key residues for the high MR selectivity of finerenone. Moreover, we showed that, in contrast to spironolactone, which activates the S810L mutant MR responsible for a severe form of early onset hypertension, finerenone displays strict antagonistic properties. Aldosterone-dependent phosphorylation and degradation of MR are inhibited by both finerenone and spironolactone. However, automated quantification of MR subcellular distribution demonstrated that finerenone delays aldosterone-induced nuclear accumulation of MR more efficiently than spironolactone. Finally, chromatin immunoprecipitation assays revealed that, as opposed to spironolactone, finerenone inhibits MR, steroid receptor coactivator-1, and RNA polymerase II binding at the regulatory sequence of the SCNN1A gene and also remarkably reduces basal MR and steroid receptor coactivator-1 recruitment, unraveling a specific and unrecognized inactivating mechanism on MR signaling. Overall, our data demonstrate that the highly potent and selective MR antagonist finerenone specifically impairs several critical steps of the MR signaling pathway and therefore represents a promising new generation MR antagonist.     

E2F factors rate controls the dual role of CDE/E2F composite element: a model of E2F-regulated gene expression in plant development

The promoters of several E2F-regulated genes identified in plants contain a variety of E2F motifs, notably a composite element consisting of a "CDE-like element" C/GGCGG on one strand, described as repressor in animals, associated with an E2F element on the complementary strand. This detailed study throughout plant development using ribonucleotide reductase promoters, allows us to propose a model, where E2F and composite elements play a dual role. Such regulation is mainly conditioned by the availability of E2F factors in tissues and during the cell cycle in tobacco.