Regulation of activities of steroid hormone receptors by tibolone and its primary metabolites.

This work was undertaken (i) to study deeply the estrogen, androgen and progestative activities of tibolone and its metabolites (ii) to determine whether tibolone and its metabolites present glucocorticoid or mineralocorticoid activity. For this purpose, we used human cell lines bearing a luciferase gene with a responsive element under the control of human estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) or androgen receptor (AR) or chimeric Gal4 fusion with progesterone receptor (PR), glucocorticoid receptor (GR) or mineralocorticoid receptor (MR). The major tibolone metabolites, the two hydroxymetabolites, bind and activate ER with a preference for ERalpha. Tibolone and the Delta(4)-tibolone are agonists for AR and PR and surprisingly 3alpha- and 3beta-OH-tibolone are antagonists for them. Moreover we showed for the first time that tibolone and its primary metabolites are GR and MR antagonists with a stronger affinity for MR than for GR. In conclusion, tibolone by these actions on different receptors and by this capacity to transform in different metabolites, has more complex effects than initially supposed.

Identification of steroid ligands able to inactivate the mineralocorticoid receptor harboring the S810L mutation responsible for a severe form of hypertension.

The ability of steroid ligands to inactivate the human mineralocorticoid receptor (MR(WT)) has been shown to be due to their inability to contact Asn770, a residue of the H3 helix involved in stabilizing contacts with the H11-H12 loop region. However, all steroid ligands that display antagonist properties when bound to MR(WT), have been shown to activate a mutant receptor (MR(L810)) associated with a severe form of hypertension. Biochemical studies revealed that S810L mutation induces a change in the receptor conformation and increases the steroid-receptor complexes stability. From a three-dimensional model of the MR ligand-binding domain, it is likely that the S810L mutation causes a steric hindrance between the side chains of Leu810 (H5) and Gln776 (H3) that provokes a bending of the H3 helix. As a consequence, the positioning of MR(WT) antagonists within the ligand-binding cavity is modified in such a way that they can activate the mutant MR(L810). The results from biochemical studies also revealed that 5alpha-pregnan-20-one, 4,9-androstadiene-3,17-dione and RU486, unable to bind MR(WT), acted as potent MR(L810) antagonists.

Mutation of the androgen receptor at amino acid 708 (Gly-->Ala) abolishes partial agonist activity of steroidal antiandrogens.

Mutation of a single amino acid in the ligand-binding domain (LBD) of the human androgen receptor (hAR) can induce functional abnormalities in androgen binding, stabilization of active conformation, or interaction with coactivators. The Gly708Ala and Gly708Val substitutions are associated with partial and complete androgen insensitivity syndromes, respectively. In this work, we introduced Ala, Val, and aromatic Phe mutations at position 708 on helix H3 of the hAR-LBD and tested the functional and structural consequences on hAR activity in the presence of steroidal or nonsteroidal agonists and antagonists. The residues involved in the specific recognition of these androgen ligands were identified and analyzed in the light of in vitro biological experiments and the 3D hAR-LBD structure. Our study demonstrated that the Gly708Ala mutation influenced the agonist versus antagonist activity of the ligands and confirmed the crucial role of this residue within the ligand-binding pocket (LBP) in the modulation of androgen agonists. The Gly708Ala mutation transformed the antiandrogen cyproterone acetate (CPA), a partial agonist, into a pure antiandrogen, and the pure nonsteroidal antiandrogen hydroxyflutamide in a partial agonist. From the docking studies, we suggest that CPA acts on AR through the novel mechanism called "passive antagonism".