Post-translational modification of steroid receptors allows fine-tuning different properties of this family of proteins, including stability, activation, or interaction with co-regulators. Recently, a novel effect of phosphorylation on steroid receptor biology was described. Phosphorylation of human mineralocorticoid receptor (MR) on Ser-843, a residue placed on the ligand binding domain, lowers affinity for agonists, producing inhibition of gene transactivation. We now show that MR inhibition by phosphorylation occurs even at high agonist concentration, suggesting that phosphorylation may also impair coupling between ligand binding and receptor activation. Our results demonstrate that agonists are able to induce partial nuclear translocation of MR but fail to produce transactivation due at least in part to impaired co-activator recruitment. The inhibitory effect of phosphorylation on MR acts in a dominant-negative manner, effectively amplifying its functional effect on gene transactivation.Steroid receptors (SRs) 3 are part of the nuclear receptor superfamily of ligand-dependent transcription factors that modulate gene transcription in response to changes in steroid hormone levels (1). SRs present a modular architecture, with three well defined domains: an NH 2 -terminal activation domain (NTD), a central DNA binding domain, and a COOHterminal ligand binding domain (LBD). Generally, the apo receptor resides in the cytosol forming a heterocomplex with other proteins including chaperones such as heat-shock protein 90 (Hsp90). Ligand binding alters SR conformation, releasing it from the complex and promoting translocation to the nucleus, where it binds specific DNA sequences as a dimer and alters transcription of target genes by recruiting transcriptional co-regulators (2). Post-translational modifications, including phosphorylation, ubiquitylation, sumoylation, and acetylation, modulate SR stability and different steps on the activation pathway, including receptor dimerization, DNA binding, and interaction with co-regulators (3).Until recently, it was widely assumed that SR ligand affinity is an intrinsic property defined by the structure of the LBD and not subject to modulation by post-translational modifications. However, Shibata et al. (4) have shown that regulated phosphorylation of Ser-843 in the LBD of human mineralocorticoid receptor (MR), a canonical member of the SR subfamily of nuclear receptors, impairs its ability to mediate gene transactivation by lowering ligand affinity. MR is closely related to the glucocorticoid receptor (GR) and can be activated under physiological conditions by mineralocorticoids such as aldosterone and glucocorticoids such as cortisol or corticosterone. MR has a wide variety of physiological and pathophysiological functions, with a prominent role in regulating transepithelial ion and fluid transport, which is essential for extracellular volume homeostasis and, therefore, blood pressure control. Phosphorylation of MR Ser-843 is restricted to intercalated cells of the distal ...

The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily that transduces the biological effects of corticosteroids. Its best-characterized role is to enhance transepithelial sodium reabsorption in response to increased aldosterone levels. In addition, MR participates in other aldosterone- or glucocorticoid-controlled processes such as cardiovascular homeostasis, adipocyte differentiation or neurogenesis, and regulation of neuronal activity in the hippocampus. Like other steroid receptors, MR forms cytosolic heterocomplexes with heat shock protein (Hsp) 90), Hsp70, and other proteins such as immunophilins. Interaction with Hsp90 is thought to maintain MR in a ligand-binding competent conformation and to regulate ligand-dependent and -independent nucleocytoplasmatic shuttling. It has previously been shown that acetylation of residue K295 in Hsp90 regulates its interaction with the androgen receptor and glucocorticoid receptor (GR). In this work we hypothesized that Hsp90 acetylation provides a regulatory step to modulate MR cellular dynamics and activity. We used Hsp90 acetylation mimic mutant K295Q or nonacetylatable mutant K295R to examine whether MR nucleocytoplasmatic shuttling and gene transactivation are affected. Furthermore, we manipulated endogenous Hsp90 acetylation levels by controlling expression or activity of histone deacetylase 6 (HDAC6), the enzyme responsible for deacetylation of Hsp90-K295. Our data demonstrates that HDAC6-mediated Hsp90 acetylation regulates MR cellular dynamics but it does not alter its function. This stands in contrast with the down-regulation of GR by HDAC6, suggesting that Hsp90 acetylation may play a role in balancing relative MR and GR activity when both factors are co-expressed in the same cell.

Aldosterone binds to the mineralocorticoid receptor (MR) and exerts pleiotropic effects beyond enhancing renal sodium reabsorption. Excessive mineralocorticoid signaling is deleterious during the evolution of cardiac failure, as evidenced by the benefits provided by adding MR antagonists (MRA) to standard care in humans. In animal models of cardiovascular diseases, MRA reduce cardiac fibrosis. Interestingly diuretics such as torasemide also appear efficient to improve cardiovascular morbidity and mortality, through several mechanisms. Among them, it has been suggested that torasemide could block aldosterone binding to the MR. To evaluate whether torasemide acts as a MRA in cardiomyocytes, we compared its effects with a classic MRA such as spironolactone. We monitored ligand-induced nuclear translocation of MR-GFP and MR transactivation activity in the cardiac-like cell line H9C2 using a reporter gene assay and known endogenous aldosterone-regulated cardiac genes. Torasemide did not modify MR nuclear translocation. Aldosterone-induced MR transactivation activity was reduced by the MRA spironolactone, not by torasemide. Spironolactone blocked the induction by aldosterone of endogenous MR-responsive genes (Sgk-1, PAI-1, Orosomucoid-1, Rgs-2, Serpina-3, Tenascin-X), while torasemide was ineffective. These results show that torasemide is not an MR antagonist; its association with MRA in heart failure may however be beneficial, through actions on complementary pathways.

The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) has an essential role in aldosterone target tissues, conferring aldosterone selectivity for the mineralocorticoid receptor (MR) by converting 11β-hydroxyglucocorticoids to inactive 11-ketosteroids. Congenital deficiency of 11β-HSD2 causes a form of salt-sensitive hypertension known as the syndrome of apparent mineralocorticoid excess. The disease phenotype, which ranges from mild to severe, correlates well with reduction in enzyme activity. Furthermore, polymorphisms in the 11β-HSD2 coding gene (HSD11B2) have been linked to high blood pressure and salt sensitivity, major cardiovascular risk factors. 11β-HSD2 expression is controlled by different factors such as cytokines, sex steroids, or vasopressin, but posttranslational modulation of its activity has not been explored. Analysis of 11β-HSD2 sequence revealed a consensus site for conjugation of small ubiquitin-related modifier (SUMO) peptide, a major posttranslational regulatory event in several cellular processes. Our results demonstrate that 11β-HSD2 is SUMOylated at lysine 266. Non-SUMOylatable mutant K266R showed slightly higher substrate affinity and decreased Vmax, but no effects on protein stability or subcellular localization. Despite mild changes in enzyme activity, mutant K266R was unable to prevent cortisol-dependent MR nuclear translocation. The same effect was achieved by coexpression of wild-type 11β-HSD2 with sentrin-specific protease 1, a protease that catalyzes SUMO deconjugation. In the presence of 11β-HSD2-K266R, increased nuclear MR localization did not correlate with increased response to cortisol or increased recruitment of transcriptional coregulators. Taken together, our data suggests that SUMOylation of 11β-HSD2 at residue K266 modulates cortisol-mediated MR nuclear translocation independently of effects on transactivation.

The mineralocorticoid receptor (MR) is a nuclear receptor that transduces the biological effects of the corticosteroid hormone aldosterone, playing a key role in the regulation of transepithelial Na+ reabsorption, extracellular volume and blood pressure. MR forms cytosolic heterocomplexes with heat‐shock protein 90 (Hsp90), Hsp70 and other proteins such as immunophilins. Interaction with Hsp90 is essential to maintain MR in a ligand‐binding competent conformation and to regulate nucleocytoplasmatic shuttling. Acetylation of residue K295 in Hsp90 regulates its interaction with androgen and glucocorticoid receptors. In this work we hypothesized that Hsp90 acetylation may provide a regulatory step to modulate MR cellular dynamics and activity. We used Hsp90 acetylation mimic mutant K295Q or non‐acetylable mutant K295R to examine MR ligand‐dependent and independent nucleocytoplasmatic shuttling and gene transactivation function in cell models with different endogenous levels of Hsp90 expression. Furthermore, we manipulated endogenous Hsp90 acetylation levels by controlling expression or activity of histone deacetylase 6 (HDAC6), the enzyme responsible for deacetylation of Hsp90‐K295. Taken together, our data demonstrates that Hsp90 expression level and acetylation‐dependent activity are essential for regulating MR subcellular localization, ligand‐induced nuclear translocation and gene transactivation. Grant Funding Source: Supported by grants BFU2010‐16625 and CSD‐2008‐00005 from MINECO, Spain