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Mineralocorticoid receptors: mechanisms of ligand- and tissue-specific activation
thesisposted on 14.02.2017, 02:49 by Yang, Jun
The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors. It is critical for controlling sodium and potassium transport in the kidney but is also present in the heart, blood vessel walls, hippocampus and adipose tissue. Pathological activation of the MR in the heart causes cardiac fibrosis and failure which are ameliorated by MR antagonists. However, the clinical use of MR antagonists is limited by the renal side effect of hyperkalemia. A tissue-specific MR modulator that selectively antagonises the cardiac MR would be of significant therapeutic benefit. In vivo and in vitro studies have shown that MR activation, rather than elevated plasma aldosterone per se, mediates vascular inflammation and cardiac fibrosis, suggesting a novel mode of activation of the MR in heart failure. It is well known that aside from the physiological ligand, aldosterone, the MR can also be activated by the glucocorticoid hormones cortisol (in humans) and corticosterone (in rodents), which bind the MR with equal high affinity to aldosterone. Given that cortisol circulates at concentrations 100 – 1000 fold higher than aldosterone, it is likely to be the predominant ligand of the cardiac MR which lacks the cortisol-inactivating enzyme 11β hydroxysteroid dehydrogenase (11βHSD2). Our current understanding of the molecular mechanisms for ligand- and tissue-specific activation of the MR is limited. Studies from other nuclear receptors indicate a role for coregulator proteins. Coregulators are known to be critical for nuclear receptor-mediated gene expression. They are diverse in their structure and function, and a subset of these proteins may confer specificity to MR-mediated responses. It is currently unknown whether different physiological ligands can induce distinct MR conformations to permit differential coregulator recruitment and ligand-specific gene regulation. Furthermore, an understanding of the ligand- and tissue-specific signaling pathways of the MR is undermined by the limited repertoire of established MR coregulators. In the current study, M13 phage display was used to screen vast phage peptide libraries for MR-binding peptides in the presence of different ligands. Identification of ligand-selective MR-interacting peptides provides proof of concept that the MR does undergo ligand-specific alterations in conformation leading to differential peptide recruitment. Furthermore, a consensus motif, MPxLxxLL, was noted amongst MR-interacting peptides. Gem-associated protein 4 (Gemin4) was found to contain this motif and was characterised using transactivation assays, gene expression studies and immunofluorescence as a novel cell- and gene-specific corepressor of the MR. T7 phage display was then used to screen phage libraries containing tissue-specific cDNA libraries for biologically relevant MR-interacting proteins. Three novel coactivators were identified for the MR, i.e. eukaryotic elongation factor 1A1 (EEF1A1), structure-specific recognition protein 1 (SSRP1), and x-ray repair cross-complementing protein 6 (XRCC6). These proteins were found to regulate MR-mediated gene transcription in a cell-, ligand- and promoter-specific manner. Results of the current study not only expand the current repertoire of MR coregulators, but also provide novel insight for the mechanisms by which the MR may be able to modulate gene expression in a ligand- or tissue-selective manner. This in turn opens up the exciting possibility of designing therapeutic agents that target MR activity by modulating the interaction between the MR and specific coregulators so as to better separate desirable therapeutic efficacy from undesirable adverse effects.