The mineralocorticoid receptor (MR) is a transcription factor expressed in many cells that regulates the expression of genes in involved myriad cell type-specific functions. Inappropriate activation of the MR causes hypertension and pathological tissue remodeling even in conditions of normal aldosterone levels. Activity of the MR is regulated by post-translational modifications including phosphorylation of serines and threonines. Phosphorylation activates or suppresses MR activity depending upon the amino acid involved. The kinase ULK1 was recently reported to phosphorylate serine 843 of the MR, thereby suppressing its activity, and to be expressed with the MR only in intercalated cells of the distal nephron. Our own specific antibodies reveal more extensive co-expression of ULK1 and MR in the kidney. ULK1 expression is widespread in the body and in multiple cell lines. Other kinases also have the potential to phosphorylate the MR at S843, thus inhibit MR activity. The MR has similar affinity for aldo, cortisol or corticosterone. Specificity for aldo is conferred by conversion of cortisol and corticosterone to the inactive cortisone and 11-dehydrocorticosterone by the 11?-hydroxysteroid dehydrogenase 2 (11-HSD2), however this enzyme is not present in most non-epithelial aldo target tissues. Cortisol and corticosterone are also converted to 20?-dihydro-cortisol and -corticosterone by carbonyl reductase 1 (Cbr1). We that found the 20?-dihydro metabolites do not activate the MR, thus providing an alternative mechanism for MR specificity for aldo where 11-HSD2 is not expressed. Hypotheses: 1) Phosphorylation of the MR at serine 843 is an important negative regulator of MR action that occurs in multiple tissues. 2) Reduction of the 20-keto of corticosterone and cortisol by carbonyl reductase 1 regulates ligand selectivity for the MR by converting corticosterone and cortisol into the inactive metabolites 20?-dihydrocorticosterone (20?-DHC) and 20?-dihydrocortisol. Both potentially modulate MR activity.
Specific Aim 1 : Study the role of phosphorylation of the MR at serine 843 (human) on genomic and non- genomic activity of the MR. Study the effects of over-expression and suppression of several kinases, ULK1, ULK2, TBK1, NEK2 and PAK1, with putative ability to phosphorylate the MR at S843 in cells expressing an MR reporter gene system. Study the distribution and co-localization of the MR with ULK1, ULK2 and other kinases that catalyze S843 phosphorylation in the kidney, heart, vessels and brain.
Specific Aim 2 : Study the role of the 20-keto reduction of cortisol and corticosterone on MR ligand selectivity. Determine the dynamics of the 20-keto reduction of corticosterone and cortisol by the human and rat Cbr1 in cells. Measure the conversion of corticosterone to 20?-dihydrocorticosterone in various tissues including in micropunches from specific areas of the rat brain where we demonstrate that Cbr1 and MR are co-expressed by immunofluorescent histochemistry. Measure plasma levels of corticosterone and 20?-DHC in plasma and tissues of rats fed different amounts of salt. The mechanisms for the cell-specific modulation of MR activity will never be known if not explored and are crucial for the development of rational therapy with minimal side effects for inappropriate MR activation.
Aldosterone is a hormone that acts through the mineralocorticoid receptor to promote the retention of sodium and water in the kidney and maintain normal blood pressure. The mineralocorticoid receptor also can bind the more abundant hormones cortisol and corticosterone. These competing hormones are degraded within cells by an enzyme 11?-hydroxysteroid dehydrogenase 2 (11-HSD2) allowing the specific action of aldosterone. We have evidence that another enzyme the carbonyl reductase 1 can also inactivate them and contribute to the specificity of aldosterone in tissues where the 11-HSD2 is not present. Mineralocorticoid receptor activity is increased or decreased by adding phosphate groups to different parts of the molecule. We plan to study how the addition of a phosphate to position 843, which decreases its action, is regulated by studying the enzymes that add and remove the phosphate.