Abstract 09: Mtor Inhibition Decreases Ligand-induced Mineralocorticoid Receptor Activation

Abstract

Introduction: ULK1 phosphorylates the MR at S843, decreasing its ligand binding and transcriptional activity. Angiotensin II-induced mTOR phosphorylation of ULK1 inactivates ULK1, preventing its phosphorylation of MR. Aim: Further elucidate the role of mTOR in the regulation of MR transcriptional activity. Methods: M1 mouse cortical collecting duct cells stably transduced with the rat MR cDNA and a MMTV- Gaussia luciferase reporter gene, were incubated with an mTOR activator and several inhibitors, +/- aldosterone or corticosterone. Similar studies were done after lentiviral transduction of CRISPR/gRNA for raptor and rictor genes or mutated MR (mu/S843A) cDNA. Results: mTOR inhibition significantly decreased ligand activation of the MR reporter gene, while the mTOR activator MHY1485 had no effect suggesting that mTOR is tonically active. MR activation induced by aldosterone and corticosterone was also decreased by CRISPR/gRNA gene knockdown of raptor and rictor, the adaptors of mTOR complex 1 and 2, respectively, supporting a role for mTOR. The mTOR inhibitor AZD8055 (AZD) reduced phospho-ULK1 and attenuated ligand-mediated MR transactivation in a dose-dependent manner. The ULK1 inhibitor MRT68921 increased MR transactivation. We speculated that mTOR decreased ULK1 activity by phosphorylating it, thereby preventing ULK1 phosphorylation of the MR at Serine (S843). However, when M1 cells were transduced with an MR cDNA in which S843 was replaced with Alanine that cannot be phosphorylated, ligand-induced activation of the mu/S843A MR was still decreased by AZD, but unchanged by MRT68921. This suggests that mTOR has an additional effect on MR activity unrelated to ULK1 activity. AZD also decreased P70S6K and AKT phosphorylation in these cells. Conclusions: mTOR phosphorylation of ULK1 prevents its phosphorylation of the MR and reduction of MR transcriptional activity. mTOR inhibitors and deletion of raptor and rictor decreased MR transcriptional activity. mTOR has additional positive effects on MR activity possibly related to its phosphorylation of AKT and P70S6K. Inhibition of mTOR action may be a useful target for mitigating excessive MR activation.