Abstract P102: Inhibition Of Mtor Decreases Angiotensin Ii-induced Steroidogenesis In Hac15 Cells

A recent study reported that mTOR inhibition suppressed plasma aldosterone in mice but not humans. We investigated the effects and molecular mechanisms of mTOR inhibition on steroidogenesis using the human adrenocarcinoma HAC15. Methods: HAC15 cells were incubated with an mTOR activator and several inhibitors including AZD8055 (AZD) at concentrations of 0 to 1000 nmol/L +/- 100 nmol/L Angiotensin II (AngII) to stimulate steroid synthesis. The raptor and rictor genes were deleted by lentiviral mediated transduction of CRISPR/gRNA. Their presence was demonstrated by Western blotting and immunocytochemical staining. Aldo and cortisol synthesis were measured by ELISA; steroidogenic enzymes, p70S6K and AKT phophorylation by western blot. Results: The raptor and rictor, adaptor proteins of mTOR complex 1 and 2, were demonstrated by Western blotting and IHC staining in HAC15 cells. The mTOR inhibitors decreased AngII aldosterone synthesis; AZD was the most effective. The mTOR activator MHY did not stimulate aldosterone synthesis. AZD significantly suppressed both Ang II-induced aldo and cortisol formation in a dose-dependent manner without altering cell proliferation and viability. AZD did not alter forskolin-induced aldo production, suggesting that AZD inhibition of aldo biosynthesis is adenylate cyclase-independent and is specific to the AngII signaling pathway. AZD dose-dependently suppressed AngII-induced steroidogenic enzymes including 3β-hydroxysteroid dehydrogenase-type 2, CYP17A1, StAR, and CYP11B2 protein levels. p70S6K and AKT phosphorylation levels were inhibited by AZD. As mTOR exerts its effects by forming complexes with adaptor proteins raptor and rictor, we studied the role of these individual complexes further. CRISPR/ gRNA-mediated raptor and rictor knockdown in HAC15 cells significantly decreased AngII-induced steroidogenic enzymes levels and aldo and cortisol production. Conclusion: These results suggest that mTOR signaling has a role in transducing the AngII signal for aldo and cortisol biosynthesis and that inhibition of mTOR is a therapeutic option for conditions associated with excessive RAS-mediated steroid synthesis.