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Updated: Aug 23, 2025
Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions
Published on: March 12, 2019
Mammalian Target of Rapamycin Inhibition Decreases Angiotensin II-Induced Steroidogenesis in HAC15 Human
Yusuf Ali1,2, Elise P Gomez-Sanchez2, Celso E Gomez-Sanchez1,2
1G. V. (Sonny) Montgomery, VA Medical Center, Jackson, MS, USA.
Background:
Mammalian target of rapamycin (mTOR) inhibitors suppress adrenal cortical carcinoma cell proliferation and cortisol production; the relationship between mTOR and aldosterone production has not been examined.
Methods:
HAC15 cells were incubated with an mTOR activator and several inhibitors including AZD8055 (AZD) in the presence and absence of angiotensin II (AngII). The expression of rapamycin-sensitive adapter protein of mTOR (Raptor) and rapamycin-insensitive companion of mTOR (Rictor), adaptor proteins of mTOR complex 1 and 2, respectively, were studied in the HAC15 cells and deleted by CRISPR/gRNA.
Results:
The mTOR inhibitors decreased aldosterone induced by AngII. Inhibition of mTOR by AZD significantly suppressed AngII-induced aldosterone and cortisol formation in a dose-dependent manner, whereas the mTOR activator MHY had no effect. AZD did not alter forskolin-induced aldosterone production showing that it is specific to the AngII signaling pathway. AngII-mediated ERK and mTOR activation were suppressed by AZD, along with a concomitant dose-dependent reduction of AngII-induced steroidogenic enzymes including steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase-type 2, CYP17A1, and aldosterone synthase protein. Furthermore, mTOR components ribosomal protein S6 kinase (P70S6K) and protein kinase B phosphorylation levels were decreased by AZD. As mTOR exerts its main effects by forming complexes with adaptor proteins Raptor and Rictor, the roles of these individual complexes were studied. We found an increase in the phosphorylation of Raptor and Rictor by AngII and that their CRISPR/gRNA-mediated knockdown significantly attenuated AngII-induced aldosterone and cortisol production.
Conclusion:
mTOR signaling has a critical role in transducing the AngII signal initiating aldosterone and cortisol synthesis in HAC15 cells and that inhibition of mTOR could be a therapeutic option for conditions associated with excessive renin-angiotensin system-mediated steroid synthesis.
Insights
Mammalian target of rapamycin (mTOR) inhibitors reduce aldosterone and cortisol production by suppressing angiotensin II signaling in adrenal cells. This suggests mTOR inhibition may treat conditions with excessive steroid synthesis.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Signaling
Background:
- Mammalian target of rapamycin (mTOR) inhibitors are known to reduce adrenal cortical carcinoma cell proliferation and cortisol production.
- The specific role of mTOR in aldosterone production, however, remained unexamined.
Purpose of the Study:
- To investigate the role of mTOR signaling in aldosterone and cortisol production stimulated by angiotensin II (AngII) in HAC15 cells.
- To evaluate the potential of mTOR inhibition as a therapeutic strategy for conditions involving excessive steroid synthesis.
Main Methods:
- HAC15 cells were treated with an mTOR activator (MHY) and inhibitors (AZD8055), with or without AngII.
- Expression and function of mTOR components (Raptor, Rictor) were analyzed, including knockdown via CRISPR/gRNA.
- Effects on steroidogenic enzyme expression and signaling pathways (ERK, P70S6K, protein kinase B) were assessed.
Main Results:
- mTOR inhibition by AZD8055 significantly suppressed AngII-induced aldosterone and cortisol production in a dose-dependent manner.
- AZD8055 specifically targeted the AngII pathway, not forskolin-induced production, and reduced AngII-mediated ERK and mTOR activation.
- Knockdown of mTOR components Raptor and Rictor attenuated AngII-induced steroid production, implicating mTOR complexes in this process.
Conclusions:
- mTOR signaling is crucial for mediating AngII-induced aldosterone and cortisol synthesis in HAC15 cells.
- Inhibiting mTOR presents a potential therapeutic approach for disorders characterized by excessive renin-angiotensin system-mediated steroid synthesis.
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