Mammalian Target of Rapamycin Inhibition Decreases Angiotensin II-Induced Steroidogenesis in HAC15 Human Adrenocortical Carcinoma Cells

Abstract

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.

Synthesis of aldosterone occurs in the zona glomerulosa (ZG) of the adrenal gland and is regulated primarily by the renin–angiotensin system (RAAS). This involves a rate-limiting transfer of cholesterol from outer to the inner mitochondrial membrane through the steroidogenic acute regulatory (StAR) protein where the CYP11A1 catalyzes the formation of pregnenolone; leaving the mitochondria, the 3β-hydroxysteroid dehydrogenase-type 2 (3β-HSD2) converts pregnenolone to progesterone, which is then hydroxylated to deoxycorticosterone by CYP21A2. Finally, aldosterone is biosynthesized through a series of reactions in the mitochondria mediated by CYP11B2, the sole enzyme unique to the formation of aldosterone (1). Inappropriate activation of the RAAS leads to aldosterone excess that is responsible for sodium and fluid retention, vascular dysfunction, myocardial fibrosis, and cardiac arrhythmias, all of which contribute to the development of hypertension and pathological renal and cardiovascular remodeling. In the normal adrenal gland, most enzymes are common to the ZG and zona fasciculata (ZF); however, the CYP17A1 and CYP11B1, required for cortisol synthesis, are in the ZF. Cortisol secretion in vivo is under the regulation of the hypothalamic-pituitary axis through ACTH (2).

The complex mechanisms involved in the regulation of aldosterone production remain incompletely understood. Aldosterone production in the ZG cells is stimulated by the hormones angiotensin II (AngII) and ACTH, as well as paracrine factors including growth factors, neuropeptides, and neurotransmitters (3). AngII binding to the AngII type-I receptor induces aldosterone biosynthesis by activating several intracellular signaling cascades, including ERK, mitogen-activated protein kinase, calcium/calmodulin dependent kinases, and protein kinase C (1). ACTH activates the cAMP-PKA-CREB signaling cascade by binding to melanocortin 2 receptor (MC2R) (3). The mechanistic target of rapamycin (mTOR) is a serine-threonine protein kinase of the phosphatidylinositol 3-kinase/protein kinase B (AKT) (PI3-K/Akt) signaling pathway that acts as a gatekeeper of cell metabolism and growth and receives signals from sensors detecting intracellular nutrients levels, several growth factors, and cell stress. mTOR regulates myriad physiological functions, including transcription and translation of genes; cell cycle progression; and cell differentiation, motility, metabolism, apoptosis, and autophagy (4–7). mTOR is composed of 2 distinct molecular complexes, mTOR complex 1 (mTORC1), containing rapamycin-sensitive adapter protein of mTOR (Raptor), which is essential for mTORC1 activity, and mTOR complex 2 (mTORC2), containing rapamycin-insensitive companion of mTOR (Rictor), which is required for mTORC2 activity, along with other proteins (8, 9).

Excessive activation of the PI3-K/AKT/mTOR signaling occurs in aldosterone-producing adenomas and increases aldosterone secretion and plasma aldosterone level is positively correlated with AKT and mTOR activation (10). Inhibition of mTOR was reported to decrease proliferation and cortisol production by cultured human adrenocortical carcinoma (ACC) cells (11) and Trinh et al (12) demonstrated that mTORC1 inhibition suppressed plasma aldosterone levels in mice. Plasma aldosterone levels were not decreased in humans receiving an mTORC1 inhibitor, despite a reduction in blood pressure and increased renin levels (12).

We have investigated the impact of mTOR inhibition on adrenal steroidogenesis and molecular mechanisms involved using HAC15 human ACC cells (13). HAC15 cells and the parent cell line H295R are the only available immortal cell models of the human adrenal cortex that express all enzymes necessary to synthesize both aldosterone and cortisol in response to AngII (14). HAC15 cells do not express the MC2R accessory protein necessary to respond to ACTH stimulation of steroidogenesis; the adenylyl cyclase activator forskolin is used to stimulate steroid secretion through cAMP bypassing the MC2R-MC2R accessory protein pathway (15–17). As mTOR exerts its effects by forming2 distinct complexes with adaptor proteins Raptor and Rictor, we also studied the roles of these individual complexes.

Materials and Methods

Chemicals and Antibodies

Angiotensin II, forskolin, and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The mTOR activator MHY1435 and its inhibitors AZD8055 (AZD), MK-8669, GDC-0349, CZ-415, Rapamycin, CC-223, and Oxa-01 were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F12) (1:1) medium was purchased from ThermoFisher (thermofisher.com), Fetalgro serum from Rocky Mountain Biologicals (rmbio.com), and Trypsin (0.25%) from ThermoFisher (Thermofisher.com). The primary antibodies used are given in Table 1. The Westview horseradish peroxidase (HRP) conjugated anti-rabbit (cat no. WB-1000, RRID:AB_2336860) and anti-mouse were from Vector Laboratories (cat. no. WB-2000, RRID:AB_2336861). CRISPR/gRNA Raptor (Raptor CRISPR guide RNA 6_pLentiCRISPR v2, cat no. SC1678) and Rictor (Rictor CRISPR guide RNA 1_pLentiCRISPR v2, cat. no. SC1678) were obtained from GenScript USA Inc (genscript.com). Intracellular calcium was measured using a kit from AAT Bioquest, Calbryte™520 (aatbio.com).

Table 1.

Antibodies used in the study

Peptide/protein target	Developer or manufacturer, catalog no.	Species/clonality	Dilution used	RRID
Aldosterone	In-house, Aldo AB A2E11 (18)	Mouse, monoclonal	15 000	AB_2892670
CYP11B2	In-house, CYP11B2 (19)	Mouse, monoclonal	10 000	AB_2650562
CYP17A1	In-house, Gomez-Sanchez CE	Mouse, monoclonal	2000	AB_2895091
3β-HSD2	In-house, Clone 6 (20)	Mouse, monoclonal	2000	AB_2868546
StAR	Cell Signaling Technology, 8449	Rabbit, monoclonal	1000	AB_10889737
phospho-mTOR	Cell Signaling Technology, 8450	Rabbit, monoclonal	2000	AB_2262884
phospho-ERK	Cell Signaling Technology, 9101	Rabbit, monoclonal	1000	AB_331646
ERK	Cell Signaling Technology, 9102	Rabbit, monoclonal	1000	AB_330744
phospho-P70S6K	Cell Signaling Technology, 97596	Rabbit, monoclonal	1000	AB_2800283
P70S6K	Cell Signaling Technology, 2708	Rabbit, monoclonal	1000	AB_390722
phospho-AKT	Cell Signaling Technology, 9611	Rabbit, monoclonal	1000	AB_330302
AKT	Cell Signaling Technology, 9272	Rabbit, monoclonal	1000	AB_329827
phospho-Raptor	Cell Signaling Technology, 2083	Rabbit, monoclonal	1000	AB_2249475
Raptor	Cell Signaling Technology, 2280	Rabbit, monoclonal	1000	AB_561245
phospho-Rictor	Cell Signaling Technology, 3806	Rabbit, monoclonal	1000	AB_10557237
Rictor	Cell Signaling Technology, 2114	Rabbit, monoclonal	1000	AB_2179963
mTOR	Proteintech, 66888-1-Ig	Mouse, monoclonal	10 000	AB_2882219
β-actin	Proteintech, HRP-60008	Mouse, monoclonal	20 000	AB_2819183

Abbreviations: 3β-HSD2, 3β-hydroxysteroid dehydrogenase-type 2; AKT, protein kinase B; HRP, horseradish peroxidase; mTOR, mechanistic target of rapamycin; Raptor, rapamycin-sensitive adapter protein of mTOR; Rictor, rapamycin-insensitive companion of mTOR; StAR, steroidogenic acute regulatory protein.

Cell Culture

The HAC15 human ACC cell line (13, 14) was kindly provided by Dr. William Rainey (University of Michigan, Ann Arbor, MI, USA). The cells were cultured in DMEM/F12 (1:1) medium supplemented with 3% Fetalgro serum and maintained at 37 °C in a humidified atmosphere containing air and carbon dioxide (95%/5%, vol/vol). Lentivirus were produced as previously described (21).

Silencing of Raptor and Rictor

Raptor and Rictor genes in HAC15 cells were silenced with lentivirus containing CRISPR/gRNA-Raptor or Rictor. Briefly, cells were cultured on a 12-well plate until about 50% confluent and then transduced with the virus using 8 µg/mL of polybrene and 400 µg/mL of poloxamer 407, then spinoculated at 2000g for 2 hours. Transduced cells were selected with 1 µg/mL of puromycin. Seventy-two hours later, the cells were serum starved overnight and then left untreated or treated with 100 nmol/L AngII for 24 hours. Western blot assays were performed to confirm the stable deletion of the targeted proteins.

Steroid Production Analyses

HAC15 cells were cultured in 96-well plates until about 80% confluent. The media was then replaced with reduced-serum media (0.1% Fetalgro serum) and incubated for 24 hours (serum starvation). The media was then replaced with the reduced-serum medium containing vehicle (0.1% dimethyl sulfoxide), with and without AngII, with and without mTOR activator or inhibitors. In some experiments, cells in which CRISPR/gRNA-mediated gene knockdown of Raptor or Rictor had been done were treated with AngII. After 24 hours of incubation, the supernatants were collected and stored at −80 °C until further analysis. Aldosterone and cortisol levels were measured by ELISA using antibodies developed in our laboratory (18, 22). Steroid levels were normalized to the total protein content of the respective sample, which was measured using Precision Red Advanced Protein Assay kit (Cytoskeleton, Inc, cytoskeleton.com) according to the manufacturer's protocol.

Western Blotting

HAC15 cells were cultured in 12-well plates until subconfluent, incubated overnight in 0.1% low-serum medium, and then treated with the agents as indicated in the low-serum medium. Western blotting for protein levels was performed as described recently (23). Briefly, the samples were separated by SDS-polyacrylamide gel (4-15%) and transferred to polyvinylidene difluoride membrane (EMD Millipore, Darmstadt, Germany). The membranes were blocked in 1% nonfat dry milk for 45 minutes and then probed with antibodies against StAR (rabbit, 1:1000 dilution), 3β-HSD2 (mouse, 1:2000), CYP17A1 (mouse, 1:2000), CYP11B2 (mouse, 1:10 000), pERK and ERK (rabbit, 1:1000), pmTOR (rabbit, 1:2000), mTOR (mouse, 1:10 000), pRaptor and Raptor (rabbit, 1:1000), pRictor and Rictor (rabbit, 1:1000), pAKT and AKT (rabbit, 1:1000), pP70S6K1 and P70S6K1 (rabbit, 1:1000) in 1% nonfat dry milk, overnight at 4°C with constant rocking. Membranes were then incubated with the appropriate Westview anti-rabbit or anti-mouse HRP conjugated secondary antibody (1:20 000) for 1 hour at room temperature. Chemiluminescence was performed for visualization using a luminol reagent (24). Protein bands were imaged with a ChemiDoc™ imager (Bio-Rad, USA). The membranes were stripped and reincubated with a HRP conjugated anti-β-actin antibody (mouse, 1:20 000) for protein normalization. The quantification of signal densities from triplicate wells was performed by Image J software (National Institutes of Health, USA).

Immunocytochemistry

HAC15 cells were grown on coverslips in a 24-well plate for 48 hours, washed with PBS, and fixed with 4% paraformaldehyde in PBS for 20 minutes. After another washing, the cells were treated with 0.5% Triton X-100 in PBS solution for 3 minutes with gentle shaking and then incubated with 1% skim milk at reverse transcription (RT) for 30 minutes. After washing, the cells were incubated with Raptor and Rictor primary antibodies (1:500 dilution each) for 2 hours at RT, then incubated for 1 hour at RT with fluorescent secondary antibody (Alexa fluor-594 conjugated anti-Rabbit IgG at 1:500 dilution). Coverslips were mounted on slides using 20% glycerol in PBS. Bound antibodies were visualized by EVOS™ 5000 microscope (Invitrogen).

Cell Proliferation Assay

Cell proliferation was evaluated using the 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) cell proliferation assay kit (American Type Culture Collection, Manassas, VA, USA) according to the manufacturer's instructions. Briefly, HAC15 cells were seeded on 96-well plates and treated with AngII and AZD as indicated. Cells in which CRISPR/gRNA-mediated gene knockdown of Raptor or Rictor had been done were treated with AngII, then incubated with activated-XTT solution (50 µL) for 4 hours. The absorbance was measured at a wavelength of 475nm and 660 nm using a BMG FLUOstar Omega Reader (BMG Labtech, Ortenberg, Germany).

Intracellular Calcium Measurement

HAC15 cells were cultured as above in 96-well plates and incubated with vehicle or AngII 10 nM overnight and different concentrations of AZD (0.03, 0.1, and 0.3 μM). Intracellular calcium was then measured using a Calbryte™520 kit from AAT Bioquest (AATbio.com).

Statistical Analysis

Results were expressed as mean ± SEM. Differences between single data set and grouped data set were analyzed by 1-way and 2-way ANOVA, respectively, followed by Bonferroni's post hoc test for multiple comparisons. P < 0.05 was considered statistically significant. Graphs were generated and statistical analyses were performed using GraphPad/Prism version 6 for Windows software (GraphPad Software, La Jolla, CA, USA).

Results

mTOR Inhibition Attenuated AngII-induced Aldosterone and Cortisol Production in HAC15 Cells

AngII 100 nmol/L treatment markedly increased aldosterone production in HAC15 cells, and this increase was significantly attenuated, but not abolished, by all the mTOR inhibitors tested including 100 nmol/L of AZD, MK, GDC, or CZ; 2 µmol/L of Rapamycin; or 1 µmol/L of CC223 and Oxa01 (Fig. 1A). The dual mTORC1 and mTORC2 inhibitor AZD was the most effective and was used in the remaining studies. The effects of graded mTOR inhibition on the production of aldosterone and cortisol stimulated by AngII or forskolin are shown in Fig. 1B, 1C, and 1D. The dose response curves in Fig. 1B indicate that inhibition of mTOR by AZD treatment (0-1000 nmol/L) did not alter the basal aldosterone levels. However, AZD at 30 nmol/L and above dose-dependently suppressed aldosterone synthesis induced by AngII. The mTOR activator MHY (25) had no effect on basal or AngII stimulation. AZD also significantly inhibited AngII-induced increases of cortisol synthesis in a dose-dependent manner (Fig. 1C). The inhibitory effect of AZD on steroid synthesis occurred without affecting survival and proliferation of cells (data not shown). Forskolin-induced aldosterone production was not altered by AZD (Fig. 1D).

Figure 1.

Inhibition of mTOR decreased AngII-induced aldosterone and cortisol synthesis in HAC15 cells. (A) Effect of a panel of mTOR inhibitors on AngII-induced aldosterone production. (B) Dose-dependent effects of mTOR activator MHY and inhibitor AZD on AngII-induced aldosterone production. (C) Dose-dependent effect of AZD on AngII-induced cortisol production. (D) Dose-dependent effect of AZD on Forskolin-induced aldosterone production. Results are shown as mean ± SEM (n = 4). †P < 0.01 vs nontreated Cont; *P < 0.05 and **P < 0.01 vs AngII. AngII, angiotensin II; AZD, AZD8055; Cont, control; mTOR, mechanistic target of rapamycin.

mTOR Inhibition Attenuated AngII-induced StAR Protein and Steroidogenic Enzymes Expression Levels in HAC15 Cells

To determine whether inhibition in aldosterone production were driven by a change in steroidogenic enzymes, we analyzed the effect of mTOR inhibition on the enzymes involved in adrenal steroidogenesis by assessing protein expression levels by Western blot analysis. Consistent with the inhibitory effect on aldosterone and cortisol production, mTOR inhibition by AZD (at 30, 100, and 300 nmol/L) dose-dependently suppressed AngII-induced expression levels of StAR protein (Fig. 2A) and the expression of the steroidogenic enzymes 3β-HSD2, CYP17A1, and CYP11B2 (Fig. 2B, 2C, 2D, respectively).

Figure 2.

Inhibition of mTOR with AZD dose-dependently blunted the AngII stimulation of (A) StAR protein, (B) 3β-HSD2, (C) CYP17A1, and (D) CYP11B2 expression in HAC15 cells. Results are shown as mean ± SEM (n = 4). †P < 0.01 vs Cont; *P < 0.05 and **P< 0.01 vs AngII.

Abbreviations: 3β-HSD2, 3β-hydroxysteroid dehydrogenase-type 2; AngII, angiotensin II; AZD, AZD8055; Cont, control; mTOR, mechanistic target of rapamycin; StAR, steroidogenic acute regulatory protein.

mTOR Inhibition by AZD Reduced Phosphorylated ERK, mTOR, P70S6K, and AKT in HAC15 Cells

To determine mechanisms by which mTOR inhibition reduces AngII-stimulated steroid production, the AngII-response pathway in adrenal steroidogenesis was assessed by Western blot analysis (3, 26). Cells were treated with AZD (0, 0.03 and 0.3 µmol/L) overnight and then stimulated with 100 nmol/L AngII for 5 minutes. As shown in Fig. 3A, pERK levels were very low without AngII and were not significantly affected by AZD. Incubation of the cells with AngII markedly increased the phosphorylation of ERK, and this was significantly attenuated by inhibition of mTOR in a dose-dependent fashion. pERK level was normalized with the inner control β-actin, because the total ERK levels in our experiments could hardly be detected after AngII stimulation.

Figure 3.

Effect of mTOR inhibition on AngII-induced ERK, mTOR, AKT, and P70S6K phosphorylation in HAC15 cells. (A) Representative immunoblot for ERK phosphorylation. (B) Representative immunoblot for mTOR phosphorylation. (C) Representative immunoblots for P70S6K and (D) AKT phosphorylation. Results are shown as mean ± SEM (n = 4). †P < 0.01 vs Cont; *P < 0.05 and **P< 0.01 vs AngII.

Abbreviations: AKT, protein kinase B; AngII, angiotensin II; Cont, control; mTOR, mechanistic target of rapamycin.

AZD significantly decreased basal, as well as AngII-induced mTOR phosphorylation in HAC15 cells in a dose-dependent fashion, though the inhibition was greater in the AngII-stimulated cells (Fig. 3B). AZD at 100 nmol/L significantly decreased phosphorylation of the signaling molecules of mTOR pathway ribosomal protein S6 kinase (P70S6K) (Fig. 3C) and AKT (Fig. 3D), substrates of mTORC1 and mTORC2, respectively.

AngII Increased Phosphorylation of Raptor and Rictor in HAC15 Cells

The expression of Raptor and Rictor, adaptor proteins of mTOR complex 1 and 2, respectively, were detected by immunocytochemical staining (Fig. 4A) and Western blotting (Fig. 4B and 4C) in HAC15 cells. AngII significantly increased the phosphorylation levels of Raptor and Rictor as shown in Fig. 4B and 4C, respectively.

Figure 4.

AngII increased Raptor and Rictor phosphorylation levels in HAC15 cells. (A) Immunocytochemical detection of Raptor and Rictor expressed in cultured HAC15 cells. The primary antibody was omitted for the negative control. (B) Raptor and (C) Rictor phosphorylation levels induced by AngII as analyzed by Western blot. Results are shown as mean ± SEM (n = 4). †P < 0.05 vs Cont.

Abbreviations: AngII, angiotensin II; Cont, control; Raptor, rapamycin-sensitive adapter protein of mTOR; Rictor, rapamycin-insensitive companion of mTOR.

Raptor and Rictor CRISPR/gRNA Knockdown of RAPTOR and RICTOR Attenuated AngII-induced Production of Aldosterone, Cortisol, StAR Protein, and Steroidogenic Enzymes in HAC15 Cells

Lentiviral delivery of CRISPR/gRNA to HAC15 cells significantly decreased Raptor and Rictor protein expression levels as determined by Western blot analysis (Fig. 5A and 5B). Loss of either gene significantly inhibited AngII-induced increases in aldosterone and cortisol production without significantly altering unstimulated steroid synthesis (Fig. 5C and 5D).

Figure 5.

Effects of CRISPR/gRNA reduction of Raptor and Rictor on AngII-induced aldosterone and cortisol production in HAC15 cells. Western blot analysis of Raptor (A) and Rictor (B) protein levels in HAC15 cells expressing CRISPR/gRNA-Raptor and CRISPR/gRNA-Rictor. β-actin was used as a loading control. Reduction of Raptor and Rictor significantly reduced AngII-induced aldosterone (C) and cortisol (D) production. Results represent at least 3 independent experiments. Results are shown as mean ± SEM (n = 6). †P < 0.01 vs Cont; *P < 0.05 vs AngII.

Abbreviations: AngII, angiotensin II; Cont, control; Raptor, rapamycin-sensitive adapter protein of mTOR; Rictor, rapamycin-insensitive companion of mTOR.

Consistent with the effect of pharmacological mTOR inhibition on steroidogenesis, silencing of Raptor and Rictor genes significantly suppressed both basal and AngII-induced upregulation of StAR protein, 3β-HSD2, CYP17A1, and CYP11B2 (Fig. 6A-6D, respectively) and had no influence on cell proliferation (data not shown).

Figure 6.

Effects of CRISPR/gRNA reduction of Raptor and Rictor on AngII-induced protein expression of StAR protein and steroidogenic enzymes in HAC15 cells. Western blot analysis for StAR protein (A) and steroidogenic enzymes such as 3β-HSD2, CYP17A1, and CYP11B2 (B-D, respectively) protein expression levels. Results are shown as mean ± SEM (n = 4). †P < 0.05 vs Cont; *P < 0.05 vs AngII.

Abbreviations: 3β-HSD2, 3β-hydroxysteroid dehydrogenase-type 2; AngII, angiotensin II; Cont, control; Raptor, rapamycin-sensitive adapter protein of mTOR; Rictor, rapamycin-insensitive companion of mTOR; StAR, steroidogenic acute regulatory protein.

P70S6K Phosphorylation is Inhibited But AKT Phosphorylation Is Increased by Raptor and Rictor CRISPR/gRNA Knockdown in HAC15 Cells

Representative immunoblots for P70S6K and AKT phosphorylation are shown in Fig. 7A and 7B, respectively. Consistent with the effects of mTOR inhibition by AZD, CRISPR/gRNA knockdown of Raptor or Rictor produced a significant decrease in P70S6K phosphorylation levels in HAC15 cells. In contrast, basal and AngII-induced AKT phosphorylation was significantly increased by the loss of either component (Fig. 7B).

Figure 7.

Effects of CRISPR/gRNA reduction of Raptor and Rictor on P70S6K (A) and AKT (B) phosphorylation in HAC15 cells. β-actin was used as a loading control. (C) Intracellular calcium increased with stimulation of AngII and this increase was inhibited with 0.1 and 0.3 μM of AZD. Results are shown as mean ± SEM (n = 4). †P < 0.05 vs Cont; *P < 0.05 vs AngII.

Abbreviations: AKT, protein kinase B; AngII, angiotensin II; AZD, AZD8055; Cont, control; Raptor, rapamycin-sensitive adapter protein of mTOR; Rictor, rapamycin-insensitive companion of mTOR.

Effect of AZD8055 on control and AngII stimulated HAC15 cells

AngII increased intracellular calcium, which was significantly inhibited with 0.1 and 0.3 μM of AZD8055 (Fig 7C).

Discussion

AZD is a potent and specific dual mTOR kinase inhibitor acting on both mTORC1 and mTORC2 and their downstream substrates that result in the inhibition of growth of tumor cell lines both in vitro and in vivo in mice (27). The mTOR inhibitor rapamycin decreased proliferation and aldosterone secretion in NCI-H295R cells (11). However, specific mechanisms for mTOR signaling in the regulation of adrenal steroidogenesis, particularly in response to AngII, was not examined. The present study confirms that mTOR signaling is an important component of the regulation of steroidogenesis by the RAAS in HAC15 human ACC cells and provides evidence for the mechanisms involved. The pharmacological inhibition of mTOR suppressed AngII-induced increase in the enzymes required for the synthesis of aldosterone and cortisol in a dose-dependent manner. Deletion of either of its components RICTOR or RAPTOR using CRISPR technology also suppressed AngII-induced increase in proteins required for steroidogenesis and aldosterone and cortisol synthesis in these cells.

Aldosterone overproduction is a driver in pathological cardiovascular and renal remodeling. It acts through mineralocorticoid receptor (MR), and MR antagonists (MRA) improve such debilitating conditions (28–30). Notwithstanding their documented efficacy in the clinical setting, because of the broad range of homeostatic effects mediated by the MR, MRA can trigger adverse effects including hyperkalaemia, which limit their clinical use, particularly in hypertensive individuals with chronic kidney disease and heart failure (31). In addition, MRA do not distinguish between MR in aldosterone target cells and MR that are normally activated by glucocorticoids. Thus, alternative approaches to control adrenal steroid hypersecretion must be explored. Targeting the mTOR pathway could be such a strategy. The mTOR inhibitor sirolimus reduced cell survival and cortisol secretion in cultured primary cells of human adrenocortical tumors (32). Brooks et al demonstrated that inhibition of mTOR activity exerted a beneficial outcome comparable to that of MR blockade in reducing aldosterone-dependent worsening of renal function with minimal side effects in mice (33). Inhibition of mTORC1 was recently demonstrated to reduce plasma aldosterone levels in mice but only in some humans (12).

Our studies confirm and extend the information about the role of mTOR in adrenal steroidogenesis. mTOR inhibition had no effect on basal aldosterone levels but attenuated AngII-stimulated aldosterone production, while the mTOR activator MHY had no effect on either basal or AngII-stimulated steroid synthesis, suggesting that mTOR is tonically active in these cells. Our findings that the inhibition of mTOR did not alter aldosterone production induced by forskolin, an adenylate cyclase activator, indicates that the inhibition of aldosterone synthesis by the loss of mTOR function is cAMP/adenylyl cyclase-independent and is related to the AngII signaling pathway. The marked reduction of AngII-induced increases in StAR, 3β-HSD2, CYP17A1, and CYP11B2 protein production by mTOR inhibitors and deletion of Raptor and Rictor confirms the action of mTOR.

Heightened mTOR activity may be causal in some steroid-producing adrenal neoplasms. De Martino et al described an increase in the major components of the mTOR signaling pathway in primary cultures of normal and pathological human adrenals; however, mTOR inhibition reduced proliferation and cortisol production only in some ACC cells (32) and patients (34). mTOR was found to be involved in the neoplastic process in primary cells from about a third of 57 human adrenocortical carcinomas, as well as in the H295R (35). The HAC15 was derived from the same human adrenal carcinoma cell line H295R (13). Very recent transcriptomic and bioinformatic analyses revealed a high mTORC1 signaling in aldosterone-producing adenomas and adjacent ZG cells (36).

Activation of AngII-ERK axis is a widely accepted signal transduction cascade for adrenal steroidogenesis (26, 37). Both ERK and mTOR have key roles in the regulation of cell survival, differentiation, proliferation, and metabolism. In addition to the signaling response of their individual components, the ERK and mTOR pathways interact extensively and modulate one another (38, 39). Our findings represented in Fig. 5 showing that mTOR inhibition decreased AngII-induced ERK and mTOR phosphorylation, as well as that of P70S6K and AKT, is consistent with this interaction in the HAC15 cells and indicate that mTOR and AngII signaling synergize to increase StAR protein and requisite enzymes for aldosterone and cortisol synthesis.

We extended our findings by analyzing the involvement of the 2 major components of mTOR signaling and found comparable increases in the phosphorylation levels of Raptor and Rictor by AngII treatment. mTORC1 (Raptor) and mTORC2 (Rictor), the 2 functional complexes forming mTOR, occasionally differ in action. For instance, activation of mTORC1, but not mTORC2, was largely responsible for osteoblast cytoprotection (25).

mTORC1 and mTORC2 activities can be evaluated based on the phosphorylation levels of their downstream substrates P70S6K and AKT (33, 40). Both pharmacological inhibition of mTOR by AZD and knockdown of RAPTOR or RICTOR significantly inhibited phospho-P70S6K levels but had a variable effect on AKT. Phosphorylation of AKT was inhibited by the dual mTOR inhibitor AZD; however, its phosphorylation increased greatly when either complex was silenced separately. This discrepancy was not surprising since there appears an escape pathway resulting from a negative feedback loop for AKT activation following depletion of 1 of the 2 complexes (8). In conclusion, we have demonstrated that one of the mechanisms by which AngII stimulates steroidogenesis is by activating mTOR signaling in which both mTORC1 and mTORC2 are involved. Inhibition of the mTOR pathway may be a viable strategy to treat excessive steroid production due to unregulated AngII signaling.

Significance of the Study

What was already known:

• mTOR and AngII interact.

• mTORC1 inhibition reduced proliferation and cortisol formation in ACC cells.

What is new in this study:

• Suppression of mTOR action by pharmacological inhibition or gene silencing of either RICTOR or RAPTOR in HAC15 human ACC cells:

  • ○ suppresses ERK-dependent AngII-induced aldosterone and cortisol production

  • ○ attenuates increased expression of StAR protein and key steroidogenic enzymes as well as intracellular calcium levels in response to AngII.

• mTORC1 (Raptor) and mTORC2 (Rictor) participate in AngII mediated steroidogenesis.

• mTOR is not involved in basal synthesis of aldosterone or cortisol synthesis.

• mTOR is not involved in steroidogensis through the cAMP-PKA-CREB signaling cascade stimulated by ACTH.

Summary

The present study revealed that AngII phosphorylation of mTOR and activation of the mTOR pathway is responsible for a significant proportion of AngII-induced aldosterone and cortisol production in HAC15 human adrenocortical carcinoma cells. Interference with mTOR activity significantly decreased AngII-induced phosphorylation of mTOR, ERK, and P70S6K and significantly suppressed the AngII-induced increase in the cholesterol transfer protein StAR and enzymes required for adrenal steroidogenesis. mTOR interference had no effect on basal aldosterone or cortisol synthesis by HAC15 cells or on forskolin-induced steroidogensis. The latter suggests that mTOR is not involved in the adenylate cyclase stimulation of adrenal steroidogenesis produced by ACTH. (The MC2R to which ACTH binds in normal adrenocortical cells is not present in HAC15 cells. Our data suggest that existing mTOR inhibitors may be useful in treating conditions of excessive adrenal cortical steroid production due to inappropriate RAAS activation.)

Abbreviations

3β-HSD2

3β-hydroxysteroid dehydrogenase-type 2

ACC

adrenocortical carcinoma

AKT

protein kinase B

AngII

angiotensin II

AZD

AZD8055

HRP

horseradish peroxidase

MC2R

melanocortin 2 receptor

MR

mineralocorticoid receptor

MRA

MR antagonists

mTOR

mechanistic target of rapamycin

mTORC1

mTOR complex 1

mTORC2

mTOR complex 2

P70S6K

ribosomal protein S6 kinase

RAAS

renin–angiotensin system

Raptor

rapamycin-sensitive adapter protein of mTOR

Rictor

rapamycin-insensitive companion of mTOR

RT

reverse transcription

StAR

steroidogenic acute regulatory protein

ZF

zona fasciculata

ZG

zona glomerulosa

Data Availability

All data generated or analyzed in this study are included in this manuscript.