ENDOCRINE SOCIETY
OXFORD
Mammalian Target of Rapamycin Inhibition Decreases Angiotensin II-Induced Steroidogenesis in HAC15 Human Adrenocortical Carcinoma Cells
Yusuf Ali, 1,2[D Elise P. Gomez-Sanchez,2[D and Celso E. Gomez-Sanchez 1,2iD
1G. V. (Sonny) Montgomery, VA Medical Center, Jackson, MS, USA
2Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS, USA Correspondence: Celso E. Gomez-Sanchez, MD, University of Mississippi Medical Center, 2500 N State St, Jackson, MS 39216, USA. Email: cgomez-sanchez@umc.edu.
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 (AnglI). 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 AnglI-induced steroidogenic enzymes including steroidogenic acute regulatory protein, 3-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 AnglI 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.
Key Words: aldosterone, angiotensin II, mTOR, Raptor, Rictor, steroidogenesis
Abbreviations: 30-HSD2, 3ß-hydroxysteroid dehydrogenase-type 2; ACC, adrenocortical carcinoma; AKT, protein kinase B; AnglI, 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.
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 3ß-hydroxysteroid dehydrogenase-type 2 (3ß-HSD2) converts pregnenolone to progesterone, which is then hydroxylated to deoxycorticosterone by CYP21A2. Finally, aldosterone is bio- synthesized through a series of reactions in the mitochondria mediated by CYP11B2, the sole enzyme unique to the forma- tion 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 car- diovascular remodeling. In the normal adrenal gland, most en- zymes 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 al- dosterone production remain incompletely understood. Aldosterone production in the ZG cells is stimulated by the hormones angiotensin II (AngII) and ACTH, as well as para- crine 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 melano- cortin 2 receptor (MC2R) (3). The mechanistic target of rapa- mycin (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 metab- olism and growth and receives signals from sensors detecting intracellular nutrients levels, several growth factors, and cell stress. mTOR regulates myriad physiological functions, includ- ing transcription and translation of genes; cell cycle progres- sion; and cell differentiation, motility, metabolism, apoptosis, and autophagy (4-7). mTOR is composed of 2 distinct molecu- lar 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 oc- curs in aldosterone-producing adenomas and increases aldos- terone 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 ad- renal steroidogenesis and molecular mechanisms involved us- ing 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 re- sponse 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 path- way (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 pri- mary 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™M520 (aatbio.com).
Cell Culture
The HAC15 human ACC cell line (13, 14) was kindly pro- vided 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 °℃ 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 un- treated or treated with 100 nmol/L AngII for 24 hours. Western blot assays were performed to confirm the stable de- letion 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 (se- rum 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 ℃ until further analysis. Aldosterone and corti- sol 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 manu- facturer’s protocol.
Western Blotting
HAC15 cells were cultured in 12-well plates until subconflu- ent, 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 sepa- rated 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), 3B-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
| 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 |
| 3ß-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 |
| ß-actin | Proteintech, HRP-60008 | Mouse, monoclonal | 20 000 | AB_2819183 |
Abbreviations: 3B-HSD2, 3ß-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.
A
B
MHY
Aldosterone (pg/mg protein)
1200
Cont
Angll
Aldsoterone (pg/mg protein)
MHY+Angll
+
2000
AZD
AZD+Angll
1000
+
T
800
1500
*
600
**
**
*
**
**
**
1000
*
400
**
**
500
200
I
0
Non treated
AZD8055
MK8669
GDC0349
CZ415
Rapamycin
CC223
0
Oxa01
0
1
10
100
1000
(MHY/AZD nmol/L)
C
D
Cortisol (pg/mg protein)
15000
Cont
Aldosterone (pg/mg protein)
300
Cont
12000
+
Angll
Forskolin
+
9000
*
200
*
*
**
6000
**
100
3000
*
0
0
1
10
100
1000
0
AZD (nmol/L)
0
1
10
100
1000
AZD (nmol/L)
Rictor (rabbit, 1:1000), pAKT and AKT (rabbit, 1:1000), pP70S6K1 and P70S6K1 (rabbit, 1:1000) in 1% nonfat dry milk, overnight at 4 ℃ 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™M imager (Bio-Rad, USA). The membranes were stripped and reincubated with a HRP conjugated anti-ß-actin antibody (mouse, 1:20 000) for protein normal- ization. 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% paraformalde- hyde 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 pri- mary antibodies (1:500 dilution each) for 2 hours at RT, then
incubated for 1 hour at RT with fluorescent secondary anti- body (Alexa fluor-594 conjugated anti-Rabbit IgG at 1:500 dilution). Coverslips were mounted on slides using 20% gly- cerol in PBS. Bound antibodies were visualized by EVOS™M 5000 microscope (Invitrogen).
Cell Proliferation Assay
Cell proliferation was evaluated using the 2,3-Bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox- anilide 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 in- dicated. Cells in which CRISPR/gRNA-mediated gene knock- down 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 in- cubated with vehicle or AngII 10 nM overnight and different
A
Angli 100 nmol/L
Angli 100 nmol/L
8
+
StAR 28 kDa
StAR/ ß actin Fold over cont
6
4
2
**
**
**
0
3
B
+
3ßHSD2 48 kDa
3ßHSD2/ ß actin Fold over cont
2
H*
1
**
**
0
1.5
C
CYP17A1 48 kDa
CYP17A1/ß actin Fold over cont
1.0
**
**
0.5
**
0.0
D
CYP11B2 55 kDa
10
CYP11B2/ ß actin Fold over cont
+
8
6
**
4
ß actin 45 kDa
**
2
0 0.03 0.1 0.3 0 0.03 0.1 0.3
0
0
0.03 0.1
0.3
0
0.03
0.1
0.3
AZD (mol/L)
AZD (µmol/L)
A
Angli 100 nmol/L
15
PERK 42/44 kDa
Angli 100 nmol/L
ERK 42/44 kDa
pERK/ ß actin Fold over cont
+
10
*
**
5
ß actin 45 kDa
0 0 0.1 0.1 0.3 0.3 0 0 0.1 0.1 0.3 0.3 AZD (µmol/L)
0
0
0.1
0.3
0
AZD (umol/L)
0.1
0.3
B
Angll 100 nmol/L
2.0
Angll 100 nmol/L
+
P-mTOR 289 kDa
P-mTOR/ mTOR Fold over cont
1.5
*
1.0
*
mTOR 289 kDa
*
+
0.5
**
0 0.03 0.1 0.3 0 0.03 0.1 0.3
AZD (umol/L)
0.0
0
0.03
0.1
0.3
0
AZD (umol/L)
0.03
0.1
0.3
C
1.5
D
pAKT
1.5
pP70S6K 70 kDa
Fold over cont
Fold over cont
1.0
60 kDa
1.0
P70S6K
0.5
AKT
0.5
+
Cont
AZD
70 kDa
0.0
+
Cont
AZD
60 kDa
0.0
Cont
AZD
Cont
AZD
concentrations of AZD (0.03, 0.1, and 0.3 µM). Intracellular calcium was then measured using a Calbryte™M520 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 consid- ered statistically significant. Graphs were generated and stat- istical 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 pmol/L of Rapamycin; or 1 pmol/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).
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 ef- fect of mTOR inhibition on the enzymes involved in adrenal ster- oidogenesis 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 30-HSD2, CYP17A1, and CYP11B2 (Fig. 2B, 2C, 2D, respectively).
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
A
Cont
Raptor
Rictor
B
pRaptor 150 kDa
C
pRictor 200 kDa
Raptor 150 kDa
Rictor 200 kDa
Cont
Angll
Cont
Angll
Fold over cont
2.5
+
2.0
Fold over cont
2.0
+
1.5
1.5
1.0
1.0
0.5
0.0
0.5
Cont
Angil
0.0
Cont
Angll
pathway in adrenal steroidogenesis was assessed by Western blot analysis (3, 26). Cells were treated with AZD (0, 0.03 and 0.3 pmol/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 significant- ly attenuated by inhibition of mTOR in a dose-dependent fash- ion. pERK level was normalized with the inner control ß-actin, because the total ERK levels in our experiments could hardly be detected after AngII stimulation.
AZD significantly decreased basal, as well as AngII-induced mTOR phosphorylation in HAC15 cells in a dose-dependent fash- ion, though the inhibition was greater in the AngII-stimulated cells (Fig. 3B). AZD at 100 nmol/L significantly decreased phosphoryl- ation of the signaling molecules of m TOR pathway ribosomal pro- tein 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 im- munocytochemical 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.
Raptor and Rictor CRISPR/gRNA Knockdown of RAPTOR and RICTOR Attenuated AnglI-induced Production of Aldosterone, Cortisol, StAR Protein, and Steroidogenic Enzymes in HAC15 Cells
Lentiviral delivery of CRISPR/gRNA to HAC15 cells signifi- cantly 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 in- creases in aldosterone and cortisol production without signifi- cantly altering unstimulated steroid synthesis (Fig. 5C and 5D).
Consistent with the effect of pharmacological mTOR inhib- ition on steroidogenesis, silencing of Raptor and Rictor genes significantly suppressed both basal and AngII-induced upre- gulation of StAR protein, 36-HSD2, CYP17A1, and CYP11B2 (Fig. 6A-6D, respectively) and had no influence on cell proliferation (data not shown).
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 phosphor- ylation 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 de- crease 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).
A
Raptor 150 kDa
B
Rictor 200 kDa
ß actin 45 kDa
ß actin 45 kDa
Cont
Crispr/gRNA-Rp
Cont
Crispr/gRNA-Rc
1.5
1.5-
Fold over cont
1.0
Fold over cont
1.0
0.5
0.5
+
+
0.0
Cont
CRISPR/gRNA-Rp
0.0
Cont
CRISPR/gRNA-Rc
Aldosterone (pg/mg protein)
D
3000
Angli 100 nM
20000
Angli 100 nM
+
Cortisol (pg/mg protein)
+
2000
15000
*
*
10000
*
*
1000
5000
0
Rp
-
Rc
Rp
Rc
0
-
-
Rp
Rc
-
Rp
Rc
Crispr/gRNA
Crispr/gRNA
Effect of AZD8055 on control and AngII stimulated HAC15 cells
AngII increased intracellular calcium, which was significantly inhibited with 0.1 and 0.3 uM of AZD8055 (Fig 7C).
Discussion
AZD is a potent and specific dual mTOR kinase inhibitor act- ing 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 in- hibitor rapamycin decreased proliferation and aldosterone secretion in NCI-H295R cells (11). However, specific mecha- nisms for mTOR signaling in the regulation of adrenal steroi- dogenesis, particularly in response to AngII, was not examined. The present study confirms that mTOR signaling is an important component of the regulation of steroidogene- sis by the RAAS in HAC15 human ACC cells and provides evi- dence for the mechanisms involved. The pharmacological inhibition of mTOR suppressed AngII-induced increase in the enzymes required for the synthesis of aldosterone and cor- tisol 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 syn- thesis in these cells.
Aldosterone overproduction is a driver in pathological car- diovascular and renal remodeling. It acts through mineralocor- ticoid 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 hyperkalemia, 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 hypersecre- tion 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 ef- fects in mice (33). Inhibition of mTORC1 was recently
A
Angll 100 nM
8
Angll 100 nM
StAR/ ß actin Fold over cont
+
6
StAR 28 kDa
4
*
*
2
+
+
0
B
2.0-
+
*
3HSD2 48 kDa
3@HSD2/ ß actin Fold over cont
1.5
*
1.0
0.5
0.0
1.5-
C
CYP17A1 48 kDa
CYP17A1/ ß actin Fold over cont
1.0
T
+
+
*
*
0.5
0.0
CYP11B2 50 kDa
CYP11B2/ ß actin Fold over cont
20
+
D
15
10
*
5
*
0
ß actin 45 kDa
Rp
-
Rc
-
Rp
Rc
Crispr/gRNA
-
Rp
Rc
-
Rp
Rc
Crispr/gRNA
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 for- skolin, an adenylate cyclase activator, indicates that the inhib- ition 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, 36-HSD2, CYP17A1, and CYP11B2 protein production by mTOR inhibitors and dele- tion 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 signal- ing 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 com- ponents, 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 signal- ing synergize to increase StAR protein and requisite enzymes for aldosterone and cortisol synthesis.
A
Angli 100 nM
B
Angli 100 nM
pP70S6K
pAKT
70 kDa
60kDa
P70S6K
AKT
70 kDa
60kDa
ß actin
ß actin
45kDa
45kDa
1.5
5
Fold over cont
Fold over cont
4
*
1.0
*
T
T
+
+
3
+
*
*
2
+
0.5
1
0.0
0
Rp
Rc
Rp
Rc
Rp
Rc
Rp
Rc
Crispr/gRNA
Crispr/gRNA
C
Angll 10 nmol/L
Intracellular calcium Fold over cont
1.5
+
*
*
1.0
0.5
0.0
0
0.03
0.1
0.3
0
0.03
0.1
0.3
AZD (µmol/L)
We extended our findings by analyzing the involvement of the 2 major components of mTOR signaling and found com- parable increases in the phosphorylation levels of Raptor and Rictor by AngII treatment. mTORC1 (Raptor) and mTORC2 (Rictor), the 2 functional complexes forming mTOR, occa- sionally 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 in- hibited by the dual mTOR inhibitor AZD; however, its phos- phorylation 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 steroi- dogenesis 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 inhib- ition or gene silencing of either RICTOR or RAPTOR in HAC15 human ACC cells:
o suppresses ERK-dependent AngII-induced aldoster- one and cortisol production
o attenuates increased expression of StAR protein and key steroidogenic enzymes as well as intracellular cal- cium 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 car- cinoma 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 sug- gests 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 ex- isting mTOR inhibitors may be useful in treating conditions of excessive adrenal cortical steroid production due to inappro- priate RAAS activation.)
Acknowledgments
Research reported in this publication was supported by National Heart, Lung and Blood Institute Grant R01 HL144847 (CEGS), the National Institute of General Medical Sciences Grant U54 GM115428 (CEGS), and the Department of Veteran Affairs BX00468 (CEGS). The con- tent is solely the responsibility of the authors and does not ne- cessarily represent the official views of the National Institutes of Health or the Department of Veteran Affairs.
Data Availability
All data generated or analyzed in this study are included in this manuscript.
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