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ORIGINAL ARTICLE
Modulation of Calcium Signaling on Demand to Decipher the Molecular Mechanisms of Primary Aldosteronism
Bakhta Fedlaoui, Teresa Cosentino, Zeina R. Al Sayed®, Rita Alexandre CoelhoD, Isabelle Giscos-Douriez, Nicolo FaeddaD, May Fayad@D, Jean-Sebastien Hulot®D, Christopher J. Magnus@D, Scott M. Sternson@D, Simon Travers-Allard D, Stephanie Baron®D, David PentonD, Fabio L. Fernandes-Rosa, Maria-Christina ZennaroD, Sheerazed BoulkrounD
BACKGROUND: Primary aldosteronism is the most common form of secondary hypertension. The most frequent genetic cause of aldosterone-producing adenomas is somatic mutations in the potassium channel KCNJ5. They affect the ion selectivity of the channel, with sodium influx leading to cell membrane depolarization and activation of calcium signaling, the major trigger for aldosterone biosynthesis.
METHODS: To investigate how KCNJ5 mutations lead to the development of aldosterone-producing adenomas, we established an adrenocortical cell model in which sodium entry into the cells can be modulated on demand using chemogenetic tools [H295R-S2 a7-5HT3-R (o.7-5HT3 receptor) cells]. We investigated their functional and molecular characteristics with regard to aldosterone biosynthesis and cell proliferation.
RESULTS: A clonal cell line with stable expression of the chimeric a7-5HT3-R in H295R-S2 (human adrenocortical carcinoma cell line, Strain 2) cells was obtained. Increased sodium entry through a7-5HT3-R upon stimulation with uPSEM-817 (uPharmacologically Selective Effector Molecule-817) led to cell membrane depolarization, opening of voltage-gated Ca2+ channels, and increased intracellular Ca2+ concentrations, resulting in the stimulation of CYP11B2 expression and increased aldosterone biosynthesis. Increased intracellular sodium influx did not increase proliferation but rather induced apoptosis. RNA sequencing and steroidome analyses revealed unique profiles associated with Na+ entry, with only partial overlap with Ang Il (angiotensin II) or potassium-induced changes.
CONCLUSIONS: H295R-S2 a7-5HT3-R cells are a new model reproducing the major features of cells harboring KCNJ5 mutations. Increased expression of CYP11B2 and stimulation of the mineralocorticoid biosynthesis pathway are associated with a decrease of cell proliferation and an increase of apoptosis, indicating that additional events may be required for the development of aldosterone-producing adenomas. (Hypertension. 2025;82:716-732. DOI: 10.1161/HYPERTENSIONAHA.124.23295.) . Supplement Material.
GRAPHIC ABSTRACT: A graphic abstract is available for this article.
Key Words: adrenal cortex calcium signaling cell proliferation mutation potassium channels sodium
T he adrenal gland consists of 2 distinct regions: the outer adrenal cortex and the inner adrenal medulla. The adrenal cortex is further divided into 3 zones, the zona glomerulosa (ZG), zona fasciculata, and zona reticularis, each specialized in hormone production due
to the expression of specific enzymes. Among these hormones, aldosterone, produced by the adrenal ZG, plays an important role in regulating salt and fluid bal- ance, thereby controlling arterial blood pressure. As a key component of the renin-angiotensin-aldosterone system,
Correspondence to: Sheerazed Boulkroun, INSERM, U970, Paris Cardiovascular Research Center-PARCC, 56, rue Leblanc, 75015 Paris, France. Email
Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/HYPERTENSIONAHA.124.23295.
@ 2025 American Heart Association, Inc.
Hypertension is available at www.ahajournals.org/journal/hyp
NOVELTY AND RELEVANCE
What Is New?
Herein, we have developed a novel cell model in which intracellular Ca2+ concentration can be modulated on demand, through modulation of sodium entry, repro- ducing the major characteristics of cells harboring a KCNJ5 mutation.
We demonstrate that Na+ entry into the cells results, on one hand, in cell membrane depolarization, Ca2+ influx, activation of Ca2+ signaling leading to increased expres- sion of CYP11B2, and stimulation of mineralocorticoid biosynthesis and, on the other hand, in a decrease in cell proliferation and an increase in apoptosis.
We show that Na+ influx leads to a specific tran- scriptome signature that may explain some of the peculiar features of aldosterone-producing adenoma carrying KCNJ5 mutations compared with other genetic abnormalities.
What Is Relevant?
While the role of KCNJ5 mutations in promoting autonomous aldosterone biosynthesis has been clearly established, their role in promoting abnor- mal cell proliferation remains to be established. Our study demonstrates that KCNJ5 mutations are not able to promote both autonomous aldosterone production and cell proliferation, suggesting that additional events may be required for adenoma development.
Clinical/Pathophysiological Implications?
This cellular model offers valuable new insights into the mechanisms leading to primary aldosteronism development, revealing novel processes involved in the disease and paving the way for new therapeutic approaches in primary aldosteronism.
| Nonstandard Abbreviations and Acronyms | |
| a7-5HT3-R | a7-5HT3 receptor |
| Ang II | angiotensin II |
| APA | aldosterone-producing adenoma |
| AT1R | Ang II type 1 receptor |
| AUC | area under the curve |
| Dab2 | disabled-2 |
| GIRK4 | G-protein-gated inwardly rectifying K+ channel 4 |
| NF-KB | nuclear factor-KB |
| PA | primary aldosteronism |
| TNFa | tumor necrosis factor-a |
| ZG | zona glomerulosa |
its production is mainly stimulated by Ang II (angiotensin II), which increases in response to volume depletion, and elevated plasma potassium (K+) levels.1,2 Ang II signals through AT1R (Ang II type 1 receptor) and activates, via Gaq, an entire signaling cascade that leads to the release of Ca2+ from the endoplasmic reticulum. The stimulation by K+ and to a less extent by Ang II results in ZG cell membrane depolarization and opening of voltage-gated calcium (Ca2+) channels, leading to an increased intra- cellular Ca2+ concentration. The activation of Ca2+ sig- naling triggers a phosphorylation cascade that leads to increased transcription of CYP11B2, coding for aldoste- rone synthase and aldosterone biosynthesis.1
Deregulation of the mechanisms regulating adrenal aldosterone biosynthesis results in primary aldosteronism
(PA). PA is the leading cause of secondary hypertension, affecting ~5% to 10% of patients with hypertension and up to 20% of those with treatment resistant hyperten- sion.3-6 It is characterized by hypertension with elevated aldosterone levels, low plasma renin concentration, increased aldosterone-to-renin ratio, and is often asso- ciated with hypokalemia. The main subtypes of PA are bilateral adrenal hyperplasia and aldosterone-producing adenomas (APAs), accounting together for 95% of cases. PA is associated with an increased risk of car- diometabolic and renal complications7 due to the major adverse effects of aldosterone excess; therefore, early diagnosis and appropriate treatment of PA are essential.
In the past decade, research has uncovered mutations in ion channels (KCNJ5 [potassium inwardly rectifying chan- nel subfamily J member 5],8 CACNA1D,9,10 CACNA1H,11,12 CLCN2,13,14 and SLC30A115) and pumps (ATP1A19,16 and ATP2B316), as principal causes of APAs and familial forms of PA. These mutations enhance either directly or indirectly intracellular Ca2+ concentrations, the main trigger for aldo- sterone biosynthesis. Notably, in the case of KCNJ5, which encodes GIRK4 (G-protein-gated inwardly rectifying K+ channel 4), the majority of mutations cluster near the chan- nel’s ion-selective filter, leading to a loss of selectivity for K+ ions in favor of an intracellular sodium (Na+) influx.8 These mutations lead to cell membrane depolarization, the open- ing of voltage-gated Ca2+ channels, an increase of intra- cellular Ca2+ concentration, activation of Ca2+ signaling pathways, and ultimately an increase of CYP11B2 expres- sion and aldosterone biosynthesis.8
While the role of KCNJ5 mutations in autonomous aldo- sterone production has been well established, their role in modulating cell proliferation is still under debate.17-20 The
first objective of our study was, therefore, to investigate the role of KCNJ5 mutations in regulating proliferative processes that could lead to adenoma development. The second objective was to determine whether changes in intracellular sodium balance could induce specific intra- cellular transcriptional profiles that could explain the peculiar pathological and biological features of adenoma carrying KCNJ5 mutations. For this purpose, we used chemogenetic tools, which allow to manipulate specific ion fluxes via modified ion channels with pharmacologi- cally selective properties, known as PSAM (pharmaco- logically selective actuator modules). PSAM, comprising mutated ligand-binding domains and selective ionic pore domains, respond to PSEM (pharmacologically selec- tive effector molecules), inducing channel opening and allowing specific ions to flow through the activated chan- nel.21,22 Here, we used second-generation chemogenetic tools to establish a human adrenal cell model in which we could modulate sodium entry on demand, by introducing the a7-5HT3 chimeric receptor. This chimeric receptor consists of the ligand-binding domain of the a7 nicotinic acetylcholine receptor and the ionic pore domain of the serotonin receptor type 3. Importantly, the ligand-binding domain has been mutated to bind only the pharmacologi- cally selective effector molecules and not the endogenous ligand.21 Activation of a7-5HT3-R (a7-5HT3 receptor) by uPSEM-817 (uPharmacologically Selective Effector Molecule-817) was used to study the effect of Na+ entry into the cells, mimicking molecular abnormalities observed in the presence of KCNJ5 mutations. To determine the functional and molecular characteristics of this new cell model, we assessed changes in membrane potential, intracellular Ca2+ concentration, and impact on cell prolif- eration. Additionally, we conducted RNA-sequencing and steroidomic analyses following treatment with uPSEM- 817, Ang II, and K+.
METHODS
Data Availability
Expanded Materials and Methods are available in the Supplemental Material.
The data, analytic methods, and study materials that support the findings of this study are available from the corresponding author upon reasonable request.
Cell Culture and Electroporation
The H295R-S2 (human adrenocortical carcinoma cell line, Strain 2) cell line, a subclone of the H295R human adreno- cortical carcinoma cell, was kindly provided by Dr William E. Rainey23 and cultured in complete medium containing DMEM/ Eagle F12 medium (1:1; GIBCO, Life Technologies) supple- mented with 2% Ultroser G (Sartorius, France), 1% insulin/ transferrin/selenium premix (BD Biosciences), 7.5 mmol/L HEPES (GIBCO), 1% penicillin and streptomycin (GIBCO, Life Technologies) and 20 mg/mL G418 (Thermo Fisher Scientific).
Cells were maintained at 37 ℃ under a humid atmosphere of 95% air and 5% CO2.
The a7-5HT3 chimeric receptor consists of the ligand- binding domain of the nicotinic receptor fused with the ionic pore domain of the serotonin receptor type 3. The sequence has been inserted into the pcDNA3.1 vector.21
Five million H295R-S2 cells were seeded into a 100-mm tissue culture dish. After 24 hours, the cells were trypsinized, counted, and 3.106 cells resuspended in 100 uL Nucleofector R solution and electroporated with 3 µg of plasmid (pcDNA3 containing or not containing «7-5HT3-R cDNA) using the Amaxa Nucleofector Kit R (Lonza) according to the manufac- turer’s instructions. After electroporation, the mixed popula- tions were amplified under selection pressure with G418. Pure clones were isolated by picking clones after limited dilution of the cells to isolate 1 cell at an optimum distance from the other in a 200-mm diameter plate. Colonies were, afterward, isolated in wells and amplified for further characterization.
Statistics
The number of independent experiments (n) refers to the number of cells or dishes studied to calculate mean±SEM or median. The measurements were performed at different days and from different cell preparations using different cell pas- sages to ensure the reproducibility of the experiments. For patch-clamp experiments, each cell was analyzed separately.
Data were analyzed in the Prism10 software (GraphPad, San Diego, CA) using the appropriate statistical tests as indi- cated in the text. The normality of the data distribution was checked using the Shapiro-Wilk test. Quantitative variables were reported as mean±SEM when a gaussian distribution was pres- ent or as medians and interquartile ranges when no gaussian distribution was present. Pairwise comparisons were conducted using unpaired t tests for normally distributed data and Mann- Whitney U tests for non-normally distributed data. For multiple comparisons, ANOVA followed by Bonferroni was applied when data presented a gaussian distribution while Kruskal-Wallis fol- lowed by Dunn test was used when non-gaussian distribution was present. P<0.05 was considered significant.
RESULTS
Characterization of a Cell Model Expressing the a7-5HT3-R
To assess the impact of modulating intracellular Na+ con- centration on adrenal cell function, we established stable expression of the chimeric a7-5HT3-R in H295R-S2 cells. This receptor was formed by combining the extra- cellular ligand-binding domain of the a7 nicotinic acetyl- choline receptor and the ion pore domain of the serotonin receptor 5HT3.21 Expression of a7-5HT3-R was inves- tigated by quantitative reverse transcription PCR (RT- qPCR)24 and found exclusively in cells transfected with the vector containing its genetic sequence (Figure S1A) but not in control cells transfected with an empty vector. Upon treatment with 12 mmol/L of K+, both cell lines exhibited a rapid increase in intracellular Ca2+ concen- tration (Figure S1B). However, treatment with different
concentrations of varenicline, a nicotinic receptor par- tial agonist and cholinergic agonist, resulted in a dose- dependent increase in intracellular Ca2+ concentration in cells expressing the a7-5HT3-R only (Figure S1B), indicating a specific effect on the chimeric receptor. This was associated with an increase in CYP11B2 mRNA expression for varenicline concentrations ranging from 10-8 to 10-5 mol/L (Figure S1C).
To further characterize the cells expressing a7-5HT3-R, monoclonal cell populations were obtained via limiting dilution under antibiotic selection pressure. Among the 19 monoclonal cell populations selected, 6 showed a7-5HT3-R mRNA expression (Figure S1D), and in-depth characterization was performed on 3 of them, designated as clones 17, 24, and 42 (Figure 1). The expression of a7-5HT3-R mRNA was not statisti- cally different between clones 17, 24, and 42 (Figure 1A).
Bright-field images revealed no discernible varia- tions in cell morphology between control cells and the 3 selected clones (Figure 1B). Examination of the cel- lular localization of «7-5HT3-R using a-bungarotoxin, a peptide binding to the a-subunit of the nicotinic acetyl- choline receptor, showed its presence on the cell surface of clones 17, 24, and 42 but not in control cells (Fig- ure 1B). To ensure that the expression of a7-5HT3-R did
not induce structural changes, we studied the expres- sion of markers of cytoskeletal organization and of ZG cells. Phalloidin staining revealed similar actin organiza- tion in cells expressing an empty vector (control cells) or expressing a7-5HT3-R (Figure 1B). A similar staining was observed for Dab2 (disabled-2; Figure 1B), a protein marker of ZG cells expressed at the cell membrane.
Varenicline is a nicotinic receptor partial agonist and a cholinergic agonist, which may have nonspecific effects in our cell model. To avoid this, we compared the effect of varenicline to those of a drug, uPSEM-817, specifi- cally designed to bind to a7-5HT3-R. Similar responses were obtained following uPSEM-817 and varenicline treatment in intracellular Ca2+ responses, both as traces (Figure S2A and S2C) and maximum of activation (Fig- ure S2B and S2D).
a7-5HT3-R Activation by uPSEM-817 Induces Cell Membrane Depolarization and Increases Intracellular Ca2+ Content via Na+ Influx
Perforated patch-clamp recordings were conducted to assess the electrophysiological properties of 2 of the 3 clones expressing a7-5HT3-R and selected for fur- ther investigations (clones 17 and 42) in comparison
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to control cells. Control cells expressing an empty vector, as well as clones 17 and 42, displayed similar membrane hyperpolarization with membrane poten- tials of -60.83±1.2, -61.73±1.16, and -63.27±1.26,
respectively (Figure 2A), indicating that o.7-5HT3-R was not leaking ions.
All the clones responded to induction by Ang II, exhib- iting membrane depolarization upon the application of
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10-8 mol/L Ang II (AEm, 48.26±3.52, 51.81±3.73, and 46.00±3.51 for control cells; clones 17 and 42, respectively; Figure 2B). However, only clones 17 and 42 were responsive to 10-7 mol/L (AEm, 41.58±4.12 and 41.49±2.13 for clones 17 and 42, respectively) and 10-5 mol/L of uPSEM-817 (AEm, 49.03±5.51 and 47.19±2.24 for clones 17 and 42, respectively). The lower concentration of 10-9 mol/L uPSEM-817 did not consistently induce membrane depolarization (Figure 2B).
Electrophysiological measurements were per- formed using high-throughput automated patch-clamp to determine whether activation of a7-5HT3-R by uPSEM-817 increased Na+ current. Repetitive stimula- tions using a voltage ramp from -100 to +20 mV that was used to calculate the reversal potential (ERev; Fig- ure S3A), a surrogate of the resting membrane voltage, confirmed that uPSEM-817 induced depolarization of clones 24 and 42 compared with vehicle-treated cells (Figure S3B). Inward currents were measured at -100 mV as they correspond to positive charges (eg, Na+ ions) entering the cells. Treatment with 10-6 mol/L of uPSEM-817 under extracellular Na+ concentrations of 80 or 132.5 mmol/L activated a statistically significant inward current in clone 42 and a similar trend in clone 24 (Figure S3C). These results are consistent with an increase of Na+ permeability when cells were treated with uPSEM-817.
To determine whether the membrane depolarization triggered by uPSEM-817 correlated with an increase in intracellular Ca2+ concentration, «7-5HT3-R-express- ing cells were exposed to concentrations of uPSEM- 817 ranging from 10-9 to 10-5 mol/L, as well as 12 mmol/L K+ and 10-8 mol/L Ang Il as positive controls (Figure 2C through 2E). The results are presented for clone 17 (Figure 2C through 2E), and similar results were obtained for clone 42 (Figure S4). Application of Ang II or K+ led to a rapid increase in intracellular Ca2+ levels followed by a rapid decline without returning to baseline levels (Figure 2C; Figure S4A), occurring after similar response latency (data not shown). The maxi- mum of activation (Figure 2D; Figure S4B) was sig- nificantly increased when cells were treated with Ang II or K+. The areas under the curve (AUCs) at the peak (between 0 and 360 s; Figure 2E, left; Figure S4C) and at the steady-state response (between 0 and 700 s; Figure 2E, right; Figure S4D) were also significantly increased after K+ or Ang II treatment, these increases being more important when cells were treated with K+. Treatment with uPSEM-817 ranging from 10-9 to 10-5 mol/L also resulted in a similar rapid increase in intra- cellular Ca2+ content; this increase was more modest at 10-9 mol/L uPSEM-817 compared with higher con- centrations of the compound. Interestingly, the decline following the peak was slower with uPSEM-817 compared with Ang II or K+ (Figure 2C; Figure S4A).
Maximum activation (Figure 2D; Figure S4B) and AUC at the peak (Figure 2E, left; Figure S4C) and at steady state (Figure 2E, right; Figure S4D) were lower in response to 10-9 mol/L of uPSEM-817 than with 10-7 and 10-5 mol/L uPSEM-817. Despite variations in Ca2+ content, while the maximum activations were compa- rable when cells were treated with Ang II, K+, and 10-7 and 10-5 mol/L uPSEM-817, the AUCs at the steady state were significantly higher when cells were treated with 10-7 and 10-5 mol/L uPSEM-817 compared with Ang Il and K+. The sustained higher Ca2+ content when cells were treated with uPSEM-817 suggests the acti- vation of different mechanisms compared with Ang II and K+.
a7-5HT3-R Activation by uPSEM-817 Activates Steroidogenesis
The steroidome of clones 17, 24, and 42 was deter- mined using the cell supernatant after 8 hours (Figure S5A and S5B) or 24 hours (Figure S5C and S5D) of treatment with 10-8 mol/L Ang Il or 12 mmol/L K+ as a positive control (Figure S5A and S5C) and uPSEM- 817 ranging from 10-9 to 10-5 mol/L (Figure S5B and S5D), and the results are presented separately for each clone (Figure S5). Interestingly, despite similar modifica- tion in intracellular Ca2+ concentration, different profiles were obtained for the 3 different clones in response to Ang II, K+, and uPSEM-817. However, they all con- verge toward a stimulation of steroid biosynthesis in response to uPSEM-817. Among the 19 steroids mea- sured, only 14 were detectable after 8 and 24 hours of treatment. After 8 hours of treatment, an overall activa- tion of steroid biosynthesis was observed in response to Ang II and K+ (Figure S5A; Table). Similarly, uPSEM- 817 at concentrations from 10-7 to 10-5 mol/L led to an increase in steroid biosynthesis, with no significant changes observed at 10-9 and 10-8 mol/L of uPSEM- 817 (Figure S5B; Table). Interestingly, the increases in certain steroids due to uPSEM-817 were less pro- nounced compared with those induced by Ang II and K+. After 24 hours of treatment, a significant decrease in the concentration of 2 steroid precursors, pregnenolone and progesterone, was observed in response to Ang II and K+, while deoxycorticosterone, 17-hydroxyprogesterone, 17-hydroxypregnenolone, pregnenolone, and proges- terone concentrations remained elevated in response to uPSEM-817 (Table). Aldosterone biosynthesis was highly stimulated by Ang II and K+ and to a lesser extent by uPSEM-817. While no effect was observed after 8 hours of treatment, after 24 hours, the concentration of 18-oxocortisol was significantly increased in response to K+, and a trend was also observed in response to Ang II and for all the concentrations of uPSEM-817. The concentrations of 18-hydroxycortisol were undetectable in all tested conditions.
| Characteristics | Time | Ang II (10-8 mol/L) | K+ (12 mmol/L) | PSEM (10-9 mol/L) | PSEM (10-8 mol/L) | PSEM (10-7 mol/L) | PSEM (10-6 mol/L) | PSEM (10-5 mol/L) |
|---|---|---|---|---|---|---|---|---|
| Pregnenolone | 8 h | 2.622±0.176* | 2.458±0.203* | 1.071±0.025 | 1.035±0.024 | 1.269±0.031* | 1.254±0.037+ | 1.489±0.066* |
| 24 h | 0.441±0.023* | 0.813±0.038 | 1.031±0.036 | 1.045±0.041 | 1.485±0.206 | 1.419±0.157# | 1.643±0.065+ | |
| Progesterone | 8 h | 4.005±0.366* | 3.112±0.296§ | 1.167±0.020 | 1.150±0.016 | 1.422±0.065+ | 1.453±0.065* | 1.652±0.077* |
| 24 h | 0.330±0.022* | 0.533±0.036* | 1.058±0.058 | 1.002±0.056 | 1.165±0.045 | 1.115±0.052 | 1.196±0.048# | |
| 11-deoxycorticosterone | 8 h | 4.100±0.478+ | 3.811±0.350§ | 1.025±0.046 | 0.981±0.060 | 1.272±0.049+ | 1.241±0.039# | 1.378±0.060§ |
| 24 h | 0.409±0.044* | 0.828±0.051 | 1.055±0.040 | 1.012±0.024 | 1.219±0.041§ | 1.197±0.056§ | 1.349±0.048* | |
| 18-hydroxy 11-deoxycorticosterone | 8 h | 2.761±0.205+ | 2.585±0.238§ | 1.122±0.019 | 1.114±0.023 | 1.260±0.055+ | 1.353±0.064* | 1.455±0,038* |
| 24 h | 2.467±0.193* | 2.235±0.198* | 0.982±0.038 | 0.973±0.034 | 1.234±0.041# | 1.271±0.081 | 1.404±0,101§ | |
| Corticosterone | 8 h | 2.515±0.179* | 2.424±0.245* | 1.108±0.035 | 1.148±0.036 | 1.294±0.074 | 1.090±0.187 | 1.021±0.188 |
| 24 h | 2.329±0.268* | 1.983±0.183§ | 0.984±0.051 | 1.002±0.055 | 1.169±0.033 | 1.174±0.032§ | 1.302±0.026* | |
| 18-hydroxycortisol | 8 h | 0.794±0.090 | 0.921±0.112 | 0.944±0.057 | 0.754±0.126 | 0.910±0.053 | 0.894±0.037 | 1314±0.249 |
| 24 h | 2.200±0.575 | 8.049±2.958§ | 2.203±0.557 | 2.241±0.595 | 3.047±1.206 | 2.126±0.691 | 2.086±0.622 | |
| 18-oxocortisol | 8 h | 1.046±0.094 | 1.131±0.126 | 0.937±0.043 | 0.798±0.075 | 0.929±0.028 | 1.068±0.102 | 1.633±0.355 |
| 24 h | 2.200±0.575 | 8.049±2.958§ | 2.203±0.557 | 2.241±0.595 | 3.047±1.206 | 2.126±0.691 | 2.086±0.622 | |
| 18-hydroxycorticosterone | 8 h | 1.927±0.172+ | 2.088±0.156* | 1.125±0.033 | 1.125±0.052 | 1.231±0.051+ | 1.003±0.108 | 0.835±0.063 |
| 24 h | 3.496±0.400* | 2.456±0.341§ | 0.989±0.074 | 1.004±0.094 | 1.177±0.157 | 0.891±0.102 | 0.818±0.085 | |
| Aldosterone | 8 h | 2.454±0.238§ | 2.580±0.273+ | 0.914±0.040 | 0.927±0,038 | 1.024±0.054 | 1.187±0.063 | 1.388±0.062 |
| 24 h | 12.13±4.007* | 6.046±1.657§ | 0.946±0.043 | 1.035±0.082 | 1.495±0.211 | 1.459±0.138§ | 1.436±0.135§ | |
| 17-hydroxyprogesterone | 8 h | 2.761±0.351* | 2.535±0.242+ | 1.187±0.027# | 1.141±0.035 | 1.334±0.059* | 1.325±0.051* | 1.589±0.041* |
| 24 h | 0.243±0.009* | 0.896±0.034§ | 1.052±0.038 | 0.975±0.041 | 1.068±0.034 | 1.049±0.032 | 1.230±0.030§ | |
| 11-deoxycortisol | 8 h | 1.586±0.147+ | 1.706±0.105+ | 1.026±0.033 | 0.995±0.056 | 1.087±0.038 | 1.064±0.035 | 1.079±0.039 |
| 24 h | 1.065±0.046 | 0.626±0.064* | 1.016±0.025 | 0.985±0.030 | 1.096±0.041 | 1.073±0.052 | 1.060±0.046 | |
| Cortisol | 8 h | 1.093±0.061 | 1.066±0.056 | 1.119±0.082 | 1.157±0.058 | 1.126±0.081 | 1.051±0.057 | 1.003±0.055 |
| 24 h | 2.541±0.310* | 1.923±0.251§ | 0.974±0.033 | 1.034±0.041 | 1.161±0.080 | 1.194±0.095 | 1.199±0.076 | |
| 21-deoxycortisol | 8 h | 1.546±0.135# | 1.443±0.139 | 1.046±0.074 | 1.059±0.146 | 0.968±0.143 | 0.926±0.163 | 1.863±0.548 |
| 24 h | 0.852±0.121 | 1.954±0.115* | 1.151±0.120 | 1.107±0.098 | 1.345±0.162 | 1.195±0.073 | 0.941±0.122 | |
| Delta-4- androstenedione | 8 h | 1.450±0.123 | 1.541±0.131§ | 1.001±0.033 | 0.978±0.085 | 1.113±0.039 | 1.069±0.034 | 1.246±0.038§ |
| 24 h | 0.482±0.046* | 1.146±0.055 | 1.049±0.032 | 1.003±0.027 | 1.098±0.047 | 0.985±0.039 | 1.104±0.041 |
Results are expressed as fold induction over untreated cells expressing a7-5HT3-R (set as 1, not shown) and represent meantSEM of the 3 clones, compared with ANOVA followed by Bonferroni when data presented for gaussian distribution and Kruskal-Wallis followed by Dunn test for non-gaussian distribution. Cells treated with Ang Il and K+ and cells treated with uPSEM-817 (PSEM) were analyzed separately. n=9 per condition except for 18-oxocortisol (n=6) and 18-hydroxycortisol (n=6). a7-5HT3-R indicates @7-5HT3 receptor; Ang Il, angiotensin II; H295R-S2, human adrenocortical carcinoma cell line, strain 2; PSEM, pharmacologically selective effec- tor molecule; and uPSEM-817, uPharmacologically Selective Effector Molecule-817.
*P≤0.0001, tP<0.001, +P<0.05, SP≤0.01.
T- and L-Type Channels Are Both Involved in Ca2+ Entry Into the Cells in Response to uPSEM-817
Both T- and L-type voltage-gated Ca2+ channels have been shown to be involved in aldosterone biosynthesis regulation by modulating intracellular Ca2+ concentra- tions.25,26 We evaluated the effect of a 2-hour pretreat- ment with 10-6 mol/L of the L-type Ca2+ channel blocker nifedipine or 10-6 mol/L of the T-type Ca2+ channel blocker mibefradil on the modulation of intracellular Ca2+ content in response to 10 8 mol/L Ang II, 12 mmol/L K+, and increasing concentrations of uPSEM-817 (Fig- ure 3A through 3C). Interestingly, pretreatment of the cells with nifedipine or mibefradil partially abolished Ca2+
entry into the cells after treatment with K+ and uPSEM- 817, as revealed by lower peak (Figure 3A and 3B) and decreased AUC (representing the modifications of intracellular Ca2+ concentrations, illustrating activation of Ca2+ signaling; Figure 3C). In contrast, nifedipine and mibefradil had no effect on the initial entry of Ca2+ into the cell in response to Ang II (Figure 3B), and only nife- dipine pretreatment led to a significant decrease in the AUC (Figure 3A through 3C). These results indicate that both T- and L-type Ca2+ channels are mobilized to allow Ca2+ entry into the cells in response to K+ and uPSEM- 817, a mobilization that appears to be greater than in response to Ang II.
To determine whether the differences observed in the modulation of intracellular Ca2+ concentration in response
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Figure 3. Inhibition of Ca2+ entry using nifedipine or mibefradil.
A, Representative traces of intracellular Ca2+ responses to 10-8 mol/L Ang Il (angiotensin II), 12 mmol/L K+, and 10-9, 10-7, and 10-5 mol/L uPSEM-817 (uPharmacologically Selective Effector Molecule-817) after pretreatment with nifedipine (left) or mibefradil (right; clone 17); n=6. B, Determination of the maximum Fura-2 (ratiometric fluorescent dye which bind to free intracellular Ca2+) ratio 340/380 nm in response to uPSEM-817, Ang II, and K+ after pretreatment with nifedipine (light grey) or mibefradil (dark grey) or without pretreatment (white; clone 17); n=6. C, Illustration of [Ca2+], signaling, illustrated by the determination of the area under the curve assessed for 360 s, in response to uPSEM-817, Ang II, and K+ after pretreatment with nifedipine (light grey) or mibefradil (dark grey) or without pretreatment (white; clone 17); n=6. D, Patch-clamp recordings of cells treated with uPSEM-817 and nifedipine or mibefradil (clone 17). n=11 to 17; P values were determined by 1-way ANOVA followed by Bonferroni post hoc comparison tests,*P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. CCB indicates Ca2+ channel blocker.
to Ca2+ channel blockers and uPSEM-817 cotreatment were due to differences in cell membrane depolariza- tion, we performed patch-clamp analyses (Figure 3D).
Before the administration of 10-7 mol/L uPSEM-817, we treated each patched cell with 10-6 mol/L mibefradil or with 10-6 mol/L nifedipine. These blockers were applied
locally using an ejector solution. The results displayed a range of responses among cells: in certain instances, the blockers effectively suppressed depolarization, return- ing the membrane potential to a state near its baseline (AEM, ~0); while in other cases, the membrane remained depolarized, albeit to a lesser degree than when exposed exclusively to 10-7 mol/L uPSEM-817 (Figure 3D).
a7-5HT3-R Activation by uPSEM-817 Led to a Decrease in Cell Proliferation and an Increase of Apoptosis
To evaluate the impact of elevated intracellular Ca2+ concentration on cell proliferation, cells were treated with 12 mmol/L K+ or 10-9 to 10-5 mol/L uPSEM-817 for 24 and 72 hours. The number of viable proliferat- ing cells was determined using a colorimetric method. Although following 24 hours of treatment, cell prolifera- tion remained unaffected by K+ or uPSEM-817 treat- ment, a decrease in cell proliferation was observed after 72 hours of treatment with the higher concentration of uPSEM-817 (Figure 4A).
To explore the impact on cell cycle progression of Ang II, K+, and uPSEM-817 treatment, we assessed the cell cycle phase distribution using propidium iodide staining and cytometric analysis at 8, 24, 48, and 72 hours. Treatment with Ang II for 8 hours resulted in a reduction in the percentage of cells in the G1 phase and in an increase in the percentage of cells in G2 com- pared with untreated cells suggesting an increase in cell proliferation (Figure 4B). After 24 hours of Ang II treat- ment, an increase in the percentage of cells in the G1 phase and a decrease in the S phase was also observed. Longer exposure to Ang II had no effect on the distribu- tion of cells across different phases of the cell cycle. In response to K+ treatment, a similar pattern was observed with a decrease in the percentage of cells in G1 and an increase in G2 after 8 hours. While no alterations in cell distribution were noted after 24 hours of K+ treat- ment, an increase in the percentage of cells in G1 and a decrease in the percentage of cells in G2 was observed (Figure 4B). Conversely, treatment with uPSEM-817 did not affect cell distribution among the different phases compared with untreated cells (Figure 4C). We assessed the effect of uPSEM-817 on apoptosis by determining the proportion of cells in the sub-G1 phase. The sub-G1 phase reflects DNA fragmentation, which occurs in the late stage of apoptosis. Similar to what was observed for cell proliferation results, no effect was observed when cells were treated with uPSEM-817, Ang II, and K+ for 24 hours (Figure 4D, left). At 72 hours, a sig- nificant increase in the percentage of cells in the sub- G1 phase was observed in response to K+, Ang II, and uPSEM-817 (10-7 mol/L; Figure 4D, right), indicating an increase in cell apoptosis.
a7-5HT3-R Activation by uPSEM-817 Results in the Activation of Specific Pathways
To gain insight into the impact of modulating intracel- lular Na+ concentration in cells expressing a7-5HT3-R, we conducted RNA-sequencing analysis on these cells (clones 17, 24, and 42) following treatment with 10-7 mol/L uPSEM-817 for 8 hours (Figures S6 and S7) and 24 hours (Figure 5; Figure S8). Hierarchical clus- tering effectively segregated cells treated for 8 and 24 hours from untreated cells (Figures S6A, S7A, and S8A; Figure 5A). After 8 hours of treatment, we identi- fied 28 differentially expressed genes with a fold change of at least 0.5; 18 upregulated genes (64.29%) and 10 downregulated genes (35.71%; Table S2). Gene ontol- ogy analyses unveiled 18 specific enriched biological processes in uPSEM-817-treated cells (Figure S6B; Table S3), primarily related to Ca2+ ion transport and sig- naling pathways, cell adhesion, and aldosterone synthe- sis and secretion. Following 24 hours of treatment, 30 genes were differentially expressed with a fold change of at least 0.5, with 22 (73.33%) genes upregulated and 8 (26.67%) genes downregulated (Figure 5B; Table S4).27,28 Enriched pathways included protein regulation of GTPase activity, Ras (Rat sarcoma virus) protein signal transduction, and aldosterone synthesis and regulation (Figure 5B; Table S5). These analyses were completed by using the gene set enrichment analysis and Hallmark database that provides information on the most universal cellular mechanisms (Figures S7 and S8). Using a false discovery rate <25%, we identified 6 differentially regu- lated pathways after 8 hours of treatment with pharma- cologically selective effector molecules and 7 after 24 hours. After 8 hours of treatment, TNFa (tumor necrosis factor-a) signaling via NF-KB (nuclear factor-KB) and coagulation pathway were found enriched in basal con- dition (Figure S7B), whereas Kras (v-Ki-ras2 Kirstenb rat sarcoma viral oncogene homolog) signaling, heme metabolism, UV response, and interferon-a response were found to be enriched in response to uPSEM-817 (Figure S7C). After 24 hours of treatment (Figure S8), only TNFa signaling via the NF-KB pathway (Figure S8B) was enriched in basal condition, and Myc proto- oncogene targets, oxidative phosphorylation, DNA repair apical surface, unfolded protein response, and G2/M checkpoint pathways were enriched after uPSEM-817 treatment (Figure S8C).
To identify genes and pathways specifically regulated by modulation of intracellular Na+ concentration, we also performed RNA sequencing on a7-5HT3-R-expressing cells treated with 12 mmol/L K+ and 10-8 mol/L Ang II for 8 and 24 hours (Figures S6 and S9; Figure 5). After 8 hours of treatment, 4932 genes were differentially expressed in response to Ang II and 728 in response to K+ (Figure S6C and S6G; Tables S6 and S7). Gene ontol- ogy analyses27,28 for 8 hours of Ang II treatment revealed
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Figure 4. Effect of uPSEM-817 on cell proliferation and apoptosis.
A, Cell viability was measured on cells treated for 24 or 72 hours with 12 mmol/L K+ or uPSEM-817 (uPharmacologically Selective Effector Molecule-817) by MTS (3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium) assay (clone 17). B, Cell cycle distribution measured by FACS (Fluorescence-Activated Cell Sorting) using propidium iodide in response to 10-8 mol/L Ang II (angiotensin II) and 12 mmol/L K+ (top), and 10-9, 10-7, and 10-5 mol/L uPSEM-817 (bottom; clone 17). C, Apoptosis determined by the proportion of cells in the sub-G1 phase (clone 17). D, Proportion of cells in the sub-G1 phase after 24 hours (left) or 72 hours (right) in response to 12 mmol/L K+, 10-8 mol/L Ang II, and 10-9, 10-7, and 10-5 mol/L uPSEM-817. n=6 (cell cycle), n=12 (proliferation); P values were determined by 1-way ANOVA followed by Bonferroni post hoc comparison tests,*P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.
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enrichment in 390 different pathways (Table S8), with significant involvement in the regulation of reticulum activity, negative regulation of cell growth, and aldoste- rone, cortisol, and parathyroid hormone synthesis and secretion (Figure S6D). Eight hours of treatment with K+ resulted in enrichment of 211 pathways (Table S9),
including membrane depolarization during action poten- tial, Ca2+ and Na+ ion transport, and circadian rhythm (Figure S6H). Interestingly, both Ang Il and K+ treatment led to enrichment in positive regulation of transcription and MAPK (Mitogen-Activated Protein Kinase) signaling pathways. Among the 28 differentially expressed genes
in response to uPSEM-817, 17 were common to Ang II (OLFML2B, RGPD6, LMOD1, MAT1A, MC2R, CA2, CAC- NA1C, EGFR, ETV5, TNXB, PFKP, antisense to WASF3, antisense to ERVW-1 and PEX1, PPP2R1B, LYPLAL1- AS1, LONRF2, and ST6GALNAC6; Figure S6E) and 12 to K+ (OLFML2B, LMOD1, MAT1A, MC2R, CA2, CACNA1C, ATP8B4, ETV5 PFKP, PPP2R1B, LYPLAL1-AS1, and LONRF2; Figure S6I). Among the 18 enriched pathways in response to uPSEM-817, 9 were commonly enriched in response to Ang II (Figure S6F) and 5 in response to K+ (Figure S6J). Most importantly, 11 genes were spe- cifically regulated by uPSEM-817 when compared with Ang II (HPX, EPHA10, ATP8B4, HHIP, MICAL1, RBMS2, E9PAM4, H7C2Y5, TP53TG3B, DUXAP8, and RNVU1- 7) and 16 when compared with K+ (HPX, antisense to WASF3, EPHA10, HHIP, ST6GALNAC6, antisense to ERVW-1 and PEX1, EGFR, MICAL1, TNXB, RGPD6, RBMS2, E9PAM4, H7C2Y5, TP53TG3B, DUXAP8, and RNVU1-7). This allowed us to define a specific Na+-induced signature composed of 10 genes (HPX, EPHA10, HHIP, MICAL1, RBMS2, E9PAM4, H7C2Y5, TP53TG3B, DUXAP8, and RNVU1-7; Figure S10).
After 24 hours of treatment, 589 genes were sta- tistically differentially expressed in response to Ang II (Figure 5C and 5D; Table S10). Among them, 12 were common to uPSEM-817 (SOBP, ADRA2A, ETV5, anti- sense to DLGAP1, VSIR, GCNA, FCHSD1, RAPGEFL1, PPP2R1B, CYP11B2, LYPLAL1-AS1, and PRSS53); 146 pathways were specifically enriched in response to Ang II and 2 (Cushing syndrome and aldosterone synthesis and secretion) were commonly enriched in response to Ang II and uPSEM-817 (Figure S9B; Table S11). Interestingly, enriched pathways included those associated with cell adhesion and extracellular matrix organization (Figure S9A). K+ treatment for 24 hours led to the regulation of 798 genes (Figure S7A; Figure 5F; Table S12); among them, 12 were common to uPSEM- 817 (KLF10, ADRA2A, C9ORF72, MC2R, ETV5, RAP- GEF4, VSIR, GCNA, FCHSD1, PPP2R1B, CYP11B2, and LYPLAL1-AS1). Similarly, enriched pathways included those associated with cell adhesion and extracellular matrix organization (Figure S9C; Table S13). Five of the 8 enriched pathways identified in response to uPSEM- 817 treatment were also commonly enriched in response to K+ (Figure S9D). These common pathways involved positive regulation of GTPase activity, Cushing syndrome, negative regulation of cell migration, Ras protein signal transduction, and aldosterone synthesis and secretion. Notably, Cushing syndrome and aldosterone biosynthesis and secretion pathways were also commonly enriched between uPSEM-817 and Ang II. We were able to define a 24-hour sodium-specific signature composed by 14 genes (FMNL1-AS1, IDI2-AS1, WHAMMP1, MUSTN1- ITIH4 readthrough, NFKBIZ, KCNQ1OT1, SH3BP5-AS1, CDK10, PTPRU, IL17RD, TMEM67, MAZ-AS, DOP1B, and 1 unknown sequence; Figure S11).
The list of differentially expressed genes was com- pared with their expression in 11 control adrenals and 123 APAs retrieved from transcriptomic data29 (Tables S14 and S15). Of these 123 APAs, 50 carried a muta- tion in the KCNJ5 gene and 73 in another APA driver gene; the comparison was, therefore, made between the 11 control adrenals, the 50 APAs with a mutation in the KCNJ5 gene, and the 73 APAs without a muta- tion in the KCNJ5 gene. After 8 hours of treatment with 10-7 mol/L of uPSEM-817, among the 55 identified genes that were significantly differentially expressed without consideration of the fold change, the expres- sion of 49 genes was found in our transcriptomic data from control adrenals and APAs and could be retrieved. In these data, no expression was detected for 5 genes, and of the remaining 44 genes, 13 were also found to be differentially expressed in APAs compared with con- trol adrenals (Table S12), 2 only in APAs with a KCNJ5 mutation (HPX and WHRN) and 3 in APAs without KCNJ5 mutation (ETV5, GPAM, and OLFML2B). Inter- estingly, among the 13 genes differentially expressed in APAs independently of the mutational status, 4 were found to be upregulated (CACNA1C, ATP2B2, VDR, and EPHA10) and 1 downregulated (RGPD6) in both APAs and a7-5HT3-R cells, whereas 6 genes were found to be upregulated in a7-5HT3-R cells but downregulated in APAS (PPP2R1B, LONRF2, ATP8B4, TNXB, ITGA7, and HSPA12A), independently of the mutational status and 2 downregulated in response to uPSEM-817 but upregulated in all APAs (TMEM200A and ELL2). Simi- larly, after 24 hours of treatment, among the 73 genes identified, genes that were significantly differentially expressed regardless of the fold change, the expression of 64 genes could be retrieved from our transcriptomic data. No expression was found for 10 genes, and among the remaining 54 genes, 15 were also found to be differ- entially expressed in APAs compared with control adre- nals (Table S13). Among these 15 genes, 3 were found to be commonly upregulated in both o7-5HT3-R cells and APAs (FCHSD1, CACNA1C, and TTN1) and 1 down- regulated (KLF10) whereas 2 were found to be upregu- lated in a7-5HT3-R cells and downregulated in APAs, independently of the mutational status (PPP2R1B and NEAT1), and 4 downregulated in a7-5HT3-R cells and upregulated in all APAs (STMN3, FIBCD1, FSCN1, and CCDC71L). Interestingly, 3 genes were found to be significantly regulated only in APAs without KCNJ5 mutations, 2 downregulated in a7-5HT3-R cells but upregulated in APAs (ETV5 and CEP170), and 1 down- regulated in both (NR2F2); 2 genes were found to be significantly regulated only in APAs with KCNJ5 muta- tions, 1 downregulated in a7-5HT3-R cells but upregu- lated in APAs (NFKBI2), and 1 downregulated in both (ABHD2). Interestingly, none of the genes commonly regulated in a7-5HT3-R cells and APAs belongs to the Na+ signature defined after 8 and 24 hours of treatment,
suggesting that they are probably involved in the early phase of APA development.
DISCUSSION
Somatic KCNJ5 mutations are the most frequent genetic abnormalities found in APAs with a prevalence between 43% and 75% of cases. APAs with KCNJ5 mutations are more frequent in women and at younger age29,30 and are characterized by hybrid steroid production and larger adenoma size.31 Expression of mutated KCNJ5 in adre- nocortical cells leads to increased aldosterone produc- tion without increasing cell proliferation,17,19 raising the question as to whether those mutations are sufficient to lead to both increased aldosterone production and ade- noma formation. Here, we describe the development of an adrenocortical cell model that recapitulates the main features of KCNJ5 mutations, by modulating on demand sodium entry into the cells using chemogenetic tools. Increased sodium entry through the chimeric a7-5HT3-R upon stimulation with uPSEM-817 led to cell membrane depolarization, opening of voltage-gated Ca2+ channels and increased intracellular Ca2+ concentrations, resulting in the stimulation of CYP11B2 expression and increased aldosterone biosynthesis.
The steroid response of H295R-S2 a7-5HT3-R cells to Ang Il is similar to that described in H295R cells,32 with an early and transitory increase of pregnenolone, progesterone, and 11-deoxycorticosterone and a late and sustained production of aldosterone and corticos- terone.32 In contrast, sodium entry into the cells induced by uPSEM-817 led to a specific steroid profile only par- tially overlapping with that induced by Ang II and K+. In particular, we observed a prolonged induction of early steroid precursors and deoxycorticosterone, suggesting an action on both early and late steps of aldosterone bio- synthesis, as well as a delayed and smaller induction of aldosterone biosynthesis; there was no induction of glu- cocorticoid biosynthesis in H295R-S2 a7-5HT3-R cells. An increase of 18-oxocortisol and 18-hydroxycortisol was also observed in response to uPSEM-817 in 2 of the 3 clones, suggesting production of hybrid steroids in response to increased intracellular Na+ concentra- tion. This is consistent with results reported for the overexpression of KCNJ5 harboring the p.Tyr158Ala mutation in HAC15 (Human Adrenocortical Carcinoma cell line) cells resulting in a significant increase of both 18-hydroxycortisol and 18-oxocortisol.18,33 However, despite similar depolarization of the cells in response to uPSEM-817 or Ang II, our data suggest a delayed activa- tion of mineralocorticoid biosynthesis in response to the modulation of intracellular Na+ concentration compared with Ang II and K+ that could be due to the activation of specific signaling pathways. This hypothesis is supported by our finding of a specific gene expression signature associated with Na+ influx into the cells.
Interestingly, gene expression analysis revealed expression of KCNJ5 in H295R-S2 a7-5HT3-R cells (Figure S12A), which remains unchanged after uPSEM- 817 treatment. In this context, Na+ may act as an activa- tor for the KCNJ5 channel34 by binding to the C-terminal part of the channel, near a region also sensitive to PtdIns(4,5)P2, enhancing the sensitivity of the channels to PtdIns(4,5)P2.35 The interaction of PtdIns(4,5)P2 with the C-terminal part of GIRK4 has been shown to regu- late its opening by stabilizing the structure of the pore.35 The resulting K+ extrusion may attenuate the activa- tion of Ca2+ signaling and explain the delayed increase in aldosterone biosynthesis observed in response to uPSEM-817.
If the role of KCNJ5 mutations in inducing aldoste- rone biosynthesis has been clearly established, their role in promoting abnormal cell proliferation is still a matter of debate. In our model, we demonstrated that Na+ entry into the cells lead to decreased cell proliferation and increased apoptosis. This is in accordance with previous studies showing that expression of KCNJ5 mutants in adrenocortical cells resulted in decreased cell prolifera- tion associated in some cases with increased apopto- sis.18,20 Choi et al8 postulated that the activation of Ca2+ signaling induced by KCNJ5 mutations is responsible for both increased aldosterone biosynthesis and cell proliferation, and indeed some studies also reported an association between KCNJ5 mutations and larger adenoma size29,30 and a positive correlation between the expression of Ki67, a marker of cell proliferation, and the diameter of APA harboring KCNJ5 mutations.2º Specific factors overexpressed in APA harboring KCNJ5 muta- tions36,37 have been suggested to possibly counteract the pro-apoptotic effect of these mutations,20 suggesting the existence of compensatory mechanisms maintaining cell proliferation over long term. In particular, expression of TDGF1 and VSNL1, 2 genes with anti-apoptotic prop- erties, was found to be upregulated in KCNJ5 mutated APAs.38,39 However, in our cell model, the expression of VSNL1 was not modified (Figure S12B) and TDGF1 was not expressed at all. The increased expression of these genes may be a later event secondary to the development of an APA to compensate for Na+-induced cell death. Moreover, signaling pathway analyses revealed down- regulation of genes involved in the G2/M checkpoint pathway and in the progression through the cell division cycle, probably contributing to the decrease of cell pro- liferation observed in our model. Alternatively, our data suggest that other mechanisms may be responsible for abnormal cell proliferation, leading to the development of APA. These include the presence of 2 hits, 1 responsible for cell proliferation and the other for autonomous aldo- sterone production. Recently, we and others identified risk alleles associated to PA in genome-wide association studies. Within the identified genetic loci, some genes appear to modulate adrenal cortex homeostasis and may
affect cell proliferation, eventually generating a propitious environment leading to the occurrence of somatic muta- tions.40,41 The 2 models are not mutually exclusive and may be linked together by mechanisms that remain to be identified. Finally, in FH-III (Familial Hyperaldosteronism type III) it cannot be ruled out that Na+ entry induced by KCNJ5 mutations may have an impact on cell prolifera- tion during adrenal development, which could explain the bilateral hyperplasia observed in severe cases.8,19
Ang II and potassium both stimulate aldosterone biosynthesis by activating Ca2+ signaling, but through different mechanisms, resulting in only a partial over- lap in the genes they induce. Similarly, uPSEM-817, by increasing intracellular Na+ levels, induces membrane depolarization, which like potassium, leads to opening of voltage-gated Ca2+ channels and increase of intra- cellular Ca2+ concentration. However, uPSEM-817 also has unique effects, which account for the partial overlap in the differentially expressed genes induced by these 3 compounds. Gene expression profiles allowed us to identify a sodium-induced gene signature composed of 10 genes after 8 hours of treatment with uPSEM-817 (Figure S9) and 14 genes after 24 hours of treatment (Figure S10). Interestingly, among these lists of genes, some are involved in cell cycle regulation, proliferation, and apoptosis. Hence, the expression of RBMS2 and PTPRU, 2 genes involved in the control of cell cycle progression42 and cell proliferation,43,44 respectively, was decreased after 8 or 24 hours while the expression of CDK10 (Cyclin Dependent Kinase 10), a Cdc2 (Cell division control protein 2)-related kinase involved in the regulation of the G2/M phase of the cell cycle, was increased. However, the role of CDK10 in regulating cell proliferation is not clear, as some studies suggest a role of CDK10 in cell proliferation activation,45-47 while others report a tumor suppressor role for CDK10 through inhi- bition of cell proliferation.48,49 The identification of these genes in the Na+ signature supports our results showing an inhibitory effect of uPSEM-817 on cell proliferation. Among the genes belonging to the Na+ signature after 8 hours of treatment with uPSEM-817, HPX expression was found to be upregulated. HPX encodes hemopexin, a protein with high binding affinity for heme. Interestingly, CYP11B2 and other cytochrome P450 enzymes use heme as a cofactor required for their activity. The avail- ability of heme has been shown to affect aldosterone and corticosterone biosynthesis in rats.5º Our transcriptomic data revealed a significant increase of HPX expression in APAs harboring a KCNJ5 mutation,29 and a recent study also reported increased expression of HPX associated with CpG hypomethylation51 in APAs compared with the adjacent adrenal gland, suggesting a role of HPX in APA development potentially through the regulation of aldo- sterone biosynthesis. Similarly, the expression of MC2R, the melanocortin 2 receptor, was found to be increased in APAs associated with CpG hypomethylation.51 Gene
expression profiling of H295R-S2 a7-5HT3-R cells treated with Ang II, K+, or uPSEM-817 also revealed a significant increase of MC2R expression; and a previous study reported higher expression of MC2R in APAs com- pared with control adrenals.52 Finally, a recent work of our laboratory shows that expression of MC2R in APAs was more frequently found in regions expressing CYP11B2,53 suggesting a potential role in regulating aldosterone bio- synthesis. Moreover, the existence of a continuum of PA and dysregulated aldosterone production, prominently influenced by ACTH (adrenocorticotropic hormone), has recently been described.54
In conclusion, we demonstrate that H295R-S2 a7-5HT3-R cells are a new cell model in which intracel- lular Ca2+ concentration can be modulated on demand, reproducing the major features of cells harboring KCNJ5 mutations. The stimulation of Na+ entry into the cells leads to cell membrane depolarization, Ca2+ entry into the cells, activation of Ca2+ signaling, increased expression of CYP11B2, and stimulation of the mineralocorticoid biosynthesis pathway. This is associated with a decrease of cell proliferation and an increase of apoptosis, indi- cating that additional events may be required for the development of APA. RNA sequencing revealed that Na+ entry into the cells is responsible for a specific transcrip- tomic signature that may explain some of the features of APA carrying KCNJ5 mutations. This cellular model thus provides important new insight into the mechanisms leading to PA development, uncovering new mechanisms involved in the disease, thereby paving the way for new therapeutic approaches in PA.
The strengths of our study are multiple. First, the model provides a novel and highly valuable tool for studying the early events that occur following a mutational event lead- ing to Na+ entry into the cells, such as mutations affect- ing KCNJ58 but also mutations affecting SLC30A1,15 ATP1A1,9,16 and MCOLN3.55 Second, the cell model allows following dynamic changes in cell proliferation and function and to explore molecular mechanisms and specific transcriptional changes induced by mutations in PA. Importantly, these events cannot be studied in human tissues from patients with APAs, as they only provide a snapshot at a given moment of the development of the disease. Indeed, we were able to show that while modi- fication of steroid biosynthesis occurs rapidly, alterations in the cellular sodium balance result in reduced cell pro- liferation and increased apoptosis, in accordance with data from the literature.18 However, we cannot exclude the possibility that, at a later stage of development of APA, the constitutive activation of Ca2+ signaling might stimulate cell proliferation. Another significant finding of our study was the identification of specific pathways and signatures associated with the modification of the Na+ influx. While some pathways, such as aldosterone biosynthesis and secretion, calcium signaling, and cell proliferation, were further explored, others remain to be
investigated and will be the scope of future research. Moreover, this innovative cellular model offers a valuable tool for evaluating the molecular effects of novel thera- peutic strategies for PA, particularly the application of novel aldosterone synthase inhibitors.
Limitations of our study are related to possible biases in interpreting proliferation studies in our model. Indeed, these cells are generated from H295R-S2 cells, which derive from an adrenocortical adenoma carrying TP53 (Tumor protein P53) and ß-catenin mutations. Because ß-catenin signaling is known to promote cell prolifera- tion in various tumors, this model may be suboptimal for investigating positive effects on proliferation. Neverthe- less, we have demonstrated that the activation of Ca2+ signaling, through modulation of Na+ entry into the cells, is achievable within this context. Therefore, the pres- ence of ß-catenin mutations does not pose an obstacle to these specific investigations. An additional limitation may be the lack of the microenvironment of steroido- genic cells; further studies in integrated mouse models will be necessary to establish the natural history of PA development.
PERSPECTIVES
Using a chemogenetic approach allowing the modula- tion of sodium influx on demand, we have generated a new cell model that replicates the main characteristics of KCNJ5-mutated APAs and provides new insights into the mechanisms responsible for their development. Our results show that modulation of intracellular ionic balance leads to cell membrane depolarization, activa- tion of Ca2+ signaling, and enhanced steroid biosynthe- sis, associated with a decrease in cell viability and an increase in apoptosis, indicating that additional events may be required for the development of an APA with a KCNJ5 mutation.
This innovative model could be used to test the molec- ular impact of new treatments for PA in vitro. Additionally, applying this chemogenetic approach could also enable the development of a new inducible mouse model of PA, providing a valuable tool for dissecting the mechanisms underlying APA development and for evaluating new therapeutic strategies.
ARTICLE INFORMATION
Received May 06, 2024; accepted January 27, 2025.
Affiliations
Université Paris Cité, INSERM, PARCC (Paris Cardiovascular Research Center), France (B.F., T.C., Z.R.A.S., I.G .- D., N.F., M.F., J .- S.H., S.T .- A., F.L.F .- R., M .- C.Z., S. Boulkroun). Electrophysiology Facility, University of Zurich, Switzerland (R.A.C., D.P.). CIC1418 and DMU CARTE (J .- S.H.), Service de Physiologie (S.T .- A., S. Baron), and Service de Génétique (M .- C.Z.), Assistance Publique Hôpitaux de Paris, Hôpital Européen Georges Pompidou, France. Department of Neurosci- ences, Howard Hughes Medical Institute, University of California San Diego (C.J.M., S.M.S.).
Sources of Funding
This work was funded through institutional support from INSERM (Institut Na- tional de la Santé et de la Recherche Médicale), by Agence Nationale pour la Recherche (ANR-18-CE93-0003-01), Fondation pour la Recherche Médicale (EQU201903007864), the European Union Horizon 2020 research and in- novation program under the Marie Sklodowska-Curie grant agreement number 954798 (MINDSHIFT Innovative Training Network), for which M .- C. Zennaro is the principal investigator, and by a grant from the Leducq Foundation (18CVD05) for J .- S. Hulot. Research in the laboratory of D. Penton was financed by the Swiss National Foundation for Science (CRSII-222773) and by the University Research Priority Program (URPP) of the University of Zurich ITINERARE-Innovative Ther- apies in Rare Diseases.
Disclosures
None.
Supplemental Material
Supplemental Materials and Methods Tables S1-S15 Figures S1-S13
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