Endocrinology
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CACNA1HM1549V mutant calcium channel causes autonomous aldosterone production in HAC15 cells and is inhibited by Mibefradil
Esther N. Reimer1, Gudrun Walenda1, Eric Seidel1, and Ute I. Scholl1
1Department of Nephrology, Medical School, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany.
We recently demonstrated that a recurrent gain-of-function mutation in a T-type calcium channel, CACNA1HM1549V, causes a novel Mendelian disorder featuring early-onset primary aldosteronism and hypertension. This variant was found independently in five families. CACNA1HM1549V leads to impaired channel inactivation and activation at more hyperpolarized potentials, inferred to cause increased calcium entry. We here aimed to study the effect of this variant on aldosterone production.
We heterologously expressed empty vector, CACNA1HWT and CACNA1HM1549V in the aldosterone- producing adrenocortical cancer cell line H295R and its subclone HAC15. Transfection rates, ex- pression levels and subcellular distribution of the channel were similar between CACNA1HWT and CACNA1HM1549V. We measured aldosterone production by ELISA and CYP11B2 (aldosterone syn- thase) expression by real-time PCR. In unstimulated cells, transfection of CACNA1HWT led to a 2-fold increase in aldosterone levels compared to vector-transfected cells. Expression of CACNA1HM1549V caused a 7-fold increase in aldosterone levels. Treatment with angiotensin II or increased extra- cellular potassium levels further stimulated aldosterone production in both CACNA1HWT- and CACNA1HM1549V-transfected cells. Similar results were obtained for CYP11B2 expression. Inhibition of CACNA1H channels with the T-type calcium channel blocker Mibefradil completely abrogated the effects of CACNA1HWT and CACNA1HM1549V on CYP11B2 expression.
These results directly link CACNA1HM1549V to increased aldosterone production. They suggest that calcium channel blockers may be beneficial in the treatment of a subset of patients with primary aldosteronism. Such blockers could target CACNA1H or both CACNA1H and the L-type calcium channel CACNA1D that is also expressed in the adrenal gland and mutated in patients with primary aldosteronism.
P rimary aldosteronism (PA) (1) is the most common cause of secondary hypertension, with a prevalence of up to 11% in hypertension referral centers (2). PA is due to autonomous production of the steroid hormone aldo- sterone, which is physiologically produced in the zona glomerulosa, mainly in response to volume depletion (via angiotensin II) or hyperkalemia. Aldosterone increases re- nal and intestinal salt (re)absorption and potassium se- cretion. Excess production leads to hypertension and op- tional hypokalemia. The main causes of PA are bilateral
adrenal hyperplasia, also known as idiopathic hyperaldo- steronism, and aldosterone-producing adenomas. Famil- ial hyperaldosteronism (FH) is rare, and all solved forms are inherited in an autosomal-dominant fashion (3). These include (a) glucocorticoid-remediable aldosteronism (GRA or FH-I) (4), with crossing-over events between CYP11B2 (aldosterone synthase) and CYP11B1 (11ß-hy- droxylase) (4), (b) FH-III, with mutations in the inward rectifier potassium channel KCNJ5 (5-7), and (c) a syn- drome of PA, seizures, and neurologic abnormalities
Abbreviations:
Copyright @ 2016 by the Endocrine Society
caused by mutations in the voltage-gated L-type calcium channel CACNA1D (8).
Recently, by sequencing the exomes of 40 children with PA diagnosed at age 10 years or below, we discovered the identical, novel heterozygous germline mutation (M1549V) in the CACNA1H gene in five families (9). CACNA1H is expressed in the adrenal gland and encodes a T-type calcium channel that is activated by small depo- larizing potentials. Electrophysiological studies in HEK293 cells demonstrated that the mutation leads to reduced channel inactivation and a shift of activation to less depolarized potentials. These effects were inferred to cause increased calcium influx in adrenal glomerulosa cells.
We here express CACNA1HWT and M1594V in the hu- man adrenocortical cancer cell line H295R and its sub- clone HAC15 and demonstrate that CACNA1HM1594V increases aldosterone production and expression of the aldosterone synthase gene CYP11B2. We further show that these effects are abolished by treatment with the T- type calcium channel blocker Mibefradil, suggesting that similar compounds may be useful in the treatment of pa- tients with CACNA1HM1549V.
Materials and Methods
Cell culture and reagents
HAC15 cells were cultured at 37°℃ and 5% CO2 in DMEM: F12 (1:1, Gibco, Life Technologies, Grand Island, NY) supple- mented with 5% Cosmic Calf Serum (Hyclone Laboratories, Logan, UT), 1% Penicillin/Streptomycin, 1% Insulin-Transfer- rin-Selenium, 1% nonessential amino acids and 0.1% lipid mix- ture (all Gibco). NCI-H295R cells were cultured in DMEM/ Ham’s F12 medium (Gibco) supplemented with 2.5% Ultroser G (Pall Biosepra, Cergy, France), 1% ITS+ Premix (Corning, Low- ell, MA) and 1% Penicillin/Streptomycin.
Angiotensin II and Mibefradil (Sigma-Aldrich, St. Louis, MO) were dissolved in PBS, and cell culture grade KCl (Sigma- Aldrich) in H2O.
Plasmids
CACNA1HWT and CACNA1HM1549V cDNAs were sub- cloned from pCMV6-Entry (Origene, Rockville, MD) (9) into pCMV6-AC-IRES-GFP-Puro (Origene) using MluI and RsrII (NEB, Beverly, MA). Plasmids were purified using the EndoFree Maxi Kit (Qiagen, Hilden, Germany).
Transfection, Treatment and Harvesting for ELISA and qPCR
Two million HAC15 cells were transfected with 1 µg DNA using the Amaxa Cell Line Nucleofector Kit R and the Nucleo- fector I (program X-05) (Lonza, Cologne, Germany) according to the manufacturer’s instructions. Transfection was confirmed by fluorescence microscopy. 24 hours after transfection, Cosmic
Calf Serum concentration was reduced to 0.1%. 48 hours after transfection, the medium was changed to serum-deprived me- dium containing either 10 nM angiotensin II, 10 µM Mibefradil, 14 mM K+ (total, by addition of KCI) or a vehicle control (PBS (Biochrom, Berlin, Germany)). 72 hours after transfection, cells and/or supernatants were harvested for aldosterone EIA and iso- lation of protein or RNA.
Immunofluorescent analysis and flow cytometry of transiently transfected cells
Three million H295R cells were transfected with 3 µg plas- mid DNA using the Nucleofector I (kit R, program P-20), fol- lowed by 30 minutes recovery in RPMI 1640 medium. For im- munofluorescence, 5 × 105 cells were seeded onto Nunc Lab- Tek Chamber Slides (Thermo Fisher Scientific, Waltham, MA) in 1.5 mL H295R culture medium. Three days after transfection, cells were fixed with 4% PFA in PBS with 4% sucrose and stained with primary antibodies rabbit @-Cav3.2 (#ACC-025, Alomone, Jerusalem, Israel, 1:500) or mouse ANTI-FLAG M2 (#F3165, Sigma-Aldrich, 1:200). Secondary antibodies were Goat anti- Rabbit IgG (H+L) Alexa Fluor 647 conjugate, and Goat anti- Mouse IgG (H+L) Alexa Fluor 633 conjugate (#A-21 052 and #A-21 245, both ThermoFisher, 1:250). Images were acquired with a LSM 510 Meta (Zeiss, Oberkochen, Germany) and pro- cessed using the manufacturer’s software.
For flow cytometry, cells were isolated by trypsination 48 hours after transfection. All stainings were performed in regular medium. The live cell population was identified after propidium iodide (Sigma-Aldrich, 5 ng/uL) staining, using the PerCP-Cy5.5 channel of a FACSCanto II Flow Cytometer (BD Biosciences, San Jose, CA). For FLAG analysis, cells were fixed (4% PFA, 10 minutes), permeabilized (0.1% Saponin (Sigma-Aldrich), 5% FCS/PBS, 5 minutes) and stained with mouse ANTI-FLAG M2 (#F3165, Sigma-Aldrich; 1:200; 60 minutes on ice), followed by Alexa Fluor 633 conjugate (#A-21 245, Thermo Fisher Scien- tific; 1:100; 60 minutes on ice). Alexa Fluor 633 was measured using the APC channel and GFP using the FITC channel. GFP fluorescence of unfixed transfected cells was recorded as control. Results were analyzed using the FlowJo software.
RNA isolation and real-time PCR
Total RNA was harvested using the RNeasy Mini Kit (Qia- gen) according to the manufacturer’s instructions. 200 ng RNA was transcribed using the Quantitect Reverse Transcription Kit (Qiagen). Expression levels of CYP11B2 and GAPDH were quantified in a 7300 Real-Time PCR System (Applied Biosys- tems, Foster City, CA) using the Taqman Gene Expression Mas- ter Mix (Applied Biosystems), published primers and probes for CYP11B2 (10) and a commercial assay for GAPDH (#HS02758991_g1, Applied Biosystems). Samples were ana- lyzed in triplicates. AACT values were calculated by normaliza- tion of ACT values, with average ACT values of PBS-treated empty vector-transfected cells as a reference.
Aldosterone EIA and protein
Aldosterone concentrations were determined using the Aldo- sterone EIA Kit (#10004377, Cayman, Ann Arbor, MI) accord- ing to the manufacturer’s instructions. Two dilutions were mea- sured in duplicates each. For values outside the assay range, both duplicates were removed from the analysis. Total protein levels
were determined in duplicates from cell lysates using the Micro BCA Protein Assay kit (Thermo Scientific, Rockford, IL).
Statistical analysis
Data were analyzed using the Prism software (GraphPad, San Diego, CA). P values were determined via unpaired, two-tailed Student’s t test in Prism (ELISA, qPCR) or via one-way analysis of variance (ANOVA) in the Microsoft Excel analysis tool (flow cytometry).
Results
The human NCI-H295R cell line, derived from a female patient with adrenocortical carcinoma (11, 12), is a com- monly used model of aldosterone production. We used electroporation to transfect H295R cells with FLAG- tagged CACNA1HWT and CACNA1HM1594V in a vector containing the IRES sequence and GFP (Figures 1-2). Transfection rates were assessed by flow cytometry (Fig- ure 1A). The number of GFP-positive cells was > 12%, and no significant differences were observed between empty vector, CACNA1HWT and CACNA1HM1594V. Similarly, we assessed the number of FLAG-positive cells, which was about 20% in both CACNA1HWT- and CACNA1HM1594V-transfected cells, with very little un- specific staining in the empty vector control. We then as- sessed median FLAG (approximated by AF633 fluores- cence) and GFP expression within four bins of GFP expression after staining. There was no significant differ- ence between CACNA1HWT and CACNA1HM1549V re- garding GFP or AF633 fluorescence, suggesting that ex- pression levels are similar (Figure 1B, Supplemental Table 1). Next, we assessed the subcellular distribution of CACNA1HWT and CACNA1HM1594V. By immunofluo- rescence with a native CACNA1H antibody, endogenous expression was detected in empty vector-transfected cells, consistent with previous studies using both electrophysi- ology and real-time PCR (13, 14). Confocal microscopy localized most the channels to intracellular compartments (Figure 2A). Transfection of CACNA1H substantially in- creased expression levels without changing the expression pattern, with similar results for CACNA1HWT and CACNA1HM1594V. Immunofluorescence with an anti- FLAG antibody specifically stained heterologously trans- fected cells (Figure 2B).
To assess effects of CACNA1HWT and CACNA1HM1594V on aldosterone production, we trans- fected HAC15 cells, a subclone of NCI-H295R that has previously been used to assess the effect of mutant chan- nels on aldosterone production (15-18). Cells were serum- starved, and aldosterone levels were measured in the su- pernatant by ELISA (Figure 3 A). Compared with empty
vector-transfected cells, CACNA1HWT-expressing cells showed a 1.96-fold increase in aldosterone levels (23.93 ± 1.08 pg/µg protein vs 12.23 ± 0.53 pg/µg protein, P < .0001, N = 4 independent transfections for all ELISA re- sults). Importantly, expression of CACNA1HM1594V in- creased aldosterone production even further (3.70-fold of CACNA1HWT, 88.58 ±5.57 pg/µg protein, P <. 0001), consistent with a gain of function (Figure 3A). Because no angiotensin II was administered to the serum-starved cells, the conditions resemble the in vivo situation in PA, with suppressed renin and angiotensin levels. The results dem- onstrate that CACNA1HM1549V can cause autonomous aldosterone production in the absence of depolarizing stimuli.
To examine the effects of such depolarizing stimuli on CACNA1HWT and CACNA1HM1594V expressing cells,
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we incubated cells with 10 nM angiotensin II, a concen- tration previously shown to increase aldosterone produc- tion (19). Angiotensin II led to significant increases in al- dosterone production in all groups (P < . 0001 for all). Angiotensin II-stimulated CACNA1HM1549V-transfected cells produced significantly more aldosterone than angio- tensin II-stimulated CACNA1HWT-transfected cells (203.71 ± 18.47 pg/µg protein vs 153.07 ± 9.63 pg/µg protein, P = . 018), but the relative increase in response to angiotensin II was smaller for CACNA1HM1549V than for CACNA1HWT (2.30-fold vs 6.40-fold).
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Next, we examined the effect of increased extracellular potassium levels. After stimulation with 14 mM K+ (19), aldosterone production increased in cells transfected with empty vector, CACNA1HWT (both P < . 0001) and CACNA1HM1594V (P = . 0015), with a smaller relative increase for CACNA1HM1549V (1.50-fold vs 4.58-fold for CACNA1HWT).
Lastly, we investigated whether the gain-of-function effects of the CACNA1HM1549V variant can be blocked by pharmacological intervention. Treatment with Mibe- fradil, a potent CACNA1H blocker (20), significantly de- creased aldosterone production in empty vector-trans- fected cells (5.43 ± 0.17 pg/µg protein vs 12.23 ± 0.53 pg/µg protein in untreated cells, P < . 0001), in line with published data from cultured bovine glomerulosa cells (21). More importantly, Mibefradil significantly reduced the increased aldosterone production caused by transfec- tion of CACNA1HWT (9.51 ± 0.73 pg/ug protein vs 23.93 ± 1.08 pg/µg protein in untreated cells, P <. 0001) and CACNA1HM1549V (16.63 ± 1.32 pg/µg protein vs 88.58 ± 5.57 pg/µg protein, P < . 0001).
unstimulated
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| Peptide/ protein target | Antigen sequence (if known) | Name of Antibody | Manufacturer, catalog #, and/or name of individual providing the antibody | Species raised in; monoclonal or polyclonal | Dilution used |
|---|---|---|---|---|---|
| Cav3.2 | CHVEGPQERARVAHS | Anti- | Alomone, #ACC-025 | rabbit, | 0.388 888 |
| Cav3.2 | polyclonal | 889 | |||
| FLAG | DYKDDDDK | ANTI- | Sigma-Aldrich, #F3165 | mouse, | 0.180 555 |
| FLAG M2 | monoclonal | 556 | |||
| Mouse IgG | Goat | ThermoFisher, #A-21 052 | goat, polyclonal | 0.215 277 | |
| anti- | 778 | ||||
| Mouse lgG (H + L) Secondary Antibody, Alexa Fluor | |||||
| 633 conjugate | |||||
| Rabbit | Goat | ThermoFisher, #A-21 245 | goat, polyclonal | 0.215 277 | |
| IgG | anti- | 778 | |||
| Rabbit lgG (H + L) Secondary Antibody, Alexa Fluor 647 conjugate |
Next, we assessed expression levels of CYP11B2 (al- dosterone synthase), the rate-limiting enzyme in aldoste- rone biosynthesis, by real-time PCR (Figure 3B).
Transfection of CACNA1HWT increased CYP11B2 levels by a factor of 2.49 compared to cells transfected with empty vector. Transfection of CACNA1HM1549V further increased levels by a factor of 7.10 compared to CACNA1HWT (P =. 0072), again compatible with a gain- of-function effect of the mutant channel. Similar to the results of aldosterone ELISAs, treatment with angiotensin II further stimulated CYP11B2 expression, with a larger relative effect on CACNA1HWT (9.12-fold, P = . 0048) than on CACNA1HM1549V (2.30-fold, P = . 0107). The stimulatory effect of treatment with KCl on CYP11B2 expression was larger than the effect on aldosterone in the supernatant. Treatment of CACNA1HWT or CACNA1HM1549V-transfected cells with Mibefradil sup- pressed CYP11B2 expression to the same levels observed in empty vector-transfected cells.
Discussion
We here show that CACNA1HM1549V causes autonomous aldosterone production by stimulation of CYP11B2 expression.
In line with previous reports (22, 23), CACNA1H channels were found in intracellular compartments. Mod- ulation of channel trafficking may play a role in the reg- ulation of calcium entry.
Remarkably, the gain-of-function effect of CACNA1HM1549V is more pronounced in the absence of
stimulatory factors (angiotensin II and increased extracel- lular potassium concentration) than in their presence, sug- gesting that CACNA1HM1549V activates the same path- ways that are upregulated by physiological stimulation of aldosterone production. This is interesting with regard to salt intake. Whereas aldosterone production physiologi- cally decreases on a high-salt diet, our in vitro data suggest that aldosterone levels will remain elevated in subjects with CACNA1HM1549V variant, leading to volume ex- pansion and hypertension. Conversely, a low-salt diet may prevent the manifestation of hypertension and explain in- complete penetrance of the condition (9).
The magnitude of the effect on aldosterone production observed in HAC15 cells could be an underestimate com- pared to the in vivo situation because CACNA1HM1549V appears to cause microscopic adrenal hyperplasia, in- creasing not only the aldosterone production per cell, but also the number of aldosterone-producing cells (9). Low transient transfection efficiencies are common for large cDNAs (~7 kb for CACNA1H) and may lead to an un- derestimation of aldosterone production. The rate of FLAG positive cells could be higher than that of GFP pos- itive cells due to lower expression of GFP downstream of IRES (24). Preferential damage of transfected cells during staining may also play a role since the number of GFP positive cells was higher in unfixed cells (Supplemental Figure 1).
The effects on aldosterone production are mediated by increased expression of aldosterone synthase, likely via increased calcium signaling. Hyperplasia is probably sim- ilarly mediated by chronically increased calcium signaling
(25). We observed slight differences in the magnitude of the effects between aldosterone levels in the supernatant (Figure 3A) and CYP11B2 expression levels (Figure 3B). This is likely due to the shorter half-life of CYP11B2 mRNA (about 3 hours in H295R cells (26)) compared to aldosterone in the supernatant. A delay in the reduction of aldosterone production explains why treatment with Mibefradil abrogates the effect of CACNA1HWT and CACNA1HM1549V transfection on CYP11B2 expression, but not on aldosterone levels. Similar explanations apply to the differential stimulatory effect of angiotensin II and KCl on CYP11B2 expression levels vs aldosterone levels. CYP11B2 expression has been shown to peak about 12 hours after addition of angiotensin II and decrease there- after. In contrast, potassium stimulation causes increasing CYP11B2 levels even after 40 hours (27). At 24 hours, CYP11B2 expression and promoter activity were shown to be lower after angiotensin II stimulation than after po- tassium stimulation, yet total aldosterone in the superna- tant was higher after angiotensin II stimulation than after potassium stimulation (27, 28), in line with our data.
It is interesting to note that transfection of CACNA1HWT leads to an increase in aldosterone pro- duction even in the absence of depolarizing stimuli such as angiotensin II or elevated extracellular potassium concen- trations and that Mibefradil decreases the aldosterone production of empty vector-transfected cells. Our results suggest that the HAC15 membrane potential is at least temporarily depolarized enough to allow for activation of endogeneous and heterologously expressed CACNA1H (threshold-60 mV) (29). In native glomerulosa cells, such potentials are reached during spontaneous membrane os- cillations (30). Prior studies have demonstrated that Mibe- fradil and the dual L-type/T-type calcium channel blocker Efonidipine can abrogate angiotensin II and potassium- induced aldosterone production, demonstrating that cal- cium influx through T-type channels is essential for the response to these stimuli (31).
Even though Mibefradil was withdrawn from the mar- ket due to drug interactions (32), the sustained response to Mibefradil in our experiments suggests that T-type cal- cium channel inhibitors may be a useful treatment option for patients with CACNA1H gain-of-function mutations. Treatment of hypertensive patients with 100 and 200 mg Mibefradil daily leads to mean plasma concentrations of ~2 µM and ~3 MM, respectively (33). The 10 AM con- centration in our study will achieve virtually complete channel inhibition, whereas 3 pM concentrations result in ~80% channel inhibition (20). Whether T-type calcium channel inhibitors would be useful for the treatment of PA in the general population, however, is doubtful given the lack of effect on aldosterone levels (34, 35), which could
be due to overlapping function of CACNA1D (8, 36). Combined L- and T-type channel blockers may be more promising (37, 38), but further studies are warranted.
Acknowledgments
We thank Drs. Rainer Haas, Ron-Patrick Cadeddu and Stefanie Geyh (Heinrich Heine University Düsseldorf) for providing the Amaxa Nucleofector, Dr. Häussinger (Düsseldorf) for providing the FACSCanto II Flow Cytometer, Dr. William Rainey (Uni- versity of Michigan) for his kind gift of the HAC15 cell line and Dr. Matthias Haase (Düsseldorf) for the H295R cell line. Mi- croscopy was performed at the Düsseldorf Center for Advanced imaging.
Address all correspondence and requests for reprints to: Cor- responding author and person to whom reprint requests should be addressed: Ute I Scholl, M.D., Department of Nephrology, Medical School, Heinrich Heine University, Moorenstraße 5, 40 225 Düsseldorf, Germany, Phone: +49-211-81-10 845, Fax: +49-211-81-015-04 086, Email:
ute.scholl@med.uni-duesseldorf.de.
Disclosure Summary: The authors have nothing to disclose.
This work was supported by Grant and fellowship support: NRW-Rückkehrerprogramm and Junges Kolleg der Nordrhein- Westfälischen Akademie der Wissenschaften und der Künste (both Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, Germany, to UIS).
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