Role of Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase in Adrenal Aldosterone Production

Abstract

There is considerable evidence supporting the role of calcium signaling in adrenal regulation of both aldosterone synthase (CYP11B2) and aldosterone production. However, there have been no studies that investigated the role played by the Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK) in adrenal cells. In this study we investigated the role of CaMKK in adrenal cell aldosterone production. To determine the role of CaMKK, we used a selective CaMKK inhibitor (STO-609) in the HAC15 human adrenal cell line. Cells were treated with angiotensin II (Ang II) or K⁺ and evaluated for the expression of steroidogenic acute regulatory protein and CYP11B2 (mRNA/protein) as well as aldosterone production. We also transduced HAC15 cells with lentiviral short hairpin RNAs of CaMKK1 and CaMKK2 to determine which CaMKK plays a more important role in adrenal cell regulation of the calcium signaling cascade. The CaMKK inhibitor, STO-609, decreased aldosterone production in cells treated with Ang II or K⁺ in a dose-dependent manner. STO-609 (20μM) also inhibited steroidogenic acute regulatory protein and CYP11B2 mRNA/protein induction. CaMKK2 knockdown cells showed significant reduction of CYP11B2 mRNA induction and aldosterone production in cells treated with Ang II, although there was no obvious effect in CaMKK1 knockdown cells. In immunohistochemical analysis, CaMKK2 protein was highly expressed in human adrenal zona glomerulosa with lower expression in the zona fasciculata. In conclusion, the present study suggests that CaMKK2 plays a pivotal role in the calcium signaling cascade regulating adrenal aldosterone production.

There is growing evidence that chronic inappropriate elevations in circulating aldosterone cause renal, cardiovascular, cerebrovascular, and other pathologic complications (1, 2). This inappropriate elevation, also known as primary aldosteronism is the most common cause of endocrine hypertension and occurs in 8% of the hypertensive population (3–5). Primary aldosteronism can be caused by aldosterone-producing adenomas (APAs) or bilateral hyperplasia (4). Recent studies have shown that most APA results from the disruption of intracellular calcium homeostasis and of the normal structure of the adrenal (6–10).

Adrenal production of aldosterone relies acutely on increased expression of steroidogenic acute regulatory protein (StAR), whereas the overall capacity to produce aldosterone relies on aldosterone synthase (CYP11B2) (11). In vitro studies have defined the intracellular signals involving angiotensin II (Ang II)-directed expression of CYP11B2 (12, 13). The angiotensin II type 1 receptor couples to several signaling pathways in zona glomerulosa (ZG) cells, including activation of phospholipase C, resulting in increased levels of intracellular calcium and diacylglycerol (14); these second messengers activate calmodulin and protein kinase C, respectively. On the other hand, K⁺ increases calcium through activation of voltage-sensitive L- and T-type calcium channels, resulting in the influx of calcium from extracellular sources (15). Both Ang II and K⁺ share calcium signaling as a key regulator of aldosterone production.

The key role of calcium signaling is further supported by human adrenal gene mutations that cause aldosterone excess, through the disruption of calcium signaling, resulting in a major dysregulation of aldosterone production (6–10). In most cells, the calcium signaling cascade includes Ca²⁺/calmodulin-dependent protein kinase (CaMK)I, CaMKII, and CaMKIV as well as their upstream regulators CaMK kinase (CaMKK)1 and CaMKK2. Previous studies suggest that either CaMKI or CaMKIV are responsible for adrenal aldosterone production (16). Despite the important role of calcium signaling, there have been no studies that investigated the role played by CaMKK in adrenal cells. In the present study, we investigated the role of CaMKK in adrenal cell aldosterone production.

Materials and Methods

Cell culture

The human adrenocortical cell line HAC15 (17) was cultured in DME/F12 medium (Invitrogen), 10% cosmic calf serum (Hyclone), and antibiotics. Cells were plated in 48-well plates at a density of 100 000 per well and incubated at 37°C for 2 days. One day before the experiment, cells were changed to a low serum experimental medium (DME/F12 medium with 1% cosmic calf serum and antibiotics). The next morning, cells were treated in the same low serum experimental medium for the indicated times. For the pharmacological studies with a selective inhibitor of CaMKK (STO-609) (Abcam) (18) or 22(R)-hydroxycholesterol (22OHC) (Sigma-Aldrich), cells were preincubated with inhibitor for 30 minutes before addition of agonist.

RNA isolation and quantitative real-time RT-PCR (qPCR)

Total RNA was extracted from cells using RNeasy mini kits (QIAGEN). The purity and integrity of the RNA were checked spectroscopically using a Nano Drop spectrometer (Nano Drop Technologies). Total RNA was reverse transcribed using the High-capacity cDNA Archive kit (Applied Biosystems). PCRs were performed in the ABI StepOnePlus Real-Time PCR systems (Applied Biosystems). Primer and probe mixtures for the amplification of the CaMKK1 (Hs01039863_m1), CaMKK2 (Hs00198032_m1), and peptidylprolyl isomerase A (Hs99999901_m1) target sequences were purchased from Applied Biosystems. CYP11B2 primer/probe mix was prepared as described previously (19). Peptidylprolyl isomerase A transcript was used for normalization of sample loading. Relative quantification was determined using the comparative threshold cycle method (20).

Steroid assay

Aldosterone was measured in cell culture medium using an aldosterone RIA (Coat-A-Count kit; Siemens Healthcare). Briefly, 200 μL of experimental media or standards were assayed in aldosterone antibody-coated tubes, and 1 mL of ¹²⁵I labeled aldosterone was added. After 18 hours of incubation at room temperature, tubes were decanted thoroughly and counted in a γ counter.

Protein extraction and Western blot analysis

Cells were lysed in mammalian protein extraction reagent (Pierce Chemical Co). The protein content of samples was determined by the bicinchoninic acid protein assay using the micro bicinchoninic acid protocol (Pierce Chemical Co). For Western blot analysis, equal amounts of protein samples were incubated with reducing buffer and boiled in lithium dodecyl sulfate sample buffer. The reaction mix was separated on 10% Bis-Tris gels and transferred to polyvinylidene difluoride membranes (Invitrogen). The membranes were incubated with StAR antibody (rabbit, 1:2500; kindly provided by Dr D.B. Hales, Southern Illinois University School of Medicine), CYP11B2 antibody (mouse, 1:500; kindly provided by Dr C.E. Gomez-Sanchez, University of Mississippi Medical Center) (21), CaMKK antibody (mouse, 1:1000, C46920; BD Transduction Laboratories), or β-actin antibody (mouse, 1:10 000, A5441; Sigma-Aldrich) followed by secondary antibody incubation at room temperature. Signals were detected with the enhanced electrochemiluminescence kit (Pierce Chemical Co). The signal density of each protein band was analyzed for quantification using ImageJ software (National Institutes of Health).

Knockdown of CaMKK1 and CaMKK2

The expression of CaMKK1 and CaMKK2 was silenced in HAC15 cells using GIPZ Lentiviral short hairpin RNA (shRNA) technology (Thermo Scientific): CaMKK1 (RHS4430-200178021), TTGGAAAGGACTTTCATTG; and CaMKK2 (RHS4430-200224471), TGCATAGTAGGTATTGTCA.

Lentiviral particles containing scrambled (control), CaMKK1, and CaMKK2 shRNAs were transduced into HAC15 cells (multiplicity of infection 5.0) with 6 μg/mL of polybrene (EMD Millipore Corp). Transduced cells were then selected by 10 μg/mL of puromycin (Sigma-Aldrich). The concentration of puromycin was determined according to a puromycin selectivity test with HAC15 cells. After the antibiotic selection, cells were plated at a density of 100 000 per well and treated with or without aldosterone agonist and then analyzed.

Immunohistochemistry

After obtaining informed consent from the families, normal human adrenal samples were collected from renal transplantation donors at Georgia Regents University. Use of these samples was approved by the Institutional Review Board of the University of Michigan. Immunohistochemical staining of normal human adrenals was performed using antibodies for CaMKK2 (rabbit, 1:100, HPA017389; Sigma-Aldrich) and CYP11B2 (mouse, 1:100; kindly provided by Dr C.E. Gomez-Sanchez) (21) on formalin-fixed paraffin-embedded sections as described previously (22).

Statistical analysis

Results are given as mean ± SEM. Individual experiments were repeated at least 3 times. The data were analyzed and compared with control values using the t test with the SigmaPlot 12.5 software package (Systat Software, Inc). One-way ANOVA methods were applied for samples with variables totaling 3 or more groups. The results were significantly different when the P < .05.

Results

Effect of CaMKK inhibitor on adrenal cell aldosterone production

To determine the role of CaMKK in the regulation of adrenal aldosterone production, we used a CaMKK inhibitor (STO-609) in HAC15 cells treated with aldosterone agonists (10nM Ang II or 18mM K⁺). STO-609 demonstrated dose-dependent inhibition of aldosterone production in 24 hours of treatment with Ang II or K⁺, at 3μM–20μM and 10μM–30μM, respectively (Figure 1, A and B). STO-609 (20μM) also significantly inhibited CYP11B2 induction in mRNA (% inhibition after a 24-h treatment was 58% vs Ang II [P < .01] and 50% vs K⁺ [P < .05]) (Figure 2A). Using Western blot analysis, STO-609 was found to significantly inhibit protein levels for StAR (% inhibition after and 8-h treatment was 64% vs Ang II [P < .001] and 48% vs K⁺ [P < .05]) and CYP11B2 (% inhibition after a 24-h treatment was 76% vs Ang II [P < .001] and 58% vs K⁺ [P < .05]) (Figure 2, B–D).

Figure 1.

Effect of CaMKK inhibitor on adrenal cell aldosterone production. HAC15 cells were treated with Ang II (A) or K⁺ (B) for 24 hours with or without CaMKK inhibitor (STO-609). HAC15 cells were preincubated with STO-609 for 30 minutes followed by aldosterone agonists. Media content of aldosterone were measured by RIA. Results represent the mean ± SEM of data from at least 3 independent experiments. Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test. *, P < .001 vs basal; #, P < .05 vs K⁺; ##, P < .01 vs Ang II or K⁺; ###, P < .001 vs Ang II.

Figure 2.

Effect of CaMKK inhibitor on StAR and CYP11B2 induction. A, CYP11B2 mRNA expression was measured by qPCR. The experiments were performed in triplicate and results represent the mean ± SEM of data from at least 3 independent experiments. Statistical analyses were performed using one-way ANOVA followed by Dunnett's post hoc test. B, Protein expression of StAR and CYP11B2 was examined by Western blotting using specific antibodies for StAR and CYP11B2. Results are representative of those obtained from at least 3 individual experiments. β-Actin was used as a loading control. StAR (C) and CYP11B2 (D) protein expression was quantified. Results represent the mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by Student-Newman-Keuls post hoc test. *, P < .05 vs basal; **, P < .01 vs basal; ***, P < .001 vs basal; #, P < .05 vs K⁺; ##, P < .01 vs Ang II or K⁺; ###, P < .001 vs Ang II.

As a test for drug toxicity, we incubated HAC15 cells with STO-609 and 22OHC (Supplemental Figure 1). The addition of the cell permeant 22OHC increased aldosterone production by 5.3-fold (P < .05 vs basal). STO-609 treatment (6 h) did not decrease 22OHC metabolism to aldosterone. This finding suggest that STO-609 inhibition of aldosterone (Figure 1) was not due to toxicity or an off target direct inhibition of a steroidogenic enzyme.

Effect of CaMKK knockdown on adrenal cell regulation of the calcium signaling cascade

Selective shRNAs for CaMKK1 and CaMKK2 were used to further examine the pharmacological findings. After antibiotic selection with puromycin (10 μg/mL), almost all HAC15 cells expressed transduction marker, green fluorescent protein (Figure 3A). Successful knockdown was confirmed by qPCR for mRNA (Figure 3B) and Western blot analysis for protein levels (Figure 3C). CaMKK1 and CaMKK2 mRNA were knocked down by 84% and 94%, respectively, compared with control cells with scrambled shRNA (Figure 3B). Western blot analysis with a CaMKK antibody, which recognizes C-terminal sequences common to CaMKK1 and CaMKK2 (23), also showed inhibition of CaMKK1 and CaMKK2 expression in protein levels (Figure 3C). In qPCR, CaMKK2 knockdown cells, but not CaMKK1 knockdown, significantly decreased CYP11B2 mRNA induction in cells treated with Ang II compared with those in cells with scrambled shRNA (24-h treatment, P < .05 vs scrambled with Ang II) (Figure 3D). Only CaMKK2 knockdown cells showed significant reduction in aldosterone production after a 24-hour treatment with Ang II (P < .05 vs scrambled with Ang II) (Figure 3E).

Figure 3.

Adrenal cell knockdown of CaMKKs. A, Expression of green fluorescent protein in HAC15 cells transduced with lentiviral shRNA. Scale bars, 400 μm. B, Knockdown of CaMKKs mRNA. Results are given as mean ± SEM. Statistical analyses were performed using the t test. ***, P < .001 vs scrambled. C, Western blot analysis of expression of CaMKK1 and CaMKK2 protein in knockdown cells. D, Measure of CYP11B2 mRNA after treatment with Ang II. Experiments were performed in triplicate. Results represent the mean ± SEM of data from 3 independent experiments. Statistical analyses were performed using one-way ANOVA followed by Dunnett's post hoc test. #, P < .05 vs scrambled with Ang II; ##, P < .01 vs CaMKK1 shRNA with Ang II. E, Aldosterone production after treatment with Ang II. Experiments were performed in triplicate. Results were normalized to protein concentration and shown as fold change over basal conditions of cells with scrambled shRNA. Results represent the mean ± SEM of data from 3 independent experiments. Statistical analyses were performed using one-way ANOVA followed by Student-Newman-Keuls post hoc test. #, P < .05 vs scrambled or CaMKK1 shRNA with Ang II.

Localization and expression of CaMKK2 in human adrenal cortex

To evaluate the expression profile of CaMKK2 in human adrenal, we performed immunohistochemical analysis. We stained 4 sets of normal human adrenals for CaMKK2 and CYP11B2. Both CaMKK2 and CYP11B2 were highly expressed in human adrenal ZG with lower expression in the zona fasciculata (Figure 4).

Figure 4.

Expression of CaMKK2 and CYP11B2 in human adrenal cortex. A, Human adrenal stained with hematoxylin-eosin (HE). B, Adrenal immunostaining for CaMKK2. C, Adrenal immunostaining for CYP11B2. Results are representative of those obtained from 4 normal human adrenals. Morphological zones of the adrenal cortex are indicated. Cap, capsule; ZF, zona fasciculata. Scale bars, 200 μm.

Discussion

Ca²⁺ signaling controls many cellular functions through the CaMK cascade (24). In the adrenal, this cascade is involved in normal aldosterone production (16, 25, 26) as well as in pathological settings, specifically APA harboring somatic mutations that disrupt cellular calcium homeostasis (6–10). CaMKK consists of unique N- and C-terminal domains and a central kinase domain that is followed by a regulatory domain composed of overlapping autoinhibitory and calmodulin-binding regions (27). Once Ca²⁺/calmodulin binds and activates CaMKK, CaMKK phosphorylate and activate CaMKI and CaMKIV (27, 28). Members of this cascade respond to elevation of intracellular Ca²⁺ levels and are abundant in many tissues. CaMKK and CaMKIV localize both to the nucleus and to the cytoplasm, whereas CaMKI is mainly cytoplasmic (24). Both CaMKI and CaMKIV regulate transcription through phosphorylation of several transcription factors, including cAMP response element-binding protein. Therefore, we hypothesized that CaMKK/CaMK cascade is the major regulator for Ang II and K⁺ induction of StAR and CYP11B2 expression and aldosterone production.

As a pharmacological approach, we used a CaMKK inhibitor STO-609 to define the involvement of CaMKK in adrenal aldosterone production. STO-609 is a selective and cell-permeable CaMKK inhibitor (18). STO-609 has an in vitro IC₅₀ of 0.32μM and 0.11μM for CaMKK1 and CaMKK2, respectively, whereas a 100- to 250-fold higher concentration (26μM) inhibits CaMKII by only 50% with essentially no effects on CaMKI, CaMKIV, PKA, PKC, or Erk1 (18, 29). In the present study, STO-609 inhibited aldosterone production in cells treated with Ang II or K⁺ in a dose dependent manner. STO-609 (20μM) also blocked StAR protein and CYP11B2 mRNA/protein induction. Furthermore, the conversion of 22OHC to aldosterone was not inhibited by STO-609, suggesting that the inhibitory effect of STO-609 on aldosterone production was not due to cellular toxicity.

Two CaMKK isoforms (CaMKK1 and CaMKK2) have been identified in mammals (30, 31). In addition to the brain, where both CaMKKs are highly expressed, CaMKK1 mRNA is found in thymus and spleen, whereas CaMKK2 is present at lower levels in all tissues that express CaMKIV (32). Although derived from distinct genes, rat CaMKKs are 80% similar and either CaMKK can phosphorylate and activate CaMKI and CaMKIV in vitro (32). In the present study, we have used selective shRNAs for CaMKK1 and CaMKK2 to extend the pharmacological findings. Interestingly, CaMKK2 knockdown cells, but not CaMKK1 knockdown cells, showed significant reduction in CYP11B2 mRNA induction and aldosterone production after treatment with Ang II. We however did not see significant effects on StAR protein induction in CaMKK1 or CaMKK2 knockdown cells treated with Ang II (K. Nanba and W.E.R., unpublished data). Of note, the adrenal cell model used in this study has high basal levels of StAR and the induction seen by Ang II and K⁺ is limited. This may influence the ability of this model to examine the acute regulatory step. Alternatively, this might indicate that CaMKK is more involved in chronic step rather than acute step in the calcium signaling pathway. In immunohistochemical analysis, both CaMKK2 and CYP11B2 were highly expressed in ZG with lower expression in the zona fasciculata. Collectively, our results suggest that CaMKK2 plays a more important role in adrenal cell regulation of the Ca²⁺ signaling cascade (Figure 5). Although the inhibition of CYP11B2 mRNA induction and aldosterone production in CaMKK2 knockdown cells treated with Ang II was statistically significant when compared with those in control cells and CaMKK1 knockdown cells, the effect was not remarkable. One possibility would be the compensation of the phosphorylation ability by CaMKK1. Another possibility might be that CaMKI and/or CaMKIV can be somehow activated by Ca²⁺/calmodulin in the absence of CaMKKs.

Figure 5.

Schematic presentation of calcium signaling cascade regulating aldosterone production indicating the proposed involvement of CaMKK2 in the cascade. AT1-R, angiotensin II type 1 receptor.

In conclusion, the present study suggests that CaMKK, especially CaMKK2, plays an important role in aldosterone production in human adrenal. Our findings improve the understanding of the adrenal cell regulatory systems for aldosterone production and aid in finding new therapeutic targets that could directly block abnormal adrenal cell aldosterone production in hyperaldosteronism.