GSTA1 Expression Is Correlated With Aldosterone Level in KCNJ5-Mutated Adrenal Aldosterone-Producing Adenoma

GSTA1 Expression Is Correlated With Aldosterone Level in KCNJ5-Mutated Adrenal... Abstract Context KCNJ5 mutation is a major cause of aldosterone-producing adenomas (APAs). The development of APA apart from KCNJ5 mutation is less investigated. Objective To investigate other mechanisms affecting aldosterone secretion apart from KCNJ5. Patients and Methods Six pairs of KCNJ5-mutated, high and low aldosterone–secreting APAs, five non–KCNJ5-mutated APAs, and four normal adrenal glands were assayed by Affymetrix GeneChip Human Transcriptome Array 2.0. A total of 113 APA samples were investigated to explore the expression of glutathione-S-transferase A1 (GSTA1). H295R cells were used to verify the function of GSTA1. Results GSTA1 was the top gene downregulated in high-aldosterone KCNJ5-mutated APAs. GSTA1 was also downregulated in KCNJ5-mutated APAs compared with wild-type KCNJ5 APAs. Accordingly, mutant KCNJ5 decreased GSTA1 messenger RNA and protein expression levels. GSTA1 overexpression suppressed aldosterone secretion whether in wild-type or mutant KCNJ5 H295R cells. Adding ethacrynic acid or silencing of GSTA1 increased aldosterone secretion by increasing reactive oxygen species (ROS), superoxide, H2O2 levels, and Ca2+ influx. The expression of the transcription factors NR4A1, NR4A2, and CAMK1 and intracellular Ca2+ were significantly upregulated by GSTA1 inhibition. The reduced form of NAD phosphate oxidase inhibitor or H2O2 scavenger or blocking calmodulin or calcium channels could significantly reduce aldosterone secretion in GSTA1-inhibited cells. Conclusions (1) GSTA1 expression is reversely correlated with aldosterone level in KCNJ5-mutated APAs, (2) GSTA1 regulates aldosterone secretion by ROS and Ca2+ signaling, and (3) KCNJ5 mutation downregulates GSTA1 expression, and overexpression of GSTA1 reverses increased aldosterone in KCNJ5-mutated adrenal cells. As discussed in the study by Rossi et al. (1), 60 years ago, Conn and Lewis (2) stated that aldosterone-producing adenomas (APAs) constituted 5% of all hypertensive patients (1, 2). APA is found in 30% to 40% of patients with primary aldosteronism. Primary aldosteronism is characterized by inappropriately high aldosterone levels, suppressed plasma renin concentrations, elevated blood pressure, and hypokalemia (3). Increased aldosterone is the actual cause of these syndromes, which ultimately results in other symptoms. Even under physiological conditions, increased aldosterone levels increase predisposition to high blood pressure (4, 5). Choi et al. (6) first reported on the somatic and germline mutations of the KCNJ5 gene in APAs. Subsequent studies found that somatic gene mutations of ATP1A1, ATP2B3, CACNA1D, CACNA1H, CTNNB1, and ARMC5 constitute a small proportion of APA cases (7–11). KCNJ5-mutated adrenal cells resemble zona fasciculata (ZF), whereas ATP1A1 and CACNA1D-mutated cells resemble zona glomerulosa (ZG) (12). Several studies used transcriptome and microRNA microarray analyses to compare KCNJ5-mutated and non–KCNJ5-mutated APAs, but found limited differentially expressed genes or microRNAs between the two groups (13–15). Boulkroun et al. (14) posited that KCNJ5 mutation does not activate unique signaling cascades, but is one of several different mechanisms mediating APA development. The KCNJ5 mutation rate is reported higher in Asian countries than in other countries (16–19). Our previous study found KCNJ5 mutations in 86 out of 114 patients with APA (75.4%). Serum aldosterone levels were significantly higher in KCNJ5-mutated than in non–KCNJ5-mutated samples. However, we did not find that all patients with KCNJ5-mutated APAs had higher serum aldosterone levels. Therefore, KCNJ5-mutated samples are highly heterogeneous. To further investigate the mechanism affecting aldosterone level, 12 KCNJ5-mutated APAs, 6 of which had high aldosterone levels (HALD-mKCNJ5) and 6 had low aldosterone levels (LALD-mKCNJ5), and 5 non–KCNJ5-mutated APAs (wild-type KCNJ5 [wtKCNJ5]) were selected. Microarray analysis was performed to identify other potential factors possibly affecting aldosterone secretion. We found differentially expressed genes or noncoding RNAs (ncRNAs) between the HALD-mKCNJ5 and LALD-mKCNJ5 groups. The Gene Ontology (GO) and pathway analysis between HALD-mKCNJ5 and LALD-mKCNJ5 APAs revealed that glutathione metabolism was the most different pathway. Glutathione-S-transferase A1 (GSTA1) was the most differentially expressed gene and downregulated by 9.56-fold in the HALD-mKCNJ5 group compared with the LALD-mKCNJ5 group. GSTA1, a member of the GST family, is an intracellular detoxification enzyme (20). Oxidative stress was also implicated in aldosterone secretion (21–24). These possible links initiated our investigation of the pathological role of GSTA1 in KCNJ5-mutated and non–KCNJ5-mutated adrenal cells. We report that GSTA1 played an important role in regulating aldosterone synthesis in H295R cells. Materials and Methods Patients and tumor samples We analyzed the medical records of 113 patients who received surgery at Chinese People’s Liberation Army General Hospital and collected tissue samples. The inclusion criteria included functional and clinical diagnosis of APA and single and unilateral adrenal adenoma. The diagnosis criteria was provided in the Supplemental Methods, following previously described procedures (21). All multiple or bilateral adrenal adenomas were excluded. The 11 normal adrenal tissues were obtained from the resected adrenals in patients receiving radical nephrectomy suspected of adrenal invasion. For the KCNJ5-nonmutation group, the presence of mutation in the other known genes (ATP1A1, ATP2B3, CACNA1D, CACNA1H, and CTNNB1) was excluded. Written informed consent for a tumor-oriented study was obtained from all patients prior to sample collection, and the study was approved by the Protection of Human Subjects. Microarray assay Microarray assay was performed with the GeneChip Human Transcriptome Array 2.0 by OE BioTech (Affymetrix, Shanghai, China). A total of 17 APA samples and 4 normal adrenal tissues were assayed, including 6 mKCNJ5 samples with high aldosterone levels (median: 34.9 ng/dL; range 34.5 to 42.8 ng/dL), 6 mKCNJ5 samples with low aldosterone levels (median: 21.0 ng/dL; range 11.9 to 23.9 ng/dL), 5 nonmutated KCNJ5 samples (median: 22.1 ng/dL; range 14.1 to 25.2 ng/dL), and 4 normal adrenal tissues. The detailed procedure is shown in the Supplemental Methods. RNA extraction and real-time polymerase chain reaction The detailed procedure is shown in the Supplemental Methods. The primer sequences are shown in Supplemental Table 1. Immunohistochemistry and Western blot All of the antibodies used in the experiment included rabbit anti-GSTA1 antibodies (1:200 dilution; Abcam), mouse anti–β-actin, goat anti-CYP11B2 (sc-47655; Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-mouse immunoglobulin G (IgG)–horseradish peroxidase (HRP), goat anti-rabbit IgG-HRP, and rabbit anti-goat IgG-HRP (ZSGB-BIO, Beijing, China). The detailed procedure is shown in the Supplemental Methods. Cell culture, plasmid construction, small interfering RNA knockdown, and lentiviral production All of the detailed procedures are shown in the Supplemental Methods. The human adrenocortical cell line H295R was bought from the Cell Resource Center (Institute of Basic Medicine Sciences, Chinese Academy of Medical Sciences, Beijing, China). The small interfering RNA (siRNA) targeting GSTA1 was designed and synthesized by Shanghai GenePharma Co. Ltd. (Shanghai, China). The three siRNA sequences are shown in Supplemental Table 1. Immunofluorescence The detailed procedure is shown in the Supplemental Methods. Measurement of membrane voltage, reactive oxygen species, reduced NAD oxidase activity, H2O2, and intracellular Ca2+ Reactive oxygen species (ROS) measurement was performed with the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, Shanghai, China). The reduced form of NAD phosphate oxidase (Nox) activity was measured as intracellular superoxide generation. Intracellular superoxide levels were measured with a Superoxide Assay Kit (S0060; Beyotime Biotechnology). H2O2 was measured with a commercially available Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188; Molecular Probes, Invitrogen, Carlsbad, CA). Intracellular Ca2+ concentration was measured by Fluo-4 am (F14201; Invitrogen). The membrane voltage was measured by DiSBAC4 (3) (D8189; Sigma-Aldrich, St. Louis, MO). The detailed procedure is shown in the Supplemental Methods. Data analysis All of the experiments were performed in triplicate. All data were expressed as mean ± standard error. Statistical significance was evaluated by an unpaired t test. Correlations between two variables were analyzed by linear regression. Continuous data were examined for normality using the Kolmogorov-Smirnov test. All of the comparisons were considered to be statistically significant at P < 0.05. Statistical analyses were performed by SPSS 19.0 (SPSS Inc., Chicago, IL). Results Clinical features of patients with APA included in the study The clinical features of the 113 patients with APA are shown in Supplemental Table 2. There were 87 patients with KCNJ5 mutation and 26 patients without KCNJ5 mutation. Further sequencing of KCNJ5 nonmutation samples showed one patient with ATP1A1 mutation and one with CACNA1D mutation. The comparison results showed younger age, higher preoperative systolic blood pressure, lower preoperative potassium level, and higher recumbent serum aldosterone level in KCNJ5-mutated than in KCNJ5-nonmutated samples. The clinical features of the 17 patients are shown in Table 1. The median age of the patients was 47 years (36 to 62 years old), with a male/female ratio of 8:9. All of the patients were diagnosed according to the criteria shown in the Supplemental Methods. Three cases exhibited KCNJ5 G151R mutations, and three exhibited L168R mutations in the KCNJ5-mutated, high and low aldosterone-secreting groups, respectively. The KCNJ5-mutated high, low, and non–KCNJ5-mutated groups had average recumbent serum aldosterone levels of 36.2 ng/dL, 18.9 ng/dL, and 20.2 ng/dL, respectively. All of the KCNJ5-mutated samples showed ZF-like features, whereas the five non–KCNJ5-mutated samples showed ZG–like features. The ZF-like adrenal cells showed a large clear cell with slightly stained cytoplasma, whereas the ZG-like adrenal cells showed a small compact cell with densely stained cytoplasm (Supplemental Fig. 1A). Table 1. Clinical Features of the Patients With APA Involved in the Microarray Analysis Group  Patient Number  Mutation Type  Sex  Age (y)  Recumbent ALD (ng/dL)  Upright ALD (ng/dL)  ARR  Serum K+ (mmol/L)  BP (mm Hg)  mKCNJ5, high ALD  1  G151R  M  48  35.1  39.8  172.1  2.4  180/120    2  G151R  F  56  42.8  51.2  173.6  1.78  160/100    3  G151R  M  62  34.5  37.0  383.3  2.17  230/110    4  L168R  M  44  34.7  44.2  230. 1  2.28  220/120    5  L168R  F  38  34.5  34.0  346.5  2.6  180/110    6  L168R  F  47  35.6  34.1  173.8  1.8  170/110  mKCNJ5, low ALD  7  L168R  F  49  23.9  26.1  133. 1  2.61  170/110    8  L168R  M  61  22.2  15.3  51.9  2.1  160/100    9  L168R  F  39  15.9  16.8  67.7  2.76  170/100    10  G151R  M  54  13.6  16.3  82.9  2.9  150/90    11  G151R  M  42  20.1  27.8  73.9  2.6  160/100    12  G151R  F  43  21.9  24.5  56.6  2.7  160/100  wtKCNJ5  13  None  F  51  22.3  26.9  137.3  2.8  140/90    14  None  M  39  17.3  19.0  32.3  3.46  150/110    15  None  F  36  14.1  20.7  42.2  2.08  160/120    16  None  F  52  25.2  29.3  170.0  2.43  180/120    17  None  M  42  22.1  25.3  128.6  2.21  180/110  Group  Patient Number  Mutation Type  Sex  Age (y)  Recumbent ALD (ng/dL)  Upright ALD (ng/dL)  ARR  Serum K+ (mmol/L)  BP (mm Hg)  mKCNJ5, high ALD  1  G151R  M  48  35.1  39.8  172.1  2.4  180/120    2  G151R  F  56  42.8  51.2  173.6  1.78  160/100    3  G151R  M  62  34.5  37.0  383.3  2.17  230/110    4  L168R  M  44  34.7  44.2  230. 1  2.28  220/120    5  L168R  F  38  34.5  34.0  346.5  2.6  180/110    6  L168R  F  47  35.6  34.1  173.8  1.8  170/110  mKCNJ5, low ALD  7  L168R  F  49  23.9  26.1  133. 1  2.61  170/110    8  L168R  M  61  22.2  15.3  51.9  2.1  160/100    9  L168R  F  39  15.9  16.8  67.7  2.76  170/100    10  G151R  M  54  13.6  16.3  82.9  2.9  150/90    11  G151R  M  42  20.1  27.8  73.9  2.6  160/100    12  G151R  F  43  21.9  24.5  56.6  2.7  160/100  wtKCNJ5  13  None  F  51  22.3  26.9  137.3  2.8  140/90    14  None  M  39  17.3  19.0  32.3  3.46  150/110    15  None  F  36  14.1  20.7  42.2  2.08  160/120    16  None  F  52  25.2  29.3  170.0  2.43  180/120    17  None  M  42  22.1  25.3  128.6  2.21  180/110  Abbreviations: ALD, aldosterone; ARR, aldosterone renin ratio; BP, blood pressure; F, female; M, male; mKCNJ5, KCNJ5 mutation. View Large Microarray results Compared with normal adrenal glands, the APA group had a total of 2017 upregulated messenger RNAs (mRNAs) or ncRNAs and 2192 downregulated mRNA or ncRNAs (fold change >2; P < 0.05). The top differentially expressed genes are shown in Supplemental Table 3. The unsupervised clustering analysis of 17 APAs revealed that there were no tendencies to separate KCNJ5- mutated and -nonmutated APAs. However, five of the six HALD-mKCNJ5 APAs were clustered into one group, suggesting possible different gene expression patterns in these samples (Fig. 1A). The GO biological process, GO cell component, GO molecular function, and pathway analysis results of the 4209 genes are shown in Supplemental Tables 4–7. Figure 1. View largeDownload slide Microarray results. (A) The unsupervised cluster analysis of the 17 APAs and 4 normal adrenal tissues. Unsupervised clustering analysis revealed that there was no tendency to separate KCNJ5 mutated and nonmutated APAs, although five of the six HALD-mKCNJ5 APAs were clustered into one group. (B) GSTA1, GSTA2, GSTA5, GSTK1, and MGST2 were downregulated in APA compared with normal adrenal tissue. NA, normal adrenal gland. Figure 1. View largeDownload slide Microarray results. (A) The unsupervised cluster analysis of the 17 APAs and 4 normal adrenal tissues. Unsupervised clustering analysis revealed that there was no tendency to separate KCNJ5 mutated and nonmutated APAs, although five of the six HALD-mKCNJ5 APAs were clustered into one group. (B) GSTA1, GSTA2, GSTA5, GSTK1, and MGST2 were downregulated in APA compared with normal adrenal tissue. NA, normal adrenal gland. Compared with the LALD-mKCNJ5 group, the HALD-mKCNJ5 group had 359 differentially expressed mRNAs or ncRNAs (fold change >1.5; P < 0.05), including 128 downregulated and 231 upregulated mRNAs or ncRNAs. The top differentially expressed genes are shown in Supplemental Table 8. The GO biological process, GO cell component, GO molecular function, and pathway analysis results of the 359 genes are shown in Supplemental Tables 9–12. GSTA1 was downregulated in the HALD-mKCNJ5 group, with a 9.56-fold change compared with the LALD-mKCNJ5 group. Compared with the normal adrenal group, GSTA1 was downregulated in the APA group, with a 15.4-fold change. GSTA2, GSTA3, and GSTA5 were also downregulated in the HALD-mKCNJ5 group compared with the LALD-mKCNJ5 group (Supplemental Fig. 1C–1E). GSTA1, GSTA2, GSTA5, GSTK1, and MGST2 were downregulated in APA compared with normal adrenal tissue (Fig. 1B). GSTA1 expression is correlated with aldosterone levels and KCNJ5 mutation status Of the 113 APA tissues, 87 had KCNJ5 mutations, including 49 with G151R mutation, 34 with L168R mutation, 2 with T158A mutation, and 2 with insertion mutation and duplication mutation. The 87 KCNJ5-mutated tissues were divided into the LALD-mKCNJ5 group and HALD-mKCNJ5 group according to recumbent serum aldosterone with the median criteria of 24.6 ng/dL. The 26 wtKCNJ5 tissues were divided into high- and low-aldosterone groups according to the same cutoff value. As shown in Fig. 2A, GSTA1 mRNA expression was significantly lower in the HALD-mKCNJ5 group for both KCNJ5-mutated (P < 0.001) and KCNJ5-nonmutated APA tissues (P = 0.013). When the samples were divided according to KCNJ5 mutation status, GSTA1 mRNA expression was lowest in the KCNJ5-mutated groups, higher in the wtKCNJ5 groups, and highest in the normal adrenal gland group (Fig. 2B). Furthermore, GSTA1 mRNA expression was significantly and inversely correlated with recumbent serum aldosterone in both the KCNJ5-mutated group (r = −0.6311; P < 0.0001) and KCNJ5-nonmutated group (r = −0.5918; P = 0.0014) (Fig. 2C and Supplemental Fig. 1B). It was also significantly inversely correlated with the upright serum aldosterone in the KCNJ5-mutated group (r = −0.6082; P < 0.0001) (Supplemental Fig. 1F). Quantitative real-time polymerase chain reaction showed that GSTA2, GSTA3, and GSTA5 were also downregulated in the HALD-mKCNJ5 APA group compared with the LALD-mKCNJ5 and normal adrenal gland groups (Supplemental Fig. 1C–1E). Figure 2. View largeDownload slide GSTA1 mRNA and protein expression levels in mKCNJ5, wtKCNJ5, and normal adrenal gland. (A) GSTA1 mRNA expression was significantly lower in the HALD-mKCNJ5 group for both KCNJ5-mutated tissues (P < 0.001) and KCNJ5-nonmutated tissues (P = 0.013). (B) GSTA1 mRNA expression was lowest in the KCNJ5-mutated groups, higher in the wtKCNJ5 groups, and highest in the normal adrenal gland group. (C) GSTA1 mRNA expression was significantly and inversely correlated with recumbent serum aldosterone (r = −0.6311; P < 0.0001). (D) Western blot showed that GSTA1 protein expression was significantly downregulated in the KCNJ5-mutated APA group and less expressed in the HALD-mKCNJ5 APA than in the LALD-mKCNJ5 APA group. Ins.Dup., insertion and duplication mutation; Non., KCNJ5 nonmutated. ***P < 0.001. Figure 2. View largeDownload slide GSTA1 mRNA and protein expression levels in mKCNJ5, wtKCNJ5, and normal adrenal gland. (A) GSTA1 mRNA expression was significantly lower in the HALD-mKCNJ5 group for both KCNJ5-mutated tissues (P < 0.001) and KCNJ5-nonmutated tissues (P = 0.013). (B) GSTA1 mRNA expression was lowest in the KCNJ5-mutated groups, higher in the wtKCNJ5 groups, and highest in the normal adrenal gland group. (C) GSTA1 mRNA expression was significantly and inversely correlated with recumbent serum aldosterone (r = −0.6311; P < 0.0001). (D) Western blot showed that GSTA1 protein expression was significantly downregulated in the KCNJ5-mutated APA group and less expressed in the HALD-mKCNJ5 APA than in the LALD-mKCNJ5 APA group. Ins.Dup., insertion and duplication mutation; Non., KCNJ5 nonmutated. ***P < 0.001. Western blot showed that GSTA1 protein expression was significantly downregulated in the KCNJ5-mutated group and less expressed in the HALD-mKCNJ5 than in the LALD-mKCNJ5 group (Fig. 2D). Immunohistochemistry showed that the GSTA1 protein was less expressed in KCNJ5-mutated than in wtKCNJ5 APAs or nonfunctional adrenal adenomas. However, GSTA1 expression was nearly undetectable in HALD-mKCNJ5 APAs, whereas a slight and focal staining of GSTA1 was observed in the LALD-mKCNJ5 APAs (Supplemental Fig. 2A). In normal adrenal tissue, GSTA1 was much significantly stained in the ZF, zona reticularis, and medulla, but was less expressed in the ZG (Supplemental Fig. 2B). Mutant KCNJ5 decreases GSTA1 expression H295R cells transfected with mutated KCNJ5-G151R or L168R also reduced GSTA1 mRNA expression by 60.8% and 56.1%, respectively (Fig. 3A). The change in mRNA levels was accompanied by increased CYP11B2 and aldosterone secretion in KCNJ5-mutated adrenal cells (data not shown). Both Western blot analysis and immunofluorescence showed that GSTA1 protein expression was lower in KCNJ5-mutated cells (Fig. 3C, 3G, and 3H). However, GSTA1 expression was increased after addition different concentrations of aldosterone, so KCNJ5 should not decrease GSTA1 expression via the oversecretion of aldosterone. Figure 3. View largeDownload slide The effect of KCNJ5 mutation on GSTA1 expression and function. (A) H295R cells transfected with mutated KCNJ5-G151R or L168R reduced GSTA1 mRNA expression by 60.8% and 56.1%, respectively. (B, D, and E) GSTA1 overexpression significantly reduced CYP11B2 activity by 42.5%, CYP11B2 expression by 53.4%, and aldosterone secretion by 27.9% in KCNJ5-mutated cells. Adding EA slightly increased CYP11B2 activity by 109.0%, CYP11B2 expression by 112.6%, and aldosterone secretion by 103.9%. (C) Immunofluorescence of GSTA1 protein in control, wtKCNJ5, and KCNJ5-mutated adrenal cells. GSTA1 expression levels localized in the cytoplasm and nucleus were downregulated in G151R- and L168R-mutated KCNJ5 cells. (F) KCNJ5 mutation increased the intracellular Ca2+ (indicated by Fluo-4 am fluorescence), which could be suppressed by overexpression of GSTA1. (G and H) Western blot analysis and immunofluorescence showed that GSTA1 protein expression was lower in KCNJ5-mutated cells. DAPI, 4′,6-diamidino-2-phenylindole; NC, negative control. *P < 0.05; ***P < 0.001. Figure 3. View largeDownload slide The effect of KCNJ5 mutation on GSTA1 expression and function. (A) H295R cells transfected with mutated KCNJ5-G151R or L168R reduced GSTA1 mRNA expression by 60.8% and 56.1%, respectively. (B, D, and E) GSTA1 overexpression significantly reduced CYP11B2 activity by 42.5%, CYP11B2 expression by 53.4%, and aldosterone secretion by 27.9% in KCNJ5-mutated cells. Adding EA slightly increased CYP11B2 activity by 109.0%, CYP11B2 expression by 112.6%, and aldosterone secretion by 103.9%. (C) Immunofluorescence of GSTA1 protein in control, wtKCNJ5, and KCNJ5-mutated adrenal cells. GSTA1 expression levels localized in the cytoplasm and nucleus were downregulated in G151R- and L168R-mutated KCNJ5 cells. (F) KCNJ5 mutation increased the intracellular Ca2+ (indicated by Fluo-4 am fluorescence), which could be suppressed by overexpression of GSTA1. (G and H) Western blot analysis and immunofluorescence showed that GSTA1 protein expression was lower in KCNJ5-mutated cells. DAPI, 4′,6-diamidino-2-phenylindole; NC, negative control. *P < 0.05; ***P < 0.001. GSTA1 overexpression inhibits CYP11B2 expression and aldosterone synthesis GSTA1 overexpression significantly increased GSTA1 mRNA and protein expression levels (Fig. 4A and Supplemental Fig. 3B). Immunofluorescence showed enhanced fluorescence of the cytoplasm and nucleus (Supplemental Fig. 3C). GSTA1 overexpression decreased aldosterone secretion, CYP11B2 mRNA expression, and activity compared with the vector control without (decreased by 33.7%, 44.1%, and 37.0%) or with 10 nM angiotensin II stimulation (decreased by 45.6%, 67.9%, and 56.7%) (Fig. 4D and 4G and Supplemental Fig. 4A). GSTA1 mRNA expression in H295R cells was downregulated to 29.1% by angiotensin II (10 nM) stimulation (Fig. 4C). Figure 4. View largeDownload slide GSTA1 function in H295R adrenal cells. (A) GSTA1 overexpression significantly increased GSTA1 mRNA expression compared with the untreated group and control vector (P < 0.001). (B) The three siRNAs knocked down GSTA1 mRNA expression by 87.9%, 82.6%, and 76.5% compared with their nontargeting siRNA controls. (C) GSTA1 mRNA expression in H295R cells was downregulated to 29.1% by angiotensin II (Ang II; 10 nM) stimulation. (D and G) GSTA1 overexpression decreased aldosterone secretion and CYP11B2 mRNA expression compared with the vector control without (decreased by 33.7% and 44.1%) or with 10 nM angiotensin II stimulation (decreased by 45.6% and 67.9%). (E and H) GSTA1 siRNAs increased aldosterone secretion by 1.86-, 1.54-, and 1.73-fold and CYP11B2 mRNA expression by 3.43-, 2.46-, and 3.02-fold compared with the untreated group. (F and I) Incubating H295R cells with 30 μM EA (GSTA1 inhibitor) increased CYP11B2 mRNA levels and aldosterone secretion by 8.06- and 2.16-fold, respectively. The increase varied with different EA densities. NC, negative control. DMSO, dimethyl sulfoxide. *P < 0.05; **P < 0.01; ***P < 0.001. Figure 4. View largeDownload slide GSTA1 function in H295R adrenal cells. (A) GSTA1 overexpression significantly increased GSTA1 mRNA expression compared with the untreated group and control vector (P < 0.001). (B) The three siRNAs knocked down GSTA1 mRNA expression by 87.9%, 82.6%, and 76.5% compared with their nontargeting siRNA controls. (C) GSTA1 mRNA expression in H295R cells was downregulated to 29.1% by angiotensin II (Ang II; 10 nM) stimulation. (D and G) GSTA1 overexpression decreased aldosterone secretion and CYP11B2 mRNA expression compared with the vector control without (decreased by 33.7% and 44.1%) or with 10 nM angiotensin II stimulation (decreased by 45.6% and 67.9%). (E and H) GSTA1 siRNAs increased aldosterone secretion by 1.86-, 1.54-, and 1.73-fold and CYP11B2 mRNA expression by 3.43-, 2.46-, and 3.02-fold compared with the untreated group. (F and I) Incubating H295R cells with 30 μM EA (GSTA1 inhibitor) increased CYP11B2 mRNA levels and aldosterone secretion by 8.06- and 2.16-fold, respectively. The increase varied with different EA densities. NC, negative control. DMSO, dimethyl sulfoxide. *P < 0.05; **P < 0.01; ***P < 0.001. siRNA of GSTA1 or GSTA1 inhibitor and increased CYP11B2 expression and aldosterone synthesis Compared with the nontargeting, siRNA control, the three siRNAs knocked down GSTA1 mRNA expression by 87.9%, 82.6%, and 76.5% (Fig. 4B), reducing protein levels by 64.5%, 51.2%, and 54.2%, respectively (Supplemental Fig. 3A). Immunofluorescence showed that cytoplasmic and nuclear GSTA1 expression levels were significantly reduced (Supplemental Fig. 3C). siRNA of GSTA1 increased CYP11B2 activity, mRNA, protein, and aldosterone by 2.21-, 2.95-, 3.28-, and 1.70-fold, respectively (Fig. 4E and 4H and Supplemental Figs. 3B and 4B). A total of 30 μM ethacrynic acid (EA) increased CYP11B2 activity, CYP11B2 mRNA levels, and aldosterone secretion by 3.84-, 8.06-, and 2.16-fold, respectively. The increase varied with different EA densities (Fig. 4F and 4I and Supplemental Fig. 4C). The function of GSTA1 in KCNJ5-mutated H295R cells Furthermore, we investigated the function of GSTA1 in KCNJ5-mutated cells. GSTA1 overexpression significantly reduced CYP11B2 activity by 42.5%, CYP11B2 expression by 53.4%, and aldosterone secretion by 27.9% in KCNJ5-mutated cells. However, adding EA only slightly increased CYP11B2 activity by 1.09%, CYP11B2 expression by 112.6%, and aldosterone secretion by 103.9% (Fig. 3B, 3D, and 3E). Furthermore, KCNJ5 mutation increased accumulation of DiSBAC4 (3), an indicator of higher plasma membrane voltage and the intracellular Ca2+ (indicated by Fluo-4 am fluorescence), which could be partly suppressed by GSTA1 overexpression (Fig. 3F and Supplemental Figs. 5 and 7). However, GSTA1 change had no impact on expressions of other steroid synthesis genes. KCNJ5 mutation significantly increased CYP11B1 expression and cortisol production, but GSTA1 change also had no impact on cortisol production (Supplemental Fig. 4E and 4F). GSTA1 regulates aldosterone synthesis through ROS, superoxide, and H2O2 production Three GSTA1 siRNAs and inhibition significantly increased 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence by 2.23-, 2.69-, 2.34-, and 4.23-fold (P < 0.001), respectively. The addition of 20 μM VAS-2870 and 350 U/mL polyethylene glycol–catalase (PEG-Cat) after EA inhibition decreased fluorescence to 34.2% and 31.2% (P < 0.001), respectively (Fig. 5A and 5G). Figure 5. View largeDownload slide GSTA1 regulates aldosterone synthesis through the production of ROS, superoxide, and H2O2. (A and G) Three GSTA1 siRNAs and inhibition significantly increased DCFH-DA fluorescence by 2.23-, 2.69-, 2.34-, and 4.23-fold (P < 0.001). The addition of 20 μM VAS-2870 and 350 U/mL PEG-Cat after EA inhibition decreased fluorescence to 34.2% and 31.2% (P < 0.001), respectively. (B) GSTA1 siRNAs or 30 μM EA significantly increased superoxide production by 2.33-, 2.65-, 2.82- (P < 0.05), and 4.41-fold (P < 0.001). The addition of 20 μM VAS-2870 and 350 U/mL PEG-Cat after EA inhibition decreased superoxide production to 61.0% and 38.8% (P < 0.001), respectively. (C) GSTA1 overexpression significantly decreased H2O2 production to 66.9% (P < 0.05), whereas GSTA1 siRNAs or 30 μM EA significantly increased H2O2 production. Three GSTA1 siRNAs and EA inhibition significantly increased the H2O2 by 1.59-, 1.41-, 1.68- (P < 0.05), and 3.47-fold (P < 0.001). Adding VAS-2870 or PEG-Cat to the EA group significantly attenuated the increase in expression levels (decreased by 48.1% and 42.7%). (D–F) EA-mediated GSTA1 inhibition significantly increased CYP11B2 activity, CYP11B2 mRNA expression, and aldosterone secretion. Adding 20 μM VAS-2870, 350 U/mL PEG-Cat, 200 μM N-acetyl-l-cysteine (NAC), 10 μM l-NG-nitro-l-arginine methyl ester (L-NAME), 30 μM tocopherol, or 100 μM MnTMPyP significantly reduced this increase. (G) DCFH-DA fluorescence images in various conditions. DMSO, dimethyl sulfoxide. *P < 0.05; **P < 0.01; ***P < 0.001. Figure 5. View largeDownload slide GSTA1 regulates aldosterone synthesis through the production of ROS, superoxide, and H2O2. (A and G) Three GSTA1 siRNAs and inhibition significantly increased DCFH-DA fluorescence by 2.23-, 2.69-, 2.34-, and 4.23-fold (P < 0.001). The addition of 20 μM VAS-2870 and 350 U/mL PEG-Cat after EA inhibition decreased fluorescence to 34.2% and 31.2% (P < 0.001), respectively. (B) GSTA1 siRNAs or 30 μM EA significantly increased superoxide production by 2.33-, 2.65-, 2.82- (P < 0.05), and 4.41-fold (P < 0.001). The addition of 20 μM VAS-2870 and 350 U/mL PEG-Cat after EA inhibition decreased superoxide production to 61.0% and 38.8% (P < 0.001), respectively. (C) GSTA1 overexpression significantly decreased H2O2 production to 66.9% (P < 0.05), whereas GSTA1 siRNAs or 30 μM EA significantly increased H2O2 production. Three GSTA1 siRNAs and EA inhibition significantly increased the H2O2 by 1.59-, 1.41-, 1.68- (P < 0.05), and 3.47-fold (P < 0.001). Adding VAS-2870 or PEG-Cat to the EA group significantly attenuated the increase in expression levels (decreased by 48.1% and 42.7%). (D–F) EA-mediated GSTA1 inhibition significantly increased CYP11B2 activity, CYP11B2 mRNA expression, and aldosterone secretion. Adding 20 μM VAS-2870, 350 U/mL PEG-Cat, 200 μM N-acetyl-l-cysteine (NAC), 10 μM l-NG-nitro-l-arginine methyl ester (L-NAME), 30 μM tocopherol, or 100 μM MnTMPyP significantly reduced this increase. (G) DCFH-DA fluorescence images in various conditions. DMSO, dimethyl sulfoxide. *P < 0.05; **P < 0.01; ***P < 0.001. GSTA1 siRNAs or 30 μM EA significantly increased superoxide production by 2.33-, 2.65-, 2.82- (P < 0.05), and 4.41-fold (P < 0.001). The addition of 20 μM VAS-2870 and 350 U/mL PEG-Cat after EA inhibition decreased superoxide production to 61.0% and 38.8% (P < 0.001), respectively (Fig. 5B). GSTA1 overexpression significantly decreased H2O2 production to 66.9% (P < 0.05), whereas GSTA1 siRNAs or 30 μM EA significantly increased H2O2 production. Three GSTA1 siRNAs and EA inhibition significantly increased the H2O2 by 1.59-, 1.41-, 1.68- (P < 0.05), and 3.47-fold (P < 0.001). Adding VAS-2870 or PEG-Cat to the EA group significantly attenuated the increase in expression levels (decreased by 48.1% and 42.7%) (Fig. 5C). EA-mediated GSTA1 inhibition significantly increased CYP11B2 activity, CYP11B2 mRNA expression, and aldosterone secretion. Adding 20 μM VAS-2870, 350 U/mL PEG-Cat, 200 μM N-acetyl-l-cysteine, 10 μM l-NG-nitro-l-arginine methyl ester, 30 μM tocopherol, or 100 μM MnTMPyP significantly reduced this increase (Fig. 5D–5F). Ca2+-dependent signaling is involved in GSTA1 regulation of aldosterone synthesis Ca2+-dependent signaling has been shown to directly cause the oversecretion of aldosterone. Therefore, we investigated whether inhibiting GSTA1 affected the Ca2+/calmodulin-dependent protein kinase signaling pathway. Our results showed that GSTA1 overexpression significantly downregulated CAMK1, NR4A1, and NR4A2 expression levels. GSTA1 inhibition significantly increased CAMK1, NR4A1, and NR4A2 expression. Blocking calmodulin by calmidazolium (2.5 μM) and W-7 (30 μM) and blocking calcium channels by nifedipine (10 μM) significantly reduced CYP11B2 activity, CYP11B2 expression, and aldosterone secretion in GSTA1-inhibited cells. However, the difference in expression levels was less notable in the control cells (Supplemental Fig. 6E–6G). At the same time, the intracellular Ca2+ significantly increased after GSTA1 inhibition (Supplemental Figs. 6D and 8). Discussion At our medical center, KCNJ5-mutated APAs account for as much as 80% of all cases. The mean aldosterone level was high in patients with KCNJ5 mutations. Although comparative transcriptomic analyses have been conducted with KCNJ5 mutants or nonmutant APAs (13–15), aldosterone levels have not been compared. We selected and analyzed the differentially expressed genes between HALD-mKCNJ5 and LALD-mKCNJ5 APAs. Both GO and pathway analysis showed that glutathione metabolism was the top different pathway. GSTA1, GSTA2, GSTA3, and GSTA5 were all downregulated in HALD-mKCNJ5 compared with LALD-mKCNJ5. Therefore, the GSTA family genes may significantly regulate aldosterone in KCNJ5-mutated APAs. Recently, Zhou et al. (25) reported that GSTA1 was the most differentially expressed between ZG and ZF and between APA and ZG. In addition, its expression may be regulated by the NRF2-mediated oxidative stress response pathway. However, there was no further detailed functional investigation about GSTA1 (25). Sarkar et al. (26) found that GSTA1 is highly expressed in cortisol-producing adrenocortical adenomas compared with normal adrenal glands. GST inhibition interfered with cellular proliferation. Another study investigated the role of the GSTA family in steroidogenesis, which is regulated by steroidogenic factor 1. GSTA1 and GSTA3 interactions exhibit steroid isomerase activities and are involved in steroidogenesis (27). Unlike the high GSTA1 expression in cortisol-producing adenoma, GSTA1 was less expressed in APA tissue than in the normal adrenal tissue. Moreover, GSTA1 expression was lower in KCNJ5-mutated than in non–KCNJ5-mutated APAs and lowest in HALD-mKCNJ5 cases. GSTA1 expression was inversely correlated with recumbent and upright aldosterone levels. Western blot and immunohistochemistry staining showed lower protein expression in KCNJ5-mutated APAs and even lower expression in HALD-mKCNJ5 APAs. We observed that GSTA1 expression was absent in the ZG of the normal adrenal gland, whereas it was higher in other zones, which was consistent with another study (28). Our key finding was that GSTA1 overexpression suppressed aldosterone secretion, whereas silencing or inhibiting GSTA1 increased aldosterone secretion. This increase in expression level was probably caused by the parallel increase in CYP11B2 expression and activity. The decrease in GSTA1 mRNA levels after stimulation with angiotensin II corresponded with this result. GSTA1 was mostly related to the detoxification of various xenobiotics and products of oxidative stress. Therefore, the downregulation of GSTA1 resulted in the accumulation of ROS and H2O2, which then led to the excess secretion of aldosterone. Silencing or inhibiting GSTA1 significantly increased DCFH-DA fluorescence, Nox activity, and H2O2 production, whereas GSTA1 overexpression in H295R decreased fluorescence less drastically. These results may be explained by H295R cells expressing GSTA1 protein. Adding Nox inhibitor (VAS-2870), H2O2 scavenger (PEG-Cat), and other antioxidants significantly reduced aldosterone secretion in cells with EA. However, the reduction was less noteworthy without EA, supporting that increased aldosterone secretion was mediated by ROS. Immunofluorescence staining of GSTA1 in nucleus might be nonspecific. Because the GSTA1 was stained in the nucleus using immunofluorescence in the siRNA transfection group or mKCNJ5 group. In the immunohistochemistry staining, GSTA1 was expressed in the cytoplasm but not in the nucleus. Other studies also showed that GSTA1 is abundantly distributed in the cytoplasm of adrenal cells (26) and other tumor cells (29). Oxidative stress has been associated with aldosteronism. Garrido and Griendling (30) reported that angiotensin II could stimulate Nox and produce ROS, which further result in hypertension and various complications. In addition, Rajamohan et al. (22) showed that angiotensin II increased CYP11B2 expression and aldosterone secretion by Nox-H2O2 signaling. Calo et al. (23) reported oxidative related proteins were increasingly expressed in APA including p22(phox), Nox, transforming growth factor-β, plasminogen activator inhibitor-1, and heme oxygenase-1. The increased plasma Nox activity and urinary excretion of isoprostanes showed increased oxidative stress in patients with primary aldosteronism (24). Geng et al. (21) showed nox2-induced oxidative stress might play a critical role in regulated aldosterone secretion. These results showed that oxidative stress is an important pathogenic mechanism in aldosteronism. Conversely, aldosterone can also induce oxidative stress in many ways. Other studies showed that aldosterone could increase Nox activity, resulting in ROS production and leading to myocyte apoptosis and glomerular diseases (31, 32). Our study and previous results suggested that the interaction between GSTA1 and oxidative stress might be one important reason for pathological autonomous aldosterone hypersecretion in APAs. In contrast, other steroidogenic organs need antioxidants to defend against oxidative stress to synthetize steroid. Otherwise, these ROS can cause mitochondrial DNA damage and cell apoptosis. Therefore, unlike in APAs, several studies showed antioxidants were increasingly expressed in such organs as the corpus luteum, adrenal cortex fasciculata, and reticularis (33–35). As Ca2+ influx is the common signal and direct cause of aldosterone oversecretion, we investigated the effect of GSTA1 inhibition on intracellular Ca2+. Similar to KCNJ5 mutation, GSTA1 inhibition increased intracellular Ca2+ and the expression of calmodulin-related genes, including CAMK1, NR4A1, and NR4A2. The pharmacological blockage of Ca2+ channels or calmodulin significantly inhibited the EA-induced increase of aldosterone, but not the control cells. These results suggested that GSTA1 also functions through Ca2+-dependent signaling. Previously, the cross talk between calcium and ROS signaling has been extensively studied (36, 37). Therefore, we suppose that GSTA1 interact with both signals to stimulate aldosterone secretion. GSTA1 mRNA and protein expressions were significantly downregulated by G151R or L168R KCNJ5 mutation. The addition of aldosterone did not decrease GSTA1 expression, which suggested that KCNJ5 mutation did not regulate GSTA1 via aldosterone levels. Furthermore, GSTA1 overexpression significantly reduced CYP11B2 expression, activity, and aldosterone secretion in KCNJ5-G151R H295R cells. GSTA1 inhibition showed little effect because of the low GSTA1 expression in mKCNJ5 H295R cells. Other factors might increase GSTA1 expression and thereby lower aldosterone levels in some mKCNJ5 APAs. We also tried to detect the methylation status of GSTA1 by methylmion-specific polymerase chain reaction after adding aldosterone or not. The primers were designed for the CpG island on the upstream of the promoter. We found that there was apparent methylation in the 5′-untranslated region and promoter region of GSTA1 gene. However, no marked difference was found between the group with aldosterone and without. The explorations of GSTA1 epigenetic change should be further investigated. Although we identified the critical role of the GSTA1 gene in regulating aldosterone secretion in KCNJ5-mutated H295R cells. There were some limitations. First, given that the definition of high or low aldosterone secretion is flexible, we selected values approaching the extreme values for microarray analysis and used correlation analysis to verify the results. Second, the mechanisms under mutated KCNJ5 on GSTA1 were not fully elucidated. Third, we only divided APAs into KCNJ5 mutation and nonmutation groups because the KCNJ5 mutation accounted for a large proportion, and few other mutations were identified. In conclusion, we identified a series of transcript differences between the HALD-mKCNJ5 and LALD-mKCNJ5 APAs. The GSTA1 gene is the most downregulated in HALD-mKCNJ5 and inversely correlated with aldosterone level. It may be an important factor affecting aldosterone secretion in adrenal cells. Abbreviations: APA aldosterone-producing adenoma DCFH-DA 2,7-dichlorodihydrofluorescein diacetate EA ethacrynic acid GO Gene Ontology GSTA1 glutathione-S-transferase A1 HRP horseradish peroxidase mRNA messenger RNA ncRNA noncoding RNA Nox reduced form of NAD phosphate oxidase PEG-Cat polyethylene glycol–catalase ROS reactive oxygen species siRNA small interfering RNA wtKCNJ5 wild-type KCNJ5 ZF zona fasciculata ZG zona glomerulosa. Acknowledgments Financial Support: This work was financially supported by National Natural Science Foundation of China Grants 81670726 and 81770790. Disclosure Summary: The authors have nothing to disclose. References 1. Rossi GP, Bernini G, Caliumi C, Desideri G, Fabris B, Ferri C, Ganzaroli C, Giacchetti G, Letizia C, Maccario M, Mallamaci F, Mannelli M, Mattarello MJ, Moretti A, Palumbo G, Parenti G, Porteri E, Semplicini A, Rizzoni D, Rossi E, Boscaro M, Pessina AC, Mantero F; PAPY Study Investigators. 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Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

GSTA1 Expression Is Correlated With Aldosterone Level in KCNJ5-Mutated Adrenal Aldosterone-Producing Adenoma

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Endocrine Society
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Copyright © 2018 Endocrine Society
ISSN
0021-972X
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1945-7197
D.O.I.
10.1210/jc.2017-00950
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Abstract

Abstract Context KCNJ5 mutation is a major cause of aldosterone-producing adenomas (APAs). The development of APA apart from KCNJ5 mutation is less investigated. Objective To investigate other mechanisms affecting aldosterone secretion apart from KCNJ5. Patients and Methods Six pairs of KCNJ5-mutated, high and low aldosterone–secreting APAs, five non–KCNJ5-mutated APAs, and four normal adrenal glands were assayed by Affymetrix GeneChip Human Transcriptome Array 2.0. A total of 113 APA samples were investigated to explore the expression of glutathione-S-transferase A1 (GSTA1). H295R cells were used to verify the function of GSTA1. Results GSTA1 was the top gene downregulated in high-aldosterone KCNJ5-mutated APAs. GSTA1 was also downregulated in KCNJ5-mutated APAs compared with wild-type KCNJ5 APAs. Accordingly, mutant KCNJ5 decreased GSTA1 messenger RNA and protein expression levels. GSTA1 overexpression suppressed aldosterone secretion whether in wild-type or mutant KCNJ5 H295R cells. Adding ethacrynic acid or silencing of GSTA1 increased aldosterone secretion by increasing reactive oxygen species (ROS), superoxide, H2O2 levels, and Ca2+ influx. The expression of the transcription factors NR4A1, NR4A2, and CAMK1 and intracellular Ca2+ were significantly upregulated by GSTA1 inhibition. The reduced form of NAD phosphate oxidase inhibitor or H2O2 scavenger or blocking calmodulin or calcium channels could significantly reduce aldosterone secretion in GSTA1-inhibited cells. Conclusions (1) GSTA1 expression is reversely correlated with aldosterone level in KCNJ5-mutated APAs, (2) GSTA1 regulates aldosterone secretion by ROS and Ca2+ signaling, and (3) KCNJ5 mutation downregulates GSTA1 expression, and overexpression of GSTA1 reverses increased aldosterone in KCNJ5-mutated adrenal cells. As discussed in the study by Rossi et al. (1), 60 years ago, Conn and Lewis (2) stated that aldosterone-producing adenomas (APAs) constituted 5% of all hypertensive patients (1, 2). APA is found in 30% to 40% of patients with primary aldosteronism. Primary aldosteronism is characterized by inappropriately high aldosterone levels, suppressed plasma renin concentrations, elevated blood pressure, and hypokalemia (3). Increased aldosterone is the actual cause of these syndromes, which ultimately results in other symptoms. Even under physiological conditions, increased aldosterone levels increase predisposition to high blood pressure (4, 5). Choi et al. (6) first reported on the somatic and germline mutations of the KCNJ5 gene in APAs. Subsequent studies found that somatic gene mutations of ATP1A1, ATP2B3, CACNA1D, CACNA1H, CTNNB1, and ARMC5 constitute a small proportion of APA cases (7–11). KCNJ5-mutated adrenal cells resemble zona fasciculata (ZF), whereas ATP1A1 and CACNA1D-mutated cells resemble zona glomerulosa (ZG) (12). Several studies used transcriptome and microRNA microarray analyses to compare KCNJ5-mutated and non–KCNJ5-mutated APAs, but found limited differentially expressed genes or microRNAs between the two groups (13–15). Boulkroun et al. (14) posited that KCNJ5 mutation does not activate unique signaling cascades, but is one of several different mechanisms mediating APA development. The KCNJ5 mutation rate is reported higher in Asian countries than in other countries (16–19). Our previous study found KCNJ5 mutations in 86 out of 114 patients with APA (75.4%). Serum aldosterone levels were significantly higher in KCNJ5-mutated than in non–KCNJ5-mutated samples. However, we did not find that all patients with KCNJ5-mutated APAs had higher serum aldosterone levels. Therefore, KCNJ5-mutated samples are highly heterogeneous. To further investigate the mechanism affecting aldosterone level, 12 KCNJ5-mutated APAs, 6 of which had high aldosterone levels (HALD-mKCNJ5) and 6 had low aldosterone levels (LALD-mKCNJ5), and 5 non–KCNJ5-mutated APAs (wild-type KCNJ5 [wtKCNJ5]) were selected. Microarray analysis was performed to identify other potential factors possibly affecting aldosterone secretion. We found differentially expressed genes or noncoding RNAs (ncRNAs) between the HALD-mKCNJ5 and LALD-mKCNJ5 groups. The Gene Ontology (GO) and pathway analysis between HALD-mKCNJ5 and LALD-mKCNJ5 APAs revealed that glutathione metabolism was the most different pathway. Glutathione-S-transferase A1 (GSTA1) was the most differentially expressed gene and downregulated by 9.56-fold in the HALD-mKCNJ5 group compared with the LALD-mKCNJ5 group. GSTA1, a member of the GST family, is an intracellular detoxification enzyme (20). Oxidative stress was also implicated in aldosterone secretion (21–24). These possible links initiated our investigation of the pathological role of GSTA1 in KCNJ5-mutated and non–KCNJ5-mutated adrenal cells. We report that GSTA1 played an important role in regulating aldosterone synthesis in H295R cells. Materials and Methods Patients and tumor samples We analyzed the medical records of 113 patients who received surgery at Chinese People’s Liberation Army General Hospital and collected tissue samples. The inclusion criteria included functional and clinical diagnosis of APA and single and unilateral adrenal adenoma. The diagnosis criteria was provided in the Supplemental Methods, following previously described procedures (21). All multiple or bilateral adrenal adenomas were excluded. The 11 normal adrenal tissues were obtained from the resected adrenals in patients receiving radical nephrectomy suspected of adrenal invasion. For the KCNJ5-nonmutation group, the presence of mutation in the other known genes (ATP1A1, ATP2B3, CACNA1D, CACNA1H, and CTNNB1) was excluded. Written informed consent for a tumor-oriented study was obtained from all patients prior to sample collection, and the study was approved by the Protection of Human Subjects. Microarray assay Microarray assay was performed with the GeneChip Human Transcriptome Array 2.0 by OE BioTech (Affymetrix, Shanghai, China). A total of 17 APA samples and 4 normal adrenal tissues were assayed, including 6 mKCNJ5 samples with high aldosterone levels (median: 34.9 ng/dL; range 34.5 to 42.8 ng/dL), 6 mKCNJ5 samples with low aldosterone levels (median: 21.0 ng/dL; range 11.9 to 23.9 ng/dL), 5 nonmutated KCNJ5 samples (median: 22.1 ng/dL; range 14.1 to 25.2 ng/dL), and 4 normal adrenal tissues. The detailed procedure is shown in the Supplemental Methods. RNA extraction and real-time polymerase chain reaction The detailed procedure is shown in the Supplemental Methods. The primer sequences are shown in Supplemental Table 1. Immunohistochemistry and Western blot All of the antibodies used in the experiment included rabbit anti-GSTA1 antibodies (1:200 dilution; Abcam), mouse anti–β-actin, goat anti-CYP11B2 (sc-47655; Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-mouse immunoglobulin G (IgG)–horseradish peroxidase (HRP), goat anti-rabbit IgG-HRP, and rabbit anti-goat IgG-HRP (ZSGB-BIO, Beijing, China). The detailed procedure is shown in the Supplemental Methods. Cell culture, plasmid construction, small interfering RNA knockdown, and lentiviral production All of the detailed procedures are shown in the Supplemental Methods. The human adrenocortical cell line H295R was bought from the Cell Resource Center (Institute of Basic Medicine Sciences, Chinese Academy of Medical Sciences, Beijing, China). The small interfering RNA (siRNA) targeting GSTA1 was designed and synthesized by Shanghai GenePharma Co. Ltd. (Shanghai, China). The three siRNA sequences are shown in Supplemental Table 1. Immunofluorescence The detailed procedure is shown in the Supplemental Methods. Measurement of membrane voltage, reactive oxygen species, reduced NAD oxidase activity, H2O2, and intracellular Ca2+ Reactive oxygen species (ROS) measurement was performed with the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, Shanghai, China). The reduced form of NAD phosphate oxidase (Nox) activity was measured as intracellular superoxide generation. Intracellular superoxide levels were measured with a Superoxide Assay Kit (S0060; Beyotime Biotechnology). H2O2 was measured with a commercially available Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188; Molecular Probes, Invitrogen, Carlsbad, CA). Intracellular Ca2+ concentration was measured by Fluo-4 am (F14201; Invitrogen). The membrane voltage was measured by DiSBAC4 (3) (D8189; Sigma-Aldrich, St. Louis, MO). The detailed procedure is shown in the Supplemental Methods. Data analysis All of the experiments were performed in triplicate. All data were expressed as mean ± standard error. Statistical significance was evaluated by an unpaired t test. Correlations between two variables were analyzed by linear regression. Continuous data were examined for normality using the Kolmogorov-Smirnov test. All of the comparisons were considered to be statistically significant at P < 0.05. Statistical analyses were performed by SPSS 19.0 (SPSS Inc., Chicago, IL). Results Clinical features of patients with APA included in the study The clinical features of the 113 patients with APA are shown in Supplemental Table 2. There were 87 patients with KCNJ5 mutation and 26 patients without KCNJ5 mutation. Further sequencing of KCNJ5 nonmutation samples showed one patient with ATP1A1 mutation and one with CACNA1D mutation. The comparison results showed younger age, higher preoperative systolic blood pressure, lower preoperative potassium level, and higher recumbent serum aldosterone level in KCNJ5-mutated than in KCNJ5-nonmutated samples. The clinical features of the 17 patients are shown in Table 1. The median age of the patients was 47 years (36 to 62 years old), with a male/female ratio of 8:9. All of the patients were diagnosed according to the criteria shown in the Supplemental Methods. Three cases exhibited KCNJ5 G151R mutations, and three exhibited L168R mutations in the KCNJ5-mutated, high and low aldosterone-secreting groups, respectively. The KCNJ5-mutated high, low, and non–KCNJ5-mutated groups had average recumbent serum aldosterone levels of 36.2 ng/dL, 18.9 ng/dL, and 20.2 ng/dL, respectively. All of the KCNJ5-mutated samples showed ZF-like features, whereas the five non–KCNJ5-mutated samples showed ZG–like features. The ZF-like adrenal cells showed a large clear cell with slightly stained cytoplasma, whereas the ZG-like adrenal cells showed a small compact cell with densely stained cytoplasm (Supplemental Fig. 1A). Table 1. Clinical Features of the Patients With APA Involved in the Microarray Analysis Group  Patient Number  Mutation Type  Sex  Age (y)  Recumbent ALD (ng/dL)  Upright ALD (ng/dL)  ARR  Serum K+ (mmol/L)  BP (mm Hg)  mKCNJ5, high ALD  1  G151R  M  48  35.1  39.8  172.1  2.4  180/120    2  G151R  F  56  42.8  51.2  173.6  1.78  160/100    3  G151R  M  62  34.5  37.0  383.3  2.17  230/110    4  L168R  M  44  34.7  44.2  230. 1  2.28  220/120    5  L168R  F  38  34.5  34.0  346.5  2.6  180/110    6  L168R  F  47  35.6  34.1  173.8  1.8  170/110  mKCNJ5, low ALD  7  L168R  F  49  23.9  26.1  133. 1  2.61  170/110    8  L168R  M  61  22.2  15.3  51.9  2.1  160/100    9  L168R  F  39  15.9  16.8  67.7  2.76  170/100    10  G151R  M  54  13.6  16.3  82.9  2.9  150/90    11  G151R  M  42  20.1  27.8  73.9  2.6  160/100    12  G151R  F  43  21.9  24.5  56.6  2.7  160/100  wtKCNJ5  13  None  F  51  22.3  26.9  137.3  2.8  140/90    14  None  M  39  17.3  19.0  32.3  3.46  150/110    15  None  F  36  14.1  20.7  42.2  2.08  160/120    16  None  F  52  25.2  29.3  170.0  2.43  180/120    17  None  M  42  22.1  25.3  128.6  2.21  180/110  Group  Patient Number  Mutation Type  Sex  Age (y)  Recumbent ALD (ng/dL)  Upright ALD (ng/dL)  ARR  Serum K+ (mmol/L)  BP (mm Hg)  mKCNJ5, high ALD  1  G151R  M  48  35.1  39.8  172.1  2.4  180/120    2  G151R  F  56  42.8  51.2  173.6  1.78  160/100    3  G151R  M  62  34.5  37.0  383.3  2.17  230/110    4  L168R  M  44  34.7  44.2  230. 1  2.28  220/120    5  L168R  F  38  34.5  34.0  346.5  2.6  180/110    6  L168R  F  47  35.6  34.1  173.8  1.8  170/110  mKCNJ5, low ALD  7  L168R  F  49  23.9  26.1  133. 1  2.61  170/110    8  L168R  M  61  22.2  15.3  51.9  2.1  160/100    9  L168R  F  39  15.9  16.8  67.7  2.76  170/100    10  G151R  M  54  13.6  16.3  82.9  2.9  150/90    11  G151R  M  42  20.1  27.8  73.9  2.6  160/100    12  G151R  F  43  21.9  24.5  56.6  2.7  160/100  wtKCNJ5  13  None  F  51  22.3  26.9  137.3  2.8  140/90    14  None  M  39  17.3  19.0  32.3  3.46  150/110    15  None  F  36  14.1  20.7  42.2  2.08  160/120    16  None  F  52  25.2  29.3  170.0  2.43  180/120    17  None  M  42  22.1  25.3  128.6  2.21  180/110  Abbreviations: ALD, aldosterone; ARR, aldosterone renin ratio; BP, blood pressure; F, female; M, male; mKCNJ5, KCNJ5 mutation. View Large Microarray results Compared with normal adrenal glands, the APA group had a total of 2017 upregulated messenger RNAs (mRNAs) or ncRNAs and 2192 downregulated mRNA or ncRNAs (fold change >2; P < 0.05). The top differentially expressed genes are shown in Supplemental Table 3. The unsupervised clustering analysis of 17 APAs revealed that there were no tendencies to separate KCNJ5- mutated and -nonmutated APAs. However, five of the six HALD-mKCNJ5 APAs were clustered into one group, suggesting possible different gene expression patterns in these samples (Fig. 1A). The GO biological process, GO cell component, GO molecular function, and pathway analysis results of the 4209 genes are shown in Supplemental Tables 4–7. Figure 1. View largeDownload slide Microarray results. (A) The unsupervised cluster analysis of the 17 APAs and 4 normal adrenal tissues. Unsupervised clustering analysis revealed that there was no tendency to separate KCNJ5 mutated and nonmutated APAs, although five of the six HALD-mKCNJ5 APAs were clustered into one group. (B) GSTA1, GSTA2, GSTA5, GSTK1, and MGST2 were downregulated in APA compared with normal adrenal tissue. NA, normal adrenal gland. Figure 1. View largeDownload slide Microarray results. (A) The unsupervised cluster analysis of the 17 APAs and 4 normal adrenal tissues. Unsupervised clustering analysis revealed that there was no tendency to separate KCNJ5 mutated and nonmutated APAs, although five of the six HALD-mKCNJ5 APAs were clustered into one group. (B) GSTA1, GSTA2, GSTA5, GSTK1, and MGST2 were downregulated in APA compared with normal adrenal tissue. NA, normal adrenal gland. Compared with the LALD-mKCNJ5 group, the HALD-mKCNJ5 group had 359 differentially expressed mRNAs or ncRNAs (fold change >1.5; P < 0.05), including 128 downregulated and 231 upregulated mRNAs or ncRNAs. The top differentially expressed genes are shown in Supplemental Table 8. The GO biological process, GO cell component, GO molecular function, and pathway analysis results of the 359 genes are shown in Supplemental Tables 9–12. GSTA1 was downregulated in the HALD-mKCNJ5 group, with a 9.56-fold change compared with the LALD-mKCNJ5 group. Compared with the normal adrenal group, GSTA1 was downregulated in the APA group, with a 15.4-fold change. GSTA2, GSTA3, and GSTA5 were also downregulated in the HALD-mKCNJ5 group compared with the LALD-mKCNJ5 group (Supplemental Fig. 1C–1E). GSTA1, GSTA2, GSTA5, GSTK1, and MGST2 were downregulated in APA compared with normal adrenal tissue (Fig. 1B). GSTA1 expression is correlated with aldosterone levels and KCNJ5 mutation status Of the 113 APA tissues, 87 had KCNJ5 mutations, including 49 with G151R mutation, 34 with L168R mutation, 2 with T158A mutation, and 2 with insertion mutation and duplication mutation. The 87 KCNJ5-mutated tissues were divided into the LALD-mKCNJ5 group and HALD-mKCNJ5 group according to recumbent serum aldosterone with the median criteria of 24.6 ng/dL. The 26 wtKCNJ5 tissues were divided into high- and low-aldosterone groups according to the same cutoff value. As shown in Fig. 2A, GSTA1 mRNA expression was significantly lower in the HALD-mKCNJ5 group for both KCNJ5-mutated (P < 0.001) and KCNJ5-nonmutated APA tissues (P = 0.013). When the samples were divided according to KCNJ5 mutation status, GSTA1 mRNA expression was lowest in the KCNJ5-mutated groups, higher in the wtKCNJ5 groups, and highest in the normal adrenal gland group (Fig. 2B). Furthermore, GSTA1 mRNA expression was significantly and inversely correlated with recumbent serum aldosterone in both the KCNJ5-mutated group (r = −0.6311; P < 0.0001) and KCNJ5-nonmutated group (r = −0.5918; P = 0.0014) (Fig. 2C and Supplemental Fig. 1B). It was also significantly inversely correlated with the upright serum aldosterone in the KCNJ5-mutated group (r = −0.6082; P < 0.0001) (Supplemental Fig. 1F). Quantitative real-time polymerase chain reaction showed that GSTA2, GSTA3, and GSTA5 were also downregulated in the HALD-mKCNJ5 APA group compared with the LALD-mKCNJ5 and normal adrenal gland groups (Supplemental Fig. 1C–1E). Figure 2. View largeDownload slide GSTA1 mRNA and protein expression levels in mKCNJ5, wtKCNJ5, and normal adrenal gland. (A) GSTA1 mRNA expression was significantly lower in the HALD-mKCNJ5 group for both KCNJ5-mutated tissues (P < 0.001) and KCNJ5-nonmutated tissues (P = 0.013). (B) GSTA1 mRNA expression was lowest in the KCNJ5-mutated groups, higher in the wtKCNJ5 groups, and highest in the normal adrenal gland group. (C) GSTA1 mRNA expression was significantly and inversely correlated with recumbent serum aldosterone (r = −0.6311; P < 0.0001). (D) Western blot showed that GSTA1 protein expression was significantly downregulated in the KCNJ5-mutated APA group and less expressed in the HALD-mKCNJ5 APA than in the LALD-mKCNJ5 APA group. Ins.Dup., insertion and duplication mutation; Non., KCNJ5 nonmutated. ***P < 0.001. Figure 2. View largeDownload slide GSTA1 mRNA and protein expression levels in mKCNJ5, wtKCNJ5, and normal adrenal gland. (A) GSTA1 mRNA expression was significantly lower in the HALD-mKCNJ5 group for both KCNJ5-mutated tissues (P < 0.001) and KCNJ5-nonmutated tissues (P = 0.013). (B) GSTA1 mRNA expression was lowest in the KCNJ5-mutated groups, higher in the wtKCNJ5 groups, and highest in the normal adrenal gland group. (C) GSTA1 mRNA expression was significantly and inversely correlated with recumbent serum aldosterone (r = −0.6311; P < 0.0001). (D) Western blot showed that GSTA1 protein expression was significantly downregulated in the KCNJ5-mutated APA group and less expressed in the HALD-mKCNJ5 APA than in the LALD-mKCNJ5 APA group. Ins.Dup., insertion and duplication mutation; Non., KCNJ5 nonmutated. ***P < 0.001. Western blot showed that GSTA1 protein expression was significantly downregulated in the KCNJ5-mutated group and less expressed in the HALD-mKCNJ5 than in the LALD-mKCNJ5 group (Fig. 2D). Immunohistochemistry showed that the GSTA1 protein was less expressed in KCNJ5-mutated than in wtKCNJ5 APAs or nonfunctional adrenal adenomas. However, GSTA1 expression was nearly undetectable in HALD-mKCNJ5 APAs, whereas a slight and focal staining of GSTA1 was observed in the LALD-mKCNJ5 APAs (Supplemental Fig. 2A). In normal adrenal tissue, GSTA1 was much significantly stained in the ZF, zona reticularis, and medulla, but was less expressed in the ZG (Supplemental Fig. 2B). Mutant KCNJ5 decreases GSTA1 expression H295R cells transfected with mutated KCNJ5-G151R or L168R also reduced GSTA1 mRNA expression by 60.8% and 56.1%, respectively (Fig. 3A). The change in mRNA levels was accompanied by increased CYP11B2 and aldosterone secretion in KCNJ5-mutated adrenal cells (data not shown). Both Western blot analysis and immunofluorescence showed that GSTA1 protein expression was lower in KCNJ5-mutated cells (Fig. 3C, 3G, and 3H). However, GSTA1 expression was increased after addition different concentrations of aldosterone, so KCNJ5 should not decrease GSTA1 expression via the oversecretion of aldosterone. Figure 3. View largeDownload slide The effect of KCNJ5 mutation on GSTA1 expression and function. (A) H295R cells transfected with mutated KCNJ5-G151R or L168R reduced GSTA1 mRNA expression by 60.8% and 56.1%, respectively. (B, D, and E) GSTA1 overexpression significantly reduced CYP11B2 activity by 42.5%, CYP11B2 expression by 53.4%, and aldosterone secretion by 27.9% in KCNJ5-mutated cells. Adding EA slightly increased CYP11B2 activity by 109.0%, CYP11B2 expression by 112.6%, and aldosterone secretion by 103.9%. (C) Immunofluorescence of GSTA1 protein in control, wtKCNJ5, and KCNJ5-mutated adrenal cells. GSTA1 expression levels localized in the cytoplasm and nucleus were downregulated in G151R- and L168R-mutated KCNJ5 cells. (F) KCNJ5 mutation increased the intracellular Ca2+ (indicated by Fluo-4 am fluorescence), which could be suppressed by overexpression of GSTA1. (G and H) Western blot analysis and immunofluorescence showed that GSTA1 protein expression was lower in KCNJ5-mutated cells. DAPI, 4′,6-diamidino-2-phenylindole; NC, negative control. *P < 0.05; ***P < 0.001. Figure 3. View largeDownload slide The effect of KCNJ5 mutation on GSTA1 expression and function. (A) H295R cells transfected with mutated KCNJ5-G151R or L168R reduced GSTA1 mRNA expression by 60.8% and 56.1%, respectively. (B, D, and E) GSTA1 overexpression significantly reduced CYP11B2 activity by 42.5%, CYP11B2 expression by 53.4%, and aldosterone secretion by 27.9% in KCNJ5-mutated cells. Adding EA slightly increased CYP11B2 activity by 109.0%, CYP11B2 expression by 112.6%, and aldosterone secretion by 103.9%. (C) Immunofluorescence of GSTA1 protein in control, wtKCNJ5, and KCNJ5-mutated adrenal cells. GSTA1 expression levels localized in the cytoplasm and nucleus were downregulated in G151R- and L168R-mutated KCNJ5 cells. (F) KCNJ5 mutation increased the intracellular Ca2+ (indicated by Fluo-4 am fluorescence), which could be suppressed by overexpression of GSTA1. (G and H) Western blot analysis and immunofluorescence showed that GSTA1 protein expression was lower in KCNJ5-mutated cells. DAPI, 4′,6-diamidino-2-phenylindole; NC, negative control. *P < 0.05; ***P < 0.001. GSTA1 overexpression inhibits CYP11B2 expression and aldosterone synthesis GSTA1 overexpression significantly increased GSTA1 mRNA and protein expression levels (Fig. 4A and Supplemental Fig. 3B). Immunofluorescence showed enhanced fluorescence of the cytoplasm and nucleus (Supplemental Fig. 3C). GSTA1 overexpression decreased aldosterone secretion, CYP11B2 mRNA expression, and activity compared with the vector control without (decreased by 33.7%, 44.1%, and 37.0%) or with 10 nM angiotensin II stimulation (decreased by 45.6%, 67.9%, and 56.7%) (Fig. 4D and 4G and Supplemental Fig. 4A). GSTA1 mRNA expression in H295R cells was downregulated to 29.1% by angiotensin II (10 nM) stimulation (Fig. 4C). Figure 4. View largeDownload slide GSTA1 function in H295R adrenal cells. (A) GSTA1 overexpression significantly increased GSTA1 mRNA expression compared with the untreated group and control vector (P < 0.001). (B) The three siRNAs knocked down GSTA1 mRNA expression by 87.9%, 82.6%, and 76.5% compared with their nontargeting siRNA controls. (C) GSTA1 mRNA expression in H295R cells was downregulated to 29.1% by angiotensin II (Ang II; 10 nM) stimulation. (D and G) GSTA1 overexpression decreased aldosterone secretion and CYP11B2 mRNA expression compared with the vector control without (decreased by 33.7% and 44.1%) or with 10 nM angiotensin II stimulation (decreased by 45.6% and 67.9%). (E and H) GSTA1 siRNAs increased aldosterone secretion by 1.86-, 1.54-, and 1.73-fold and CYP11B2 mRNA expression by 3.43-, 2.46-, and 3.02-fold compared with the untreated group. (F and I) Incubating H295R cells with 30 μM EA (GSTA1 inhibitor) increased CYP11B2 mRNA levels and aldosterone secretion by 8.06- and 2.16-fold, respectively. The increase varied with different EA densities. NC, negative control. DMSO, dimethyl sulfoxide. *P < 0.05; **P < 0.01; ***P < 0.001. Figure 4. View largeDownload slide GSTA1 function in H295R adrenal cells. (A) GSTA1 overexpression significantly increased GSTA1 mRNA expression compared with the untreated group and control vector (P < 0.001). (B) The three siRNAs knocked down GSTA1 mRNA expression by 87.9%, 82.6%, and 76.5% compared with their nontargeting siRNA controls. (C) GSTA1 mRNA expression in H295R cells was downregulated to 29.1% by angiotensin II (Ang II; 10 nM) stimulation. (D and G) GSTA1 overexpression decreased aldosterone secretion and CYP11B2 mRNA expression compared with the vector control without (decreased by 33.7% and 44.1%) or with 10 nM angiotensin II stimulation (decreased by 45.6% and 67.9%). (E and H) GSTA1 siRNAs increased aldosterone secretion by 1.86-, 1.54-, and 1.73-fold and CYP11B2 mRNA expression by 3.43-, 2.46-, and 3.02-fold compared with the untreated group. (F and I) Incubating H295R cells with 30 μM EA (GSTA1 inhibitor) increased CYP11B2 mRNA levels and aldosterone secretion by 8.06- and 2.16-fold, respectively. The increase varied with different EA densities. NC, negative control. DMSO, dimethyl sulfoxide. *P < 0.05; **P < 0.01; ***P < 0.001. siRNA of GSTA1 or GSTA1 inhibitor and increased CYP11B2 expression and aldosterone synthesis Compared with the nontargeting, siRNA control, the three siRNAs knocked down GSTA1 mRNA expression by 87.9%, 82.6%, and 76.5% (Fig. 4B), reducing protein levels by 64.5%, 51.2%, and 54.2%, respectively (Supplemental Fig. 3A). Immunofluorescence showed that cytoplasmic and nuclear GSTA1 expression levels were significantly reduced (Supplemental Fig. 3C). siRNA of GSTA1 increased CYP11B2 activity, mRNA, protein, and aldosterone by 2.21-, 2.95-, 3.28-, and 1.70-fold, respectively (Fig. 4E and 4H and Supplemental Figs. 3B and 4B). A total of 30 μM ethacrynic acid (EA) increased CYP11B2 activity, CYP11B2 mRNA levels, and aldosterone secretion by 3.84-, 8.06-, and 2.16-fold, respectively. The increase varied with different EA densities (Fig. 4F and 4I and Supplemental Fig. 4C). The function of GSTA1 in KCNJ5-mutated H295R cells Furthermore, we investigated the function of GSTA1 in KCNJ5-mutated cells. GSTA1 overexpression significantly reduced CYP11B2 activity by 42.5%, CYP11B2 expression by 53.4%, and aldosterone secretion by 27.9% in KCNJ5-mutated cells. However, adding EA only slightly increased CYP11B2 activity by 1.09%, CYP11B2 expression by 112.6%, and aldosterone secretion by 103.9% (Fig. 3B, 3D, and 3E). Furthermore, KCNJ5 mutation increased accumulation of DiSBAC4 (3), an indicator of higher plasma membrane voltage and the intracellular Ca2+ (indicated by Fluo-4 am fluorescence), which could be partly suppressed by GSTA1 overexpression (Fig. 3F and Supplemental Figs. 5 and 7). However, GSTA1 change had no impact on expressions of other steroid synthesis genes. KCNJ5 mutation significantly increased CYP11B1 expression and cortisol production, but GSTA1 change also had no impact on cortisol production (Supplemental Fig. 4E and 4F). GSTA1 regulates aldosterone synthesis through ROS, superoxide, and H2O2 production Three GSTA1 siRNAs and inhibition significantly increased 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence by 2.23-, 2.69-, 2.34-, and 4.23-fold (P < 0.001), respectively. The addition of 20 μM VAS-2870 and 350 U/mL polyethylene glycol–catalase (PEG-Cat) after EA inhibition decreased fluorescence to 34.2% and 31.2% (P < 0.001), respectively (Fig. 5A and 5G). Figure 5. View largeDownload slide GSTA1 regulates aldosterone synthesis through the production of ROS, superoxide, and H2O2. (A and G) Three GSTA1 siRNAs and inhibition significantly increased DCFH-DA fluorescence by 2.23-, 2.69-, 2.34-, and 4.23-fold (P < 0.001). The addition of 20 μM VAS-2870 and 350 U/mL PEG-Cat after EA inhibition decreased fluorescence to 34.2% and 31.2% (P < 0.001), respectively. (B) GSTA1 siRNAs or 30 μM EA significantly increased superoxide production by 2.33-, 2.65-, 2.82- (P < 0.05), and 4.41-fold (P < 0.001). The addition of 20 μM VAS-2870 and 350 U/mL PEG-Cat after EA inhibition decreased superoxide production to 61.0% and 38.8% (P < 0.001), respectively. (C) GSTA1 overexpression significantly decreased H2O2 production to 66.9% (P < 0.05), whereas GSTA1 siRNAs or 30 μM EA significantly increased H2O2 production. Three GSTA1 siRNAs and EA inhibition significantly increased the H2O2 by 1.59-, 1.41-, 1.68- (P < 0.05), and 3.47-fold (P < 0.001). Adding VAS-2870 or PEG-Cat to the EA group significantly attenuated the increase in expression levels (decreased by 48.1% and 42.7%). (D–F) EA-mediated GSTA1 inhibition significantly increased CYP11B2 activity, CYP11B2 mRNA expression, and aldosterone secretion. Adding 20 μM VAS-2870, 350 U/mL PEG-Cat, 200 μM N-acetyl-l-cysteine (NAC), 10 μM l-NG-nitro-l-arginine methyl ester (L-NAME), 30 μM tocopherol, or 100 μM MnTMPyP significantly reduced this increase. (G) DCFH-DA fluorescence images in various conditions. DMSO, dimethyl sulfoxide. *P < 0.05; **P < 0.01; ***P < 0.001. Figure 5. View largeDownload slide GSTA1 regulates aldosterone synthesis through the production of ROS, superoxide, and H2O2. (A and G) Three GSTA1 siRNAs and inhibition significantly increased DCFH-DA fluorescence by 2.23-, 2.69-, 2.34-, and 4.23-fold (P < 0.001). The addition of 20 μM VAS-2870 and 350 U/mL PEG-Cat after EA inhibition decreased fluorescence to 34.2% and 31.2% (P < 0.001), respectively. (B) GSTA1 siRNAs or 30 μM EA significantly increased superoxide production by 2.33-, 2.65-, 2.82- (P < 0.05), and 4.41-fold (P < 0.001). The addition of 20 μM VAS-2870 and 350 U/mL PEG-Cat after EA inhibition decreased superoxide production to 61.0% and 38.8% (P < 0.001), respectively. (C) GSTA1 overexpression significantly decreased H2O2 production to 66.9% (P < 0.05), whereas GSTA1 siRNAs or 30 μM EA significantly increased H2O2 production. Three GSTA1 siRNAs and EA inhibition significantly increased the H2O2 by 1.59-, 1.41-, 1.68- (P < 0.05), and 3.47-fold (P < 0.001). Adding VAS-2870 or PEG-Cat to the EA group significantly attenuated the increase in expression levels (decreased by 48.1% and 42.7%). (D–F) EA-mediated GSTA1 inhibition significantly increased CYP11B2 activity, CYP11B2 mRNA expression, and aldosterone secretion. Adding 20 μM VAS-2870, 350 U/mL PEG-Cat, 200 μM N-acetyl-l-cysteine (NAC), 10 μM l-NG-nitro-l-arginine methyl ester (L-NAME), 30 μM tocopherol, or 100 μM MnTMPyP significantly reduced this increase. (G) DCFH-DA fluorescence images in various conditions. DMSO, dimethyl sulfoxide. *P < 0.05; **P < 0.01; ***P < 0.001. GSTA1 siRNAs or 30 μM EA significantly increased superoxide production by 2.33-, 2.65-, 2.82- (P < 0.05), and 4.41-fold (P < 0.001). The addition of 20 μM VAS-2870 and 350 U/mL PEG-Cat after EA inhibition decreased superoxide production to 61.0% and 38.8% (P < 0.001), respectively (Fig. 5B). GSTA1 overexpression significantly decreased H2O2 production to 66.9% (P < 0.05), whereas GSTA1 siRNAs or 30 μM EA significantly increased H2O2 production. Three GSTA1 siRNAs and EA inhibition significantly increased the H2O2 by 1.59-, 1.41-, 1.68- (P < 0.05), and 3.47-fold (P < 0.001). Adding VAS-2870 or PEG-Cat to the EA group significantly attenuated the increase in expression levels (decreased by 48.1% and 42.7%) (Fig. 5C). EA-mediated GSTA1 inhibition significantly increased CYP11B2 activity, CYP11B2 mRNA expression, and aldosterone secretion. Adding 20 μM VAS-2870, 350 U/mL PEG-Cat, 200 μM N-acetyl-l-cysteine, 10 μM l-NG-nitro-l-arginine methyl ester, 30 μM tocopherol, or 100 μM MnTMPyP significantly reduced this increase (Fig. 5D–5F). Ca2+-dependent signaling is involved in GSTA1 regulation of aldosterone synthesis Ca2+-dependent signaling has been shown to directly cause the oversecretion of aldosterone. Therefore, we investigated whether inhibiting GSTA1 affected the Ca2+/calmodulin-dependent protein kinase signaling pathway. Our results showed that GSTA1 overexpression significantly downregulated CAMK1, NR4A1, and NR4A2 expression levels. GSTA1 inhibition significantly increased CAMK1, NR4A1, and NR4A2 expression. Blocking calmodulin by calmidazolium (2.5 μM) and W-7 (30 μM) and blocking calcium channels by nifedipine (10 μM) significantly reduced CYP11B2 activity, CYP11B2 expression, and aldosterone secretion in GSTA1-inhibited cells. However, the difference in expression levels was less notable in the control cells (Supplemental Fig. 6E–6G). At the same time, the intracellular Ca2+ significantly increased after GSTA1 inhibition (Supplemental Figs. 6D and 8). Discussion At our medical center, KCNJ5-mutated APAs account for as much as 80% of all cases. The mean aldosterone level was high in patients with KCNJ5 mutations. Although comparative transcriptomic analyses have been conducted with KCNJ5 mutants or nonmutant APAs (13–15), aldosterone levels have not been compared. We selected and analyzed the differentially expressed genes between HALD-mKCNJ5 and LALD-mKCNJ5 APAs. Both GO and pathway analysis showed that glutathione metabolism was the top different pathway. GSTA1, GSTA2, GSTA3, and GSTA5 were all downregulated in HALD-mKCNJ5 compared with LALD-mKCNJ5. Therefore, the GSTA family genes may significantly regulate aldosterone in KCNJ5-mutated APAs. Recently, Zhou et al. (25) reported that GSTA1 was the most differentially expressed between ZG and ZF and between APA and ZG. In addition, its expression may be regulated by the NRF2-mediated oxidative stress response pathway. However, there was no further detailed functional investigation about GSTA1 (25). Sarkar et al. (26) found that GSTA1 is highly expressed in cortisol-producing adrenocortical adenomas compared with normal adrenal glands. GST inhibition interfered with cellular proliferation. Another study investigated the role of the GSTA family in steroidogenesis, which is regulated by steroidogenic factor 1. GSTA1 and GSTA3 interactions exhibit steroid isomerase activities and are involved in steroidogenesis (27). Unlike the high GSTA1 expression in cortisol-producing adenoma, GSTA1 was less expressed in APA tissue than in the normal adrenal tissue. Moreover, GSTA1 expression was lower in KCNJ5-mutated than in non–KCNJ5-mutated APAs and lowest in HALD-mKCNJ5 cases. GSTA1 expression was inversely correlated with recumbent and upright aldosterone levels. Western blot and immunohistochemistry staining showed lower protein expression in KCNJ5-mutated APAs and even lower expression in HALD-mKCNJ5 APAs. We observed that GSTA1 expression was absent in the ZG of the normal adrenal gland, whereas it was higher in other zones, which was consistent with another study (28). Our key finding was that GSTA1 overexpression suppressed aldosterone secretion, whereas silencing or inhibiting GSTA1 increased aldosterone secretion. This increase in expression level was probably caused by the parallel increase in CYP11B2 expression and activity. The decrease in GSTA1 mRNA levels after stimulation with angiotensin II corresponded with this result. GSTA1 was mostly related to the detoxification of various xenobiotics and products of oxidative stress. Therefore, the downregulation of GSTA1 resulted in the accumulation of ROS and H2O2, which then led to the excess secretion of aldosterone. Silencing or inhibiting GSTA1 significantly increased DCFH-DA fluorescence, Nox activity, and H2O2 production, whereas GSTA1 overexpression in H295R decreased fluorescence less drastically. These results may be explained by H295R cells expressing GSTA1 protein. Adding Nox inhibitor (VAS-2870), H2O2 scavenger (PEG-Cat), and other antioxidants significantly reduced aldosterone secretion in cells with EA. However, the reduction was less noteworthy without EA, supporting that increased aldosterone secretion was mediated by ROS. Immunofluorescence staining of GSTA1 in nucleus might be nonspecific. Because the GSTA1 was stained in the nucleus using immunofluorescence in the siRNA transfection group or mKCNJ5 group. In the immunohistochemistry staining, GSTA1 was expressed in the cytoplasm but not in the nucleus. Other studies also showed that GSTA1 is abundantly distributed in the cytoplasm of adrenal cells (26) and other tumor cells (29). Oxidative stress has been associated with aldosteronism. Garrido and Griendling (30) reported that angiotensin II could stimulate Nox and produce ROS, which further result in hypertension and various complications. In addition, Rajamohan et al. (22) showed that angiotensin II increased CYP11B2 expression and aldosterone secretion by Nox-H2O2 signaling. Calo et al. (23) reported oxidative related proteins were increasingly expressed in APA including p22(phox), Nox, transforming growth factor-β, plasminogen activator inhibitor-1, and heme oxygenase-1. The increased plasma Nox activity and urinary excretion of isoprostanes showed increased oxidative stress in patients with primary aldosteronism (24). Geng et al. (21) showed nox2-induced oxidative stress might play a critical role in regulated aldosterone secretion. These results showed that oxidative stress is an important pathogenic mechanism in aldosteronism. Conversely, aldosterone can also induce oxidative stress in many ways. Other studies showed that aldosterone could increase Nox activity, resulting in ROS production and leading to myocyte apoptosis and glomerular diseases (31, 32). Our study and previous results suggested that the interaction between GSTA1 and oxidative stress might be one important reason for pathological autonomous aldosterone hypersecretion in APAs. In contrast, other steroidogenic organs need antioxidants to defend against oxidative stress to synthetize steroid. Otherwise, these ROS can cause mitochondrial DNA damage and cell apoptosis. Therefore, unlike in APAs, several studies showed antioxidants were increasingly expressed in such organs as the corpus luteum, adrenal cortex fasciculata, and reticularis (33–35). As Ca2+ influx is the common signal and direct cause of aldosterone oversecretion, we investigated the effect of GSTA1 inhibition on intracellular Ca2+. Similar to KCNJ5 mutation, GSTA1 inhibition increased intracellular Ca2+ and the expression of calmodulin-related genes, including CAMK1, NR4A1, and NR4A2. The pharmacological blockage of Ca2+ channels or calmodulin significantly inhibited the EA-induced increase of aldosterone, but not the control cells. These results suggested that GSTA1 also functions through Ca2+-dependent signaling. Previously, the cross talk between calcium and ROS signaling has been extensively studied (36, 37). Therefore, we suppose that GSTA1 interact with both signals to stimulate aldosterone secretion. GSTA1 mRNA and protein expressions were significantly downregulated by G151R or L168R KCNJ5 mutation. The addition of aldosterone did not decrease GSTA1 expression, which suggested that KCNJ5 mutation did not regulate GSTA1 via aldosterone levels. Furthermore, GSTA1 overexpression significantly reduced CYP11B2 expression, activity, and aldosterone secretion in KCNJ5-G151R H295R cells. GSTA1 inhibition showed little effect because of the low GSTA1 expression in mKCNJ5 H295R cells. Other factors might increase GSTA1 expression and thereby lower aldosterone levels in some mKCNJ5 APAs. We also tried to detect the methylation status of GSTA1 by methylmion-specific polymerase chain reaction after adding aldosterone or not. The primers were designed for the CpG island on the upstream of the promoter. We found that there was apparent methylation in the 5′-untranslated region and promoter region of GSTA1 gene. However, no marked difference was found between the group with aldosterone and without. The explorations of GSTA1 epigenetic change should be further investigated. Although we identified the critical role of the GSTA1 gene in regulating aldosterone secretion in KCNJ5-mutated H295R cells. There were some limitations. First, given that the definition of high or low aldosterone secretion is flexible, we selected values approaching the extreme values for microarray analysis and used correlation analysis to verify the results. Second, the mechanisms under mutated KCNJ5 on GSTA1 were not fully elucidated. Third, we only divided APAs into KCNJ5 mutation and nonmutation groups because the KCNJ5 mutation accounted for a large proportion, and few other mutations were identified. In conclusion, we identified a series of transcript differences between the HALD-mKCNJ5 and LALD-mKCNJ5 APAs. The GSTA1 gene is the most downregulated in HALD-mKCNJ5 and inversely correlated with aldosterone level. It may be an important factor affecting aldosterone secretion in adrenal cells. 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Mar 1, 2018

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