Effect of KCNJ5 mutations on gene expression in aldosterone-producing adenomas and adrenocortical cells

Abstract

Context: Primary aldosteronism is a heterogeneous disease that includes both sporadic and familial forms. A point mutation in the KCNJ5 gene is responsible for familial hyperaldosteronism type III. Somatic mutations in KCNJ5 also occur in sporadic aldosterone producing adenomas (APA).

Objective: The objective of the study was to define the effect of the KCNJ5 mutations on gene expression and aldosterone production using APA tissue and human adrenocortical cells.

Methods: A microarray analysis was used to compare the transcriptome profiles of female-derived APA samples with and without KCNJ5 mutations and HAC15 adrenal cells overexpressing either mutated or wild-type KCNJ5. Real-time PCR validated a set of differentially expressed genes. Immunohistochemical staining localized the KCNJ5 expression in normal adrenals and APA.

Results: We report a 38% (18 of 47) prevalence of KCNJ5 mutations in APA. KCNJ5 immunostaining was highest in the zona glomerulosa of NA and heterogeneous in APA tissue, and KCNJ5 mRNA was 4-fold higher in APA compared with normal adrenals (P < 0.05). APA with and without KCNJ5 mutations displayed slightly different gene expression patterns, notably the aldosterone synthase gene (CYP11B2) was more highly expressed in APA with KCNJ5 mutations. Overexpression of KCNJ5 mutations in HAC15 increased aldosterone production and altered expression of 36 genes by greater than 2.5-fold (P < 0.05). Real-time PCR confirmed increases in CYP11B2 and its transcriptional regulator, NR4A2.

Conclusions: KCNJ5 mutations are prevalent in APA, and our data suggest that these mutations increase expression of CYP11B2 and NR4A2, thus increasing aldosterone production.

Fig. 1.

A, Heat map representation of the 10 genes with the highest differential expression in female APA with or without KCNJ5 mutations. Genes were selected based on a significance of P < 0.05 and a differential expression of at least 2.5-fold. Heat map data are presented as log₂ of the signal intensity value. Absolute fold change (FC) is also provided. B, Validation of microarray using real-time PCR. Four genes were selected to confirm microarray analysis by using real-time PCR on a larger subset of RNA samples from women with APA (13 KCNJ5^WT and 20 KCNJ5 mutant APA). Comparison of APA with and without KCNJ5 mutations demonstrated a significant up-regulation of CYP11B2 (3.1-fold change), RELN (2.5-fold change), and HTR2B (2.2-fold change) but a significant down-regulation of TSPAN12 (−5.1-fold change). Data are presented as normalized (cyclophylin) transcript fold change with each bar representing the mean ± se. *, P < 0.05 vs. APA with wild-type KCNJ5.

Fig. 2.

A, Heat map representation of differentially expressed genes in HAC15 adrenal cells overexpressing either KCNJ5 mutations (p.G151R and p.L168R) or KCNJ5^WT. Genes were selected based on a significance of P < 0.05 and a differential expression of at least 2.0-fold. Heat map data are presented as log₂ of the intensity value. Absolute fold change is also provided. Samples are HAC15 cells transfected with pcDNA3.1/KCNJ5^G151R (1 and 2); pcDNA3.1/KCNJ5^L168R (3 and 4); and pcDNA3.1/KCNJ5^WT (5 and 6). B, Validation of up-regulated genes using real-time PCR. CYP11B2 was up-regulated 6.3 ± 0.6-fold and 7.2 ± 1.4-fold in HAC15 cells overexpressing KCNJ5^G151R or KCNJ5^L168R, respectively, compared with HAC15 cells overexpressing KCNJ5^WT. NR4A2 was markedly up-regulated 98 ± 28-fold and 38 ± 6-fold in HAC15 cells overexpressing KCNJ5^L168R or KCNJ5^G151R, respectively, vs. KCNJ5^WT, with a relative fold change L168R/G151R of 2.6 (P < 0.05). No differences were observed between the effects of the G151R and L168R mutations on the gene expression levels of CYP11B1, CYP11B2, and NR4A3 in HAC15 cells. No statistically significant difference was observed between HAC15 cells overexpressing KCNJ5^WT and pcDNA3.1 mock-transfected cells for any of the four selected genes. Each bar represents the mean ± sd of relative fold change of gene expression in five independent experiments. Each assay was performed in triplicate, and PPAI (cyclophylin) was used as endogenous control. *, P < 0.05 compared with WT; **, P < 0.05 compared with WT and G151R.