Hypomethylation of CYP 11 B 2 in Aldosterone-Producing Adenoma

Primary aldosteronism (PA) is the most common cause of secondary hypertension and occurs by an excessive and autonomous production of aldosterone independent of the renin–angiotensin system. Patients with PA have an increased risk of cardiovascular and cerebrovascular complications compared with those with essential hypertension. Therefore, the elucidation of the molecular mechanisms of aldosterone production is crucially important for the development of therapeutic agents for PA. The 2 most common forms of PA are aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism, also called as bilateral adrenal zona glomerulosa hyperplasia. Aldosterone is produced from the precursor cholesterol via a series of steroidogenic enzymes, and APA has higher levels of aldosterone synthase (CYP11B2) that catalyzes the final steps of aldosterone biosynthesis. Ca signaling is upregulated in APA, and it plays a pivotal role in the activation of CYP11B2 transcription. Recently, exome sequencing analyses demonstrated that 50% to 80% of APA harbored somatic mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, or CTNNB1. KCNJ5, ATP1A1, ATP2B3, and CACNA1D mutations lead to an increased intracellular Ca concentration, an activation of Ca signaling, and an increase in CYP11B2 transcription. High CYP11B2 expression levels were found in APA with somatic CTNNB1 mutations that activated Wnt signal. Epigenetic regulation, such as DNA methylation as well as intracellular signaling cascades, is important for tumor progression, cell survival, DNA damage repair, and hormone secretion via their influence of gene transcription. DNA methylation by DNA methyltransferase enzymes occurs at the cytosine of 5′-cytosine-guanine-3′ dinucleotides (CpG), where the methylation of the promoter region typically acts to repress the gene transcription. Previous reports demonstrated that methylation status might be regulated in APA, and a methylation site at the CYP11B2 promoter might be Abstract—The purpose of this study was to evaluate the DNA methylation levels of steroidogenic enzyme genes in aldosterone-producing adenoma (APA) and the effects of gene mutations in APA on the DNA methylation levels. DNA methylation array analysis was conducted using nonfunctioning adrenocortical adenoma (n=12) and APA (n=35) samples, including some with a KCNJ5 mutation (n=21), an ATP1A1 mutation (n=5), and without the known mutations (n=9). The quantitative polymerase chain reaction assay was performed for the detection of CYP11B2 and CYP11B1 expression levels in nonfunctioning adrenocortical adenoma and APA. We introduced the KCNJ5 T158A mutation using lentivirus delivery in the human adrenocortical 15 cell line, and analyzed the effects of the mutation on DNA methylation levels. We analyzed the 83 presumed DNA methylation sites of steroidogenic enzymes. In APA, we found 7 hypomethylated sites in CYP11B2 and 1 hypomethylated and 6 hypermethylated sites in CYP11B1. There were no differences in the steroidogenic enzymes gene DNA methylation of peripheral leukocytes between nonfunctioning adrenocortical adenoma and APA. No CYP11B2 methylation level was associated with CYP11B2 transcription levels in APA. All methylation sites, except for a CYP11B2 region, showed no difference among APAs with or without gene mutations. Human adrenocortical 15 cells with the KCNJ5 mutation showed no changes in CYP11B2 or CYP11B1 methylation levels compared with control cells. We demonstrated that CYP11B2 in APA was extensively hypomethylated, and CYP11B2 methylation in the region with hypomethylation was not induced by KCNJ5 or ATP1A1 mutations that cause aldosterone overproduction in APA and a KCNJ5 mutation human adrenocortical 15 cells. (Hypertension. 2016;68:1432-1437. DOI: 10.1161/HYPERTENSIONAHA.116.08313.) • Online Data Supplement