Aldosterone production is initiated by angiotensin II stimulation and activation of intracellular Ca signaling. In aldosterone-producing adenoma (APA) cells, the activation of intracellular Ca signaling is independent of the renin-angiotensin-aldosterone systems. The purpose of our study was to clarify molecular mechanisms of aldosterone production related to Ca signaling. Transcriptome analysis revealed that the gene encoding calneuron 1 had the strongest correlation with (aldosterone synthase) among genes encoding Ca-binding proteins in APA. modulation and synthetic or fluorescent compounds were used for functional studies in human adrenocortical carcinoma (HAC15) cells. expression was 4.4-fold higher in APAs than nonfunctioning adrenocortical adenomas. CALN1 expression colocalized with CYP11B2 expression as investigated using immunohistochemistry in APA and zona glomerulosa of male rats fed by a low-salt diet. CALN1 expression was detected in the endoplasmic reticulum (ER) by using GFP-fused CALN1, CellLight ER-RFP, and the corresponding antibodies. -overexpressing HAC15 cells showed increased Ca in the ER and cytosol fluorescence-based studies. Aldosterone production was potentiated in HAC15 cells by expression, and dose-responsive inhibition with TMB-8 showed that CALN1-mediated Ca storage in ER involved sarcoendoplasmic reticulum calcium transport ATPase. The silencing of decreased Ca in ER, and abrogated angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells. Increased CALN1 expression in APA was associated with elevated Ca storage in ER and aldosterone overproduction. Suppression of CALN1 expression prevented angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells, suggesting that CALN1 is a potential therapeutic target for excess aldosterone production.

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 non-functioning adrenocortical adenoma (NF, n=12), and APA (n=35) samples including some with a KCNJ5 mutation (n=21), an ATP1A1 mutation (n=5), and without known mutations (n=9). The quantitative polymerase chain reaction assay was performed for the detection of CYP11B2 and CYP11B1 expression levels in NF and APA. We introduced the KCNJ5 T158A mutation using lentivirus delivery in the human adrenocortical cell line (HAC15), 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 seven hypo-methylated sites in CYP11B2 and one hypo-methylated six hyper-methylated sites in CYP11B1. There were no differences in the steroidogenic enzymes gene DNA methylation of peripheral leucocytes between NF 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. HAC15 cells with the KCNJ5 mutation showed no changes in CYP11B2 or CYP11B1 methylation levels compared to control cells. We demonstrated that CYP11B2 in APA was extensively hypo-methylated, and CYP11B2 methylation in the region with hypo-methylation was not induced by KCNJ5 or ATP1A1 mutations that cause aldosterone over-production in APA and a KCNJ5 mutation HAC15 cells.