Role of ANO4 in regulation of aldosterone secretion in the zona glomerulosa of the human adrenal gland.

BACKGROUND: Cell origin of aldosterone-producing adenomas, a major cause of hypertension, is unknown. A less common subtype of these adenomas, composed of cells resembling zona glomerulosa, have mutations in genes ATP1A1 and CACNA1D. To understand whether the adenomas originate from zona glomerulosa, we carried out a microarray analysis comparing transcriptomes of zona glomerulosa, zona fasciculata, and tumour in human adrenal tissue, and investigated the functional role of genes upregulated in the zona glomerulosa. METHODS: Using a microarray analysis (Affymetrix, High Wycombe, UK), we compared transcriptomes of zona glomerulosa, zona fasciculata, and tumour obtained by laser capture microdissection of 14 patients with aldestosterone adenomas and seven with phaeochromocytoma. One of the most zona glomerulosa-selective genes was ANO4, a member of the anoctamin family. Subcellular localisation was observed by immunofluorescence microscopy of transfected HEK293 cells. Yellow fluorescent protein-based assay was performed to detect ANO4 activity as a calcium-activated chloride channel. H295R cells were transfected by ANO4 to measure aldosterone and CYP11B2 expression. FINDINGS: Microarray analysis revealed 28 genes that were at least five times overexpressed in zona glomerulosa compared with zona fasciculata. ANO4 was 19·9 times higher in zona glomerulosa than in zona fasciculata (p=6·6 × 10(-24)). Haemagglutinin-tagged ANO4 was localised to the plasma membrane of transfected HEK293 cells. In response to increased intracellular calcium, ANO4-transfected cells triggered a lower flow of iodide than did other anoctamins. ANO4 overexpression in H295R cells increased aldosterone secretion from mean 0·9 pmol/µg protein (SE 0·2) to 1·1 (0·1), whereas CYP11B2 mRNA expression increased five times. INTERPRETATION: We show that ANO4 is one of the most highly expressed genes in zona glomerulosa of the human adrenal gland. When overexpressed in vitro, it increases aldosterone production. FUNDING: British Heart Foundation.