ANO4 (Anoctamin 4) Is a Novel Marker of Zona Glomerulosa That Regulates Stimulated Aldosterone Secretion

Abstract

Microarray comparison of the transcriptomes of human adrenal zona glomerulosa (ZG) and zona fasciculata found several ZG-specific genes that negatively regulate aldosterone secretion. The third and most significantly upregulated ZG-gene (19.9-fold compared with zona fasciculata, P=6.58×10^-24) was ANO4, a putative Ca²⁺-activated chloride channel. We have investigated the role of ANO4 in human adrenal, and whether it functions like the prototype anoctamin, ANO1. We evaluated ANO4 mRNA and protein expression in human adrenal by qPCR and immunohistochemistry, compared the effects of ANO4 and ANO1 overexpression on baseline and stimulated aldosterone secretion and cell proliferation in H295R cells, and analyzed ANO4 activity as a Ca²⁺-activated chloride channel in comparison with other anoctamins by a fluorescence-based functional assay. The expression of ANO4 in ZG was confirmed by qPCR as 23.21-fold upregulated compared with zona fasciculata (n=18; P=4.93×10^-7). Immunohistochemistry found cytoplasmic, ZG-selective expression of ANO4 (anoctamin 4) protein. ANO4 overexpression in H295R cells attenuated calcium-mediated aldosterone secretion and cell proliferation in comparison to controls. The latter effects were in a different direction to those of ANO1. The functional assay showed that, in contrast to ANO1, ANO4 expression results in low levels of calcium-dependent anion transport. In conclusion, ANO4 is one of the most highly expressed genes in ZG. It attenuates stimulated aldosterone secretion and cell proliferation. Although belonging to a family of Ca²⁺-activated chloride channels, it does not generate significant plasma membrane chloride channel activity.

Keywords: Zona glomerulosa; aldosterone; anoctamins; cell proliferation; chloride channel.

Figure 1.

Immunohistochemistry of ANO4 (anoctamin 4) in formalin-fixed paraffin-embedded human normal adrenal cortex. ANO4 is selectively expressed in zona glomerulosa (ZG) cells. The staining shows a diffuse cytoplasmic pattern. A–C, Shows 4×, 10×, and 20× magnification, respectively. These images are representative of 6 observations. C indicates capsule; M, medulla; and ZF, zona fasciculata.

Figure 2.

Effect of ANO4 overexpression on basal aldosterone production. Overexpression of ANO4 in H295R cells increased ANO4 mRNA by 12.6-fold (A), and increased NR4A2 (B), and CYP11B2 (C) mRNA levels but did not affect aldosterone production (D) in comparison with control (EV=empty vector). Similarly, silencing ANO4 for 48 h with small interfering RNA significantly reduced ANO4 (anoctamin 4) protein expression (C) but did not affect aldosterone secretion (D). Data are shown in geometric mean values ±SEM and are representative of 4 experiments.

Figure 3.

Effect of ANO4 knockdown on basal aldosterone production. Silencing ANO4 for 48 h with small interfering RNA significantly reduced ANO4 (anoctamin 4) protein expression (A) and NR4A2 (B), and CYP11B2 (C) mRNA levels but did not affect aldosterone secretion (D). Data are shown in geometric mean values ±SEM and are representative of 4 experiments.

Figure 4.

Effect of ANO4 (anoctamin 4) overexpression in H295R on ionomycin (A) and ATP-stimulated aldosterone secretion. Ionomycin increased aldosterone secretion by 2.7-fold (vs basal conditions) in controls, and only 1.2-fold in ANO4-transfected cells (*P<0.05, A). ATP increased aldosterone secretion by 1.5-fold (vs basal aldosterone) in controls, while it had no effect in ANO4-transfected cells (*P<0.05), B). Data are shown in geometric mean values+SEM.

Figure 5.

Representative traces (left) and summary of data (right) from experiments carried out with the HS-YFP (halide-sensitive yellow fluorescent protein) assay on HEK-293 cells transiently transfected with ANO1, ANO2, ANO4 (anoctamin 4), and ANO5 or empty plasmids (null). Traces show the cell fluorescence decrease following I⁻ addition (arrow) with or without ionomycin (1 or 10 µmol/L). The bar graph reports the maximal quenching rate (QR) for the indicated conditions. Data are from 6 to 13 experiments. Asterisks (*P<0.05; ***P<0.001) indicate a statistically significant difference vs cells transfected with empty vectors. Hash signs indicate a statistically significant difference between indicated groups of data (#P<0.05; ###P<0.001).