DACH1, a zona glomerulosa selective gene in the human adrenal, activates transforming growth factor-β signaling and suppresses aldosterone secretion

Abstract

Common somatic mutations in CACNAID and ATP1A1 may define a subgroup of smaller, zona glomerulosa (ZG)-like aldosterone-producing adenomas. We have therefore sought signature ZG genes, which may provide insight into the frequency and pathogenesis of ZG-like aldosterone-producing adenomas. Twenty-one pairs of zona fasciculata and ZG and 14 paired aldosterone-producing adenomas from 14 patients with Conn's syndrome and 7 patients with pheochromocytoma were assayed by the Affymetrix Human Genome U133 Plus 2.0 Array. Validation by quantitative real-time polymerase chain reaction was performed on genes >10-fold upregulated in ZG (compared with zona fasciculata) and >10-fold upregulated in aldosterone-producing adenomas (compared with ZG). DACH1, a gene associated with tumor progression, was further analyzed. The role of DACH1 on steroidogenesis, transforming growth factor-β, and Wnt signaling activity was assessed in the human adrenocortical cell line, H295R. Immunohistochemistry confirmed selective expression of DACH1 in human ZG. Silencing of DACH1 in H295R cells increased CYP11B2 mRNA levels and aldosterone production, whereas overexpression of DACH1 decreased aldosterone production. Overexpression of DACH1 in H295R cells activated the transforming growth factor-β and canonical Wnt signaling pathways but inhibited the noncanonical Wnt signaling pathway. Stimulation of primary human adrenal cells with angiotensin II decreased DACH1 mRNA expression. Interestingly, there was little overlap between our top ZG genes and those in rodent ZG. In conclusion, (1) the transcriptome profile of human ZG differs from rodent ZG, (2) DACH1 inhibits aldosterone secretion in human adrenals, and (3) transforming growth factor-β signaling pathway is activated in DACH1 overexpressed cells and may mediate inhibition of aldosterone secretion in human adrenals.

Keywords: DACH1 protein, human; aldosterone; hyperaldosteronism; transforming growth factor beta; zona glomerulosa.

Figure 1.

The heat map and dendrogram of unsupervised clustering of (A) 293 genes differentially expressed in zona glomerulosa (ZG) compared with zona fasciculata (ZF) and (B) 210 genes differentially expressed in aldosterone-producing adenoma (APA) compared with ZG. Unsupervised cluster analysis separated ZG, ZF, and APA from patients with a KCNJ5 somatic mutation from those without. Red and green indicate high and low expression, respectively. The purple, blue, and grey boxes in the dendogram indicate the tissue came from a patient with Conn’s syndrome with a somatic KCNJ5 mutation in their APA, without a KCNJ5 mutation, or from a patient with pheochromocytoma, respectively.

Figure 2.

Immunohistochemistry (IHC) of DACH1 in formalin-fixed paraffin-embedded human adrenal sections. A, IHC of DACH1 in adjacent adrenal glands. DACH1 is highly expressed in zona glomerulosa (ZG) cell’s nuclei. Pictures are representative of 13 adrenals from 10 patients with aldosterone-producing adenomas (APA) and 3 with pheochromocytomas. B, IHC of DACH1 in APAs. ZG-like APAs appeared to have similar staining of DACH1 to their adjacent zona glomerulosa. Left, Scanned images. Right, Zoomed insets of left panel. C, Colocalization of DACH1 cytoplasmic staining with PCP4 in cell clusters. D, Hematoxylin and eosin (H&E) stain and IHC of DACH1, CYP11B1 (as a zona fasciculata [ZF] marker), and ZG markers, CYP11B2, PCP4, and KCNJ5, in serial adrenal sections from a patient with Conn’s syndrome.

Figure 3.

Effect of DACH1 on aldosterone production. Silencing of DACH1 in H295R cells increased aldosterone production compared with control (nontargeting), whereas overexpression of DACH1 decreased aldosterone production compared with control vector. A–C, H295R cells were treated with vehicle control (untreated), nontargeting siRNA or Si-DACH1 for 48 h after which 24-h supernatant was collected for aldosterone measurement. D–F, H295R cells were transfected with either control vector, DACH1 706aa or DACH1 709aa—24-h supernatant was collected for aldosterone measurement after 48 h. mRNA expression of DACH1 (A and D) and (C and F) mRNA expression of CYP11B2 after transfection of either siRNAs (A and C) or overexpression vectors (D and F).

Figure 4.

DACH1 regulates transforming growth factor-β (TGF-β) and Wnt signaling activity. A, Overexpression of DACH1 in H295R cells increased TGF-β signaling activity. H295R cells were cotransfected with either a negative control or a SMAD reporter and with control vector, DACH1 706aa or 709aa, to measure DACH1’s effect on TGF-β signaling activity; n=10. B, The effect of DACH1 on increasing TGF-β signaling activity in H295R cells was additive to the effect of TGF-β1 exposure. H295R cells were cotransfected with a SMAD reporter and control vector, DACH1 706aa or 709aa, and treated with varying TGF-β1 concentration; n=9. C, TCF/LEF reporter assay showed upregulation of Wnt signaling activity in H295R cells overexpressing DACH1. H295R cells were cotransfected with either a negative control or a TCF/LEF reporter, and with control vector, DACH1 706aa or 709aa, to measure DACH1’s effect on Wnt signaling activity; n=13. D, TCF/LEF reporter assay showed downregulation of Wnt signaling activity in DACH1-silenced H295R cells. H295R cells were cotransfected with either a negative control or a TCF/LEF reporter, and with either a nontargeting siRNA or a siRNA specific for DACH1 (Si-DACH1), to measure endogenous DACH1’s effect on Wnt signaling activity; n=4.