Colocalization of Wnt/β-Catenin and ACTH Signaling Pathways and Paracrine Regulation in Aldosterone-producing Adenoma

Abstract

Context Aldosterone-producing adenomas (APA) are a common cause of primary aldosteronism. Despite the discovery of somatic mutations in APA and characterization of multiple factors regulating adrenal differentiation and function, the sequence of events leading to APA formation remains to be determined. Objective We investigated the role of Wnt/β-catenin and ACTH signaling, as well as elements of paracrine regulation of aldosterone biosynthesis in adrenals with APA and their relationship to intratumoral heterogeneity and mutational status. Design We analyzed expression of CYP11B2, CYP17A1, β-catenin, MC2R, pCREB, Tryptase, S100, CD34 by multiplex immunofluorescence and IHC guided RT-qPCR. Setting 11 adrenals with APA and one with micronodular hyperplasia from patients with PA were analysed. Main Outcome Measure(s) Localization of CYP11B2, CYP17A1, β-catenin, MC2R, pCREB, Tryptase, S100, CD34 in APA and aldosterone producing cell clusters (APCC). Results Immunofluorescence revealed abundant mast cells and a dense vascular network in APA, independent of mutational status. Within APA, mast cells were localized in areas expressing CYP11B2 and were rarely co-localized with nerve fibers, suggesting that their degranulation is not controlled by innervation. In these same areas, ß-catenin was activated, suggesting a zona glomerulosa cell identity. In heterogeneous APA with KCNJ5 mutations, MC2R and VEGFA expression was higher in areas expressing CYP11B2. A similar pattern was observed in APCC, with high expression of CYP11B2, activated β-catenin, and numerous mast cells. Conclusions Our results suggest that aldosterone producing structures in adrenals with APA share common molecular characteristics and cellular environment, despite different mutation status, suggesting common developmental mechanisms.

Transcriptomics, Epigenetics, and Metabolomics of Primary Aldosteronism

“…Another study found transitional structures with a combination of subcapsular APM-like structure and an inner APA-like microstructure without well-defined borders, characterized by the presence of KCNJ5 and ATP1A1 mutations [ 35 ]. Recently, two further studies documented that APMs in the adrenals of patients with APAs carried mutations predominantly in CACNA1D , but also in KCNJ5, ATP1A1 , CACNA1H , PRKACA and CTNNB1, weakening the hypothesis that KCNJ5 mutations do not correlate with the APM-APA transition theory [ 36 , 37 ]. The presence of somatic mutations in APMs suggests that co-driver mutations are necessary in order to promote APA formation.…”

Vascular and hormonal interactions in the adrenal gland

“…Recently, we have investigated the relationship between different signaling pathways and components of the microenvironment in adrenals with APA. We have applied multiplex immunofluorescence and multispectral image analysis to investigate the colocalization of proteins involved in aldosterone (CYP11B2) and cortisol (CYP11B1/CYP17A1) biosynthesis, markers of Wnt/β-catenin (β-catenin) and ACTH/cAMP/PKA (MC2R, pCREB) signaling, as well as paracrine pathways of the tumor microenvironment (Tryptase, S100) and vascularization (CD34) ( 100 ). Our results show a dense vascularization in APA, which is independent of the somatic mutation status of the tumor.…”

Pathophysiology of bilateral hyperaldosteronism

“…The synthesis of aldosterone in APM was further determined by mass spectrometry-based imaging studies [66,67]. Both APA and APM have elevated Wnt/β-catenin activity and alterations in CYP11B2 methylation, which may influence their production of aldosterone [68 ▪▪ ,69 ▪ ]. A recent study demonstrated the presence of significant amount of mast cells in APMs as well as in APAs [68 ▪▪ ].…”

“…Both APA and APM have elevated Wnt/β-catenin activity and alterations in CYP11B2 methylation, which may influence their production of aldosterone [68 ▪▪ ,69 ▪ ]. A recent study demonstrated the presence of significant amount of mast cells in APMs as well as in APAs [68 ▪▪ ]. Degranulation of mast cells can stimulate aldosterone production through the local release of serotonin and activation of the 5-HT4 receptor [70,71].…”

“…Degranulation of mast cells can stimulate aldosterone production through the local release of serotonin and activation of the 5-HT4 receptor [70,71]. The presence of mast cells in APM suggests a potential paracrine regulation of aldosterone production [68 ▪▪ ]. Finally, it is worth noting that APM likely have significant variation in their ability to produce aldosterone, progress to adenomas, or cause disease.…”