Background: The mechanisms causing hypercortisolism in primary macronodular adrenocortical hyperplasia (PMAH) are not fully clarified. The participation of ectopic receptors and autocrine/paracrine regulation of intra-adrenal ACTH in hyperplastic tissue have been considered. Additionally, germline ARMC5 mutations have been described as main cause of PMAH. So far, the functional study that analyzed the role of ARMC5 used the adrenocortical carcinoma cell line, H295R. Therefore, we propose a more suitable model in cell cultures obtained from nodules of PMAH.
Aim: To investigate the role of ARMC5 related to PMAH.
Methods: Cell cultures obtained from nodules of 13 unrelated patients with PMAH ARMC5 mutated or not were analyzed for: i) Morphological and functional characterization; ii) ARMC5 sequencing; iii) ARMC5 silencing through shRNA in non-mutated PMAH cells for steroidogenic and proliferative functional studies; iv) ARMC5 overexpression through transient transfection in mutated PMAH cells and viability analyses.
Results: i) The cell cultures demonstrating presence of steroidogenic cells, ectopic/aberrant receptors and functional intra-adrenal ACTH. ii) ARMC5 mutations were located mostly in exons 1, 2 and 3, responsible for protein-protein interactions of ARM repeat family; ARMC5 germline mutations identified in seven samples were associated or not to somatic mutations or LOH. In six samples, ARMC5 mutation was not identified; iii) ARMC5 silencing in non-mutated PMAH cell cultures decreased StAR, CYP17A1, CYP11A1, NR5A1 and MC2R, increased CCNE1 mRNA expression and its proliferative capacity, but without interference in cell viability. iv) ARMC5 overexpression induced apoptosis and necrosis after 8h in PMAH mutated cell cultures, decreasing cell viability.
Conclusions: We confirm the role of ARMC5 as a pro-apoptotic protein and its importance in the steroidogenesis related to PMAH, as previously described in H295R cells. We also report for the first time the involvement of ARMC5 in proliferation control and cell cycle regulation of PMAH cell cultures, which needs to be further explored.
20 - 23 May 2017
European Society of Endocrinology