Endocrine Abstracts

Introduction: Adrenal Cushing due to bilateral multiple adrenal tumors known as Primary Pigmented Nodular Adrenocortical Disease (PPNAD) can be observed in the multiple neoplasia syndrome Carney Complex or as an isolated disease. In both situations germline inactivating mutations of PRKAR1A (regulatory subunit R1A of PKA) can be observed. The loss of PKA R1A results in an increased PKA activity. Comparison of the transcriptome of PPNAD and stably transfected H295R adrenal cortical cells with and without inactivation of PRKAR1A (H295R/TR/shPRKAR1A) identified a gene whose expression is decreased following the loss of PRKAR1A expression: KCTD20 (potassium channel tetramerization domain containing). This work aims to understand the role of KCTD20 in endocrine hyperactivity and tumor development of PPNADs and Carney Complex.

Methods: H295R, HEK293 cells and PPNAD primary cell culture, were used. The transcriptional regulation of KCTD20 by PKA R1A (siRNA interference) (RNA, protein) was analysed. The role of KCTD20 was evaluated through its inactivation by silencing RNA (siKCTD20) and its overexpression (vector-KCTD20) to investigate the effects on PKA R1A alteration concerning cell proliferation/apoptosis (MTT/annexin V, flow cytometer), cell signalling pathways (PKA, AKT, intracellular calcium), and steroidogenesis. KCTD20 intracellular localisation was analysed by immunofluorescence. KCTD20 protein contains a BTB domain. The immunoprecipitation of KCTD20 and mass spectrometry analyses was used to identify the partners of KCTD20 and to precise its role.

Results: In H295R, the decrease in KCTD20 expression after PRKAR1A invalidation is independent of PKA activity. Overexpression of KCTD20 increases apoptosis and decreases cell proliferation. Invalidation of KCTD20 protects against apoptosis (P<0.01), increases the activity of the Star-Luc reporter (P<0.001), the expression of Star (P=0.01) and CYP11B1 genes (P=0.05), and cortisol production in H295R cells (P<0.05). These effects seem independent of PKA activity. Invalidation of KCTD20 results in membrane depolarization in response to KCL and increases intracellular calcium (P<0.001). Flag-KCTD20 exhibited a cytosolic and spot-like distribution in transiently transfected H295R and Hela cells. Culline 3 was identified as a potential partner of KCTD20. In PPNAD from patients with PRKAR1A mutations in primary cell culture, the overexpression of KCTD20 decreases the CYP11B1 gene (RT/PCR).

Conclusion: PKA R1A acts on KCTD20 via a PKA independent pathway. KCTD20 may play a role in adrenal Cushing by mechanisms independent of PKA activity.