Mitochondrial dynamics regulation by Angiotensin II involves Mitofusin 2 induction as a novel mechanism in aldosterone synthesis in H295R cells

CASTILLO, ANA FERNANDA; PODEROSO, CECILIA; HELFENBERGER, KATIA E.; HERRERA, LUCÍA; FIORE, ANA

Munich

Congreso; 18th Adrenal Cortex Conference; 2018

Adrenal Cortex Conference

In steroid-producing cells, cholesterol transport from the outer to the inner mitochondrial membrane is the first and rate-limiting step for the synthesis of all steroid hormones. Cholesterol can be transported into mitochondria by specific mitochondrial protein carriers like the Steroidogenic Acute Regulatory (StAR) protein. We have shown in MA-10 Leydig cells that the activity of StAR protein is dependent on phosphorylation by ERK, specifically located in mitochondria, along with PKA and MEK, the upstream activator of ERK. This cascade is activated in mitochondria by cAMP-dependent transduction pathways.Mitochondria are dynamic organelles that undergo replication, mitophagy and morphology changes. Mitochondrial fusion and fission (known as dynamics) allow these processes. Mitofusin (Mfn) 1 and 2 are GTPases implicated in the regulation of fusion, while Dynamin-related protein 1 (Drp1) is the major regulator of mitochondrial fission. Despite the importance of mitochondrial dynamics in several neurological and endocrine disorders, little is known about fusion/fission under hormone-regulation in steroidogenic tissues. We have previously described that LH/CG stimulates mitochondrial fusion and Mfn2 expression in MA-10 Leydig cells. However, mitochondrial dynamics have not been extensively studied in other steroidogenic tissues like adrenal cortex, where aldosterone is produced. Aldosterone is the major mineralocorticoid involved in maintaining fluid and electrolyte balance in all mammals. In the present work, we aimed to study the role of Ang II and cAMP in mitochondrial dynamics and the involvement of this process in the regulation of aldosterone synthesis. To accomplish these objectives, we used the H295R adrenocortical human cell line, which maintains steroidogenic function and is widely used to study adrenocortical function. We demonstrate here that Ang II and cAMP stimulation promoted a shift from punctuated to tubular/elongated (fusion) mitochondrial shape, observed by the use of a recombinant fluorescent mitochondrial protein (mtYFP) and confocal microscopy, and by electronic microscopy at the ultrastructural level. Mfn2 expression was upregulated after Ang II or cAMP stimulation assessed by qPCR and immunoblot, and mitochondrial Drp1 decreased with Ang II in a time-dependent manner. We observed that inhibition of Mfn2 expression by knockdown (siRNA) was sufficient to impair steroid biosynthesis (without siRNA-Mfn2: C: 0.20±0.06; Ang II: 0.48±0.10; cAMP: 0.70±0.13; with siRNA-Mfn2: C: 0.24±0.08; Ang II: 0.29±0.07; cAMP: 0.50±0.10; ng/ml aldosterone P