Angiotensin II Regulates Mitochondrial mTOR Pathway Activity Dependent on Acyl-CoA Synthetase 4 in Adrenocortical Cells.

Two well-known protein complexes in mammalian cells, mTOR type 1 and type 2 (mTORC1/2) are involved in several cellular processes such as protein synthesis, cell proliferation, and commonly dysregulated in cancer. An acyl-CoA synthetase type 4 (ACSL4) is one of the most recently mTORC1/2 regulators described, in breast cancer cells. The expression of ACSL4 is hormone-regulated in adrenocortical cells and required for steroid biosynthesis. mTORC1/2 have been reported to be crucial in the proliferation of human adrenocortical tumor cells H295R and interestingly reported at several subcellular locations, which has brought cell biology to the vanguard of the mTOR signaling field. In the present work, we study the regulation of mTORC1/2 activation by angiotensin II (Ang II)-the trophic hormone for adrenocortical cells-the subcellular localization of mTORC1/2 signaling proteins and the role of ACSL4 in the regulation of this pathway, in H295R cells. Ang II promotes activation by phosphorylation of mTORC1/2 pathway proteins in a time-dependent manner. Mitochondrial pools of ribosomal protein S6, protein kinase B (Akt) in threonine 308, and serine 473 and Rictor are phosphorylated and activated. Glycogen synthase kinase type 3 (GSK3) is phosphorylated and inactivated in mitochondria, favoring mTORC1 activation. Epidermal growth factor, a classic mTORC1/2 activator, promoted unique activation kinetics of mTORC1/2 pathway, except for Akt phosphorylation. Here, we demonstrate that ACSL4 is necessary for mTORC1/2 effectors phosphorylation and H295R proliferation, triggered by Ang II. Ang II promotes activation of mitochondrial mTORC1/2 signaling proteins, through ACSL4, with a direct effect on adrenocortical cellular proliferation.

Angiotensin II stimulation promotes mitochondrial fusion as a novel mechanism involved in protein kinase compartmentalization and cholesterol transport in human adrenocortical cells.

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 protein (StAR). StAR is phosphorylated by mitochondrial ERK in a cAMP-dependent transduction pathway to achieve maximal steroid production. Mitochondria are highly dynamic organelles that undergo replication, mitophagy and morphology changes, all processes allowed by mitochondrial fusion and fission, known as mitochondrial dynamics. Mitofusin (Mfn) 1 and 2 are GTPases involved in the regulation of fusion, while dynamin-related protein 1 (Drp1) is the major regulator of mitochondrial fission. Despite the role of mitochondrial dynamics in neurological and endocrine disorders, little is known about fusion/fission in steroidogenic tissues. In this context, the present work aimed to study the role of angiotensin II (Ang II) in protein subcellular compartmentalization, mitochondrial dynamics and the involvement of this process in the regulation of aldosterone synthesis. We demonstrate here that Ang II stimulation promoted the recruitment and activation of PKCε, ERK and its upstream kinase MEK to the mitochondria, all of them essential for steroid synthesis. Moreover, Ang II prompted a shift from punctate to tubular/elongated (fusion) mitochondrial shape, in line with the observation of hormone-dependent upregulation of Mfn2 levels. Concomitantly, mitochondrial Drp1 was diminished, driving mitochondria toward fusion. Moreover, Mfn2 expression is required for StAR, ERK and MEK mitochondrial localization and ultimately for aldosterone synthesis. Collectively, this study provides fresh insights into the importance of hormonal regulation in mitochondrial dynamics as a novel mechanism involved in aldosterone production.

Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells.

Activation of ERK1/2 implies the phosphorylation of tyrosine (pTyr) and threonine (pThr) by MEK1/2; both reactions were thought to be cytoplasmic, promoting ERK to reach the nucleus where it activates several transcription factors. In addition, H2O2 concentrations are known to modulate ERK intracellular translocation, which impacts on cellular proliferation. In this context, the objective of this work was to study the sequence of ERK phosphorylation under two redox conditions and to analyze a putative mitochondrial contribution to this process, in LP07 murine lung cells. A time-course of H2O2 administration was used and ERK phosphorylation was analyzed in cytosol, mitochondria and nuclei. At 1µM H2O2, a proliferative redox stimulus, immunoblot revealed a fast and transient increase in cytosol pTyr and a sustained increase in mitochondrial pTyr content. The detection for pThr/pTyrERK (2pERK) showed in cytosol a marked increase at 5 minutes with a fast dephosphorylation after that time, for both H2O2 concentrations. However, at 50 µM H2O2, an anti-proliferative condition, 2pERK was gradually retained in mitochondria. Interestingly, these results were confirmed by in vivo experiments using mice treated with a highly oxidizing agent [H2O2]. By the use of two ERK2 mutant constructions, where Tyr and Thr were replaced by alanine, we confirmed that 2pERK relied almost completely on pThr183. Confocal microscopy confirmed ERK subcellular distribution dependence on the incidence of cytosolic pTyr and mitochondrial pThr at 1µM H2O2. This work shows for the first time, both in vitro and in vivo, an ERK cycle involving a cross-talk between cytosol and mitochondria phosphorylation events, which may play a significant role in cell cycle progression, proliferation or differentiation under two different redox conditions.

The role of mitochondrial fusion and StAR phosphorylation in the regulation of StAR activity and steroidogenesis.

The steroidogenic acute regulatory (StAR) protein regulates the rate-limiting step in steroidogenesis, i.e. the delivery of cholesterol from the outer (OMM) to the inner (IMM) mitochondrial membrane. StAR is a 37-kDa protein with an N-terminal mitochondrial targeting sequence that is cleaved off during mitochondrial import to yield 30-kDa intramitochondrial StAR. StAR acts exclusively on the OMM and its activity is proportional to how long it remains on the OMM. However, the precise fashion and the molecular mechanism in which StAR remains on the OMM have not been elucidated yet. In this work we will discuss the role of mitochondrial fusion and StAR phosphorylation by the extracellular signal-regulated kinases 1/2 (ERK1/2) as part of the mechanism that regulates StAR retention on the OMM and activity.