MEHP INDUCES ALTERATION OF MITOCHONDRIAL FUNCTION AND INHIBITION OF STEROID BIOSYNTHESIS IN MA-10 MOUSE TUMOR LEYDIG CELLS

The first and rate-limiting step in the biosynthesis of steroid hormones is the transfer of cholesterol into mitochondria, which is facilitated by the steroidogenic acute regulatory (StAR) protein. Recent study of Leydig cell function has focused on the mechanisms regulating steroidogenesis; however, few investigations have examined the importance of mitochondria in this process. The purpose of this investigation was to determine which aspects of mitochondrial function are necessary for acute cAMP-stimulated Leydig cell steroidogenesis. MA-10 cells were treated with 8-bromoadenosine 3′,5′-cyclic monophosphate (cAMP) and different site-specific agents that disrupt mitochondrial function, and the effects on acute cAMP-stimulated progesterone synthesis, StAR mRNA and protein, mitochondrial membrane potential (Δψm), and ATP synthesis were determined. cAMP treatment of MA-10 cells resulted in significant increases in both cellular respiration and Δψm. Dissipating Δψm with carbonyl cyanide m-chlorophenyl hydrazone resulted in a profound reduction in progesterone synthesis, even in the presence of newly synthesized StAR protein. Preventing electron transport in mitochondria with antimycin A significantly reduced cellular ATP, potently inhibited steroidogenesis, and reduced StAR protein levels. Inhibiting mitochondrial ATP synthesis with oligomycin reduced cellular ATP, inhibited progesterone synthesis and StAR protein, but had no effect on Δψm. Disruption of intramitochondrial pH with nigericin significantly reduced progesterone production and StAR protein but had minimal effects on Δψm. 22(R)-hydroxycholesterol-stimulated progesterone synthesis was not inhibited by any of the mitochondrial reagents, indicating that neither P450 side-chain cleavage nor 3β-hydroxysteroid dehydrogenase activity was inhibited. These results indicate that Δψm, mitochondrial ATP synthesis, and mitochondrial pH are all required for acute steroid biosynthesis. These results suggest that mitochondria must be energized, polarized, and actively respiring to support Leydig cell steroidogenesis, and alterations in the state of mitochondria may be involved in regulating steroid biosynthesis.

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Synergistic Activation of Steroidogenic Acute Regulatory Protein Expression and Steroid Biosynthesis by Retinoids: Involvement of cAMP/PKA Signaling

Endocrinology ◽

10.1210/en.2013-1694 ◽

2014 ◽

Vol 155 (2) ◽

pp. 576-591 ◽

Cited By ~ 27

Author(s):

Pulak R. Manna ◽

Andrzej T. Slominski ◽

Steven R. King ◽

Cloyce L. Stetson ◽

Douglas M. Stocco

Keyword(s):

Retinoic Acid ◽

Leydig Cells ◽

Binding Protein ◽

Regulatory Protein ◽

Liver X Receptor ◽

Steroidogenic Acute Regulatory Protein ◽

Type I ◽

Steroid Biosynthesis ◽

Steroidogenic Acute Regulatory ◽

Star Gene

Both retinoic acid receptors (RARs) and retinoid X receptors (RXRs) mediate the action of retinoids that play important roles in reproductive development and function, as well as steroidogenesis. Regulation of steroid biosynthesis is principally mediated by the steroidogenic acute regulatory protein (StAR); however, the modes of action of retinoids in the regulation of steroidogenesis remain obscure. In this study we demonstrate that all-trans retinoic acid (atRA) enhances StAR expression, but not its phosphorylation (P-StAR), and progesterone production in MA-10 mouse Leydig cells. Activation of the protein kinase A (PKA) cascade, by dibutyrl-cAMP or type I/II PKA analogs, markedly increased retinoid-responsive StAR, P-StAR, and steroid levels. Targeted silencing of endogenous RARα and RXRα, with small interfering RNAs, resulted in decreases in 9-cis RA-stimulated StAR and progesterone levels. Truncation of and mutational alterations in the 5′-flanking region of the StAR gene demonstrated the importance of the −254/−1-bp region in retinoid responsiveness. An oligonucleotide probe encompassing an RXR/liver X receptor recognition motif, located within the −254/−1-bp region, specifically bound MA-10 nuclear proteins and in vitro transcribed/translated RXRα and RARα in EMSAs. Transcription of the StAR gene in response to atRA and dibutyrl-cAMP was influenced by several factors, its up-regulation being dependent on phosphorylation of cAMP response-element binding protein (CREB). Chromatin immunoprecipitation studies revealed the association of phosphorylation of CREB, CREB binding protein, RXRα, and RARα to the StAR promoter. Further studies elucidated that hormone-sensitive lipase plays an important role in atRA-mediated regulation of the steroidogenic response that involves liver X receptor signaling. These findings delineate the molecular events by which retinoids influence cAMP/PKA signaling and provide additional and novel insight into the regulation of StAR expression and steroidogenesis in mouse Leydig cells.

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Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

PLoS ONE ◽

10.1371/journal.pone.0022822 ◽

2011 ◽

Vol 6 (8) ◽

pp. e22822 ◽

Cited By ~ 16

Author(s):

Ana Fernanda Castillo ◽

Jinjiang Fan ◽

Vassilios Papadopoulos ◽

Ernesto J. Podestá

Keyword(s):

Leydig Cells ◽

Antisense Transcript ◽

Regulatory Protein ◽

Steroidogenic Acute Regulatory Protein ◽

Mouse Tumor ◽

Natural Antisense Transcript ◽

Steroidogenic Acute Regulatory

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Acetamiprid inhibits testosterone synthesis by affecting the mitochondrial function and cytoplasmic adenosine triphosphate production in rat Leydig cells†

Biology of Reproduction ◽

10.1093/biolre/iox007 ◽

2017 ◽

Vol 96 (4) ◽

pp. 936-936 ◽

Cited By ~ 9

Author(s):

Deying Kong ◽

Jidong Zhang ◽

Xiaohui Hou ◽

Shibin Zhang ◽

Jun Tan ◽

...

Keyword(s):

Adenosine Triphosphate ◽

Mitochondrial Function ◽

Leydig Cells ◽

Adenosine Triphosphate Production ◽

Testosterone Synthesis

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Role of Constitutive STAR in Leydig Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22042021 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2021 ◽

Cited By ~ 1

Author(s):

Melanie Galano ◽

Yuchang Li ◽

Lu Li ◽

Chantal Sottas ◽

Vassilios Papadopoulos

Keyword(s):

Leydig Cells ◽

Lipid Droplets ◽

Regulatory Protein ◽

Translocator Protein ◽

Mouse Tumor ◽

Liquid Chromatography Mass Spectrometry ◽

Wild Type ◽

Cell Functions ◽

Steroidogenic Acute Regulatory

Leydig cells contain significant amounts of constitutively produced steroidogenic acute regulatory protein (STAR; STARD1). Hormone-induced STAR plays an essential role in inducing the transfer of cholesterol into the mitochondria for hormone-dependent steroidogenesis. STAR acts at the outer mitochondrial membrane, where it interacts with a protein complex, which includes the translocator protein (TSPO). Mutations in STAR cause lipoid congenital adrenal hyperplasia (lipoid CAH), a disorder characterized by severe defects in adrenal and gonadal steroid production; in Leydig cells, the defects are seen mainly after the onset of hormone-dependent androgen formation. The function of constitutive STAR in Leydig cells is unknown. We generated STAR knockout (KO) MA-10 mouse tumor Leydig cells and showed that STAR KO cells failed to form progesterone in response to dibutyryl-cAMP and to TSPO drug ligands, but not to 22(R)-hydroxycholesterol, which is a membrane-permeable intermediate of the CYP11A1 reaction. Electron microscopy of STAR KO cells revealed that the number and size of lipid droplets were similar to those in wild-type (WT) MA-10 cells. However, the density of lipid droplets in STAR KO cells was drastically different than that seen in WT cells. We isolated the lipid droplets and analyzed their content by liquid chromatography–mass spectrometry. There was a significant increase in cholesteryl ester and phosphatidylcholine content in STAR KO cell lipid droplets, but the most abundant increase was in the amount of diacylglycerol (DAG); DAG 38:1 was the predominantly affected species. Lastly, we identified genes involved in DAG signaling and lipid metabolism which were differentially expressed between WT MA-10 and STAR KO cells. These results suggest that constitutive STAR in Leydig cells is involved in DAG accumulation in lipid droplets, in addition to cholesterol transport. The former event may affect cell functions mediated by DAG signaling.

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Dynamic Remodeling of Membranes and Their Lipids during Acute Hormone-Induced Steroidogenesis in MA-10 Mouse Leydig Tumor Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22052554 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2554

Author(s):

Sathvika Venugopal ◽

Melanie Galano ◽

Rachel Chan ◽

Esha Sanyal ◽

Leeyah Issop ◽

...

Keyword(s):

Lipid Composition ◽

Leydig Cells ◽

Hormone Treatment ◽

Mouse Tumor ◽

Hormone Activity ◽

Lipid Trafficking ◽

Cellular Processes ◽

Leydig Tumor Cells ◽

Induced Changes ◽

Camp Stimulation

Lipids play essential roles in numerous cellular processes, including membrane remodeling, signal transduction, the modulation of hormone activity, and steroidogenesis. We chose steroidogenic MA-10 mouse tumor Leydig cells to investigate subcellular lipid localization during steroidogenesis. Electron microscopy showed that cAMP stimulation increased associations between the plasma membrane (PM) and the endoplasmic reticulum (ER) and between the ER and mitochondria. cAMP stimulation also increased the movement of cholesterol from the PM compared to untreated cells, which was partially inhibited when ATPase family AAA-domain containing protein 3 A (ATAD3A), which functions in ER and mitochondria interactions, was knocked down. Mitochondria, ER, cytoplasm, PM, PM-associated membranes (PAMs), and mitochondria-associated membranes (MAMs) were isolated from control and hormone-stimulated cells. Lipidomic analyses revealed that each isolated compartment had a unique lipid composition, and the induction of steroidogenesis caused the significant remodeling of its lipidome. cAMP-induced changes in lipid composition included an increase in phosphatidylserine and cardiolipin levels in PAM and PM compartments, respectively; an increase in phosphatidylinositol in the ER, mitochondria, and MAMs; and a reorganization of phosphatidic acid, cholesterol ester, ceramide, and phosphatidylethanolamine. Abundant lipids, such as phosphatidylcholine, were not affected by hormone treatment. Our data suggested that PM–ER–mitochondria tethering may be involved in lipid trafficking between organelles and indicated that hormone-induced acute steroid production involves extensive organelle remodeling.

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