Abstract
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.
Highlights
Steroidogenesis begins with the transport of cholesterol from intracellular stores into the mitochondria
These results suggest that constitutive steroidogenic acute regulatory protein (STAR) in Leydig cells is involved in DAG
In addition to characterizing the responsiveness of our MA-10 cells, these data show that, in Leydig cells, hormonal stimulation parallels an increase in STAR expression and in steroid production, but that STAR expression and low levels of steroid production are present under basal conditions
Summary
Steroidogenesis begins with the transport of cholesterol from intracellular stores into the mitochondria. This is the hormone-sensitive and rate-limiting step. The steroidogenic acute regulatory protein (STAR; STARD1) plays a critical role in cholesterol transport to the mitochondria for steroidogenesis. In Leydig cells, STAR is constitutively expressed under basal conditions, independent of hormonal stimulation, which parallels the formation of low steroid levels [1]. Hormonal stimulation causes a rapid induction of STAR, which is coupled to an increase in cholesterol transfer to mitochondria and increased steroid formation [2]. It has been suggested that STAR along with other cytosolic and outer mitochondrial membrane proteins such as the translocator protein (TSPO; 18 kDa) form the transduceosome complex upon hormonal stimulation to move cholesterol to the inner mitochondrial membrane, where the CYP11A1 enzyme resides and converts cholesterol into pregnenolone [3]
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