Identification of Sec23ip, Part of 14-3-3γ Protein Network, as a Regulator of Acute Steroidogenesis in MA-10 Leydig Cells.

Testosterone production occurs in the Leydig cells of the testes and is essential for virilization, development, reproduction, and quality of life. Although the steroidogenic proteins involved in cholesterol conversion to testosterone (T) are well characterized, the causes of reduced T during fetal, neonatal, and adult life remain uncertain. It is well established that normal cellular function is achieved through fine-tuning of multiple rather than single protein networks. Our objective was to use mass spectrometry (MS)-based proteomics to identify which cellular pathways, other than the steroidogenic machinery, influence testosterone production in MA-10 mouse tumor Leydig cells. The 14-3-3 family of scaffolds mediate protein-protein interactions facilitating the crosstalk between protein networks. We previously showed that in MA-10 cells, 14-3-3γ is a critical regulator of steroidogenesis. Therefore, identifying proteins that interact with 14-3-3γ during steroidogenesis could provide clues into the other networks involved. Using liquid chromatography (LC)-MS, we identified 688 proteins that interact with 14-3-3γ and thus potentially impact MA-10 cell steroidogenesis. The identified proteins belong to multiple protein networks, including endoplasmic reticulum-Golgi cargo sorting and vesicle biogenesis, micro ribonucleic acid-induced gene silencing, inflammation, and vesicle trafficking, to name a few. We found that silencing one of the candidates, Sec23ip, a protein known to be involved in vesicle trafficking, resulted in decreased steroidogenesis. We further showed that in Sec23ip-silenced MA-10 cells, cholesterol mobilization from the cytoplasmic membrane to mitochondria is impaired. Taken together these data suggest that Sec23ip is involved in cholesterol trafficking to supply cholesterol for acute steroidogenesis through its interactions with 14-3-3γ.

Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells.

Hormone-sensitive acute steroid biosynthesis requires trafficking of cholesterol from intracellular sources to the inner mitochondrial membrane. The precise location of the intracellular cholesterol and its transport mechanism are uncertain. Perfringolysin O, produced by Clostridium perfringens, binds cholesterol. Its fourth domain (D4) retains cholesterol-binding properties but not cytotoxicity. We transfected steroidogenic MA-10 cells of mouse Leydig cell tumors with the mCherry-D4 plasmid. Tagged D4 with fluorescent proteins enabled us to track cholesterol. The staining was primarily localized to the inner leaflet of the plasma membrane and was partially released upon treatment with dibutyryl-cAMP (Bt2cAMP), a cAMP analog. Inhibitors of cholesterol import into mitochondria blocked steroidogenesis and prevented release of D4 (and presumably cholesterol) from the plasma membrane. We conclude that the bulk of the steroidogenic pool of cholesterol, mobilized by Bt2cAMP for acute steroidogenesis, originates from the plasma membrane. Treatment of the cells with steroid metabolites, 22(R)-hydroxycholesterol and pregnenolone, also reduced D4 release from the plasma membrane, perhaps evidence for a feedback effect of elevated steroid formation on cholesterol release. Interestingly, D4 staining was localized to endosomes during Bt2cAMP stimulation suggesting that these organelles are on the route of cholesterol trafficking from the plasma membrane to mitochondria. Finally, D4 was expressed in primary rat Leydig cells with a lentivirus and was released from the plasma membrane following Bt2cAMP treatment. We conclude that the plasma membrane is the source of cholesterol for steroidogenesis in these cells as well as in MA-10 cells.