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.
From the Research Institute of the McGill University Health Centre and the Department of Medicine, McGill University, Montreal H4A 3J1, Canada.
Sathvika
S
Venugopal
From the Research Institute of the McGill University Health Centre and the Department of Medicine, McGill University, Montreal H4A 3J1, Canada.
Daniel Benjamin
DB
Martinez-Arguelles
From the Research Institute of the McGill University Health Centre and the Department of Medicine, McGill University, Montreal H4A 3J1, Canada.
Seimia
S
Chebbi
the Lipid Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan.|INSERM U1060, Université Lyon 1, INSA Lyon, 69621 Villeurbanne, France.
Françoise
F
Hullin-Matsuda
the Lipid Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan.|INSERM U1060, Université Lyon 1, INSA Lyon, 69621 Villeurbanne, France.|UMR 7213 CNRS, University of Strasbourg, 67401 Illkirch, France, and.
Toshihide
T
Kobayashi
From the Research Institute of the McGill University Health Centre and the Department of Medicine, McGill University, Montreal H4A 3J1, Canada, vpapadop@usc.edu.|the Departments of Pharmacology and Therapeutics and.|Biochemistry, McGill University, Montreal H3G 1Y6, Canada.
Vassilios
V
Papadopoulos
D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics; D001693:Biological Transport, Active / Q000187:drug effects / Q000502:physiology; D003994:Bucladesine / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000235:genetics; D006888:Hydroxycholesterols / Q000378:metabolism*; D007985:Leydig Cells / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D011284:Pregnenolone / Q000378:metabolism*; D051381:Rats
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D006888:Hydroxycholesterols; C003585:22-hydroxycholesterol; D003994:Bucladesine; C020452:Clostridium perfringens theta-toxin; D011284:Pregnenolone
cholesterol; lipid transport; mitochondria; plasma membrane; steroidogenesis; testis