Investigations into the phenotypic expression of endogenous benzodiazepine-like molecules in Leydig cells and implications for their role in steroid biosynthesis

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 358-380-Steroid Hormone Biosynthesis & Metabolism
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-371
Andrew Stephen Midzak*1, Zoe Brewster2 and Vassilios Papadopoulos2
1McGill University, Montreal, Canada, 2McGill University Health Centre, Montreal, QC, Canada
Steroid biosynthesis is performed by highly specialized cell types able to metabolize cholesterol to steroid hormones. These cells are defined by expression of the mitochondrial cytochrome P450 enzyme CYP11A1, which cleaves the aliphatic hydrocarbon tail of cholesterol to yield the steroid pregnenolone, the metabolic precursor of all other vertebrate steroids. The principal regulation of CYP11A1 is dependent on metabolic access to cholesterol, facilitated by the translocation of cholesterol across the double membranes of the mitochondria. This process is critically dependent upon the mitochondrial translocator protein (18kDa; TSPO), which was originally named the peripheral benzodiazepine receptor for its ability to bind psychoactive benzodiazepine (BZP) drugs outside of the central nervous system. The TSPO BZP binding site appears critical for steroidogenesis, as binding at this site by drugs or the endogenous protein diazepam binding inhibitor (DBI) stimulates cholesterol translocation into mitochondria and steroid synthesis, and inhibition of DBI expression reduces steroid synthesis. While DBI is an acknowledged protein ligand of TSPO, observations have indicated the presence in mammalian cells of drug-like molecules, including morphine alkaloids and benzodiazepines, raising the possibility of additional layers of complexity characterizing TSPO function and steroidogenesis. To investigate possibility of endogenous BZPs in steroidogenic cells, we probed the hormone-responsive MA-10 mouse tumor Leydig cells with antibodies specifically targeted against BZPs. The presence of BZPs was confirmed by immunocytochemical analysis of these cells;  these findings were not due to cross-reactivity of the antibodies used with protein epitopes, as immunoblot analysis of MA-10 cellular lysates did not present any signal. Interestingly, analysis of BZP levels in a panel of cell lines derived from a spectrum of steroidogenic and non-steroidogenic tissues indicated that BZP levels were highest in steroidogenic cells. Chronicstimulation of MA-10 cells for twenty-four hours with human chorionic gonadotropin (hCG) increased BZP levels over 1.5-fold, indicating that benzodiazepine production was hormonally regulated. In summary, this work provides novel evidence for the presence of hormonally regulated benzodiazepines in steroidogenic tissues, opening investigations into novel mechanisms of steroidogenic control.

Nothing to Disclose: ASM, ZB, VP

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Sources of Research Support: This work was supported bythe Canadian Institutes of Health Research MOP102647 (V.P.), and a Canada Research Chair in Biochemical Pharmacology (V.P.). A.M.was supported in part by a postdoctoral fellowship from the LeFonds de la Recherche du Québec-Santé. The Research Instituteof MUHC was supported by a Center grant from Le Fonds de larecherche du Québec-Santé.