J Biol Chem, Vol. 273, Issue 10, 5478-5483, March 6, 1998

Effects of Peripheral-type Benzodiazepine Receptor Antisense Knockout on MA-10 Leydig Cell Proliferation and Steroidogenesis

Ela Kelly-Hershkovitz, Ronit Weizman^§¶, Ilana Spanier, Svetlana Leschiner, Michal Lahav, Gary Weisinger^**, and Moshe Gavish

From the Departments of  Pharmacology,  Endocrinology, and ^** Clinical Biochemistry, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 31096 Haifa, Israel, the  Rappaport Family Institute for Research in the Medical Sciences, 31096 Haifa, Israel, the ^§ Tel Aviv Community Mental Health Center, 97197 Tel Aviv, Israel, and the ^¶ Sackler Faculty of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel

The peripheral-type benzodiazepine receptor (PBR) is not only widely expressed throughout the body, but it is also genetically conserved from bacteria to humans. Many functions have been attributed to it, but its primary role remains a puzzle. In the current study, we stably transfected cultures of MA-10 Leydig cells with either control or 18-kDa PBR antisense knockout plasmids. The antisense knockout vector was driven by the human enkephalin promoter, which contains two cAMP response elements, such that cAMP treatment of transfected cells could superinduce 18-kDa PBR antisense RNA transcription and, hence, down-regulate endogenous 18-kDa PBR mRNA levels. Control and knockout MA-10 cell lines were then compared at the level of receptor binding, thymidine incorporation, and steroid biosynthesis. Eighteen-kilodalton PBR knockout reduced the maximal binding capacity of tritium-labeled PBR ligands, and the affinity of receptors to the ligands remained unaltered. Additionally, 24-h accumulation of progesterone was lower in the knockout cells. Exposure of the two cell types to 8-bromo-cAMP resulted in a robust increase in steroid production. However, a complex pattern of steroid accumulation was observed, in which further progestin metabolism was indicated. The later decline in accumulated progesterone as well as the synthesis of androstenedione were different in the two cell types. At the level of cell proliferation, reduction of 18-kDa PBR mRNA showed no effect. Thus, we conclude that the 18-kDa PBR may have a more important role in steroidogenesis than in proliferation in this Leydig cell line.