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

Abstract

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.

Current status and future perspectives: TSPO in steroid neuroendocrinology

“…Answer: The aforementioned study on TSPO knockdown in MA-10 Leydig cells did not observe differences in acute production of PROG (Kelly-Hershkovitz et al 1998). They observed that in TSPO antisense 'knockout' cells, PROG levels decreased by about 20% and only at a late time point (24 h), leading to an interpretation that TSPO may play a role in PROG metabolism rather than synthesis (Kelly-Hershkovitz et al 1998).…”

“…They observed that in TSPO antisense 'knockout' cells, PROG levels decreased by about 20% and only at a late time point (24 h), leading to an interpretation that TSPO may play a role in PROG metabolism rather than synthesis (Kelly-Hershkovitz et al 1998). They concluded that 'further studies are needed to confirm the involvement of the 18-kDa PBR subunit in MA-10 Leydig cell steroid biosynthesis' (Kelly-Hershkovitz et al 1998).…”

“…They observed that in TSPO antisense 'knockout' cells, PROG levels decreased by about 20% and only at a late time point (24 h), leading to an interpretation that TSPO may play a role in PROG metabolism rather than synthesis (Kelly-Hershkovitz et al 1998). They concluded that 'further studies are needed to confirm the involvement of the 18-kDa PBR subunit in MA-10 Leydig cell steroid biosynthesis' (Kelly-Hershkovitz et al 1998). This study was perhaps the very first indication that the assumptions underlying TSPO function may be incorrect, but there 231:1 was a key limitation, in that knockdown estimated by PK11195 or Ro5-4864 binding was only at 50% (KellyHershkovitz et al 1998).…”

“…A study performed in 1998 that examined a stable knockdown of gene expression to decrease TSPO protein levels in clones of MA-10 Leydig cells (Kelly-Hershkovitz et al 1998) has often been cited to indicate that TSPO was involved in the steroidogenic machinery. Advances in research of STAR mechanism of action resulted in the development of OMM TOM20-STAR fusion constructs that showed maximal hormone production (Bose et al 2002).…”

Mitochondrial regulation of mineralocorticoid biosynthesis by calcium and the StAR protein

“…While it was believed initially that transmembrane cholesterol transfer could be facilitated during the importation of StAR mediated by its N-terminal addressing sequence, the subsequent discovery that all the mutations that inactivate StAR are located at its C-terminal end (61) and that the biologically active domain of StAR resides in its C-terminus (68), strongly suggested that additional components(s) participate in StAR action. The peripheral-type benzodiazepine receptor has been proposed as one such partner (69,70).…”