TARGETED DISRUPTION OF THE PERIPHERAL-TYPE BENZODIAZEPINE RECEPTOR GENE INHIBITS STEROIDOGENESIS IN THE R2C LEYDIG TUMOR-CELL LINE

To evaluate the role of the mitochondrial peripheral-type benzodiazepi ne receptor (PER) in steroidogenesis, we developed a molecular approac h based on the disruption of the PER gene, by homologous recombination , in the constitutive steroid producing R2C rat Leydig tumor cell line , Inactivation of one allele of the PER gene resulted in the suppressi on of PER mRNA and ligand binding expression, Immunoblot and electron microscopic immunogold labeling analyses confirmed the absence of the 18-kDa PER protein in the selected clone, Although mitochondria from t he PER-negative cells contained high levels of the constitutively expr essed 30-kDa steroidogenic activity regulator protein, these cells pro duced minimal amounts of steroids compared with normal cells (5%). Mor eover, mitochondria from PER-negative cells failed to produce pregneno lone when supplied with exogenous cholesterol, Addition of the hydroso luble cholesterol derivative, 22R-hydroxycholesterol, increased steroi d production by the PBR-negative R2C cells, indicating that the choles terol transport mechanism was impaired. Stable transfection of the PER -negative R2C Leydig cells with a vector containing the PER cDNA resul ted in the recovery of the steroidogenic function of the cells. These data demonstrate that PER is an indispensable element of the steroidog enic machinery, where it mediates the delivery of the substrate choles terol to the inner mitochondrial side chain cleavage cytochrome P-450.