Comprehension of the molecular systems underlying the control of steroid hormone biosynthesis is essential for the study and treatment of a multitude of physiological disorders. Trophic hormones acutely stimulate steroid production by acting on the rate-determining step of steroidogenesis, the transport of the substrate, cholesterol, from intracellular stores to the inner mitochondrial membrane. There, cholesterol win be metabolized by the cytochrome P-450 side chain cleavage (P-450scc). We identified an intrinsic outer mitochondrial membrane protein participating in cholesterol transport. This protein is known as the peripheral-type benzodiazepine receptor(s) (PBR). We then identified the polypeptide diazepam binding inhibitor (DBI), also called endozepine, as the endogenous ligand for the receptor. Although we and others have now established that PBR activation by drug ligands or DBI leads to increased cholesterol transport in all steroidogenic tissues, direct evidence that PBR and/or DBI mediate the hormone-induced stimulation of cholesterol delivery to P-450scc is missing. Our goal is to understand the role of PBR and DBI in the trophic hormone stimulation of Leydig cell steroidogenesis.
Specific aims : We propose: 1) to investigate the hormonal regulation of PBR, 2) to examine the structural/functional organization of the receptor, 3) to investigate the role of DBI in the steroidogenic process, and 4) to examine the function of PBR and DBI in the context of the whole animal using transgenic mouse technology. Our model systems are the MA-10 hormone-responsive, Leydig cell line, the R2C Leydig cell line expressing constitutive steroidogenesis, and purified rat Leydig cells. The effect of LH/hCG on PBR, the mechanism(s) by which LH acts, as well as the mediator(s) of its action will be examined. We have cloned, expressed, and purified recombinant PBR and we demonstrated that the Leydig cell receptor is a multimeric complex of the 18,000 PBR protein and the voltage-dependent anion channel (VDAC). The characteristics of the PBR- VDAC association and its role in Leydig cell steroidogenesis will be examined. Purification and characterization of native PBR and its associated proteins will be undertaken. The role of the 18,000 PBR protein in steroidogenesis will be evaluated using gene targeting to introduce a null mutation in the PBR gene and examine its effect on hormone-stimulated steroid biosynthesis. The role of DBI in LH-stimulated steroidogenesis win be examined by manipulating the cellular levels of DBI. Moreover, the role of DBI as an activator of mitochondrial steroidogenesis acting via PBR and/or as a direct activator of P-450scc win be investigated. Transgenic mice overexpressing PBR and DBI win be also created in order to define the role of these proteins in physiologic and pathologic processes. We believe that these studies will uncover novel aspects concerning the molecular mechanisms which mediate hormone- regulated steroidogenesis.
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