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 will be metabolized to pregnenolone. We demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) mediates the transport of cholesterol from the outer to the inner mitochondrial membrane and the subsequent steroid biosynthesis. It is our hypothesis that PBR functions as a cholesterol binding protein and channel, thus allowing this steroid precursor to get into the outer membrane and cross from the outer to the inner mitochondrial membrane through the intermembrane contact sites. Therefore, the structure/state and levels of this lipid channel protein will determine the amount of cholesterol available for testosterone synthesis and consequently testicular function and fertility. In the first aim, we will examine the structure/function relationship of PBR. We were able to reconstitute a functional recombinant PBR that binds cholesterol with nM affinity and identified in vitro conditions that mimic the hormone-induced changes in PBR. Molecular (deletion mutations and site-directed mutagenesis), functional (cholesterol & drug ligand binding, cholesterol transport), structural (electron microscopy & two dimensional crystallization), and computational (molecular modeling and simulations) will be undertaken in order to define the structure of the receptor and its function as a channel for cholesterol (lipophorin). The function of PBR will be also examined in the second aim in cell and animal models where PBR levels are decreased leading to decreased testosterone production. Peroxisome proliferators (PPs), which are extensively used in humans, exert anti-androgenic activity and trigger testicular atrophy in rodents. PP hypolipidemic drugs and phthalate ester plasticizers inhibit Leydig cell steroidogenesis and PBR expression in vitro and in vivo. Based on preliminary studies we propose that the effect of PPs on PBR expression might be due to PP-activated receptor (PPAR)-mediated indirect transrepression of AP-1/CRE activity and/or to the activation of a novel PP-activated transcription factor (PPAF) and/or to the inhibition of binding of SRY/SOX-like or Nkx-like proteins to SRY- and Nkx-binding sites residing within the PBR promotor. To ascertain the in vitro effects of PPs on PBR gene transcription, the effect of PPs will be examined in in vivo experiments where the PBR upstream region(s), defined in the in vitro studies, will be used to drive the expression of the eGFP reporter gene in transgenic mice. Based on the in vivo data, the transcriptional mechanisms described above will be investigated in detail. It is our goal to elucidate the mode of action and define the health hazard risk of PPs on testicular function.
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