Studies over the last funding period were directed to elucidating the function and molecular regulation of the ABC transporter, Mrp4. The current proposal represents an evolution of this work, based upon recent studies from this laboratory using the Mrp4-/- mouse we developed, and using our anti-Mrp4 antibodies, we determined that Mrp4 is highly expressed in both human and murine Leydig cells in the testes. As the Leydig cells are responsible for supplying systemic testosterone we evaluated the Mrp4-/- mice for serum levels of testosterone despite the fact that testosterone is not a substrate for Mrp4. We found that as Mrp4 gene copy decreases there are corresponding reductions in serum testosterone that are directly associated with reduced expression of Mrp4 in Leydig cells. We hypothesized that elevated prostaglandins (PG) (a known Mrp4 substrate) might be responsible for this effect because PG inhibit testosterone synthesis. The enzyme responsible for formation of PG, COX-2 is expressed in Leydig cells and COX-2 inhibition increases systemic testosterone levels. In adult Mrp4-/- mice the level of testosterone is restored and PG levels are reduce in the Mrp4-/-. These findings led us to hypothesize that, in the testes, compensatory pathways re-activated testosterone synthesis in the absence of Mrp4. Our preliminary data shows, in testes, upregulation of genes directly linked to PG metabolism (although COX-2 is unchanged, however one gene encoding a protein known to bind PG (FABP4) is upregulated). These findings in this model system are of human relevance because chemotherapeutic agents (e.g., imatinib, ganciclovir) and food components (polyphenols) that inhibit Mrp4 cause acute reduction in testosterone and reproductive effects. We hypothesize they produce these effects by interfering with Mrp4 transport. Our in vivo model will allow us to directly test how they impair testosterone biosynthesis. These findings raise the intriguing possibility that, in the absence of Mrp4, alternate pathways can be activated in Leydig cells to restore testosterone biosynthesis. The objectives of the proposed studies are to: i) to elucidate how post-puberty Mrp4-/- mice achieve normal testosterone levels;ii) determine if Mrp4-/- vs. Mrp4 +/+ have altered differentiation status;iii) evaluate the role of fatty-acid binding protein 4 (FABP4), a protein known to bind PG, in regulating testosterone biosynthesis in model systems;iv) determine if chemotherapeutic agents and xenobiotics known to alter testosterone levels do so by interfering with Mrp4 function. The proposed studies are supported by preliminary data from the Mrp4-/- and Mrp4+/+ mice and are of direct human relevance.
A number of drugs and xenobiotics can alter testosterone levels leading to untoward effects. We have used a genetic model system to identify a novel transport system that regulates testosterone levels. Our studies are of direct human relevance because xenobiotic and drug interference with this transport system may account for reduced testosterone levels in humans. These studies may lead to therapeutic approaches to prevent this unfortunate side effect.
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