MRP4 in Drug Sensitivity and Physiology

St. Jude Children'S Research Hospital, Memphis TN

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Abstract

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 the cell 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 in pre-puberty Mrp4-/- mice had dramatically reduced levels of systemic testosterone and impaired gametogenesis. We reasoned 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. Given that chemotherapeutic agents (e.g., imatinib, ganciclovir) that inhibit Mrp4 cause acute reduction in testosterone and reproductive effects, we hypothesized that drugs and food components (polyphenols) that inhibit Mrp4 produce these effects by interfering with Mrp4 transport, which leads to elevated PG and impaired testosterone biosynthesis. In post-puberty (adult) Mrp4-/- mice the level of testosterone and gamete production is restored. 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 encodes a protein known to bind PG (FABP) is upregulated). These findings raise the intriguing possibility that, in the absence of Mrp4, alternate pathways are activated in the testes to restore testosterone biosynthesis. The objectives of the proposed studies are to: i) explore the gene expression patterns underlying how post-puberty Mrp4-/- mice achieve normal testosterone levels; ii) determine if Mrp4-/- vs. Mrp4+/+ have altered testes maturation in pre- to post puberty iii) evaluate the role of fatty-acid binding protein 4, 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 because xenobiotic and drug interference with this transport system may account for reduced testosterone levels in humans.