Several classes of environmental and therapeutic chemicals are recognized for their capacity to induce expression of batteries of mammalian genes. For substances undergoing biotransformation, it is likely that an individual's capacity for tolerance to chemical injury, or metabolic clearance, will be determined in part by prior exposures to inducing agents, and the magnitude of inductive response mounted within potential target cells and tissues. Phenobarbital (pB) is a prototype agent for a variety of xenobiotics that exhibit profound inductive effects on a variety of biotransformation enzyme genes, including the cytochrome P450 monooxygenases (P45Os). The highly homologous (approximately 97% identical) rat P45Os, CYP2B1 and CYP2B2, have been studied extensively with respect to PB modulatory effects. Each of these genes are markedly activated in the liver by PB treatment and exhibit strict tissue and developmental-specific programs. Although it was demonstrated previously that PB stimulates these genes at the transcriptional level, relatively little is known regarding the underlying molecular mechanisms governing the PB induction response or cell-specific expression of P450 genes. The overall hypothesis being tested with the proposed and anticipated future studies is that cooperative interactions between several arrayed nuclear regulatory factors, including the CAAT enhancer binding proteins (C/EBP- family), hepatic nuclear factor proteins (HNF-family), and the Ets- protooncogene products, dictate the overall regulatory potential of PB- inducible genes. The current application will focus on the region from - 800 to -2500 of the CYP2B1 and 2B2 genes, shown to contain core recognition sequences and possess protein interactions within a clustered array of regulatory sites. Transgenic mouse models, as well as a battery of in vitro methods, such as gel shift and gel supershift assays, DNA transfection of cultured hepatocytes, laser-activated fluorescence cytometry, and nucleosomal structure determinations, will be deployed to thoroughly test individual aspects of this hypothesis. Preliminary data collected in the laboratory indicates that PB may exert its nuclear effects on hepatocytes through interaction with established signal transduction pathways. Thus, a specific hypothesis that will be tested with the proposed research is that C/EBP- and Ets-proteins modulate PB responsiveness of the liver-selective CYP2B1/2 genes via the multifunctional Ca2+-calmodulin dependent protein kinase II, and the mitogen-activated protein (MAP) kinase signal transduction pathways. The results generated from the proposed studies should substantially enhance our understanding of the molecular events responsible for PB induction, and pave the way for future experiments to ascertain the genetics, and toxicological and pharmacological ramifications of this response in human populations.
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