The pregnane X receptor (PXR) is recognized as a master regulator on the expression of chemical elimina- tion genes such as cytochrome P450 3A4 (CYP3A4), the most abundant CYP enzyme that is responsible for the metabolism of more than 50% of therapeutic agents. Studies on the modulators for PXR signaling have linked DEC transcription factors to cytokine-suppressed expression of PXR and CYP3A. Proinflammatory :ytokines (e.g., IL-6) markedly increase the levels of DEC1 and DEC2, and simultaneously suppress the expression of PXR and CYP3A. Co-transfection of DECs effectively represses PXR and CYP3A promoters. Studies on the genomic basis for CYP3A4 transcription have established that PXR transactivates the CYP3A4 promoter through two regions: the proximal and distal regions. PXR is physically present in both regions through a PXR element, however, deletion of the distal region no longer responds to PXR. The proposed studies are designed to test the hypotheses that the distal and proximal regions coordinately support PXR-directed displacement/recruitment of corepressor and coactivator, and that PXR and CYP3A genes are sequence-specific targets of DEC transcription factors.
The specific aims of the proposed studies are: (1) to define the differential roles of the distal and proximal regions in PXR-directedtranscription, and (2) to elucidate the mechanisms on DECs-repressed expression of PXR and CYP3A. To determine the region- specific recruitment of co-regulators, ChIP will be performed for PXR, corepressor SMRT and coactivator SRC-1 (found to regulate PXR-directed transcription). The important residues in PXR to mediate the interac- tions will be established. To elucidate the molecular basis for .DECs-repressed expression of PXR and CYP3A, lentiviral transduction will be performed to up-regulate or knockdown DECs, and the effect on PXR and CYP3A expression will be determined. A set of experiments will be conducted to locate in the PXR and CYP3A promoters DMA sequences that support DECs-repression. PXR-directed induction and cytokines- mediated suppression of drug-metabolizing enzymes are two major determinants on the elimination of chemicals. Therefore, these studies will contribute significantly to our basic understanding of how PXR, CYP3A and DECs, as a group of structurally distinct but functionally related proteins, are involved in pharma- cologic determination and xenobiotic detoxication/bioactivation. Lay Relevance: Two or more drugs concurrently administered may interfere with each other. Bacterial/viral infection lowers the capacity of drug-elimination. Results from the proposed studies will provide important information on how to prevent drug incompatibility and adjust dosage during infection.
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