Genomic and Proteomic Approaches to Identify Mechanism of Regulation of Cytochrome P450 3A Enzymes

Abstract

BackgroundCytochrome P450 (CYP) 3A enzymes are the most abundant enzymes in human liver, and are involved in the metabolism of majority of the drugs. Changes in expression of these enzymes are associated with drug‐drug interactions, failure of therapy and adverse drug reactions. Therefore, in order to explain the variability in efficacy and toxicity due to drug‐drug interactions of substrate drugs, it is crucial to understand the complete mechanism of CYP3A regulation. To date, basal transcription factors (TFs), nuclear receptors (PXR, CAR etc.), kinases and phosphatases are known play a role. However, the exact mechanisms of epigenetic changes, involvement of miRNAs etc. are unknown. Two major effectors that modulate CYP3A in opposite direction are known. The first one is PXR (pregnane X receptor), a nuclear receptor that binds directly to CYP3A promoter and induces it; the second one is represented by LPS (liposaccharide), which induces cytokines that suppress CYP3A levels.ObjectiveTo enhance our understanding of CYP3A regulation by elucidating the competing mechanisms of its two major modulators, PXR and LPS‐induced cytokines.MethodsC57BL/6 mice were pre‐treated with corn oil (CO) or pregnenolone‐16alpha‐carbonitrile [PCN (mPXR activator)], followed by saline (Sal) or LPS. Hepatic gene expression was evaluated using Illumina MouseWG‐6 v2.0 expression bead chip. Data was normalized using the Lumi package and analyzed using the R statistical system. Analysis of transcription factors in up‐ or down‐ regulated genes was performed using the hypergeometric distribution (p<0.05). Global enrichment of transcription factor targets was evaluated via Gene Set Enrichment Analysis (GSEA). Protein lysates were prepared at the CPRIT Cancer Proteomics and Metabolomics center, Houston, TX for RPPA analysis.ResultsFive hundred and sixty‐two TFs were significantly downregulated with PCN and 472 TFs were significantly downregulated with LPS as compared to CO/Sal. In PCN/LPS group, 536 TFs were differentially expressed (UR: 35, DR: 501) as compared to CO/Sal. Using TRANSFAC based motif analysis, we identified TFs which are altered by PCN or LPS and might bind to CYP3A4 promotor sequence such as FOXO4, LEF1, PEA3, HNF3 etc. LPS and PCN lead to opposite effects on targets of epigenetic regulators such as HDAC1 and HDAC3, and similar effects on EZH2 targets. HDAC4 protein is also downregulated with combined treatment of PCN and LPS in RPPA analysis. Apart from that, LPS and PCN lead to opposite regulation of miR‐150 targets, and similar and strong regulation was seen with miR‐124, miR‐200 family, and miR‐181 targets. PXR also attenuated the pro‐inflammatory pathways induced by LPS, and this anti‐inflammatory effect of PXR may have an indirect effect on CYP3A regulation during inflammation.ConclusionsNovel regulators that are involved in both upregulation and downregulation of CYP3A were identified. Ultimately, identification of these regulators will lead to the development of targeted strategies to treat disorders due to altered drug metabolism.