Direct Transcriptional Regulation of Human Hepatic Cytochrome P450 3A4 (CYP3A4) by Peroxisome Proliferator–Activated Receptor Alpha (PPARα)

Abstract

The nuclear receptor peroxisome proliferator-activated receptor (PPAR)a is known primarily as a regulator of fatty acid metabolism, energy balance, and inflammation, but evidence suggests a wider role in regulating the biotransformation of drugs and other lipophilic chemicals. We investigated whether PPARa directly regulates the transcription of cytochrome P450 3A4, the major human drug-metabolizing enzyme. Using chromatin immunoprecipitation in human primary hepatocytes as well as electrophoretic mobility shift and luciferase reporter-gene assays, we identified three functional PPARa-binding regions (PBR-I, -II, and -III) within ∼12 kb of the CYP3A4 upstream sequence. Furthermore, a humanized CYP3A4/3A7 mouse model showed in vivo induction of CYP3A4 mRNA and protein by [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (WY14,643) in liver but not in intestine, whereas hepatic occupancy of PBRs by PPARa was ligand independent. Using lentiviral gene knock-down and treatment with WY14,643 in primary human hepatocytes, PPARa was further shown to affect the expression of a distinct set of CYPs, including 1A1, 1A2, 2B6, 2C8, 3A4, and 7A1, but not 2C9, 2C19, 2D6, or 2E1. Interestingly, the common phospholipid 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (16:0/18:1-PC), previously proposed to reflect nutritional status and shown to be a specific endogenous ligand of PPARa, induced CYP3A4 (up to 4-fold) and other biotransformation genes in hepatocytes with similar selectivity and potency as WY14,643. These data establish PPARa as a direct transcriptional regulator of hepatic CYP3A4. This finding warrants investigation of both known and newly developed PPARa-targeted drugs for their drug-drug interaction potential. Furthermore, our data suggest that nutritional status can influence drug biotransformation capacity via endogenous phospholipid signaling.

Activating and Inhibitory Functions of WNT/β-Catenin in the Induction of Cytochromes P450 by Nuclear Receptors in HepaRG Cells

“…Transcriptional activation of other drug biotransformation genes by PPARa has already been reported for the fatty acid v-hydroxylase, CYP4A11 (Johnson et al, 2002;Wanders et al, 2011), several UDP-glucuronosyltransferases (Barbier et al, 2003;Senekeo-Effenberger et al, 2007), and some drug transporters (Cheng et al, 2005;Moffit et al, 2006). Our comparison of ligand-induced gene expression changes mediated by PPARa and the two prototypic xenosensors PXR and CAR further emphasizes the similar target profiles of these three nuclear receptors with respect to the regulation of drug biotransformation genes in the liver (Thomas et al, 2013). Thus, PPARa is a relatively novel player in the regulation of drugmetabolizing enzymes, and PPARa activity in linking regulatory pathways of intermediary and xenobiotic metabolism warrants further study.…”

“…This nuclear receptor acts as a lipid sensor to control the expression of gene networks involved in lipid and energy homeostasis, adipocyte differentiation, and inflammatory responses (Lalloyer and Staels, 2010;Wahli and Michalik, 2012). We recently demonstrated that PPARa directly regulates the transcription of CYP3A4 (Klein et al, 2012;Thomas et al, 2013). Transcriptional activation of other drug biotransformation genes by PPARa has already been reported for the fatty acid v-hydroxylase, CYP4A11 (Johnson et al, 2002;Wanders et al, 2011), several UDP-glucuronosyltransferases (Barbier et al, 2003;Senekeo-Effenberger et al, 2007), and some drug transporters (Cheng et al, 2005;Moffit et al, 2006).…”

“…Lipid-and xenobiotic-sensing nuclear receptors pregnane X receptor (PXR, NR1I2), constitutive androstane receptor (CAR, NR1I3), and aryl hydrocarbon receptor (AhR), together with the liverenriched transcription factor peroxisome proliferator-activated receptor (PPAR) a (NR1C1) form networks of transcription factors that coordinately regulate hepatic expression of the majority of drug-metabolizing P450s, phase II enzymes, and transporters in response to xenobiotic exposure (Handschin and Meyer, 2005;Pascussi et al, 2008;Pelkonen et al, 2008;Thomas et al, 2013). Recent research elucidated extensive crosstalk between these receptors and other nuclear receptors and transcription factors, linking xenobiotic metabolism to the homeostasis of lipids, bile acids, glucose, and other endogenous processes (Moreau et al, 2008;Gao and Xie, 2012).…”

No Activation of Human Pregnane X Receptor by Hyperforin-Related Phloroglucinols

“…The use of human hepatocytes for research was approved by the local ethics committees of Berlin and Regensburg, and written informed consent was obtained from all patients. Hepatocytes from one female and two male donors were isolated and cultured essentially as described by Thomas et al (2013). Cells were treated for 24 hours with 10 mM rifampicin, 1 or 5 mM hyperforin, Hyp1, Hyp5, Hyp7, Hyp8, Hyp9, or Hyp10 and 50 mM Hyp2, Hyp3, Hyp4, Hyp6, or the vehicles 0.5% DMSO or 0.5% ethanol for 24 hours.…”

The virtual liver: state of the art and future perspectives

“…Vertical integration of computational models across different levels of biological organization has been used, for example, to simulate the effect of acetaminophen at the cellular scale (Krauss et al 2012;Diaz Ochoa et al 2013) or to describe the impact of steatosis on drug pharmacokinetics (Schwen et al 2014). In future, integrated computational models are generally expected to play an increasingly important role for integration and analysis of experimental data in drug development and toxicology (Magdy et al 2013;Godoy et al 2013;Zanger and Schwab 2013;Thomas et al 2013;Mielke et al 2011;Schug et al 2013;Heise et al 2012). …”