A Transcriptional Regulatory Network Containing Nuclear Receptors and Long Noncoding RNAs Controls Basal and Drug-Induced Expression of Cytochrome P450s in HepaRG Cells

Abstract

Cytochrome P450 (P450) enzymes are responsible for biotransformation of xenobiotics, including environmental toxicants and drugs. Expression of P450s can directly affect drug metabolism, resulting in various outcome in either therapeutic efficacy or adverse effects. Nuclear receptors are a class of transcription factors that can regulate expression of P450s at both basal and drug‐induced levels. Some long non‐coding RNAs (lncRNAs) near a transcription factor are found to participate in the regulatory functions of the transcription factors in either promotion or repression manner. The aim of this study is to determine whether there is a transcriptional regulatory network containing nuclear receptors and lncRNAs controlling both basal and drug‐induced expression of P450s in liver cells. Four nuclear receptors, hepatocyte nuclear factor 1a (HNF1a), hepatocyte nuclear factor 4a (HNF4a), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) as well as two lncRNAs, HNF1a‐antisense 1 (HNF1a‐AS1) and HNF4a‐antisense 1 (HNF4a‐AS1) were examined in control of basal and drug‐induced expression of 8 different P450 enzymes. Small interfering RNAs (siRNAs) were applied to knock down the nuclear receptors and lncRNAs in HepaRG cells with or without treatment of phenobarbital or rifampicin. Expression of the nuclear receptors, lncRNAs, and P450s was measured by RT‐PCR and Western blots. Knocking down of HNF1a or HNF4a affected basal expression of numerous P450s as well as CAR and PXR, and also impaired phenobarbital or rifampicin induced expression of several P450s, which may be due to decreased CAR/PXR expression. Moreover, HNF4a knockdown was able to decrease HNF1a expression and showed more potent effect than HNF1a knockdown. Knocking down of CAR or PXR only affected basal and induced expression of their target P450s without affecting expression of HNF1a or HNF4a. These data suggested the existence of a regulatory network in controlling P450 expression in an order through HNF4a to HNF1a to CAP/PXR. Expression of HNF1a‐AS1 and HNF4a‐AS1 is largely dependent on expression of their nearby coding gene HNF1a and HNF4a. Moreover, HNF4a knockdown depleted expression of both lncRNAs, but HNF1a knockdown only depleted HNF1a‐AS1 expression. Knocking down of HNF1a‐AS1 showed a similar regulation trend on the P450s compared to HNF1a or HNF4a knockdown, indicating involvement of this lncRNA in the regulation of P450s. However, knocking down of HNF4a‐AS1 showed an opposite regulation trend compared to HNF1a or HNF4a knockdown, indicating that HNF4a‐AS1 may play an inhibitory role in the HNF4a function. Altogether, out study concludes that there is a transcription regulatory network among the different nuclear receptors and lncRNAs in the regulation of both basal and drug‐induced expression of P450s in liver cells. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of Hepatic Long Noncoding RNAs by Pregnane X Receptor and Constitutive Androstane Receptor Agonists in Mouse Liver

“…As a first step to predict the function of lncRNAs with regard to influencing the transcriptional output of PCGs, the genomic locations of PCN-and TCPOBOP-regulated lncRNAs relative to the PCGs were determined using PAVIS, and the lncRNA-PCG gene pairs were defined if they met all the following criteria: 1) the lncRNA gene overlaps with or is within 5 kb upstream of TSS or 1 kb downstream of TTS of any PCG, and 2) both the lncRNA and the proximal PCG were differentially expressed by PCN or TCPOBOP exposure (average FPKM .1 and P , 0.05). These criteria were set based on the assumption that although exceptions may exist, lncRNAs produced locally around the neighboring PCGs have a more spatial advantage in influencing the transcriptional output of these PCGs compared with lncRNAs produced in distal regions (Ørom et al, 2010;Kim et al, 2012;Villegas and Zaphiropoulos, 2015;Engreitz et al, 2016;Chen et al, 2018).…”

“…Further mechanistic investigations are needed using lncRNA knockdown approach to validate the dependency of lncRNAs in the transcriptional/translational output of the paired PCGs. In humans, a recent study demonstrated that knocking down the neighboring lncRNAs produced from the antisense strand of the transcription factors HNF1a and HNF4a affected the expression of P450s in HepaRG cells (Chen et al, 2018). These observations have demonstrated the critical role of lncRNAs may play in modulating PCG expression.…”

“…In addition, small interfering RNA (siRNA) knockdown of the lncRNA gene HNF4a-AS1 increased the mRNA expression of PXR as well as the expression of six P450s (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2E1, and CYP3A4). This indicated that there is a complex network between transcription factors and lncRNAs that regulate the expression of P450s (Chen et al, 2018).…”

“…In addition to modulating intermediary metabolism in liver, lncRNAs are suggested to modulate hepatic xenobiotic biotransformation. For example, two lncRNAs that are transcribed from the antisense strands of the DNA encoding the hepatocyte nuclear factor 1a (HNF1a) and HNF4a are important in regulating the major drug-metabolizing cytochrome P450s (P450s) in human liver cancer-derived hepatic stem cell line (HepaRG) cells (Chen et al, 2018). Specifically, short hairpin RNA (shRNA) knockdown of the lncRNA gene HNF1a antisense 1 (AS-1) decreased the mRNA expression of PXR and CAR, as well as the expression of seven major P450s (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, and CYP3A4) in hepaRG cells.…”

Effect of panaxytriol on cytochrome P450 3A4 via the pregnane X receptor regulatory pathway

“…Similar to the other nuclear receptors, PXR also includes DNA‐binding domain and ligand‐binding domain (Buchman, Chai, & Chen, ). PXR can be activated by combining with a large number of ligands for various structural and chemical properties (Banerjee, Robbins, & Chen, ), including steroids (Chianese, Sepe, & Limongelli, ), rifampicin (Kodama, Yamazaki, & Negishi, ), bile acid (Wang, Wang, & Lu, ), dexamethasone (Ponce‐Ruiz, Rojas‐García, & Barrón‐Vivanco, ), phenobarbital (Chen, Bao, & Piekos, ), HIV protease inhibitor ritonavir (Liu, Xu, & Hui, ), hyperforin (Taneja, Chu, & Maturu, ), and so forth. HIV protease inhibitor ritonavir regulated the expression of CYP3A via inducing the PXR target gene (Liu et al, ).…”