CCAAT/Enhancer-binding Protein α (C/EBPα) and Hepatocyte Nuclear Factor 4α (HNF4α) Synergistically Cooperate with Constitutive Androstane Receptor to Transactivate the Human Cytochrome P450 2B6 (CYP2B6) Gene

Abstract

The transcription of tissue-specific and inducible genes is usually subject to the dynamic control of multiple activators. Dedifferentiated hepatic cell lines lose the expression of tissue-specific activators and many characteristic hepatic genes, such as drug-metabolizing cytochrome P450. Here we demonstrate that by combining adenoviral vectors for CCAAT/ enhancer-binding protein ␣ (C/EBP␣), hepatocyte nuclear factor 4␣ (HNF4␣), and constitutive androstane receptor, the CYP2B6 expression and inducibility by CITCO are restored in human hepatoma HepG2 cells at levels similar to those in cultured human hepatocytes. Moreover, several other phase I and II genes are simultaneously activated, which suggests that this is an effective approach to endow dedifferentiated human hepatoma cells with a particular metabolic competence and response to inducers. In order to gain insight into the molecular mechanism, we examined the cooperation of these three transcription factors on the CYP2B6 5-flanking region. We show new CYP2B6-responsive sequences for C/EBP␣ and HNF4␣ and a novel synergistic regulatory mechanism whereby C/EBP␣, HNF4␣, and constitutive androstane receptor bind and cooperate through proximal and distal response elements to confer a maximal level of expression. The results obtained from human liver also suggest that important differences in the expression and binding of C/EBP␣ and HNF4␣ could account for the large interindividual variability of the hepatic CYP2B6 enzyme, which metabolizes commonly used drugs.Cytochromes P450 (CYPs) 3 are involved in the oxidative metabolism of drugs, carcinogens, and environmental pollutants to more polar metabolites, thereby facilitating their excretion and preventing these potentially harmful compounds from accumulating. In addition, many CYPs participate in the metabolism and conversion of a diverse range of endogenous compounds, including steroid hormones, bile acids, fatty acids, and prostaglandins (1, 2).CYP2B6 accounts for 2-10% of the total CYP content in the human liver, where it plays an important role in the metabolism of an increasing number of clinically important drugs (3). These include anti-neoplastics like cyclophosphamide, ifosfamide, and tamoxifen (4, 5); the anti-malarial artemisinin (6); the antiretrovirals nevirapine (7) and efavirenz (8); anesthetics like propofol (9) and ketamine (10); the anti-Parkinsonian selegiline (11); the anti-epileptic mephobarbital (12); and the anti-depressant bupropion, which is now the most commonly used probe drug for CYP2B6 (13). Moreover, CYP2B6 plays a key role in the metabolism of many environmental pollutants, which can serve as substrates, inhibitors, and/or inducers of CYP2B6 and can cause metabolic interactions between environmental chemicals and clinical drugs (14).A 20 -250-fold interindividual variation in CYP2B6 expression has been demonstrated, which is presumably due to polymorphisms and the induction of transcription by xenobiotics. These interindividual differences may result in variable systemic exposure to...

p38 MAP Kinase Links CAR Activation and Inactivation in the Nucleus via Phosphorylation at Threonine 38

“…dmd.aspetjournals.org looping, possibly enabling the promoter to recruit RNA polymerase II for effective promoter activation Negishi, 2008, 2009). In addition to early growth response 1, various other transcription factors were shown to interact with and activate the CYP2B promoters together with CAR, such as peroxisome proliferator-activated receptor -g coactivator-1 alpha (PGC-1a), steroid receptor coactivator-1 (SRC-1), HNF-4a, and CCAAT/enhancer-binding protein alpha (Shiraki et al, 2003;Li et al, 2008;Benet et al, 2010). Since p38 MAPK can activate these factors (Lim et al, 1998;Knutti et al, 2001;Guo et al, 2007;Qiao et al, 2006;Fernandez-Marcos and Auwerx, 2011;Antoon et al, 2012), it may enable CAR to bind to PBREM and to loop the promoter by phosphorylating one or more of them.…”

From differential induction of UDP-glucuronosyltransferases in rat liver to characterization of responsible ligand-activated transcription factors, and their multilevel crosstalk in humans

“…Interestingly, when hepatic cell lines were supplemented with C/EBP-, HNF4α-and CAR-expressing vectors, expression of CYP2B6 and UGT1A1 was synergistically enhanced [26]. These findings underline the importance of cooperation between liver-enriched transcription factors and LATFs.…”

Deciphering the roles of the constitutive androstane receptor in energy metabolism

“…The outcome of crosstalk is either stimulatory or inhibitory. The transcriptional factors that potentiate the activity of CAR include HNF4α, CCAATenhancer-binding protein α (C/EBPα), activating transcription factor 5 (ATF5), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and hypoxia-inducible factor 1 (Hif1) [64][65][66][67] . Much of the crosstalk occurs between specific genes.…”

“…Nevertheless, with the help of additional factors, crosstalk between CAR and other transcriptional factors will be facilitated and will exhibit synergistic effects [67,[69][70][71] . However, several CAR inhibitory factors, such as sterol regulatory elementbinding protein 1 (SREBP1), liver X receptor (LXR), farnesoid X receptor (FXR), estrogen receptor (ER), and histone deacetylase 1 (HDAC1), also exist [72][73][74][75][76] .…”