Novel CAR-mediated Mechanism for Synergistic Activation of Two Distinct Elements within the Human Cytochrome P450 2B6 Gene in HepG2 Cells

Swales, Karen E.; Kakizaki, Satoru; Yamamoto, Yukio; Inoue, Kaoru; Kobayashi, Kaoru; Negishi, Masahiko

doi:10.1074/jbc.m411318200

Cited by 51 publications

(72 citation statements)

References 46 publications

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“…PB, TCPOBOP, and ITS liquid media supplement were obtained from Sigma (St. Louis, MO); HGF was obtained from Chemicon (Temecula, CA); U0126 was obtained from Promega (Madison, WI); EGF and U0124 were obtained from Calbiochem (San Diego, CA); anti-rabbit IgG was obtained from Santa Cruz biotechnology (Santa Cruz, CA); anti-ERK1/2 and anti-phospho-ERK1/2 were obtained from Cell Signaling Technology (Danvers, MA); androstenol was obtained from Steraloids (Newport, RI). All plasmids used were previously constructed; pcDNA 3 mCAR, PBREM-tk-luciferase reporter, and Ϫ1.8-kb CYP2B6 promoter in pGL3, phRL-tk Zelko et al, 2001;Kobayashi et al, 2003;Swales et al, 2005).…”

Section: Methodsmentioning

confidence: 99%

Extracellular Signal-Regulated Kinase Is an Endogenous Signal Retaining the Nuclear Constitutive Active/Androstane Receptor (CAR) in the Cytoplasm of Mouse Primary Hepatocytes

Koike¹,

Moore²,

Negishi³

2007

Mol Pharmacol

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The nuclear receptor constitutive active/androstane receptor (CAR) is sequestered in the cytoplasm of liver cells before its activation by therapeutic drugs and xenobiotics such as phenobarbital (PB) and 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in mouse liver, the regulatory mechanism of which remains poorly understood. Given the finding that epidermal growth factor repressed PB activation of CAR-mediated transcription (Mol Pharmacol 65:172-180, 2004), here we investigated the regulatory role of hepatocyte growth factor (HGF)-mediated signal in sequestering CAR in the cytoplasm of mouse primary hepatocytes. HGF treatment effectively repressed the induction of endogenous CYP2b10 gene by PB and TCPOBOP in mouse primary hepatocytes. On the other hand, inhibition by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) of an HGF downstream kinase mitogenactivated protein kinase kinase (MEK) induced the Cyp2b10 gene and up-regulated the CAR-regulated promoter activity in the absence of TCPOBOP. HGF treatment increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in the cytosol, thus decreasing the TCPOBOP-induced nuclear accumulation of CAR. In contrast, U0126 dephosphorylated ERK1/2 and increased nuclear CAR accumulation in the absence of TCPOBOP. These results are consistent with the conclusion that the HGF-dependent phosphorylation of ERK1/2 is the endogenous signal that sequesters CAR in the cytoplasm of mouse primary hepatocytes.The nuclear constitutive active/androstane receptor CAR is a xeno-sensing transcription factor that regulates numerous hepatic genes in response to a large group of chemicals and therapeutic drugs. Phenobarbital (PB) represents this group of xenobiotics; it not only induces drug metabolism and secretion but also elicits pleiotropic effects on liver functions. These effects include metabolism and secretion of endobiotics (such as bilirubin), and changes in energy metabolism, cell growth, cell-cell communication, and tumor promotion (Honkakoski and Negishi, 1998a). Activation of CAR by drugs such as PB is paramount to elicit these effects by up-or down-regulating the genes that encode the key proteins and enzymes for these liver functions (Kodama and Negishi, 2006). As the function of CAR has expanded, deciphering the molecular mechanism of its activation by drugs is an urgent subject of current investigations.Mouse hepatocytes retain CAR in the cytoplasm, thus making the nuclear translocation an initial step of its acti-

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Section: Methodsmentioning

confidence: 99%

Extracellular Signal-Regulated Kinase Is an Endogenous Signal Retaining the Nuclear Constitutive Active/Androstane Receptor (CAR) in the Cytoplasm of Mouse Primary Hepatocytes

Koike¹,

Moore²,

Negishi³

2007

Mol Pharmacol

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“…Ym17 cells, a stable cell line that expresses mCAR tagged with V5-His, were established from HepG2 cells (Swales et al, 2005). HepG2 and Ym17 cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 g/ml streptomycin).…”

Section: Methodsmentioning

confidence: 99%

The Antiapoptotic Factor Growth Arrest and DNA-Damage-Inducible 45 β Regulates the Nuclear Receptor Constitutive Active/Androstane Receptor-Mediated Transcription

Yamamoto

Negishi

2008

Drug Metab Dispos

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ABSTRACT:The nuclear receptor constitutive active/androstane receptor (CAR) up-regulated expression of the apoptotic growth arrest and DNA-damage-inducible 45 ␤ (GADD45B) gene in HepG2 cells. Overexpression of GADD45B augmented CAR-mediated induction of the human CYP2B gene by the CAR activator 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and coactivated CARdependent transcription of the NR1-luciferase reporter gene. Small interfering RNA knockdown of GADD45B resulted in repression of both the induction and the coactivation. Induction of the mouse Cyp2b10 gene by TCPOBOP was profoundly attenuated in the primary hepatocytes prepared from GADD45B-knockout mice compared with those from wild-type mice. Because CAR is a key transcription factor that activates the genes that encode for xenobiotic metabolizing enzymes and transporters, GADD45B, acting as a CAR coactivator and coregulating CAR target genes, may be involved in hepatic drug metabolism and excretion of xenobiotics.

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“…More recently, the activation of PBREM by xenobiotics has been seen to be synergistically increased by co-treatment with the protein phosphatase inhibitor okadaic acid (OA) (24). Deletion assays delineate the OA-responsive activity to a proximal 50 bp (Ϫ217 to Ϫ268 bp) sequence in the CYP2B6 promoter (24,25).…”

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confidence: 99%

“…Deletion assays delineate the OA-responsive activity to a proximal 50 bp (Ϫ217 to Ϫ268 bp) sequence in the CYP2B6 promoter (24,25). In this OA-responsive module, EGR1 (early growth response 1) and HNF4␣ (hepatocyte nuclear factor 4␣) bind and determine the CAR-mediated induction of the human CYP2B6 gene.…”

mentioning

confidence: 99%

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

Benet

Lahoz

Guzmán

et al. 2010

Journal of Biological Chemistry

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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...

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Novel CAR-mediated Mechanism for Synergistic Activation of Two Distinct Elements within the Human Cytochrome P450 2B6 Gene in HepG2 Cells

Cited by 51 publications

References 46 publications

Extracellular Signal-Regulated Kinase Is an Endogenous Signal Retaining the Nuclear Constitutive Active/Androstane Receptor (CAR) in the Cytoplasm of Mouse Primary Hepatocytes

Extracellular Signal-Regulated Kinase Is an Endogenous Signal Retaining the Nuclear Constitutive Active/Androstane Receptor (CAR) in the Cytoplasm of Mouse Primary Hepatocytes

The Antiapoptotic Factor Growth Arrest and DNA-Damage-Inducible 45 β Regulates the Nuclear Receptor Constitutive Active/Androstane Receptor-Mediated Transcription

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

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