The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene

The endogenous CYP2B6 gene becomes phenobarbital (PB) inducible in androstenol-treated HepG2 cells either transiently or stably transfected with a nuclear receptor CAR expression vector. The PB induction mediated by CAR is regulated by a conserved 51-base pair element called PB-responsive enhancer module (PBREM) that has now been located between -1733 and -1683 bp in the gene's 5'-flanking region. An in vitro translated CAR acting as a retinoid X receptor alpha heterodimer binds directly to the two nuclear receptor sites NR1 and NR2 within PBREM. In a stably transfected HepG2 cell line, both PBREM and NR1 are activated by PB and PB-type compounds such as chlorinated pesticides, polychlorinated biphenyls and chlorpromazine. In addition to PBREM, CAR also transactivates the steroid/rifampicin-response element of the human CYP3A4 gene in HepG2 cells. Thus, activation of the repressed nuclear receptor CAR appears to be a versatile mediator that regulates PB induction of the CYP2B and other genes.     

Human constitutive androstane receptor mediates induction of CYP2B6 gene expression by phenytoin

Compared with its rodent orthologs, little is known about the chemical specificity of human constitutive androstane receptor (hCAR) and its regulation of hepatic enzyme expression. Phenytoin (PHY), a widely used antiepileptic drug, is a potent inducer of CYP2B6 in primary human hepatocytes, but does not activate human pregnane X receptor (PXR) significantly in cell-based transfection assays at the same concentrations associated with potent induction of CYP2B6. Based on this observation, we hypothesized that PHY may be a selective activator of hCAR. In primary human hepatocytes, expression of CYP2B6 reporter genes containing phenobarbital-responsive enhancer module (PBREM) or PBREM/xenobiotic-responsive enhancer module (XREM) response elements were activated up to 14- and 28-fold, respectively, by 50 microm PHY. By contrast, parallel experiments in HepG2 cell lines co-transfected with an hPXR expression vector did not show increased reporter activity. These results indicated that a PXR-independent pathway, which is retained in primary hepatocytes, is responsible for PHY induction of CYP2B6. Further experiments revealed that PHY effectively translocates hCAR from the cytoplasm into the nucleus in both primary human hepatocytes and CAR(-/-) mice. Compared with vehicle controls, PHY administration significantly increased CYP2B6 reporter gene expression, when this reporter construct was delivered together with hCAR expression vector into CAR(-/-) mice. However, PHY did not increase reporter gene expression in CAR(-/-) mice in the absence of hCAR vector, implying that CAR is essential for mediating PHY induction of CYP2B6 gene expression. Taken together, these observations demonstrate that, in contrast to most of the known CYP2B6 inducers, PHY is a selective activator of CAR in humans.     

Regulation of cytochrome P450 2C9 expression in primary cultures of human hepatocytes

Cytochrome P450 2C9 (CYP2C9) expression is regulated by multiple nuclear receptors including the constitutive androstane receptor (CAR) and pregnane X receptor (PXR). We compared coregulation of CYP2C9 with CYP2B6 and CYP3A4, prototypical target genes for human CAR and PXR using human hepatocyte cultures treated for three days with the PXR activators clotrimazole, rifampin, and ritonavir; the CAR/PXR activator phenobarbital (PB); and the CAR‐selective agonists CITCO, (6‐(4‐chlorophenyl)imidazo[2,1‐β][1,3]thiazole‐5‐carbaldehyde‐O‐(3,4‐dichlorobenzyl)oxime) and phenytoin. Clotrimazole, rifampin, ritonavir, phenytoin, and phenobarbital induced CYP2C9 consistent with previous findings for CYP3A4. We observed EC₅₀ values of 519 μM (phenobarbital), 11 μM (phenytoin), and 0.75 μM (rifampin), similar to those for CYP3A4 induction. Avasimibe, a potent PXR activator, produced nearly identical concentration‐dependent CYP2C9 and CYP3A4 activity profiles and EC₅₀ values. In 17 donors, rifampin increased mean basal CYP2C9 activity from 59 ± 43 to 143 ± 68 pmol/mg protein/min; fold induction ranged from 1.4‐ to 6.4‐fold. Enzyme activity and mRNA measurements after rifampin, CITCO and PB treatment demonstrated potency and efficacy consistent with CYP2C9 regulation being analogous to CYP3A4 rather than CYP2B6. We demonstrate that hepatic CYP2C9 is differentially regulated by agonists of CAR and PXR, and despite sharing common regulatory mechanisms with CYP3A4 and CYP2B6; this enzyme exhibits an induction profile more closely aligned with that of CYP3A4. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:43–58, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20264     

Impaired nuclear translocation of CAR in hepatic preneoplastic lesions: association with an attenuated CYP2B induction by phenobarbital

Phenobarbital (PB) induction of CYP2B, a representative target gene of constitutive androstane receptor (CAR), has been observed to be attenuated in preneoplastic lesions of rat liver; however, molecular basis for this attenuation is poorly understood. In this report, we provide evidence indicating that the CAR expressed in the hepatic preneoplastic lesions of rats and mice was resistant to nuclear translocation and transactivation of the PB-responsive enhancer module upon PB treatment. These observations suggest that the attenuation of the induction of CYP2B by PB in hepatic preneoplastic lesions is evidently a consequence of impaired nuclear translocation of CAR.     

Glucocorticoid receptor-interacting protein 1 mediates ligand-independent nuclear translocation and activation of constitutive androstane receptor in vivo

Phenobarbital (PB) induction of CYP2B genes is mediated by translocation of the constitutively active androstane receptor (CAR) to the nucleus. Interaction of CAR with p160 coactivators and enhancement of CAR transactivation by the coactivators have been shown in cultured cells. In the present studies, the interaction of CAR with the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) was examined in vitro and in vivo. Binding of GRIP1 to CAR was shown by glutathione S-transferase (GST) pull-down and affinity DNA binding. N- or C-terminal fragments of GRIP1 that contained the central receptor-interacting domain bound to GST-CAR, but the presence of ligand increased the binding to GST-CAR of only the fragments containing the C-terminal region. In gel shift analysis, binding to CAR was observed only with GRIP1 fragments containing the C-terminal region, and the binding was increased by a CAR agonist and decreased by a CAR antagonist. Expression of GRIP1 enhanced CAR-mediated transactivation in cultured hepatic-derived cells 2-3-fold. In hepatocytes transfected in vivo, expression of exogenous GRIP1 alone induced transactivation of the CYP2B1 PB-dependent enhancer 15-fold, whereas CAR expression alone resulted in only a 3-fold enhancement in untreated mice. Remarkably, CAR and GRIP1 together synergistically transactivated the enhancer about 150-fold, which is approximately equal to activation by PB treatment. In PB-treated mice, expression of exogenous CAR alone had little effect, expression of GRIP1 increased transactivation about 2-fold, and with CAR and GRIP, a 4-fold activation was observed. In untreated mice, expression of GRIP resulted in nuclear translocation of green fluorescent protein-CAR. These results strongly suggest that a p160 coactivator functions in CAR-mediated transactivation in vivo in response to PB treatment and that the synergistic activation of CAR by GRIP in untreated animals results from both nuclear translocation and activation of CAR.     

Phosphorylation/Dephosphorylation steps are crucial for the induction of CYP2B1 and CYP2B2 gene expression by phenobarbital.

The effects of several protein kinase activators and protein phosphatase inhibitors on the phenobarbital (PB)-induced gene expression of CYP2B1 and CYP2B2 (CYP2B1/2B2) in adult rat hepatocytes were investigated. Insulin, epidermal growth factor, interleukin 6, cAMP, phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, vanadate, and okadaic acid were found to suppress the induction of CYP2B1/2B2 at mRNA and protein levels in hepatocytes. cAMP and vanadate completely suppressed the induction of CYP2B1/2B2 gene expression in both rat hepatocytes and liver. The addition of genistein to vanadate-treated hepatocytes partially recovered the induction of CYP2B1/2B1 gene expression by PB. These results of the present study demonstrate that phosphorylation/dephosphorylation steps are crucial for the induction of CYP2B1/2B2 gene expression by PB.     

Phenobarbital-Responsive Nuclear Translocation of the Receptor CAR in Induction of the CYP2B Gene

The constitutively active receptor (CAR) transactivates a distal enhancer called the phenobarbital (PB)-responsive enhancer module (PBREM) found in PB-inducible CYP2B genes. CAR dramatically increases its binding to PBREM in livers of PB-treated mice. We have investigated the cellular mechanism of PB-induced increase of CAR binding. Western blot analyses of mouse livers revealed an extensive nuclear accumulation of CAR following PB treatment. Nuclear contents of CAR perfectly correlate with an increase of CAR binding to PBREM. PB-elicited nuclear accumulation of CAR appears to be a general step regulating the induction of CYP2B genes, since treatments with other PB-type inducers result in the same nuclear accumulation of CAR. Both immunoprecipitation and immunohistochemistry studies show cytoplasmic localization of CAR in the livers of nontreated mice, indicating that CAR translocates into nuclei following PB treatment. Nuclear translocation of CAR also occurs in mouse primary hepatocytes but not in hepatocytes treated with the protein phosphatase inhibitor okadaic acid. Thus, the CAR-mediated transactivation of PBREM in vivo becomes PB responsive through an okadaic acid-sensitive nuclear translocation process.     

Induction of cytochrome P4502B: Role of regulatory elements and nuclear receptors

Cytochrome P450 of the 2B subfamily is easily induced by many xenobiotics. In spite of intensive investigations, the molecular mechanisms of regulation of the CYP2B genes are not clear. The nuclear receptor CAR is shown to play a crucial role in the activation of CYP2B genes by xenobiotics, but many problems of CAR activation in different animal species and humans remain unsolved. This review focuses on signaling pathways involved in the control of CYP2B gene expression in mammals.     

Repression of CAR-mediated transactivation of CYP2B genes by the orphan nuclear receptor, short heterodimer partner (SHP)

The induction of CYP2B gene expression by phenobarbital (PB) is mediated by the translocation of the constitutive androstane receptor (CAR) from the cytoplasm to the nucleus. The CAR/RXR heterodimer binds to two DR-4 sites in a complex phenobarbital responsive unit (PBRU) in the CYP2B gene. The short heterodimer partner (SHP), an orphan nuclear receptor that lacks a conventional DNA binding domain, was initially identified by its interaction with CAR. We have examined the role of SHP in CAR-mediated transactivation of the CYP2B gene. Coexpression of SHP inhibited the transactivation of the CYP2B gene by CAR in cultured hepatoma cells and the p160 coactivator GRIP1 reversed the inhibition. The interaction of CAR with SHP was confirmed by GST pulldown experiments. SHP did not block the binding of either CAR/RXR to the PBRU or binding of GRIP1 to the CAR/RXR complex in gel mobility shift assays, but slightly increased CAR/RXR binding and slightly altered the mobility of the CAR/RXR/GRIP1 complex, suggesting an interaction of SHP with these complexes. The presence of SHP in the complexes, however, could not be detected in an antibody supershift assay. Recombinant corepressors mSin3A, SMRT, and HDAC1, but not NCoR1, interacted with GST-SHP but each of these corepressors in liver nuclear extracts bound to GST-SHP. SMRT and NCoR1 inhibited CAR-mediated activation independent of SHP, but mSin3A and HDAC1 had little effect alone, and were additive with SHP. These studies demonstrate that SHP does not inhibit CAR-mediated trans-activation by interfering with DNA binding or by competition with GRIP1. Instead, SHP may either inhibit recruitment of other coactivators by GRIP1 or actively recruit corepressors directly to the CAR/RXR/PBRU complex.     

Insulin‐mediated modulation of cytochrome P450 gene induction profiles in primary rat hepatocyte cultures

In this investigation, we examined the effects of insulin on gene induction responsiveness in primary rat hepatocytes. Cells were cultured for 72 hours either in the absence or presence of 1 μM insulin and then exposed to increasing concentrations of phenobarbital (PB; 0.01–3.5 mM). Culturing in the absence of insulin produced 1.5–2‐fold increases in the induction magnitude of CYP2B1 and CYP2B2 mRNA expression resulting from PB exposures, without altering the bell‐shaped dose‐response curve characteristic of this agent. However, for the CYP3A1 gene, insulin removal led to a pronounced shift in both the PB‐induction magnitude and dose‐response relationships of the induction response, with higher levels of CYP3A1 expression resulting from exposures to lower concentrations of inducer. Insulin removal also reduced the time required to attain maximal induction of CYP2B1/2 and CYP3A1 gene expression. The insulin effects were not specific for PB induction, as insulin deprivation similarly enhanced both dexamethasone‐ and β‐naphthoflavone‐inducible CYP3A1 and CYP1A1 expression profiles, respectively. In contrast, the level of albumin mRNA expression was reduced considerably in cells deprived of insulin. We conclude that insulin is an important regulator of inducible and liver‐specific gene expression in primary rat hepatocytes. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 1–9, 1999