No abstract

Both the human pregnane X receptor (hPXR) and constitutive androstane receptor (hCAR) are capable of regulating CYP3A4 and CYP2B6 gene expression. However, the majority of currently identified CYP3A4 and CYP2B6 inducers are confirmed activators of hPXR but not hCAR. To compare these receptors with respect to their chemical selectivities, 16 drugs known to induce CYP3A4 and/or CYP2B expression were evaluated for relative activation of hPXR versus hCAR. Because of the high basal but low chemical-induced activation of hCAR in immortalized cells, alternative methods were used to evaluate hCAR activation potential. Thirteen of the 16 compounds were classified as moderate to strong hPXR activators. In contrast, carbamazepine (CMZ), efavirenz (EFV), and nevirapine (NVP) were classified as negligible or weak hPXR activators at concentrations associated with efficacious CYP2B6 reporter or endogenous gene induction in primary human hepatocytes, suggesting potential activation of hCAR. Subsequent experiments demonstrated that these three drugs efficiently induced nuclear accumulation of in vivo-transfected enhanced yellow fluorescent protein-hCAR and significantly increased expression of a CYP2B6 reporter gene when hCAR was expressed in CAR Ϫ/Ϫ mice. In addition, using a recently identified, chemically responsive splice variant of hCAR (hCAR3), the hCAR activation profiles of the 16 compounds were evaluated. By combining results from the hPXR-and hCAR3-based reporter gene assays, these inducers were classified as hPXR, hCAR, or hPXR/hCAR dual activators. Our results demonstrate that CMZ, EFV, and NVP induce CYP2B6 and CYP3A4 preferentially through hCAR and that hCAR3 represents a sensitive tool for in vitro prediction of chemical-mediated human CAR activation.CYP3A4 and CYP2B6 are induced at the mRNA, protein, and activity levels by the same compounds, including rifampin, phenobarbital, clotrimazole, cyclophosphamide, calcium channel antagonists, HMG-CoA reductase inhibitors, and thiazolidinediones (Drocourt et al., 2001;Kocarek et al., 2002;Lindley et al., 2002;Sahi et al., 2003;Faucette et al., 2004). Coinduction of these enzymes is mediated through transcriptional activation of the corresponding genes by the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which are capable of binding to the same response elements in the promoter regions of the CYP3A4 and CYP2B6 genes (Goodwin et al

Hepatocellular carcinoma (HCC) is known to progress through a step often called tumor promotion. Phenobarbital (PB) is the prototype of nongenotoxic cacinogens that promote HCC in rodents. The molecular target of PB to elicit the promotion has been the subject of intense investigations over the last 30 years since it was discovered. The nuclear receptor constitutive active/androstane receptor (CAR) is activated by PB as well as by various other xenobiotics such as therapeutic drugs and environmental pollutants. CAR activation results in the transcriptional induction of numerous hepatic genes including those that encode xenobiotic-metabolizing enzymes such as a set of cytochrome P450s. In addition to PB, many CAR activators are nongenotoxic carcinogens, but the role of CAR in liver tumor promotion remains unexplored. Using Car ؊/؊ mice, we have here examined tumor promotion by chronic treatment with PB in drinking water after tumor initiation with a single dose of the genotoxic carcinogen diethylnitrosamine. None of the Car ؊/؊ mice developed either eosinophilic foci or advanced liver tumors, whereas all Car ؉/؉ mice developed HCC and/or adenoma by 39 weeks. The results indicate that CAR is the molecular target of promotion by PB and that activation of this receptor is an essential requirement for liver tumor development.

Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr52, which then promoted the dephosphorylation of CAR at Thr38 by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.