Originally published In Press as doi:10.1074/jbc.M400580200 on April 28, 2004
J. Biol. Chem., Vol. 279, Issue 28, 29295-29301, July 9, 2004
Human Constitutive Androstane Receptor Mediates Induction of CYP2B6 Gene Expression by Phenytoin*
Hongbing Wang
,
Stephanie Faucette,
Rick Moore
,
Tatsuya Sueyoshi,
Masahiko Negishi¶, and
Edward LeCluyse||
From the
Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina at Chapel Hill, North Carolina 27599 and the Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
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 µM 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.
Received for publication, January 20, 2004
, and in revised form, April 19, 2004.
* This work was supported by National Institutes of Health Grant DK061652. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence may be addressed: Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709. Tel.: 919-541-2404; Fax: 919-541-0696; E-mail: negishi{at}niehs.nih.gov. || To whom correspondence may be addressed: Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Tel.: 919-966-9104; Fax: 919-966-0197; E-mail: ed_lecluyse{at}unc.edu.
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Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
