Journal of Biological Chemistry
Volume 278, Issue 16, 18 April 2003, Pages 14146-14152
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ENZYME CATALYSIS AND REGULATION
A Novel Distal Enhancer Module Regulated by Pregnane X Receptor/Constitutive Androstane Receptor Is Essential for the Maximal Induction of CYP2B6 Gene Expression*

https://doi.org/10.1074/jbc.M212482200Get rights and content
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CYP2B6 plays an important role in the metabolism of a variety of structurally unrelated xenobiotics, including the anticancer drugs cyclophosphamide and ifosfamide. Previous studies have shown that the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are involved in the transcriptional regulation of CYP2B genes through the phenobarbital-responsive enhancer module (PBREM). However, for humanCYP2B6 the relatively weak response of the PBREM to PXR and CAR activation in transfection assays fails to describe the potent induction observed in primary human hepatocyte cultures. In this report, a novel nuclear receptor response module located −8.5 kilobases upstream from the CYP2B6 encoding region is described. Several potential PXR/CAR binding motifs were identified within the distal regulatory cluster. In electrophoretic mobility shift assays, one DR4 motif showed the strongest binding to both PXR and CAR. Transient transfection assays in HepG2 cells demonstrated that the novel distal response cluster could be activated by PXR and CAR. In primary human hepatocytes, both PBREM and the distal responsive element were activated individually by endogenous nuclear receptors upon exposure to prototypical inducers. However, in both HepG2 cells and primary human hepatocytes maximal reporter activation was observed in a construct containing both PBREM and the distal responsive element. In mouse tail-vein injection experiments, a construct containing both the distal responsive element and the proximal PBREM exhibited a strong synergistic expression in phenobarbital-treated mice. These results show that a novel xenobiotic-responsive enhancer module in the distal region of the CYP2B6 promoter (CYP2B6-XREM) together with the PBREM mediates optimal drug-induced expression ofCYP2B6.

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Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M212482200

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Wang, H., Faucette, S. R., Gilbert, D., Jolley, S. L., Sueyoshi, T., Negishi, M., and LeCluyse, E. L. (2003) Drug Metab. Dispos., in press.

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This work was supported in part by National Institutes of Health Grants P30 ES10126 and P30 DK34987.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.