Abstract
Cytochrome P450 3A4 plays an outstanding role in the metabolism of clinically used drugs and shows a marked interindividual variability in expression even in the absence of inducing agents. Thus, regulation of basal expression contributes considerably to variability. The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) was previously shown to be associated with basal hepatic CYP3A4 expression. As how HNF4α regulates basal expression of CYP3A4 still remains elusive, we systematically screened 12.5 kilobase pairs (kb) of the CYP3A4 5′ upstream region for activation by the receptor in the human intestinal cell line LS174T. In this study, we newly identified two widely separated regions mediating the activation by HNF4α: a far distal region at -9.0 kb and the proximal promoter region at ∼-0.2 kb. By gel shift experiments and transient transfections, we characterized direct repeat (DR) 1-type motifs in both regions as functional HNF4α response elements. Cooperation of the two regions was shown to be required for maximal activation by HNF4α. The effect of HNF4α was antagonized by chicken ovalbumin upstream promoter transcription factor II, which was shown to bind to one of the DR1 motifs. Furthermore, activation of CYP3A4 via the DR1 element in the proximal promoter depends on an additional, yet unknown, factor, which is binding at ∼-189 base pairs. Physiological relevance of this position for activation by HNF4α in vivo is suggested by the presence of a binding activity in small intestine similar to that in LS174T cells. In summary, we here have elucidated a molecular mechanism of direct regulation of CYP3A4 by HNF4α, which is probably specific for the intestine.
Footnotes
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This work was supported by grants from Deutsche Forschungsgemeinschaft (Bu 1249/1), the German Federal Ministry for Education and Research (Network Program Hepatosys), and the Robert Bosch Foundation (Germany).
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doi:10.1124/dmd.106.013565.
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ABBREVIATIONS: PXR, pregnane X receptor; CAR, constitutive androstane receptor; HNF, hepatocyte nuclear factor; DR, direct repeat; XREM, xenobiotic-responsive enhancer module; CLEM4, constitutive liver enhancer module of CYP3A4; PCR, polymerase chain reaction; kb, kilobase pair(s); ER, everted repeat; bp, base pair(s); COUP-TF, chicken ovalbumin upstream promoter transcription factor; RT, reverse transcriptase.
- Received October 26, 2006.
- Accepted March 1, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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