Received for publication October 27, 2006.
Revised March 1, 2007.
Accepted for publication March 1, 2007.

Molecular mechanism of basal CYP3A4 regulation by hepatocyte nuclear factor 4 alpha: evidence for direct regulation in the intestine

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 HNF4 was previously shown to be associated with basal hepatic CYP3A4 expression. As it still remains elusive how HNF4 regulates basal expression of CYP3A4, we systematically screened 12.5 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 HNF4: a far distal region at -9.0 kb and the proximal promoter region around -0.2 kb. By gel shift experiments and transient transfections, we characterized DR1 type motifs in both regions as functional HNF4 response elements. Cooperation of the two regions was shown to be required for maximal activation by HNF4. The effect of HNF4 was antagonized by COUP-TFII, 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 around position -189 bp. Physiological relevance of this position for activation by HNF4 in vivo is suggested by the presence of a similar binding activity in small intestine as in LS174T cells. In summary, we here have elucidated a molecular mechanism of direct regulation of CYP3A4 by HNF4, which is probably specific for the intestine.

Key words: CYP3A, cytochrome P450, gene regulation, nuclear receptors

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