Molecular Mechanism of Basal CYP3A4 Regulation by Hepatocyte Nuclear Factor 4α: Evidence for Direct Regulation in the Intestine

Abstract

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 4␣ (HNF4␣) was previously shown to be associated with basal hepatic CYP3A4 expression. As how HNF4␣ 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 HNF4␣: a far distal region at ؊9.0 kb and the proximal promoter region at ϳ؊0.2 kb. By gel shift experiments and transient transfections, we characterized direct repeat (DR) 1-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 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 ϳ؊189 base pairs. Physiological relevance of this position for activation by HNF4␣ 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 HNF4␣, which is probably specific for the intestine.

Intestinal cell-specific vitamin D receptor (VDR)-mediated transcriptional regulation of CYP3A4 gene

“…In addition, we should consider involvement of tissue-enriched transcription factors such as liver-enriched transcription factors (LEFTs -HNF4α, HNF1α, C/EBPα and β etc.) in CYP3A4 transactivation [3,4,13].…”

“…an unknown RE 5 LCA prER6 DR3 = CLEM4 no effect of eNR3A4 DR3 prER6 + CLEM4 2 no effect of eNR3A4 1 the same pattern for LS174T, HCT-8 and HT-29 intestinal cell lines, CLEM4 has a modulatory effect together with pER6 and DR3 sites; for CACO-2 cells-DR3 prER6 CLEM4, no effect of eNR3A4 2 cooperation, minimal individual effects of the response elements 3 In HepG2, relative fold induction is not affected by any response element mutation due to the parallel decrease in basal activation. 4 for Mz-Hep-1 cells…”

“…The CYP3A4 5'-flanking region is 35.8 kb [2], but only 13 kb have been systematically analyzed for regulation in hepatocyte or intestinal cells [3,4].…”

Role of Human Pregnane X Receptor in Tamoxifen- and 4-Hydroxytamoxifen-Mediated CYP3A4 Induction in Primary Human Hepatocytes and LS174T Cells

“…In a previous study, Tirona et al (2003) suggested a critical role of HNF4␣ in the PXR-and CAR-mediated transcriptional activation of CYP3A4 in Caco-2 cells. Furthermore, Tegude et al (2007) underscored the role of HNF4␣ in directly regulating CYP3A4 in LS174T cells. Thus, it is likely that in addition to GR␣, reduced expression of other transcription factors, such as HNF4␣ observed in LS174T cells, and/or interaction with CAR may also contribute to the apparent lack of CYP3A4 induction by tamoxifen/4OHT in these cells.…”

“…Furthermore, we observed that both compounds activated the human pregnane X receptor (PXR) in cellbased reporter assays (Desai et al, 2002). It is well established that in addition to PXR, several other tissue-specific factors and nuclear receptors impact the transcriptional regulation of CYP3A4 (Pascussi et al, 2003a,b;Tegude et al, 2007). As such, the expression of PXR and other receptors, such as glucocorticoid receptor (GR) ␣, and transcriptional factors, such as hepatic nuclear factor (HNF) 4␣, impact the regulation of PXR target genes (Pascussi et al, 2000;Li and Chiang, 2006).…”