Rifampicin Induction of Cyp3a4 Requires Pregnane X Receptor Cross Talk with Hepatocyte Nuclear Factor 4 and Coactivators, and Suppression of Small Heterodimer Partner Gene Expression

Bile acids and drugs activate pregnane X receptor (PXR) to induce CYP3A4, which is the predominant cytochrome P450 enzyme expressed in the liver and intestine and plays a critical role in detoxifying bile acids and drugs, and protecting against cholestasis. The aim of this study is to investigate the molecular mechanism of PXR cross talk with other nuclear receptors and coactivators in regulating human CYP3A4 gene transcription. Rifampicin dose dependently induced the CYP3A4 but inhibited small heterodimer partner (SHP) mRNA expression levels in primary human hepatocytes. Rifampicin strongly stimulated PXR and hepatocyte nuclear factor 4 (HNF4 ) interaction, and CYP3A4 reporter activity, which was further stimulated by peroxisome proliferators-activated receptor co-activator 1 (PGC-1 ) and steroid receptor coactivator-1 (SRC-1) but inhibited by SHP. Mutation of the putative HNF4 binding site in the distal xenobiotic responsive element module did not affect CYP3A4 basal promoter activity and synergistic stimulation by PXR and HNF4 . Chromatin immunoprecipitation assays revealed that rifampicin-activated PXR recruited HNF4 and SRC-1 to the CYP3A4 chromatin. On the other hand, SHP reduced PXR recruitment of HNF4 and SRC-1 to the CYP3A4 chromatin. The human SHP promoter was stimulated by HNF4 and PGC-1 . Upon activation by rifampicin, PXR inhibited SHP promoter activity. Results suggest that PXR strongly induces CYP3A4 gene transcription by interacting with HNF4 , SRC-1, and PGC-1 . PXR concomitantly inhibits SHP gene transcription and maximizes the PXR induction of the CYP3A4 gene in human livers. Drugs targeted to PXR may be developed for treating cholestatic liver diseases induced by bile acids and drugs. CYP3A4 is the most abundant cytochrome P450 enzyme expressed in the liver and small intestine and plays a crucial role in metabolism and detoxification of xenobiotics and endobiotics (Guengerich, 1999). CYP3A4 has very broad substrate specificity and metabolizes over 50% of clinical drugs. The expression levels of CYP3A4 vary widely among individuals (Lehmann et al., 1998). Compounds that activate human PXR (NR1I2) also induce CYP3A4 and drug metabolism activity (Bertilsson et al., 1998; Kliewer et al., 1998; Lehmann et al., 1998; Goodwin et al., 2002). An antibiotic, rifampicin, is a highly efficacious human PXR agonist (Kliewer et al., 2002) and has been used to treat pruritus of primary biliary cirrhosis patients for many years, although its molecular mechanism of drug action is not known (Bachs et al., 1989; Hofmann, 2002). The ligand-activated PXR forms a heterodimer with RXR and binds to the AGGTCA-like direct repeats spaced by 3, 4, or 5 bases (DR3, DR4, and DR5), and the everted repeats separated by 6 or 8 bases (ER6 and ER8) located in the PXR target genes (Kliewer et al., 1998). In the human CYP3A4 promoter, PXR binds to an ER6 located in the proximal promoter and a DR3 and ER6 in the distal xenobiotic responsive enhancer module (XREM) located approximately 8 kilobases upstream of the transcription start site (Bertilsson et al., 1998; Lehmann et al., 1998; Goodwin et al., 1999). The maximal induction of the CYP3A4 gene by PXR requires both the proximal ER6 and the distal XREM (Goodwin et al., 1999). Lithocholic acid (LCA) is the most efficacious bile acid that activates PXR and induces CYP3A4 to catalyze 6-hydroxylation of LCA to hyodeoxycholic acid (Staudinger et al., 2001; Xie et al., 2001). Feeding LCA causes liver damage in Pxr null mice, but transgenic mice expressing a human PXR are protected from bile acid toxicity and cholestasis (Staudinger et al., 2001). PXR inhibits cholesterol 7 -hydroxylase (CYP7A1), the regulatory gene in bile acid synthesis, but induces organic anion transport protein 2, a sinusoidal Na independent bile acid transporter (Staudinger et al., 2001). Nuclear receptors interact with their coactivators such as steroid receptor coactivator-1 (SRC-1) or corepressors and activate or inhibit their target genes. We reported previously that rifampicin-activated PXR interacts with hepatocyte nuclear factor 4 (HNF4 ) and blocks HNF4 recruitment of peroxisome proliferator-activated receptor This work was supported by National Institutes of Health Grants DK44442 and