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Constitutive androstane receptor (CAR; NR1I3) controls the metabolism and elimination of endogenous and exogenous toxic compounds by up-regulating a battery of genes. In this work, we analyzed the expression of human CAR (hCAR) in normal liver during development and in hepatocellular carcinoma (HCC) and investigated the effect of hepatocyte nuclear factor 4alpha isoforms (HNF4alpha1 and HNF4alpha7) on the hCAR gene promoter. By performing functional analysis of hCAR 5'-deletions including mutants, chromatin immunoprecipitation in human hepatocytes, electromobility shift and cotransfection assays, we identified a functional and species-conserved HNF4alpha response element (DR1: ccAGGCCTtTGCCCTga) at nucleotide -144. Both HNF4alpha isoforms bind to this element with similar affinity. However, HNF4alpha1 strongly enhanced hCAR promoter activity whereas HNF4alpha7 was a poor activator and acted as a repressor of HNF4alpha1-mediated transactivation of the hCAR promoter. PGC1alpha stimulated both HNF4alpha1-mediated and HNF4alpha7-mediated hCAR transactivation to the same extent, whereas SRC1 exhibited a marked specificity for HNF4alpha1. Transduction of human hepatocytes by HNF4alpha7-expressing lentivirus confirmed this finding. In addition, we observed a positive correlation between CAR and HNF4alpha1 mRNA levels in human liver samples during development, and an inverse correlation between CAR and HNF4alpha7 mRNA levels in HCC. These observations suggest that HNF4alpha1 positively regulates hCAR expression in normal developing and adult livers, whereas HNF4alpha7 represses hCAR gene expression in HCC.

During the inflammatory response, intrahepatic cholestasis and decreased drug metabolism are frequently observed. At the hepatic level, the orphan nuclear constitutive androstane receptor (CAR) (NR1I3) controls phase I (cytochrome P450 [CYP] 2B and CYP3A), phase II (UGT1A1), and transporter (SLC21A6, MRP2) genes involved in drug metabolism and bilirubin clearance in response to xenobiotics such as phenobarbital or endobiotics such as phenobarbital or endobiotics such as bilirubin. We investigated the negative regulation of CAR, a glucocorticoid-responsive gene, via proinflammatory cytokine interleukin 1β (IL-1β) and lipopolysaccharides (LPSs) in human hepatocytes. We show that IL-1β decreases CAR expression and decreases phenobarbital- or bilirubin-mediated induction of CYP2B6, CYP2C9, CYP3A4, UGT1A1, GSTA1, GSTA2, and SLC21A6 messenger RNA. This occurs via nuclear factor kB (NF-kB) p65 activation, which interferes with the enhancer function of the distal glucocorticoid response element that we have identified recently in the CAR promoter. We demostrate that: (1) LPSs, IL-1β, or overexpression of p65Re1A inhibit glucocorticoid receptor (GR)-mediated CAR transactivation; (2) these suppressive effects can be blocked both by pyrrolidine dithiocarbamate, an inhibitor of NF-kB activation, or by overexpression of SRIkBα, a NF-kB repressor; and (3)the GR agonist dexamethasone induces histone H4 acetylation at the proximal CAR promoter region, whereas LPSs and IL-1β inhibit this acetylation as assessed via chromatin immunoprecipitation assay. In conclusion, GR/NF-kB interaction affects CAR gene transcription through chromatin remodeling and provide a mechanistic explanation for the long-standing observation that inflammation and sepsis inhibit drug metabolism while inducing intrahepatic cholestasis or hyperbilirubinemia. Supplementary material for this article can be found on the Hepatology website (http:/interscience.wiley.com/jpages/0270–9139/suppmat/index.html). (Hepatology 2004;40:951-960).