Functional inhibitory cross-talk between constitutive androstane receptor and hepatic nuclear factor-4 in hepatic lipid/glucose metabolism is mediated by competition for binding to the DR1 motif and to the common coactivators, GRIP-1 and PGC-1alpha

The role of the constitutive androstane receptor (CAR) in xenobiotic metabolism by inducing expression of cytochromes P450 is well known, but CAR has also been implicated in the down-regulation of key genes involved in bile acid synthesis, gluconeogenesis, and fatty acid beta-oxidation by largely unknown mechanisms. Because a key hepatic factor, hepatic nuclear factor-4 (HNF-4), is crucial for the expression of many of these genes, we examined whether CAR could suppress HNF-4 transactivation. Expression of CAR inhibited HNF-4 transactivation of CYP7A1, a key gene in bile acid synthesis, in HepG2 cells, and mutation of the DNA binding domain of CAR impaired this inhibition. Gel shift assays revealed that CAR competes with HNF-4 for binding to the DR1 motif in the CYP7A1 promoter. TCPOBOP, a CAR agonist that increases the interaction of CAR with coactivators, potentiated CAR inhibition of HNF-4 transactivation. Furthermore, inhibition by CAR was reversed by expression of increasing amounts of GRIP-1 or PGC-1alpha, indicating that CAR competes with HNF-4 for these coactivators. Treatment of mice with phenobarbital or TCPOBOP resulted in decreased hepatic mRNA levels of the reported genes down-regulated by CAR, including Cyp7a1 and Pepck. In vivo recruitment of endogenous CAR to the promoters of Cyp7a1 and Pepck was detected in mouse liver after phenobarbital treatment, whereas association of HNF-4 and coactivators, GRIP-1, p300, and PGC-1alpha, with these promoters was significantly decreased. Our data suggest that CAR inhibits HNF-4 activity by competing with HNF-4 for binding to the DR1 motif and to the common coactivators, GRIP-1 and PGC-1alpha, which may be a general mechanism by which CAR down-regulates key genes in hepatic lipid and glucose metabolism.     

Localization of estradiol-responsive region in the phenobarbital-responsive enhancer module of mouse Cyp2b-10 gene

The mouse Cyp2b-10 gene is inducible by treatment with estradiol as well as so-called phenobarbital (PB)-like inducers. To identify 5'-flanking elements responsible for induction by estradiol, we carried out reporter gene assays using a primary mouse hepatocyte culture system. Cyp2b-10 gene-driven luciferase activities were induced by estradiol as well as PB in this system. Deletion analysis demonstrated that the sequence contained within the region from -2331 bp to -2281 bp was responsible for the estradiol-induced luciferase activity. This region corresponds to the core element of PB-responsive enhancer module (PBREM). Several nucleotide mutations in the putative binding sites of the PBREM core element showed that the NR1 site was required for estradiol induction, and the same element was required for PB induction. These results indicate that estradiol induces Cyp2b-10 gene expression via PBREM.