Repression of CAR-mediated transactivation of CYP2B genes by the orphan nuclear receptor, short heterodimer partner (SHP)

The induction of CYP2B gene expression by phenobarbital (PB) is mediated by the translocation of the constitutive androstane receptor (CAR) from the cytoplasm to the nucleus. The CAR/RXR heterodimer binds to two DR-4 sites in a complex phenobarbital responsive unit (PBRU) in the CYP2B gene. The short heterodimer partner (SHP), an orphan nuclear receptor that lacks a conventional DNA binding domain, was initially identified by its interaction with CAR. We have examined the role of SHP in CAR-mediated transactivation of the CYP2B gene. Coexpression of SHP inhibited the transactivation of the CYP2B gene by CAR in cultured hepatoma cells and the p160 coactivator GRIP1 reversed the inhibition. The interaction of CAR with SHP was confirmed by GST pulldown experiments. SHP did not block the binding of either CAR/RXR to the PBRU or binding of GRIP1 to the CAR/RXR complex in gel mobility shift assays, but slightly increased CAR/RXR binding and slightly altered the mobility of the CAR/RXR/GRIP1 complex, suggesting an interaction of SHP with these complexes. The presence of SHP in the complexes, however, could not be detected in an antibody supershift assay. Recombinant corepressors mSin3A, SMRT, and HDAC1, but not NCoR1, interacted with GST-SHP but each of these corepressors in liver nuclear extracts bound to GST-SHP. SMRT and NCoR1 inhibited CAR-mediated activation independent of SHP, but mSin3A and HDAC1 had little effect alone, and were additive with SHP. These studies demonstrate that SHP does not inhibit CAR-mediated trans-activation by interfering with DNA binding or by competition with GRIP1. Instead, SHP may either inhibit recruitment of other coactivators by GRIP1 or actively recruit corepressors directly to the CAR/RXR/PBRU complex.     

Phenobarbital-Responsive Nuclear Translocation of the Receptor CAR in Induction of the CYP2B Gene

The constitutively active receptor (CAR) transactivates a distal enhancer called the phenobarbital (PB)-responsive enhancer module (PBREM) found in PB-inducible CYP2B genes. CAR dramatically increases its binding to PBREM in livers of PB-treated mice. We have investigated the cellular mechanism of PB-induced increase of CAR binding. Western blot analyses of mouse livers revealed an extensive nuclear accumulation of CAR following PB treatment. Nuclear contents of CAR perfectly correlate with an increase of CAR binding to PBREM. PB-elicited nuclear accumulation of CAR appears to be a general step regulating the induction of CYP2B genes, since treatments with other PB-type inducers result in the same nuclear accumulation of CAR. Both immunoprecipitation and immunohistochemistry studies show cytoplasmic localization of CAR in the livers of nontreated mice, indicating that CAR translocates into nuclei following PB treatment. Nuclear translocation of CAR also occurs in mouse primary hepatocytes but not in hepatocytes treated with the protein phosphatase inhibitor okadaic acid. Thus, the CAR-mediated transactivation of PBREM in vivo becomes PB responsive through an okadaic acid-sensitive nuclear translocation process.