CONSTITUTIVE ACTIVE RECEPTOR-MEDIATED INDUCTION BY 1 , 4-BIS [ 2-( 3 , 5-DICHLOROPYRIDYLOXY ) ] BENZENE OF THE CYP 2 B 10 GENE IN MOUSE PRIMARY HEPATOCYTES

The nuclear orphan receptor constitutive active receptor (CAR) can be activated to induce CYP2B genes by the potent phenobarbital-type inducer 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in which the receptor forms a heterodimer with the retinoid X receptor (RXR) and binds to a conserved enhancer element NR1. Effects of retinoic acids on the activation of CAR were examined. Treatment with 9-cisor all-trans-retinoic acid markedly repressed TCPOBOP induction of CYP2B10 mRNA in mouse primary hepatocytes. Both retinoic acids also repressed TCPOBOP-induced NR1 enhancer activity in both transfected hepatocytes and HepG2 cells. Moreover, coexpression of the retinoic acid receptor (RAR) increased the repression in the cotransfected HepG2 cells, whereas that of RXR decreased the repression. Thus, the increased heterodimerization of RXR with RAR by retinoic acid treatment seemed to reduce the RXR available for CAR heterodimerization, resulting in the repression of CAR activity. This type of nuclear receptor signaling may play an important role as a modulator in the CYP2B regulation. Cytochromes P450 (P450s) play a major role in the detoxification and activation of therapeutic drugs and xenochemicals. Inducible gene transcription by exposure to xenochemicals is characteristic for P450s and increases the metabolic capabilities against drug and chemical toxicity (Nebert and Gonzalez, 1987; Waxman and Azaroff, 1992). Phenobarbital (PB) is the prototype for a large number of structurally diverse chemicals that induce P450s and other xenochemical-metabolizing enzymes (Waxman and Azaroff, 1992; Honkakoski et al., 1998a). Nuclear orphan receptors have recently emerged as important P450 gene regulators and their cross talk becomes an important focus for understanding the induction mechanisms. The nuclear orphan receptor CAR was originally characterized as a receptor that activates an empirical set of retinoic acid response elements without the presence of retinoic acids (Baes et al., 1994; Choi et al., 1997). The first gene identified as a direct target of CAR is the CYP2B genes of mouse, rat, and human, which are induced in response to PB treatment (Honkakoski et al., 1998a; Sueyoshi et al., 1999). Treatment with PB translocates CAR from the cytoplasm into the nucleus of mouse livers and primary hepatocytes (Kawamoto et al., 1999). Forming a heterodimer with RXR in the nucleus, CAR binds to the NR1 site within the conserved 51-bp PBREM (phenobarbital-responsive enhancer module) found in the mouse and human CYP2B genes (Honkakoski et al., 1998a,b; Kawamoto et al., 1999; Sueyoshi et al., 1999). CAR dimerizes with RXR in the absence of retinoic acids (Baes et al., 1994; Choi et al., 1997). A role for retinoic acids in the CAR/RXR heterodimer remains an intriguing question that may be critical for modulating P450 induction. Yamada et al. (2000) have recently reported that 9-cisand all-trans-retinoic acids markedly repressed CYP2B1/2 induction by PB in rat primary hepatocytes. In the present studies, the effects of retinoic acids on CARmediated induction of the CYP2B gene has been further examined using CAR-transfected HepG2 cells and/or mouse primary hepatocytes. The experimental considerations will be presented to suggest that retinoic acids decrease availability of RXR for CAR by heterodimerization, promoting interactions with other receptors, such as the retinoic acid receptor (RAR). Experimental Procedures Materials. The following chemicals were purchased from Sigma Chemical Co. (St. Louis, MO): 9-cis-retinoic acid and all-trans-retinoic acid. Stock solutions of retinoic acids were prepared in dimethyl sulfoxide (DMSO). 1,4-bis[2-(3,5-Dichloropyridyloxy)]benzene (TCPOBOP) was synthesized using the method of Kende et al. (1985). The expression plasmids RSV-RAR and pCMV-RXR were kindly provided by Ronald Evans (The Salk Institute for Biological Studies, La Jolla, CA). (NR1)5-tk (thymidine kinase)-luciferase plasmid was constructed by cloning quintuple NR1 sequences in front of the tk-luciferase promoter (at the BglII site), as described previously (Sueyoshi et Satoru Kakizaki is a JSPS (Japan Society for the Promotion of Science Research) fellow. Sohrab Karami is a recipient of a fellowship from Centrala Studie Nämnden International, Sweden. 1 Abbreviations used are: P450, cytochrome P450; CAR, constitutive active receptor; PB, phenobarbital; RXR, retinoid X receptor; PBREM, phenobarbitalresponsive enhancer module; RAR, retinoid acid receptor; DMSO, dimethyl sulfoxide; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; tk, thymidine kinase; RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mCAR, mouse CAR; RA, retinoic