FACTORS INVOLVED IN THE INDUCTION OF THE CYP2B SUBFAMILY P450: SEARCH FOR THE TARGET OF INDUCERS USING PHOTOAFFINITY PROBE AND THE ROLE OF STEROLS IN THE INDUCTION

To clarify the mechanism of phenobarbital-mediated increase in the CYP2B subfamily, we tried to detect target(s) of the inducer using azido-PB (AZPB), a photoaffinity ligand, as the probe. Immunoblot experiments with anti-AZPB antibody showed that binding of AZPB to proteins in rat liver microsomes the molecular weight of which is around 50 kDa is specifically competed by PB. Liver cytosol did not have any such proteins. When purified P450s were used as the target for AZPB, the binding was competed by PB most markedly in the CYP2B subfamily. The binding of AZPB to purified CYP2B2 was effectively lowered by 2, 2', 4, 4', 5, 5'-hexachlorobiphenyl, one of the potent inducers of the CYP2B subfamily, as well as PB. The other inducers for other subfamilies such as dexamethasone and β-naphthotlavone were without the effect. These results support the idea that the target of inducers mediating CYP2B induction is the CYP2B itself. To search the endogenous mediators which play a role in the CYP2B induction, we examined the role of steroid hormones and certain sterols in cultured rat hepatocytes. As the results, testosterone and the metabolites did not have ability to induce CYP2B proteins and to suppress PB-mediated induction. On the other hand, estradiol caused marked induction of the CYP2B 1/2. The role of estradiol was partially supported by experiments using aromatase-deficient mice: constitutive expression of the Cyp2b 10 in the knockout mice is less than the wild-type mice. The role of cholesterol and androstenol in the regulation of the rat CYP2B will also be presented.