Novel CAR-mediated mechanism for synergistic activation of two distinct elements within the human cytochrome P4502B6 gene in HepG2 cells

The constitutive active receptor (CAR) regulates the induction of the cytochrome P450 2B6 (CYP2B6) gene by phenobarbital-type inducers, such as 1,4 bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) via the distal phenobarbital-responsive enhancer module (PBREM, at -1732/-1685 bp). Activation of the PBREM by TCPOBOP generated a 10-fold induction of CYP2B6 mRNA in HepG2 cells stably expressing mouse CAR (Ym17). Co-treatment with the protein phosphatase inhibitor okadaic acid (OA) synergistically increased this induction over 100-fold without directly activating CAR or the PBREM. Although OA synergy required the presence of PBREM, deletion assays delineated the OA-responsive activity to a proximal 24-bp (-256/-233) sequence (OARE) in the CYP2B6 promoter. CAR did not directly bind to the OARE in electrophoretic mobility shift assays. However, both DNA affinity and chromatin immunoprecipitation assays showed a significant increase in CAR association with the OARE after co-treatment with TCPOBOP and OA, indicating the indirect binding of CAR to the OARE. The two cis-acting elements, the distal PBREM and the proximal OARE, within the chromatin structure are both regulated by CAR in response to TCPOBOP and OA, respectively, to maximally induce the CYP2B6 promoter. This functional interaction between the two sites expands the current understanding of the mechanism of CAR-mediated inducible transcription.