Transcriptional regulation of CYP2B1 induction in primary rat hepatocyte cultures: repression by epidermal growth factor is mediated via a distal enhancer region

Abstract

Phenobarbital (PB) alters expression of numerous hepatic genes, including genes involved in xenobiotic metabolism. Phenobarbital-dependent induction of cytochrome P-450 2B1 (CYP2B1) is subject to regulation by cytokines [e.g., by epidermal growth factor (EGF)], hormones [e.g., by growth hormone (GH)], or the cellular redox status. To investigate mechanisms involved in regulation of CYP2B1 transcription, we performed promoter activation studies using primary rat hepatocyte cultures transiently transfected with individual CYP2B1 promoter-luciferase reporter gene constructs. The 2679-bp native 5'-flanking region of the CYP2B1 gene conferred reporter gene activation by PB and the potent PB-like inducer permethrin (PM). Furthermore, this region mediated EGF- and GH-dependent repression of gene activation by PB-like inducers. A wide promoter mapping strategy with constructs bearing internal CYP2B1 promoter deletions led to identification of a distal responsive CYP2B1 enhancer region at -2230 to -2170, encompassing the section equivalent to the 51-bp PB-responsive enhancer module situated in the distal mouse Cyp2b10-5'-flanking region. The distal CYP2B1 enhancer region conferred gene activation by PM, repression of PM-dependent activation by EGF, and enhancement of activation by the antioxidant N-acetylcysteine (NAC). Mutational analyses of the region at -2230 to -2170 suggested that the mechanisms of PB-dependent induction of CYP2B1 and the modulating effects by EGF or NAC are closely related.