Transcriptional Regulation ofCYP2B1Induction 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 promoterluciferase reporter gene constructs. The 2679-bp native 5Ј-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 Ϫ2230 to Ϫ2170, encompassing the section equivalent to the 51-bp PB-responsive enhancer module situated in the distal mouse Cyp2b10-5Ј-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 Ϫ2230 to Ϫ2170 suggested that the mechanisms of PB-dependent induction of CYP2B1 and the modulating effects by EGF or NAC are closely related.

Extracellular Signal-Regulated Kinase Is an Endogenous Signal Retaining the Nuclear Constitutive Active/Androstane Receptor (CAR) in the Cytoplasm of Mouse Primary Hepatocytes

“…This finding explains why EGF repressed the CAR-mediated activation of CYP2B genes in rat primary hepatocytes (Bauer et al, 2004). It also agrees with the observation that U0126 augmented the induction of the CYP2B gene by PB in rat primary hepatocytes (Joannard et al, 2006).…”

“…These facts indicate that cellular signals are involved in regulating the cytoplasmic retention and nuclear translocation of CAR. Two observations providing insight into such a signal have been reported: the attenuation by epidermal growth factor (EGF) of CAR-mediated trans-activation of PBREM by PB and the augmentation by the MEK inhibitor U0126 of PB induction of the CYP2B gene in rat primary hepatocytes (Bauer et al, 2004;Joannard et al, 2006). MEK is a downstream protein kinase of EGF signaling and activates extracellular signal-regulated kinase (ERK).…”

The Nrf2 Activator Oltipraz Also Activates the Constitutive Androstane Receptor

“…The involvement of the extracellular signal-regulated kinase (ERK) in the repression of CAR nuclear translocation is of particular interest. Previous work has shown that epidermal growth factor is able to attenuate CAR-mediated induction of CYP2B by PB treatment (Bauer et al, 2004). Inhibitors of mitogenactivated protein kinase kinase 1/2 (a kinase downstream of epidermal growth factor signaling) have been shown to potentiate the induction CYP2B by PB (Joannard et al, 2006).…”

Phenobarbital Meets Phosphorylation of Nuclear Receptors

“…How is this dephosphorylation regulated? First, investigators found that growth factors such as EGF, hepatocyte growth factor, and insulin repress CAR activation and nuclear translocation in rat and/or mouse primary hepatocytes (Bauer et al, 2004;Koike et al, 2007;Yasujima et al, 2016). Therefore, CAR is, in principle, a cell signal-regulated nuclear receptor (Fig.…”