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

Abstract

The nuclear receptor constitutive active/androstane receptor (CAR) is sequestered in the cytoplasm of liver cells before its activation by therapeutic drugs and xenobiotics such as phenobarbital (PB) and 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in mouse liver, the regulatory mechanism of which remains poorly understood. Given the finding that epidermal growth factor repressed PB activation of CAR-mediated transcription (Mol Pharmacol 65:172-180, 2004), here we investigated the regulatory role of hepatocyte growth factor (HGF)-mediated signal in sequestering CAR in the cytoplasm of mouse primary hepatocytes. HGF treatment effectively repressed the induction of endogenous CYP2b10 gene by PB and TCPOBOP in mouse primary hepatocytes. On the other hand, inhibition by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) of an HGF downstream kinase mitogenactivated protein kinase kinase (MEK) induced the Cyp2b10 gene and up-regulated the CAR-regulated promoter activity in the absence of TCPOBOP. HGF treatment increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in the cytosol, thus decreasing the TCPOBOP-induced nuclear accumulation of CAR. In contrast, U0126 dephosphorylated ERK1/2 and increased nuclear CAR accumulation in the absence of TCPOBOP. These results are consistent with the conclusion that the HGF-dependent phosphorylation of ERK1/2 is the endogenous signal that sequesters CAR in the cytoplasm of mouse primary hepatocytes.The nuclear constitutive active/androstane receptor CAR is a xeno-sensing transcription factor that regulates numerous hepatic genes in response to a large group of chemicals and therapeutic drugs. Phenobarbital (PB) represents this group of xenobiotics; it not only induces drug metabolism and secretion but also elicits pleiotropic effects on liver functions. These effects include metabolism and secretion of endobiotics (such as bilirubin), and changes in energy metabolism, cell growth, cell-cell communication, and tumor promotion (Honkakoski and Negishi, 1998a). Activation of CAR by drugs such as PB is paramount to elicit these effects by up-or down-regulating the genes that encode the key proteins and enzymes for these liver functions (Kodama and Negishi, 2006). As the function of CAR has expanded, deciphering the molecular mechanism of its activation by drugs is an urgent subject of current investigations.Mouse hepatocytes retain CAR in the cytoplasm, thus making the nuclear translocation an initial step of its acti-

Transdifferentiated rat pancreatic progenitor cells (AR42J-B13/H) respond to phenobarbital in a rat hepatocyte-specific manner.

“…This could indicate that in human primary hepatocytes EGF is acting as a negative regulator of PB-mediated gene expression, which is similar to reports in primary hepatocytes in the rat (Kawamura et al 1999) and mouse (Koike et al 2007). …”

“…In primary rat hepatocytes, Egf has been reported to inhibit PB-induced Cyp2b1/2 mRNA, while Hgf did not (Kawamura et al 1999). However, in mouse primary hepatocytes both Egf and Hgf appear to be inhibitory to the PB response (Koike et al 2007). It would be interesting to interrogate this system further and establish the effect of growth factors on Cyp2b1/2 expression in this cell line.…”

Dephosphorylation of Threonine 38 Is Required for Nuclear Translocation and Activation of Human Xenobiotic Receptor CAR (NR1I3)

“…Thus, more work is needed to establish the molecular mechanism by which threonine 38 can be dephosphorylated after PB treatment. Recently, the growth factor-MEK-ERK1/2 pathway has been characterized as a cellular signal to sequester CAR in the cytoplasm; the inactivation of ERK1/2 by the MEK inhibitor U0126 spontaneously accumulates CAR in the nucleus of mouse primary hepatocytes (13). Our recent study has found that ERK1/2 specifically interacts with the phosphorylated form of CAR (data not shown), suggesting that ERK1/2 may be a signal that enables CAR to be phosphorylated.…”

“…Available evidence, however, has suggested that the underlying mechanism should be a cell signal-mediated regulation; protein phosphatase 2A, for instance, may be involved in the PB-induced nuclear translocation in the mouse hepatocytes (11,12). Moreover, growth factors, such as epidermal growth factor and hepatocyte growth factor, repress CAR nuclear translocation, and the U0126 inhibition of the MEK-ERK pathway spontaneously translocated CAR into the nucleus and activated the target gene Cyp2b10 without drug exposures (13). Neither the type of kinase that phosphorylates CAR nor the residue of CAR that can be phosphorylated by this kinase, however, has been identified to this time.…”

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.…”