Aryl Hydrocarbon Receptor Activation and Cytochrome P450 1A Induction by the Mitogen-Activated Protein Kinase Inhibitor U0126 in Hepatocytes

Abstract

The aryl hydrocarbon receptor (AhR) is involved in various processes such as cytochrome P450 (P450) 1A induction after xenobiotic exposure. It is also considered to play a major role in cell proliferation and differentiation. Recent evidences have suggested a cross-talk between AhR functions and the mitogen-activated protein kinase (MAPK) cascade. We now report that 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a specific inhibitor of MAPK kinase (MEK) MEK1/2, elicits a marked increase in CYP1A1 expression at both mRNA and protein levels associated with a significant increase of enzyme activity in primary rat hepatocytes and a human hepatoma cell line. This induction occurred independently of MEK/extracellular signal-regulated kinase (ERK) activation and in the absence of ERK1 and ERK2 expression. The effect of U0126 was mediated by its ability to transactivate xenobiotic responsive element (XRE)-driven genes, as demonstrated by transfection assays with an XRE-driven luciferase construct in the human B16A2 hepatoma cell line. CYP1A1 modulation was abolished by a cotreatment with resveratrol, an established AhR antagonist, arguing for AhR activation by U0126. Such an effect was demonstrated by direct in vitro ligand binding competition assays using rabbit liver cytosol, showing that this compound binds AhR with an EC 50 ϭ 25 ϫ 10 Ϫ6 M. Moreover, we demonstrated that U0126 is a substrate for several P450s including human CYP1A2, -1A1, and -1B1. We conclude that the widely used specific inhibitor of MEK/ ERK, U0126, also acts as a potent AhR activator and an inducer of related genes. Such effects on the AhR may have an impact on biological functions attributed previously to MAPK inhibition.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAH) are potent inducers of several genes, including some encoding "Phase I" and "Phase II" xenobiotic-metabolizing enzymes. These enzymes include cytochrome P450 (P450), glutathione transferases, NADPH: quinone reductases, and UDP-glucuronosyl transferases.TCDD and PAH effects are mediated by activation of the aryl hydrocarbon receptor (AhR), a cytosolic protein that forms complexes with two 90-kDa heat shock proteins and some other proteins. After ligand binding, the AhR is translocated and localized in the nucleus followed by an heterodimerization with the AhR nuclear translocator (Arnt) protein and acts as a transcriptional factor. Several reports have also shown constitutive activation of the AhR in the absence of exogenous ligand under certain conditions (Singh et al

Activation of the Aryl Hydrocarbon Receptor by the Calcium/Calmodulin-Dependent Protein Kinase Kinase Inhibitor 7-Oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic Acid (STO-609)

“…Such data suggest that STO-609 may constitute a relatively potent AhR ligand. In contrast, much higher EC 50 s have been reported for indole-3-carbinol (60 ϫ 10 Ϫ6 M), resveratrol (6 ϫ 10 Ϫ6 M), U0126 (25 ϫ 10 Ϫ6 M), and 7-ketocholesterol (7 ϫ 10 Ϫ6 M), using the same in vitro approach (Casper et al, 1999;Savouret et al, 2001), leading to the classification of these compounds as weak AhR ligands (Andrieux et al, 2004). Moreover, it is noteworthy that the chemical structure of STO-609 reveals some similarities with that of TCDD, especially the presence of aromatic rings (Fig.…”

“…To determine whether STO-609 may directly activate AhR via an interaction with its ligand-binding domain, we performed ligand binding competition assays using rabbit liver cytosol as source of AhR (Andrieux et al, 2004). Our results show that STO-609 displaced [1,6-3 H]TCDDspecific binding with an EC 50 of 4.3 ϫ 10 Ϫ8 M (Fig.…”

“…The AhR binding competition assay was performed with rabbit liver cytosol as the AhR source as previously reported (Casper et al, 1999;Andrieux et al, 2004). In brief, 1 ml of 0.8 mg of cytosol prepared in 20 mM HEPES, pH 7.6, containing 1.5 mM EDTA, 10% glycerol (v/v), 20 mM KCl, and 5 mM CaCl 2 , was incubated with various concentrations of STO-609 or the PAH benzo(a)pyrene (used as positive control), dissolved in DMSO/ethanol [50/50 (v/v)], in the presence of 2 nM [1,6-3 H]TCDD for 4 h at 4°C.…”

“…Therefore, such data obtained with a CaMKK inhibitor suggest that CaMKK activation is likely not involved in the Ca 2ϩ / CaMKI␣/AhR signaling pathway. In addition, they highlight the fact that some kinase inhibitors are AhR ligands, as already described for the MAPK inhibitors U0126, PD98059, and SP600125 (Reiners et al, 1998;Joiakim et al, 2003;Andrieux et al, 2004;Dvorak et al, 2008), which may require caution for the use of these compounds as specific kinase inhibitors.…”

Modulation of CYP1A1 by PKC Inhibitors and TPA Pre-Treatments in MH1C1 Rat Hepatoma Cells Exposed to 3 -Methylcholanthrene

“…Several studies indicate that PKC plays an important role in cellular communication by relaying signaling events upstream of phospholipid hydrolysis to downstream kinases such as MAP kinase and AP-1 complex. Interactions between MAP kinase activity and regulation of the AhR-Arnt heterodimer complex -CYP1A1 induction [29][30][31][32][33][34] have been reported and AP-1 activity has been shown to be induced by AhR ligands [35,36].…”