The Glycogen Synthase Kinase Inhibitor 3-( 2 , 4-Dichlorophenyl )-4-( 1-methyl-1 H-indol-3-yl )-1 H-pyrrole-2 , 5-dione ( SB 216763 ) Is a Partial Agonist of the Aryl Hydrocarbon Receptor

Kinase inhibitors are frequently used tools in signal transduction research. 3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1Hpyrrole-2,5-dione (SB216763), a potent inhibitor of glycogen synthase kinase 3 (GSK3 ), is frequently used to activate -catenin signaling by mimicking the action of Wnt molecules. -Catenin is a crucial player in the regulation of hepatic drug metabolism. Thus, it is of particular importance to know whether the tools used to study the effects of -catenin signaling may affect the respective drugmetabolizing target enzymes in an unwanted manner. In this study, we show that SB216763 is able to induce cytochrome P450 1a1 (Cyp1a1) expression in a dose-dependent manner in mouse hepatoma cells. Moreover, SB216763 is able to inhibit Cyp1a1 induction by the prototype aryl hydrocarbon receptor (AhR) ligand 2,3,7,8tetrachloro-p-dibenzodioxin. Cyp1a1 induction by SB216763 is independent of GSK3 and the -catenin pathway. Instead, SB216763 induces Cyp1a1 by activation of AhR-mediated transcription. The present results suggest that SB216763 acts as a partial agonist of the AhR. A variety of cellular signal transduction pathways are regulated by phosphorylation/dephosphorylation events, and a huge number of small molecule kinase inhibitors has been developed allowing for the blockade or activation of distinct signaling cascades. Besides the fact that many kinase inhibitors—often acting in an ATP-competitive manner—do not provide satisfactory specificity toward only one single kinase (Davies et al., 2000; Bain et al., 2007), there are also several reports which demonstrate that some of these molecules can act through additional mechanisms that are different from kinase inhibition. An example of this difference is the interaction with nuclear receptors such as the xenobiotic sensor aryl hydrocarbon receptor (AhR), which is a crucial player in the expression of various drug-metabolizing enzymes. In the absence of a ligand, the AhR is complexed by heat shock proteins in the cytosol. Upon binding of a ligand, mostly a planar hydrophobic molecule, the AhR translocates to the nucleus, releases the chaperones, and dimerizes with the Arnt protein. The AhR/Arnt heterodimer then binds to so-called dioxin response elements (DREs) on the DNA and activates transcription of target genes such as cytochrome P450 1a1 (Cyp1a1). For a recent review on AhR-dependent signal transduction, see Beischlag et al. (2008). The widely used kinase inhibitors U0126, PD98059, and SP600125 are ligands and partial agonists of the AhR (Reiners et al., 1998; Joiakim et al., 2003; Andrieux et al., 2004; Dvorak et al., 2008). Several other kinase inhibitors also affect the process of AhR activation by direct interaction with the AhR or by more complex indirect mechanisms, as reviewed in Braeuning (2009). Because most of the kinase inhibitors available from commercial suppliers have never been tested for a possible interplay with xenobiotic sensors, one might overlook such nonkinase-related properties and attribute the observed effects to the respective kinase-dependent pathway. We were therefore interested to determine whether the glycogen synthase kinase 3 (GSK3 ) inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3yl)-1H-pyrrole-2,5-dione (SB216763) (Fig. 1A) (Coghlan et al., 2000) would also interact with AhR-dependent signal transduction. GSK3 , a decisive player in the Wnt/ -catenin signaling pathway, is a core component of the multiprotein complex that phosphorylates -catenin, thus marking it for proteasomal degradation (for review, see Willert and Nusse, 1998; Lustig and Behrens, 2003). Upon binding of Wnt molecules to Frizzled receptors, GSK3 activity is inhibited. -Catenin is no longer degraded, accumulates in the cytosol, and translocates to the nucleus, where it binds to T cell factor (TCF) transcription factors and activates the transcription of target genes. Inhibitors of GSK3 , such as LiCl (Stambolic et al., 1996) and SB216763, are commonly used to mimic the action of Wnt molecules and to activate -catenin. The activity of the Wnt/ -catenin signaling pathway has been recently linked to the expression of different drug-metabolizing enzymes: various cytochrome P450 (P450) isoenzymes, among them the AhR-inducible Cyp1a isoforms, are overexpressed in tumors with activated -catenin and can be induced by treatment of primary hepatocytes with activators of -catenin signaling (Loeppen et al., 2005; Stahl et al., 2005; Hailfinger et al., 2006; Braeuning et al., This work was supported by the Deutsche Forschungsgemeinschaft [Grant