Mechanisms of activation of the aryl hydrocarbon receptor by novel inducers of the CYP1A1 gene

Abstract

The aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that is responsible for the activation of several response genes, of which the best characterised is the CYP1A1 gene. The prototype high affinity ligand for the AhR is the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The present study was undertaken in an attempt to elucidate the mechanism of activation of the CYP1A1 gene by several compounds that recently have been reported to be capable of increasing CYP1A1 levels in vivo or in different cellular systems, but are not conventional AhR ligands.

The transcriptional activation of the CYP1A1 gene by benzimidazoles like omeprazole (OME), thiabendazole, carbendazim and 2-mercapto-5-metoxy-benzimidazole (MMB), as well as the quinoline compound primaquine (PRQ) was shown to involve the AhR. However, rat, mouse and human hepatoma cell lines responded differently to CYP1A1 induction, suggesting that the determinants for AhR-activation differ between species or cell types. The magnitude and duration of induction was much less pronounced as compared to TCDD, indicating that these compounds are weak activators of the AhR. Both MMB and PRQ were capable of transforming cytosolic AhR to a DNA-binding form, as well as of displacing AhRbound [3 H]TCDD in vitro, indicating that they are low affinity ligands for the Ahr. In contrast, OME was neither capable of transforming AhR to a DNA-bound complex, nor to compete for specific [3 H]TCDD-binding, although the potency of OME and MMB to e.g. cause Gal4-AhR activation in rat H411E cells was similar.

The involvement of protein kinases in AhR signalling was investigated by using various protein kinase inhibitors. In transfected H411E or HepG2 cells, inhibition of OME- or TCDDdependent Gal4-AhR activation was obtained by using c-src kinase inhibitors as well as by co-transfection of a dominant negative c-src-plasmid, indicating that AhR function is regulated by c-src activity. Three tyrosine kinase inhibitors, genistein, tyrphostin AG17 and tyrphostin AG879, selectively blocked OME- but not TCDD-mediated AhR activation in H411E or HepG2 cells. These data strongly support the action of a tyrosine kinase dependent pathway in OME-mediated activation of the Ahr, which is different from that of TCDD.

When several different residues in the ligand-binding domain of the AhR was subjected to site-directed mutagenesis, a selective and complete loss of Gal4-AhR activation by OME, MMB and PRQ, but not by TCDD, was revealed by mutation of Tyr 320 to Phe. Competition of AhR-bound [3 H]TCDD by PRQ was completely abolished in extracts form HEK293 cells expressing the mutant Gal4-Y32OF protein, while TCDD-binding was unaffected. These findings indicate that different residues in the ligand-binding pocket of the AhR are important for recognition of low and high affinity ligands, alternatively, phosphorylation of Tyr 320 is critical for activation of the receptor by weak AhR activators, without having a great impact on the activation by high affinity ligands.

In summary, two alternative ways of activation of the AhR by novel CYP1A1 inducers have been demonstrated in this study; i) direct low affinity binding and activation of the receptor by MMB and PRQ, and ii) indirect activation of the receptor in a tyrosine kinase dependent pathway by OME, presumably by a ligand-independent mechanism.