Signal Transduction-mediated Activation of the Aryl Hydrocarbon Receptor in Rat Hepatoma H4IIE Cells

Abstract

We have investigated mechanisms of omeprazole (OME)-mediated induction of CYP1A1 and CYP3A, using the rat hepatoma H4IIE cell line, in comparison with mechanisms exerted by traditional aryl hydrocarbon receptor (AhR) ligands such as benso(a)pyrene (B(a)P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). OME did not bind specifically to AhR, and it could not activate the AhR complex in rat cytosol to a xenobioticresponsive element (XRE)-binding form in vitro. Genistein, a tyrosine kinase inhibitor, and daidzein, an inhibitor of casein kinase II, efficiently inhibited OMEmediated but not B(a)P-or TCDD-mediated induction of CYP1A1, as monitored at the transcriptional, mRNA, and protein levels as well as by analysis of activation of XRE-luciferase reporter constructs transfected into H4IIE cells. The protease inhibitor N ␣ -p-tosyl-L-lysine chloromethyl ketone (TLCK) and lavendustin A also had similar OME-specific effects. In addition, insulin pretreatment caused an almost complete inhibition of OMEdependent CYP1A1 induction but only partially affected TCDD and B(a)P-mediated induction of CYP1A1. Staurosporine, an inhibitor of protein kinase C, impaired the induction by both B(a)P and OME. OME caused an approximately 2-fold increase in the level of CYP3A expression, but all inhibitors used were ineffective in preventing this induction. Gel shift analysis with radiolabeled XRE and specific peptide antibodies toward AhR and aryl hydrocarbon receptor nuclear translocator protein (Arnt) revealed an OME-mediated translocation of the AhR⅐Arnt complex into the nuclei. Genistein inhibited the specific nuclear XRE binding caused by OME, but it potentiated the formation of the TCDD-induced XRE⅐AhR complex. Although daidzein was able to effectively inhibit the OME-stimulated CYP1A1 gene transcription, it did not influence the OMEdependent AhR⅐XRE complex formation. The data are consistent with a mechanism for OME-mediated induction of CYP1A1 that involves activation of the AhR complex via intracellular signal transduction systems and that is distinct from induction mediated by AhR ligands.

Influence of Omeprazole on Multidrug Resistance Protein 3 Expression in Human Liver

“…First, activation of the Ah receptor pathway mediates CYP1A1 induction by omeprazole (Daujat et al, 1992;Backlund et al, 1997;Dzeletovic et al, 1997). Second, dioxinresponsive elements to which the Ah receptor/Arnt heterodimer may bind have been identified in the 5Ј-flanking region of human MRP3 (Takada et al, 2000).…”

Different Structural Requirements of the Ligand Binding Domain of the Aryl Hydrocarbon Receptor for High- and Low-Affinity Ligand Binding and Receptor Activation

“…1A for structures) cause induction of the CYP1A1 gene in rat hepatoma H4IIE cells (Backlund et al, 1997(Backlund et al, , 1999Werlinder et al, 2001). In the case of OME, it has been shown that this benzimidazole is not capable of transformation of the AhR, in cytosol from H4IIE cells or mouse Hepa1 cells (Backlund et al, 1997;Dzeletovic et al, 1997).…”

Microbial Metabolites of Omeprazole Activate Murine Aryl Hydrocarbon Receptor In Vitro and In Vivo

“…When OME was treated before cytosol isolation, a reduction in 2,3,7,8-tetrachlorodibenzo-p-dioxin binding to AhR was observed in rabbit hepatocytes (Lesca et al, 1995). In the electrophoresis mobility shift assay, OME failed to shift the AhR/Arnt complex signal when it was added to the cytosol isolated from human tumor cells (Daujat et al, 1992;Backlund et al, 1997). In contrast, cytosol from cells pretreated with OME showed a retardation band specific to the AhR/Arnt complex (Quattrochi and Tukey, 1993).…”

“…Nevertheless, similar to the typical AhR ligands, OME induces AhR/Arnt translocation into the nucleus, which then binds to the XRE sequence required for CYP1A1 induction (Quattrochi and Tukey, 1993;Yoshinari et al, 2008). Several studies reported that OME-mediated AhR activation occurred via a protein tyrosine kinase pathway in both rat and human hepatoma cells (Backlund et al, 1997;Kikuchi et al, 1998;Lemaire et al, 2004;Backlund and Ingelman-Sundberg, 2005). Thus, the molecular mechanisms of OME-mediated AhR activation are not completely understood.…”