ABSTRACT
Pregnane X receptor (PXR) has been shown to form a heterodimer with retinoid X receptor alpha (RXRalpha) and to bind to the distal nuclear receptor-binding element 1 and an everted repeat separated by six nucleotides in the proximal promoter of the CYP3A4 gene. In the present study, a new rifampicin-responsive region, located at -7.6 kilobases upstream from the transcription initiation site, has been identified using reporter assays in HepG2 cells. This region contains a cluster of possible nuclear receptor-binding half-sites, AG(G/T)TCA-like sequence. Of these putative half-sites, we focused six half-sites and termed them alpha-eta half-sites. Introduction of a mutation into either an alpha or beta half-site of CYP3A4 reporter genes almost completely diminished the rifampicin-induced transcription. In electrophoretic mobility shift assays, PXR/RXRalpha heterodimer bound to the direct repeat separated by four nucleotides (DR4) formed with alpha and beta half-sites. HepG2-based transactivation assays with the reporter gene constructs with or without mutations in the PXR binding element(s) demonstrated that this DR4 motif is essential for the transcriptional activation not only by rifampicin but also by various human PXR activators. In addition, reporter assays performed in human hepatocytes and mice with adenoviruses expressing luciferase derived from various CYP3A4 reporter genes and that expressing human PXR supported the results of experiments in HepG2 cells. These results suggest the obligatory role of the newly identified direct repeat separated by four nucleotides-type PXR binding element of the CYP3A4 gene for xenobiotic induction of CYP3A4.
Subject(s)
Cytochrome P-450 CYP3A/genetics , Response Elements/genetics , Transcriptional Activation/genetics , Xenobiotics/pharmacology , Animals , Base Sequence , Cell Line , Cells, Cultured , Cytochrome P-450 CYP3A/biosynthesis , Female , Humans , Male , Mice , Mice, Inbred ICR , Middle Aged , Molecular Sequence Data , Protein Binding/drug effects , Protein Binding/genetics , Response Elements/drug effects , Transcriptional Activation/drug effectsABSTRACT
An orally active antiallergic agent, M50367, skews the Th1/Th2 balance toward Th1 dominance by suppressing naive Th cell differentiation into Th2 cells in vitro. Administration results in the suppression of IgE synthesis and peritoneal eosinophilia in vivo. In this report, we determined that M50354 (an active metabolite of M50367) was a ligand for the aryl hydrocarbon receptor (AhR); the immunological effects of this compound on in vitro Th1/Th2 differentiation from naive Th cells and Th1/Th2 balance in vivo were manifested through binding to AhR. These effects were completely abolished in AhR-deficient mice. AhR expression in the naive Th cell was significantly up-regulated by costimulation of TCR and CD28. Suppression of naive Th cell differentiation into Th2 cells via binding of M50354 to AhR was associated with inhibition of GATA-3 expression in Th cells. In addition, forced expression of a constitutively active form of AhR or activation of AhR by the addition of representative ligands suppressed naive Th cell differentiation into Th2 cells. Based on these results, we conclude that AhR functions as a modulator of the in vivo Th1/Th2 balance through activation in naive Th cells.
Subject(s)
Receptors, Aryl Hydrocarbon/metabolism , Signal Transduction/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Animals , Anti-Allergic Agents/pharmacology , Benzimidazoles/metabolism , Benzimidazoles/pharmacology , Blotting, Western , Cell Differentiation/immunology , Cytokines/drug effects , Cytokines/immunology , Cytokines/metabolism , Electrophoretic Mobility Shift Assay , Mice , Mice, Mutant Strains , Rats , Receptors, Aryl Hydrocarbon/immunology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Th1 Cells/cytology , Th1 Cells/drug effects , Th2 Cells/cytology , Th2 Cells/drug effects , Th2 Cells/metabolism , TransfectionABSTRACT
In an assay system using a human CYP3A4 reporter constructed with the promoter (+11 nt to -362 nt) and enhancer (-7.2 knt to -7.8 knt) regions including everted repeat separated by six nucleotides (ER-6) and direct repeat separated by three nucleotides (DR-3) motifs, the CYP3A4 transactivation was detected without overexpression of any nuclear receptors in rifampicin-treated HepG2 cells. Overexpression of human pregnane X receptor (hPXR) enhanced the transactivation. Rat CYP3A1 reporter constructed with the promoter region (+31 nt to -171 nt) including both DR-3 and ER-6 motifs was, however, not transactivated in rifampicin-treated cells, even after overexpression of hPXR. Although overexpression of retinoid X receptor alpha (RXRalpha) had no clear effect for both CYP3A reporters, co-expression of apolipoprotein AI regulatory protein-1 (ARP-1) with hPXR resulted in the rifampicin-induced transactivation of the CYP3A1 reporter. A truncated CYP3A4 reporter retaining the both motifs showed the rifampicin-induced transactivation by overexpression of hPXR and ARP-1, while the transactivation in hPXR-overexpressed cells was not observed. These results support the idea that a nuclear receptor other than RXRalpha may play a role in the CYP3A transactivation together with hPXR. The present study also suggests the involvement of a novel cis-element in the hPXR-mediated CYP3A4 transactivation.
